WO2023077147A2 - Vaccins à lymphocytes t pour patients présentant une immunité humorale réduite - Google Patents

Vaccins à lymphocytes t pour patients présentant une immunité humorale réduite Download PDF

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WO2023077147A2
WO2023077147A2 PCT/US2022/079034 US2022079034W WO2023077147A2 WO 2023077147 A2 WO2023077147 A2 WO 2023077147A2 US 2022079034 W US2022079034 W US 2022079034W WO 2023077147 A2 WO2023077147 A2 WO 2023077147A2
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cell
virus
antigen
cells
subject
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PCT/US2022/079034
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WO2023077147A3 (fr
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Thomas S. Kupper
Matthew Cardinal
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Pellis Therapeutics, Inc.
The Brigham And Women's Hospital, Inc.
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    • 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
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • 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
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae

Definitions

  • the invention is generally in the field of vaccines and specifically in the area of using non-replicating or replication-impaired pox virus, perferably administered by scarification or mechanical disruption, for inducing protective immunity to infectious diseases or cancer antigens in patients with reduced humoral immunity.
  • Anti-CD20 monoclonal antibodies are used to deplete B cells and were initially developed to treat B cell proliferative disorders, including non-Hodgkin’s lymphoma (NHL) and chronic lymphocytic leukemia (CLL).
  • Anti-CD20 mAbs have subsequently been tested and used in the treatment of the autoimmune disorder rheumatoid arthritis (RA) based on the rationale that the removal of the autoantibody-producing or T cell- activating B cells would lead to clinical improvement (Edwards JC and Cambridge G. Rheumatology. 40(2):205-211 (2001)).
  • the first anti-CD20 mAh developed as a therapeutic agent is rituximab, a murine-human chimeric anti-CD20 mAh. It was introduced in 1997 and has since gained FDA approval for the treatment of NHL, CLL, RA, granulomatosis with polyangiitis (GPA), and microscopic polyangiitis. It was also found in some studies to be effective in treatment of other autoimmune conditions including SLE, relapsing-remitting MS (RRMS), and pemphigus (Paran D, Naparstek Y. Israel Med Assoc J. 2015;17(2):98-103; Wang HH, et al., Acta Derm Venereol. 2015;95(8):928-932; and Hauser SL, et al., N Engl J Med. 2008;358(7):676-688).
  • Vaccination is known to reduce rates of hospital admissions due to infections, emergency room visits, and the rate of invasive infectious diseases in patients with autoimmune disease.
  • anti-CD20 therapy is associated with reduced immunological response to vaccines.
  • antibody responses can be impaired up to 6 months after anti-CD20 therapy, with reduction in B cellular immunity in parallel with depleted B-cell pools. It has been proposed that vaccine response is blunted until B cells repopulate. Repopulation kinetics varies among the anti- CD20 agents, ranging from 24 weeks to 35 weeks for rituximab, 40 weeks for ofatumumab, and 72 weeks for ocrelizumab.
  • T cell-mediated immunity may provide a robust and long- lasting immunity conferred by vaccines especially in those with a reduced number of peripheral B cells such as those undergoing anti-CD20 therapy.
  • current vaccine strategies do attempt to induce TCMI, and many vaccines based on inactivated viruses have proven ineffective in producing TCMI.
  • TCMI T cell-mediated immunity
  • live, modified, non-replicating or replication-impaired vaccinia viruses can deliver one or more exogenous antigens to elicit or stimulate an antigen-specific T cell mediated immunity (TCMI) to the one or more exogenous antigens when administered to disrupted epithelial tissue in a subject.
  • TCMI T cell mediated immunity
  • the TCMI provides a robust and protective immunity against vaccines especially in patients with reduced humoral immunity such as those undergoing anti-CD20 therapy.
  • compositions and methods for inducing or stimulating a T cell mediated immune response to a T cell antigen in epithelial tissues of a human subject include administering to disrupted epithelial tissue of the subject a non-replicating or replication-impaired poxvirus, expressing or including one or more T cell antigens.
  • the intact, non-replicating or replication-impaired poxvirus is derived by natural or artificial modification of a wild-type poxvirus.
  • Exemplary poxviruses include orthopox, suipox, avipox, capripox, leporipox, parapoxvirus, molluscpoxvirus, and yatapoxvirus.
  • a preferred orthopox virus is a vaccinia virus.
  • Exemplary vaccinia viruses include Modified Vaccinia Ankara (MV A), Wyeth strain, WR strain, NYCBH strain, ACAM2000, Lister strain, LC16m8, Elstree-BNm, Copenhagen strain, and Tiantan strain.
  • a preferred vaccinia virus is Modified Vaccinia Ankara (MVA).
  • the vaccine is administered via epithelial tissue, for example, skin, lung, oral mucosa, gastrointestinal tract, rectal or vaginal mucosa.
  • epithelial tissue is skin tissue.
  • epithelial skin tissue is disrupted through the epidermal layer, and nonreplicating or replication-impaired poxvirus including one or more T cell antigens are administered to the disrupted epidermis.
  • the epithelial tissue is mechanically disrupted by a scarification needle, a needle, an abrader, or microneedles. In some embodiments, the epithelial tissue is disrupted essentially at the same time as the administration of the poxvirus. In other embodiments, the epithelial tissue is disrupted prior to or immediately after administration of the poxvirus composition.
  • the methods are particularly suited for inducing or stimulating a T cell mediated immune response to a T cell antigen in epithelial tissues in a subject having a reduced number of peripheral B cells in the blood, for example, less than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, compared to a healthy control.
  • the subject is undergoing a B cell depletion therapy or has completed a B cell depletion therapy but not yet have B cells repopulated in the blood.
  • the patients are undergoing or having completed anti-CD20 therapy using one or more antibodies of rituximab, ocrelizumab, veltuzumab, obinutuzumab, and ofatumumab.
  • the subject in need is one undergoing or having completed anti-CD20 therapy using rituximab.
  • the subject undergoing or having completed anti-CD20 therapy has been diagnosed with and/or is suffering from one or more conditions or disorders of B cell-derived malignancies, autoimmune diseases, metabolic disorders, myocardial disorders, and vascular disorders.
  • the subject has one or more conditions or disorders of non- Hodgkin’ s lymphoma and chronic lymphocytic leukemia; or one or more conditions or disorders of multiple sclerosis, N-methyl-D-aspartate receptor encephalitis, myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis, myelin-oligodendrocyte glycoprotein spectrum disorder, and neuromyelitis optica spectrum disorder.
  • the subject has or is at risk of developing cancer
  • the T cell antigen is a tumor- associated antigen (TAA), a tumor- specific antigen (TSA), or a tissue- specific antigen.
  • TAA tumor-associated antigen
  • TSA tumor-specific antigen
  • An exemplary T cell antigen is derived from, or raises an immune response against a cancer selected from the group consisting of melanoma, squamous cell carcinoma, basal cell carcinoma, Merkel cell carcinoma, adenexal carcinoma, cutaneous T or B cell lymphoma, sarcomas, adenocarcinoma, prostate adenocarcinoma, prostatic intraepithelial neoplasia, squamous cell lung carcinoma, lung adenocarcinoma, small cell lung carcinoma, ovarian cancer of epithelial origin, colorectal adenocarcinoma and leiomyosarcoma, stomach adenocarcinoma and leiomyosarcom
  • the subject has or is at risk of developing a viral, bacterial, fungal, or protozoal infection
  • the T cell antigen is a viral, bacterial, fungal, or protozoal antigen.
  • An exemplary T cell antigen is derived from, or raises a protective immune response against a pathogen including HIV, influenza, dengue, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, human papilloma virus, Ebola, Marburg, Rabies, Hanta virus infection, West Nile virus, SARS-like Coronaviruses, other beta coronaviruses, Herpes simplex virus 1 and2, Varicella-zoster virus, Epstein- Barr virus, Human herpesvirus, Alpha viruses, and St.
  • the T cell antigen is derived from, or raises a protective immune response against a SARS-like coronavirus including SARS-Cov-2 virus. In other embodiments, the T cell antigen is derived from, or raises a protective immune response against an influenza A, B or C virus.
  • the methods include administering the poxvirus co-expressing one or more T cell antigens which are polypeptides.
  • An exemplary polypeptide is an HLA-restricted peptide, such as an epitope for an MHC class I peptide from the sequence of proteins derived from the pathogens above.
  • the methods also include administering or co-expressing co-stimulatory molecule, growth factor, adjuvant and/or cytokine, before, at the same time, or after the antigen, at the same or a distant site.
  • co-stimulatory molecules, growth factors, adjuvants and/or cytokines include IL-1, IL-2, IL-7, IL-12, IL-15, IL-18, IL-23, IL-27, B7-2, B7-H3, CD40, CD40L, ICOS-ligand, OX-40L, 4-1BBL, GM-CSF, SCF, FGF, Flt3-ligand, and CCR4.
  • Epidermal immunization with live, modified, non-replicating or replication-impaired vaccines such as replication-deficient poxviruses (e.g., Modified Vaccinia Ankara; MV A), administered using mechanical disruption of the skin, generates a stronger immune response and stronger protection of the immunized host at a much lower dose compared to vaccines delivered via other injection routes.
  • the vaccine composition is typically administered to induce a primary response. However, in some case, this response will be boosted by one or more subsequent administrations.
  • the methods include repeating the step of administering the intact, non-replicating or replication-impaired poxvirus to epithelial tissue that has been mechanically disrupted; and typically, the second administration is carried out at a time of one, two, three, four, five, six, seven, eight, nine, or ten days or weeks or months after the first administration.
  • composition is typically provided as a lyophilized powder in a sterile sealed vial. This may be rehydrated at the time of administration using sterile saline or water.
  • a kit includes a device for disrupting a subject’s epidermis and the live, modified, non-replicating or replication-impaired virus.
  • the vaccine construct may be incorporated into microneedles, or other mechanical disrupter.
  • Figure 1 is a flowchart of an assay for vaccination by skin immunization in mice with recombinant modified vaccinia Ankara including influenza nucleoprotein (MVA NP ), immunity after day 40 post- vaccine (d40), lethal H1N1 influenza challenge with the HINl/Puerto Rico/8 (PR8) mouse influenza virus, and subsequent survival and weight-loss statistics for the study.
  • VMA NP influenza nucleoprotein
  • d40 immunity after day 40 post- vaccine
  • PR8 lethal H1N1 influenza challenge with the HINl/Puerto Rico/8
  • Figures 2A and 2B are graphs showing % of initial body weight over time and percent survival in C57BL/6J mice (B6 mice) after lethal H1N1 influenza challenge with the H1N1 Puerto Rico 8 (PR8) influenza virus at 2x10 1.6 EID50 of PR8.
  • Figure 2A shows % of initial body weight over time (d) for each of Naive control ( ⁇ ); modified vaccinia Ankara virus including influenza nucleoprotein delivered once via skin scarification (MVA-NP s.s. one time; ⁇ ) in mice treated with a control antibody; and modified vaccinia Ankara virus including influenza nucleoprotein delivered once via skin scarification (MVA-NP s.s. one time; ⁇ ) in mice treated with anti-CD20 antibody; respectively.
  • Figure 2B shows percent survival over time for each of Naive control (green); modified vaccinia Ankara virus including influenza nucleoprotein delivered once via skin scarification (MVA-NP s.s. one time; red) in mice treated with a control antibody; and modified vaccinia Ankara virus including influenza nucleoprotein delivered once via skin scarification (MVA-NP s.s. one time; blue) in mice treated with anti-CD20 antibody, respectively.
  • Figures 3 A and 3B are graphs showing % of initial body weight over time and percent survival in BALB/c mice after lethal H1N1 influenza challenge with the HINl/Puerto Rico/8 (PR8) influenza virus at 2x10 1.6 EID50 of PR8.
  • Figure 3A shows % of initial body weight over time (d) for each of Naive control ( ⁇ ); modified vaccinia Ankara virus including influenza nucleoprotein delivered once via skin scarification (MVA-NP s.s. one time; ⁇ ) in mice treated with a control antibody; and modified vaccinia Ankara virus including influenza nucleoprotein delivered once via skin scarification (MVA-NP s.s. one time; ⁇ ) in mice treated with anti-CD20 antibody; respectively.
  • Figure 3B shows percent survival over time for each of Naive control (green); modified vaccinia Ankara virus including influenza nucleoprotein delivered once via skin scarification (MVA-NP s.s. one time; red) in mice treated with a control antibody; and modified vaccinia Ankara virus including influenza nucleoprotein delivered once via skin scarification (MVA-NP s.s. one time; blue) in mice treated with anti-CD20 antibody, respectively.
  • the blue lines for the anti CD20 overlap at 100% with the control antibody, and thus the two individual lines are tough to see.
  • non-replicating or “replication-impaired” poxvirus refers to a poxvirus that is not capable of replication to any significant extent in the majority of normal mammalian cells or normal primary human cells.
  • significant extent means a replication capability of 75% or less as compared to wild-type vaccinia virus in standardized assays.
  • the poxvirus has a replication capability of 65%, 55%, 45%, 35%, 25%, or 15% compared to wild-type vaccinia virus.
  • modified virus refers to a poxvirus that has been altered in some way that changes one or more characteristics of the modified virus compared to the wild-type virus. These changes may have occurred naturally or through engineering.
  • the modified virus is altered to include an antigen(s) that are immunogenic (i.e., induce an immune response in a host).
  • Antigens include, for example, cancer antigens or microbial antigens.
  • immunological refers to the development of a beneficial humoral (antibody mediated) and/or a cellular (mediated by antigen- specific T cells or their secretion products) response directed against an immunogen in a recipient patient.
  • Such a response can be an active response induced by administration of immunogen or a passive response induced by administration of antibody or primed T- cells.
  • a cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II MHC molecules to activate antigen- specific CD4 + T helper cells and/or CD8 + cytotoxic T cells.
  • the response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils, or other components of innate immunity.
  • the presence of a cell-mediated immunological response can be determined by proliferation assays (CD4 + T cells) or CTL (cytotoxic T lymphocyte) assays.
  • proliferation assays CD4 + T cells
  • CTL cytotoxic T lymphocyte
  • T cell antigen refers to a protein or fragment thereof which can be processed into a peptide that can bind to either Class I MHC, Class II MHC, non-classical MHC, or CD1 family molecules (collectively antigen presenting molecules), and in this combination can engage a T cell receptor on a T cell.
  • a T cell mediated immune response is a response that occurs as a result of recognition of a T cell antigen bound to an antigen presenting molecule on the cell surface of an antigen presenting cell, coupled with other interactions between costimulatory molecules on the T cell and APC. This response serves to induce T cell proliferation, anatomic migration, and production of effector molecules, including cytokines and other factors that can injure cells.
  • B cell antigen refers to a protein, glycoprotein, carbohydrate, or lipid that can bind to cell surface antibody and can generate the production of soluble antibodies.
  • a humoral immune response is the generation of an immune response that leads to high and sustained levels of circulating antibodies.
  • treat or “treatment” of a disease, disorder or condition refer to improving one or more symptoms or the general condition of a subject having the disease. Treating the disease or condition includes ameliorating at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected, such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis.
  • “treating” the cancer refers to inhibiting proliferation or metastasis of a cancer or tumor cells.
  • treatment leads to stasis, partial or complete remission of a tumor or inhibition of metastatic spreading of the tumor.
  • “treating” the infectious disease means reducing the load of the infectious agent in the subject.
  • the load is viral load
  • reducing the viral load means, for example, reducing the number of cells infected with influenza virus or coronavirus, reducing the rate of replication of influenza virus or coronavirus, reducing the number of new virions produced or reducing the number of total viral genome copies in a cell, as compared to an untreated subject.
  • the load is influenza virus, or coronavirus, as compared to an untreated subject, or as compared to a healthy, uninfected subject.
  • the term “protect” or “protection of’ a subject from developing a disease or from becoming susceptible to an infection means to partially or fully protect a subject from disease, infection and/or symptoms.
  • to “fully protect” means that a treated subject does not develop a disease or infection caused by an agent such as an influenza virus, or coronavirus.
  • to “partially protect” as used herein means that a certain subset of subjects may be fully protected from developing a disease or infection after treatment, or that the subject does not develop a disease or infection with the same severity as an untreated subject.
  • an amount of an agent is therapeutically effective if it is sufficient to alleviate one or more symptoms of a disorder, disease, or condition being treated, or to otherwise provide a desired pharmacologic and/or physiologic effect.
  • the precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease or disorder being treated, as well as the route of administration and the pharmacokinetics of the agent being administered.
  • subject-dependent variables e.g., age, immune system health, etc.
  • the disease or disorder being treated as well as the route of administration and the pharmacokinetics of the agent being administered.
  • One of ordinary skill in the art may empirically determine the effective amount of a particular compound without necessitating undue experimentation.
  • pharmaceutically acceptable refers to compositions, polymers, and other materials and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to pharmaceutically acceptable materials, compositions, or vehicles, such as a liquid or solid filler, diluent, solvent or encapsulating material involved in carrying or transporting any subject composition, from one organ, or portion of the body, to another organ, or portion of the body.
  • pharmaceutically acceptable salt is art- recognized, and includes relatively non-toxic, inorganic and organic acid addition salts of compounds.
  • pharmaceutically acceptable salts include those derived from mineral acids, such as hydrochloric acid and sulfuric acid, and those derived from organic acids, such as ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.
  • suitable inorganic bases for the formation of salts include the hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, and zinc.
  • Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts.
  • the class of such organic bases may include mono-, di-, and trialkylamines, such as methylamine, dimethylamine, and triethylamine; mono-, di- or trihydroxyalkylamines such as mono-, di-, and triethanolamine; amino acids, such as arginine and lysine; guanidine; N- methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; and N-benzylphenethylamine.
  • biodegradable generally refers to a material that will degrade or erode under physiological conditions to smaller units or chemical species that are capable of being metabolized, eliminated, or excreted by the body.
  • the degradation time of a material is a function of composition and morphology of the material.
  • inhibitor or “reduce” generally mean to reduce or decrease in activity and quantity. This can be a complete inhibition or reduction in activity or quantity, or a partial inhibition or reduction. Inhibition or reduction can be compared to a control or to a standard level. Inhibition can be 5, 10, 25, 50, 75, 80, 85, 90, 95, 99, or 100%, or an integer there between. In some embodiments, the inhibition and reduction are compared at nucleic acid, protein, cell, tissue and/or organ levels.
  • prevention means to administer a composition or method to a subject or a system at risk for or having a predisposition for one or more symptom caused by a disease or disorder, to decrease the likelihood the subject will develop one or more symptoms of the disease or disorder, or to reduce the severity, duration, or time of onset of one or more symptoms of the disease or disorder.
  • bioactive agent and “active agent”, as used interchangeably include, physiologically or pharmacologically active substances that act locally or systemically in the body.
  • a bioactive agent is a substance used for the treatment (e.g., therapeutic agent), prevention (e.g., prophylactic agent), diagnosis (e.g., diagnostic agent), cure or mitigation of disease or illness, a substance which affects the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment or processed.
  • protein polypeptide or “peptide” refer to a natural or synthetic molecule including two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.
  • polynucleotide or “nucleic acid” or “nucleic acid sequence” refers to a natural or synthetic molecule including two or more nucleotides linked by a phosphate group at the 3’ position of one nucleotide to the 5’ end of another nucleotide.
  • the polynucleotide is not limited by length, and thus the polynucleotide can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • compositions of replication-deficient poxvirus vectors modified to include or encode one or more exogenous antigens can provide long-term, cross-protective T-Cell mediated immunity to the antigen, when administered to epithelial tissue or subepithelial tissue like dermis and lamina basement that has been disrupted.
  • Compositions of poxviral vaccines encoding or including exogenous antigens for inducing broadly cross-protective T-cell mediated immunity to the vaccine antigens are described.
  • the compositions include a pox viral vector, the vector encoding or including one or more T-cell antigens.
  • one or more additional molecules enhances or induces the immune response in the recipient when co- administered with the poxviral vaccine.
  • Exemplary additional molecules include co-stimulatory molecules, growth factors, and cytokines.
  • the compositions include a pharmaceutically acceptable excipient for administration into the body by skin scarification.
  • modified, non-replicating or replication impaired vaccinia virus vaccines encode or include one or more exogenous T-cell antigens derived from an influenza virus and/or a coronavirus. Expression of the influenza virus and/or coronavirus antigens within the recipient provides protective T cell immunity to infection by influenza viruses and/or a coronaviruses having diverse genetic backgrounds.
  • compositions include a non-replicating or replication-impaired poxvirus encoding or including one or more exogenous antigens.
  • Several different replication-deficient or live, modified, non-replicating or replication-impaired vectors can be used. Because poxviruses have a large genome, they can readily be used to deliver a wide range of genetic material including multiple genes (i.e., act as a multivalent vector). The sizes of the poxvirus genomes range between about 130-300 kbp with up to 300 genes, depending on the strain of the virus. Therefore, it is possible to insert large fragments of foreign DNA into these viruses and yet maintain stability of the viral genome. The viruses have a low replicative efficiency in the host cell, which prevents sustained replication and infection of other cells.
  • Poxviruses are useful vectors for a range of uses, for example vaccines to generate immune responses, for the development of new vaccines, for delivery of desired proteins and for gene therapy.
  • the advantages of poxvirus vectors include: (i) ease of generation and production, (ii) the large size of the genome permitting insertion of multiple genes (i.e., as a multivalent vector), (iii) efficient delivery of genes to multiple cell types, including antigen-presenting cells, (iv) high levels of protein expression, (v) optimal presentation of antigens to the immune system, and (vi) the ability to elicit cell-mediated immune responses as well as antibody responses, (vii) the ability to use combinations of poxviruses from different genera, as they are not immunologically cross-reactive and (viii) the long-term experience gained with using this vector in humans as a smallpox vaccine.
  • Poxviruses can be genetically engineered to contain and express foreign DNA with or without impairing the ability of the virus to replicate.
  • Such foreign DNA can encode a wide range of proteins, such as antigens that induce protection against one or more infectious agents, immune modulating proteins such as co-stimulatory molecules, or enzymatic proteins.
  • recombinant vaccinia viruses have been engineered to express antigens of herpesvirus, hepatitis B, rabies, influenza, human immunodeficiency virus (HIV), and other viruses (Kieny et al., Nature 312:163-6 (1984); Smith et al., Nature 302: 490-5 (1983); Smith et al., Proc. Natl. Acad. Sci.
  • Poxviruses are well-known cytoplasmic viruses.
  • the genetic material expressed by such viral vectors typically remains in the cytoplasm and does not have the potential for inadvertent integration of the genetic material into host cell genes, unless specific steps are taken.
  • the poxvirus vector will not result in having long-term persistence in other cells within the host.
  • the vector and the transformed cells will not adversely affect cells in the host animal at locations distant from the target cell.
  • retrovirus vectors including lentiviral vectors
  • adenoviral vectors adenoviral vectors
  • adeno-associated virus vectors the large genome of poxviruses enables large genes to be inserted into pox-based vectors.
  • the intact non-replicating or replication- impaired poxvirus is derived by natural or artificial modification of a wildtype poxvirus.
  • poxviruses for use as viral vectors encoding or expressing T cell antigens include orthopox, suipox, avipox, capripox, leporipox, parapoxvirus, molluscpoxvirus, and yatapoxvirus.
  • a preferred orthopox virus is a vaccinia virus.
  • Exemplary vaccinia viruses include Modified Vaccinia Ankara (MVA), Wyeth strain, WR strain, NYCBH strain, ACAM2000, Lister strain, LC16m8, Elstree-BNm, Copenhagen strain, and Tiantan strain.
  • a preferred vaccinia virus is Modified Vaccinia Ankara (MVA).
  • modified poxvirus refers to a poxvirus that has been altered in some way that changes one or more characteristics of the modified virus compared to the wild-type virus. These changes may have occurred naturally or through engineering.
  • the sizes of the poxvirus genomes range between about 130-300 kbp with up to 300 genes, depending on the strain of the virus.
  • non-replicating poxvirus refers to a poxvirus that is not capable of replication to any significant extent in the majority of normal mammalian cells or normal primary human cells.
  • significant extent means a replication capability of 75% or less as compared to wild-type vaccinia virus in standardized assays.
  • the poxvirus has a replication capability of 65%, 55%, 45%, 35%, 25%, or 15% compared to wild-type vaccinia virus.
  • the virus has a replication capability 10% or less, 5% or less, or 1% or less compared to wild-type virus.
  • Nonreplicating viruses are 100% replication deficient in normal primary human cells.
  • the replication deficient, or non-replicating, or replication-impaired poxviruses are intact and viable particles, as opposed to virus that has been physically or chemically inactivated, for example, by exposure to formalin or P-propiolactone, to destroy infectivity.
  • Viral replication assays are known in the art, and can be performed for vaccinia viruses on e.g. primary keratinocytes, and are described in Liu et al. J.Virol. 2005, 79:12, 7363-70.
  • Viruses which are non-replicating or replication-impaired may have become so naturally (i.e., they may be isolated as such from nature) or artificially e.g., by breeding in vitro or by genetic manipulation, for example deletion of a gene which is critical for replication.
  • the modified poxvirus may also have altered characteristics concerning aspects of the viral life cycle, such as target cell specificity, route of infection, rate of infection, rate of replication, rate of virion assembly and/or rate of viral spreading.
  • the modified poxvirus is a Vaccinia virus (VV).
  • VV Vaccinia virus
  • Vaccinia virus (VV) is the prototype of the genus Orthopoxvirus. It is a double- stranded DNA (deoxyribonucleic acid) virus that has a broad host range under experimental conditions (Fenner et al. Orthopoxviruses. San Diego, Calif.: Academic Press, Inc., 1989; Damaso et al., Virology 277:439- 49 (2000)).
  • a number of poxviruses have been developed as live attenuated vaccinia virus strains, including Modified Vaccinia Ankara (MVA) and Wyeth (Cepko et al., Cell 37:1053 1062 (1984); Morin et al., Proc. Natl. Acad. Sci. USA 84:4626 4630 (1987); Lowe et al., Proc. Natl. Acad. Sci. USA, 84:3896 3900 (1987); Panicali & Paoletti, Proc. Natl. Acad. Sci. USA, 79:4927 4931(1982); Mackett et al., Proc. Natl. Acad. Sci.
  • Attenuated vaccinia virus strains include WR strain, NYCBH strain, ACAM2000, Lister strain, LC16m8, Elstree-BNm, Copenhagen strain, and Tiantan strain.
  • WR strain NYCBH strain
  • ACAM2000 Lister strain
  • LC16m8 Elstree-BNm
  • Copenhagen strain Copenhagen strain
  • Tiantan strain Tiantan strain.
  • the modified poxvirus is a Modified vaccinia Ankara (MVA) virus, or a derivative thereof.
  • Modified vaccinia virus Ankara, and derivative strains have been generated by long-term serial passages of the Ankara strain of vaccinia virus (CVA) on chicken embryo fibroblasts (for review see Mayr, A., et al., Infection, 3:6-14 (1975).
  • the MVA virus itself may be obtained from a number of public repository sources. For example, MVA was deposited in compliance with the requirements of the Budapest Treaty at CNCM (Institut Pasteur, Collection Nationale de Cultures Microorganisms, 25, rue du Dondel Roux, 75724 Pans Cedex 15) on Dec. 15, 1987 under Depositary No.
  • MVA virus was deposited in compliance with the Budapest Treaty at the European Collection of Cell Cultures (ECACC) (CAMR, Porton Down, Salisbury, SP4 OJG, UK) on Jan. 27, 1994, under Depository No. V94012707) (U.S. Patent No. 6,440,422 and United States patent publication number 2003/0013190). Also, United States patent publication number 2003/0013190 further discloses particular MVA strains deposited at the ECACC under Depository No. 99101431, and ECACC provisional accession number 01021411. Commercially available are THERION-MVA, THERION PRIFREE vectors and THERION M-SERIES vectors (Therion Biologies Corporation, MA).
  • MVA was generated by 516 serial passages on chicken embryo fibroblasts of the Ankara strain of vaccinia virus (CVA) (Mayr, A., et al. Infection 3, 6-14 [1975]). As a consequence of these long-term passages, about 31 kilobases of the genomic sequence were deleted from the virus (deletion I, II, III, IV, V, and VI) and, therefore, the resulting MVA virus was described as being highly host cell restricted to avian cells (Meyer, H. et al., J. Gen. Virol. 72, 1031-1038 [1991]). It was shown in a variety of animal models that the resulting MVA was significantly avirulent (Mayr, A. & Danner, K.
  • the genome of both wild type VV and MVA have both been sequenced, it is possible to clone viruses that bear some resemblance to MVA with regard to replication properties, but are genetically distinct from MVA. These may serve the same purpose, or may be more immunogenic than MVA while being just as safe by virtue of their replication deficiency. Changes in the virus include, for example, alterations in the gene expression profile of the virus.
  • the modified virus may express genes or portions of genes that encode peptides or polypeptides that are foreign to the poxvirus, i.e., would not be found in a wild-type virus.
  • These foreign, heterologous or exogenous peptides or polypeptides can include sequences that are immunogenic such as, for example, viral antigens, or antigenic sequences derived from viruses other than the viral vector.
  • the genetic material may be inserted at an appropriate site within the virus genome for the recombinant virus to remain viable, i.e., the genetic material may be inserted at a site in the viral DNA (e.g., non-essential site in the viral DNA) to ensure that the recombinant virus retains the ability to infect foreign cells and to express DNA, while maintaining the desired immunogenicity and diminished virulence.
  • MV A contains 6 natural deletion sites which have been demonstrated to serve as insertion sites. See, for example, U.S.
  • genes that code for desired antigens are inserted into the genome of a poxvirus in such a manner as to allow them to be expressed by that virus along with the expression of the normal complement of parent virus proteins.
  • Modified vaccinia virus vectors encode or include one or more exogenous T-cell antigens, inserted as genes for expression within the viral vector.
  • the inserted gene(s) are operably linked to one or more of a promoter, an enhancer, and a transcriptional regulatory element (TRE).
  • TRE transcriptional regulatory element
  • the inserted gene(s) encoding antigens may be operably linked to a promoter to express the inserted gene.
  • Promoters are well known in the art and can readily be selected depending on the host and the cell type one wishes to target.
  • pox viral promoters may be used, such as the vaccinia 7.5K, 40K, fowlpox.
  • enhancer elements can also be used in combination to increase the level of expression.
  • inducible promoters which are also well known in the art, may be used.
  • Representative poxvirus promoters include an entomopox promoter, an avipox promoter, or an orthopox promoter such as a vaccinia promoter, e.g., HH, 1 IK or Pi.
  • a vaccinia promoter e.g., HH, 1 IK or Pi.
  • the Pi promoter from the Ava I H region of vaccinia, is described in Wachsman et al. , J. of lnf. Dis. 155, 1188-1197 (1987).
  • This promoter is derived from the Ava I H (Xho I G) fragment of the L-variant WR vaccinia strain, in which the promoter directs transcription from right to left.
  • the map location of the promoter is approximately 1.3 Kbp (kilobase pair) from the 5' end of Ava IH, approximately 12.5 Kbp from the 5' end of the vaccinia genome, and about 8.5 Kbp 5' of the Hind III C/N junction.
  • the Hind III H promoter also "HH" and "H6” herein sequence is an up-stream of open reading frame H6 by Rosel et al., J. Virol. 60, 436-449 (1986).
  • the 11K promoter is as described by Wittek, J. Virol.
  • the promoter is modulated by an external factor or cue, allowing control of the level of polypeptide being produced by the vectors by activating that external factor or cue.
  • heat shock proteins are proteins encoded by genes in which the promoter is regulated by temperature.
  • the promoter of the gene which encodes the metal-containing protein metallothionine is responsive to Cd + ions. Incorporation of this promoter or another promoter influenced by external cues also makes it possible to regulate the production of the polypeptides including antigen.
  • the nucleic acid encoding at least one gene of interest encoding, e.g., an antigen is operably linked to an "inducible" promoter.
  • Inducible systems allow careful regulation of gene expression. See, Miller and Whelan, Human Gene Therapy, 8:803-815 (1997).
  • the phrase "inducible promoter” or “inducible system” as used herein includes systems wherein promoter activity can be regulated using an externally delivered agent. Such systems include, for example, systems using the lac repressor from E. coll as a transcription modulator to regulate transcription from lac operator-bearing mammalian cell promoters (Brown et al.
  • a "transcriptional regulatory element” or “TRE” is introduced for regulation of the gene of interest.
  • a TRE is a polynucleotide sequence, preferably a DNA sequence, that regulates transcription of an operably-linked polynucleotide sequence by an RNA polymerase to form RNA.
  • a TRE increases transcription of an operably linked polynucleotide sequence in a host cell that allows the TRE to function.
  • the TRE includes an enhancer element and/or viral promoter element, which may or may not be derived from the same gene.
  • the promoter and enhancer components of a TRE may be in any orientation and/or distance from the coding sequence of interest, and include multimers of the foregoing, as long as the desired, transcriptional activity is obtained.
  • an "enhancer" for regulation of the gene of interest is provided.
  • An enhancer is a polynucleotide sequence derived from a gene which increases transcription of a gene which is operably linked to a promoter to an extent which is greater than the transcription activation effected by the promoter itself when operably-linked to the gene, i.e. it increases transcription from the promoter.
  • a regulatory element such as a TRE or an enhancer generally depends upon the presence of transcriptional regulatory factors and/or the absence of transcriptional regulatory inhibitors.
  • Transcriptional activation can be measured in a number of ways known in the art, but is generally measured by detection and/or quantification of mRNA or the protein product of the coding sequence under control of (i.e., operatively linked to) the regulatory element.
  • the regulatory element can be of varying lengths, and of varying sequence composition.
  • Basal levels are generally the level of activity, if any, in a non-target cell, or the level of activity (if any) of a reporter construct lacking the TRE or enhancer of interest as tested in a target cell type.
  • Certain point mutations within sequences of TREs decrease transcription factor binding and gene activation.
  • One of skill in the art would recognize that some alterations of bases in and around known the transcription factor binding sites are more likely to negatively affect gene activation and cell-specificity, while alterations in bases which are not involved in transcription factor binding are not as likely to have such effects.
  • Certain mutations also increase TRE activity. Testing of the effects of altering bases may be performed in vitro or in vivo by any method known in the art, such as mobility shift assays, or transfecting vectors containing these alterations in TRE functional and TRE non-functional cells. Additionally, one of skill in the art would recognize that point mutations and deletions can be made to a TRE sequence without altering the ability of the sequence to regulate transcription.
  • Antigens are compounds that are specifically bound by antibodies or T lymphocyte antigen receptors.
  • T cell antigens are recognized by the TCR in the context of MHC I or II. These can be CD8 or CD4 effector T cells, as well as Tfh which help B cells make antibodies.
  • a T cell antigen can be any protein or peptide, but it is recognized by the B cell receptor/antibody alone, no MHC required. T cell antigens stimulate production of or are recognized by antibodies. Sometimes antigens are part of the host itself and can result in an autoimmune disease when the body attacks the self-antigens.
  • An immunogen is an antigen (or adduct) that is able to trigger a humoral (innate) or cell-mediated immune response.
  • an antigen binds the highly variable immunoreceptor products (B cell receptor or T cell receptor) once these have been generated.
  • Immunogens are those antigens, termed immunogenic, capable of inducing an immune response.
  • an immunogen is necessarily an antigen, but an antigen may not necessarily be an immunogen.
  • any of the antigens can also be an immunogenic (i.e., an immunogen).
  • antigens are selected or designed for immune stimulation or immune tolerance, of B- cells and/or T-cells, with or without the context of an MHC complex.
  • antigens are selected or designed for immune stimulation or immune tolerance, predominantly of T-cells.
  • the antigens are those suitable for MHC complex presentation by APC such as dendritic cells at or around the site of administration.
  • T cells respond to threats in an antigen- specific manner using T cell receptors (TCRs) that recognize short peptide antigens presented on major histocompatibility complex (MHC) proteins.
  • TCRs T cell receptors
  • MHC major histocompatibility complex
  • the TCR-peptide-MHC interaction mediated between a T cell and its target cell dictates its function and thereby influences its role in disease.
  • the antigen is a T cell antigen.
  • the T cell antigen is one that requires processing such as proteolytic cleavage by antigen-presenting cell before it can be recognized by the T lymphocytes.
  • Antigens include, for example, proteins, nucleic acids, lipids, and polysaccharides.
  • B cell antigens can be peptides, proteins, polysaccharides, saccharides, lipids, nucleic acids, small molecules (alone or with a hapten) or combinations thereof.
  • T cell antigens are typically proteins or peptides.
  • the T cell antigen is a polypeptide T cell receptors recognize peptides (or lipids in the case of CD1). They do not directly recognize nucleotides, only peptides from proteins encoding by nucleotides (since all proteins come from RNA which is transcribed from DNA, that’s pretty much everything)
  • the antigen can be derived from a virus, bacterium, parasite, plant, protozoan, fungus, tissue or transformed cell such as a cancer or leukemic cell and immunogenic component thereof, e.g., cell wall components or molecular components thereof.
  • the antigens can be allergens or environmental antigens or tumor antigens.
  • the antigen can be associated with one or more diseases or conditions such as infectious diseases, autoimmune diseases, and cancer.
  • Suitable antigens are known in the art and are available from commercial government and scientific sources.
  • the antigens can be purified or partially purified polypeptides derived from tumors or viral or bacterial sources.
  • the antigens can be recombinant polypeptides produced by expressing DNA or mRNA encoding the polypeptide antigen in a heterologous expression system.
  • Antigens can be provided as single antigens or can be provided in combination. Antigens can also be provided as complex mixtures of polypeptides or nucleic acids.
  • the antigen is a viral antigen.
  • a viral antigen can be isolated from any virus.
  • the antigen is a natural viral capsid structure or portion thereof, or a composite of a structure of multiple strains of the virus.
  • the antigen is a bacterial antigen. Bacterial antigens can originate from any bacteria.
  • the antigen is a parasite antigen.
  • the antigen is an allergen or environmental antigen.
  • allergens and environmental antigens include but are not limited to, an antigen derived from naturally occurring allergens such as pollen allergens (tree-, herb, weed-, and grass pollen allergens), insect allergens (inhalant, saliva and venom allergens), animal hair and dandruff allergens, and food allergens.
  • the antigen is a self-antigen such as in immune tolerance applications for auto-immune or related disorders such as Multiple Sclerosis.
  • the antigen is a tumor antigen.
  • Exemplary tumor antigens include a tumor-associated or tumor-specific antigen.
  • the antigens are those in an approved vaccines that are designed to elicit an immune response to protect again a particular pathogen.
  • Vaccines can elicit a response based ono wholepathogen vaccines such as inactivated viruses, live-attenuated viruses, and chimeric vaccine; subunit vaccines such as protein subunit vaccines, peptide vaccines, virus-like particles (VLPs), and recombinant proteins; and nucleic acid-based vaccines such as DNA plasmid vaccines, mRNA vaccines, and recombinant vector vaccines utilizing viral expression vectors.
  • Exemplary vaccines include Adenovirus Type 4 and Type 7 Vaccine, ERVEBO® (Ebola Zaire Vaccine, Live), DENGVAXIA® (Dengue Tetravalent Vaccine, Live), DAPTACEL® (Diphtheria and Tetanus Toxoids and Acellular Pertussis Vaccine), M-M-R II® (Measles, Mumps, and Rubella Virus Vaccine Live), TRUMENBA® (Meningococcal Group B Vaccine), POLIOVAX® (Poliovirus Vaccine Inactivated), IMOVAX® (Rabies Vaccine), RABAVERT® (Rabies Vaccine), ROTARIX® (Rotavirus Vaccine, Live), JYNNEOS® (Smallpox and Monkeypox Vaccine, Live), TYPHIM Vi® (Typhoid Vi Polysaccharide Vaccine), and YF-VAX® (Yello
  • COVID- 19 vaccines include Pfizer- BioNTech COVID-19 vaccine, Modema CO VID- 19 vaccine, Oxford/AstraZeneca CO VID-19 vaccine, Russia's Sputnik V COVID-19 vaccine, and Chinese Sinopharm CO VID- 19 vaccine.
  • the antigen is a viral antigen isolated from a virus including, but not limited to, a virus from any of the following viral families: Arenaviridae, Arterivirus, Astroviridae, Baculoviridae, Badnavirus, Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Capillovirus, Carlavirus, Caulimovirus, Circoviridae, Closterovirus, Comoviridae, Coronaviridae (e.g., Coronavirus, such as severe acute respiratory syndrome (SARS) virus), Corticoviridae, Cystoviridae, Deltavirus, Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virus and Ebola virus (e.g., Zaire, Reston, Ivory Coast, or Sudan strain)), Flaviviridae, (e.g., Hepatitis C virus, Dengue virus 1, Dengue virus
  • Viral antigens can be derived from a particular strain such as a papilloma virus, a herpes virus, e.g., herpes simplex 1 and 2; a hepatitis virus, for example, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the delta hepatitis D virus (HDV), hepatitis E virus (HEV) and hepatitis G virus (HGV), the tick-borne encephalitis viruses; parainfluenza, varicella-zoster, cytomeglavirus, Epstein-Barr, rotavirus, rhinovirus, adenovirus, coxsackieviruses, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, and lymphocytic choriomeningitis.
  • a hepatitis virus for example, hepatitis A virus (HAV), hepati
  • Exemplary viral antigens include influenza virus hemagglutinin (Genbank accession No. JO2132; Air, 1981, Proc. Natl. Acad. Sci. USA 78:7639-7643; Newton et al., 1983, Virology 128:495-501), influenza virus neuraminidase, PB1, PB2, PA, NP, Mi, M2, NSi, NS2)) of Influenza virus; E1A, E1B, E2, E3, E4, E5, LI, L2, L3, L4, L5 of Adenovirus; Pneumonoviridae (e.g., pneumovirus, human respiratory syncytial virus): Papovaviridae (polyomavirus and papillomavirus): El, E2, E3, E4, E5a, E5b, E6, E7, E8, LI, L2; Human respiratory syncytial virus: human respiratory syncytial virus: G glycoprotein (Genbank accession no.
  • RSV-viral proteins e.g., RSV F glycoprotein
  • Dengue virus core protein, matrix protein or other protein of Dengue virus (Genbank accession no. M19197; Hahn et al., 1988, Virology 162:167-180); Measles: measles virus hemagglutinin (Genbank accession no.
  • Herpesviridae e.g., herpes simplex virus 1, herpes simplex virus 2, herpes simplex virus 5, and herpes simplex virus 6: herpes simplex virus type 2 glycoprotein gB (Genbank accession no.
  • HIV ribonucleotide reductase
  • a-TIF ICP4, ICP8, 1CP35
  • LAT-related proteins gB, gC, gD, gE, gH, gl, gj, and dD antigens
  • Lentivirus e.g., human immunodeficiency virus 1 and human immunodeficiency virus 2: envelope glycoproteins of HIV I (Putney et al., 1986, Science 234:1392-1395)
  • Picomavindae e.g., enterovirus, rhinovirus, hepato
  • Hep B Surface Antigen gp27S, gp36S, gp42S, p22c, pol, x
  • Additional viruses include Ebola, Marburg, Rabies, Hanta virus infection, West Nile virus, SARS-like Coronaviruses, Varicella- zoster virus, Epstein-Barr virus, Alpha viruse, St. Louis encephalitis.
  • Adenovirdiae (mastadenovirus and aviadeno virus), Leviviridae (levivirus, enterobacteria phase MS2, allolevirus), Poxyiridae (e.g., chordopoxyirinae, parapoxvirus, avipoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus, molluscipoxvirus, and entomopoxyirinae), Papovaviridae (polyomavirus and papillomavirus); Paramyxoviridae (paramyxovirus, parainfluenza virus 1), Mobillivirus (measles virus), Rubulavirus (mumps virus), metapneumovirus (e.g., avian pneumovirus and human metapneumovirus); Pseudorabies: pseudorabies virus g50 (gpD), pseudorabies virus II (gpB), pseudorabies virus gill
  • Exemplary swine viruses include swine rotavirus glycoprotein 38, swine parvovirus capsid protein, Serpulina hydodysenteriae protective antigen, bovine viral diarrhea glycoprotein 55, neonatal calf diarrhea virus (Matsuno and Inouye, 1983, Infection and Immunity 39:155), hog cholera virus, African swine fever virus, swine influenza including antigens such as swine flu hemagglutinin and swine flu neuraminidase.
  • Exemplary equine viruses include equine influenza virus or equine herpesvirus: equine influenza virus type A/ Alaska 91 neuraminidase, equine influenza virus type A/Miami 63 neuraminidase, equine influenza virus type A/Kentucky 81 neuraminidase, equine herpesvirus type 1 glycoprotein B, and equine herpesvirus type 1 glycoprotein D, Venezuelan equine encephalomyelitis virus (Mathews and Roehrig, 1982, J. Immunol. 129:2763).
  • Exemplary cattle viruses include bovine respiratory syncytial virus or bovine parainfluenza virus: bovine respiratory syncytial virus attachment protein (BRSV G), bovine respiratory syncytial virus fusion protein (BRSV F), bovine respiratory syncytial virus nucleocapsid protein (BRSV N), bovine parainfluenza virus type 3 fusion protein, and bovine parainfluenza virus type 3 hemagglutinin neuraminidase), bovine viral diarrhea virus glycoprotein 48 or glycoprotein 53, infectious bovine rhinotracheitis virus: infectious bovine rhinotracheitis virus glycoprotein E or glycoprotein G, foot and mouth disease virus, punta toro virus (Dalrymple et al., 1981, in Replication of Negative Strand Viruses, Bishop and Compans (eds.), Elsevier, N.Y., p. 167).
  • BRSV G bovine respiratory syncytial virus attachment protein
  • BRSV F bovine respiratory syncytial virus
  • the antigen is a viral antigen isolated from an Orthomyxoviridae (e.g., Influenza virus A and B and C), or Coronaviridae (e.g., Coronavirus, such as severe acute respiratory syndrome (SARS) virus).
  • Orthomyxoviridae e.g., Influenza virus A and B and C
  • Coronaviridae e.g., Coronavirus, such as severe acute respiratory syndrome (SARS) virus.
  • SARS severe acute respiratory syndrome
  • the T cell antigen is an influenza virus antigen.
  • Influenza Virus antigens can be derived from a particular influenza clade or strain, or can be synthetic antigens, designed to correspond with highly conserved epitopes amongst multiple different influenza virus strains.
  • influenza viruses There are four types of influenza viruses: A, B, C and D.
  • Human influenza A and B viruses cause seasonal epidemics of disease.
  • Influenza A viruses are the only influenza viruses known to cause flu pandemics, i.e., global epidemics of flu disease.
  • Influenza type C infections generally cause mild illness and are not thought to cause human flu epidemics
  • Influenza D viruses primarily affect cattle and are not known to infect or cause illness in people (see w.w.w.cdc.gov/flu/about/viruses/types.htm).
  • the influenza A virion is studded with glycoprotein spikes of hemagglutinin (HA) and neuraminidase (NA), in a ratio of approximately four to one, projecting from a host cell-derived lipid membrane ( Figure 1).
  • HA hemagglutinin
  • NA neuraminidase
  • a smaller number of matrix (M2) ion channels traverse the lipid envelope, with an M2: HA ratio on the order of one M2 channel per 101-102 HA molecules.
  • the envelope and its three integral membrane proteins HA, NA, and M2 overlay a matrix of Ml protein, which encloses the virion core.
  • the nuclear export protein also called nonstructural protein 2, NS2
  • RNP ribonucleoprotein
  • NP nucleoprotein
  • PB1, PB2, and PA ribonucleoprotein
  • the organization of the influenza B virion is similar, with four envelope proteins: HA, NA, and, instead of M2, NB and BM2. Therefore, in some embodiments, the T cell antigen is derived from one or more of the HA, NA, M2, NS2, NB, PB1, PB2, PA or NP genes of any influenza A or B virus.
  • the T cell antigen is derived from the NP gene of an influenza A or B virus (Bouvier and Palese P, Vaccine. 2008;26 Suppl 4(Suppl 4):D49-D53. doi:10.1016/j.vaccine.2008.07.039).
  • influenza A and B virus genomes each include eight negativesense, single-stranded viral RNA (vRNA) segments, while influenza C virus has a seven-segment genome.
  • the eight segments of influenza A and B viruses (and the seven segments of influenza C virus) are numbered in order of decreasing length.
  • segments 1, 3, 4, and 5 encode just one protein per segment: the PB2, PA, hemagglutinin (HA) and nucleoprotein (NP) proteins.
  • All influenza viruses encode the polymerase subunit PB1 on segment 2; in some strains of influenza A virus, this segment also codes for the accessory protein PB1-F2, a small, 87-amino acid protein with pro-apoptotic activity, in a +1 alternate reading frame.
  • segment 6 of the influenza A virus encodes only the NA protein
  • that of influenza B virus encodes both the NA protein and, in a -1 alternate reading frame, the NB matrix protein, which is an integral membrane protein corresponding to the influenza A virus M2 protein.
  • Segment 7 of both influenza A and B viruses code for the Ml matrix protein.
  • the M2 ion channel is also expressed from segment 7 by RNA splicing, while influenza B virus encodes its BM2 membrane protein in a +2 alternate reading frame.
  • both influenza A and B viruses possess a single RNA segment, segment 8, from which they express the interferon- antagonist NS1 protein and, by mRNA splicing, the NEP/NS2, which is involved in viral RNP export from the host cell nucleus, (see Figure 1).
  • Influenza A viruses are divided into subtypes based on hemagglutinin (H) and neuraminidase (N) proteins on the surface of the virus. There are 18 different hemagglutinin subtypes and 11 different neuraminidase subtypes (Hl through Hl 8, and N1 through Nil, respectively). Therefore, in some embodiments, the T cell antigen is derived from the HA gene of an influenza virus influenza from any one or more of the Hl through Hl 8 subtypes. In other embodiments, the T cell antigen is derived from the NA gene of an influenza virus from any one or more of the N1 through Nil subtypes.
  • the T cell antigen is derived from, or provides T cell immunity to an A(H1N1) influenza virus, or an A(H3N2) influenza virus.
  • the T cell antigen is conserved amongst, and/or provides T cell immunity to all A(H1N1) influenza viruses.
  • the T cell antigen is conserved amongst, and/or provides T cell immunity to all A(H3N2) influenza viruses.
  • the T cell antigen is conserved amongst, and/or provides T cell immunity to both A(H1N1) influenza viruses and A(H3N2) influenza viruses.
  • Influenza A viruses are further classified into multiple subtypes (e.g., H1N1, or H3N2), while influenza B viruses are classified into one of two lineages: B/Yamagata and B/Victoria. Both influenza A and B viruses can be further classified into specific clades and sub-clades. Clades and sub-clades can be alternatively called “groups” and “sub-groups,” respectively.
  • An influenza clade or group is a further subdivision of influenza viruses (beyond subtypes or lineages) based on the similarity of their HA gene sequences.
  • Clades and subclades are shown on phylogenetic trees as groups of viruses that usually have similar genetic changes (i.e., nucleotide or amino acid changes) and have a single common ancestor represented as a node in the tree. Clades and sub-clades that are genetically different from others are not necessarily antigenically different (i.e., viruses from a specific clade or subclade may not have changes that impact host immunity in comparison to other clades or sub-clades). In some embodiments, the T cell antigen is conserved amongst, and/or provides T cell immunity to two or more influenza viruses within the same subtype and/or sub-clade.
  • the T cell antigen is conserved amongst, and/or provides T cell immunity to two or more influenza viruses within different subtypes and/or sub-clades. In some embodiments, the T cell antigen is conserved amongst, and/or provides T cell immunity to all influenza viruses within the same subtype and/or sub-clade. In preferred embodiments, the T cell antigen is conserved amongst, and/or provides T cell immunity to multiple influenza viruses within different subtypes and/or sub-clades.
  • H1N1 viruses are related to the pandemic 2009 H1N1 virus that emerged in spring of 2009 and caused a flu pandemic (See w.w.w.cdc.gov/flu/about/viruses/types.htm). This virus is known as “A(HlNl)pdmO9 virus,” or “2009 H1N1,” and continued to circulate seasonally from 2009 to 2021. These H1N1 viruses have undergone relatively small genetic changes and changes to their antigenic properties over time.
  • the T cell antigen is derived from and/or provides T cell immunity to all currently circulating H1N1 influenza viruses. In some embodiments, the T cell antigen is derived from and/or provides T cell immunity to all currently circulating H3N2 influenza viruses. In preferred embodiments, the T cell antigen is derived from and/or provides T cell immunity to all currently circulating H1N1 influenza viruses and H3N2 influenza viruses. In some embodiments, the T cell antigen is derived from an Influenza A virus NP gene, or an Influenza A virus NP gene expression product.
  • Influenza B viruses are classified into two lineages: B/Yamagata and B/Victoria. Influenza B viruses are further classified into specific clades and sub-clades. Influenza B viruses change more slowly in terms of genetic and antigenic properties than influenza A viruses. Surveillance data from recent years shows co-circulation of influenza B viruses from both lineages in the United States and around the world with. Therefore, in some embodiments, the T cell antigen is derived from and/or provides T cell immunity to influenza B viruses. In some embodiments, the T cell antigen is derived from and/or provides T cell immunity to all currently circulating influenza B viruses. In some embodiments, the T cell antigen is derived from an Influenza B virus NP gene, or an Influenza B virus NP gene expression product.
  • the T cell antigen is derived from and/or provides T cell immunity to B/Yamagata and B/Victoria influenza viruses. In other embodiments, the T cell antigen is derived from and/or provides T cell immunity to one or more H1N1 influenza virus, and to one or more influenza B virus. In other embodiments, the T cell antigen is derived from and/or provides T cell immunity to one or more H3N2 influenza virus, and to one or more influenza B virus. In other embodiments, the T cell antigen is derived from and/or provides T cell immunity to one or more H1N1 influenza virus, to one or more H3N2 influenza virus, and to one or more influenza B virus.
  • Exemplary T Cell antigens include influenza virus hemagglutinin (Genbank accession No. JO2132; Air, 1981, Proc. Natl. Acad. Sci. USA 78:7639-7643; Newton et al., 1983, Virology 128:495-501), influenza virus neuraminidase, PB1, PB2, PA, NP, Mi, M2, NSi, NS2)) of Influenza virus; swine influenza including antigens such as swine flu hemagglutinin and swine flu neuraminidase.
  • influenza virus hemagglutinin Genbank accession No. JO2132; Air, 1981, Proc. Natl. Acad. Sci. USA 78:7639-7643; Newton et al., 1983, Virology 128:495-501
  • influenza virus neuraminidase PB1, PB2, PA, NP, Mi, M2, NSi, NS2
  • Exemplary equine viruses include equine influenza virus or equine herpesvirus: equine influenza virus type A/ Alaska 91 neuraminidase, equine influenza virus type A/Miami 63 neuraminidase, equine influenza virus type A/Kentucky 81 neuraminidase.
  • Exemplary cattle viruses include bovine parainfluenza virus type 3 fusion protein, and bovine parainfluenza virus type 3 hemagglutinin neuraminidase).
  • NP Influenza Nucleoprotein
  • the antigen includes the NP gene of H1N1 2009 pandemic influenza virus.
  • An exemplary nucleic acid sequence for an H1N1 subtype influenza virus nucleoprotein (NP) gene is the Influenza A virus (A/California/07/2009(H1N1)) segment 5 nucleocapsid protein (NP) gene, set forth in GenBank accession number FJ969536.1 (SEQ ID NO:1):
  • GAGCATCCCA GTGCTGGGAA GGACCCTAAG AAAACAGGAG
  • Derivatives of SEQ ID NO:2 are also described. Derivatives include changes to one or more amino acids within SEQ ID NO:2, for example, by substitution, deletion or addition of one or more single amino acids at any position within SEQ ID NO:2.
  • Exemplary T-Cell epitopes for the H1N1 influenza virus nucleoprotein (NP) include 5-100 contiguous amino acid residues from SEQ ID NO:2, or derivatives of SEQ ID NO:2.
  • Exemplary T- Cell antigens against the H1N1 influenza virus nucleoprotein (NP) include 5- 50 contiguous amino acid residues in length from SEQ ID NO:2, or from derivatives of SEQ ID NO:2.
  • T-Cell antigens from the H1N1 influenza virus nucleoprotein include 5-10, 10- 15, 15-20, 20-35 or more than 35 contiguous amino acid residues from SEQ ID NO:2, or from derivatives of SEQ ID NO:2.
  • An exemplary nucleic acid sequence for an H3N2 subtype influenza virus nucleoprotein (NP) gene is the Influenza A virus (strain A/X-31 H3N2) segment 5 nucleocapsid protein (NP) gene, set forth in GenBank accession number AB036779 (SEQ ID NOG): AGCAAAAGCA GGGTAGATAA TCACTCACTG AGTGACATCA AAATCATGGC GTCTCAAGGC ACCAAACGAT CTTACGAACA GATGGAGACT GATGGAGAAC GCCAGAATGC CACTGAAATC AGAGCATCCG TCGGAAAAAT GATTGGTGGA ATTGGACGAT TCTACATCCA AATGTGCACC GAACTCAAAC TCAGTGATTA TGAGGGACGG TTGATCCAAA ACAGCTTAAC AATAGAGAGA ATGGTGCTCT CTGCTTTTGA CGAAAGGAGA AATAAATACC TTGAAGAACA TCCCAGTGCG GGGAAAGATC CTAAGAAAAC TG
  • Derivatives of SEQ ID NO:3 are also described. Derivatives include changes to one or more nucleic acids within SEQ ID NO: 3, for example, by substitution, deletion or addition of one or more single nucleic acids at any position within SEQ ID NO:3.
  • An exemplary amino acid sequence for an H3N2 subtype influenza virus nucleoprotein (NP) gene expression product is the nucleocapsid protein (NP) from (strain A/X-31 H3N2), set forth in GenBank accession number AB036779.1 (SEQ ID NO:4): MASQGTKRSY EQMETDGERQ NATEIRASVG KMIGGIGRFY IQMCTELKLS DYEGRLIQNS LTIERMVLSA FDERRNKYLE EHPSAGKDPK KTGGPIYRRV NGKWMRELIL YDKEEIRRIW RQANNGDDAT AGLTHMMIWH SNLNDATYQR TRALVRTGMD PRMCSLMQGS TLPRRSGAAG AAVKGVGTMV MELVRMIKRG INDRNFWRGE NGRKTRIAYE RMCNILKGKF QTAAQKAMMD QVRESRNPGN AEFEDLTFLA RSALILRGSV AHKSCLPACV YGPAVASG
  • Derivatives of SEQ ID NO:4 are also described. Derivatives include changes to one or more amino acids within SEQ ID NO:4, for example, by substitution, deletion or addition of one or more single amino acids at any position within SEQ ID NO:4.
  • Exemplary T-Cell epitopes for the H3N2 influenza virus nucleoprotein (NP) include 5-100 contiguous amino acid residues from SEQ ID NO:4, or from derivatives of SEQ ID NO:4.
  • Exemplary T-Cell antigens against the H3N2 influenza virus nucleoprotein (NP) include 5-50 contiguous amino acid residues in length from SEQ ID NO:4, or from derivatives of SEQ ID NO:4.
  • T-Cell antigens from the H3N2 influenza virus nucleoprotein include 5-10, 10-15, 15-20, 20-35 or more than 35 contiguous amino acid residues from SEQ ID NO:4, or from derivatives of SEQ ID NO:4.
  • influenza virus T Cell antigen is conserved amongst multiple different strains of influenza viruses such as the conserved influenza proteins like Ml, M2, PB1 etc. b. Coronavirus antigens
  • the antigen is derived from one or more coronavirus.
  • the coronaviruses (order Nidovirales, family Coronaviridae, and genus Coronavirus) are a diverse group of large, enveloped, positive- stranded RNA viruses that cause respiratory and enteric diseases in humans and other animals.
  • Coronaviruses typically have narrow host specificity and can cause severe disease in many animals, and several viruses, including infectious bronchitis virus, feline infectious peritonitis virus, and transmissible gastroenteritis virus, are significant veterinary pathogens.
  • Human coronaviruses are found in both group 1 (HCoV-229E) and group 2 (HCoV-OC43) and are historically responsible for -30% of mild upper respiratory tract illnesses.
  • RNA viruses At -30,000 nucleotides, their genome is the largest found in any of the RNA viruses.
  • groups 1 and 2 contain mammalian viruses, while group 3 contains only avian viruses.
  • group 3 contains only avian viruses.
  • coronaviruses are classified into distinct species by host range, antigenic relationships, and genomic organization.
  • the genomic organization is typical of coronaviruses, with the characteristic gene order (5’-replicase [rep], spike [S], envelope [E], membrane [M], nucleocapsid [N]-3 ’ ) and short untranslated regions at both termini.
  • SARS-CoV rep gene which includes approximately two-thirds of the genome, encodes two polyproteins (encoded by ORFla and ORFlb) that undergo co-translational proteolytic processing. There are four open reading frames (ORFs) downstream of rep that are predicted to encode the structural proteins, S, E, M, and N, which are common to all known coronaviruses. i. SARS-CoV-2
  • the antigen is an antigen from a severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) betacoronavirus of the subgenus Sarbecovirus.
  • SARS-CoV-2 viruses share approximately 79% genome sequence identity with the SARS-CoV virus identified in 2003.
  • An exemplary nucleic acid sequence for the SARS-CoV-2 ORFla/b gene is set forth in GenBank accession number MN908947.3. The genome organization of SARS-CoV-2 viruses is shared with other betacoronaviruses; six functional open reading frames (ORFs) are arranged in order from 5’ to 3’: replicase (ORFla/ORFlb), spike (S), envelope (E), membrane (M) and nucleocapsid (N). In addition, seven putative ORFs encoding accessory proteins are interspersed between the structural genes.
  • the T cell antigen includes one or more SARS-CoV-2 antigens from one or more of the genes encoding structural (S, E, M, N), or non-structural (NSPs) SARS-CoV-2 proteins.
  • the T cell antigen includes one or more SARS-CoV-2 genes or gene expression products with selected epitopes in the SARS-CoV- 2 genome that are conserved amongst multiple different coronaviruses.
  • the antigen includes one or more CD 8 T cell peptides targeting one or more structural (S, M, N) and non-structural (NSPs) SARS-CoV-2 proteins with selected epitopes in conserved regions of the SARS-CoV-2 genome, eliciting T cell mediate immune response specific to the one or more SARS-CoV-2 antigens.
  • Exemplary antigens include, but are not limited to the peptides listed in Table 1, and/or variants thereof. Table 1.
  • Coronavirus Antigens ii. SARS-CoV-2 N on-structural proteins (nsp)
  • the T cell antigen is derived from, or raises a T cell immune response to, one or more SARS-CoV-2 non- structural proteins (nsp).
  • nsps non-structured proteins
  • ORFla open-reading frame la
  • ORFlab genes which are translated as polyproteins followed by proteolytic cleavage.
  • These subunit proteins form the multi-subunit polymerase complex, which enable the transcription and replication of the viral genome.
  • nsp 12 is the catalytic subunit with RNA dependent RNA polymerase (RdRp) activity.
  • RdRp RNA dependent RNA polymerase
  • nspl2-nsp7-nsp8 subcomplex is thus defined as the minimal core components for mediating coronavirus RNA synthesis.
  • severealothernsp subunits arerequiredtoassembleintoaholoenzymecomplex,includingthe nsp10,nsp!3,nsp14andnsp!6(Peng,etal.,CellReports,31(11),107774, june16,2020).
  • GCCCTTGCAC CTAATATGAT GGTAACAAAC AATACCTTCA CACTCAAAGG CGGTGCACCA
  • AAAACTTTGC AACCAGTATC TGAATTACTT ACACCACTGG GCATTGATTT AGATGAGTGG
  • TATTGTTCTT TCTACCCTCC AGATGAGGAT GAAGAAGAAG GTGATTGTGA AGAAGAAGAG
  • GGTAGTACAT TTATTAGTGA TGAAGTTGCG AGAGACTTGT CACTACAGTT TAAAAGACCA
  • GAAAACATGA CACCCCGTGA CCTTGGTGCT TGTATTGACT GTAGTGCGCG TCATATTAAT
  • TTCAGTAACT CAGGTTCTGA TGTTCTTTAC CAACCACCAC AAACCTCTAT CACCTCAGCT
  • ATGCGTATTA TGACATGGTT GGATATGGTT GATACTAGTT TGTCTGGTTT TAAGCTAAAA
  • AATGCTAACC AAGTCATCGT CAACAACCTA GACAAATCAG CTGGTTTTCC ATTTAATAAA
  • GTAGAAAACC CTCACCTTAT GGGTTGGGAT TATCCTAAAT GTGATAGAGC CATGCCTAAC
  • AATATCTCAG ATGAGTTTTC TAGCAATGTT GCAAATTATC AAAAGGTTGG TATGCAAAAG TATTCTACAC TCCAGGGACC ACCTGGTACT GGTAAGAGTC ATTTTGCTAT TGGCCTAGCT CTCTACTACC CTTCTGCTCG CATAGTGTAT ACAGCTTGCT CTCATGCCGC TGTTGATGCA CTATGTGAGA AGGCATTAAA ATATTTGCCT ATAGATAAAT GTAGTAGAAT TATACCTGCA CGTGCTCGTG TAGAGTGTTT TGATAAATTC AAAGTGAATT CAACATTAGA ACAGTATGTC TTTTGTACTG TAAATGCATT GCCTGAGACG ACAGCAGATA TAGTTGTCTT TGATGAAATT TCAATGGCCA CAAATTATGA TTTGAGTGTT GTCAATGCCA GATTACGTGC TAAGCACTAT GTGTACATTG GCGACCCTGC TCAATTACCT GCACCACGCA CATTGCTAAC TAAGGGCACA
  • GTTAGTGCTA AACCACCGCC TGGAGATCAA TTTAAACACC TCATACCACT TATGTACAAA
  • AAAGGTTTAC AACCATCTGT AGGTCCCAAA CAAGCTAGTC TTAATGGAGT CACATTAATT
  • AAAGAAGGTC AAATCAATGA TATGATTTTA TCTCTTCTTA GTAAAGGTAG ACTTATAATT
  • DerivativesofSEQ ID NO:5 are alsodescribed.Derivativesinclude changestooneormorenucleicacidswithinSEQ ID NO:5,forexample,by substitution,deletionoradditionofoneormoresinglenucleicacidsatany positionwithinSEQ ID NO:5.
  • VDNTNLHTQL VDMSMTYGQQ FGPTYLDGAD VTKIKPHVNH EGKTFFVLPS
  • VKTQFNYFKK VDGI IQQLPE TYFTQSRDLE DFKPRSQMET DFLELAMDEF
  • EKGRLIIREN NRWVSSDIL VNN Denvatives of SEQ ID NO:6 are also described.
  • Derivatives include changes to one or more amino acids within SEQ ID NO:6, for example, by substitution, deletion or addition of one or more single amino acids at any position within SEQ ID NO:6.
  • Exemplary T-Cell antigens from the SARS- COV-2 virus include 5-5,000 contiguous amino acid residues in length from SEQ ID NO:6, or from derivatives of SEQ ID NO:6.
  • T-Cell antigens from the SARS-COV-2 virus include 5-10, 10-15, 15-20, 20-35 or more than 35 contiguous amino acid residues from SEQ ID NO: 6
  • a T cell antigen for the SARS-COV-2 virus Nspl2 includes five or more contiguous amino acid residues from positions 4393-5324 of SEQ ID NO:6, or from derivatives of SEQ ID NO:6.
  • a T cell antigen for the SARS-COV-2 virus Nspl3 includes five or more contiguous amino acid residues from positions 5325-5925 of SEQ ID NO:6, or from derivatives of SEQ ID NO:6.
  • a T cell antigen for the SARS-COV-2 virus Nspl4 includes five or more contiguous amino acid residues from positions 5926-6798 of SEQ ID NO:6, or from derivatives of SEQ ID NO:6.
  • a T cell antigen for the SARS-COV-2 virus Nspl5 includes five or more contiguous amino acid residues from positions 6457-6798 of SEQ ID NO:6, or from derivatives of SEQ ID NO:6.
  • a T cell antigen for the SARS-COV-2 virus Nspl6 includes five or more contiguous amino acid residues from positions 6799-7096 of SEQ ID NO:6, or from derivatives of SEQ ID NO:6.
  • the viral gene segment encoding a T cell antigen for the SARS-COV-2 virus includes the entire length of NSP1-14, or segments thereof, especially Nspl2 and Nspl3, or Nspl2 and Nspl4, or Nspl2 and Nspl6, or Nspl3 and Nspl4, or Nspl3 and Nspl6, or Nspl4 and Nspl6, or the entire length of Nspl2, Nspl3 and Nspl4.
  • GenBank accession number KF963214.1 GenBank accession number KF963214.1 (SEQ ID NO:7): TCAAAAGATA CTAATTTTTT AAACGGGTTC GGGGTGCGAG TGTAGATGCC CGTCTCGTAC CCTGCGCCAG TGGTTTATCT ACTGATGTAC AATTAAGGGC ATTTGATATT TACAATGCTA GTGTTGCTGG CATTGGTTTA CATTTAAAAG
  • CAGTATGATT TTACTGATTA CAAGCTTGAA TTGTTTAATA AGTATTTTAA
  • DerivativesofSEQ ID NO:7 are alsodescribed.Derivativesinclude changestooneormorenucleicacidswithinSEQ ID NO:7,forexample,by substitution,deletionoradditionofoneormoresinglenucleicacidsatany positionwithinSEQ ID NO:7.
  • Derivatives of SEQ ID NO:8 are also described. Derivatives include changes to one or more amino acids within SEQ ID NO:8, for example, by substitution, deletion or addition of one or more single amino acids at any position within SEQ ID NO:8.
  • Exemplary T-Cell antigens for the SARS- COV-2 virus nspl2 include amino acid fragments of 5-100 residues in length from SEQ ID NO: 8, or from derivatives of SEQ ID NO: 8.
  • the antigen is derived from, or raises a T cell immune response to, one or more of the S (spike) gene, nspl2 gene, nspl3 gene, nspl4 gene, or nspl6 gene of SARS-COV-2.
  • two or more antigens are derived from two or more of the S (spike) gene, nspl2 gene, nspl3 gene, nspl4 gene, or nspl6 gene of SARS-COV-2.
  • T-Cell antigens are derived from, or raise a T cell immune response to, one or more of the SARS-COV-2 peptides, such as the virus nsplO gene product, the nspll gene product, the nspl2 gene product, the nspl3 gene product, the nspl4 gene product, or the nspl6 gene product.
  • the virus nsplO gene product such as the virus nsplO gene product, the nspll gene product, the nspl2 gene product, the nspl3 gene product, the nspl4 gene product, or the nspl6 gene product.
  • two or more T-Cell antigens are derived from, or raise a T cell immune response to two or more of the SARS- COV-2 virus nsplO gene product, the nspll gene product, the nspl2 gene product, the nspl3 gene product, the nspl4 gene product, or the nspl6 gene product.
  • the antigen is a bacterial antigen.
  • Bacterial antigens can originate from bacteria such as Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Hemophilus influenza type B (HTB), Hyphomicrobium, Legionella, Leptspirosis, Listeria, Meningococcus A, B and C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas, Phodospirillum, Ricket
  • the antigenic or immunogenic protein fragment or epitope is derived from a pathogenic bacteria such as Anthrax; Chlamydia: Chlamydia protease-like activity factor (CPAF), major outer membrane protein (MO MP); Mycobacteria; Legioniella: Legionella peptidoglycan-associated lipoprotein (PAL), mip, flagella, OmpS, hsp60, major secretory protein (MSP); Diptheria: diptheria toxin (Audibert et al., 1981, Nature 289:543); Streptococcus 24M epitope (Beachey, 1985, Adv. Exp. Med. Biol.
  • a pathogenic bacteria such as Anthrax; Chlamydia: Chlamydia protease-like activity factor (CPAF), major outer membrane protein (MO MP); Mycobacteria; Legioniella: Legionella peptidoglycan-associated lipoprotein (PAL), mip, flagella, OmpS, hs
  • LcrV pestis low calcium response protein V
  • Fl Fl and Fl-V fusion protein
  • Francisella tularensis Rickettsia typhi
  • Treponema pallidum Salmonella: SpaO and Hl a
  • OMPs outer membrane proteins
  • Pseudomonas P.aeruginosa OMPs, PcrV, OprF, OprI, PilA and mutated ToxA.
  • the antigenic or immunogenic protein fragment or epitope is derived from a fungus, including, but not limited to,
  • Coccidioides immitis Coccidioides Ag2/Pral06, Prp2, phospholipase (P1b), alpha-mannosidase (Amnl), aspartyl protease, Gell;
  • Blastomyces dermatitidis Blastomyces dermatitidis surface adhesin WI-1;
  • Cryptococcus neoformans Cryptococcus neoformans GXM and its Peptide mimotopes, and mannoproteins, Cryptosporidiums surface proteins gpl5 and gp40, Cp23 antigen, p23; Candida spp. inclduing C. albicans. C. glabrata. C. parapsilosis. C. dubliniensis. C. krusei. and others;
  • Aspergillus species Aspergillus Asp f 16 , Asp f 2, Der p 1, and Fel d 1, rodlet A, PEP2, Aspergillus HSP90, 90-kDa catalase.
  • the antigenic or immunogenic protein fragment or epitope is derived from a pathogenic protozoan.
  • exemplary protozoa or protozoan antigens include:
  • Plasmodium falciparum Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium apical membrane antigen 1 (AMA1), 25-kDa sexual-stage protein (Pfs25), erythrocyte membrane protein 1 (PfEMPl) circumsporozoite protein (CSP), Merozoite Surface Protein- 1 (MSP1);
  • AMA1 apical membrane antigen 1
  • Pfs25 25-kDa sexual-stage protein
  • PfEMPl erythrocyte membrane protein 1
  • CSP circumsporozoite protein
  • MSP1 Merozoite Surface Protein- 1
  • Leishmania species Leishmania cysteine proteinase type III (CPC)
  • Trypanosome species African and American: T. pallidum outer membrane lipoproteins, Trypanosome beta-tubulin (STIB 806), microtubule- associate protein (MAP pl5), cysteine proteases (CPs)
  • Cryptosporidiums isospora species; Naegleria fowleri ; Acanthamoeba species; Balamuthia mandrillaris;
  • Pneumocystis carinii Pneumocystis carinii major surface glycoprotein (MSG), p55 antigen;
  • Schistosomiasis Schistosomiasis mansoni Sml4, 21.7 and SmFim antigen, Tegument Protein Sm29, 26kDa GST, Schistosoma japonicum, SjCTPI, SjC23, Sj22.7, or SjGST-32
  • Toxoplasmosis gondii surface antigen 1 (TgSAGl), protease inhibitor- 1 (TgPI-1), surface-associated proteins MIC2, MIC3, ROP2, GRA1-GRA7. 5. Cancer Antigens
  • the antigen is a cancer antigen or a nucleic acid or vector thereof encoding a cancer antigen.
  • a cancer antigen is an antigen that is typically expressed preferentially by cancer cells (i.e., it is expressed at higher levels in cancer cells than on non-cancer cells; cancer- associated antigen) and in some instances it is expressed solely by cancer cells (cancer-specific antigen).
  • Cancer antigen may be expressed within a cancer cell or on the surface of the cancer cell.
  • Exemplary cancer antigens include tumor-associated antigens (TAAs), tumor specific antigens (TSAs), tissue- specific antigens, viral tumor antigens, cellular oncogene proteins, and/or tumor- associated differentiation antigens.
  • TAAs can serve as targets for the host immune system and elicit responses which result in tumor destruction.
  • This immune response is mediated primarily by lymphocytes; T cells in general and class I MHC-restricted cytotoxic T lymphocytes in particular play a central role in tumor rejection.
  • the cloning of TAAs for cancer immunotherapy is described e.g. in Boon, T., et al., (1994) Annu. Rev.
  • T-cell activation is often potentiated by providing a suitable immunomodulator, for example a T-cell co-stimulatory factor such as those of the B7 gene family.
  • a suitable immunomodulator for example a T-cell co-stimulatory factor such as those of the B7 gene family. See e.g., Greenberg, P. D. (1991) in Advances in Immunology, Vol. 49 (Dixon, D. J., ed.), pp. 281 355, Academic Press, Inc., Orlando, Fla.; Fox B. A. et al. (1990) J. Biol. Response Mod. 9:499 511.
  • vaccinia viruses for anti-tumor immunotherapy has been described in Hu, S. L., Hellstrom, I., and Hellstrom K. E. (1992) in Vaccines: New Approaches to Immunological Problems (R. W. Ellis, ed) pp. 327 343, Butterworth- Heinemann, Boston.
  • Anti-tumor responses have been elicited using recombinant pox viruses expressing TAAs such as carcinoembryonic antigen (CEA) and prostate specific antigen (PSA).
  • CEA carcinoembryonic antigen
  • PSA prostate specific antigen
  • Cancer antigens include, but are not limited to, melanoma TAAs such as MART-1 (Kawakami et al., J. Exp. Med. 180:347-352, 1994), MAGE-1, MAGE-3, GP-100, (Kawakami et al., Proc. Nat'l. Acad. Sci. U.S.A. 91:6458-6462, 1994), tyrosinase (Brichard et al. J. Exp. Med.
  • TAAs such as MUC-1, MUC-2, MUC-3, MUC-4, MUC-18, the point mutated ras oncogene and the point mutated p53 oncogenes (pancreatic cancer), PSA (prostate cancer), c-erb/B2 (breast cancer), KS 1/4 pan- carcinoma antigen (Perez and Walker, 1990, J. Immunol. 142:3662-3667; Bumal, 1988, Hybridoma 7(4):407-415), ovarian carcinoma antigen (CA125) (Yu et al., 1991, Cancer Res. 51 (2) :468-475), prostatic acid phosphate (Tailor et al., 1990, Nucl.
  • PSA prostate specific antigen
  • PSA prostate specific antigen
  • melanoma- associated antigen p97 Estin et al., 1989, J. Natl. Cancer Instit. 81(6):445- 446)
  • melanoma antigen gp75 Vijayasardahl et al., 1990, J. Exp. Med.
  • HMW-MAA high molecular weight melanoma antigen
  • CEA carcinoembryonic antigen
  • TAG-72 Yokata et al., 1992, Cancer Res.
  • melanoma specific antigens such as ganglioside GD2 (Saleh et al., 1993, J. Immunol., 151, 3390-3398), ganglioside GD3 (Shitara et al., 1993, Cancer Immunol. Immunother. 36:373-380), ganglioside GM2 (Livingston et al., 1994, J. Clin. Oncol. 12:1036-1044), ganglioside GM3 (Hoon et al., 1993, Cancer Res.
  • tumor-specific transplantation type of cell-surface antigen such as virally induced tumor antigens including T-antigen DNA tumor viruses and Envelope antigens of RNA tumor viruses, bladder tumor oncofetal antigen (Hellstrom et al., 1985, Cancer. Res. 45:2210-2188), differentiation antigen such as human lung carcinoma antigen L6, L20 (Hellstrom et al., 1986, Cancer Res. 46:3917-3923), antigens of fibrosarcoma, human leukemia T cell antigen- Gp37 (Bhattacharya-Chatterjee et al., 1988, J. of Immuno specifically.
  • TSTA tumor-specific transplantation type of cell-surface antigen
  • neoglycoprotein neoglycoprotein
  • sphingolipids breast cancer antigen such as EGFR (Epidermal growth factor receptor), HER2 antigen (pl 85 HER2 ), polymorphic epithelial mucin (PEM) (Hilkens et al., 1992, Trends in Bio. Chem. Sci.
  • malignant human lymphocyte antigen- APO- 1 (Bernhard et al., 1989, Science 245:301-304), differentiation antigen (Feizi, 1985, Nature 314:53-57) such as I antigen found in fetal erythrocytes, primary endoderm, I antigen found in adult erythrocytes, preimplantation embryos, I(Ma) found in gastric adenocarcinomas, Ml 8, M39 found in breast epithelium, SSEA-1 found in myeloid cells, VEP8, VEP9, Myl, VIM-D5, D156-22 found in colorectal cancer, TRA-1-85 (blood group H), C14 found in colonic adenocarcinoma, F3 found in lung adenocarcinoma, AH6 found in gastric cancer, Y hapten, LeY found in embryonal carcinoma cells, TL5 (blood group A), EGF receptor found in A431 cells, Ei series (blood group B) found in pancre
  • the antigen is a neoantigen or a patient- specific antigen.
  • neoantigen or a patient-specific antigen.
  • Recent technological improvements have made it possible to identify the immune response to patient-specific neoantigens that arise as a consequence of tumor- specific mutations, and emerging data indicate that recognition of such neoantigens is a major factor in the activity of clinical immunotherapies (Schumacher and Schreidber, Science, 348(6230):69-74 (2015).
  • Neoantigen load provides an avenue to selectively enhance T cell reactivity against this class of antigens.
  • TAAs tumor-associated antigens
  • TAAs tumor-associated antigens
  • TAAs include cancer-testis antigens and differentiation antigens, and even though self-antigens have the benefit of being useful for diverse patients, expanded T cells with the high-affinity TCR (T-cell receptor) needed to overcome the central and peripheral tolerance of the host, which would impair anti-tumor T-cell activities and increase risks of autoimmune reactions.
  • the antigen is recognized as “non-self” by the host immune system, and preferably can bypass central tolerance in the thymus.
  • examples include pathogen-associated antigens, mutated growth factor receptor, mutated K-ras, or idiotype-derived antigens. Somatic mutations in tumor genes, which usually accumulate tens to hundreds of fold during neoplastic transformation, could occur in protein-coding regions. Whether missense or frameshift, every mutation has the potential to generate tumor- specific antigens. These mutant antigens can be referred to as “cancer neoantigens” Ito, et al., Cancer Neoantigens: A Promising Source of Immunogens for Cancer Immunotherapy.
  • Neoantigen-based cancer vaccines have the potential to induce more robust and specific anti-tumor T-cell responses compared with conventional shared-antigen-targeted vaccines.
  • MPS massively parallel sequencing
  • the composition including an intact, non- replicating or replication-impaired poxvirus and one or more T cell antigens is administered in combination with one or more molecules that enhance or induce a T cell-mediated immune response within the recipient.
  • exemplary molecules include cytokines and co-stimulatory molecules.
  • the composition administered to the subject, or co-expressed within the subject includes a co-stimulatory molecule, a growth factor, or a cytokine.
  • exemplary molecules include IL-1, IL-2, IL-7, IL-12, IL-15, IL-18, IL-23, IL-27, B7-2, B7-H3, CD40, CD40L, ICOS- ligand, OX-40L, 4-1BBL, GM-CSF, SCF, FGF, Fantigen-ligand, and CCR4.
  • the one or more additional molecules is administered to the subject before, at the same time, and/or after the poxvirus and T cell antigen is administered, the one or more additional molecules may be administered to the subject to the same site that the T cell antigen is administered, or at a distant site.
  • the compositions also include one or more cytokines or co-stimulatory molecules.
  • the one or more cytokines or co-stimulatory molecules is co-administered with the antigen and the replication-deficient or non-replicating viral vectors.
  • the one or more cytokines or co-stimulatory molecules is encoded within the replication-deficient or non-replicating viral vectors.
  • the one or more cytokines or co-stimulatory molecules is encoded in a separate vector.
  • cytokines or co-stimulatory molecules such as interleukin (IL) (e.g., IL-2, IL-4, IL-10, IL-12), an interferon (IFN) (e.g., IFN- ⁇ ), granulocyte macrophage colony stimulating factor (GM-CSF) or an accessory molecule (e.g., ICAM-1) or co-stimulatory molecules, e.g., B7.1, B7.2, may be used as adjuvants.
  • IL interleukin
  • IFN interferon
  • GM-CSF granulocyte macrophage colony stimulating factor
  • accessory molecule e.g., ICAM-1
  • co-stimulatory molecules e.g., B7.1, B7.2
  • one or more of cytokines, co-stimulatory and other immunomodulatory molecules can be co-administered via co-insertion of the genes encoding the molecules into the replication-deficient or non- replicating viral vectors or a second vector which is admixed with the recombinant virus expressing the antigen.
  • one or more of cytokines, co-stimulatory and other immunomodulatory molecules can be administered separately at the same site or different site, or systemically to the host. It may be desirable to administer a substantially pure preparation of the immunomodulator to boost efficacy of the host immune response to the T cell antigen.
  • costimulatory molecules include, but are not limited to, B7-1, B7-2, ICAM-1, CD40, CD40L, LFA-3, CD72, OX40L (with or without 0X40).
  • cytokines and growth factors include but are not limited to: granulocyte macrophage-colony stimulating factor (GM- CSF), granulocyte-colony stimulating factor (G-CSF), macrophage-colony stimulating factor (M-CSF), tumor necrosis factors (TNF ⁇ and TNFp), transforming growth factors (TGF ⁇ and TGF ⁇ ), insulin-like growth factors (IGF-I and IGF-II), growth hormone, interleukins 1 to 15 (IL-1 to IL-15), interferons ⁇ , ⁇ , ⁇ (IFN- ⁇ IFN- ⁇ and IFN- ⁇ ), brain-derived neurotrophic factor, neurotrophins 3 and 4, hepatocyte growth factor, erythropoietin, EGF- like mitogens, TGF-like growth factors
  • the co-stimulatory molecule, growth factor, adjuvant or cytokine is IL-1, IL-2, IL-4, IL-7, IL1-9, IL-12, IL-15, IL-18, IL-23, IL-27, IL-31, IL-33, B7-1, B7-2, B7-H3, LFA-3, B7- H3, CD40, CD40L, ICOS-ligand, OX-40L, 4-1BBL, GM-CSF, SCF, FGF, Fantigen- ligand, CCR4, QS-7, QS-17, QS-21, CpG oligonucleotides, ST- 246, AS-04, LT R192G mutant, Montanide ISA 720, heat shock proteins, synthetic mycobacterial cordfactor (CAF01), Lipid A mimetics, Salmonella enterica serovar Typhimurium flagellin (FliC), Montanide 720, Levamisole (LMS), Imi
  • poxviruses expressing B7-1, ICAM-1, and LFA-3 also known as TRICOM, are provided that induce activation of both CD4 + and CD8 + T cells.
  • TRICOM poxviruses expressing B7-1, ICAM-1, and LFA-3
  • TRICOM also known as TRICOM
  • 0X40 is a primary co-stimulator of T cells that have encountered antigen, rather than naive T cells, and promotes T-cell expansion after T cell tolerance is induced.
  • OX40L plays a role during T cell activation by a) sustaining the long-term proliferation of CD4 + and CD8 + T cells, b) enhancing the production of Thl cytokines such as IL-2, IGN-y, and TNF- ⁇ from both CD4 + and CD8 + T cells without changing IL-4 expression, c) protecting T cells from apoptosis.
  • the combination of B7-1, ICAM-1, LFA-3, and OX40L enhances initial activation and then further potentiates sustained activation of naive and effector T cells.
  • Adjuvants can also be administered.
  • the poxvirus also contains OX40L.
  • Other useful adjuvants that can be administered separately from the poxvirus are, for example, RIBI Detox (Ribi Immunochemical), QS21 (Aquila), incomplete Freund's adjuvant.
  • compositions including poxviruses and T cell antigens are administered in the form of a composition including one or more other pharmaceutically acceptable carriers, including any suitable diluent or excipient.
  • the pharmaceutically acceptable carrier does not itself induce a physiological response, e.g., an immune response does not result in any adverse or undesired side effects and/or does not result in undue toxicity.
  • Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof. Additional examples of pharmaceutically acceptable carriers, diluents, and excipients are provided in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J., current edition).
  • compositions include a conventional adjuvant such as alum (e.g., aluminum hydroxide, aluminum phosphate); saponins purified from the bark of the Q. saponana tree such as QS21 (a glycolipid that elutes in the 21st peak with HPLC fractionation; Antigenics, Inc., Worcester, Mass.); poly[di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus Research Institute, USA), Fantigen ligand, Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, Wash.), ISCOMS (immunostimulating complexes which contain mixed saponins, lipids and form virus-sized particles with pores that can hold antigen; CSL, Melbourne, Australia), Pam3Cys, SB-AS4 (SmithKline Beecham adjuvant system #4 which contains alum and MPL; SBB, Belgium), non-ionic block copolymers that form micelles such as CRL 1005
  • Adjuvants may be TLR ligands.
  • Adjuvants that act through TLR3 include without limitation double- stranded RNA.
  • Adjuvants that act through TLR4 include without limitation derivatives of lipopolysaccharides such as monophosphoryl lipid A (MPLA; Ribi ImmunoChem Research, Inc., Hamilton, Mont.) and muramyl dipeptide (MDP; Ribi) andthreonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland).
  • Adjuvants that act through TLR5 include without limitation flagellin.
  • Adjuvants that act through TLR7 and/or TLR8 include single- stranded RNA, oligoribonucleotides (ORN), synthetic low molecular weight compounds such as imidazoquinolinamines (e.g., imiquimod (R-837), resiquimod (R-848)).
  • Adjuvants acting through TLR9 include DNA of viral or bacterial origin, or synthetic oligodeoxynucleotides (ODN), such as CpG ODN.
  • Another adjuvant class is phosphorothioate containing molecules such as phosphorothioate nucleotide analogs and nucleic acids containing phosphorothioate backbone linkages.
  • the adjuvant can also be oil emulsions (e.g., Freund's adjuvant); saponin formulations; virosomes and viral-like particles; bacterial and microbial derivatives; immunostimulatory oligonucleotides; ADP- ribosylating toxins and detoxified derivatives; alum; BCG; mineralcontaining compositions (e.g., mineral salts, such as aluminium salts and calcium salts, hydroxides, phosphates, sulfates, etc.); bioadhesives and/or mucoadhesives; microparticles; liposomes; polyoxyethylene ether and polyoxyethylene ester formulations; polyphosphazene; muramyl peptides; imidazoquinolone compounds; and surface active substances (e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).
  • Adjuvants may also include immunomodulators such as cytokines, interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., interferon-gamma), macrophage colony stimulating factor, and tumor necrosis factor.
  • immunomodulators such as cytokines, interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., interferon-gamma), macrophage colony stimulating factor, and tumor necrosis factor.
  • the compositions include one or more pharmaceutically acceptable carriers, or excipients, or preservatives.
  • Pharmaceutically acceptable carriers include compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio, in accordance with the guidelines of agencies such as the Food and Drug Administration.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, diluents, preservatives, binders, stabilizers, a mixture or polymer of sugars (lactose, sucrose, dextrose, etc.), salts, and combinations thereof.
  • compositions may be administered in combination with one or more physiologically or pharmaceutically acceptable carriers, thickening agents, co-solvents, adhesives, antioxidants, buffers, viscosity and absorption enhancing agents and agents capable of adjusting osmolarity of the formulation.
  • physiologically or pharmaceutically acceptable carriers such as thickening agents, co-solvents, adhesives, antioxidants, buffers, viscosity and absorption enhancing agents and agents capable of adjusting osmolarity of the formulation.
  • the compositions may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, dyes, pH buffering agents, or preservatives.
  • compositions including effective amounts of the composition, and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or earners.
  • Such compositions include diluents such as stenle water, buffered saline of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and optionally, additives such as antioxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives and bulking substances (e.g., lactose, mannitol).
  • non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.
  • the pharmaceutical composition for cells is a saline solution, preferably a buffered saline solution phosphate buffered saline or sterile saline, or tissue culture medium.
  • the DNA donor vector contains the following elements: (i) a prokaryotic origin of replication, so that the vector may be amplified in a prokaryotic host; (ii) a gene encoding a marker which allows selection of prokaryotic host cells that contain the vector (e.g., a gene encoding antibiotic resistance); (iii) at least one gene encoding a desired protein located adjacent to a transcriptional promoter capable of directing the expression of the gene; and (iv) DNA sequences homologous to the region of the parent virus genome where the foreign gene(s) will be inserted, flanking the construct of element (iii).
  • a prokaryotic origin of replication so that the vector may be amplified in a prokaryotic host
  • a gene encoding a marker which allows selection of prokaryotic host cells that contain the vector e.g., a gene encoding antibiotic resistance
  • Genes encoding an antigen of interest can be amplified by cloning the gene into a bacterial host.
  • various prokaryotic cloning vectors can be used. Examples are plasmids pBR322 and pEMBL.
  • the cloned genes can be excised from the prokaryotic cloning vector by restriction enzyme digestion.
  • the DNA fragment carrying the cloned gene can be modified as needed, for example, to make the ends of the fragment compatible with the insertion sites of the poxvirus vectors, then purified prior to insertion into these vectors at restriction endonuclease cleavage sites (cloning sites).
  • Foreign genes for insertion into the genome of a virus in expressible form can be obtained using conventional techniques for isolating a desired gene.
  • the genes encoding an antigen of interest may be isolated from the genomic DNA; for organisms with RNA genomes, the desired gene may be isolated from cDNA copies of the genome.
  • restriction maps of the genome are available, strategies can be designed for cleaving genomic DNA by restriction endonuclease digestion to yield DNA fragments that contain the gene of interest.
  • the DNA sequence of the gene is known, the gene can be synthesized by any of the conventional techniques for polymerase chain reaction or synthesis of deoxyribonucleic acids (e.g., the phosphate or phosphite-triester techniques).
  • nucleic acids are provided that express antigenic domains rather than the entire protein. These fragments may be of any length sufficient to be immunogenic or antigenic. Fragments may be at least four amino acids long, preferably 5-9 amino acids, but may be longer, such as e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500 amino acids long or more, or any length in between.
  • Epitopes that induce a protective immune response to a pathogen such as bacteria, viruses, fungi, or protozoa or to a cancer antigen may be combined with heterologous gene sequences that encode proteins with immunomodulating activities, such as cytokines, interferon type 1, gamma interferon, colony stimulating factors, interleukin- 1, -2, -4, -5, -6, -12.
  • immunomodulating activities such as cytokines, interferon type 1, gamma interferon, colony stimulating factors, interleukin- 1, -2, -4, -5, -6, -12.
  • At least one nucleic acid fragment encoding a gene is inserted into a viral vector.
  • at least two and up to about ten different nucleic acids encoding different genes are inserted into the viral vector.
  • multiple immunogenic fragments or subunits of various proteins may be used. For example, several different epitopes from different sites of a single protein or from different proteins of the same species, or from a protein ortholog from different species may be expressed.
  • the DNA gene sequence to be inserted into the virus can be placed into a plasmid, e.g., an E. coll plasmid construct, into which DNA homologous to a section of DNA such as that of the poxvirus has been inserted.
  • a plasmid e.g., an E. coll plasmid construct
  • the DNA gene sequence to be inserted is ligated to a promoter.
  • the promoter-gene linkage is positioned in the plasmid construct so that the promoter-gene linkage is flanked on both ends by DNA homologous to a DNA sequence flanking a region of pox DNA which is the desired insertion region.
  • the resulting plasmid construct is then amplified by growth within E. coli bacteria and isolated.
  • the plasmid also contains an origin of replication such as the E. coli origin of replication, and a marker such as an antibiotic resistance gene for selection and propagation in E. coli.
  • the isolated plasmid containing the DNA gene sequence to be inserted is transfected into a cell culture, e.g., chick embryo fibroblasts, along with the poxvirus. Recombination between homologous pox DNA in the plasmid and the viral genome respectively results in a poxvirus modified by the presence of the promoter-gene construct in its genome, at a site which does not affect virus viability.
  • the gene(s) is inserted into a site or region (insertion region) in the virus which does not significantly affect viability of the resultant recombinant virus, e.g., intragenic regions between viral genes, preferably non-essential viral genes.
  • a site or region in the virus which does not significantly affect viability of the resultant recombinant virus, e.g., intragenic regions between viral genes, preferably non-essential viral genes.
  • the skilled artisan can readily identify such regions in a virus by, for example, testing segments of virus DNA for regions that allow recombinant formation without seriously affecting virus viability of the recombinant.
  • insertion regions include, for example, BamHI J (Jenkins, et al., AIDS Research and Human-Retroviruses 7:991-998 (1991)) the EcoRI-Hindlll fragment, BamHI fragment, EcoRV-HindHIII fragment, BamHI fragment and the Hindlll fragment set forth in EPO Application No. 0308 220 Al. (Calvert, et al., J.
  • donor plasmids for the introduction of multiple foreign genes into poxvirus are described, for example, in WO91/19803, the techniques of which are incorporated herein by reference.
  • all DNA fragments for construction of the donor vector including fragments containing transcriptional promoters and fragments containing sequences homologous to the region of the parent virus genome into which foreign genes are to be inserted, can be obtained from genomic DNA or cloned DNA fragments.
  • the donor plasmids can be mono-, di-, or multivalent (i.e. , can contain one or more inserted foreign gene sequences).
  • the donor vector may contain an additional gene which encodes a marker which will allow identification of recombinant viruses containing inserted foreign DNA.
  • marker genes can be used to permit the identification and isolation of recombinant viruses. These include, for example, genes that encode antibiotic or chemical resistance (e.g., see Spyropoulos et al., J. Virol., 62:1046 (1988); Falkner and Moss, J. Virol., 62:1849 (1988); Franke et al., Mol. Cell. Biol., 5:1918 (1985), as well as genes such as the E. coli lacZ gene, that permit identification of recombinant viral plaques by colorimetric assay (Panicali et al., Gene, 47:193 199 (1986)).
  • Homologous recombination between donor plasmid DNA and viral DNA in an infected cell results in the formation of recombinant viruses that incorporate the desired elements.
  • Appropriate host cells for in vivo recombination are generally eukaryotic cells that can be infected by the virus and transfected by the plasmid vector. Examples of such cells suitable for use with a poxvirus are chick embryo dermal (CED) cells, HuTK143 (human) cells, and CV-1 and BSC-40 (both monkey kidney) cells. Infection of cells with poxvirus and transfection of these cells with plasmid vectors is accomplished by techniques standard in the art (Panicali and Paoletti, U.S. Pat. No.
  • the donor DNA can be directly ligated into the parental virus genome at a unique restriction site (Scheiflinger, et al. (1992) Proc. Natl. Acad. Sci. (USA) 89:9977 9981).
  • recombinant viral progeny can be identified by several techniques well known in the art. For example, if the DNA donor vector is designed to insert foreign genes into the parent virus thymidine kinase (TK) gene, viruses containing integrated DNA will be TK’ and can be selected on this basis (Mackett et al., Proc. Natl. Acad. Sci. USA, 79:7415 (1982)). Alternatively, co-integration of a gene encoding a marker or indicator gene with the foreign gene(s) of interest, as described above, can be used to identify recombinant progeny.
  • One preferred indicator gene is the E. coli lacZ gene: recombinant viruses expressing - galactosidase can be selected using a chromogenic substrate for the enzyme (Panicali et al., Gene, 47:193 (1986)).
  • the cells used for virus/vaccine production may be cell lines, i.e., cells that grow continuously in vitro, either as single-cell suspension culture in bioreactors or as a monolayer on a cellsupport surface of tissue culture flasks or roller-bottles.
  • Primary animal cells may be used for the manufacture of vaccines.
  • chordopoxvirinae, in particular MVA are amplified in cell cultures of primary or secondary chicken embryo fibroblasts (CEF). The cells are obtained from embryos of chicken eggs that are incubated for 10 to 12 days. The cells of the embryos are then dissociated and purified.
  • primary CEF cells can either be used directly or after one further cell passage as secondary CEF cells.
  • the primary or secondary CEF cells are infected with the MVA.
  • the infected cells are incubated for 2-3 days at 37°C. (see, e.g., Meyer, H. et al. 1991; J. of General Virology 72, 1031-1038; Sutter et al. 1994, Vaccine, Vol. 12, No. 11, 1032-1040).
  • CEF cells are often used since many virus vaccines are made by attenuating the virulent disease-causing virus by serially passaging in CEF cells.
  • Attenuated viruses such as MVA are preferably not propagated on human cells since there is a concern that the viruses might become replication competent in cells of human origin. Viruses that have regained the ability to replicate in human cells represent a health risk if administered to humans, in particular if the individuals are immune compromised. For this reason, some attenuated viruses, such as MVA, are strictly manufactured from CEF cells, if intended for human use. Moreover, CEF cells are used for those viruses that grow only in these cells, for example avian viruses such as avipox viruses, canary pox virus, ALVAC, Fowl pox virus and NYVAC. Replication defective or deficient typically apply to diploid mammalian cells or human cells, and not for example duck embryo fibroblasts. For example, MVA can be grown in DEF but not mammalian cells
  • host cells such as epidermal epithelial cells, fibroblasts, or dendritic cells, infected with the recombinant viruses express the antigen(s) and may additionally express the immunostimulatory molecule(s).
  • the antigen may be expressed at the cell surface of the infected host cell.
  • the immunostimulatory molecule may be expressed at the cell surface or may be actively secreted by the host cell.
  • both the antigen and/or the immunostimulatory molecule may provide the necessary MHC restricted peptide to specific immunosurveilling T cells and the appropriate signal to the T cell in the skin to aid in antigen recognition and proliferation or clonal expansion of antigen specific T cells.
  • the overall result may be an upregulation of the immune system.
  • the upregulation of the immune response is an increase in antigen specific T-helper lymphocytes and/or cytotoxic lymphocytes, including for example, Thl or Th2 CD4 + T-helper cell- mediated or CD8 + cytotoxic T-lymphocytes, which are able to kill or inhibit the growth of a disease causing agent (such as a cancer cell) or a cell infected with a disease causing agent (such as a cell infected with a virus, a bacteria, a fungus, or a protozoa.
  • the immune stimulation may also involve an antibody response including generations of one or more antibody classes, such as IgM, IgG, and/or IgA.
  • a variety of methods well known in the art can be used to assay the expression of the polypeptide encoded by the inserted gene(s). These methods include, for example, black plaque assay (an in situ enzyme immunoassay performed on viral plaques), Western blot analysis, radioimmunoprecipitation (RIP A), enzyme immunoassay (EIA), or functional assay such as CTL assay.
  • black plaque assay an in situ enzyme immunoassay performed on viral plaques
  • Western blot analysis Western blot analysis
  • EIA enzyme immunoassay
  • functional assay such as CTL assay.
  • Methods for inducing or stimulating a protective, broadly cross- reactive T cell mediated immune response to a T cell antigen have been established.
  • the methods deliver vaccine compositions including intact, non-replicating or replication-impaired poxviruses expressing one or more T cell antigens to an epithelial tissue that has been mechanically disrupted.
  • Suitable epithelial tissue includes skin, lung, oral mucosa, gastrointestinal tract, and reproductive mucosa.
  • the vaccine composition is administered in an amount sufficient to stimulate a protective immune response in the recipient.
  • the epithelial tissue is epidermis which is mechanically disrupted by a scarification needle, an abrader, or microneedles.
  • the epidermis is typically mechanically disrupted essentially at the same time as or before the administration of the composition.
  • the methods include administering or co-expressing a co-stimulatory molecule, a growth factor, an adjuvant and/or a cytokine, before, at the same time, or after the antigen, at the same or a distant site.
  • Suitable stimulatory molecules, growth factors, adjuvants and cytokines include IL-1, IL-2, IL-7, IL-12, IL-15, IL-18, IL-23, IL-27, B7-2, B7-H3, CD40, CD40L, ICOS-ligand, OX-40L, 4-1BBL, GM-CSF, SCF, FGF, Flt3-ligand, and CCR4.
  • the methods repeat the step of administering the vaccine composition to epithelial tissue that has been mechanically disrupted.
  • the second administration is carried out at a time of one, two, three, four, five, six, seven, eight, nine, or ten days or weeks after the first administration.
  • the methods provide protective immunity against the vaccinating antigen(s) and/or the original host where the vaccinating antigen is sourced.
  • TRM skin resident memory T cells
  • the methods have been developed based on the discovery that protective immune memory is mediated in large part by TRM that have accumulated over time in epithelial tissues.
  • the body has several epithelial surfaces that interface with the environment. The most accessible is skin, but continuous with skin is the oropharyngeal mucosal epithelium, the female reproductive epithelium, and the large and complex epithelial tissues that line the respiratory and gastrointestinal tracts.
  • a subject in need of treatment is a subject having or at risk of having cancer or a subject having or at risk of having an infection (e.g., a subject having or at risk of contracting a viral, bacterial, fungal, or protozoal infection).
  • the methods effectively induce T cell mediated immunity.
  • the methods are particularly suited for those having reduced or compromised humoral immunity such as those undergoing anti-CD20 therapy.
  • a subject having cancer is a subject that has detectable cancerous cells.
  • “Cancer” as used herein refers to an uncontrolled growth of cells which interferes with the normal functioning of the bodily organs and systems.
  • a subject at risk of developing a cancer is one who has a higher- than-normal probability of developing cancer.
  • These subjects include, for instance, subjects having a genetic abnormality, the presence of which has been demonstrated to have a correlative relation to a higher likelihood of developing a cancer.
  • These subjects also include subjects exposed to cancer causing agents (i.e., carcinogens) such as tobacco, asbestos, or other chemical toxins, or subjects who have previously been treated for cancer and are in apparent remission.
  • Other patients include those whose cancers have been resected surgically or removed with radiation therapy, so no apparent or detectable cancer remains. These patients often relapse, so the inability to detect cancer cells does not mean they do not have these cells at very low numbers.
  • a subject having an infection is a subject that has been exposed to an infectious microorganism and has acute or chronic detectable levels of the microorganism in his/her body or has signs and symptoms of the infectious microorganism.
  • Methods of assessing and detecting infections in a subject are known by those of ordinary skill in the art.
  • a subject at risk of having an infection is a subject that may be expected to come in contact with an infectious microorganism. Examples of such subjects are medical workers or those traveling to parts of the world where the incidence of infection is high.
  • the subject is at an elevated risk of an infection because the subject has one or more risk factors to have an infection.
  • risk factors to have an infection include, for example, immunosuppression, immunocompromised, age, trauma, bums (e.g., thermal burns), surgery, foreign bodies, cancer, newborns especially newborns born prematurely.
  • the degree of risk of an infection depends on the multitude and the severity or the magnitude of the risk factors that the subject has.
  • Risk charts and prediction algorithms are available for assessing the risk of an infection in a subject based on the presence and severity of risk factors. Other methods of assessing the risk of an infection in a subject are known by those of ordinary skill in the art.
  • the subject who is at an elevated risk of an infection may be an apparently healthy subject.
  • An apparently healthy subject is a subject who has no signs or symptoms of disease.
  • viruses that can be treated by the described methods, or for which the described methods confer protection include, but are not limited to, HIV, influenza, dengue, Hepatitis A virus, Hepatitis B vims, Hepatitis C virus, Human papilloma vims, Ebola, Marburg, Rabies, Hanta vims infection, West Nile virus, SARS-like Coronavimses, Herpes simplex vims (HSV1 and HSV2), Varicella- zoster virus, Epstein-Barr vims, Human herpesvirus 8, Alpha vimses, St. Louis encephalitis.
  • the vimses that can be treated by the described methods, or for which the described methods confer protection are coronaviruses.
  • the virus is SARS-CoV-2.
  • enteroviruses including, but not limited to, viruses that the family picornaviridae, such as polio virus, Coxsackie virus, echo virus
  • rotaviruses adenovirus
  • adenovirus hepatitis virus
  • viruses that have been found in humans include, but are not limited to, retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviviridae (e.g., encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e (e
  • Bacterial infections or diseases that can be treated or prevented are caused by bacteria including, but not limited to, Mycobacterium tuberculosis, Salmonella typhi, Bacillus anthracis, Yersinia perstis, Francisella tularensis, Legionella, Chlamydia, Rickettsia typhi, and Treponema pallidum.
  • Other bacteria that may be treated or for which the methods described herein confer protection include, but are not limited to, Pasteurella species, Staphylococci species, and Streptococcus species.
  • Gram negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas species, and Salmonella species.
  • infectious bacteria include, but are not limited to, Helicobacter pyloris, Borelia burgdorferi, Mycobacteria sps (e.g., M. avium, M. intracellulare, M. kansaii, M.
  • Fungal diseases that can be treated or prevented using the poxviruses and methods described herein include, but are not limited to, Coccidioides immitis, Blastomyces dermatitidis, Cryptococcus neoformans, Candida albicans and other Candida species, Aspergillus species.
  • Other fungi that may be treated or for which the methods described herein confer protection include, but are not limited to: Histoplasma capsulatum, Coccidioides immitis, and Chlamydia trachomatis.
  • Protozoal diseases or infections that can be treated or prevented unclude but are not limited to, Malaria (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae), Leishmania species, Trypanosome species (African and American), cryptosporidiums, isospora species, Naegleria fowleri, Acanthamoeba species, Balamuthia mandrillaris, Toxoplasma gondii, and Pneumocystis carinii.
  • Cancers or tumors which may be treated or prevented, but are not limited to, melanoma, cutaneous squamous cell carcinoma, basal cell carcinoma, breast cancer, prostate adenocarcinoma, prostatic intraepithelial neoplasia, squamous cell lung carcinoma, lung adenocarcinoma, small cell lung carcinoma, ovary cancer of epithelial origin, colorectal adenocarcinoma and leiomyosarcoma, stomach adenocarcinoma and leiomyosarcoma, hepatocellular carcinoma, cholangiocarcinoma, ductal adenocarcinomas of pancreas, endocrine pancreatic tumors, renal cell carcinoma, transitional cell carcinoma of kidney and bladder, bladder squamous cell carcinoma, papillary thyroid cancer, follicular thyroid cancer, brain cancers (astrocytoma, glioblastoma multiforme).
  • BCDTs B cell depletion therapies
  • B cells along with T cells, form the core of the adaptive arm of the immune system. They are generated continually and throughout the life of the organism in the bone marrow, a primary lymphoid tissue, from haematopoietic stem cell progenitors that progress through sequential developmental steps. Each developing B cell expresses a unique B cell receptor (BCR), which is composed of two identical heavy chain proteins and two identical light chain proteins. In both humans and rodents, once a developing B cell expresses a correctly assembled BCR on its cell surface, and that BCR has been confirmed to not be autoreactive, the IgM+ immature B cell exits the bone marrow, enters the blood and migrates to the spleen. It should be noted at some point that survival without detectable B cells is not only possible but frequent. In contrast, life without T cells (even CD4 T cells) renders the host susceptible to opportunistic infections which may be deadly.
  • GC B cells Due to their vigorous proliferation and the accompanying mutagenesis program, GC B cells can be vulnerable to cancerous transformation. For this reason, reagents that bind surface glycoproteins expressed by B cells such as CD20 have been successfully used to target B cell lymphomas for depletion, removing both cancerous and non-cancerous CD20+ B cells from the patient (Maloney, D. G. et al., Blood 84, 2457-2466 (1994).). Given the excellent safety profile of many B cell depletion therapies (BCDTs) in cancer (van Vollenhoven, R. F., et al., J. Rheumatol. 42, 1761-1766 (2015)), these drugs were reimagined for use in autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and multiple sclerosis (MS).
  • SLE systemic lupus erythematosus
  • RA rheumatoid
  • CD20 is a transmembrane calcium channel implicated in B cell activation, proliferation, and differentiation (Cheson BD and Leonard JP. N Engl J Med. 2008;359(6):613-626). It is present on the surface of B cells in the late pre-B cell through mature memory B cell stages. Therefore, anti- CD20 mAbs target B cells in this intermediate stage of development, sparing early pre-B cells and plasma cells, thus allowing for retention of long-term immune memory and B cell reconstitution following depletion. Due to the maintenance of antibody production by plasma cells, administration of anti- CD20 mAbs almost completely depletes peripheral B cells, but antibody levels are not dramatically reduced (DiLillo DJ, et al., J Immunol.
  • B cell depletion therapy may stem from the loss of other prominent B cells functions such as antigen presentation, production of inflammatory cytokines, activation of T cells, and creation of ectopic lymphoid follicles (Engel P, et al., Pharmacol Rev. 2011 ;63(1): 127- 156).
  • B lymphocytes or B cells are a central component of adaptive immunity and are a requisite for the secretion of antibodies against “non-self” antigens.
  • B -cell-depleting therapies do not eliminate B-cell immunity completely.
  • Anti-CD20 therapy results in depletion of circulating CD20-positive B cells in the periphery, but not hematopoietic stem cells in the bone marrow or antibody -producing long- lived plasma cells that lack CD20. Therefore, humoral immunity to childhood vaccines, such as tetanus or meningitis, is preserved during therapy with anti-CD20 therapy.
  • anti-CD 20 therapy both rituximab and ocrelizumab
  • anti-CD 20 therapy both rituximab and ocrelizumab
  • Similar blunting in immune response has been reported with respect to pneumococcus, haemophilus influenza B, hepatitis B, and tetanus toxoid in the context of anti-CD20 therapy (Richi P. et al., Clin Rheumatol. 2020; 39: 2751-2756).
  • mRNA messenger RNA
  • MS multiple sclerosis
  • a subject in need of treatment is a subject with a reduced number of peripheral B cells in the blood, for example, those undergoing B cell depletion therapy (BCDT) or those having completed a cycle of BCDT but prior to B cells repopulate.
  • BCDTs include those targeting CD20, CD 19 and BAFF.
  • the subject is one undergoing anti-CD20 therapy or those having recently completed one or more cycles of anti-CD20 therapy but prior to B cells repopulate.
  • a subject in need of treatment is a subject with a reduced number of peripheral B cells in the blood, for example, less than 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, compared to a healthy control.
  • a subject in need of treatment is a subject with CD19 + B cells less than 0.5%, 1%, 2%, 3%, 4%, or 5% of the total lymphocytes in the blood after rituximab treatment.
  • the subject undergoing BCDT has B cell- derived malignancies such as non-Hodgkin’s lymphoma (NHL) and chronic lymphocytic leukemia (CLL).
  • B cell- derived malignancies such as non-Hodgkin’s lymphoma (NHL) and chronic lymphocytic leukemia (CLL).
  • the subject undergoing BCDT has a systemic autoimmune disease such as multiple sclerosis, N-methyl-D-aspartate receptor (NMD AR) encephalitis, myasthenia gravis, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), myelin-oligodendrocyte glycoprotein (MOG) spectrum disorder (MOGSD), and neuromyelitis optica spectrum disorder (NMOSD).
  • a systemic autoimmune disease such as multiple sclerosis, N-methyl-D-aspartate receptor (NMD AR) encephalitis, myasthenia gravis, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), myelin-oligodendrocyte glycoprotein (MOG) spectrum disorder (MOGSD), and neuromyelitis optica spectrum disorder (NMOSD).
  • a systemic autoimmune disease such as multiple sclerosis, N-methyl-D-aspart
  • the subject undergoing BCDT has one or more of metabolic, myocardial, and vascular disorders including insulin resistance, myocardial infarction, myocardial injury, aneurysm, and atherosclerosis.
  • the subject undergoing BCDT has pemphigus vulgaris, a severe and rare autoimmune disease that results in blisters on the skin and mucosal membranes.
  • the subject undergoing BCDT has a neurological autoimmune disease such as those diagnosed by, or linked to, abnormal antibody levels in the cerebrospinal fluid (CSF).
  • CSF cerebrospinal fluid
  • the subject undergoing BCDT has MS or anti-MOG (myelin- oligodendrocyte glycoprotein) spectrum disorder.
  • the vaccine composition is administered to a subject during the course of BCDT (e.g., anti-CD20 therapy), or within a week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more than 12 months after having completed BCDT (e.g., anti-CD20 therapy).
  • BCDT e.g., anti-CD20 therapy
  • Modified, replication-deficient or non-replicating viral vectors expressing one or more exogenous T-cell antigens in an amount sufficient to elicit or stimulate the immune response against the antigen are administered to mechanically disrupted epidermal tissue of the subject.
  • Intranasal vaccine will generate a robust population of lung TRM, and skin TRM(Pan et al., 2021 NPJ vaccines) but relatively few liver, or GI tract T RM - Intraperitoneal administration does not generate large populations of skin or lung T RM . Intramuscular administration is ineffective at generating T RM in most tissues.
  • the adaptive immune system through the generation and maintenance of protective immunologic memory, permits people to live amongst a plethora of pathogens for many decades.
  • the body has several epithelial surfaces that interface with the environment. The most accessible is skin, but continuous with skin is the oropharyngeal mucosal epithelium, the female reproductive epithelium, and the large and complex epithelial tissues that line the respiratory and gastrointestinal tracts. For pathogens to gain access to blood and internal tissues, they must infect and then breach one or more of these epithelial barriers. While each of these epithelial tissues is structurally different and employs different innate immune defenses, the adaptive immune system mediates lifelong protection against pathogen attack, through T cell memory and B cell antibody production.
  • T RM skin resident T cells
  • T RM rather than antibody or TCM recruited from blood, provide principal protection against viral challenge, even many months after the initial infection.
  • Similar populations of T cells have been identified in lung (Connor et al. Eur J Immunol. 2010 Sep;40(9):2482- 92) and GI tract (Masopust et al. J Exp Med. 2010 Mar 15;207(3):553-64. Epub 2010 Feb 15) after infection.
  • These resident TEM/ TRM include largely of T cells that were originally (as naive T cells) activated in lymph nodes draining that tissue (Liu et al., Immunity. 2006;25(3):511-20), and thus represent a resident army of tissue specific T cells specific for tissue selective pathogens.
  • tissue specific T cells specific for tissue selective pathogens.
  • T cell recruitment into extranodal peripheral tissues is a highly regulated process controlled by the sequential interactions of adhesion molecules and chemokine receptors that are differentially expressed on various T cell subsets and their target.
  • Human skin-homing T cells express cutaneous lymphocyte antigen (CLA) which binds to skin microvasculature- expressed E-selectin, typically in combination with CCR4 whose ligand CCL17 (TARC) is constitutively expressed on skin endothelium.
  • CLA cutaneous lymphocyte antigen
  • TARC ligand CCL17
  • Guthoming T cells express ⁇ 4 ⁇ 7 integrin and chemokine receptor CCR9, to which the corresponding ligands MadCAM- 1 and CCL25 (TECK) are expressed on the endothelium and epithelium of the small intestine Following VACV skin scarification, the skin homing molecules E- and P- selectin ligands (E-lig and P-lig, the functional murine equivalents of human CLA) are strongly upregulated on antigen-specific CD8 T cells between the 3 rd and 10 th cell divisions in the regional LN draining the scarified site.
  • E-lig and P-lig the functional murine equivalents of human CLA
  • Subsets of proliferating VV specific CD8 T cells leave the draining inguinal node after as few as three cell divisions and migrate through blood to skin (TEM), or to other LN (TCM), respectively.
  • TEM skin
  • TCM LN
  • vaccinia- specific TCM cells continue to proliferate, in the absence of continued antigen receptor stimulation, and acquire homing receptors consistent with the regional drainage of the LN they have migrated to; e.g., ⁇ 4 ⁇ 7-integrin in mesenteric LN.
  • generalized CD 8 T cell mediated immunity to a local challenge is acquired by systemic dissemination of activated T cells from the local draining LN.
  • the tissue homing properties of these cells then are imprinted in the respective LN environments to which they disseminate.
  • Modes of vaccine delivery play a critical role in the generation of T RM -
  • Different modes of vaccine delivery can generate similar levels of antibody and T CM , but vastly different levels of T RM .
  • the presence or absence of T RM is reflected in resistance to infection, and demonstrates clearly that simply sampling blood for antibody titers and/or memory T cell abundance and function as a surrogate for protective immunity falls far short of this goal and may even be misleading.
  • Skin scarification with VACV is far and away the most effective way of generating both optimal TRM populations as well as TCM, not only in skin, but also in distant epithelial tissues. Mimicking infection through an epithelial tissue is essential to generating the robust protective immunity that the vertebrate immune system has, over millions of years, evolved to generate.
  • T RM significant populations of T RM can be readily demonstrated in human skin.
  • Normal skin of healthy adults contains approximately 20 billion memory T cells, nearly twice the number of T cells that are present in the entire circulation.
  • these skin homing/resident T cells are all CD45RO + memory cells, predominantly ⁇ TCR + , CLA + CCR4 + , and express some CCR6 and some CCR8 and CXCR6, and a subset of about 15-20% express both CD62L and CCR7.
  • skin TRM are 80% TEM and 20% TCM.
  • TRM TNF ⁇ and IL-17 producing T cells, as well as T cells that produce IFN- ⁇ ; relatively few cells that produce IL-4 are present.
  • TCR V ⁇ antibody FACS analysis indicates that these TRM are highly diverse, and all VP families are represented. Nearly half of these TRM expressed the activation markers CD25 (at intermediate levels) and CD69. TRM are also much more responsive to stimulation than cells from the blood.
  • An analogous population of TRM resides in human lung. These tissue resident cells are also exclusively CD45RO+, and do not bear either skin (CLA/CCR4 co expression) or gut ( ⁇ 4 ⁇ 7 integrin) homing markers, but express integrin ⁇ 4 ⁇ 7.
  • TRM Repetitive encounter with antigen through a peripheral tissue generates increasing numbers of TRM that accumulate in peripheral tissues over months, years, and decades in humans.
  • the distribution of TRM after immunization can be modified by administering mediators that have been shown to alter the expression of T cell homing markers. For example, retinoic acid has been shown to enhance the expression of gut homing markers, while reducing skin homing markers.
  • Antigens from a pathogen encountered through infected skin are presented by skin-derived dendritic cells in lymph nodes draining skin, and activate naive T cells to proliferate and force their differentiation into skin homing T cells as well as central memory T cells. These skin homing T cells rapidly traffic to skin, seeding both infected skin and normal skin. A subset of these T cells then takes up residence in skin for years to decades. This occurs repeatedly throughout childhood, adolescence, and adulthood. For antigens encountered multiple times over the years, an amplified population of central memory T cells can be activated in lymph node, and these in turn can differentiate into skin homing T cells and seed skin in even greater numbers.
  • the skin contains polyfunctional CD45RO+/ CLA+/CCR4+ TRM enriched for common skin pathogens, such as C albicans, S aureus, Trichophyton species, P.acnes, P.ovale, HSV, as well as VACV and Yellow Fever (in vaccinated individuals), but not for common gut or lung pathogens.
  • the frequency of TRM responsive to these microorganisms will vary from individual to individual, but should be significantly higher than the frequency of responsive T CM in peripheral blood, and even higher than the frequency of responsive naive T cells.
  • the lung should contain polyfunctional CD45RO+/VLA-1+ TRM enriched for common lung pathogens, for example, Influenza virus, M pneumonia, S.pneumonia, Adenovirus species, Rhinovirus species
  • the gastrointestinal tract should contain polyfunctional CD45RO/ ⁇ 4 ⁇ 7+ T RM enriched for those specific for rotavirus, norovirus, E. coli, enteroviruses, H. pylori, calciviruses.
  • the blood contains TCM and TEM specific for all antigens found in skin, lung, and colon, as well as endogenous viruses such as CMV and EBV. Most antigen reactive T cells will be non-polyfunctional TCM.
  • vaccines to infectious diseases would have to be evaluated not based solely on their ability to generate a neutralizing antibody response, but rather their capacity to generate populations of antigen specific TEM and TCM that would migrate to relevant tissues and become TRM.
  • Vaccines to prevent the emergence or recurrence of human cancers would have to similarly be modified so as to generate optimal populations of TRM that would be deployed to the relevant tissue.
  • the HPV vaccine for cervical cancer might be modified to deliver TRM to reproductive mucosa.
  • Immunologically mediated diseases that involve peripheral tissues, including skin (e.g., psoriasis), lung (e.g., asthma), and gut (e.g., inflammatory bowel disease) would have to be studied to fully establish the role of TRM in these processes.
  • the implication of TRM playing a major role in disease activity, or the tendency for these diseases to chronically recur, would lead to treatment strategies that might be very different from those currently employed.
  • Modified poxviruses are administered by mechanical disruption of the epidermis (e.g., by skin scarification, scratching, abrading or superficial cutting).
  • Methods and devices for disrupting the skin and for depositing a substance into the epidermis of the skin are known in the art. Examples of devices for disrupting the skin include a scarification needle, a hypodermic needle, or an abrader.
  • the device is incorporated as part of a sterile vial containing lyophilized vaccine, which is rehydrated either from a separate source of diluent or by perforating a membrane separating the vaccine from the diluent.
  • the vaccine is administered using a microneedle array.
  • the microneedle length and diameter can be modified to control where the virus is administered into the epidermis. These are typically in the range of 150-1500 micrometers.
  • the microneedles can be used to deliver vaccine from a reservoir of the microneedles can contain antigen encapsulated or entrapped into a polymeric or sugar matrix, so that the needles break off in the epidermis where the antigen is released by degradation and/or dissolution of the matrix.
  • the device mechanically disrupts the epidermis (e.g., abrader) by moving or rubbing over the epidermal tissue such as the skin.
  • a chemical abrader is used, such as a surfactant to disrupt the mucosal layer in the lung, gastrointestinal tract, or reproductive tract. It is preferred that the minimum amount of abrasion/mechanical disruption to produce the desired result be used. Determination of the appropriate amount of abrasion/mechanical disruption for a selected poxvirus and/or composition thereof is within the ordinary skill in the art.
  • the viral vaccine may be applied in dry form to the abrading surface of the abrading device prior to application.
  • a reconstituting liquid is applied to the skin at the delivery site and the viral vaccine-coated abrading device is applied to the skin at the site of the reconstituting liquid. It is then moved or rubbed over the skin so that the poxvirus (composition) becomes dissolved in the reconstituting liquid on the surface of the skin and is delivered simultaneously with abrasion.
  • a reconstituting liquid may be contained in the device (e.g., a scarification needle, a hypodermic needle, or an abrader) and released to dissolve the viral vaccine as the device is applied to the skin for mechanical disruption of the epidermis.
  • Certain viral vaccines may also be coated on the device (e.g., abrading device) in the form of a gel.
  • the devices disrupt the skin to penetrate the epidermis without penetrating the dermis.
  • the devices disrupt the skin to penetrate the epidermis and the upper dermis is also involved.
  • the compositions are administered to one or more specific layers of the skin.
  • the compositions are administered to the epidermis. Suitable layers of the epidermis include the stratum corneum, stratum lucidum, stratum granulosum, stratum pinosum, and stratum basale.
  • the compositions are administered to the dermis.
  • the compositions are administered to the subcutis, or to a combination of the epidermis, dermis and/or subcutis.
  • the vaccine composition to be administered using the methods described herein may be applied to the skin prior to abrading, simultaneous with abrading, or post-abrading.
  • the device In order to achieve the desired mechanical disruptions of the epidermis, the device should be moved across a subject's skin at least once.
  • the subject's skin may be disrupted in alternating directions.
  • the surface of the device may be coated with the viral vaccine.
  • the length and thickness of the microprotrusions are selected based on the particular substance being administered and the thickness of the epidermis in the location where the device is to be applied.
  • the microprotrusions penetrate the epidermis without piercing or passing through the entire dermis. In some embodiments, the protrusions penetrate the stratum corneum substantially without piercing or passing through the entire epidermis.
  • the device is moved about 1 to 2 centimeters (cm). In some embodiments, the device is moved to produce a mechanically disrupted epidermal site having a surface area of about 4 cm 2 to about 300 cm 2 . The extent of the mechanical disruption of the epidermis is dependent on the pressure applied during movement and the number of repetitions with the device.
  • devices such as a scarification needle or an abrader for accurately targeting the epidermal space are provided. These devices may have solid or hollow microprotrusions.
  • the microprotrusions can have a length up to about 1500 microns. In some embodiments, the microprotrusions have a length of about 200 to 1500 microns. In some embodiments, the microprotrusions have a length of about 300 to 1000 microns, or in the range of about 400 to 800 microns.
  • Microneedle devices for administration of vaccine are available from BioSeren Tach Inc and described in U.S. Patent No. 6,334,856. Skin patches are described in U.S. Patent No. 6,706,693. These devices can be made of a biocompatible polymer or metal.
  • MEMS microelectromechanical
  • Dosage units of reagents for providing a T cell-mediated immune response to an exogeneous antigen in a subject with reduced humoral immunity by mechanical disruption of an epidermal tissue are also provided.
  • the dosage units include an effective amount for inducing or stimulating a protective T cell mediated immune response to a T cell antigen in epithelial tissues such as skin, lung, oral mucosa, gastrointestinal tract, and reproductive mucosa.
  • compositions are typically administered to a subject in need thereof in an effective amount.
  • the compositions can be administered in a dosage sufficient reduce or prevent one or more symptoms of the cancer or an infectious disease, or to otherwise provide a desired pharmacologic and/or physiologic effect.
  • the symptom may be physical or biological.
  • the symptom may be physical, such as tumor burden, or biological such as proliferation of cancer cells.
  • the amount is effective to increase the killing of tumor cells or inhibit proliferation or metastasis of tumor cells.
  • the amount is effective to reduce tumor burden.
  • the amount is effective to reduce or prevent at least one comorbidity of a cancer or infection.
  • the precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, clinical symptoms etc.). Persons of ordinary skill can determine optimum dosages, dosing methodologies and repetition rates, which may vary depending on the relative potency of individual vaccines and can generally be estimated based on EC50s found to be effective in ex vivo assay and in in vivo animal models.
  • an immune response to the antigen can be generated by administering between about 100-fold to about a 100-fold less pfu (plaque forming units) of the viral vector, when applied by mechanical disruption of the epidermis compared to conventional injection routes.
  • a specific immune response to the antigen can be generated by administering between about 90-fold, 80-fold, 70-fold, 60-fold, 50-fold, 40-fold, 30-fold, 20-fold, 10-fold, 5 -fold less pfu of the viral vaccine when applied by mechanical disruption of the epidermis compared to conventional injection routes such as intramuscular.
  • a single deposition of recombinant viral vaccine is required to elicit a long- lasting, potent antigen-specific immune response in the subject.
  • the disclosed compositions are administered on a dosage schedule, for example, an initial administration of a vaccine with subsequent booster administrations.
  • the composition is administered 2, 3, 4, or more times, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days, weeks, months, or years apart.
  • Dosage regimens or cycles of the compositions and/or additional agents can be completely or partially overlapping, or can be sequential.
  • a second dose of the vaccine is administered anywhere from two weeks to one year, preferably from one to six months, after the initial administration.
  • a third dose may be administered after the second dose and from three months to two years, or even longer, preferably 4 to 6 months, or 6 months to one year after the initial administration.
  • the boosting antigen may be administered using the same vaccine, or as a whole protein, an immunogenic peptide fraction of the protein, another recombinant viral vector, or DNA encoding the protein or peptide.
  • different viral vaccines are used.
  • vaccinia may be followed by an avipox such as fowlpox, or vice versa.
  • no booster immunization is required.
  • the intact, nonreplicating or replication-deficient poxvirus is only given with the initial administration. In other embodiments, the intact, non-replicating or replication-deficient poxvirus is only given with one or more subsequent booster administrations.
  • compositions including an intact, nonreplicating or replication-deficient poxvirus and a T cell antigen can be compared to a control.
  • suitable controls are known in the art and include, for example, untreated cells or an untreated subject.
  • a typical control is a comparison of a condition or symptom of a subject prior to and after administration of the targeted agent.
  • the condition or symptom can be a biochemical, molecular, physiological, or pathological readout.
  • the effect of the composition on a particular symptom, pharmacologic, or physiologic indicator can be compared to an untreated subject, or the condition of the subject prior to treatment.
  • the symptom, pharmacologic, or physiologic indicator is measured in a subject prior to treatment, and again one or more times after treatment is initiated.
  • the control is a reference level, or average determined based on measuring the symptom, pharmacologic, or physiologic indicator in one or more subjects that do not have the disease or condition to be treated (e.g., healthy subjects).
  • the effect of the treatment is compared to a conventional treatment that is known the art.
  • kits including one or more containers filled with one or more of the following components: a live, modified, nonreplicating or replication-impaired virus including an antigen and optionally including a co- stimulatory molecule, either in dried form (e.g. lyophilized), as a salt, or in a solution, optionally a second virus and/or a co-stimulatory molecule, either in dried form (e.g. lyophilized), as a salt, or in a solution, optionally a solution or gel to dissolve or admix the virus(es), and optionally an adjuvant.
  • the kits additionally contain a device for disrupting the epidermis.
  • kit can be instructions on how to use the kit and optionally a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • in vitro assays may be used to determine the occurrence of an immune response. Examples of such in vitro assays include ELISA assays and cytotoxic T cell (CTL) assays.
  • the immune response is measured by detecting and/or quantifying the relative amount of an antibody, which specifically recognizes an antigen in the sera of a subject who has been treated by administering the live, modified, non-replicating or replication-impaired poxvirus including the antigen, relative to the amount of the antibody in an untreated subject.
  • monoclonal antibodies in an immunoassay is preferred because of the ability to produce them in large quantities and the homogeneity of the product.
  • the preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be achieved by techniques which are well known to those who are skilled in the art.
  • ELISA assays may be used to determine the level of isotype specific antibodies using methods known in the art.
  • CTL assays can be used to determine the lytic activity of CTLs, measuring specific lysis of target cells expressing a certain antigen.
  • Immune- assays may be used to measure the activation (e.g., degree of activation) of sample immune cells.
  • Sample immune cells refer to immune cells contained in samples from any source, including from a human patient, human donor, animal, or tissue cultured cell line.
  • the immune cell sample can be derived from peripheral blood, lymph nodes, bone marrow, thymus, any other tissue source including in situ or excised tumor, or from tissue or organ cultures.
  • the sample may be fractionated or purified to generate or enrich a particular immune cell subset before analysis.
  • the immune cells can be separated and isolated from their source by standard techniques.
  • Immune cells include both non-resting and resting cells, and cells of the immune system that may be assayed, including, but not limited to, B lymphocytes, T lymphocytes, natural killer (NK) cells, lymphokine- activated killer (LAK) cells, monocytes, macrophages, neutrophils, granulocytes, mast cells, platelets, Langerhans cells, stem cells, dendritic cells, and peripheral blood mononuclear cells.
  • Immune cell activity that may be measured include, but is not limited to (1) cell proliferation by measuring the cell or DNA replication; (2) enhanced cytokine production, including specific measurements for cytokines, such as ⁇ lFN, GM-CSF, or TNF-alpha, IFN-alpha, IL-6, IL- 10, IL-12; (3) cell mediated target killing or lysis; (4) cell differentiation; (5) immunoglobulin production; (6) phenotypic changes; (7) production of chemotactic factors or chemotaxis, meaning the ability to respond to a chemotactin with chemotaxis; (8) immunosuppression, by inhibition of the activity of some other immune cell type; (9) chemokine secretion such as IP- 10; (10) expression of costimulatory molecules (e.g., CD80, CD 86) and maturation molecules (e.g., CD83), (12) upregulation of class II MHC expression; and (13) apoptosis, which refers to fragmentation of activated immune cells under certain circumstances
  • reporter molecules may be used.
  • a reporter molecule is a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative.
  • the most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e., radioisotopes) and chemiluminescent molecules.
  • an enzyme immunoassay an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan.
  • Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta- galactosidase and alkaline phosphatase, amongst others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a Auorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labeled antibody is added to the first antibody-antigen complex, allowed to bind, and then the excess reagent is washed away.
  • a solution containing the appropriate substrate is then added to the complex of antibody-antigen- antibody.
  • the substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of antigen which was present in the sample.
  • Auorescent compounds such as Auorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity.
  • the Auorochrome-labeled antibody When activated by illumination with light of a particular wavelength, the Auorochrome-labeled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope.
  • the Auorescent labeled antibody is allowed to bind to the first antibody- antigen complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the Auorescence observed indicates the presence of the antigen of interest.
  • Activated immune cell proliferation is intended to include increase in cell number, cell growth, cell division, or cell expansion, as measured by cell number, cell weight, or by incorporation of radiolabelled nucleic acids, amino acids, proteins, or other precursor molecules.
  • DNA replication is measured by incorporation of radioisotope labels.
  • cultures of stimulated immune cells can be measured by DNA synthesis by pulse-labeling the cultures with tritiated thymidine ( 3 H-Tdr), a nucleoside precursor that is incorporated into newly synthesized DNA. Thymidine incorporation provides a quantitative measure of the rate of DNA synthesis, which is usually directly proportional to the rate of cell division.
  • the amount of 3 H-labeled thymidine incorporated into the replicating DNA of cultured cells is determined by scintillation counting in a liquid scintillation spectrophotometer. Scintillation counting yields data in counts per minute (cpm) which may then be used as a standard measure of immune cell responsiveness. The cpm in resting immune cell cultures may be either subtracted from or divided into cpm of the primed immune cells, which will yield a stimulation index ratio.
  • Flow cytometry can also be used to measure proliferation by measuring DNA with light scatter, Coulter volume and fluorescence, all of which are techniques that are well known in the art.
  • a measure of immune cell stimulation is the ability of the cells to secrete cytokines, lymphokines, or other growth factors.
  • Cytokine production including specific measurements for cytokines, such as ylFN, GM-CSF, or TNF-alpha, may be made by radioimmunoassay (RIA), enzyme-linked immunoabsorbent assay (EEISA), bioassay, or measurement of messenger RNA levels.
  • RIA radioimmunoassay
  • EEISA enzyme-linked immunoabsorbent assay
  • bioassay or measurement of messenger RNA levels.
  • a monoclonal antibody to the cytokine to be measured is used to specifically bind to and thus identify the cytokine.
  • Immunoassays are well known in the art and can include both competitive assays and immunometric assays, such as forward sandwich immunoassays, reverse sandwich immunoassays and simultaneous immunoassays.
  • the sample-containing cytokine is incubated with the cytokine-specific monoclonal antibody under conditions and for a period of time sufficient to allow the cytokines to bind to the monoclonal antibodies.
  • the specific concentrations of antibodies, the temperature and time of incubation, as well as other such assay conditions can be varied, depending upon various factors including the concentration of cytokine in the sample, the nature of the sample, and the like. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
  • Immunode-mediated target cell lysis Another type of indicator for degree of immune cell activation is immune cell-mediated target cell lysis, which is meant to encompass any type of cell killing, including cytotoxic T lymphocyte activity, apoptosis, and the induction of target lysis by molecules secreted from non-resting immune cells stimulated to activity.
  • Cell-mediated lympholysis techniques typically measure the ability of the stimulated immune cells to lyse 51 Cr-labeled target cells. Cytotoxicity is measured as a percentage of 51 Cr released in specific target cells compared to percentage of 51 Cr released from control target cells. Cell killing may also be measured by counting the number of target cells, or by quantifying an inhibition of target cell growth.
  • Cell Differentiation Assay Another indicator of immune cell activity is immune cell differentiation and maturation.
  • Cell differentiation may be assessed in several different ways. One such method is by measuring cell phenotypes. The phenotypes of immune cells and any phenotypic changes can be evaluated by flow cytometry after immunofluorescent staining using monoclonal antibodies that will bind membrane proteins characteristic of various immune cell types.
  • a second means of assessing cell differentiation is by measuring cell function. This may be done biochemically, by measuring the expression of enzymes, mRNA's, genes, proteins, or other metabolites within the cell, or secreted from the cell. Bioassays may also be used to measure functional cell differentiation.
  • Immune cells express a variety of cell surface molecules which can be detected with either monoclonal antibodies or polyclonal antisera. Immune cells that have undergone differentiation or activation can also be enumerated by staining for the presence of characteristic cell surface proteins by direct immunofluorescence in fixed smears of cultured cells. Mature B cells can be measured in immunoassays, for example, by cell surface antigens including CD 19 and CD20 with monoclonal antibodies labeled with fluorochromes or enzymes may be used to these antigens. B cells that have differentiated into plasma cells can be enumerated by staining for intracellular immunoglobulins by direct immunofluorescence in fixed smears of cultured cells.
  • Immunoglobulin Production Assay B cell activation results in small, but detectable, quantities of polyclonal immunoglobulins. Following several days of culture, these immunoglobulins may be measured by radioimmunoassay or by enzyme-linked immunosorbent assay (ELISA) methods.
  • ELISA enzyme-linked immunosorbent assay
  • B cells that produce immunoglobulins can also be quantified by the reversed hemolytic plaque assay.
  • erythrocytes are coated with goat or rabbit anti-human immunoglobulins. These immunoglobulins are mixed with the activated immunoglobulin-producing lymphocytes and semisolid agar, and complement is added. The presence of hemolytic plaques indicates that there are immunoglobulin-producing cells.
  • Chemotactic Factor Assay Chemotactic factors are molecules which induce or inhibit immune cell migration into or out of blood vessels, tissues or organs, including cell migration factors.
  • the chemotactic factors of immune cells can be assayed by flow cytometry using labeled monoclonal antibodies to the chemotactic factor or factors being assayed.
  • Chemotactic factors may also be assayed by ELISA or other immunoassays, bioassays, messenger RNA levels, and by direct measurements, such as cell counting, of immune cell movements in specialized migration chambers.
  • Addback Assays When added to fresh peripheral blood mononuclear cells, autologous ex vivo activated cells exhibit an enhanced response to a "recall” antigen, which is an antigen to which the peripheral blood mononuclear cells had previously been exposed. Primed or stimulated immune cells should enhance other immune cells response to a "recall” antigen when cultured together. These assays are termed “helper” or “addback” assays.
  • primed or stimulated immune cells are added to untreated, usually autologous immune cells to determine the response of the untreated cells. The added primed cells may be irradiated to prevent their proliferation, simplifying the measurement of the activity of the untreated cells. These assays may be particularly useful in evaluating cells for blood exposed to virus.
  • the addback assays can measure proliferation, cytokine production, and target cell lysis as described herein.
  • the method(s) for treating or preventing cancer described herein may be used in combination with one or more anticancer agents.
  • the method(s) for treating or preventing infections (or diseases described herein may be used in combination with one or more anti-bacterial agents, anti-viral agents, antifungal agents, or anti-protozoal agents.
  • Example 1 Protection of CD20-treated mice against lethal influenza A virus challenge by vaccination with MVA-Influenza Nuclear Protein (NP)
  • mice (10/group) were treated with anti-CD20 intraperitoneal (i.p.) or isotype matched control (100 pg per mouse) at day -2 and q2 weeks throughout the experiment. At day 0, they were immunized once via skin scarification (s.s.) with influenza MVA-NP and at Day 40, they were given a lethal challenge intranasal (i.n.) inoculation of PR8 (2xl0 5 pfu).
  • i.p. intraperitoneal
  • isotype matched control 100 pg per mouse

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Abstract

Selon l'invention, l'administration de poxvirus non réplicatif ou à réplication déficiente intacts, en combinaison avec un antigène de lymphocyte T, à un tissu épithélial interrompu induit une immunité à médiation assurée par les lymphocytes T spécifiques d'antigène chez un sujet présentant une immunité humorale réduite, par exemple les sujets auxquels sont administrées des thérapies d'appauvrissement en lymphocytes B. L'invention concerne également des compositions et des méthodes pour induire des réponses immunitaires à médiation par les lymphocytes T à l'antigène dans les tissus épithéliaux d'un sujet. Les méthodes comprennent l'administration à un tissu épithélial interrompu de poxvirus non réplicatifs ou à réplication déficiente intacts comprenant un ou plusieurs antigènes de lymphocytes T. Le tissu épithélial peut être mécaniquement interrompu par un dispositif tel qu'une aiguille de scarification ou un dispositif d'abrasion avant, en même temps, ou immédiatement après l'administration de la composition vaccinale.
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