Patent Grant
12150986
Pathogen infections, including influenza and HIV viral infections, have a massive impact on global health. Vaccines have been developed for many pathogens, including both influenza and HIV viral infections, however they have failings and limitations. Thus, a novel vaccine strategy must be engineered and evaluated.
Disclosed herein are new and improved HSV-2-based vaccines for various antigenic targets, including influenza and HIV.
A process is provided for producing a vaccine vector directed against a heterologous antigen, the process comprising:
Also provided is a recombinant herpes simplex virus-2 (HSV-2) having a genome encoding a heterologous antigen made by the process described herein.
Also provided is a recombinant herpes simplex virus-2 (HSV-2) having (i) a complete deletion of an HSV-2 glycoprotein D-encoding gene (referred to as ΔgD-2) in the genome thereof and (ii) (a) encoding: a promoter, a heterologous antigen signal sequence, a heterologous antigen or (b) encoding: a promoter, a heterologous antigen.
Also provided is a recombinant herpes simplex virus-2 (HSV-2) having (i) a partial deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof and (ii) (a) encoding, in order, an HSV-2 gD signal sequence, a heterologous antigen, an HSV-2 gD transmembrane domain, optionally an HSV-2 gD cytosolic domain, but not encoding an HSV-2 gD extracellular domain, or (b) encoding, in order, an HSV-2 gD signal sequence, a heterologous antigen, and transmembrane cytoplasmic tail of HSV-2 gD.
Also provided is an isolated cell comprising therein a recombinant HSV-2 virus as described herein, wherein the cell is not present in a human being.
Also provided is a vaccine composition comprising the recombinant HSV-2 virus as described herein.
Also provided is a pharmaceutical composition comprising a recombinant HSV-2 virus as described herein, and a pharmaceutically acceptable carrier.
Also provided is a method of eliciting and/or enhancing an immune response in a subject comprising administering to the subject an amount of (i) a recombinant HSV-2 virus as described herein; (ii) a vaccine as described herein; or (iii) a pharmaceutical composition as described herein, in an amount effective to elicit and/or enhance an immune response in a subject.
Also provided is a method of treating or reducing the likelihood of an influenza infection in a subject comprising administering to the subject an amount of (i) a recombinant HSV-2 virus as described herein; (ii) a vaccine as described herein; or (iii) a pharmaceutical composition as described herein, in an amount effective to treat or reduce the likelihood of an influenza infection in a subject.
Also provided is a method of treating or reducing the likelihood of an HIV infection in a subject comprising administering to the subject an amount of (i) a recombinant HSV-2 virus as described herein; (ii) a vaccine as described herein; or (iii) a pharmaceutical composition as described herein, in an amount effective to treat or reduce the likelihood of an HIV infection in a subject.
Also provided is a method of vaccinating a subject for influenza infection comprising administering to the subject an amount of (i) a recombinant HSV-2 virus as described herein; (ii) a vaccine as described herein; or (iii) a pharmaceutical composition as described herein, in an amount effective to vaccinate a subject for influenza infection.
Also provided is a method of vaccinating a subject for HIV infection comprising administering to the subject an amount of (i) a recombinant HSV-2 virus as described herein; (ii) a vaccine as described herein; or (iii) a pharmaceutical composition as described herein, in an amount effective to vaccinate a subject for HIV infection.
Also provided is a method of quantitating a rate or amount of antibody-dependent cell-mediated killing (ADCK) in a population of cells comprising infecting a plurality of cells of the population of cells with a fluorescent protein-expressing recombinant HSV-2 that comprises a genome deleted for the gene encoding HSV-2 gD, under conditions permitting expression of the fluorescent protein in the cells, contacting the plurality of infected cells with an antibody-containing solution and a population of immune cells, and quantitating at one or more time points the amount of cells exhibiting fluorescent protein fluorescence and, optionally, one or more markers, so as to quantitate over time the amount of live infected cells, so as to thereby quantitating the rate or amount of ADCK in the population of cells.
A process is provided for producing a vaccine vector directed against a heterologous antigen, the process comprising:
In embodiments, the promoter of the promoter-FP construct is a heterologous promoter.
A process is also provided for producing a vaccine vector directed against an antigen, the process comprising:
A variety of promoters can be employed. Heterologous promoters are preferred including those with high efficiency. Such promoters include the CMV promoter, including the cytomegalovirus major immediate-early promoter. The human elongation factor-1 alpha (EF-1 alpha) constitutive promoter is of human origin and can be used to drive ectopic gene expression in vitro and in vivo. A variety of PEF-1 alpha may be used. The human EF1α gene sequence is known in the art, for example see NCBI accession No. J04617. Other heterologous promoters known in the art and usable in the invention include, but are not limited to, CMV enhancer fused to the chicken beta-actin promoter (CAG), mouse cytomegalovirus (mouse CMV), Chinese hamster elongation factor-1α (CHEF-1α), and phosphoglycerate kinase (PGK).
In embodiments of the processes, the host cell is a complementing cell, for example a cell that phenotypically complements for HSV-1 glycoprotein D. In embodiments of the processes, the host cell is a VD60 cell, which phenotypically complements HSV-1 gD. In embodiments, the vaccine vector produced is genotypically deleted for HSV-2 gD and is phenotypically complemented for HSV-1 gD on a lipid bilayer thereof. In embodiments, the vaccine vector produced does not genotypically encode any HSV gD.
In embodiments, the host cell is co-transfected with (i) the HSV-2 genome of a) and (ii) a linear DNA fragment encoding, in order, (i) HSV-2 gD signal sequence, the heterologous antigen, HSV-2 gD transmembrane domain, HSV-2 gD cytosolic domain, but not encoding a HSV-2 gD extracellular domain, or (ii) HSV-2 gD signal sequence, the heterologous antigen, and transmembrane cytoplasmic tail of HSV-2 gD.
In embodiments, the host cell is co-transfected with (i) the HSV-2 genome of a) and (ii) linear DNA fragments encoding, in order, (i) a promoter, the heterologous antigen, and optionally a poly-A signal.
In embodiments, the co-transfecting is effected by electroporation.
Examples of nucleic acid-encodable fluorescent proteins for use in the invention include red, far red, yellow, green, orange, cyan or photo switchable fluorescent protein. Examples of such proteins are well known in the art. Supplies include Molecular Probes (ThermoFisher USA) and Takara (USA). In an embodiment, the fluorescent protein is Red Fluorescent Protein. Fluorescent proteins with an excitation range of 554-584 nanometers (nm) and an emission range of 562-610 nm are preferred. Examples include Red Fluorescent Protein, mCherry, mTomato, J-Red and mOrange. RFP has an excitation of 556 nm and emission of 584 nm. Alternatively, firefly luciferase or nano-luciferase can be used.
In embodiments, the antigen is not an HSV-2 antigen, i.e. it is a heterologous antigen. As used herein, an antigen is heterologous when it is heterologous relative to HSV-2, i.e. is not naturally found on or in a wildtype HSV-2.
The heterologous antigen can be derived from a living organism, comprising for example, a virus, a bacteria, a parasite, a human cell, an animal cell, or a combination thereof. The heterologous antigen can be a surface protein or a non-surface protein.
The virus can be a pathogenic virus, examples of which include cytomegalovirus (CMV), coxsackie virus, Crimean-Congo hemorrhagic fever virus, chikungunya virus, dengue virus, Dhori virus, Eastern equine encephalitis (EEE) virus, ebola virus, Epstein Barr virus (EBV), hepatitis virus, herpesvirus, human immunodeficiency (HIV) virus, human papilloma virus, human SARS corona virus, human T lymphotropic virus (HTLV), influenza virus, measles virus, mumps virus, Norwalk virus, rabies virus, rotavirus, rubella virus, severe fever with thrombocytopenia syndrome (SFTS) virus, respiratory syncytial virus (RSV), varicella zoster virus, Western equine encephalitis virus, West Nile virus, yellow fever virus, Zika virus, or a combination thereof.
The bacteria can be a pathogenic bacteria, examples of which include Bacillus sp., Baronella sp., Bordatella sp., Borelli asp., Brucella sp., Campylobacter sp., Chlamydia sp., Clostridium sp., Corynebacterium sp., Enterococcus sp., Escherichia sp., Haemophilis sp., Helicobacter sp., Legionella sp., Leptospira sp., Listeria sp., Mycobacterium sp., Mycoplasma sp., Neisseria sp., Rickettsia sp., Pseudomonas sp., Salmonella sp., Shigella sp., Staphylococcus sp., Streptococcus sp., Treponema sp., Vibrio sp., Yersinia sp., or a combination thereof.
The parasite can be a pathogenic parasite, examples of which include Acanthamoeba spp., Balamuthia spp., Babesia sp., Balantidium coli, Blastocystic sp., Cryptospiridium sp., Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia, Isospora bello, Leishmania sp., Naegleria foweri, Plasmodium sp., Rhinosporidium seeberi, Sarcocystis sp., Toxoplasma gondii, Trichomonas sp., Trypanosoma sp., or a combination thereof.
The human cell or animal cell can be, for example, a cancer cell.
In embodiments, the heterologous antigen is an influenza antigen. In embodiments, the heterologous antigen is an influenza hemagglutinin (HA) antigen. In embodiments, the HA antigen is a full-length HA extracellular domain or is a HA stalk domain.
In embodiments, the heterologous antigen is an HIV antigen. In embodiments, the HIV antigen is an Env gp145.
In embodiments, the heterologous antigen is under control of an upstream CMV promoter and has a downstream SV40 poly-A signal. The SV40 poly-A signal is known in the art. It promotes polyadenylation and transcription termination.
In embodiments, the promoter is a promoter of Elongation Factor 1□ gene (PEF1α) and wherein PEF1α and FP are fused together (PEF1α-FP).
In embodiments, the nucleic acid is codon-optimized for expression. See, for example, Table 4 in the examples hereinbelow.
Also provided is a vaccine vector made by the process described herein.
Also provided is a recombinant herpes simplex virus-2 (HSV-2) having a genome encoding a heterologous antigen made by the process described herein.
Also provided is a recombinant herpes simplex virus-2 (HSV-2) having (i) a complete deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof and (ii) (a) encoding: a promoter, a heterologous antigen signal sequence, and a heterologous antigen or (b) encoding: a promoter and a heterologous antigen.
Also provided is a recombinant herpes simplex virus-2 (HSV-2) having (i) a partial deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof and (ii) (a) encoding, in order, an HSV-2 gD signal sequence, a heterologous antigen, an HSV-2 gD transmembrane domain, optionally an HSV-2 gD cytosolic domain, but not encoding an HSV-2 gD extracellular domain, or (b) encoding, in order, an HSV-2 gD signal sequence, a heterologous antigen, cytosolic domain of HSV-2 gD.
Also provided is a recombinant herpes simplex virus-2 (HSV-2) having (i) a partial deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof and (ii) (a) encoding, in order, an HSV-2 gD signal sequence, a heterologous antigen, an HSV-2 gD transmembrane domain, an HSV-2 gD cytosolic domain, but not encoding an HSV-2 gD extracellular domain, or (b) encoding, in order, an HSV-2 gD signal sequence, a heterologous antigen, and transmembrane cytoplasmic tail of HSV-2 gD.
In embodiments, the recombinant HSV-2 further comprises a parasitic surface glycoprotein on a lipid bilayer thereof, wherein the parasite is a parasite of a mammal.
In an embodiment, the HSV-2 glycoprotein D-encoding gene is an HSV-2 US6 gene. (For example, see Dolan et al. J Virol. 1998 March; 72(3): 2010-2021. (PMCID: PMC109494) “The Genome Sequence of Herpes Simplex Virus Type 2” for HSV-2 genome and US6 gene, hereby incorporated by reference in its entirety). In embodiments, the HSV-2 glycoprotein D-encoding gene is equivalent of an HSV-2 US6 gene. Such equivalents are easily identifiable by those of skill in the art using readily available sequencing and alignment tools.
In embodiments, the heterologous antigen is an influenza antigen. In embodiments, the heterologous antigen is an influenza hemagglutinin (HA) antigen.
In embodiments, the HA antigen is a full-length HA extracellular domain or is a HA stalk. In embodiments, the full-length HA includes a HA signal sequence. In embodiments, the HA is an HA of a human influenza A or human influenza B. Examples of the hemagglutinin gene (“HA”) include GenBank V01098.1; NCBI Reference Sequence: NP_040980.1. HA gene sequences, and the mature hemagglutinin peptide sequences, are well known in the art, and multiple HA sequences are available to those skilled in the art at the NCBI database. In addition, one skilled in the art can readily identify the commonly discussed stalk, extracellular domain and other regions of hemagglutinin. In addition, seasonal influenza virus strain sequences, including the HA sequence, are routinely sequenced and identified in the art.
Other influenza genes, as heterologous antigens, that can be added to ΔgD-2 by allelic exchange with B3×2.8 (ΔgD-2::PEF1α-RFP) include neuraminidase (NA), matrix protein 1 (M1), influenza A virus (IAV) matrix protein 2 (M2), influenza B virus (IBV) matrix protein 2 (M2), nucleoprotein (NP), and influenza B virus NB. A combination comprising at least one of the foregoing can also be used. The corresponding modified headless HA gene for each strain can be added as well as a version of each headless antigen where a trimerization domain has been added to increase stability. Table 1 provides non-limiting influenza examples of other such antigen genes. Other examples include inserting the HA gene from A/Vietnam/1203/04, the HA, headless HA, NA, M1, M2, NP, and NB genes from, e.g., B/Yamagata/16/1988 and B/Victoria/2/1987 strains.
One method for generating the recombinant HSV-2 gD −/− viral vector expressing HIV or Influenza A virus antigens is as follows:
In embodiments, the heterologous antigen is an HIV antigen. In embodiments, the HIV antigen is an HIV-1 or HIV-2 antigen. For example, the heterologous antigen is an HIV-1 antigen. In embodiments, the HIV is a C-subtype. In embodiments, the HIV antigen is an Env, Pol, Gag, or Nef. In an embodiment, the HIV antigen is an Env antigen. In embodiments, the HIV antigen is a C-subtype Env antigen. In embodiments, the antigen is an Env gp145. In embodiments, the heterologous antigen is a fully intact membrane-proximal external region (MPER). In embodiments, the heterologous antigen is extended by a polylysine tail. In embodiments, the heterologous antigen is not extended by a polylysine tail. HIV gp145 Env protein sequences are readily identifiable by alignment tools, and are routine to sequence. In embodiments, the ectodomain of HIV Env gp145 is fused with the signal peptide and transmembrane cytoplasmic tail of HSV gD.
Also provided is a cell comprising therein a recombinant virus as described herein, wherein the cell is not present in a human being.
Also provided is a vaccine composition comprising a recombinant virus as described herein. In embodiments, the vaccine comprises an adjuvant which is not derived from the HSV-2. Adjuvants are well known in the art and include alum, oil-in-water or water-in-oil emulsions, aluminum salts such as aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate, and monophosphoryl lipid A. In embodiments, the vaccine does not comprise an adjuvant.
Also provided is a pharmaceutical composition comprising a recombinant HSV-2 virus as described herein, and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well-known in the art.
Also provided is a method of eliciting and/or enhancing an immune response in a subject, the method comprising administering to the subject an amount of (i) a recombinant virus as described herein; (ii) a vaccine as described herein; or (iii) a pharmaceutical composition as described herein, in an amount effective to elicit and/or enhance immune response in a subject.
Also provided is a method of eliciting and/or enhancing an immune response in a subject against a pathogen expressing a heterologous antigen, the method comprising administering to the subject an amount of (i) a recombinant virus as described herein; (ii) a vaccine as described herein; or (iii) a pharmaceutical composition as described herein, in an amount effective to elicit and/or enhance immune response in a subject, wherein the recombinant virus of (i), (ii) or (iii) expresses the heterologous antigen.
Also provided is a method of treating or reducing the likelihood of an influenza infection in a subject, the method comprising administering to the subject an amount of (i) a recombinant virus as described herein; (ii) a vaccine as described herein; or (iii) a pharmaceutical composition as described herein, in an amount effective to treat or reduce the likelihood of an influenza infection in a subject. For treating or reducing likelihood of influenza, the heterologous antigen is an influenza HA antigen. Also provided is a method of vaccinating a subject for influenza infection comprising administering to the subject an amount of (i) a recombinant virus as described herein; (ii) a vaccine as described herein; or (iii) a pharmaceutical composition as described herein, in an amount effective to vaccinate a subject for influenza infection.
Also provided is a method of treating or reducing the likelihood of an HIV infection in a subject, the method comprising administering to the subject an amount of (i) a recombinant virus as described herein; (ii) a vaccine as described herein; or (iii) a pharmaceutical composition as described herein, in an amount effective to treat or reduce the likelihood of an HIV infection in a subject. For treating HIV or reducing likelihood of HIV, the heterologous antigen is an HIV antigen. Also provided is a method of vaccinating a subject for HIV infection comprising administering to the subject an amount of (i) a recombinant virus as described herein; (ii) a vaccine as described herein; or (iii) a pharmaceutical composition as described herein, in an amount effective to vaccinate a subject for HIV infection.
Also provided is a method of eliciting and/or enhancing an immune response in a subject, the method comprising administering to the subject an amount of a recombinant herpes simplex virus-2 (HSV-2) made by a process described herein and comprising (i) a complete deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof and (ii) encoding a promoter, a influenza hemagglutinin (HA) antigen signal sequence, and an HA antigen in an amount effective to elicit and/or enhance an immune response in a subject.
Also provided is a method of treating or reducing the likelihood of an influenza infection in a subject, the method comprising administering to the subject an amount of a recombinant herpes simplex virus-2 (HSV-2) made by a process described herein and comprising (i) a complete deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof and (ii) encoding a promoter, a influenza hemagglutinin (HA) antigen signal sequence, and an HA antigen in an amount effective to treat or reduce the likelihood of an influenza infection in a subject.
Also provided is a method of vaccinating a subject for influenza infection, the method comprising administering to the subject an amount of a recombinant herpes simplex virus-2 made by a process described herein and comprising (i) a complete deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof and (ii) encoding a promoter, a influenza hemagglutinin (HA) antigen signal sequence, and an HA antigen in an amount effective to vaccinate a subject for influenza infection.
In embodiments, the HA antigen is a full-length HA extracellular domain.
In embodiments, the methods further comprise, subsequent to an initial administration of the recombinant herpes simplex virus-2 encoding a full-length HA extracellular domain, administering one or more amounts of a recombinant herpes simplex virus-2 having (i) a complete deletion of an HSV-2 glycoprotein D-encoding gene in the genome thereof and (ii) encoding a promoter, a HA antigen signal sequence, and an HA stalk, but not encoding a full-length HA.
In an embodiment of the methods or processes, the HSV-2 glycoprotein D comprises the amino acid sequence set forth in SEQ ID NO:1:
In an embodiment, the HSV-2 in which the HSV-2 glycoprotein D-encoding gene is deleted is an HSV-2 having a genome (prior to the deletion) as set forth in one of the following Genbank listed sequences: HSV-2(G) (KU310668), HSV-2(4674) (KU310667), B3×1.1 (KU310657), B3×1.2 (KU310658), B3×1.3 (KU310659), B3×1.4 (KU310660), B3×1.5 (KU310661), B3×2.1 (KU310662), B3×2.2 (KU310663), B3×2.3 (KU310664), B3×2.4 (KU310665), B3×2.5 (KU310666). These sequences are hereby incorporated by reference.
A cell is provided comprising therein a recombinant HSV-2 genome as described herein.
Also provided is a vaccine composition comprising the recombinant HSV-2 virus as described herein. In an embodiment, the vaccine comprises an immunological adjuvant. In an embodiment, the vaccine does not comprise an immunological adjuvant. In an embodiment of the vaccine, compositions or pharmaceutical compositions described herein comprising a recombinant HSV-2, the HSV-2 is live-attenuated.
Also provided is a composition comprising the recombinant HSV-2 virus as described herein, wherein the genome of the virus or virion comprises at least a deletion of a second gene, wherein the second gene is necessary for HSV-2 viral replication or virulence.
A pharmaceutical composition comprising the recombinant HSV-2 virus as described herein, and a pharmaceutically acceptable carrier.
In an embodiment, the composition or pharmaceutical composition or vaccine is formulated so that it is suitable for subcutaneous administration to a human subject. In an embodiment, the composition or pharmaceutical composition or vaccine is formulated so that it is suitable for oral administration to a human subject. In an embodiment, the composition or pharmaceutical composition or vaccine is formulated so that it is suitable for intravaginal administration to a human subject. In an embodiment, the composition or pharmaceutical composition or vaccine is formulated so that it is suitable for intra-muscular, intra-nasal, or mucosal administration to a human subject. In embodiments of the methods herein, and embodiments of the composition or pharmaceutical composition or vaccine formulations herein, administration can be auricular, buccal, conjunctival, cutaneous, subcutaneous, endocervical, endosinusial, endotracheal, enteral, epidural, via hemodialysis, interstitial, intrabdominal, intraamniotic, intra-arterial, intra-articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intracisternal, intracorneal, intracoronary, intradermal, intradiscal, intraductal, intraepidermal, intraesophagus, intragastric, intravaginal, intragingival, intraileal, intraluminal, intralesional, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intraepicardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumor, intratympanic, intrauterine, intravascular, intravenous, intraventricular, intravesical, intravitreal, laryngeal, nasal, nasogastric, ophthalmic, oral, oropharyngeal, parenteral, percutaneous, periarticular, peridural, rectal, inhalationally, retrobulbar, subarachnoid, subconjuctival, sublingual, submucosal, topically, transdermal, transmucosal, transplacental, transtracheal, ureteral, urethral, or vaginal. A combination comprising at least one of the foregoing routes of administration can also be used.
Also provided is a method of eliciting an immune response in a subject, the method comprising administering to the subject an amount of (i) the recombinant HSV-2 virus as described herein in an amount effective to elicit an immune response in a subject.
In an embodiment, the HSV-2 glycoprotein D-encoding gene is an HSV-2 US6 gene. In an embodiment, the HSV-2 recombinant virus encodes a heterologous surface glycoprotein. In an embodiment the heterologous surface glycoprotein is an HSV-1 gD. In an embodiment, the HSV-2 recombinant virus comprises a non-genotypically encoded HSV-1 gD also encodes a heterologous surface glycoprotein that is not a herpesvirus glycoprotein and/or is and not involved in herpesviridae infection, and which is encoded by a transgene that has been inserted into the genome of the recombinant HSV-2. In an embodiment, the genome of the recombinant HSV-2 does not encode any herpes virus gD. In an embodiment, the surface glycoprotein is present on a lipid bilayer of the virus by way of infecting a cell with a recombinant HSV-2 having a deletion of an HSV-2 glycoprotein D-encoding gene, wherein the cell is or has been transfected to express the surface glycoprotein on a cell membrane thereof, and wherein the recombinant HSV-2 comprising the surface glycoprotein present on a lipid bilayer is produced from the cell. In an embodiment the host cell is a HSV-1 gD complementing cell. In an embodiment, the host cell encodes an HSV-1 gD under the endogenous gene promoter. In an embodiment the host cell is a HSV-1 gD complementing VD60 cell. (See, e.g., Ligas et al., J Virol. 1988 May; 62(5):1486-94, hereby incorporated by reference).
Also provided is a cell comprising therein a recombinant virus as described herein, wherein the cell is not present in a human being.
A vaccine composition comprising a recombinant virus as described herein. In an embodiment of the vaccine composition, the vaccine composition comprises an immunological adjuvant.
Also provided is a composition comprising a recombinant virus as described herein, wherein the genome of the virus comprises at least a deletion of a second gene, wherein the second gene is necessary for HSV-2 viral replication.
In an embodiment, the recombinant virus as described herein does not comprise a deletion of a second gene.
Reducing the likelihood of a viral infection is understood to mean amelioration of the extent of development of the relevant disease or chances of infection in a subject treated with the virus, vaccine or compositions described herein, as compared to an untreated subject.
In an embodiment of the methods herein for immunizing, vaccinating or eliciting an immune response, passive transfer of the virus or the antibodies or immune factors induced thereby may be effected from one subject to another. The relevant product may be treated after obtention from one subject before administration to a second subject. In a preferred embodiment of the inventions described herein, the subject is a mammalian subject. In an embodiment, the mammalian subject is a human subject.
In an embodiment, vaccinating a subject with an antigen elicits a humoral immune response to that antigen in the subject. A vaccinated individual is usually able to mount a more efficacious immune response to a subsequent challenge from a pathogen comprising that antigen than they would be able to prior to vaccination.
In an embodiment of the methods described herein, the subject has not yet been infected with influenza virus. In an embodiment of the methods described herein, the subject has not yet been infected with HIV. In an embodiment of the methods described herein, the subject has been infected with influenza virus. In an embodiment of the methods described herein, the subject has been infected with HIV.
In embodiments, the influenza infection is a human influenza A infection. In embodiments, the influenza infection is a human influenza B infection. In embodiments, the HIV infection is an HIV-1 infection. In embodiments, the HIV infection is an HIV-2 infection.
“Codon optimization” is defined as modifying a nucleic acid sequence for enhanced expression in the cells of interest, e.g. human, by replacing at least one, more than one, or a significant number, of codons of the native sequence with codons that are more frequently or most frequently used in the genes of that vertebrate. Various species exhibit particular bias for certain codons of a particular amino acid. In one aspect, the present invention relates to codon optimized inserts, nucleic acids or vectors, or host cells comprising such.
Also provided is a method of quantitating a rate or amount of antibody-dependent cell-mediated killing (ADCK) in a population of cells comprising infecting a plurality of cells of the population of cell with a fluorescent protein-expressing recombinant HSV-2 that comprises a genome deleted for the gene encoding HSV-2 gD, under conditions permitting expression of the fluorescent protein in the cells, contacting the plurality of infected cells with an antibody-containing solution and a population of immune cells, and quantitating at one or more time points the amount of cells exhibiting fluorescent protein fluorescence and, optionally, one or more markers, so as to quantitate over time the amount of live infected cells, so as to thereby quantitating the rate or amount of ADCK in the population of immune cells.
Also provided is a method of quantitating a rate or amount of antibody-dependent cell-mediated killing (ADCK) in a population of cells comprising infecting a plurality of cells of the population of cells with a fluorescent protein-expression expressing recombinant HSV-2 that comprises a genome deleted for the gene encoding HSV-2 gD, under conditions permitting expression of the fluorescent protein in the cells, contacting the plurality of infected cells with an antibody-containing solution, and quantitating at one or more time points the amount of cells exhibiting fluorescent protein fluorescence and, optionally, one or more markers, so as to quantitate over time the amount of live infected cells, so as to thereby quantitating the rate or amount of ADCK in the population of cells.
In embodiments, the recombinant HSV-2 is made by a process as described herein.
In embodiments, the method is performed in vitro. In embodiments, the population of immune cells comprises a population of macrophages. In embodiments, the macrophages are human. In embodiments, the antibody-containing solution comprises serum. In embodiments, the fluorescent protein is as described hereinabove. In an embodiment, the fluorescent protein is Red Fluorescent Protein. In an embodiment, only the cells quantitated as RFPhigh are considered live. In an embodiment, the plurality of cells are RFPhigh if they express RFP above the mean intensity of an RFp-expressing cell of the infected population of cells. In embodiments, the method is performed with the population of immune cells present at effector:target ratio of 5:1 or greater. In embodiments, the method is performed with the population of immune cells present at effector:target ratio of 10:1 or greater.
In embodiments, the amount of cells exhibiting fluorescent protein fluorescence and, optionally, one or more markers, is measured by fluorescence-activated cell sorting (FACS). In embodiments, the amount of cells exhibiting fluorescent protein fluorescence and, optionally, one or more markers, is measured by fluorescence spectrometer, fluorescence microplate reader, and fluorescence microscopy or fluorescence plate reader. In embodiments, the one or more markers comprise a cell membrane marker and/or a live/dead marker.
In embodiments, the method further comprises quantitating at one or more time points the amount of cells exhibiting fluorescent protein fluorescence and, optionally, one or more markers, in a control population of infected cells otherwise identical, but not contacted with an antibody-containing solution and comparing the amount or rate quantitated to that quantitated for the population of cells contacted with the antibody-containing solution.
In other embodiments of the invention relating to assays for ADCK, instead of a fluorescent protein the marker in the recombinant HSV-2 ΔgD-2 can be a beta galactosidase or an alkaline phosphatase. Thus, the methods, processes and compositions disclosed herein can comprise, mutatis mutandis, recombinant HSV-2 ΔgD-2 comprising a nucleic acid in the genome thereof encoding beta galactosidase or an alkaline phosphatase.
“And/or” as used herein, for example, with option A and/or option B, encompasses the separate embodiments of (i) option A, (ii) option B, and (iii) option A plus option B.
All combinations of the various elements described herein are within the scope of the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
This invention may be better understood from the Examples, which follow.
An engineered HSV-2 virus that replaces the gD gene (ΔgD-2) with a gene that strongly expresses the red fluorescent protein (RFP) was constructed. Once achieved, this provided an improved screen for identifying and obtaining new recombinants. The ΔgD-2::RFP recombinant was made from a ΔgD-2 that showed protection. The red fluorescent ΔgD-2:RFP, referred to herein also as B3×2.8 or ΔgD-2::PEF1α-RFP, retains the capacity of the original ΔgD-2 to elicit protective immunity against HSV infection but also appears to have unaltered in vitro replication kinetics. Moreover, ΔgD-2::RFP possesses significant advantages over HSV-2 ΔgD::GFP virus for making recombinants. Since the RFP gene is fused to a highly efficient promoter, for example promoter of the EF1α (Elongation Factor 1 α) gene, its expression is easily detected using a fluorescent microscope. In addition, visualization of red fluorescence in cells infected with ΔgD-2::RFP does not have the background found with the green fluorescent ΔgD-2. This property enables unambiguous identification of recombinant viruses in which the ΔgD-2::PEF1α-RFP allele is replaced by other allelic exchange substrates.
Construction of the HSV-2 ΔgD-−/+gD-1 virus (hereafter called “ΔgD-2”) using HSV-2(G) as the backbone was accomplished as detailed below. To make the allelic exchange constructs, an expression construct was generated by Gibson cloning into an E. coli plasmid, cloning the expression construct in between the SpeI and BclI sites of a shuttle (for example, cosmid pYUB2169) that contains 12 kb of the HSV-2(G) virus spanning from US1 To US9, electroporated the Pad restriction fragment from the pYUB2169 recombinant alongside ΔgD-2::RFP DNA into VD60 cells, and screening the resulting plaques for loss of red fluorescence. (See
This system was used to generate two sets of recombinant ΔgD-2 strains containing hemagglutinin (HA) antigens derived from the influenza A virus (IAV) strain A/Puerto Rico/8/1934 (PR8). The first set of recombinants contain chimeric gD::HA genes recombined into the US6 region of ΔgD-2 through the use of, for example, pYUB2169. These recombinant genes were made up of the extracellular domain of PR8 HA fused to the transmembrane, cytosolic, and signal sequence domains of HSV-2 (G) gD. The second set of recombinants contain modified or unmodified PR8 HA genes fused to an upstream CMV promoter and downstream SV40 polyadenylation signal. These cassettes were inserted into the US6 region of ΔgD-2 using a modified pYUB2169 identified as pBRL812. Each set of recombinants contained three type of HA genes:
FL HA nOP—Full-length extracellular domain hemagglutinin, non-codon optimized.
HL HA nOP—Headless extracellular domain hemagglutinin, non-codon optimized.
HL HA OPT—Headless extracellular domain hemagglutinin, codon-optimized.
The following constructs were made:
ΔgD-2::RFP (ΔgD-2::PEF1α-RFP) was constructed by (1) introducing a kanamycin marker in RFP plasmid ptwB, (2) amplifying the kan marker and RFP (dTomato) with oligos having ˜50 bp homologous sequence upstream and downstream of US6, (3) recombining in DY331 cells, then adding the PCR product followed by transformation with cosmid pYUB2156. (4) The kan marker was removed by restriction digest and ligation. (5) HSV2(G) genomic DNA and pYUB2167 were co-transfected in VD60 cells to generate ΔgD-2::RFP by allelic exchange.
ΔgD-2 was obtained by co-transfection of cosmid pYUB2163 with the ΔgD-2::RFP genome in VD60 cells to generate the ΔgD-2 genome by allelic exchange. This genome is free of all markers and antibiotic resistance genes. The resultant unmarked virus was sorted based on lack of RFP expression. RFP-negative plaques were purified three times and the lack of the RFP gene was verified by PCR and sequencing.
ΔgD-2::FL HA nOP was constructed by (1) Gibson cloning the cytosolic, transmembrane, and signal sequence domains of HSV-2 (G) gD to the extracellular domain of custom synthesized PR8 HA (Genscript, Piscataway, NJ) in the pYUB2169 plasmid and (2) co-transfecting the resultant plasmid, pBJJ1, with the ΔgD-2:RFP genome in VD60 cells to generate the desired recombinant by allelic exchange.
ΔgD-2::HL HA nOP was constructed similarly to ΔgD-2::FL HA nOP, but using a custom synthesized PR8 HA gene where the HA1 head domain was replaced with 4 glycine residues (Genscript). The plasmid used for transfection was called pBJJ2.
ΔgD-2::HL HA OPT was constructed similarly to ΔgD-2::HL HA nOP, but using a custom synthesized PR8 HA gene where the HA1 head domain was replaced with 4 glycine residues and every codon which had less than 9.5% representation in HSV-2 (G) gD for its cognate amino acid was replaced with the codon most abundant in HSV-2 (G) gD (Genscript). The plasmid used for transfection was called pBJJ4.
ΔgD-2::PCMV-FL HA nOP was constructed by (1) restriction cloning the full length PR8 HA gene (Genscript) in between the xbaI and HindIII restriction sites of pEGFP-N1 (Addgene, Cambridge, MA) (2) restriction cloning the PCMV-FL HA nOP::SV40 polyA cassette from the resultant plasmid into the SpeI and BclI sites of pBRL812. The resulting plasmid, pJHA1, was cut with PacI and AsiSI and co-transfected with the ΔgD-2::RFP genome in VD60 cells to generate the desired recombinant by allelic exchange.
ΔgD-2::PCMV-HL HA nOP was constructed similarly to ΔgD-2::PCMV-FL HA nOP but using a custom synthesized PR8 HA gene where the HA1 head domain was replaced with 4 glycine residues (Genscript). The plasmid used for transfection was called pJHA2.
ΔgD-2::PCMV-HL HA OPT was constructed similarly to ΔgD-2::PCMV-FL HA nOP but using a custom synthesized PR8 HA gene where the HA1 head domain was replaced with 4 glycine residues and every codon which had less than 9.5% representation in HSV-2 (G) gD for its cognate amino acid was replaced with the codon most abundant in HSV-2 (G) gD (Genscript). The plasmid used for transfection was called pJHA4.
Whole genome sequencing was used to verify the deletion of US6. To test immunogenicity, mice were vaccinated on Day 0 (d0) and d21, then sera was taken on d40. ΔgD-2 elicited anti-HSV antibodies that are similar to those obtained with ΔgD-2::GFP (See
Mechanistically, it seems that the most important correlates of protection are ADCC and ADCP. Sera from ΔgD-2 vaccinated mice induced significantly more ADCC/ADCP immunity than sera from ΔgD-2::GFP vaccinated mice (
ΔgD-2 vaccination protects mice against challenge with a wild-type HSV-2 4674, which is a clinical isolate obtained from the virology laboratory at Montefiore Hospital, Bronx, NY, as well as the GFP-marked construct. Mice were vaccinated with either the wild-type HSV-2 or the GFP-marked construct virus on d0 and d21. Twenty-one days after boost, mice were challenged subcutaneously with 10× LD90 HSV-2 4674 and followed for skin lesion changes, presence of virus in the dorsal root ganglia, body weight, and survival. The unmarked virus behaved nearly identically by all measures (
HSV-1(17) (Brown et al., 1973), HSV-2(G) (Ejercito et al., 1968), HSV-1(F) (Ejercito et al., 1968), and HSV-2(333)ZAG (Nixon et al., 2013), a recombinant virus expressing the green fluorescent protein (GFP) were propagated on Vero cells. HSV-2 4674 (Nixon et al., 2013) was propagated on HaCAT cells. VD60 cells (Vero cells encoding gD-1 under the endogenous gene promoter (Ligas and Johnson, 1988)) were passaged in DMEM supplemented with 10% fetal bovine serum. Stocks of HSV-2 ΔgD-2 (ΔgD−/+gD-1) virus were propagated on complementing VD60 cells and titered on VD60 and Vero cells. Concentrated viral stocks were stored at −80° C. and diluted in PBS to the desired concentration when needed.
Construction of ΔgD-2::GFP. Plasmid pcDNA3-eGFP (13031; Addgene, Cambridge, MA, USA) was used as a template to PCR amplify the pCMV-eGFP-Neor and OriE-Ampr regions flanked by Van91I restriction enzyme sites. The pCMV-eGFP-Neor region was PCR-amplified using primers Fwd-pCMV and Rev-NeoR-Term (see Table 3 for a list of primers). The OriE-Ampr region was PCR amplified using primers Fwd-Origin and Rev-AmpR.
In parallel, genomic regions flanking the left and right of the US6 gene (gD) in HSV-2 were PCR amplified using purified viral DNA (HSV-2 strain 4674) as a template and primers LL-V91I-US6 plus LR-V91I-US6 for the left homology arm and primers RL-V91I-US6 and RR-V91I-US6 for the right homology arm (see Table 3 for sequence alignment). All four PCR fragments were gel purified, digested with Van91I (Fermentas Molecular Biology Tools, Thermo Scientific, West Palm Beach, FL, USA), ligated with Quick-Ligase [New England Biolabs (NEB), Ipswich, Massachusetts., USA], and transformed into NEB 5-α competent cells.
The resulting plasmid (eKO2-US6) was sequence verified and extracted from E. coli using an endotoxin-free miniprep kit (MO-BIO Laboratories, Carlsbad, CA, USA). HSV-2 DNA (1 μg) was co-transfected with 100 ng of eKO2-US6 into VD60 cells using Effectene (Qiagen, Valencia, CA, USA), according to the manufacturer recommendations.
At 4 d after transfection, plates were screened for green plaques and supernatants were collected and overlaid on fresh VD60 cells for 1 h, then washed and covered with 4% low-melting agarose prepared in Optimem (Invitrogen, Carlsbad, CA, USA). Single green fluorescent plaques were picked and purified 3 times using this method. Viral stocks were grown on VD60 cells, and noncomplemented virus was generated by harvesting infected cell lysates from Vero cells, as described for HSV-1 gD-deletion viruses (Ligas, 1988).
Genotypic confirmation of the gD deletion in ΔgD-2::GFP was performed by PCR. A primer set was used to confirm the presence of wild-type (WT) and ΔgD-2 virus DNA in the samples (primers RL-V91I-US6 and RL-V91IUS6), while another set of primers (Neo-Out and US8-Out) was used to amplify a DNA region comprising eKO2-US6 and the genomic target region. To confirm deletion of gD expression, Vero or VD60 cells were infected with parental WT or ΔgD-2 virus (grown on VD60 cells and thus competent for entry) at a multiplicity of infection (MOI) of 10 plaque-forming units (PFU)/cell (based on VD60 titer). After 1-h incubation, cells were washed twice with PBS, incubated in Optimem for 48 h at 37° C., and harvested and evaluated for gD expression by Western blot.
Sequence homologies for the PCR fragments used to construct the eKO2-US6 plasmid. Searching the published Addgene pcDNA3-eGFP (id:13031, 6159 bp) sequence for the pCMV-eGFP-Neomycin resistance primers using Blast2 yields a 1819 bp fragment:
Searching the pcDNA3-eGFP sequence for the pUC Origin-Ampicillin resistance primers yields a 1754 bp fragment:
Searching the blast.ncbi.nlm.nih.gov/Blast.cgi database for Left US6 homology arm of Herpes simplex virus (type 2) (taxid:10310) returned sequence from the Human HSV-2 strain HG52, complete genome:
BLAST for Left-US6 homology arm yields a 991 bp homologous region, 1029 bp fragment:
BLAST for Right-US6 homology arm yields a 1072 bp homologous region, 1110 bp fragment:
When synthesizing HA genes, versions were designed that replaced codons in PR8 HA that had less than 9.5% representation in HSV-2 (G) gD with codons for their respective amino acid that had the highest representation in HSV-2 (G) gD.
The presence of chimeric gD::HA in recombinant viruses was verified by PCR (see
Mice that were vaccinated with ΔgD-2::FL HA nOP and ΔgD-2::HL HA OPT are fully protected against HSV-2 challenge but do not form anti-HA IgGs (see
Construction of ΔgD-2::PCMV-HA viruses and HA expression. It was investigated if it would be more immunogenic to insert non-chimeric HA genes into the ΔgD genome in cassettes containing a constitutive promoter and poly-adenylation sequence. To accomplish this, the synthesized PR8 HA genes were restriction cloned into expression plasmid pEGFP-N1 (Addgene). HA genes were inserted in between xbaI and HindIII sites and replaced EGFP in the plasmid.
Mice were vaccinated with ΔgD-2::PCMV-FL HA nOP. The mice were prime vaccinated with 5×106 PFU of each virus (See
ΔgD-2::PCMV-HA recombinant viruses all express HA. As shown in
To determine if the insertion of full-length PR8 HA into ΔgD-2 would introduce a novel route of cell entry, Vero cells were infected with 0.01 MOI of ΔgD-2::P-CMV-FL HA nOP or ΔgD-2::PEF1α-RFP. At 120 hours post-infection, there were no signs of productive infection. Additionally, 5 C57BL/6 mice were given subcutaneous injections with 5×106 PFU of ΔgD-2::PCMV-FL HA nOP and monitored daily for one week. They showed no signs of disease or distress. Additionally, an in vitro assay was conducted in which non-complementing Vero cells were infected with titrations of either ΔgD-2::PCMV-FL HA nOP and ΔgD-2::PEF1α-RFP. At 70 hours post-infection, the cells were fixed and stained for HSV-2 glycoprotein B (gB) Staining shows the presence of isolated infected cells at a 10-3 dilution of each virus, but no evidence of cell-to-cell viral spread (See
Designing the HIV constructs to express in HSV-2 ΔgD virus: Producing a vaccine vector in which a glycoprotein antigen from HIV would be expressed from the ADCC-inducing HSV-2 □gD vector was investigated. The rationale for using this vector is that it elicits non-neutralizing, ADCC-inducing IgG antibodies against HSV, and this type of immune response has been correlated with protection in the only HIV vaccine trial to show any efficacy thus far (Haynes, Gilbert et al. 2012). For the glycoprotein antigen, the transmitted/founder clone of Env gp145 was chosen, which lacks the cytoplasmic tail, from donor CH505 in the CHAVI001 acute HIV-1 infection cohort, as this well-characterized HIV-1 clade C glycoprotein is thought to be representative of those that pass the bottleneck of infection in a region of high HIV prevalence (Liao, Lynch et al. 2013). HIV Env is not expressed particularly well in the context of natural infection and often not expressed well exogenously, so steps were investigated to enhance antigen expression in our construct. Env gp145 was found to be incorporated more efficiently into virus like particles (VLPs) than full length, and replacing the Env signal peptide and transmembrane domains with corresponding domains from host proteins or other viral glycoproteins increased incorporation into VLPs further (Wang, Liu et al. 2007). To provide efficient incorporation of HIV Env into HSV VLPs, a chimera of the ectodomain of HIV Env with the signal peptide and transmembrane cytoplasmic tail of HSV-2 gD was constructed. The signal peptide of HSV2 gD is 25 residues in length and the ectodomain is 306 residues total (Eisenberg, Long et al. 1984, Nicola, Willis et al. 1996). The allelic exchange construct was generated by Gibson assembly (Gibson, Young et al. 2009). Briefly, oligonucleotide primers were synthesized to amplify from HSV2 strain G genomic DNA arms of homologous sequence ˜800 bp 5′ to the 25th codon of HSV2 US6 and ˜800 bp 3′ to the 306th codon of HSV2 US6. The insert was amplified from the CH505 TF gp145 expression plasmid HV1300631 (gift of Huaxin Liao, Duke University) with primers that encompassed the 30th codon to codon 680. The fragments were cloned into pUC19 between EcoRI and BamHI restriction sites.
Functional in vitro macrophage antibody-dependent cell-mediated killing (ADCK) assay using an RFP-expressing HSV-2 ΔgD strain (as made in Example 1): The single-cycle herpes simplex virus type 2 (HSV-2) strain deleted in glycoprotein D (ΔgD-2) elicits sterilizing anti-HSV immunity by inducing antibodies that bind and activate Fcγ-receptors (FcγRs). Murine FcγRIV is highly activated in the presence of serum from vaccinated mice and is expressed on macrophages, monocytes, and neutrophils. The precise mechanisms of cell killing through FcγRs are not well understood and additional tools are needed. FcγR-binding antibodies mediate killing of HSV-infected cells by binding antigens on infected cell and then binding and activating FcγRs on innate leukocytes. This precipitates antibody-dependent cell-mediated cytotoxicity and phagocytosis (ADCC and ADCP), here referred to as antibody-dependent cell-mediated killing (ADCK). Current assays face many limitations including, but not limited to, inflexible target and effector cell lines, artificial antigen presentation systems, indirect or separate outputs for ADCC and ADCP, and the use of cumbersome radioactive isotopes.
To overcome these restrictions, a quantitative in vitro assay was constructed to study ADCK in response to anti-HSV antibodies. A ΔgD-2 variant that highly expresses the gene for red fluorescent protein (rfp) was used to mark infection. RFP and cell viability markers were then used to identify live-infected target cells, the decreasing proportion of which was determined to be the result of cell killing. FACS analysis was used to quantify the decreasing proportion of live-infected target cells after co-culture with macrophages.
Using the new assay, it was shown that serum from mice vaccinated with ΔgD-2 induces significant amounts of ADCK by both immortalized macrophage cell lines and bone marrow derived macrophages (BMDMs) from both mice and guinea pigs. Live-imaging of the assay using Raw macrophages shows that killing readouts cannot easily be attributed to cytotoxicity or phagocytosis, but that the processes can occur simultaneously, underlining the importance of using an assay which measures both. Additionally, ADCK was eliminated when BMDMs from FcγR−/− mice were used, indicating that the primary readout is FcγR dependent and that the assay is amenable to studying ADCK in different knockout mouse strains. This assay allows for the precise study of ADCK and its associated genes and FcγRs in different species and animal models.
Traditionally, ADCC was measured by cumbersome 51Cr release assays, but a number of FACS methods have been developed to measure ADCC in response to different antigens and antibodies. The collective term for these methods is Rapid fluorometric ADCC (RFADCC). In these assays, target cells are stained with a persistent membrane die and a live-dead marker that dissipates upon the initiation of apoptosis. ADCC activity is then measured as the decrease in proportion of membrane dye+ cells that are also live-dead+. In similar assays, ADCP is measured by the proportion of macrophages that are marked by the phagocytosis of fluorescent cells. However, as the data herein show, ADCC and ADCP cannot be cleanly separated by these methods, so they are referred to collectively in this text as antibody-dependent cell-mediated killing (ADCK). The prior art assays have some additional drawbacks, as they are usually restricted in their choice of antibody or effector cell. A similar assay was used in Petro et al. 2015, but the ability of HSV to infect, replicate in, and kill both target and effector cells limited the utility of the assay for studying immune cells and their FcγRs (Petro et al., 2015).
ΔgD-2 is a single-cycle virus in non-complementing cells and was recently shown not to induce dendritic cell death in vitro. It was investigated whether a brightly fluorescent ΔgD-2 strain would allow for precise investigation of the cellular mechanisms of ADCK. An RFP expressing ΔgD-2 (ΔgD-2::RFP) strain was constructed and developed with an RFADCK assay that precisely measures the effector activity of macrophages on HSV-2 ΔgD-2:RFP infected cells in vitro. The assay was validated for both immortalized and primary cell lines using J774 cells, Raw 264.7 macrophages, and bone marrow-derived macrophages (BMDMs). Unlike current RFADCC assays, this method simulates an infectious environment, measures both ADCC and ADCP, and is profoundly flexible, allowing for the use of polyclonal animal sera and different cell lines and mouse strains.
Cells Lines: Vero (CCL-81; ATCC, Manassas, VA) and VD60 cells (Vero cells containing multiple copies of gD-1 under the endogenous gene promoter) were passaged in DMEM (Thermo Fisher Scientific, Waltham, MA) supplemented with 5% fetal bovine serum (FBS, Gemini Bio-Products, West Sacramento, CA) and 10 U/ml penicillin, and 10 μg/ml streptomycin sulfate (Thermo Fisher Scientific). Bone-marrow derived macrophages (BMDMs) were obtained as previously described from C57B1/6 mice. Bone marrow precursors were stored in DMEM supplemented with 50% FBS and 10% DMSO (Sigma-Aldrich, St. Louis, MO) if not used immediately. Mouse BMDMs were differentiated using the supernatant from L929 cell cultures (ATCC). Guinea pig BMDMs were differentiated using recombinant human M-CSF (BioLegend, San Diego, CA). Raw 264.7, J774.1, and HEK 293 cells (ATCC) were passaged in DMEM supplemented with 10% FBS and 1% Pen-strep.
Creation of ΔgD-2::RFP and Virus Propagation. VD60 cells were co-transfected with HSV-2 ΔgD:GFP (ΔgD-2) genomic DNA and cosmid DNA containing 40 kB of the HSV-2 genome in which the US6 gene was replaced with tdtomato downstream of an EF1α promoter. Resultant virus was plaque purified three times using RFP expression as a marker of homologous recombination. The purified ΔgD-2::RFP virus was verified by PCR and sequencing.
ΔgD-2 and ΔgD-2::RFP were propagated on VD60 cell, which complement the gD deletion and allow for multiple rounds of replication. All viral strains were titered by serial dilution and propagation on their respective cell types.
Animals: Female C57BL/6 mice, aged 4-6 weeks, obtained from Jackson Laboratory (JAX, Bar Harbor, ME) were used to obtain serum. Male C57BL/6 mice, aged 4-6 weeks, were obtained from JAX and bone marrow cell suspensions were isolated by flushing their femurs with DMEM supplemented with 10% PBS and 1% Pen-strep. Female Hartley guinea pigs, aged 5-6 weeks, were purchased from Charles River laboratories (Wilmington, MA). Bone marrow cell suspensions were isolated by flushing femurs and tibias with DMEM supplemented with 10% FBS and 1% Pen-strep. Vaccinations were administered by subcutaneous injection. All procedures were approved by the Albert Einstein College of Medicine Institutional Animal Care and Use Committee
Immunization: Mice or guinea pigs, aged 6-8 weeks, were prime vaccinated subcutaneously with 5×106 plaque forming units (PFU) of HSV-2 ΔgD::RFP or an equal volume of VD60 cell lysate in phosphate-buffered saline (PBS) to a total volume of 200 μL. Animals were boosted with the same dose 21 days later.
Antibody-dependent cell killing assay: Bone Marrow Derived Macrophages (BMDMs), J774.1 macrophages, and Raw 264.7 macrophages were incubated in LPS (Sigma-Aldrich) for 12 hours prior to co-culture with HEK 293 cells (ATCC). HEK 293 cells were double stained with PKH67 membrane (Sigma-Aldrich) and Tag-it Violet™ (Biolegend) dyes according to manufacturer's instructions. HEK cells were infected with HSV-2 ΔgD:: RFP at a MOI of 3 in serum free DMEM 4 hours before co-culture. The infection media was removed after 3.5 hours and was replaced with a 1:5 dilution of heat-inactivated mouse or guinea pig serum collected at day 40 from mice prime-boost injected as described previously with either HSV-2 ΔgD:: RFP or VD60 cell lysate. HEK 293T cells were incubated in serum for 30 minutes at 37° C. and then added to macrophage cultures in 96-well tissue culture plates (Corning Inc, New York City, NY). The co-cultures were incubated for 12 hours, then fixed and analyzed by flow cytometry on a LSRII (BD Biosciences, Franklin Lakes, NJ). For live-imaging experiments, Raw 264.7 macrophages and HEK 293 cells were co-cultured at a ratio of 10:1 on glass-bottom 96-well plates (Matrical Bioscience, Spokane WA) and imaged with an inverted NIKON Eclipse TiE microscope using NIS Elements software with deconvolution.
Statistical Analyses: Data was compiled in GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). Statistical analysis using the tests noted in the figures were also done using GraphPad Prism. Statistical significance is indicated in figures as *, p<0.5; ***, p<0.001.
Inserting rfp into US6 of HSV-2 ΔgD does not alter viral growth phenotype. Vero cells were infected with HSV-2 ΔgD-2::GFP at a multiplicity of infection (MOI) of 1 to determine the kinetics of GFP expression. Fluorescent microscopy was used to visualize GFP expression, which was low even at 24 hours post-infection (
Serum from ΔgD-2::RFP vaccinated mice induces significant ADCK in the RFADCK assay. In order to measure the levels of ADCK activity elicited by serum from ΔgD-2::RFP vaccinated mice, an in vitro protocol was developed based on previous RFADCC assays. Target HEK 293T cells were stained with a membrane dye and a live-dead marker and infected with ΔgD-2::RFP at an MOI of 3. Infected target cells were incubated for 3.5 hours before infection media was removed and media or mouse serum was added for an additional 30-minute incubation. The infected HEK 293T cells were co-cultured for 12 hours with J774.1 murine macrophage cells which had been stimulated with LPS 12 hours prior. Based on previously reported assays, we used an effector to target cell ratio of 10:1. At this ratio, serum from ΔgD-2::RFP vaccinated mice induced killing of approximately 70.5% of highly infected (RFPhigh) target cells (
ADCK is significantly increased in the presence of serum from mice vaccinated with ΔgD-2::RFP compared to serum from mice vaccinated with a control cell lysate. Infected HEK 293 cells were incubated with serum from mice vaccinated with either ΔgD-2::RFP or a control VD60 cell lysate to determine whether killing mediated by different immortalized macrophage cells lines was caused by antigen-specific ADCK. For each treatment group, the proportion of RFPhigh cells after 16 hours of co-culture was compared to that of parallel assays lacking serum. In cultures containing J774.1 macrophages, serum from ΔgD-2::RFP vaccinated mice induced significantly more killing of RFPhigh cells than serum from VD60 mock-vaccinated mice (p<0.001;
Tracking of infected cells by live-imaging of ADCK assay shows that cytotoxicity and phagocytosis are not mutually exclusive. ADCK assay was carried out as described above with the exceptions that glass-bottom 96-well plates were used and the effector cells were Raw 264.7 murine macrophage cells which have been shown to carry out similar amounts of ADCC and ADCP. The assays were imaged on a deconvolution microscope at 60× magnification approximately every 15 minutes for 24 hours. Infected cells are RFP+. Uninfected HEK293 cells are highly Mem+ and Live/Dead+. Macrophages are unstrained.
ADCK activity by bone marrow-derived macrophages is FcγR-dependent. RFADCK assays were performed with bone marrow-derived macrophages (BMDMs) from wild-type and FcγR−/− mice to determine whether observed ADCK was FcγR dependent. ADCK activity was defined as the decrease in RFPhigh cells compared to parallel cultures with serum from VD60 lysate mock-vaccinated mice. At an effector to target cell ratio of 10:1, wild-type BMDMs killed significantly more infected target cells than FcγR−/− BMDMs in the presence of serum from HSV-2 ΔgD-2::RFP vaccinated mice (p<0.05;
RFADCK using guinea pig bone marrow-derived macrophages recapitulates murine results. To determine the ability of another species' effector cells to carry out ADCK in our assay, guinea pig BMDMs were co-cultured with ΔgD-2::RFP infected HEK 293 cells in the presence of serum from ΔgD-2 vaccinated, VD60 lysate mock vaccinated, or naïve guinea pigs. The killing of RFPhigh cells in parallel co-cultures containing serum from naïve animals was used as the baseline. In the presence of serum from ΔgD-2 vaccinated animals, guinea pig BMDMs carried out significant ADCK (p<0.001;
Anti-PR8 IgG Isotype ELISA. Mice were subcutaneously injected with ΔgD-2::RFP, ΔgD-2 expressing full-length PR8 hemagglutinin (FL nOP, (β3χ4), ΔgD-2 expressing headless PR8 hemagglutinin (HL nOP, β3χ5), or ΔgD-2 expressing headless PR8 hemagglutinin codon-optimized for HSV-2 (G) (HL OPT, β3χ6) or mock vaccinated with VD60 cell lysate. 5 mice/group were prime-boost vaccinated with each treatment separately. (See
Mice vaccinated with ΔgD-2::HAPR8 are fully protected from challenge with PR8. Mice were prime-boost vaccinated subcutaneously 3 weeks apart with 5×106 PFU of ΔgD-2::RFP or ΔgD-2::HAPR8 or mock vaccinated with VD60 cell lysate. The mice were bled one-week post-boost and serum neutralization titer was measured against A/Puerto Rico/1934/8 IAV (PR8). (See
Recombinant gD-2::HAPR8 expresses high levels of PR8 protein. To assess expression, ΔgD-2::RFP gDNA was co-transfected into VD60 cells alongside an HA expression cassette containing the hemagglutinin (HA) gene from IAV H1N1 strain A/Puerto Rico/1934/8 (PR8) downstream of PCMV and upstream of a poly-adenylation signal as illustrated in
Mice immunized with gD-2::HAPR8 develop high titers of functional and isotype switched anti-PR8 antibodies. Mice were prime-boost vaccinated 21 days apart with either VD60 cell lysate, ΔgD-2::RFP vector, or ΔgD-2::HAPR8 (5 mice per group). At day 28 post-prime vaccination, serum was collected for analysis. Anti-PR8 antibodies were measured by ELISA against purified HA PR8 protein. Mice immunized with ΔgD-2::HAPR8 developed isotype switch anti-PR8 HA antibodies that were predominantly IgG2c and IgG2b. (See
Mice immunized with gD-2::HAPR8 develop protection against IAV challenge. Mice were prime-boost immunized 21 days apart with VD60 cell lysate, ΔgD-2, or ΔgD-2::HAPR8, bled at day 28 post-prime, and challenged intranasally 14 days later with a 6× LD50 of IAV. Mice were sacrificed after reaching 70% of starting weight and neutralization titers were measured using microneutralization assays against the respective strains. As shown in
Mice immunized with ΔgD-2::HAPR8 develop fully protective ADCC immunity against HSV-2. Mice were prime-boost vaccinated 21 days apart with either VD60 cell lysate, ΔgD-2, or ΔgD-2::HAPR8 (5 mice per group). In
mFcγRIV ADCC reporter bioassay response. Serum samples collected from mice vaccinated with ΔgD-2::HA, ΔgD-2::RFP, and inactivated A/Puerto Rico/8/1934 H1N1 virus (PR8) were serially diluted and subjected to mFcγRIV ADCC reporter bioassay. Madin-Darby Canine Kidney (MDCK) cells infected with PR8 virus were used as target cells and mFcγRIV expressing Jurkat T cells were used as effector cells. The target cells were incubated with the serially diluted serum samples and effector cells. Bio-Glo™ Reagent was added, and luminescence was measured. The HSV-2 ΔgD::HA vaccinated mice showed significantly higher activation of mFcγRIV receptors at each dilution tested in comparison to mice vaccinated with ΔgD-2::RFP and inactivated PR8 virus. (See
Cloning of HIV-1 Env in pBkk412 plasmid containing HSV-2 genes. The full length HIV-1 Env (Clade C) and Rev genes, together with partial Nef gene, were PCR amplified from the plasmid ZM109F.PB4 (
The PCR fragment product, which also contained bGH polyA signal and T7 promoter, was cloned into the multiple cloning site (MCS) of the pBkk412 plasmid under control of CMV promoter, using RE sbf I and blp I. (See
Stable, competent E.coli cells were transformed and plated on Agar+ carbenicillin. Colonies were screened for the presence of the insert using PCR (
Once expression of the HIV proteins in transfected cells is confirmed, cells will be co-transfected with the linear recombinant plasmid and the HSV ΔgD-2 negative virus (e.g., ΔgD-2::RFP, ΔgD-2::GFP) and selected for recombinants that express the HIV protein and do not express the GFP or RFP marker. Expression of the HIV protein(s) by the new recombinant virus will be assessed by Western blot or flow cytometry (FACS). Mice will be immunized intramuscularly (prime and boost) and serum collected and screen for the presence of HIV-specific antibodies. The functionality (e.g., neutralizing and non-neutralizing functions) of the Abs will also be measured. A humanized mouse model which renders mice susceptible to infection with HIV, will also be used to assess whether the vaccine protects the mice from HIV infection.
Set forth below are some embodiments of the process, the recombinant virus, and the methods disclosed herein.
Throughout this application various publications are referred to. Full citations for these references may be found at the end of the specification. The disclosures of these publications, and all patents, patent application publications and books referred to herein, are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
One skilled in the art will readily appreciate that the specific methods and results discussed hereinabove are merely illustrative of the invention as described more fully in the claims that follow thereafter.
This application is a continuation of U.S. application Ser. No. 17/051,992, filed on Oct. 30, 2020, which is a National Stage application of PCT/US2019/030259, filed on May 1, 2019, which claims the benefit of U.S. Provisional Application No. 62/665,050, filed on May 1, 2018, both of which are incorporated by reference herein in their entirety.
This invention was made with government support under grant numbers AI117321 and AI007501 awarded by the National Institutes of Health. The government has certain rights in the invention.
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| 20230293674 A1 | Sep 2023 | US |
| Number | Date | Country | |
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| 62665050 | May 2018 | US |
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| Parent | 17051992 | US | |
| Child | 18071109 | US |
