Patent Application
20210024584
The invention relates to genetically modified arenaviruses suitable as vaccines against neoplastic diseases or cancer. The invention also relates to pharmaceutical compositions and methods for the treatment or prevention of certain infections causing neoplastic diseases or cancer, such as infections with oncogenic viruses. Specifically, provided herein are pharmaceutical compositions, vaccines, and methods of treating or preventing diseases and conditions caused by and associated with infections with Human Papillomavirus (HPV), such as cervical cancer, anogenital cancer, head and neck cancer and skin cancers. Also provided herein are immunotherapies for the treatment of a neoplastic disease, such as a neoplastic disease caused by infection with oncogenic viruses.
2.1 Medical Need
Neoplastic disease, such as cancer, can be caused by infectious agents, such as viruses, or so-called oncogenic viruses. Oncogenic viruses can be DNA viruses, such as Adenovirus, RNA viruses, such as Hepatitis C virus, or retroviruses, such as Human T-lymphotropic virus.
Human papillomavirus (HPV) is a DNA virus from the papillomavirus family, which has been found to be associated with several types of cancer. Although most HPV infections are subclinical and cause no physical symptoms, subclinical infections can become clinical and cause benign papillomas (such as warts or squamous cell papilloma), or cancers in certain populations. Over 170 HPV types have been identified and are referred to by number (Bzhalava et al., 2013, Virology 445 (1-2): 224-31). There is currently no cure for HPV infections.
About a dozen HPV types (including types 16, 18, 31, and 45) are called “high-risk” types because they can lead to cervical cancer, anal cancer, vulvar cancer, vaginal cancer, and penile cancer (Parkin et al., 2002, CA Cancer J Clin 2005;55:74-108). It is estimated that 99.7% of all cervical cancers are caused by high-risk oncogenic HPV types (Ault, 2006, Infectious Diseases in Obstetrics and Gynecology 2006: 1-5), including HPV type 16 and HPV type 18, which together account for about 70% of cervical cancers (See World Health Organization's website on HPV and cervical cancer, and the Center for Disease Control's “Pink Book” on HPV). Several types of HPV, in particular type 16, have also been found to be associated with HPV-positive oropharyngeal cancer (OSCC), a form of head and neck cancer (D'Souza et al., 2007, N. Engl. J. Med. 356 (19): 1944-56). Overall, HPV type 16 is the most problematic genotype associated with at least half of all cervical cancers and the great majority (approximately 90%) of the HPV-associated cancers at other anogenital sites and the oral cavity (Peng et al., 2014, Cell Biosci., 4(1):11).
It is estimated that in 2002 about 5.2% of all new cancers worldwide (561,200 new cancer cases) were attributable to HPV, making HPV one of the most important infectious causes of cancer (Parkin, 2006, Int. J. Cancer 118 (12): 3030-44). Cervical cancer is the second most lethal form of cancer in women worldwide, with nearly half a million women diagnosed each year (Parkin et al., 2005, CA Cancer J Clin; 55:74-108).
In developed countries, effective national programs for cytologic (Pap) screening for the precursor lesion, high-grade cervical intraepithelial neoplasia (CIN), have been established. Such cytologic screening is usually followed by ablation of preinvasive lesions by conization or loop electrosurgical excision procedure (LEEP), which has reduced the incidence of cervical cancer by approximately 70-80% in the US, such that there are now approximately 5000 cervical cancer deaths each year (Roden et al., 2006, Nat Rev Cancer; 6:753-763). In cases where cervical cancer has already established, the primary treatment is radical hysterectomy and surgical debulking, followed by chemoradiation therapy. Even after undergoing this conventional therapy, which has significant unwanted side effects, patients with advanced cervical carcinoma still have a poor prognosis. Therefore, novel therapeutics specifically targeting cancerous cells while leaving normal cells unaffected, are still urgently needed for the treatment of established cervical cancer.
In addition, therapeutic vaccines would also be valuable to ensure viral clearance in patients with persistent HPV infection, which presents a necessary, though not sufficient cause of uterine cervical carcinoma, both squamous cell carcinoma and adenocarcinoma (zur Hausen et al., 2002, Nature Rev Cancer 2002;2:342-350; Schiffman et al., 1993, J Natl Cancer Inst, 85:958-964; Walboomers et al., 1999, J Pathol, 189:12-19). Molecular testing for oncogenic HPV infection has recently been licensed as an adjunct to cytologic screening (Schiffman et al., 2007, Lancet, 370:890-907), and patients tested positive for HPV infection could significantly benefit from therapeutic vaccination.
2.2 HPV Vaccines
Two prophylactic multivalent HPV L1 virus-like particle (VLP) vaccines, i.e., Gardasil® and Cervarix®, preventing oncogenic HPV infection (Roden et al., 2006, Nat Rev Cancer, 6:753-763), HPV related cervical neoplasia, and genital warts, have been approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA). These vaccines are believed to prevent HPV related disease by induction of neutralizing antibody responses, but they do not, however, alter the course of pre-existing HPV infections (Hung et al., 2008, Expert Opin Biol Ther., 8(4): 421-439). Thus, there is still a compelling medical need for the development of effective immunotherapeutics that could be used for therapeutic elimination of chronic HPV infection as well as for treatment of established HPV-related cancers.
The HPV early proteins (E1-E7) are expressed throughout the viral life cycle, are only present in infected cells, and are involved in regulation of disease progression. Proteins E6 and E7 are known to act as oncogenes that promote tumor growth and malignant transformation. The expression of these viral oncoproteins has been reported to be necessary to maintain the transformed phenotype of cervical cancer cells (Goodwin et al., 2000, Proc Natl Acad Sci USA 97:12513-12518; Goodwin et al., 2001, Cell Growth Differ., 12:525-534).
2.3 Recombinant LCMV Expressing Genes of Interest
The generation of recombinant negative-stranded RNA viruses expressing foreign genes of interest has been pursued for a long time. Different strategies have been published for other viruses (Garcia-Sastre et al., 1994, J Virol 68(10): 6254-6261; Percy et al., 1994, J Virol 68(7): 4486-4492; Flick and Hobom, 1999, Virology 262(1): 93-103; Machado et al., 2003, Virology 313(1): 235-249). In the past it has been shown that it is possible to introduce additional foreign genes into the genome of bi-segmented LCMV particles (Emonet et al., 2009, PNAS, 106(9):3473-3478). Two foreign genes of interest were inserted into the bi-segmented genome of LCMV, resulting in tri-segmented LCMV particles (r3LCMV) with two S segments and one L segment. In the tri-segmented virus, published by Emonet et al., (2009), both NP and GP were kept in their respective natural position in the S segment and thus were expressed under their natural promoters in the flanking UTR.
2.4 Replication-deficient Arenavirus Vectors
The use of infectious, replication-deficient arenaviruses as vectors for the expression of antigens has been reported (see Flatz et. al., 2010, Nat. Med., 16(3):339-345; Flatz et al., 2012, J. Virol., 86(15), 7760-7770). These infectious, replication-deficient arenaviruses can infect a host cell, i.e., attach to a host cell and release their genetic material into the host cell. However, they are replication-deficient, i.e., the arenavirus is unable to produce further infectious progeny particles in a non-complementing cell, due to a deletion or functional inactivation of an open reading frame (ORF) encoding a viral protein, such as the GP protein. Instead, the ORF is substituted with a nucleotide sequence of an antigen of interest. In Flatz 2010, the authors used infectious, replication-deficient arenaviruses as vectors to express OVA (SIINFEKL epitope). In Flatz 2012, the authors used replication deficient arenaviruses as vectors to express HIV/SIV Env.
Provided herein are infectious arenavirus vectors, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, and a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment to treat or prevent a neoplastic disease, such as a neoplastic disease caused by infection with oncogenic viruses.
3. SUMMARY OF THE INVENTION
Provided herein is an arenavirus viral vector having a first nucleotide sequence encoding an antigen of an oncogenic virus, or an antigen of a tumor-associated virus. In certain embodiments, the oncogenic virus or tumor-associated virus is not cytomegalo virus, Hepatitis B virus, or Hepatitis C virus. In certain embodiments, the viral vector is an infectious, replication-deficient arenavirus viral vector, which can be a bi-segmented or a tri-segmented arenavirus viral vector. In certain embodiments, the viral vector is a tri-segmented arenavirus viral vector, which can be replication-competent or replication-deficient. Thus, in certain embodiments, the tri-segmented arenavirus viral vector is a replication-competent tri-segmented arenavirus viral vector. In certain embodiments, the tri-segmented arenavirus viral vector is a replication-deficient tri-segmented arenavirus viral vector.
Also provided herein are arenaviruses with rearrangements of the ORFs in their genomes. In particular, provided herein is an arenavirus genomic segment that has been engineered to carry an arenavirus ORF in a position other than the wild-type position and a first nucleotide sequence encoding an antigen of an oncogenic virus, or an antigen of a tumor-associated virus. In certain embodiments, the oncogenic virus or tumor-associated virus is not cytomegalo virus, Hepatitis B virus, or Hepatitis C virus.
Still further provided herein is an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, and a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment comprising a first nucleotide sequence encoding an oncogenic virus antigen, wherein the oncogenic virus is human papillomavirus (HPV), Kaposi's sarcoma-associated herpesvirus, Epstein-Barr virus, Merkel cell polyomavirus, or human T-lymphotropic virus. In particular, provided herein is an arenavirus viral vector or an arenavirus genomic segment comprising a nucleotide sequence encoding a HPV antigen as described herein, including Section 3.1.
In certain embodiments, an arenavirus viral vector as provided herein is infectious, i.e., is capable of entering into or injecting its genetic material into a host cell. In certain more specific embodiments, an arenavirus viral vector as provided herein is infectious, i.e., is capable of entering into or injecting its genetic material into a host cell followed by amplification and expression of its genetic information inside the host cell. In certain embodiments, the arenavirus viral vector provided herein is an infectious, replication-deficient arenavirus viral vector engineered to contain a genome with the ability to amplify and express its genetic information in infected cells but unable to produce further infectious progeny particles in normal, not genetically engineered cells. In certain embodiments, the infectious arenavirus viral vector is replication-competent and able to produce further infectious progeny particles in normal, not genetically engineered cells.
Also provided herein are immunotherapies for the treatment of a neoplastic disease, such as a neoplastic disease caused by infection with oncogenic viruses, such as those caused by human papillomavirus (HPV), Kaposi's sarcoma-associated herpesvirus, Epstein-Barr virus, Merkel cell polyomavirus, or human T-lymphotropic virus. In certain embodiments, the immunotherapies are for the treatment of neoplastic diseases cause by HPV and/or treatment of an HPV infection. Such immunotherapies include administration of an arenavirus viral vector described herein, a pharmaceutical composition comprising an arenavirus viral vector as described herein, an immunogenic composition comprising an arenavirus viral vector as described herein, or a vaccine comprising an arenavirus viral vector as described herein to a subject.
3.1 Oncogenic Virus Antigens and HPV Antigens
In certain embodiments, an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, and a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment provided herein comprises a first nucleotide sequence encoding a first HPV antigen.
In certain embodiments, the first nucleotide sequence further encodes a second HPV antigen.
In certain embodiments, the arenavirus viral vector or arenavirus genomic segment provided herein further comprises a second nucleotide sequence encoding a second HPV antigen.
In certain embodiments, the first and/or second nucleotide sequence encodes multiple HPV antigens. In a more specific embodiment, the arenavirus viral vector or arenavirus genomic segment comprising a first nucleotide sequence provided herein encodes two, three, four, five, six, seven, eight, nine, ten or more HPV antigens. In another specific embodiment, the arenavirus viral vector or arenavirus genomic segment comprising a second nucleotide sequence provided herein encodes two, three, four, five, six, seven, eight, nine, ten or more HPV antigens. In still another embodiment, the arenavirus viral vector or arenavirus genomic segment comprising a first and a second nucleotide sequence wherein the first nucleotide sequence encodes two, three, four, five, six, seven, eight, nine, ten or more HPV antigens and the second nucleotide sequence encodes two, three, four, five, six, seven, eight, nine, ten or more HPV antigens.
In certain embodiments, the first antigen is selected from the group consisting of HPV protein E1, HPV protein E2, HPV protein E3, HPV protein E4, HPV protein E5, HPV protein E6, HPV protein E7, HPV protein L1 and HPV protein L2.
In certain embodiments, the second antigen is selected from the group consisting of HPV protein E1, HPV protein E2, HPV protein E3, HPV protein E4, HPV protein E5, HPV protein E6, HPV protein E7, HPV protein L1 and HPV protein L2.
In certain embodiments, the first and/or second antigen is an antigen of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV73, or HPV82.
In certain embodiments, the first antigen is an HPV16 antigen, and the second antigen is an HPV18 antigen.
In certain embodiments, the arenavirus viral vector or arenavirus genomic segment provided herein further encodes two, three, four, five or more HPV antigens. In a specific embodiment, the arenavirus viral vector or arenavirus genomic segment encodes one, two, or three HPV16 antigens and one, two or three HPV18 antigens. In an even more specific embodiment, the arenavirus viral vector or arenavirus genomic segment encodes two HPV16 antigens and two HPV18 antigens. In certain embodiments, these HPV antigens are selected from the groups consisting of:
an HPV16 protein E6, or an antigenic fragment thereof;
an HPV16 protein E7, or an antigenic fragment thereof;
an HPV18 protein E6, or an antigenic fragment thereof; and
an HPV18 protein E7, or an antigenic fragment thereof.
In certain embodiments, the first antigen is selected from the group consisting of:
an HPV16 protein E6, or an antigenic fragment thereof;
an HPV16 protein E7, or an antigenic fragment thereof;
an HPV18 protein E6, or an antigenic fragment thereof; and
an HPV18 protein E7, or an antigenic fragment thereof.
In certain embodiments, the first and the second antigens are selected from the group consisting of:
an HPV16 protein E6, or an antigenic fragment thereof;
an HPV16 protein E7, or an antigenic fragment thereof;
an HPV18 protein E6, or an antigenic fragment thereof; and
an HPV18 protein E7, or an antigenic fragment thereof, wherein the first and the second antigen are not the same.
In certain specific embodiments, the arenavirus viral vector or arenavirus genomic segment provided herein further encodes HPV protein E7 fused to HPV protein E6 fused to HPV protein E6 fused to HPVprotein E7, wherein one HPV protein E7 is from strain HPV16 and the other is from strain HPV18 and one HPV protein E6 is from strain HPV 18 and the other is from strain HPV18.
In certain embodiments, the first or second antigen is HPV protein E7 with a mutation in the Rb binding site.
In certain embodiments, the first or second antigen is HPV protein E7 with mutations in the Rb binding site and the zinc finger motif.
In certain embodiments, the first or second antigen is HPV protein E6 with a mutation in the zinc binding domain.
In certain embodiments, the first or second antigen is HPV protein E6 with mutations in the zinc finger motif.
In certain embodiments, the first and the second antigen are fused directly to each other.
In certain embodiments, the first and the second antigen are fused to each other via a peptide linker.
In certain embodiments, the first and the second antigen are separated from each other via a self-cleaving peptide.
In certain embodiments, the self-cleaving peptide is Porcine teschovirus-1 2A peptide, Thosea asigna virus 2A peptide, or Foot-and-mouth disease virus 2A peptide.
In certain embodiments, the arenavirus viral vector or arenavirus genomic segment provided herein further comprises a third nucleotide sequence encoding an immunomodulatory peptide, polypeptide, or protein.
In certain embodiments, the immunomodulatory peptide, polypeptide, or protein is selected from the group consisting of:
Calreticulin (CRT), or a fragment thereof;
Ubiquitin or a fragment thereof;
Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), or a fragment thereof;
Invariant chain (CD74) or an antigenic fragment thereof;
Mycobacterium tuberculosis Heat shock protein 70 or an antigenic fragment thereof;
Herpes simplex virus 1 protein VP22 or an antigenic fragment thereof;
CD40 ligand or an antigenic fragment thereof;
Fms-related tyrosine kinase 3 (Flt3) ligand or an antigenic fragment thereof.
In certain embodiments, the immunomodulatory peptide, polypeptide, or protein is selected from the group consisting of:
Calreticulin (CRT), or a fragment thereof;
Ubiquitin or a fragment thereof; and
Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), or a fragment thereof.
In certain embodiments, the immunomodulatory peptide, polypeptide, or protein is directly fused to the first antigen, or is fused to the first antigen through a peptide linker.
In certain embodiments, the immunomodulatory peptide, polypeptide, or protein is directly fused to the second antigen, or is fused to the second antigen through a peptide linker.
In certain embodiments, the first antigen and the immunomodulatory peptide, polypeptide, or protein are separated from each other via a self-cleaving peptide.
In certain embodiments, the second antigen and the immunomodulatory peptide, polypeptide, or protein are separated from each other via a self-cleaving peptide.
In certain embodiments, the self-cleaving peptide is Porcine teschovirus-1 2A peptide, Thosea asigna virus 2A peptide, or Foot-and-mouth disease virus 2A peptide.
In certain embodiments, the arenavirus viral vector or arenavirus genomic segment provided herein further comprises a nucleotide sequence encoding a human tyrosinase secretion signal, a human growth hormone secretion signal, or a tissue plasminogen activator signal sequence.
In certain embodiments, the resulting fusion protein is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10.
In certain embodiments, the resulting fusion protein is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the fusion protein of amino acid sequence of SEQ ID NO: 34.
In certain embodiments, the resulting fusion protein is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the fusion protein of amino acid sequence of SEQ ID NO: 36.
In certain embodiments, the resulting fusion protein is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the fusion protein of amino acid sequence of SEQ ID NO: 38.
In certain embodiments, the arenavirus viral vector or arenavirus genomic segment provided herein comprises a nucleic acid sequence encoding an HPV16 E7/E6 polypeptide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:10.
In certain embodiments, the arenavirus viral vector or arenavirus genomic segment provided herein comprises a nucleic acid sequence encoding an HPV16 E7/HPV18 E6 polypeptide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to amino acids 17-263 of the amino acid sequence of SEQ ID NO:34.
In certain embodiments, the arenavirus viral vector or arenavirus genomic segment provided herein comprise a nucleic acid sequence encoding an HPV18 E7/HPV16 E6 polypeptide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to amino acids 17-270 of the amino acid sequence of SEQ ID NO:36.
In certain embodiments, the arenavirus viral vector or arenavirus genomic segment provided herein comprise a nucleic acid sequence encoding an HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 polypeptide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to amino acids 17-516 of the amino acid sequence of SEQ ID NO:38.
3.2 Replication-Deficient Arenavirus
In certain embodiments, the viral vector provided herein is an infectious, replication-deficient arenavirus viral vector.
In certain embodiments, provided herein is an infectious, replication-deficient arenavirus viral vector comprising a first nucleotide sequence encoding an antigen of an oncogenic virus, or an antigen of a tumor-associated virus, wherein the oncogenic virus or tumor-associated virus is not cytomegalo virus, Hepatitis B virus, or Hepatitis C virus.
In certain embodiments, provided herein is an infectious, replication-deficient arenavirus viral vector comprising a first nucleotide sequence encoding an antigen of an oncogenic virus, wherein the oncogenic virus is human papillomavirus, Kaposi's sarcoma-associated herpesvirus, Epstein-Barr virus, Merkel cell polyomavirus, or human T-lymphotropic virus.
In certain embodiments, provided herein is an infectious, replication-deficient arenavirus viral vector engineered to contain a genome with the ability to amplify and express its genetic information in infected cells but unable to produce further infectious progeny particles in normal, not genetically engineered cells, wherein one arenavirus open reading frame is at least partially removed and replaced by a first nucleotide sequence encoding a first HPV antigen.
In certain embodiments, provided herein is an infectious, replication-deficient arenavirus viral vector engineered to contain a genome with the ability to amplify and express its genetic information in infected cells but unable to produce further infectious progeny particles in normal, not genetically engineered cells, wherein one arenavirus open reading frame is at least partially removed or functionally inactivated and wherein the genome of the arenaviral vector encodes HPV16 E6 or antigenic fragment thereof, HPV16 E7 or antigenic fragment thereof, HPV18 E6 or antigenic fragment thereof, and HPV18 E7 or antigenic fragment thereof.
In certain embodiments, the arenaviral vector encodes an HPV16 E6/E7 fusion protein and an HPV 18 E6/E7 fusion protein. In certain embodiments, the arenaviral vector encodes an HPV16 E7/HPV18 E6 fusion protein. In certain embodiments, the arenaviral vector encodes an HPV18 E7/HPV16 E6 fusion protein. In certain embodiments, the arenaviral vector encodes an HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein.
In certain embodiments, the HPV16 E6 or antigenic fragment thereof, HPV16 E7 or antigenic fragment thereof, HPV18 E6 or antigenic fragment thereof, and HPV18 E7 or antigenic fragment thereof are encoded by one, two, three, or four heterologous nucleotide sequences.
In certain embodiments, the vector encodes a signal peptide fused to one or more of HPV16 E6 or antigenic fragment thereof, HPV16 E7 or antigenic fragment thereof, HPV18 E6 or antigenic fragment thereof, and HPV18 E7 or antigenic fragment thereof.
In certain embodiments, the vector encodes a peptide linker between two or more of HPV16 E6 or antigenic fragment thereof, HPV16 E7 or antigenic fragment thereof, HPV18 E6 or antigenic fragment thereof, and HPV18 E7 or antigenic fragment thereof.
In certain embodiments, the vector further comprises a nucleotide sequence encoding an immunomodulatory peptide, polypeptide, or protein. In particular, in certain embodiments, the arenavirus comprises a nucleotide sequence encoding a HPV antigen as described herein, including Section 3.1.
In certain embodiments, the vector encodes a peptide linker between an HPV antigen (HPV16 E6, HPV16 E7, HPV18 E6, HPV18 E7) or antigenic fragment thereof as described herein, including Section 3.1 and the immunomodulatory peptide, polypeptide, or protein.
In certain embodiments, the arenavirus is lymphocytic choriomeningitis virus (LCMV) or Junin virus.
In certain embodiments, the genomic information encoding the infectious, replication-deficient arenavirus viral vector is derived from the lymphocytic choriomeningitis virus Clone 13 strain or MP strain.
In certain embodiments, the viral vector is engineered to contain a genome with the ability to amplify and express its genetic information in infected cells but unable to produce further infectious progeny particles in normal, not genetically engineered cells, wherein one arenavirus open reading frame is functionally inactivated.
In certain embodiments, an infectious, replication-deficient arenavirus viral vector provided herein includes a viral vector wherein a viral open reading frame (“ORF”) that encodes the glycoprotein (“GP”), nucleoprotein (“NP”), matrix protein Z (“Z protein”) or RNA dependent RNA polymerase L (“L protein”) of the arenavirus is removed or functionally inactivated.
In certain embodiments, at least one of the four ORFs encoding GP, NP, Z protein, and L protein is removed or functionally inactivated.
In certain embodiments, at least one of the four ORFs encoding GP, NP, Z protein and L protein is removed and replaced with a heterologous ORF from an organism other than an arenavirus. In other embodiments, only one of the four ORFs encoding GP, NP, Z protein and L protein is removed and replaced with a heterologous ORF from an organism other than an arenavirus. In a more specific embodiment, the ORF encoding GP is removed and replaced with a heterologous ORF from an organism other than an arenavirus. In other embodiments, the ORF encoding NP is removed and replaced with a heterologous ORF from an organism other than an arenavirus. In some embodiments, the ORF encoding the Z protein is removed and replaced with a heterologous ORF from an organism other than an arenavirus. In other embodiments, the ORF encoding the L protein is removed and replaced with a heterologous ORF from an organism other than an arenavirus.
In certain embodiments, the heterologous ORF encodes a reporter protein. In some embodiments, the heterologous ORF encodes an antigen derived from an infectious organism, tumor, or allergen. In other embodiments, the heterologous ORF encoding an antigen is selected from human papillomavirus (HPV) antigens, human immunodeficiency virus antigens, hepatitis C virus antigens, hepatitis B surface antigen, varizella zoster virus antigens, cytomegalovirus antigens, mycobacterium tuberculosis antigens, and tumor associated antigens.
In certain embodiments, the growth or infectivity of the infectious, replication-deficient arenavirus viral vector is not affected by the heterologous ORF from an organism other than an arenavirus.
3.3 Arenavirus Genomic Segments
In certain embodiments, provided herein are arenaviruses with rearrangements of the ORFs in their genomes. In particular, provided herein is an arenavirus genomic segment that has been engineered to carry an arenavirus ORF in a position other than the wild-type position and a first nucleotide sequence encoding an antigen of an oncogenic virus, or an antigen of a tumor-associated virus as described herein. In certain embodiments, the oncogenic virus or tumor-associated virus is not cytomegalo virus, Hepatitis B virus, or Hepatitis C virus.
In certain embodiments, provided herein is an arenavirus genomic segment that has been engineered to carry an arenavirus ORF in a position other than the wild-type position and a first nucleotide sequence encoding an oncogenic virus antigen, wherein the oncogenic virus is human papillomavirus (HPV), Kaposi's sarcoma-associated herpesvirus, Epstein-Barr virus, Merkel cell polyomavirus, or human T-lymphotropic virus. In particular, in certain embodiments, the arenavirus genomic segment comprises a nucleotide sequence encoding a HPV antigen as described herein, including Section 3.1.
In certain embodiments, the arenavirus genomic segment is selected from the group consisting of:
In certain embodiments, the arenavirus 3′ UTR is the 3′ UTR of the arenavirus S segment or the arenavirus L segment. In certain embodiments, the arenavirus 5′ UTR is the 5′ UTR of the arenavirus S segment or the arenavirus L segment.
In certain embodiments, the arenavirus genomic segment is derived from lymphocytic choriomeningitis virus (“LCMV”) or Junin virus. In particular embodiments, the arenavirus genomic segment is derived from LCMV. The LCMV can be MP strain, Armstrong strain, or Armstrong Clone 13 strain. In particular embodiments, the arenavirus genomic segment is derived from Junin virus. The Junin virus can be Junin virus vaccine Candid #1, or Junin virus vaccine XJ Clone 3 strain.
Also provided herein, is an arenavirus viral vector comprising the arenavirus genomic segment and a second arenavirus genomic segment so that the arenavirus viral vector comprises an S segment and an L segment.
In certain embodiments, the arenavirus viral vector is infectious and replication-competent. In some embodiments, the arenavirus viral vector is attenuated. In other embodiments, the arenavirus viral vector is infectious but unable to produce further infectious progeny in non-complementing cells.
In certain embodiments, the arenavirus viral vector is derived from lymphocytic choriomeningitis virus (“LCMV”) or Junin virus. In particular embodiments, the arenavirus viral vector is derived from LCMV. The LCMV can be MP strain, Armstrong strain, or Armstrong Clone 13 strain. In particular embodiments, the arenavirus viral vector is derived from Junin virus. The Junin virus can be Junin virus vaccine Candid #1, or Junin virus vaccine XJ Clone 3 strain.
In certain embodiments, an arenavirus viral vector or an arenavirus genomic segment provided herein includes a viral vector wherein a viral open reading frame (“ORF”) that encodes the glycoprotein (“GP”), nucleoprotein (“NP”), matrix protein Z (“Z protein”) or RNA dependent RNA polymerase L (“L protein”) of the arenavirus is removed or functionally inactivated.
In certain embodiments, at least one of the four ORFs encoding GP, NP, Z protein, and L protein is removed or functionally inactivated.
In certain embodiments, at least one of the four ORFs encoding GP, NP, Z protein and L protein is removed and replaced with a heterologous ORF from an organism other than an arenavirus. In other embodiments, only one of the four ORFs encoding GP, NP, Z protein and L protein is removed and replaced with a heterologous ORF from an organism other than an arenavirus. In a more specific embodiment, the ORF encoding GP is removed and replaced with a heterologous ORF from an organism other than an arenavirus. In other embodiments, the ORF encoding NP is removed and replaced with a heterologous ORF from an organism other than an arenavirus. In some embodiments, the ORF encoding the Z protein is removed and replaced with a heterologous ORF from an organism other than an arenavirus. In other embodiments, the ORF encoding the L protein is removed and replaced with a heterologous ORF from an organism other than an arenavirus.
In certain embodiments, the heterologous ORF encodes a reporter protein. In some embodiments, the heterologous ORF encodes an antigen derived from an infectious organism, tumor, or allergen. In other embodiments, the heterologous ORF encoding an antigen is selected from human papillomavirus (HPV) antigens, human immunodeficiency virus antigens, hepatitis C virus antigens, hepatitis B surface antigen, varizella zoster virus antigens, cytomegalovirus antigens, mycobacterium tuberculosis antigens, and tumor associated antigens.
In certain embodiments, the growth or infectivity of the arenavirus viral vector is not affected by the heterologous ORF from an organism other than an arenavirus.
3.4 Tri-Segmented Arenavirus Viral Vectors
In certain embodiments, the viral vector provided herein is a tri-segmented arenavirus viral vector having a first nucleotide sequence encoding an antigen of an oncogenic virus, or an antigen of a tumor-associated virus. In certain embodiments, the oncogenic virus or tumor-associated virus is not cytomegalo virus, Hepatitis B virus, or Hepatitis C virus. In certain embodiments, the tri-segmented arenavirus viral vector is a replication-competent tri-segmented arenavirus viral vector. In certain embodiments, the tri-segmented arenavirus viral vector is a replication-deficient tri-segmented arenavirus viral vector. The tri-segmented arenavirus viral vector provided herein also includes a first nucleotide sequence encoding an oncogenic virus antigen, wherein the oncogenic virus is human papillomavirus (HPV), Kaposi's sarcoma-associated herpesvirus, Epstein-Barr virus, Merkel cell polyomavirus, or human T-lymphotropic virus. In particular, in certain embodiments, the tri-segmented arenavirus viral vector comprises a nucleotide sequence encoding a HPV antigen as described herein, including Section 3.1.
In particular embodiments, the tri-segmented arenavirus viral vector comprises one L segment and two S segments or two L segments and one S segment that do not recombine into a replication-competent bi-segmented arenavirus particle. The tri-segmented arenavirus viral vectors described herein have improved genetic stability and lasting transgene expression. Accordingly, in certain embodiments, propagation of the tri-segmented arenavirus viral vector does not result in a replication-competent bi-segmented viral viral vector after 70 days of persistent infection in mice lacking type I interferon receptor, type II interferon receptor and recombination activating gene 1 (RAG1) and having been infected with 104 PFU of the tri-segmented arenavirus viral vector. Moreover, in certain embodiments, inter-segmental recombination of the two S segments or two L segments, uniting two arenavirus ORFs on only one instead of two separate segments, abrogates viral promoter activity.
In certain embodiments, provided herein is a tri-segmented arenavirus viral vector comprising one L segment and two S segments and a first nucleotide sequence encoding an antigen of an oncogenic virus, or an antigen of a tumor-associated virus, wherein the oncogenic virus or tumor-associated virus is not cytomegalo virus, Hepatitis B virus, or Hepatitis C virus, and wherein propagation of the tri-segmented arenavirus viral vector does not result in a replication-competent bi-segmented viral vector after 70 days of persistent infection in mice lacking type I interferon receptor, type II interferon receptor and recombination activating gene 1 (RAG1) and having been infected with 104 PFU of the tri-segmented arenavirus viral vector. The tri-segmented arenavirus viral vector also includes a first nucleotide sequence encoding an oncogenic virus antigen, wherein the oncogenic virus is is human papillomavirus (HPV), Kaposi's sarcoma-associated herpesvirus, Epstein-Barr virus, Merkel cell polyomavirus, or human T-lymphotropic virus. In particular, in certain embodiments, the tri-segmented arenavirus viral vector comprises a nucleotide sequence encoding a HPV antigen as described herein, including Section 3.1.
In certain embodiments, provided herein is a tri-segmented arenavirus viral vector comprising two L segments and one S segment and a first nucleotide sequence encoding an antigen of an oncogenic virus, or an antigen of a tumor-associated virus, wherein the oncogenic virus or tumor-associated virus is not cytomegalo virus, Hepatitis B virus, or Hepatitis C virus, and wherein propagation of the tri-segmented arenavirus viral vector does not result in a replication-competent bi-segmented viral vector after 70 days of persistent infection in mice lacking type I interferon receptor, type II interferon receptor and recombination activating gene 1 (RAG1) and having been infected with 104 PFU of the tri-segmented arenavirus viral vector. The tri-segmented arenavirus viral vector also includes a first nucleotide sequence encoding an oncogenic virus antigen, wherein the oncogenic virus is human papillomavirus (HPV), Kaposi's sarcoma-associated herpesvirus, Epstein-Barr virus, Merkel cell polyomavirus, or human T-lymphotropic virus. In particular, in certain embodiments, the tri-segmented arenavirus viral vector comprises a nucleotide sequence encoding a HPV antigen as described herein, including Section 3.1.
Also provided herein is a tri-segmented arenavirus viral vector comprising an arenavirus genomic segment described herein and a second arenavirus genomic segment described herein so that the arenavirus viral vector comprises an S segment and an L segment.
In certain embodiments, the tri-segmented arenavirus viral vector comprising one L segment and two S segments provided herein includes an arenavirus viral vector wherein one of the two S segments is selected from the group consisting of:
In certain embodiments, the tri-segmented arenavirus viral vector comprising two L segments and one S segment provided herein includes an arenavirus viral vector wherein one of the two L segments is selected from the group consisting of:
In certain embodiments, the tri-segmented arenavirus viral vector 3′ UTR is the 3′ UTR of the arenavirus S segment or the arenavirus L segment. In other embodiments, the tri-segmented arenavirus viral vector 5′ UTR is the 5′ UTR of the arenavirus S segment or the arenavirus L segment.
In certain embodiments, the two S segments comprise (i) one or two heterologous ORFs from an organism other than an arenavirus; or (ii) one or two duplicated arenavirus ORFs; or (iii) one heterologous ORF from an organism other than an arenavirus and one duplicated arenavirus ORF.
In certain embodiments, the two L segments comprise (i) one or two heterologous ORFs from an organism other than an arenavirus; or (ii) one or two duplicated arenavirus ORFs; or (iii) one heterologous ORF from an organism other than an arenavirus and one duplicated arenavirus ORF.
In certain embodiments, the heterologous ORF encodes an antigen derived from an infectious organism, tumor, or allergen. In other embodiments, the heterologous ORF encoding an antigen is selected from human papillomavirus (HPV) antigens, human immunodeficiency virus antigens, hepatitis C virus antigens, hepatitis B surface antigen, varizella zoster virus antigens, cytomegalovirus antigens, mycobacterium tuberculosis antigens, and tumor associated antigens.
In certain embodiments, at least one heterologous ORF encodes a fluorescent protein. In other embodiments the fluorescent protein is a green fluorescent protein (GFP) or red fluorescent protein (RFP).
In certain embodiments, the tri-segmented arenavirus viral vector comprises all four arenavirus ORFs. In some embodiments the tri-segmented arenavirus viral vector is infectious and replication-competent.
In certain embodiments, the tri-segmented arenavirus viral vector lacks one or more of the four arenavirus ORFs. In other embodiments, the tri-segmented arenavirus viral vector is infectious but unable to produce further infectious progeny in non-complementing cells.
In certain embodiments, the tri-segmented arenavirus viral vector lacks one of the four arenavirus ORFs, wherein the tri-segmented arenavirus viral vector is infectious but unable to produce further infectious progeny in non-complementing cells.
In some embodiments, the tri-segmented arenavirus viral vector lacks the GP ORF.
In a further aspect, provided herein is a tri-segmented arenavirus viral vector comprising one L segment and two S segments. In certain embodiments, a first S segment is engineered to carry an ORF encoding GP in a position under control of an arenavirus 3′ UTR and an ORF encoding a first HPV antigen in a position under control of an arenavirus 5′ UTR. In some embodiments, a second S segment is engineered to carry an ORF encoding the NP in a position under control of an arenavirus 3′ UTR and an ORF encoding a second HPV antigen in a position under control of an arenavirus 5′ UTR.
In yet another aspect, provided herein, is a tri-segmented arenavirus viral vector comprising one L segment and two S segments. In certain embodiments, a first S segment is engineered to carry an ORF encoding GP in a position under control of an arenavirus 5′ UTR and an ORF encoding a first HPV antigen in a position under control of an arenavirus 3′ UTR. In some embodiments, a second S segment is engineered to carry an ORF encoding NP in a position under control of an arenavirus 5′ UTR and an ORF encoding a second HPV antigen in a position under control of an arenavirus 3′ UTR.
In certain embodiments, the first antigen is an HPV16 antigen, and the second antigen is an HPV18 antigen.
In certain embodiments, the tri-segmented arenavirus viral vector provided herein encodes one, two, or three HPV16 antigens and one, two or three HPV18 antigens.
In certain embodiments, the tri-segmented arenavirus viral vector provided herein encodes two HPV16 antigens and two HPV18 antigens, wherein the antigens are selected from the group consisting of:
an HPV16 protein E6, or an antigenic fragment thereof;
an HPV16 protein E7, or an antigenic fragment thereof;
an HPV18 protein E6, or an antigenic fragment thereof; and
an HPV18 protein E7, or an antigenic fragment thereof.
In certain embodiments, the first and the second antigens are selected from the group consisting of:
an HPV16 protein E6, or an antigenic fragment thereof;
an HPV16 protein E7, or an antigenic fragment thereof;
an HPV18 protein E6, or an antigenic fragment thereof; and
an HPV18 protein E7, or an antigenic fragment thereof, and wherein the first and the second antigen are not the same.
In certain embodiments, the first antigen is selected from the group consisting of:
an HPV16 protein E6, or an antigenic fragment thereof;
an HPV16 protein E7, or an antigenic fragment thereof;
an HPV18 protein E6, or an antigenic fragment thereof; and
an HPV18 protein E7, or an antigenic fragment thereof.
In certain embodiments, the HPV antigen is an HPV16 E7/E6 polypeptide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:10.
In certain embodiments, the HPV antigen is an HPV16 E7/HPV18 E6 polypeptide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to amino acids 17-263 of the amino acid sequence of SEQ ID NO:34.
In certain embodiments, the HPV antigen is an HPV18 E7/HPV16 E6 polypeptide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to amino acids 17-270 of the amino acid sequence of SEQ ID NO:36.
In certain embodiments, the HPV antigen is an HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 polypeptide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to amino acids 17-516 of the amino acid sequence of SEQ ID NO:38.
In certain embodiments, the tri-segmented arenavirus viral vector is infectious and replication-competent. In some embodiments, the arenavirus viral vector is attenuated. In other embodiments, the tri-segmented arenavirus viral vector is infectious but unable to produce further infectious progeny in non-complementing cells.
In certain embodiments, the tri-segmented arenavirus viral vector has the same tropism as the bi-segmented arenavirus particle. In other embodiments, the tri-segmented arenavirus viral vector is replication deficient.
In certain embodiments, the tri-segmented arenavirus viral vector is derived from lymphocytic choriomeningitis virus (“LCMV”) or Junin virus. In particular embodiments, the tri-segmented arenavirus viral vector is derived from LCMV. The LCMV can be MP strain, Armstrong strain, or Armstrong Clone 13 strain. In particular embodiments, the tri-segmented arenavirus viral vector is derived from Junin virus. The Junin virus can be Junin virus vaccine Candid #1, or Junin virus vaccine XJ Clone 3 strain.
3.5 Nucleic Acids, Host Cells and Methods of Generating Viral Vectors
In certain embodiments, provided herein is an isolated nucleic acid, including a cDNA, wherein the nucleic acid encodes a viral vector as described above. In certain embodiments, provided herein is an expression vector comprising such a nucleic acid. Also provided herein is a host cell comprising such a nucleic acid or such an expression vector.
In certain embodiments, provided herein is an isolated cDNA of an arenavirus genomic segment provided herein. Also provided herein, is a DNA expression vector comprising the cDNA of an arenavirus genomic segment provided herein.
Still further provided herein is a host cell comprising an arenavirus genomic segment provided herein, a cDNA of the arenavirus genomic segment, or the vector comprising a cDNA of the arenavirus genomic segment.
In certain embodiments, provided herein is a method for generating an infectious, replication-deficient arenavirus viral vector comprising:
Also provided herein is a method of producing the arenavirus genomic segment. In certain embodiments, the method comprises transcribing the cDNA of the arenavirus genomic segment.
Also provided herein is a method of generating the arenavirus viral vector. In certain embodiments the method of generating the arenavirus viral vector comprises:
In certain embodiments, the transcription of the L segment and the S segment is performed using a bidirectional promoter.
In certain embodiments, the method further comprises transfecting into a host cell one or more nucleic acids encoding an arenavirus polymerase. In yet more specific embodiments, the polymerase is the L protein. In other embodiments, the method further comprises transfecting into the host cell one or more nucleic acids encoding the NP.
In certain embodiments, transcription of the L segment, and the S segment are each under the control of a promoter selected from the group consisting of:
Also provided herein is a method of generating the tri-segmented arenavirus viral vector. In certain embodiments the method of generating the tri-segmented arenavirus viral vector comprises:
Also provided herein is a method of generating the tri-segmented arenavirus viral vector. In certain embodiments the method of generating the tri-segmented arenavirus viral vector comprises:
In certain embodiments, the transcription of the one L segment and two S segment is performed using a bidirectional promoter. In some embodiments, the transcription of the two L segments and one S segment is performed using a bidirectional promoter.
In certain embodiments, the method further comprises transfecting into a host cell one or more nucleic acids encoding an arenavirus polymerase. In yet more specific embodiments, the polymerase is the L protein. In other embodiments, the method further comprises transfecting into the host cell one or more nucleic acids encoding the NP protein.
In certain embodiments, transcription of the one L segment, and two S segments are each under the control of a promoter selected from the group consisting of:
In certain embodiments, transcription of the two L segments, and one S segment are each under the control of a promoter selected from the group consisting of:
3.6 Pharmaceutical Compositions, Vaccines and Methods of Treatment
In certain embodiments, provided herein is a pharmaceutical composition comprising an arenavirus viral vector as described herein and a pharmaceutically acceptable carrier.
In certain embodiments, provided herein is an immunogenic composition comprising an arenavirus viral vector as described herein and a pharmaceutically acceptable carrier.
In certain embodiments, provided herein is a vaccine comprising an arenavirus viral vector as described herein and a pharmaceutically acceptable carrier.
Still further provided herein is a method of treating or preventing a human papillomavirus infection in a patient. In certain embodiments, the method comprises administering to the patient an arenavirus viral vector as described herein, an pharmaceutical composition as described herein, an immunogenic composition as described herein, or a vaccine as described herein.
In certain embodiments, the method results in a reduction of pre-exisiting HPV titer in the patient.
In certain embodiments, the method induces an antigen specific CD8+ T-cell response in the patient.
In certain embodiments, the HPV infection is symptomatic.
In certain embodiments, the HPV infection is asymptomatic.
In certain embodiments, the method reduces the severity or frequency of, or prevents manifestations of the HPV infection.
In certain embodiments, the manifestation is selected from the group consisting of: cervical cancer, anal cancer, vulvar cancer, vaginal cancer, penile cancer, HPV-positive oropharyngeal cancer (OSCC), common warts, plantar warts, subungual or periungual warts, genital warts, condylomata acuminata or venereal warts, respiratory papillomatosis, and epidermodysplasia verruciformis.
In certain embodiments, provided herein is a method of treating or preventing a human papillomavirus infection in a patient, wherein said method comprises administering to the patient a first viral vector as described herein, a first pharmaceutical composition as described herein, a first immunogenic composition as described herein, or a first vaccine as described herein, and administering to the patient a second viral vector as described herein, a second pharmaceutical composition as described herein, a second immunogenic composition as described herein, or a second vaccine as described herein.
In certain embodiments, provided herein is a method of inducing an immune response in a subject. Such a method can comprise administering to the patient a first arenavirus viral vector described herein, and administering to the patient, after a period of time, a second, different arenavirus viral vector as described herein.
In certain embodiments, the first viral vector, the first pharmaceutical composition, the first immunogenic composition, or the first vaccine, and the second viral vector, the second pharmaceutical composition, the second immunogenic composition, or the second vaccine, are homologous (e.g., derived from the same virus).
In certain embodiments, the first viral vector, the first pharmaceutical composition, the first immunogenic composition, or the first vaccine, and the second viral vector, the second pharmaceutical composition, the second immunogenic composition, or the second vaccine, are heterologous (e.g., derived from the different viruses).
In certain embodiments, the first viral vector, the first pharmaceutical composition, the first immunogenic composition, or the first vaccine, is derived from LCMV, and the second viral vector, the second pharmaceutical composition, the second immunogenic composition, or the second vaccine, is derived from Junin virus.
In certain embodiments, the first viral vector, the first pharmaceutical composition, the first immunogenic composition, or the first vaccine, is derived from Junin virus, and the second viral vector, the second pharmaceutical composition, the second immunogenic composition, or the second vaccine, is derived from LCMV.
In certain embodiments, the first arenavirus viral vector and the second arenavirus viral vector express the same antigen. In certain embodiments, the first arenavirus viral vector and the second arenavirus viral vector express different antigens.
3.7 Conventions and Abbreviations
SEQ ID NO: 1 Lymphocytic choriomeningitis virus segment S, complete sequence. The genomic segment is RNA, the sequence in SEQ ID NO:1 is shown for DNA; however, exchanging all thymidines (“T”) in SEQ ID NO:1 for uridines (“U”) provides the RNA sequence.
SEQ ID NO: 2 Lymphocytic choriomeningitis virus clone 13 segment S, complete sequence (GenBank: DQ361065.2). The genomic segment is RNA, the sequence in SEQ ID NO: 2 is shown for DNA; however, exchanging all thymidines (“T”) in SEQ ID NO: 2 for uridines (“U”) provides the RNA sequence.
SEQ ID NO: 3 Lymphocytic choriomeningitis virus clone 13 segment L, complete sequence (GenBank: DQ361066.1). The genomic segment is RNA, the sequence in SEQ ID NO: 3 is shown for DNA; however, exchanging all thymidines (“T”) in SEQ ID NO: 3 for uridines (“U”) provides the RNA sequence.
SEQ ID NO: 4 Lymphocytic choriomeningitis strain MP segment L, complete sequence. The genomic segment is RNA, the sequence in SEQ ID NO: 4 is shown for DNA; however, exchanging all thymidines (“T”) in SEQ ID NO: 4 for uridines (“U”) provides the RNA sequence.
SEQ ID NO: 5 Lymphocytic choriomeningitis strain MP segment S, complete sequence. The genomic segment is RNA, the sequence in SEQ ID NO: 5 is shown for DNA; however, exchanging all thymidines (“T”) in SEQ ID NO:5 for uridines (“U”) provides the RNA sequence.
SEQ ID NO: 6 Amino acid sequence of the NP protein of the MP strain of LCMV.
SEQ ID NO: 7 Amino acid sequence of the GP protein of the MP strain of LCMV.
SEQ ID NO: 8 Amino acid sequence of the L protein of the MP strain of LCMV.
SEQ ID NO: 9 Amino acid sequence of the Z protein of the MP strain of LCMV.
SEQ ID NO: 10 Amino acid sequence of HPV16 E7/E6 fusion protein with mutations in Rb binding site and zinc finger motifs.
SEQ ID NO: 11 Amino acid sequence of HPV16 E7/E6 fusion protein with mutations in Rb binding site and zinc finger motifs, linked to mouse Calreticulin.
SEQ ID NO: 12 Amino acid sequence of HPV16 E7/E6 fusion protein with mutations in Rb binding site and zinc finger motifs, linked to mouse Ubiquitin.
SEQ ID NO: 13 Amino acid sequence of HPV16 E7/E6 fusion protein with mutations in Rb binding site and zinc finger motifs, co-expressed with mouse GM-CSF, separated by a nucleotide sequence that encodes a self-cleaving peptide (2A peptide).
SEQ ID NO: 14 Nucleotide sequence encoding HPV16 E7/E6 fusion protein with mutations in Rb binding site and zinc finger motifs.
SEQ ID NO: 15 Nucleotide sequence encoding HPV16 E7/E6 fusion protein with mutations in Rb binding site and zinc finger motifs, linked to mouse Calreticulin.
SEQ ID NO: 16 Nucleotide sequence encoding HPV16 E7/E6 fusion protein with mutations in Rb binding site and zinc finger motifs, linked to mouse Ubiquitin.
SEQ ID NO: 17 Nucleotide sequence encoding HPV16 E7/E6 fusion protein with mutations in Rb binding site and zinc finger motifs, co-expressed with mouse GM-CSF, separated by a nucleotide sequence that encodes a self-cleaving peptide (2A peptide).
SEQ ID NO: 18 GSG.
SEQ ID NO: 19 Junin virus Candid#1 L segment.
SEQ ID NO: 20 Junin virus Candid#1 S segment.
SEQ ID NO: 21 Nucleotide sequence of HK1-E7E6-GMCSF
SEQ ID NO: 22 Amino acid sequence of E7E6-GMCSF antigen
SEQ ID NO: 23 Nucleotide sequence of HK1-E7E6-VP22
SEQ ID NO: 24 Amino acid sequence of E7E6-VP22 antigen
SEQ ID NO: 25 Nucleotide sequence of HK1-E7E6-CD40L
SEQ ID NO: 26 Amino acid sequence of E7E6-CD40L antigen
SEQ ID NO: 27 Nucleotide sequence of HK1-Flt3L-E7E6
SEQ ID NO: 28 Amino acid sequence of Flt3L-E7E6 antigen
SEQ ID NO: 29 Nucleotide sequence of HK1-Flt3L-E7E6shuffle
SEQ ID NO: 30 Amino acid sequence of Flt3L-E7E6shuffle antigen
SEQ ID NO: 31 Nucleotide sequence of HK1-li-E7E6
SEQ ID NO: 32 Amino acid sequence of li-E7E6 antigen
SEQ ID NO: 33 Nucleotide sequence encoding a HPV16E7-HPV18E6 fusion protein having an N-terminal VSVG signal sequence and a C-terminal GSG linker followed by a self-cleaving peptide (2A peptide from Porcine Teschovirus) and the CDS for human GM-CSF.
SEQ ID NO: 34 Amino acid sequence of a HPV16E7-HPV18E6 fusion protein having an N-terminal VSVG signal sequence and a C-terminal GSG linker followed by a self-cleaving peptide (2A peptide from Porcine Teschovirus) and the CDS for human GM-CSF.
SEQ ID NO: 35 Nucleotide sequence encoding a HPV18E7-HPV16E6 fusion protein having an N-terminal VSVG signal sequence and a C-terminal GSG linker followed by a self-cleaving peptide (2A peptide from Porcine Teschovirus) and the CDS for human GM-CSF.
SEQ ID NO: 36 Amino acid sequence of a HPV18E7-HPV16E6 fusion protein having an N-terminal VSVG signal sequence and a C-terminal GSG linker followed by a self-cleaving peptide (2A peptide from Porcine Teschovirus) and the CDS for human GM-CSF
SEQ ID NO: 37 Nucleotide sequence encoding a HPV16E7-HPV18E6 HPV16E6-HPV18E7 fusion protein having an N-terminal VSVG signal sequence and a C-terminal GSG linker followed by a self-cleaving peptide (2A peptide from Porcine Teschovirus) and the CDS for human GM-CSF.
SEQ ID NO: 38 Amino acid sequence of a HPV16E7-HPV18E6 HPV16E6-HPV18E7 fusion protein having an N-terminal VSVG signal sequence and a C-terminal GSG linker followed by a self-cleaving peptide (2A peptide from Porcine Teschovirus) and the CDS for human GM-CSF.
SEQ ID NO: 39 Nucleotide sequence of a tri-segmented r3LCMVart-based vector expressing HPV16 E7E6 fusion protein: S segment 1 (containing GP).
SEQ ID NO: 40 Nucleotide sequence of a tri-segmented r3LCMVart-based vector expressing HPV16 E7E6 fusion protein: S segment 2 (containing GP).
SEQ ID NO: 41 Nucleotide sequence of a tri-segmented r3LCMVart-based vector expressing HPV16 E7E6 fusion protein: L segment.
SEQ ID NO: 42 Nucleotide sequence of a tri-segmented r3JUNVart-based vector expressing the HPV16 E7E6 fusion protein: S segment 1 (containing NP).
SEQ ID NO: 43 Nucleotide sequence of a tri-segmented r3JUNVart-based vector expressing the HPV16 E7E6 fusion protein: S segment 2 (containing GP).
SEQ ID NO: 44 Nucleotide sequence of a tri-segmented r3JUNVart-based vector expressing the HPV16 E7E6 fusion protein: L segment .
Provided herein are methods and compositions for the prevention or treatment of diseases and conditions associated with neoplastic disease, such as cancer. Provided herein are methods and compositions for the treatment or prevention of diseases and conditions associated with neoplastic disease, such as cancer, using vaccines. Specifically, provided herein are arenavirus viral vectors, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, and a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, for use as vaccines for the prevention or treatment of diseases and conditions caused by tumor-associated viruses. Such vaccines can be an infectious, replication-deficient arenavirus, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment expressing an antigen of a tumor-associated virus.
Provided herein are methods and compositions for the prevention or treatment of neoplastic disease, such as cancer. Provided herein are methods and compositions for the treatment or prevention of neoplastic disease, such as cancer, using vaccines. Specifically, provided herein are infectious, replication-deficient arenavirus viral vectors, replication-competent tri-segmented arenavirus viral vectors, replication-deficient tri-segmented arenavirus viral vectors, or arenavirus genomic segments for use as vaccines for the prevention or treatment of neoplastic disease, such as cancer. More specifically, these vaccines can be used for the prevention or treatment of cancer caused by infection with oncogenic viruses, such as human papillomavirus (HPV). Such vaccines can be infectious, replication-deficient arenaviruses, replication-competent tri-segmented arenavirus viral vectors, replication-deficient tri-segmented arenavirus viral vectors, or arenavirus genomic segments expressing an antigen of an oncogenic virus, such as HPV.
In certain specific embodiments, provided herein is a genetically modified arenavirus, wherein the arenavirus:
i) is infectious;
ii) cannot form infectious progeny virus in a non-complementary cell (i.e., a cell that does not express the functionality that is missing from the replication-deficient arenavirus and causes it to be replication-deficient);
iii) is capable of replicating its genome and expressing its genetic information; and
iv) encodes an antigen of an oncogenic virus, such as an HPV virus, or a fragment thereof, alone or in combination with an immunomodulatory peptide, polypeptide, or protein.
In certain specific embodiments, the arenavirus for use with the methods and compositions provided is a genetically engineered lymphocytic choriomeningitis virus (LCMV) or is a genetically engineered Junin virus. In certain specific embodiments, an LCMV or a Junin virus is genetically modified by a functional inactivation (e.g., deletion) of an open reading frame (ORF) such that the resulting virus cannot produce further infectious progeny virus particles in non-complementing cells, i.e., a cell that does not provide the functionally inactivated ORF in trans. The resulting infectious replication-deficient LCMV or Junin virus can be used as a vector to express an antigen of an oncogenic virus, such as HPV. The generation and propagation of arenavirus vectors for use with the compositions and methods provided herein is described in more detail in Sections 6.1, 6.2, 6.3 and 6.4.
The arenavirus vectors provided herein are genetically engineered to comprise a heterologous nucleotide sequence, which expresses a heterologous peptide or protein. In certain embodiments, the heterologous sequence encodes a tumor antigen. In certain embodiments, the heterologous sequence encodes an antigen of an oncogenic virus. In certain specific embodiments, the heterologous sequence encodes an HPV antigen. In certain specific embodiments, the heterologous sequence encodes two, three, four or more antigens of one or more oncogenic viruses. In certain embodiments, an arenavirus vector for use with the present methods encodes also an immunomodulatory peptide or protein. In certain embodiments, the arenavirus vector also encodes a signal peptide or protein. Without being bound by theory, such a signal peptide facilitates the transport of a protein (e.g., an HPV antigen and/or an immunomodulatory protein or peptide) outside the cell in which the antigen and/or immunomodulatory protein or peptide was expressed. The heterologous sequences for use with the compositions and methods provided herein are described in more detail in Section 6.5.
Pharmaceutical compositions, immunogenic compositions, and vaccines comprising the arenavirus vectors provided herein are described in Section 6.6.
Methods of use of the arenavirus vectors for the prevention or treatment of neoplastic disease, e.g., non-malignant neoplasm or cancer, are provided herein. Specifically, provided herein are methods for preventing or treating cancer in a subject comprising administering to the subject one or more arenaviruses expressing an HPV antigen or a fragment thereof. In a specific embodiment, provided herein are methods for preventing or treating cancer in a subject comprising administering to the subject one or more arenaviruses expressing an HPV antigen or a fragment thereof, alone or in combination with one or more of an immunomodulatory peptide, polypeptide, or protein, a linker, or a signal sequence. In certain embodiments, immunization with an arenavirus that expresses an HPV antigen or a fragment thereof, as described herein provides a cytotoxic T-cell response. In certain embodiments, a second or third immunization can be administered for a boosting effect. In certain embodiments, the second or third immunization utilizes a homologous vector. In certain embodiments, the second or third immunization utilizes a heterologous vector. In certain embodiments, the first immunization utilizes an Old World arenavirus vector, and the second immunization utilizes an Old World arenavirus vector. In certain embodiments, the first immunization utilizes an Old World arenavirus vector, and the second immunization utilizes an New World arenavirus vector. In certain embodiments, the first immunization utilizes an New World arenavirus vector, and the second immunization utilizes an Old World arenavirus vector. In certain embodiments, the first immunization utilizes an New World arenavirus vector, and the second immunization utilizes an New World arenavirus vector. A more detailed description of methods of treatment and/or prevention of neoplastic disease using an arenavirus as described herein is provided in Section 6.7.
6.1 Replication Defective Arenavirus Vectors
Infectious, replication-deficient viruses as described herein can be produced as described in International Patent Application Publication No. WO 2009/083210 (application number PCT/EP2008/010994), which is incorporated by reference herein in its entirety.
Arenaviruses for use with the methods and compositions provided herein can be Old World viruses, for example Lassa virus, Lymphocytic choriomeningitis virus (LCMV), Mobala virus, Mopeia virus, or Ippy virus, or New World viruses, for example Amapari virus, Flexal virus, Guanarito virus, Junin virus, Latino virus, Machupo virus, Oliveros virus, Parana virus, Pichinde virus, Pirital virus, Sabia virus, Tacaribe virus, Tamiami virus, Bear Canyon virus, or Whitewater Arroyo virus.
A genetically modified arenavirus described herein is infectious, i.e., it can attach to a host cell and release its genetic material into the host cell. A genetically modified arenavirus described herein is replication-deficient, i.e., the arenavirus is unable to produce further infectious progeny particles in a non-complementing cell. In particular, the genome of the arenavirus is modified (e.g., by deletion or functional inactivation of an open reading frame or another genetic element of the virus genome that is required for the generation of an infectious particle) such that a virus carrying the modified genome can no longer produce infectious progeny viruses in a non-complementing cell. A non-complementing cell is a cell that does not provide the functionality that has been eliminated from the replication-deficient arenavirus by modification of its genome. For example, if the open reading frame encoding the GP protein has been deleted or functionally inactivated, a non-complementing cell does not provide the GP protein. However, a genetically modified arenavirus as provided herein is capable of producing infectious progeny viruses in complementing cells. Complementing cells are cells that provide the functionality that has been eliminated from the replication-deficient arenavirus by modification of its genome. For example, if the open reading frame encoding the GP protein is deleted or functionally inactivated, a complementing cell does provide the GP protein. Expression of the complementing functionality (e.g., the GP protein) can be accomplished by any method known to the skilled artisan (e.g., transient or stable transfection, using a suitable expression vector).
A genetically modified arenavirus as described herein can amplify and express its genetic information in a cell that has been infected by the virus. Specifically, as described herein, the genetically modified arenavirus can amplify and express its genetic information in a complementing cell or a non-complementing cell. A genetically modified infectious, replication-deficient arenavirus as provided herein comprises a heterologous nucleotide sequence that encodes antigens of interest, an immunomodulatory peptide, polypeptide, or protein , a signal sequence, and/or a linker. Such sequences and their arrangement are described in Section 6.5.
In certain embodiments, the open reading frame (ORF) that encodes the glycoprotein (GP) gene of the arenavirus is deleted to generate a replication-deficient arenavirus for use with the compositions and methods provided herein. A heterologous nucleotide sequence (Section 6.5) is inserted in place of the deleted ORF. Thus, in certain embodiments, a genetically modified arenavirus viral vector provided herein comprises a genomic segment that a) has a deletion or functional inactivation of an open reading frame that is present in the wild type form of the genomic segment; and b) encodes one or more antigens of an oncogenic virus (e.g., HPV E6, HPV E7, and/or HPV E6/E7 fusion protein), and/or an immunomodulatory peptide, polypeptide, or protein.
Generally, arenavirus viral vectors can be recombinantly produced by standard reverse genetic techniques as described for LCMV (Flatz, et al., 2006
Proc Natl Acad Sci USA 103:4663-4668; Sanchez et al., 2006, Virology 350:370; Ortiz-Riano, et al., 2013 J Gen Virol. 94:1175-88). Infectious, replication-deficient virus vectors as described herein can be produced as described in International Patent Application Publication No. WO 2009/083210 (application number PCT/EP2008/010994), which is incorporated by reference herein in its entirety. The genome of the rescued virus is modified as described in Section 6.5. These modifications can be: i) one or more, e.g., two, three or four, of the four arenavirus open reading frames (glycoprotein (GP); nucleoprotein (NP); matrix protein Z; RNA-dependent RNA polymerase L) are removed or functionally inactivated to prevent formation of infectious particles in non-complementing cells, albeit still allowing gene expression in arenavirus vector-infected host cells; and ii) a nucleic acid that encodes one or more of an heterologous antigen, an immunomodulatory peptide, polypeptide, or protein, a signal sequence, or a linker can be introduced.
Owing to the removal or functional inactivation of one or more of the viral genes in arenavirus vectors (here, deletion of the glycoprotein, GP, will be taken as an example), arenavirus vectors can be generated and expanded in cells providing in trans the deleted viral gene(s), e.g., the GP in the present example. Such a complementing cell line, henceforth referred to as C-cells, is generated by transfecting a mammalian cell line such as BHK-21, HEK293, VERO or other (here HEK293 will be taken as an example) with one or more plasmid(s) for expression of the viral gene(s) of interest (complementation plasmid, referred to as C-plasmid). The C-plasmid(s) express the viral gene(s) deleted in the arenavirus vector to be generated under control of one or more expression cassettes suitable for expression in mammalian cells, e.g., a mammalian polymerase II promoter such as the CMV or EF1 alpha promoter with a polyadenylation signal. In addition, the complementation plasmid features a mammalian selection marker, e.g., puromycin resistance, under control of an expression cassette suitable for gene expression in mammalian cells, e.g., polymerase II expression cassette as above, or the viral gene transcript(s) are followed by an internal ribosome entry site, such as the one of encephalomyocarditis virus, followed by the mammalian resistance marker. For production in E. coli, the plasmid additionally features a bacterial selection marker, such as an ampicillin resistance cassette.
For generation of C-cells, cells that can be used, e.g., BHK-21, HEK293, MC57G, are kept in culture and are transfected with the C-plasmid(s) using any of the commonly used strategies such as calcium-phosphate, liposome-based protocols, or electroporation. A few days later, the suitable selection agent, e.g., puromycin, is added in titrated concentrations. Surviving clones are isolated and subcloned following standard procedures, and high-expressing C-cell clones are identified using Western blot or flow cytometry procedures with antibodies directed against the viral protein(s) of interest. As an alternative to the use of stably transfected C-cells, transient transfection of normal cells can complement the missing viral gene(s) in each of the steps where C-cells will be used below. In addition, a helper virus can be used to provide the missing functionality in trans. In other certain embodiments, other methods known in the art can be used for the generation of stable cell lines e.g., lentivirus transduction.
For generation of arenavirus vectors, plasmids that can be used can be of two types: i) Two plasmids, referred to as TF-plasmids for expressing intracellularly in C-cells the minimal transacting factors of the arenavirus, the vector is derived from e.g., NP and L proteins of LCMV or Junin virus in the present example; and ii) Plasmids, referred to as GS-plasmids, for expressing intracellularly in C-cells the arenavirus vector genome segments, e.g., the segments with designed modifications. TF-plasmids express the NP and L proteins of the respective arenavirus vector under control of an expression cassette suitable for protein expression in mammalian cells, typically e.g., a mammalian polymerase II promoter such as the CMV or EFlalpha promoter, either one of them preferentially in combination with a polyadenylation signal. From the GS-plasmids the small (S) and the large (L) genome segments of the vector are transcribed. Typically, polymerase I-driven expression cassettes or T7 bacteriophage RNA polymerase (T7-) driven expression cassettes can be used, the latter preferentially with a 3′-terminal ribozyme for processing of the primary transcript to yield the correct end. In the case of using a T7-based system, expression of T7 in C-cells must be provided by either including in the recovery process an additional expression plasmid, constructed analogously to TF-plasmids, providing T7, or C-cells are constructed to additionally express T7 in a stable manner. In certain embodiments, TF and GS plasmids can be the same, i.e. the genome sequence and transacting factors can be transcribed by T7, polI and polII promoters from one plasmid.
For recovering of the arenavirus vector, the following procedures can be used. First day: C-cells, typically 80% confluent in M6-well plates, are transfected with a mixture of the two TF-plasmids plus the two GS-plasmids. In certain embodiments, the TF and GS plasmids can be the same, i.e., the genome sequence and transacting factors can be transcribed by T7, polI and polII promoters from one plasmid. For this one can exploit any of the commonly used strategies such as calcium-phosphate, liposome-based protocols or electroporation. In another embodiment, C-cells, e.g., P5A3 cells, can also be cultured in suspension and transfected at a defined cell density.
3-5 days later: The culture supernatant (arenavirus vector preparation) is harvested, aliquoted and stored at 4° C., −20° C. or −80° C. depending on how long the arenavirus vector should be stored prior to use. Then the arenavirus vector preparation's infectious titer is assessed by an immunofocus assay on C-cells. In another embodiment, 3-5 days later, the transfected cells and supernatant are transferred to a larger culture flask. 3 days later, the culture supernatant (arenavirus vector preparation) is harvested, aliquoted and stored at 4° C., −20° C. or at −80° C. depending on how long the arenavirus vector should be stored prior to use. Then, the arenavirus vector preparation's infectious titer is assessed by an immunofocus assay on C-cells.
Once generated from cDNA, the infectious, replication-deficient arenaviruses provided herein can be propagated in complementing cells. Complementing cells are cells that provide the functionality that has been eliminated from the infectious, replication-deficient arenavirus by modification of its genome (e.g., if the open reading frame encoding the GP protein is deleted or functionally inactivated, a complementing cell does provide the GP protein).
Provided herein are compositions and methods for the expression of a heterologous antigen in a cell culture wherein the cell culture is infected with an infectious, replication-deficient arenavirus expressing a heterologous sequence. When used for expression of a heterologous sequence in cultured cells, the following two procedures can be used:
i) The cell type of interest is infected with the arenavirus vector preparation described herein at a multiplicity of infection (MOI) of one or more, e.g., two, three or four, resulting in production of the heterologous sequence in all cells already shortly after infection.
ii) Alternatively, a lower MOI can be used and individual cell clones can be selected for their level of virally driven heterologous sequence expression. Subsequently individual clones can be expanded infinitely owing to the non-cytolytic nature of arenavirus vectors. Irrespective of the approach, the heterologous sequence can subsequently be collected (and purified) either from the culture supernatant or from the cells themselves, depending on the properties of the heterologous sequence produced. However, the compositions and methods provided herein are not limited to these two strategies, and other ways of driving expression of heterologous sequence using infectious, replication-deficient arenaviruses as vectors may be considered.
Alternatively, a rescue system consisting of three plasmids can be used: (1) the first plasmid expresses the protein NP by transcription via Polymerase II and subsequent translation in transfected cells; (2) the second plasmid gives rise to the (negative-stranded) L-Segment of the LCMV genome by transcription via Polymerase I as well as the L protein by transcription via Polymerase II from the same template in the opposite direction of the Polymerase I promoter; (3) the third plasmid gives rise to the S-segment of the LCMV genome (encoding the antigen coding sequence instead of the LCMV glycoprotein) via transcription by Polymerase I. 3 μg of each plasmid is used for electroporation of C-cells, followed by seeding of cells in 6-well plates and incubation at 37° C. After incubation, cells and supernatant from transfections are combined with freshly seeded C-cells, and vectors are harvested and cleared from cells & debris at a defined timepoint post infection. Once the vector has been generated, a nucleic acid encoding an antigen of an oncogenic virus and/or an immunomodulatory peptide, polypeptide, or protein (see Section 6.5) can be inserted into a plasmid from which a genomic segment of an infectious replication-deficient vector is transcribed by any technique known to the skilled artisan.
Owing to the removal or functional inactivation of one or more of the viral genes in arenavirus vectors (here deletion of the glycoprotein, GP, will be taken as an example) arenavirus vectors can be generated and expanded in cells that provide the deleted or functionally inactivated viral gene(s) (e.g., the GP) in trans. The resulting virus itself is infectious but is unable to produce further infectious progeny particles in non-complementing cells due to the lack of the deleted or functionally inactivated viral gene(s) (e.g., the GP). The complementing cell can provide the missing functionality either by stable transfection, transient transfection, or by infection with a helper virus that expresses the missing functionality.
In certain embodiments, the complementing cell provides the viral gene that has been deleted or functionally inactivated from the arenavirus vector genome. In a specific embodiment, the complementing cell provides the viral gene from a viral strain that is the same as the viral strain that was used to generate the genome of the arenavirus vector. In another embodiment, the complementing cell provides the viral gene from a viral strain that is different from the viral strain that was used to generate the genome of the arenavirus vector. For example, the viral gene provided in the complementing cell is obtained from the MP strain of LCMV and encodes a protein having the amino acid sequence of SEQ ID NO: 6, 7, 8, or 9.
In a specific embodiment, the complementing cell provides the GP of the MP strain of LCMV and the arenavirus vector comprises an ORF of a human HPV antigen as described herein in place of the ORF encoding the GP protein. In an even more specific embodiment, the complementing cell provides the GP of the MP strain of LCMV and the arenavirus vector is obtained from LCMV Clone 13 and comprises an ORF of a human HPV antigen as described herein in place of the ORF encoding the GP protein. In an even more specific embodiment, the GP protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7.
6.2 Arenaviruses with an Open Reading Frame in a Non-natural Position
Provided herein are arenaviruses with rearrangements of their ORFs. In certain embodiments, such arenaviruses are replication-competent and infectious. In certain embodiments, such arenaviruses are replication-deficient and infectious. Genomic sequences of such arenaviruses are provided herein. In one aspect, provided herein is an arenavirus genomic segment, wherein the arenavirus genomic segment is engineered to carry an arenavirus ORF in a position other than the position in which the respective gene is found in viruses isolated from the wild. In one embodiment, the arenavirus viral vector is LCMV. In another aspect, an arenavirus genomic segment as provided herein comprises a heterologous nucleotide sequence that encodes antigens of interest, an immunomodulatory peptide, polypeptide, or protein , a signal sequence, and/or a linker. Such sequences and their arrangement are described in Section 6.5.
The wild-type arenavirus genomic segments and ORFs are known in the art. In particular, the arenavirus genome consists of an S segment and an L segment. The S segment carries the ORFs encoding the GP and the NP. The L segment encodes the L protein and the Z protein. Both segments are flanked by the respective 5′ and 3′ UTRs.
In certain embodiments, an arenavirus genomic segment can be engineered to carry two or more arenavirus ORFs in a position other than the wild-type position. In other embodiments, the arenavirus genomic segment can be engineered to carry two arenavirus ORFs, or three arenavirus ORFs, or four arenavirus ORFs in a position other than the wild-type position.
In certain embodiments, the open reading frame (ORF) that encodes the glycoprotein (“GP”), nucleoprotein (“NP”), matrix protein Z (“Z protein”) or RNA dependent RNA polymerase L (“L protein”) of the arenavirus is removed (e.g. deleted) to generate a replication-deficient arenavirus for use with the compositions and methods provided herein. A heterologous nucleotide sequence (Section 6.5) can be inserted in place of the deleted arenavirus ORF. Thus, in certain embodiments, an arenavirus genomic segment provided herein comprises a genomic segment that a) has a deletion or functional inactivation of an open reading frame that is present in the wild type form of the genomic segment; and b) encodes one or more antigens of an oncogenic virus (e.g., HPV E6, HPV E7, and/or HPV E6/E7 fusion protein), and/or an immunomodulatory peptide, polypeptide, or protein.
In certain embodiments, an arenavirus genomic segment provided herein can be:
In certain embodiments, the ORF that is in the non-natural position of the arenavirus genomic segment described herein can be under the control of an arenavirus 3′ UTR or an arenavirus 5′ UTR. In more specific embodiments, the arenavirus 3′ UTR is the 3′ UTR of the arenavirus S segment. In another specific embodiment, the arenavirus 3′ UTR is the 3′UTR of the arenavirus L segment. In more specific embodiments, the arenavirus 5′ UTR is the 5′ UTR of the arenavirus S segment. In other specific embodiments, the 5′ UTR is the 5′ UTR of the L segment.
In other embodiments, the ORF that is in the non-natural position of the arenavirus genomic segment described herein can be under the control of the arenavirus conserved terminal sequence element (the 5′- and 3′-terminal 19-20-nt regions) (see e.g., Perez & de la Torre, 2003, J Virol. 77(2): 1184-1194).
In certain embodiments, the ORF that is in the non-natural position of the arenavirus genomic segment can be under the control of the promoter element of the 5′ UTR (see e.g., Albarino et al., 2011, J Virol., 85(8):4020-4). In another embodiment, the ORF that is in the non-natural position of the arenavirus genomic segment can be under the control of the promoter element of the 3′ UTR (see e.g., Albarino et al., 2011, J Virol., 85(8):4020-4). In more specific embodiments, the promoter element of the 5′ UTR is the 5′ UTR promoter element of the S segment or the L segment. In another specific embodiment, the promoter element of the 3′ UTR is the 3′ UTR the promoter element of the S segment or the L segment.
In certain embodiments, the ORF that is in the non-natural position of the arenavirus genomic segment can be under the control of a truncated arenavirus 3′ UTR or a truncated arenavirus 5′ UTR (see e.g., Perez & de la Torre, 2003, J Virol. 77(2): 1184-1194; Albarino et al., 2011, J Virol., 85(8):4020-4). In more specific embodiments, the truncated 3′ UTR is the 3′ UTR of the arenavirus S segment or L segment. In more specific embodiments, the truncated 5′ UTR is the 5′ UTR of the arenavirus S segment or L segment.
Also provided herein, is an arenavirus viral vector comprising a first genomic segment that has been engineered to carry an ORF in a position other than the wild-type position of the ORF and a second arenavirus genomic segment so that the arenavirus viral vector comprises an S segment and an L segment. In specific embodiments, the ORF in a position other than the wild-type position of the ORF is one of the arenavirus ORFs.
In certain specific embodiments, the arenavirus viral vector can comprise a full complement of all four arenavirus ORFs. In specific embodiments, the second arenavirus genomic segment has been engineered to carry an ORF in a position other than the wild-type position of the ORF. In another specific embodiment, the second arenavirus genomic segment can be the wild-type genomic segment (i.e., comprises the ORFs on the segment in the wild-type position) .
In certain embodiments, the first arenavirus genomic segment is an L segment and the second arenavirus genomic segment is an S segment. In other embodiments, the first arenavirus genomic segment is an S segment and the second arenavirus genomic segment is an L segment.
Non-limiting examples of the arenavirus viral vector comprising a genomic segment with an ORF in a position other than the wild-type position of the ORF and a second genomic segment are illustrated in Table 1
In certain embodiments, provided herein is an arenavirus genomic segment that can be suitable for use as a vaccine and methods of using such arenavirus genomic segment in a vaccination and treatment or prevention of, for example, infections and cancers. For example, in certain embodiments, an arenavirus genomic segment provided herein with a heterologous nucleotide sequence that encodes antigens of interest, an immunomodulatory peptide, polypeptide, or protein , a signal sequence, and/or a linker can be used as a vaccine in the methods provided herein or as a component of compositions provided herein. More detailed description of the methods of using the arenavirus genomic segment described herein is provided in Section 6.7.
In certain embodiments, provided herein is an arenavirus genomic segment that can be suitable for use as a pharmaceutical composition and methods of using such arenavirus genomic segment in a vaccination and treatment or prevention of, for example, infections or cancers. For example, in certain embodiments, an arenavirus genomic segment provided herein with a heterologous nucleotide sequence that encodes antigens of interest, an immunomodulatory peptide, polypeptide, or protein, a signal sequence, and/or a linker can be used in the methods provided herein or as a component of compositions provided herein. More detailed description of the methods of using the arenavirus genomic segment described herein is provided in Section 6.7.
Also provided herein, is a cDNA of the arenavirus genomic segment engineered to carry an ORF in a position other than the wild-type position of the ORF. In more specific embodiments, provided herein is a cDNA or a set of cDNAs of an arenavirus genome as set forth in Table 1.
In certain embodiments, a cDNA of the arenavirus genomic segment that is engineered to carry an ORF in a position other than the wild-type position of the ORF is part of or incorporated into a DNA expression vector. In a specific embodiment, a cDNA of the arenavirus genomic segment that is engineered to carry an ORF in a position other than the wild-type position of the ORF is part of or incorporated into a DNA expression vector that facilitates production of an arenavirus genomic segment as described herein. In another embodiment, a cDNA described herein can be incorporated into a plasmid. More detailed description of the cDNAs or nucleic acids and expression systems are provided is Section 6.8 Techniques for the production of a cDNA are routine and conventional techniques of molecular biology and DNA manipulation and production. Any cloning technique known to the skilled artesian can be used. Such techniques are well known and are available to the skilled artesian in laboratory manuals such as, Sambrook and Russell, Molecular Cloning: A laboratory Manual, 3rdedition, Cold Spring Harbor Laboratory N.Y. (2001).
In certain embodiments, the cDNA of the arenavirus genomic segment that is engineered to carry an ORF in a position other than the wild-type position of the ORF is introduced (e.g., transfected) into a host cell. Thus, in some embodiments provided herein, is a host cell comprising a cDNA of the arenavirus genomic segment that is engineered to carry an ORF in a position other than the wild-type position of the ORF (i.e., a cDNA of the genomic segment). In other embodiments, the cDNA described herein is part of or can be incorporated into a DNA expression vector and introduced into a host cell. Thus, in some embodiments provided herein is a host cell comprising a cDNA described herein that is incorporated into a vector. In other embodiments, the arenavirus genomic segment described herein is introduced into a host cell.
In certain embodiments, described herein is a method of producing the arenavirus genomic segment, wherein the method comprises transcribing the cDNA of the arenavirus genomic segment. In certain embodiments, a viral polymerase protein can be present during transcription of the arenavirus genomic segment in vitro or in vivo.
In certain embodiments, transcription of the arenavirus genomic segment is performed using a bi-directional promoter. In other embodiments, transcription of the arenavirus genomic segment is performed using a bi-directional expression cassette (see e.g., Ortiz-Riaño et al., 2013, J Gen Virol., 94(Pt 6): 1175-1188). In more specific embodiments the bi-directional expression cassette comprises both a polymerase I and a polymerase II promoter reading from opposite sides into the two termini of the inserted arenavirus genomic segment, respectively. In yet more specific embodiments the bi-directional expression cassette with pol-I and pol-II promoters read from opposite sides into the L segment and S segment
In other embodiments, transcription of the cDNA of the arenavirus genomic segment described herein comprises a promoter. Specific examples of promoters include an RNA polymerase I promoter, an RNA polymerase II promoter, an RNA polymerase III promoter, a T7 promoter, an SP6 promoter or a T3 promoter.
In certain embodiments, the method of producing the arenavirus genomic segment can further comprise introducing into a host cell the cDNA of the arenavirus genomic segment. In certain embodiments, the method of producing the arenavirus genomic segment can further comprise introducing into a host cell the cDNA of the arenavirus genomic segment, wherein the host cell expresses all other components for production of the arenavirus genomic segment; and purifying the arenavirus genomic segment from the supernatant of the host cell. Such methods are well-known to those skilled in the art.
Provided herein are cell lines, cultures and methods of culturing cells infected with nucleic acids, vectors, and compositions provided herein. More detailed description of nucleic acids, vector systems and cell lines described herein is provided in Section 6.8.
In certain embodiments, the arenavirus viral vector as described herein results in an infectious and replication-competent arenavirus viral vector. In specific embodiments, the arenavirus viral vector described herein is attenuated. In a particular embodiment, the arenavirus viral vector is attenuated such that the virus remains, at least partially, able to spread and can replicate in vivo, but can only generate low viral loads resulting in subclinical levels of infection that are non-pathogenic. Such attenuated viruses can be used as an immunogenic composition. Provided herein, are immunogenic compositions that comprise an arenavirus with an ORF in a non-natural position as described in Section 6.6.
In certain embodiments, provided herein is an arenavirus viral vector that can be suitable for use as a vaccine and methods of using such arenavirus viral vector in a vaccination and treatment or prevention of, for example, infections and cancers. For example, in certain embodiments, an arenavirus viral vector provided herein with a heterologous nucleotide sequence that encodes antigens of interest, an immunomodulatory peptide, polypeptide, or protein , a signal sequence, and/or a linker can be used as a vaccine in the methods provided herein or as a component of compositions provided herein. More detailed description of the methods of using the arenavirus viral vector described herein is provided in Section 6.7.
In certain embodiments, provided herein is an arenavirus viral vector that can be suitable for use as a pharmaceutical composition and methods of using such arenavirus viral vector in a vaccination and treatment or prevention of, for example, infections or cancers. For example, in certain embodiments, an arenavirus viral vector provided herein with a heterologous nucleotide sequence that encodes antigens of interest, an immunomodulatory peptide, polypeptide, or protein, a signal sequence, and/or a linker can be used in the methods provided herein or as a component of compositions provided herein. More detailed description of the methods of using the arenavirus viral vector described herein is provided in Section 6.7.
(a) Replication-Deficient Arenavirus Particle with an Open Reading Frame in a Non-Natural Position
In certain embodiments, provided herein is an arenavirus viral vector in which (i) an ORF is in a position other than the wild-type position of the ORF; and (ii) an ORF encoding GP, NP, Z protein, and L protein has been removed (e.g., deleted) or functionally inactivated such that the resulting virus cannot produce further infectious progeny virus particles. An arenavirus viral vector comprising a genetically modified genome in which one or more ORFs has been deleted or functionally inactivated can be produced in complementing cells (i.e., cells that express the arenavirus ORF that has been deleted or functionally inactivated). The genetic material of the resulting arenavirus viral vector can be transferred upon infection of a host cell into the host cell, wherein the genetic material can be expressed and amplified. In addition, the genome of the genetically modified arenavirus viral vector described herein can encode a heterologous ORF from an organism other than an arenavirus viral vector.
In certain embodiments, at least one of the four ORFs encoding GP, NP, Z protein, and L protein is removed and replaced with a heterologous ORF from an organism other than an arenavirus. In another embodiment, at least one ORF, at least two ORFs, at least three ORFs, or at least four ORFs encoding GP, NP, Z protein and L protein can be removed and replaced with a heterologous ORF from an organism other than an arenavirus, including a heterologous ORF as described in Section 6.5. In specific embodiments, only one of the four ORFs encoding GP, NP, Z protein, and L protein is removed and replaced with a heterologous ORF from an organism other than an arenavirus viral vector, including a heterologous ORF as described in Section 6.5. In more specific embodiments, the ORF that encodes GP of the arenavirus genomic segment is removed. In another specific embodiment, the ORF that encodes the NP of the arenavirus genomic segment is removed. In more specific embodiments, the ORF that encodes the Z protein of the arenavirus genomic segment is removed. In yet another specific embodiment, the ORF encoding the L protein is removed.
Thus, in certain embodiments, the arenavirus viral vector provided herein comprises a genomic segment that (i) is engineered to carry an arenavirus ORF in a non-natural position; (ii) an ORF encoding GP, NP, Z protein, or L protein is removed; (iii) the ORF that is removed is replaced with a heterologous ORF from an organism other than an arenavirus, including a heterologous ORF as described in Section 6.5.
In certain embodiments, the heterologous ORF is 8 to 100 nucleotides in length, 15 to 100 nucleotides in length, 25 to 100 nucleotides in length, 50 to 200 nucleotide in length, 50 to 400 nucleotide in length, 200 to 500 nucleotide in length, or 400 to 600 nucleotides in length, 500 to 800 nucleotide in length. In other embodiments, the heterologous ORF is 750 to 900 nucleotides in length, 800 to 1000 nucleotides in length, 850 to 1000 nucleotides in length, 900 to 1200 nucleotides in length, 1000 to 1200 nucleotides in length, 1000 to 1500 nucleotides or 1200 to 1500 nucleotides in length, 1500 to 2000 nucleotides in length, 1700 to 2000 nucleotides in length, 2000 to 2300 nucleotides in length, 2200 to 2500 nucleotides in length, 2500 to 3000 nucleotides in length, 3000 to 3200 nucleotides in length, 3000 to 3500 nucleotides in length, 3200 to 3600 nucleotides in length, 3300 to 3800 nucleotides in length, 4000 nucleotides to 4400 nucleotides in length, 4200 to 4700 nucleotides in length, 4800 to 5000 nucleotides in length, 5000 to 5200 nucleotides in length, 5200 to 5500 nucleotides in length, 5500 to 5800 nucleotides in length, 5800 to 6000 nucleotides in length, 6000 to 6400 nucleotides in length, 6200 to 6800 nucleotides in length, 6600 to 7000 nucleotides in length, 7000 to 7200 nucleotides in lengths, 7200 to 7500 nucleotides in length, or 7500 nucleotides or more in length. In some embodiments, the heterologous ORF encodes a peptide or polypeptide that is 5 to 10 amino acids in length, 10 to 25 amino acids in length, 25 to 50 amino acids in length, 50 to 100 amino acids in length, 100 to 150 amino acids in length, 150 to 200 amino acids in length, 200 to 250 amino acids in length, 250 to 300 amino acids in length, 300 to 400 amino acids in length, 400 to 500 amino acids in length, 500 to 750 amino acids in length, 750 to 1000 amino acids in length, 1000 to 1250 amino acids in length 1250 to 1500 amino acids in length, 1500 to 1750 amino acids in length, 1750 to 2000 amino acids in length, 2000 to 2500 amino acids in length, or more than 2500 or more amino acids in length. In some embodiments, the heterologous ORF encodes a polypeptide that does not exceed 2500 amino acids in length. In specific embodiments the heterologous ORF does not contain a stop codon. In certain embodiments, the heterologous ORF is codon-optimized. In certain embodiments the nucleotide composition, nucleotide pair composition or both can be optimized. Techniques for such optimizations are known in the art and can be applied to optimize a heterologous ORF.
Any heterologous ORF from an organism other than an arenavirus may be included in an arenavirus genomic segment. In one embodiment, the heterologous ORF encodes a reporter protein. More detailed description of reporter proteins are described in Section 6.5. In another embodiment, the heterologous ORF encodes an antigen for an infectious pathogen or an antigen associated with any disease that is capable of eliciting an immune response. In specific embodiments the antigen is derived from an infectious organism, a tumor (i.e., cancer), or an allergen. More detailed description on heterologous ORFs is described in Section 6.5.
In certain embodiments, the growth and infectivity of the arenavirus viral vector is not affected by the heterologous ORF from an organism other than an arenavirus.
Techniques known to one skilled in the art may be used to produce an arenavirus viral vector comprising an arenavirus genomic segment engineered to carry an arenavirus ORF in a position other than the wild-type position. For example, reverse genetics techniques may be used to generate such arenavirus viral vector. In other embodiments, the replication-deficient arenavirus viral vector (i.e., the arenavirus genomic segment engineered to carry an arenavirus ORF in a position other than the wild-type position, wherein an ORF encoding GP, NP, Z protein, L protein, has been deleted) can be produced in a complementing cell.
In certain embodiments, the arenavirus genomic segment or the arenavirus viral vector using according to the present application can be Old World Viruses, for example, LCMV.
In certain embodiments, the present application relates to the arenavirus viral vector as described herein suitable for use as a vaccine and methods of using such arenavirus viral vector in a vaccination and treatment or prevention of, for example, infections or cancers. More detailed description of the methods of using the arenavirus viral vector described herein is provided in Section 6.7.
In certain embodiments, provided herein is a kit comprising, in one or more containers, one or more cDNAs described herein. In a specific embodiment, a kit comprises, in one or two or more containers, an arenavirus genomic segment or an arenavirus viral vector as described herein. The kit may further comprise one or more of the following: a host cell suitable for rescue of the arenavirus genomic segment or the arenavirus viral vector, reagents suitable for transfecting plasmid cDNA into a host cell, a helper virus, plasmids encoding viral proteins and/or one or more primers specific for an modified arenavirus genomic segment or arenavirus viral vector or cDNAs of the same.
In certain embodiments, the present application relates to the arenavirus viral vector as described herein suitable for use as a pharmaceutical composition and methods of using such arenavirus viral vector in a vaccination and treatment or prevention of, for example, infections and cancers. More detailed description of the methods of using the arenavirus viral vector described herein is provided in Section 6.7.
6.3 Tri-Segmented Arenavirus Viral Vector
Provided herein are tri-segmented arenavirus viral vectors with rearrangements of their ORFs.
In one aspect, the tri-segmented arenavirus viral vector as provided herein comprises a heterologous nucleotide sequence that encodes antigens of interest, an immunomodulatory peptide, polypeptide, or protein , a signal sequence, and/or a linker. Such sequences and their arrangement are described in Section 6.5.
In another aspect, provided herein is a tri-segmented arenavirus viral vector comprising one L segment and two S segments or two L segments and one S segment. In certain embodiments, the tri-segmented arenavirus viral vector does not recombine into a replication-competent bi-segmented arenavirus particle. In specific embodiments, the tri-segmented arenavirus viral vector comprises an ORF in a position other than the wild-type position of the ORF. In yet another specific embodiment, the tri-segmented arenavirus viral vector comprises all four arenavirus ORFs. Thus, in certain embodiments, the tri-segmented arenavirus viral vector is replication-competent and infectious. In other embodiments, the tri-segmented arenavirus viral vector lacks one of the four arenavirus ORFs. Thus, in certain embodiments, the tri-segmented arenavirus viral vector is infectious but is replication-deficient (i.e., unable to produce further infectious progeny in non-complementing cells).
In certain embodiments, the ORF encoding GP, NP, Z protein, or the L protein of the tri-segmented arenavirus viral vector described herein can be under the control of an arenavirus 3′ UTR or an arenavirus 5′ UTR. In more specific embodiments, the tri-segmented arenavirus 3′ UTR is the 3′ UTR of an arenavirus S segment(s). In another specific embodiment, the tri-segmented arenavirus 3′ UTR is the 3′ UTR of a tri-segmented arenavirus L segment(s). In more specific embodiments, the tri-segmented arenavirus 5′ UTR is the 5′ UTR of an arenavirus S segment(s). In other specific embodiments, the 5′ UTR is the 5′ UTR of the L segment(s).
In other embodiments, the ORF encoding GP, NP, Z protein, or the L protein of tri-segmented arenavirus viral vector described herein can be under the control of the arenavirus conserved terminal sequence element (the 5′- and 3′-terminal 19-20-nt regions) (see e.g., Perez & de la Torre, 2003, J Virol. 77(2): 1184-1194).
In certain embodiments, the ORF encoding GP, NP, Z protein or the L protein of the tri-segmented arenavirus viral vector can be under the control of the promoter element of the 5′ UTR (see e.g., Albarino et al., 2011, J Virol., 85(8):4020-4). In another embodiment, the ORF encoding GP, NP Z protein, L protein of the tri-segmented arenavirus viral vector can be under the control of the promoter element of the 3′ UTR (see e.g., Albarino et al., 2011, J Virol., 85(8):4020-4). In more specific embodiments, the promoter element of the 5′ UTR is the 5′ UTR promoter element of the S segment(s) or the L segment(s). In another specific embodiment, the promoter element of the 3′ UTR is the 3′ UTR the promoter element of the S segment(s) or the L segment(s).
In certain embodiments, the ORF that encoding GP, NP, Z protein or the L protein of the tri-segmented arenavirus viral vector can be under the control of a truncated arenavirus 3′ UTR or a truncated arenavirus 5′ UTR (see e.g., Perez & de la Torre, 2003, J Virol. 77(2): 1184-1194; Albarino et al., 2011, J Virol., 85(8):4020-4). In more specific embodiments, the truncated 3′ UTR is the 3′ UTR of the arenavirus S segment or L segment. In more specific embodiments, the truncated 5′ UTR is the 5′ UTR of the arenavirus S segment(s) or L segment(s).
Also provided herein, is a cDNA of the tri-segmented arenavirus viral vector. In more specific embodiments, provided herein is a DNA nucleotide sequence or a set of DNA nucleotide sequences encoding a tri-segmented arenavirus viral vector as set forth in Table 2 or Table 3.
In certain embodiments, the nucleic acids encoding the tri-segmented arenavirus genome are part of or incorporated into one or more DNA expression vectors. In a specific embodiment, nucleic acids encoding the genome of the tri-segmented arenavirus viral vector are part of or incorporated into one or more DNA expression vectors that facilitate production of a tri-segmented arenavirus viral vector as described herein. In another embodiment, a cDNA described herein can be incorporated into a plasmid. More detailed description of the cDNAs and expression systems are provided is Section 6.8. Techniques for the production of a cDNA routine and conventional techniques of molecular biology and DNA manipulation and production. Any cloning technique known to the skilled artesian can be used. Such techniques are well known and are available to the skilled artesian in laboratory manuals such as, Sambrook and Russell, Molecular Cloning: A laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory N.Y. (2001).
In certain embodiments, the cDNA of the tri-segmented arenavirus is introduced (e.g., transfected) into a host cell. Thus, in some embodiments provided herein, is a host cell comprising a cDNA of the tri-segmented arenavirus viral vector (i.e., a cDNA of the genomic segments of the tri-segmented arenavirus viral vector). In other embodiments, the cDNA described herein that is part of or can be incorporated into a DNA expression vector and introduced into a host cell. Thus, in some embodiments provided herein is a host cell comprising a cDNA described herein that is incorporated into a vector. In other embodiments, the tri-segmented arenavirus genomic segments (i.e., the L segment and/or S segment or segments) described herein is introduced into a host cell.
In certain embodiments, described herein is a method of producing the tri-segmented arenavirus viral vector, wherein the method comprises transcribing the cDNA of the tri-segmented arenavirus viral vector. In certain embodiments, a viral polymerase protein can be present during transcription of the tri-segmented arenavirus viral vector in vitro or in vivo. In certain embodiments, transcription of the arenavirus genomic segment is performed using a bi-directional promoter.
In other embodiments, transcription of the arenavirus genomic segment is performed using a bi-directional expression cassette (see e.g., Ortiz-Riaño et al., 2013, J Gen Virol., 94(Pt 6): 1175-1188). In more specific embodiments the bi-directional expression cassette comprises both a polymerase I and a polymerase II promoter reading from opposite sides into the two termini of the inserted arenavirus genomic segment, respectively.
In other embodiments, transcription of the cDNA of the arenavirus genomic segment described herein comprises a promoter. Specific examples of promoters include an RNA polymerase I promoter, an RNA polymerase II promoter, an RNA polymerase III promoter, a T7 promoter, an SP6 promoter, or a T3 promoter.
In certain embodiments, the method of producing the tri-segmented arenavirus viral vector can further comprise introducing into a host cell the cDNA of the tri-segmented arenavirus viral vector. In certain embodiments, the method of producing the tri-segmented arenavirus viral vector can further comprise introducing into a host cell the cDNA of the tri-segmented arenavirus viral vector, wherein the host cell expresses all other components for production of the tri-segmented arenavirus viral vector; and purifying the tri-segmented arenavirus viral vector from the supernatant of the host cell. Such methods are well-known to those skilled in the art.
Provided herein are cell lines, cultures and methods of culturing cells infected with nucleic acids, vectors, and compositions provided herein. More detailed description of nucleic acids, vector systems and cell lines described herein is provided in Section 6.8.
In certain embodiments, the tri-segmented arenavirus viral vector as described herein results in a infectious and replication-competent arenavirus viral vector. In specific embodiments, the arenavirus viral vector described herein is attenuated. In a particular embodiment, the tri-segmented arenavirus viral vector is attenuated such that the virus remains, at least partially, replication-competent and can replicate in vivo, but can only generate low viral loads resulting in subclinical levels of infection that are non-pathogenic. Such attenuated viruses can be used as an immunogenic composition.
In certain embodiments, the tri-segmented arenavirus viral vector has the same tropism as the bi-segmented arenavirus particle.
Also provided herein is a kit comprising, in one or more containers, one or more cDNAs described herein. In a specific embodiment, a kit comprises, in one or two or more containers a tri-segmented arenavirus viral vector as described herein. The kit may further comprise one or more of the following: a host cell suitable for rescue of the tri-segmented arenavirus viral vector, reagents suitable for transfecting plasmid cDNA into a host cell, a helper virus, plasmids encoding viral proteins and/or one or more oligonucleotide primers specific for a modified arenavirus genomic segment or arenavirus viral vector or nucleic acids encoding the same.
Also provided herein, are immunogenic compositions that comprise the tri-segmented arenavirus viral vector as described in Section 6.6.
In certain embodiments, provided herein is a tri-segmented arenavirus viral vector that can be suitable for use as a vaccine and methods of using such arenavirus viral vector in a vaccination and treatment or prevention of, for example, infections and cancers. For example, in certain embodiments, a tri-segmented arenavirus viral vector provided herein with rearrangements of it ORF's and a heterologous nucleotide sequence that encodes antigens of interest, an immunomodulatory peptide, polypeptide, or protein , a signal sequence, and/or a linker can be used as a vaccine in the methods provided herein or as a component of compositions provided herein. More detailed description of the methods of using the tri-segmented arenavirus viral vector described herein is provided in Section 6.7.
In certain embodiments, provided herein is a tri-segmented arenavirus viral vector that can be suitable for use as a pharmaceutical composition and methods of using such arenavirus viral vector in a vaccination and treatment or prevention of, for example, infections or cancers. For example, in certain embodiments, a tri-segmented arenavirus viral vector provided herein with rearrangements of it ORF's and a heterologous nucleotide sequence that encodes antigens of interest, an immunomodulatory peptide, polypeptide, or protein , a signal sequence, and/or a linker can be used in the methods provided herein or as a component of compositions provided herein. More detailed description of the methods of using the arenavirus viral vector described herein is provided in Section 6.7.
(a) Tri-Segmented Arenavirus Viral Vector Comprising One L Segment and Two S Segments
In one aspect, provided herein is a tri-segmented arenavirus viral vector comprising one L segment and two S segments. In certain embodiments, propagation of the tri-segmented arenavirus viral vector comprising one L segment and two S segments does not result in a replication-competent bi-segmented viral vector. In specific embodiments, propagation of the tri-segmented arenavirus viral vector comprising one L segment and two S segments does not result in a replication-competent bi-segmented viral particle after at least 10 days, at least 20 days, at least 30 days, at least 40 days, at least 50 days, at least 60 days, at least 70 days, at least 80 days, at least 90 days, or at least 100 days of persistent infection in mice lacking type I interferon receptor, type II interferon receptor and recombination activating gene (RAG1), and having been infected with 104 PFU of the tri-segmented arenavirus viral vector. In other embodiments, propagation of the tri-segmented arenavirus viral vector comprising one L segment and two S segments does not result in a replication-competent bi-segmented viral vector after at least 10 passages, at least 20 passages, at least 30 passages, at least 40 passages, or at least 50 passages.
In one aspect, the tri-segmented arenavirus viral vector comprising one L segment and two S segments further comprises a heterologous nucleotide sequence that encodes antigens of interest, an immunomodulatory peptide, polypeptide, or protein , a signal sequence, and/or a linker. Such sequences and their arrangement are described in Section 6.5.
The tri-segmented arenavirus viral vector with all viral genes in their respective wild-type position is known in the art (e.g., Emonet et al., 2011 J. Virol., 85(4):1473; Popkin et al., 2011, J. Virol, 85(15):7928). In particular, the tri-segmented arenavirus genome consists of one L segment and two S segments, in which a heterologous ORF (Section 6.5) is inserted into one position on each S segment. More specifically, one S segment encodes GP and an HPV antigen, respectively. The other S segment encodes an HPV antigen and NP, respectively. The L segment encodes the L protein and Z protein. All segments are flanked by the respective 5′ and 3′ UTRs.
In certain embodiments, inter-segmental recombination of the two S segments of the tri-segmented arenavirus viral vector, provided herein, that unities the two arenaviral ORFs on one instead of two separate segments results in a non functional promoter (i.e., a genomic segment of the structure: 5′ UTR-5′ UTR or a 3′ UTR-3′ UTR), wherein each UTR forming one end of the genome is an inverted repeat sequence of the other end of the same genome.
In certain embodiments, the tri-segmented arenavirus viral vector comprising one L segment and two S segments has been engineered to carry an arenavirus ORF in a position other than the wild-type position of the ORF. In other embodiments, the tri-segmented arenavirus viral vector comprising one L segment and two S segments has been engineered to carry two arenavirus ORFs, or three arenavirus ORFs, or four arenavirus ORFs, or five arenavirus ORFs, or six arenavirus ORFs in a position other than the wild-type position. In specific embodiments, the tri-segmented arenavirus viral vector comprising one L segment and two S segments comprises a full complement of all four arenavirus ORFs. Thus, in some embodiments, the tri-segmented arenavirus viral vector is an infectious and replication-competent tri-segmented arenavirus viral vector. In specific embodiments, the two S segments of the tri-segmented arenavirus viral vector have been engineered to carry one of their ORFs in a position other than the wild-type position. In more specific embodiments, the two S segments comprise a full complement of the S segment ORF's. In certain specific embodiments, the L segment has been engineered to carry an ORF in a position other than the wild-type position or the L segment can be the wild-type genomic segment.
In certain embodiments, one of the two S segments can be:
In certain embodiments, the tri-segmented arenavirus viral vector comprising one L segment and two S segments can comprise a duplicate ORF (i.e., two wild-type S segment ORFs e.g., GP or NP). In specific embodiments, the tri-segmented arenavirus viral vector comprising one L segment and two S segments can comprise one duplicate ORF (e.g., (GP, GP)) or two duplicate ORFs (e.g., (GP, GP) and (NP, NP)).
Table 2A, below, is an illustration of the genome organization of a tri-segmented arenavirus viral vector comprising one L segment and two S segments, wherein intersegmental recombination of the two S segments in the tri-segmented arenavirus genome does not result in a replication-competent bi-segmented viral vector and abrogates arenaviral promoter activity (i.e., the resulting recombined S segment is made up of two 3′UTRs instead of a 3′ UTR and a 5′ UTR).
In certain embodiments, the IGR between position one and position two can be an arenavirus S segment or L segment IGR; the IGR between position two and three can be an arenavirus S segment or L segment IGR; and the IGR between the position five and six can be an arenavirus L segment IGR. In a specific embodiment, the IGR between position one and position two can be an arenavirus S segment IGR; the IGR between position two and three can be an arenavirus S segment IGR; and the IGR between the position five and six can be an arenavirus L segment IGR. In certain embodiments, other combinations are also possible. For example, a tri-segmented arenavirus viral vector comprising one L segment and two S segments, wherein intersegmental recombination of the two S segments in the tri-segmented arenavirus genome does not result in a replication-competent bi-segmented viral vector and abrogates arenaviral promoter activity (i.e., the resulting recombined S segment is made up of two 5′UTRs instead of a 3′ UTR and a 5′ UTR).
In certain embodiments, intersegmental recombination of an S segment and an L segment in the tri-segmented arenavirus viral vector comprising one L segment and two S segments, restores a functional segment with two viral genes on only one segment instead of two separate segments. In other embodiments, intersegmental recombination of an S segment and an L segment in the tri-segmented arenavirus viral vector comprising one L segment and two S segments does not result in a replication-competent bi-segmented viral particle.
Table 2B, below, is an illustration of the genome organization of a tri-segmented arenavirus viral vector comprising one L segment and two S segments, wherein intersegmental recombination of an S segment and an L segment in the tri-segmented arenavirus genome does not result in a replication-competent bi-segmented viral particle and abrogates arenaviral promoter activity (i.e., the resulting recombined segment is made up of two 3′UTRs instead of a 3′ UTR and a 5′ UTR).
In certain embodiments, the IGR between position one and position two can be an arenavirus S segment or L segment IGR; the IGR between position two and three can be an arenavirus S segment or L segment IGR; and the IGR between the position five and six can be an arenavirus L segment IGR. In a specific embodiment, the IGR between position one and position two can be an arenavirus S segment IGR; the IGR between position two and three can be an arenavirus S segment IGR; and the IGR between the position five and six can be an arenavirus L segment IGR. In certain embodiments, other combinations are also possible. For example, a tri-segmented arenavirus viral vector comprising one L segment and two S segments, wherein intersegmental recombination of the two S segments in the tri-segmented arenavirus genome does not result in a replication-competent bi-segmented viral particle and abrogates arenaviral promoter activity (i.e., the resulting recombined S segment is made up of two 5′UTRs instead of a 3′ UTR and a 5′ UTR).
In certain embodiments, one skilled in the art could construct an arenavirus genome with an organization as illustrated in Table 2A or 2B and as described herein, and then use an assay as described in Section 6.9 to determine whether the tri-segmented arenavirus viral vector is genetically stable, i.e., does not result in a replication-competent bi-segmented viral particle as discussed herein.
(b) Tri-Segmented Arenavirus Viral Vector Comprising Two L Segments and One S Segment
In one aspect, provided herein is a tri-segmented arenavirus viral vector comprising two L segments and one S segment. In certain embodiments, propagation of the tri-segmented arenavirus viral vector comprising two L segments and one S segment does not result in a replication-competent bi-segmented viral particle. In specific embodiments, propagation of the tri-segmented arenavirus viral vector comprising two L segments and one S segment does not result in a replication-competent bi-segmented viral particle after at least 10 days, at least 20 days, at least 30 days, at least 40 days, or at least 50 days, at least 60 days, at least 70 days, at least 80 days, at least 90 days, at least 100 days of persistent in mice lacking type I interferon receptor, type II interferon receptor and recombination activating gene (RAG1), and having been infected with 104 PFU of the tri-segmented arenavirus viral vector. In other embodiments, propagation of the tri-segmented arenavirus viral vector comprising two L segments and one S segment does not result in a replication-competent bi-segmented viral particle after at least 10 passages, 20 passages, 30 passages, 40 passages, or 50 passages.
In one aspect, the tri-segmented arenavirus viral vector comprising two L segments and one S segment further comprises a heterologous nucleotide sequence that encodes antigens of interest, an immunomodulatory peptide, polypeptide, or protein , a signal sequence, and/or a linker. Such sequences and their arrangement are described in Section 6.5.
In certain embodiments, inter-segmental recombination of the two L segments of the tri-segmented arenavirus viral vector, provided herein, that unities the two arenaviral ORFs on one instead of two separate segments results in a non functional promoter (i.e., a genomic segment of the structure: 5′ UTR-5′ UTR or a 3′ UTR-3′ UTR), wherein each UTR forming one end of the genome is an inverted repeat sequence of the other end of the same genome.
In certain embodiments, the tri-segmented arenavirus viral vector comprising two L segments and one S segment has been engineered to carry an arenavirus ORF in a position other than the wild-type position of the ORF. In other embodiments, the tri-segmented arenavirus viral vector comprising two L segments and one S segment has been engineered to carry two arenavirus ORFs, or three arenavirus ORFs, or four arenavirus ORFs, or five arenavirus ORFs, or six arenavirus ORFs in a position other than the wild-type position. In specific embodiments, the tri-segmented arenavirus viral vector comprising two L segments and one S segment comprises a full complement of all four arenavirus ORFs. Thus, in some embodiments, the tri-segmented arenavirus viral vector is an infectious and replication-competent tri-segmented arenavirus viral vector. In specific embodiments, the two L segments of the tri-segmented arenavirus viral vector have been engineered to carry one of their ORFs in a position other than the wild-type position. In more specific embodiments, the two L segments comprise a full complement of the L segment ORF's. In certain specific embodiments, the S segment has been engineered to carry one of their ORFs in a position other than the wild-type position or the S segment can be the wild-type genomic segment.
In certain embodiments, one of the two L segments can be:
In certain embodiments, the tri-segmented arenavirus viral vector comprising two L segments and one S segment can comprise a duplicate ORF (i.e., two wild-type L segment ORFs e.g., Z protein or L protein). In specific embodiments, the tri-segmented arenavirus viral vector comprising two L segments and one S segment can comprise one duplicate ORF (e.g., (Z protein, Z protein)) or two duplicate ORFs (e.g., (Z protein, Z protein) and (L protein, L protein)).
Table 3, below, is an illustration of the genome organization of a tri-segmented arenavirus viral vector comprising two L segments and one S segment, wherein intersegmental recombination of the two L segments in the tri-segmented arenavirus genome does not result in a replication-competent bi-segmented viral vector and abrogates arenaviral promoter activity (i.e., the putatively resulting recombinant L segment would be made up of two 3′UTRs instead of a 3′ UTR and a 5′ UTR). Based on Table 3 similar combinations could be predicted for generating an arenavirus viral vector made up of two 5′ UTRs instead of a 3′ UTR and a 5′ UTR.
In certain embodiments, the IGR between position one and position two can be an arenavirus S segment or L segment IGR; the IGR between position two and three can be an arenavirus S segment or L segment IGR; and the IGR between the position five and six can be an arenavirus S or L segment IGR. In a specific embodiment, the IGR between position one and position two can be an arenavirus L segment IGR; the IGR between position two and three can be an arenavirus L segment IGR; and the IGR between the position five and six can be an arenavirus S segment IGR. In certain embodiments, other combinations are also possible.
In certain embodiments, intersegmental recombination of an L segment and an S segment from the tri-segmented arenavirus viral vector comprising two L segments and one S segment restores a functional segment with two viral genes on only one segment instead of two separate segments. In other embodiments, intersegmental recombination of an L segment and an S segment in the tri-segmented arenavirus viral vector comprising two L segments and one S segment does not result in a replication-competent bi-segmented viral particle.
Table 3B, below, is an illustration of the genome organization of a tri-segmented arenavirus viral vector comprising two L segments and one S segment, wherein intersegmental recombination of an L segment and an S segment in the tri-segmented arenavirus genome does not result in a replication-competent bi-segmented viral particle and abrogates arenaviral promoter activity (i.e., the resulting recombined segment is made up of two 3′UTRs instead of a 3′ UTR and a 5′ UTR).
In certain embodiments, the IGR between position one and position two can be an arenavirus S segment or L segment IGR; the IGR between position two and three can be an arenavirus S segment or L segment IGR; and the IGR between the position five and six can be an arenavirus S segment or L segment IGR. In a specific embodiment, the IGR between position one and position two can be an arenavirus L segment IGR; the IGR between position two and three can be an arenavirus L segment IGR; and the IGR between the position five and six can be an arenavirus S segment IGR. In certain embodiments, other combinations are also possible.
In certain embodiments, one skilled in the art could construct an arenavirus genome with an organization as illustrated in Table 3A or 3B and as described herein, and then use an assay as described in Section 6.9 to determine whether the tri-segmented arenavirus viral vector is genetically stable, i.e., does not result in a replication-competent bi-segmented viral vector as discussed herein.
(c) Replication-Deficient Tri-Segmented Arenavirus Viral Vector
In certain embodiments, provided herein is a tri-segmented arenavirus viral vector in which (i) an ORF is in a position other than the wild-type position of the ORF; and (ii) an ORF encoding GP, NP, Z protein, or L protein has been removed or functionally inactivated such that the resulting virus cannot produce further infectious progeny virus particles (i.e., is replication defective). In certain embodiments, the third arenavirus segment can be an S segment. In other embodiments, the third arenavirus segment can be an L segment. In more specific embodiments, the third arenavirus segment can be engineered to carry an ORF in a position other than the wild-type position of the ORF or the third arenavirus segment can be the wild-type arenavirus genomic segment. In yet more specific embodiments, the third arenavirus segment lacks an arenavirus ORF encoding GP, NP, Z protein, or the L protein.
In one aspect, the replication-deficient tri-segmented arenavirus viral vector provided herein further comprises a heterologous nucleotide sequence that encodes antigens of interest, an immunomodulatory peptide, polypeptide, or protein, a signal sequence, and/or a linker. Such sequences and their arrangement are described in Section 6.5.
In certain embodiments, a tri-segmented genomic segment could be a S or a L segment hybrid (i.e., a genomic segment that can be a combination of the S segment and the L segment). In other embodiments, the hybrid segment is an S segment comprising an L segment IGR. In another embodiment, the hybrid segment is an L segment comprising an S segment IGR. In other embodiments, the hybrid segment is an S segment UTR with an L segment IGR. In another embodiment, the hybrid segment is an L segment UTR with an S segment IGR. In specific embodiments, the hybrid segment is an S segment 5′ UTR with an L segment IGR or an S segment 3′ UTR with an L segment IGR. In other specific embodiments, the hybrid segment is an L segment 5′ UTR with an S segment IGR or an L segment 3′ UTR with an S segment IGR.
A tri-segmented arenavirus viral vector comprising a genetically modified genome in which one or more ORFs has been removed (e.g., deleted) or functionally inactivated can be produced in complementing cells (i.e., cells that express the arenavirus ORF that has been removed or functionally inactivated). The genetic material of the resulting arenavirus viral vector can be transferred upon infection of a host cell into the host cell, wherein the genetic material can be expressed and amplified. In addition, the genome of the genetically modified arenavirus viral vector described herein can encode a heterologous ORF from an organism other than an arenavirus viral vector.
In certain embodiments, at least one of the four ORFs encoding GP, NP, Z protein, and L protein is removed and replaced with a heterologous ORF from an organism other than an arenavirus. In another embodiment, at least one ORF, at least two ORFs, at least three ORFs, or at least four ORFs encoding GP, NP, Z protein and L protein can be removed and replaced with a heterologous ORF from an organism other than an arenavirus. In specific embodiments, only one of the four ORFs encoding GP, NP, Z protein, and L protein is removed and replaced with a heterologous ORF from an organism other than an arenavirus viral vector. In more specific embodiments, the ORF that encodes GP of the arenavirus genomic segment is removed. In another specific embodiment, the ORF that encodes the NP of the arenavirus genomic segment is removed. In more specific embodiments, the ORF that encodes the Z protein of the arenavirus genomic segment is removed. In yet another specific embodiment, the ORF encoding the L protein is removed.
In certain embodiments, provided herein is a tri-segmented arenavirus viral vector comprising one L segment and two S segments in which (i) an ORF is in a position other than the wild-type position of the ORF; and (ii) an ORF encoding GP or NP has been removed or functionally inactivated, such that the resulting virus is replication-deficient and not infectious. In a specific embodiment, one ORF is removed and replaced with a heterologous ORF from an organism other than an arenavirus. In another specific embodiment, two ORFs are removed and replaced with heterologous ORFs from an organism other than an arenavirus. In other specific embodiments, three ORFs are removed and replaced with heterologous ORFs from an organism other than an arenavirus. In specific embodiments, the ORF encoding GP is removed and replaced with a heterologous ORF from an organism other than an arenavirus. In other specific embodiments, the ORF encoding NP is removed and replaced with a heterologous ORF from an organism other than an arenavirus. In yet more specific embodiments, the ORF encoding NP and the ORF encoding GP are removed and replaced with one or two heterologous ORFs from an organism other than an arenavirus viral vector. Thus, in certain embodiments the tri-segmented arenavirus viral vector comprises (i) one L segment and two S segments; (ii) an ORF in a position other than the wild-type position of the ORF; (iii) one or more heterologous ORFs from an organism other than an arenavirus.
In certain embodiments, provided herein is a tri-segmented arenavirus viral vector comprising two L segments and one S segment in which (i) an ORF is in a position other than the wild-type position of the ORF; and (ii) an ORF encoding the Z protein, and/or the L protein has been removed or functionally inactivated, such that the resulting virus replication-deficient and not infectious. In a specific embodiment, one ORF is removed and replaced with a heterologous ORF from an organism other than an arenavirus. In another specific embodiment, two ORFs are removed and replaced with a heterologous ORF from an organism other than an arenavirus. In specific embodiments, the ORF encoding the Z protein is removed and replaced with a heterologous ORF from an organism other than an arenavirus. In other specific embodiments, the ORF encoding the L protein is removed and replaced with a heterologous ORF from an organism other than an arenavirus. In yet more specific embodiments, the ORF encoding the Z protein and the ORF encoding the L protein is removed and replaced with a heterologous ORF from an organism other than an arenavirus viral vector. Thus, in certain embodiments the tri-segmented arenavirus viral vector comprises (i) two L segments and one S segment; (ii) an ORF in a position other than the wild-type position of the ORF; (iii) a heterologous ORF from an organism other than an arenavirus.
Thus, in certain embodiments, the tri-segmented arenavirus viral vector provided herein comprises a tri-segmented arenavirus viral vector (i.e., one L segment and two S segments or two L segments and one S segment) that i) is engineered to carry an ORF in a non-natural position; ii) an ORF encoding GP, NP, Z protein, or L protein is removed); and iii) the ORF that is removed is replaced with one or more heterologous ORFs from an organism other than an arenavirus (Section 6.5).
In certain embodiments, the heterologous ORF is 8 to 100 nucleotides in length, 15 to 100 nucleotides in length, 25 to 100 nucleotides in length, 50 to 200 nucleotide in length, 50 to 400 nucleotide in length, 200 to 500 nucleotide in length, or 400 to 600 nucleotides in length, 500 to 800 nucleotide in length. In other embodiments, the heterologous ORF is 750 to 900 nucleotides in length, 800 to 100 nucleotides in length, 850 to 1000 nucleotides in length, 900 to 1200 nucleotides in length, 1000 to 1200 nucleotides in length, 1000 to 1500 nucleotides or 1200 to 1500 nucleotides in length, 1500 to 2000 nucleotides in length, 1700 to 2000 nucleotides in length, 2000 to 2300 nucleotides in length, 2200 to 2500 nucleotides in length, 2500 to 3000 nucleotides in length, 3000 to 3200 nucleotides in length, 3000 to 3500 nucleotides in length, 3200 to 3600 nucleotides in length, 3300 to 3800 nucleotides in length, 4000 nucleotides to 4400 nucleotides in length, 4200 to 4700 nucleotides in length, 4800 to 5000 nucleotides in length, 5000 to 5200 nucleotides in length, 5200 to 5500 nucleotides in length, 5500 to 5800 nucleotides in length, 5800 to 6000 nucleotides in length, 6000 to 6400 nucleotides in length, 6200 to 6800 nucleotides in length, 6600 to 7000 nucleotides in length, 7000 to 7200 nucleotides in lengths, 7200 to 7500 nucleotides in length, or 7500 nucleotides or more in length. In some embodiments, the heterologous ORF encodes a peptide or polypeptide that is 5 to 10 amino acids in length, 10 to 25 amino acids in length, 25 to 50 amino acids in length, 50 to 100 amino acids in length, 100 to 150 amino acids in length, 150 to 200 amino acids in length, 200 to 250 amino acids in length, 250 to 300 amino acids in length, 300 to 400 amino acids in length, 400 to 500 amino acids in length, 500 to 750 amino acids in length, 750 to 1000 amino acids in length, 1000 to 1250 amino acids in length 1250 to 1500 amino acids in length, 1500 to 1750 amino acids in length, 1750 to 2000 amino acids in length, 2000 to 2500 amino acids in length, or more than 2500 amino acids in length. In some embodiments, the heterologous ORF encodes a polypeptide that does not exceed 2500 amino acids in length. In specific embodiments the heterologous ORF does not contain a stop codon. In certain embodiments, the heterologous ORF is codon-optimized. In certain embodiments the nucleotide composition, nucleotide pair composition or both can be optimized. Techniques for such optimizations are known in the art and can be applied to optimize a heterologous ORF.
Any heterologous ORF from an organism other than an arenavirus may be included in the tri-segmented arenavirus viral vector. Thus, in certain embodiments, a tri-segmented arenavirus viral vector provided herein comprises a) a deletion or functional inactivation of an open reading frame that is present in the wild type arenavirus; and b) encodes one or more antigens of an oncogenic virus (e.g., HPV E6, HPV E7, and/or HPV E6/E7 fusion protein), and/or an immunomodulatory peptide, polypeptide, or protein. More detailed description on heterologous ORFs is described in Section 6.5.
In one embodiment, the heterologous ORF encodes a reporter protein. More detailed description of reporter proteins are described in Section 6.5.
In certain embodiments, the growth and infectivity of the arenavirus viral vector is not affected by the heterologous ORF from an organism other than an arenavirus.
Techniques known to one skilled in the art may be used to produce an arenavirus viral vector comprising an arenavirus genomic segment engineered to carry an arenavirus ORF in a position other than the wild-type position. For example, reverse genetics techniques may be used to generate such arenavirus viral vector. In other embodiments, the replication-deficient arenavirus viral vector (i.e., the arenavirus genomic segment engineered to carry an arenavirus ORF in a position other than the wild-type position, wherein an ORF encoding GP, NP, Z protein, L protein, has been deleted) can be produced in a complementing cell.
In certain embodiments, the tri-segmented arenavirus viral vector used according to the present application can be Old World viruses, for example, LCMV.
6.4 Generation of an Arenavirus Viral Vector and a Tri-Segmented Arenavirus Viral Vector
Generally, arenavirus viral vectors can be recombinantly produced by standard reverse genetic techniques as described for LCMV (see Flatz et al., 2006, Proc Natl Acad Sci USA 103:4663-4668; Sanchez et al., 2006, Virology 350:370; Ortiz-Riano et al., 2013, J Gen Virol. 94:1175-88, which are incorporated by reference herein). To generate the arenavirus viral vectors provided herein, these techniques can be applied as described below. The genome of the viruses can be modified as described in Sections 6.2 or 6.3.
(a) Non-Natural Position Open Reading Frame
The generation of an arenavirus viral vector comprising a genomic segment that has been engineered to carry a viral ORF in a position other than the wild-type position of the ORF can be recombinantly produced by any reverse genetic techniques known to one skilled in the art.
Infectious and Replication-Competent Arenavirus Viral Vector
In certain embodiments, the method of generating the arenavirus viral vector comprises (i) transfecting into a host cell the cDNA of the first arenavirus genomic segment; (ii) transfecting into a host cell the cDNA of the second arenavirus genomic segment; (iii) transfecting into a host cell plasmids expressing the arenavirus' minimal trans-acting factors NP and L; (iv) maintaining the host cell under conditions suitable for virus formation; and (v) harvesting the arenavirus viral vector. In certain more specific embodiments, the cDNA is comprised in a plasmid.
Once generated from cDNA, arenavirus viral vectors (i.e., infectious and replication-competent) can be propagated. In certain embodiments, the arenavirus viral vector can be propagated in any host cell that allows the virus to grow to titers that permit the uses of the virus as described herein. In one embodiment, the host cell allows the arenavirus viral vector to grow to titers comparable to those determined for the corresponding wild-type.
In certain embodiments, the arenavirus viral vector may be propagated in host cells. Specific examples of host cells that can be used include BHK-21, HEK 293, VERO or other. In a specific embodiment, the arenavirus viral vector may be propagated in a cell line.
In certain embodiments, the host cells are kept in culture and are transfected with one or more plasmid(s). The plasmid(s) express the arenavirus genomic segment(s) to be generated under control of one or more expression cassettes suitable for expression in mammalian cells, e.g., consisting of a polymerase I promoter and terminator.
Plasmids that can be used for the generation of the arenavirus viral vector can include: i) a plasmid encoding the S genomic segment e.g., pol-I S, ii) a plasmid encoding the L genomic segment e.g., pol-I L. In certain embodiments, the plasmid encoding an arenavirus polymerase that direct intracellular synthesis of the viral L and S segments can be incorporated into the transfection mixture. For example, a plasmid encoding the L protein and/or a plasmid encoding NP (pC-L and pC-NP, respectively) can be present. The L protein and NP are the minimal trans-acting factors necessary for viral RNA transcription and replication. Alternatively, intracellular synthesis of viral L and S segments, together with NP and L protein can be performed using an expression cassette with pol-I and pol-II promoters reading from opposite sides into the L and S segment cDNAs of two separate plasmids, respectively.
In certain embodiments, the arenavirus genomic segments are under the control of a promoter. Typically, RNA polymerase I-driven expression cassettes, RNA polymerase II-driven cassettes or T7 bacteriophage RNA polymerase driven cassettes can be used. In certain embodiments, the plasmid(s) encoding the arenavirus genomic segments can be the same, i.e., the genome sequence and transacting factors can be transcribed by a promoter from one plasmid. Specific examples of promoters include an RNA polymerase I promoter, an RNA polymerase II promoter, an RNA polymerase III promoter, a T7 promoter, an SP6 promoter or a T3 promoter.
In addition, the plasmid(s) can feature a mammalian selection marker, e.g., puromycin resistance, under control of an expression cassette suitable for gene expression in mammalian cells, e.g., polymerase II expression cassette as above, or the viral gene transcript(s) are followed by an internal ribosome entry site, such as the one of encephalomyocarditis virus, followed by the mammalian resistance marker. For production in E. coli, the plasmid additionally features a bacterial selection marker, such as an ampicillin resistance cassette.
Transfection of a host cell with a plasmid(s) can be performed using any of the commonly used strategies such as calcium-phosphate, liposome-based protocols or electroporation. A few days later the suitable selection agent, e.g., puromycin, is added in titrated concentrations. Surviving clones are isolated and subcloned following standard procedures, and high-expressing clones are identified using Western blot or flow cytometry procedures with antibodies directed against the viral protein(s) of interest.
For recovering the arenavirus viral vector described herein, the following procedures are envisaged. First day: cells, typically 80% confluent in M6-well plates, are transfected with a mixture of the plasmids, as described above. For this one can exploit any commonly used strategies such as calcium-phosphate, liposome-based protocols or electroporation.
3-5 days later: The cultured supernatant (arenavirus vector preparation) is harvested, aliquoted and stored at 4° C., −20° C., or −80° C., depending on how long the arenavirus vector should be stored prior use. The arenavirus vector preparation's infectious titer is assessed by an immunofocus assay. Alternatively, the transfected cells and supernatant may be passaged to a larger vessel (e.g., a T75 tissue culture flask) on day 3-5 after transfection, and culture supernatant is harvested up to five days after passage.
The present application furthermore provides expression of a heterologous ORF, wherein a plasmid encoding the genomic segment is modified to incorporate a heterologous ORF. More detailed description on heterologous ORFs is described in Section 6.5. The heterologous ORF can be incorporated into the plasmid using restriction enzymes.
(ii) Infectious, Replication-Deficient Arenavirus Viral Vector
Infectious, replication-deficient arenavirus viral vectors can be rescued as described above. However, once generated from cDNA, the infectious, replication-deficient arenaviruses provided herein can be propagated in complementing cells. Complementing cells are cells that provide the functionality that has been eliminated from the replication-deficient arenavirus by modification of its genome (e.g., if the ORF encoding the GP protein is deleted or functionally inactivated, a complementing cell does provide the GP protein).
Owing to the removal or functional inactivation of one or more of the ORFs in arenavirus vectors (here deletion of the glycoprotein, GP, will be taken as an example), arenavirus vectors can be generated and expanded in cells providing in trans the deleted viral gene(s), e.g., the GP in the present example. Such a complementing cell line, henceforth referred to as C-cells, is generated by transfecting a cell line such as BHK-21, HEK 293, VERO or other with one or more plasmid(s) for expression of the viral gene(s) of interest (complementation plasmid, referred to as C-plasmid). The C-plasmid(s) express the viral gene(s) deleted in the arenavirus vector to be generated under control of one or more expression cassettes suitable for expression in mammalian cells, e.g., a mammalian polymerase II promoter such as the EFlalpha promoter with a polyadenylation signal. In addition, the complementation plasmid features a mammalian selection marker, e.g., puromycin resistance, under control of an expression cassette suitable for gene expression in mammalian cells, e.g., polymerase II expression cassette as above, or the viral gene transcript(s) are followed by an internal ribosome entry site, such as the one of encephalomyocarditis virus, followed by the mammalian resistance marker. For production in E. coli, the plasmid additionally features a bacterial selection marker, such as an ampicillin resistance cassette.
Cells that can be used, e.g., BHK-21, HEK 293, MC57G or other, are kept in culture and are transfected with the complementation plasmid(s) using any of the commonly used strategies such as calcium-phosphate, liposome-based protocols or electroporation. A few days later the suitable selection agent, e.g., puromycin, is added in titrated concentrations. Surviving clones are isolated and subcloned following standard procedures, and high-expressing C-cell clones are identified using Western blot or flow cytometry procedures with antibodies directed against the viral protein(s) of interest. As an alternative to the use of stably transfected C-cells transient transfection of normal cells can complement the missing viral gene(s) in each of the steps where C-cells will be used below. In addition, a helper virus can be used to provide the missing functionality in trans.
Plasmids can be of two types: i) two plasmids, referred to as TF-plasmids for expressing intracellularly in C-cells the minimal transacting factors of the arenavirus, is derived from e.g., NP and L proteins of LCMV in the present example; and ii) plasmids, referred to as GS-plasmids, for expressing intracellularly in C-cells the arenavirus vector genome segments, e.g., the segments with designed modifications. TF-plasmids express the NP and L proteins of the respective arenavirus vector under control of an expression cassette suitable for protein expression in mammalian cells, typically e.g., a mammalian polymerase II promoter such as the CMV or EF 1 alpha promoter, either one of them preferentially in combination with a polyadenylation signal. GS-plasmids express the small (S) and the large (L) genome segments of the vector. Typically, polymerase I-driven expression cassettes or T7 bacteriophage RNA polymerase (T7−) driven expression cassettes can be used, the latter preferentially with a 3′-terminal ribozyme for processing of the primary transcript to yield the correct end. In the case of using a T7-based system, expression of T7 in C-cells must be provided by either including in the recovery process an additional expression plasmid, constructed analogously to TF-plasmids, providing T7, or C-cells are constructed to additionally express T7 in a stable manner. In certain embodiments, TF and GS plasmids can be the same, i.e., the genome sequence and transacting factors can be transcribed by T7, polI and polII promoters from one plasmid.
For recovering of the arenavirus vector, the following procedures can be used. First day: C-cells, typically 80% confluent in M6-well plates, are transfected with a mixture of the two TF-plasmids plus the two GS-plasmids. In certain embodiments, the TF and GS plasmids can be the same, i.e., the genome sequence and transacting factors can be transcribed by T7, polI and polII promoters from one plasmid. For this one can exploit any of the commonly used strategies such as calcium-phosphate, liposome-based protocols or electroporation.
3-5 days later: The culture supernatant (arenavirus vector preparation) is harvested, aliquoted and stored at 4° C., −20° C. or −80° C. depending on how long the arenavirus vector should be stored prior to use. Then the arenavirus vector preparation's infectious titer is assessed by an immunofocus assay on C-cells. Alternatively, the transfected cells and supernatant may be passaged to a larger vessel (e.g., a T75 tissue culture flask) on day 3-5 after transfection, and culture supernatant is harvested up to five days after passage.
Also provided herein is expression of an antigen in a cell culture, wherein the cell culture is infected with an infectious, replication-deficient arenavirus expressing an antigen. When used for expression of an antigen in cultured cells, the following two procedures can be used:
i) The cell type of interest is infected with the arenavirus vector preparation described herein at a multiplicity of infection (MOI) of one or more, e.g., two, three or four, resulting in production of the antigen in all cells already shortly after infection.
ii) Alternatively, a lower MOI can be used and individual cell clones can be selected for their level of virally driven antigen expression. Subsequently individual clones can be expanded infinitely owing to the non-cytolytic nature of arenavirus vectors. Irrespective of the approach, the antigen can subsequently be collected (and purified) either from the culture supernatant or from the cells themselves, depending on the properties of the antigen produced. However, the invention is not limited to these two strategies, and other ways of driving expression of antigen using infectious, replication-deficient arenaviruses as vectors may be considered.
(b) Generation of a Tri-Segmented Arenavirus Viral Vector
A tri-segmented arenavirus viral vector can be recombinantly produced by reverse genetic techniques known in the art, for example as described by Emonet et al., 2008, PNAS, 106(9):3473-3478; Popkin et al., 2011, J. Virol., 85 (15):7928-7932, which are incorporated by reference herein. The generation of the tri-segmented arenavirus viral vector provided herein can be modified as described in Section 6.3.
Infectious and Replication-Competent Tri-Segmented Arenavirus Viral Vector
In certain embodiments, the method of generating the tri-segmented arenavirus viral vector comprises (i) transfecting into a host cell the cDNAs of the one L segment and two S segments or two L segments and one S segment; (ii) transfecting into a host cell plasmids expressing the arenavirus' minimal trans-acting factors NP and L; (iii) maintaining the host cell under conditions suitable for virus formation; and (iv) harvesting the arenavirus viral vector.
Once generated from cDNA, the tri-segmented arenavirus viral vector (i.e., infectious and replication-competent) can be propagated. In certain embodiments tri-segmented arenavirus viral vector can be propagated in any host cell that allows the virus to grow to titers that permit the uses of the virus as described herein. In one embodiment, the host cell allows the tri-segmented arenavirus viral vector to grow to titers comparable to those determined for the corresponding wild-type.
In certain embodiments, the tri-segmented arenavirus viral vector may be propagated in host cells. Specific examples of host cells that can be used include BHK-21, HEK 293, VERO or other. In a specific embodiment, the tri-segmented arenavirus viral vector may be propagated in a cell line.
In certain embodiments, the host cells are kept in culture and are transfected with one or more plasmid(s). The plasmid(s) express the arenavirus genomic segment(s) to be generated under control of one or more expression cassettes suitable for expression in mammalian cells, e.g., consisting of a polymerase I promoter and terminator.
In specific embodiments, the host cells are kept in culture and are transfected with one or more plasmid(s). The plasmid(s) express the viral gene(s) to be generated under control of one or more expression cassettes suitable for expression in mammalian cells, e.g., consisting of a polymerase I promoter and terminator.
Plasmids that can be used for generating the tri-segmented arenavirus comprising one L segment and two S segments can include: i) two plasmids each encoding the S genome segment e.g., pol-I S, ii) a plasmid encoding the L genome segment e.g., pol-I L. Plasmids needed for the tri-segmented arenavirus comprising two L segments and one S segments are: i) two plasmids each encoding the L genome segment e.g., pol-L, ii) a plasmid encoding the S genome segment e.g., pol-I S.
In certain embodiments, plasmids encoding an arenavirus polymerase that direct intracellular synthesis of the viral L and S segments can be incorporated into the transfection mixture. For example, a plasmid encoding the L protein and a plasmid encoding NP (pC-L and pC-NP, respectively). The L protein and NP are the minimal trans-acting factors necessary for viral RNA transcription and replication. Alternatively, intracellular synthesis of viral L and S segments, together with NP and L protein can be performed using an expression cassette with pol-I and pol-II promoters reading from opposite sides into the L and S segment cDNAs of two separate plasmids, respectively.
In addition, the plasmid(s) features a mammalian selection marker, e.g., puromycin resistance, under control of an expression cassette suitable for gene expression in mammalian cells, e.g., polymerase II expression cassette as above, or the viral gene transcript(s) are followed by an internal ribosome entry site, such as the one of encephalomyocarditis virus, followed by the mammalian resistance marker. For production in E. coli, the plasmid additionally features a bacterial selection marker, such as an ampicillin resistance cassette.
Transfection of BHK-21 cells with a plasmid(s) can be performed using any of the commonly used strategies such as calcium-phosphate, liposome-based protocols or electroporation. A few days later the suitable selection agent, e.g., puromycin, is added in titrated concentrations. Surviving clones are isolated and subcloned following standard procedures, and high-expressing clones are identified using Western blot or flow cytometry procedures with antibodies directed against the viral protein(s) of interest.
Typically, RNA polymerase I-driven expression cassettes, RNA polymerase II-driven cassettes or T7 bacteriophage RNA polymerase driven cassettes can be used, the latter preferentially with a 3′-terminal ribozyme for processing of the primary transcript to yield the correct end. In certain embodiments, the plasmids encoding the arenavirus genomic segments can be the same, i.e., the genome sequence and transacting factors can be transcribed by T7, polI and polII promoters from one plasmid.
For recovering the arenavirus the tri-segmented arenavirus vector, the following procedures are envisaged. First day: cells, typically 80% confluent in M6-well plates, are transfected with a mixture of the plasmids, as described above. For this one can exploit any commonly used strategies such as calcium-phosphate, liposome-based protocols or electroporation.
3-5 days later: The cultured supernatant (arenavirus vector preparation) is harvested, aliquoted and stored at 4° C., −20° C., or −80° C., depending on how long the arenavirus vector should be stored prior use. The arenavirus vector preparation's infectious titer is assessed by an immunofocus assay. Alternatively, the transfected cells and supernatant may be passaged to a larger vessel (e.g., a T75 tissue culture flask) on day 3-5 after transfection, and culture supernatant is harvested up to five days after passage.
The present application furthermore relates to expression of a heterologous ORF, wherein a plasmid encoding the genomic segment is modified to incorporated a heterologous ORF. More detailed description on heterologous ORFs is described in Section 6.5. The heterologous ORF can be incorporated into the plasmid using restriction enzymes.
(ii) Infectious, Replication-Deficient Tri-Segmented Arenavirus Viral Vector
Infectious, replication-deficient tri-segmented arenavirus viral vectors can be rescued as described above. However, once generated from cDNA, the infectious, replication-deficient arenaviruses provided herein can be propagated in complementing cells. Complementing cells are cells that provide the functionality that has been eliminated from the replication-deficient arenavirus by modification of its genome (e.g., if the ORF encoding the GP protein is deleted or functionally inactivated, a complementing cell does provide the GP protein).
Owing to the removal or functional inactivation of one or more of the ORFs in arenavirus vectors (here deletion of the glycoprotein, GP, will be taken as an example), arenavirus vectors can be generated and expanded in cells providing in trans the deleted viral gene(s), e.g., the GP in the present example. Such a complementing cell line, henceforth referred to as C-cells, is generated by transfecting a mammalian cell line such as BHK-21, HEK 293, VERO or other (here BHK-21 will be taken as an example) with one or more plasmid(s) for expression of the viral gene(s) of interest (complementation plasmid, referred to as C-plasmid). The C-plasmid(s) express the viral gene(s) deleted in the arenavirus vector to be generated under control of one or more expression cassettes suitable for expression in mammalian cells, e.g., a mammalian polymerase II promoter such as the CMV or EFlalpha promoter with a polyadenylation signal. In addition, the complementation plasmid features a mammalian selection marker, e.g., puromycin resistance, under control of an expression cassette suitable for gene expression in mammalian cells, e.g., polymerase II expression cassette as above, or the viral gene transcript(s) are followed by an internal ribosome entry site, such as the one of encephalomyocarditis virus, followed by the mammalian resistance marker. For production in E. coli,the plasmid additionally features a bacterial selection marker, such as an ampicillin resistance cassette.
Cells that can be used, e.g., BHK-21, HEK 293, MC57G or other, are kept in culture and are transfected with the complementation plasmid(s) using any of the commonly used strategies such as calcium-phosphate, liposome-based protocols or electroporation. A few days later the suitable selection agent, e.g., puromycin, is added in titrated concentrations. Surviving clones are isolated and subcloned following standard procedures, and high-expressing C-cell clones are identified using Western blot or flow cytometry procedures with antibodies directed against the viral protein(s) of interest. As an alternative to the use of stably transfected C-cells transient transfection of normal cells can complement the missing viral gene(s) in each of the steps where C-cells will be used below. In addition, a helper virus can be used to provide the missing functionality in trans.
Plasmids of two types can be used: i) two plasmids, referred to as TF-plasmids for expressing intracellularly in C-cells the minimal transacting factors of the arenavirus, is derived from e.g., NP and L proteins of LCMV in the present example; and ii) plasmids, referred to as GS-plasmids, for expressing intracellularly in C-cells the arenavirus vector genome segments, e.g., the segments with designed modifications. TF-plasmids express the NP and L proteins of the respective arenavirus vector under control of an expression cassette suitable for protein expression in mammalian cells, typically e.g., a mammalian polymerase II promoter such as the CMV or EF 1 alpha promoter, either one of them preferentially in combination with a polyadenylation signal. GS-plasmids express the small (S) and the large (L) genome segments of the vector. Typically, polymerase I-driven expression cassettes or T7 bacteriophage RNA polymerase (T7−) driven expression cassettes can be used, the latter preferentially with a 3′-terminal ribozyme for processing of the primary transcript to yield the correct end. In the case of using a T7-based system, expression of T7 in C-cells must be provided by either including in the recovery process an additional expression plasmid, constructed analogously to TF-plasmids, providing T7, or C-cells are constructed to additionally express T7 in a stable manner. In certain embodiments, TF and GS plasmids can be the same, i.e., the genome sequence and transacting factors can be transcribed by T7, polI and polII promoters from one plasmid.
For recovering of the arenavirus vector, the following procedures can be used. First day: C-cells, typically 80% confluent in M6-well plates, are transfected with a mixture of the two TF-plasmids plus the two GS-plasmids. In certain embodiments, the TF and GS plasmids can be the same, i.e., the genome sequence and transacting factors can be transcribed by T7, polI and polII promoters from one plasmid. For this one can exploit any of the commonly used strategies such as calcium-phosphate, liposome-based protocols or electroporation.
3-5 days later: The culture supernatant (arenavirus vector preparation) is harvested, aliquoted and stored at 4° C., −20° C. or −80° C. depending on how long the arenavirus vector should be stored prior to use. Then the arenavirus vector preparation's infectious titer is assessed by an immunofocus assay on C-cells. Alternatively, the transfected cells and supernatant may be passaged to a larger vessel (e.g., a T75 tissue culture flask) on day 3-5 after transfection, and culture supernatant is harvested up to five days after passage.
The invention furthermore relates to expression of an antigen in a cell culture wherein the cell culture is infected with an infectious, replication-deficient tri-segmented arenavirus expressing a antigen. When used for expression of a CMV antigen in cultured cells, the following two procedures can be used:
i) The cell type of interest is infected with the arenavirus vector preparation described herein at a multiplicity of infection (MOI) of one or more, e.g., two, three or four, resulting in production of the antigen in all cells already shortly after infection.
ii) Alternatively, a lower MOI can be used and individual cell clones can be selected for their level of virally driven antigen expression. Subsequently individual clones can be expanded infinitely owing to the non-cytolytic nature of arenavirus vectors. Irrespective of the approach, the antigen can subsequently be collected (and purified) either from the culture supernatant or from the cells themselves, depending on the properties of the antigen produced. However, the invention is not limited to these two strategies, and other ways of driving expression of CMV antigen using infectious, replication-deficient arenaviruses as vectors may be considered.
6.5 Heterologous Sequences
(a) Oncogenic Virus Antigens and HPV Antigens
In certain embodiments, a heterologous sequence encompassed by an arenavirus viral vector described herein encodes an antigen. In certain embodiments, an oncogenic virus antigen for use with the methods and compositions described herein is an antigen of a DNA virus, an RNA virus or of a retrovirus. In certain, more specific embodiments, the antigen itself is oncogenic.
In certain embodiments, the heterologous nucleotide sequence is derived from an oncogenic virus.
In certain embodiments, an antigen for use with the methods and compositions described herein can be an antigen of any oncogenic virus excluding Hepatitis B virus antigen and Hepatitis C virus antigen.
In certain embodiments, oncogenic virus antigens are antigens of human papillomavirus, antigens of Kaposi's sarcoma-associated herpesvirus, such as latency-associated nuclear antigen, antigens of Epstein-Barr virus, such as EBV-EA, EBV-MA, or EBV-VCA, antigens of Merkel cell polyomavirus, such as MCV T antigen, or antigens of human T-lymphotropic virus, such as HTLV-1 Tax antigen.
In certain specific embodiments, antigens for use with the methods and compositions described herein are HPV antigens.
In certain embodiments, any strain of human HPV or any clinical isolate of human HPV can be used to obtain the heterologous sequence for generation of the arenaviruses for the use with the compositions and methods described herein . In certain embodiments, the heterologous sequence is obtained from, and encodes an antigen of, an HPV strain, such as strains including HPV genotype 1 (HPV1), HPV genotype 2 (HPV2), HPV genotype 3 (HPV3), HPV genotype 4 (HPV4), HPV genotype 6 (HPV6), HPV genotype 7 (HPV7), HPV genotype 8 (HPV8), HPV genotype 10 (HPV10), HPV genotype 11 (HPV11), HPV genotype 13 (HPV13), HPV genotype 16 (HPV16), HPV genotype 18 (HPV18), HPV genotype 22 (HPV22), HPV genotype 26 (HPV26), HPV genotype 31 (HPV31), HPV genotype 32 (HPV32), HPV genotype 33 (HPV33), HPV genotype 35 (HPV35), HPV genotype 39 (HPV39), HPV genotype 42 (HPV42), HPV genotype 44 (HPV44), HPV genotype 45 (HPV45), HPV genotype 51 (HPV51), HPV genotype 52(HPV52), HPV genotype 53 (HPV53), HPV genotype 56 (HPV56), HPV genotype 58 (HPV58), HPV genotype 59 (HPV59), HPV genotype 60 (HPV60), HPV genotype 63 (HPV63), HPV genotype 66 (HPV66), HPV genotype 68 (HPV68), HPV genotype 73 (HPV73), or HPV genotype 82 (HPV82), or other genotypes. In certain embodiments, strains include “high-risk” genotypes of HPV, such as HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV73, and HPV82.
In certain embodiments, the antigen can be a papillomavirus antigen ortholog, e.g., a mammalian (i.e., non-human primate, pig, dog, cat, or horse) papillomavirus antigen.
In certain embodiments, an open reading frame (ORF) of an arenavirus is deleted and replaced with a heterologous sequence encoding an antigen of an oncogenic virus.
More specifically, one or more, e.g., two, three, or four, of the four arenavirus ORFs (glycoprotein (GP); nucleoprotein (NP); matrix protein Z; RNA-dependent RNA polymerase L) are removed or mutated to prevent formation of infectious particles in normal cells, albeit still allowing gene expression in arenavirus vector-infected cells. A heterologous sequence, such as foreign nucleic acids coding for one or more proteins can be introduced. These foreign nucleic acids are transcribed from one or more, e.g., two or three of the four arenavirus promoters 5′ UTR and 3′ UTR of the S segment, and 5′ UTR and 3′ UTR of the L segment, or from additionally introduced promoter sequences that can be read by the viral RNA-dependent RNA polymerase, by cellular RNA polymerase I, RNA polymerase II, or RNA polymerase III, such as duplications of viral promoter sequences that are naturally found in the viral UTRs, the 28S ribosomal RNA promoter, the beta-actin promoter, or the 55 ribosomal RNA promoter, respectively. The ribonucleic acids coding for proteins or modulating host gene expression are transcribed and translated either by themselves or as read-through by fusion to arenavirus protein ORFs. Expression of proteins in the host cell may be enhanced by introducing in the viral transcript sequence at the appropriate place(s) one or more, e.g., two, three or four, internal ribosome entry sites.
In certain embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous sequence encoding a DNA virus antigen, an RNA virus antigen, or a retrovirus antigen.
In certain, more specific embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous sequence encoding any oncogenic virus antigen excluding Hepatitis B virus antigens and Hepatitis C virus antigens.
In certain embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous sequence encoding an antigen of an oncogenic virus, such as antigens of human papillomavirus, antigens of Kaposi's sarcoma-associated herpesvirus, such as latency-associated nuclear antigen, antigens of Epstein-Barr virus, such as EBV-EA, EBV-MA, or EBV-VCA, antigens of Merkel cell polyomavirus, such as MCV T antigen, or antigens of human T-lymphotropic virus, such as HTLV-1 Tax antigen.
In certain embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous sequence encoding one or more HPV antigens.
In certain embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous sequence encoding an antigen of any strain of HPV or any clinical isolate of HPV. Such strains include HPV genotype 1 (HPV1), HPV genotype 2 (HPV2), HPV genotype 3 (HPV3), HPV genotype 4 (HPV4), HPV genotype 6 (HPV6), HPV genotype 7 (HPV7), HPV genotype 8 (HPV8), HPV genotype 10 (HPV10), HPV genotype 11 (HPV11), HPV genotype 13 (HPV13), HPV genotype 16 (HPV16), HPV genotype 18 (HPV18), HPV genotype 22 (HPV22), HPV genotype 26 (HPV26), HPV genotype 31 (HPV31), HPV genotype 32 (HPV32), HPV genotype 33 (HPV33), HPV genotype 35 (HPV35), HPV genotype 39 (HPV39), HPV genotype 42 (HPV42), HPV genotype 44 (HPV44), HPV genotype 45 (HPV45), HPV genotype 51 (HPV51), HPV genotype 52 (HPV52), HPV genotype 53 (HPV53), HPV genotype 56 (HPV56), HPV genotype 58 (HPV58), HPV genotype 59 (HPV59), HPV genotype 60 (HPV60), HPV genotype 63 (HPV63), HPV genotype 66 (HPV66), HPV genotype 68 (HPV68), HPV genotype 73 (HPV73), or HPV genotype 82 (HPV82), or other genotypes. In certain embodiments, strains include “high-risk” genotypes of HPV, such as HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV73, or HPV82.
In certain embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous sequence encoding an antigen of any strain of HPV or any clinical isolate of HPV, wherein the amino acid sequence of the antigen is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of an antigen, for example E6 and/or E7 antigen of HPV genotype 1 (HPV1), HPV genotype 2 (HPV2), HPV genotype 3 (HPV3), HPV genotype 4 (HPV4), HPV genotype 6 (HPV6), HPV genotype 7 (HPV7), HPV genotype 8 (HPV8), HPV genotype 10 (HPV10), HPV genotype 11 (HPV11), HPV genotype 13 (HPV13), HPV genotype 16 (HPV16), HPV genotype 18 (HPV18), HPV genotype 22 (HPV22), HPV genotype 26 (HPV26), HPV genotype 31 (HPV31), HPV genotype 32 (HPV32), HPV genotype 33 (HPV33), HPV genotype 35 (HPV35), HPV genotype 39 (HPV39), HPV genotype 42 (HPV42), HPV genotype 44 (HPV44), HPV genotype 45 (HPV45), HPV genotype 51 (HPV51), HPV genotype 52 (HPV52), HPV genotype 53 (HPV53), HPV genotype 56 (HPV56), HPV genotype 58 (HPV58), HPV genotype 59 (HPV59), HPV genotype 60 (HPV60), HPV genotype 63 (HPV63), HPV genotype 66 (HPV66), HPV genotype 68 (HPV68), HPV genotype 73 (HPV73), or HPV genotype 82 (HPV82), or other genotypes. In certain embodiments, strains include “high-risk” genotypes of HPV, such as HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV73, or HPV82.
In certain embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous sequence encoding one or more HPV antigens. In certain embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous sequence encoding an early (E) or late (L) protein of HPV. In certain embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous sequence encoding an HPV protein E1, HPV protein E2, HPV protein E3, HPV protein E4, HPV protein E5, HPV protein E6, HPV protein E7, HPV protein L1 or HPV protein L2. In certain embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous sequence encoding a fusion protein of two, three, four, five, or more of HPV protein E1, HPV protein E2, HPV protein E3, HPV protein E4, HPV protein E5, HPV protein E6, HPV protein E7, HPV protein L1 or HPV protein L2. In certain specific embodiments, the heterologous sequence is HPV protein E6 fused to HPV protein E7. In certain specific embodiments, the heterologous sequence is HPV protein E7 fused to HPV protein E6 fused to HPV protein E6 fused to HPVprotein E7, wherein one HPV protein E7 is from strain HPV16 and the other is from strain HPV18 and one HPV protein E6 is from strain HPV 18 and the other is from strain HPV18. In certain specific embodiments, the heterologous sequence is a shuffled sequence of HPV protein E6 fused to HPV protein E7. In certain specific embodiments, the sequence of HPV protein E6 fused to HPV protein E7 is expressed with protein E7 upstream of protein E6. In certain specific embodiments, the sequence of HPV protein E6 fused to HPV protein E7 is expressed with protein E6 upstream of protein E7. In certain embodiments, the E7 protein has mutations in the Rb binding site and the zinc finger motif. In certain embodiments, the E6 protein has mutations in the zinc finger motifs.
In certain embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous sequence encoding an antigen that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of an antigen of a HPV protein E1, HPV protein E2, HPV protein E3, HPV protein E4, HPV protein E5, HPV protein E6, HPV protein E7, HPV protein L1 or HPV protein L2. In certain embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous sequence encoding an antigen that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of HPV protein E6 fused to HPV protein E7. In certain embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous sequence encoding an antigen that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a shuffled sequence of HPV protein E6 fused to HPV protein E7. In certain specific embodiments, the HPV protein E6 sequence fused to HPV protein E7 sequence is expressed with protein E7 sequence upstream of protein E6 sequence. In certain specific embodiments, the HPV protein E6 sequence fused to HPV protein E7 sequence is expressed with the protein E6 sequence upstream of the protein E7 sequence. In certain embodiments, the E7 protein sequence has mutations in the Rb binding site and the zinc finger motif. In certain embodiments, the E6 protein sequence has mutations in the zinc finger motifs.
In certain embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous nucleotide sequence encoding an antigen, for example an HPV protein E6 and/or E7 antigen. HPV protein E6 is an oncoprotein. For example, it has been reported that protein E6 binds to tumor suppressor p53 and causes proteasomal degradation of p53 (Ganguly et al., 2009, J. Biosci. 34(1), 113-123). HPV protein E7 is also an oncoprotein. For example, it has been shown that E7 binds to the retinoblastoma protein (pRb), which is a tumor suppressor protein, and inactivates its function (Ganguly et al., 2009, J. Biosci. 34(1), 113-123).
In certain embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous nucleotide sequence encoding an antigen, for example an HPV protein E6 and/or E7 antigen, or a fragment thereof. In certain embodiments, the E6 protein fragment is an N-terminal truncated fragment. In certain embodiments, the E6 protein fragment is a C-terminal truncated fragment. In certain embodiments, the E6 protein fragment is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 158 amino acids in length. In certain embodiments, the E7 protein fragment is an N-terminal truncated fragment. In certain embodiments, the E7 protein fragment is an C-terminal truncated fragment. In certain embodiments, the E7 protein fragment is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 98 amino acids in length. In certain embodiments, the E7 protein fragment has mutations in the Rb binding site and the zinc finger motif. In certain embodiments, the E6 protein fragment has mutations in the zinc finger motifs.
In certain embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous nucleotide sequence encoding HPV16 protein E6, HPV16 protein E7, HPV18 protein E6, and HPV18 protein E7. In certain embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous nucleotide sequence encoding HPV16 protein E6 or an antigenic fragment thereof, HPV16 protein E7 or an antigenic fragment thereof, HPV18 protein E6 or an antigenic fragment thereof, and HPV18 protein E7 or an antigenic fragment thereof. In certain embodiments, one, two, three or all four of HPV16 protein E6 or antigenic fragment thereof, HPV16 protein E7 or antigenic fragment thereof, HPV18 protein E6 or antigenic fragment thereof, and HPV18 protein E7 or antigenic fragment thereof, can be shuffled sequences. Each one of HPV16 protein E6 or antigenic fragment thereof, HPV16 protein E7 or antigenic fragment thereof, HPV18 protein E6 or antigenic fragment thereof, and HPV18 protein E7 or antigenic fragment thereof, can be directly fused to one or two different sequences of HPV16 protein E6 or antigenic fragment thereof, HPV16 protein E7 or antigenic fragment thereof, HPV18 protein E6 or antigenic fragment thereof, and HPV18 protein E7 or antigenic fragment thereof. Each one of HPV16 protein E6 or antigenic fragment thereof, HPV16 protein E7 or antigenic fragment thereof, HPV18 protein E6 or antigenic fragment thereof, and HPV18 protein E7 or antigenic fragment thereof, can be fused to one or two different sequences of HPV16 protein E6 or antigenic fragment thereof, HPV16 protein E7 or antigenic fragment thereof, HPV18 protein E6 or antigenic fragment thereof, and HPV18 protein E7 or antigenic fragment thereof, via a linker or self-cleaving peptide. Each one of HPV16 protein E6 or antigenic fragment thereof, HPV16 protein E7 or antigenic fragment thereof, HPV18 protein E6 or antigenic fragment thereof, and HPV18 protein E7 or antigenic fragment thereof, can be fused to one or two different sequences of HPV16 protein E6 or antigenic fragment thereof, HPV16 protein E7 or antigenic fragment thereof, HPV18 protein E6 or antigenic fragment thereof, and HPV18 protein E7 or antigenic fragment thereof. The sequence of HPV16 protein E6 or antigenic fragment thereof, HPV16 protein E7 or antigenic fragment thereof, HPV18 protein E6 or antigenic fragment thereof, and HPV18 protein E7 or antigenic fragment thereof, can be arranged in any manner known to the skilled artisan, e.g., each one of HPV16 protein E6 or antigenic fragment thereof, HPV16 protein E7 or antigenic fragment thereof, HPV18 protein E6 or antigenic fragment thereof, and HPV18 protein E7 or antigenic fragment thereof, can be upstream or downstream of a different one of HPV16 protein E6 or antigenic fragment thereof, HPV16 protein E7 or antigenic fragment thereof, HPV18 protein E6 or antigenic fragment thereof, and HPV18 protein E7 or antigenic fragment thereof. Each one of HPV16 protein E6 or antigenic fragment thereof, HPV16 protein E7 or antigenic fragment thereof, HPV18 protein E6 or antigenic fragment thereof, and HPV18 protein E7 or antigenic fragment thereof, can be fused to a signal peptide. In certain more specific embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous nucleotide sequence encoding an HPV16 E6/HPV16 E7 fusion protein or antigenic fragment thereof, or an HPV16 E6/HPV18 E6 fusion protein or antigenic fragment thereof, or an HPV16 E6/HPV18 E7 fusion protein or antigenic fragment thereof, or an HPV16 E7/HPV18 E6 fusion protein or antigenic fragment thereof, or an HPV16 E7/HPV18 E7 fusion protein or antigenic fragment thereof, or an HPV18 E6/HPV18 E7 fusion protein or antigenic fragment thereof. In certain more specific embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous nucleotide sequence encoding two fusion proteins, wherein the first fusion protein is an HPV16 E6/HPV16 E7 fusion protein or antigenic fragment thereof, or an HPV16 E6/HPV18 E6 fusion protein or antigenic fragment thereof, HPV16 E6/HPV18 E7 fusion protein or antigenic fragment thereof, or an HPV16 E7/HPV18 E6 fusion protein or antigenic fragment thereof, HPV16 E7/HPV18 E7 fusion protein or antigenic fragment thereof, or an HPV18 E6/HPV18 E7 fusion protein or antigenic fragment thereof, and the second fusion protein is a different fusion protein selected from an HPV16 E6/HPV16 E7 fusion protein or antigenic fragment thereof, or an HPV16 E6/HPV18 E6 fusion protein or antigenic fragment thereof, HPV16 E6/HPV18 E7 fusion protein or antigenic fragment thereof, or an HPV16 E7/HPV18 E6 fusion protein or antigenic fragment thereof, HPV16 E7/HPV18 E7 fusion protein or antigenic fragment thereof, or an HPV18 E6/HPV18 E7 fusion protein or antigenic fragment thereof. In certain specific embodiments, the heterologous nucleotide sequence further encodes an immunomodulatory peptide, polypeptide, or protein. In certain specific embodiments, the heterologous nucleotide sequence further encodes a signal sequence (e.g. derived from VSVG).
In certain embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous nucleotide sequence encoding an HPV16 E6/E7 fusion protein and an HPV18 E6/E7 fusion protein. In certain embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous nucleotide sequence encoding shuffled sequence of an HPV16 E6/E7 fusion protein and a shuffled sequence of an HPV18 E6/E7 fusion protein. In certain specific embodiments, the heterologous nucleotide sequence encodes an HPV16 E6/E7 fusion protein and an HPV18 E6/E7 fusion protein that are directly fused to each other. In certain specific embodiments, the heterologous sequence encodes an HPV16 E6/E7 fusion protein and an HPV18 E6/E7 fusion protein that are fused to each other via a peptide linker or self-cleaving peptide. In certain specific embodiments, the heterologous sequence encodes an HPV16 E6/E7 fusion protein located upstream of the HPV18 E6/E7 fusion protein. In certain specific embodiments, the heterologous nucleotide sequence encodes an HPV16 E6/E7 fusion protein located downstream of the HPV18 E6/E7 fusion protein. In certain specific embodiments, the heterologous nucleotide sequence encodes an HPV16 E6/E7 fusion protein fused to a signal peptide. In certain specific embodiments, the heterologous nucleotide sequence encodes an HPV18 E6/E7 fusion protein fused to a signal peptide. In certain specific embodiments, the heterologous nucleotide sequence further encodes an immunomodulatory peptide, polypeptide, or protein.
In certain embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous sequence encoding an antigen that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of HPV16 E6/E7 fusion protein and an antigen that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of HPV18 E6/E7 fusion protein.
In certain embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous sequence encoding an antigen that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a shuffled sequence of an HPV16 E6/E7 fusion protein. In certain embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous sequence encoding an antigen that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a shuffled sequence of an HPV18 E6/E7 fusion protein.
In certain specific embodiments, the E6 protein fragment of the HPV16 E6/E7 fusion protein or the HPV18 E6/E7 fusion protein is an N-terminal truncated fragment. In certain embodiments, the E6 protein fragment is a C-terminal truncated fragment. In certain embodiments, the E6 protein fragment is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 158 amino acids in length. In certain embodiments, the E7 protein fragment of the HPV16 E6/E7 fusion or the HPV18 E6/E7 fusion is an N-terminal truncated fragment. In certain embodiments, the E7 protein fragment is an C-terminal truncated fragment. In certain embodiments, the E7 protein fragment is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 98 amino acids in length.
In certain embodiments, the heterologous nucleotide sequence encoding the HPV16 E6/E7 fusion protein and the heterologous nucleotide sequence encoding the HPV18 E6/E7 fusion protein are on the same position of the viral genome. In certain embodiments, the heterologous nucleotide sequence encoding the HPV16 E6/E7 fusion protein and the heterologous nucleotide sequence encoding the HPV18 E6/E7 fusion protein are on different positions of the viral genome. In certain embodiments, the heterologous nucleotide sequence encoding the HPV16 E6/E7 fusion protein and the heterologous nucleotide sequence encoding the HPV18 E6/E7 fusion protein are expressed on the same virus. In certain embodiments, the heterologous nucleotide sequence encoding the HPV16 E6/E7 fusion protein and the heterologous nucleotide sequence encoding the HPV18 E6/E7 fusion protein are expressed on different viruses.
In certain specific embodiments, the heterologous nucleotide sequence of HPV16 protein E6 fused to protein E7 is expressed with protein E7 upstream of protein E6. In certain specific embodiments, the heterologous nucleotide sequence of HPV18 protein E6 fused to protein E7 is expressed with protein E6 upstream of protein E7. In certain embodiments, the E7 protein of the HPV16 E6/E7 fusion protein or the HPV18 E6/E7 fusion protein has mutations in the Rb binding site and the zinc finger motif. In certain embodiments, the E6 protein of the HPV16 E6/E7 fusion protein or the HPV18 E6/E7 fusion protein has mutations in the zinc finger motifs.
In certain embodiments, the heterologous sequence encoding the antigen of an oncogenic virus further encodes a signal peptide. More specifically, the heterologous sequence encodes an antigen that is fused to the signal peptide such that the resulting expression product is secreted from the cell in which it is expressed. Such a signal peptide can be fused to the N-terminus or the C-terminus of the antigen. Any signal peptide known to the skilled artisan can be used with the compositions and methods provided herein. Specifically, the signal peptide is a signal peptide of a human secreted protein. More specifically, the signal peptide is a human tyrosinase secretion signal, a human growth hormone secretion signal, a human tissue plasminogen activator signal sequence, or a VSVG signal sequence.
The heterologous nucleotide sequence can encode more than one antigen. In certain embodiments, the heterologous nucleotide sequence encodes two, three, four, five, or more antigens of one or more different oncogenic viruses. Specifically, the heterologous nucleotide sequence can encode a first antigen of one strain of HPV and a second antigen that is the analogous antigen from a different strain of HPV. For example, the heterologous nucleotide sequence can encode protein E6 from one strain of HPV (e.g., strain HPV 16), and protein E6 from another strain (e.g., strain HPV 18), and/or protein E7 from one strain of HPV (e.g., strain HPV 16), and protein E7 from another strain (e.g., strain HPV 18). In certain embodiments, the heterologous nucleotide sequence encodes two, three, four, five, or more different antigens of the same oncogenic virus, or of one or more different oncogenic viruses. Specifically, the heterologous nucleotide sequence can encode a first antigen of one strain of HPV and a second different antigen that is the analogous antigen from the same strain or a different strain of HPV. For example, the heterologous nucleotide sequence can encode protein E6 from one strain of HPV (e.g., strain HPV 16), and protein E7 from the same strain or another strain (e.g., strain HPV 18). As another example, the heterologous nucleotide sequence can encode protein E6 from two strains of HPV (e.g., strain HPV 16 and 18), and protein E7 from the two strains (e.g., strain HPV 16 and 18).
In certain embodiments, the heterologous sequence encoding the antigen of an oncogenic virus further encodes a linker or a self-cleaving peptide. The linker or self-cleaving peptide is useful for the simultaneous expression of two or more genes. More specifically, the heterologous sequence encodes an antigen that is fused to another antigen or an immunomodulatory peptide, polypeptide, or protein, either directly or fused through a linker sequence. In another specific embodiment, the heterologous sequence encodes an antigen linked to another antigen or an immunomodulatory peptide, polypeptide, or protein, through a self-cleaving peptide. Such a linker or self-cleaving peptide can be fused to the N-terminus or the C-terminus of the antigen. Any linker peptide or self-cleaving peptide known to the skilled artisan can be used with the compositions and methods provided herein. Any number of antigens or immunomodulatory peptides, polypeptides, or proteins can be fused or linked in this manner. For example, in one specific embodiment, the first HPV antigen is directly fused to a second HPV antigen, or is fused to the second antigen through a peptide linker. In another specific embodiment, the second HPV antigen is directly fused to a third HPV antigen, or is fused to the third antigen through a peptide linker. In another specific embodiment, the first HPV antigen and the second HPV antigen are separated from each other via a self-cleaving peptide. In another specific embodiment, the second HPV antigen and the third HPV antigen are separated from each other via a self-cleaving peptide.
In certain embodiments, the ORFs encoding two, three, four, or more HPV antigens described herein are transcribed as a single transcript. In certain embodiments, the ORFs encoding the HPV antigens on that transcript are separated by a nucleic acid encoding a self-cleaving peptide or a ribosome-skipping sequence. In certain embodiments, the self-cleaving peptide can be obtained from a 2A protein from a member of the virus family Picornaviridae. In certain specific embodiments, the self-cleaving peptide is obtained from (or derived from) Porcine teschovirus-1 2A, Thosea asigna virus 2A, Foot-and-mouth disease virus 2A peptide, or equine rhinitis A virus 2A peptide. In certain specific embodiments, the 2A peptide obtained from (or derived from) the porcine teschovirus-1 2A has the highest cleavage efficiency. In certain embodiments, the 2A peptide has a high cleavage efficiency in combination with the HPV antigens described herein upstream or downstream of the 2A peptide.
In certain embodiments, the ORFs encoding two, three, four, or more HPV antigens are separated by a ribosome-skipping sequence. In more specific embodiments, the ribosome-skipping sequence is a cis-acting hydrolase element sequence.
In certain embodiments, the ORFs encoding two, three, four, or more HPV antigens are separated by a self-cleaving protease obtained from (or derived from) tobacco etch viruses (TEVs) of the Potyviridae family.
In certain embodiments, a Gly-Ser-Gly, NDAQAPKS or a SDRYLNRRA linker is inserted at the N-terminus and/or C-terminus of the 2A peptide. In more specific embodiments, the Gly-Ser-Gly, NDAQAPKS or a SDRYLNRRA linker is inserted at the N-terminus of the 2A peptide. In more specific embodiments, the Gly-Ser-Gly, NDAQAPKS or a SDRYLNRRA linker is inserted at the C-terminus of the 2A peptide. In certain embodiments, the Gly-Ser-Gly, NDAQAPKS or a SDRYLNRRA linker improves the efficiency of cleavage by the 2A peptide.
In certain embodiments, the ORFs encoding two, three, four, or more HPV antigens are separated by an internal ribosome entry site. In certain embodiments, the internal ribosome entry site functions under the control of an upstream promoter. In certain embodiments the internal ribosome entry site is obtained from (or derived from) the encephalomyocarditis virus.
In certain embodiments, the ORFs encoding two, three, four, or more HPV antigens are separated by a 2A peptide and a furin cleavage site. In certain embodiments, the 2A peptide is flanked by a furin cleavage site. In certain embodiments, the furin cleavage site is located between an ORF encoding an HPV antigen and the 2A peptide. In certain embodiments, the furin cleavage site is added upstream of the 2A peptide. In certain embodiments, the furin cleavage site is added downstream of the 2A peptide. In certain embodiments, the furin cleavage site is located in the vector with the ORFs encoding two, three, or four, or more HPV antigens, a self-cleaving peptide, and combinations thereof. In certain embodiments, the furin cleavage site consensus sequence is R—X—K—/R—R. In a more specific embodiment the furin cleavage site is cleaved by the furin protein in the trans golgi network. In another embodiment, the furin cleavage site removes the 2A peptide sequence. In yet another embodiment, the furin cleavage site removes the self-cleaving peptide sequence at the C-terminus. For example, see Fang et al., 2007, Molecular Therapy 15(6):1153-1159.
In certain embodiments, the ORFs encoding two, three, or four, or more HPV antigens are separated by the 2A peptide and a tag. In certain embodiments, the tag is linked to the 2A peptide. In certain embodiments, the tag is located between the 2A peptide and the furin cleavage site. In certain embodiments, the tag is located at the C-terminus or N-terminus of the downstream ORF encoding the HPV antigen. In certain embodiments, the tag is located at the C-terminus or N-terminus of the upstream ORF encoding the HPV antigen. In certain embodiments the tag is located in the vector with the ORFs encoding two, three, four, or more HPV antigens, a 2A peptide, a furin cleavage site, or a combination thereof. In certain embodiments the tag is a peptide tag. In more specific embodiments the tag is a V5 amino acid tag.
In certain embodiments, the ORFs encoding two, three, four, or five or more HPV antigens are separated by the 2A peptide and a spacer sequence. In certain embodiments, the spacer sequence is located upstream of the 2A peptide. In certain embodiments, the spacer sequence is located between the ORFs encoding the HPV antigens. In certain embodiments, the spacer sequence is located between the upstream of the 2A peptide and the tag. In certain embodiments, the spacer sequence is located between the upstream 2A peptide and the downstream furin cleavage site. In certain embodiments the spacer sequence is located in the vector with the ORFs encoding HPV antigens, a self-cleaving peptide, a furin cleavage site, a tag or a combination thereof. In certain embodiments, the spacer sequence increases cleavage efficiency.
In certain embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous sequence encoding one, two, three, or four, or more HPV antigens.
(b) Immunomodulatory Sequences
In certain embodiments, antigens for use with the methods and compositions described herein are administered together with an immunomodulatory element, e.g., an immunomodulatory peptide, polypeptide, or protein.
In certain embodiments, the heterologous nucleotide sequence encompassed by an infectious replication-deficient arenavirus further encodes an immunomodulatory peptide, polypeptide, or protein. The immunomodulatory peptide, polypeptide, or protein can be Calreticulin (CRT), or a fragment thereof, Ubiquitin or a fragment thereof, Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), or a fragment thereof, Invariant chain (CD74) or an antigenic fragment thereof, Mycobacterium tuberculosis Heat shock protein 70 or an antigenic fragment thereof, Herpes simplex virus 1 protein VP22 or an antigenic fragment thereof; CD40 ligand or an antigenic fragment thereof; or Fms-related tyrosine kinase 3 (Flt3) ligand or an antigenic fragment thereof.
In certain embodiments, the sequence encoding the immunomodulatory peptide, polypeptide, or protein and the sequence encoding an antigen are on the same position of the viral genome. For example, the sequence encoding the immunomodulatory peptide, polypeptide, or protein and the sequence encoding an antigen are located in place of the functionally inactivated, e.g., deleted, ORF of the infectious, replication-deficient arenavirus. In certain embodiments, the sequence encoding the immunomodulatory peptide, polypeptide, or protein and the sequence encoding an antigen are on different positions of the viral genome. In certain embodiments, the sequence encoding the immunomodulatory peptide, polypeptide, or protein and the sequence encoding a first antigen are located on different genomic segments of the infectious, replication-deficient arenavirus.
In certain embodiments, an ORF of an arenavirus is deleted and replaced with a heterologous sequence encoding one, two, three, or four, or more HPV antigens and an immunomodulatory peptide, polypeptide, or protein.
In certain embodiments, the heterologous sequence encoding the immunomodulatory peptide, polypeptide, or protein, further encodes a signal peptide. More specifically, the heterologous sequence encodes an immunomodulatory peptide, polypeptide, or protein that is fused to the signal peptide such that the resulting expression product is secreted from the cell in which it is expressed. Such a signal peptide can be fused to the N-terminus or the C-terminus of the immunomodulatory peptide, polypeptide, or protein. Any signal peptide known to the skilled artisan can be used with the compositions and methods provided herein. Specifically, the signal peptide is a signal peptide of a human secreted protein. More specifically, the signal peptide is a human tyrosinase secretion signal, a human growth hormone secretion signal, or a tissue plasminogen activator signal sequence.
In certain embodiments, the heterologous sequence encoding the immunomodulatory peptide, polypeptide, or protein, further encodes a linker or a self-cleaving peptide. More specifically, the heterologous sequence encodes an immunomodulatory peptide, polypeptide, or protein, which is fused to an antigen or another immunomodulatory peptide, polypeptide, or protein, either directly or fused through a linker sequence. In another specific embodiment, the heterologous sequence encodes an immunomodulatory peptide, polypeptide, or protein, linked to an antigen or another immunomodulatory peptide, polypeptide, or protein, through a self-cleaving peptide. Such a linker or self-cleaving peptide can be fused to the N-terminus or the C-terminus of the immunomodulatory peptide, polypeptide, or protein. Any linker peptide or self-cleaving peptide known to the skilled artisan can be used with the compositions and methods provided herein. Any number of immunomodulatory peptides, polypeptides, or proteins, can be fused or linked in this manner to an antigen or another immunomodulatory peptide, polypeptide, or protein. For example, in one specific embodiment, the immunomodulatory peptide, polypeptide, or protein is directly fused to a first antigen, or is fused to the first antigen through a peptide linker. In another specific embodiment, the immunomodulatory peptide, polypeptide, or protein is directly fused to a second antigen, or is fused to the second antigen through a peptide linker. In another specific embodiment, the first antigen and the immunomodulatory peptide, polypeptide, or protein are separated from each other via a self-cleaving peptide. In another specific embodiment, the second antigen and the immunomodulatory peptide, polypeptide, or protein are separated from each other via a self-cleaving peptide.
In certain embodiments, the ORFs encoding two, three, or four, or more HPV antigens and the immunomodulatory peptide, polypeptide, or protein are transcribed as a single transcript. In certain embodiments, the ORFs encoding the HPV antigens and the immunomodulatory sequence on that transcript are separated by a nucleic acid encoding a self-cleaving peptide or a ribosome-skipping sequence. In certain embodiments, the self-cleaving peptide can be obtained from a 2A protein from a member of the virus family Picornaviridae. In certain specific embodiments, the self-cleaving peptide is obtained from (or derived from) Porcine teschovirus-1 2A peptide, Thoseaasignavirus 2A peptide, Foot-and-mouth disease virus 2A peptide, or equine rhinitis A virus 2A peptide. In certain specific embodiments, the 2A peptide obtained from (or derived from) the porcine teschovirus-1 2A has the highest cleavage efficiency. In certain embodiments, the 2A peptide has a high cleavage efficiency in combination with the HPV antigens described herein upstream or downstream of the 2A peptide.
In certain embodiments, the ORFs encoding two, three, or four, or more HPV antigens and the immunomodulatory peptide, polypeptide, or protein are separated by a ribosome-skipping sequence. In more specific embodiments, the ribosome-skipping sequence is a cis-acting hydrolase element sequence.
In certain embodiments, the ORFs encoding two, three, or four, or more HPV antigens and the immunomodulatory peptide, polypeptide, or protein are separated by a self-cleaving protease obtained from (or derived from) tobacco etch viruses (TEVs) of the Potyviridae family.
In certain embodiments, a Gly-Ser-Gly, NDAQAPKS or a SDRYLNRRA linker is inserted at the N-terminus and/or C-terminus of the 2A peptide. In more specific embodiments, the Gly-Ser-Gly, NDAQAPKS or a SDRYLNRRA linker is inserted at the N-terminus of the 2A peptide. In more specific embodiments, the Gly-Ser-Gly, NDAQAPKS or a SDRYLNRRA linker is inserted at the C-terminus of the 2A peptide. In certain embodiments, the Gly-Ser-Gly, NDAQAPKS or a SDRYLNRRA linker improves the efficiency of cleavage by the 2A peptide.
In certain embodiments, the ORFs encoding two, three, or four, or more HPV antigens and the immunomodulatory peptide, polypeptide, or protein are separated by an internal ribosome entry site. In certain embodiments, the internal ribosome entry site functions under the control of an upstream promoter. In certain embodiments the internal ribosome entry site is obtained from (or derived from) the encephalomyocarditis virus.
In certain embodiments the ORFs encoding two, three, or four, or more HPV antigens and the immunomodulatory peptide, polypeptide, or protein are separated by a 2A peptide and a furin cleavage site. In certain embodiments, the 2A peptide is flanked by a furin cleavage site. In certain embodiments, the furin cleavage site is located between an ORF encoding an HPV antigen and the 2A peptide. In certain embodiments the furin cleavage site is added upstream of the 2A peptide. In certain embodiments the furin cleavage site is added downstream of the 2A peptide. In certain embodiments, the furin cleavage site is located in the vector with the ORFs encoding two, three, or four, or more HPV antigens, a self-cleaving peptide, and combinations thereof. In certain embodiments, the furin cleavage site consensus sequence is R—X—K—/R—R. In a more specific embodiment the furin cleavage site is cleaved by the furin protein in the trans golgi network. In another embodiment the furin cleavage site removes the 2A peptide sequence. In yet another embodiment the furin cleavage site removes the self-cleaving peptide sequence at the C-terminus. For example, see Fang et al., Molecular Therapy, 2007; 15(6):1153-1159.
In certain embodiments, the ORFs encoding two, three, or four, or more HPV antigens and the immunomodulatory peptide, polypeptide, or protein are separated by the 2A peptide and a tag. In certain embodiments, the tag is linked to the 2A peptide. In certain embodiments, the tag is located between the 2A peptide and the furin cleavage site. In certain embodiments the tag is located at the C-terminus or N-terminus of the downstream ORF encoding the HPV antigen. In certain embodiments the tag is located at the C-terminus or N-terminus of the upstream ORF encoding the HPV antigen. In certain embodiments the tag is located in the vector with the ORFs encoding two, three, four, or more HPV antigens, a 2A peptide, a furin cleavage site, or a combination thereof. In certain embodiments the tag is a peptide tag. In more specific embodiments the tag is a V5 amino acid tag.
In certain embodiments, the ORFs encoding two, three, four, or five or more HPV antigens and the immunomodulatory peptide, polypeptide, or protein are separated by the 2A peptide and a spacer sequence. In certain embodiments, the spacer sequence is located upstream of the 2A peptide. In certain embodiments, the spacer sequence is located between the ORFs encoding the HPV antigens. In certain embodiments, the spacer sequence is located between the upstream of the 2A peptide and the tag. In certain embodiments, the spacer sequence is located between the upstream 2A peptide and the downstream furin cleavage site. In certain embodiments the spacer sequence is located in the vector with the ORFs encoding HPV antigens, a self-cleaving peptide, a furin cleavage site, a tag or a combination thereof. In certain embodiments, the spacer sequence increases cleavage efficiency.
In certain embodiments, the ORFs encoding two, three, four, or five, or more HPV antigens and the immunomodulatory peptide, polypeptide, or protein are separated by a nucleotide sequence that encodes: a self-cleaving peptide, an amino acid sequence that leads to release of the upstream amino acid sequence by “ribosome skipping,” or a sequence element leading to binding of the ribosome and translation of the downstream sequence such as “internal ribosome entry sites” (IRES).
(c) Illustrative Insertions
In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by a nucleic acid sequence encoding one, two, three, or four, or more HPV antigens described herein and an immunomodulatory peptide, polypeptide, or protein. In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by a nucleic acid sequence encoding two, three, or four, or more HPV antigens described herein, separated from the immunomodulatory peptide, polypeptide, or protein by self-cleaving peptides or ribosome-skipping sequences. In certain embodiments, the self-cleaving peptide (or the ribosome-skipping sequence) can be obtained from a 2A protein from a member of the virus family Picornaviridae. In certain specific embodiments, the self-cleaving peptide (or the ribosome-skipping sequence) is obtained from (or derived from) Porcine teschovirus-1 2A, Thoseaasignavirus 2A, or Foot-and-mouth disease virus 2A peptide.
In certain embodiments, the heterologous nucleotide sequence encodes one or more of:
an HPV antigen or an antigenic fragment thereof;
an HPV16 protein E6, or an antigenic fragment thereof;
an HPV16 protein E7, or an antigenic fragment thereof;
an HPV18 protein E6, or an antigenic fragment thereof;
an HPV18 protein E7, or an antigenic fragment thereof;
an HPV16 protein E6/protein E7 fusion protein or an antigenic fragment thereof;
a shuffled HPV16 protein E6/protein E7 fusion protein or an antigenic fragment thereof;
an HPV16 protein E6/HPV18 protein E7 fusion protein or an antigenic fragment thereof;
an HPV16 protein E7/HPV18 protein E6 fusion protein or an antigenic fragment thereof;
an HPV18 protein E6/HPV16 protein E7 fusion protein or an antigenic fragment thereof; or
an HPV18 protein E7/HPV16 protein E6 fusion protein or an antigenic fragment thereof.
In certain embodiments, the heterologous nucleotide sequence further encodes, or the infectious, replication deficient arenavirus genome further comprises a second heterologous nucleotide sequence that encodes
Calreticulin (CRT), or a fragment thereof;
Ubiquitin or a fragment thereof;
Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), or a fragment thereof;
Invariant chain (CD74) or an antigenic fragment thereof;
Mycobacterium tuberculosis Heat shock protein 70 or an antigenic fragment thereof;
Herpes simplex virus 1 protein VP22 or an antigenic fragment thereof;
CD40 ligand or an antigenic fragment thereof; or
Fms-related tyrosine kinase 3 (Flt3) ligand or an antigenic fragment thereof.
In certain embodiments, the Calreticulin protein fragment is an N-terminal truncated fragment. In certain embodiments, the Calreticulin protein fragment is a C-terminal truncated fragment. In certain embodiments, the Calreticulin protein fragment is at least 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, or 417 amino acids in length. In certain embodiments, the Ubiquitin protein fragment is an N-terminal truncated fragment. In certain embodiments, the Ubiquitin protein fragment is a C-terminal truncated fragment. In certain embodiments, the Ubiquitin protein fragment is at least 10, 20, 30, 40, 50, 60, 70, or 76 amino acids in length. In certain embodiments, the GM-CSF protein fragment is an N-terminal truncated fragment. In certain embodiments, the GM-CSF protein fragment is a C-terminal truncated fragment. In certain embodiments, the GM-CSF protein fragment is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 127 amino acids in length. In certain embodiments, the Invariant chain (CD74) protein fragment is an N-terminal truncated fragment. In certain embodiments, the Invariant chain (CD74) protein fragment is a C-terminal truncated fragment. In certain embodiments, the Invariant chain (CD74) protein fragment is at least 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, or 232 amino acids in length. In certain embodiments, the Mycobacterium tuberculosis Heat shock protein 70 protein fragment is an N-terminal truncated fragment. In certain embodiments, the Mycobacterium tuberculosis Heat shock protein 70 protein fragment is a C-terminal truncated fragment. In certain embodiments, the Mycobacterium tuberculosis Heat shock protein 70 protein fragment is at least 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, or 701 amino acids in length. In certain embodiments, the Herpes simplex virus 1 protein VP22 protein fragment is an N-terminal truncated fragment. In certain embodiments, the Herpes simplex virus 1 protein VP22 protein fragment is a C-terminal truncated fragment. In certain embodiments, the Herpes simplex virus 1 protein VP22 protein fragment is at least 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, or 301 amino acids in length. In certain embodiments, the CD40 ligand protein fragment is an N-terminal truncated fragment. In certain embodiments, the CD40 ligand protein fragment is a C-terminal truncated fragment. In certain embodiments, the CD40 ligand protein fragment is at least 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, or 261 amino acids in length. In certain embodiments, the Fms-related tyrosine kinase 3 (Flt3) ligand protein fragment is an N-terminal truncated fragment. In certain embodiments, the Fms-related tyrosine kinase 3 (Flt3) ligand protein fragment is a C-terminal truncated fragment. In certain embodiments, the Fms-related tyrosine kinase 3 (Flt3) ligand protein fragment is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 153 amino acids in length.
In specific embodiments, the heterologous nucleotide sequence encodes HPV 16 protein E6, or a fragment thereof. In more specific embodiments, the antigen encoded by the heterologous nucleotide sequence is the HPV 16 protein E6 with one or more mutation(s) in the zinc finger motif(s). A mutation in the zinc finger motif prevents binding to Tumor protein p53. Tumor protein p53 has an anticancer function, because it can activate DNA repair proteins when DNA has sustained damage, because it can arrest growth by holding the cell cycle at the G1/S regulation point on DNA damage recognition, and because it can initiate apoptosis if DNA damage proves to be irreparable. In specific embodiments, the antigen is the HPV 16 protein E7, or a fragment thereof. In more specific embodiments, the antigen is the HPV 16 protein E7 with one or more mutation(s) in the Rb binding site and the zinc finger motif. The mutation prevents binding to retinoblastoma protein (pRb). Oncogenic proteins bind and inactivate pRb, which can lead to cancer because one function of pRb is to prevent excessive cell growth by inhibiting cell cycle progression until a cell is ready to divide. In specific embodiments, the antigen is the HPV 18 protein E6, or a fragment thereof. In more specific embodiments, the antigen is the HPV 18 protein E6 with one or more mutation(s) in the zinc finger motif. In specific embodiments, the antigen is the HPV 18 protein E7, or a fragment thereof. In more specific embodiments, the antigen is the HPV 18 protein E7 with one or more mutation(s) in the Rb binding site and the zinc finger motif
In certain embodiments, the antigen is an HPV16 protein E7/E6 fusion protein, an HPV18 protein E7/E6 fusion protein, an HPV16 protein E7/HPV18 protein E6 fusion protein, or an HPV18 protein E7/HPV 16 protein E6 fusion protein, wherein the E6 protein has one or more mutation(s) in the zinc finger motif, and the protein E7 has one or more mutation(s) in the Rb binding site and the zinc finger motif.
In certain embodiments, the antigen is the HPV16 protein E7/E6 fusion protein, HPV18 protein E7/E6 fusion protein, HPV16 protein E7/HPV18 protein E6 fusion protein, or HPV18 protein E7/HPV 16 protein E6 fusion protein, expressed together with an immunomodulatory peptide, polypeptide, or protein, wherein the immunomodulatory peptide, polypeptide, or protein is Calreticulin (CRT), or a fragment thereof; Ubiquitin or a fragment thereof; Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), or a fragment thereof; Invariant chain (CD74) or an antigenic fragment thereof; Mycobacterium tuberculosis Heat shock protein 70 or an antigenic fragment thereof; Herpes simplex virus 1 protein VP22 or an antigenic fragment thereof; CD40 ligand or an antigenic fragment thereof; or Fms-related tyrosine kinase 3 (Flt3) ligand or an antigenic fragment thereof, wherein the E6 protein has one or more mutation(s) in the zinc finger motif and the protein E7 has one or more mutation(s) in the Rb binding site and the zinc finger motif.
In one embodiment, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding an HPV antigen. In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding antigen that is a fragment of at least at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 158 amino acids of a gene product of a gene of HPV 16 protein E6 or a fragment thereof. In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding antigen that is a fragment of at least at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 98 amino acids of a gene product of a gene of HPV 16 protein E7 or a fragment thereof. In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding antigen that is a fragment of at least at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 158 amino acids of a gene product of a gene of HPV 18 protein E6 or a fragment thereof. In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding antigen that is a fragment of at least at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 98 amino acids of a gene product of a gene of HPV 18 protein E7 or a fragment thereof
In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding an antigen that is a fusion protein between HPV 16 protein E6 and HPV 16 protein E7. . In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding an antigen that is a fusion protein between HPV 16 protein E7 and HPV 18 protein E6. In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding an antigen that is a fusion protein between HPV 18 protein E7 and HPV 16 protein E6. In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding an antigen that is a fusion protein between HPV 18 protein E6 and HPV 18 protein E7. In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid seqeucnes encoding an antigen that is a fusion protein of HPV16 E7, HPV18 E6, HPV16 E6 and HPV18 E7. In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding an antigen that is at least 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1000 or more amino acids long. In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14.
In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding an antigen that is a fusion protein between HPV 16 protein E6 and HPV16 protein E7, and Calreticulin. In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding an antigen that is at least 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 500, 600, or at least 676 amino acids long. In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:15.
In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding an antigen that is a fusion protein between HPV 16 protein E6 and HPV 16 protein E7, and Ubiquitin. In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding an antigen that is at least 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, or at least 332 amino acids long. In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:16.
In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding an antigen that is a fusion protein between HPV 16 protein E6 and HPV 16 protein E7, and GM-CSF, separated by a nucleotide sequence that encodes a self-cleaving peptide (2A peptide). In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding an antigen that is at least 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, or at least 383 amino acids long. In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:17.
In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding an antigen that is a fusion protein between HPV 16 protein E7 and HPV 18 protein E6, having an N-terminal VSVG signal sequence and a C-terminal peptide linker followed by a nucleotide sequence that encodes a self-cleaving peptide (2A peptide) and GM-CSF. In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding an antigen that is at least 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, or at least 428 amino acids long. In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:33.
In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding an antigen that is a fusion protein between HPV 18 protein E7 and HPV 16 protein E6, having an N-terminal VSVG signal sequence and a C-terminal peptide linker followed by a nucleotide sequence that encodes a self-cleaving peptide (2A peptide) and GM-CSF. In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding an antigen that is at least 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, or at least 435 amino acids long. In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:35.
In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding an antigen that is a fusion protein between HPV 16 protein E7, HPV 18 protein E6, HPV 16 protein E6 and HPV 18 protein E7, having an N-terminal VSVG signal sequence and a C-terminal peptide linker followed by a nucleotide sequence that encodes a self-cleaving peptide (2A peptide) and GM-CSF. In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequences encoding an antigen that is at least 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or at least 681 amino acids long. In certain embodiments, the ORF that encodes the glycoprotein of the arenavirus is substituted by nucleic acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:37.
The immunomodulatory peptides, polypeptides, or proteins presented in these illustrative examples are murine sequences. Analogous constructs encoding the human sequences would be generated for human vaccine development.
In other embodiments, the arenavirus genomic segment or arenavirus viral vector described herein further comprises a reporter protein. The reporter protein is capable of expression at the same time as the antigen described herein. Ideally, expression is visible in normal light or other wavelengths of light. In certain embodiments, the intensity of the effect created by the reporter protein can be used to directly measure and monitor the arenavirus particle or tri-segmented arenavirus particle.
Reporter genes would be readily recognized by one skilled in the art. In certain embodiments, the arenavirus particle is a fluorescent protein. In other embodiments, the reporter gene is GFP. GFP emits bright green light when exposed to UV or blue like. Non-limiting examples of reporter proteins include various enzymes, such as, but not to β-galactosidase, chloramphenicol acetyltransferase, neomycin phosphotransferase, luciferase or RFP.
6.6 Immunogenic Compositions and Vaccines
Provided herein are vaccines, immunogenic compositions, and pharmaceutical compositions comprising an arenavirus viral vector as described herein, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment as described herein. Such vaccines and pharmaceutical compositions can be formulated according to standard procedures in the art. Such compositions can be used in methods of treatment and prevention of disease.
In a specific embodiment, the compositions described herein are used in the treatment of subjects infected with, or susceptible to, an infection with HPV or reactivation of HPV. In another specific embodiment, the compositions provided herein can be used to induce an immune response in a host to whom the composition is administered. The immunogenic compositions described herein can be used as vaccines and can accordingly be formulated as pharmaceutical compositions. In a specific embodiment, the immunogenic compositions described herein are used in the prevention or treatment of infection of subjects (e.g., human subjects) by HPV or reactivation of HPV in subjects (e.g., human subjects).
In certain embodiments, the compositions provided herein further comprise a pharmaceutically acceptable excipient. In certain embodiments, such an immunogenic composition further comprises an adjuvant. An adjuvant can also be administered in combination with, but separate from, an arenavirus viral vector as described herein (including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment as described herein) before, concomitantly with, or after administration of said arenavirus viral vector. In some embodiments, the term “adjuvant” refers to a compound that when administered in conjunction with or as part of a composition described herein augments, enhances and/or boosts the immune response to an arenavirus viral vector as described herein, but when the compound is administered alone does not generate an immune response to the arenavirus viral vector. In some embodiments, the adjuvant generates an immune response to the arenavirus viral vector and does not produce an allergy or other adverse reaction. Adjuvants can enhance an immune response by several mechanisms including, e.g., lymphocyte recruitment, stimulation of B and/or T cells, and stimulation of macrophages. When a vaccine or immunogenic composition as provided herein comprises adjuvants or is administered together with one or more adjuvants, the adjuvants that can be used include, but are not limited to, mineral salt adjuvants or mineral salt gel adjuvants, particulate adjuvants, microparticulate adjuvants, mucosal adjuvants, and immunostimulatory adjuvants. Examples of adjuvants include, but are not limited to, aluminum salts (alum) (such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate), 3 De-O-acylated monophosphoryl lipid A (MPL) (see GB 2220211), MF59 (Novartis), AS03 (GlaxoSmithKline), ASO4 (GlaxoSmithKline), polysorbate 80 (Tween 80; ICL Americas, Inc.), imidazopyridine compounds (see International Application No. PCT/US2007/064857, published as International Publication No. WO2007/109812), imidazoquinoxaline compounds (see International Application No. PCT/US2007/064858, published as International Publication No. WO2007/109813) and saponins, such as QS21 (see Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, N.Y., 1995)); U.S. Pat. No. 5,057,540). In some embodiments, the adjuvant is Freund's adjuvant (complete or incomplete). Other adjuvants are oil in water emulsions (such as squalene or peanut oil), optionally in combination with immune stimulants, such as monophosphoryl lipid A (see Stoute et al., 1997, N. Engl. J. Med. 336, 86-91).
The compositions comprise an arenavirus described herein, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, and a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment described herein, alone or together with a pharmaceutically acceptable carrier. Suspensions or dispersions of genetically engineered arenaviruses, especially isotonic aqueous suspensions or dispersions, can be used. The pharmaceutical compositions may be sterilized and/or may comprise excipients, e.g., preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, salts for regulating osmotic pressure and/or buffers and are prepared in a manner known per se, for example by means of conventional dispersing and suspending processes. In certain embodiments, such dispersions or suspensions may comprise viscosity-regulating agents. The suspensions or dispersions are kept at temperatures around 2-8° C., or preferentially for longer storage may be frozen and then thawed shortly before use. For injection, the vaccine or immunogenic preparations may be formulated in aqueous solutions, preferably in physiologically compatible buffers. The solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
In certain embodiments, the compositions described herein additionally comprise a preservative,. In other embodiments, the pharmaceutical compositions described herein do not comprise a preservative.
The pharmaceutical compositions comprise from about 103 to about 1011 focus forming units of the genetically engineered arenaviruses. Unit dose forms for parenteral administration are, for example, ampoules or vials, e.g., vials containing from about 103 to 1010 focus forming units (e.g., focus forming units in a complementing cell line) or 105 to 1015 physical particles of genetically engineered arenaviruses.
In another embodiment, a vaccine or immunogenic composition provided herein is formulated suitable for administration to a subject by, including but not limited to, oral, intradermal, intramuscular, intraperitoneal, intravenous, topical, subcutaneous, percutaneous, intranasal and inhalation routes, and via scarification (scratching through the top layers of skin, e.g., using a bifurcated needle). Specifically, subcutaneous, intramuscular or intravenous routes can be used. In one aspect, the vaccine or immunogenic composition is formulated for intravenous administration to a subject.
For administration intranasally or by inhalation, the preparation for use provided herein can be conveniently formulated in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflators may be formulated containing a powder mix of the compound and asuitable powder base such as lactose or starch.
The dosage of the active ingredient depends upon the type of vaccination and upon the subject, and their age, weight, individual condition, the individual pharmacokinetic data, and the mode of administration.
Provided herein is also a process and a use of genetically engineered arenaviruses for the manufacture of vaccines in the form of pharmaceutical preparations, which comprise genetically engineered arenaviruses as active ingredient. The pharmaceutical compositions as provided herein are prepared in a manner known per se, for example by means of conventional mixing and/or dispersing processes.
6.7 Methods of Treatment
Provided herein are methods for the treatment and/or prevention of neoplastic disease, such as cancer. These methods comprise administration to a subject in need of treatment and/or prevention of neoplastic disease, such as cancer, an effective amount of an arenavirus as described herein (see Sections 6.1, 6.2, 6.3 and 6.4). Also provided herein are methods for the treatment and/or prevention of an infection with an oncogenic virus, wherein the method comprises administration to a subject in need of treatment and/or prevention of an infection with an oncogenic virus an effective amount of an arenavirus that expresses at least one antigen of the oncogenic virus. Such oncogenic viruses can be human papillomavirus, Kaposi's sarcoma-associated herpesvirus, Epstein-Barr virus, Merkel cell polyomavirus, or human T-lymphotropic virus. Such antigens of oncogenic viruses can be antigens of human papillomavirus, Kaposi's sarcoma-associated herpesvirus, Epstein-Barr virus, Merkel cell polyomavirus, or human T-lymphotropic virus.
In one embodiment, provided herein are methods of treating and/or preventing an HPV infection in a subject comprising administering to the subject an arenavirus expressing an HPV antigen as described herein (see Sections 6.1, 6.2, 6.3, 6.4 and 6.5). In a specific embodiment, a method for treating and/or preventing an HPV infection comprises administering to a subject in need thereof an effective amount of an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, and a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing at least one HPV antigen described herein. The subject can be a mammal, such as, but not limited to a human being, a mouse, a rat, a guinea pig, a domesticated animal, such as, but not limited to, a cow, a horse, a sheep, a pig, a goat, a cat, a dog, a hamster, a donkey. In a specific embodiment, the subject is a human.
In another embodiment, provided herein are methods for inducing an immune response against HPV infection or its manifestation in a subject comprising administering to the subject an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen or a composition thereof.
In another embodiment, the subjects to whom an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen described herein or a composition thereof is administered have, are susceptible to, or are at risk for an HPV infection or reactivation. In another specific embodiment, the subjects to whom an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen described herein or a composition thereof is administered are infected with, are susceptible to, or are at risk for, an infection with HPV or reactivation with HPV.
In another embodiment, the subjects to whom an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen described herein or a composition thereof is administered are suffering from, are susceptible to, or are at risk for, an infection with HPV in the keratinocytes of the skin or the mucous membrane. In a specific embodiment, the subjects to whom an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen described herein or a composition thereof is administered are suffering from, are susceptible to, or are at risk for, an infection with HPV in one or more organs of the body, including but not limited to the skin, uterus, genitalia, areas of the respiratory tract.
In another embodiment, the subjects to whom an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, an replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen described herein or a composition thereof is administered to are suffering from symptoms including but not limited to cervical cancer, anal cancer, vulvar cancer, vaginal cancer, penile cancer, HPV-positive oropharyngeal cancer (OSCC), common warts, plantar warts, subungual or periungual warts, genital warts, condylomata acuminata or venereal warts, respiratory papillomatosis, and epidermodysplasia verruciformis.
In another embodiment, an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, an replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen as described herein or a composition thereof is administered to a subject of any age group suffering from, are susceptible to, or are at risk for, an infection with HPV. In a specific embodiment, an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen as described herein or a composition thereof is administered to a subject with a compromised immune system, a pregnant subject, a subject undergoing an organ or bone marrow transplant, a subject taking immunosuppressive drugs, a subject undergoing hemodialysis, a subject who has cancer, or a subject who is suffering from, is susceptible to, or is at risk for, an infection with HPV or reactivation of HPV. In a more specific embodiment, an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen as described herein or a composition thereof is administered to a subject with a compromised immune system due to HIV infection, who is suffering from, is susceptible to, or is at risk for, an infection with HPV or reactivation of HPV.
In another embodiment, an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen described herein or a composition thereof is administered to subjects with a heightened risk of disseminated HPV infection.
In another embodiment, an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen as described herein or a composition thereof is administered to a subject having a dormant infection with HPV. In a specific embodiment, an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment expressing an HPV antigen described herein or a composition thereof is administered to a subject having a dormant infection with HPV, which can reactivate upon immune system compromise. Thus, provided herein is a method for preventing reactivation of HPV.
In another embodiment, an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen described herein or a composition thereof is administered to subjects infected with, or at risk of infection with, one or more genotypes of HPV. In certain embodiments, one or more of those genotypes include HPV genotype 1 (HPV1), HPV genotype 2 (HPV2), HPV genotype 3 (HPV3), HPV genotype 4 (HPV4), HPV genotype 6 (HPV6), HPV genotype 7 (HPV7), HPV genotype 8 (HPV8), HPV genotype 10 (HPV10), HPV genotype 11 (HPV11), HPV genotype 13 (HPV13), HPV genotype 16 (HPV16), HPV genotype 18 (HPV18), HPV genotype 22 (HPV22), HPV genotype 26 (HPV26), HPV genotype 31 (HPV31), HPV genotype 32 (HPV32), HPV genotype 33 (HPV33), HPV genotype 35 (HPV35), HPV genotype 39 (HPV39), HPV genotype 42 (HPV42), HPV genotype 44 (HPV44), HPV genotype 45 (HPV45), HPV genotype 51 (HPV51), HPV genotype 52 (HPV52), HPV genotype 53 (HPV53), HPV genotype 56 (HPV56), HPV genotype 58 (HPV58), HPV genotype 59 (HPV59), HPV genotype 60 (HPV60), HPV genotype 63 (HPV63), HPV genotype 66 (HPV66), HPV genotype 68 (HPV68), HPV genotype 73 (HPV73), or HPV genotype 82 (HPV82), or other genotypes.
In another embodiment, an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen described herein or a composition thereof is administered to subjects infected with, or at risk of infection with, one or more “high-risk” genotypes of HPV, such as HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV73, and HPV82.
In another embodiment, administering an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen as described herein or a composition thereof to subjects confer cell-mediated immunity (CMI) against an infection with HPV or reactivation of HPV. Without being bound by theory, in another embodiment, an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen as described herein or a composition thereof infects and expresses antigens of interest in antigen presenting cells (APC) of the host (e.g., macrophages) for direct presentation of antigens on Major Histocompatibility Complex (MHC) class I. In another embodiment, administering an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen as described herein or a composition thereof to subjects induces IFN-γ and CD8+ T cell responses (IFN-γ is produced by CD8+ T cells) of high magnitude to treat or prevent an infection with HPV or reactivation of HPV.
In another embodiment, administering an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen described herein or a composition thereof reduces the risk that an individual will develop an infection with HPV or reactivation of HPV by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the risk of developing an infection with HPV or reactivation of HPV in the absence of such treatment.
In another embodiment, administering an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen described herein or a composition thereof reduces the symptoms or manifestations of an infection with HPV or reactivation of HPV by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the manifestation of the symptoms of an infection HPV or reactivation of HPV in the absence of such treatment.
Manifestations of HPV infections include but are not limited to cervical cancer, anal cancer, vulvar cancer, vaginal cancer, penile cancer, HPV-positive oropharyngeal cancer (OSCC), common warts, plantar warts, subungual or periungual warts, genital warts, condylomata acuminata or venereal warts, respiratory papillomatosis, and epidermodysplasia verruciformis.
In another embodiment, administering an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen described herein or a composition thereof in subjects with immature neonatal immune system induces cell-mediated immunity (CMI) response against an infection with HPV or reactivation of HPV by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to CMI response against an infection with HPV or reactivation of HPV in the absence of such a treatment.
In another embodiment, administering an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen described herein or a composition thereof in subjects induces an HPV antigen specific immune response resulting in an increased amount of antigen-specific CD8+ T cells detected in peripheral blood. In certain embodiments, administering an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen described herein or a composition thereof in subjects induces an increase of HPV antigen specific CD8+ T-cells, wherein the HPV antigen specific CD8+ T-cells comprise approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40% or 50% of the total CD8+ T-cell population. In certain embodiments, the percentage of HPV antigen specific CD8+ T-cells can be determined through any method known to the skilled artisan, such as through a tetramer staining assay.
Changes in cell-mediated immunity (CMI) response function against an infection with HPV or reactivation of HPV induced by administering an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen described herein or a composition thereof in subjects can be measured by any assay known to the skilled artisan including, but not limited to flow cytometry (see, e.g., Perfetto et al., 2004, Nat Rev Immun., 4(8):648-55), lymphocyte proliferation assays (see, e.g., Bonilla et al., 2008, Ann Allergy Asthma Immunol., 101:101-4; and Hicks et al., 1983, Am J Clin Pathol., 80:159-63), assays to measure lymphocyte activation including determining changes in surface marker expression following activation of measurement of cytokines of T lymphocytes (see, e.g., Caruso et al., 1997, Cytometry, 27:71-6), ELISPOT assays (see, e.g., Czerkinsky et al., 1983, J Immunol Methods., 65:109-121; and Hutchings et al., 1989, J Immunol Methods, 120:1-8), or Natural killer cell cytotoxicity assays (see, e.g., Bonilla et al., 2005, Ann Allergy Asthma Immunol. May; 94(5 Suppl 1):S1-63). (a) Combination Therapy
In one embodiment, provided herein are methods of treating and/or preventing an HPV infection in a subject comprising administering to the subject two or more arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen as described herein. See Sections 6.1 to 6.5. In specific embodiments, a method for treating and/or preventing an HPV infection comprises administering a first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, and a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen as described herein, e.g., in which the ORF encoding the GP of the S genomic segment is substituted with a nucleotide sequence encoding the HPV antigen, wherein the HPV antigen can be but is not limited to:
an HPV16 protein E6, or an antigenic fragment thereof;
an HPV16 protein E7, or an antigenic fragment thereof;
an HPV18 protein E6, or an antigenic fragment thereof; or
an HPV18 protein E7, or an antigenic fragment thereof.
and a second arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing a HPV antigen as described herein, e.g., in which the ORF encoding the GP of the S genomic segment is substituted with a nucleotide sequence encoding the HPV antigen, wherein the HPV antigen can be but is not limited to:
an HPV16 protein E6, or an antigenic fragment thereof;
an HPV16 protein E7, or an antigenic fragment thereof;
an HPV18 protein E6, or an antigenic fragment thereof; or
an HPV18 protein E7, or an antigenic fragment thereof.
In certain embodiments, provided herein are methods for treating and/or preventing an infection comprising administering two arenavirus viral vector constructs, or two arenavirus genomic segments, expressing an HPV antigen as described herein. In a specific embodiment, the two arenavirus viral vectors, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, or replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, express a different HPV antigen. In other embodiments, the two arenavirus viral vector constructs, or arenavirus genomic segments, have different arenaviral backbones. In yet other embodiments, the two arenavirus viral vector constructs, or arenavirus genomic segments, express different HPV antigens and have different arenaviral backbones.
In certain embodiments, provided herein are methods for treating and/or preventing an HPV infection comprising administering three or more arenavirus viral vector constructs, or arenavirus genomic segments, expressing an HPV antigen as described herein. In another embodiment, provided herein are methods for treating/and or preventing an infection comprising administering four or more arenavirus viral vector constructs or arenavirus genomic segments, five or more arenavirus viral vector constructs or arenavirus genomic segments, six or more arenavirus viral vector constructs or arenavirus genomic segments, or seven arenavirus viral vector constructs or arenavirus genomic segments, each expressing an HPV antigen as described herein. In certain embodiments, each of the different arenavirus viral vectors expresses a different HPV antigen as described herein. In certain embodiments, the arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, is derived from LCMV. In certain embodiments, the arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, is derived from Junin virus. In certain embodiments, the arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, is derived from a combination of LCMV and Junin virus.
In certain specific embodiments, administration of an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing HPV16 protein E7/E6 fusion protein and an immunomodulatory peptide, polypeptide, or protein elicits a greater antigen specific CD8+ T-cell response than administration of an arenavirus viral vector, including an infectious, replication-deficient arenavirus arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV16 protein E7/E6 fusion protein alone. In certain specific embodiments, administration of an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing HPV16 protein E7/E6 fusion protein and GM-CSF elicits a greater antigen specific CD8+ T-cell response than administration of an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing HPV16 protein E7/E6 fusion protein alone. In certain specific embodiments, administration of an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing HPV16 protein E7/E6 fusion protein and GM-CSF elicits an antigen specific CD8+ T-cell response that is 10%, 50%, 100%, 150%, or 200% greater than the antigen specific CD8+ T-cell response to administration of an arenavirus viral vector, including infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing HPV16 protein E7/E6 fusion protein alone.
In certain specific embodiments, administration of an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing HPV16 E7/HPV18 E6 fusion protein and an immunomodulatory peptide, polypeptide, or protein elicits a greater antigen specific CD8+ T-cell response than administration of an arenavirus viral vector, including an infectious, replication-deficient arenavirus arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV16 E7/HPV18 E6 fusion protein alone. In certain specific embodiments, administration of an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing HPV16 E7/HPV18 E6 fusion protein and GM-CSF elicits a greater antigen specific CD8+ T-cell response than administration of an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing HPV16 E7/HPV18 E6 fusion protein alone. In certain specific embodiments, administration of an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing HPV16 E7/HPV18 E6 fusion protein and GM-CSF elicits an antigen specific CD8+ T-cell response that is 10%, 50%, 100%, 150%, or 200% greater than the antigen specific CD8+ T-cell response to administration of an arenavirus viral vector, including infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing HPV16 E7/HPV18 E6 fusion protein alone
In certain specific embodiments, administration of an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing HPV18 E7/HPV16 E6 fusion protein and an immunomodulatory peptide, polypeptide, or protein elicits a greater antigen specific CD8+ T-cell response than administration of an arenavirus viral vector, including an infectious, replication-deficient arenavirus arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV18 E7/HPV16 E6 fusion protein alone. In certain specific embodiments, administration of an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing HPV18 E7/HPV16 E6 fusion protein and GM-CSF elicits a greater antigen specific CD8+ T-cell response than administration of an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing HPV18 E7/HPV16 E6 fusion protein alone. In certain specific embodiments, administration of an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing HPV18 E7/HPV16 E6 fusion protein and GM-CSF elicits an antigen specific CD8+ T-cell response that is 10%, 50%, 100%, 150%, or 200% greater than the antigen specific CD8+ T-cell response to administration of an arenavirus viral vector, including infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing HPV18 E7/HPV16 E6 fusion protein alone
In certain specific embodiments, administration of an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein and an immunomodulatory peptide, polypeptide, or protein elicits a greater antigen specific CD8+ T-cell response than administration of an arenavirus viral vector, including an infectious, replication-deficient arenavirus arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein alone. In certain specific embodiments, administration of an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein and GM-CSF elicits a greater antigen specific CD8+ T-cell response than administration of an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein alone. In certain specific embodiments, administration of an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein and GM-CSF elicits an antigen specific CD8+ T-cell response that is 10%, 50%, 100%, 150%, or 200% greater than the antigen specific CD8+ T-cell response to administration of an arenavirus viral vector, including infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein alone
In specific embodiments, the HPV antigens as described herein are expressed together with signal peptides and/or linkers as described herein. In specific embodiments the HPV antigens and immunomodulatory peptides, polypeptides, or proteins as described herein are expressed together with signal peptides and/or linkers as described herein.
In another embodiment, the vector generated to encode one or more HPV antigens as described herein of the first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, may be based on LCMV Clone 13 or LCMV MP strain. (See, e.g., Section 6.8).
In another embodiment, the vector generated to encode one or more HPV antigens as described herein of the second arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, may be based on LCMV Clone 13 or LCMV MP strain. (See, e.g., Section 6.8).
In another embodiment, the vector generated to encode one or more HPV antigens as described herein of the first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, may be based on Junin virus.
In another embodiment, the vector generated to encode one or more HPV antigens as described herein of the second arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, may be based on Junin virus.
(b) Treatment Regimens
The HPV antigens can be any HPV antigen as described herein. Without being limited by theory, administration of a first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, and subsequently of a second arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, results in a prime-boost effect.
In certain embodiments, provided herein are methods for treating and/or preventing an infection comprising administering two or more arenavirus vector constructs each expressing the same or a different HPV antigen sequentially. The time interval between each administration can be about 1 week, about 2 weeks, about 3 week, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 18 months, or about 24 months.
In certain embodiments, the first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, and the second arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, are homologous. In certain embodiments, the first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, and the second arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, are heterologous.
In certain specific embodiments, the first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, is an Old World arenavirus, and the second arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, is an Old World arenavirus. In certain specific embodiments, the first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, is an Old World arenavirus, and the second arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, is a New World arenavirus. In certain specific embodiments, the first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, is a New World arenavirus, and the second arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, is an New World arenavirus. In certain specific embodiments, the first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, is an New World arenavirus, and the second arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, is an Old World arenavirus.
In certain specific embodiments, the first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, is derived from LCMV, and the second arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, is derived from LCMV. In certain specific embodiments, the first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, is derived from LCMV, and the second arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, is derived from Junin virus. In certain specific embodiments, the first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, is derived from Junin virus, and the second arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, is derived from Junin virus. In certain specific embodiments, the first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, arenavirus is derived from Junin virus, and the second arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, is derived from LCMV.
In certain embodiments, provided herein is a method of treating and/or preventing, a neoplastic disease, such as cancer, wherein a first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, is administered first as a “prime,” and a second arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, is administered as a “boost.” The first and the second arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, can express the same or different tumor antigens. The tumor antigen can be an antigen of human papillomavirus, antigen of Kaposi's sarcoma-associated herpesvirus, such as latency-associated nuclear antigen, antigen of Epstein-Barr virus, such as EBV-EA, EBV-MA, or EBV-VCA, antigen of Merkel cell polyomavirus, such as MCV T antigen, or antigen of human T-lymphotropic virus, such as HTLV-1 Tax antigen. The tumor antigen can also be Alphafetoprotein (AFP), Carcinoembryonic antigen (CEA), CA-125, MUC-1, Epithelial tumor antigen (ETA), Tyrosinase, Melanoma-associated antigen (MAGE), or abnormal products of ras, and p53. The neoplastic disease can be a disease associated with benign neoplasms, such as uterine fibroids and melanocytic nevi, potentially malignant neoplasms, such as carcinoma in situ, or malignant neoplasms, such as cancer. In certain specific embodiments, the “prime” administration is performed with an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, and a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, derived from LCMV, and the “boost” is performed with an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, derived from Junin virus. In certain specific embodiments, the “prime” administration is performed with an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, derived from Junin virus, and the “boost” is performed with an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, derived from LCMV.
In certain embodiments, administering a first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen or a fragment thereof, followed by administering a second arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen or a fragment thereof results in a greater antigen specific CD8+ T cell response than administering a single arenavirus viral vector expressing an HPV antigen or a fragment thereof. In certain specific embodiments, administering a first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV16 E7/E6 fusion protein, followed by administering a second arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV16 E7/E6 fusion protein results in a greater antigen specific CD8+ T cell response than administering a single arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, and a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV16 E7/E6 fusion protein. In certain embodiments, the antigen specific CD8+ T cell count increases by 50%, 100%, 150% or 200% after the second administration compared to the first administration. In certain embodiments, administering a third arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV16 E7/E6 fusion protein results in a greater antigen specific CD8+ T cell response than administering two consecutive arenavirus viral vectors expressing an HPV16 E7/E6 fusion protein. In certain embodiments, the antigen specific CD8+ T cell count increases by about 50%, about 100%, about 150%, about 200% or about 250% after the third administration compared to the first administration (See
In certain embodiments, provided herein are methods for treating and/or preventing an infection comprising administering two or more arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, wherein the two or more arenavirus viral vectors are homologous, and wherein the time interval between each administration is about 1 week, about 2 weeks, about 3 week, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 18 months, or about 24 months.
In certain embodiments, administering a first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen or a fragment thereof and a second, heterologous, arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen or a fragment thereof elicits a greater CD8+ T cell response than administering a first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen or a fragment thereof and a second, homologous, arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen or a fragment thereof.
In certain specific embodiments, the first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV16 E7/E6 fusion protein, an HPV16 E7/HPV18 E6 fusion protein, an HPV18 E7/HPV16 E6 fusion protein or an HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein is LCMV, and the second, homologous, arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV16 E7/E6 fusion protein, an HPV16 E7/HPV18 E6 fusion protein, an HPV18 E7/HPV16 E6 fusion protein or an HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein is LCMV. In certain specific embodiments, the first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV16 E7/E6 fusion protein, an HPV16 E7/HPV18 E6 fusion protein, an HPV18 E7/HPV16 E6 fusion protein or an HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein is Junin virus, and the second, homologous, arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV16 E7/E6 fusion protein, an HPV16 E7/HPV18 E6 fusion protein, an HPV18 E7/HPV16 E6 fusion protein or an HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein is Junin virus.
In certain specific embodiments, the first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV16 E7/E6 fusion protein, an HPV16 E7/HPV18 E6 fusion protein, an HPV18 E7/HPV16 E6 fusion protein or an HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein is LCMV, and the second, heterologous, arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV16 E7/E6 fusion protein, an HPV16 E7/HPV18 E6 fusion protein, an HPV18 E7/HPV16 E6 fusion protein or an HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein is Junin virus. In certain specific embodiments, the first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV16 E7/E6 fusion protein, an HPV16 E7/HPV18 E6 fusion protein, an HPV18 E7/HPV16 E6 fusion protein or an HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein is Junin virus, and the second, heterologous, arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV16 E7/E6 fusion protein, an HPV16 E7/HPV18 E6 fusion protein, an HPV18 E7/HPV16 E6 fusion protein or an HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein is LCMV.
In certain specific embodiments, administering a first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV16 E7/E6 fusion protein, an HPV16 E7/HPV18 E6 fusion protein, an HPV18 E7/HPV16 E6 fusion protein or an HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein and a second, heterologous, arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV16 E7/E6 fusion protein, an HPV16 E7/HPV18 E6 fusion protein, an HPV18 E7/HPV16 E6 fusion protein or an HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein thereof elicits a greater CD8+ T cell response than administering a first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, arenavirus expressing an HPV16 E7/E6 fusion protein, an HPV16 E7/HPV18 E6 fusion protein, an HPV18 E7/HPV16 E6 fusion protein or an HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein and a second, homologous, arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV16 E7/E6 fusion protein, an HPV16 E7/HPV18 E6 fusion protein, an HPV18 E7/HPV16 E6 fusion protein or an HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein. In certain specific embodiments, administering a first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV16 E7/E6 fusion protein, an HPV16 E7/HPV18 E6 fusion protein, an HPV18 E7/HPV16 E6 fusion protein or an HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein and a second, heterologous, arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV16 E7/E6 fusion protein, an HPV16 E7/HPV18 E6 fusion protein, an HPV18 E7/HPV16 E6 fusion protein or an HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein thereof elicits a CD8+ T cell response that is about 20%, about 40%, about 60%, about 80%, about 100%, about 120%, about 140%, about 160%, about 180%, or about 200% greater than administering a first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV16 E7/E6 fusion protein, an HPV16 E7/HPV18 E6 fusion protein, an HPV18 E7/HPV16 E6 fusion protein or an HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein and a second, homologous, arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV16 E7/E6 fusion protein, an HPV16 E7/HPV18 E6 fusion protein, an HPV18 E7/HPV16 E6 fusion protein or an HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein (See
In certain embodiments, provided herein are methods for treating and/or preventing an infection comprising administering two or more arenavirus vector constructs, wherein the two or more arenavirus vector constructs are heterologous, and wherein the time interval between each administration is about 1 week, about 2 weeks, about 3 week, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 18 months, or about 24 months.
In yet another embodiment, provided herein is the combined use of the arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen described herein and one or more replication-deficient virus vectors. In a more specific embodiment the replication-deficient virus vector is selected from the group comprising of poxviruses, adenoviruses, alphaviruses, herpes simplex viruses, paramyxoviruses, rhabdoviruses, poliovirus, adeno-associated virus, and sendai virus, and mixtures thereof. In a specific embodiment, the poxvirus is a modified vaccine Ankara.
In yet another embodiment, provided herein is the combined use of the arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen described herein and one or more replication-deficient virus vectors expressing an HPV antigen. In a more specific embodiment the replication-deficient virus vector is selected from the group comprising of poxviruses, adenoviruses, alphaviruses, herpes simplex viruses, paramyxoviruses, rhabdoviruses, poliovirus, adeno-associated virus, and sendai virus, and mixtures thereof. In a specific embodiment, the poxvirus is a modified vaccine Ankara.
In another embodiment, the first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen as described herein is administered before or after the second arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen as described herein. For example the first arenavirus viral vector expressing an HPV antigen is administered around 30-60 minutes before or after the first administration of the second arenavirus viral vector.
In another embodiment, the first arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing a vaccine antigen is administered before the second arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing a vaccine antigen. In certain embodiments there is a period of about 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year between the administration of the first arenavirus viral vector and the second arenavirus viral vector.
In another embodiment, two arenavirus viral vectors, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or two arenavirus genomic segments, are administered in a treatment regime at molar ratios ranging from about 1:1 to 1:1000, in particular including: 1:1 ratio, 1:2 ratio, 1:5 ratio, 1:10 ratio, 1:20 ratio, 1:50 ratio, 1:100 ratio, 1:200 ratio, 1:300 ratio, 1:400 ratio, 1:500 ratio, 1:600 ratio, 1:700 ratio, 1:800 ratio, 1:900 ratio, 1:1000 ratio.
In another embodiment, the subjects to whom two or more arenavirus viral vectors, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or two or more arenavirus genomic segments, expressing an HPV antigen described herein are administered have, are susceptible to, or are at risk for an HPV infection or reactivation. In another embodiment, the subjects to whom two or more arenavirus viral vectors, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or two or more arenavirus genomic segments, expressing an HPV antigen described herein are administered are infected with, are susceptible to, or are at risk for, an infection with HPV or reactivation with HPV.
The subjects who can be treated with the methods provided herein are susceptible to, or are at risk for an HPV infection or reactivation.
In another embodiment, said two or more arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, expressing an HPV antigen as described herein further express at least another immunostimulatory peptide, polypeptide or protein. In certain embodiments, the immunostimulatory peptide, polypeptide or protein is Calreticulin (CRT), or a fragment thereof; Ubiquitin or a fragment thereof; Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), or a fragment thereof; Invariant chain (CD74) or an antigenic fragment thereof; Mycobacterium tuberculosis Heat shock protein 70 or an antigenic fragment thereof; Herpes simplex virus 1 protein VP22 or an antigenic fragment thereof, CD40 ligand or an antigenic fragment thereof; or Fms-related tyrosine kinase 3 (Flt3) ligand or an antigenic fragment thereof.
Heterologous prime-boost methods with arenavirus viral vectors, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segments, wherein the two arenavirus viral vectors are derived from different arenaviruses (e.g., LCMV and Junin virus) are also provided. These arenavirus viral vectors can express an antigen, such as an antigen of an oncogenic virus, or an antigen of a tumor-associated virus. In specific embodiments, the oncogenic virus is human papillomavirus, Kaposi's sarcoma-associated herpesvirus, Epstein-Barr virus, Merkel cell polyomavirus, or human T-lymphotropic virus.
6.8 Nucleic Acids, Vector Systems and Cell Lines
In one embodiment, described herein is a nucleic acid sequence encoding the large genomic segment (L segment) of an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, and a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, described herein, in which one ORF of the genomic segment is deleted or functionally inactivated, and the genomic segment comprises a heterologous nucleotide sequence as described in Section 6.5, such as a heterologous nucleotide sequence encoding an HPV antigen.
In one embodiment, described herein is a nucleic acid sequence that encodes the short genomic segment (S segment) of an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, and a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, described herein, in which one ORF of the genomic segment is deleted or functionally inactivated and wherein the short genomic segment comprises a nucleotide sequence encoding an HPV antigen. In another embodiment, described herein is a nucleic acid sequence that encodes the short genomic segment (S segment) of an arenavirus viral vector, including an infectious, replication-deficient arenavirus viral vector, a replication-competent tri-segmented arenavirus viral vector, and a replication-deficient tri-segmented arenavirus viral vector, or an arenavirus genomic segment, described herein, in which the ORF of the glycoprotein gene is deleted or functionally inactivated and wherein the short genomic segment comprises a heterologous nucleotide sequence encoding an HPV antigen. In certain, more specific embodiments, the HPV antigen is an antigen as described in Section 6.5.
In certain embodiments, the nucleic acid sequences provided herein can be derived from a particular strain of LCMV. Strains of LCMV include Clone 13, MP strain, Arm CA 1371, Arm E-250, WE, UBC, Traub, Pasteur, 810885, CH-5692, Marseille #12, HP65-2009, 200501927, 810362, 811316, 810316, 810366, 20112714, Douglas, GR0, SN05, CABN and their derivatives. In specific embodiments, the nucleic acid is derived from LCMV Clone 13. In other specific embodiments, the nucleic acid is derived from LCMV MP strain or Junin virus.
In a more specific embodiment, provided herein is a nucleic acid encoding an arenavirus genomic segment comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. In another embodiment, provided herein is a nucleic acid that encodes an arenavirus genomic segment comprising (i) a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the sequence of nucleotide 1639 to 3315 of SEQ ID NO: 1; and (ii) a heterologous nucleotide sequence encoding an HPV antigen.
In another embodiment, provided herein is a nucleic acid that encodes an arenavirus genomic segment comprising (i) a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the sequence of nucleotide 1639 to 3315 of SEQ ID NO: 1; (ii) a heterologous nucleotide sequence encoding an HPV antigen; and (iii) a heterologous nucleotide sequence encoding an immunomodulatory peptide, polypeptide, or protein.
In another embodiment, provided herein is a nucleic acid that encodes an arenavirus genomic segment comprising (i) a nucleotide sequence encoding an expression product whose amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence encoded by 1639 to 3315 of SEQ ID NO: 1; and (ii) a heterologous nucleotide sequence encoding an HPV antigen.
In another embodiment, provided herein is a nucleic acid that encodes an arenavirus genomic segment comprising (i) a nucleotide sequence encoding an expression product whose amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence encoded by 1639 to 3315 of SEQ ID NO: 1; (ii) a heterologous nucleotide sequence encoding an HPV antigen; and (iii) a heterologous nucleotide sequence encoding an immunomodulatory peptide, polypeptide, or protein.
In another embodiment, provided herein is a nucleic acid that encodes an arenavirus genomic segment comprising (i) a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the sequence of nucleotide 1640 to 3316 of SEQ ID NO: 2; and (ii) a heterologous nucleotide sequence encoding an HPV antigen.
In another embodiment, provided herein is a nucleic acid that encodes an arenavirus genomic segment comprising (i) a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the sequence of nucleotide 1640 to 3316 of SEQ ID NO: 2; and (ii) a heterologous nucleotide sequence encoding an HPV antigen; and (iii) a heterologous nucleotide sequence encoding an immunomodulatory peptide, polypeptide, or protein.
In another embodiment, provided herein is a nucleic acid that encodes an arenavirus genomic segment comprising (i) a nucleotide sequence encoding an expression product whose amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence encoded by 1640 to 3316 of SEQ ID NO: 2; and (ii) a heterologous nucleotide sequence encoding an HPV antigen.
In another embodiment, provided herein is a nucleic acid that encodes an arenavirus genomic segment comprising (i) a nucleotide sequence encoding an expression product whose amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence encoded by 1640 to 3316 of SEQ ID NO: 2; (ii) a heterologous nucleotide sequence encoding an HPV antigen, and (iii) a heterologous nucleotide sequence encoding an immunomodulatory peptide, polypeptide, or protein.
In another embodiment, provided herein are nucleic acids that encode an arenavirus genomic segment comprising (i) a nucleotide sequence encoding at least one self-cleaving peptide or ribosome-skipping sequence; and (ii) a nucleotide sequence encoding two, three, or four, or more HPV antigens. In specific embodiments, the nucleotide sequence encoding a self-cleaving peptide encodes Teschovirus 2A. In certain embodiments, provided herein are nucleic acids that encode two, three, four, or more HPV antigens separated by one or more nucleotide sequences encoding self-cleaving peptides or ribosome-skipping sequences (e.g., T2A). In certain embodiments, provided herein are nucleic acids that encode a HPV16 E6/E7 fusion protein and a HPV18 E6/E7 fusion protein, separated by one or more nucleotide sequences encoding self-cleaving peptides or ribosome-skipping sequences.
In another embodiment, provided herein are nucleic acids that encode an arenavirus genomic segment comprising (i) a nucleotide sequence encoding at least one self-cleaving peptide or ribosome-skipping sequence; (ii) a heterologous nucleotide sequence encoding two, three, or four, or more HPV antigens; and (iii) a heterologous nucleotide sequence encoding an immunomodulatory peptide, polypeptide, or protein. In specific embodiments, the nucleotide sequence encoding a self-cleaving peptide encodes Teschovirus 2A. In certain embodiments, provided herein are nucleic acids that encode two, three, four, or five HPV antigens separated by one or more nucleotide sequences encoding self-cleaving peptides or ribosome-skipping sequences (e.g., T2A). In certain embodiments, provided herein are nucleic acids that encode HPV16 E6/E7 fusion protein and HPV18 E6/E7 fusion protein, and one or more immunomodulatory peptides, polypeptides, or proteins, separated from the HPV16 E6/E7 fusion protein by one or more nucleotide sequences encoding self-cleaving peptides or ribosome-skipping sequences. In other embodiments, provided herein are nucleic acids that encode HPV16 E6/E7 fusion protein and HPV18 E6/E7 fusion protein, and one or more immunomodulatory peptides, polypeptides, or proteins, separated from the HPV18 E6/E7 fusion protein by one or more nucleotide sequences encoding self-cleaving peptides or ribosome-skipping sequences. In certain embodiments, provided herein are nucleic acids that encode an HPV16 E7/HPV18 E6 fusion protein, and one or more immunomodulatory peptides, polypeptides, or proteins, separated from the HPV16 E7/HPV18 E6 fusion protein by one or more nucleotide sequences encoding self-cleaving peptides or ribosome-skipping sequences. In other embodiments, provided herein are nucleic acids that encode a HPV16 E7/HPV18 E6 fusion protein, and one or more immunomodulatory peptides, polypeptides, or proteins, separated from the HPV16 E7/HPV18 E6 fusion protein by one or more nucleotide sequences encoding self-cleaving peptides or ribosome-skipping sequences. In certain embodiments, provided herein are nucleic acids that encode a HPV18 E7/HPV16 E6 fusion protein, and one or more immunomodulatory peptides, polypeptides, or proteins, separated from the HPV18 E7/HPV16 E6 fusion protein by one or more nucleotide sequences encoding self-cleaving peptides or ribosome-skipping sequences. In other embodiments, provided herein are nucleic acids that encode a HPV18 E7/HPV16 E6 fusion protein, and one or more immunomodulatory peptides, polypeptides, or proteins, separated from the HPV18 E7/HPV16 E6 fusion protein by one or more nucleotide sequences encoding self-cleaving peptides or ribosome-skipping sequences. In certain embodiments, provided herein are nucleic acids that encode a HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein, and one or more immunomodulatory peptides, polypeptides, or proteins, separated from the HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein by one or more nucleotide sequences encoding self-cleaving peptides or ribosome-skipping sequences. In other embodiments, provided herein are nucleic acids that encode HPV16 E7/HPV18 E6/HPV16 E6/HPV18 E7 fusion protein, and one or more immunomodulatory peptides, polypeptides, or proteins, separated from the HPV16 E7/HPV18 E6 HPV16 E6/HPV18 E7 fusion protein by one or more nucleotide sequences encoding self-cleaving peptides or ribosome-skipping sequences.
In another embodiment, provided herein are nucleic acids that encode an arenavirus genomic segment comprising (i) a nucleotide sequence encoding at least one self-cleaving peptide (or ribosome-skipping sequence); (ii) a heterologous nucleotide sequence encoding two, three, or four, or more HPV antigens; (iii) a heterologous nucleotide sequence encoding an immunomodulatory peptide, polypeptide, or protein, and (iv) a nucleotide sequence encoding a signaling sequence including a secretion signal from human tyrosinase, including a secretion signal from human growth hormone, signal sequence of tissue plasminogen activator.
In one embodiment, described herein is a vector system comprising one or more vectors that together encode the genome of an infectious, replication-deficient arenavirus particle described herein. Specifically, provided herein is a vector system wherein one or more vectors encode two arenavirus genomic segments, namely an L segment and an S segment, of an infectious, replication-deficient arenavirus described herein. Such a vector system can encode (on one or more separate DNA molecules):
an arenavirus S genomic segment that is modified such that an arenavirus particle carrying this modified S genomic segment cannot produce infectious progeny virus particles and an arenavirus L genomic segment that comprises a nucleotide sequence encoding (in sense or antisense) an HPV antigen;
an arenavirus L genomic segment that is modified such that an arenavirus particle carrying this modified L genomic segment cannot produce infectious progeny virus particles and an arenavirus S genomic segment that comprises a nucleotide sequence encoding (in sense or antisense) an HPV antigen;
an arenavirus S genomic segment that is modified such that an arenavirus particle carrying this modified S genomic segment cannot produce infectious progeny virus particles and wherein the arenavirus S genomic segment comprises a heterologous nucleotide sequence encoding (in sense or antisense) an HPV antigen and a wild type arenavirus L genomic segment; or
an arenavirus L genomic segment that is modified such that an arenavirus particle carrying this modified L genomic segment cannot produce infectious progeny virus particles and wherein the arenavirus L genomic segment comprises a heterologous nucleotide sequence encoding (in sense or antisense) an HPV antigen and a wild type arenavirus S genomic segment.
In certain embodiments, described herein is cDNA of an arenavirus (e.g., LCMV or Junin virus) genomic segment in which the ORF encoding the GP of the S genomic segment is substituted with a heterologous nucleotide sequence encoding:
an HPV16 protein E6, or an antigenic fragment thereof;
an HPV16 protein E7, or an antigenic fragment thereof;
an HPV18 protein E6, or an antigenic fragment thereof;
an HPV18 protein E7, or an antigenic fragment thereof;
an HPV16 protein E6/protein E7 fusion protein or an antigenic fragment thereof;
a shuffled HPV16 protein E6/protein E7 fusion protein or an antigenic fragment thereof;
an HPV18 protein E6/protein E7 fusion protein or an antigenic fragment thereof; or
a shuffled HPV18 protein E6/protein E7 fusion protein or an antigenic fragment thereof.
In certain embodiments, described herein is cDNA of an arenavirus (e.g., LCMV or Junin virus) genomic segment in which the ORF encoding the GP of the S genomic segment is substituted with a heterologous nucleotide sequence encoding:
an HPV16 protein E6, or an antigenic fragment thereof;
an HPV16 protein E7, or an antigenic fragment thereof;
an HPV18 protein E6, or an antigenic fragment thereof;
an HPV18 protein E7, or an antigenic fragment thereof;
an HPV16 protein E6/protein E7 fusion protein or an antigenic fragment thereof;
a shuffled HPV16 protein E6/protein E7 fusion protein or an antigenic fragment thereof;
an HPV18 protein E6/protein E7 fusion protein or an antigenic fragment thereof; or
a shuffled HPV18 protein E6/protein E7 fusion protein or an antigenic fragment thereof
and an immunomodulatory peptide, polypeptide, or protein, or a fragment thereof.
In certain embodiments, the heterologous nucleotide sequence further encodes, or the infectious, replication deficient arenavirus genome further comprises a second heterologous nucleotide sequence that encodes
Calreticulin (CRT), or a fragment thereof;
Ubiquitin or a fragment thereof;
Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), or a fragment thereof;
Invariant chain (CD74) or an antigenic fragment thereof;
Mycobacterium tuberculosis Heat shock protein 70 or an antigenic fragment thereof;
Herpes simplex virus 1 protein VP22 or an antigenic fragment thereof;
CD40 ligand or an antigenic fragment thereof; or
Fms-related tyrosine kinase 3 (Flt3) ligand or an antigenic fragment thereof.
In certain embodiments, described herein is a nucleic acid sequence encoding an arenavirus (e.g., LCMV or Junin virus) genomic segment in which the ORF encoding the GP of the S genomic segment is substituted with a heterologous nucleotide sequence encoding one or more HPV antigen sequences (e.g., one or more of those listed in the above paragraph), separated by nucleotide sequences encoding a self-cleaving peptide (or ribosome-skipping sequences). In specific embodiments, the nucleotide sequences encoding a self-cleaving peptide encode Teschovirus 2A.
In another embodiment, provided herein is a cell wherein the cell comprises a nucleic acid or a vector system described above in this section. Cell lines derived from such cells, cultures comprising such cells, and methods of culturing such cells infected are also provided herein. In certain embodiments, provided herein is a cell wherein the cell comprises a nucleic acid encoding the large genomic segment (L segment) of an infectious, replication-deficient arenavirus described herein, in which one ORF of the genomic segment is deleted or functionally inactivated, and the genomic segment comprises a nucleotide sequence encoding an HPV antigen.
In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes the short genomic segment (S segment) of an infectious, replication-deficient arenavirus described herein, in which one ORF of the genomic segment is deleted or functionally inactivated and wherein the short genomic segment comprises a heterologous nucleotide sequence encoding HPV 16 protein E6 or an antigenic fragment thereof.
In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes the short genomic segment (S segment) of an infectious, replication-deficient arenavirus described herein, in which one ORF of the genomic segment is deleted or functionally inactivated and wherein the short genomic segment comprises a heterologous nucleotide sequence encoding HPV 16 protein E7 or an antigenic fragment thereof.
In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes the short genomic segment (S segment) of an infectious, replication-deficient arenavirus described herein, in which one ORF of the genomic segment is deleted or functionally inactivated and wherein the short genomic segment comprises a heterologous nucleotide sequence encoding HPV 18 protein E6 or an antigenic fragment thereof.
In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes the short genomic segment (S segment) of an infectious, replication-deficient arenavirus described herein, in which one ORF of the genomic segment is deleted or functionally inactivated and wherein the short genomic segment comprises a heterologous nucleotide sequence encoding HPV 18 protein E7 or an antigenic fragment thereof.
In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes the short genomic segment (S segment) of an infectious, replication-deficient arenavirus described herein, in which one ORF of the genomic segment is deleted or functionally inactivated and wherein the short genomic segment comprises a heterologous nucleotide sequence encoding an HPV16 E7/E6 fusion protein or an antigenic fragment thereof.
In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes the short genomic segment (S segment) of an infectious, replication-deficient arenavirus described herein, in which one ORF of the genomic segment is deleted or functionally inactivated and wherein the short genomic segment comprises a heterologous nucleotide sequence encoding an HPV18 E7/E6 fusion protein or an antigenic fragment thereof.
In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes the short genomic segment (S segment) of an infectious, replication-deficient arenavirus described herein, in which one ORF of the genomic segment is deleted or functionally inactivated and wherein the short genomic segment comprises a heterologous nucleotide sequence encoding an HPV16 E7/E6 fusion protein or an antigenic fragment thereof, and an HPV18 E7/E6 fusion protein or an antigenic fragment thereof.
In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes the short genomic segment (S segment) of an infectious, replication-deficient arenavirus described herein, in which one ORF of the genomic segment is deleted or functionally inactivated and wherein the short genomic segment comprises a heterologous nucleotide sequence encoding an HPV16 E7/E6 fusion protein or an antigenic fragment thereof and encoding Calreticulin, or an immunomodulatory fragment thereof.
In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes the short genomic segment (S segment) of an infectious, replication-deficient arenavirus described herein, in which one ORF of the genomic segment is deleted or functionally inactivated and wherein the short genomic segment comprises a heterologous nucleotide sequence encoding an HPV16 E7/E6 fusion protein or an antigenic fragment thereof, and an HPV18 E7/E6 fusion protein or an antigenic fragment thereof, and encoding Calreticulin, or an immunomodulatory fragment thereof.
In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes the short genomic segment (S segment) of an infectious, replication-deficient arenavirus described herein, in which one ORF of the genomic segment is deleted or functionally inactivated and wherein the short genomic segment comprises a heterologous nucleotide sequence encoding an HPV16 E7/E6 fusion protein or an antigenic fragment thereof and encoding Ubiquitin, or an immunomodulatory fragment thereof.
In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes the short genomic segment (S segment) of an infectious, replication-deficient arenavirus described herein, in which one ORF of the genomic segment is deleted or functionally inactivated and wherein the short genomic segment comprises a heterologous nucleotide sequence encoding an HPV16 E7/E6 fusion protein or an antigenic fragment thereof, and an HPV18 E7/E6 fusion protein or an antigenic fragment thereof, and encoding Ubiquitin, or an immunomodulatory fragment thereof.
In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes the short genomic segment (S segment) of an infectious, replication-deficient arenavirus described herein, in which one ORF of the genomic segment is deleted or functionally inactivated and wherein the short genomic segment comprises a heterologous nucleotide sequence encoding an HPV16 E7/E6 fusion protein or an antigenic fragment thereof and encoding GM-CSF, or an immunomodulatory fragment thereof. In other, more specific embodiments, the short genomic segment comprises a heterologous nucleotide sequence encoding an HPV16 E7/E6 fusion protein, GM-CSF, and a self-cleaving peptide. In certain embodiments, the self-cleaving peptide is 2A peptide.
In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes HPV16 protein E6, HPV16 protein E7, HPV18 protein E6, and HPV18 protein E7. In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes HPV16 protein E6 or an antigenic fragment thereof, HPV16 protein E7 or an antigenic fragment thereof, HPV18 protein E6 or an antigenic fragment thereof, and HPV18 protein E7 or an antigenic fragment thereof. In certain embodiments, one, two, three or all four of HPV16 protein E6 or antigenic fragment thereof, HPV16 protein E7 or antigenic fragment thereof, HPV18 protein E6 or antigenic fragment thereof, and HPV18 protein E7 or antigenic fragment thereof, can be shuffled sequences. Each one of HPV16 protein E6 or antigenic fragment thereof, HPV16 protein E7 or antigenic fragment thereof, HPV18 protein E6 or antigenic fragment thereof, and HPV18 protein E7 or antigenic fragment thereof, can be directly fused to one or two different sequences of HPV16 protein E6 or antigenic fragment thereof, HPV16 protein E7 or antigenic fragment thereof, HPV18 protein E6 or antigenic fragment thereof, and HPV18 protein E7 or antigenic fragment thereof. Each one of HPV16 protein E6 or antigenic fragment thereof, HPV16 protein E7 or antigenic fragment thereof, HPV18 protein E6 or antigenic fragment thereof, and HPV18 protein E7 or antigenic fragment thereof, can be fused to one or two different sequences of HPV16 protein E6 or antigenic fragment thereof, HPV16 protein E7 or antigenic fragment thereof, HPV18 protein E6 or antigenic fragment thereof, and HPV18 protein E7 or antigenic fragment thereof, via a linker or self-cleaving peptide. Each one of HPV16 protein E6 or antigenic fragment thereof, HPV16 protein E7 or antigenic fragment thereof, HPV18 protein E6 or antigenic fragment thereof, and HPV18 protein E7 or antigenic fragment thereof, can be fused to one or two different sequences of HPV16 protein E6 or antigenic fragment thereof, HPV16 protein E7 or antigenic fragment thereof, HPV18 protein E6 or antigenic fragment thereof, and HPV18 protein E7 or antigenic fragment thereof. The sequence of HPV16 protein E6 or antigenic fragment thereof, HPV16 protein E7 or antigenic fragment thereof, HPV18 protein E6 or antigenic fragment thereof, and HPV18 protein E7 or antigenic fragment thereof, can be arranged in any manner known to the skilled artisan, e.g., each one of HPV16 protein E6 or antigenic fragment thereof, HPV16 protein E7 or antigenic fragment thereof, HPV18 protein E6 or antigenic fragment thereof, and HPV18 protein E7 or antigenic fragment thereof, can be upstream or downstream of a different one of HPV16 protein E6 or antigenic fragment thereof, HPV16 protein E7 or antigenic fragment thereof, HPV18 protein E6 or antigenic fragment thereof, and HPV18 protein E7 or antigenic fragment thereof. Each one of HPV16 protein E6 or antigenic fragment thereof, HPV16 protein E7 or antigenic fragment thereof, HPV18 protein E6 or antigenic fragment thereof, and HPV18 protein E7 or antigenic fragment thereof, can be fused to a signal peptide. In certain other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes HPV16 E6/HPV16 E7 fusion protein or antigenic fragment thereof, or an HPV16 E6/HPV18 E6 fusion protein or antigenic fragment thereof, HPV16 E6/HPV18 E7 fusion protein or antigenic fragment thereof, or an HPV16 E7/HPV18 E6 fusion protein or antigenic fragment thereof, HPV16 E6/HPV18 E7 fusion protein or antigenic fragment thereof, or an HPV18 E6/HPV18 E7 fusion protein or antigenic fragment thereof. In certain other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes two fusion proteins, wherein the first fusion protein is an HPV16 E6/HPV16 E7 fusion protein or antigenic fragment thereof, or an HPV16 E6/HPV18 E6 fusion protein or antigenic fragment thereof, HPV16 E6/HPV18 E7 fusion protein or antigenic fragment thereof, or an HPV16 E7/HPV18 E6 fusion protein or antigenic fragment thereof, HPV16 E6/HPV18 E7 fusion protein or antigenic fragment thereof, or an HPV18 E6/HPV18 E7 fusion protein or antigenic fragment thereof, and the second fusion protein is a different fusion protein selected from an HPV16 E6/HPV16 E7 fusion protein or antigenic fragment thereof, or an HPV16 E6/HPV18 E6 fusion protein or antigenic fragment thereof, HPV16 E6/HPV18 E7 fusion protein or antigenic fragment thereof, or an HPV16 E7/HPV18 E6 fusion protein or antigenic fragment thereof, HPV16 E6/HPV18 E7 fusion protein or antigenic fragment thereof, or an HPV18 E6/HPV18 E7 fusion protein or antigenic fragment thereof. In certain other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that further encodes an immunomodulatory peptide, polypeptide, or protein.
In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes an HPV16 E6/E7 fusion protein and an HPV18 E6/E7 fusion protein. In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes a shuffled sequence of an HPV16 E6/E7 fusion protein and a shuffled sequence of an HPV18 E6/E7 fusion protein. In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes an HPV16 E6/E7 fusion protein and an HPV18 E6/E7 fusion protein that are directly fused to each other. In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes an HPV16 E6/E7 fusion protein and an HPV18 E6/E7 fusion protein that are fused to each other via a peptide linker or self-cleaving peptide. In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes an HPV16 E6/E7 fusion protein located upstream of the HPV18 E6/E7 fusion protein. In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes HPV16 E6/E7 fusion protein located downstream of the HPV18 E6/E7 fusion protein. In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes an HPV16 E6/E7 fusion protein that is fused to a signal peptide. In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes HPV18 E6/E7 fusion protein fused to a signal peptide. In certain specific embodiments, the heterologous nucleotide sequence further encodes an immunomodulatory peptide, polypeptide, or protein.
In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes the short genomic segment (S segment) of an infectious, replication-deficient arenavirus described herein, in which one ORF of the genomic segment is deleted or functionally inactivated and wherein the short genomic segment comprises a heterologous nucleotide sequence encoding an HPV16 E7/E6 fusion protein or an antigenic fragment thereof, and an HPV18 E7/E6 fusion protein or an antigenic fragment thereof, and encoding GM-CSF, or an immunomodulatory fragment thereof. In other, more specific embodiments, the short genomic segment comprises a heterologous nucleotide sequence encoding an HPV16 E7/E6 fusion protein or an antigenic fragment thereof, and an HPV18 E7/E6 fusion protein or an antigenic fragment thereof, and encoding GM-CSF, and a self-cleaving peptide. In certain embodiments, the self-cleaving peptide is 2A peptide.
In other embodiments, provided herein is a cell wherein the cell comprises a nucleic acid sequence that encodes the short genomic segment (S segment) of an infectious, replication-deficient arenavirus described herein, in which one ORF of the genomic segment is deleted or functionally inactivated and wherein the short genomic segment comprises a heterologous nucleotide sequence encoding one or more of HPV antigens separated by one or more self-cleaving peptides (or ribosome-skipping sequences). In specific embodiments, the one or more self-cleaving peptides are T2A peptides.
In another embodiment, provided herein is a cell wherein the cell comprises two nucleic acids or a vector system described herein. Cell lines derived from such cells, cultures comprising such cells, and methods of culturing such cells infected are also provided herein.
In certain embodiments, provided herein is a nucleic acid comprising a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4 or SEQ ID NO: 5. In certain embodiments, provided herein is an expression vector comprising a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4 or SEQ ID NO: 5. In certain embodiments, provided herein is a host cell comprising a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4 or SEQ ID NO: 5.
In certain embodiments, provided herein is a nucleic acid comprising a nucleotide sequence encoding an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6, 7, 8, or 9. In certain embodiments, provided herein is an expression vector comprising a nucleotide sequence encoding an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6, 7, 8, or 9. In certain embodiments, provided herein is a host cell comprising a nucleotide sequence that encodes an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6, 7, 8, or 9.
In certain embodiments, provided herein is an isolated protein comprising an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6, 7, 8, or 9. In certain embodiments, provided herein is a host cell that expresses a protein comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6, 7, 8, or 9. In certain embodiments, the host cell is cultured in cell culture medium.
In certain embodiments, provided herein is a nucleic acid comprising a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2 or SEQ ID NO: 3. In certain embodiments, provided herein is an expression vector comprising a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2 or SEQ ID NO: 3. In certain embodiments, provided herein is a host cell comprising a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2 or SEQ ID NO: 3.
In certain embodiments, provided herein are cDNAs comprising or consisting of the arenavirus genomic segment or the tri-segmented arenavirus viral vector as described in Section 6.2 and Section 6.3, respectively.
(a) Non-Natural Position Open Reading Frame
In one embodiment, provided herein are nucleic acids that encode an arenavirus genomic segment as described in Section 6.2. In more specific embodiments, provided herein is a DNA nucleotide sequence or a set of DNA nucleotide sequences as set forth in Table 1. Host cells that comprise such nucleic acids are also provided Section 6.2.
In specific embodiments, provided herein is a cDNA of the arenavirus genomic segment engineered to carry an ORF in a position other than the wild-type position of the ORF, wherein the arenavirus genomic segment encodes a heterologous ORF as described in Section 6.5.
In one embodiment, provided herein is a DNA expression vector system that encodes the arenavirus genomic segment engineered to carry an ORF in a position other than the wild-type position of the ORF. Specifically, provided herein is a DNA expression vector system wherein one or more vectors encodes two arenavirus genomic segments, namely, an L segment and an S segment, of an arenavirus viral vector described herein. Such a vector system can encode (one or more separate DNA molecules).
In another embodiment, provided herein is a cDNA of the arenavirus S segment that has been engineered to carry an ORF in a position other than the wild-type position is part of or incorporated into a DNA expression system. In other embodiments, a cDNA of the arenavirus L segment that has been engineered to carry an ORF in a position other than the wild-type position is part of or incorporated into a DNA expression system. In certain embodiments, is a cDNA of the arenavirus genomic segment that has been engineered to carry (i) an ORF in a position other than the wild-type position of the ORF; and (ii) and ORF encoding GP, NP, Z protein, or L protein has been removed and replaced with a heterologous ORF from an organism other than an arenavirus.
In certain embodiments, the cDNA provided herein can be derived from a particular strain of LCMV. Strains of LCMV include Clone 13, MP strain, Arm CA 1371, Arm E-250, WE, UBC, Traub, Pasteur, 810885, CH-5692, Marseille #12, HP65-2009, 200501927, 810362, 811316, 810316, 810366, 20112714, Douglas, GRO1, SN05, CABN and their derivatives. In specific embodiments, the cDNA is derived from LCMV Clone 13. In other specific embodiments, the cDNA is derived from LCMV MP strain.
In certain embodiments, the vector generated to encode an arenavirus viral vector or a tri-segmented arenavirus viral vector as described herein may be based on a specific strain of LCMV. Strains of LCMV include Clone 13, MP strain, Arm CA 1371, Arm E-250, WE, UBC, Traub, Pasteur, 810885, CH-5692, Marseille #12, HP65-2009, 200501927, 810362, 811316, 810316, 810366, 20112714, Douglas, GRO1, SN05, CABN and their derivatives. In certain embodiments, an arenavirus viral vector or a tri-segmented arenavirus viral vector as described herein may be based on LCMV Clone 13. In other embodiments, the vector generated to encode an arenavirus viral vector or a tri-segmented arenavirus viral vector as described herein LCMV MP strain. The sequence of the S segment of LCMV Clone 13 is listed as SEQ ID NO: 2. In certain embodiments, the sequence of the S segment of LCMV Clone 13 is the sequence set forth in SEQ ID NO: 1. The sequence of the L segment of LCMV Clone 13 is listed as SEQ ID NO: 5. The sequence of the S segment of LCMV strain MP is listed as SEQ ID NO: 53. The sequence of the L segment of LCMV strain MP is listed as SEQ ID NO: 4.
In another embodiment, provided herein is a cell, wherein the cell comprises a cDNA or a vector system described above in this section. Cell lines derived from such cells, cultures comprising such cells, methods of culturing such cells infected are also provided herein. In certain embodiments, provided herein is a cell, wherein the cell comprises a cDNA of the arenavirus genomic segment that has been engineered to carry an ORF in a position other than the wild-type position of the ORF. In some embodiments, the cell comprises the S segment and/or the L segment.
(b) Tri-Segmented Arenavirus Viral Vector
In one embodiment, provided herein are nucleic acids that encode a tri-segmented arenavirus viral vector as described in Section 6.3. In more specific embodiments, provided herein is a DNA nucleotide sequence or a set of DNA nucleotide sequences, for example, as set forth in Table 2 or Table 3. Host cells that comprise such nucleic acids are also provided Section 6.3. In specific embodiments, provided herein are nucleic acids that encode a tri-segmented arenavirus viral vector as described, wherein the tri-segmented arenavirus viral vector encodes a heterologous ORF as described in Section 6.5.
In specific embodiments, provided herein is a cDNA consisting of a cDNA of the tri-segmented arenavirus viral vector that has been engineered to carry an ORF in a position other than the wild-type position of the ORF. In other embodiments, is a cDNA of the tri-segmented arenavirus viral vector that has been engineered to (i) carry an arenavirus ORF in a position other than the wild-type position of the ORF; and (ii) wherein the tri-segmented arenavirus viral vector encodes a heterologous ORF as described in Section 6.3.
In one embodiment, provided herein is a DNA expression vector system that together encodes the tri-segmented arenavirus viral vector as described herein. Specifically, provided herein is a DNA expression vector system wherein one or more vectors encode three arenavirus genomic segments, namely, one L segment and two S segments or two L segments and one S segment of a tri-segmented arenavirus viral vector described herein. Such a vector system can encode (one or more separate DNA molecules).
In another embodiment, provided herein is a cDNA of the arenavirus S segment(s) that has been engineered to carry an ORF in a position other than the wild-type position, and is part of or incorporated into a DNA expression system. In other embodiments, a cDNA of the arenavirus L segment(s) that has been engineered to carry an ORF in a position other than the wild-type position is part of or incorporated into a DNA expression system. In certain embodiments, is a cDNA of the tri-segmented arenavirus viral vector that has been engineered to carry (i) an ORF in a position other than the wild-type position of the ORF; and (ii) an ORF encoding GP, NP, Z protein, or L protein has been removed and replaced with a heterologous ORF from an organism other than an arenavirus.
In certain embodiments, the cDNA provided herein can be derived from a particular strain of LCMV. Strains of LCMV include Clone 13, MP strain, Arm CA 1371, Arm E-250, WE, UBC, Traub, Pasteur, 810885, CH-5692, Marseille #12, HP65-2009, 200501927, 810362, 811316, 810316, 810366, 20112714, Douglas, GRO1, SN05, CABN and their derivatives. In specific embodiments, the cDNA is derived from LCMV Clone 13. In other specific embodiments, the cDNA is derived from LCMV MP strain.
In certain embodiments, the vector generated to encode an arenavirus viral vector or a tri-segmented arenavirus viral vector as described herein may be based on a specific strain of LCMV. Strains of LCMV include Clone 13, MP strain, Arm CA 1371, Arm E-250, WE, UBC, Traub, Pasteur, 810885, CH-5692, Marseille #12, HP65-2009, 200501927, 810362, 811316, 810316, 810366, 20112714, Douglas, GRO1, SN05, CABN and their derivatives. In certain embodiments, an arenavirus viral vector or a tri-segmented arenavirus viral vector as described herein may be based on LCMV Clone 13. In other embodiments, the vector generated to encode an arenavirus viral vector or a tri-segmented arenavirus viral vector as described herein LCMV MP strain. The sequence of the S segment of LCMV Clone 13 is listed as SEQ ID NO: 2. In certain embodiments, the sequence of the S segment of LCMV Clone 13 is the sequence set forth in SEQ ID NO: 1. The sequence of the L segment of LCMV Clone 13 is listed as SEQ ID NO: 5. The sequence of the S segment of LCMV strain MP is listed as SEQ ID NO: 53. The sequence of the L segment of LCMV strain MP is listed as SEQ ID NO: 4.
In another embodiment, provided herein is a cell, wherein the cell comprises a cDNA or a vector system described above in this section. Cell lines derived from such cells, cultures comprising such cells, methods of culturing such cells infected are also provided herein. In certain embodiments, provided herein is a cell, wherein the cell comprises a cDNA of the tri-segmented arenavirus viral vector. In some embodiments, the cell comprises the S segment and/or the L segment.
6.9 Assays
Assay for Measuring Arenavirus Vector Infectivity: Any assay known to the skilled artisan can be used for measuring the infectivity of an arenavirus vector preparation. For example, determination of the virus/vector titer can be done by a “focus forming unit assay” (FFU assay). In brief, complementing cells, e.g., HEK 293 cells expressing LCMV GP protein, are plated and inoculated with different dilutions of a virus/vector sample. After an incubation period, to allow cells to form a monolayer and virus to attach to cells, the monolayer is covered with Methylcellulose. When the plates are further incubated, the original infected cells release viral progeny. Due to the Methylcellulose overlay the spread of the new viruses is restricted to neighboring cells. Consequently, each infectious particle produces a circular zone of infected cells called a Focus. Such Foci can be made visible and by that countable using antibodies against LCMV-NP and a HRP-based color reaction. The titer of a virus/vector can be calculated in focus-forming units per milliliter (FFU/mL).
To determine the infectious titer (FFU/mL) of transgene-carrying vectors this assay is modified by the use of the respective transgene-specific antibody instead of anti-LCMV-NP antibody.
Serum ELISA: Determination of the humoral immune response upon vaccination of animals (e.g., mice, guinea pigs) can be done by antigen-specific serum ELISA's (enzyme-linked immunosorbent assays). In brief, plates are coated with antigen (e.g., recombinant protein), blocked to avoid unspecific binding of antibodies and incubated with serial dilutions of sera. After incubation, bound serum-antibodies can be detected, e.g., using an enzyme-coupled anti-species (e.g., mouse, guinea pig)-specific antibody (detecting total IgG or IgG subclasses) and subsequent color reaction. Antibody titers can be determined as, e.g., endpoint geometric mean titer.
Neutralizing Assay in ARPE-19 cells: Determination of the neutralizing activity of induced antibodies in sera is performed with the following cell assay using ARPE-19 cells from ATCC and a GFP-tagged virus. In addition supplemental guinea pig serum as a source of exogenous complement is used. The assay is started with seeding of 6.5×103 cells/well (50 μl/well) in a 384 well plate one or two days before using for neutralization. The neutralization is done in 96-well sterile tissue culture plates without cells for 1 h at 37° C. After the neutralization incubation step the mixture is added to the cells and incubated for additional 4 days for GFP-detection with a plate reader. A positive neutralizing human sera is used as assay positive control on each plate to check the reliability of all results. Titers (EC50) are determined using a 4 parameter logistic curve fitting. As additional testing the wells are checked with a fluorescence microscope.
Plaque Reduction Assay: In brief, plaque reduction (neutralization) assays for guinea pig cytomegalovirus are performed by use of an isolate of GPCMV tagged with green fluorescent protein, 5% rabbit serum was used as a source of exogenous complement, and plaques were enumerated by fluorescence microscopy. Neutralization titers were defined as the highest dilution of serum that resulted in a 50% reduction in plaques, compared with that in control (pre-immune) serum samples.
Neutralization Assay in guinea pig lung fibroblast (GPL) cells: In brief, serial dilutions of test and control (pre-vaccination) sera were prepared in GPL complete media with supplemental rabbit serum (1%) as a source of exogenous complement. The dilution series spanned 1:40 through 1:5120. Serum dilutions were incubated with eGFP tagged virus (100-200 pfu per well) for 30 min at 37° C., and then transferred to 12-well plates containing confluent GPL cells. Samples were processed in triplicate. After 2 hours incubation at 37° C. the cells were washed with PBS, re-fed with GPL complete media and incubated at 37° C./5% CO2 for 5 days. Plaques were visualized by fluorescence microscopy, counted, and compared to control wells. That serum dilution resulting in a 50% reduction in plaque number compared to controls was designated as the neutralizing titer.
qPCR: LCMV RNA genomes are isolated using QIAamp Viral RNA mini Kit (QIAGEN), according to the protocol provided by the manufacturer. LCMV RNA genome equivalents are detected by quantitative PCR carried out on an StepOnePlus Real Time PCR System (Applied Biosystems) with SuperScript® III Platinum® One-Step qRT-PCR Kit (Invitrogen) and primers and probes (FAM reporter and NFQ-MGB Quencher) specific for part of the LCMV NP coding region. The temperature profile of the reaction is : 30 min at 60° C., 2 min at 95° C., followed by 45 cycles of 15 s at 95° C., 30 s at 56° C. RNA is quantified by comparison of the sample results to a standard curve prepared from a log10 dilution series of a spectrophotometrically quantified, in vitro-transcribed RNA fragment, corresponding to a fragment of the LCMV NP coding sequence containing the primer and probe binding sites.
Western Blotting: Infected cells grown in tissue culture flasks or in suspension are lysed at indicated timepoints post infection using RIPA buffer (Thermo Scientific) or used directly without cell-lysis. Samples are heated to 99° C. for 10 minutes with reducing agent and NuPage LDS Sample buffer (NOVEX) and chilled to room temperature before loading on 4-12% SDS-gels for electrophoresis. Proteins are blotted onto membranes using Invitrogens iBlot Gel transfer Device and visualized by Ponceau staining. Finally, the preparations are probed with an primary antibodies directed against proteins of interest and alkaline phosphatase conjugated secondary antibodies followed by staining with 1-Step NBT/BCIP solution (INVITROGEN).
MHC-Peptide Multimer Staining Assay for Detection of Antigen-Specific CD8+ T-cell proliferation: Any assay known to the skilled artisan can be used to test antigen-specific CD8+ T-cell responses. For example, the MHC-peptide tetramer staining assay can be used (see, e.g., Altman et al., 1996, Science; 274:94-96; and Murali-Krishna et al., 1998, Immunity, 8:177-187). Briefly, the assay comprises the following steps, a tetramer assay is used to detect the presence of antigen specific T-cells. In order for a T-cell to detect the peptide to which it is specific, it must both recognize the peptide and the tetramer of MHC molecules custom made for an antigen specific T-cell (typically fluorescently labeled). The tetramer is then detected by flow cytometry via the fluorescent label.
ELISPOT Assay for Detection of Antigen-Specific CD4+ T-cell Proliferation: Any assay known to the skilled artisan can be used to test antigen-specific CD4+ T-cell responses. For example, the ELISPOT assay can be used (see, e.g., Czerkinsky et al., 1983, J Immunol Methods.; 65:109-121; and Hutchings et al., 1989, J Immunol Methods.; 120:1-8). Briefly, the assay comprises the following steps: An immunospot plate is coated with an anti-cytokine antibody. Cells are incubated in the immunospot plate. Cells secrete cytokines and are then washed off. Plates are then coated with a second biotyinlated-anticytokine antibody and visualized with an avidin-HRP system.
Intracellular Cytokine Assay for Detection of Functionality of CD8+ and CD4+ T-cell Responses: Any assay known to the skilled artisan can be used to test the functionality of CD8+ and CD4+ T cell responses. For example, the intracellular cytokine assay combined with flow cytometry can be used (see, e.g., Suni et al., 1998, J Immunol Methods.; 212:89-98; Nomura et al., 2000, Cytometry; 40:60-68; and Ghanekar et al., 2001, Clinical and Diagnostic Laboratory Immunology; 8:628-63). Briefly, the assay comprises the following steps: activation of cells via specific peptides or protein, an inhibition of protein transport (e.g., brefeldin A) is added to retain the cytokines within the cell. After washing, antibodies to other cellular markers can be added to the cells. Cells are then fixed and permeabilized. The anti-cytokine antibody is added and the cells can be analyzed by flow cytometry.
Assay for Confirming Replication-Deficiency of Viral Vectors: Any assay known to the skilled artisan that determines concentration of infectious and replication-competent virus particles can also be used as a to measure replication-deficient viral particles in a sample. For example, FFU assays with non-complementing cells can be used for this purpose.
Furthermore, plaque-based assays are the standard method used to determine virus concentration in terms of plaque forming units (PFU) in a virus sample. Specifically, a confluent monolayer of non-complementing host cells is infected with the virus at varying dilutions and covered with a semi-solid medium, such as agar to prevent the virus infection from spreading indiscriminately. A viral plaque is formed when a virus successfully infects and replicates itself in a cell within the fixed cell monolayer (see, e.g., Kaufmann, S. H.; Kabelitz, D. (2002). Methods in Microbiology Vol.32:Immunology of Infection. Academic Press. ISBN 0-12-521532-0). Plaque formation can take 3-14 days, depending on the virus being analyzed. Plaques are generally counted manually and the results, in combination with the dilution factor used to prepare the plate, are used to calculate the number of plaque forming units per sample unit volume (PFU/mL). The PFU/mL result represents the number of infective replication-competent particles within the sample.
Assay for Expression of Viral Antigen Any assay known to the skilled artisan can be used for measuring expression of viral antigens. For example, FFU assays can be performed. For detection, mono- or polyclonal antibody preparation(s) against respective viral antigens are used (transgene-specific FFU). Furthermore, Western Blotting can be performed.
Animal Models The safety, tolerance and immunogenic effectiveness of vaccines comprising of an infectious, replication-deficient arenavirus expressing an HPV antigen described herein or a composition thereof can be tested in animals models. In certain embodiments, the animal models that can be used to test the safety, tolerance and immunogenic effectiveness of the vaccines and compositions thereof used herein include mouse, guinea pig, rat, and monkey. In a preferred embodiment, the animal models that can be used to test the safety, tolerance and immunogenic effectiveness of the vaccines and compositions thereof used herein include mouse.
7. EXAMPLES
These examples demonstrate that arenavirus-based vector technology can be successfully used to develop new vaccines against infection with HPV by including antigens into the arenavirus vector, and that administration of such vaccines can induce antigen-specific CD8+ T cell responses of high magnitude to control HPV infection.
7.1 Design of Arenavirus Vector Genome
Referring to established approaches (U.S. Patent Application Publication No. US 2010/0297172 A1; and Flatz et al., 2010, Nat Med. March; 16(3): 339-345), rLCMV and rJUNV vaccine vectors were designed that express a fusion of proteins E6 and E7 of HPV type 16, a major oncogenic genotype of HPV, including mutations (Cassetti et al, 2004, Vaccine 22:520-527) to eliminate the oncogenic potential of the antigen. As the epitopes required to generate T-cell immunity targeting HPV infected cells are linear in both HPV E6 and E7, the two tumor associated antigens (TAAs) could be incorporated as a fusion protein in a single vector.
Design of rLCMV vectors expressing E7E6: For generation of rLCMV vaccine vectors expressing the E7/E6 fusion protein alone or fused to a immunomodulatory peptide, polypeptide, or protein, various rLCMV vector constructs were designed.
The following sequences are illustrative amino acid sequences and nucleotide sequences that can be used with the methods and compositions described herein. In some instances a DNA sequence is used to describe the RNA sequence of a viral genomic segment. The RNA sequence can be readily deduced from the DNA sequence. Exemplary sequences are:
7.2 Vector Characterization
In order to analyze replication of the generated vectors, growth curves were performed using suspension HEK 293 cells expressing LCMV GP. Respective cells were infected with individual E7/E6 vectors (HK1-E7E6, HK1-E7E6-CRT, HK1-E7E6-Ub and HK1-E7E6-GMCSF) at multiplicity of infection (MOI) of 0.001, or a control vector expressing the green-fluorescent-protein (HK1-GFP). Samples were drawn every 24 hours and analyzed by Focus Forming Units (FFU) Assay. As shown in
(a) Transgene Expression
Western blot experiments confirmed presence of the HPV E7E6 antigen for all tested constructs. As shown in
(b) Immunogenicity
To investigate the ability of HK1-E7E6 to induce CD8+ T cell responses in a homologous prime-boost setting, C57BL/6 mice were vaccinated three times on days 0, 41 and 102 with HK1-E7/E6. Antigen (E7) specific CD8+ T cell responses were subsequently analyzed by tetramer staining on days 10, 38, 48, 73 and 109 of the experiment. The data obtained (
The immunogenicity of different test vaccines was subsequently compared by evaluating the induction of antigen-specific CD8+ T cell frequencies in mice upon intravenous immunization with HK1-E7E6, HK1-E7E6-CRT, HK1-E7E6-Ub and HK1-E7E6-GMCSF. A suboptimal vector dose (1×104 FFU) was used to allow for differentiation of constructs. Naive mice were used as control.
Antigen-(E7−) specific CD8+ T cell responses were subsequently analyzed by tetramer staining.
The immunogenicity of selected test vaccines was subsequently further analyzed and compared by evaluating the induction of antigen-specific CD8+ T cell responses in mice upon vaccination. C57BL/6 mice were immunized with HK1-E7E6, HK1-E7E6-GMCSF or HK1-GFP as an irrelevant negative control vector. Ad5-E7E6, a recombinant Adenovirus 5 (Ad5) vector expressing the HPV16 E7E6 fusion protein (with mutations in Rb binding site and E6 zinc binding domains), was used as a benchmark vector. Control mice received injections of 0.9% NaCl . Seven days after the second injection, splenocytes from vaccinated mice were isolated and stimulated with HPV16 E6 or HPV16 E7 peptides. The percentage of IFN-γ-producing CD8+ T cells was subsequently analyzed by double immunofluorescence assay.
This data indicated strong HPV16 E7-specific CD8+ T cell responses in groups 1, 2, 4 and 5, i.e., a strong response in mice vaccinated with HK1-E7E6, HK1-E7E6-GMCSF or Ad5-E7/E6. Weak HPV16 E6-specific CD8+ T cell responses were observed in mice immunized with HK1-E7E6-GMCSF or Ad5-E7E6, indicating weaker immunogenicity of E6 compared to E7.
To further investigate and compare the immunogenicity of test vectors encoding different immunostimulating sequences, the induction of antigen-specific CD8+ T cell frequencies was analyzed upon intravenous injection of HK1-E7E6-GMCSF, HK1-E7E6-VP22, HK1-E7E6-CD40L, HK1-Flt3L-E7E6, HK1-Flt3L-E7E6shuffle or HK1-1i-E7E6 constructs. Moreover, different vector doses were used to further investigate the dose dependency of the induced responses. Mock infected mice were used as controls. To analyze the effect of different immunization routes, one control group was injected intramuscularly with 106 FFU of HK1-E7E6-GMCSF. Frequencies of E7-specific CD8+ T cells circulating in blood were subsequently analyzed by tetramer staining (H-2Db/HPV16 E7 49-57 (RAHYNIVTF)) on days 8 and 18 of the experiment. The percentage of tetramer-binding CD8+ T cells is expressed as a percentage of the total CD8+ T cells in the test sample.
(c) Protective Efficacy
The protective efficacy of the vaccine candidates was subsequently investigated in the TC-1 model (Lin et al, 1996, Cancer Res.;56(1):21-6), which is one of the most commonly used models for developing therapeutic HPV vaccines. TC-1 tumor cells derived from mouse primary epithelial cells, co-transformed with HPV-16 E6 and E7 and c-Ha-ras oncogenes, were used in this experiment. Immunized mice were challenged by subcutaneous injection of TC-1 tumor cells after the second vaccination. A third vaccination was administered to certain treatment groups after challenge to further boost immunity. Protective efficacy was assessed by evaluating the number of tumor-free mice as well as measuring the tumor volume in the animals every 5 days. Mean tumor volumes in vaccinated animals and unvaccinated control animals were compared.
The results indicated that the observed induction of E7-specific CD8+ T cell responses correlates well with antitumor effects in the vaccinated mice. Immunization with HK1-E7E6 or HK1-E7E6-GMCSF significantly reduced the mean tumor volumes as well as the percentage of tumor-bearing mice within the experimental group. Observed results were comparable to the effects seen after vaccination with Ad5-E7E6.
(d) Therapeutic Efficacy
To further evaluate the therapeutic efficacy of the vaccine candidates, TC-1 tumor-bearing mice were vaccinated with the test vectors and frequencies of E7-specific CD8+ T cells circulating in blood were subsequently analyzed.
To also investigate the induction of anti-vector immune responses, LCMV NP specific CD8+ T cell frequencies was analyzed in the tumor-bearing mice after vaccination with the indicated test vectors. The results of this analysis are shown in
To analyze the impact of E7-specific CD8+ T cell responses on tumor control, the body weight of the vaccinated mice (data not shown) as well as the tumor volume and overall survival in the respective animals were monitored.
As a further investigation into the anti-vector immune responses, using the same methods described above, formation of E7-specific CD8+ T cells and LCMV NP specific CD8+ T cells in peripheral blood of TC-1 tumor-bearing mice were analyzed following vaccination with PBS (G1), 1×107 PFU of Ad5-E7E6 (G2) and 106 FFU HK1-E7E6 (G3). The results of this analysis are shown in
Tumor volume and overall survival were also monitored in the mice vaccinated with PBS (G1), 1×107 PFU of Ad5-E7E6 (G2) and 106 FFU HK1-E7E6 (G3).
7.3 Prime-Boost Immunization
Owing to the race against tumor growth, rapid induction of strong anti-tumor immune responses is an important challenge for successful development of cancer immunotherapies. Repeated prime-boost immunization strategies are likely necessary in order to achieve these goals. Although it has been shown that replication-deficient LCMV vectors can efficiently be re-administered in homologous prime-boost vaccination, heterologous prime-boost immunization regimens may offer distinct advantages such as to allow for shorter intervals between vaccinations, or to result in even higher efficacy. Vaccine vectors based on replication-deficient forms of various other members of the arenavirus family such as Junin virus or Mopeia virus can be used.
To investigate the ability of respective vectors to induce CD8+ T cell responses against HPV antigens, a replication-deficient glycoprotein-deficient vector based on Junin virus vaccine strain Candid#1, encoding HPV16 E7E6 fusion protein with mutations in Rb binding site and E6 zinc binding domains, was generated (rJUNV-E7E6). C57BL/6 mice were vaccinated once by intravenous injection of 105 FFU of rJUNV-E7E6 or HK1-E7E6. Eight days after immunization the induction of antigen-(E7 epitope-) specific CD8+ T cell responses was analyzed by tetramer staining from blood. Results shown in
To investigate the effect of homologous versus heterologous prime-boost immunization on the induction of antigen-specific CD8+ T cell responses, C57BL/6 mice were vaccinated on day 0 by intravenous injection of 105 FFU of either HK1-E7E6 or rJUNV-E7E6. 35 days later mice were either boosted with the respective homologous or heterologous vector (105 FFU i.v.). The induction of antigen-(E7 epitope-) specific CD8+ T cell responses was analyzed by tetramer staining on days 8, 28 and 42 of the experiment. Results in
7.4 Replication-Competent Tri-Segmented Arenavirus Viral Vectors
In an attempt to induce even stronger effector T cell responses due to the inflammation elicited by a replicating infection, replication-competent tri-segmented LCMV vectors expressing a fusion of proteins E6 and E7 of HPV type 16 were generated. The immunogenicity of the non-replicating bi-segmented vector (HK1-E7E6) and the analogous replicating, tri-segmented vector (r3LCMV-E7E6) was compared by evaluating the induction of antigen-specific CD8+ T cell responses in mice upon intravenous injection with the respective vectors. C57BL/6 mice were immunized on days 0 and 35 of the experiment with 105 FFU of r3LCMV-E7E6 or HK1-E7E6. Epitope-specific CD8+ T cells were stained using E7 epitope-loaded MHC class I tetramers in combination with anti-CD8a antibody. The frequency of E7-tetramer-binding cells within the CD8+ T cell compartment in peripheral blood was calculated.
To investigate the effect of homologous versus heterologous prime-boost immunization using replication-competent vectors, the induction of antigen-specific CD8+ T cell responses was analyzed in mice after vaccination with r3LCMV-E7E6 and an analogous replication-competent vector based on Junin Candid #1 virus (r3JUNV-E7E6) in homologous or heterologous combinations.
This application is a divisional of U.S. patent application Ser. No. 15/580,964, which is a U.S. National Stage Application under 35 U.S.C. § 371 of International Patent Application No. PCT/EP2016/063182, filed Jun. 9, 2016, which claims benefit of priority of U.S. Provisional Application No. 62/331,158, filed on May 3, 2016, U.S. Provisional Application No. 62/254,410, filed on Nov. 12, 2015, and U.S. Provisional Application No. 62/173,805, filed on Jun. 10, 2015, the entire contents of which are incorporated herein by reference. The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April 29, 2020, is named 13194-057-999_Seq_Listing.txt and is 181, 161 bytes in size.
| Number | Date | Country | |
|---|---|---|---|
| 62331158 | May 2016 | US | |
| 62254410 | Nov 2015 | US | |
| 62173805 | Jun 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15580964 | Dec 2017 | US |
| Child | 16863456 | US |
