The instant application contains a Sequence Listing which has been submitted electronically in ST26 .XML format and is hereby incorporated by reference in its entirety. Said ST26 .XML copy, created on Sep. 20, 2022, is named “065095002US” and is 473 kilobytes in size.
The invention is generally related to the field of immunomodulation, and more particularly to compositions and methods for modulating immune responses in a subject.
SARS-CoV-2 is a novel coronavirus that was first identified in Wuhan, China in December 2019 as a cause of pneumonia and has subsequently spread globally to cause the COVID-19 pandemic. According to the World Health Organization (WHO), since 2019, COVID-19 has spread globally, infected more than 519 million people, and caused at least 6 million deaths. SARS-CoV-2 initially infects the upper respiratory tract epithelium (Wolfel, R., et al., Nature, 581(7809):465 (2020)) but can progress to the lower respiratory tract to cause pneumonia and acute respiratory distress syndrome (ARDS) (Huang, C., et al., Lancet, 395(10223):497 (2020)). Since the beginning of the 2019 coronavirus disease (COVID-19) pandemic, numerous SARS-CoV-2 variants have emerged. WHO defines a SARS-CoV-2 variant of concern (VOC) as a variant that affects virus transmissibility and COVID-19 epidemiology, increases virulence and pathogenicity, or decreases the effectiveness of in-place public health measures. Current VOCs include delta and omicron, while previously circulating VOCs include alpha, beta, and gamma.
The global spread of SARS-CoV-2 prompted rapid development of prophylactic vaccines. Vaccines and antibody therapies have been introduced worldwide providing a ray of hope for the end of the COVID-19 pandemic. Currently, three vaccines are approved for use in the United States. The vaccines developed by Pfizer and Moderna are based on mRNA technology, while the vaccine produced by Johnson & Johnson (J&J) utilizes a human adenovirus type 26 vector. A vaccine produced by AstraZeneca employs a Chimpanzee adenovirus vector and is approved for use in the European Union (Ura, T., et al., Vaccine, 39(2): 197 (2021)). Since May 2022, 11 billion vaccine doses have been administered worldwide. However, SARS-CoV-2 variants have demonstrated immune escape in previously infected and fully vaccinated individuals. Compared to neutralization of alpha variant, serum from convalescent individuals was four-fold less effective against delta variant. The same study found that serum from individuals who received two doses of Pfizer's vaccine has delta-neutralizing abilities three- to five-fold lower than its alpha-neutralizing abilities. This immune escape was correlated to epitopes found on the SARS-CoV-2 spike protein (Planas, D., et al., Nature, 596(7871): 276 (2021)). Another study found that the omicron-neutralizing ability of serum from WA1-convalescent individuals is eight-fold lower than its WA1-neutralizing ability (Zhang, L., et al., Emerg Microbes Infect, 11(1): 1 (2022)). Analysis of published reports indicates that for individuals vaccinated with Pfizer, Moderna, J&J, or AstraZeneca vaccines, vaccine efficacy decreased by approximately 21 percentage points within one to six months after full vaccination. This decrease in efficacy was associated with waning immunity (Feikin, D. R., et al., Lancet, 399(10328): 924 (2022)).
Therefore, compounding waning immunity with the emergence of variants capable of immune escape, there is an urgent need for novel SARS-CoV-2 vaccine candidates with long-lasting immunity and protective against variants.
The present disclosure presents an intranasal viral-vectored vaccine against SARS-CoV-2 variants. The disclosure includes a viral expression vector having a parainfluenza virus 5 (PIV5) genome having a heterologous nucleic acid sequence with at least 98% sequence identity to SEQ ID NOs: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80, wherein the viral expression vector expresses a heterologous polypeptide comprising a coronavirus spike (S) and/or nucleocapsid (N) proteins.
In some aspects of the viral expression vector, the coronavirus S protein includes the coronavirus S protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the coronavirus N protein is the coronavirus N protein of SARS-CoV-2, a variant of interest or a variant of concern of SARS-CoV-2.
In some aspects of the viral expression vector, the coronavirus S protein is the coronavirus S protein of a SARS-CoV-2 beta variant, a SARS-CoV-2 gamma variant, a SARS-CoV-2 delta variant, or a SARS-CoV-2 omicron variant and the coronavirus N protein is the coronavirus N protein of a SARS-CoV-2 beta variant, a SARS-CoV-2 gamma variant, a SARS-CoV-2 delta variant, or a SARS-CoV-2 omicron variant.
In some aspects of the viral expression vector, the coronavirus S protein comprises the coronavirus S protein of SARS-CoV-2 and wherein the cytoplasmic tail of the coronavirus S protein has been replaced with the cytoplasmic tail of the fusion (F) protein of PINS.
In some aspects of the viral expression vector, the coronavirus S protein of a variant of SARS-CoV-2 has been inserted between the PIV5 small hydrophobic (SH) and hemagglutinin (HN) genes and the coronavirus N protein of a variant of SARS-CoV-2 has been inserted between the PIV5 HN and polymerase (L) genes.
In some aspects of the viral expression vector, the coronavirus S protein of a variant of SARS-CoV-2 has been inserted between the PIV5 HN and polymerase (L) genes.
In some aspects of the viral expression vector, the coronavirus S protein is a S protein from a variant of interest or a variant of concern of SARS-CoV-2 and the coronavirus N protein is a N protein from different variant of interest or a variant of concern of SARS-CoV-2.
In some aspects of the viral expression vector, the PIV5 small hydrophobic (SH) gene has been replaced with the coronavirus N protein of SARS-CoV-2.
In some aspects of the viral expression vector, the PIV5 genome further comprises one or more mutations comprising a mutation of the V/P gene, a mutation of the shared N-terminus of the V and P proteins, a mutation of residues 26, 32, 33, 50, 102, and/or 157 of the shared N-terminus of the V and P proteins, a mutation lacking the C-terminus of the V protein, a mutation lacking the small hydrophobic (SH) protein, a mutation of the fusion (F) protein, a mutation of the phosphoprotein (P), a mutation of the large RNA polymerase (L) protein, a mutation incorporating residues from canine parainfluenza virus, a mutation inducing apoptosis, or a combination thereof.
In some aspects of the viral expression vector, one or more mutations comprise PIV5VΔC, PIV5ΔSH, PIV5-P-S308G, or a combination thereof.
In some aspects of the viral expression vector, the heterologous polypeptide comprises a CPI V/P gene that contains mutations at amino acid residue S157 or S308, or the combination thereof, wherein serine (S) is substituted with an amino acid residue selected from a group consisting of alanine (A), asparagine (B), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), selenocysteine (U), valine (V), tryptophan (W), tyrosine (Y), and glutamine (Z).
In some aspects of the viral expression vector, the amino acid substitution at amino acid residue S157 comprises a substitution of serine (S) to phenylalanine (F) and the amino acid substitution at amino acid residue S308 comprises a substitution of serine (S) to alanine (A) or Glycine (G).
In some aspects of the viral expression vector, a viral particle comprises the viral expression vector.
The present disclosure also presents a composition comprising the viral expression vector presented above, a viral particle presented above, or a combination thereof.
In some aspects of the composition, a heterologous coronavirus spike (S) and nucleocapsid (N) proteins are expressed in a cell by contacting the cell with the composition.
The present disclosure also presents a method of inducing an immune response in a subject having coronavirus disease 2019 (COVID-19) to coronavirus spike (S) and nucleocapsid (N) proteins, the method comprising administering the composition presented above to the subject, wherein the immune response comprises a humoral immune response and/or a cellular immune response.
In some aspects of the method, the subject is vaccinated against COVID-19), the method comprising administering the composition to the subject.
In some aspects of the method, the composition is administered intranasally, intramuscularly, topically, or orally.
The present disclosure also presents a method of inducing in a subject an immune response comprising administering a PIV-5 booster vaccine composition comprising a viral expression vector or a viral particle having a PIV5 genome comprising a heterologous nucleic acid sequence with at least 98% sequence identity to SEQ ID NOs: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80, wherein said subject has previously received a primary vaccination against SARS-COV-2.
To facilitate an understanding of the principles and features of the various embodiments of the disclosure, various illustrative embodiments are explained herein. Although exemplary embodiments of the disclosure are explained in detail, it is to be understood that other embodiments are contemplated. Accordingly, it is not intended that the disclosure is limited in its scope to the details of construction and arrangement of components set forth in the description or examples. The disclosure is capable of other embodiments and of being practiced or carried out in various ways.
In describing the exemplary embodiments, specific terminology will be resorted to for the sake of clarity. As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, reference to a component is intended also to include composition of a plurality of components. References to a composition containing “a” constituent is intended to include other constituents in addition to the one named.
Ranges may be expressed herein as from “about” or “approximately” or “substantially” one particular value and/or to “about” or “approximately” or “substantially” another particular value. When such a range is expressed, other exemplary embodiments include from the one particular value and/or to the other particular value.
Similarly, as used herein, “substantially free” of something, or “substantially pure”, and like characterizations, can include both being “at least substantially free” of something, or “at least substantially pure”, and being “completely free” of something, or “completely pure.”
The terms “patient”, “individual”, “subject”, and “animal” are used interchangeably herein and refer to mammals, including, without limitation, human and veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models. In a preferred embodiment, the subject is a human.
As used herein, the term “combination” of a PIV5-based AVLP composition as described herein and at least a second pharmaceutically active ingredient means at least two, but any desired combination of compounds can be delivered simultaneously or sequentially (e.g., within a 24-hour period). It is contemplated that when used to treat various diseases, the compositions and methods of the present disclosure can be utilized with other therapeutic methods/agents suitable for the same or similar diseases. Such other therapeutic methods/agents can be co-administered (simultaneously or sequentially, in any order) to generate additive or synergistic effects. Suitable therapeutically effective dosages for each agent may be lowered due to the additive action or synergy. Also, two or more embodiments of the disclosure may be also co-administered to generate additive or synergistic effects.
The term “coronavirus” refers to a group of related RNA viruses that cause diseases in mammals and birds. In humans, these viruses cause respiratory tract infections that can range from mild to lethal. Mild illnesses include some cases of the common cold (which is caused also by certain other viruses, predominantly rhinoviruses), while more lethal varieties can cause SARS, MERS, and COVID-19. There are presently no vaccines or antiviral drugs to prevent or treat human coronavirus infections.
The term “SARS” or “severe acute respiratory syndrome” refers to a viral respiratory disease of zoonotic origin that surfaced in the early 2000s caused by severe acute respiratory syndrome coronavirus (SARS-CoV or SARS-CoV-1), the first-identified strain of the SARS coronavirus species severe acute respiratory syndrome-related coronavirus (SARS-CoV). The syndrome caused the 2002-2004 SARS outbreak. In 2019, its successor, the related virus strain Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), was discovered.
The term “Covid-19” or “Coronavirus disease 2019” refers to a severe acute respiratory syndrome (SARS) caused by a virus known as SARS-Coronavirus 2 (SARS-CoV2).
As described herein, the term “vaccinating” designates typically the sequential administration of one or more antigens to a subject, to produce and/or enhance an immune response against the antigen(s). The sequential administration includes a priming immunization followed by one or several boosting immunizations.
Within the context of the present invention, the term “pathogen” refers to any agent that can cause a pathological condition. Examples of “pathogens” include, without limitation, cells (e.g., bacteria cells, diseased mammal cells, cancer mammal cells), fungus, parasites, viruses, prions or toxins. Preferred pathogens are infectious pathogens. In a particular embodiment, the infectious pathogen is a virus, such as the coronaviruses.
An antigen, as used therein, designates any molecule which can cause a T-cell or B-cell immune response in a subject. An antigen specific for a pathogen is, typically, an element obtained or derived from said pathogen, which contains an epitope, and which can cause an immune response against the pathogen. Depending on the pathogenic agent, the antigen may be of various nature, such as a (poly)peptide, protein, nucleic acid, lipid, cell, etc. Live weakened forms of pathogens (e.g., bacteria, viruses), or killed or inactivated forms thereof may be used as well, or purified material therefrom such as proteins, peptides, lipids, etc. The antigen may be naturally-occurring or artificially created. It may be exogenous to the treated mammal, or endogenous (e.g., tumor antigens). The antigen may be produced by techniques known per se in the art, such as for instance synthetic or recombinant technologies, or enzymatic approaches.
In a particular embodiment, the antigen is a protein, polypeptide and/or peptide. The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residues may be modified or non-naturally occurring residues, such as an artificial chemical mimetic of a corresponding naturally occurring amino acid. It should be understood that the term “protein” also includes fragments or variants of different antigens, such as epitope-containing fragments, or proteins obtained from a pathogen and subsequently enzymatically, chemically, mechanically or thermally modified.
A “therapeutically effective amount” means the amount of a compound (e.g., a PIV5-based composition as described herein) that, when administered to a subject for treating a state, disorder or condition, is sufficient to effect such treatment. The “therapeutically effective amount” will vary depending on the compound or bacteria administered as well as the disease and its severity and the age, weight, physical condition and responsiveness of the mammal to be treated.
The phrase “pharmaceutically acceptable”, as used in connection with compositions of the disclosure, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., a human). Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.
The term “pharmaceutically acceptable composition” as used herein refers to a composition comprising at least one compound as disclosed herein formulated together with one or more pharmaceutically acceptable carriers.
The term “administration” refers to the introduction of an amount of a predetermined substance into a patient by a certain suitable method. The composition disclosed herein may be administered via any of the common routes, as long as it is able to reach a desired tissue, for example, but is not limited to, inhaling, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, or intrarectal administration. However, since peptides are digested upon oral administration, active ingredients of a composition for oral administration should be coated or formulated for protection against degradation in the stomach.
The term “dose” means a single amount of a compound or an agent that is being administered thereto; and/or “regimen: which means a plurality of pre-determined doses that can be different in amounts or similar, given at various time intervals, which can be different or similar in terms of duration. In some embodiments, a regimen also encompasses a time of a delivery period (e.g., agent administration period, or treatment period). Alternatively, a regimen is a plurality of predetermined plurality pre-determined vaporized amounts given at pre-determined time intervals.
The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.
The terms “treat” or “treatment” of a state, disorder or condition include: (1) preventing or delaying the appearance of at least one clinical or sub-clinical symptom of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or sub-clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.
As used herein, the term “parainfluenza virus 5” (PIV5) includes, for example and not limitation, strains KNU-11, CC-14, D277, 1168-1, and 08-1990. Non-limiting examples of PIV5 genomes are listed in GenBank Accession Nos. NC_006430.1, AF052755.1, KC852177.1, KP893891.1, KC237065.1, KC237064.1 and KC237063.1, which are hereby incorporated by reference.
By “comprising” or “containing” or “including” is meant that at least the named compound, element, particle, or method step is present in the composition or article or method, but does not exclude the presence of other compounds, materials, particles, method steps, even if the other such compounds, material, particles, method steps have the same function as what is named.
The mention of one or more method steps does not preclude the presence of additional method steps or intervening method steps between those steps expressly identified. Similarly, it is also to be understood that the mention of one or more components in a composition does not preclude the presence of additional components than those expressly identified.
The materials described as making up the various elements of the disclosure are intended to be illustrative and not restrictive. Many suitable materials that would perform the same or a similar function as the materials described herein are intended to be embraced within the scope of the disclosure. Such other materials not described herein can include, but are not limited to, for example, materials that are developed after the time of the development of the disclosure.
The disclosure provides PIV5-based compositions, systems and methods for their use in multiple applications including functional genomics, drug discovery, target validation, protein production (e.g., therapeutic proteins, vaccines, monoclonal antibodies), gene therapy, and therapeutic treatments such as cancer therapy.
I. SARS-CoV-2
With the present invention, constructs of the parainfluenza virus type-5 (PIV5) virus expressing the SARS-CoV-2 envelope spike (S) and nucleocapsid (N) protein have been generated for use as vaccines against COVID. These constructs demonstrate effectiveness as vaccines, with single dose intranasal immunization inducing sterilizing immunity in ferrets and cats.
Coronavirus disease 2019 (COVID-19) is a newly emerging infectious disease currently spreading across the world. It is caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Zhu et al., 2020, N Engl J Med; 382:727-733). SARS-CoV-2 was first identified in Wuhan, China in December 2019, and has subsequently spread globally to cause the COVID-19 pandemic. The virus has infected more than 221 million persons world-wide, caused more than 4,574,000 deaths as of Sep. 8, 2021, and is poised to continue to spread in the absence of herd immunity. While vaccines and antibody therapies have been introduced worldwide, the emergence of multiple viral variants which are rapidly replacing the original virus identified in Wuhan has allowed for immune escape in vaccinated populations, presenting a need for improved vaccine efficacy.
SARS-CoV-2 is a single-stranded RNA-enveloped virus belonging to the B coronavirus family (Lu et al., 2020, Lancet; 395:565-74). An RNA-based metagenomic next-generation sequencing approach has been applied to characterize its entire genome, which is 29,881 nucleotides (nt) in length (GenBank Sequence Accession MN908947) encoding 9860 amino acids (Chen et al., 2020, Emerg Microbes Infect; 9:313-9). Full-genome sequenced genomes available at GenBank include isolate 2019-nCoV WHU01 (GenBank accession number MN988668) and NC_045512 for SARS-CoV-2, both isolates from Wuhan, China, and at least seven additional sequences (MN938384.1, MN975262.1, MN985325.1, MN988713.1, MN994467.1, MN994468.1, and MN997409.1) which are >99.9% identical and are hereby incorporate by reference.
1. SARS-CoV-2 Variants
Since SARS-CoV-2 was first identified in 2019, multiple genetic variants of SARS-CoV-2 have been emerging and circulating around the world. Viral mutations and variants in the United States are routinely monitored through sequence-based surveillance, laboratory studies, and epidemiological investigations. The US government SARS-CoV-2 Interagency Group (SIG) developed a Variant Classification scheme that defines three classes of SARS-CoV-2 variants: variant of interest, variant of concern and variant of high consequence.
a. Variant of Interest
A SARS-CoV-2 variant of interest is a variant with specific genetic markers that have been associated with changes to receptor binding, reduced neutralization by antibodies generated against previous infection or vaccination, reduced efficacy of treatments, potential diagnostic impact, or predicted increase in transmissibility or disease severity.
A variant of interest might require one or more appropriate public health actions, including enhanced sequence surveillance, enhanced laboratory characterization, or epidemiological investigations to assess how easily the virus spreads to others, the severity of disease, the efficacy of therapeutics and whether currently approved or authorized vaccines offer protection. The growing list variants of interest that are being monitored and characterized include, but are not limited to, Eta, Iota, Kappa, Lambda and Mu.
b. Variant of Concern
A SARS-CoV-2 variant of concern is a variant for which there is evidence of an increase in transmissibility, more severe disease (e.g., increased hospitalizations or deaths), significant reduction in neutralization by antibodies generated during previous infection or vaccination, reduced effectiveness of treatments or vaccines, or diagnostic detection failures. Possible attributes of a variant of concern include evidence of impact on diagnostics, treatments, or vaccines, widespread interference with diagnostic test targets, evidence of substantially decreased susceptibility to one or more class of therapies, evidence of significant decreased neutralization by antibodies generated during previous infection or vaccination, evidence of reduced vaccine-induced protection from severe disease, evidence of increased transmissibility and evidence of increased disease severity.
Variants of concern might require one or more appropriate public health actions, such as notification to WHO under the International Health Regulations, reporting to CDC, local or regional efforts to control spread, increased testing, or research to determine the effectiveness of vaccines and treatments against the variant. Based on the characteristics of the variant, additional considerations may include the development of new diagnostics or the modification of vaccines or treatments. The growing list of variants of concern that are being closely monitored and characterized, includes, but is not limited to, Alpha, Beta, Delta, and Gamma.
c. Variant of High Consequence
A SARS-CoV-2 variant of high consequence has clear evidence that prevention measures or medical countermeasures (MCMs) have significantly reduced effectiveness relative to previously circulating variants. Possible attributes of a variant of high consequence include a demonstrated failure of diagnostic test targets, evidence to suggest a significant reduction in vaccine effectiveness, a disproportionately high number of vaccine breakthrough cases, or very low vaccine-induced protection against severe disease, significantly reduced susceptibility to multiple Emergency Use Authorization (EUA) or approved therapeutics and more severe clinical disease and increased hospitalizations.
A variant of high consequence would require notification to WHO under the International Health Regulations, reporting to CDC, an announcement of strategies to prevent or contain transmission, and recommendations to update treatments and vaccines. Currently, there are no SARS-CoV-2 variants that rise to the level of high consequence.
II. Parainfluenza Virus 5 (PIV5)
Parainfluenza virus 5 (PIV5), a negative-stranded RNA virus, is a member of the Rubulavirus genus of the family Paramyxoviridae which includes many important human and animal pathogens such as mumps virus, human parainfluenza virus type 2 and type 4, Newcastle disease virus, Sendai virus, HPIV3, measles virus, canine distemper virus, rinderpest virus and respiratory syncytial virus. PIV5 was previously known as Simian Virus-5 (SV5). Although PIV5 is a virus that infects many animals and humans, no known symptoms or diseases in humans have been associated with PIV5. Unlike most paramyxoviruses, PIV5 infect normal cells with little cytopathic effect. As a negative stranded RNA virus, the genome of PIV5 is very stable. As PIV5 does not have a DNA phase in its life cycle and it replicates solely in cytoplasm, PIV5 is unable to integrate into the host genome. Therefore, using PIV5 as a vector avoids possible unintended consequences from genetic modifications of host cell DNAs. PIV5 can grow to high titers in cells, including Vero cells which have been approved for vaccine production by WHO and FDA. Thus, PIV5 presents many advantages as a vaccine vector.
A PIV5-based vaccine vector of the present invention may be based on any of a variety of wild type, mutant, or recombinant (rPIV5) strains. Wild type strains include, but are not limited to, the PIV5 strains W3A, WR (ATCC® Number VR-288TM), canine parainfluenza virus strain 78-238 (ATCC number VR-1573) (Evermann et al., 1980, J Am Vet Med Assoc; 177:1132-1134; and Evermann et al., 1981, Arch Virol; 68:165-172), canine parainfluenza virus strain D008 (ATCC number VR-399) (Binn et al., 1967, Proc Soc Exp Biol Med; 126:140-145), MIL, DEN, LN, MEL, cryptovirus, CPI+, CPI−, H221, 78524, T1 and SER. See, for example, Chatziandreou et al., 2004, J Gen Virol; 85(Pt 10):3007-16; Choppin, 1964, Virology: 23:224-233; and Baumgartner et al., 1987, Intervirology; 27:218-223. Additionally, PIV5 strains used in commercial kennel cough vaccines, such as, for example, BI, FD, Merck, and Merial vaccines, may be used.
III. PIV5 CPI Vectored SARS-CoV-2 Constructs
A PIV5 vaccine vector of the present invention may be constructed using any of a variety of methods, including, but not limited to, the reverse genetics system described in more detail in He et al. (Virology; 237(2):249-60, 1997). PIV5 encodes eight viral proteins. Nucleocapsid protein (NP), phosphoprotein (P) and large RNA polymerase (L) protein are important for transcription and replication of the viral RNA genome. The V protein plays important roles in viral pathogenesis as well as viral RNA synthesis. The fusion (F) protein, a glycoprotein, mediates both cell-to-cell and virus-to-cell fusion in a pH-independent manner that is essential for virus entry into cells. The structures of the F protein have been determined and critical amino acid residues for efficient fusion have been identified. The hemagglutinin-neuraminidase (HN) glycoprotein is also involved in virus entry and release from the host cells. The matrix (M) protein plays an important role in virus assembly and budding. The hydrophobic (SH) protein is a 44-residue hydrophobic integral membrane protein and is oriented in membranes with its N terminus in the cytoplasm. For reviews of the molecular biology of paramyxoviruses see, for example, Whelan et al., 2004, Curr Top Microbiol Immunol; 283:61-119; and Lamb & Parks, (2006). Paramyxoviridae: the viruses and their replication. In Fields Virology, 5th edn, pp. 1449-1496. Edited by D. M. Knipe & P. M. Howley. Philadelphia, Pa.: Lippincott Williams & Wilkins.
Previously, recombinant PIV5 viruses expressing foreign genes from numerous pathogens, including Influenza, Rabies, Respiratory Syncytial Virus, Tuberculosis, Burkholderia, and MERS-CoV have been generated and tested as vaccine candidates (Li, Z., et al., J Virol, 87(1):354 (2013); Chen, Z., et al., J Virol, 87(6): 2986 (2013); Wang, D., et al., J Virol, 91(11) (2017); Chen, Z., et al., Vaccine, 33(51):7217 (2015); Lafontaine, E. R., et al., Vaccine X., 1:100002 (2018); Li, K., et al., mBio, 11(2) (2020)). Because it actively replicates in the respiratory tract following intranasal immunization, PIV5-vectored vaccines can generate mucosal immunity that includes antigen-specific IgA antibodies and long-lived IgA plasma cells (Wang, D., et al., J Virol, 91(11) (2017). Xiao, P., et al., Front Immunol., 12:623996 (2021)). Recently a PIV5-vectored vaccine expressing the spike protein from SARS-CoV-2 Wuhan (WA1; CVXGA1) has been shown to be efficacious in mice and ferrets. A single, intranasal dose of CVXGA1 induced WA1-neutralizing antibodies and protected K18-hACE2 mice against lethal infection with SARS-CoV-2 WA1. Furthermore, a single, intranasal dose of CVXGA1 protected ferrets from infection with SARS-CoV-2 WA1 and blocked transmission to cohoused naïve ferrets (An, D., et al., Sci Adv, 7(27) (2021)). While these studies determined its efficacy against SARS-CoV-2 WA1, further studies were necessary to establish its efficacy against SARS-CoV-2 variants.
1. PIV5-Based Vaccine Vectors Encoding the SARS-CoV-2 Spike (S) Protein
With the PIV5-based vaccine vectors of the present invention, a heterologous nucleotide sequence encoding the spike (S) protein of a coronavirus, including, but not limited to, the S protein of SARS-CoV-2, is inserted in the PIV5 genome. Coronavirus entry into host cells is mediated by the transmembrane S glycoprotein (Tortorici and Veesler, 2019, Adv Virus Res; 105:93-116). As the coronavirus S glycoprotein is surface-exposed and mediates entry into host cells, it is the main target of neutralizing antibodies upon infection and the focus of therapeutic and vaccine design. The spike S protein of SARS-CoV-2 is composed of two subunits, S1 and S2. The S1 subunit contains a receptor-binding domain that recognizes and binds to the host receptor angiotensin-converting enzyme 2, while the S2 subunit mediates viral cell membrane fusion by forming a six-helical bundle via the two-heptad repeat domain. (Huang et al., 2020, Acta Pharmacol Sinica; 0:1-9 (available on the worldwide web at doi.org/10.1038/s41401-020-0485-4).
The total length of SARS-CoV-2 S is 1273 amino acids (aa) and consists of a signal peptide (amino acids 1-13) located at the N-terminus, the S1 subunit (14-685 residues), and the S2 subunit (686-1273 residues); the last two regions are responsible for receptor binding and membrane fusion, respectively. In the S1 subunit, there is an N-terminal domain (14-305 residues) and a receptor-binding domain (RBD, 319-541 residues); the fusion peptide (FP) (788-806 residues), heptapeptide repeat sequence 1 (HR1) (912-984 residues), HR2 (1163-1213 residues), TM domain (1213-1237 residues), and cytoplasm domain (1237-1273 residues) comprise the S2 subunit (Xia et al., 2020, Cell Mol Immunol; 17:765-7).
In some PIV5-based vaccine vectors of the present invention, the heterologous nucleotide sequence encoding the spike (S) protein of a coronavirus, including, but not limited to, the S protein of SARS-CoV-2, has been modified so that the cytoplasmic tail of the coronavirus S protein has been replaced with the cytoplasmic tail of the fusion (F) protein of PIV5. An example of such a PIV5 construct includes the PIV5 construct CVX-GA1, also referred to herein as CVXGA1, CVX-UGA1, pDA27, or DA27. CVXGA1, recombinant PIV5 expressing S from SARS-CoV-2 WA1, is currently under phase 1 clinical trial in the US (ClinicalTrials.gov. Phase 1 Study of Intranasal PIV5-vectored COVID-19 Vaccine Expressing SARS-CoV-2 Spike Protein in Healthy Adults (CVXGA1-001) 2021).
In some PIV5-based vaccine vectors of the present invention, the heterologous nucleotide sequence encoding the coronavirus S protein, including but not limited to, the S protein of SARS-CoV-2, has been modified so that the S protein includes an amino acid substitution at amino acid residue W886 and/or F888. In some aspects, the amino acid substitution at amino acid residue W886 includes a substitution of tryptophan (W) to arginine (R) and/or the amino acid substitution at amino acid residue W888 includes a substitution of phenylalanine (F) to arginine (R).
In some PIV5-based vaccine vectors of the present invention, the heterologous nucleotide sequence encoding the spike (S) protein of a coronavirus, including, but not limited to, the S protein of SARS-CoV-2, includes both a modification so that the cytoplasmic tail of the coronavirus S protein has been replaced with the cytoplasmic tail of the fusion (F) protein of PIV5 and includes an amino acid substitution at amino acid residue W886 and/or F888. In some aspects, the amino acid substitution at amino acid residue W886 includes a substitution of tryptophan (W) to arginine (R) and/or the amino acid substitution at amino acid residue W888 includes a substitution of phenylalanine (F) to arginine (R). An example of such a PIV5 construct includes the PIV5 construct CVX-GA2, also referred to herein as CVXGA2 or CVX-UGA2.
The heterologous nucleotide sequence encoding the coronavirus S protein, including but not limited to the S protein of SARS-CoV-2, may be inserted in any of a variety of locations in the PIV5 genome.
In some embodiments, the heterologous nucleotide sequence encoding the coronavirus S protein, including but not limited to the S protein of SARS-CoV-2, may be inserted between the small hydrophobic protein (SH) gene and the hemagglutinin-neuraminidase (HN) gene of the PIV5 genome.
The heterologous nucleotide sequence encoding the coronavirus S protein, including but not limited to the S protein of SARS-CoV-2, may be inserted between the hemagglutinin-neuraminidase (HN) and large RNA polymerase protein (L) gene of the PIV5 genome. In some embodiments, the heterologous nucleotide sequence is not inserted at a location between the hemagglutinin-neuraminidase (HN) and large RNA polymerase protein (L) gene of the PIV5 genome. In some embodiments, the heterologous nucleotide sequence is inserted at a location other than between the hemagglutinin-neuraminidase (FIN) and large RNA polymerase protein (L) gene of the PIV5 genome.
The heterologous nucleotide sequence encoding the coronavirus S protein, including but not limited to the S protein of SARS-CoV-2, may be inserted between the nucleocapsid protein (NP) gene and the V/P gene of the PIV5 genome.
The heterologous nucleotide sequence encoding the coronavirus S protein, including but not limited to the S protein of SARS-CoV-2, may be inserted between the M gene and the F gene of the PIV5 genome.
The heterologous nucleotide sequence encoding the coronavirus S protein, including but not limited to the S protein of SARS-CoV-2, may be inserted between the F gene and the SH gene of the PIV5 genome.
The heterologous nucleotide sequence encoding the coronavirus S protein, including but not limited to the S protein of SARS-CoV-2, may be inserted between the VP gene and the matrix protein (M) gene of the PIV5 genome.
The heterologous nucleotide sequence encoding the coronavirus S protein, including but not limited to the S protein of SARS-CoV-2, may be inserted between the leader sequence and the nucleocapsid protein (NP) gene of the PIV5 genome.
The heterologous nucleotide sequence encoding the coronavirus S protein, including but not limited to the S protein of SARS-CoV-2, may be inserted immediately downstream of the leader sequence of the PIV5 genome.
The heterologous nucleotide sequence encoding the coronavirus S protein, including but not limited to the S protein of SARS-CoV-2, may be inserted to replace all or part of a PIV5 gene within the PIV5 genome. For example, the heterologous nucleotide sequence may replace the F, HN, or SH gene of the PIV5 genome. A heterologous nucleotide sequence may be inserted within a PIV5 gene, resulting in the expression of a chimeric polypeptide. For example, the heterologous nucleotide sequence may be inserted within the SH gene nucleotide sequence, within the NP gene nucleotide sequence, within the V/P gene nucleotide sequence, within the M gene nucleotide sequence, within the F gene nucleotide sequence, within the HN gene nucleotide sequence, and/or within the L gene nucleotide sequence of a PIV5 genome.
The heterologous nucleotide sequence encoding the coronavirus S protein may be produced by inserting the coronavirus S protein gene from different variants into PIV5 canine parainfluenza virus (CPI) vector: CVXGA1, 3, 4, 5, 6, 13, 14 and 16.2. PIV5-based vaccine vectors encoding the SARS-CoV-2 spike (S) and nucleocapsid (N) proteins
PIV5 canine parainfluenza virus (CPI) vaccine vectors encoding SARS-COV-2 variants of concern or variants of interest are disclosed herein. The PIV5-based vaccine vectors may comprise inserting the Spike (S) protein gene from different variants into PIV5 CPI vector: CVXGA1, 3, 4, 5, 6, 13, 14 and 16.
To improve vaccine efficacy, the SARS-CoV-2 nucleocapsid (N) may be inserted between the HN and L gene junction in addition to the SARS-CoV-2 spike (S) inserted at the SH and HN junction. In studies disclosed herein, the expression of both the S and N proteins of SARS-CoV-2 have been shown to offer additional protection for the vaccine especially the cellular immune responses offered by the N protein.
Since the CPI-S+N has reduced titer/yield, the construct may be further modified by moving the SARS-CoV-2 N protein gene to the PIV5 CPI SH gene location as SH deletion has been shown not impacting virus growth. CVXGA7 was constructed to have the N and S genes from SARS-CoV-2 inserted into the CPIΔSH backbone to produce CPIΔSH-N+S.
The mutations in the V/P gene, S157F and S308A, have been shown previously to increase viral polymerase activities and improve viral titer/yield (Timani K A, Sun D, Sun M, et al. A single amino acid residue change in the P protein of parainfluenza virus 5 elevates viral gene expression. J Virol. 2008; 82(18):9123-9133. doi:10.1128/JVI.00289-08; Sun D, Luthra P, Li Z, He B. PLK1 down-regulates parainfluenza virus 5 gene expression. PLoS Pathog., 5(7):e1000525 (2009)). Mutations S157F and S308A are herein introduced into the CPI V/P gene to produce CVXGA10 (CPI-S-PLK) and CVXGA12 (CPIΔSH-S-PLK) viruses.
A PIV5 viral vaccine of the present invention may also have a mutation, alteration, or deletion in one or more of these eight proteins of the PIV5 genome. For example, a PIV5 viral expression vector may include one or more mutations, including, but not limited to any of those described herein. In some aspects, a combination of two or more (two, three, four, five, six, seven, or more) mutations may be advantageous and may demonstrated enhanced activity.
A mutation includes, but is not limited to, a mutation of the V/P gene, a mutation of the shared N-terminus of the V and P proteins, a mutation of residues 26, 32, 33, 50, 102, and/or 157 of the shared N-terminus of the V and P proteins, a mutation lacking the C-terminus of the V protein, a mutation lacking the small hydrophobic (SH) protein, a mutation of the fusion (F) protein, a mutation of the phosphoprotein (P), a mutation of the large RNA polymerase (L) protein, a mutation incorporating residues from canine parainfluenza virus, and/or a mutation that enhances syncytial formation.
A mutation may include, but is not limited to, rPIV5-V/P-CPI-, rPIV5-CPI-, rPIV5-CPI+, rPIV5V ΔC, rPIV-Rev, rPIV5-RL, rPIV5-P-S157A, rPIV5-P-S308A, rPIV5-L-A1981D and rPIV5-F-5443P, rPIV5-MDA7, rPIV5 ΔSH-CPI-, rPIV5 ΔSH-Rev, and combinations thereof.
PIV5 can infect cells productively with little cytopathic effect (CPE) in many cell types. In some cell types, PIV5 infection causes formation of syncytia, i.e., fusion of many cells together, leading to cell death. A mutation may include one or more mutations that promote syncytia formation (see, for example Paterson et al., 2000, Virology; 270:17-30).
The V protein of PIV5 plays a critical role in blocking apoptosis induced by virus. Recombinant PIV5 lacking the conserved cysteine-rich C-terminus (rPIV5V ΔC) of the V protein induces apoptosis in a variety of cells through an intrinsic apoptotic pathway, likely initiated through endoplasmic reticulum (ER)-stress (Sun et al., 2004, J Virol; 78:5068-5078). Mutant recombinant PIV5 with mutations in the N-terminus of the V/P gene products, such as rPIV5-CPI-, also induce apoptosis (Wansley and Parks, 2002, J Virol; 76:10109-10121). A mutation includes, but is not limited to, rPIV5 ΔSH, rPIV5-CPI-, rPIV5VΔC, and combinations thereof.
In some embodiments, PIV5-based vaccine vectors of the present invention are chosen from Table 4.
Also included in the present invention are virions and infectious viral particles that include a PIV5 genome including a heterologous nucleotide sequence encoding a coronavirus S protein, including but not limited to the S protein of SARS-CoV-2.
Also included in the present invention are compositions including one or more of the PIV5 viral constructs or virions, as described herein. Such a composition may include a pharmaceutically acceptable carrier. As used, a pharmaceutically acceptable carrier refers to one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal. Such a carrier may be pyrogen free. The present invention also includes methods of making and using the viral vectors and compositions described herein.
The compositions of the present disclosure may be formulated in pharmaceutical preparations in a variety of forms adapted to the chosen route of administration. One of skill will understand that the composition will vary depending on mode of administration and dosage unit.
The agents of this invention can be administered in a variety of ways, including, but not limited to, intravenous, topical, oral, intranasal, subcutaneous, intraperitoneal, intramuscular, and intratumor deliver. In some aspects, the agents of the present invention may be formulated for controlled or sustained release. One advantage of intranasal immunization is the potential to induce a mucosal immune response.
IV. PIV5 Vaccine Virus Sequences
The vaccine virus sequences from the constructs in Table 1 are listed below. The inserted N of SARS-CoV-2 sequences are lower cased and underlined and the inserted S of SARS-CoV-2 sequences are in the lower cased sequences:
1. CVL34
The nucleic acid sequence for CVL34 is:
cccaggattactttcggaggaccaagcgatagcaccgggagcaac
cagaatggagagcggagcggagcaagatccaagcagagacggccc
cagggcctgccaaacaataccgcatcctggttcaccgccctgaca
cagcacggcaaggaggacctgaagtttccaaggggacagggagtg
cctatcaacaccaatagctcccctgacgatcagatcggctactat
aggagggcaacaaggagaatcaggggaggcgacggcaagatgaag
gatctgagcccacgctggtacttctactatctgggaaccggacct
gaggcaggcctgccatatggcgccaacaaggacggaatcatctgg
gtggcaaccgagggcgccctgaacacaccaaaggatcacatcggc
acaagaaatcccgccaacaatgcagcaatcgtgctgcagctgcca
cagggaaccacactgcccaagggcttttacgcagagggctctcgg
ggaggcagccaggcatctagcagatcctctagccggagcagaaac
tcctctaggaattccaccccaggaagctccaggggcacatcccct
gcccgcatggcaggaaacggaggcgacgccgccctggccctgctg
ctgctggatcgcctgaatcagctggagtccaagatgtctggcaag
ggacagcagcagcagggacagaccgtgacaaagaagtccgccgcc
gaggcctctaagaagccaaggcagaagcgcaccgccacaaaggcc
tacaacgtgacccaggccttcggcaggcgcggaccagagcagaca
cagggcaattttggcgaccaggagctgatcaggcagggaaccgat
tataagcactggcctcagatcgcccagttcgccccatctgccagc
gccttctttggcatgtctagaatcggcatggaggtgacccccagc
ggcacatggctgacctacacaggcgccatcaagctggacgataag
gaccctaacttcaaggatcaggtcatcctgctgaacaagcacatc
gacgcctataagacctttccccctacagagcccaagaaggacaag
aagaagaaggccgatgagacacaggccctgcctcagaggcagaag
aagcagcagaccgtgacactgctgccagccgccgatctggacgat
ttctccaaacagctgcagcagagcatgtccagtgccgactccacc
caggcttgatgaTGAGGTACCTGCGGCCGCCTAAGGTCGACTCAT
2. CVL44
The nucleic acid sequence for CVL44 is:
3. CVL48
The nucleic acid sequence for CVL48 is:
4. CVL49
The nucleic acid sequence for CVL49 is:
5. CVL50
The nucleic acid sequence for CVL50 is:
6. CVL52
The nucleic acid sequence for CVL52 is:
accagaggaacgcacccaggattactttcggaggaccaagcgatagcaccgggagcaaccagaatgg
agagcggagcggagcaagatccaagcagagacggccccagggcctgccaaacaataccgcatcct
ggttcaccgccctgacacagcacggcaaggaggacctgaagtttccaaggggacagggagtgccta
tcaacaccaatagctcccctgacgatcagatcggctactataggagggcaacaaggagaatcaggg
gaggcgacggcaagatgaaggatctgagcccacgctggtacttctactatctgggaaccggacc
tgaggcaggcctgccatatggcgccaacaaggacggaatcatctgggtggcaaccgagggcgccctg
aacacaccaaaggatcacatcggcacaagaaatcccgccaacaatgcagcaatcgtgctgcagc
tgccacagggaaccacactgcccaagggcttttacgcagagggctctcggggaggcagccaggcatct
agcagatcctctagccggagcagaaactcctctaggaattccaccccaggaagctccaggggcacatcc
cctgcccgcatggcaggaaacggaggcgacgccgccctggccctgctgctgctggatcgcctgaatcag
ctggagtccaagatgtctggcaagggacagcagcagcagggacagaccgtgacaaagaagtccgccgc
cgaggcctctaagaagccaaggcagaagcgcaccgccacaaaggcctacaacgtgacccaggcctt
cggcaggcgcggaccagagcagacacagggcaattttggcgaccaggagctgatcaggcaggga
accgattataagcactggcctcagatcgcccagttcgccccatctgccagcgccttctttggcatgtcta
gaatcggcatggaggtgacccccagcggcacatggctgacctacacaggcgccatcaagctggac
gataaggaccctaacttcaaggatcaggtcatcctgctgaacaagcacatcgacgcctataagaccttt
ccccctacagagcccaagaaggacaagaagaagaaggccgatgagacacaggccctgcctcagaggcagaa
gaagcagcagaccgtgacactgctgccagccgccgatctggacgatttctccaaacagctgcagcagag
catgtccagtgccgactccacccaggcttgaCGTACGACCTGCTATAGGCTATCCACTGCATCATCTC
7. CVL53
The nucleic acid sequence for CVL53 is:
cagaaccagaggaacgcacccaggattactttcggaggaccaagcgatagcaccgggagcaaccagaat
ggagagcggagcggagcaagatccaagcagagacggccccagggcctgccaaacaataccgcatcctggt
tcaccgccctgacacagcacggcaaggaggacctgaagtttccaaggggacagggagtgcctatcaac
accaatagctcccctgacgatcagatcggctactataggagggcaacaaggagaatcaggggaggcgac
ggcaagatgaaggatctgagcccacgctggtacttctactatctgggaaccggacctgaggcaggcc
atcacatcggcacaagaaatcccgccaacaatgcagcaatcgtgctgcagctgccacagggaaccacac
tgcccaagggcttttacgcagagggctctcggggaggcagccaggcatctagcagatcctctagccgg
agcagaaactcctctaggaattccaccccaggaagctccaggggcacatcccctgcccgcatggca
ggaaacggaggcgacgccgccctggccctgctgctgctggatcgcctgaatcagctggagtccaagatg
tctggcaagggacagcagcagcagggacagaccgtgacaaagaagtccgccgccgaggcctctaagaa
gccaaggcagaagcgcaccgccacaaaggcctacaacgtgacccaggccttcggcaggcgcggacc
agagcagacacagggcaattttggcgaccaggagctgatcaggcagggaaccgattataagcactggc
ctcagatcgcccagttcgccccatctgccagcgccttctttggcatgtctagaatcggcatggaggtga
cccccagcggcacatggctgacctacacaggcgccatcaagctggacgataaggaccctaacttcaa
ggatcaggtcatcctgctgaacaagcacatcgacgcctataagacctttccccctacagagcccaaga
aggacaagaagaagaaggccgatgagacacaggccctgcctcagaggcagaagaagcagcagaccgt
gacactgctgccagccgccgatctggacgatttctccaaacagctgcagcagagcat
gtccagtgccgactccacccaggcttgaGCTAGCCTCCTGCCATACTTCCTACTCACA
8. CVL54
The nucleic acid sequence for CVL54 is:
cagaaccagaggaacgcacccaggattactttcggaggaccaagcgatagcaccgggagcaaccagaatg
gagagcggagcggagcaagatccaagcagagacggccccagggcctgccaaacaataccgcatcctggttc
accgccctgacacagcacggcaaggaggacctgaagtttccaaggggacagggagtgcctatcaacaccaa
tagctcccctgacgatcagatcggctactataggagggcaacaaggagaatcaggggaggcgacggcaa
gatgaaggatctgagcccacgctggtacttctactatctgggaaccggacctgaggcaggcctgc
catatggcgccaacaaggacggaatcatctgggtggcaaccgagggcgccctgaacacaccaaagga
tcacatcggcacaagaaatcccgccaacaatgcagcaatcgtgctgcagctgccacagggaaccacactg
cccaagggcttttacgcagagggctctcggggaggcagccaggcatctagcagatcctctagccggagc
agaaactcctctaggaattccaccccaggaagctccaggggcacatcccctgcccgcatggcaggaaa
cggaggcgacgccgccctggccctgctgctgctggatcgcctgaatcagctggagtccaagatgtc
tggcaagggacagcagcagcagggacagaccgtgacaaagaagtccgccgccgaggcctctaagaa
gccaaggcagaagcgcaccgccacaaaggcctacaacgtgacccaggccttcggcaggcgcggacca
gagcagacacagggcaattttggcgaccaggagctgatcaggcagggaaccgattataagcact
ggcctcagatcgcccagttcgccccatctgccagcgccttctttggcatgtctagaatcggcatgg
aggtgacccccagcggcacatggctgacctacacaggcgccatcaagctggacgataaggaccct
aacttcaaggatcaggtcatcctgctgaacaagcacatcgacgcctataagacctttccccctacaga
gcccaagaaggacaagaagaagaaggccgatgagacacaggccctgcctcagaggcagaagaagcag
cagaccgtgacactgctgccagccgccgatctggacgatttctccaaacagctgcagcagagcatgtc
cagtgccgactccacccaggcttgaGCTAGCCTCCTGCCATACTTCCTACTCACA
9. CVL55
The nucleic acid sequence for CVL55 is:
10. CVL58
The nucleic acid sequence for CVL58 is:
11. CVL59
The nucleic acid sequence for CVL58 is:
12. CVL60
The nucleic acid sequence for CVL60 is:
13. CVL64
The nucleic acid sequence for CVL64 is:
15. CVL65
The nucleic acid sequence for CVL65 is:
cagaaccagaggaacgcacccaggattactttcggaggaccaagcgatagcaccgggagcaaccagaatg
gagagcggagcggagcaagatccaagcagagacggccccagggcctgccaaacaataccgcatcctgg
ttcaccgccctgacacagcacggcaaggaggacctgaagtttccaaggggacagggagtgcctatcaa
caccaatagctcccctgacgatcagatcggctactataggagggcaacaaggagaatcaggggaggcgac
ggcaagatgaaggatctgagcccacgctggtacttctactatctgggaaccggacctgaggcaggcct
gccatatggcgccaacaaggacggaatcatctgggtggcaaccgagggcgccctgaacacaccaaagga
tcacatcggcacaagaaatcccgccaacaatgcagcaatcgtgctgcagctgccacagggaaccacact
gcccaagggcttttacgcagagggctctcggggaggcagccaggcatctagcagatcctctagccggagc
agaaactcctctaggaattccaccccaggaagctccaggggcacatcccctgcccgcatggcaggaa
acggaggcgacgccgccctggccctgctgctgctggatcgcctgaatcagctggagtccaagatgtctgg
caagggacagcagcagcagggacagaccgtgacaaagaagtccgccgccgaggcctctaagaagccaagg
cagaagcgcaccgccacaaaggcctacaacgtgacccaggccttcggcaggcgcggaccagagca
gacacagggcaattttggcgaccaggagctgatcaggcagggaaccgattataagcactggcctca
gatcgcccagttcgccccatctgccagcgccttctttggcatgtctagaatcggcatggaggtgac
ccccagcggcacatggctgacctacacaggcgccatcaagctggacgataaggaccctaacttcaagg
atcaggtcatcctgctgaacaagcacatcgacgcctataagacctttccccctacagagcccaagaa
ggacaagaagaagaaggccgatgagacacaggccctgcctcagaggcagaagaagcagcagaccgtga
cactgctgccagccgccgatctggacgatttctccaaacagctgcagcagagcatgtccagtgc
cgactccacccaggcttgaGCTAGCCTCCTGCCATACTTCCTACTCACATCATA
16. CVL67
The nucleic acid sequence for CVL67 is:
17. CVL80
The nucleic acid sequence for CVL80 is:
18. CVL86
The nucleic acid sequence for CVL86 is:
19. CVL108
The nucleic acid sequence for CVL108 is:
20. CVL111
The nucleic acid sequence for CVL111 is:
21. CVL113
The nucleic acid sequence for CVL116 is:
Also included in the present invention are methods of making and using PIV5 viral expression vectors, including, but not limited to any of those described herein.
For example, the present invention includes methods of expressing a coronavirus S protein, including but not limited to the S protein of SARS-CoV-2, in a cell by contacting or infection the cell with a PIV5 viral expression vector, viral particle, or composition as described herein.
The present invention includes methods of inducing an immune response in a subject to a coronavirus S protein, including but not limited to the S protein of SARS-CoV-2, by administering a viral expression vector, viral particle, or composition as described herein to the subject. The immune response may include a humoral immune response and/or a cellular immune response. The immune response may enhance an innate and/or adaptive immune response.
The present invention includes methods expressing a heterologous coronavirus S protein, including but not limited to the S protein of SARS-CoV-2, in a subject by administering a viral expression vector, viral particle, or composition as described herein to the subject.
The present invention includes methods expressing a heterologous coronavirus S protein, including but not limited to the S protein of SARS-CoV-2 alpha, gamma, delta, and omicron strains, in a subject by administering a viral expression vector, viral particle, or composition as described herein to the subject.
The disclosure can be used in gene therapy and/or therapeutic approaches for the treatment of disease which involve the increase or decrease of a nucleotide sequence of interest in a host-cell. In these embodiments, the expressible heterologous nucleotide sequence may be derived from a mammalian genome. It may be particularly useful in some embodiments to have the expressible heterologous nucleotide sequence derived from a human genome, wherein expression of the wild-type RNA and/or protein can produce therapeutic effects in a patient. For example, the expressible heterologous nucleotide sequence can encode CFTR, NeuroD1, Cas9 and Guide RNAs, or any other such sequence. In other embodiments, the heterologous nucleotide sequence encodes a secreted protein.
In other embodiments, the expressible heterologous nucleotide sequence responds to positive selection stimuli. In other embodiments, the expressible heterologous nucleotide sequence also responds to negative selection stimuli. In further embodiments, it may be useful for the polynucleotide sequences to further comprise a reporter gene. For example, the report gene can be a luciferase or green fluorescent protein.
In some embodiments, AVLP expresses one or more nucleotide sequences (e.g., siRNAs) that modify the translation and/or transcription of a host-cell nucleotide sequence of interest within a host cell. In some embodiments, transcription and/or translation of the expressible heterologous nucleotide sequence is modified so that its nucleotide sequence is codon degenerated with respect to the endogenous gene in a cell. Additionally, the expressible heterologous nucleotide sequence can be modified so that it co-expresses inhibitory or silencing sequences capable of inhibiting or silencing a host-cell nucleotide sequence of interest within a host cell.
In other embodiments, the expressible heterologous nucleotide of interest generates a product that stabilizes host-cell RNA nucleotide sequences. Such a product can be inducible or continually expressed. For example, the 3′ RhoB untranslated region (UTR) can stabilize target RNAs that express either toxic proteins or other proteins of interest in response to serum. Another example is linking the eotaxin 3′ untranslated region to the target gene of interest, which normally has a low half-life, but is stabilized with the addition of TNF-alpha and IL-4 to the cells. Alternatively, sequences contained in 16 mer sequence in the 5′ coding region of CYP2E 1 and CYP2B 1 mRNA destabilizes target RNAs in the presence of insulin. Upon the removal of insulin the target RNAs are stabilized and the proteins can be expressed (Trong et al., Biochem J., Dec. 23, 2004).
Further non-limiting examples of expressible heterologous sequences that can be used in the invented compositions and methods include sequences can produce proteins, including, for example, e.g., interferons (alpha, beta, gamma, epsilon), erythropoietin, Factor VIII, clotting factors, antibodies and fragments thereof (e.g., including single chain, Fab, and humanized), insulin, chemokines, cytokines, growth factors, angiogenesis modulatory factors, apoptosis modulatory factors, e.g., Growth Factors, including, e.g., Amphiregulin, B-lymphocyte stimulator, Interleukin 16 (IL16), Thymopoietin, TRAIL, Apo-2, Pre B cell colony enhancing factor, Endothelial differentiation-related factor 1 (EDF1), Endothelial monocyte activating polypeptide II, Macrophage migration inhibitory factor MIF, Natural killer cell enhancing factor (NKEFA), Bone morphogenetic protein 8 (osteogenic protein 2), Bone morphogenic protein 6, Connective tissue growth factor (CTGF), CGI-149 protein (neuroendocrine differentiation factor), Cytokine A3 (macrophage inflammatory protein 1-alpha), Glialblastoma cell differentiation-related protein (GBDR1), Hepatoma-derived growth factor, Neuromedin U-25 precursor, any tumor gene, oncogene, proto-oncogene or cell modulating gene (which can be found at condor.bcm.tmc.edu/oncogene), Vascular endothelial growth factor (VEGF), Vascular endothelial growth factor B (VEGF-B), T-cell specific RANTES precursor, Thymic dendritic cell-derived factor 1; Receptors, such as Activin A receptor, type II (ACVR2), β-signal sequence receptor (SSR2), CD14 monocyte LPS receptor, CD36 (collagen type 1/thrombospondin receptor)-like 2, CD44R (Hermes antigen gp90 homing receptor), G protein coupled receptor 9, Chemokine CXC receptor 4, Colony stimulating factor 2 receptor β(CSF2RB), FLT-3 receptor tyrosine kinase, Similar to transient receptor potential C precursor, Killer cell lectin-like receptor subfamily B, Low density lipoprotein receptor gene, low-affinity Fc-gamma receptor IIC, MCP-1 receptor, Monocyte chemoattractant protein 1 receptor (CCR2), Nuclear receptor subfamily 4, group A, member 1, Orphan G protein-coupled receptor GPRC5D, Peroxisome proliferative activated receptor gamma, Pheromore related-receptor (rat), Vasopressin-activated calcium mobilizing putative receptor, Retinoic×receptor, Toll-like receptor 6, Transmembrane activator and CAML interactor (TACI), B cell maturation peptide (BCMA), CSF-1 receptor, Interferon (α, β and gamma) receptor 1 (IFNAR1). Pathways that can be modulated to increase antibody production include, e.g., ubiquitin/proteosome; telomerase; FGFR3; and Mcd-1, etc.
In certain embodiments of the disclosure, AVLP compositions can be utilized to prepare antigenic preparations that be used as vaccines. Any suitable antigen(s) can be prepared in accordance with the disclosure, including antigens obtained from prions, viruses, mycobacterium, protozoa (e.g., Plasmodium falciparum (malaria)), trypanosomes, bacteria (e.g., Streptococcus, Neisseria, etc.), etc.
Host cells can be transfected with single AVLP particles containing one or more heterologous polynucleotide sequences, or with a plurality of AVLP particles, where each comprises the same or different heterologous polynucleotide sequence(s). For example, a multi-subunit antigen (including intracellular and cell-surface multi-subunit components) can be prepared by expressing the individual subunits on separate vectors, but infecting the same host cell with all the vectors, such that assembly occurs within the host cell.
Vaccines often contain a plurality of antigen components, e.g., derived from different proteins, and/or from different epitopic regions of the same protein. For example, a vaccine against a viral disease can comprise one or more polypeptide sequences obtained from the virus which, when administered to a host, elicit an immunogenic or protective response to viral challenge.
As mentioned, the disclosure can also be utilized to prepare polypeptide multimers, e.g., where an antigenic preparation is produced which is comprised of more than one polypeptide. For instance, virus capsids can be made up of more than one polypeptide subunit. By transducing a host cell with vectors carrying different viral envelope sequences, the proteins, when expressed in the cell, can self-assemble into three-dimensional structures containing more than one protein subunit (e.g., in their native configuration).
In further embodiments, the expressible heterologous nucleotide sequence is derived from another virus, other than PIV5. For example, the heterologous nucleotide sequence may encode (from any strain) influenza HA, RSV F, HIV Gag and/or Env, etc. Such embodiments can be useful for developing vaccines and/or methods of vaccination. The examples given here are non-limiting, as it will be understood by those in the art that nucleotide sequences from a variety of pathogenic agents (including also bacteria, parasites, etc.) may be desirable to use for an AVLP vaccine composition and/or method of vaccination.
Examples of viruses to which vaccines can be produced in accordance with the disclosure include, e.g., coronaviruses, orthomyxoviruses, influenza virus A (including all strains varying in their HA and NA proteins, such as (non-limiting examples) H1N1, H1N2, H2N2, H3N2, H7N7, and H3N8); influenza B, influenza C, thogoto virus (including Dhori, Batken virus, SiAR 126 virus), and isavirus (e.g., infectious salmon anemia virus) and the like. These include coronaviruses isolated or transmitted from all species types, including isolates from invertebrates, vertebrates, mammals, humans, non-human primates, monkeys, pigs, cows, and other livestock, birds, domestic poultry such as turkeys, chickens, quail, and ducks, wild birds (including aquatic and terrestrial birds), reptiles, etc. These also include existing strains which have changed, e.g., through mutation, antigenic drift, antigenic shift, recombination, etc., especially strains which have increased virulence and/or interspecies transmission (e.g., human-to-human).
I. Administration by Vaccination
The present invention includes methods of vaccinating a subject by administering a viral expression vector, viral particle, or composition as described herein to the subject.
The disclosure provides vaccines against all coronaviruses, including existing subtypes, derivatives thereof, and recombinants thereof, such as subtypes and recombinants which have the ability to spread from human-to-human. Various isolates have been characterized, especially for SARS-COV-2.
The disclosure also provides methods for producing AVLP compositions. Examples of host cells which can be utilized to produce AVLP compositions, include, any mammalian or human cell line or primary cell. Non-limiting examples include, e.g., 293, HT1080, Jurkat, and SupT1 cells. Other examples are CHO, 293, Hela, Vero, L929, BHK, NIH 3T3, MRC-5, BAE-1, HEP-G2, NSO, U937, Namalwa, HL60, WEHI 231, YAC 1, U 266B1, SH-SY5Y, CHO, e.g., CHO-K1 (CCL-61), 293 (e.g., CRL-1573). Cells are cultured under conditions effective to produce transfection and expression. Such conditions include, e.g., the particular milieu needed to achieve protein production. Such a milieu, includes, e.g., appropriate buffers, oxidizing agents, reducing agents, pH, co-factors, temperature, ion concentrations, suitable age and/or stage of cell (such as, in particular part of the cell cycle, or at a particular stage where particular genes are being expressed) where cells are being used, culture conditions (including cell media, substrates, oxygen, carbon dioxide, glucose and other sugar substrates, serum, growth factors, etc.).
The disclosure also provides various treatment methods involving delivering AVLP to host cells in vivo. In some embodiments, AVLP is delivered into a subject for treating or preventing coronaviruses. In other embodiments, AVLP is delivered into a subject for treating or preventing SARS-COV-2 alpha, delta, omicron strains, or variants thereof or eliciting an immune response to SARS-COV-2 in a subject.
It is contemplated that when used to treat various diseases, the compositions and methods of the disclosure can be combined with other therapeutic agents suitable for the same or similar diseases. Also, two or more embodiments of the disclosure may be also co-administered to generate additive or synergistic effects. When co-administered with a second therapeutic agent, the embodiment of the disclosure and the second therapeutic agent may be simultaneously or sequentially (in any order). Suitable therapeutically effective dosages for each agent may be lowered due to the additive action or synergy.
As a non-limiting example, the disclosure can be combined with other therapies that block inflammation through (e.g., via inhibition, reduction and/or blockage of ILL INFα/β, IL6, TNF, L13, IL23, etc.). In some embodiments, AVLP compositions and methods disclosed herein are useful to enhance the efficacy of vaccines directed to SARS-COV-2 infections. The compositions and methods of the disclosure can be administered to a subject either simultaneously with or before (e.g., 1-30 days before) a reagent (including but not limited to small molecules, antibodies, or cellular reagents) that acts to elicit an immune response (e.g., to treat cancer or an infection). The compositions and methods of the disclosure can be also administered in combination with an anti-tumor antibody or an antibody directed at a pathogenic antigen or allergen.
The pharmaceutical compositions of the invention can be readily employed in a variety of therapeutic or prophylactic applications, e.g., for treating SARS-COV-2 infection or eliciting an immune response to SARS-COV-2 in a subject. In various embodiments, the vaccine compositions can be used for treating or preventing infections caused by a pathogen from which the displayed immunogen polypeptide in the PIV5-based vaccine is derived. Thus, the vaccine compositions of the invention can be used in diverse clinical settings for treating or preventing infections caused by various viruses. As exemplification, a SARS-COV-2 PIV-5-based vaccine composition can be administered to a subject to induce an immune response to SARS-COV-2, e.g., to induce production of broadly neutralizing antibodies to the virus. For subjects at risk of developing an SARS-COV-2 infection, a vaccine composition of the invention can be administered to provide prophylactic protection against viral infection. Therapeutic and prophylactic applications of vaccines derived from the other immunogens described herein can be similarly performed. Depending on the specific subject and conditions, pharmaceutical compositions of the invention can be administered to subjects by a variety of administration modes known to the person of ordinary skill in the art, for example, topical, oral, intranasal, intramuscular, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, or parenteral routes. In some aspects, administration is to a mucosal surface. A vaccine may be administered by mass administration techniques such as by placing the vaccine in drinking water or by spraying the animals' environment. When administered by injection, the immunogenic composition or vaccine may be administered parenterally. Parenteral administration includes, for example, administration by intravenous, subcutaneous, intramuscular, or intraperitoneal injection.
1. Vaccination by Inhalation
In one embodiment, the disclosed PIV-5 vaccine compositions are formulated to allow intranasal administration. Intranasal compositions may comprise an inhalable dry powder pharmaceutical formulation comprising a therapeutic agent, wherein the therapeutic agent is present as a freebase or as a mixture of a salt and a freebase. Pharmaceutical formulations disclosed herein can be formulated as suitable for airway administration, for example, nasal, intranasal, sinusoidal, peroral, and/or pulmonary administration. Typically, formulations are produced such that they have an appropriate particle size for the route, or target, of airway administration. As such, the formulations disclosed herein can be produced so as to be of defined particle size distribution.
For example, the particle size distribution for a salt form of a therapeutic agent for intranasal administration can be between about 5 μm and about 350 More particularly, the salt form of the therapeutic agent can have a particle size distribution for intranasal administration between about 5μ to about 250 μm, about 10 μm to about 200 μm, about 15 μm to about 150 μm, about 20 μm to about 100 μm, about 38 μm to about 100 μm, about 53 μm to about 100, about 53 μm to about 150 μm, or about 20 μm to about 53 μm. The salt form of the therapeutic agent in the pharmaceutical compositions of the invention can a particle size distribution range for intranasal administration that is less than about 200 μm. In other embodiments, the salt form of the therapeutic agent in the pharmaceutical compositions has a particle size distribution that is less than about 150 μm, less than about 100 μm, less than about 53 μm, less than about 38 μm, less than about 20 μm, less than about 10 μm, or less than about 5 μm. The salt form of the therapeutic agent in the pharmaceutical compositions of the invention can have a particle size distribution range for intranasal administration that is greater than about 5 μm, greater than about 10 μm, greater than about 15 μm, greater than about 20 greater than about 38 μm, less than about 53 μm, less than about 70 μm, greater than about 100 or greater than about 150 μm.
Additionally, the salt form of the therapeutic agent in the pharmaceutical compositions of the invention can have a particle size distribution range for pulmonary administration between about 1 μm and about 10 μm. In other embodiments for pulmonary administration, particle size distribution range is between about 1 μm and about 5 or about 2 μm and about 5 In other embodiments, the salt form of the therapeutic agent has a mean particle size of at least 1 μm, at least 2 μm, at least 3 μm, at least 4 μm, at least 5 μm, at least 10 at least 20 μm, at least 25 μm, at least 30 μm, at least 40 μm, at least 50 μm, at least 60 at least 70 μm, at least 80 μm, at least 90 μm, or at least 100 μm.
In some embodiments the disclosed cannabinoid compositions include one or more cannabinoids or pharmaceutically acceptable derivatives or salts thereof, a propellant, an alcohol, and a glycol and/or glycol ether. The alcohol may be a monohydric alcohol or a polyhydric alcohol, and is preferably a monohydric alcohol. Monohydric alcohol has a lower viscosity than a glycol or glycol ether. Accordingly, the composition is able to form droplets of a smaller diameter in comparison to compositions in which the monohydric alcohol is not present. The present inventors have surprisingly found that a specific ratio of monohydric alcohol to glycol or glycol ether results in a composition with a desired combination of both long term stability (for example the composition remains as a single phase for at least a week at a temperature of 2-40° C.) and small droplet size.
2. Pulmonary Compositions
One embodiment provides a formulation and method for treating SARS-COV-2 in the pulmonary system by inhalation or pulmonary administration. The diffusion characteristics of the particular drug formulation through the pulmonary tissues are chosen to obtain an efficacious concentration and an efficacious residence time in the tissue to be treated. Doses may be escalated or reduced or given more or less frequently to achieve selected blood levels. Additionally, the timing of administration of administration and amount of the formulation is preferably controlled to optimize the therapeutic effects of the administered formulation on the tissue to be treated and/or titrate to a specific blood level.
Diffusion through the pulmonary tissues can additionally be modified by various excipients that can be added to the formulation to slow or accelerate the absorption of drugs into the pulmonary tissues. For example, the drug may be combined with surfactants such as the phospholipids, dimyristoylphosphatidyl choline, and of administration dimyristoylphosphatidyl glycerol. The drugs may also be used in conjunction with bronchodilators that can relax the bronchial airways and allow easier entry of the antineoplastic drug to the lung. Albuterol is an example of the latter with many others known in the art. Further, the drug may be complexed with biocompatible polymers, micelle forming structures or cyclodextrins.
Particle size for the aerosolized drug used in the present examples was measured at about 1.0-5.0 μm with a GSD less than about 2.0 for deposition within the central and peripheral compartments of the lung. As noted elsewhere herein particle sizes are selected depending on the site of desired deposition of the drug particles within the respiratory tract.
Aerosols useful in the invention include aqueous vehicles such as water or saline with or without ethanol and may contain preservatives or antimicrobial agents such as benzalkonium chloride, paraben, and the like, and/or stabilizing agents such as polyethyleneglycol.
Powders useful in the invention include formulations of the neat drug or formulations of the drug combined with excipients or carriers such as mannitol, lactose, or other sugars. The powders used herein are effectively suspended in a carrier gas for administration. Alternatively, the powder may be dispersed in a chamber containing a gas or gas mixture which is then inhaled by the patient.
An agent of the present disclosure may be administered at once or may be divided into a number of multiple doses to be administered at intervals of time. For example, agents of the invention may be administered repeatedly, e.g., at least 2, 3, 4, 5, 6, 7, 8, or more times, or may be administered by continuous infusion. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that any concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions and methods.
In some therapeutic embodiments, an “effective amount” of an agent is an amount that results in a reduction of at least one pathological parameter. Thus, for example, in some aspects of the present disclosure, an effective amount is an amount that is effective to achieve a reduction of at least about 10%, at least about 15%, at least about 20%, or at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%, compared to the expected reduction in the parameter in an individual not treated with the agent.
In some aspects, any of the PIV5-based constructs and methods described in WO 2013/112690 and WO 2013/112720 (which is hereby incorporated by reference herein in its entirety) may be used in the present invention.
As used herein, the term “subject” represents an organism, including, for example, a mammal. A mammal includes, but is not limited to, a human, a non-human primate, and other non-human vertebrates. A subject may be an “individual,” “patient,” or “host.” Non-human vertebrates include livestock animals (such as, but not limited to, a cow, a horse, a goat, and a pig), a domestic pet or companion animal, such as, but not limited to, a dog or a cat, and laboratory animals. Non-human subjects also include non-human primates as well as rodents, such as, but not limited to, a rat or a mouse. Non-human subjects also include, without limitation, poultry, horses, cows, pigs, goats, dogs, cats, guinea pigs, hamsters, mink, and rabbits.
As used herein “in vitro” is in cell culture and “in vivo” is within the body of a subject. As used herein, “isolated” refers to material that has been either removed from its natural environment (e.g., the natural environment if it is naturally occurring), produced using recombinant techniques, or chemically or enzymatically synthesized, and thus is altered “by the hand of man” from its natural state.
I. Booster Vaccines
The present disclosure provides for the administration of a booster PIV-5 vaccine for use in such a method for inducing in a human subject an immune response, wherein said subject has previously received a primary vaccination against SARS-COV-2.
The method of booster vaccination according to the disclosure comprises the step of administering the vaccine composition to the subject.
The immune response induced by the vaccine composition of the disclosure or by the method of the disclosure is preferably a humoral response, especially a response comprising the production of neutralizing antibodies against the COVID-19 virus, i.e. a neutralizing antibody response.
Exemplary Embodiments of the present disclosure include, but are not limited to, the following.
Embodiment 1: A viral expression vector comprising a parainfluenza virus 5 (PINS) genome having a heterologous nucleic acid sequence with at least 98% sequence identity to SEQ ID NOs: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80, wherein the viral expression vector expresses a heterologous polypeptide comprising a coronavirus spike (S) and/or nucleocapsid (N) proteins.
Embodiment 2: The viral expression vector of Embodiment 1, wherein the coronavirus S protein is a coronavirus S protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a variant of interest or a variant of concern of SARS-CoV-2 and the coronavirus N protein is the coronavirus N protein of SARS-CoV-2, a variant of interest or a variant of concern of SARS-CoV-2.
Embodiment 3: The viral expression vector of Embodiment 1, wherein the coronavirus S protein is the coronavirus S protein of a SARS-CoV-2 beta variant, a SARS-CoV-2 gamma variant, a SARS-CoV-2 delta variant, or a SARS-CoV-2 omicron variant and the coronavirus N protein is the coronavirus N protein of a SARS-CoV-2 beta variant, a SARS-CoV-2 gamma variant, a SARS-CoV-2 delta variant, or a SARS-CoV-2 omicron variant.
Embodiment 4: The viral expression vector of Embodiment 1, wherein the coronavirus S protein comprises the coronavirus S protein of SARS-CoV-2 and wherein the cytoplasmic tail of the coronavirus S protein has been replaced with the cytoplasmic tail of the fusion (F) protein of PIV5.
Embodiment 5: The viral expression vector of Embodiment 1, wherein the coronavirus S protein of a variant of SARS-CoV-2 has been inserted between the PIV5 small hydrophobic (SH) and hemagglutinin (HN) genes and the coronavirus N protein of a variant of SARS-CoV-2 has been inserted between the PIV5 HN and polymerase (L) genes.
Embodiment 6: The viral expression vector of any one of Embodiments 1, wherein the coronavirus S protein of a variant of SARS-CoV-2 has been inserted between the PIV5 HN and polymerase (L) genes.
Embodiment 7: The viral expression vector of Embodiment 1, wherein the coronavirus S protein is a S protein from a variant of interest or a variant of concern of SARS-CoV-2 and the coronavirus N protein is a N protein from different variant of interest or a variant of concern of SARS-CoV-2.
Embodiment 8: The viral expression vector of Embodiment 1, wherein the PIV5 small hydrophobic (SH) gene has been replaced with the coronavirus N protein of SARS-CoV-2.
Embodiment 9: The viral expression vector of Embodiment 1, wherein the PIV5 genome further comprises one or more mutations comprising a mutation of the V/P gene, a mutation of the shared N-terminus of the V and P proteins, a mutation of residues 26, 32, 33, 50, 102, and/or 157 of the shared N-terminus of the V and P proteins, a mutation lacking the C-terminus of the V protein, a mutation lacking the small hydrophobic (SH) protein, a mutation of the fusion (F) protein, a mutation of the phosphoprotein (P), a mutation of the large RNA polymerase (L) protein, a mutation incorporating residues from canine parainfluenza virus, a mutation inducing apoptosis, or a combination thereof.
Embodiment 10: The viral expression vector of Embodiment 9, wherein the one or more mutations comprise PIV5VΔC, PIV5ΔSH, PIV5-P-S308G, or a combination thereof.
Embodiment 11: The viral expression vector of Embodiment 1, wherein the heterologous polypeptide comprises a CPI V/P gene that contains mutations at amino acid residue S157 or S308, or the combination thereof, wherein serine (S) is substituted with an amino acid residue selected from a group consisting of alanine (A), asparagine (B), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), selenocysteine (U), valine (V), tryptophan (W), tyrosine (Y), and glutamine (Z).
Embodiment 12: The viral expression vector of Embodiment 11, wherein the amino acid substitution at amino acid residue S157 comprises a substitution of serine (S) to phenylalanine (F) and the amino acid substitution at amino acid residue S308 comprises a substitution of serine (S) to alanine (A) or Glycine (G).
Embodiment 13: The viral expression vector of Embodiment 1, wherein a viral particle comprises the viral expression vector.
Embodiment 14: A composition comprising the viral expression vector of Embodiment 1 or the viral particle of Embodiment 13 or a combination thereof.
Embodiment 15: The composition of Embodiment 14, wherein a heterologous coronavirus spike (S) and nucleocapsid (N) proteins are expressed in a cell by contacting the cell with the composition.
Embodiment 16: A method of inducing an immune response in a subject having coronavirus disease 2019 (COVID-19) to coronavirus spike (S) and nucleocapsid (N) proteins, the method comprising administering the composition of Embodiment 14 to the subject, wherein the immune response comprises a humoral immune response and/or a cellular immune response.
Embodiment 17: The method of Embodiment 16, wherein the subject is vaccinated against COVID-19), the method comprising administering the composition to the subject.
Embodiment 18: The method of Embodiment 16, wherein the composition is administered intranasally, intramuscularly, topically, or orally.
Embodiment 19: A method of inducing in a subject an immune response comprising administering a PIV-5 booster vaccine composition comprising a viral expression vector or a viral particle having a PIV5 genome comprising a heterologous nucleic acid sequence with at least 98% sequence identity to SEQ ID NOs: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80, wherein said subject has previously received a primary vaccination against SARS-COV-2.
The term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements.
The words “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.
The terms “comprises” and variations thereof do not have a limiting meaning where these terms appear in the description and claims.
Unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one.
Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.
For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
The description exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.
All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.
The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.
Materials:
The SARS-CoV-2 vaccine candidates generated are described herein.
CVL34 for CVXGA2: The N gene of SARS-CoV-2 Wuhan strain was amplified by PCR, inserted into CPI containing the S gene of SARS-CoV-2 Wuhan strain by Gibson assembly. The recombinant virus was rescued on 293-T serum free cells, which contains the S gene of SARS-CoV-2 Wuhan strain between SH and HN, the N gene of SARS-CoV-2 Wuhan strain between HN and L of CPI.
CVL44 for CVXGA3: The S gene of SARS-CoV-2 Beta variant was amplified by PCR, inserted into CPI backbone by Gibson assembly. The recombinant virus was rescued on 293-T serum free cells, which contains the S gene of SARS-CoV-2 Beta variant between SH and HN of CPI.
CVL48 for CVXGA4: The S gene of SARS-CoV-2 Alpha variant was digested from a plasmid containing the gene generated from Genscript, inserted into CPI backbone by Gibson assembly. The recombinant virus was rescued on 293-T serum free cells, which contains the S gene of SARS-CoV-2 Alpha variant between SH and HN of CPI.
CVL49 for CVXGA5: The S gene of SARS-CoV-2 Gamma variant was digested from a plasmid containing the gene generated from Genscript, inserted into CPI backbone by Gibson assembly. The recombinant virus was rescued on 293-T serum free cells, which contains the S gene of SARS-CoV-2 Gamma variant between SH and HN of CPI.
CVL50 for CVXGA6: The S gene of SARS-CoV-2 Epsilon variant was amplified by PCR, inserted into CPI backbone by Gibson assembly. The recombinant virus was rescued on Vero serum free cells, which contains the S gene of SARS-CoV-2 Epsilon variant between SH and HN of CPI.
CVL52 for CVXGA7: The N gene of SARS-CoV-2 Wuhan strain was amplified by PCR, inserted into CVL44 backbone by Gibson assembly. The recombinant plasmid contains the N gene of SARS-CoV-2 Wuhan strain and the S gene of SARS-CoV-2 Beta variant between F and HN, and lacking the CPI SH gene. The plasmid construct is being generated.
CVL53 for CVXGA8: The N gene of SARS-CoV-2 Wuhan strain was amplified by PCR, inserted into CPI backbone by Gibson assembly. The recombinant virus was rescued on 293-T serum free cells, which contains the N gene of SARS-CoV-2 Wuhan strain between F and HN, and the SH gene deletion of CPI.
CVL54 for CVXGA9: The S gene of SARS-CoV-2 Beta variant was amplified by PCR, inserted into CVL53 backbone by Gibson assembly. The recombinant plasmid contains the N gene of SARS-CoV-2 Wuhan strain between F and HN, the S gene of SARS-CoV-2 Beta variant between HN and L, and the SH gene deletion of the CPI backbone. The plasmid construct is being generated.
CVL55 for CVXGA10: CPI-V/P gene containing the S157F and S308A mutations (PLK mutations) was amplified by PCR, inserted into CVL44 backbone by Gibson assembly. The recombinant virus was rescued on 293-T serum free cells, which contains the S gene of SARS-CoV-2 Beta variant between SH and HN, and the S157F and S308A mutations in CPI-V/P gene.
CVL58 for CVXGA11: CPI with the SH gene deletion (CPIΔSH) was obtained by PCR amplification and Gibson assembly in CVL44 backbone. The recombinant plasmid containing the S gene of SARS-CoV-2 Beta variant between F and HN, and the CPI SH gene deletion, was obtained. Virus rescue is being rescued.
CVL59 for CVXGA12: CPI lacking SH gene (CPIΔSH) was obtained by PCR amplification and Gibson assembly in CVL55 backbone. The recombinant plasmid has been obtained, which contains the S gene of SARS-CoV-2 Beta variant between F and HN, CPI SH gene deletion, and S157F and S308A mutations in CPI-V/P gene.
CVL60 for CVXGA13: The S gene of SARS-CoV-2 Delta variant was digested from a plasmid containing the gene generated from Genscript, then inserted into CPI backbone by Gibson assembly. The recombinant CVGA13 virus was rescued in Vero serum free Vero cells, which contains the S gene of SARS-CoV-2 Delta variant between SH and HN.
CVL64 for CVXGA26: The S gene of SARS-CoV-2 Lambda variant was digested from a plasmid containing the gene generated in-house, then inserted into CPI backbone by Gibson assembly. The recombinant plasmid contains the S gene of SARS-CoV-2 Lambda variant between SH and HN. The plasmid construct is being generated.
CVL65 for CVXGA27: The S gene of SARS-CoV-2 Delta variant was amplified by PCR and inserted into CVL54 backbone by Gibson assembly. The recombinant virus will contain N gene of SARS-CoV-2 Wuhan strain between F and HN, S gene of SARS-CoV-2 Delta variant between HN and L, and lacking SH gene of CPI backbone. The plasmid DNA construct will be generated.
CVL67 for CVXGA28: The S gene of SARS-CoV-2 Mu variant will be digested from a plasmid containing the gene generated in-house and inserted into CPI backbone by Gibson assembly. The recombinant virus will contain S gene of SARS-CoV-2 Mu variant between SH and HN. The primers have been ordered for making the plasmid construct.
CVL80 for CVXGA14: The S gene of SARS-CoV-2 Omicron BA.1 variant was generated by Genscript, then inserted into CPI backbone by Gibson assembly. The recombinant plasmid contains the S gene of SARS-CoV-2 Omicron BA.1 variant between SH and HN. The recombinant virus is obtained.
CVL86 for CVXGA15: The 5′ end of S gene of SARS-CoV-2 Omicron BA.2 variant was generated by Genscript, 3′ end of S gene was amplified by mutagenesis of the PCR reactions and inserted into CPI backbone by Gibson assembly. The recombinant plasmid contains the S gene of SARS-CoV-2 Omicron BA.2 variant between the SH and HN genes. The recombinant virus rescue is ongoing.
CVL108 for CVXGA20: The S gene of SARS-CoV-2 Omicron BA.5 variant was amplified and inserted into CVL86 backbone to replace the S gene of BA.2 variant by Gibson assembly. The recombinant plasmid contains the S gene of SARS-CoV-2 Omicron BA.5 variant between the SH and HN genes of PIV5. The recombinant virus is obtained.
CVL111 for CVXGA23: The 5′ end of S gene of SARS-CoV-2 Omicron BA.2.75 variant was generated by Genscript, and 3′ end of S gene was amplified from CVL86 plasmid and inserted into CVL6 backbone to replace the S gene of Wuhan strain by Gibson assembly. The recombinant plasmid will contain S gene of SARS-CoV-2 Omicron BA.2.75 variant between the SH and HN genes of PIV5.
CVL113 for CVXGA25: The PIV5 F and HN genes was deleted from CVL54 backbone by Gibson assembly. The recombinant plasmid will contain CPI backbone without F and HN genes, and S gene of SARS-CoV-2 Wuhan strain between HN and L genes of PIV5. The recombinant plasmid construction is ongoing. The recombinant virus will only contain the glycoprotein from the SARS-COV-2 virus. This virus can serve either as the vaccine strain and/or the virus for microneutralization assay to measure S-specific antibody response.
The primers used in cDNA cloning of the CPI-S vaccine candidates are listed in Table 1.
Five candidate vaccines based on the PIV5 CPI vector were evaluated in this study (Table 2). CVXGA1, 2, 3, and 5 have a SARS-CoV-S gene inserted between the PIV5 small hydrophobic (SH) and hemagglutinin (HN) genes. CVXGA2 has an additional insert, the SARS-CoV-2 nucleoprotein (N), inserted between the PIV5 HN and polymerase (L) genes (
Study Outline.
Forty male, 5-7 week-old Syrian hamsters were used in this study (N=8 per vaccine group). The hamsters were intranasally immunized with 100 μL of WT PIV5 CPI virus, CVXGA1, CVXGA2, and CVXGA3 of approximately 1×105 plaque-forming units (PFU). For CVXGA5, hamsters received 100 uL of approximately 5×102 PFU (Table 2). At 36 days post-immunization (dpi), four hamsters from each group were challenged with 103 PFU of SARS-CoV-2 Wuhan strain (WA1), and the remaining four hamsters from each group were challenged with 103 PFU SARS-CoV-2 alpha variant (CA; BEI NR54011). Following challenge infection, the hamster weights were monitored for 5 days, the hamster lungs were harvested, and SARS-CoV-2 viral burden was quantified via plaque assay and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR).
Determination of infectious SARS-CoV-2 viral burden in hamster lungs post-challenge. Hamster lung homogenates were serially diluted in Dulbecco's modified Eagle medium (DMEM)+2% fetal bovine serum (FBS)+1% antibiotic/antimycotic and added to 12-well plates of Vero E6 cells. At 1 hour post-infection, the inoculum was removed, and a methylcellulose overlay (500 mL Opti-MEM+0.8% methylcellulose+2% FBS+1% antibiotic/antimycotic) was added to the wells. The plates were incubated for 3 days, the overlay was removed, and the cells were fixed with 40% acetone/60% methanol. The cells were stained with crystal violet, the number of plaques were counted and viral titers are expressed as log 10 PFU/mL.
Quantification of SARS-CoV-2 RNA load in hamster lungs post-challenge by qRT-PCR. For the standard curves, SARS CoV-2 challenge virus at concentrations of 2×107 PFU/mL and 4×108 PFU/mL for WA1 and CA1 respectively, was inactivated by 1:10 diluted Trizol reagent (ThermoFisher Scientific) with the genetic material preserved. Hamster lung homogenate was also treated with 1:10 diluted Trizol. Using a Qiagen Viral RNA extraction kit, RNA was extracted from 140 μL inactivated virus and lung homogenate and eluted in 15 buffer. WA1 and CA1 RNA was serial-diluted 1:10 in sterile water to serve as the standard curves. For the qRT-PCR reactions, 5 μL eluted RNA was mixed with 5 μL Thermofisher TaqPath 1-Step qPCR Master Mix (cat #A15300), 1.5 μL 2019-nCoV CDC EUA Authorized qPCR Probe Assay primer/probe mix, IDT (cat #10006770), and 8.5 μL water. qRT-PCR thermocycler settings were used as described on “CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel . . . Instructions for Use” page 26. To generate the standard curves, the viral titer was plotted on the x-axis and the cycle threshold (CT) value was plotted on the y-axis.
Results.
Hamster weight changes post-challenge infection. Following challenge with either WA1 or CA1/alpha SARS-CoV-2 variants, the hamster weights were monitored daily. For hamsters challenged with the WA1 variant, those immunized with CVXGA1, CVXGA2, CVXGA3, or CVXGA5 had minimal weight loss and resumed weight gaining within 5 dpc. However, the hamsters immunized with WT PIV5 CPI strain had weight loss and did not regain weight by the end of the study at 5 dpc (
CVXGA2 encoding both the S and N protein from SARS-CoV-2 Wuhan strain had the best weight gain after the viral challenge, indicating additonal protection offered by the inserted N gene in the vaccine candidate. In addition, the vaccine dose of CVXGA5 was approx. 100-fold lower than the other vaccine viruses, yet it offered protection as good as CVXGA1 and CVXGA3.
SARS-CoV-2 challenge virus titer in the lung. At 5 dpc, SARS-CoV-2 viral burden in the hamster lungs was quantified. Hamsters immunized with WT PIV5 CPI had an average titer of 2.5×104 and 6.2×104 PFU/mL following challenge with WA1 and CA1/alpha, respectively. In contrast, none of the CVXGA-immunized hamsters had detectable infectious virus in their lungs (
Quantitation of SARS-CoV-2 RNA in the vaccinated hamster lungs following challenge infection. SARS-CoV2 challenge virus RNA load in the hamster lungs were quantitated by qRT-PCR. Note that a lower cycle threshold (CT) value corresponds to a higher viral titer. Hamsters immunized with WT PIV5 CPI and challenged with WA1, had CT values between 14.7 and 18.2, which equates to 8.9×103 and 6.5×102 PFU/rxn (
In order to protect against SARS-CoV-2 variants, recombinant PIV5 CPI-vectored candidate vaccines expressing the S protein alone or with S and N from SARS-CoV-2 Wuhan strain, beta, and gamma variants have been developed. All four CVXGA candidate vaccines offered significant protection from weight loss not only from homologous but also heterologous SARS-CoV-2 challenge virus infection. Furthermore, no infectious SARS-CoV-2 live virus was detected in the lungs of CVXGA-immunized hamster 5 dpc, which was confirmed by the low/no viral RNA burden by qRT-PCR assay. These results indicate that PIV5 CPI vectored CVXGA candidate SARS-CoV-2 vaccines are effective in providing protection against SARS-CoV-2 variants.
Materials and methods.
Two candidate SARS-CoV-2 vaccine viruses based on the PIV5 CPI vector were evaluated in this study. CVXGA 1 and 3 have the SARS-CoV-S gene from variants Wuhan (WA1) and California/alpha (CA1/alpha), respectively, inserted between the PIV5 small hydrophobic (SH) and hemagglutinin (HN) genes. BLB-201 has the fusion (F) gene from respiratory syncytial virus (RSV) in the same location as the control (
Study Outline.
Twelve AGMs were used in this study with 4 per vaccine group. The AGMs were intranasally immunized with 106 PFU/AGM of vaccine virus. Serum was collected prior to dosing (Day −1) and 28 days post-immunization (dpi) to quantify the anti-S IgG antibody titer via enzyme-linked immunosorbent assay (ELISA). Blood was collected at −1, 7, 14, and 28 dpi to quantify the CoV-S-specific cellular response. At 40 dpi, the mice were challenged with 1.5×106 PFU SARS-CoV-2 CA1/alpha variant. At 2, 4, and 7 days post-challenge (dpc), AGM nasal washes (NW) and bronchoalveolar lavage (BAL) were collected, and SARS-CoV-2 viral burden was quantified via plaque assay and qRT-PCR (
ELISA to quantify anti-SARS-Cov-2-S IgG antibodies. For the enzyme-linked immunosorbent assay (ELISA), Immulon 2HB 96-well microtiter plates were coated with 100 μL/well purified SARS-CoV-2 S protein at 1 μg/mL. Serum samples were diluted 3-fold in KPL Wash Solution (SeraCare Life Sciences, Inc., Milford, Mass.) supplemented with 0.5% bovine serum albumin (BSA) and 5% nonfat milk. Serum samples were incubated at 100 μL/well for 1 h. After washing with KPL Wash Solution three times, the plates were incubated for 1 h with a 1:1000 dilution of horseradish peroxidase-labelled goat anti-monkey IgG antibody (Southern Biotech, Birmingham, Ala.) in KPL Wash Solution supplemented with 0.5% BSA and 5% nonfat milk. After the plates were washed three times, antibody-antigen binding was realized using KPL SureBlue Reserve TMB Microwell Peroxidase Substrate (SeraCare Life Sciences, Inc.) and a Glomax 96 microplate luminometer (Promega) set at OD450. An antibody titer was calculated as the highest serum dilution at which the OD450 was greater than two standard deviations above the mean OD450 of naïve serum.
ELISPOT to quantify SARS-CoV-2-S-specific cellular response. SARS-CoV-2-specific peripheral blood mononuclear cells (PBMCs) in blood were quantified by ELISpot assay at days −1, 7, 14, and 28 dpi. The number of spots was reported as the number of IFN-γ spots per 2×105 PBMCs.
Plaque assays to determine infectious SARS-CoV-2 viral burden in AGM NW and BAL post-challenge. NW and BAL were serially diluted in Dulbecco's modified Eagle medium (DMEM)+2% fetal bovine serum (FBS)+1% antibiotic/antimycotic and added to 12-well plates of Vero E6 cells. At 1 hour post-infection, the inoculum was removed, and a methylcellulose overlay (500 mL Opti-MEM+0.8% methylcellulose+2% FBS+1% antibiotic/antimycotic) was added to the wells. The plates were incubated for 3 days, the overlay was removed, and the cells were fixed with 40% acetone/60% methanol. The cells were stained with crystal violet, the number of plaques were counted, and viral titers are expressed as log 10 PFU/mL.
Quantification of SARS-CoV-2 RNA in AGM NW and BAL by qRT-PCR. AGM NW and BAL samples were inactivated by 1:10 diluted Trizol reagent (ThermoFisher Scientific) with the genetic material preserved. Using a Qiagen Viral RNA extraction kit, RNA was extracted from 140 μL inactivated sample and eluted in 15 μL buffer. For the qRT-PCR reactions, 5 μL eluted RNA was mixed with 5 μL Thermofisher TaqPath 1-Step qPCR Master Mix (cat #A15300), 1.5 μL 2019-nCoV CDC EUA Authorized qPCR Probe Assay primer/probe mix, IDT (cat #10006770), and 8.5 μL water. qRT-PCR thermocycler settings were used as described on “CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel . . . Instructions for Use” page 26.
Results.
SARS-CoV-2-S-specific cellular response. Following immunization, blood was collected at days −1, 7, 14, and 28 to quantify the SARS-CoV-2-S-specific cellular response. For all groups, PBMCs collected at day −1 failed to respond to SARS-CoV-2-S stimulation. Following immunization, AGMs immunized with CVXGA1 and CVXGA3 generated a cellular response that is SARS-CoV-2 S protein-specific. No SARS-CoV-2-specific cellular response was detected in the BLB control animals (group 6), the cellular responses from animals immunized with CVXGA1 and CVXGA3 increased at each time point post-immunization, Days 7, 14 and 28 (
SARS-CoV-2-S IgG antibody response. At −1 and 28 dpi, anti-SARS-CoV-2-S IgG antibodies were quantified. Interestingly, two CVXGA3- and three CVXGA1-immunized AGMs had detectable anti-S antibodies prior to immunization. For all except one CVXGA1-immunized AGM, the anti-S antibody titers increased from −1 to 28 dpi to over 103. For all CVXGA3-immunized AGMs, the anti-S antibody titers increased from −1 to 28 dpi to over 104. One AGM, who had detectable antibodies prior to immunization with CVXGA1, did not have an increase in its anti-S IgG antibody titer (
Infectious SARS-CoV-2 challenge virus titer in NW and BAL of vaccinated AGM. At 2, 4, and 7 dpc, NW and BAL were collected and infectious SARS-CoV-2 viral burden was quantified. In the NW, only one AGM, immunized with vector control, had detectable infectious virus (
Quantitation of SARS-CoV-2 RNA in the vaccinated AGM NW and BAL following challenge infection. At 2, 4, and 7 dpc, NW and BAL were collected to quantitate SARS-CoV2 challenge virus RNA load by qRT-PCR. Note that a lower cycle threshold (CT) value corresponds to a higher viral titer. AGMs immunized with vector control had CT values corresponding to infectious virus in their BAL at all time points. For CVXGA1-immunized AGMs, three of four at 2 dpc and one of two at 3 dpc had CT values corresponding to infectious virus in their BAL. For CVXGA3, two immunized AGMs had CT values corresponding to infectious virus in their BAL at 2 dpc (
Both CVXGA1 and CVXGA3 candidate vaccines induced anti-SARS-CoV-2 S IgG antibodies and SARS-CoV-2 S-specific cellular responses. Furthermore, no infectious SARS-CoV-2 live virus was detected in the NW of CVXGA-immunized AGMs 2, 4, or 7 dpc. For CVXGA3-immunized AGMs, no infectious virus was detected in the BAL. These results were confirmed by the low/no viral RNA burden by qRT-PCR assay, indicating that PIV5 CPI vectored CVXGA candidate SARS-CoV-2 vaccines are effective in providing protection against SARS-CoV-2 alpha variant. CVXGA3 appears to provide a better protection against SARS-CoV-2 alpha variant challenge infection.
Materials and Methods.
Cells: Vero E6 cells were maintained in Dulbecco's modified Eagle media (DMEM) supplemented with 5% fetal bovine serum (FBS) plus 100 IU/mL penicillin and 100 ug/mL streptomycin (1% P/S; Mediatech Inc, Manassas, Va., USA). Serum-free (SF) Vero cells were maintained in VP-SFM (ThermoFisher Scientific) plus 4 mM GlutaMax (Gibco). Vero-TEMPRSS cells were obtained from Dr. Jeff Hogan, University of Georgia, and maintained in DMEM+10% FBS+1 mg/mL G418. All cells were incubated at 37° C., 5% CO2.
Plasmids and virus rescue: The construction of a plasmid encoding for PIV5 antigenome and generation of recombinant PIV5 has already been described (He, B., et al., Virology, 237(2):249-60 (1997)). To construct plasmids encoding for the antigenome of CVXGA1, CVXGA3, CVXGA5, CVXGA13, and CVXGA14, the Spike (S) genes from SARS-CoV-2 WA1, alpha, gamma, delta, and omicron, respectively, were placed into an additional open reading frame (ORF) between the PIV5 SH and HN genes. The S cytoplasmic tail was replaced by the PIV5 fusion (F) protein cytoplasmic tail. To construct CVXGA2, the SARS-CoV-2 WA1 nucleoprotein (N) gene was placed into an additional ORF between the PIV5 HN and L genes of CVXGA1. Primer sequences are available upon request. To generate recombinant PIV5 viruses CVXGA1, CVXGA2, CVXGA3, CVXGA5, CVXGA13, and CVXGA14, the Neon Transfection System (Invitrogen) was used to electroporate SF Vero cells with 1.9 μg pPIV5-NP, 0.6 μg pPIV5-P, 3.1 μg pPIV5-L, 3.1 μg pT7-polymerase, and 6.3 μg antigenome. Recovered virus was amplified in SF Vero cells, single viral isolates were obtained by limiting dilution or plaque purification, and the viral genomes were verified by RT-PCR and Sanger sequencing.
Virus propagation: The recombinant PIV5 viruses were propagated in SF Vero cells. The cells were infected at a multiplicity of infection (MOI) 0.001 PFU in VP-SFM+4 mM GlutaMax. One hour post-infection, the media was replaced with fresh VP-SFM 4 mM GlutaMax. After 5 to 7 days of incubation at 37° C. with 5% CO2, the media was collected and centrifuged at 1500 rpm for 10 mins to pellet cell debris. The supernatant was mixed with 0.1 volume of 10× sucrose-phosphate-glutamate (SPG) buffer or 10×SPG+10% Arginine, aliquotted, flash-frozen in liquid nitrogen, and stored at −80° C. The PIV5 virus stocks were titrated via immunostain in Vero cells.
The SARS-CoV-2 viruses were propagated in Vero cells with DMEM+1% FBS+1× P/S. WA1 (BEI NR-52281) and alpha variant (BEI NR-54011) were obtained from BEI Resources. The delta variant was provided by Dr. Michael Gale, University of Washington. The omicron variant was provided by Dr. Jeff Hogan, University of Georgia.
Immunofluorescence assay (IFA): To detect PIV5 V/P, SARS-CoV-2 S, and SARS-CoV-2 N, immunofluorescence assays were performed. Vero cells were infected at MOI 0.01 with PIV5, CVXGA1, CVXGA2, CVXGA3, CVXGA5, CVXGA13, or CVXGA14. 3 days post-infection, the cells were fixed with 80% methanol. The cells were incubated with mouse anti-PIV5 V/P, rabbit anti-SARS-CoV-2 S (Sino Biological catalog no. 40150-R007), or SARS-CoV-2 N (ProSci catalog no. 35-579) antibodies at 1:500 in PBS+3% BSA for 1 hr. Next, the cells were washed with PBS and incubated with goat a-mouse Cy3 (KPL) or goat a-rabbit Cy3 (KPL) at 1:500 in PBS+3% BSA for 30 mins. The cells were washed with PBS and imaged with an EVOS M5000 microscope (Thermo Fisher Scientific).
Results
A PIV5-vectored vaccine for SARS-CoV-2 was previously generated by inserting the SARS-CoV-2 WA1 S gene between the PIV5 SH and HN genes (CVXGA1). Additionally, the cytoplasmic tail of the S protein was replaced with the cytoplasmic tail from the PIV5 F protein. A single, intranasal dose of CVXGA1 was shown to protect K18-hACE2 mice from lethal infection with the WA1 strain, the initial circulating strain in the US, and blocks contact transmission in ferrets (An, D., et al., Sci Adv, 7(27) (2021)). To determine whether expressing the N protein of SARS-CoV2 as an additional antigen enhances protection afforded by the S antigen alone, PIV5 expressing both S and N (CVXGA2) was generated. During the study period, SARS-CoV 2 variants of concern (VOC) emerged and some of them became dominant strains circulating in different regions at different times. Thus, PIV5-vectored vaccine candidates expressing S from SARS-CoV-2 VOC were generated in a similar manner as CVXGA1 (
The vaccine viruses were recovered by electroporating the plasmid containing the PIV5 genome with the SARS-CoV-2 antigen along with plasmids encoding for PIV5 NP, P, and L proteins as well as T7 polymerase. The recovered viral genomes were confirmed with RT-PCR and sequencing. To confirm antigen expression, Vero cells were infected at MOI 0.01 with PIV5, CVXGA1, CVXGA2, CVXGA3, CVXGA5, CVXGA13, or CVXGA14 and assayed for immunofluorescence with WA1 S-specific antibody. As expected, S expression was detected in cells infected with CVXGA1, 2, 3, 5, 13 and 14. Additionally, SARS-CoV-2 N expression was only detected in cells infected with CVXGA2 (
Materials and Methods.
Hamsters: Five-to-seven-week-old Golden Syrian hamsters were obtained from Charles River Laboratories. The hamsters were single-housed in biosafety level 2 facilities with ad libitum access to food and water. Pre-challenge procedures were performed at the University of Georgia Biological Sciences Animal Facility. The hamsters were transferred to the University of Georgia Animal Health Research Center for the challenge and post-challenge procedures. The hamsters were anesthetized for immunization, blood collection, and challenge by intraperitoneal injection of 100 μL ketamine/acepromazine cocktail. All experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee at the University of Georgia.
Immunization and challenge of hamsters: The hamsters were anesthetized by intraperitoneal injection of 100 μL ketamine/acepromazine cocktail. To administer intranasal immunizations, anesthetized hamsters were placed on their backs, a pipette was used to dispense 100 μL inoculum onto their noses (50 μL each nostril), and the inoculum was allowed to drain into their respiratory tracts. They were recovered on heating pads.
A Covid-19 mRNA vaccine was obtained from a clinical site after it was thawed and stored at −80° C. 2 μg mRNA vaccine in 50 μL was administered via intramuscular injection.
For study AE19, hamsters (n=8) received a single intranasal immunization of 100 μL of 105 plaque-forming units (PFU) PIV5, CVXGA1, CVXGA2, CVXGA3, or CVXGA5. At 28 dpi, blood was collected from the hamster saphenous vein for serological analysis. At 36 days post immunization (dpi), the hamsters were anesthetized, four hamsters were challenged with 103 PFU of SARS-CoV-2 Wuhan strain (WA1), and the remaining four hamsters were challenged with 103 PFU SARS-CoV-2 alpha variant (CA; BEI NR54011). Challenge virus was delivered intranasally with a Teleflex MAD device. Following challenge infection, the hamster weights were monitored for 5 days, the hamster lungs were harvested and homogenized in approximately 1.7 mL sterile PBS, and SARS-CoV-2 viral burden was quantified via plaque assay and real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR).
For study AE23, hamsters received intramuscular immunizations of 100 μL PBS (n=20, group 1) or 2 μg mRNA COVID vaccine (n=20, group 2). At 21 dpi, hamsters that received the mRNA vaccine were boosted with the mRNA vaccine again. At 28 dpi, blood was collected for serological analysis. At 35 dpi following initial immunization, hamsters that received PBS during the first immunization received either 100 μL PBS i.n. (n=5, group 1A), 100 μL 3×105 PFU CVXGA1 (n=5, group 1B), 100 μL 2×105 PFU CVXGA3 (n=5, group 1C), or 100 μL 1.5×105 PFU CVXGA13 (n=5, group 1D). Group 2 hamsters that received two doses of mRNA received 100 μL PBS (n=4, group 2A), 100 μL 3×105 PFU CVXGA1 (n=4, group 2B), 100 μL 2×105 PFU CVXGA3 (n=4, group 2C), 100 μL 1.5×105 PFU CVXGA13 (n=4, group 2D), or a third dose of mRNA (n=4, group 2E). Hamsters were anesthetized for intranasal immunizations but not intramuscular injections. Blood was collected at 54 dpi. At 63 dpi, the hamsters were challenged with 104 PFU SARS-CoV-2 delta variant. Following challenge infection, hamster weights were monitored for 5 days, the hamster lungs were harvested, and SARS-CoV-2 viral burden was quantified via plaque assay and RT-qPCR.
For study AE24, hamsters received 100 μL PBS i.n. (n=5, Group 1), 2 μg mRNA COVID vaccine (n=25, Group 2), or 100 μL 7×104 PFU CVXGA1 (n=10, Groups 3 & 4). At 29 dpi, hamsters that received the mRNA vaccine were boosted with the mRNA vaccine again and group 3 hamsters received another dose of CVXGA1. At 91 dpi following initial immunization, hamsters who received two doses of mRNA received 2 μg mRNA vaccine (n=5, Group 2A), 100 μL 7×104 PFU CVXGA1 (n=5, Group 2B), 100 μL 103 PFU CVXGA13 (n=5, Group 2C), 100 μL PBS i.n. (n=5, Group 2D), or 100 μL 104 PFU CVXGA14 (n=5, Group 2E). Hamsters were anesthetized for intranasal immunizations but not intramuscular injections. Blood was collected at 36 and 108 dpi. At 116 dpi, the hamsters were challenged with 104 PFU SARS-CoV-2 delta variant. Following challenge infection, hamster weights were monitored for 5 days, the hamster lungs were harvested, and SARS-CoV-2 viral burden was quantified via plaque assay and RT-qPCR.
All animal experiments were performed according to the protocols approved by the Institutional Animal Care and Use Committee at the University of Georgia.
Enzyme-linked immunosorbent assay (ELISA): To quantify the anti-SARS-CoV-2 S and RBD humoral response, hamster serum was analyzed via ELISA. Immulon® 2HB 96-well microtiter plates were coated with 100 μL SARS-CoV-2 S or RBD at 1 μg/mL. For all ELISAs, plates were coated with SARS-CoV-2 S and RBD from the WA1 strain. S and RBD were purified as described previously. (An, D., et al., Sci Adv, 7(27) (2021)). Hamster serum was serial diluted two-fold and incubated on the plates for 2 hrs. Horseradish peroxidase-labelled goat anti-mouse IgG secondary antibody (Southern Biotech, Birmingham, Ala.) was diluted 1:2000 and incubated on the wells for 1 hr. The plates were developed with KPL SureBlue Reserve TMB Microwell Peroxidase Substrate (SeraCare Life Sciences, Inc., Milford, Mass.), and OD450 values were detected with a BioTek Epoch Microplate Spectrophotometer (BioTek, Winooski, Vt.). Antibody titers were calculated as log 10 of the highest serum dilution at which the OD450 was greater than two standard deviations above the mean OD450 of naïve serum.
Results.
Golden Syrian hamsters are susceptible to SARS-CoV 2 infection. (Chan, J. F., et al., Clin Infect Dis, 71(9):2428-2446 (2020)) To test efficacy of new PIV5-vectored COVID-19 vaccines in hamsters, their ability to induce an immune response in hamsters was examined. Golden Syrian hamsters were immunized with a single, intranasal dose of 105 plaque-forming units (PFU) PIV5 vector, CVXGA1, CVXGA2, CVXGA3, or 5×102 PFU CVXGA5 (
Materials and methods.
Neutralization assays: To quantify the SARS-CoV-2-neutralizing antibodies generated by the hamsters, microneutralization assays were performed. Hamster serum was heat-inactivated at 56° C. for 45 mins and serially diluted two-fold. The serum was mixed 1:1 with 6×103 focus-forming units (FFU)/mL SARS-CoV-2 WA1, delta, or omicron. The serum/virus mixture was incubated at 37° C. for 1 hr before being incubated on 96-wells of Vero cells for WA1 or Vero TEMPRSS2 cells for delta and omicron. One hour post-infection, a methylcellulose overlay (Dulbecco's modified Eagle medium (DMEM)+5% fetal bovine serum (FBS)+1% penicillin/streptomycin+1% methylcellulose) was added on top of the serum/virus mixture. The plates were incubated at 37° C., 5% CO2 for 24 hrs. The methylcellulose overlay was removed, the wells were washed with PBS, and the cells were fixed with 60% methanol/40% acetone. The number of infected cells were quantified via immunostain. Neutralization titers were calculated as log 10 of the highest serum dilution at which the virus infectivity was reduced by at least 50%.
Plaque assay for infectious virus: Following euthanasia, whole hamster lungs were removed, added to approximately 1.7 mL sterile PBS, and homogenized. To quantify infectious SARS-CoV-2, plaque assays were performed with lungs from AE19 and AE23, and FFU assays were performed with lungs from AE24.
For the plaque assays, lung homogenates were serially diluted in Dulbecco's modified Eagle medium (DMEM)+2% fetal bovine serum (FBS)+1% antibiotic/antimycotic and added to 12-well plates of Vero E6 cells for SARS-CoV-2 WA1 and alpha variant or Vero TEMPRSS2 cells for delta and omicron variants. At 1 hour post-infection, the inoculum was removed, and a methylcellulose overlay (500 mL Opti-MEM+0.8% methylcellulose+2% FBS+1% antibiotic/antimycotic) was added to the wells. The plates were incubated for 3 days, the overlay was removed, and the cells were fixed with 60% methanol/40% acetone. The cells were stained with crystal violet, the number of plaques were counted, and viral titers are expressed as PFU/mL of lung homogenate.
qPCR: SARS-CoV-2 viral RNA levels were quantified by RT-qPCR. SARS-CoV-2 virus was inactivated by mixing 100 μL lung homogenate with 900 μL TRIzol (Invitrogen). Using a QIAgen RNA extraction kit, RNA was extracted from 140 μL homogenate/TRIzol and eluted in 15 μL of elution buffer, of which 5 μL was used in the qRT-PCR reaction. qRT-PCR was performed according to the protocol described in the “CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel . . . Instructions for Use” (page 26) with Applied Biosystems TaqPath One Step RT qPCR Master Mix and SARS-CoV-2 Research Use Only qPCR Primer and Probe Kit primer/probe mix N1. SARS-CoV-2 viral RNA was extracted from SARS-CoV-2 WA1, alpha variant, and delta variant viruses of known titers. RNA was extracted from 140 μL virus and eluted in 15 μL of elution buffer. The viral RNA was serially diluted 10-fold and 5 μL from each dilution was used in the RT-qPCR assay. To generate a standard curve, the viral titer was plotted on the x-axis and the CT value was plotted on the y-axis. The standard curve was used to calculate the CT value that corresponds to 1 PFU/rxn. The standard curve was also used to calculate PFU/rxn from the CT values of homogenized lung RNA samples. The CT value of RNA extracted from sterile elution buffer was designated the PCR negative cutoff
Results.
To assess the efficacy of PIV5-vectored SARS-CoV-2 vaccines against homologous and heterologous challenges, CVXGA-immunized hamsters were challenged with either 103 PFU SARS-CoV-2 WA1 (USA-WA01/2020) or alpha variant (CA; BEI NR54011) at 36 dpi (
To quantify viral burden in the lungs, hamster lungs were harvested and homogenized 5 dpc. Infectious virus was quantified by plaque assay. Hamsters immunized with PIV5 vector had infectious virus of over 4 log 10 PFU/mL lung homogenate following challenge with WA1 or alpha variant, while no infectious WA1 or alpha variant was detected in hamsters immunized with CVXGA1, CVXGA2, CVXGA3, or CVXGA5 (
Results.
As of May 2022, delta variant is one of two VOCs circulating in the United States. Therefore, we assessed the efficacy of our primary vaccine candidate, CVXGA1, against heterologous challenge with delta variant and test a vaccine expressing S from delta variant against homologous challenge. Hamsters received a single, intranasal dose of PBS or 105 PFU CVXGA1, CVXGA3, or CVXGA13. 19 dpi, blood was collected and serum anti-SARS-CoV-2 WA1 S and -RBD IgG antibodies were quantified via ELISA. Immunization with CVXGA1, CVXGA3, and CVXGA13 elicited anti-S antibodies with mean titers of over 10,000 (
At 28 dpi, the hamsters were intranasally challenged with 104 PFU SARS-CoV-2 delta variant and their weights were monitored for 5 days. Beginning at 2 dpc, hamsters immunized with PBS experienced weight loss that steadily declined until the study was terminated. In contrast, hamsters immunized with CVXGA1, CVXGA3, or CVXGA13 experienced weight gain after 2 days post-challenge. While not statistically significant, hamsters immunized with CVXGA13 had greater weight gain than hamsters immunized with CVXGA1 or CVXGA3, indicating that PIV5 expressing S from SARS-CoV-2 delta variant protected hamsters best from weight loss following homologous challenge with delta variant (
Results.
As of May, 2022, 67 percent of individuals in the United States are fully vaccinated against COVID-19. However, data suggests that vaccine-induced immunity wanes over time and is less effective against SARS-CoV-2 variants (Planas, D., et al., Nature, 596(7871): 276 (2021); Zhang, L., et al., Emerg Microbes Infect, 11(1): 1 (2022); Feikin, D. R., et al., Lancet, 399(10328): 924 (2022)). To assess the efficacy of CVXGA1, we compared one (1×CVXGA1) and two (2×CVXGA1) intranasal doses of CVXGA1 to two intramuscular doses of a mRNA COVID-19 vaccine (2× mRNA) (
To compare longevity of antibody responses in the hamsters, 79 days after boost (day 108 after initial immunization) sera were collected. Anti-S ELISA titers dropped over this time period by 56.5, 56.5, and 24.3 percent for 2× mRNA, 2×CVXGA1, and 1×CVXGA1, respectively (
87 days after the second immunization, the hamsters were challenged with delta variant and the hamster weights were monitored for five days. Compared to hamsters who were immunized with PBS, hamsters who received two doses of CVXGA1 had significant weight gain following challenge. In contrast, hamsters who received two doses of mRNA vaccine did not experience weight gain that was statistically significant (
Results.
Due to large populations having already immunized with COVID-19 vaccines, we investigated the use of CVXGA vaccines as a boost (
Twenty-five days following the boost, the hamsters were challenged with delta variant. Hamsters who received intranasal boosts of CVXGA14 or CVXGA1 had the best weight gain compared to hamsters received PBS, no boost, or an mRNA boost. Interestingly, hamsters who received CVXGA14 experienced higher weight gain than hamsters who received CVXGA13 (homologous delta S antigen) (
A number of novel SARS-COV-2 vaccine candidates were rescued as described below and summarized in
SARAS-COV-2 variants representing variants of concern or variants of interest were produced by inserting the Spike (S) protein gene from different variants into PIV5 canine parainfluenza virus (CPI) vector: CVXGA1, 3, 4, 5, 6, 13, 14, 15, 20, 23, 26, and 28.
To improve vaccine efficacy, the N protein gene of SARS-CoV-2 was inserted between the HN and L gene junction in addition to the S inserted at the SH and HN junction, the resulting virus is named as CVXGA2. The expression of both the S and N proteins of SARS-CoV-2 is expected to offer additional protection by eliciting both antibody and cellular immune responses offered by the N protein. However, CVXGA2's titer is low, further improvement is therefore explored in this disclosure.
The mutations in the V/P gene, S157F and 5308A, have been shown previously to increase viral polymerase activities and improve viral titer or yield (Timani K A, et al., J Virol., 82(18):9123(2008); Sun D, et al., PLoS Pathog., 5(7):e1000525 (2009)). We therefore introduced S157F and 5308A into the CPI V/P gene to produce CVXGA10 (CPI-S-PLK) and CVXGA12 (CPIΔSH-S-PLK) viruses.
The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.
This application is the United States non-provisional patent application of U.S. Provisional Application Ser. No. 63/246,161, filed Sep. 20, 2021, and of U.S. Provisional Application Ser. No. 63/365,934, filed Jun. 6, 2022, each of which are hereby incorporated by this reference in their entireties.
Number | Date | Country | |
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63246161 | Sep 2021 | US | |
63365934 | Jun 2022 | US |