WO2024197451A1

WO2024197451A1 - Homologous and heterologous therapeutic vaccination strategies for cancer treatment

Info

Publication number: WO2024197451A1
Application number: PCT/CN2023/083679
Authority: WO
Inventors: William Wei-Guo JIA; Xiaohu Liu; Zhibin Yu; Yanal M. Murad; Jun Ding; Kuan Zhang; Yue GONG; Fujun HOU; Guoyu LIU; Dmitry V. CHOULJENKO; Delwar ZAHID; Jiang Xu
Original assignee: Virogin Biotech (Shanghai) Ltd.; Virogin Biotech Canada Ltd.
Priority date: 2023-03-24
Filing date: 2023-03-24
Publication date: 2024-10-03
Also published as: WO2024198943A1

Abstract

Compositions and methods are provided for eliciting an immune response in a subject, by (a) administering to a subject a first vaccine, wherein the first vaccine includes a macromolecule or an oncolytic virus capable of inducing an immune response in a subject, and (b) administering a second vaccine, wherein the second vaccine includes an oncolytic virus. Within preferred embodiments, the immune response is used to treat a patient having cancer.

Description

HOMOLOGOUS AND HETEROLOGOUS THERAPEUTIC VACCINATION STRATEGIES FOR CANCER TREATMENT

FIELD OF THE INVENTION

The present invention relates generally to cancer therapeutic strategies using a first vaccine to prime the immune response, followed by a second vaccine to boost the immune response.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The contents of the electronic sequence listing (VIRO421P1_SEQ_LISTING; Size: 18.6 bytes; created on February 16, 2023, and updated on March 24, 2023) is herein incorporated by reference in its entirety.

BACKGROUND

Cancers are a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. It is one of the leading causes of death, causing an estimated 10 million worldwide deaths in 2020. In 2021, there were an estimated 1.9 million new cases in the United States alone, and over 600,000 deaths. Although there are hundreds of forms of cancer, the most common types occur in the breast, lung, colorectal region, prostate, stomach and liver.

Numerous therapies have been developed to treat cancer, including for example, chemotherapy (use of cytotoxic drugs to kill the cancer) , immunotherapy, surgical removal of the cancer, and radiation therapy. Nevertheless, a “cure” for cancer has remained elusive.

Multiple therapeutic methods have been developed in order to prevent cancer occurrence or to treat cancer with vaccines. (see, e.g., patent nos. AU2019226125B2; CN108430456B; CN102892777B; and US8895514B2; as well as “Immune System and Chronic Diseases” 2020; Volume 2020; Article ID 5825401 -https: //doi. org/10.1155/2020/5825401, “Cancer Vaccines: Toward the Next Breakthrough in Cancer Immunotherapy, by Igarashi and Saada) .

Systemically delivered tumor vaccines typically carry tumor antigens which are processed by professional antigen presenting cells (APC) to primeT cells and B cells in lymphatic tissues. However, immunosuppressive conditions within the tumor microenvironment (TME) limit the infiltration of immune cells and inhibit tumor cell killing efficacy induced by tumor antigen-specific T cells. Efficacy is further limited by low levels of tumor antigen expression in many tumors.

The present invention overcomes shortcomings of current tumor vaccines, and further provides additional unexpected benefits.

SUMMARY

Briefly stated, the invention relates to compositions and methods for treating cancer by administering a prime vaccine to induce a systemic immune response, followed by an oncolytic virus (OV) designed to create a stronger intratumoral immune response.

In one embodiment, methods are provided for eliciting an immune response in a subject, comprising administering to a subject a first vaccine, wherein the first vaccine comprises a macromolecule capable of inducing an immune response in said subject, and a second vaccine, wherein the second vaccine comprises an oncolytic virus which induces an immune response against a Tumor Antigen and/or a Non-Tumor Associated Antigen.

In a further embodiment, the prime vaccine may comprise one or more tumor antigens (TAs) or tumor-associated antigens (TAAs) to induce a tumor-specific systemic immune response (see, e.g., FIG. 1) . In another embodiment, the prime vaccine may comprise one or more non-tumor antigens (NTAs) such as pathogen-derived antigens (e.g. HPV antigens, HBV antigens, CMV antigens, etc. ) to induce a non-tumor-specific systemic immune response. Alternatively, the prime vaccine may comprise one or more immunogenic non-tumor antigens co-expressed or fused with one or more tumor antigens.

Within various embodiments the tumor antigen and/or tumor-associated antigen and/or non-tumor antigen in the prime vaccine may also be fused to, or co-expressed with, an APC-targeting protein or peptide.

In one embodiment, the prime vaccine may comprise purified protein (tumor antigen and/or tumor-associated antigen and/or non-tumor antigen, either alone or fused to, or co-administered with, a professional-APC-targeting protein or peptide) . In another embodiment, the prime vaccine may comprise mRNA encoding the tumor antigen and/or tumor-associated antigen and/or non-tumor antigen, either alone or fused to a professional-APC-targeting protein or peptide and encapsulated within lipid nanoparticles (LNP) .

Within various embodiments the OV can be intratumorally administered after systemic injection of the prime vaccine. Alternatively, the OV may be used as the prime vaccine by administering the OV systemically, intravenously, intramuscularly, intradermally, or subcutaneously. If an OV is used as the prime vaccine, the same OV can also be intratumorally administered after injection of the prime vaccine (homologous prime-boost) . Within other embodiments, different OVs may be used as the prime vaccine and for intratumoral injection (heterologous prime-boost) as long as both OVs express the same tumor antigen and/or tumor-associated antigen and/or non-tumor antigen. Alternatively, if an OV is used as the prime and/or the boost vaccine, the OV in question may not be modified to express any specific antigens, instead leveraging OV-mediated lysis of infected tumor cells in situ to release a plethora of novel antigens specific to the treated tumor. The OV may be engineered to express one or more proteins such as IL-12, IL-15, IL-15 receptor alpha subunit, or IL-18 to enhance immune cell activation in addition to (or instead of) expressing a non-tumor antigen and/or tumor antigen (FIG. 3) . Tumor antigen delivered by OV (or released by OV-mediated tumor lysis) within the tumor should promote more effective targeting and killing of tumor cells by tumor antigen-specific T cells, particularly in tumors with low or heterogenous tumor antigen expression. The non-tumor antigen-induced non-specific immune response should synergistically alter the tumor microenvironment by upregulating innate immune cells and immunostimulatory cytokines, leading to an active anti-tumor immune response at the OV-injected tumor sites and enhancing tumor cell killing which releases additional tumor antigens that may be recognized by the immune system.

In lieu of, or in addition to, a prime vaccine, existing anti-pathogen immunity may be activated and redirected to promote tumor clearance by engineering an OV that expresses, or displays on its surface, or otherwise elicits an immune response to the appropriate non-tumor antigen. To date, more than 60%of the global population has been vaccinated against SARS-CoV-2. In a preferred embodiment, the OV-encoded non-tumor antigen comprises the SARS-CoV-2 S protein receptor binding domain (RBD) , and the OV comprises herpes simplex virus type 1 (HSV-1) . When the engineered oncolytic HSV-1 expressing the SARS-CoV-2 S protein RBD is administered into the tumor, the expression of SARS-CoV-2 S protein RBD will awaken existing immunity against SARS-CoV-2 to enhance the therapeutic effects of the OV. T cells specific to SARS-CoV-2 will be attracted to the tumor site and activated, leading to destruction of tumor cells that present the RBD epitope and secretion of proinflammatory cytokines such as interferon gamma, which will result in bystander effects including TCR-independent and cell contact independent cytotoxicity. The oncolytic HSV-1 may also be engineered to express cytokine adjuvants such as IL-12, IL-15, IL- 15 receptor alpha subunit, or IL-18 to further enhance immune cell activation and tumor cell killing. In other embodiments, the OV-encoded non-tumor antigen may comprise one or more antigens derived from other common viral or bacterial pathogens for which vaccines are widely available such as influenza virus, poliovirus, coronavirus, varicella-zoster virus, human papillomavirus, hepatitis A virus, hepatitis B virus, dengue virus, rotavirus, adenovirus, yellow fever virus, variola virus, Japanese encephalitis virus, rabies virus, measles virus, mumps virus, rubella virus, Neisseria meningitidis, Streptococcus pneumoniae, Mycobacterium tuberculosis, Clostridium tetani, Salmonella enterica, Bacillus anthracis, Vibrio cholerae, Yersinia pestis, Bordetella pertussis, Corynebacterium diphtheriae, Haemophilus influenzae, etc., in order to activate and redirect existing immunity against said pathogens to promote tumor destruction.

The details of one or more embodiments are set forth in the description below. The features illustrated or described in connection with one exemplary embodiment may be combined with the features of other embodiments. Thus, any of the various embodiments described herein can be combined to provide further embodiments. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications as identified herein to provide yet further embodiments. Other features, objects and advantages will be apparent from the description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Features of the present disclosure, its nature and various advantages over the current state of the art will be apparent from the accompanying drawings and the following detailed description of various embodiments.

FIG. 1 is a diagrammatic illustration of a prime vaccine eliciting a tumor antigen-specific and/or non-tumor antigen-specific systemic immune response.

FIG. 2 is a diagrammatic illustration of enhanced antitumor immune response stemming from the use of an oncolytic virus armed with a tumor antigen.

FIG. 3 is a diagrammatic illustration depicting intratumoral administration of an antigen-armed recombinant oncolytic virus (i.e., an oncolytic virus that expresses one or more tumor-associated or non-tumor-associated antigens) after administration of a prime vaccine.

FIGs. 4A and 4B are diagrammatic illustrations of existing anti-pathogen immunity being used to fight cancer. FIG. 4A depicts administration of a cancer vaccine, and FIG. 4B depicts the recall of existing anti-viral immunity being used to target cancer.

FIG. 5 is a diagrammatic illustration of the genome structure of the recombinant HSV-1 named VG2044, highlighting modified regions of the genome.

FIG. 6 is a diagrammatic illustration of the genome structure of the recombinant HSV-1 named VG22401, highlighting modified regions of the genome.

FIGs. 7A and 7B depict ELISA-based quantification of anti-HER2 antibodies in the serum of mice doubly immunized with VG2044, VG161, or purified HER2 protein. FIG. 7A depicts the relative amounts of anti-HER2 antibodies in the serum of immunized mice, and FIG. 7B depicts the titer of anti-HER2 antibodies in the serum of immunized mice after serial dilution.

FIG. 8 depicts the results of a mouse IFN-γ ELISPOT assay performed on splenocytes isolated from mice doubly immunized with VG2044, VG161, or purified HER2 protein and stimulated using either inactivated HSV-1, CT26 tumor cells, or CT26 tumor cells stably expressing HER2.

FIG. 9 is a diagrammatic illustration of the design of an experiment to study the immunogenicity of VG2044 and VG2062 used for two-dose subcutaneous immunization in an immunocompetent BALB/c mouse model.

FIGs. 10A and 10B depict ELISA-based quantification of anti-gD (FIG. 10A) and anti-HER2 (FIG. 10B) antibodies in the serum of mice doubly immunized with VG2044 or VG2062.

FIG. 11 depicts the results of a mouse IFN-γ ELISPOT assay performed on splenocytes isolated from mice doubly immunized with VG2044 or VG2062 and stimulated using HER2 PepMix.

FIG. 12 is a diagrammatic illustration of the design of an experiment to study the antitumor efficacy of VG2044 or vehicle control used for two-dose subcutaneous immunization followed by intratumoral inoculation in a CT26-HER2 tumor-bearing immunocompetent BALB/c mouse model.

FIGs. 13A, 13B, 13C, and 13D depict tumor volumes from CT26-HER2 tumor-bearing immunocompetent mice that were treated with two-dose subcutaneous immunization of VG2044 followed by intratumoral inoculation of vehicle control (FIG. 13A) , CT26-HER2 tumor-bearing immunocompetent mice that were treated with two-dose subcutaneous immunization of VG2044 followed by intratumoral inoculation of VG2044 (FIG. 13B) , CT26-HER2 tumor-bearing immunocompetent mice that were treated with two-dose subcutaneous immunization of vehicle control followed by intratumoral inoculation of vehicle control (FIG. 13C) , or CT26-HER2 tumor-bearing immunocompetent mice that were treated with two-dose subcutaneous immunization of vehicle control followed by intratumoral inoculation of VG2044 (FIG. 13D) .

FIG. 14 depicts a survival curve from CT26-HER2 tumor-bearing immunocompetent mice that were treated with two-dose subcutaneous immunization of VG2044 or vehicle control followed by intratumoral inoculation of either VG2044 or vehicle control.

FIG. 15 depicts the results of a mouse IFN-γ ELISPOT assay performed on splenocytes stimulated using HER2 PepMix that were isolated from CT26-HER2 tumor-bearing immunocompetent mice that were treated with two-dose subcutaneous immunization of VG2044 or vehicle control followed by intratumoral inoculation of either VG2044 or vehicle control.

FIG. 16 is a diagrammatic illustration of the design of an experiment to study the antitumor efficacy of VG2044, VG2062, VG22401, and vehicle control used for two-dose subcutaneous immunization followed by intratumoral inoculation in a CT26-HER2 tumor-bearing immunocompetent BALB/c mouse model.

FIGs. 17A, 17B, and 17C depict tumor volumes from CT26-HER2 tumor-bearing immunocompetent mice that were treated with two-dose subcutaneous immunization of VG2062, followed by intratumoral inoculation of VG2062 (FIG. 17A) , CT26-HER2 tumor-bearing immunocompetent mice that were treated with two-dose subcutaneous immunization of VG2044 followed by intratumoral inoculation of VG2044 (FIG. 17B) , or CT26-HER2 tumor-bearing immunocompetent mice that were treated with two-dose subcutaneous immunization of VG22401 followed by intratumoral inoculation of VG22401 (FIG. 17C) .

FIG. 18 depicts a panel of 13 different mRNA vaccine constructs encoding the human HER2 extracellular domain.

FIGs. 19A and 19B depict results of an experiment to evaluate the anti-HER2 immune response after intramuscular or intravenous immunization with the HR1 mRNA construct. FIG. 19A is a diagrammatic illustration of the timeline of intramuscular or intravenous immunization with HR1 LNPs. FIG. 19B depicts granzyme B and interferon-γexpression in splenocytes isolated from mice 14 days after initial immunization and treated with HER2 ECD protein for 24 hours prior to intracellular cytokine staining (ICS) .

FIGs. 20A, 20B, 20C, 20D, and 20E depict results of an experiment to evaluate the efficacy of immunization with lipid nanoparticles (LNPs) loaded with the mRNA vaccine construct HR13 against challenge with HER2-positive tumor cell lines in immunocompetent BALB/c mice. FIG. 20A is a diagrammatic illustration of the timeline of immunization with HR13 LNPs and subsequent subcutaneous or intravenous challenge with CT26-HER2 or 4T1-HER2 tumor cells. FIG. 20B depicts granzyme B and interferon-γ expression in splenocytes isolated from mice 2 weeks after the second immunization and treated with HER2 ECD protein for 24 hours prior to intracellular cytokine staining (ICS) . FIG. 20C depicts tumor volumes from CT26-HER2 or 4T1-HER2 subcutaneous tumor-bearing immunocompetent mice that were treated with 3 doses of HR13 LNPs. FIG. 20D depicts survival curves of mice challenged intravenously with CT26-HER2 or 4T1-HER2 tumor cells that were treated with 3 doses of HR13 LNPs. FIG. 20E depicts H&E staining of lung tissues extracted from mice that were challenged intravenously with CT26-HER2 or 4T1-HER2 tumor cells and treated with 3 doses of HR13 LNPs.

FIGs. 21A, 21B, and 21C depict results of an experiment to evaluate the antitumor efficacy of a prime injection comprising LNPs loaded with HR13 followed by boosting via intratumoral injection of the oncolytic virus VG401 in immunocompetent BALB/c mice bearing HER2-positive tumors. FIG. 21A is a diagrammatic illustration of the timeline of intramuscular immunization with HR13 LNPs and subsequent intratumoral injection of VG401 in immunocompetent BALB/c mice bearing HER2-positive CT26 tumors. FIG. 21B depicts tumor volumes from CT26-HER2 subcutaneous tumor-bearing immunocompetent mice that were treated with either placebo, 3 doses or HR13 LNPs, or 3 doses of HR13 LNPs followed by a single intratumoral injection of VG401 OV. FIG. 21C depicts flow cytometric analysis of tumor samples to evaluate infiltration of cytotoxic T lymphocytes into the tumor microenvironment in CT26-HER2 tumors from mice that were treated with either placebo, 3 doses or HR13 LNPs, or 3 doses of HR13 LNPs followed by a single intratumoral injection of VG401 OV.

Corresponding reference numerals indicate corresponding parts throughout the drawings.

DETAILED DESCRIPTION OF THE INVENTION

Definitions:

As used herein, the term “antigen” refers to a molecule capable of being bound by an antibody or T-cell receptor. An antigen is additionally capable of inducing an immune response (e.g., either humoral or cellular) in a subject.

The term “immunological protein” refers to one or more proteins or peptides that comprises one or more antigenic sequences. Representative examples of immunological proteins include tumor antigens and non-tumor antigens, as is discussed in more detail below.

The term “antigen presenting cell targeted protein” or “APC-targeted protein” refers to proteins or peptide sequences which are known to associate with cell-surface receptors on macrophages and dendritic cells. For example, viral surface proteins from dengue virus, human immunodeficiency virus (HIV) , simian immunodeficiency virus (SIV) , hepatitis C virus (HCV) , cytomegalovirus (CMV) , West Nile virus (WNV) , influenza A virus (IAV) , Ebola virus, Marburg, and SARS-CoV can bind to C-type lectin receptors (CLR) such as DC-specific ICAM-grabbing non-integrin (DC-SIGN) , which plays a role in viral uptake by dendritic cells and promotes MHC-I exogenous presentation of viral antigens.

The term “oncolytic virus” refers generally to any virus capable of replicating in and killing tumor cells. Representative examples of oncolytic virus include without limitation, adenovirus, herpes simplex virus (HSV) , Newcastle disease virus, poxvirus, myxoma virus, rhabdovirus, picornavirus, influenza virus, coxsackievirus and parvovirus. In preferred embodiments, the oncolytic virus is a vaccinia virus (e.g. Copenhagen, Western Reserve, Wyeth strain) , reovirus, rhabdovirus (e.g., vesicular stomatitis virus (VSV) ) . The term “recombinant oncolytic virus” refers to an oncolytic virus that has been recombinantly or genetically engineered, e.g., to produce or express one or more proteins, or, immunological protein as described herein.

The term “tumor antigen” refers to antigens that are presented by MHC class I or class II molecules on the surface of tumor cells. Antigens which are found only on tumor cells are referred to as “Tumor Specific Antigens” or “TSAs” , while antigens that are presented by both tumor cells and normal cells are referred to as “Tumor-Associated Antigens” or “tumor antigens” . Representative examples of tumor antigens include, but are not limited to AIM-2, AIM-3, ART1, ART4, BAGE, β1, 6-N, β-catenin, B-cyclin, BM11, BRAF, BRAP, C13orf24, C6orf153, C9orf112, CA-125, CABYR, CASP-8, cathepsin B, Cav-1, CD74, CDK-1, CEAmidkin, COX-2, CRISP3, CSAG2, CTAG2, CYNL2, DHFR, E-cadherin, EGFRvIII, EphA2/Eck, ESO-1, EZH2, Fra-1/Fosl 1, FTHL17, GAGE1, Ganglioside/GD2, GLEA2, Glil, GnT-V, GOLGA, gp75, gplOO, HER-2, HSPH1, IL13Ralpha, IL13Ralpha2, ING4, Ki67, KIAA0376, Ku70/80, LDHC, LICAM, Livin, MAGE-A1, MAGE-2, MAGE-A3, MAGE-B6, MAPPK1, MART-1, MICA, MRP-3, MUC-1, MUM-1, Nestin, NKTR, NLRP4, NSEP1, NY-ES-01, OLIG2, p53, PAP, PBK, PRAME, PROX1, PSA, PSCA, PSMA, ras, RBPSUH, RTN4, SART1, SART2, SART3, SOX10, SOX11, SOX2, SPANXA1, SSX2, SSX4, SSX5, Survivin, TNKS2, TPR, TRP-1, TRP-2, TSGA10, TSSK6, TULP2, Tyrosinase, U2AF1L, UPAR, WT-1, XAGE2, and ZNF165.

The term “Non-Tumor Antigen” or “Non-Tumor Associated Antigen” refers to antigens which are not typically found in cancer cells, or, found natively in the human body. Representative examples include antigens from infectious or pathogenic agents (e.g., from bacteria, viruses or fungi) .

Briefly stated, compositions and methods are provided for eliciting an immune response in a subject, comprising administering to a subject a first vaccine, wherein the first vaccine comprises a macromolecule capable of inducing an immune response in said subject, and a second vaccine, wherein the second vaccine comprises an oncolytic virus which induces an immune response against a Tumor Antigen and/or a Non-Tumor Associated Antigen.

Within certain embodiments the first vaccine is an oncolytic virus (e.g., a recombinant oncolytic virus) . Within other embodiments, the second vaccine comprises a wild-type or a recombinant oncolytic virus. If the virus is a recombinant \oncolytic virus, it can express one or more immunological proteins (e.g., Tumor antigens, tumor-associated antigens, non-tumor antigens, and fused proteins comprising APC targeting peptides) .

Within preferred embodiments of the invention compositions and methods are provided for treating cancer by using a first vaccine (the “prime” vaccine) to induce a systemic immune response, followed by administering an oncolytic virus (OV) designed to create a stronger intratumoral immune response. In one embodiment, the prime vaccine may comprise one or more tumor antigens to induce a tumor-specific systemic immune response as shown in FIG. 1. More specifically, FIG. 1 depicts the systemic administration of a prime vaccine (10) , eliciting an immune response due to APC ( “Antigen Presenting Cells” ) targeting a tumor antigen (20) , in a lymphatic organ.

Within other embodiments, the prime vaccine may comprise a wild-type oncolytic virus or a recombinant oncolytic virus (OV) that expresses or otherwise elicits an immune response to one or more tumor antigens such as HER2 in order to induce a tumor-specific immune response in addition to virus-mediated tumor cell lysis (FIG. 2) . The prime vaccine may be administered systemically, intravenously, intramuscularly, intradermally, or subcutaneously, and it may be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times, followed by intratumoral injection of the same OV in order to enhance the intratumoral immune response. Alternatively, different wild-type OVs or recombinant OVs may be used as the prime vaccine and for intratumoral injection as long as said recombinant OVs express the same tumor antigen.

In another embodiment, the prime vaccine may comprise one or more non-tumor antigens (non-tumor antigen) such as pathogen-derived antigens (e.g. HPV antigens, HBV antigens, CMV antigens, etc. ) to induce a non-tumor-specific systemic immune response. Alternatively, the prime vaccine may comprise one or more immunogenic non-tumor antigen co-expressed or fused with tumor antigen. The rationale for using non-tumor antigen instead of, or in addition to tumor antigen, is twofold. First, the immunogenic nature of the non-tumor antigen generates a strong innate immune response, resulting in increased proinflammatory cytokine production, natural killer cell (NK cell) activation, improved immune cell migration, and enhanced maturation and activation of professional APCs. Secondly, non-tumor antigen-specific T cells that enter the TME can specifically target cancer cells infected with a recombinant oncolytic virus engineered to express the same non-tumor antigen, leading to more effective tumor lysis and release of additional tumor antigens. The recombinant oncolytic virus may also be engineered to express the same tumor antigen used in the prime vaccine in order to counteract the issue of heterogeneity and plasticity of tumor antigen expression in malignant cells.

The tumor antigen and/or non-tumor antigen in the prime vaccine may also be fused to, or coexpressed with, a professional-APC-targeting protein or peptide. Briefly, many proteins or peptides are known to associate with cell-surface receptors on macrophages and dendritic cells. For example, viral surface proteins from dengue virus, human immunodeficiency virus (HIV) , simian immunodeficiency virus (SIV) , hepatitis C virus (HCV) , cytomegalovirus (CMV) , West Nile virus (WNV) , influenza A virus (IAV) , Ebola virus, Marburg, and SARS-CoV can bind to C-type lectin receptors (CLR) such as DC-specific ICAM-grabbing non-integrin (DC-SIGN) which plays a role in viral uptake by dendritic cells and promotes MHC-I exogenous presentation of viral antigens.

In one embodiment, the prime vaccine may comprise purified protein (tumor antigen and/or non-tumor antigen, either alone or fused to, or coadministered with, a professional-APC-targeting protein or peptide) . In another embodiment, the prime vaccine may comprise mRNA encoding the tumor antigen and/or non-tumor antigen, either alone or fused to a professional-APC-targeting protein or peptide and encapsulated within lipid nanoparticles (LNP) . In some embodiments, the prime vaccine may be administered along with a pharmaceutically acceptable carrier.

Within one embodiment, the OV is intratumorally administered after systemic (e.g., subcutaneous, intravenous, intramuscular, or intradermal) delivery of the prime vaccine. The OV may be engineered to express cytokine adjuvants such as IL-12, IL-15, IL-15 receptor alpha subunit, or IL-18 to enhance immune cell activation in addition to expressing the non-tumor antigen and/or tumor antigen as shown in FIG. 3. More specifically, FIG. 3. depicts the intratumoral administration of an AgX (antigen X) -armed OV following the prime vaccine, wherein “antigen X” may comprise tumor antigen and/or non-tumor antigen. Injected OV directly induces lysis of infected cells leading to release of additional tumor antigens and causes localized tumor inflammation, promoting infiltration of both tumor antigen-specific and non-specific immune cells into the tumor microenvironment (e.g., CD8+ T-cells. Tumor antigen-specific T cells target and kill cancer cells, while non-specific immune cells alter the tumor microenvironment by releasing proinflammatory cytokines and attracting active APCs. The OV may express multiple payloads, including tumor antigen and/or non-tumor antigen to activate tumor antigen-specific and non-tumor antigen-specific immune responses that directly lead to T cell mediated cytotoxicity. Other payloads may include a suite of adjuvant cytokines designed to enhance immune cell activation and maturation, such as IL-12, IL-15, IL-15 receptor alpha subunit, or IL-18. Alternatively, the OV may be kept in a wild-type configuration without any payloads or engineered to express multiple payloads without including a tumor antigen payload or a non-tumor antigen payload. If the OV lacks an exogenous antigen payload, the suite of immunogenic viral proteins naturally expressed by the OV may act as suitable non-tumor associated antigens for the purpose of immune activation.

Within further embodiments of the invention OV-delivered tumor antigen within the tumor promotes more effective targeting and killing of tumor cells by tumor antigen-specific T cells, particularly in tumors with low or heterogenous tumor antigen expression. The non-tumor antigen-induced non-specific immune response should synergistically alter the tumor microenvironment by upregulating innate immune cells and immunostimulatory cytokines, leading to an active anti-tumor immune response at the OV-injected tumor sites and enhancing tumor cell killing which releases additional tumor antigens that may be recognized by the immune system.

Within yet other embodiments, in lieu of, or in addition to, a prime vaccine, existing anti-pathogen immunity may be activated and redirected to promote tumor clearance by engineering an OV that expresses, or displays on its surface, or, otherwise elicits an immune response to the appropriate non-tumor antigen. For example, more than 60%of the global population has been vaccinated against SARS-CoV-2. In a preferred embodiment, the OV-encoded non-tumor antigen comprises the SARS-CoV-2 S protein receptor binding domain (RBD) , and the OV comprises herpes simplex virus type 1 (HSV-1) . When the engineered oncolytic HSV-1 expressing the SARS-CoV-2 S protein RBD is administered into the tumor, the expression of SARS-CoV-2 S protein RBD will awaken existing immunity against SARS-CoV-2 to enhance the therapeutic effects of the OV. T cells specific to SARS-CoV-2 will be attracted to the tumor site and activated, leading to destruction of tumor cells that present the RBD epitope and secretion of proinflammatory cytokines such as interferon gamma, which will result in bystander effects including TCR-independent and cell contact-independent cytotoxicity. The oncolytic HSV-1 may also be engineered to express cytokine adjuvants such as IL-12, IL-15, IL-15 receptor alpha subunit, or IL-18 to further enhance immune cell activation and tumor cell killing. In other embodiments, the OV-encoded non-tumor antigen may comprise one or more antigens derived from common viral or bacterial pathogens for which vaccines are widely available such as influenza virus, poliovirus, coronavirus, varicella-zoster virus, human papillomavirus, hepatitis A virus, hepatitis B virus, dengue virus, rotavirus, adenovirus, yellow fever virus, variola virus, Japanese encephalitis virus, rabies virus, measles virus, mumps virus, rubella virus, Neisseria meningitidis, Streptococcus pneumoniae, Mycobacterium tuberculosis, Clostridium tetani, Salmonella enterica, Bacillus anthracis, Vibrio cholerae, Yersinia pestis, Bordetella pertussis, Corynebacterium diphtheriae, Haemophilus influenzae, etc., in order to activate and redirect existing vaccine-mediated immunity against said pathogens to promote tumor destruction.

One representative example of such methods is diagrammatically illustrated in FIG. 4. Briefly, a cancer vaccine may be constructed by encapsulating mRNA encoding tumor antigen within lipid nanoparticles (RNA-LPX) for systemic or intramuscular delivery (FIG. 4A) , but efficacy is generally limited to the subset of tumor cells expressing the tumor antigen, and is further limited by the immunosuppressive tumor microenvironment. More preferably however, priming with vaccine against a common pathogen such as the ubiquitous mRNA vaccines encoding the SARS-CoV-2 Spike protein enables recall of anti-SARS-CoV-2 immunity in the context of cancer treatment by intratumorally administering OV expressing the SARS-CoV-2 S protein RBD (FIG. 4B) . Tumor clearance is facilitated by activation of T cells specific to SARS-CoV-2, triggering a cascade of proinflammatory cytokine production and inflammation that results in additional tumor cell death via the bystander effect.

In order to further an understanding of the various embodiments herein, the following sections are provided to more specifically describe various embodiments of the invention: A. Oncolytic viruses; B. Herpes Virus Constructs; C. Immunological Proteins; D. Therapeutic Compositions; and E. Administration.

A. Oncolytic viruses

As noted above, the present invention provides oncolytic viruses which, optionally, may be engineered to express and/or secrete one or more immunological proteins. Briefly, an “oncolytic virus” is a virus that is capable of replicating in and killing tumor cells. Within certain embodiments the virus can be engineered in order to more selectively target tumor cells. Representative examples of oncolytic viruses include without limitation, adenovirus, coxsackievirus, H-1 parvovirus, herpes simplex virus (HSV) , influenza virus, measles virus, Myxoma virus, Newcastle disease virus, parvovirus picornavirus, reovirus, rhabdovirus (e.g. vesicular stomatitis virus (VSV) ) , paramyxovirus such as Newcastle disease virus, picornavirus such as poliovirus or Seneca valley virus, pox viruses such as vaccinia virus (e.g. Copenhagen, Indiana Western Reserve, and Wyeth strains) , reovirus, or retrovirus such as murine leukemia virus. Further representative examples are described in: US 8,147,822 and 9,045,729 (oncolytic rhabdovirus /VSV) ; US 9,272,008 (oncolytic Measles virus) ; US Patent Nos. 7,223,593, 7,537,924, 7,063,835, 7,063,851, 7,118,755, 8,216,564, 8,277,818, and 8,680,068 (oncolytic herpes virus vectors) ; and US 8,980,246 (oncolytic vaccinia virus) , all of which are incorporated by reference in their entirety.

Within certain embodiments of the invention, the oncolytic virus is Herpes Simplex virus (e.g., HSV-1 or HSV-2) . Briefly, Herpes Simplex Virus (HSV) 1 and 2 are members of the Herpesviridae family, which infects humans. The HSV genome contains two unique regions, which are designated unique long (U_L) and unique short (U_S) region. Each of these regions is flanked by a pair of inverted terminal repeat sequences. There are about 75 known open reading frames. The viral genome can be engineered to develop recombinant oncolytic viruses for use in e.g., cancer therapy. Tumor-selective replication of HSV may be conferred by mutation of the HSV ICP34.5 (also called γ34.5) gene. HSV contains two copies of ICP34.5. Mutants inactivating one or both copies of the ICP34.5 gene are known to lack neurovirulence, i.e. be avirulent/non-neurovirulent and be oncolytic. Tumor selective replication of HSV may also be conferred by controlling expression of key viral genes such as ICP27 and/or ICP4.

Within other embodiments of the invention oHSV are provided which have one or more mutations or deletions in one or more genes that are involved in immune downregulation in infected cells, e.g., by controlling or otherwise modulating the antiviral interferon response. Hence, within preferred embodiments either ICP34.5 and/or ICP0 can be modified (e.g., by mutation or deletion) to decrease Type I interferon signaling. Suitable oncolytic HSV may be derived from either HSV-1 or HSV-2, including any laboratory strain or clinical isolate. In some embodiments, the oHSV may be or may be derived from one of laboratory strains HSV-1 strain 17, HSV-1 strain F, or HSV-2 strain HG52. In other embodiments, it may be of or derived from non-laboratory strain JS-1. Other suitable HSV-1 viruses include HrrR3 (Goldstein and Weller, J. Virol. 62, 196-205, 1988) , G2O7 (Mineta et al. Nature Medicine. 1 (9) : 938-943, 1995; Kooby et al. The FASEB Journal, 13 (11) : 1325-1334, 1999) ; G47Delta (Todo et al. Proceedings of the National Academy of Sciences. 2001; 98 (11) : 6396-6401) ; HSV 1716 (Mace et al. Head &Neck, 2008; 30 (8) : 1045-1051; Harrow et al. Gene Therapy. 2004; 11 (22) : 1648-1658) ; HF10 (Nakao et al. Cancer Gene Therapy. 2011; 18 (3) : 167-175) ; NV1020 (Fong et al. Molecular Therapy, 2009; 17 (2) : 389-394) ; T-VEC (Andtbacka et al. Journal of Clinical Oncology, 2015: 33 (25) : 2780-8) ; J100 (Gaston et al. PloS one, 2013; 8 (11) : e81768) ; M002 (Parker et al. Proceedings of the National Academy of Sciences, 2000; 97 (5) : 2208-2213) ; NV1042 (Passer et al. Cancer Gene Therapy. 2013; 20 (1) : 17-24) ; G2O7-IL2 (Carew et al. Molecular Therapy, 2001; 4 (3) : 250-256) ; rQNestin34.5 (Kambara et al. Cancer Research, 2005; 65 (7) : 2832-2839) ; G47Δ-mIL-18 (Fukuhara et al. Cancer Research, 2005; 65 (23) : 10663-10668) ; and those vectors which are disclosed in PCT applications PCT/US2017/030308 entitled “HSV Vectors with Enhanced Replication in Cancer Cells” , and PCT/US2017/018539 entitled “Compositions and Methods of Using Stat1/3 Inhibitors with Oncolytic Herpes Virus” , all of the above of which are incorporated by reference in their entirety.

As disclosed herein, the oHSV vector may be a recombinant oHSV which includes one or more immunological protein expression cassettes that include genes or nucleotide sequences that encode an immunological protein (e.g., a tumor antigen or non-tumor antigen) under the control of a heterologous promoter. In one embodiment, the immunological protein expression cassette carried by the oncolytic HSV is controlled by a strong constitutive promoter such as the CMV promoter or the EF1α promoter or, alternatively, by a tumor-specific promoter. In other embodiments, the immunological protein may be fused directly to a structural protein within the oHSV vector, such as a capsid protein, a tegument protein, or an envelope protein.

The oHSV vector may have at least one γ34.5 gene that is modified with miRNA target sequences in its 3’ UTR as disclosed herein; there are no unmodified γ34.5 genes in the vector. In some embodiments, the oHSV has two γ34.5 genes modified with miRNA target sequences; in other embodiments, the oHSV has only one γ34.5 gene, and it is modified with miRNA target sequences. In some embodiments, the modified γ34.5 gene (s) are constructed in vitro and inserted into the oHSV vector as replacements for one or more native viral gene (s) . When the modified γ34.5 gene is a replacement of only one of the two native γ34.5 gene copies, the other native γ34.5 gene copy is deleted. As used herein, the term “deleted” means inactivated by full or partial deletion, unless otherwise indicated. Either of the two native γ34.5 gene copies can be deleted. Alternatively, both copies of the native γ34.5 gene can be deleted. In one embodiment, the terminal repeat, which comprises one copy of the γ34.5 gene, ICP0 gene, and ICP4 gene, is deleted and the remaining copy of the native γ34.5 gene is also deleted. In another embodiment, the internal repeat, which is identical to the terminal repeat and comprises one copy of the γ34.5 gene, ICP0 gene, and ICP4 gene, is deleted instead of the terminal repeat and the remaining copy of the native γ34.5 gene is also deleted. In another embodiment, the internal repeat long (IRL) or terminal repeat long (TRL) region comprising one copy of the γ34.5 gene and ICP0 gene is deleted and the remaining copy of the native γ34.5 gene is also deleted. In another embodiment, the internal repeat short (IRS) or terminal repeat short (TRS) region comprising one copy of ICP4 is deleted and both copies of the native γ34.5 gene are also deleted. Alternatively, if the terminal repeat, internal repeat, terminal repeat long, internal repeat long, terminal repeat short, or internal repeat short region is deleted, any remaining functional copies of the γ34.5 gene are modified with miRNA target sequences. As discussed herein, the γ34.5 gene may comprise additional changes, such as having an exogenous promoter or modifications to the 5’-untranslated region (5’-UTR) or to the 3’-untranslated region (3’-UTR) .

The oHSV may have additional mutations, which may include disabling mutations (e.g., deletions, substitutions, insertions) , which may affect the virulence of the virus or its ability to replicate. For example, mutations may be made in any one or more of ICP6, ICPO, ICP4, ICP27, ICP47, ICP24, ICP56, ICP34.5, and LAT. Preferably, a mutation in one of these genes (optionally in both copies of the gene where appropriate) leads to an inability (or reduction of the ability) of the HSV to express the corresponding functional polypeptide. In some embodiments, the promoter of a viral gene may be substituted with a promoter that is selectively active in target cells or inducible upon delivery of an inducer or inducible upon a cellular event or particular environment. In other embodiments, the promoter of a viral gene may be substituted with a promoter of a different viral gene that reduces the risk of recombination event in the repetitive and non-unique region of the native, or natural, promoter.

In certain embodiments, the expression of ICP4 or ICP27 is controlled by an exogenous (i.e., heterologous) promoter, e.g., a tumor-specific promoter. Exemplary tumor-specific promoters include survivin, CEA, CXCR4, PSA, ARR2PB, or telomerase; other suitable tumor-specific promoters may be specific to a single tumor type and are known in the art. Other elements may also be present. In some cases, an enhancer such as NFkB/oct4/sox2 enhancer is present. As well, the 5’ UTR may be exogenous, such as a 5’ UTR from growth factor genes such as FGF.

The oHSV may also have genes and nucleotide sequences that are non-HSV in origin. For example, a sequence that encodes a prodrug, a sequence that encodes a cytokine or other immune stimulating factor, a tumor-specific promoter, an inducible promoter, an enhancer, a sequence homologous to a host cell, among others may be in the oHSV genome. Exemplary sequences encode IL12, IL15, IL15 receptor alpha subunit, OX40L, PD-L1 blocker or a PD-1 blocker. For sequences that encode a product, they are operatively linked to a promoter sequence and other regulatory sequences (e.g., enhancer, polyadenylation signal sequence) necessary or desirable for expression.

The regulatory region of viral genes may be modified to comprise response elements that affect expression. Exemplary response elements include response elements for NF-κB, Oct-3/4-SOX2, enhancers, silencers, cAMP response elements, CAAT enhancer binding sequences, and insulators. Other response elements may also be included. A viral promoter may be replaced with a different promoter. The choice of the promoter will depend upon a number of factors, such as the proposed use of the HSV vector, treatment of the patient, disease state or condition, and ease of applying an inducer (for an inducible promoter) . For treatment of cancer, generally when a promoter is replaced it will be with a cell-specific or tissue-specific or tumor-specific promoter. Tumor-specific, cell-specific and tissue-specific promoters are known in the art. Other gene elements may be modified as well. For example, the 5’ UTR of the viral gene may be replaced with an exogenous UTR.

Within certain embodiments of the invention the oncolytic Herpes Virus is as described in PCT/US2017/044993; PCT/US2018/061687; PCT/US2019/063838; PCT/US2022/021798, and US20170319639A1, all of which are incorporated by reference in their entirety.

B. Oncolytic Herpes Virus Vectors

Within preferred embodiments of the invention, the oncolytic virus is a genetically engineered herpes virus construct such as VG401, VG22401, VG2046, VG22403, VG22407, VG22408, VG22409, or VG22410. VG2044 is an alternative name for VG401 and may be used interchangeably. Briefly, VG401 is a recombinant HSV-1 that uses tumor-associated enhancers inserted within the promoter-regulatory region of ICP27 and deletion of the key neurovirulence factor ICP34.5 to ensure patient safety and facilitate virus replication in tumor cells. VG22401, VG2046, VG22403, VG22407, VG22408, VG22409, and VG22410 employ a novel non-attenuated platform that utilizes both transcriptional and translational dual-regulation (TTDR) of key viral genes to limit virus replication to tumor cells and enhance tumor-specific virulence without compromising safety. The TTDR platform incorporates transcriptional regulation of the key HSV gene transactivator ICP27 using a tumor-specific promoter and translational regulation of the major neurovirulence determinant ICP34.5 via inclusion of tandem microRNA binding sites in the 3’-UTR of ICP34.5 with the binding sites comprising multiple copies of DNA sequences that are complementary to microRNAs which are present at relatively high concentrations in normal cells but are downregulated in cancer cells. In addition, VG22401, VG401, VG2046, VG22403, VG22407, VG22408, VG22409, and VG22410 express a payload cassette composed of IL12, IL15 and IL15 alpha receptor subunit. Expression of the cytokine payload is controlled by a cytomegalovirus (CMV) promoter in VG401 and VG22401, and by an EF1a promoter in VG2046, VG22403, VG22407, VG22408, VG22409, and VG22410. Expression of the cytokine payload may alternatively be controlled by a tumor-specific promoter such as the CEA or CXCR4 promoter for selective expression in tumor cells. Both VG22401 and VG401 also incorporate an expression cassette for the extracellular domain of HER2 driven by the EF1a promoter and inserted between HSV-1 genes US1 and US2, while VG22403, VG22407, VG22408, VG22409, and VG22410 contain a non-tumor antigen payload comprising the SARS-CoV-2 S protein receptor binding domain (RBD) either as an expression cassette driven by the CMV promoter and inserted between HSV-1 genes US1 and US2 or fused to an HSV-1 capsid, tegument, or envelope protein. Additionally, the HSV-1 gene encoding glycoprotein B (gB) in VG2046, VG22401, VG22403, VG22407, VG22408, VG22409, and VG22410 was truncated to facilitate virus spread throughout the tumor via enhanced fusogenicity. The gene encoding ICP34.5 is inactivated in VG401, but in VG22401, VG2046, VG22403, VG22407, VG22408, VG22409, and VG22410 the expression of ICP34.5 is post-transcriptionally regulated. Briefly, in wild-type HSV-1, there are 2 copies of the ICP34.5 gene located within the internal and terminal repeat regions of the viral genome. In the recombinant oncolytic HSV named in this application, including VG401, VG22401, VG2046, VG22403, VG22407, VG22408, VG22409, and VG22410 the terminal repeat region containing one copy of ICP34.5, ICP0, and ICP4 has been entirely or partially deleted to create more space for payload insertion. In VG401, the remaining single copy of ICP34.5 was also inactivated via deletion, but in VG22401, VG2046, VG22403, VG22407, VG22408, VG22409, and VG22410 the remaining single copy of the gene encoding ICP34.5 was modified by insertion of multiple copies of binding domains for miR124 (also referred to as miR-124) and miR143 (also referred to as miR-143) in the 3’-UTR region to regulate the expression of ICP34.5 post-transcriptionally.

ICP34.5 is encoded by the HSV late gene g-34.5. It is well known for its function of suppressing anti-viral immunity of host cells, particularly neuronal cells, to cause neurotoxicity. To abolish the functions of ICP34.5 in neurons and other normal cells while retaining its activity in tumor cells for robust replication, instead of deleting the gene or using a specific promoter to control the expression of ICP34.5 to target specific tumors, VG22401, VG2046, VG22403, VG22407, VG22408, VG22409, and VG22410 use microRNAs as a post- transcriptional control to achieve differential expression of ICP34.5 in tumor cells. Briefly, miRNAs are ～22 nucleotides, noncoding small RNAs coded by miRNA genes, which are transcribed by RNA polymerase II to produce primary miRNA (pri-miRNA) . Mature single-stranded (ss) miRNA forms the miRNA-associated RNA-induced silencing complex (miRISC) . miRNA in miRISC may influence gene expression by binding to the 3′-untranslated region (3′-UTR) in the target mRNA. This region consists of sequences recognized by miRNA. If the complementarity of the miRNA: mRNA complex is perfect, the mRNA is degraded by Ago2, a protein belonging to the Argonaute family. However, if the complementarity is not perfect, the translation of the target mRNA is suppressed.

miRNAs are expressed differentially in a tissue specific fashion. One of the examples is miR-124. While the precursors of miR-124 from different species are different, the sequences of mature miR-124 in human, mice, rats are completely identical. MiR-124 is the most abundantly expressed miRNA in neuronal cells and is highly expressed in the immune cells and organs (Qin et al., 2016, miRNA-124 in immune system and immune disorders. Frontiers in Immunology, 7 (OCT) , 1–8) . Another example of differential expression of miRNA is miR143 (Lagos-Quinona et al., 2002, Identification of tissue-specific MicroRNAs from mouse. Current Biology, 12 (9) , 735–739) . MiR-143 is constitutively expressed in normal tissues but significantly downregulated in cancer cells (Michael et al., 2003, Reduced Accumulation of Specific MicroRNAs in Colorectal Neoplasia. Molecular Cancer Research, 1 (12) , 882–891) .

The 3’ UTR region of the ICP34.5 gene in VG22401, VG2046, VG22403, VG22407, VG22408, VG22409, and VG22410 contains multiple copies of binding sites that are completely complementary to miR-124 and miR-143 (see e.g., U.S. Publication No. 2020/0171110A1 with respect to miR-124 and miR-143, which is incorporated by reference in its entirety) . Binding of miR-124 and miR-143 to the 3’ UTR of ICP34.5 mRNA causes degradation of the mRNA; therefore, the gene is post-transcriptionally downregulated in normal cells but not tumor cells. This design allows differential expression of ICP34.5 in tumor cells.

In one embodiment, five tandem copies of binding sites for both miR-124 and miR-143 are inserted into the 3’-UTR of ICP34.5. In other embodiments, any suitable number of miR binding sites in any suitable arrangement may be used. In certain embodiments, the miR binding sites may be separated by a region of spacer DNA of any suitable length. In preferred embodiments, the length of the spacer DNA ranges from 1bp to 27bp. Within further embodiments there is no spacer which separates miR binding sites.

Within certain embodiments of the present invention, the oncolytic virus is a genetically engineered herpes virus construct such as VG161. Description of VG161 can be found in US patent application Ser. No. 62/369, 646, filed on Aug. 01, 2016, and entitled “Oncolytic herpes simplex virus vectors expressing immune system-stimulatory molecules, ” commonly assigned with the present application and incorporated herein in its entirety.

Transcriptional Control HSV-1 viral replication depends on a cascade of expression of viral genes, with expression of immediate early genes (particularly ICP4 and ICP27) controlling subsequent expression of viral early genes and late genes that govern the lytic replication cycle of the virus. Deletion of ICP4 or ICP27 results in complete abrogation of viral replication and a significant reduction in viral gene expression, which makes ICP4 and ICP27 excellent targets for tumor specific regulation in oncolytic HSV.

While ICP4 is a major transcriptional factor for viral gene expression, ICP27 is a multi-functional protein that regulates transcription of many virus genes. ICP27 functions in all stages of mRNA biogenesis from transcription, RNA processing and export through to translation. ICP27 has also been implicated in nuclear protein quality control, cell cycle control, activation of stress signaling pathways and prevention of apoptosis.

In VG22401, VG2046, VG22403, VG22407, VG22408, VG22409, and VG22410, the native promoter of ICP27 is replaced with a tumor-specific CXCR4 promoter to facilitate virus replication in CXCR4-positive tumor cells. In VG401, the native promoter-regulatory region of ICP27 is modified by insertion of NFkB response elements and OCT4/SOX4 enhancer elements to promote increased expression of ICP27 in tumor cells while retaining native ICP27 promoter function. In other embodiments, the native ICP27 promoter may be replaced with a different tumor-specific promoter such as the RAN promoter or CEA promoter.

Cytokine Payload All recombinant oncolytic HSV named in this application, including VG401, VG22401, VG2046, VG22403, VG22407, VG22408, VG22409, and VG22410 co-express IL12 (SEQ ID NO: 9) , IL15 (SEQ ID NO: 10) and IL15 receptor alpha subunit (SEQ ID NO: 11) to further stimulate an immunomodulatory response. Expression of IL12 promotes polarization of antigen exposed T cells towards an inflammatory and anti-tumor T_H1 phenotype, while IL-15 activates NK cells to further increase tumor killing and activation of antigen presenting cells. In addition to IL15 expression, the mutant viruses also express IL15Rα to further enhance immune stimulation.

Transcription of IL-12, IL-15, and IL-15Rα can, for example, be driven by a cytomegalovirus (CMV) promoter, although an alternative promoter such as the EF1a promoter may also be used. Expression of IL-12, IL-15, and IL-15Rα may alternatively be controlled by a tumor-specific promoter such as the CEA or CXCR4 (SEQ ID NO: 13) promoter for expression in tumor cells. The IL-12, IL-15, and IL-15Rα polypeptides are linked with 2A self-cleaving peptides (SEQ ID NO: 12) (Z. Liu et al., 2017, Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Scientific Reports, 7 (1) , 1–9) that generate the 3 individual proteins through a mechanism of ribosomal skipping during translation.

Antigen Payload The recombinant oncolytic HSV may be engineered to contain an antigen payload comprising one or more tumor antigens and/or one or more tumor-associated antigens and/or one or more non tumor antigens. The antigen payload may comprise an expression cassette for one or more antigens that can be secreted outside the infected cell. The one or more antigens in the antigen payload may be fused to a copy of an immunogenic viral protein such as the HSV surface glycoprotein D (gD) to further stimulate the immune response. Alternatively, the antigen payload may be directly fused to an HSV surface protein such as glycoprotein D, glycoprotein B, glycoprotein G, or glycoprotein C to allow for display of the antigen payload on the viral surface. The antigen payload may also be directly fused to an HSV capsid protein such as VP26 to allow for display of the antigen payload on naked capsids which make up a sizable proportion of purified virus particles. In other embodiments, the antigen payload may be fused to a protein such as the HSV protein VP22 or a fragment thereof which can facilitate trafficking of the antigen payload to nearby uninfected cells. In yet other embodiments, direct fusion of the antigen payload to a viral protein may be used in conjunction with an expression cassette for one or more antigens that can be secreted outside the infected cell.

More specifically, VG2044 and VG22401 encode a secretable tumor-associated antigen payload comprising an expression cassette for the extracellular and transmembrane domains of HER2 driven by an EF1a promoter and inserted between the viral genes US1 and US2. The amino acid sequence of HER2 extracellular and transmembrane domains from the antigen expression cassette located between viral genes US1 and US2 in VG2044 and VG22401 is provided in SEQ ID NO: 1.

The VG22403 virus encodes a secretable non tumor antigen payload comprising an expression cassette for SARS-CoV-2 S protein receptor binding domain (RBD) inserted between the viral genes US1 and US2 and driven by the CMV promoter. The amino acid sequence of SARS-CoV-2 S protein receptor binding domain and its secretory signal peptide from the antigen expression cassette located between viral genes US1 and US2 in VG22403 is provided in SEQ ID NO: 3.

The VG22407 virus encodes a secretable non tumor antigen payload comprising an expression cassette for SARS-CoV-2 S protein RBD fused to a fragment of HSV-1 glycoprotein D (gD) inserted between the viral genes US1 and US2 and driven by the CMV promoter. The amino acid sequence of SARS-CoV-2 S protein receptor binding domain fused to a fragment of HSV-1 gD from the antigen expression cassette located between viral genes US1 and US2 in VG22407, along with its secretory signal peptide and linker peptide separating the SARS-CoV-2 S protein receptor binding domain from the fragment of HSV-1 gD, is provided in SEQ ID NO: 4.

The VG22408 virus comprises a non tumor antigen payload comprising the SARS-CoV-2 S protein RBD directly fused to the N-terminus of the HSV-1 gene encoding VP22. The amino acid sequence of SARS-CoV-2 S protein RBD alone in this virus is provided in SEQ ID NO. 2. The above sequence is inserted immediately following the VP22 gene start codon and is separated from the rest of VP22 using the linker sequence SSGGGSGSGGSG (SEQ ID NO: 5) .

The VG22409 virus comprises a non tumor antigen payload comprising the SARS-CoV-2 S protein RBD directly fused to the N-terminus of the HSV-1 gene encoding VP26. The amino acid sequence of SARS-CoV-2 S protein RBD alone in this virus is provided in SEQ ID NO. 2. The above sequence is inserted between amino acids 4 and 5 of the HSV-1 gene encoding VP26 and is separated from the main body of VP26 via the linker SSGGGSGSGGSG (SEQ ID NO: 5) .

The VG22410 virus comprises a non-tumor antigen payload comprising the SARS-CoV-2 S protein RBD directly fused to the N-terminus of the HSV-1 gene encoding glycoprotein C (gC) . The amino acid sequence of SARS-CoV-2 S protein RBD alone in this virus is provided in SEQ ID NO. 2. The above sequence is inserted between amino acids 19 and 20 of the HSV-1 gene encoding glycoprotein C and is separated from the main body of glycoprotein C via the linker SGGGGSGGGGSGGGGS (SEQ ID NO: 7) .

Truncated Glycoprotein B (gB) HSV-1 fusion is a crucial step of infection. It is dependent on four essential viral glycoproteins (gB, gD, gH, and gL) , which mediate entry into host cells by merging the viral envelope with a host cell membrane. The core fusion protein is glycoprotein B (gB) , a 904-residue glycosylated transmembrane protein encoded by the UL27 gene of HSV-1. Multiple types of mutations within the cytoplasmic domain of gB have yielded a hyperfusogenic phenotype, increasing cell-cell fusion (Chowdary &Heldwein, 2010, Syncytial Phenotype of C-Terminally Truncated Herpes Simplex Virus Type 1 gB Is Associated with Diminished Membrane Interactions. Journal of Virology, 84 (10) , 4923–4935) . In one embodiment, gB may be modified by truncating C-terminal amino acids 877 to 904 from the full-length protein.

Modified ICP47 Promoter In the HSV genome, the promoter controlling expression of the US12 gene, which encodes ICP47, is identical to the promoter controlling expression of the US1 gene, which is located approximately 13k base pairs from the US12 gene. In addition, large regions of the native ICP47 promoter include repetitive sequences that may facilitate spurious homologous recombination events. Thus, replacement of the native ICP47 promoter with a heterologous (e.g., exogenous) promoter is predicted to improve viral genomic stability.

In HSV, both ICP27 and ICP47 are encoded by immediate early genes, expressed very early after infection, and share many regulatory elements. To reduce the risk of homologous recombination while maintaining a natural expression pattern, the VG401 construct replaces the native ICP47 promoter with the ICP27 promoter.

In some embodiments, the native ICP27 promoter includes the entire sequence of DNA located between the coding regions of UL53 (gK) and UL54 (ICP27) . In one embodiment, the ICP27 promoter includes the 538bp sequence set forth in SEQ ID NO: 6.

In other embodiments, the ICP27 promoter sequence may be 90%, 80%, 70%, 60%, or 50%identical to the ICP27 promoter sequence of any known human herpes virus 1 strain, e.g., human herpes virus 1 strain 17 (NCBI reference sequence NC_001806.2) .

Within further embodiments of the invention short hairpin RNA (shRNA) mediated gene silencing may be utilized to reduce or eliminate expression of the broadly expressed tumor associated antigen (i.e., the second Tumor antigen) such as TfR1 from infected cells in order to extend the length of productive viral infection by protecting infected cells from premature death via retargeted immune cells. To that end, shRNA targeting the second Tumor antigen (e.g., TfR1 or GLUT1) may also be expressed by the same recombinant oncolytic virus that is engineered to express the immunological protein.

As an alternative to using a recombinant oncolytic virus to express and secrete the immunological protein, in some embodiments the immunological protein may be encoded within an mRNA molecule that is encapsulated within lipid nanoparticles and injected into the tumor for internalization and translation. Tumor-specific expression of the immunological protein from said mRNA may be achieved by adding miRNA target sequences to the 3’-end and/or the 5’-end of said mRNA, wherein said miRNA target sequences are recognized by miRNAs that are less abundant in the targeted tumor cells compared to normal cells.

List of abbreviations CEA = carcinoembryonic antigen; CXCR4 = C-X-C Motif Chemokine Receptor 4; gB = glycoprotein B; HSV-1 = herpes simplex virus-1; ICP0 = infected cell polypeptide 0; ICP27 = infected cell polypeptide 27; ICP47 = infected cell polypeptide 47; ICP34.5 = infected cell polypeptide 34.5; IL = interleukin; miR = microRNA; RA = receptor alpha; TRL = terminal repeat long; TRS = terminal repeat short; IRL = internal repeat long; IRS =internal repeat short; UL = unique long; US = unique short; CEACAM6 = carcinoembryonic antigen-related cell adhesion molecule 6; EF1α = elongation factor 1-alpha.

C. Alternative Compositions and Methods related to Immunological Proteins

As noted herein, the present invention provides a variety of Immunological Proteins, as well as sequences which encode such proteins and methods for producing such proteins both in vivo and in vitro.

Within one embodiment of the invention, isolated nucleic acid molecules are provided which encode the immunological proteins. As utilized herein the term “isolated” refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring) , and thus is altered “by the hand of man” from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide. The term “isolated” does not refer to genomic or cDNA libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots) , sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. However, a nucleic acid contained in a clone that is a member of a library (e.g., a genomic or cDNA library) that has not been isolated from other members of the library (e.g., in the form of a homogeneous solution containing the clone and other members of the library) or a chromosome removed from a cell or a cell lysate (e.g., a “chromosome spread” , as in a karyotype) , or a preparation of randomly sheared genomic DNA or a preparation of genomic DNA cut with one or more restriction enzymes is not “isolated” for the purposes of this invention. As discussed further herein, isolated nucleic acid molecules according to the present invention may be produced naturally, recombinantly, or synthetically.

The immunological proteins of the present invention may also be contained within an expression cassette. As used herein the term “expression cassette” is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. Within preferred embodiments the expression cassette comprises a promoter which is operably linked to a nucleic acid sequence encoding the immunological protein.

Within further embodiments of the invention the expression cassette is introduced into a vector that facilitate entry into a host cell and maintenance of the expression cassette in the host cell. Numerous such vectors are commonly used and well known to those of skill in the art, including for example, those which are available from Invitrogen, Stratagene, Clontech and others.

D. THERAPEUTIC COMPOSITIONS

Therapeutic compositions are provided that may be used to prevent, treat, or ameliorate the effects of a disease, such as, for example, cancer. More particularly, therapeutic compositions are provided comprising at least one recombinant oncolytic virus and/or immunological protein as described herein.

In certain embodiments, the compositions will further comprise a pharmaceutically acceptable carrier. The phrase “pharmaceutically acceptable carrier” is meant to encompass any carrier, diluent or excipient that does not interfere with the effectiveness of the biological activity of the recombinant oncolytic virus or immunological protein and that is not toxic to the subject to whom it is administered (see generally Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins; 21st ed. (May 1, 2005 and in The United States Pharmacopoeia: The National Formulary (USP 40 –NF 35 and Supplements) .

In the case of a recombinant oncolytic virus and immunological proteins as described herein, non-limiting examples of suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions (such as oil /water emulsions) , various types of wetting agents, sterile solutions, and others. Additional pharmaceutically acceptable carriers include gels, bioabsorbable matrix materials, implantation elements containing the recombinant oncolytic virus, or any other suitable vehicle, delivery or dispensing means or material (s) . Such carriers can be formulated by conventional methods and can be administered to the subject at an effective dose. Additional pharmaceutically acceptable excipients include, but are not limited to, water, saline, polyethylene glycol, hyaluronic acid and ethanol. Pharmaceutically acceptable salts can also be included therein, e.g., mineral acid salts (such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like) and the salts of organic acids (such as acetates, propionates, malonates, benzoates, and the like) . Such pharmaceutically acceptable (pharmaceutical-grade) carriers, diluents and excipients that may be used to deliver the oHSV to a cancer cell will preferably not induce an immune response in the individual (subject) receiving the composition (and will preferably be administered without undue toxicity) .

The compositions provided herein can be provided at a variety of concentrations. For example, dosages of recombinant oncolytic virus can be provided which range from about 10⁴ pfu to about 10¹⁰ pfu. Within further embodiments, the dosage can range from about 10⁶ pfu to about 10⁷ pfu, or from about 10⁷ pfu to about 10⁸ pfu, or from about 10⁸ pfu to 10⁹ pfu, and may be administered as a single dose or as multiple doses spread out over time. Doses may be administered daily, weekly, biweekly, monthly, or bimonthly, and dosage frequency may be cyclical, with each cycle comprising a repeating dosage pattern (e.g. once a week or biweekly dose administration for about 4 weeks comprising one cycle, repeating for up to about 24 cycles) . Within other embodiments of the invention, the virus can be provided in ranges from about 5x10⁴ pfu/kg to about 2x10⁹ pfu/kg for intravenous delivery in humans. For intratumoral injection, the preferred dosage can range from about 10⁶ pfu to about 10⁹ pfu per dose (with an injectable volume which ranges from about 0.1 mL to about 5 mL) .

Within certain embodiments of the invention, lower or higher dosages than standard may be utilized. Hence, within certain embodiments less than about 10⁶ pfu or more than about 10⁹ pfu can be administered to a patient.

Within certain embodiments of the invention, lower dosages than standard may be utilized. Hence, within certain embodiments less than about 10⁶ pfu/ml (with up to 4 ml being injected into a patient every 2 –3 weeks) can be administered to a patient.

The compositions may be stored at a temperature conducive to stable shelf-life and includes room temperature (about 20℃) , 4℃, -20℃, -80℃, and in liquid N₂. Because compositions intended for use in vivo generally do not have preservatives, storage will generally be at colder temperatures. Compositions may be stored dry (e.g., lyophilized) or in liquid form.

E. ADMINISTRATION

In addition to the compositions described herein, various methods of using such compositions to treat or ameliorate cancer are provided, comprising the step of administering to a subject a first vaccine, wherein the first vaccine comprises a macromolecule capable of inducing an immune response in said subject, and a second vaccine, wherein the second vaccine comprises an oncolytic virus which induces an immune response against a Tumor Antigen and/or a Non-Tumor Associated Antigen. Within various embodiments of the invention, the first vaccine, second vaccine, or both may be administered once or, multiple times.

The terms “effective dose” and “effective amount” refers to amounts of the recombinant oncolytic virus that is sufficient to effect treatment of a targeted cancer, e.g., amounts that are effective to reduce a targeted tumor size or load, or otherwise hinder the growth rate of targeted tumor cells. More particularly, such terms refer to amounts of recombinant oncolytic virus that is effective, at the necessary dosages and periods of treatment, to achieve a desired result. For example, in the context of treating a cancer, an effective amount of the compositions described herein is an amount that induces remission, reduces tumor burden, and/or prevents tumor spread or growth of the cancer. Effective amounts may vary according to factors such as the subject’s disease state, age, gender, and weight, as well as the pharmaceutical formulation, the route of administration, and the like, but can nevertheless be routinely determined by one skilled in the art.

The therapeutic compositions are administered to a subject diagnosed with cancer or is suspected of having a cancer. Subjects may be human or non-human animals. As noted above, within certain embodiments of the invention a first or prime vaccine is administered to a subject in order to induce a first immune response, followed by a second or boost vaccine to further enhance the immune response. Within various embodiments the first vaccine may comprises purified immunological proteins (e.g., tumor antigens, non-tumor antigens, or APC-fused tumor or non-tumor antigens) , or, sequences which encode such immunological proteins (e.g., mRNA which encodes such proteins and is encapsulated (e.g., in a liposome or suitable lipid nanoparticle ( “LNP” ) ) for administration) . Within certain embodiments the vaccine (e.g., prime vaccine) can be administered along with a pharmaceutically acceptable carrier. In other embodiments, the vaccine (e.g., prime vaccine) can be administered along with a pharmaceutically acceptable adjuvant such as aluminum, AS01, AS04, CpG 1018, Matrix-M, or MF59. The vaccine (e.g., prime vaccine or boost vaccine) may be administered once or multiple times (e.g., twice, three times, four times, five, times, six times, seven times, eight times, nine times or ten times) . Within certain embodiments, either the first or second vaccine is administered in a series of anywhere between 2 to 10 injections, separated by a time period of anywhere between 1 day to 1 year between injections. Within preferred embodiments of the invention the first vaccine is administered systemically, and the second vaccine is administered intratumorally.

The compositions are used to treat cancer. The terms “treat” or “treating” or “treatment, ” as used herein, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total) , whether detectable or undetectable. The terms “treating” and “treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

Representative forms of cancer include carcinomas, leukemias, lymphomas, myelomas and sarcomas. Representative forms of leukemias include acute myeloid leukemia (AML) and representative forms of lymphoma include B cell lymphomas. Further examples include, but are not limited to cancer of the bile duct, brain (e.g., glioblastoma) , breast, cervix, colorectal, CNS (e.g., acoustic neuroma, astrocytoma, craniopharyogioma, ependymoma, glioblastoma, hemangioblastoma, medulloblastoma, menangioma, neuroblastoma, oligodendroglioma, pinealoma and retinoblastoma) , endometrial lining, hematopoietic cells (e.g., leukemias and lymphomas) , kidney, larynx, lung, liver, oral cavity, ovaries, pancreas, prostate, skin (e.g., melanoma and squamous cell carcinoma) , GI (e.g., esophagus, stomach, and colon) and thyroid. Cancers can comprise solid tumors (e.g., sarcomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma and osteogenic sarcoma) , be diffuse (e.g., leukemia’s ) , or some combination of these (e.g., a metastatic cancer having both solid tumors and disseminated or diffuse cancer cells) . Cancers can also be resistant to conventional treatment (e.g. conventional chemotherapy and/or radiation therapy) . Benign tumors and other conditions of unwanted cell proliferation may also be treated.

Particularly preferred cancers to be treated include cancers that overexpress one or more tumor-associated antigens. Representative examples of tumor-associated antigens include, but are not limited to AIM-2, AIM-3, ART1, ART4, BAGE, β1, 6-N, β-catenin, B-cyclin, BM11, BRAF, BRAP, C13orf24, C6orf153, C9orf112, CA-125, CABYR, CASP-8, cathepsin B, Cav-1, CD74, CDK-1, CEAmidkin, COX-2, CRISP3, CSAG2, CTAG2, CYNL2, DHFR, E-cadherin, EGFRvIII, EphA2/Eck, ESO-1, EZH2, Fra-1/Fosl 1, FTHL17, GAGE1, Ganglioside/GD2, GLEA2, Glil, GnT-V, GOLGA, gp75, gplOO, HER-2 (HER2) , HSPH1, IL13Ralpha, IL13Ralpha2, ING4, Ki67, KIAA0376, Ku70/80, LDHC, LICAM, Livin, MAGE-A1, MAGE-2, MAGE-A3, MAGE-B6, MAPPK1, MART-1, MICA, MRP-3, MUC-1, MUM-1, Nestin, NKTR, NLRP4, NSEP1, NY-ES-01, OLIG2, p53, PAP, PBK, PRAME, PROX1, PSA, PSCA, PSMA, ras, RBPSUH, RTN4, SART1, SART2, SART3, SOX10, SOX11, SOX2, SPANXA1, SSX2, SSX4, SSX5, Survivin, TNKS2, TPR, TRP-1, TRP-2, TSGA10, TSSK6, TULP2, Tyrosinase, U2AF1L, UPAR, WT-1, XAGE2, and ZNF165. Cancers that do not overexpress one or more of the commonly recognized tumor-associated antigens listed above may also be treated due to OV-mediated lytic destruction of tumor cells releasing novel tumor-associated antigens to potentiate enhanced immune response against the tumor.

Another preferred category of cancers to be treated comprises cancers associated with oncogenic infectious agents. Representative viral examples of oncogenic infectious agents include, but are not limited to human papillomavirus, Epstein-Barr virus, hepatitis B virus, hepatitis C virus, human T-cell lymphoma virus, Kaposi’s sarcoma virus, Merkel cell polyomavirus. Representative bacterial examples of oncogenic infectious agents include, but are not limited to bacteria in the genus Helicobacter, Mycoplasma, Clostridium, Chlamydia, Treponema, Neisseria, Borrelia, Bacteroides, and Salmonella. Representative parasitic examples of oncogenic infectious agents include, but are not limited to Clonorchis sinensis, Schistosoma haematobium, Opithorchis viverrini, Toxoplasma gondii, Cryptosporidium parvum, Trichomonas vaginalis, Strongyloides stercoralis, Plasmodium falciparum, and Trypanosoma cruzi.

The recombinant oncolytic viruses and immunological proteins described herein may be given by a route that is, for example, oral, topical, parenteral, systemic, intravenous, intramuscular, intraocular, intrathecal, intratumoral, subcutaneous, intradermal, or transdermal. Within certain embodiments the recombinant oncolytic virus may be delivered by a cannula, by a catheter, or by direct injection. The site of administration may be intra-tumor or at a site distant from the tumor. The route of administration will often depend on the type of cancer being targeted.

The optimal or appropriate dosage regimen of the recombinant oncolytic virus and immunological protein is readily determinable within the skill of the art, by the attending physician based on patient data, patient observations, and various clinical factors, including for example a subject’s size, body surface area, age, gender, and the particular recombinant oncolytic virus being administered, the time and route of administration, the type of cancer being treated, the general health of the patient, and other drug therapies to which the patient is being subjected. According to certain embodiments, treatment of a subject using the recombinant oncolytic virus described herein may be combined with additional types of therapy, such as radiotherapy or chemotherapy using, e.g., a chemotherapeutic agent such as etoposide, ifosfamide, adriamycin, vincristine, doxycycline, and others.

Recombinant herpes simplex viruses described herein may be formulated as medicaments and pharmaceutical compositions for clinical use and may be combined with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The formulation will depend, at least in part, on the route of administration. Suitable formulations may comprise the virus and inhibitor in a sterile medium. The formulations can be fluid, gel, paste or solid forms. Formulations may be provided to a subject or medical professional.

A therapeutically effective amount is preferably administered. This is an amount that is sufficient to show benefit to the subject. The actual amount administered, and the time-course of administration will depend at least in part on the nature of the cancer, the condition of the subject, site of delivery, and other factors.

Within yet other embodiments of the invention the oncolytic virus can be administered by a variety of methods, e.g., intratumorally, intravenously, subcutaneously, intramuscularly, intradermally, transdermally, or, after surgical resection of a tumor.

The following are some exemplary numbered embodiments of the present disclosure.

1) A method of eliciting an immune response in a subject, comprising administering to a subject a first vaccine, wherein the first vaccine comprises a macromolecule capable of inducing an immune response in said subject, and a second vaccine, wherein the second vaccine comprises an oncolytic virus which induces an immune response against a Tumor Antigen and/or a Non-Tumor Associated Antigen.

2) The method of embodiment 1 wherein said first vaccine induces an immune response against a cancer.

3) The method of embodiment 1 wherein said first vaccine induces an immune response against a non-human protein.

4) The method of embodiment 1 wherein said first vaccine induces an immune response against a non-tumor associated antigen derived from a virus selected from the group consisting of adenovirus, coronavirus, cytomegalovirus, dengue virus, hepatitis virus (e.g., hepatitis A and/or B) , herpes simplex virus, human papilloma virus, influenza virus, Japanese encephalitis virus, measles virus, mumps virus, poliovirus, rotavirus, rabies virus, rubella virus, varicella-zoster virus, variola virus, and yellow fever virus.

5) The method of embodiment 1 wherein said first vaccine induces an immune response against a non-tumor associated antigen derived from a bacterium selected from the group consisting of Neisseria meningitidis, Streptococcus pneumoniae, Mycobacterium tuberculosis, Clostridium tetani, Salmonella enterica, Bacillus anthracis, Vibrio cholerae, Yersinia pestis, Bordetella pertussis, Corynebacterium diphtheriae, and Haemophilus influenzae.

6) The method of embodiment 1, wherein said first vaccine is a protein vaccine, or, a nucleic acid vaccine.

7) The method of embodiment 5, wherein said first vaccine is an mRNA vaccine, wherein the mRNA vaccine encodes a tumor antigen and/or a non-tumor associated antigen.

8) The method of embodiment 6 or 7, wherein said first vaccine is encapsulated in lipid nanoparticles.

9) The method of embodiment 1 wherein said first vaccine is an oncolytic virus.

10) The method of embodiment 1, wherein said first vaccine elicits an immune response to one or more immunologically active proteins.

11) The method of embodiment 1, wherein said first vaccine is a recombinant oncolytic virus.

12) The method of embodiment 11 wherein said recombinant oncolytic virus has an inactivation or a regulation of a gene involved with Type 1 interferon signaling.

13) The method of embodiment 12 wherein said inactivation is a mutation, a partial deletion, or, a full deletion of the gene involved with Type 1 interferon signaling.

14) The method of embodiment 12 wherein said regulation is an insertion of an miRNA binding site into the 3’ UTR of the gene involved with Type 1 interferon signaling.

15) The method of embodiment 12 wherein said mutation or inactivation is in the ICP34.5 or ICP0 gene.

16) The method of any one of embodiments 1 to 15, wherein said first vaccine induces an immune response to a Tumor Antigen, and/or against a Non-Tumor Associated Antigen.

17) The method of embodiment 16, wherein said Tumor Antigen or Non-Tumor Associated Antigen is fused to an APC Targeting Peptide.

18) The method of embodiment 1, wherein said first vaccine is administered once.

19) The method of embodiment 1, wherein said first vaccine is administered multiple times.

20) The method of embodiment 19, wherein each dose of said first vaccine is separated by more than 12 hours or more than 1 day.

21) The method of embodiment 1, wherein said the second vaccine is an oncolytic virus that elicits an immune response to one or more immunologically active proteins.

22) The method of embodiment 1, wherein said second vaccine is a recombinant oncolytic virus.

23) The method of embodiment 21 or 22 wherein said second vaccine is a virus selected from the group consisting of adenovirus, parvovirus, vaccinia virus, reovirus, herpes simplex virus, measles virus, coxsackievirus, fowl pox virus, Sendai virus, echovirus, poliovirus, poxvirus, picornavirus, alphavirus, Semliki forest virus, Sindbis virus, myxoma virus, retrovirus, paramyxovirus, rhabdovirus, Newcastle disease virus, Maraba virus, and vesicular stomatitis virus.

24) The method of embodiment 21 or 22 wherein said second vaccine is a chimeric virus comprising elements from one or more viruses selected from the group consisting of adenovirus, parvovirus, vaccinia virus, reovirus, herpes simplex virus, measles virus, coxsackievirus, fowl pox virus, Sendai virus, echovirus, poliovirus, poxvirus, picornavirus, alphavirus, Semliki forest virus, Sindbis virus, myxoma virus, retrovirus, paramyxovirus, rhabdovirus, Newcastle disease virus, Maraba virus, and vesicular stomatitis virus.

25) The method of embodiment 21 or 22 wherein said second vaccine is a Herpes Simplex virus.

26) The method of embodiment 25 wherein said recombinant oncolytic virus has an inactivation or regulation of a gene involved with Type 1 interferon signaling.

27) The method of embodiment 26 wherein said inactivation is a mutation, a partial deletion, or, a full deletion of the gene involved with Type 1 interferon signaling.

28) The method of embodiment 26 wherein said regulation is an insertion of an miRNA binding site into the 3’ UTR of the gene involved with Type 1 interferon signaling.

29) The method of embodiment 1, wherein said second vaccine is administered once.

30) The method of embodiment 1, wherein said second vaccine is administered multiple times.

31) The method of embodiment 30, wherein each dose of said second vaccine is separated by more than 12 hours or more than 1 day.

32) The method of embodiment 1 wherein said first vaccine is administered systemically, intravenously, intramuscularly, intradermally, or, subcutaneously.

33) The method of embodiment 1 wherein said second vaccine is administered intratumorally.

34) The method of embodiment 1 wherein said oncolytic virus expresses one or more immunologically active proteins.

35) The method of embodiments 10, 21 or 34 wherein said immunologically active protein is selected from the group consisting of IL-12, IL-15, IL-15 receptor alpha, and IL-18.

36) The method of embodiment 1 wherein said first vaccine is derived from an oncolytic virus, and, said second vaccine is derived from the same type of oncolytic virus.

37) The method of embodiment 1 wherein said first vaccine is derived from an oncolytic virus, and said second vaccine is derived from a different type of oncolytic virus.

38) The method of embodiment 36 or 37, wherein said first vaccine is derived from the group consisting of adenovirus, parvovirus, vaccinia virus, reovirus, herpes simplex virus, measles virus, coxsackievirus, fowl pox virus, Sendai virus, echovirus, poliovirus, poxvirus, picornavirus, alphavirus, Semliki forest virus, Sindbis virus, myxoma virus, retrovirus, paramyxovirus, rhabdovirus, Newcastle disease virus, Maraba virus, vesicular stomatitis virus,

39) The method of embodiment 36 or 37 wherein said second vaccine is a chimeric virus comprising elements from one or more viruses selected from the group consisting of adenovirus, parvovirus, vaccinia virus, reovirus, herpes simplex virus, measles virus, coxsackievirus, fowl pox virus, Sendai virus, echovirus, poliovirus, poxvirus, picornavirus, alphavirus, Semliki forest virus, Sindbis virus, myxoma virus, retrovirus, paramyxovirus, rhabdovirus, Newcastle disease virus, Maraba virus, and vesicular stomatitis virus.

40) The method of embodiment 36 or 37, wherein said first vaccine induces an immune response against an antigen from a virus selected from the group consisting of an adenovirus, coronavirus, cytomegalovirus, dengue virus, hepatitis virus (e.g., hepatitis A and/or B) , herpes simplex virus, human papilloma virus, influenza virus, Japanese encephalitis virus, measles virus, mumps virus, poliovirus, rotavirus, rabies virus, rubella virus, varicella-zoster virus, variola virus, and yellow fever virus.

41) The method of embodiment 36 or 37, wherein said first vaccine induces an immune response against an antigen from a bacterium selected from the group consisting of Neisseria meningitidis, Streptococcus pneumoniae, Mycobacterium tuberculosis, Clostridium tetani, Salmonella enterica, Bacillus anthracis, Vibrio cholerae, Yersinia pestis, Bordetella pertussis, Corynebacterium diphtheriae, and Haemophilus influenzae.

42) A method of eliciting a heterologous immune response in a subject, comprising administering a vaccine to a subject, wherein the vaccine elicits an immune response to a Non-Tumor Associated Antigen to which the subject has already been exposed.

43) The method of embodiment 42 wherein said Non-Tumor Associated Antigen is derived from a virus.

44) The method of embodiment 42 wherein said Non-Tumor Associated Antigen is an antigen from a virus selected from the group consisting of an adenovirus, coronavirus, cytomegalovirus, dengue virus, hepatitis virus (e.g., hepatitis A and/or B) , herpes simplex virus, human papilloma virus, influenza virus, Japanese encephalitis virus, measles virus, mumps virus, poliovirus, rotavirus, rabies virus, rubella virus, varicella-zoster virus, variola virus, and yellow fever virus.

45) The method of embodiment 43 wherein said Non-tumor Associated Antigen is the SARS-CoV-2 S protein receptor binding domain.

46) The method of embodiment 42 wherein said Non-Tumor Associated Antigen is an antigen from a bacterium selected from the group consisting of Neisseria meningitidis, Streptococcus pneumoniae, Mycobacterium tuberculosis, Clostridium tetani, Salmonella enterica, Bacillus anthracis, Vibrio cholerae, Yersinia pestis, Bordetella pertussis, Corynebacterium diphtheriae, and Haemophilus influenzae.

47) The method of embodiment 42 wherein said exposure is due to prior vaccination.

48) The method of embodiment 42 wherein said exposure is due to prior infection.

49) The method according to embodiment 42 wherein said Non-Tumor Associated Antigen is administered systemically or intratumorally.

50) The method according to embodiment 42 wherein said vaccine is a recombinant oncolytic virus.

51) The method according to embodiment 50 wherein said vaccine is a genetically engineered Herpes Simplex virus.

52) The method according to embodiment 51 wherein said Herpes Simplex virus expresses one or more immunologically active proteins.

53) The method according to embodiment 52 wherein said immunologically active protein is selected from the group consisting of IL-12, IL-15, IL-15 receptor alpha, and IL-18.

54) A recombinant oncolytic virus which expresses an antigen derived from a coronavirus.

55) The recombinant oncolytic virus according to embodiment 54, wherein said recombinant oncolytic virus is a Herpes Simplex virus.

56) The recombinant oncolytic virus according to embodiment 54, wherein said antigen is the SARS-CoV-2 S protein receptor binding domain.

57) The recombinant oncolytic virus according to embodiment 54, wherein said recombinant oncolytic virus further expresses an immunologically active protein.

58) The recombinant oncolytic virus according to embodiment 57 wherein said immunologically active protein is selected from the group consisting of IL-12, IL-15, IL-15 receptor alpha, and IL-18.

59) A pharmaceutical composition, comprising the recombinant oncolytic virus according to any one of embodiments 54 to 58, and a pharmaceutically acceptable excipient.

60) A method of treating cancer, comprising administering the pharmaceutical composition according to embodiment 59 to a subject having a cancer.

61) The method according to embodiment 60 wherein said pharmaceutical composition is administered systemically, or, intratumorally.

62) A kit for the treatment of cancer, comprising a first composition comprising a first vaccine, according to any one of embodiments 1 to 41, and a second composition comprising a second vaccine, according to any one of embodiments 1 to 41.

The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

It is also to be understood that as used herein and in the appended claims, the singular forms “a, ” “an, ” and “the” include plural reference unless the context clearly dictates otherwise, the term “X and/or Y” means “X” or “Y” or both “X” and “Y, ” and the letter “s” following a noun designates both the plural and singular forms of that noun. In addition, where features or aspects of the invention are described in terms of Markush groups, it is intended, and those skilled in the art will recognize, that the invention embraces and is also thereby described in terms of any individual member and any subgroup of members of the Markush group, and Applicants reserve the right to revise the application or claims to refer specifically to any individual member or any subgroup of members of the Markush group.

It is to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. It is further to be understood that unless specifically defined herein, the terminology used herein is to be given its traditional meaning as known in the relevant art.

Reference throughout this specification to “one embodiment” or “an embodiment” and variations thereof means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

As used in this specification and the appended claims, the singular forms “a, ” “an, ” and “the” include plural referents, i.e., one or more, unless the content and context clearly dictates otherwise. It should also be noted that the conjunctive terms, “and” and “or” are generally employed in the broadest sense to include “and/or” unless the content and context clearly dictates inclusivity or exclusivity as the case may be. Thus, the use of the alternative (e.g., "or" ) should be understood to mean either one, both, or any combination thereof of the alternatives. In addition, the composition of “and” and “or” when recited herein as “and/or” is intended to encompass an embodiment that includes all of the associated items or ideas and one or more other alternative embodiments that include fewer than all of the associated items or ideas.

Unless the context requires otherwise, throughout the specification and claims that follow, the word “comprise” and synonyms and variants thereof such as “have” and “include, ” as well as variations thereof such as “comprises” and “comprising” are to be construed in an open, inclusive sense, e.g., “including, but not limited to. ” The term "consisting essentially of" limits the scope of a claim to the specified materials or steps, or to those that do not materially affect the basic and novel characteristics of the claimed invention.

Any headings used within this document are only being utilized to expedite its review by the reader, and should not be construed as limiting the invention or claims in any manner. Thus, the headings and Abstract of the Disclosure provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

Where a range of values is provided herein, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

For example, any concentration range, percentage range, ratio range, or integer range provided herein is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer) , unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated. As used herein, the term "about" means ± 20%of the indicated range, value, or structure, unless otherwise indicated.

All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, are incorporated herein by reference, in their entirety. Such documents may be incorporated by reference for the purpose of describing and disclosing, for example, materials and methodologies described in the publications, which might be used in connection with the presently described invention. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such patents, publications, scientific articles, web sites, electronically available information, and other referenced materials or documents.

The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate any referenced publication by virtue of prior invention.

In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Furthermore, the written description portion of this patent includes all claims. Furthermore, all claims, including all original claims as well as all claims from any and all priority documents, are hereby incorporated by reference in their entirety into the written description portion of the specification, and Applicants reserve the right to physically incorporate into the written description or any other portion of the application, any and all such claims. Thus, for example, under no circumstances may the patent be interpreted as allegedly not providing a written description for a claim on the assertion that the precise wording of the claim is not set forth in haec verba in written description portion of the patent.

The claims will be interpreted according to law. However, and notwithstanding the alleged or perceived ease or difficulty of interpreting any claim or portion thereof, under no circumstances may any adjustment or amendment of a claim or any portion thereof during prosecution of the application or applications leading to this patent be interpreted as having forfeited any right to any and all equivalents thereof that do not form a part of the prior art.

Other nonlimiting embodiments are within the following claims. The patent may not be interpreted to be limited to the specific examples or nonlimiting embodiments or methods specifically and/or expressly disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.

The Examples and preparations provided below further illustrate and exemplify the compounds of the present invention and methods of preparing such compounds. It is to be understood that the scope of the present invention is not limited in any way by the scope of the following Examples and preparations. In the following Examples, molecules with a single chiral center, unless otherwise noted, exist as a racemic mixture. Those molecules with two or more chiral centers, unless otherwise noted, exist as a racemic mixture of diastereomers. Single enantiomers/diastereomers may be obtained by methods known to those skilled in the art. The starting materials and various reactants utilized or referenced in the examples may be obtained from commercial sources, or are readily prepared from commercially available organic compounds, using methods well-known to one skilled in the art.

EXAMPLES

Overview: All viral mutagenesis can be performed in Escherichia coli using standard lambda Red-mediated recombineering techniques implemented on the viral genome cloned into a bacterial artificial chromosome (BAC) (see generally: Tischer BK, Smith GA, Osterrieder N. Methods Mol Biol. 2010; 634: 421-30. doi: 10.1007/978-1-60761-652-8_30. PMID: 20677001; Tischer BK, von Einem J, Kaufer B, and Osterrieder N., BioTechniques 40: 191-197, Feb. 2006 (including the Supplementary Material, doi: 10.2144/000112096; and Tischer BK, Smith, GA and Osterrieder N. Chapter 30, Jeff Braman (ed. ) , In Vitro Mutagenesis Protocols: Third Edition, Methods in Molecular Biology, vol. 634, doi: 10.1007/978-1-60761-652-8_30, Springer Science+Business Media, LLC 2010) .

BAC recombineering requires the presence of exogenous BAC DNA within the viral genome to facilitate mutagenesis in E. coli. The BAC sequence is inserted either between viral genes such as the HSV genes US1/US2, UL3/UL4 and/or UL50/UL51, or into the thymidine kinase (TK) gene, which can disrupt expression of native TK. TK-deficient viral vectors may include an expression cassette for a copy of the native viral thymidine kinase (TK) gene under the control of a constitutive promoter inserted into a non-coding region of the viral genome. Alternatively, TK function may be restored by removing the exogenous BAC sequences via homologous recombination to reconstitute the native TK gene sequence. Presence of a functional TK gene enhances virus safety by rendering the virus sensitive to common treatment with guanosine analogues, such as ganciclovir and acyclovir.

Example 1

Engineering recombinant HSV-1 expressing SARS-CoV-2 S protein RBD

Objective: To construct a series of recombinant HSV-1 expressing SARS-CoV-2 S protein RBD.

Procedure: A series of HSV-1 mutants are created using VG2046 virus as the backbone. Briefly, the VG2046 parental virus encodes a cytokine expression cassette comprising IL-12, IL-15, and the IL-15 receptor alpha subunit inserted between the viral genes UL3 and UL4. VG22403 virus encodes an expression cassette for SARS-CoV-2 S protein RBD inserted between the viral genes US1 and US2 and driven by the CMV promoter. VG22407 virus encodes an expression cassette for SARS-CoV-2 S protein RBD fused to HSV-1 glycoprotein D (gD) inserted between the viral genes US1 and US2 and driven by the CMV promoter. VG22408 virus fuses the SARS-CoV-2 S protein RBD to the N-terminus of the HSV-1 gene encoding VP22. VG22409 virus fuses the SARS-CoV-2 S protein RBD to the N-terminus of the HSV-1 gene encoding VP26. VG22410 virus fuses the SARS-CoV-2 S protein RBD to the N-terminus of the HSV-1 gene encoding glycoprotein C (gC) .

Example 2

Engineering recombinant HSV-1 expressing HER2

Objective: To construct a recombinant HSV-1 expressing HER2.

Procedure: The recombinant HSV-1 mutant VG2044 (FIG. 5) was constructed by deleting most of the terminal repeat region of the HSV-1 genome that contains one copy of ICP34.5, ICP0 and ICP4. The remaining copy of ICP34.5 was inactivated by deletion. VG2044 was further modified by inserting an NFkB response element (SEQ ID NO: 14) and OCT4/SOX2 enhancer (SEQ ID NO: 15) into the promoter-regulatory region of ICP27. VG2044 also expresses a potent immunomodulatory payload, consisting of IL-12, IL-15, and IL 15Rα, which is controlled by a cytomegalovirus (CMV) promoter. Finally, VG2044 encodes an expression cassette for the extracellular and transmembrane domains of HER2 controlled by the EF1αpromoter.

VG22401 is a conditionally replicating oncolytic HSV-1 that also encodes an expression cassette for the extracellular and transmembrane domains of HER2 controlled by the EF1α promoter and incorporates a deletion of the terminal repeat region of the HSV-1 genome that contains one copy of ICP34.5, ICP0 and ICP4 (FIG. 6) . The remaining copy of ICP34.5 is regulated in a tumor-specific fashion via an insertion in its 3’UTR region containing multiple copies of binding domains for miRNA miR-124 and miR-143, which are highly expressed in neurons and normal tissues but not in tumor cells. VG22401 is further modified by replacing the native viral promoter for the essential viral gene UL54 which encodes ICP27 (infected cell polypeptide 27) with a tumor selective C-X-C Motif Chemokine Receptor 4 (CXCR4) promoter and replacing the native viral promoter for the viral gene US12 which encodes ICP47 (infected cell polypeptide 47) with the native viral promoter for the HSV-1 gene UL54. VG22401 also expresses a potent immunomodulatory payload, consisting of IL-12, IL-15, and IL 15Rα, which is controlled by a cytomegalovirus (CMV) promoter. Finally, VG22401 has a glycoprotein B (gB) truncation to enhance fusogenic activity, to facilitate virus spread within the tumor microenvironment.

Example 3

In vivo testing of recombinant HSV-1 expressing SARS-CoV-2 S protein RBD in tumor-bearing mice

Objective: To utilize a recombinant HSV-1 expressing SARS-CoV-2 S protein RBD to activate and redirect existing immunity against SARS-CoV-2 for the treatment of cancer.

Procedure: Two studies are performed, one to examine the mode of action and one to examine treatment efficacy. Initially, mice are immunized (primed) with lipid nanoparticles (LNP) loaded with mRNA encoding the SARS-CoV-2 S protein, followed by bilateral implantation of A20 murine B-cell lymphoma tumors and treatment with oncolytic HSV-1 expressing the SARS-CoV-2 S protein RBD (boost) .

For the mode of action (MOA) study, 32 BALB/c immunocompetent mice are randomized into 8 groups of 4 mice per group. Half of the mice (4 groups) are primed at day 0 with 30μg/mouse of lipid nanoparticles (LNP) loaded with mRNA encoding the SARS-CoV-2 S protein, followed by an identical booster dose at day 14. The remaining 4 groups serve as controls and are injected with 30μg/mouse of empty lipid nanoparticles at day 0 and day 14. At day 28, all mice are implanted with A20 murine B-cell lymphoma cells in both left and right flanks. Treatment commences between days 38 and 48 after tumors reached a volume of approximately 100 mm³. Tumors on the right flank are treated with a single intratumoral injection (1x10⁷ PFU/mouse) of either vehicle, VG2046, VG22403, or VG22407 (2 groups of mice per treatment, one group from the S-protein LNP primed cohort and one group from the control LNP primed cohort) . Mice are euthanized 3-5 days after virus treatment, and blood serum, spleen, and both injected and non-injected tumors are collected for analysis.

For the efficacy study, 40 BALB/c immunocompetent mice are randomized into 8 groups of 5 mice per group. Half of the mice (4 groups) are primed at day 0 with 30μg/mouse of lipid nanoparticles (LNP) loaded with mRNA encoding the SARS-CoV-2 S protein, followed by an identical booster dose at day 14. The remaining 4 groups serve as controls and are injected with 30μg/mouse of empty lipid nanoparticles at day 0 and day 14. At day 28, all mice are implanted with A20 murine B-cell lymphoma cells in both left and right flanks. Treatment commences between days 38 and 48 after tumors reached a volume of approximately 100 mm³. Tumors on the right flank are treated with a single intratumoral injection (1x10⁷ PFU/mouse) of either vehicle, VG2046, VG22403, or VG22407 (2 groups of mice per treatment, one group from the S-protein LNP primed cohort and one group from the control LNP primed cohort) . Mice are euthanized 3-5 days after virus treatment, and blood serum, spleen, and both injected and non-injected tumors are collected for analysis.

Results: Testing is performed to determine if treatment with oncolytic HSV-1 expressing the SARS-CoV-2 S protein RBD can elicit anti-SARS-CoV-2 immunity. Abscopal effects on untreated contralateral tumors and survival of mice treated with either backbone virus or virus expressing the SARS-CoV-2 S protein RBD is also compared.

Example 4

Detection of anti-HER2 immune response in immunized mice

Objective: To measure the extent of humoral and cellular immune responses in mice doubly immunized with VG2044.

Procedure: Mice were subcutaneously immunized twice at two week intervals with 1x10⁷ PFU of either VG2044 or VG161. Immunization with 5 μg of HER2 protein with CpG adjuvant was used as a positive control. Mice were sacrificed one week after the second immunization, and samples of blood and spleen cells were collected for ELISA and ELISPOT to evaluate the immune response.

Anti-HER2 IgG were measured using ELISA. Briefly, a 96-well plate was coated overnight with HER2 recombinant protein. The following day, serially diluted serum collected from immunized mice was applied to the plate for 1 hour. The plate was washed with PBS, and a secondary horseradish peroxidase (HRP) -conjugated anti-mouse IgG was applied to the plate (1: 1000 dilution) for 1 hour of incubation at room temperature. The plate was then washed with PBS and the binding of anti-HER2 antibodies was detected using a biotinylated monoclonal antibody, streptavidin, and 3, 3, 5, 5-Tetramethylbenzidine (TMB) substrate. Color development was stopped by adding 1M H2SO4. Absorbance measurements were collected at 450 nm and 570 nm wavelengths via microplate reader.

The mouse IFN-γ ELISPOT assay was performed by isolating spleen cells from immunized mice and adding the isolated splenocytes to each well of an ELISPOT plate (100,000 cells/well) , followed by stimulation overnight with either CT26 or CT26-HER2 cells (5000 cells/well) to detect CT26-and HER2-specific responses. Spleen cells were also stimulated with inactivated HSV-1 virus to demonstrate T-cell response to the virus. Results were expressed as the number of spots per well.

Results: Anti-HER2 antibodies were measured in the serum of immunized mice by ELISA, revealing that mice treated with VG2044 and its HER2 payload were able to generate a large amount of anti-HER2 antibodies equivalent to positive control mice treated with purified HER2 protein plus CpG adjuvant (FIG. 7A, FIG. 7B) . By contrast, mice treated with VG161 or with vehicle control generated negligible levels of anti-HER2 antibodies.

The ELISPOT results demonstrated that HSV-1 is highly immunogenic, as evidenced by dramatically increased IFN-γ production by splenocytes isolated from mice immunized with VG2044 and stimulated with inactivated HSV-1 (FIG. 8) . Furthermore, mice immunized with VG2044 exhibited increased T-cell response against CT26 cells expressing HER2 protein when compared to mice treated with purified HER2 protein vaccine.

Example 5

Immunogenicity study in immunocompetent mouse model

Objective: To evaluate the immunogenicity of VG2044 virus and VG2062 virus homologous prime-boost in immunocompetent BALB/c mice. The growth of VG2044 is attenuated due to deletion of ICP34.5, while VG2062 is a non-attenuated virus that employs a transcriptional and translational dual regulation (TTDR) strategy to control production of HSV-1 proteins ICP27 and ICP34.5 through the use of a tumor-specific promoter and tissue-specific microRNA regulation, respectively.

Procedure: BALB/c mice were subcutaneously immunized twice at 14-day intervals with 1x10⁷ PFU/mouse in 100μL total volume of VG2062 virus, VG2044 virus, or vehicle control (FIG. 9) . Animals were euthanized on day 35 post treatment initiation, with their spleens and blood serum harvested and stored at 4℃.

Anti-gD IgG in immunized mice were measured using ELISA. Briefly, a 96-well plate was coated overnight with gD recombinant protein. The next day, serially diluted serum collected from immunized mice was applied to the plate for 1 hour. The plate was washed with PBS, and a secondary horseradish peroxidase (HRP) -conjugated anti-mouse IgG was added to the plate (1: 1000 dilution) . The plate was incubated at room temperature for 1 hour, then washed with PBS. The binding of anti-HER2 antibodies was detected via a biotinylated monoclonal antibody, streptavidin, and 3, 3, 5, 5-Tetramethylbenzidine (TMB) substrate. Color development was stopped by adding 1M H2SO4. Absorbance measurements were collected at 450 nm and 570 nm wavelengths using a microplate reader.

The mouse IFNγ ELISPOT assay was performed by isolating the spleen cells from immunized mice and adding them to each well of an ELISPOT plate (100,000 cells/well) , followed by stimulation overnight with HER2 PepMix (JPT) (1 μg/well) to detect HER2-specific responses. Non-stimulated spleen cells were also used to measure the background stimulation signal. Results were expressed as the number of spots per PepMix-stimulated well subtracted from the non-stimulated well.

Results: ELISA was performed to measure anti-HER2 and anti-gD responses. gD is a ubiquitous immunogenic HSV surface glycoprotein, while only VG2044 encodes a HER2 expression cassette. Correspondingly, anti-HER2 antibodies were detected only in mice immunized with VG2044, while anti-gD antibodies were detected in both VG2044-and VG2062-treated mice (FIG. 10) .

ELISPOT assay was used to evaluate the cellular immune response against HER2, with splenocytes isolated from mice immunized with VG2044 showing elevated IFNγproduction after stimulation with HER2 PepMix compared to splenocytes from mice treated with VG2062 or with vehicle control (FIG. 11) .

Example 6

Efficacy study in immunocompetent CT26-HER2 mouse tumor model

Objective: To evaluate the efficacy of VG2044 homologous prime-boost vaccination strategy in the context of immunocompetent mice bearing CT26 tumors that stably express HER2.

Procedure: CT26 mouse tumor cells were transfected with HER2 lentivirus to generate a cell line stably expressing HER2 (CT26-HER2) . 40 immunocompetent BALB/c mice were randomized into four groups consisting of 10 mice per group. Two groups of mice were subcutaneously immunized twice at 14-day intervals with either VG2044 virus or with vehicle control (FIG. 12) . A dose of 5x10⁶ PFU/mouse in 100μL total volume was used for each immunization. CT26-HER2 (200μL) cells in raw medium were inoculated subcutaneously into the right and left flanks of the mice at 28 days post treatment initiation, and the resulting tumors were measured until they reached a volume of 100 mm³. At 40 days post treatment initiation, tumors located in the right flank were injected once intratumorally with either vehicle control or with VG2044 (1x10⁷ PFU/mouse in 50μL total volume) as depicted in FIG. 12, and the tumor volumes were measured and recorded until scheduled euthanasia. Tumors, spleens, and blood serum were harvested and stored at 4℃.

A mouse IFNγ ELISPOT assay was performed using splenocytes isolated from each treatment group by adding splenocytes to each well of an ELISPOT plate (100,000 cells/well) , followed by stimulation overnight with HER2 PepMix (JPT) (1 μg/well) to detect HER2-specific responses. Non-stimulated spleen cells were also used to measure the background stimulation signal. Results were expressed as the number of spots per PepMix-stimulated well subtracted from the non-stimulated well.

Results: Mice that have been primed twice with VG2044 and then intratumorally injected with VG2044 showed the best antitumor efficacy and a pronounced abscopal effect, with 4 out of 10 animals achieving a complete response on the injected side and 3 out of 10 animals showing a complete response on the non-injected side (FIG. 13) . In terms of survival, preimmunization with VG2044 offered a statistically significant advantage regardless of which virus was used for intratumoral injection (FIG. 14) . However, the survival advantage was most prominent in the group that was both preimmunized with VG2044 and treated intratumorally with VG2044. Splenocytes isolated from this group of mice also yielded the highest levels of IFNγ after stimulation with HER2 PepMix compared to splenocytes isolated from any of the other treatment groups (FIG. 15) .

Example 7

Efficacy studies in immunocompetent CT26-HER2 mouse tumor model

Objective: To evaluate the efficacy of VG2044, VG2062, and VG22401 homologous prime-boost vaccination strategies in the context of immunocompetent mice bearing CT26 tumors that stably express HER2. The growth of VG2044 is attenuated due to deletion of ICP34.5, while VG2062 is a non-attenuated virus that employs a transcriptional and translational dual regulation (TTDR) strategy to control production of HSV-1 proteins ICP27 and ICP34.5 through the use of a tumor-specific promoter and tissue-specific microRNA regulation, respectively. VG22401 is a non-attenuated TTDR virus similar to the VG2062 virus that further incorporates an expression cassette for the extracellular and transmembrane domains of HER2 controlled by the EF1α promoter.

Procedure: 40 immunocompetent BALB/c mice were randomized into four groups consisting of 10 mice per group. One group of mice was subcutaneously immunized twice at 14-day intervals with either VG2044 virus, VG2062 virus, VG22401 virus, or with vehicle control (FIG. 16) . A dose of 5x10⁶ PFU/mouse in 100μL total volume was used for each immunization. CT26-HER2 (200μL) cells in raw medium were inoculated subcutaneously into the right and left flanks of the mice at 28 days post treatment initiation, and the resulting tumors were measured until they reached a volume of 100 mm³. At 38 days post treatment initiation, tumors located in the right flank were injected once intratumorally with the same virus that was used for pre-immunization (1x10⁷ PFU/mouse in 50μL total volume) as depicted in FIG. 16, and the tumor volumes were measured and recorded until scheduled euthanasia. Tumors, spleens, and blood serum were harvested and stored at 4℃.

Results: Surprisingly, mice that have been primed twice with VG2044 and then intratumorally injected with VG2044 showed better antitumor efficacy and abscopal effect than mice that were immunized and intratumorally injected with the non-attenuated VG22401 virus (FIG. 17) , suggesting that ICP34.5 deletion may be beneficial in the context of eliciting antitumor immunity via an OV-based tumor vaccine. Half of the mice treated with VG2044 achieved a partial response on both the injected and the non-injected side, compared with 4/10 partial response on the injected side and 2/10 partial response on the non-injected side for mice treated with VG22401. ICP34.5 plays a role in controlling Type I interferon signaling, so a similarly beneficial effect for future recombinant HSV tumor vaccines may also be observed after modifying or deleting the gene encoding ICP0 which is also involved in immune downregulation in infected cells, including dampening the Type I interferon response.

Example 8

Engineering mRNA constructs encoding HER2

Objective: To engineer a panel of mRNA vaccine constructs encoding the HER2 antigen.

Procedure: A panel of 13 different mRNA vaccine constructs (HR1, HR2, HR3, HR4, HR5, HR6, HR7, HR8, HR9, HR10, HR11, HR12, and HR13) encoding the human HER2 extracellular domain was created as depicted in FIG. 18. Each of the 13 different mRNA vaccine constructs was loaded into lipid nanoparticles (LNPs) for use in all experiments. The prefix “LNP- “was added to the corresponding mRNA vaccine construct name when said mRNA vaccine construct was loaded into LNPs. All experiments where an mRNA vaccine construct was used for immunization utilized the LNP-loaded version of the mRNA vaccine construct even if the “LNP- “prefix was omitted from the construct name in the figure or description of the experiment. The preferred embodiment is construct 13, also known as HR13 (LNP-HR13 when loaded into LNPs) , which contains the human HER2 extracellular domain (HER2 ECD) and kinase-dead intracellular domain (X ICD) fused with the MHC1 trafficking domain (MITD) signal peptide and the MITD transmembrane domain.

Example 9

Evaluation of anti-HER2 immune response to intramuscularly or intravenously administered HR1

Objective: To evaluate the anti-HER2 immune response after immunization with the HR1 mRNA construct administered either intramuscularly or intravenously.

Procedure: 8-week-old female BALB/c mice were immunized 3 times with lipid nanoparticles (LNPs) loaded with HR1 (20μg/dose) via intramuscular or intravenous injection at days 0, 3, and 7 as depicted in FIG. 19A. At 14 days after start of immunization, splenocytes were isolated and treated with HER2 ECD protein for 24 hours before intracellular cytokine staining (ICS) to detect levels of granzyme B and interferon-γ.

Results: Three rounds of immunization with HR1 (LNP-HR1) successfully induced anti-HER2 ECD cellular immune responses in mice immunized through both intramuscular and intravenous routes. However, intramuscular immunization elicited a more robust T cell response, especially CD8+ T cell activity, when compared to mice immunized intravenously (FIG. 19B) .

Example 10

Prophylactic efficacy of HR13 against tumor challenge in an immunocompetent mouse model

Objective: To evaluate the efficacy of immunization with HR13 against challenge with HER2-positive tumor cell lines in immunocompetent BALB/c mice.

Procedure: 8-week-old female BALB/c mice were immunized 3 times with lipid nanoparticles (LNPs) loaded with HR13 (20μg/dose) via intramuscular injection at days 0, 3, and 7 as depicted in FIG. 20A. At 14 days after start of immunization, the mice were challenged either subcutaneously or intravenously with CT26-HER2 or 4T1-HER2 tumor cells that stably express HER2. Splenocytes were isolated from mice 2 weeks after the second immunization and treated with HER2 ECD protein for 24 hours before intracellular cytokine staining (ICS) to detect levels of granzyme B and interferon-γ. Lung tissue was isolated from mice challenged with intravenously injected tumor cells and subjected to hematoxylin and eosin (H&E) staining to visualize lung metastases.

Results: Three rounds of immunization with HR13 LNPs induced high levels of HER2 ECD-specific T cells as detected by intracellular cytokine staining. More than 10%of CD8+ T cells expressed the effector cytokines granzyme B and IFNγ after stimulation with HER2 ECD, suggesting the formation of memory T cells (FIG. 20B) . When challenged with the human HER2-positive tumor cell line CT26-HER2, LNP-HR13-immunized mice can completely reject subcutaneous tumor growth (FIG. 20C) . Growth of subcutaneously inoculated 4T1-HER2 tumors was merely delayed instead of rejected, likely due to confirmed poor expression of HER2 in 4T1-HER2 tumor tissues. LNP-HR13 immunization was highly protective in a murine lung metastasis model where mice were injected with CT26-HER2 or 4T1-HER2 cells intravenously. LNP-HR13-immunized mice survived much longer (FIG. 20D) and exhibited minimal tumor load in lung tissues (FIG. 20E) . Overall, these results suggest that HR13 LNPs can induce a robust and functional anti-HER2 immune response, specifically a cytotoxic T cell response which provides effective protection against HER2-positive tumors.

Example 11

Antitumor efficacy of prime-boost strategy using HR13 in combination with oncolytic virus

Objective: The tumor microenvironment is known to contain a multitude of immunosuppressive factors that restrict the function of cytotoxic T lymphocytes. Intratumoral delivery of oncolytic viruses may help to remodel the tumor microenvironment by promoting immune cell infiltration and activation of cytotoxic T lymphocytes to promote tumor-specific cell destruction. This study was designed to evaluate the antitumor efficacy of a prime injection comprising LNPs loaded with HR13 followed by boosting via intratumoral injection of the oncolytic virus VG401 which encodes HER2 in addition to a suite of immunomodulatory cytokines.

Procedure: 8-week-old female BALB/c mice were immunized 3 times with lipid nanoparticles (LNPs) loaded with HR13 (20μg/dose) via intramuscular injection at days 0, 5, and 12 as depicted in FIG. 21A. At 12 days after start of immunization, 1x10⁷ PFU/mouse of VG401 was intratumorally administered concurrent with the third dose of HR13. At the start of immunization (day 0) , mice were also subcutaneously implanted with 1x10⁶ of CT26-HER2 cells that stably express HER2. Tumor samples were retained for flow cytometry to evaluate immune cell infiltration in the tumor microenvironment.

Results: Mice treated with LNP-HR13 alone showed a statistically significant reduction in tumor volume by 15 days post treatment initiation, but antitumor efficacy decreased in later time points (FIG. 21B) . However, the combination of LNP-HR13 and VG401 showed more promise in retarding the growth of established tumors (FIG. 21B) and promoting CD8+ T cell infiltration into the tumor microenvironment (FIG. 21C) than LNP-HR13 alone, suggesting that a heterologous prime-boost combination of LNP-encapsulated mRNA encoding a tumor antigen and OV expressing the same tumor antigen can act synergistically to promote anti-tumor immunity and clearance of established tumors.

All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Such documents may be incorporated by reference for the purpose of describing and disclosing, for example, materials and methodologies described in the publications, which might be used in connection with the presently described invention. The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate any referenced publication by virtue of prior invention.

Claims

A method of eliciting an immune response in a subject, comprising administering to a subject a first vaccine, wherein the first vaccine comprises a macromolecule capable of inducing an immune response in said subject, and a second vaccine, wherein the second vaccine comprises an oncolytic virus which induces an immune response against a Tumor Antigen and/or a Non-Tumor Associated Antigen.
The method of claim 1 wherein said first vaccine induces an immune response against a cancer.
The method of claim 1 wherein said first vaccine induces an immune response against a non-human protein.
The method of claim 1 wherein said first vaccine induces an immune response against a non-tumor associated antigen derived from a virus selected from the group consisting of adenovirus, coronavirus, cytomegalovirus, dengue virus, hepatitis virus (e.g., hepatitis A and/or B) , herpes simplex virus, human papilloma virus, influenza virus, Japanese encephalitis virus, measles virus, mumps virus, poliovirus, rotavirus, rabies virus, rubella virus, varicella-zoster virus, variola virus, and yellow fever virus.
The method of claim 1 wherein said first vaccine induces an immune response against a non-tumor associated antigen derived from a bacterium selected from the group consisting of Neisseria meningitidis, Streptococcus pneumoniae, Mycobacterium tuberculosis, Clostridium tetani, Salmonella enterica, Bacillus anthracis, Vibrio cholerae, Yersinia pestis, Bordetella pertussis, Corynebacterium diphtheriae, and Haemophilus influenzae.
The method of claim 1, wherein said first vaccine is a protein vaccine, or, a nucleic acid vaccine.
The method of claim 5, wherein said first vaccine is an mRNA vaccine, wherein the mRNA vaccine encodes a tumor antigen and/or a non-tumor associated antigen.
The method of claim 6 or 7, wherein said first vaccine is encapsulated in lipid nanoparticles.
The method of claim 1 wherein said first vaccine is an oncolytic virus.
The method of claim 1, wherein said first vaccine elicits an immune response to one or more immunologically active proteins.
The method of claim 1, wherein said first vaccine is a recombinant oncolytic virus.
The method of claim 11 wherein said recombinant oncolytic virus has an inactivation or a regulation of a gene involved with Type 1 interferon signaling.
The method of claim 12 wherein said inactivation is a mutation, a partial deletion, or, a full deletion of the gene involved with Type 1 interferon signaling.
The method of claim 12 wherein said regulation is an insertion of an miRNA binding site into the 3’ UTR of the gene involved with Type 1 interferon signaling.
The method of claim 12 wherein said mutation or inactivation is in the ICP34.5 or ICP0 gene.
The method of any one of claims 1 to 15, wherein said first vaccine induces an immune response to a Tumor Antigen, and/or against a Non-Tumor Associated Antigen.
The method of claim 16, wherein said Tumor Antigen or Non-Tumor Associated Antigen is fused to an APC Targeting Peptide.
The method of claim 1, wherein said first vaccine is administered once.
The method of claim 1, wherein said first vaccine is administered multiple times.
The method of claim 19, wherein each dose of said first vaccine is separated by more than 12 hours or more than 1 day.
The method of claim 1, wherein said the second vaccine is an oncolytic virus that elicits an immune response to one or more immunologically active proteins.
The method of claim 1, wherein said second vaccine is a recombinant oncolytic virus.
The method of claim 21 or 22 wherein said second vaccine is a virus selected from the group consisting of adenovirus, parvovirus, vaccinia virus, reovirus, herpes simplex virus, measles virus, coxsackievirus, fowl pox virus, Sendai virus, echovirus, poliovirus, poxvirus, picornavirus, alphavirus, Semliki forest virus, Sindbis virus, myxoma virus, retrovirus, paramyxovirus, rhabdovirus, Newcastle disease virus, Maraba virus, and vesicular stomatitis virus.
The method of claim 21 or 22 wherein said second vaccine is a chimeric virus comprising elements from one or more viruses selected from the group consisting of adenovirus, parvovirus, vaccinia virus, reovirus, herpes simplex virus, measles virus, coxsackievirus, fowl pox virus, Sendai virus, echovirus, poliovirus, poxvirus, picornavirus, alphavirus, Semliki forest virus, Sindbis virus, myxoma virus, retrovirus, paramyxovirus, rhabdovirus, Newcastle disease virus, Maraba virus, and vesicular stomatitis virus.
The method of claim 21 or 22 wherein said second vaccine is a Herpes Simplex virus.
The method of claim 25 wherein said recombinant oncolytic virus has an inactivation or regulation of a gene involved with Type 1 interferon signaling.
The method of claim 26 wherein said inactivation is a mutation, a partial deletion, or, a full deletion of the gene involved with Type 1 interferon signaling.
The method of claim 26 wherein said regulation is an insertion of an miRNA binding site into the 3’ UTR of the gene involved with Type 1 interferon signaling.
The method of claim 1, wherein said second vaccine is administered once.
The method of claim 1, wherein said second vaccine is administered multiple times.
The method of claim 30, wherein each dose of said second vaccine is separated by more than 12 hours or more than 1 day.
The method of claim 1 wherein said first vaccine is administered systemically, intravenously, intramuscularly, intradermally, or, subcutaneously.
The method of claim 1 wherein said second vaccine is administered intratumorally.
The method of claim 1 wherein said oncolytic virus expresses one or more immunologically active proteins.
The method of claims 10, 21 or 34 wherein said immunologically active protein is selected from the group consisting of IL-12, IL-15, IL-15 receptor alpha, and IL-18.
The method of claim 1 wherein said first vaccine is derived from an oncolytic virus, and, said second vaccine is derived from the same type of oncolytic virus.
The method of claim 1 wherein said first vaccine is derived from an oncolytic virus, and said second vaccine is derived from a different type of oncolytic virus.
The method of claim 36 or 37, wherein said first vaccine is derived from the group consisting of adenovirus, parvovirus, vaccinia virus, reovirus, herpes simplex virus, measles virus, coxsackievirus, fowl pox virus, Sendai virus, echovirus, poliovirus, poxvirus, picornavirus, alphavirus, Semliki forest virus, Sindbis virus, myxoma virus, retrovirus, paramyxovirus, rhabdovirus, Newcastle disease virus, Maraba virus, vesicular stomatitis virus,
The method of claim 36 or 37 wherein said second vaccine is a chimeric virus comprising elements from one or more viruses selected from the group consisting of adenovirus, parvovirus, vaccinia virus, reovirus, herpes simplex virus, measles virus, coxsackievirus, fowl pox virus, Sendai virus, echovirus, poliovirus, poxvirus, picornavirus, alphavirus, Semliki forest virus, Sindbis virus, myxoma virus, retrovirus, paramyxovirus, rhabdovirus, Newcastle disease virus, Maraba virus, and vesicular stomatitis virus.
The method of claim 36 or 37, wherein said first vaccine induces an immune response against an antigen from a virus selected from the group consisting of an adenovirus, coronavirus, cytomegalovirus, dengue virus, hepatitis virus (e.g., hepatitis A and/or B) , herpes simplex virus, human papilloma virus, influenza virus, Japanese encephalitis virus, measles virus, mumps virus, poliovirus, rotavirus, rabies virus, rubella virus, varicella-zoster virus, variola virus, and yellow fever virus.
The method of claim 36 or 37, wherein said first vaccine induces an immune response against an antigen from a bacterium selected from the group consisting of Neisseria meningitidis, Streptococcus pneumoniae, Mycobacterium tuberculosis, Clostridium tetani, Salmonella enterica, Bacillus anthracis, Vibrio cholerae, Yersinia pestis, Bordetella pertussis, Corynebacterium diphtheriae, and Haemophilus influenzae.
A method of eliciting a heterologous immune response in a subject, comprising administering a vaccine to a subject, wherein the vaccine elicits an immune response to a Non-Tumor Associated Antigen to which the subject has already been exposed.
The method of claim 42 wherein said Non-Tumor Associated Antigen is derived from a virus.
The method of claim 42 wherein said Non-Tumor Associated Antigen is an antigen from a virus selected from the group consisting of an adenovirus, coronavirus, cytomegalovirus, dengue virus, hepatitis virus (e.g., hepatitis A and/or B) , herpes simplex virus, human papilloma virus, influenza virus, Japanese encephalitis virus, measles virus, mumps virus, poliovirus, rotavirus, rabies virus, rubella virus, varicella-zoster virus, variola virus, and yellow fever virus.
The method of claim 43 wherein said Non-tumor Associated Antigen is the SARS-CoV-2 S protein receptor binding domain.
The method of claim 42 wherein said Non-Tumor Associated Antigen is an antigen from a bacterium selected from the group consisting of Neisseria meningitidis, Streptococcus pneumoniae, Mycobacterium tuberculosis, Clostridium tetani, Salmonella enterica, Bacillus anthracis, Vibrio cholerae, Yersinia pestis, Bordetella pertussis, Corynebacterium diphtheriae, and Haemophilus influenzae.
The method of claim 42 wherein said exposure is due to prior vaccination.
The method of claim 42 wherein said exposure is due to prior infection.
The method according to claim 42 wherein said Non-Tumor Associated Antigen is administered systemically or intratumorally.
The method according to claim 42 wherein said vaccine is a recombinant oncolytic virus.
The method according to claim 50 wherein said vaccine is a genetically engineered Herpes Simplex virus.
The method according to claim 51 wherein said Herpes Simplex virus expresses one or more immunologically active proteins.
The method according to claim 52 wherein said immunologically active protein is selected from the group consisting of IL-12, IL-15, IL-15 receptor alpha, and IL-18.
A recombinant oncolytic virus which expresses an antigen derived from a coronavirus.
The recombinant oncolytic virus according to claim 54, wherein said recombinant oncolytic virus is a Herpes Simplex virus.
The recombinant oncolytic virus according to claim 54, wherein said antigen is the SARS-CoV-2 S protein receptor binding domain.
The recombinant oncolytic virus according to claim 54, wherein said recombinant oncolytic virus further expresses an immunologically active protein.
The recombinant oncolytic virus according to claim 57 wherein said immunologically active protein is selected from the group consisting of IL-12, IL-15, IL-15 receptor alpha, and IL-18.
A pharmaceutical composition, comprising the recombinant oncolytic virus according to any one of claims 54 to 58, and a pharmaceutically acceptable excipient.
A method of treating cancer, comprising administering the pharmaceutical composition according to claim 59 to a subject having a cancer.
The method according to claim 60 wherein said pharmaceutical composition is administered systemically, or, intratumorally.
A kit for the treatment of cancer, comprising a first composition comprising a first vaccine, according to any one of claims 1 to 41, and a second composition comprising a second vaccine, according to any one of claims 1 to 41.