WO2023283601A1 - Vaccins contre des agents pathogènes intracellulaires et leurs procédés de production - Google Patents

Vaccins contre des agents pathogènes intracellulaires et leurs procédés de production Download PDF

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Publication number
WO2023283601A1
WO2023283601A1 PCT/US2022/073512 US2022073512W WO2023283601A1 WO 2023283601 A1 WO2023283601 A1 WO 2023283601A1 US 2022073512 W US2022073512 W US 2022073512W WO 2023283601 A1 WO2023283601 A1 WO 2023283601A1
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variant
immunogenic fragment
polypeptide
immunogenic
vaccine
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PCT/US2022/073512
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English (en)
Inventor
Agustin FERNANDEZ
Edward GERSHBURG
Paul Predki
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Rational Vaccines, Inc.
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Priority to EP22838579.5A priority Critical patent/EP4366769A1/fr
Publication of WO2023283601A1 publication Critical patent/WO2023283601A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16634—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to vaccines for intracellular pathogens, including herpes simplex virus type 2 (HSV-2), and related methods and compositions.
  • HSV-2 herpes simplex virus type 2
  • HSV-2 herpes simplex virus type 2
  • HSV-2 is a DNA virus that often results in skin lesions and is characterized by latent and recurrent infections. HSV-2 can manifest as a cluster of small fluid-filled blisters that rupture and form painful sores, taking several weeks to heal, i.e., an outbreak.
  • the virus can exist in nerve cells for the lifetime of the infected subject and reactivate at irregular intervals.
  • HSV-2 infected individuals live with genital herpes disease that recurs once every 3-12 months. Even in the absence of actual ulcers, the virus can be produced and spread to new individuals at a rate of ⁇ 20 million per year. Currently, there is no cure for HSV-2 infection.
  • HSV-2 Treatment options for HSV-2 symptoms are limited, and it is highly desirable to develop pharmaceutical compositions that inhibit or counteract infection by HSV-2.
  • An effective vaccine may be used to elicit an enhanced immune response against HSV-2, thereby preventing initial infection, eliminating recurrence of outbreaks, and/or preventing viral shedding.
  • a consensus is that the optimal vaccine should engage all the respective arms of the immune response, including Thl, Th2 and Thl7, along with the presence of neutralizing antibodies and mucosal antibodies (IgA).
  • Viral vaccines can generally be divided into three groups: 1) live, attenuated, 2) inactivated, and 3) subunit.
  • Subunit vaccines containing specific antigens are regarded as one of the safest types of vaccines.
  • the efficiency of the subunit vaccine is often lower than live, attenuated vaccines, and many subunit vaccines lack the ability to generate effective and long-lasting immune responses. Therefore, new and improved compositions of subunit vaccines, including an improved HSV-2 subunit vaccine, are desirable.
  • proteomics serves as a promising strategy in the development of new subunit vaccines, especially when combined with immunomics (search of immunogenic proteins) and vaccinomics (characterization of host response to immunization). Therefore, new and improved methods of using proteomics to identify protein components in subunit vaccines that deliver both safe and effective treatments are highly desirable.
  • the present invention discloses novel methods of identifying protein components for use in a vaccine against an intracellular pathogen, as well as related vaccines and their uses.
  • the disclosure provides a method of identifying one or more protein components for use in a vaccine against an intracellular pathogen, comprising: a) contacting a cell lysate prepared from cells infected with an intracellular pathogen, with serum obtained from blood of a subject previously infected by the intracellular pathogen, under conditions and for a time sufficient for antibodies against the intracellular pathogen present in the serum to bind intracellular pathogen antigens in the cell lysate, thereby forming a mixture comprising antibody/antigen complexes; b) separating the antibody/antigen complexes from the mixture, optionally by immunoprecipitation; c) identifying, and optionally quantifying, antigens present in the antibody/antigen complexes, wherein the antigens identified in c) are protein components that may be used in a vaccine against the intracellular pathogen, thereby identifying one or more protein components for use in a vaccine against the intracellular pathogen.
  • the vaccine may comprise the full length antigen identified in c) or an immunogenic fragment, variant, or epitope thereof, or a combination of full length antigens and immunogenic fragments, variants, or epitopes thereof.
  • the method further comprises d) contacting each of a plurality of populations of PBMCs isolated from the blood of the subject with one or more proteins selected from a library of proteins; and e) measuring the cellular immune response of each of the populations of PBMCs contacted with the one or more protein, thereby identifying proteins that invoke a cellular immune response, wherein the proteins that invoke the cellular immune response are protein components that may be used in the vaccine against the intracellular pathogen.
  • the library of proteins comprises or consists of antigens or immunogenic fragments, variants, or epitopes thereof identified in the antibody/antigen complexes in step c).
  • the library of proteins comprises polypeptides and/or peptides not identified in step c).
  • the polypeptides and/or peptides are selected from a library of random and/or known polypeptides and/or peptides, e.g. a library of random known polypeptides or peptides.
  • the polypeptides and/or peptides are selected from a library of immunogenic polypeptides or fragments thereof, or peptides, e.g., polypeptides or peptides comprising known and/or computer-generated sequences.
  • the library of proteins comprises a combination of antigens or immunogenic fragments or epitopes thereof identified in the antibody/antigen complexes in step c) and other immunogenic polypeptides, e.g., peptides selected from the library of random or known polypeptides or peptides or the library of immunogenic peptide or polypeptides or fragments thereof.
  • the pathogen is a Herpes Simplex Virus 1 (HSV-1), a Herpes Simplex Virus 2 (HSV-2), or aSARS-CoV-2 virus.
  • HSV-1 Herpes Simplex Virus 1
  • HSV-2 Herpes Simplex Virus 2
  • SARS-CoV-2 virus a Herpes Simplex Virus 2 virus
  • the host cells are Vero cells, L7 cells,
  • the vaccine comprises more than one protein components.
  • the subject is a human.
  • the serum comprises at least one IgG that can bind to at least one antigen of the pathogen.
  • the antibody/antigen complex is separated from the mixture by immunoprecipitation.
  • the antigens or immunogenic fragments thereof in the antibody/antigen complexes are identified and/or quantified by mass spectrometry.
  • the PBMCs comprise T-cells that recognize the pathogen.
  • the cellular immune response is measured by ELISPOT or flow cytometry.
  • the method further comprises f) selecting one or more identified protein components for use in a vaccine against an intracellular pathogen.
  • the protein components are selected based on the amount of each antigen or immunogenic fragment thereof identified in the antibody/antigen complexes of the subject.
  • the protein components are selected based on the magnitude of the cellular immune response invoked in step e).
  • the protein components are selected based on the amount of each antigen or immunogenic fragment thereof identified in the antibody/antigen complexes of the subject and the magnitude of the cellular immune response invoked in step e).
  • one or more protein components for use in the vaccine against the intracellular pathogen are identified for a plurality of subjects previously infected with the intracellular pathogen.
  • step a) of the method comprises separately contacting each of a plurality of cell lysates prepared from cells infected with an intracellular pathogen with the serum obtained from blood of each of a plurality of subjects previously infected by the intracellular pathogen, under conditions and for a time sufficient for antibodies against the intracellular pathogen present in the serum to bind intracellular pathogen antigens in the cell lysate, thereby forming a plurality of mixtures comprising antibody/antigen complexes.
  • step a) of the method comprises contacting a cell lysate prepared from cells infected with an intracellular pathogen, with a serum composition comprising a mixture of serum obtained from blood of a plurality of subjects previously infected by the intracellular pathogen, under conditions and for a time sufficient for antibodies against the intracellular pathogen present in the serum to bind intracellular pathogen antigens in the cell lysate, thereby forming a mixture comprising antibody/antigen complexes.
  • the method further comprises selecting one or more protein components for use in a vaccine.
  • the protein components are selected based on the amount of each antigen or immunogenic fragment thereof identified in the plurality of subjects.
  • the protein components are selected based on the magnitude of the cellular immune response invoked in step e).
  • the protein components are selected based on the frequency at which each antigen or immunogenic fragment thereof is identified amongst the plurality of subjects.
  • the protein components are selected based on the amount of each antigen or immunogenic fragment thereof identified in the plurality of subjects and the magnitude of the cellular immune response invoked in step e).
  • the protein components are selected based on the amount of each antigen or immunogenic fragment thereof identified in the plurality of subjects and the frequency at which each antigen or immunogenic fragment thereof is identified amongst the plurality of subjects. In some embodiments, the protein components are selected based on the magnitude of the cellular immune response invoked in step e) and the frequency at which each antigen or immunogenic fragment thereof is identified amongst the plurality of subjects. In some embodiments, the protein components are selected based on the amount of each antigen or immunogenic fragment thereof identified in the plurality of subjects, the magnitude of the cellular immune response invoked in step e), and the frequency at which each antigen or immunogenic fragment thereof is identified amongst the plurality of subjects.
  • the disclosure provides a method of producing a vaccine against an intracellular pathogen, said method comprising preparing a vaccine comprising one or more isolated protein components identified or selected according to the method of any one of the methods of the disclosure.
  • each of the protein components comprises an antigen, or immunogenic fragment thereof, identified in at least 50% of the subjects, and the combination of protein components represents at least 50% of all antigens identified in the subjects, or immunogenic fragments thereof.
  • each of the protein components comprises an antigen, or immunogenic fragment thereof, identified in at least 0.5% of the antibody/antigen complexes, and the combination of protein components represents at least 5% of all antigens identified in the antibody/antigen complexes, or immunogenic fragments thereof.
  • each of the protein components comprises an antigen, or immunogenic fragment thereof, identified in at least 50% of the subjects and in at least 0.5% of the antibody/antigen complexes, and the combination of protein components represents at least 50% of all antigens identified in the subjects and at least 5% of all antigens identified in the antibody/antigen complexes, or immunogenic fragments thereof.
  • each of the protein components comprises an antigen, or immunogenic fragment thereof, identified in at least 0.5% of the antibody/antigen complexes, and the combination of protein components represents at least 10% of all antigens identified in the antibody/antigen complexes, or immunogenic fragments thereof.
  • each of the protein components comprises an antigen, or immunogenic fragment thereof, identified in at least 50% of the subjects and in at least 0.5% of the antibody/antigen complexes, and the combination of protein components represents at least 50% of all antigens identified in the subjects and at least 10% of all antigens identified in the antibody/antigen complexes, or immunogenic fragments thereof.
  • each of the protein components comprises an antigen, or immunogenic fragment thereof, identified in at least 1% of the antibody/antigen complexes, and the combination of protein components represents at least 5% of all antigens identified in the antibody/antigen complexes, or immunogenic fragments thereof.
  • each of the protein components comprises an antigen, or immunogenic fragment thereof, identified in at least 50% of the subjects and in at least 1% of the antibody/antigen complexes, and the combination of protein components represents at least 50% of all antigens identified in the subjects and at least 5% of all antigens identified in the antibody/antigen complexes, or immunogenic fragments thereof.
  • each of the protein components comprises an antigen, or immunogenic fragment thereof, identified in at least 1% of the antibody/antigen complexes, and the combination of protein components represents at least 10% of all antigens identified in the antibody/antigen complexes, or immunogenic fragments thereof.
  • each of the protein components comprises an antigen, or immunogenic fragment thereof, identified in at least 50% of the subjects and in at least 1% of the antibody/antigen complexes, and the combination of protein components represents at least 50% of all antigens identified in the subjects and at least 10% of all antigens identified in the antibody/antigen complexes, or immunogenic fragments thereof.
  • the protein components are recombinantly produced in cells, optionally E. coli, yeast, insect, or mammalian cells.
  • the vaccine further comprises one or more adjuvants.
  • the method further comprises measuring the efficacy of the vaccine by measuring the immune response to the vaccine elicited in an animal model.
  • the disclosure provides a vaccine produced according to any one of the methods of the disclosure.
  • the disclosure provides a method of treating, suppressing, inhibiting, or preventing an intracellular pathogen infection in a subject, the method comprising administering to said subject an effective amount of a vaccine comprising isolated immunogenic polypeptides of the intracellular pathogen that were identified according to a method disclosed herein.
  • the present invention also provides a novel approach for inducing a protective immune response against HSV-2 infections and for treating HSV-2 infections.
  • the disclosure provides a Herpes Simplex Virus 2 (HSV-2) vaccine comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 isolated polypeptides selected from the group consisting of: i. a polypeptide comprising DBP or an immunogenic fragment or variant thereof, ii. a polypeptide comprising MCP or an immunogenic fragment or variant thereof, iii. a polypeptide comprising gB or an immunogenic fragment or variant thereof, iv. a polypeptide comprising RIR1 or an immunogenic fragment or variant thereof, v.
  • HSV-2 Herpes Simplex Virus 2
  • a polypeptide comprising TRX1 or an immunogenic fragment or variant thereof vi. a polypeptide comprising UL25 or an immunogenic fragment or variant thereof, vii. a polypeptide comprising UL26 or an immunogenic fragment or variant thereof, viii. a polypeptide comprising TRX2 or an immunogenic fragment or variant thereof, ix. a polypeptide comprising gD or an immunogenic fragment or variant thereof, x. a polypeptide comprising gC or an immunogenic fragment or variant thereof, xi. a polypeptide comprising VP22 or an immunogenic fragment or variant thereof, xii. a polypeptide comprising ICP4 or an immunogenic fragment or variant thereof, xiii.
  • the HSV-2 vaccine comprises 13 proteins or immunogenic fragments thereof, e g., DBP, MCP, gB, RIR1, TRX1, UL25, UL26, TRX2, gD, gC, VP22, UL12, and ICP4, TRX1, or an immunogenic fragment or variant of any of the foregoing.
  • the HSV-2 vaccine comprises all 15 proteins or immunogenic fragments thereof, e.g., DBP, MCP (e.g., MCP (l-560aa) and/or MCP (561-1373aa)), gB, RIR1, TRX1, UL25, UL26, TRX2, gD, gC, VP22, UL12, ICP4, UL42, and gG, or an immunogenic fragment or variant of any of the foregoing.
  • the vaccine comprises: i) three or more of: DBP, MCP (l-560aa), MCP (561-1373aa), gB (30-730aa), gC (28-447aa), and R1R1; ii) four or more of DBP, MCP (l-560aa), MCP (561-1373aa), gB (30-730aa), gC (28-447aa), and R1R1; iii) DBP, MCP (l-560aa and 561-1373aa), gB (30-730aa), gC (28-447aa), and R1R1; iv) three or more of: UL37, UL47, NEC2, LTPD (l-933aa), and LTPD (934-2079aa), andNECl; or DBP, MCP, gB, RIR1, TRX1, UL25, UL26, TRX2, gD, gC, VP22, UL12
  • the sequence of DBP is at least 95% identical to SEQ ID NO. 1
  • the sequence of MCP comprises a region that is at least 95% identical to SEQ ID NO. 2 or SEQ ID NO. 3
  • the sequence of gB is at least 95% identical to SEQ ID NO. 4
  • the sequence of gC is at least 95% identical to SEQ ID NO. 5
  • the sequence of R1R1 is at least 95% identical to SEQ ID NO. 6
  • the sequence of TRX1 is at least 95% identical to SEQ ID NO. 7
  • the sequence of ICP4 is at least 95% identical to SEQ ID NO. 8
  • the sequence of TRX2 is at least 95% identical to SEQ ID NO. 9
  • the sequence of UL42 is at least 95% identical to SEQ ID NO.
  • the sequence of gG is at least 95% identical to SEQ ID NO. 11
  • the sequence of VP22 is at least 95% identical to SEQ ID NO. 12
  • the sequence of gD is at least 95% identical to SEQ ID NO. 13
  • the sequence of UL12 is at least 95% identical to SEQ ID NO. 14
  • the sequence of UL25 is at least 95% identical to SEQ ID NO:23
  • the sequence of UL26 is at least 95% identical to SEQ ID NO:24.
  • the vaccine further comprises at least one isolated polypeptide selected from the group consisting of: a) a polypeptide comprising UL54 or an immunogenic fragment or variant thereof, b) a polypeptide comprising gE or an immunogenic fragment or variant thereof, c) a polypeptide comprising UL37 or an immunogenic fragment or variant thereof, d) a polypeptide comprising UL47 or an immunogenic fragment or variant thereof, e) a polypeptide comprising NEC2 or an immunogenic fragment or variant thereof, f) a polypeptide comprising LTPD or an immunogenic fragment or variant thereof, and g) a polypeptide comprising NEC1 or an immunogenic fragment or variant thereof.
  • the composition further comprises all 7 proteins: UL54, gE, UL37, UL47, NEC2, LTPD, NEC.
  • the sequence of UL54 is at least 95% identical to SEQ ID NO. 15
  • the sequence of gE is at least 95% identical to SEQ ID NO. 16
  • the sequence of UL37 is at least 95% identical to SEQ ID NO. 17
  • the sequence of UL47 is at least 95% identical to SEQ ID NO. 18
  • the sequence of NEC2 is at least 95% identical to SEQ ID NO. 19
  • the sequence of LTPD comprises a region at least 95% identical to SEQ ID NO. 20 or SEQ ID NO:21
  • the sequence of NEC 1 is at least 95% identical to SEQ ID NO. 22.
  • the vaccine comprises DBP or an immunogenic fragment or variant thereof.
  • the vaccine comprises an immunogenic fragment of MCP or an immunogenic variant thereof.
  • the immunogenic fragment of MCP comprises amino acids 1-560 or amino acids 561-1373 of MCP or an immunogenic variant thereof.
  • the vaccine comprises an immunogenic fragment of LTPD or an immunogenic fragment or variant thereof.
  • the immunogenic fragment of LTPD comprises amino acids 1-933 or amino acids 934-2079 of LTPD or an immunogenic variant thereof.
  • the vaccine comprises: i) a polypeptide comprising DBP or an immunogenic fragment or variant thereof, ii) a polypeptide comprising MCP or an immunogenic fragment or variant thereof, iii) a polypeptide comprising gB or an immunogenic fragment or variant thereof, iv) a polypeptide comprising RIR1 or an immunogenic fragment or variant thereof, v) a polypeptide comprising TRX1 or an immunogenic fragment or variant thereof, vi) a polypeptide comprising UL25 or an immunogenic fragment or variant thereof, vii) a polypeptide comprising UL26 or an immunogenic fragment or variant thereof, viii) a polypeptide comprising TRX2 or an immunogenic fragment or variant thereof, ix) a polypeptide comprising gD or an immunogenic fragment or variant thereof, x) a polypeptide comprising gC or an immunogenic fragment or variant thereof, xi) a polypeptide comprising
  • the disclosure provides a Herpes Simplex Virus 2 (HSV-2) vaccine that comprises at least 10 isolated polypeptides selected from the group consisting of i) a polypeptide comprising DBP or an immunogenic fragment or variant thereof, ii) a polypeptide comprising MCP or an immunogenic fragment or variant thereof, iii) a polypeptide comprising gB or an immunogenic fragment or variant thereof, iv) a polypeptide comprising RIR1 or an immunogenic fragment or variant thereof, v) a polypeptide comprising TRX1 or an immunogenic fragment or variant thereof, vi) a polypeptide comprising UL25 or an immunogenic fragment or variant thereof, vii) a polypeptide comprising UL26 or an immunogenic fragment or variant thereof, viii) a polypeptide comprising TRX2 or an immunogenic fragment or variant thereof, ix) a polypeptide comprising gD or an immunogenic fragment or variant thereof, x) a polypeptide comprising HSV-2 (HSV-2)
  • the HSV-2 vaccine further comprises at least one isolated polypeptide selected from the group consisting of: a) a polypeptide comprising ICP4 or an immunogenic fragment or variant thereof, b) a polypeptide comprising gH or an immunogenic fragment or variant thereof, c) a polypeptide comprising UL12 or an immunogenic fragment or variant thereof, d) a polypeptide comprising gE or an immunogenic fragment or variant thereof, e) a polypeptide comprising UL42 or an immunogenic fragment or variant thereof, f) a polypeptide comprising gG or an immunogenic fragment or variant thereof, g) a polypeptide comprising UL50 or an immunogenic fragment or variant thereof, h) a polypeptide comprising UL54 or an immunogenic fragment or variant thereof, i) a polypeptide comprising UL40 or an immunogenic fragment or variant thereof, j) a polypeptide comprising UL48 or an immunogenic fragment or variant thereof, k)
  • the disclosure provides a Herpes Simplex Virus 2 (HSV-2) vaccine comprising a combination of immunogenic HSV-2 polypeptides or fragments or variants thereof, wherein at least 30% of the anti-HSV-2 antibodies identified in serum of HSV- 2-infected individuals specifically bind the polypeptides in the combination, according to Table 1
  • HSV-2 Herpes Simplex Virus 2
  • the composition further comprises one or more adjuvants.
  • said one or more adjuvants comprises QS-21.
  • the disclosure provides a method of treating, suppressing, inhibiting, or preventing an intracellular pathogen infection in a subject, the method comprising administering to said subject an effective amount of any one of the vaccines of the disclosure.
  • said intracellular pathogen is HSV-2.
  • said HSV- 2 infection is a primary HSV-2 infection, a recurrence following a primary HSV-2 infection, a genital HSV-2 infection, or an oral HSV-2 infection.
  • Said vaccine can be administered before exposure to said intracellular pathogen or after exposure to said intracellular pathogen.
  • the administration of an effective amount of any one of the vaccines disclosed herein generates an immune response against the intracellular pathogen in the subject.
  • said immune response comprises a cellular immune response, optionally mediated by
  • said immune response comprises a humoral response, optionally comprising the induction of neutralizing antibodies.
  • said immune response comprises a cellular immune response, optionally mediated by and a humoral response, optionally comprising the induction of neutralizing antibodies.
  • the vaccine is administered intradermally, mucosally, intramuscularly, subcutaneously, sublingually, rectally, or vaginally.
  • the disclosure provides a method of generating an immune response to an intracellular pathogen in a subject, the method comprising administering to the subject an effective amount of the vaccine of any of the vaccines disclosed herein.
  • the immune response comprises a cellular immune response, optionally mediated by
  • the immune response comprises a humoral response, optionally comprising the induction of neutralizing antibodies.
  • the immune response comprises a cellular immune response, optionally mediated by and ahumoral response, optionally comprising the induction of neutralizing antibodies.
  • the vaccine is administered intradermally, mucosally, intramuscularly, subcutaneously, sublingually, rectally, or vaginally.
  • FIG. 1 shows hair loss and erythema (HLE) observed in mice at the indicated days infection (DPI) following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control).
  • DPI days infection
  • results from the infected control are the top line
  • results from RVx-PM-3 are the line below
  • results from RVx-PM-2 are on the x-axis.
  • the total number of mice with HLE following each treatment or no treatment is shown in the table.
  • FIG. 2 shows disease symptoms of mice as analyzed using a score system (provided by Rational Vaccines) ranging from 0 (No Disease) to 5 (Death).
  • the graph shows the mean disease score for mice following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control).
  • the table summarizes these results.
  • FIG. 3 shows the number of animals with disease symptoms of mice following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control).
  • the graph shows the percent of mice that were disease positive following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control).
  • the table summarizes these results.
  • FIG. 4 shows survival of mice at the indicated days post-infection (DPI) following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control).
  • DPI 20 on the graph the top line is RVx-PM-2, the middle line is RVx-PM-3, and the bottom line is infected control.
  • the table summarizes these results.
  • FIG. 5 shows the mean DPI of onset of HLE symptoms for the infected ontrol mice and the mice vaccinated with RVx-PM-3. The table summarizes these results.
  • FIG. 6 shows viral titers at the indicated DPI (D2, D4, D6, and D8) following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control).
  • DPI D2, D4, D6, and D8
  • RVx-PM-2 RVx-PM3
  • RVx-PM-3 no vaccine treatment
  • FIG. 7 summarizes acute viral titers at the indicated DPI (D2, D4, D6, and D8) following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control).
  • FIG. 8 shows the percentage of mice with a positive viral swab sample at the indicated DPI (D2, D4, D6, and D8) following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control).
  • DPI D2, D4, D6, and D8
  • RVx-PM-2 RVx-PM3
  • RVx-PM-3 no vaccine treatment
  • FIG. 9 summarizes the number of virus positive animals at the indicated DPI (D2, D4, D6, and D8) following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control).
  • FIG. 10 shows the viral burden in the dorsal root ganglion (DRG) of surviving mice at 21 DPI following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control). The table summarizes these results.
  • FIG. 11 shows the percentage of mice with virus detected in the DRG 21 DPI following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control). The table summarizes these results.
  • FIG. 12 shows the viral burden in the spinal cord (SC) of surviving mice at 21 DPI following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control).
  • the table summarizes these results.
  • FIG. 13 shows the percentage of mice with virus detected in the SC 21 DPI following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control). The table summarizes these results.
  • “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In any embodiment discussed in the context of a numerical value used in conjunction with the term “about,” it is specifically contemplated that the term about can be omitted.
  • An “increased” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times ( e.g ., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 2.1, 2.2, 2.3, 2.4, etc.) greater than an amount or level described herein.
  • An “increase” may be an increase of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, at least 2-fold, at least 5-fold, or at least 10-fold.
  • a “decreased” or “reduced” or “lesser” amount is typically a “statistically significant” amount, and may include a decrease that is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) less than an amount or level described herein, for example an amount that is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of an amount or level described herein.
  • a decrease may be a decrease of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, least 80%, at least 90%, or about 100%.
  • an increase or decrease is relative to a value determined prior to infection or prior to treatment.
  • an increase or decrease is relative to a predetermined value, e.g., an average value obtained from numerous subjects, e.g., prior to infection or prior to treatment.
  • a “composition” can comprise an active agent, e.g., an immunogenic polypeptide, and a carrier, inert or active, e.g., a pharmaceutically acceptable carrier, diluent or excipient.
  • a composition may be a pharmaceutical composition.
  • the compositions are sterile, substantially free of endotoxins or non-toxic to recipients at the dosage or concentration employed.
  • mammal and “subject” includes human and non-human mammals, such as, e.g., a human, mouse, rat, rabbit, monkey, cow, hog, sheep, horse, dog, and cat.
  • HSV-2 refers, in one embodiment, to a Herpes Simplex Virus-2.
  • the term refers to an HSV-2333 strain.
  • the term refers to a 2.12 strain.
  • the term refers to an HG-52 strain.
  • the term refers to an MS strain.
  • the term refers to a G strain.
  • the term refers to a 186 strain.
  • the term refers to any other HSV-2 strain known in the art.
  • Treating” or “treatment” as used herein covers the treatment of the disease, injury, or condition of interest, e.g. , HSV-2 infection, in a biological material, e.g. , a mammal, preferably a human, having the disease or condition of interest, and includes: (i) preventing or inhibiting the disease, injury, or condition from occurring in a biological material, e.g., mammal, in particular, when such mammal is predisposed to the condition but has not yet been diagnosed as having it; (ii) reducing the severity or duration of the disease, injury or condition, e.g., when it occurs, e.g., in a mammal predisposed to the condition; (iii) inhibiting the disease, injury, or condition, i.e., arresting its development; (iv) relieving the disease, injury, or condition, i.e., causing regression of the disease or condition; or (v) relieving the symptoms resulting from the disease, injury, or condition
  • prevention includes inhibiting or impeding the onset or progression of a disease or injury or reducing the amount of injury or damage caused by a disease or injury.
  • disease includes inhibiting or impeding the onset or progression of a disease or injury or reducing the amount of injury or damage caused by a disease or injury.
  • condition may be used interchangeably.
  • isolated refers to proteins, glycoproteins, peptide derivatives or fragments or polynucleotides that are independent from its natural location. Viral components that are independently obtained through recombinant genetics means typically leads to products that are relatively purified. It is understood that the vaccines and immunogenic compositions disclosed herein comprise one or more isolated polypeptides, i.e., these polypeptides are not present within their natural location, e.g., within a pathogen. Thus, the vaccines and immunogenic compositions disclosed herein do not include live or dead pathogen or mature virion.
  • a “polypeptide” includes proteins, fragments of proteins, variants of proteins, and peptides, whether isolated from natural sources, produced by recombinant techniques or chemically synthesizes. Polypeptides of the invention typically comprise at least about 8 amino acids.
  • a polynucleotide or polypeptide has a certain “percent sequence identity” or “percent identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the worldwide web at ncbi.nlm.nih.gov/BLAST/. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA, a wholly owned subsidiary of Oxford Molecular Group, Inc.
  • GCG Genetics Computing Group
  • antibody is used in the broadest sense and specifically includes, for example, single monoclonal antibodies, polyclonal antibodies, antibody compositions with poly epitope specificity, and fragments of antibodies.
  • Antigen refers to proteins, glycoproteins or derivative or fragments that can contain one or more epitopes (linear, conformation, sequential, T-cell) which can elicit an immune response. Antigens can be separated in isolated viral proteins or peptide derivatives. As used herein, the terms “antigen”, “pathogen antigen”, and “intracellular pathogen antigen” may be used interchangeably.
  • Protein component is used in the broadest sense and specifically includes, for example, proteins, polypeptides, peptides, immunogenic protein fragments, antigens and epitopes.
  • Immunogen refers to the entire group of polypeptides that are: (a) full length antigen, (2) immunogenic fragments of the antigen, (3) immunogenic variants of the full-length antigen or variants of an immunogenic fragment, (4) chimeric fusions thereof comprising portions of a different polypeptide, and (5) conjugates thereof.
  • the proteins for use in a vaccine include a polypeptide comprising any of an immunogenic fragment thereof or a variant thereof capable of inducing an immune response specific for the protein.
  • immunogenic fragment refers to fragments or portions of proteins that elicit an antibody response or a cellular cytotoxic response that retains specificity for (cross reactivity with) the full-length protein.
  • immunogenic fragments, and variants thereof comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 48 or 50 contiguous amino acids of the antigen.
  • the immunogenic fragment may comprise any number of contiguous amino acids between the aforementioned such that, for example, an immunogenic fragment is between about 6-10, 10-15, 15-20, 20-30, 30-40, 40-50, 50-60, 60- 70, 70-80, 80-90, 90-100, or more contiguous amino acids of an immunogenic polypeptide.
  • the immunogenic fragment elicits an immune response at least 50%, at least 75%, or at least 90% as strong as the immune response elicited by the full protein.
  • a “variant” refers to a molecule having one or more amino acid modification, e.g., substitutions, deletions, or additions, as compared to the indicated amino acid sequence, yet preferably retaining the ability to be recognized by an immune cell.
  • One method for determining whether a molecule can be recognized by an immune cell is the proliferation assay described in D. M. Koelle et al., 1994, J. Virol. 68(5):2803-2810.
  • immunogenic variants retain at least 90% amino acid identity over at least 10 contiguous amino acids of the antigen, or at least 85% amino acid identity over at least 15 contiguous amino acids of the antigen (e.g. an envelope protein or non-envelope structural protein).
  • variants of antigens or sequences disclosed herein may have, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to a reference sequence.
  • an immunogenic variant has at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over the full length of a particular antigen.
  • the variant is a naturally occurring variant.
  • the variant is an engineered variant.
  • immune response refers to a subject’s response by the immune system to immunogens (i.e., antigens) that the subject’s immune system recognizes as foreign.
  • Immune responses include both cell-mediated immune responses (responses mediated by antigen-specific T cells and non-specific cells of the immune system — humoral immune responses (responses mediated by antibodies).
  • the term “immune response” encompasses both the initial “innate immune responses” to an immunogen, as well as memory responses that are a result of “acquired immunity”.
  • Immuno enhancing refers to a significant boost in the level and breath of the innate and/or acquired immune response to a given pathogen following administration of a vaccine of the present invention relative to the level of innate and acquired immunity when a vaccine of the present invention has not been administered.
  • Subunit refers to isolated and optionally purified proteins, e.g., HSV-2 proteins, that are compositions of a vaccine.
  • the subunit vaccine is preferably free from mature virions, cells, or lysate of cells or virions.
  • Viral antigens included in a subunit vaccine can be produced using standard recombinant genetics techniques and synthetic methods, e.g., recombinant expression, and with standard isolation and purification protocols.
  • “Pharmaceutically acceptable carrier” includes any material which, when combined with an active ingredient, allow the ingredient to retain biological activity and is non-reactive with the subject’s immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline.
  • “Therapeutically effective amount” refers to any amount of the HSV-2 vaccine that is effective in preventing, treating or ameliorating a disease caused by the HSV-2 pathogen associated with the immunogen administered in the composition of the present invention.
  • a therapeutically effective amount of the HSV-2 vaccine may be an amount effective to treat an HSV-2-infected subject, or an amount effective to prophylactically prevent a non-HSV-2- infected subject from being infected by HSV-2.
  • “protective immune response” it is meant that the immune response is associated with prevention, treating, impeding, or amelioration of a disease. Complete prevention is not required, though is encompassed by the present invention.
  • an effective amount is an amount sufficient to significantly reduce the frequency of outbreaks in an HSV- 2-infected subject. In certain embodiments, an effective amount is an amount sufficient to significantly reduce the duration of one or more outbreaks in an HSV-2-infected subject.
  • an effective amount is an amount sufficient to impede, e.g., inhibit or reduce, an HSV-2 infection or a primary HSV-2 infection.
  • the terms “impeding a HSV-2 infection” and “impeding a primary HSV-2 infection” refer, in one embodiment, to decreasing the titer of infectious virus by at least 90%. In another embodiment, the titer is decreased by at least 50%. In another embodiment, the titer is decreased by at least 55%. In another embodiment, the titer is decreased by at least 60%. In another embodiment, the titer is decreased by at least 65%. In another embodiment, the titer is decreased by at least 70%. In another embodiment, the titer is decreased by at least 75%.
  • the titer is decreased by at least 80%. In another embodiment, the titer is decreased by at least 85%. In another embodiment, the titer is decreased by at least 92%. In another embodiment, the titer is decreased by at least 95%. In another embodiment, the titer is decreased by at least 96%. In another embodiment, the titer is decreased by at least 97%. In another embodiment, the titer is decreased by at least 98%. In another embodiment, the titer is decreased by at least 99%. In another embodiment, the titer is decreased by over 99%.
  • DBP refers to the HSV Major DNA-binding Protein encoded by the UL29 gene (UniProt accession number P89452; SEQ ID NO:l). DBP is a single-stranded DNA-binding protein required for DNA replication, it participates in the opening of the viral DNA origin to initiate replication by interacting with the origin-binding protein. As used herein, DBP would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • MCP refers to the HSV Major Capsid Protein encoded by the UL19 gene (UniProt accession number W0NW54; SEQ IDNOs:2 and 3; SEQ ID NO:2 is amino acids 1-560 (MCP- 1), and SEQ ID NO:3 is amino acids 5611-1373 (MCP-2)). MCP self-assembles to form a capsid in the nucleus of the infected host cell. The capsid, once formed, will be packaged with virus DNA and transported to the cytoplasm of the host cell.
  • MCP would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • gB refers to the HSV envelope glycoprotein B encoded by the UL27 gene (UniProt accesssion number P08666;; SEQ ID NO:4). gB contains multiple transmembrane segments and is essential for viral entry into host cells. As used herein, gB would encompass the full- length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • gC refers to the HSV envelope glycoprotein C encoded by the UL44 gene (UniProt accession number Q89730; SEQ ID NO: 5). gC mediates viral attachment to host cells and acts to modulate complement activation in the innate immune response. As used herein, gC would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • RIR1 refers to the HSV ribonucleoside-di phosphate reductase large subunit encoded by the UL39 gene (UniProt accession number P89462; SEQ ID NO:6). RIR1 provides the precursors necessary for DNA synthesis and catalyzes the biosynthesis of deoxyribonucleotides from the corresponding ribonucleotides. As used herein, RIR1 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • ICP4 refers to the HSV major viral transcription factor ICP4 encoded by the gene RSI (UniProt accession number P90493; SEQ ID NO:8). It plays an essential role in the regulation of viral gene expression by both activating and repressing host RNA polymerase II- mediated transcription. As used herein, ICP4 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • [00100] refers to the HSV triplex capsid protein 2 encoded by the gene UL18
  • TRX2 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL42 refers to the HSV DNA polymerase processivity factor encoded by the gene UL42 (UniProt accession number P89463; SEQ ID NO: 10).
  • UL42 plays an essential role in viral DNA replication by acting as the polymerase accessory subunit.
  • UL42 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • gG refers to the HSV envelope glycoprotein G encoded by the gene US4 (UniProt accession number P13290; SEQ ID NO: 11). gG is a chemokine-binding protein that inhibits neutrophils chemotaxis. As used herein, gG would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • VP22 refers to the HSV tegument protein VP22 encoded by the gene UL49 (UniProt accession number P89449; SEQ ID NO: 12). VP22 plays different roles during the time course of infection. It participates in both the accumulation of viral mRNAs and viral protein translation at late time of infection. As used herein, VP22 would encompass the full- length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • gD refers to the HSV envelope glycoprotein encoded by the US6 gene (UniProt accession number Q69467; SEQ ID NO: 13).
  • the gD glycoprotein is a multifunction protein that helps to define viral host tropism.
  • gD would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL12 refers to the HSV alkaline nuclease encoded by the gene UL12 (UniProt accession number P89435; SEQ ID N014). UL12 plays a role in processing non linear or branched viral DNA intermediates in order to promote the production of mature packaged unit- length linear progeny viral DNA molecules. As used herein, UL12 would encompass the full- length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL25 refers to the human herpes virus 2 capsid vertex component 2 encoded by the gene UL25 (UniProt accession number D6PUY5; SEQ ID NO:23). As used herein, UL25 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL26 refers to the human herpes virus 2 capsid vertex component 2 encoded by the gene UL26 (UniProt accession number Q69527; SEQ ID NO:24). As used herein, UL26 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL54 refers to the HSV mRNA export factor encoded by the gene UL54 (UniProt accession number P28276; SEQ ID NO: 15). It is a multifunctional regulator of the expression of viral genes that contributes to the shutoff of host protein synthesis and mediates nuclear export of viral mRNAs. As used herein, UL54 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • gE refers to HSV envelope glycoprotein encoded by the US 8 gene (UniProt accession number P89475; SEQ ID NO: 16). The gE glycoprotein has been shown to form a heterodimer with gl and functions in virion transport and modulating host defense. As used herein, gE would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL37 refers to the HSV inner tegument protein encoded by the gene UL37 (UniProt accession number P89460; SEQ ID NO: 17). UL37 plays an essential role in cytoplasmic secondary envelopment during viral egress. As used herein, UL37 would encompass the full- length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL47 refers to the HSV tegument protein encoded by the gene UL47 (UniProt accession number P89467; SEQ ID NO: 18). The tegument protein binds to various RNA transcripts and plays a role in the attenuation of selective viral and cellular mRNA degradation by modulating the activity of host shutoff RNase UL41. As used herein, UL47 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • NEC2 refers to the Nuclear Egress Protein 2 encoded by the gene UL34 (UniProt accession number P89457; SEQ ID NO: 19). Nuclear Egress Proteins play an essential role in virion nuclear egress, the first step of virion release from an infected cell. As used herein, NEC2 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • LTPD refers to the Large Tegument Protein Deneddylase encoded by the gene UL36 (UniProt accession number P89459; SEQ ID NOS: 20 and 21; SEQ ID NO:21 includes amino acids 1-933 (LTPD-1), and SEQ ID NO:21 includes amino acids 934-2079 (LTPD-2)).
  • LTPD plays multiple roles in the viral cycle, including viral entry, routing of the capsid, and stabilizing nuclear CRL substrates.
  • LTPD would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • NEC1 refers to the Nuclear Egress Protein 1 encoded by the gene UL31 (UniProt accession number P89454; SEQ ID NO:22). Nuclear Egress Proteins play an essential role in virion nuclear egress, the first step of virion release from an infected cell. As used herein, NEC1 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • gH refers to the HSV envelope glycoprotein H encoded by the UL22 gene (UniProt accession number G9I243; SEQ ID NO:25). gH is required for the fusion of viral and plasma membranes leading to virus entry into the host cell. As used herein, gH would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL50 refers to the HSV deoxyuridine 5 '-triphosphate nucleotidohydrolase encoded by the UL50 gene (UniProt accession number G9I273; SEQ ID NO:26). UL50 is involved in nucleotide metabolism: produces dUMP and decreases the intracellular concentration of dUTP to avoid uracil incorporation into viral DNA. As used herein, UL50 would encompass the full- length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL40 refers to the HSV ribonucleoside-diphosphate reductase small subunit encoded by the UL40 gene (UniProt accession number B9X2I1; SEQ ID NO:27). UL40 provides the precursors necessary for viral DNA synthesis; it allows virus growth in dividing cells, as well as reactivation from latency in infected hosts. As used herein, UL40 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL48 refers to the HSV tegument protein VP16 encoded by the UL48 gene (UniProt accession number G9I270; SEQ ID NO:28). UL48 acts as a key activator of lytic infection by initiating the lytic program through the assembly of the transcriptional regulatory VP 16- induced complex composed of VP16 and two cellular factors, HCFC1 and POU2F 1. As used herein, UL48 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • TK refers to the HSV thymidine kinase encoded by the TK gene (UniProt accession number Q6L709; SEQ ID NO:29). TK catalyzes the transfer of the gamma-phosphate group of ATP to thymidine to generate dTMP in the salvage pathway of pyrimidine synthesis. As used herein, TK would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL35 refers to the HSV small capsomere-interacting protein encoded by the UL35 gene (UniProt accession number G9I257; SEQ ID NO:30). UL35 participates in the assembly of the infectious particles by decorating the outer surface of the capsid shell and thus forming a layer between the capsid and the tegument. As used herein, UL35 would encompass the full- length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL21 refers to the HSV tegument protein encoded by the UL21 gene (UniProt accession number G9I242; SEQ ID NO:31). UL21 may participate in DNA packaging/capsid maturation events. As used herein, UL21 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL1 refers to the HSV envelope glycoprotein L encoded by the UL1 gene (UniProt accession number G9I222; SEQ ID NO:32).
  • the heterodimer glycoprotein H-gly coprotein L is required for the fusion of viral and plasma membranes leading to virus entry into the host cell.
  • UL1 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • the disclosure provides methods for identifying one or more protein components for use in a vaccine against an intracellular pathogen.
  • the methods include proteomic methods as well as methods based on cellular immune response.
  • the method comprises: a) contacting a cell lysate prepared from cells infected with an intracellular pathogen, with serum obtained from blood of a subject previously infected by the intracellular pathogen, under conditions and for a time sufficient for antibodies against the intracellular pathogen present in the serum to bind intracellular pathogen antigens in the cell lysate, thereby forming a mixture comprising antibody/antigen complexes; and b) identifying, and optionally quantifying, antigens present in the antibody/antigen complexes, thereby identifying one or more protein components for use in a vaccine against the intracellular pathogen.
  • the antibody/antigen complexes are separated from the mixture, optionally by immunoprecipitation, before the antigens are identified and/or quantified.
  • the cell lysate may be prepared from host cells infected with the intracellular pathogen, e.g., live virus.
  • the host cells are Vero cells, L7 cells, LLC- or MDCK cells.
  • the host cells may be infected with the intracellular pathogen, e.g., live virus, by contacting the host cells with the intracellular pathogen, e.g., live virus, under suitable conditions and for a sufficient time for infection to occur, as known in the art.
  • the host cells are contacted with the intracellular pathogen, e.g., live virus, in liquid suspension.
  • the host cells are contacted with the intracellular pathogen, e.g., live virus, while adhered to a solid support.
  • the inoculation density of the cells is between about 10,000 cells/cm 2 to about 150,000 cells/cm 2 . In certain embodiments, the inoculation density of the cells is about 10,000 cells/cm 2 , about 20,000 cells/cm 2 , about 30,000 cells/cm 2 , about 40,000v, about 50,000v, about 60,000 cells/cm 2 , about 70,000v, about 80,000 cells/cm 2 , about 90,000 cells/cm 2 or about 100,000 cells/cm 2 .
  • the Multiplicity of Infection (MOI) by a live virus is between 0.00001 to 10 PFU/cell or between 0.005 and 2.0 PFU/cell. In one embodiment, the MOI is about 0.005 PFU/cell. In one embodiment, the MOI is about 2 PFU/cell. In some embodiments, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of the host cells are infected by the intracellular pathogen, e.g., live virus.
  • the intracellular pathogen is a virus, a bacterium, e.g., a gram negative or gram positive bacterium, a fungus (e.g. ayeast), or protozoa.
  • the virus is aHerpes Simplex Virus 1 (HSV-1), aHerpes Simplex Virus 2 (HSV-2), or a SARS- CoV-2 virus.
  • HSV-1 Herpes Simplex Virus 1
  • HSV-2 Herpes Simplex Virus 2
  • SARS- CoV-2 virus aHerpes Simplex Virus 1
  • the infected host cells are cultured in a culture media for a period of time, e.g., to allow propagation of additional pathogens.
  • the cells may be cultured in suspension or while adhered to a solid support.
  • the time period may range depending upon the amount of virus to be produced.
  • the time period is from about 10 hours to about 100 hours, e.g., about 10 hours, about 16 hours, about 20 hours, about 30 hours, about 40 hours, about 50 hours, about 60 hours, about 70 hours, about 80 hours, about 90 hours, or about 100 hours.
  • the time period is about 48 hours to about 96 hours.
  • the time period is about 60 hours.
  • the host cells are seeded to a solid platform after virus infection and cultured for the aforementioned time period. In some embodiments, the cells are incubated at about 34° C.
  • the host cells may be harvested.
  • host cells adhered to a solid support are harvested by contacting them with a solution comprising EDTA, trypsin, or a combination thereof.
  • said solution comprises about 5 mM EDTA in phosphate buffered saline (PBS).
  • host cells are harvested by scraping or other types of physical removal. In certain embodiments, scraping may occur after contact with EDTA, trypsin, or a combination thereof.
  • the harvested host cells are separated from the solution and/or culture media by centrifugation.
  • the separated host cells are resuspended in a PBS (or DPBS) buffer, and a sample of the resuspended cells is used to calculate total number of cells.
  • cells are centrifuged and resuspended in PBS (or DPBS) buffer to reach a final cell concentration of 6-8 million cells per mL.
  • host cells may be lysed to release intracellular pathogen antigens.
  • Host cells may be lysed by any means or method known in the art, such as sonication, homogenization, chemical lysis, or a combination thereof. In some embodiments, cells are
  • serum obtained from blood of a subject previously infected by the intracellular pathogen may be contacted with the composition comprising lysed cells and released antigens, under conditions and for a time sufficient for antibodies against the intracellular pathogen present in the serum to bind intracellular pathogen antigens in the cell lysate, thereby forming a mixture comprising antibody/antigen complexes.
  • the serum comprises at least one IgG that can bind to at least one antigen of the pathogen.
  • the subject is a human subject.
  • the serum is incubated with the cell lysate for a period from about 60 minutes to about four hours, or about 120 minutes to about 150 minutes. In certain embodiments, the serum is incubated with the cell lysate at about 4 °C.
  • one or more protein components for use in the vaccine against the intracellular pathogen is identified for a plurality of subjects previously infected with the intracellular pathogen.
  • the number of subjects is between 2 and 100. In certain embodiments, the number of subjects is at least 10.
  • a plurality of serum compositions is obtained from the plurality of subjects previously infected with the intracellular pathogen by any means or method known in the art. In one example, each serum composition of the plurality of serum compositions is separately contacted with the composition comprising lysed cells and released antigens. In another example, the serum compositions obtained from different subjects are pooled, and the pooled serum composition is contacted with the composition comprising lysed cells and released antigens.
  • the serum comprises at least one IgG that can bind to at least one antigen of the pathogen.
  • the subjects are human subjects.
  • the serum is incubated with the cell lysate for a period from about 60 minutes to about four hours, or about 120 minutes to about 150 minutes. In certain embodiments, the serum is incubated with the cell lysate at about 4 °C.
  • the antibody/antigen complexes may be separated from the mixture.
  • the antibody/antigen complexes are separated by immunoprecipitation.
  • immunoprecipitation is preformed using Dynabeads Protein G (Invitrogen #10004D) according to manufacturer’s protocols.
  • the antigens present in the complexes may be identified.
  • the antigens are identified by mass spectrometry.
  • the antigens are quantified by mass spectrometry.
  • the antigens are identified by mass spectrometry and quantified by mass spectrometry.
  • identification and/or quantification by mass spectrometry comprises calculating normalized spectral abundance factors for each antigen.
  • identification and/or quantification by mass spectrometry comprises multiple reaction monitoring (MRM).
  • MRM multiple reaction monitoring
  • the method is used to identify antigens present in the complexes.
  • immunogenic fragments of the antigens may be identified by methods known in the art, for example, by synthesizing a series of overlapping peptides covering the entirety of an identified antigen, and then testing the peptides for their ability to be bound by antibodies in the sera of an infected subject. In certain embodiments, such immunogenic fragments are used in vaccines.
  • one or more protein components for use in a vaccine against the intracellular pathogen are selected.
  • the protein components are selected based on the amount of each identified in the antibody/antigen complexes of the subject.
  • the amount of each antigen refers to the percentage of the total antibody/antigen complexes for which the antigen was identified that contained the particular antigen or immunogenic fragment thereof, e.g., for a single subject.
  • any of the vaccines disclosed herein may comprise an immunogenic fragment or epitope of an identified antigen, instead of (or in addition to) the full length antigen polypeptide.
  • immunogenic fragments or peptide epitopes thereof may be advantageous for testing in certain cell-based assays of immune response disclosed herein.
  • the method further comprises testing the identified antigens, or immunogenic fragments or epitopes thereof, to determine which ones invoke a cellular immune response.
  • samples of peripheral blood mononuclear cells (PBMCs) isolated from the blood of the subject are each contacted with one or more of the identified antigen or immunogenic fragment or epitope thereof under conditions and for a time sufficient to allow a cellular immune response to occur, according to methods known in the art.
  • PBMCs peripheral blood mononuclear cells
  • the cellular immune response of each of the populations of PBMCs contacted with the identified antigen or immunogenic fragment or epitope thereof may be measured, thereby identifying the antigens or immunogenic fragments thereof that invoke a cellular immune response.
  • the cellular response is measured using an ELISPOT assay.
  • the cellular response is measured using an assays are well known in the art, and are described, for example, in Posavad et al. (Detailed Characterization of T Cell Responses to HSV-2 in Immune Seronegative Persons, J Immunol. 2010 March 15; 184(6): 3250-3259).
  • a cellular response is determined to be significant if the spot forming cell (SFC)/well is 3-4 times greater than the mean SFC/well for control wells.
  • the cellular response is measured by measuring antigen- specific T cell responses by MHC multimer flow cytometry.
  • assays are well known in the art, and are described, for example, in Zhu et al. (Virus-specific CD8+ T cells accumulate near sensory nerve endings in genital skin during subclinical HSV-2 reactivation, J Exp Med. 2007 March 19; 204(3): 595-603).
  • the method further comprises testing a library of polypeptides and/or peptides to determine which ones invoke a cellular immune response.
  • the library comprises antigens, or immunogenic fragments thereof, identified according to the method disclosed above.
  • the library of proteins comprises antigens, or immunogenic fragments thereof, identified in the antibody/antigen complexes according to the methods disclosed herein.
  • the library of proteins comprises polypeptides and/or peptides not identified in the antibody/antigen complexes.
  • the polypeptides and/or peptides are selected from a library of random and/or known polypeptides and/or peptides, e.g.
  • the polypeptides and/or peptides are selected from a library of immunogenic polypeptides or peptides comprising known and/or generated sequences.
  • the library of proteins comprises antigens or immunogenic fragments thereof identified in the antibody/antigen complexes in step c) and other immunogenic polypeptides, e.g., peptides selected from the library of random or known peptides.
  • the library of proteins is recombinantly produced and purified according to any means or method known in the art.
  • the library comprises epitopes of antigens of the intracellular pathogen. In some embodiments, different epitopes from the same antigen are used.
  • samples of peripheral blood mononuclear cells (PBMCs) isolated from the blood of the subject are each contacted with one or more polypeptide or peptide of the library under conditions and for a time sufficient to allow a cellular immune response to occur, as known in the art.
  • PBMCs peripheral blood mononuclear cells
  • the cellular immune response of each of the populations of PBMCs contacted with the polypeptide or peptide is measured, thereby identifying the polypeptides or peptides that invoke a cellular immune response.
  • the protein components for use in the vaccine are selected based on the magnitude of the cellular immune response invoked by a protein in the library. In some embodiments, the protein components are selected based on the amount of each antigen or immunogenic fragment thereof identified in the antibody/antigen complexes of the subject and the magnitude of the cellular immune response invoked.
  • antigens or immunogenic fragment thereof for use in a vaccine are identified from the blood of a plurality of patients infected with the intracellular pathogen. This can allow the identification of antigens or immunogenic fragments thereof that are common amongst different patients, which may be beneficial to include in the vaccine.
  • one or more protein components for use in a vaccine against the intracellular pathogen are selected.
  • the protein components are selected based on the amount of each antigen or immunogenic fragment thereof identified in a patient, or a plurality of patients. In the context of a plurality of patients, the amount may be the average of the amount present in each patient.
  • an antigen or immunogenic fragment thereof is selected as a protein component of the vaccine, if the antigen or immunogenic fragment thereof is present in at least at least 0.1%, at least 0.3%, at least 0.5%, at least 0.7%, at least 0.9%, at least 1%, at least 1.2%, at least 1.4%, at least 1.6%, at least 1.8%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, or at least 20%, or at least 30% of the antibody/antigen complexes examined.
  • an antigen or immunogenic fragment thereof is selected as a protein component of the vaccine, if the antigen or immunogenic fragment thereof is present in at least at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, or at least 20%, or at least 30% of the antibody/antigen complexes examined.
  • the protein components are selected based on the magnitude of the cellular immune response.
  • an antigen or immunogenic fragment thereof is selected as a protein component of the vaccine, if the antigen or immunogenic fragment thereof (or polypeptide or peptide) invokes a positive cellular immune response greater than a control composition that does not invoke a significant immune response.
  • the protein components are selected based on the frequency at which each antigen is identified amongst the plurality of subjects.
  • an antigen, or immunogenic fragment or epitope thereof is selected as a protein component of the vaccine, if antibodies directed against the antigen are present in at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the patients’ sera.
  • the protein components are selected based on the amount of antibodies directed against each antigen identified in the subject or plurality of subjects and the magnitude of the cellular immune response invoked.
  • an antigen or immunogenic fragment or epitope thereof is selected as a protein component of the vaccine, if the antigen is present in at least 0.1%, at least 0.3%, at least 0.5%, at least 0.7%, at least 0.9%, at least 1%, at least 1.2%, at least 1.4%, at least 1.6%, at least 1.8%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, or at least 20%, or at least 30% of the antibody/antigen complexes examined, and if the antigen or immunogenic fragment or epitope thereof (or polypeptide or peptide thereof (or polypeptide or peptide
  • the antigen or immunogenic fragment thereof is present in at least at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, or at least 20%, or at least 30% of the antibody/antigen complexes examined.
  • the protein components are selected based on the amount of antibodies directed to each antigen identified in the subject or plurality of subjects and the frequency at which each antigen is identified amongst the plurality of subjects.
  • an antigen, or immunogenic fragment or epitope thereof is selected as a protein component of the vaccine, if the antigen is present in at least 0.1%, at least 0.3%, at least 0.5%, at least 0.7%, at least 0.9%, at least 1%, at least 1.2%, at least 1.4%, at least 1.6%, at least 1.8%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, or at least 20%, or at least 30% of the antibody/ antigen complexes examined, and if antibodies directed to the antigen are present in at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the patients’ sera.
  • the antigen is present in at least at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, or at least 20%, or at least 30% of the antibody/antigen complexes examined.
  • the protein components are selected based on the magnitude of the cellular immune response invoked and the frequency at which each antigen is identified amongst the plurality of subjects.
  • an antigen, or immunogenic fragment or epitope thereof is selected as a protein component of the vaccine, if the antigen or immunogenic fragment or epitope thereof (or polypeptide or peptide) invokes a cellular immune response at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold greater than a control polypeptide that does not invoke a significant immune response, and if antibodies directed to the antigen arepresent in at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the patients’ sera.
  • the protein components are selected based on the amount of antibodies directed to each antigen identified in the plurality of subjects, the magnitude of the cellular immune response invoked by the antigen, or immunogenic fragment or epitope thereof, and the frequency at which antibodies directed to each antigen are identified amongst the plurality of subjects.
  • an antigen or immunogenic fragment, or epitope thereof is selected as a protein component of the vaccine, if the antigen is present in at least 0.1%, at least 0.3%, at least 0.5%, at least 0.7%, at least 0.9%, at least 1%, at least 1.2%, at least 1.4%, at least 1.6%, at least 1.8%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, or at least 20%, or at least 30% of the antibody/antigen complexes examined, and if the antigen, or immunogenic fragment or epitope thereof (or polypeptide or peptide), invokes a positive cellular immune response greater than a control composition that does not invoke a significant immune response, and if antibodies directed to the antigen or immunogenic fragment thereof is present in
  • an antibody is directed to an antigen if the antibody specifically binds to the antigen.
  • an antibody “specifically binds” or “preferentially binds” to an antigen, if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances, e.g., at least 5-fold, at least 10- fold, at least 100-fold, at least 1000-fold greater affinity or avidity.
  • the disclosure provides a method of producing a vaccine against an intracellular pathogen, comprising preparing a vaccine composition comprising one or more protein components identified according to any one of the methods disclosed herein.
  • the vaccine composition comprises at least 5, at least 10, at least 15, or at least 20 antigens, or antigenic fragments, variants, or epitopes thereof, identified according to a method disclosed herein.
  • the vaccine composition comprises less than 100, less than 50, less than 40, less than 30, less than 20, or less than 10 antigens, or antigenic fragments, variants, or epitopes thereof, identified according to a method disclosed herein.
  • the vaccine composition does not comprise the pathogen from which the vaccine composnents were identified, e.g., dead, live, or attenuated pathogen.
  • the protein components are recombinantly produced in cells, optionally E. coli, yeast, insect, or mammalian cells. Such methods of recombinant production of protein are well known in the art, and persons of skill in the art would readily select and use appropriate production methods.
  • the protein components are purified or isolated, e.g., the protein components are separate from or isolated from the pathogen from which they were identified.
  • the vaccine compositions of the disclosure confer protective immunity, allowing a vaccinated subject to exhibit delayed onset of symptoms or reduced severity of symptoms as the result of exposure to the vaccine (e.g., a memory response).
  • the reduction in severity of symptoms is at least 25%, 40%, 50%, 60%, 70%, 80% or 90%.
  • Some vaccinated individuals may display no symptoms upon contact with the pathogen or even no infection by the pathogen.
  • the duration of protective immunity is preferably as long as possible.
  • vaccine formulations produce protective immunity lasting six months, one year, two years, five years, ten years, twenty years or a lifetime.
  • Mucosal immunity is primarily the result of secretory IgA (SIGA) antibodies on mucosal Surfaces of the respiratory, gastrointestinal, and genitourinary tracts.
  • SIGA antibodies are generated after a series of events mediated by antigen processing cells, B and T lymphocytes, that result in SIGA production by B lymphocytes on mucosa-lined tissues of the body.
  • Humoral immunity is typically the result of IgG anti bodies and IgM antibodies in serum.
  • the IgG titer can be raised by 1.5-fold, 2-fold, 3-fold, 4-fold, 5- fold, 10-fold, 20-fold, 50-fold, or 100-fold or more following administration of a vaccine formulation described herein.
  • cellular immunity can be achieved through cytotoxic T lymphocytes or through delayed-type hypersensitivity that involves macrophages and T lymphocytes, as well as other mechanisms involving T cells without a requirement for antibodies.
  • the method further comprises measuring the efficacy of the vaccine.
  • vaccine efficacy is determined by measuring the immune response to the vaccine elicited in an animal model.
  • immune response refers to a subject’s response by the immune system to immunogens (i.e., antigens) that the subject’s immune system recognizes as foreign.
  • Immune responses include both cell- mediated immune responses (e.g. responses mediated by antigen-specific T cells and non specific cells of the immune system — and humoral immune responses (responses mediated by antibodies).
  • the term “immune response” encompasses both the initial “innate immune responses” to an immunogen, as well as memory responses that are a result of “acquired immunity”.
  • suitable model systems include a guinea pig model and a mouse model. Briefly, the animals are vaccinated and then challenged with the intracellular pathogen or the vaccine is administered to already-infected animals. The response of the animals to the pathogen challenge or the vaccine is then compared with control animals, using any measure known in the art (e.g. assessment of severity of symptoms, measurement of humans. The treatment and prophylactic effects described herein may represent additional ways of determining efficacy of a vaccine. Thus, in certain embodiments, vaccine efficacy is determined by measuring immunity to the pathogen following treatment with the vaccine, e.g., by treating an animal with the vaccine and then challenging immunity by infecting the animal with the pathogen from which the vaccine is intended to confer protection.
  • vaccine efficacy can be determined by in vitro immunization of naive PBMCs, where APCs are exposed to the vaccine and then the APCs are co-cultured with naive T cells from the same donor to evaluate the primary response to immunization in a test tube.
  • vaccine efficacy can be determined by viral neutralization assays. Briefly, animals are immunized, and serum is collected on various days post immunization. Serial dilutions of serum are pre-incubated with virus during which time antibodies in the serum that are specific for the virus will bind to it. The virus/serum mixture is then added to permissive cells to determine infectivity by a plaque assay. If antibodies in the serum neutralize the virus, there are fewer plaques compared to the control group.
  • the disclosure also provides a vaccine produced according to any of the methods herein, comprising one or more protein components identified according to any one of the methods disclosed herein.
  • one or more of the protein components is selected from a full length antigen identified as described herein, or an immunogenic fragment, epitope, or variant thereof.
  • the immunogenic fragment or epitope polypeptide sequence is comprised within a larger peptide or protein sequence, e.g., a fusion protein.
  • the protein component is a variant of the full length antigen or immunogenic fragment or epitope thereof.
  • the variant has at least 80%, at least 90%, at least 95%, at least 98%, at least 99% sequence identity to the wild-type antigen, or immunogenic fragment or epitope thereof.
  • a polypeptide has a certain percent "sequence identity" to another polypeptide, meaning that, when aligned, that percentage of amino acids are the same when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi.nlm.nih.gov/BLAST/. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol.
  • GCG Genetics Computing Group
  • the gap generation penalty will generally range from 1 to 5, usually 2 to 4 and in many embodiments will be 3.
  • the gap extension penalty will generally range from about 0.01 to 0.20 and in many instances will be 0.10.
  • the program has default parameters determined by the sequences inputted to be compared. In certain embodiments, the sequence identity is determined using the default parameters determined by the program. This program is available also from Genetics Computing Group (GCG) package, from Madison, Wis., USA. Another program of interest is the FastDB algorithm. FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. Percent sequence identity is calculated by FastDB based upon the following parameters:
  • each of the protein components comprises an antigen, or immunogenic fragment, variant or epitope thereof, identified for at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the subjects. In some embodiments, each of the protein components comprises an antigen, or immunogenic fragment, variant or epitope thereof, identified for at least 50% of the subjects.
  • the combination of protein components represents at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of all antigens identified in the subjects, or immunogenic fragments, variants, or epitopes thereof. In some embodiments, the combination of protein components represents at least 50% of all antigens identified in the subjects, or immunogenic fragments, variants, or epitopes thereof.
  • each of the protein components comprises an antigen, or immunogenic fragment, variant, or epitope thereof, identified for at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the subjects, and the combination of protein components represents at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of all antigens identified in the subjects, or immunogenic fragment, variant, or epitope thereof.
  • each of the protein components comprises an antigen, or an immunogenic fragment, variant, or epitope thereof, identified in at least 0.1%, at least 0.3%, at least 0.5%, at least 0.7%, at least 0.9%, at least 1%, at least 1.2%, at least 1.4%, at least 1.6%, at least 1.8%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, or at least 20% of the antibody/antigen complexes.
  • each of the protein components comprises an antigen, or an immunogenic fragment, variant, or epitope thereof, identified in at least 0.5%, at least 1%, at least 2%, or at least 3% of the antibody/antigen complexes.
  • the combination of protein components represents at least 0.1%, at least 0.3%, at least 0.5%, at least 0.7%, at least 0.9%, at least 1%, at least 1.2%, at least 1.4%, at least 1.6%, at least 1.8%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, or at least 20% of all antigens identified in the antibody/antigen complexes, or immunogenic fragment, variant, or epitope thereof.
  • the combination of protein components represents at least 10% of all antigens identified in the antibody/antigen complexes, or
  • each of the protein components comprises an antigen, or an immunogenic fragment, variant, or epitope thereof, identified in at least 0.1%, at least 0.3%, at least 0.5%, at least 0.7%, at least 0.9%, at least 1%, at least 1.2%, at least 1.4%, at least 1.6%, at least 1.8%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, or at least 20% of the antibody/antigen complexes, and the combination of protein components represents at least 0.1%, at least 0.3%, at least 0.5%, at least 0.7%, at least 0.9%, at least 1%, at least 1.2%, at least 1.4%, at least 1.6%, at least 1.8%, at least 2%, at least 2.5%, at least 3%, at least 3.5%,
  • each of the protein components comprises an antigen, or an immunogenic fragment, variant, or epitope thereof, identified in at least 50% of the subjects and in at least 1% of the antibody/antigen complexes; and the combination of protein components represents at least 50% of all antigens, or an immunogenic fragment, variant, or epitope thereof, identified in the subjects and at least 10% of all antigens identified in the antibody/antigen complexes, or immunogenic fragments, variants, or epitopes thereof.
  • one or more of the polypeptides are immunogenic fragments of the full-length polypeptide.
  • an immunogenic fragment may consist of at least 6, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more contiguous amino acids of an immunogenic polypeptide.
  • the immunogenic fragment may comprise any number of contiguous amino acids between the aforementioned such that, for example, an immunogenic fragment is between about 6-10, 10-15, 15-20, 20-30, 30-40, 40- 50, 50-60, 60-70, 70-80, 80-90, 90-100, or more contiguous amino acids of an immunogenic polypeptide.
  • the immunogenic fragments may comprise a sufficient number of contiguous amino acids that form a linear epitope and/or may comprise a sufficient number of contiguous amino acids that permit the fragment to fold in the same (or sufficiently similar) three- dimensional conformation as the full-length polypeptide from which the fragment is derived to present a non-linear epitope or epitopes (also referred to in the art as conformational epitopes).
  • Assays for assessing whether the immunogenic fragment folds into a conformation comparable to the full-length polypeptide include, for example, the ability of the protein to react with mono- or polyclonal antibodies that are specific for native or unfolded epitopes, the retention of other ligand-binding functions, and the sensitivity or resistance of the polypeptide fragment to digestion with proteases (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, NY (2001)).
  • the three-dimensional conformation of a polypeptide fragment is sufficiently similar to the full- length polypeptide when the capability to bind and the level of binding of an antibody that specifically binds to the full-length polypeptide is substantially the same for the fragment as for the full-length polypeptide (i.e., the level of binding has been retained to a statistically, clinically, and/or biologically sufficient degree compared with the immunogenicity of the exemplary or wild-type full-length antigen).
  • the vaccine further comprises one or more adjuvants.
  • the adjuvant(s) is selected from: aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; Freund’s Incomplete Adjuvant and Complete Adjuvant;
  • the vaccine is a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers or diluents.
  • excipients, carriers or diluents used in methods of the present disclosure include, but are not limited to, water, phosphate-buffered saline, a gum, a starch (e.g. com starch, pregel etanized starch), a sugar ( e.g. lactose, mannitol, sucrose, dextrose), a cellulosic material ( e.g. microcrystalline cellulose), an acrylate ( e.g. polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.
  • the vaccine is delivered in a vesicle, e.g. a liposome.
  • pharmaceutically acceptable carriers for liquid formulations are aqueous or non-aqueous solutions, suspensions, emulsions or oils.
  • non- aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipid from milk or eggs.
  • parenteral vehicles for subcutaneous, intravenous, intraarterial, or intramuscular injection
  • parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like.
  • sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants.
  • water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.
  • oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipid from milk or eggs.
  • the vaccine compositions further comprise binders (e.g. acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxy propyl methyl cellulose, povidone, disintegrating agents (e.g. cornstarch, potato starch, alginic acid, silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g. Tris-HCL, acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g.
  • binders e.g. acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxy propyl methyl cellulose, povidone
  • disintegrating agents e.g. cornstarch, potato starch, alginic acid, silicon dioxide
  • aspartame, citric acid preservatives (e.g. Thimerosal, benzyl alcohol, parabens), lubricants (e.g. stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethyl phthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropyl cellulose, sodium 10 lauryl sulfate), polymer coatings ( e.g. poloxamers or poloxamines), coating and film forming agents ( e.g. ethyl cellulose, acrylates, polymethacrylates) and/or adjuvants.
  • lubricants e.g. stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate
  • flow-aids e.g. colloidal silicon dioxide
  • HSV-2 (herpes simplex virus type 2) is an enveloped virus. Its genome expresses over 75 different proteins, many of which are structural and are used to form the capsid and tegument, while others are part of the envelope.
  • Major capsid proteins include those expressed from open reading frames (protein names are in parentheses if the common name differs from the ORF name) UL6, UL18 (VP23), UL19 (MCP/VP5), UL35 (VP26) and UL38;
  • major tegument proteins include UL7, UL11, UL13, UL14, UL16, UL17, UL21, UL25, UL36, UL37, UL41, UL46 (VP11/12), UL47 (VP13/14), UL48 (VP16), UL49, UL51, and US11;
  • major envelope proteins include UL1 (glycoprotein L (gL)), UL10 (gM), UL20, UL22 (gH),
  • HSV-2 genome sequence is found in GenBank Accession No. NC001798.1 (incorporated herein by reference in its entirety). It is understood that the commonly used protein names may be different from the gene names, e.g., UL19 encodes MCP, but reference to the gene name herein is the same as a reference to the encoded protein. It is also understood that the exact sequence of a protein may vary from one herpesvirus to another, and thus all references to an HSV-2 protein (structural or envelope or non-envelope) encompass any such protein obtainable from any naturally occurring HSV-2. A number of HSV-2 sequences are already known and deposited in databases.
  • HSV-2 Nucleic acid encoding an HSV-2 protein with an alternative sequence can be readily isolated or amplified from one or more HSV-2 (e.g. a deposited HSV-2 or a clinical isolate) with appropriate oligonucleotide probes or primers (e.g. that specifically hybridize to a reference sequence under stringent conditions).
  • HSV-2 encodes 14 or more envelope-associated proteins, at least some of which are involved with cellular entry, including but not limited to gB, gC, gG, gD, and gE.
  • gB appears to mediate membrane fusion; gC appears to mediate viral attachment to host cells; gD appears to bind specifically to an HSV-2 receptor on cells, and gG is a chemokine-binding protein that inhibits neutrophils chemotaxis. Additionally, gE has been shown to modulate host defense.
  • HSV-2 other than envelope proteins
  • the tegument occupies the space between the capsid and the envelope.
  • Capsid proteins form a structure that surrounds the nucleic acid genome of the virion.
  • MCP the product of UL19, is the major capsid protein.
  • the cellular response involves both CD4 and CD8 T cells, cell types that play a role in combating HSV-2 infections.
  • the present disclosure provides immunogenic and pharmaceutical compositions, e.g., HSV-2 vaccines, and their use for treatment or prevention of HSV-2 infection.
  • the HSV-2 vaccines comprise immunogenic HSV-2 viral proteins or immunogenic fragments of the viral proteins, or variants of the immunogenic HSV-2 viral proteins or immunogenic fragments thereof.
  • the HSV-2 vaccines comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or at least eleven polypeptides identified in Example 1 as primary antigens, i.e., antigens selected from the group consisting of: UL29, UL19, UL27, UL39, UL38, UL25, UL26, UL18, US6, UL44, and UL49 proteins. Immunogenic fragments and variants of any of these antigens may be used.
  • the HSV-2 vaccines comprise all eleven of these antigens, or immunogenic fragments or variants thereof.
  • the HSV-2 vaccines comprise any of the following combinations of isolated polypeptides or immunogenic fragments or variants thereof:
  • the HSV-2 vaccines further comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or at least fourteen polypeptides identified in Example 1 as secondary antigens, i.e., antigens selected from the group consisting of: RSI, UL22, UL12, US8, UL42, US4, UL50, UL21, UL54, UL40, UL48, UL1, TK, and UL35. Immunogenic fragments and variants of any of these antigens may be used. In certain embodiments, the HSV-2 vaccines comprise all fourteen of these antigens, or immunogenic fragments or variants thereof.
  • the HSV-2 vaccines comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen polypeptides selected from the group consisting of: DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, UL12, UL25, and UL26. Immunogenic fragments and variants of any of these antigens may be used.
  • the HSV-2 vaccines comprises: DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, UL12, UL25, and UL26, or immunogenic antigen fragments or variants thereof.
  • the HSV- 2 vaccines comprise any of the following combinations of isolated polypeptides (or immunogenic fragments or variants thereof):
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, UL12, UL25, and UL26;
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, gD, UL12, UL25, and UL26;
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, VP22, gD, UL12, UL25, and UL26;
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, gG, VP22, gD, UL12, UL25, and UL26;
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, ICP4, UL42, gG, VP22, gD, UL12, UL25, and UL26;
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, TRX2, UL42, gG, VP22, gD, UL12, UL25, and UL26;
  • DBP DBP, MCP, gB, gC, RIR1, ICP4, TRX2, UL42, gG, VP22, gD, UL12, UL25, and UL26;
  • DBP DBP, MCP, gB, gC, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, UL12, UL25, and UL26;
  • DBP DBP, MCP, gB, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, UL12, UL25, and UL26;
  • DBP DBP, MCP, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, UL12, UL25, and UL26;
  • MCP MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, UL12, UL25, and UL26.
  • the MCP or immunogenic fragment thereof comprises one or both of MCP l-560aa and MCP 561-1373aa.
  • the gB or immunogenic fragment thereof comprises GB 30-730aa.
  • the gC or immunogenic fragment thereof comprises, gC 28-447aa.
  • the HSV-2 vaccines alternatively or further comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or at least fourteen polypeptides identified in Example 1 as secondary antigens, i.e., antigens selected
  • any of the HSV-2 vaccines comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or at least thirteen polypeptides selected from the group embodiments, the HSV-2 vaccines comprise all thirteen of these polypeptides. In some embodiments, the HSV-2 vaccines comprise any of the following combinations of polypeptides:
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, and UL12;
  • MCP MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, and UL12;
  • DBP DBP, MCP, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, and UL12;
  • DBP DBP, MCP, gB, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, and UL12;
  • DBP DBP, MCP, gB, gC, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, and UL12;
  • DBP DBP, MCP, gB, gC, RIR1, ICP4, TRX2, UL42, gG, VP22, gD, and UL12;
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, TRX2, UL42, gG, VP22, gD, and UL12;
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, ICP4, UL42, gG, VP22, gD, and UL12;
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, gG, VP22, gD, and UL12;
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, VP22, gD, and UL12;
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, gD, and UL12;
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, and UL12; or DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, and gD.
  • the MCP or immunogenic fragment thereof comprises one or both of MCP l-560aa and MCP 561-1373aa.
  • the gB or immunogenic fragment thereof comprises GB 30-730aa.
  • the gC or immunogenic fragment thereof comprises, gC 28-447aa.
  • the HSV-2 vaccines alternatively or further comprise at least one, at least two, at least three, at least four, at least five, at least six, or at least seven, polypeptides selected from the group consisting of: UL54, gE, UL37, UL47, NEC2, LTPD, and NEC1. Immunogenic fragments and variants may be used.
  • the HSV-2 vaccines comprise any of the following combinations of isolated polypeptides or immunogenic fragments or variants thereof:
  • the HSV-2 vaccines comrpise at at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, or at least 27, polypeptides selected from the group consisting of: UL37, UL36, UL6, UL47, UL10, UL46, UL45, UL34, US7, UL2, UL55, UL17, UL30, US2, US1, UL51, US9, UL13, UL53, UL31, US3, US12, UL52, UL16, UL5, UL9, and RL2. Immunogenic fragments and variants may be used.
  • the HSV-2 vaccines comprise DBP or an immunogenic fragment or variant thereof.
  • the HSV-2 vaccines comprise the following combinations of isolated polypeptides: DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, UL12; UL54, gE, UL37, UL47, NEC2, LTPD, and NEC1. Immunogenic fragments and variants may be used.
  • the HSV-2 vaccines comprise the following combinations of isolated polypeptides: DBP, MCP l-560aa, MCO 561-1373aa, gB 30-730aa, gC 28-447aa, and R1R1. Immunogenic fragments and variants may be used.
  • the HSV-2 vaccines comprise the following combinations of isolated polypeptides: UL37, UL47, MEC2, LTPD l-933aa, LTPD 934-2079aa, and NEC1. Immunogenic fragments and variants may be used.
  • the HSV-2 compositions comprise a combination of immunogenic isolated polypeptides, wherein the combination includes antigens that account for (or are specifically bound by) at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% of the anti-HSV-2 antibodies observed in the serum of infected individuals as determined in Example 1 and set forth in Table 1.
  • a combination that accounts for at least 40% of the anti-HSV-2 antibodies observed in the serum of infected individuals may include, e.g., UL29, UL19, UL27 and UL39.
  • one or more of the polypeptides are immunogenic fragments of the full length polypeptide.
  • an immunogenic fragment may consist of at least 6, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more contiguous amino acids of an immunogenic polypeptide.
  • the immunogenic fragment may comprise any number of contiguous amino acids between the aforementioned such that, for example, an immunogenic fragment is between about 6-10, 10-15, 15-20, 20-30, 30-40, 40- 50, 50-60, 60-70, 70-80, 80-90, 90-100, or more contiguous amino acids of an immunogenic polypeptide.
  • the immunogenic fragments may comprise a sufficient number of contiguous amino acids that form a linear epitope and/or may comprise a sufficient number of contiguous amino acids that permit the fragment to fold in the same ( or sufficiently similar) three- dimensional conformation as the full-length polypeptide from which the fragment is derived to present a non-linear epitope or epitopes (also referred to in the art as conformational epitopes).
  • Assays for assessing whether the immunogenic fragment folds into a conformation comparable to the full-length polypeptide include, for example, the ability of the protein to react with mono- or polyclonal antibodies that are specific for native or unfolded epitopes, the retention of other ligand-binding functions, and the sensitivity or resistance of the polypeptide fragment to digestion with proteases (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, NY (2001)).
  • the three-dimensional conformation of a polypeptide fragment is sufficiently similar to the full- length polypeptide when the capability to bind and the level of binding of an antibody that specifically binds to the full-length polypeptide is substantially the same for the fragment as for the full-length polypeptide (i.e., the level of binding has been retained to a statistically, clinically, and/or biologically sufficient degree compared with the immunogenicity of the exemplary or wild-type full-length antigen).
  • any of the HSV-2 vaccines disclosed herein comprise one or more immunogenic fragments of MCP.
  • the immunogenic fragment of MCP comprises or consists of amino acids 1-560 of MCP, or is an immunogenic fragment or variant thereof.
  • the immunogenic fragment of MCP comprises or consists of amino acids 561-1373 of MCP, or is an immunogenic fragment or variant thereof.
  • any of the HSV-2 vaccines disclosed herein comprise one or more immunogenic fragments of gB.
  • the immunogenic fragment of gB comprises or consists of amino acids 30-730 of gB, or is an immunogenic fragment or variant thereof.
  • any of the HSV-2 vaccines disclosed herein comprise one or more immunogenic fragments of gC.
  • the immunogenic fragment of gC comprises or consists of amino acids 28-447 of gC, or is an immunogenic fragment or variant thereof.
  • an HSV-2 vaccine disclosed herein comprises: DBP, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, UL12, an immunogenic fragment of MCP that comprising or consisting of amino acids 1-560 of MCP, an immunogenic fragment of MCP comprising or consisting of amino acids 561-1373 of MCP, immunogenic fragment of gB comprising or consisting of amino acids 30-730 of gB, and an immunogenic fragment of gC comprising or consisting of amino acids 28-447 of gC, or immunogenic fragments or variants of any of these polypeptides.
  • theHSV-2 vaccine further comprises an adjuvant, e.g., QS-21.
  • the HSV-2 vaccines further comprise one or more pharmaceutically acceptable excipient, diluent, or carrier.
  • an HSV-2 vaccine disclosed herein comprises: DBP, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, UL12, an immunogenic fragment of MCP that comprising or consisting of amino acids 1-560 of MCP, an immunogenic fragment of MCP comprising or consisting of amino acids 561-1373 of MCP, immunogenic fragment of gB comprising or consisting of amino acids 30-730 of gB, an immunogenic fragment of gC comprising or consisting of amino acids 28-447 of gC, UL54, gE, UL37, UL47, NEC2, LTPD, and NEC1, or immunogenic fragments or variants of any of these polypeptides.
  • theHSV-2 vaccine further comprises an adjuvant, e.g., QS-21.
  • the HSV-2 vaccines further comprise one or more pharmaceutically acceptable excipient, diluent, or carrier.
  • any of the HSV-2 vaccines disclosed herein further comprises one or more adjuvant, i.e., substances that enhance the immune response to an antigen, including any of those disclosed herein.
  • the adjuvant comprises a saponin, such as QS-21.
  • adjuvant refers to an agent that increases the immune response to an antigen (e.g., HSV-2 surface antigens).
  • adjuvants include, but are not limited to, aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; Freund’s Incomplete Adjuvant and Complete Adjuvant; Merck Adjuvant 65; an emulsion; a saponin, preferably QS- 21; a modified saponin; an unmethylated CpG dinucleotide; MF59; Montanide; AS02; AS04; ISCOM; a helper peptide; a TLR agonist; AS-2; MPL or 3d-MPL; LEIF; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; bio-degradable microspheres; monophosphoryl lipid A and quil
  • any of the HSV-2 vaccines further comprises one or more pharmaceutically acceptable excipient, diluent, or carrier.
  • any of the HSV-2 vaccines are formulated to be administered parenterally, by injection, for example, subcutaneously, intraepithelially (with or without scarification), intra-muscularly, intra-dermally, epithelially, nasally, vaginally, or orally.
  • the HSV-2 vaccines are formulated to be administered intradermally.
  • An immunogenic composition or vaccine comprising 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more polypeptides selected from: a) a polypeptide comprising DBP or an immunogenic fragment or variant thereof; b) a polypeptide comprising MCP or an immunogenic fragment or variant thereof; c) a polypeptide comprising gB or an immunogenic fragment or variant thereof; d) a polypeptide comprising gC or an immunogenic fragment or variant thereof; e) a polypeptide comprising RIR1 or an immunogenic fragment or variant thereof; f) a polypeptide comprising TRX1 or an immunogenic fragment or variant thereof; g) a polypeptide comprising ICP4 or an immunogenic fragment or variant thereof; h) a polypeptide comprising TRX2 or an immunogenic fragment or variant thereof; i) a polypeptide comprising UL42 or an immunogenic fragment
  • composition of embodiment 1, wherein DBP, MCP, gB, gC, RIR1, TRX1, ICP4 (P90493), TRX2, UL42, gG, VP22, gD, and UL12 have at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NOs 1, 2 or 3, 4, 5, 6, 7, 8 ,9, 10, 11, 12, 13, or 14, respectively.
  • composition of embodiment 1, wherein the polypeptide selected from DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, and UL12 is the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • the composition of any one of embodiments 1-3 comprising DBP or an immunogenic fragment or variant thereof.
  • the composition of embodiment 5, wherein the immunogenic fragment of MCP comprises amino acids 1-560 of MCP or an immunogenic variant thereof.
  • composition of embodiment 5, wherein the immunogenic fragment of MCP comprises amino acids 561-1373 of MCP or an immunogenic variant thereof.
  • the composition of embodiment 8, wherein the immunogenic fragment of gB comprises amino acids 30-730 of gB or an immunogenic variant thereof.
  • the composition of embodiment 10, wherein the immunogenic fragment of gC comprises amino acids 28-447 of gC or an immunogenic variant thereof.
  • composition of any one of embodiments 1-3 comprising ICP4 or an immunogenic fragment or variant thereof.
  • the composition of any one of embodiments 1-3 comprising TRX2 or an immunogenic fragment or variant thereof.
  • the composition of any one of embodiments 1-3 comprising UL42 or an immunogenic fragment or variant thereof.
  • the composition of any one of embodiments 1-3 comprising gG or an immunogenic fragment or variant thereof.
  • the composition of embodiment 17, wherein the immunogenic fragment of gG comprises amino acids 23-650 of gG or an immunogenic variant thereof.
  • composition of embodiment 20, wherein the immunogenic fragment of gD comprises amino acids 26-310 of gD or an immunogenic variant thereof.
  • the composition of any one of embodiments 1-3 comprising UL12 or an immunogenic fragment or variant thereof.
  • the composition of embodiment 1, comprising: a polypeptide comprising UL25 or an immunogenic fragment or variant thereof; a polypeptide comprising UL26 or an immunogenic fragment or variant thereof;
  • the composition of embodiment 1, wherein the composition comprises all 13 polypeptides or an immunogenic fragment or variant thereof: DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, UL12.
  • composition of any one of embodiments 1-25 further comprising 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more polypeptides selected from the group consisting of: a) a polypeptide comprising UL54 or an immunogenic fragment or variant thereof, b) a polypeptide comprising gE or an immunogenic fragment or variant thereof, c) a polypeptide comprising UL37 or an immunogenic fragment or variant thereof, d) a polypeptide comprising UL47 or an immunogenic fragment or variant thereof, e) a polypeptide comprising NEC2 (UL34) or an immunogenic fragment or variant thereof, f) a polypeptide comprising LTPD (UL36) or an immunogenic fragment or variant thereof, and g) a polypeptide comprising NEC1 (UL31) or an immunogenic fragment or variant thereof.
  • composition of embodiment 26, wherein UL54, gE, UL37, UL47, NEC2, LTPD, NEC1 have at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 15, 16, 17, 18, 19, 20 or 21, or 22, respectively.
  • the composition of embodiment 26, wherein the polypeptide selected from UL54, gE, UL37, UL47, NEC2, LTPD, and NEC1 is the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • composition of embodiment 26, wherein the immunogenic fragment of gE comprises amino acids 21-359 of gE or an immunogenic variant thereof.
  • composition of embodiment 26, wherein the immunogenic fragment of LTPD comprises amino acids 1-933 of LTPD or an immunogenic variant thereof.
  • composition of embodiment 26, wherein the immunogenic fragment of LTPD comprises amino acids 934-2079 of LTPD or an immunogenic variant thereof.
  • composition of embodiment 26, wherein the composition comprises all 7 polypeptides.
  • composition of any one of embodiments 26-32 wherein the composition comprises: a. the polypeptide comprising DBP or an immunogenic fragment or variant thereof, b. the polypeptide comprising MCP or an immunogenic fragment or variant thereof, c. the polypeptide comprising gB or an immunogenic fragment or variant thereof, d. the polypeptide comprising gC or an immunogenic fragment or variant thereof, e. the polypeptide comprising RIR1 or an immunogenic fragment or variant thereof, f. the polypeptide comprising TRX1 or an immunogenic fragment or variant thereof, g. the polypeptide comprising ICP4 or an immunogenic fragment or variant thereof, h. the polypeptide comprising TRX2 or an immunogenic fragment or variant thereof, i.
  • the polypeptide comprising UL42 or an immunogenic fragment or variant thereof j. the polypeptide comprising gG or an immunogenic fragment or variant thereof, k. the polypeptide comprising VP22 or an immunogenic fragment or variant thereof, l. the polypeptide comprising gD or an immunogenic fragment or variant thereof, m. the polypeptide comprising UL12 or an immunogenic fragment or variant thereof; n. the polypeptide comprising UL54 or an immunogenic fragment or variant thereof, o. the polypeptide comprising gE or an immunogenic fragment or variant thereof, p. the polypeptide comprising UL37 or an immunogenic fragment or variant thereof, q. the polypeptide comprising UL47 or an immunogenic fragment or variant thereof, r.
  • the composition comprises: an immunogenic fragment of MCP comprising amino acids 1-560 of MCP or an immunogenic variant thereof; an immunogenic fragment of MCP comprising amino acids 561-1373 of MCP or an immunogenic variant thereof; an immunogenic fragment of gB comprising amino acids 30-730 of gB or an immunogenic variant thereof; an immunogenic fragment of gC comprising amino acids 28-447 of gC or an immunogenic variant thereof; an immunogenic fragment of gG comprising amino acids 23-650 of gG or an immunogenic variant thereof; an immunogenic fragment of gD comprising amino acids 26-310 of gD or an immunogenic variant thereof; an immunogenic fragment of gE comprising amino acids 21-359 of gE or an immunogenic variant thereof; an immunogenic fragment of LTPD comprising amino acids 1-560 of MCP or an immunogenic variant thereof; an immunogenic fragment of MCP comprising amino acids 561-1373 of MCP or an immunogenic variant thereof; an immunogenic fragment of gB comprising amino acids 30-730 of gB or an
  • composition of embodiment 35 wherein said one or more adjuvants are selected from the list consisting of: aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; Freund’s Incomplete Adjuvant and Complete Adjuvant; Merck Adjuvant 65; an
  • composition of embodiment 36, wherein said adjuvant comprises QS-21.
  • An immunogenic composition or vaccine comprising 1 or more, 2 or more, 3 or more, 4 or more, or all five polypetpides selected from the group consisting of: a) a polypeptide comprising DBP or an immunogenic fragment or variant thereof; b) a polypeptide comprising MCP or an immunogenic fragment or variant thereof, optionally MCP-1 and/or MCP-2; c) a polypeptide comprising gB or an immunogenic fragment or variant thereof; d) a polypeptide comprising gC or an immunogenic fragment or variant thereof; and e) a polypeptide comprising RIR1 or an immunogenic fragment or variant thereof.
  • the disclosure provides methods for treatment and/or prevention of HSV-2 infection in a subject.
  • the disclosure also provides methods for generating an immune response against HSV-2 infection in a subject.
  • the methods comprise administering to the subject an effective amount of an HSV-2 vaccine disclosed herein.
  • the composition can be used as a therapeutic or prophylactic vaccine.
  • the disclosure additionally provides methods of generating an immune response against HSV-2 in the subject.
  • the methods may result in improved outcome for the treated subject, such as inhibition or reduction of acute disease, inflammation, and/or lesions, e.g., genital lesions.
  • the methods results in reduced viral titer in the treated subject, such as, e,g., reduced viral titer in neural tissues.
  • the methods inhibit death.
  • Methods of use includes prophylactic methods and treatment methods.
  • a subject may have been diagnosed as having an HSV-2 infection or considered at risk of being infected with HSV-2.
  • Patients or subjects include mammals, such as human, and other primate, bovine, equine, canine, feline, porcine, and ovine animals.
  • vaccines of the disclosure may be used for therapeutic applications.
  • a vaccine of the disclosure may be administered to a subject suffering from an intracellular pathogen, in an amount sufficient to treat the subject.
  • treating the subject may refer to delaying or reducing symptoms of the pathogen in an infected individual.
  • treating the subject refers to reducing the duration of symptoms, and/or reducing the intensity of symptoms.
  • the vaccine reduces the duration or severity of mild symptoms. In some embodiments, the vaccine reduces the duration or severity of serious symptoms.
  • the pathogen is a virus and the vaccine reduces viral shedding and therefore the transmissibility of the virus from the vaccinated patient.
  • the reductions described above are at least 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. In certain embodiments, the reductions described above include the complete cessation of symptoms and/or future outbreaks.
  • the duration of therapeutic effects of a vaccine formulation disclosed herein is preferably as long as possible. In certain embodiments, vaccine formulations produce therapeutic effects lasting one month, two months, three months, six months, one year, two years, five years, ten years, twenty years or a lifetime.
  • the HSV-2 vaccine can be administered to a subject by any method known to a person skilled in the art, such as parenterally, transmucosally, transdermally, intramuscularly, intravenously, intra-dermally, intra-nasally, subcutaneously, intra-peritonealy, intra- ventricularly, intra-cranially, or intra-vaginally.
  • HSV-2 vaccines of the instant invention are administered via intradermal injection, epidermal injection, intramuscular injection, intradermal injection, subcutaneous injection, or intra-respiratory mucosal injection.
  • the HSV-2 vaccine are administered orally, and are thus formulated in a form suitable for oral administration, i.e., as a solid or a liquid preparation.
  • suitable solid oral formulations include tablets, capsules, pills, granules, pellets and the like.
  • Suitable liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like.
  • the vaccine is formulated in a capsule.
  • compositions of the present invention comprise a hard gelating capsule.
  • the HSV-2 vaccines are administered by intravenous, arterial, or intra-muscular injection of a liquid preparation. Suitable liquid formulations include solutions, suspensions, dispersions, emulsions, oils and the like.
  • HSV- 2 vaccines are administered intravenously and are thus formulated in a form suitable for intravenous administration.
  • the HSV-2 vaccines are administered intra-arterially and are thus formulated in a form suitable for intra-arterial administration.
  • the HSV-2 vaccines are administered intramuscularly and are thus formulated in a form suitable for intra-muscular administration.
  • compositions are administered by intradermal injection and are thus formulated in a form suitable for intradermal .administration
  • the HSV-2 vaccines are administered topically to body surfaces and are thus formulated in a form suitable for topical administration.
  • Suitable topical formulations include gels, ointments, creams, lotions, drops and the like.
  • the HSV-2 vaccines is administered as a suppository, for example a rectal suppository or a urethral suppository.
  • the pharmaceutical composition is administered by subcutaneous implantation of a pellet.
  • the pellet provides for controlled release of antigen agent over a period of time.
  • the vaccine is delivered in a vesicle, e.g. a liposome.
  • carriers or diluents used in methods of the present invention include, but are not limited to, a gum, a starch (e.g. com starch, pregeletanized starch), a sugar (e.g. lactose, mannitol, sucrose, dextrose), a cellulosic material (e.g. microcrystalline cellulose), an acrylate ( e.g. poly methylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.
  • pharmaceutically acceptable carriers for liquid formulations are aqueous or non-aqueous solutions, suspensions, emulsions or oils.
  • non- aqueous solvents examples include propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipid from milk or eggs.
  • parenteral vehicles for subcutaneous, intravenous, intraarterial, or intramuscular injection
  • parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like.
  • sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants.
  • water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.
  • oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipid from milk or eggs.
  • the HSV-2 vaccines further comprise binders (e.g. acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxy propyl methyl cellulose, povidone, disintegrating agents (e.g. cornstarch, potato starch, alginic acid, silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g. Tris-HCL, acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g.
  • binders e.g. acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxy propyl methyl cellulose, povidone
  • disintegrating agents e.g. cornstarch, potato starch, alginic acid,
  • aspartame, citric acid preservatives (e.g. Thimerosal, benzyl alcohol, parabens), lubricants (e.g. stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethyl phthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropyl cellulose, sodium 10 lauryl sulfate), polymer coatings ( e.g. poloxamers or poloxamines), coating and film forming agents ( e.g. ethyl cellulose, acrylates, polymethacrylates) and/or adjuvants.
  • lubricants e.g. stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate
  • flow-aids e.g. colloidal silicon dioxide
  • the dose of HSV-2 vaccine administered to a patient should be sufficient or effective to elicit a beneficial therapeutic response in the patient over time, or to inhibit infection or disease due to infection.
  • the composition is administered to a patient in an amount sufficient to elicit an effective immune response to the specific antigens and/or to alleviate, reduce, cure or at least partially arrest symptoms and/or complications from the disease or infection.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose.”
  • Prophylaxis or treatment can be accomplished by administration at a single time point (single dose schedule) or multiple time points (multiple dose schedule).
  • the subject is provided with at least 2 doses. Administration can also be nearly simultaneous to multiple sites.
  • the HSV-2 vaccine is administered to the subject as a single dose schedule.
  • the HSV-2 vaccine is administered to the subject as a multiple dose schedule, e.g., a multiple dose schedule in which a primary course of vaccination with 1-3 separate doses elicits an immune response, and is followed by other doses given at subsequent time intervals that maintain, reinforce, and/or boost the immune response to HSV.
  • a multiple dose schedule comprises initially providing the vaccine to a subject at time 0, and then providing a second dose to the subject 1-6 weeks after the initial dose, followed by providing to the subject a third dose 4-10 weeks after the initial dose, and if needed, providing one or more subsequent dose(s) after several weeks or several months.
  • the vaccine is first provided, then a first boose is provided between about three to about four weeks later, and then a second boost is provided between about three to about four weeks following the first boost.
  • the dose will be determined by the activity of the composition produced and the condition of the patient, as well as the body weight or surface areas of the patient to be treated. effects that accompany the administration of a particular composition in a particular patient.
  • the physician In determining the effective amount of the composition to be administered in the treatment or prophylaxis of diseases such as HSV-2 infection, the physician needs to evaluate the production of an immune response against the virus, progression of the disease, and any treatment-related toxicity.
  • Methods disclosed herein may be used to prevent an initial HSV-2 infection and to treat an HSV-2 infection in a subject.
  • the present invention provides a method of treating a viral infection in a subject, the method comprising administering to the subject an effective amount of a vaccine of the present invention.
  • the infection is an HSV-2 infection.
  • the treatment alleviates or improves one or more clinical symptoms or manifestations of the infection.
  • symptoms include, but are not limited to local pain and/or burning sensation.
  • the treatment reduces the impact of a herpes outreak on a subject’s daily life.
  • Determining the effectiveness of the treatment may be determined, e.g., via patent-reported outcomes of symptoms, and patients may track symptoms and/or the effect of a herpes outbreak on daily life in a journal or diary.
  • the method may be used to treat mild or moderate to severe episodes. Mild episodes are characterized by no vesicles or appearance of singular vesicles without pain or discomfort interfering with usual daily activities. Moderate to severe episodes or outbreaks present as cluster of vesicles that are associated with pain and discomfort that interferes with usual daily activities.
  • HSV-2 vaccines prepared as discloed herein are used to treat moderate to severe episodes of HSV-2 infection.
  • the present invention provides a method of impeding, reducing, or inhibiting a primary HSV-2 infection in a subject, the method comprising the step of administering to the subject an effective amount of a vaccine of the present invention.
  • the present invention provides a method of treating an HSV- 2 infection in a subject, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the present invention provides a method of preventing or reducing the probability or likelihood of a subject becoming infected by HSV-2, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the subject has not previously been infected with HSV-2.
  • the method reduces the probability or likelihood that the subject becomes infected upon contact with an HSV-2 virus or an infected mammal, e.g., human.
  • the method reduces the probability or likelihood of reactivation of an HSV-2 virus or an infected mammal, e.g., human.
  • the present invention provides a method of inhibiting or preventing a recurrance following a primary HSV-2 infection in a subject, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • a “recurrence” also sometimes called outbreaks, episodes, or flare-ups
  • are repeat symptoms e.g., sores, blisters, patches of red skin or tiny splits
  • a recurrence may comprise reinfection of skin tissue following latent neuronal HSV-2 infection, reactivation of HSV-2 after a latency period, or symptomatic HSV-2 lesions following a non-symptomatic latency period.
  • the recurrence is a recurrence of herpes, e.g., oral or genital herpes.
  • the method reduces the number of recurrences that occur within one year, two years, or five years, e.g., by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100%.
  • the method reduces the frequency of recurrences that occur over the course of one year, two years, or five years.
  • the frequency of recurrence over the time period is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100%.
  • the frequence of recurrence is reduced to less than once per two months, less than once per four months, less than once per six months, less than once per year, less than once per two years, or less than once per five years.
  • the present invention provides a method of diminishing the severity of a recurrence of a viral infection, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the present invention provides a method of reducing the frequency of a recurrence of a viral infection, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the present invention provides any of the described methods in an infected subject.
  • the methods of the present invention may be used to treat, inhibit, or suppress a viral infection or primary or secondary symptoms related to such an infection following exposure of the subject to said virus.
  • the subject has been infected with the virus before vaccination.
  • the subject is at risk for viral infection.
  • vaccination by a method of the present invention is efficacious in treating, inhibiting, suppressing the viral infection or primary or secondary symptoms related to such an infection.
  • immune responses include both cell-mediated immune responses (responses mediated by antigen-specific T cells and non-specific cells of the immune system — and humoral immune responses (responses mediated by antibodies).
  • a mammal e.g., a human
  • a mammal is immunized with a vaccine of the invention and then boosted one or more times with the vaccine.
  • the mammal is boosted about 2 to about 4 weeks after the initial administration of the vaccine. If the mammal is to be boosted more than once, there is about a 2 to 12-week interval between boosts. In another embodiment, the mammal is boosted at about 12 weeks and about 36 weeks after the initial administration of the vaccine.
  • the dose used to boost the immune response can include one more cytokines, chemokines, or immunomodulators not present in the priming dose of the vaccine.
  • the present invention provides a method of preventing or reducing the severity or duration of HSV-2 labialis following a primary HSV-2 infection in a subject, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the duration is reduced to less than four weeks, less than three weeks, less than two weeks, less than one week, less than 3 days, or less than 1 day.
  • an infection by a HSV-2 is marked by watery blisters in the skin or mucous membranes of the mouth, lips, or genitals. Lesions heal with a scab characteristic of herpetic disease. The severity of any of these symptoms may be reduced. However, the infection is persistent and symptoms may recur periodically as outbreaks of sores near the site of original infection.
  • the present invention provides a method of preventing a recurrence of an HSV-2 infection, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the present invention provides a method of diminishing the severity of a recurrence of an HSV-2 infection, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the present invention provides a method of reducing the frequency of a recurrence of an HSV-2 infection, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the present invention provides any of the described methods in an HSV-2 infected subject.
  • the present invention provides a method of inhibiting HSV-2 replication in or HSV-2 shedding by an infected subject, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the level of shedding is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100%.
  • the present invention provides a method of vaccinating a subject against an HSV-2 infection, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the present invention provides a method of suppressing an HSV-2 infection in a subject, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the present invention provides a method of impeding an HSV-2 infection in a subject, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the present invention provides a method of impeding a primary HSV-2 infection in a subject, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the present invention provides a method of impeding neuronal HSV-2 spread in a subject, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the present invention provides a method of treating, suppressing or inhibiting an HSV-2 genital infection, e.g., genital herpes, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the present invention provides a method of treating, suppressing or inhibiting an HSV-2 oral infection, e.g., oral herpes, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the methods of the present invention may be used to treat, inhibit, or suppress an HSV-2 infection or primary or secondary symptoms related to such an infection following exposure of the subject to HSV-2.
  • the subject has been infected with HSV-2 before vaccination.
  • the subject is at risk for HSV- 2 infection.
  • vaccination by a method of the present invention is efficacious in treating, inhibiting, suppressing, etc, an HSV-2 infection or primary or secondary symptoms related to such an infection.
  • immune responses include both cell-mediated immune responses (responses mediated by antigen- specific T cells and non-specific cells of the immune system — and humoral immune responses (responses mediated by antibodies).
  • the immune response of the subject can be measured by determining the titer and/or presence of antibodies against the HSV-2 immunogen (e.g., HSV whole virus and/or an HSV surface antigen) after administration of the composition to evaluate the humoral response to the immunogen.
  • Seroconversion refers to the development of specific antibodies to an immunogen and may be used to evaluate the presence of a protective immune response.
  • Such antibody-based detection is often measured using Western blotting or enzyme- linked immunosorbent (ELISA) assays or hemagglutination inhibition assays (HAI). Persons of skill in the art would readily select and use appropriate detection methods.
  • administration of the vaccine of the invention results in generation of robust IgG antibodies that respond to all antigens of the virus.
  • administration of one or two doses of an HSV-2 vaccine according to the invention in mice can result in levels antibody that are at least about 100 to about 1000 times background, e.g., -940 times background, while unvaccinated subjects possess levels of pan-HSV-2-IgG antibody that are -1 times background.
  • Pan-HSV-2 IgG antibodies in this example may be determined by a flow cytometry -based assay.
  • HSV-2+ HSV-2- infected
  • UI uninfected
  • CFSE succinimidyl ester
  • Suspensions of -30% HSV-2 + cells and -70% UI cells are incubated with serum from HSV- 2-immunized mice or naive mice, and are fluorescently labeled with allophycocyanin (APC)- anti-mouse IgG Fc fragment secondary antibody.
  • APC allophycocyanin
  • Antibody-labeled cells are analyzed by 2- color flow cytometry.
  • Mouse serum levels of “pan-HSV-2 IgG’ ’ antibody are estimated based
  • the HSV-2 vaccines of the invention result in generation of robust neutralizing antibodies.
  • administration of one or two doses of an HSV-2 vaccine according to the invention can result in log2 neutralizing antibody titers ranging from about 3 to about 10, as determined by either the ELVIS method or PRNT assay.
  • Another method for determining the subject's immune response is to determine the cellular immune response, such as through immunogen-specific cell responses, such as cytotoxic T lymphocytes, or immunogen-specific lymphocyte proliferation assay. Additionally, challenge by the pathogen may be used to determine the immune response, either in the subject, or, more likely, in an animal model.
  • a person of skill in the art would be well versed in the methods of determining the immune response of a subject and the invention is not limited to any particular method.
  • the methods disclosed herein related to treating or preventing HSV-2 infection may be readily adapted for the treatment or prevention of infection by other intracellular pathogens, such as other viruses.
  • the disclosed methods for identifying immunogenic antigens may be used to identify immunogenic polypeptides of various intracellular pathogens, and immunogenic compositions and vaccines comprising isolated immunogenic polypeptides may be prepared as described herein for HSV-2, and used to treat or prevent infection and related disease and injury caused by the intracellular antigen from which the immunogenic polypeptides were identified.
  • any method described herein may be rewritten into Swiss-type format for the use of any agent described herein, for the manufacture of a medicament, in treating any of the disorders described herein.
  • any method described herein to be rewritten as a compound for use claim, or as a use of a compound claim.
  • Serum samples from 10 asymptomatic and 10 symptomatic HSV-2-positive individuals were incubated with whole-cell lysates made from the cells infected with HSV-2.
  • Vero cells were expanded and passaged according to standard procedure. Prior to virus infection, cells were trypsinized, suspended, and counted.
  • Virus infection and propagation The cells were seeded at 8 x 10 6 cells per dish and left to adhere for five hours. The media was removed, and the cells were then infected with the HSV-2 virus at an MOI of 2.0 pfu per cell for 30 min in 2 mL of media on a rocking platform to ensure the cells were covered with the media. Then 5 mL of media was added, and the cells were incubated for 16 hours at 34° C.
  • Cell harvesting and lysing At about 60 hours post infection (>95% of cells were infected), cells were harvested by scraping from the plate and transferred into 50 mL conical tubes. Cells were pelleted by centrifugation, culture medium was discarded, and cell pellets were resuspended in PBS (or DPBS) buffer. A sample of the resuspended cells was used to count total number of cells. Cells were centrifuged again and resuspended in PBS (or DPBS) buffer to reach cell concentration of 6 million cells per mL. Cell suspension was aliquoted at 1 mL per tube and centrifuged. Pellets were stored at -80 °C until further use. Prior to lysing, cell tubes were incubated with rotation at 4 °C for 120-150 minutes. Dynabeads Protein G
  • the resulting antigens were ranked based on their percentage of the total immune response against HSV-2 and their abundance across all samples in each group, and those representing higher percentages of the total immune response and abundance across all samples were identified as suitable antigens to include in an HSV-2 vaccine.
  • Antigens ranking 1-11 antigens in Table 1 were identified as primary antigens, antigens ranking 12-25 were identified as secondary antigens, and the rest were identified as potential antigens.
  • This Example demonstrates the efficacy of two HSV-2 subunit vaccines (RVx-PM-2 and RVx-PM-3) in a mouse immunization model followed by vaginal challenge with HSV-2 (prophylactic model). The study was designed to determine if prophylactic administration of subunit vaccines containing HSV-2 antigens identified in Example 1 would reduce acute infection compared to the unvaccinated control group.
  • RVx-PM-2 contained the following antigens: DBP, MCP (l-560aa and 561-1373aa), gB (30-730aa), gC (28-447aa), and R1R1.
  • RVx-PM-3 contained the following antigens: UL37, UL47, NEC2, LTPD (l-933aa and 934-2079aa), and NEC1.
  • the isolated polypeptides present in the subunit vaccines were produced by cloning protein encoding sequences into baculovirus and expressing the polypeptides as His-Tag and/or Flag-tag fusions in insect cells. The His and/or Flag tags were used for purification of the proteins. Certain proteins were expressed as
  • Each of the two subunit vaccines was used for a first immunization and a subsequent vaccine booster given at -42 days and -21 days prior to HSV-2 challenge.
  • the two subunit vaccines were administered subcutaneously (SQ) with adjuvant (QS-21) prepared on the day of vaccination into a stock solution (1 mg/mL) with Dulbeccos’ phosphate-buffered saline.
  • SQ subcutaneously
  • QS-21 adjuvant
  • One mouse in the RVx-PM-2 unexpectedly died when the blood sample before the second vaccine dose was being obtained.
  • Depo-Provera treatments (3 mg/mouse) were administered SQ on days -7 and -1 prior to challenge.
  • 30 ug of subunit vaccine was injected per animal per vaccination.
  • the amount of each polypeptide present in the vaccine was proportionate to the % of IgG response detected in human sera by mass spectrometry, as based on the average % abundance as shown in Table 1 of Example 1.
  • Acute disease was evaluated, beginning at 5 dpi and continued until 21 dpi. The animals were examined daily and all symptoms, as shown in Table 3, were recorded.
  • mice Symptoms [00265] The acute symptoms (5-21 dpi) of the animals in the study were analyzed using a score system ranging from 0 (No Disease) to 5 (Death). The score system and the analysis of symptoms is shown in Table 4.
  • the animals in the RVx-PM-2 group had no observable symptoms and when compared to the Infected Control group scores (18.50 ⁇ 5.4), the difference was The animals in the RVx-PM-3 group had similar scores to the Infected Control group (16.17 ⁇ 10.75), and when compared to the RVx-PM-2 group, the difference was also highly significant (p ⁇ 0.001). As shown in FIG. 3, the number of animals with symptoms was similar in the Infected Control and the group receiving RVx-PM-3 (10- 12/12). No symptoms were observed in the group receiving RVx-PM-2.
  • RVx-PM-2 was extremely effective in reducing acute disease infection and improving survival when compared to the both the Infected Control and the group receiving RVx-PM-3 in the study.
  • the RVx-PM-3 vaccine did not protect against symptoms and death, but significantly delayed the onset of HLE symptoms compared to the Infected Control group.
  • the RVx-PM-2 reduced vaginal virus replication at all time points examined.
  • the RVx-PM-3 also significantly reduced the viral titers at all time points, but did not reduce the number of mice with a positive swab sample at 2 dpi.
  • RVx-PM-2 effectively protected mice form all observable signs of disease and resulted in 100% survival during the observation period.
  • the RVx-PM-3 effectively delayed the onset of symptoms but only 25% of the mice survived.
  • a third vaccine dose may have further improved outcome in the study.
  • the present invention relates to vaccines for intracellular pathogens, including herpes simplex virus type 2 (HSV-2), and related methods and compositions.
  • HSV-2 herpes simplex virus type 2
  • HSV-2 herpes simplex virus type 2
  • HSV-2 is a DNA virus that often results in skin lesions and is characterized by latent and recurrent infections. HSV-2 can manifest as a cluster of small fluid-filled blisters that rupture and form painful sores, taking several weeks to heal, i.e., an outbreak.
  • the virus can exist in nerve cells for the lifetime of the infected subject and reactivate at irregular intervals.
  • HSV-2 infected individuals live with genital herpes disease that recurs once every 3-12 months. Even in the absence of actual ulcers, the virus can be produced and spread to new individuals at a rate of ⁇ 20 million per year. Currently, there is no cure for HSV-2 infection.
  • HSV-2 Treatment options for HSV-2 symptoms are limited, and it is highly desirable to develop pharmaceutical compositions that inhibit or counteract infection by HSV-2.
  • An effective vaccine may be used to elicit an enhanced immune response against HSV-2, thereby preventing initial infection, eliminating recurrence of outbreaks, and/or preventing viral shedding.
  • a consensus is that the optimal vaccine should engage all the respective arms of the immune response, including along with the presence of neutralizing antibodies and mucosal antibodies (IgA).
  • Viral vaccines can generally be divided into three groups: 1) live, attenuated, 2) inactivated, and 3) subunit.
  • Subunit vaccines containing specific antigens are regarded as one of the safest types of vaccines.
  • the efficiency of the subunit vaccine is often lower than live, attenuated vaccines, and many subunit vaccines lack the ability to generate effective and long-lasting immune responses. Therefore, new and improved compositions of subunit vaccines, including an improved HSV-2 subunit vaccine, are desirable.
  • proteomics serves as a promising strategy in the development of new subunit vaccines, especially when combined with immunomics (search of immunogenic proteins) and vaccinomics (characterization of host response to immunization). Therefore, new and improved methods of using proteomics to identify protein components in subunit vaccines that deliver both safe and effective treatments are highly desirable.
  • the present invention discloses novel methods of identifying protein components for use in a vaccine against an intracellular pathogen, as well as related vaccines and their uses.
  • the disclosure provides a method of identifying one or more protein components for use in a vaccine against an intracellular pathogen, comprising: a) contacting a cell lysate prepared from cells infected with an intracellular pathogen, with serum obtained from blood of a subject previously infected by the intracellular pathogen, under conditions and for a time sufficient for antibodies against the intracellular pathogen present in the serum to bind intracellular pathogen antigens in the cell lysate, thereby forming a mixture comprising antibody/antigen complexes; b) separating the antibody/antigen complexes from the mixture, optionally by immunoprecipitation; c) identifying, and optionally quantifying, antigens present in the antibody/antigen complexes, wherein the antigens identified in c) are protein components that may be used in a vaccine against the intracellular pathogen, thereby identifying one or more protein components for use in a vaccine against the intracellular pathogen.
  • the vaccine may comprise the full length antigen identified in c) or an immunogenic fragment, variant, or epitope thereof, or a combination of full length antigens and immunogenic fragments, variants, or epitopes thereof.
  • the method further comprises d) contacting each of a plurality of populations of PBMCs isolated from the blood of the subject with one or more proteins selected from a library of proteins; and e) measuring the cellular immune response of each of the populations of PBMCs contacted with the one or more protein, thereby identifying proteins that invoke a cellular immune response, wherein the proteins that invoke the cellular immune response are protein components that may be used in the vaccine against the intracellular pathogen.
  • the library of proteins comprises or consists of antigens or immunogenic fragments, variants, or epitopes thereof identified in the antibody/antigen complexes in step c).
  • the library of proteins comprises polypeptides and/or peptides not identified in step c).
  • the polypeptides and/or peptides are selected from a library of random and/or known polypeptides and/or peptides, e.g. a library of random known polypeptides or peptides.
  • the polypeptides and/or peptides are selected from a library of immunogenic polypeptides or fragments thereof, or peptides, e.g., polypeptides or peptides comprising known and/or computer-generated sequences.
  • the library of proteins comprises a combination of antigens or immunogenic fragments or epitopes thereof identified in the antibody/antigen complexes in step c) and other immunogenic polypeptides, e.g., peptides selected from the library of random or known polypeptides or peptides or the library of immunogenic peptide or polypeptides or fragments thereof.
  • the pathogen is a Herpes Simplex Virus 1 (HSV-1), a Herpes Simplex Virus 2 (HSV-2), or aSARS-CoV-2 virus.
  • HSV-1 Herpes Simplex Virus 1
  • HSV-2 Herpes Simplex Virus 2
  • SARS-CoV-2 virus a Herpes Simplex Virus 2 virus
  • the host cells are
  • the vaccine comprises more than one protein components.
  • the subject is a human.
  • the serum comprises at least one IgG that can bind to at least one antigen of the pathogen.
  • the antibody/antigen complex is separated from the mixture by immunoprecipitation.
  • the antigens or immunogenic fragments thereof in the antibody/antigen complexes are identified and/or quantified by mass spectrometry.
  • the PBMCs comprise T-cells that recognize the pathogen.
  • the cellular immune response is measured by ELISPOT or flow cytometry.
  • the method further comprises f) selecting one or more identified protein components for use in a vaccine against an intracellular pathogen.
  • the protein components are selected based on the amount of each antigen or immunogenic fragment thereof identified in the antibody/antigen complexes of the subject.
  • the protein components are selected based on the magnitude of the cellular immune response invoked in step e).
  • the protein components are selected based on the amount of each antigen or immunogenic fragment thereof identified in the antibody/antigen complexes of the subject and the magnitude of the cellular immune response invoked in step e).
  • one or more protein components for use in the vaccine against the intracellular pathogen are identified for a plurality of subjects previously infected with the intracellular pathogen.
  • step a) of the method comprises separately contacting each of a plurality of cell lysates prepared from cells infected with an intracellular pathogen with the serum obtained from blood of each of a plurality of subjects previously infected by the intracellular pathogen, under conditions and for a time sufficient for antibodies against the intracellular pathogen present in the serum to bind intracellular pathogen antigens in the cell lysate, thereby forming a plurality of mixtures comprising antibody/antigen complexes.
  • step a) of the method comprises contacting a cell lysate prepared from cells infected with an intracellular pathogen, with a serum composition comprising a mixture of serum obtained from blood of a plurality of subjects previously infected by the intracellular pathogen, under conditions and for a time sufficient for antibodies against the intracellular pathogen present in the serum to bind intracellular pathogen antigens in the cell lysate, thereby forming a mixture comprising antibody/antigen complexes.
  • the method further comprises selecting one or more protein components for use in a vaccine.
  • the protein components are selected based on the amount of each antigen or immunogenic fragment thereof identified in the plurality of subjects.
  • the protein components are selected based on the magnitude of the cellular immune response invoked in step e).
  • the protein components are selected based on the frequency at which each antigen or immunogenic fragment thereof is identified amongst the plurality of subjects.
  • the protein components are selected based on the amount of each antigen or immunogenic fragment thereof identified in the plurality of subjects and the magnitude of the cellular immune response invoked in step e).
  • the protein components are selected based on the amount of each antigen or immunogenic fragment thereof identified in the plurality of subjects and the frequency at which each antigen or immunogenic fragment thereof is identified amongst the plurality of subjects. In some embodiments, the protein components are selected based on the magnitude of the cellular immune response invoked in step e) and the frequency at which each antigen or immunogenic fragment thereof is identified amongst the plurality of subjects. In some embodiments, the protein components are selected based on the amount of each antigen or immunogenic fragment thereof identified in the plurality of subjects, the magnitude of the cellular immune response invoked in step e), and the frequency at which each antigen or immunogenic fragment thereof is identified amongst the plurality of subjects.
  • the disclosure provides a method of producing a vaccine against an intracellular pathogen, said method comprising preparing a vaccine comprising one or more isolated protein components identified or selected according to the method of any one of the methods of the disclosure.
  • each of the protein components comprises an antigen, or immunogenic fragment thereof, identified in at least 50% of the subjects, and the combination of protein components represents at least 50% of all antigens identified in the subjects, or immunogenic fragments thereof.
  • each of the protein components comprises an antigen, or immunogenic fragment thereof, identified in at least 0.5% of the antibody/antigen complexes, and the combination of protein components represents at least 5% of all antigens identified in the antibody/antigen complexes, or immunogenic fragments thereof.
  • each of the protein components comprises an antigen, or immunogenic fragment thereof, identified in at least 50% of the subjects and in at least 0.5% of the antibody/antigen complexes, and the combination of protein components represents at least 50% of all antigens identified in the subjects and at least 5% of all antigens identified in the antibody/antigen complexes, or immunogenic fragments thereof.
  • each of the protein components comprises an antigen, or immunogenic fragment thereof, identified in at least 0.5% of the antibody/antigen complexes, and the combination of protein components represents at least 10% of all antigens identified in the antibody/antigen complexes, or immunogenic fragments thereof.
  • each of the protein components comprises an antigen, or immunogenic fragment thereof, identified in at least 50% of the subjects and in at least 0.5% of the antibody/antigen complexes, and the combination of protein components represents at least 50% of all antigens identified in the subjects and at least 10% of all antigens identified in the antibody/antigen complexes, or immunogenic fragments thereof.
  • each of the protein components comprises an antigen, or immunogenic fragment thereof, identified in at least 1% of the antibody/antigen complexes, and the combination of protein components represents at least 5% of all antigens identified in the antibody/antigen complexes, or immunogenic fragments thereof.
  • each of the protein components comprises an antigen, or immunogenic fragment thereof, identified in at least 50% of the subjects and in at least 1% of the antibody/antigen complexes, and the combination of protein components represents at least 50% of all antigens identified in the subjects and at least 5% of all antigens identified in the antibody/antigen complexes, or immunogenic fragments thereof.
  • each of the protein components comprises an antigen, or immunogenic fragment thereof, identified in at least 1% of the antibody/antigen complexes, and the combination of protein components represents at least 10% of all antigens identified in the antibody/antigen complexes, or immunogenic fragments thereof.
  • each of the protein components comprises an antigen, or immunogenic fragment thereof, identified in at least 50% of the subjects and in at least 1% of the antibody/antigen complexes, and the combination of protein components represents at least 50% of all antigens identified in the subjects and at least 10% of all antigens identified in the antibody/antigen complexes, or immunogenic fragments thereof.
  • the protein components are recombinantly produced in cells, optionally E. coli, yeast, insect, or mammalian cells.
  • the vaccine further comprises one or more adjuvants.
  • the method further comprises measuring the efficacy of the vaccine by measuring the immune response to the vaccine elicited in an animal model.
  • the disclosure provides a vaccine produced according to any one of the methods of the disclosure.
  • the disclosure provides a method of treating, suppressing, inhibiting, or preventing an intracellular pathogen infection in a subject, the method comprising administering to said subject an effective amount of a vaccine comprising isolated immunogenic polypeptides of the intracellular pathogen that were identified according to a method disclosed herein.
  • the present invention also provides a novel approach for inducing a protective immune response against HSV-2 infections and for treating HSV-2 infections.
  • the disclosure provides a Herpes Simplex Virus 2 (HSV-2) vaccine comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 isolated polypeptides selected from the group consisting of: i. a polypeptide comprising DBP or an immunogenic fragment or variant thereof, ii. a polypeptide comprising MCP or an immunogenic fragment or variant thereof, iii. a polypeptide comprising gB or an immunogenic fragment or variant thereof, iv. a polypeptide comprising RIR1 or an immunogenic fragment or variant thereof, v.
  • HSV-2 Herpes Simplex Virus 2
  • a polypeptide comprising TRX1 or an immunogenic fragment or variant thereof vi. a polypeptide comprising UL25 or an immunogenic fragment or variant thereof, vii. a polypeptide comprising UL26 or an immunogenic fragment or variant thereof, viii. a polypeptide comprising TRX2 or an immunogenic fragment or variant thereof, ix. a polypeptide comprising gD or an immunogenic fragment or variant thereof, x. a polypeptide comprising gC or an immunogenic fragment or variant thereof, xi. a polypeptide comprising VP22 or an immunogenic fragment or variant thereof, xii. a polypeptide comprising ICP4 or an immunogenic fragment or variant thereof, xiii.
  • a polypeptide comprising UL12 or an immunogenic fragment or variant thereof xiv. a polypeptide comprising UL42 or an immunogenic fragment or variant thereof, xv. a polypeptide comprising gG or an immunogenic fragment or variant thereof.
  • the sequence of DBP is at least 95% identical to SEQ ID NO. 1
  • the sequence of MCP comprises a region that is at least 95% identical to SEQ ID NO. 2 or SEQ ID NO. 3
  • the sequence of gB is at least 95% identical to SEQ ID NO. 4
  • the sequence of gC is at least 95% identical to SEQ ID NO. 5
  • the sequence of R1R1 is at least 95% identical to SEQ ID NO. 6
  • the sequence of TRX1 is at least 95% identical to SEQ ID NO. 7
  • the sequence of ICP4 is at least 95% identical to SEQ ID NO. 8
  • the sequence of TRX2 is at least 95% identical to SEQ ID NO. 9
  • the sequence of UL42 is at least 95% identical to SEQ ID NO.
  • the sequence of gG is at least 95% identical to SEQ ID NO. 11
  • the sequence of VP22 is at least 95% identical to SEQ ID NO. 12
  • the sequence of gD is at least 95% identical to SEQ ID NO. 13
  • the sequence of UL12 is at least 95% identical to SEQ ID NO. 14
  • the sequence of UL25 is at least 95% identical to SEQ ID NO:23
  • the sequence of UL26 is at least 95% identical to SEQ ID NO:24.
  • the vaccine further comprises at least one isolated polypeptide selected from the group consisting of: a) a polypeptide comprising UL54 or an immunogenic fragment or variant thereof, b) a polypeptide comprising gE or an immunogenic fragment or variant thereof, c) a polypeptide comprising UL37 or an immunogenic fragment or variant thereof, d) a polypeptide comprising UL47 or an immunogenic fragment or variant thereof, e) a polypeptide comprising NEC2 or an immunogenic fragment or variant thereof, f) a polypeptide comprising LTPD or an immunogenic fragment or variant thereof, and g) a polypeptide comprising NEC1 or an immunogenic fragment or variant thereof.
  • the composition further comprises all 7 proteins: UL54, gE, UL37, UL47, NEC2, LTPD, NEC.
  • the sequence of UL54 is at least 95% identical to SEQ ID NO. 15
  • the sequence of gE is at least 95% identical to SEQ ID NO. 16
  • the sequence of UL37 is at least 95% identical to SEQ ID NO. 17
  • the sequence of UL47 is at least 95% identical to SEQ ID NO. 18
  • the sequence of NEC2 is at least 95% identical to SEQ ID NO. 19
  • the sequence of LTPD comprises a region at least 95% identical to SEQ ID NO. 20 or SEQ ID NO:21
  • the sequence of NEC 1 is at least 95% identical to SEQ ID NO. 22.
  • the vaccine comprises DBP or an immunogenic fragment or variant thereof.
  • the vaccine comprises an immunogenic fragment of MCP or an immunogenic variant thereof.
  • the immunogenic fragment of MCP comprises amino acids 1-560 or amino acids 561-1373 of MCP or an immunogenic variant thereof.
  • the vaccine comprises an immunogenic fragment of LTPD or an immunogenic fragment or variant thereof.
  • the immunogenic fragment of LTPD comprises amino acids 1-933 or amino acids 934-2079 of LTPD or an immunogenic variant thereof.
  • the vaccine comprises: i) a polypeptide comprising DBP or an immunogenic fragment or variant thereof, ii) a polypeptide comprising MCP or an immunogenic fragment or variant thereof, iii) a polypeptide comprising gB or an immunogenic fragment or variant thereof, iv) a polypeptide comprising RIR1 or an immunogenic fragment or variant thereof, v) a polypeptide comprising TRX1 or an immunogenic fragment or variant thereof, vi) a polypeptide comprising UL25 or an immunogenic fragment or variant thereof, vii) a polypeptide comprising UL26 or an immunogenic fragment or variant thereof, viii) a polypeptide comprising TRX2 or an immunogenic fragment or variant thereof, ix) a polypeptide comprising gD or an immunogenic fragment or variant thereof, x) a polypeptide comprising gC or an immunogenic fragment or variant thereof, xi) a polypeptide comprising
  • the disclosure provides a Herpes Simplex Virus 2 (HSV-2) vaccine that comprises at least 10 isolated polypeptides selected from the group consisting of i) a polypeptide comprising DBP or an immunogenic fragment or variant thereof, ii) a polypeptide comprising MCP or an immunogenic fragment or variant thereof, iii) a polypeptide comprising gB or an immunogenic fragment or variant thereof, iv) a polypeptide comprising RIR1 or an immunogenic fragment or variant thereof, v) a polypeptide comprising TRX1 or an immunogenic fragment or variant thereof, vi) a polypeptide comprising UL25 or an immunogenic fragment or variant thereof, vii) a polypeptide comprising UL26 or an immunogenic fragment or variant thereof, viii) a polypeptide comprising TRX2 or an immunogenic fragment or variant thereof, ix) a polypeptide comprising gD or an immunogenic fragment or variant thereof, x) a polypeptide comprising HSV-2 (HSV-2)
  • the HSV-2 vaccine further comprises at least one isolated polypeptide selected from the group consisting of: a) a polypeptide comprising ICP4 or an immunogenic fragment or variant thereof, b) a polypeptide comprising gH or an immunogenic fragment or variant thereof, c) a polypeptide comprising UL12 or an immunogenic fragment or variant thereof, d) a polypeptide comprising gE or an immunogenic fragment or variant thereof, e) a polypeptide comprising UL42 or an immunogenic fragment or variant thereof, f) a polypeptide comprising gG or an immunogenic fragment or variant thereof, g) a polypeptide comprising UL50 or an immunogenic fragment or variant thereof, h) a polypeptide comprising UL54 or an immunogenic fragment or variant thereof, i) a polypeptide comprising UL40 or an immunogenic fragment or variant thereof, j) a polypeptide comprising UL48 or an immunogenic fragment or variant thereof, k)
  • the disclosure provides a Herpes Simplex Virus 2 (HSV-2) vaccine comprising a combination of immunogenic HSV-2 polypeptides or fragments or variants thereof, wherein at least 30% of the anti-HSV-2 antibodies identified in serum of HSV- 2-infected individuals specifically bind the polypeptides in the combination, according to Table 1
  • HSV-2 Herpes Simplex Virus 2
  • the composition further comprises one or more adjuvants.
  • said one or more adjuvants comprises QS-21.
  • the disclosure provides a method of treating, suppressing, inhibiting, or preventing an intracellular pathogen infection in a subject, the method comprising administering to said subject an effective amount of any one of the vaccines of the disclosure.
  • said intracellular pathogen is HSV-2.
  • said HSV- 2 infection is a primary HSV-2 infection, a recurrence following a primary HSV-2 infection, a genital HSV-2 infection, or an oral HSV-2 infection.
  • Said vaccine can be administered before exposure to said intracellular pathogen or after exposure to said intracellular pathogen.
  • the administration of an effective amount of any one of the vaccines disclosed herein generates an immune response against the intracellular pathogen in the subject.
  • said immune response comprises a cellular immune response, optionally mediated by
  • said immune response comprises a humoral response, optionally comprising the induction of neutralizing antibodies.
  • said immune response comprises a cellular immune response, optionally mediated by and a humoral response, optionally comprising the induction of neutralizing antibodies.
  • the vaccine is administered intradermally, mucosally, intramuscularly, subcutaneously, sublingually, rectally, or vaginally.
  • the disclosure provides a method of generating an immune response to an intracellular pathogen in a subject, the method comprising administering to the subject an effective amount of the vaccine of any of the vaccines disclosed herein.
  • the immune response comprises a cellular immune response, optionally mediated by
  • the immune response comprises a humoral response, optionally comprising the induction of neutralizing antibodies.
  • the immune response comprises a cellular immune response, optionally mediated by and ahumoral response, optionally comprising the induction of neutralizing antibodies.
  • the vaccine is administered intradermally, mucosally, intramuscularly, subcutaneously, sublingually, rectally, or vaginally.
  • FIG. 1 shows hair loss and erythema (HLE) observed in mice at the indicated days infection (DPI) following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control).
  • DPI days infection
  • results from the infected control are the top line
  • results from RVx-PM-3 are the line below
  • results from RVx-PM-2 are on the x-axis.
  • the total number of mice with HLE following each treatment or no treatment is shown in the table.
  • FIG. 2 shows disease symptoms of mice as analyzed using a score system (provided by Rational Vaccines) ranging from 0 (No Disease) to 5 (Death).
  • the graph shows the mean disease score for mice following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control).
  • the table summarizes these results.
  • FIG. 3 shows the number of animals with disease symptoms of mice following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control).
  • the graph shows the percent of mice that were disease positive following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control).
  • the table summarizes these results.
  • FIG. 4 shows survival of mice at the indicated days post-infection (DPI) following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control).
  • DPI 20 on the graph the top line is RVx-PM-2, the middle line is RVx-PM-3, and the bottom line is infected control.
  • the table summarizes these results.
  • FIG. 5 shows the mean DPI of onset of HLE symptoms for the infected ontrol mice and the mice vaccinated with RVx-PM-3. The table summarizes these results.
  • FIG. 6 shows viral titers at the indicated DPI (D2, D4, D6, and D8) following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control).
  • DPI D2, D4, D6, and D8
  • RVx-PM-2 RVx-PM3
  • RVx-PM-3 no vaccine treatment
  • FIG. 7 summarizes acute viral titers at the indicated DPI (D2, D4, D6, and D8) following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control).
  • FIG. 8 shows the percentage of mice with a positive viral swab sample at the indicated DPI (D2, D4, D6, and D8) following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control).
  • DPI D2, D4, D6, and D8
  • RVx-PM-2 RVx-PM3
  • RVx-PM-3 no vaccine treatment
  • FIG. 9 summarizes the number of virus positive animals at the indicated DPI (D2, D4, D6, and D8) following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control).
  • FIG. 10 shows the viral burden in the dorsal root ganglion (DRG) of surviving mice at 21 DPI following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control). The table summarizes these results.
  • FIG. 11 shows the percentage of mice with virus detected in the DRG 21 DPI following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control). The table summarizes these results.
  • FIG. 12 shows the viral burden in the spinal cord (SC) of surviving mice at 21 DPI following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control).
  • the table summarizes these results.
  • FIG. 13 shows the percentage of mice with virus detected in the SC 21 DPI following treatment with RVx-PM-2 or RVx-PM3, or no vaccine treatment (infected control). The table summarizes these results.
  • “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In any embodiment discussed in the context of a numerical value used in conjunction with the term “about,” it is specifically contemplated that the term about can be omitted.
  • An “increased” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times ( e.g ., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 2.1, 2.2, 2.3, 2.4, etc.) greater than an amount or level described herein.
  • An “increase” may be an increase of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, at least 2-fold, at least 5-fold, or at least 10-fold.
  • a “decreased” or “reduced” or “lesser” amount is typically a “statistically significant” amount, and may include a decrease that is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) less than an amount or level described herein, for example an amount that is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of an amount or level described herein.
  • a decrease may be a decrease of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, least 80%, at least 90%, or about 100%.
  • an increase or decrease is relative to a value determined prior to infection or prior to treatment.
  • an increase or decrease is relative to a predetermined value, e.g., an average value obtained from numerous subjects, e.g., prior to infection or prior to treatment.
  • a “composition” can comprise an active agent, e.g., an immunogenic polypeptide, and a carrier, inert or active, e.g., a pharmaceutically acceptable carrier, diluent or excipient.
  • a composition may be a pharmaceutical composition.
  • the compositions are sterile, substantially free of endotoxins or non-toxic to recipients at the dosage or concentration employed.
  • mammal and “subject” includes human and non-human mammals, such as, e.g., a human, mouse, rat, rabbit, monkey, cow, hog, sheep, horse, dog, and cat.
  • HSV-2 refers, in one embodiment, to a Herpes Simplex Virus-2.
  • the term refers to an HSV-2333 strain.
  • the term refers to a 2.12 strain.
  • the term refers to an HG-52 strain.
  • the term refers to an MS strain.
  • the term refers to a G strain.
  • the term refers to a 186 strain.
  • the term refers to any other HSV-2 strain known in the art.
  • Treating” or “treatment” as used herein covers the treatment of the disease, injury, or condition of interest, e.g. , HSV-2 infection, in a biological material, e.g. , a mammal, preferably a human, having the disease or condition of interest, and includes: (i) preventing or inhibiting the disease, injury, or condition from occurring in a biological material, e.g., mammal, in particular, when such mammal is predisposed to the condition but has not yet been diagnosed as having it; (ii) reducing the severity or duration of the disease, injury or condition, e.g., when it occurs, e.g., in a mammal predisposed to the condition; (iii) inhibiting the disease, injury, or condition, i.e., arresting its development; (iv) relieving the disease, injury, or condition, i.e., causing regression of the disease or condition; or (v) relieving the symptoms resulting from the disease, injury, or condition
  • prevention includes inhibiting or impeding the onset or progression of a disease or injury or reducing the amount of injury or damage caused by a disease or injury.
  • disease includes inhibiting or impeding the onset or progression of a disease or injury or reducing the amount of injury or damage caused by a disease or injury.
  • condition may be used interchangeably.
  • isolated refers to proteins, glycoproteins, peptide derivatives or fragments or polynucleotides that are independent from its natural location. Viral components that are independently obtained through recombinant genetics means typically leads to products that are relatively purified. It is understood that the vaccines and immunogenic compositions disclosed herein comprise one or more isolated polypeptides, i.e., these polypeptides are not present within their natural location, e.g., within a pathogen. Thus, the vaccines and immunogenic compositions disclosed herein do not include live or dead pathogen or mature virion.
  • a “polypeptide” includes proteins, fragments of proteins, variants of proteins, and peptides, whether isolated from natural sources, produced by recombinant techniques or chemically synthesizes. Polypeptides of the invention typically comprise at least about 8 amino acids.
  • a polynucleotide or polypeptide has a certain “percent sequence identity” or “percent identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the worldwide web at ncbi.nlm.nih.gov/BLAST/. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA, a wholly owned subsidiary of Oxford Molecular Group, Inc.
  • GCG Genetics Computing Group
  • antibody is used in the broadest sense and specifically includes, for example, single monoclonal antibodies, polyclonal antibodies, antibody compositions with poly epitope specificity, and fragments of antibodies.
  • Antigen refers to proteins, glycoproteins or derivative or fragments that can contain one or more epitopes (linear, conformation, sequential, T-cell) which can elicit an immune response. Antigens can be separated in isolated viral proteins or peptide derivatives. As used herein, the terms “antigen”, “pathogen antigen”, and “intracellular pathogen antigen” may be used interchangeably.
  • Protein component is used in the broadest sense and specifically includes, for example, proteins, polypeptides, peptides, immunogenic protein fragments, antigens and epitopes.
  • Immunogen refers to the entire group of polypeptides that are: (a) full length antigen, (2) immunogenic fragments of the antigen, (3) immunogenic variants of the full-length antigen or variants of an immunogenic fragment, (4) chimeric fusions thereof comprising portions of a different polypeptide, and (5) conjugates thereof.
  • the proteins for use in a vaccine include a polypeptide comprising any of an immunogenic fragment thereof or a variant thereof capable of inducing an immune response specific for the protein.
  • immunogenic fragment refers to fragments or portions of proteins that elicit an antibody response or a cellular cytotoxic response that retains specificity for (cross reactivity with) the full-length protein.
  • immunogenic fragments, and variants thereof comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 48 or 50 contiguous amino acids of the antigen.
  • the immunogenic fragment may comprise any number of contiguous amino acids between the aforementioned such that, for example, an immunogenic fragment is between about 6-10, 10-15, 15-20, 20-30, 30-40, 40-50, 50-60, 60- 70, 70-80, 80-90, 90-100, or more contiguous amino acids of an immunogenic polypeptide.
  • the immunogenic fragment elicits an immune response at least 50%, at least 75%, or at least 90% as strong as the immune response elicited by the full protein.
  • a “variant” refers to a molecule having one or more amino acid modification, e.g., substitutions, deletions, or additions, as compared to the indicated amino acid sequence, yet preferably retaining the ability to be recognized by an immune cell.
  • One method for determining whether a molecule can be recognized by an immune cell is the proliferation assay described in D. M. Koelle et al., 1994, J. Virol. 68(5):2803-2810.
  • immunogenic variants retain at least 90% amino acid identity over at least 10 contiguous amino acids of the antigen, or at least 85% amino acid identity over at least 15 contiguous amino acids of the antigen (e.g. an envelope protein or non-envelope structural protein).
  • variants of antigens or sequences disclosed herein may have, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to a reference sequence.
  • an immunogenic variant has at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over the full length of a particular antigen.
  • the variant is a naturally occurring variant.
  • the variant is an engineered variant.
  • immune response refers to a subject’s response by the immune system to immunogens (i.e., antigens) that the subject’s immune system recognizes as foreign.
  • Immune responses include both cell-mediated immune responses (responses mediated by antigen-specific T cells and non-specific cells of the immune system — Thl, Th2, Thl7) and humoral immune responses (responses mediated by antibodies).
  • the term “immune response” encompasses both the initial “innate immune responses” to an immunogen, as well as memory responses that are a result of “acquired immunity”.
  • Immuno enhancing refers to a significant boost in the level and breath of the innate and/or acquired immune response to a given pathogen following administration of a vaccine of the present invention relative to the level of innate and acquired immunity when a vaccine of the present invention has not been administered.
  • Subunit refers to isolated and optionally purified proteins, e.g., HSV-2 proteins, that are compositions of a vaccine.
  • the subunit vaccine is preferably free from mature virions, cells, or lysate of cells or virions.
  • Viral antigens included in a subunit vaccine can be produced using standard recombinant genetics techniques and synthetic methods, e.g., recombinant expression, and with standard isolation and purification protocols.
  • “Pharmaceutically acceptable carrier” includes any material which, when combined with an active ingredient, allow the ingredient to retain biological activity and is non-reactive with the subject’s immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline.
  • “Therapeutically effective amount” refers to any amount of the HSV-2 vaccine that is effective in preventing, treating or ameliorating a disease caused by the HSV-2 pathogen associated with the immunogen administered in the composition of the present invention.
  • a therapeutically effective amount of the HSV-2 vaccine may be an amount effective to treat an HSV-2-infected subject, or an amount effective to prophylactically prevent a non-HSV-2- infected subject from being infected by HSV-2.
  • “protective immune response” it is meant that the immune response is associated with prevention, treating, impeding, or amelioration of a disease. Complete prevention is not required, though is encompassed by the present invention.
  • an effective amount is an amount sufficient to significantly reduce the frequency of outbreaks in an HSV- 2-infected subject. In certain embodiments, an effective amount is an amount sufficient to significantly reduce the duration of one or more outbreaks in an HSV-2-infected subject.
  • an effective amount is an amount sufficient to impede, e.g., inhibit or reduce, an HSV-2 infection or a primary HSV-2 infection.
  • the terms “impeding a HSV-2 infection” and “impeding a primary HSV-2 infection” refer, in one embodiment, to decreasing the titer of infectious virus by at least 90%. In another embodiment, the titer is decreased by at least 50%. In another embodiment, the titer is decreased by at least 55%. In another embodiment, the titer is decreased by at least 60%. In another embodiment, the titer is decreased by at least 65%. In another embodiment, the titer is decreased by at least 70%. In another embodiment, the titer is decreased by at least 75%.
  • the titer is decreased by at least 80%. In another embodiment, the titer is decreased by at least 85%. In another embodiment, the titer is decreased by at least 92%. In another embodiment, the titer is decreased by at least 95%. In another embodiment, the titer is decreased by at least 96%. In another embodiment, the titer is decreased by at least 97%. In another embodiment, the titer is decreased by at least 98%. In another embodiment, the titer is decreased by at least 99%. In another embodiment, the titer is decreased by over 99%.
  • DBP refers to the HSV Major DNA-binding Protein encoded by the UL29 gene (UniProt accession number P89452; SEQ ID NO:l). DBP is a single-stranded DNA-binding protein required for DNA replication, it participates in the opening of the viral DNA origin to initiate replication by interacting with the origin-binding protein. As used herein, DBP would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • MCP refers to the HSV Major Capsid Protein encoded by the UL19 gene (UniProt accession number W0NW54; SEQ IDNOs:2 and 3; SEQ ID NO:2 is amino acids 1-560 (MCP- 1), and SEQ ID NO:3 is amino acids 5611-1373 (MCP-2)). MCP self-assembles to form a capsid in the nucleus of the infected host cell. The capsid, once formed, will be packaged with virus DNA and transported to the cytoplasm of the host cell.
  • MCP would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • gB refers to the HSV envelope glycoprotein B encoded by the UL27 gene (UniProt accesssion number P08666;; SEQ ID NO:4). gB contains multiple transmembrane segments and is essential for viral entry into host cells. As used herein, gB would encompass the full- length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • gC refers to the HSV envelope glycoprotein C encoded by the UL44 gene (UniProt accession number Q89730; SEQ ID NO: 5). gC mediates viral attachment to host cells and acts to modulate complement activation in the innate immune response. As used herein, gC would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • RIR1 refers to the HSV ribonucleoside-di phosphate reductase large subunit encoded by the UL39 gene (UniProt accession number P89462; SEQ ID NO:6). RIR1 provides the precursors necessary for DNA synthesis and catalyzes the biosynthesis of deoxyribonucleotides from the corresponding ribonucleotides. As used herein, RIR1 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • ICP4 refers to the HSV major viral transcription factor ICP4 encoded by the gene RSI (UniProt accession number P90493; SEQ ID NO:8). It plays an essential role in the regulation of viral gene expression by both activating and repressing host RNA polymerase II- mediated transcription. As used herein, ICP4 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • [00100] refers to the HSV triplex capsid protein 2 encoded by the gene UL18
  • TRX2 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL42 refers to the HSV DNA polymerase processivity factor encoded by the gene UL42 (UniProt accession number P89463; SEQ ID NO: 10). UL42 plays an essential role in viral DNA replication by acting as the polymerase accessory subunit. As used herein, UL42 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • gG refers to the HSV envelope glycoprotein G encoded by the gene US4 (UniProt accession number P13290; SEQ ID NO: 11). gG is a chemokine-binding protein that inhibits neutrophils chemotaxis. As used herein, gG would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • VP22 refers to the HSV tegument protein VP22 encoded by the gene UL49 (UniProt accession number P89449; SEQ ID NO: 12). VP22 plays different roles during the time course of infection. It participates in both the accumulation of viral mRNAs and viral protein translation at late time of infection. As used herein, VP22 would encompass the full- length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • gD refers to the HSV envelope glycoprotein encoded by the US6 gene (UniProt accession number Q69467; SEQ ID NO: 13).
  • the gD glycoprotein is a multifunction protein that helps to define viral host tropism.
  • gD would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL12 refers to the HSV alkaline nuclease encoded by the gene UL12 (UniProt accession number P89435; SEQ ID N014). UL12 plays a role in processing non linear or branched viral DNA intermediates in order to promote the production of mature packaged unit- length linear progeny viral DNA molecules. As used herein, UL12 would encompass the full- length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL25 refers to the human herpes virus 2 capsid vertex component 2 encoded by the gene UL25 (UniProt accession number D6PUY5; SEQ ID NO:23). As used herein, UL25 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL26 refers to the human herpes virus 2 capsid vertex component 2 encoded by the gene UL26 (UniProt accession number Q69527; SEQ ID NO:24). As used herein, UL26 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL54 refers to the HSV mRNA export factor encoded by the gene UL54 (UniProt accession number P28276; SEQ ID NO: 15). It is a multifunctional regulator of the expression of viral genes that contributes to the shutoff of host protein synthesis and mediates nuclear export of viral mRNAs. As used herein, UL54 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • gE refers to HSV envelope glycoprotein encoded by the US 8 gene (UniProt accession number P89475; SEQ ID NO: 16). The gE glycoprotein has been shown to form a heterodimer with gl and functions in virion transport and modulating host defense. As used herein, gE would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL37 refers to the HSV inner tegument protein encoded by the gene UL37 (UniProt accession number P89460; SEQ ID NO: 17). UL37 plays an essential role in cytoplasmic secondary envelopment during viral egress. As used herein, UL37 would encompass the full- length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL47 refers to the HSV tegument protein encoded by the gene UL47 (UniProt accession number P89467; SEQ ID NO: 18). The tegument protein binds to various RNA transcripts and plays a role in the attenuation of selective viral and cellular mRNA degradation by modulating the activity of host shutoff RNase UL41. As used herein, UL47 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • NEC2 refers to the Nuclear Egress Protein 2 encoded by the gene UL34 (UniProt accession number P89457; SEQ ID NO: 19). Nuclear Egress Proteins play an essential role in virion nuclear egress, the first step of virion release from an infected cell. As used herein, NEC2 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • LTPD refers to the Large Tegument Protein Deneddylase encoded by the gene UL36 (UniProt accession number P89459; SEQ ID NOS: 20 and 21; SEQ ID NO:21 includes amino acids 1-933 (LTPD-1), and SEQ ID NO:21 includes amino acids 934-2079 (LTPD-2)).
  • LTPD plays multiple roles in the viral cycle, including viral entry, routing of the capsid, and stabilizing nuclear CRL substrates.
  • LTPD would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • NEC1 refers to the Nuclear Egress Protein 1 encoded by the gene UL31 (UniProt accession number P89454; SEQ ID NO:22). Nuclear Egress Proteins play an essential role in virion nuclear egress, the first step of virion release from an infected cell. As used herein, NEC1 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • gH refers to the HSV envelope glycoprotein H encoded by the UL22 gene (UniProt accession number G9I243; SEQ ID NO:25). gH is required for the fusion of viral and plasma membranes leading to virus entry into the host cell. As used herein, gH would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL50 refers to the HSV deoxyuridine 5 '-triphosphate nucleotidohydrolase encoded by the UL50 gene (UniProt accession number G9I273; SEQ ID NO:26). UL50 is involved in nucleotide metabolism: produces dUMP and decreases the intracellular concentration of dUTP to avoid uracil incorporation into viral DNA. As used herein, UL50 would encompass the full- length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL40 refers to the HSV ribonucleoside-diphosphate reductase small subunit encoded by the UL40 gene (UniProt accession number B9X2I1; SEQ ID NO:27). UL40 provides the precursors necessary for viral DNA synthesis; it allows virus growth in non dividing cells, as well as reactivation from latency in infected hosts. As used herein, UL40 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL48 refers to the HSV tegument protein VP16 encoded by the UL48 gene (UniProt accession number G9I270; SEQ ID NO:28). UL48 acts as a key activator of lytic infection by initiating the lytic program through the assembly of the transcriptional regulatory VP 16- induced complex composed of VP16 and two cellular factors, HCFC1 and POU2F 1. As used herein, UL48 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • TK refers to the HSV thymidine kinase encoded by the TK gene (UniProt accession number Q6L709; SEQ ID NO:29). TK catalyzes the transfer of the gamma-phosphate group of ATP to thymidine to generate dTMP in the salvage pathway of pyrimidine synthesis. As used herein, TK would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL35 refers to the HSV small capsomere-interacting protein encoded by the UL35 gene (UniProt accession number G9I257; SEQ ID NO:30). UL35 participates in the assembly of the infectious particles by decorating the outer surface of the capsid shell and thus forming a layer between the capsid and the tegument. As used herein, UL35 would encompass the full- length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL21 refers to the HSV tegument protein encoded by the UL21 gene (UniProt accession number G9I242; SEQ ID NO:31). UL21 may participate in DNA packaging/capsid maturation events. As used herein, UL21 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • UL1 refers to the HSV envelope glycoprotein L encoded by the UL1 gene (UniProt accession number G9I222; SEQ ID NO:32).
  • the heterodimer glycoprotein H-gly coprotein L is required for the fusion of viral and plasma membranes leading to virus entry into the host cell.
  • UL1 would encompass the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • the disclosure provides methods for identifying one or more protein components for use in a vaccine against an intracellular pathogen.
  • the methods include proteomic methods as well as methods based on cellular immune response.
  • the method comprises: a) contacting a cell lysate prepared from cells infected with an intracellular pathogen, with serum obtained from blood of a subject previously infected by the intracellular pathogen, under conditions and for a time sufficient for antibodies against the intracellular pathogen present in the serum to bind intracellular pathogen antigens in the cell lysate, thereby forming a mixture comprising antibody/antigen complexes; and b) identifying, and optionally quantifying, antigens present in the antibody/antigen complexes, thereby identifying one or more protein components for use in a vaccine against the intracellular pathogen.
  • the antibody/antigen complexes are separated from the mixture, optionally by immunoprecipitation, before the antigens are identified and/or quantified.
  • the cell lysate may be prepared from host cells infected with the intracellular pathogen, e.g., live virus.
  • the host cells are Vero cells, L7 cells, LLC- MK.2 cells, or MDCK cells.
  • the host cells may be infected with the intracellular pathogen, e.g., live virus, by contacting the host cells with the intracellular pathogen, e.g., live virus, under suitable conditions and for a sufficient time for infection to occur, as known in the art.
  • the host cells are contacted with the intracellular pathogen, e.g., live virus, in liquid suspension.
  • the host cells are contacted with the intracellular pathogen, e.g., live virus, while adhered to a solid support.
  • the inoculation density of the cells is between about 10,000 cells/cm 2 to about 150,000 cells/cm 2 . In certain embodiments, the inoculation density of the cells is about 10,000 cells/cm 2 , about 20,000 cells/cm 2 , about 30,000 cells/cm 2 , about 40,000v, about 50,000v, about 60,000 cells/cm 2 , about 70,000v, about 80,000 cells/cm 2 , about 90,000 cells/cm 2 or about 100,000 cells/cm 2 .
  • the Multiplicity of Infection (MOI) by a live virus is between 0.00001 to 10 PFU/cell or between 0.005 and 2.0 PFU/cell. In one embodiment, the MOI is about 0.005 PFU/cell. In one embodiment, the MOI is about 2 PFU/cell. In some embodiments, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of the host cells are infected by the intracellular pathogen, e.g., live virus. [00127] In certain embodiments, following infection, the infected host cells are cultured in a culture media for a period of time, e.g., to allow propagation of additional pathogens.
  • the cells may be cultured in suspension or while adhered to a solid support.
  • the time period may range depending upon the amount of virus to be produced. In certain embodiments, the time period is from about 10 hours to about 100 hours, e.g., about 10 hours, about 16 hours, about 20 hours, about 30 hours, about 40 hours, about 50 hours, about 60 hours, about 70 hours, about 80 hours, about 90 hours, or about 100 hours. In some embodiments, the time period is about 48 hours to about 96 hours. In some embodiments, the time period is about 60 hours.
  • the host cells are seeded to a solid platform after virus infection and cultured for the aforementioned time period. In some embodiments, the cells are incubated at about 34° C.
  • the host cells may be harvested.
  • host cells adhered to a solid support are harvested by contacting them with a solution comprising EDTA, trypsin, or a combination thereof.
  • said solution comprises about 5 mM EDTA in phosphate buffered saline (PBS).
  • host cells are harvested by scraping or other types of physical removal. In certain embodiments, scraping may occur after contact with EDTA, trypsin, or a combination thereof.
  • the harvested host cells are separated from the solution and/or culture media by centrifugation.
  • the separated host cells are resuspended in a PBS (or DPBS) buffer, and a sample of the resuspended cells is used to calculate total number of cells.
  • cells are centrifuged and resuspended in PBS (or DPBS) buffer to reach a final cell concentration of 6-8 million cells per mL.
  • serum obtained from blood of a subject previously infected by the intracellular pathogen may be contacted with the composition comprising lysed cells and released antigens, under conditions and for a time sufficient for antibodies against the intracellular pathogen present in the serum to bind intracellular pathogen antigens in the cell lysate, thereby forming a mixture comprising antibody/antigen complexes.
  • the serum comprises at least one IgG that can bind to at least one antigen of the pathogen.
  • the subject is a human subject.
  • the serum is incubated with the cell lysate for a period from about 60 minutes to about four hours, or about 120 minutes to about 150 minutes. In certain embodiments, the serum is incubated with the cell lysate at about 4 °C.
  • one or more protein components for use in the vaccine against the intracellular pathogen is identified for a plurality of subjects previously infected with the intracellular pathogen.
  • the number of subjects is between 2 and 100. In certain embodiments, the number of subjects is at least 10.
  • a plurality of serum compositions is obtained from the plurality of subjects previously infected with the intracellular pathogen by any means or method known in the art. In one example, each serum composition of the plurality of serum compositions is separately contacted with the composition comprising lysed cells and released antigens. In another example, the serum compositions obtained from different subjects are pooled, and the pooled serum composition is contacted with the composition comprising lysed cells and released antigens.
  • the serum comprises at least one IgG that can bind to at least one antigen of the pathogen.
  • the subjects are human subjects.
  • the serum is incubated with the cell lysate for a period from about 60 minutes to about four hours, or about 120 minutes to about 150 minutes. In certain embodiments, the serum is incubated with the cell lysate at about 4 °C.
  • the antibody/antigen complexes may be separated from the mixture.
  • the antibody/antigen complexes are separated by immunoprecipitation.
  • immunoprecipitation is preformed using Dynabeads Protein G (Invitrogen #10004D) according to manufacturer’s protocols.
  • the antigens present in the complexes may be identified.
  • the antigens are identified by mass spectrometry.
  • the antigens are quantified by mass spectrometry.
  • the antigens are identified by mass spectrometry and quantified by mass spectrometry.
  • identification and/or quantification by mass spectrometry comprises calculating normalized spectral abundance factors for each antigen.
  • identification and/or quantification by mass spectrometry comprises multiple reaction monitoring (MRM).
  • MRM multiple reaction monitoring
  • the method is used to identify antigens present in the complexes.
  • immunogenic fragments of the antigens may be identified by methods known in the art, for example, by synthesizing a series of overlapping peptides covering the entirety of an identified antigen, and then testing the peptides for their ability to be bound by antibodies in the sera of an infected subject. In certain embodiments, such immunogenic fragments are used in vaccines.
  • one or more protein components for use in a vaccine against the intracellular pathogen are selected.
  • the protein components are selected based on the amount of each identified in the antibody/antigen complexes of the subject.
  • the amount of each antigen refers to the percentage of the total antibody/antigen complexes for which the antigen was identified that contained the particular antigen or immunogenic fragment thereof, e.g., for a single subject.
  • any of the vaccines disclosed herein may comprise an immunogenic fragment or epitope of an identified antigen, instead of (or in addition to) the full length antigen polypeptide.
  • immunogenic fragments or peptide epitopes thereof may be advantageous for testing in certain cell-based assays of immune response disclosed herein.
  • the method further comprises testing the identified antigens, or immunogenic fragments or epitopes thereof, to determine which ones invoke a cellular immune response.
  • samples of peripheral blood mononuclear cells (PBMCs) isolated from the blood of the subject are each contacted with one or more of the identified antigen or immunogenic fragment or epitope thereof under conditions and for a time sufficient to allow a cellular immune response to occur, according to methods known in the art.
  • PBMCs peripheral blood mononuclear cells
  • the cellular immune response of each of the populations of PBMCs contacted with the identified antigen or immunogenic fragment or epitope thereof may be measured, thereby identifying the antigens or immunogenic fragments thereof that invoke a cellular immune response.
  • the cellular response is measured by measuring antigen- specific T cell responses by MHC multimer flow cytometry.
  • assays are well known in the art, and are described, for example, in Zhu et al. (Virus-specific CD8+ T cells accumulate near sensory nerve endings in genital skin during subclinical HSV-2 reactivation, J Exp Med. 2007 March 19; 204(3): 595-603).
  • the method further comprises testing a library of polypeptides and/or peptides to determine which ones invoke a cellular immune response.
  • the library comprises antigens, or immunogenic fragments thereof, identified according to the method disclosed above.
  • the library of proteins comprises antigens, or immunogenic fragments thereof, identified in the antibody/antigen complexes according to the methods disclosed herein.
  • the library of proteins comprises polypeptides and/or peptides not identified in the antibody/antigen complexes.
  • the polypeptides and/or peptides are selected from a library of random and/or known polypeptides and/or peptides, e.g.
  • the polypeptides and/or peptides are selected from a library of immunogenic polypeptides or peptides comprising known and/or computer generated sequences.
  • the library of proteins comprises antigens or immunogenic fragments thereof identified in the antibody/antigen complexes in step c) and other immunogenic polypeptides, e.g., peptides selected from the library of random or known peptides.
  • the library of proteins is recombinantly produced and purified according to any means or method known in the art.
  • the library comprises epitopes of antigens of the intracellular pathogen. In some embodiments, different epitopes from the same antigen are used.
  • samples of peripheral blood mononuclear cells (PBMCs) isolated from the blood of the subject are each contacted with one or more polypeptide or peptide of the library under conditions and for a time sufficient to allow a cellular immune response to occur, as known in the art.
  • PBMCs peripheral blood mononuclear cells
  • the cellular immune response of each of the populations of PBMCs contacted with the polypeptide or peptide is measured, thereby identifying the polypeptides or peptides that invoke a cellular immune response.
  • the protein components for use in the vaccine are selected based on the magnitude of the cellular immune response invoked by a protein in the library. In some embodiments, the protein components are selected based on the amount of each antigen or immunogenic fragment thereof identified in the antibody/antigen complexes of the subject and the magnitude of the cellular immune response invoked.
  • antigens or immunogenic fragment thereof for use in a vaccine are identified from the blood of a plurality of patients infected with the intracellular pathogen. This can allow the identification of antigens or immunogenic fragments thereof that are common amongst different patients, which may be beneficial to include in the vaccine.
  • one or more protein components for use in a vaccine against the intracellular pathogen are selected.
  • the protein components are selected based on the amount of each antigen or immunogenic fragment thereof identified in a patient, or a plurality of patients. In the context of a plurality of patients, the amount may be the average of the amount present in each patient.
  • an antigen or immunogenic fragment thereof is selected as a protein component of the vaccine, if the antigen or immunogenic fragment thereof is present in at least at least 0.1%, at least 0.3%, at least 0.5%, at least 0.7%, at least 0.9%, at least 1%, at least 1.2%, at least 1.4%, at least 1.6%, at least 1.8%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, or at least 20%, or at least 30% of the antibody/antigen complexes examined.
  • an antigen or immunogenic fragment thereof is selected as a protein component of the vaccine, if the antigen or immunogenic fragment thereof is present in at least at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, or at least 20%, or at least 30% of the antibody/antigen complexes examined.
  • the protein components are selected based on the magnitude of the cellular immune response.
  • an antigen or immunogenic fragment thereof is selected as a protein component of the vaccine, if the antigen or immunogenic fragment thereof (or polypeptide or peptide) invokes a positive cellular immune response greater than a control composition that does not invoke a significant immune response.
  • the protein components are selected based on the frequency at which each antigen is identified amongst the plurality of subjects.
  • an antigen, or immunogenic fragment or epitope thereof is selected as a protein component of the vaccine, if antibodies directed against the antigen are present in at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the patients’ sera.
  • the protein components are selected based on the amount of antibodies directed against each antigen identified in the subject or plurality of subjects and the magnitude of the cellular immune response invoked.
  • an antigen or immunogenic fragment or epitope thereof is selected as a protein component of the vaccine, if the antigen is present in at least 0.1%, at least 0.3%, at least 0.5%, at least 0.7%, at least 0.9%, at least 1%, at least 1.2%, at least 1.4%, at least 1.6%, at least 1.8%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, or at least 20%, or at least 30% of the antibody/antigen complexes examined, and if the antigen or immunogenic fragment or epitope thereof (or polypeptide or peptide thereof (or polypeptide or peptide
  • the antigen or immunogenic fragment thereof is present in at least at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, or at least 20%, or at least 30% of the antibody/antigen complexes examined.
  • the protein components are selected based on the amount of antibodies directed to each antigen identified in the subject or plurality of subjects and the frequency at which each antigen is identified amongst the plurality of subjects.
  • an antigen, or immunogenic fragment or epitope thereof is selected as a protein component of the vaccine, if the antigen is present in at least 0.1%, at least 0.3%, at least 0.5%, at least 0.7%, at least 0.9%, at least 1%, at least 1.2%, at least 1.4%, at least 1.6%, at least 1.8%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, or at least 20%, or at least 30% of the antibody/ antigen complexes examined, and if antibodies directed to the antigen are present in at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the patients’ sera.
  • the antigen is present in at least at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, or at least 20%, or at least 30% of the antibody/antigen complexes examined.
  • the protein components are selected based on the magnitude of the cellular immune response invoked and the frequency at which each antigen is identified amongst the plurality of subjects.
  • an antigen, or immunogenic fragment or epitope thereof is selected as a protein component of the vaccine, if the antigen or immunogenic fragment or epitope thereof (or polypeptide or peptide) invokes a cellular immune response at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold greater than a control polypeptide that does not invoke a significant immune response, and if antibodies directed to the antigen arepresent in at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the patients’ sera.
  • the protein components are selected based on the amount of antibodies directed to each antigen identified in the plurality of subjects, the magnitude of the cellular immune response invoked by the antigen, or immunogenic fragment or epitope thereof, and the frequency at which antibodies directed to each antigen are identified amongst the plurality of subjects.
  • an antigen or immunogenic fragment, or epitope thereof is selected as a protein component of the vaccine, if the antigen is present in at least 0.1%, at least 0.3%, at least 0.5%, at least 0.7%, at least 0.9%, at least 1%, at least 1.2%, at least 1.4%, at least 1.6%, at least 1.8%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, or at least 20%, or at least 30% of the antibody/antigen complexes examined, and if the antigen, or immunogenic fragment or epitope thereof (or polypeptide or peptide), invokes a positive cellular immune response greater than a control composition that does not invoke a significant immune response, and if antibodies directed to the antigen or immunogenic fragment thereof is present in
  • an antibody is directed to an antigen if the antibody specifically binds to the antigen.
  • an antibody “specifically binds” or “preferentially binds” to an antigen, if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances, e.g., at least 5-fold, at least 10- fold, at least 100-fold, at least 1000-fold greater affinity or avidity.
  • the disclosure provides a method of producing a vaccine against an intracellular pathogen, comprising preparing a vaccine composition comprising one or more protein components identified according to any one of the methods disclosed herein.
  • the vaccine composition comprises at least 5, at least 10, at least 15, or at least 20 antigens, or antigenic fragments, variants, or epitopes thereof, identified according to a method disclosed herein.
  • the vaccine composition comprises less than 100, less than 50, less than 40, less than 30, less than 20, or less than 10 antigens, or antigenic fragments, variants, or epitopes thereof, identified according to a method disclosed herein.
  • the vaccine composition does not comprise the pathogen from which the vaccine composnents were identified, e.g., dead, live, or attenuated pathogen.
  • the protein components are recombinantly produced in cells, optionally E. coli, yeast, insect, or mammalian cells. Such methods of recombinant production of protein are well known in the art, and persons of skill in the art would readily select and use appropriate production methods.
  • the protein components are purified or isolated, e.g., the protein components are separate from or isolated from the pathogen from which they were identified.
  • the vaccine compositions of the disclosure confer protective immunity, allowing a vaccinated subject to exhibit delayed onset of symptoms or reduced severity of symptoms as the result of exposure to the vaccine (e.g., a memory response).
  • the reduction in severity of symptoms is at least 25%, 40%, 50%, 60%, 70%, 80% or 90%.
  • Some vaccinated individuals may display no symptoms upon contact with the pathogen or even no infection by the pathogen.
  • the duration of protective immunity is preferably as long as possible.
  • vaccine formulations produce protective immunity lasting six months, one year, two years, five years, ten years, twenty years or a lifetime.
  • Mucosal immunity is primarily the result of secretory IgA (SIGA) antibodies on mucosal Surfaces of the respiratory, gastrointestinal, and genitourinary tracts.
  • SIGA antibodies are generated after a series of events mediated by antigen processing cells, B and T lymphocytes, that result in SIGA production by B lymphocytes on mucosa-lined tissues of the body.
  • Humoral immunity is typically the result of IgG anti bodies and IgM antibodies in serum.
  • the IgG titer can be raised by 1.5-fold, 2-fold, 3-fold, 4-fold, 5- fold, 10-fold, 20-fold, 50-fold, or 100-fold or more following administration of a vaccine formulation described herein.
  • cellular immunity can be achieved through cytotoxic T lymphocytes or through delayed-type hypersensitivity that involves macrophages and T lymphocytes, as well as other mechanisms involving T cells without a requirement for antibodies.
  • the method further comprises measuring the efficacy of the vaccine.
  • vaccine efficacy is determined by measuring the immune response to the vaccine elicited in an animal model.
  • immune response refers to a subject’s response by the immune system to immunogens (i.e., antigens) that the subject’s immune system recognizes as foreign.
  • Immune responses include both cell- mediated immune responses (e.g. responses mediated by antigen-specific T cells and non specific cells of the immune system — and humoral immune responses (responses mediated by antibodies).
  • the term “immune response” encompasses both the initial “innate immune responses” to an immunogen, as well as memory responses that are a result of “acquired immunity”.
  • suitable model systems include a guinea pig model and a mouse model. Briefly, the animals are vaccinated and then challenged with the intracellular pathogen or the vaccine is administered to already-infected animals. The response of the animals to the pathogen challenge or the vaccine is then compared with control animals, using any measure known in the art (e.g. assessment of severity of symptoms, measurement of humans. The treatment and prophylactic effects described herein may represent additional ways of determining efficacy of a vaccine. Thus, in certain embodiments, vaccine efficacy is determined by measuring immunity to the pathogen following treatment with the vaccine, e.g., by treating an animal with the vaccine and then challenging immunity by infecting the animal with the pathogen from which the vaccine is intended to confer protection.
  • vaccine efficacy can be determined by in vitro immunization of naive PBMCs, where APCs are exposed to the vaccine and then the APCs are co-cultured with naive T cells from the same donor to evaluate the primary response to immunization in a test tube.
  • vaccine efficacy can be determined by viral neutralization assays. Briefly, animals are immunized, and serum is collected on various days post immunization. Serial dilutions of serum are pre-incubated with virus during which time antibodies in the serum that are specific for the virus will bind to it. The virus/serum mixture is then added to permissive cells to determine infectivity by a plaque assay. If antibodies in the serum neutralize the virus, there are fewer plaques compared to the control group.
  • the disclosure also provides a vaccine produced according to any of the methods herein, comprising one or more protein components identified according to any one of the methods disclosed herein.
  • one or more of the protein components is selected from a full length antigen identified as described herein, or an immunogenic fragment, epitope, or variant thereof.
  • the immunogenic fragment or epitope polypeptide sequence is comprised within a larger peptide or protein sequence, e.g., a fusion protein.
  • the protein component is a variant of the full length antigen or immunogenic fragment or epitope thereof.
  • the variant has at least 80%, at least 90%, at least 95%, at least 98%, at least 99% sequence identity to the wild-type antigen, or immunogenic fragment or epitope thereof.
  • a polypeptide has a certain percent "sequence identity" to another polypeptide, meaning that, when aligned, that percentage of amino acids are the same when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi.nlm.nih.gov/BLAST/. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol.
  • GCG Genetics Computing Group
  • the gap generation penalty will generally range from 1 to 5, usually 2 to 4 and in many embodiments will be 3.
  • the gap extension penalty will generally range from about 0.01 to 0.20 and in many instances will be 0.10.
  • the program has default parameters determined by the sequences inputted to be compared. In certain embodiments, the sequence identity is determined using the default parameters determined by the program. This program is available also from Genetics Computing Group (GCG) package, from Madison, Wis., USA. Another program of interest is the FastDB algorithm. FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. Percent sequence identity is calculated by FastDB based upon the following parameters:
  • each of the protein components comprises an antigen, or immunogenic fragment, variant or epitope thereof, identified for at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the subjects. In some embodiments, each of the protein components comprises an antigen, or immunogenic fragment, variant or epitope thereof, identified for at least 50% of the subjects.
  • the combination of protein components represents at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of all antigens identified in the subjects, or immunogenic fragments, variants, or epitopes thereof. In some embodiments, the combination of protein components represents at least 50% of all antigens identified in the subjects, or immunogenic fragments, variants, or epitopes thereof.
  • each of the protein components comprises an antigen, or immunogenic fragment, variant, or epitope thereof, identified for at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the subjects, and the combination of protein components represents at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of all antigens identified in the subjects, or immunogenic fragment, variant, or epitope thereof.
  • each of the protein components comprises an antigen, or an immunogenic fragment, variant, or epitope thereof, identified in at least 0.1%, at least 0.3%, at least 0.5%, at least 0.7%, at least 0.9%, at least 1%, at least 1.2%, at least 1.4%, at least 1.6%, at least 1.8%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, or at least 20% of the antibody/antigen complexes.
  • each of the protein components comprises an antigen, or an immunogenic fragment, variant, or epitope thereof, identified in at least 0.5%, at least 1%, at least 2%, or at least 3% of the antibody/antigen complexes.
  • the combination of protein components represents at least 0.1%, at least 0.3%, at least 0.5%, at least 0.7%, at least 0.9%, at least 1%, at least 1.2%, at least 1.4%, at least 1.6%, at least 1.8%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, or at least 20% of all antigens identified in the antibody/antigen complexes, or immunogenic fragment, variant, or epitope thereof.
  • the combination of protein components represents at least 10% of all antigens identified in the antibody/antigen complexes, or
  • each of the protein components comprises an antigen, or an immunogenic fragment, variant, or epitope thereof, identified in at least 0.1%, at least 0.3%, at least 0.5%, at least 0.7%, at least 0.9%, at least 1%, at least 1.2%, at least 1.4%, at least 1.6%, at least 1.8%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, or at least 20% of the antibody/antigen complexes, and the combination of protein components represents at least 0.1%, at least 0.3%, at least 0.5%, at least 0.7%, at least 0.9%, at least 1%, at least 1.2%, at least 1.4%, at least 1.6%, at least 1.8%, at least 2%, at least 2.5%, at least 3%, at least 3.5%,
  • each of the protein components comprises an antigen, or an immunogenic fragment, variant, or epitope thereof, identified in at least 50% of the subjects and in at least 1% of the antibody/antigen complexes; and the combination of protein components represents at least 50% of all antigens, or an immunogenic fragment, variant, or epitope thereof, identified in the subjects and at least 10% of all antigens identified in the antibody/antigen complexes, or immunogenic fragments, variants, or epitopes thereof.
  • one or more of the polypeptides are immunogenic fragments of the full-length polypeptide.
  • an immunogenic fragment may consist of at least 6, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more contiguous amino acids of an immunogenic polypeptide.
  • the immunogenic fragment may comprise any number of contiguous amino acids between the aforementioned such that, for example, an immunogenic fragment is between about 6-10, 10-15, 15-20, 20-30, 30-40, 40- 50, 50-60, 60-70, 70-80, 80-90, 90-100, or more contiguous amino acids of an immunogenic polypeptide.
  • the immunogenic fragments may comprise a sufficient number of contiguous amino acids that form a linear epitope and/or may comprise a sufficient number of contiguous amino acids that permit the fragment to fold in the same (or sufficiently similar) three- dimensional conformation as the full-length polypeptide from which the fragment is derived to present a non-linear epitope or epitopes (also referred to in the art as conformational epitopes).
  • Assays for assessing whether the immunogenic fragment folds into a conformation comparable to the full-length polypeptide include, for example, the ability of the protein to react with mono- or polyclonal antibodies that are specific for native or unfolded epitopes, the retention of other ligand-binding functions, and the sensitivity or resistance of the polypeptide fragment to digestion with proteases (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, NY (2001)).
  • the three-dimensional conformation of a polypeptide fragment is sufficiently similar to the full- length polypeptide when the capability to bind and the level of binding of an antibody that specifically binds to the full-length polypeptide is substantially the same for the fragment as for the full-length polypeptide (i.e., the level of binding has been retained to a statistically, clinically, and/or biologically sufficient degree compared with the immunogenicity of the exemplary or wild-type full-length antigen).
  • the vaccine is a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers or diluents.
  • excipients, carriers or diluents used in methods of the present disclosure include, but are not limited to, water, phosphate-buffered saline, a gum, a starch (e.g. com starch, pregel etanized starch), a sugar ( e.g. lactose, mannitol, sucrose, dextrose), a cellulosic material ( e.g. microcrystalline cellulose), an acrylate ( e.g. polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.
  • the vaccine is delivered in a vesicle, e.g. a liposome.
  • pharmaceutically acceptable carriers for liquid formulations are aqueous or non-aqueous solutions, suspensions, emulsions or oils.
  • non- aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipid from milk or eggs.
  • parenteral vehicles for subcutaneous, intravenous, intraarterial, or intramuscular injection
  • parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like.
  • sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants.
  • water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.
  • oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipid from milk or eggs.
  • the vaccine compositions further comprise binders (e.g. acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxy propyl methyl cellulose, povidone, disintegrating agents (e.g. cornstarch, potato starch, alginic acid, silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g. Tris-HCL, acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g.
  • binders e.g. acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxy propyl methyl cellulose, povidone
  • disintegrating agents e.g. cornstarch, potato starch, alginic acid, silicon dioxide
  • aspartame, citric acid preservatives (e.g. Thimerosal, benzyl alcohol, parabens), lubricants (e.g. stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethyl phthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropyl cellulose, sodium 10 lauryl sulfate), polymer coatings ( e.g. poloxamers or poloxamines), coating and film forming agents ( e.g. ethyl cellulose, acrylates, polymethacrylates) and/or adjuvants.
  • lubricants e.g. stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate
  • flow-aids e.g. colloidal silicon dioxide
  • HSV-2 (herpes simplex virus type 2) is an enveloped virus. Its genome expresses over 75 different proteins, many of which are structural and are used to form the capsid and tegument, while others are part of the envelope.
  • Major capsid proteins include those expressed from open reading frames (protein names are in parentheses if the common name differs from the ORF name) UL6, UL18 (VP23), UL19 (MCP/VP5), UL35 (VP26) and UL38;
  • major tegument proteins include UL7, UL11, UL13, UL14, UL16, UL17, UL21, UL25, UL36, UL37, UL41, UL46 (VP11/12), UL47 (VP13/14), UL48 (VP16), UL49, UL51, and US11;
  • major envelope proteins include UL1 (glycoprotein L (gL)), UL10 (gM), UL20, UL22 (gH),
  • HSV-2 genome sequence is found in GenBank Accession No. NC001798.1 (incorporated herein by reference in its entirety). It is understood that the commonly used protein names may be different from the gene names, e.g., UL19 encodes MCP, but reference to the gene name herein is the same as a reference to the encoded protein. It is also understood that the exact sequence of a protein may vary from one herpesvirus to another, and thus all references to an HSV-2 protein (structural or envelope or non-envelope) encompass any such protein obtainable from any naturally occurring HSV-2. A number of HSV-2 sequences are already known and deposited in databases.
  • HSV-2 Nucleic acid encoding an HSV-2 protein with an alternative sequence can be readily isolated or amplified from one or more HSV-2 (e.g. a deposited HSV-2 or a clinical isolate) with appropriate oligonucleotide probes or primers (e.g. that specifically hybridize to a reference sequence under stringent conditions).
  • HSV-2 encodes 14 or more envelope-associated proteins, at least some of which are involved with cellular entry, including but not limited to gB, gC, gG, gD, and gE.
  • gB appears to mediate membrane fusion; gC appears to mediate viral attachment to host cells; gD appears to bind specifically to an HSV-2 receptor on cells, and gG is a chemokine-binding protein that inhibits neutrophils chemotaxis. Additionally, gE has been shown to modulate host defense.
  • HSV-2 other than envelope proteins
  • the tegument occupies the space between the capsid and the envelope.
  • Capsid proteins form a structure that surrounds the nucleic acid genome of the virion.
  • MCP the product of UL19, is the major capsid protein.
  • the cellular response involves both CD4 and CD8 T cells, cell types that play a role in combating HSV-2 infections.
  • the present disclosure provides immunogenic and pharmaceutical compositions, e.g., HSV-2 vaccines, and their use for treatment or prevention of HSV-2 infection.
  • the HSV-2 vaccines comprise immunogenic HSV-2 viral proteins or immunogenic fragments of the viral proteins, or variants of the immunogenic HSV-2 viral proteins or immunogenic fragments thereof.
  • the HSV-2 vaccines comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or at least eleven polypeptides identified in Example 1 as primary antigens, i.e., antigens selected from the group consisting of: UL29, UL19, UL27, UL39, UL38, UL25, UL26, UL18, US6, UL44, and UL49 proteins. Immunogenic fragments and variants of any of these antigens may be used.
  • the HSV-2 vaccines comprise all eleven of these antigens, or immunogenic fragments or variants thereof.
  • the HSV-2 vaccines comprise any of the following combinations of isolated polypeptides or immunogenic fragments or variants thereof:
  • the HSV-2 vaccines further comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or at least fourteen polypeptides identified in Example 1 as secondary antigens, i.e., antigens selected from the group consisting of: RSI, UL22, UL12, US8, UL42, US4, UL50, UL21, UL54, UL40, UL48, UL1, TK, and UL35. Immunogenic fragments and variants of any of these antigens may be used. In certain embodiments, the HSV-2 vaccines comprise all fourteen of these antigens, or immunogenic fragments or variants thereof.
  • the HSV-2 vaccines comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen polypeptides selected from the group consisting of: DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, UL12, UL25, and UL26. Immunogenic fragments and variants of any of these antigens may be used.
  • the HSV-2 vaccines comprises: DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, UL12, UL25, and UL26, or immunogenic antigen fragments or variants thereof.
  • the HSV- 2 vaccines comprise any of the following combinations of isolated polypeptides (or immunogenic fragments or variants thereof):
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, UL12, UL25, and UL26;
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, gD, UL12, UL25, and UL26;
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, VP22, gD, UL12, UL25, and UL26;
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, gG, VP22, gD, UL12, UL25, and UL26;
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, ICP4, UL42, gG, VP22, gD, UL12, UL25, and UL26;
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, TRX2, UL42, gG, VP22, gD, UL12, UL25, and UL26;
  • DBP DBP, MCP, gB, gC, RIR1, ICP4, TRX2, UL42, gG, VP22, gD, UL12, UL25, and UL26;
  • DBP DBP, MCP, gB, gC, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, UL12, UL25, and UL26;
  • DBP DBP, MCP, gB, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, UL12, UL25, and UL26;
  • DBP DBP, MCP, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, UL12, UL25, and UL26;
  • MCP MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, UL12, UL25, and UL26.
  • the MCP or immunogenic fragment thereof comprises one or both fragment thereof comprises GB 30-730aa.
  • the gC or immunogenic fragment thereof comprises, gC 28-447aa.
  • the HSV-2 vaccines alternatively or further comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or at least fourteen polypeptides identified in Example 1 as secondary antigens, i.e., antigens selected from the group consisting of: RSI, UL22, UL12, US8, UL42, US4, UL50, UL21, UL54, UL40, UL48, UL1, TK, and UL35.
  • antigens selected from the group consisting of: RSI, UL22, UL12, US8, UL42, US4, UL50, UL21, UL54, UL40, UL48, UL1, TK, and UL35.
  • the HSV-2 vaccines further comprise at least one, at least two, at least three, at least four, at least five, at least six, or at least seven, polypeptides selected from the group consisting of: UL54, gE, UL37, UL47, NEC2, LTPD, and NEC1. Immunogenic fragments and variants may be used.
  • any of the HSV-2 vaccines comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or at least thirteen polypeptides selected from the group consisting of: DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, and UL12. Immunogenic fragments and variants of any of these antigens may be used.
  • the HSV-2 vaccines comprise all thirteen of these polypeptides.
  • the HSV-2 vaccines comprise any of the following combinations of polypeptides:
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, and UL12;
  • MCP MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, and UL12;
  • DBP DBP, MCP, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, and UL12;
  • DBP DBP, MCP, gB, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, and UL12;
  • DBP DBP, MCP, gB, gC, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, and UL12;
  • DBP DBP, MCP, gB, gC, RIR1, ICP4, TRX2, UL42, gG, VP22, gD, and UL12;
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, TRX2, UL42, gG, VP22, gD, and UL12;
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, ICP4, UL42, gG, VP22, gD, and UL12;
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, gG, VP22, gD, and UL12;
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, VP22, gD, and UL12;
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, gD, and UL12;
  • DBP DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, and UL12; or DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, and gD.
  • the HSV-2 vaccines alternatively or further comprise at least one, at least two, at least three, at least four, at least five, at least six, or at least seven, polypeptides selected from the group consisting of: UL54, gE, UL37, UL47, NEC2, LTPD, and NEC1. Immunogenic fragments and variants may be used.
  • the HSV-2 vaccines comprise any of the following combinations of isolated polypeptides or immunogenic fragments or variants thereof:
  • the HSV-2 vaccines comrpise at at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, or at least 27, polypeptides selected from the group consisting of: UL37, UL36, UL6, UL47, UL10, UL46, UL45, UL34, US7, UL2, UL55, UL17, UL30, US2, US1, UL51, US9, UL13, UL53, UL31, US3, US12, UL52, UL16, UL5, UL9, and RL2. Immunogenic fragments and variants may be used.
  • the HSV-2 vaccines comprise DBP or an immunogenic fragment or variant thereof.
  • the HSV-2 vaccines comprise the following combinations of isolated polypeptides: DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, UL12; UL54, gE, UL37, UL47, NEC2, LTPD, and NEC1. Immunogenic fragments and variants may be used.
  • the HSV-2 vaccines comprise the following combinations of isolated polypeptides: UL37, UL47, MEC2, LTPD l-933aa, LTPD 934-2079aa, and NEC1. Immunogenic fragments and variants may be used.
  • the HSV-2 compositions comprise a combination of immunogenic isolated polypeptides, wherein the combination includes antigens that account for (or are specifically bound by) at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% of the anti-HSV-2 antibodies observed in the serum of infected individuals as determined in Example 1 and set forth in Table 1.
  • a combination that accounts for at least 40% of the anti-HSV-2 antibodies observed in the serum of infected individuals may include, e.g., UL29, UL19, UL27 and UL39.
  • one or more of the polypeptides are immunogenic fragments of the full length polypeptide.
  • an immunogenic fragment may consist of at least 6, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more contiguous amino acids of an immunogenic polypeptide.
  • the immunogenic fragment may comprise any number of contiguous amino acids between the aforementioned such that, for example, an immunogenic fragment is between about 6-10, 10-15, 15-20, 20-30, 30-40, 40- 50, 50-60, 60-70, 70-80, 80-90, 90-100, or more contiguous amino acids of an immunogenic polypeptide.
  • the immunogenic fragments may comprise a sufficient number of contiguous amino acids that form a linear epitope and/or may comprise a sufficient number of contiguous amino acids that permit the fragment to fold in the same ( or sufficiently similar) three- dimensional conformation as the full-length polypeptide from which the fragment is derived to present a non-linear epitope or epitopes (also referred to in the art as conformational epitopes).
  • Assays for assessing whether the immunogenic fragment folds into a conformation comparable to the full-length polypeptide include, for example, the ability of the protein to react with mono- or polyclonal antibodies that are specific for native or unfolded epitopes, the retention of other ligand-binding functions, and the sensitivity or resistance of the polypeptide fragment to digestion with proteases (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, NY (2001)).
  • the three-dimensional conformation of a polypeptide fragment is sufficiently similar to the full- length polypeptide when the capability to bind and the level of binding of an antibody that specifically binds to the full-length polypeptide is substantially the same for the fragment as for the full-length polypeptide (i.e., the level of binding has been retained to a statistically, clinically, and/or biologically sufficient degree compared with the immunogenicity of the exemplary or wild-type full-length antigen).
  • any of the HSV-2 vaccines disclosed herein comprise one or more immunogenic fragments of MCP.
  • the immunogenic fragment of MCP comprises or consists of amino acids 1-560 of MCP, or is an immunogenic fragment or variant thereof.
  • the immunogenic fragment of MCP comprises or consists of amino acids 561-1373 of MCP, or is an immunogenic fragment or variant thereof.
  • any of the HSV-2 vaccines disclosed herein comprise one or more immunogenic fragments of gB.
  • the immunogenic fragment of gB comprises or consists of amino acids 30-730 of gB, or is an immunogenic fragment or variant thereof.
  • any of the HSV-2 vaccines disclosed herein comprise one or more immunogenic fragments of gC.
  • the immunogenic fragment of gC comprises or consists of amino acids 28-447 of gC, or is an immunogenic fragment or variant thereof.
  • an HSV-2 vaccine disclosed herein comprises: DBP, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, UL12, an immunogenic fragment of MCP that comprising or consisting of amino acids 1-560 of MCP, an immunogenic fragment of MCP comprising or consisting of amino acids 561-1373 of MCP, immunogenic fragment of gB comprising or consisting of amino acids 30-730 of gB, and an immunogenic fragment of gC comprising or consisting of amino acids 28-447 of gC, or immunogenic fragments or variants of any of these polypeptides.
  • theHSV-2 vaccine further comprises an adjuvant, e.g., QS-21.
  • the HSV-2 vaccines further comprise one or more pharmaceutically acceptable excipient, diluent, or carrier.
  • an HSV-2 vaccine disclosed herein comprises: DBP, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, UL12, an immunogenic fragment of MCP that comprising or consisting of amino acids 1-560 of MCP, an immunogenic fragment of MCP comprising or consisting of amino acids 561-1373 of MCP, immunogenic fragment of gB comprising or consisting of amino acids 30-730 of gB, an immunogenic fragment of gC comprising or consisting of amino acids 28-447 of gC, UL54, gE, UL37, UL47, NEC2, LTPD, and NEC1, or immunogenic fragments or variants of any of these polypeptides.
  • theHSV-2 vaccine further comprises an adjuvant, e.g., QS-21.
  • the HSV-2 vaccines further comprise one or more pharmaceutically acceptable excipient, diluent, or carrier.
  • any of the HSV-2 vaccines disclosed herein further comprises one or more adjuvant, i.e., substances that enhance the immune response to an antigen, including any of those disclosed herein.
  • the adjuvant comprises a saponin, such as QS-21.
  • adjuvant refers to an agent that increases the immune response to an antigen (e.g., HSV-2 surface antigens).
  • adjuvants include, but are not limited to, aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; Freund’s Incomplete Adjuvant and Complete Adjuvant; Merck Adjuvant 65; an emulsion; a saponin, preferably QS- 21; a modified saponin; an unmethylated CpG dinucleotide; MF59; Montanide; AS02; AS04; ISCOM; a helper peptide; a TLR agonist; AS-2; MPL or 3d-MPL; LEIF; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; bio-degradable microspheres; monophosphoryl lipid A and quil
  • QS-21 refers to the adjuvant QS-21, a saponin with a molecular formula of It is one of the most potent immunological adjuvants that has been widely used. Studies showed that QS-21 promoted high antigen-specific antibody responses and CD8 + T- cell response in mice and favored a balanced production of both
  • any of the HSV-2 vaccines further comprises one or more pharmaceutically acceptable excipient, diluent, or carrier.
  • any of the HSV-2 vaccines are formulated to be administered parenterally, by injection, for example, subcutaneously, intraepithelially (with or without scarification), intra-muscularly, intra-dermally, epithelially, nasally, vaginally, or orally.
  • the HSV-2 vaccines are formulated to be administered intradermally.
  • An immunogenic composition or vaccine comprising 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more polypeptides selected from: a) a polypeptide comprising DBP or an immunogenic fragment or variant thereof; b) a polypeptide comprising MCP or an immunogenic fragment or variant thereof; c) a polypeptide comprising gB or an immunogenic fragment or variant thereof; d) a polypeptide comprising gC or an immunogenic fragment or variant thereof; e) a polypeptide comprising RIR1 or an immunogenic fragment or variant thereof; f) a polypeptide comprising TRX1 or an immunogenic fragment or variant thereof; g) a polypeptide comprising ICP4 or an immunogenic fragment or variant thereof; h) a polypeptide comprising TRX2 or an immunogenic fragment or variant thereof; i) a polypeptide comprising UL42 or an immunogenic fragment
  • composition of embodiment 1, wherein DBP, MCP, gB, gC, RIR1, TRX1, ICP4 (P90493), TRX2, UL42, gG, VP22, gD, and UL12 have at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NOs 1, 2 or 3, 4, 5, 6, 7, 8 ,9, 10, 11, 12, 13, or 14, respectively.
  • composition of embodiment 1, wherein the polypeptide selected from DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, and UL12 is the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • the composition of any one of embodiments 1-3 comprising DBP or an immunogenic fragment or variant thereof.
  • the composition of embodiment 5, wherein the immunogenic fragment of MCP comprises amino acids 1-560 of MCP or an immunogenic variant thereof.
  • composition of embodiment 5, wherein the immunogenic fragment of MCP comprises amino acids 561-1373 of MCP or an immunogenic variant thereof.
  • the composition of embodiment 8, wherein the immunogenic fragment of gB comprises amino acids 30-730 of gB or an immunogenic variant thereof.
  • the composition of embodiment 10, wherein the immunogenic fragment of gC comprises amino acids 28-447 of gC or an immunogenic variant thereof.
  • composition of any one of embodiments 1-3 comprising ICP4 or an immunogenic fragment or variant thereof.
  • the composition of any one of embodiments 1-3 comprising TRX2 or an immunogenic fragment or variant thereof.
  • the composition of any one of embodiments 1-3 comprising UL42 or an immunogenic fragment or variant thereof.
  • the composition of any one of embodiments 1-3 comprising gG or an immunogenic fragment or variant thereof.
  • the composition of embodiment 17, wherein the immunogenic fragment of gG comprises amino acids 23-650 of gG or an immunogenic variant thereof.
  • composition of embodiment 20, wherein the immunogenic fragment of gD comprises amino acids 26-310 of gD or an immunogenic variant thereof.
  • the composition of any one of embodiments 1-3 comprising UL12 or an immunogenic fragment or variant thereof.
  • the composition of embodiment 1, comprising: a polypeptide comprising UL25 or an immunogenic fragment or variant thereof; a polypeptide comprising UL26 or an immunogenic fragment or variant thereof;
  • the composition of embodiment 1, wherein the composition comprises all 13 polypeptides or an immunogenic fragment or variant thereof: DBP, MCP, gB, gC, RIR1, TRX1, ICP4, TRX2, UL42, gG, VP22, gD, UL12.
  • composition of any one of embodiments 1-25 further comprising 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more polypeptides selected from the group consisting of: a) a polypeptide comprising UL54 or an immunogenic fragment or variant thereof, b) a polypeptide comprising gE or an immunogenic fragment or variant thereof, c) a polypeptide comprising UL37 or an immunogenic fragment or variant thereof, d) a polypeptide comprising UL47 or an immunogenic fragment or variant thereof, e) a polypeptide comprising NEC2 (UL34) or an immunogenic fragment or variant thereof, f) a polypeptide comprising LTPD (UL36) or an immunogenic fragment or variant thereof, and g) a polypeptide comprising NEC1 (UL31) or an immunogenic fragment or variant thereof.
  • composition of embodiment 26, wherein UL54, gE, UL37, UL47, NEC2, LTPD, NEC1 have at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 15, 16, 17, 18, 19, 20 or 21, or 22, respectively.
  • the composition of embodiment 26, wherein the polypeptide selected from UL54, gE, UL37, UL47, NEC2, LTPD, and NEC1 is the full-length protein, an immunogenic fragment, a variant of an immunogenic fragment, an immunogenic variant, a fragment of an immunogenic variant, or a fusion protein formed with any of the aforementioned polypeptides.
  • composition of embodiment 26, wherein the immunogenic fragment of gE comprises amino acids 21-359 of gE or an immunogenic variant thereof.
  • composition of embodiment 26, wherein the immunogenic fragment of LTPD comprises amino acids 1-933 of LTPD or an immunogenic variant thereof.
  • composition of embodiment 26, wherein the immunogenic fragment of LTPD comprises amino acids 934-2079 of LTPD or an immunogenic variant thereof.
  • composition of embodiment 26, wherein the composition comprises all 7 polypeptides.
  • composition of any one of embodiments 26-32 wherein the composition comprises: a. the polypeptide comprising DBP or an immunogenic fragment or variant thereof, b. the polypeptide comprising MCP or an immunogenic fragment or variant thereof, c. the polypeptide comprising gB or an immunogenic fragment or variant thereof, d. the polypeptide comprising gC or an immunogenic fragment or variant thereof, e. the polypeptide comprising RIR1 or an immunogenic fragment or variant thereof, f. the polypeptide comprising TRX1 or an immunogenic fragment or variant thereof, g. the polypeptide comprising ICP4 or an immunogenic fragment or variant thereof, h. the polypeptide comprising TRX2 or an immunogenic fragment or variant thereof, i.
  • the polypeptide comprising UL42 or an immunogenic fragment or variant thereof j. the polypeptide comprising gG or an immunogenic fragment or variant thereof, k. the polypeptide comprising VP22 or an immunogenic fragment or variant thereof, l. the polypeptide comprising gD or an immunogenic fragment or variant thereof, m. the polypeptide comprising UL12 or an immunogenic fragment or variant thereof; n. the polypeptide comprising UL54 or an immunogenic fragment or variant thereof, o. the polypeptide comprising gE or an immunogenic fragment or variant thereof, p. the polypeptide comprising UL37 or an immunogenic fragment or variant thereof, q. the polypeptide comprising UL47 or an immunogenic fragment or variant thereof, r.
  • composition of embodiment 35 wherein said one or more adjuvants are selected from the list consisting of: aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; Freund’s Incomplete Adjuvant and Complete Adjuvant; Merck Adjuvant 65; an emulsion; a saponin, preferably QS-21; a modified saponin; an unmethylated CpG dinucleotide; MF59; Montanide; AS02; AS04; ISCOM; a helper peptide; a TLR agonist; AS-2; MPL or 3d-MPL; LEIF; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; bio-degradable microspheres; monophosphoryl lipid A and quil A; Glucopyranosyl Lipid A (GLA); muramyl tripeptide phosphate phosphat
  • composition of embodiment 36, wherein said adjuvant comprises QS-21.
  • An immunogenic composition or vaccine comprising 1 or more, 2 or more, 3 or more, 4 or more, or all five polypetpides selected from the group consisting of: a) a polypeptide comprising DBP or an immunogenic fragment or variant thereof; b) a polypeptide comprising MCP or an immunogenic fragment or variant thereof, optionally MCP-1 and/or MCP-2; c) a polypeptide comprising gB or an immunogenic fragment or variant thereof; d) a polypeptide comprising gC or an immunogenic fragment or variant thereof; and e) a polypeptide comprising RIR1 or an immunogenic fragment or variant thereof.
  • the disclosure provides methods for treatment and/or prevention of HSV-2 infection in a subject.
  • the disclosure also provides methods for generating an immune response against HSV-2 infection in a subject.
  • the methods comprise administering to the subject an effective amount of an HSV-2 vaccine disclosed herein.
  • the composition can be used as a therapeutic or prophylactic vaccine.
  • the disclosure additionally provides methods of generating an immune response against HSV-2 in the subject.
  • the methods may result in improved outcome for the treated subject, such as inhibition or reduction of acute disease, inflammation, and/or lesions, e.g., genital lesions.
  • the methods results in reduced viral titer in the treated subject, such as, e,g., reduced viral titer in neural tissues.
  • the methods inhibit death.
  • Methods of use includes prophylactic methods and treatment methods.
  • a subject may have been diagnosed as having an HSV-2 infection or considered at risk of being infected with HSV-2.
  • Patients or subjects include mammals, such as human, and other primate, bovine, equine, canine, feline, porcine, and ovine animals.
  • vaccines of the disclosure may be used for therapeutic applications.
  • a vaccine of the disclosure may be administered to a subject suffering from an intracellular pathogen, in an amount sufficient to treat the subject.
  • treating the subject may refer to delaying or reducing symptoms of the pathogen in an infected individual.
  • treating the subject refers to reducing the duration of symptoms, and/or reducing the intensity of symptoms.
  • the vaccine reduces the duration or severity of mild symptoms. In some embodiments, the vaccine reduces the duration or severity of serious symptoms.
  • the pathogen is a virus and the vaccine reduces viral shedding and therefore the transmissibility of the virus from the vaccinated patient.
  • the reductions described above are at least 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. In certain embodiments, the reductions described above include the complete cessation of symptoms and/or future outbreaks.
  • the duration of therapeutic effects of a vaccine formulation disclosed herein is preferably as long as possible. In certain embodiments, vaccine formulations produce therapeutic effects lasting one month, two months, three months, six months, one year, two years, five years, ten years, twenty years or a lifetime.
  • the HSV-2 vaccine can be administered to a subject by any method known to a person skilled in the art, such as parenterally, transmucosally, transdermally, intramuscularly, intravenously, intra-dermally, intra-nasally, subcutaneously, intra-peritonealy, intra- ventricularly, intra-cranially, or intra-vaginally.
  • HSV-2 vaccines of the instant invention are administered via intradermal injection, epidermal injection, intramuscular injection, intradermal injection, subcutaneous injection, or intra-respiratory mucosal injection.
  • the HSV-2 vaccine are administered orally, and are thus formulated in a form suitable for oral administration, i.e., as a solid or a liquid preparation.
  • suitable solid oral formulations include tablets, capsules, pills, granules, pellets and the like.
  • Suitable liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like.
  • the vaccine is formulated in a capsule.
  • compositions of the present invention comprise a hard gelating capsule.
  • the HSV-2 vaccines are administered by intravenous, intra arterial, or intra-muscular injection of a liquid preparation.
  • suitable liquid formulations include solutions, suspensions, dispersions, emulsions, oils and the like.
  • HSV- 2 vaccines are administered intravenously and are thus formulated in a form suitable for intravenous administration.
  • the HSV-2 vaccines are administered intra-arterially and are thus formulated in a form suitable for intra-arterial administration.
  • the HSV-2 vaccines are administered intramuscularly and are thus formulated in a form suitable for intra-muscular administration.
  • compositions are administered by intradermal injection and are thus formulated in a form suitable for intradermal .administration
  • the HSV-2 vaccines are administered topically to body surfaces and are thus formulated in a form suitable for topical administration.
  • Suitable topical formulations include gels, ointments, creams, lotions, drops and the like.
  • the HSV-2 vaccines is administered as a suppository, for example a rectal suppository or a urethral suppository.
  • the pharmaceutical composition is administered by subcutaneous implantation of a pellet.
  • the pellet provides for controlled release of antigen agent over a period of time.
  • the vaccine is delivered in a vesicle, e.g. a liposome.
  • carriers or diluents used in methods of the present invention include, but are not limited to, a gum, a starch (e.g. com starch, pregeletanized starch), a sugar (e.g. lactose, mannitol, sucrose, dextrose), a cellulosic material (e.g. microcrystalline cellulose), an acrylate ( e.g. poly methylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.
  • pharmaceutically acceptable carriers for liquid formulations are aqueous or non-aqueous solutions, suspensions, emulsions or oils.
  • non- aqueous solvents examples include propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipid from milk or eggs.
  • parenteral vehicles for subcutaneous, intravenous, intraarterial, or intramuscular injection
  • parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like.
  • sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants.
  • water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.
  • oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipid from milk or eggs.
  • the HSV-2 vaccines further comprise binders (e.g. acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxy propyl methyl cellulose, povidone, disintegrating agents (e.g. cornstarch, potato starch, alginic acid, silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g. Tris-HCL, acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g.
  • binders e.g. acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxy propyl methyl cellulose, povidone
  • disintegrating agents e.g. cornstarch, potato starch, alginic acid,
  • aspartame, citric acid preservatives (e.g. Thimerosal, benzyl alcohol, parabens), lubricants (e.g. stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethyl phthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropyl cellulose, sodium 10 lauryl sulfate), polymer coatings ( e.g. poloxamers or poloxamines), coating and film forming agents ( e.g. ethyl cellulose, acrylates, polymethacrylates) and/or adjuvants.
  • lubricants e.g. stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate
  • flow-aids e.g. colloidal silicon dioxide
  • the dose of HSV-2 vaccine administered to a patient should be sufficient or effective to elicit a beneficial therapeutic response in the patient over time, or to inhibit infection or disease due to infection.
  • the composition is administered to a patient in an amount sufficient to elicit an effective immune response to the specific antigens and/or to alleviate, reduce, cure or at least partially arrest symptoms and/or complications from the disease or infection.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose.”
  • Prophylaxis or treatment can be accomplished by administration at a single time point (single dose schedule) or multiple time points (multiple dose schedule).
  • the subject is provided with at least 2 doses. Administration can also be nearly simultaneous to multiple sites.
  • the HSV-2 vaccine is administered to the subject as a single dose schedule.
  • the HSV-2 vaccine is administered to the subject as a multiple dose schedule, e.g., a multiple dose schedule in which a primary course of vaccination with 1-3 separate doses elicits an immune response, and is followed by other doses given at subsequent time intervals that maintain, reinforce, and/or boost the immune response to HSV.
  • a multiple dose schedule comprises initially providing the vaccine to a subject at time 0, and then providing a second dose to the subject 1-6 weeks after the initial dose, followed by providing to the subject a third dose 4-10 weeks after the initial dose, and if needed, providing one or more subsequent dose(s) after several weeks or several months.
  • the vaccine is first provided, then a first boose is provided between about three to about four weeks later, and then a second boost is provided between about three to about four weeks following the first boost.
  • Methods disclosed herein may be used to prevent an initial HSV-2 infection and to treat an HSV-2 infection in a subject.
  • the present invention provides a method of treating a viral infection in a subject, the method comprising administering to the subject an effective amount of a vaccine of the present invention.
  • the infection is an HSV-2 infection.
  • the treatment alleviates or improves one or more clinical symptoms or manifestations of the infection.
  • symptoms include, but are not limited to local pain and/or burning sensation.
  • the treatment reduces the impact of a herpes outreak on a subject’s daily life.
  • Determining the effectiveness of the treatment may be determined, e.g., via patent-reported outcomes of symptoms, and patients may track symptoms and/or the effect of a herpes outbreak on daily life in a journal or diary.
  • the method may be used to treat mild or moderate to severe episodes. Mild episodes are characterized by no vesicles or appearance of singular vesicles without pain or discomfort interfering with usual daily activities. Moderate to severe episodes or outbreaks present as cluster of vesicles that are associated with pain and discomfort that interferes with usual daily activities.
  • HSV-2 vaccines prepared as discloed herein are used to treat moderate to severe episodes of HSV-2 infection.
  • the present invention provides a method of impeding, reducing, or inhibiting a primary HSV-2 infection in a subject, the method comprising the step of administering to the subject an effective amount of a vaccine of the present invention.
  • the present invention provides a method of treating an HSV- 2 infection in a subject, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the present invention provides a method of preventing or reducing the probability or likelihood of a subject becoming infected by HSV-2, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the subject has not previously been infected with HSV-2.
  • the method reduces the probability or likelihood that the subject becomes infected upon contact with an HSV-2 virus or an infected mammal, e.g., human.
  • the method reduces the probability or likelihood of reactivation of an HSV-2 virus or an infected mammal, e.g., human.
  • the present invention provides a method of inhibiting or preventing a recurrance following a primary HSV-2 infection in a subject, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • a “recurrence” also sometimes called outbreaks, episodes, or flare-ups
  • are repeat symptoms e.g., sores, blisters, patches of red skin or tiny splits
  • a recurrence may comprise reinfection of skin tissue following latent neuronal HSV-2 infection, reactivation of HSV-2 after a latency period, or symptomatic HSV-2 lesions following a non-symptomatic latency period.
  • the recurrence is a recurrence of herpes, e.g., oral or genital herpes.
  • the method reduces the number of recurrences that occur within one year, two years, or five years, e.g., by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100%.
  • the method reduces the frequency of recurrences that occur over the course of one year, two years, or five years.
  • the frequency of recurrence over the time period is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100%.
  • the frequence of recurrence is reduced to less than once per two months, less than once per four months, less than once per six months, less than once per year, less than once per two years, or less than once per five years.
  • the present invention provides a method of diminishing the severity of a recurrence of a viral infection, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the present invention provides a method of reducing the frequency of a recurrence of a viral infection, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the present invention provides any of the described methods in an infected subject.
  • the methods of the present invention may be used to treat, inhibit, or suppress a viral infection or primary or secondary symptoms related to such an infection following exposure of the subject to said virus.
  • the subject has been infected with the virus before vaccination.
  • the subject is at risk for viral infection.
  • vaccination by a method of the present invention is efficacious in treating, inhibiting, suppressing the viral infection or primary or secondary symptoms related to such an infection.
  • immune responses include both cell-mediated immune responses (responses mediated by antigen-specific T cells and non-specific cells of the immune system — and humoral immune responses (responses mediated by antibodies).
  • a mammal e.g., a human
  • a mammal is immunized with a vaccine of the invention and then boosted one or more times with the vaccine.
  • the mammal is boosted about 2 to about 4 weeks after the initial administration of the vaccine. If the mammal is to be boosted more than once, there is about a 2 to 12-week interval between boosts. In another embodiment, the mammal is boosted at about 12 weeks and about 36 weeks after the initial administration of the vaccine.
  • the dose used to boost the immune response can include one more cytokines, chemokines, or immunomodulators not present in the priming dose of the vaccine.
  • the present invention provides a method of preventing or reducing the severity or duration of HSV-2 labialis following a primary HSV-2 infection in a subject, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the duration is reduced to less than four weeks, less than three weeks, less than two weeks, less than one week, less than 3 days, or less than 1 day.
  • an infection by a HSV-2 is marked by watery blisters in the skin or mucous membranes of the mouth, lips, or genitals. Lesions heal with a scab characteristic of herpetic disease. The severity of any of these symptoms may be reduced. However, the infection is persistent and symptoms may recur periodically as outbreaks of sores near the site of original infection.
  • the present invention provides a method of preventing a recurrence of an HSV-2 infection, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the present invention provides a method of diminishing the severity of a recurrence of an HSV-2 infection, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the present invention provides a method of reducing the frequency of a recurrence of an HSV-2 infection, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the present invention provides any of the described methods in an HSV-2 infected subject.
  • the present invention provides a method of inhibiting HSV-2 replication in or HSV-2 shedding by an infected subject, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the level of shedding is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100%.
  • the present invention provides a method of vaccinating a subject against an HSV-2 infection, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the present invention provides a method of suppressing an HSV-2 infection in a subject, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the present invention provides a method of impeding an HSV-2 infection in a subject, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the present invention provides a method of impeding a primary HSV-2 infection in a subject, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the present invention provides a method of impeding neuronal HSV-2 spread in a subject, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the present invention provides a method of treating, suppressing or inhibiting an HSV-2 genital infection, e.g., genital herpes, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the present invention provides a method of treating, suppressing or inhibiting an HSV-2 oral infection, e.g., oral herpes, the method comprising the step of administering to said subject an effective amount of a vaccine of the present invention.
  • the methods of the present invention may be used to treat, inhibit, or suppress an HSV-2 infection or primary or secondary symptoms related to such an infection following exposure of the subject to HSV-2.
  • the subject has been infected with HSV-2 before vaccination.
  • the subject is at risk for HSV- 2 infection.
  • vaccination by a method of the present invention is efficacious in treating, inhibiting, suppressing, etc, an HSV-2 infection or primary or secondary symptoms related to such an infection.
  • immune responses include both cell-mediated immune responses (responses mediated by antigen- specific T cells and non-specific cells of the immune system — and humoral immune responses (responses mediated by antibodies).
  • the immune response of the subject can be measured by determining the titer and/or presence of antibodies against the HSV-2 immunogen (e.g., HSV whole virus and/or an HSV surface antigen) after administration of the composition to evaluate the humoral response to the immunogen.
  • Seroconversion refers to the development of specific antibodies to an immunogen and may be used to evaluate the presence of a protective immune response.
  • Such antibody-based detection is often measured using Western blotting or enzyme- linked immunosorbent (ELISA) assays or hemagglutination inhibition assays (HAI). Persons of skill in the art would readily select and use appropriate detection methods.
  • administration of the vaccine of the invention results in generation of robust IgG antibodies that respond to all antigens of the virus.
  • administration of one or two doses of an HSV-2 vaccine according to the invention in mice can result in levels of “pan-HSV-2 IgG” antibody that are at least about 100 to about 1000 times background, e.g., -940 times background, while unvaccinated subjects possess levels of pan-HSV-2-IgG antibody that are -1 times background.
  • Pan-HSV-2 IgG antibodies in this example may be determined by a flow cytometry -based assay.
  • HSV-2+ HSV-2- infected
  • UI uninfected
  • CFSE succinimidyl ester
  • Suspensions of -30% HSV-2 + cells and -70% UI cells are incubated with serum from HSV- 2-immunized mice or naive mice, and are fluorescently labeled with allophycocyanin (APC)- [00244]
  • Another method for determining the subject's immune response is to determine the cellular immune response, such as through immunogen-specific cell responses, such as cytotoxic T lymphocytes, or immunogen-specific lymphocyte proliferation assay. Additionally, challenge by the pathogen may be used to determine the immune response, either in the subject, or, more likely, in an animal model.
  • a person of skill in the art would be well versed in the methods of determining the immune response of a subject and the invention is not limited to any particular method.
  • the methods disclosed herein related to treating or preventing HSV-2 infection may be readily adapted for the treatment or prevention of infection by other intracellular pathogens, such as other viruses.
  • the disclosed methods for identifying immunogenic antigens may be used to identify immunogenic polypeptides of various intracellular pathogens, and immunogenic compositions and vaccines comprising isolated immunogenic polypeptides may be prepared as described herein for HSV-2, and used to treat or prevent infection and related disease and injury caused by the intracellular antigen from which the immunogenic polypeptides were identified.
  • any method described herein may be rewritten into Swiss-type format for the use of any agent described herein, for the manufacture of a medicament, in treating any of the disorders described herein.
  • any method described herein to be rewritten as a compound for use claim, or as a use of a compound claim.
  • Serum samples from 10 asymptomatic and 10 symptomatic HSV-2-positive individuals were incubated with whole-cell lysates made from the cells infected with HSV-2.
  • Vero cells were expanded and passaged according to standard procedure. Prior to virus infection, cells were trypsinized, suspended, and counted.
  • Virus infection and propagation The cells were seeded at 8 x 10 6 cells per dish and left to adhere for five hours. The media was removed, and the cells were then infected with the HSV-2 virus at an MOI of 2.0 pfu per cell for 30 min in 2 mL of media on a rocking platform to ensure the cells were covered with the media. Then 5 mL of media was added, and the cells were incubated for 16 hours at 34° C.
  • Cell harvesting and lysing At about 60 hours post infection (>95% of cells were infected), cells were harvested by scraping from the plate and transferred into 50 mL conical tubes. Cells were pelleted by centrifugation, culture medium was discarded, and cell pellets were resuspended in PBS (or DPBS) buffer. A sample of the resuspended cells was used to count total number of cells. Cells were centrifuged again and resuspended in PBS (or DPBS) buffer to reach cell concentration of 6 million cells per mL. Cell suspension was aliquoted at 1 mL per tube and centrifuged. Pellets were stored at -80 °C until further use. Prior to lysing, cell
  • the resulting antigens were ranked based on their percentage of the total immune response against HSV-2 and their abundance across all samples in each group, and those representing higher percentages of the total immune response and abundance across all samples were identified as suitable antigens to include in an HSV-2 vaccine.
  • Antigens ranking 1-11 antigens in Table 1 were identified as primary antigens, antigens ranking 12-25 were identified as secondary antigens, and the rest were identified as potential antigens.
  • This Example demonstrates the efficacy of two HSV-2 subunit vaccines (RVx-PM-2 and RVx-PM-3) in a mouse immunization model followed by vaginal challenge with HSV-2 (prophylactic model). The study was designed to determine if prophylactic administration of subunit vaccines containing HSV-2 antigens identified in Example 1 would reduce acute infection compared to the unvaccinated control group.
  • RVx-PM-2 contained the following antigens: DBP, MCP (l-560aa and 561-1373aa), gB (30-730aa), gC (28-447aa), and R1R1.
  • RVx-PM-3 contained the following antigens: UL37, UL47, NEC2, LTPD (l-933aa and 934-2079aa), and NEC1.
  • the isolated polypeptides present in the subunit vaccines were produced by cloning protein encoding sequences into baculovirus and expressing the polypeptides as His-Tag and/or Flag-tag fusions in insect cells. The His and/or Flag tags were used for purification of the proteins. Certain proteins were expressed as
  • Each of the two subunit vaccines was used for a first immunization and a subsequent vaccine booster given at -42 days and -21 days prior to HSV-2 challenge.
  • the two subunit vaccines were administered subcutaneously (SQ) with adjuvant (QS-21) prepared on the day of vaccination into a stock solution (1 mg/mL) with Dulbeccos’ phosphate-buffered saline.
  • SQ subcutaneously
  • QS-21 adjuvant
  • One mouse in the RVx-PM-2 unexpectedly died when the blood sample before the second vaccine dose was being obtained.
  • Depo-Provera treatments (3 mg/mouse) were administered SQ on days -7 and -1 prior to challenge.
  • 30 ug of subunit vaccine was injected per animal per vaccination.
  • the amount of each polypeptide present in the vaccine was proportionate to the % of IgG response detected in human sera by mass spectrometry, as based on the average % abundance as shown in Table 1 of Example 1.
  • Acute disease was evaluated, beginning at 5 dpi and continued until 21 dpi. The animals were examined daily and all symptoms, as shown in Table 3, were recorded.
  • mice receiving the RVx-PM-2 showed no signs of HLE (0/11, 0%), while in the Infected Control group 11/12 (92%) of mice exhibited HLE symptoms (p ⁇ 0.001 versus RVx-PM-2).
  • the acute symptoms (5-21 dpi) of the animals in the study were analyzed using a score system ranging from 0 (No Disease) to 5 (Death). The score system and the analysis of symptoms is shown in Table 4.
  • RVx-PM-2 was extremely effective in reducing acute disease infection and improving survival when compared to the both the Infected Control and the group receiving RVx-PM-3 in the study.
  • the RVx-PM-3 vaccine did not protect against symptoms and death, but significantly delayed the onset of HLE symptoms compared to the Infected Control group.
  • the RVx-PM-2 reduced vaginal virus replication at all time points examined.
  • the RVx-PM-3 also significantly reduced the viral titers at all time points, but did not reduce the number of mice with a positive swab sample at 2 dpi.
  • RVx-PM-2 effectively protected mice form all observable signs of disease and resulted in 100% survival during the observation period.
  • the RVx-PM-3 effectively delayed the onset of symptoms but only 25% of the mice survived.
  • a third vaccine dose may have further improved outcome in the study.

Abstract

La présente divulgation concerne des procédés d'identification de substances protéiques destinées à être utilisées dans un vaccin contre un agent pathogène intracellulaire, tels que le HSV-1, le HSV-2, ou le SARS-CoV-2, ainsi que des procédés de fabrication et des méthodes d'utilisation de compositions de vaccin comprenant les substances protéiques. La divulgation concerne également des vaccins produits selon ces procédés, notamment des vaccins HSV-2, ainsi que des méthodes d'utilisation associées pour traiter ou prévenir une infection à HSV-2.
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