AU2021254753A1 - Yeast lysate COVID-19 vaccine - Google Patents

Abstract

Yeast-based immunotherapeutic compositions comprising a SARS-CoV-2 antigen, as well as methods for stimulating an immune to SARS-CoV-2 is disclosed.

Description

YEAST LYSATE COVID-19 VACCINE

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of priority under 35 U.S.C. 119(e) to U.S. Provisional Application Serial No. 63/010,010, filed April 14, 2020. The entire disclosure of U.S. Provisional Application Serial No. 63/010,010 is incorporated herein by.

REFERENCE TO A SEQUENCE LISTING

[0002] This application contains a Sequence Listing submitted electronically as a text file by EFS-Web. The text file, named "8774-15-PCT ST25", has a size in bytes of 66000 bytes, and was recorded on April 14, 2021. The information contained in the text file is incorporated herein by reference in its entirety pursuant to 37 CFR § 1.52(e)(5).

BACKGROUND

[0003] There are numerous vaccine compositions known in the art, including orally administered vaccines, injected, and inhaled vaccines. Notably, in most cases the vaccine is either an attenuated pathogen or one or more isolated antigens from a pathogen. However, while many of the known vaccine compositions are at least somewhat effective, weakly immunogenic antigens tend to elicit insufficient immune response to produce a durable immunity. Likewise, where an individual has reduced or compromised immunity, typical antigen-based vaccines will often fail.

[0004] Protein antigens (e.g. subunit vaccines, the development of which was made possible by recombinant DNA technology), when administered without adjuvants, induce weak humoral (antibody) immunity and have therefore been disappointing to date as they exhibit only limited immunogenicity. An additional disadvantage of subunit vaccines, as well as of killed virus and recombinant live virus vaccines, is that while they appear to stimulate a strong humoral immune response when administered with adjuvants, they fail to elicit protective cellular immunity. Adjuvants are used experimentally to stimulate potent immune responses in mice, and are desirable for use in human vaccines, but few are approved for human use. Moreover, most adjuvants do not lead to induction of cytotoxic T lymphocytes (CTL). CTL are needed to kill cells that are synthesizing aberrant proteins including viral proteins and mutated "self proteins. Vaccines that stimulate CTL are being intensely studied for use against many viruses (e.g., HIV, HCV, HPV, HSY, CMV, EBY), intracellular bacteria (e.g., tuberculosis); intracellular parasites (e.g., malaria, leishmaniasis, shistosomiasis, leprosy), and all cancers (e.g., melanoma, prostate, ovarian, etc.). Thus, adjuvants are needed that stimulate CTL and cell-mediated immunity in general. [0005] Tarmogens (TARMOGEN® TARgeted MOlecular immunoGEN, Globelmmune, Inc., Louisville, Colorado) are inactivated whole recombinant yeast cells expressing disease-related “target” antigens. Immunization with Tarmogens elicits CD4+ and CD8+ T cell responses capable of eliminating tumor cells expressing class I MHC and the target antigen. The platform has been tested extensively in animal models and in >600 humans to date in FDA-regulated clinical trials (Hartley, 2015; Heery, 2015; King, 2017; Stubbs, 2001). Details of the expression system and yeast vector genetics can be found in in a published methodological review (King, 2016).

[0006] Yeast-mediated delivery of antigens into APCs concomitant with Toll-like receptor (TLR)-and mannose receptor (MR)- dependent APC activation have long been considered advantages of yeast over other vaccine platforms (Bernstein, 2008; Cereda, 2011; Remondo, 2009; Riemann, 2007). It is true that these characteristics bode well for an effective vaccine and a large body of data support the continued use of the platform in human clinical trials. The platform generated disease antigen-specific T cell responses in most patients in several cancer and infectious disease indications including chronic HBV and HCV (Boni, 2019; Gaggar, 2014; Haller, 2007; King, 2014; Lok, 2016) and clinical activity was also observed, especially against HCV. The therapeutic benefit in these trials, however, fell short of meeting clinical primary endpoints, leading to a re-assessment of the technology.

[0007] Several modifications were made to the platform to improve its potency. One such modification was to break open the yeast cells on the premise that the antigen, being trapped behind a tough chitinous cell wall, may not be readily available for rapid and efficient processing and MHC loading in APCs. This new lysed version of platform (See, e.g., U.S. Patent No. 17/047,134) is remarkably more potent, eliciting large numbers of activated T cells capable of in vivo target cell killing.

[0008] Coronaviruses (order Nidovirales, family Coronaviridae, genus Coronavirus) are enveloped positive-stranded RNA viruses that bud from the endoplasmic reticulum-Golgi intermediate compartment or the cis-Golgi network. Coronaviruses infect animals, including humans. Coronaviruses are named for the crown-like spikes on their surface and comprise four main sub-groupings known as alpha, beta, gamma, and delta., examples of which include: 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKUl (beta coronavirus)). Other human coronaviruses include MERS-CoV (the beta coronavirus that causes Middle East Respiratory Syndrome, or MERS), SARS-CoV (the beta coronavirus that causes severe acute respiratory syndrome, or SARS), and SARS-CoV2 (the novel beta coronavirus that causes coronavirus disease 2019, or COVID-19). [0009] Even though various systems and methods of vaccination are known in the art, all or almost all of them suffer from several drawbacks, including those for SARS-CoV-2. Therefore, there remains a need for compositions and methods for improved vaccine compositions and methods.

SUMMARY

[0010] One embodiment relates to a yeast immunotherapeutic composition comprising (a) a yeast vehicle or a yeast lysate prepared from a yeast, wherein the lysate lacks yeast membranes and yeast cell wall; and (b) at least one viral antigen, wherein the viral antigen is (i) expressed by the yeast vehicle; (ii) expressed by the yeast and retained in the yeast lysate; or (iii) added to the yeast vehicle or yeast lysate; wherein the at least one viral antigen is a SARS-CoV-2 antigen.

[0011] In one aspect of this embodiment, the immunotherapeutic composition further comprises a pharmaceutically acceptable excipient suitable for administration to a human. [0012] Another embodiment relates to a method to stimulate an immune response to SARS- CoV-2 in an individual comprising administering to the individual a yeast immunotherapeutic composition comprising: a) a yeast vehicle or a yeast lysate prepared from a yeast, wherein the lysate lacks yeast membranes and yeast cell wall; and (b) at least one viral antigen, wherein the viral antigen is (i) expressed by the yeast vehicle; (ii) expressed by the yeast and retained in the yeast lysate; or (iii) added to the yeast vehicle or yeast lysate; wherein the at least one viral antigen is a SARS-CoV-2 antigen; and a pharmaceutically acceptable excipient suitable for administration to the individual.

[0013] Another embodiment relates to a use of an immunotherapeutic composition comprising a yeast vehicle or yeast lysate prepared from a yeast and a SARS-CoV-2 antigen comprising at least one SARS-CoV-2 antigen to stimulate an immune response to SARS-CoV-2.

[0014] In one aspect of any of the embodiments related to a method or use related to a yeast immunotherapeutic composition described herein, the composition is formulated in a pharmaceutically acceptable excipient suitable for administration to the individual by an administration route selected from the group consisting of injection, intranasal, inhalation, oral, and combinations thereof.

[0015] In one aspect of any of the embodiments related to a method or use related to a yeast immunotherapeutic composition described herein, the individual is administered the immunotherapeutic composition in a dose amount from about 0.1 Y.U. to about 100 Y.U. [0016] In yet another aspect of any of the embodiments related to a method or use related to a yeast immunotherapeutic composition described herein, the method or use further comprises administering to the individual at least one additional dose of the immunotherapeutic composition. In one aspect, the additional dose of the immunotherapeutic composition is administered to the individual from 10 days to 52 days after the initial administration of the immunotherapeutic composition.

[0017] In any of the embodiments described above or elsewhere herein, in one aspect the SARS-CoV-2 antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO:8, and/or combinations thereof.

[0018] In any of the embodiments described above or elsewhere herein, in one aspect the SARS-CoV-2 antigen is a fusion protein. In one aspect the fusion protein has an amino acid sequence that is at least 95% identical to SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16.

[0019] In any of the embodiments described above or elsewhere herein, in one aspect the yeast vehicle or yest lysate prepared from a yeast is from a genus selected from the group consisting of: Saccharomyces, Candida , Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces and Yarrowia. In one aspect, the yeast vehicle or yeast lysate prepare from a yeast is from Saccharomyces. In yet another aspect, the yeast vehicle or yeast lysate prepare from a yeast is from Saccharomyces cerevisiae.

[0020] In any of the embodiments described above or elsewhere herein, in one aspect the yeast vehicle is a whole yeast. In one aspect, the whole yeast is heat-inactivated.

DESCRIPTION OF THE DRAWINGS

[0021] Fig. 1 shows yeast-SARS-CoVID-2 constructs disclosed herein: SARS-CoV2 spike proteins SI -SI and a lacking a c-terminal TM domain and c-terminal intraviral tail (referred to as “S1-S2” and having protein sequence SEQ ID NO: 12, and corresponding DNA sequence SEQ ID NO: 11); SARS-CoV-2 RBD protein fused to the c-terminus of the yeast cell wall protein Aga2 tail for expression (referred to as “ Aga2-RBD” and having protein sequence SEQ ID NO: 14, and corresponding DNA sequence SEQID NO: 13); SARS-CoV2 nucleocapsid fused to SARS-CoV-2 RBD (referred to as “N-RBD” and having protein sequence SEQ ID NO: 10, and corresponding DNA sequence SEQ ID NO:9); and SARS-Co2 nucleocapsid protein alone (referred to as “N” and having protein sequence SEQ ID NO: 16, and corresponding DNA sequence SEQ ID NO: 15).

[0022] Figs. 2A-2F shows results of vaccination studies. Mice were subcutaneously injected on left and right sides of the mouse such that the total vaccine dose was split between two sites. Vaccinations were administered on day 0, 24 and 42. Mice were euthanized 10 days after the first, second, or third injection and peripheral blood, spleens, and lungs harvested and processed for subsequent analysis of humoral and cellular immune responses. Serum from mice vaccinated once (1 Vac), twice (2 Vacs) or three times (3 Vacs) was assessed for the presence of Spike-specific IgG by ELISA. Individual mice are shown. YU, yeast unit; W303a, empty yeast vector lysate; SI, S1-S2, and N lysates of yeast expressing the SARS-CoV-2 SI domain, the full S ectodomain, or Nucleocapsid, respectively.

[0023] Fig. 3 shows prime boost with yeast lysates induces SARS-CoV-2 Spike specific CD8 T cell responses. Mice were subcutaneously injected on left and right sides of the mouse such that the total vaccine dose was split between two sites. Vaccinations were administered on day 0, 24 and 42. Mice were euthanized 10 days after the first, second, or third injection and peripheral blood, spleens, and lungs harvested and processed for subsequent analysis of humoral and cellular immune responses. Splenocytes were isolated 10 days after mice received a third vaccination and re-stimulated for 6 hours in vitro with Spike peptide pools or DMSO in the presence of Brefeldin A. Surface staining and intracellular cytokine staining was performed to detect IFNg and TNFa expression by antigen-specific CD8 T cells. These T cell responses were primarily detected in the SI domain of Spike suggesting that T cell epitopes are concentrated in this domain. CD4 responses (S or N) were not detected. IL4 producing T cells were not observed. Pepsets SI or S2 used for in vitro stimulation (IVS) are overlapping peptide pools spanning the SI or S2 domains of Spike. DMSO, negative control stimulation-solvent used for peptide resuspension. TNFa, tumor necrosis factor alpha; IFNg, Interferon gamma; IL4, Interleukin 4.

[0024] Fig. 4 shows the experimental protocol for a prime boost in vivo CTL assay.

[0025] Figs. 5A-5C shows in vivo CTL mediated killing of Spike peptide-pulsed targets using the experimental protocol shown in Fig. 4. Fig. 5A shows in vivo CTL mediated killing of SARS-CoV-2 Spike peptide-pulsed targets in animals immunized with COVID-19 S1-S2 yeast lysate. Figs. 5B and 5C show examples of raw flow cytometric data for naive and S1-S2 vaccinated mice respectively. Yeast lysate S1-S2 (COVID spike) or control yeast lysate (W303) were subcutaneously injected on left and right abdomen of the mouse such that the total vaccine dose was split between two sites. Vaccinations were administered on days 0 and 40, or on day 40 only. On day 47, splenocytes from naive syngeneic mice were labeled with PKH and CFSE (High CFSE labeled cells were pulsed with CFSl-5 peptide pool; low CFSE labeled cells were left unpulsed) and injected to immunized or naive control mice. Mice were euthanized 18-20 hours after target transfer, and splenocytes were isolated for flow cytometry analysis. CFSl-5 is pool of five COVID spike specific peptides. [0026] Fig. 6 shows the experimental protocol for Aga2-RBD dose response 10 to 80 YU in BALB/c mice.

[0027] Figs. 7A and 7B show subcutaneous Aga2-RBD prime plus exhibits increasing neutralization with increasing dose. BALB/cJ mice were vaccinated on days 0 and 24 with Aga2-RBD yeast lysate at 10 YU, 40 YU, and 80 YU or with recombinant COVID SI protein admixed with RIBI adjuvant (monophosphoryl Lipid A plus synthetic Trehalose Dicorynomycolate in 2% oil (squalene)-TWEEN® 80-water as a positive control (see Fig. 6). Peripheral blood was collected on day 34. Serum was diluted 1:30 and analyzed for the presence of neutralizing antibodies by CPASS™ (GENSCRIPT®) (Fig. 7 A) and the presence of trimeric spike-specific IgG by ELISA (Fig. 7B).

[0028] Fig. 8 shows the results of an ELISA-based in vitro assay used to evaluate the ACE2 binding-capacity of recombinant proteins expressed in: a negative control W303a yeast pressure lysate (pL) (empty yeast vector), W303a pL spiked with His-tagged recombinant functional trimeric Spike protein (Tri-S; positive control), V5-tagged N-RBD fusion expressing yeast pL, or His-tagged Aga2-RBD fusion-expressing yeast pL (RBD#4-pL). To evaluate the effect of various lysate processing methods on protein binding, yeast pL were left un-processed (far left 2 groups), passed through a 26Gx3/8 TB needle (middle 2 groups), or sonicated for 15 seconds (right 2 groups) and tested as a dilution series. Yeast pL suspensions were assayed undiluted or diluted at 1:10, 1:50, or 1:250 with concentration indicated by triangle on the X axis. Raw values of absorbance at 450nm (A450) is shown.

[0029] Fig. 9 shows the experimental protocol for a short interval prime plus boost intranasal vaccination with N-RBD lysate. Female BALB/c mice were inoculated with either W303a negative control yeast pL or N-RBD yeast pL at a dose of 6YU intranasally (i.n.), or 20YU subcutaneously (s.c.) on days 0 and 21. On day 28, mice were euthanized, peripheral blood, spleens and lungs harvested and processed for downstream analysis of antigen-specific immune responses via ELISA, CPASS™ (GENSCRIPT®) which is an assay that is a surrogate for virus neutralization, and ELISpot.

[0030] Figs. 10A and 10B shows intranasal N-RBD lysate vaccination induces N-specific T cell activation. Cells isolated from the spleen (Fig. 10A) and lung (Fig. 10B) were re-stimulated for 48 hours with Spike SI subunit (JPT-S1) orNucleoprotein (JPT-N) peptide pools orDMSO as a negative control. Antigen-specific IFNg production was assessed by ELISpot. Statistics generated by one-way ANOVA; *, p<0.05.

[0031] Fig. 11 shows the experimental protocol for a long-interval prime plus boost intranasal vaccination with N-RBD lysate. Female BALB/c mice received intranasal vaccinations on days 0 and 40 with the indicated dose of pressure lysates (pL) from W303a negative control yeast (group 2), W303a pL spiked with recombinant Spike SI subunit peptide (“191V”, group 1), or N-RBD fusion protein-expressing pL (groups 3-5) in a total volume of 20uL. Ten days after the homologous boost vaccination, mice were euthanized, serum was collected by RO bleed, and spleens and lungs were harvested and processed for downstream analysis.

[0032] Figs. 12A-12C show results of long-interval prime plus boost intranasal vaccination (see Fig. 11) with N-RBD lysate induces N-specific antibody production. Mice were vaccinated on days 0 and 40 with indicated yeast pL, and peripheral blood and lungs were harvested on day 50. Serum and lung homogenate were diluted 1:50 and analyzed for the presence of Nucleoprotein-specific IgG (Fig. 12A), IgGl (Fig. 12B), and IgG2a (Fig. 12C) by ELISA. Statics generated by one way ANOVA, ***, p<0.001.

[0033] Figs. 13 A and 13B show the results of an intranasal vaccination with N-RBD yeast lysate does not appear to induce neutralizing antibodies based on an undetectable or very low % inhibition in the CPASS™ assay. Mice were vaccinated on days 0 and 40 with indicated yeast pL, and peripheral blood and lungs were harvested on day 50 (see Fig. 11). Serum (Fig. 13 A) and lung (Fig. 13B) homogenate were diluted 1:30 and analyzed for the presence of neutralizing antibodies by CPASS™ (GENSCRIPT®). Given that a slight signal begins to emerge at the 6 Y.U. dose (Fig. 13 A) it is possible that testing a higher concentration of material such as 1:10 dilution of serum into the assay would produce a positive result.

[0034] Figs. 14A and 14B show the results of an intranasal vaccination with N-RBD lysate induces N-specific interferon gamma production by T cells. Mice were vaccinated on days 0 and 40 with indicated yeast pL, and spleens and lungs were harvested on day 50. Single cell suspensions from spleen and lungs were re-stimulated in vitro for 48 hours with Nucleoprotein peptide libraries (JPT) and T cell antigen-specific IFN gamma production was measured by ELISpot. Each dot represents the average of two technical replicates for each mouse. Fig. 14A shows the results for the spleen. Fig. 14B shows the results for the lung. Statistics generated by two-way ANOVA, * represents significance relative to W303a only vaccinated group ^L represents significance relative to W303a + 191V vaccinated group.

[0035] Figs. 15A and 15B: C57BL/6 mice were subcutaneously immunized with 10 YU of N lysate on days 0 and 21. One week later, splenocytes were stimulated with a pool of N peptides and Intracellular cytokine staining was conducted to evaluate levels of: CD44, TNFa, and IFNg in CD4 and in CD8 T cells (flow cytometric evaluation). Fig. 15A(data summary all mice) Shows the frequency of CD8+/CD44+ T cells that were activated to produce interferon gamma (IFNg) and tumor necrosis factor alpha (TNFa) in response to N peptide incubation. Fig 15B: Control stimulation with PMA/Ionomycin mixture (non-antigen specific mitogen).

[0036] Fig. 16 shows examples of SARS-CoV-2 proteins involved in Type 1 interferon (T1IFN) pathway inhibition. Nonstructural protein 1 (NSP1): decreased signal transducer and activator of transcription 1 (STAT1) activation. ORF3a: promotes Interferon Alpha and Beta Receptor Subunit 1 (IFNARl) degradation (inh. IFN receptor binding). ORF6: inhibits nuclear import of STAT1/STAT2/IRF9 (interferon regulatory factor 9 (IRF9)) complex; leading to reduced interferon-stimulated gene (ISG) production. A good vaccine should circumvent the TIIFN problem by generating neutralizing antibodies (nAbs) and T cells before these mechanisms kick in.

DETAILED DESCRIPTION

[0037] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry are those well-known and commonly used in the art.

[0038] All publications, patents, and published patent applications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control.

[0039] Disclosed herein are yeast-based immunotherapeutic compositions and methods and/or uses for stimulating an immune response to SARS-CoV-2. Described herein is the construction and production of novel yeast immunotherapy products, and demonstration that the yeast immunotherapy disclosed herein stimulates an immune response to SARS-CoV-2.

[0040] One embodiment relates to yeast immunotherapeutic composition comprising a yeast vehicle and/or a yeast lysate prepared from a yeast, wherein the lysate lacks yeast membranes and yeast cell walls and wherein the composition comprises at least one viral antigen that is expressed by the yeast vehicle, expressed by the yeast and retained in the yeast lysate, or added to the yeast vehicle or yeast lysate and wherein the viral antigen is a SARS-COV-2 antigen. [0041] One embodiment relates to a method to stimulate an immune response to SARS-CoV- 2 in an individual comprising administering to the individual a yeast immunotherapeutic composition comprising a yeast vehicle and/or a yeast lysate prepared from a yest, wherein the lysate lacks yeast membranes and yeast cell walls and wherein the composition comprises at least one viral antigen that is expressed by the yeast vehicle, expressed by the yeast and retained in the yeast lysate or added to the yeast vehicle or yeast lysate; wherein the viral antigen is a SARS-COV-2 antigen; and the composition comprises a pharmaceutically acceptable excipient suitable for administration to the individual.

[0042] One embodiment relates to a use of a yeast immunotherapeutic composition comprising a yeast vehicle or yeast lysate prepared from a yeast and a SARS-CoV-2 antigen comprising at least one SARS-CoV-2 antigen to stimulate an immune response to SARS-CoV-2. In one aspect, the lysate lacks yeast membranes and yeast cell walls. In one aspect, the composition comprises at least one viral antigen that is expressed by the yeast vehicle, expressed by the yeast and retained in the yeast lysate, or added to the yeast vehicle or yeast lysate. In another aspect the composition comprises a pharmaceutically acceptable excipient suitable for administration to an individual.

[0043] Also disclosed herein are methods and uses of immunotherapeutic compositions disclosed herein comprising a yeast vehicle or yeast lysate prepare from a yeast and a SARS- CoV-2 antigen comprising at least one SARS-CoV-2 antigen to treat SARS-CoV-2. In one aspect, the lysate lacks yeast membranes and yeast cell walls. In one aspect, the composition comprises at least one viral antigen that is expressed by the yeast vehicle, expressed by the yeast and retained in the yeast lysate, or added to the yeast vehicle or yeast lysate. In another aspect the composition comprises a pharmaceutically acceptable excipient suitable for administration to an individual.

[0044] Immunotherapeutic compositions of the present invention include a yeast vehicle and/or a yeast lysate. In one aspect, the yeast lysate is prepared from a yeast, wherein the lysate lacks yeast membranes and yeast cell walls. Such a yeast lysate is prepared from yeast that have been lysed, i.e., yeast in which the cell walls and membranes have been disrupted, exposing the yeast cell contents to the rest of the composition. The yeast lysates can be prepared from inactivated, such as heat inactivated, yeast or from live yeast. The yeast can contain a disease-related antigen expressed inside the yeast from a plasmid or from an integrated chromosomal allele. For example, yeast can be lysed by glass bead rupture, such as by mixing with PBS and 500 pL of acid washed 0.2 pm glass beads in a 1.5 mL total volume and vigorously shaking the mixture in a mechanical agitation machine until the cells are ruptured, such as >97% of the cells being ruptured. Alternatively, yeast can be lysed by other methods including high pressure homogenization, ultrasoni cation, and electrical, physical, chemical and enzymatic techniques. (See e.g. U. S. Patent Application No. 17/047,134).

[0045] In any of the yeast-based immunotherapy compositions used in the present invention, the following aspects related to the yeast vehicle are included in the invention. According to the present invention, a yeast vehicle is any yeast cell (e.g., a whole or intact cell) or a derivative thereof (see below) that can be used in conjunction with one or more antigens, immunogenic domains thereof or epitopes thereof in a therapeutic composition of the invention. The yeast vehicle can therefore include, but is not limited to, a live intact yeast microorganism (i.e., a yeast cell having all its components including a cell wall), a killed (dead) or inactivated intact yeast microorganism, or derivatives thereof including: a yeast spheroplast (i.e., a yeast cell lacking a cell wall), a yeast cytoplast (i.e., a yeast cell lacking a cell wall and nucleus), a yeast ghost (i.e., a yeast cell lacking a cell wall, nucleus and cytoplasm), a subcellular yeast membrane extract or fraction thereof (also referred to as a yeast membrane particle and previously as a subcellular yeast particle), any other yeast particle, or a yeast cell wall preparation.

[0046] Yeast spheroplasts are typically produced by enzymatic digestion of the yeast cell wall. Such a method is described, for example, in Franzusoff et ah, 1991, Meth. Enzymol. 194, 662- 674., incorporated herein by reference in its entirety.

[0047] Yeast cytoplasts are typically produced by enucleation of yeast cells. Such a method is described, for example, in Coon, 1978, Natl. Cancer Inst. Monogr. 48, 45-55 incorporated herein by reference in its entirety.

[0048] Yeast ghosts are typically produced by resealing a permeabilized or lysed cell and can, but need not, contain at least some of the organelles of that cell. Such a method is described, for example, in Franzusoff et ak, 1983, J. Biol. Chem. 258, 3608-3614 and Bussey et ah, 1979, Biochim. Biophys. Acta 553, 185-196, each of which is incorporated herein by reference in its entirety.

[0049] A yeast membrane particle (subcellular yeast membrane extract or fraction thereof) refers to a yeast membrane that lacks a natural nucleus or cytoplasm. The particle can be of any size, including sizes ranging from the size of a natural yeast membrane to microparticles produced by sonication or other membrane disruption methods known to those skilled in the art, followed by resealing. A method for producing subcellular yeast membrane extracts is described, for example, in Franzusoff et ak, 1991, Meth. Enzymol. 194, 662-674. One may also use fractions of yeast membrane particles that contain yeast membrane portions and, when the antigen or other protein was expressed recombinantly by the yeast prior to preparation of the yeast membrane particles, the antigen or other protein of interest. Antigens or other proteins of interest can be carried inside the membrane, on either surface of the membrane, or combinations thereof (i.e., the protein can be both inside and outside the membrane and/or spanning the membrane of the yeast membrane particle). In one embodiment, a yeast membrane particle is a recombinant yeast membrane particle that can be an intact, disrupted, or disrupted and resealed yeast membrane that includes at least one desired antigen or other protein of interest on the surface of the membrane or at least partially embedded within the membrane.

[0050] An example of a yeast cell wall preparation is isolated yeast cell walls carrying an antigen on its surface or at least partially embedded within the cell wall such that the yeast cell wall preparation, when administered to an animal, stimulates a desired immune response against a disease target.

[0051] For composition comprising lysed yeast cells, these compositions are then further treated to remove yeast membranes and yeast cell walls by any suitable method to produce a yeast lysate lacking yeast membranes and yeast cell walls. For example, yeast membranes and yeast cell walls can be removed from lysed yeast by centrifugation to produce a lysate (supernatant), which is free of cell walls and membranes, such as by centrifugation of lysed yeast for 5 minutes at 16,000 rpm, 25°C. Alternatively, lysates can be cleared of cell wall and membranous debris after rupture by means other than centrifugation. For example, filtration or treatment of cells with conA beads are alternate methods. When referring to removing yeast membranes and yeast cell walls or reference is made to a lysate lacking yeast membranes and yeast cell walls, it will be recognized that suitable processes for removal of materials may not remove 100% of the yeast membranes and yeast cell walls from a lysate. Thus, in some instances, at least about 80% of the yeast membranes and/or yeast cell walls are removed, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%.

[0052] Immunotherapeutic compositions of the present invention, in addition to a yeast lysate lacking yeast membranes and yeast cell walls, comprise at least one antigen (which term includes immunogenic domains of antigens) that is heterologous to the yeast. The heterologous antigen can have been expressed by the yeast, such as prior to lysing the yeast if the yeast vehicle is a yeast lysate or the antigen can have been combined with the yeast either before or after lysing and before or after removal of yeast membranes and yeast cell walls from a lysate. In some embodiments, the antigen is provided as a fusion protein, which can include two or more antigens. In one aspect, the fusion protein can include two or more immunogenic domains of one or more antigens, or two or more epitopes of one or more antigens.

[0053] The present invention includes the use of at least one “yeast-based immunotherapeutic composition” (which phrase may be used interchangeably with “yeast-based immunotherapy product”, “yeast-based immunotherapeutic composition”, “yeast-based composition”, “yeast- based immunotherapeutic” or “yeast-based vaccine”) which can be a yeast vehicle, or a yeast lysate that lacks yeast membranes and yeast cell walls, alone or in combination with an intact yeast-based immunotherapeutic composition, such as a TARMOGEN®. Yeast-based immunotherapeutic compositions elicit an immune response sufficient to achieve at least one therapeutic benefit in a subject. More particularly, a yeast-based immunotherapeutic composition is a composition that includes a yeast vehicle or a yeast lysate component alone or in combination with an intact yeast-based immunotherapeutic composition and can elicit or induce an immune response, such as a cellular immune response, including without limitation a T cell-mediated cellular immune response. In one aspect, the yeast-based immunotherapeutic composition useful in the invention is capable of inducing a CD8+ and/or a CD4+ T cell- mediated immune response and in one aspect, a CD8+ and a CD4+ T cell-mediated immune response. Optionally, a yeast-based immunotherapeutic composition is capable of eliciting a humoral immune response. A yeast-based immunotherapeutic composition useful in the present invention can, for example, elicit or stimulate an immune response in an individual such that the individual is treated for the disease or condition, or from symptoms resulting from the disease or condition. In one aspect, methods are disclosed to stimulate an immune response to SARS-CoV-2 comprising administering to an individual the immunotherapeutic compositions disclosed herein. In another aspect, use of an immunotherapeutic composition comprising a yeast vehicle or yeast lysate prepared from a yeast and a SARS-CoV-2 antigen comprising at least one SARS-CoV-2 antigen to stimulate an immune response to SARS-CoV- 2 is disclosed.

[0054] Yeast-based immunotherapeutic compositions of the invention may be either "prophylactic" or "therapeutic". When provided prophylactically, the immunotherapeutic compositions of the present invention are provided in advance of any symptom of a disease or condition. The prophylactic administration of the immunotherapeutic compositions serves to prevent or ameliorate or delay time to onset of any subsequent disease. When provided therapeutically, the immunotherapeutic compositions are provided at or after the onset of a symptom of disease. The term, “disease” refers to any deviation from the normal health of an animal and includes a state when disease symptoms are present, as well as conditions in which a deviation (e.g. infection, etc.) has occurred, but symptoms are not yet manifested.

[0055] Yeast lysates can be made from intact yeast-based immunotherapy compositions (i.e., TARMOGEN®. In addition, such intact yeast-based immunotherapy compositions can be combined with a yeast lysate-based composition. Such intact yeast-based immunotherapy compositions generally comprise a yeast vehicle (which does not include in this case a yeast lysate) and an antigen heterologous to the yeast.

[0056] Such intact yeast-based immunotherapy compositions, and methods of making and generally using the same, are described in detail, for example, in U.S. Patent No. 5,830,463, U.S. Patent No. 7,083,787, U.S. Patent No. 7,465,454, U.S. Patent Publication 2007-0224208, U.S. Patent Publication No. US 2008-0003239, and in Stubbs et al., Nat. Med. 7:625-629 (2001), Lu et al., Cancer Research 64:5084-5088 (2004), and in Bernstein et al., Vaccine 2008 Jan 24;26(4): 509-21, each of which is incorporated herein by reference in its entirety. These yeast-based immunotherapeutic products have been shown to elicit immune responses, including cellular and humoral immune responses. Yeast-based immunotherapeutic products are capable of killing target cells expressing a variety of antigens in vivo, in a variety of animal species, and to do so via antigen-specific, CD4+ and CD8+ mediated immune responses. Additional studies have shown that yeast are avidly phagocytosed by and directly activate dendritic cells which then present yeast-associated proteins to CD4+ and CD8+ T cells in a highly efficient manner. See, e.g., Stubbs et al. Nature Med. 5:625-629 (2001) and U.S. Patent No. 7,083,787.

[0057] Any yeast strain can be used to produce a yeast vehicle (either for production of a yeast lysate or to be used in combination with a yeast lysate). Yeast are unicellular microorganisms that belong to one of three classes: Ascomycetes, Basidiomycetes and Fungi Imperfecti. One consideration for the selection of a type of yeast for use as an immune modulator is the pathogenicity of the yeast. In one embodiment, the yeast is a non-pathogenic strain such as Saccharomyces cerevisiae. The selection of a non-pathogenic yeast strain minimizes any adverse effects to the individual to whom the yeast vehicle is administered. However, pathogenic yeast may be used if the pathogenicity of the yeast can be negated by any means known to one of skill in the art (e.g., mutant strains). In accordance with one aspect of the present invention, nonpathogenic yeast strains are used.

[0058] Preferred genera of yeast strains for production of the yeast vehicle, and/or the yeast lysate, include Saccharomyces, Candida, Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces and Yarrowia, with Saccharomyces, Candida, Hansenula, Pichia and Schizosaccharomyces being more preferred, and with Saccharomyces being particularly preferred. Preferred species of yeast strains include Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Candida albicans, Candida kejyr, Candida tropicalis, Cryptococcus laurentii, Cryptococcus neoformans, Hansenula anomala, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Kluyveromyces marxianus var. lactis, Pichia pastoris, Rhodotorula rubra, Schizosaccharomyces pombe, and Yarrowia lipolytica. It is to be appreciated that a number of these species include a variety of subspecies, types, subtypes, etc. that are intended to be included within the aforementioned species. In one aspect, yeast species used in the invention include S. cerevisiae, C. albicans, H. polymorpha, P. pastoris and S. pombe. S. cerevisiae is useful due to it being relatively easy to manipulate and being "Generally Recognized As Safe" or "GRAS" for use as food additives (GRAS, FDA proposed Rule 62FR18938, April 17, 1997). In one aspect, a yeast strain that is capable of replicating plasmids to a particularly high copy number, such as a S. cerevisiae cir° strain is useful. The S. cerevisiae strain is one such strain that is capable of supporting expression vectors that allow one or more target antigen(s) and/or antigen fusion protein(s) and/or other proteins to be expressed at high levels. In addition, any mutant yeast strains can be used, including those that exhibit reduced post-translational modifications of expressed target antigens or other proteins, such as mutations in the enzymes that extend N-linked glycosylation.

[0059] A yeast vehicle of the present invention is capable of fusing with the cell type to which the yeast vehicle and antigen/agent is being delivered, such as a dendritic cell or macrophage, thereby effecting particularly efficient delivery of the yeast vehicle, and in many embodiments, the antigen(s) or other agent, to the cell type. As used herein, fusion of a yeast vehicle with a targeted cell type refers to the ability of the yeast cell membrane, or particle thereof, to fuse with the membrane of the targeted cell type (e.g., dendritic cell or macrophage), leading to syncytia formation. As used herein, a syncytium is a multinucleate mass of protoplasm produced by the merging of cells. A number of viral surface proteins (including those of immunodeficiency viruses such as HIV, influenza virus, poliovirus and adenovirus) and other fusogens (such as those involved in fusions between eggs and sperm) have been shown to be able to effect fusion between two membranes (i.e., between viral and mammalian cell membranes or between mammalian cell membranes). For example, a yeast vehicle that produces an HIV gpl20/gp41 heterologous antigen on its surface is capable of fusing with a CD4+ T-lymphocyte. It is noted, however, that incorporation of a targeting moiety into the yeast vehicle, while it may be desirable under some circumstances, is not necessary. In the case of yeast vehicles that express antigens extracellularly, this can be a further advantage of the yeast vehicles of the present invention. In general, yeast vehicles useful in the present invention are readily taken up by dendritic cells (as well as other cells, such as macrophages). [0060] Immunotherapeutic compositions of the present invention comprise at least one antigen that is heterologous to the yeast from which the composition is formed. In one aspect the antigen is a viral antigen. In a preferred aspect, the viral antigen is a SARS-CoV-2 antigen. A SARS-CoV-2- antigen refers to a SARS-CoV-2 protein, and a variant thereof. Examples of suitable SARS-CoV-2 proteins that may be used as, or to produce, SARS-CoV-2 antigens include, but are not limited to, main protease (M^PR0, also known as Chain A 3C-like proteinase or 3C-like proteinase), SARS-CoV-2 nucleocapsid protein (N protein) (SEQ ID NO:2, corresponding DNA sequence represented by SEQ ID NO: 1), SARS-CoV-2 membrane protein (M protein), SARS-CoV-2 envelope protein (E protein), SARS-CoV-2 spike protein (S protein; SEQ ID NO:8 with corresponding DNA sequence represented by SEQ ID NO:7) which has two subunits SI (SEQ ID NO:6 with corresponding DNA sequence represented by SEQ ID NO:5) and S2 and SARS-CoV-2 Receptor Binding Domain (RBD) (SEQ ID NO:4 with corresponding DNA sequence represented by SEQ ID NO:3). In one aspect, the SARS-CoV-2 antigen comprises an amino acid sequence that is at least 85%, 86, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99% to SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or combinations thereof. In another aspect, the SARS-CoV-2 antigen comprises an amino acid sequence that is at least 95% to SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or combinations thereof. In still another aspect, the SARS-CoV-2 antigen comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8 and combinations thereof.

[0061] In still another aspect, the SARS CoV-2 antigen is a fusion protein. In one aspect, the fusion protein has an amino acid sequence that is at least 85%, 86, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 10 (N-RBD), SEQ ID NO: 12 (S1-S2), SEQ ID NO: 14 (Aga2-RBD) or SEQ ID NO: 16 (N). In another aspect, the fusion protein has an amino acid sequence that is at least 95% identical to SEQ ID NO: 10 (N-RBD), SEQ ID NO: 12 (S1-S2), SEQ ID NO: 14 (Aga2-RBD) or SEQ ID NO: 16 (N). In yet another aspect, the fusion protein has an amino acid sequence of SEQ ID NO: 10 (N-RBD), SEQ ID NO: 12 (S1-S2), SEQ ID NO: 14 (Arg2-RBD) or SEQ ID NO: 16 (N).

[0062] A SARS-CoV-2 antigen used in the disclosed methods and compositions may be a variant of a SARS-CoV-2 protein disclosed herein. The term variant refers to a protein, or fragment thereof, having an amino acids sequence that is similar, but not identical, to a referenced sequence (e.g., a SARS-CoV-2 protein sequence), wherein the activity of the variant protein is not significantly altered. These variations in sequence can be naturally occurring variations or they can be engineered through the use of technique known to those skilled in the art. Examples of suitable variations include, but are not limited to, amino acid deletions, insertions, substitutions ,and combinations thereof. [0063] According to the present invention, the general use herein of the term "antigen" refers: to any portion of a protein (peptide, partial protein, full-length protein), wherein the protein is naturally occurring or synthetically derived, to a cellular composition (whole cell, cell lysate or disrupted cells), to a microorganism or cells (whole microorganism, lysate or disrupted cells) or to a carbohydrate, or other molecule, or a portion thereof. An antigen may, in some embodiments, elicit an antigen-specific immune response (e.g., a humoral and/or a cell- mediated immune response) against the same or similar antigens that are encountered by an element of the immune system (e.g., T cells, antibodies).

[0064] An antigen can be as small as a single epitope, or larger, and can include multiple epitopes. As such, the size of an antigen can be as small as about 5-16 amino acids (e.g., a small peptide) and as large as: a domain of a protein, a partial protein (peptide or polypeptide), a full length protein, including a multimer and fusion protein, chimeric protein, or agonist protein or peptide. In addition, antigens can include carbohydrates.

[0065] When referring to stimulation of an immune response, the term “immunogen” is a subset of the term “antigen”, and therefore, in some instances, can be used interchangeably with the term "antigen". An immunogen, as used herein, describes an antigen which elicits a humoral and/or cell-mediated immune response (i.e., is immunogenic), such that administration of the immunogen to an individual in the appropriate context (e.g., as part of a yeast-based immunotherapy composition) elicits or induces an antigen-specific immune response against the same or similar antigens that are encountered by the immune system of the individual.

[0066] An “immunogenic domain” of a given antigen can be any portion, fragment or epitope of an antigen (e.g., a peptide fragment or subunit or an antibody epitope or other conformational epitope) that contains at least one epitope that acts as an immunogen when administered to an animal. For example, a single protein can contain multiple different immunogenic domains. Immunogenic domains need not be linear sequences within a protein, such as in the case of a humoral immune response.

[0067] An epitope is defined herein as a single immunogenic site within a given antigen that is sufficient to elicit an immune response. Those of skill in the art will recognize that T cell epitopes are different in size and composition from B cell epitopes, and that epitopes presented through the Class I MHC pathway differ from epitopes presented through the Class II MHC pathway. Epitopes can be linear sequence or conformational epitopes (conserved binding regions). [0068] The antigens contemplated for use in this invention include any antigen against which it is desired to elicit an immune response, and in particular, include any antigen for which a therapeutic immune response against such antigen would be beneficial to an individual. For example, the antigens can include, but are not limited to, any antigens associated with a pathogen, including viral antigens, fungal antigens, bacterial antigens, helminth antigens, parasitic antigens, ectoparasite antigens, protozoan antigens, or antigens from any other infectious agent. Antigens can also include any antigens associated with a particular disease or condition, whether from pathogenic or cellular sources, including, but not limited to, SARS- CoV-2, cancer antigens, antigens associated with an autoimmune disease (e.g., diabetes antigens), allergy antigens (allergens), mammalian cell molecules harboring one or more mutated amino acids, proteins normally expressed pre- or neo-natally by mammalian cells, proteins whose expression is induced by insertion of an epidemiologic agent (e.g. virus), proteins whose expression is induced by gene translocation, and proteins whose expression is induced by mutation of regulatory sequences. These antigens can be native antigens or genetically engineered antigens which have been modified in some manner (e.g., sequence change or generation of a fusion protein). It will be appreciated that in some embodiments (i.e., when the antigen is expressed by the yeast vehicle from a recombinant nucleic acid molecule), the antigen can be a protein or any epitope or immunogenic domain thereof, a fusion protein, or a chimeric protein, rather than an entire cell or microorganism.

[0069] In one aspect of the invention, antigens useful in one or more immunotherapy compositions of the invention include any antigens associated with a pathogen or a disease or condition caused by or associated with a pathogen. Such antigens include, but are not limited to, any antigens associated with a pathogen, including viral antigens, fungal antigens, bacterial antigens, helminth antigens, parasitic antigens, ectoparasite antigens, protozoan antigens, or antigens from any other infectious agent.

[0070] In one aspect, the antigen is from virus, including, but not limited to, coronaviruses, adenoviruses, arena viruses, bunyaviruses, coxsackie viruses, cytomegaloviruses, Epstein-Barr viruses, flaviviruses, hepadnaviruses, hepatitis viruses, herpes viruses, influenza viruses, lentiviruses, measles viruses, mumps viruses, myxoviruses, orthomyxoviruses, papilloma viruses, papovaviruses, parainfluenza viruses, paramyxoviruses, parvoviruses, picomaviruses, pox viruses, rabies viruses, respiratory syncytial viruses, reoviruses, rhabdoviruses, rubella viruses, togaviruses, and varicella viruses. Other viruses include T-lymphotrophic viruses, such as human T-cell lymphotrophic viruses (HTLVs, such as HTLV-I and HTLV-II), bovine leukemia viruses (BLVS) and feline leukemia viruses (FLVs). The lentiviruses include, but are not limited to, human (HIV, including HIV-1 or HIV-2), simian (SIV), feline (FIV) and canine (CIV) immunodeficiency viruses. In one embodiment, viral antigens include those from non-oncogenic viruses.

[0071] In some embodiments, the antigen is a fusion protein. In one aspect of the invention, fusion protein can include two or more antigens. In one aspect, the fusion protein can include two or more immunogenic domains or two or more epitopes of one or more antigens. An immunotherapeutic composition containing such antigens may provide antigen-specific immunization in a broad range of patients. For example, a multiple domain fusion protein useful in the present invention may have multiple domains, wherein each domain consists of a peptide from a particular protein, the peptide consisting of at least 4 amino acid residues flanking either side of and including a mutated amino acid that is found in the protein, wherein the mutation is associated with a particular disease or condition.

[0072] In one embodiment, fusion proteins that are used as a component of the yeast-based immunotherapeutic composition useful in the invention are produced using constructs that are particularly useful for the expression of heterologous antigens in yeast. Typically, the desired antigenic protein(s) or peptide(s) are fused at their amino-terminal end to: (a) a specific synthetic peptide that stabilizes the expression of the fusion protein in the yeast vehicle or prevents posttranslational modification of the expressed fusion protein (such peptides are described in detail, for example, in U.S. Patent Publication No. 2004-0156858 Al, published August 12, 2004, incorporated herein by reference in its entirety); (b) at least a portion of an endogenous yeast protein, wherein either fusion partner provides significantly enhanced stability of expression of the protein in the yeast and/or a prevents post-translational modification of the proteins by the yeast cells (such proteins are also described in detail, for example, in U.S. Patent Publication No. 2004-0156858 Al, supra); and/or (c) at least a portion of a yeast protein that causes the fusion protein to be expressed on the surface of the yeast (e.g., an Aga protein, described in more detail herein). In addition, the present invention includes the use of peptides that are fused to the C-terminus of the antigen-encoding construct, particularly for use in the selection and identification of the protein. Such peptides include, but are not limited to, any synthetic or natural peptide, such as a peptide tag (e.g., 6X His) or any other short epitope tag. Peptides attached to the C-terminus of an antigen according to the invention can be used with or without the addition of the N-terminal peptides discussed above. [0073] In one embodiment, a synthetic peptide useful in a fusion protein is linked to the N- terminus of the antigen, the peptide consisting of at least two amino acid residues that are heterologous to the antigen, wherein the peptide stabilizes the expression of the fusion protein in the yeast vehicle or prevents posttranslational modification of the expressed fusion protein. The synthetic peptide and N-terminal portion of the antigen together form a fusion protein that has the following requirements: (1) the amino acid residue at position one of the fusion protein is a methionine (i.e., the first amino acid in the synthetic peptide is a methionine); (2) the amino acid residue at position two of the fusion protein is not a glycine or a proline (i.e., the second amino acid in the synthetic peptide is not a glycine or a proline); (3) none of the amino acid residues at positions 2-6 of the fusion protein is a methionine (i.e., the amino acids at positions 2-6, whether part of the synthetic peptide or the protein, if the synthetic peptide is shorter than 6 amino acids, do not include a methionine); and (4) none of the amino acids at positions 2-6 of the fusion protein is a lysine or an arginine (i.e., the amino acids at positions 2-6, whether part of the synthetic peptide or the protein, if the synthetic peptide is shorter than 5 amino acids, do not include a lysine or an arginine). The synthetic peptide can be as short as two amino acids, but in one aspect, is at least 2-6 amino acids (including 3, 4, 5 amino acids), and can be longer than 6 amino acids, in whole integers, up to about 200 amino acids, 300 amino acids, 400 amino acids, 500 amino acids, or more.

[0074] The present invention includes the delivery (administration, immunization) of a composition of the invention to a subject. The administration process can be performed ex vivo or in vivo , but is typically performed in vivo. Ex vivo administration refers to performing part of the delivery step outside of the patient, such as administering a composition of the present invention to a population of cells (dendritic cells) removed from a patient under conditions such that a yeast vehicle, antigen(s) and any other agents or compositions are loaded into the cell, and returning the cells to the patient. This can include yeast vehicle (such as Tarmogens), yeast lysates or yeast lysates mixed with TARMOGEN®. The therapeutic composition of the present invention can be returned to a patient, or administered to a patient, by any suitable mode of administration.

[0075] Administration of a composition can be systemic, mucosal and/or proximal to the location of the target site. Suitable routes of administration will be apparent to those of skill in the art, depending on the type of condition to be prevented or treated, the antigen used, and/or the target cell population or tissue. Various acceptable methods of administration include, but are not limited to, intravenous administration, intranasal (i.n), inhalation (e.g., aerosol), intraperitoneal administration, intramuscular administration, intranodal administration, intracoronary administration, intraarterial administration (e.g., into a carotid artery), subcutaneous administration, retroorbital administration, transdermal delivery, intratracheal administration, intraarticular administration, intraventricular administration, intracranial, intraspinal, intraocular, aural, oral, pulmonary administration, impregnation of a catheter, direct injection into a tissue and combinations thereof. In one aspect, routes of administration include: intravenous, intranasal, inhalation, intraperitoneal, subcutaneous, intradermal, intranodal, intramuscular, transdermal, oral, intraocular, intraarticular, intracranial, and intraspinal. Parenteral delivery can include intradermal, intramuscular, intraperitoneal, intrapleural, intrapulmonary, intravenous, subcutaneous, atrial catheter and venal catheter routes. Aural delivery can include ear drops, intranasal delivery can include nose drops or intranasal injection, and intraocular delivery can include eye drops. Aerosol (inhalation) delivery can also be performed using methods standard in the art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA 189:11277-11281, 1992, which is incorporated herein by reference in its entirety). Other routes of administration that modulate mucosal immunity are useful in the treatment of viral infections. Such routes include bronchial, intradermal, intramuscular, intranasal, other inhalatory, rectal, subcutaneous, topical, transdermal, vaginal and urethral routes. In one aspect, an immunotherapeutic composition is administered subcutaneously. In yet another aspect, an immunotherapeutic composition is administered is administered intranasally. In still another aspect, an immunotherapeutic composition is administered by inhalation.

[0076] The immunotherapeutic composition of the present invention can be formulated in a pharmaceutically acceptable excipient suitable for administration to an individual or subject by an administration route selected from the group consisting of injection, intranasal, inhalation, oral, and combinations thereof.

[0077] With respect to the yeast-based immunotherapy compositions, in general, a suitable single dose is a dose that is capable of effectively providing a composition of the invention to a given cell type, tissue, or region of the patient body in an amount effective to elicit an antigen- specific immune response, when administered one or more times over a suitable time period. For example, in one embodiment, a single dose of a composition of the present invention is from about 1 x 10⁵ to about 5 x 10⁷ yeast cell equivalents per kilogram body weight of the organism being administered the composition. In one aspect, a single dose of a yeast vehicle of the present invention is from about 0.1 Y.U. (1 x 10⁶ cells) to about 200 Y.U. (2 x 10⁹ cells) per dose (i.e., per organism), including any interim dose, in increments of 0.1 x 10⁶ cells (i.e., 1.1 x 10⁶, 1.2 x 10⁶, 1.3 x 10⁶...). In one embodiment, doses include doses between 1 Y.U and 80 Y.U. and in one aspect, between 10 Y.U. and 40 Y.U. In one embodiment, the doses are administered at different sites on the individual but during the same dosing period. For example, a 40 Y.U. dose may be administered via by injecting 10 Y.U. doses to four different sites on the individual during one dosing period. In another example a 4 Y.U. dose may be administered via injecting 1 Y.U. doses to four different sites on the individual during one dosing period. In one embodiment, the doses are administered in the right and left nasal passages of the individual but during the same dosing period. For example, a 20 Y.U. dose may be administered intranasally to each nasal passage during one dosing period. In one embodiment, the dose of 1 Y.U to about 200 Y.U. includes both yeast lysates and the yeast vehicle. In one embodiment, the doses are administered in the right and left nasal passages of the individual but during the same dosing period. For example, a 1 or 20 Y.U. dose may be administered intranasally to each nasal passage during one dosing period.

[0078] "Boosters" or "boosts" of a therapeutic composition are administered, for example, when the immune response against the antigen has waned or as needed to provide an immune response or induce a memory response against a particular antigen or antigen(s). Boosters can be administered from about 10 days, 15 days, 20 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days or about 55 days after the initial or original administration. Boosters can be administered from about 1, 2, 3, 4, 5, 6, 7, or 8 weeks apart, to monthly, to bimonthly, to quarterly, to annually, to several years after the initial or original administration. In one aspect, the booster can be administered from 10 to 52 days. In yet another aspect, the booster can be administered 21 days, 24 days or 40 days after initial or original administration In one embodiment, an administration schedule is one in which from about 1 x 10⁵ to about 5 x 10⁷ yeast cell equivalents of a composition per kg body weight of the organism is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times over a time period of from weeks, to months, to years.

[0079] The immunogenic composition can be administered to the subject weekly, every other week and/or monthly. The composition can be administered once every 40 days. In still another aspect, the composition can be administered once every 60 days. In still another aspect, the composition can be administered once every 38 to 40 days.

[0080] In one aspect, inhalation of lysates for treatment of lung ailments can occur with or without mixture with intact TARMOGEN® and can be accomplished by pressurized metered dose inhalation (pMDIs), nebulizers, and dry powder inhalers (DPIs). In one aspect, inhalation of the intact TARMOGEN® alone can be accomplished by pressurized metered dose inhalation (pMDIs), nebulizers, and dry powder inhalers (DPIs).

[0081] Methods of producing yeast vehicles and/or yeast lysates and expressing, combining and/or associating yeast vehicles and/or yeast lysates with antigens and/or other proteins and/or agents of interest to produce yeast-based immunotherapy compositions are contemplated herein.

[0082] The term “yeast-lysate-antigen complex”, "yeast vehicle-antigen complex" or "yeast- antigen complex" is used generically herein to describe any association of a yeast vehicle and/or yeast lysate with an antigen, and can be used interchangeably with “yeast-based immunotherapy composition” when such composition is used to elicit an immune response as described above. Such association includes expression of the antigen by the yeast (a recombinant yeast), introduction of an antigen into a yeast, physical attachment of the antigen to the yeast, and mixing of the yeast and antigen together, such as in a buffer or other solution or formulation. These types of complexes are described in detail below. It will be recognized that yeast lysate-antigen complexes can be formed from yeast vehicle-antigen complexes by methods described herein.

[0083] In one embodiment, a yeast cell used to prepare the yeast vehicle is transfected with a heterologous nucleic acid molecule encoding a protein (e.g., the antigen or agent) such that the protein is expressed by the yeast cell. Such a yeast is also referred to herein as a recombinant yeast or a recombinant yeast vehicle. The yeast cell can then be loaded into the dendritic cell as an intact cell, or the yeast cell can be killed, or it can be derivatized such as by formation of yeast spheroplasts, cytoplasts, ghosts, or subcellular particles, any of which is followed by loading of the derivative into the dendritic cell. Yeast spheroplasts can also be directly transfected with a recombinant nucleic acid molecule (e.g., the spheroplast is produced from a whole yeast, and then transfected) in order to produce a recombinant spheroplast that expresses an antigen or other protein.

[0084] In one aspect, a yeast cell or yeast spheroplast used to prepare the yeast vehicle is transfected with a recombinant nucleic acid molecule encoding the antigen(s) or other protein such that the antigen or other protein is recombinantly expressed by the yeast cell or yeast spheroplast. In this aspect, the yeast cell or yeast spheroplast that recombinantly expresses the antigen(s) or other protein is used to produce a yeast vehicle comprising a yeast cytoplast, a yeast ghost, or a yeast membrane particle or yeast cell wall particle, or fraction thereof.

[0085] In general, the yeast vehicle and antigen(s) or other agent, can be associated by any technique described herein. In one aspect, the yeast vehicle was loaded intracellularly with the antigen(s) and/or agent(s). In another aspect, the antigen(s) and/or agent(s) was covalently or non-covalently attached to the yeast vehicle. In yet another aspect, the yeast vehicle and the antigen(s) and/or agent(s) were associated by mixing. In another aspect, and in one embodiment, the antigen(s) and/or agent(s) is expressed recombinantly by the yeast vehicle or by the yeast cell or yeast spheroplast from which the yeast vehicle was derived.

[0086] Expression of an antigen or other protein in a yeast vehicle of the present invention is accomplished using techniques known to those skilled in the art. Briefly, a nucleic acid molecule encoding at least one desired antigen or other protein is inserted into an expression vector in such a manner that the nucleic acid molecule is operatively linked to a transcription control sequence in order to be capable of effecting either constitutive or regulated expression of the nucleic acid molecule when transformed into a host yeast cell. Nucleic acid molecules encoding one or more antigens and/or other proteins can be on one or more expression vectors operatively linked to one or more expression control sequences. Particularly important expression control sequences are those which control transcription initiation, such as promoter and upstream activation sequences. Any suitable yeast promoter can be used in the present invention and a variety of such promoters are known to those skilled in the art. Promoters for expression in Saccharomyces cerevisiae include, but are not limited to, promoters of genes encoding the following yeast proteins: CUP1, alcohol dehydrogenase I (ADH1) or II (ADH2), phosphoglycerate kinase (PGK), triose phosphate isomerase (TPI), translational elongation factor EF-1 alpha (TEF2), glyceraldehyde-3 -phosphate dehydrogenase (GAPDH; also referred to as TDH3, for triose phosphate dehydrogenase), galactokinase (GAL1), galactose- 1- phosphate uridyl-transferase (GAL7), UDP-galactose epimerase (GAL 10), cytochrome cl (CYC1), Sec7 protein (SEC7) and acid phosphatase (PH05), including hybrid promoters such as ADH2/GAPDH and CYC 1 /GAL 10 promoters, and including the ADH2/GAPDH promoter, which is induced when glucose concentrations in the cell are low (e.g., about 0.1 to about 0.2 percent), as well as the CUP1 promoter and the TEF2 promoter. Likewise, a number of upstream activation sequences (UASs), also referred to as enhancers, are known. Upstream activation sequences for expression in Saccharomyces cerevisiae include, but are not limited to, the UASs of genes encoding the following proteins: PCK1, TPI, TDH3, CYC1, ADH1, ADH2, SUC2, GALl, GAL7 and GAL 10, as well as other UASs activated by the GAL4 gene product, with the ADH2 UAS being used in one aspect. Since the ADH2 UAS is activated by the ADR1 gene product, it may be preferable to overexpress the ADR1 gene when a heterologous gene is operatively linked to the ADH2 UAS. Transcription termination sequences for expression in Saccharomyces cerevisiae include the termination sequences of the a-factor, GAPDH, and CYC1 genes. [0087] Transcription control sequences to express genes in methyltrophic yeast include the transcription control regions of the genes encoding alcohol oxidase and formate dehydrogenase.

[0088] Transfection of a nucleic acid molecule into a yeast cell according to the present invention can be accomplished by any method by which a nucleic acid molecule administered into the cell and includes, but is not limited to, diffusion, active transport, bath sonication, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. Transfected nucleic acid molecules can be integrated into a yeast chromosome or maintained on extrachromosomal vectors using techniques known to those skilled in the art. Examples of yeast vehicles carrying such nucleic acid molecules are disclosed in detail herein. As discussed above, yeast cytoplast, yeast ghost, and yeast membrane particles or cell wall preparations can also be produced recombinantly by transfecting intact yeast microorganisms or yeast spheroplasts with desired nucleic acid molecules, producing the antigen therein, and then further manipulating the microorganisms or spheroplasts using techniques known to those skilled in the art to produce cytoplast, ghost or subcellular yeast membrane extract or fractions thereof containing desired antigens or other proteins.

[0089] Effective conditions for the production of recombinant yeast vehicles and expression of the antigen and/or other protein (e.g., an agent as described herein) by the yeast vehicle include an effective medium in which a yeast strain can be cultured. An effective medium is typically an aqueous medium comprising assimilable carbohydrate, nitrogen and phosphate sources, as well as appropriate salts, minerals, metals and other nutrients, such as vitamins and growth factors. The medium may comprise complex nutrients or may be a defined minimal medium. Yeast strains of the present invention can be cultured in a variety of containers, including, but not limited to, bioreactors, Erlenmeyer flasks, test tubes, microtiter dishes, and Petri plates. Culturing is carried out at a temperature, pH and oxygen content appropriate for the yeast strain. Such culturing conditions are well within the expertise of one of ordinary skill in the art (see, for example, Guthrie et al. (eds.), 1991, Methods in Enzymology, vol. 194, Academic Press, San Diego).

[0090] In some aspects of the invention, the yeast are grown under neutral pH conditions, and particularly, in a media maintained at a pH level of at least 5.5, namely the pH of the culture media is not allowed to drop below pH 5.5. In other aspects, the yeast is grown at a pH level maintained at about 5.5. In other aspects, the yeast is grown at a pH level maintained at about 5.6, 5.7, 5.8 or 5.9. In another aspect, the yeast is grown at a pH level maintained at about 6. In another aspect, the yeast is grown at a pH level maintained at about 6.5. In other aspects, the yeast is grown at a pH level maintained at about 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0. In other aspects, the yeast is grown at a pH level maintained at about 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. The pH level is important in the culturing of yeast. One of skill in the art will appreciate that the culturing process includes not only the start of the yeast culture but the maintenance of the culture as well. As yeast culturing is known to turn acidic (i.e., lowering the pH) over time, care must be taken to monitor the pH level during the culturing process. Yeast cell cultures whereby the pH level of the medium drops below 6 are still contemplated within the scope of the invention provided that the pH of the media is brought up to at least 5.5 at some point during the culturing process. As such, the longer time the yeast are grown in a medium that is at least pH 5.5 or above, the better the results will be in terms of obtaining yeast with desirable characteristics.

[0091] In one aspect, yeast is cultured such that the pH level of the medium does not drop below pH 5.5. In some cases, the drop below pH 5.5 is not more than 5 minutes. In other cases, the drop below pH 5.5 is not more than 10 minutes. In other cases, the drop below pH 5.5 is not more than 1 hour. In another aspect, yeast is cultured such that the pH level of the medium does not drop below 5.0. In some cases, the drop below pH 5.0 is not more than 5 minutes. In other cases, the drop below pH 5.0 is not more than 10 minutes, preferably 20, 30, 40, 50 or 60 minutes. In other cases, the drop below pH 5.0 is not more than 1 hour. As such, the longer time the yeast are grown in a medium that is at least pH 5.5 or above, the better the results will be in terms of obtaining yeast with desirable characteristics described infra.

[0092] In one aspect, the use of neutral pH methods to grow yeast cells means that the yeast cells are grown in neutral pH for at least 50% of the time that the yeast are in culture. It is more preferable that the yeast are grown at neutral pH for at least 60% of the time they are in culture, more preferably at least 70% of the time they are in culture, more preferably at least 80% of the time they are in culture, and most preferably at least 90% of the time they are in culture . [0093] In another aspect, growing yeast at neutral pH includes culturing yeast cells for at least five minutes at neutral pH, preferably at least 15 minutes at neutral pH, more preferably at least one hour at neutral pH, more preferably at least two hours, even more preferably, at least three hours or longer.

[0094] As used herein, the general use of the term “neutral pH” refers to a pH range between about pH 5.5 and about pH 8, and in one aspect, between about pH 6 and about 8. One of skill the art will appreciate that minor fluctuations (e.g., tenths or hundredths) can occur when measuring with a pH meter. As such, the use of neutral pH to grow yeast cells means that the yeast cells are grown in neutral pH for the majority of the time that they are in culture. The use of a neutral pH in culturing yeast promotes several biological effects that are desirable characteristics for using the yeast as vehicles for immunomodulation. In one aspect, culturing the yeast in neutral pH allows for good growth of the yeast without any negative effect on the cell generation time (e.g., slowing down the doubling time). The yeast can continue to grow to high densities without losing their cell wall pliability. In another aspect, the use of a neutral pH allows for the production of yeast with pliable cell walls and/or yeast that are sensitive to cell wall digesting enzymes (e.g., glucanase) at all harvest densities. This trait is desirable because yeast with flexible cell walls can induce unusual immune responses, such as by promoting the secretion of cytokines (e.g., interferon-g (IFN-g)) in the cells hosting the yeast. In addition, greater accessibility to the antigens located in the cell wall is afforded by such culture methods. In another aspect, the use of neutral pH for some antigens allows for release of the di-sulfide bonded antigen by treatment with dithiothreitol (DTT) that is not possible when such an antigen-expressing yeast is cultured in media at lower pH (e.g., pH 5). Finally, in another aspect, yeast cultured using the neutral pH methodologies, elicit increased production of at least THl-type cytokines including, but not limited to, IFN-g, interleukin- 12 (IL-12), and IL-2, and may also elicit increased production of other cytokines, such as proinflammatory cytokines (e.g., IL-6).

[0095] In one embodiment, control of the amount of yeast glycosylation is used to control the expression of antigens by the yeast, particularly on the surface. The amount of yeast glycosylation can affect the immunogenicity and antigenicity of the antigen expressed on the surface, since sugar moieties tend to be bulky. As such, the existence of sugar moieties on the surface of yeast and its impact on the three-dimensional space around the target antigen(s) should be considered in the modulation of yeast according to the invention. Any method can be used to reduce the amount of glycosylation of the yeast (or increase it, if desired). For example, one could use a yeast mutant strain that has been selected to have low glycosylation (e.g. mnnl, ochl and mnn9 mutants), or one could eliminate by mutation the glycosylation acceptor sequences on the target antigen. Alternatively, one could use a yeast with abbreviated glycosylation patterns, e.g. Pichia. One can also treat the yeast using methods that reduce or alter the glycosylation.

[0096] In one embodiment, as an alternative to expression of an antigen or other protein recombinantly in the yeast vehicle, a yeast vehicle is loaded intracellularly with the protein or peptide, or with carbohydrates or other molecules that serve as an antigen and/or are useful as immunomodulatory agents or biological response modifiers according to the invention. Subsequently, the yeast vehicle, which now contains the antigen and/or other proteins intracellularly, can be administered to the patient or loaded into a carrier such as a dendritic cell. Peptides and proteins can be inserted directly into yeast vehicles of the present invention by techniques known to those skilled in the art, such as by diffusion, active transport, liposome fusion, electroporation, phagocytosis, freeze-thaw cycles and bath sonication. Yeast vehicles that can be directly loaded with peptides, proteins, carbohydrates, or other molecules include intact yeast, as well as spheroplasts, ghosts or cytoplasts, which can be loaded with antigens and other agents after production. Alternatively, intact yeast can be loaded with the antigen and/or agent, and then spheroplasts, ghosts, cytoplasts, or subcellular particles can be prepared therefrom. Any number of antigens and/or other agents can be loaded into a yeast vehicle in this embodiment, from at least 1, 2, 3, 4 or any whole integer up to hundreds or thousands of antigens and/or other agents, such as would be provided by the loading of a microorganism, by the loading of a mammalian tumor cell, or portions thereof, for example. As one example, an interleukin-6 (IL-6) and/or interleukin 1 beta (ilL-lb or IL-lb)-depleting single chain variable fragment (scFv) antibody or tandem Sc-FV can be expressed inside the yeast prior to preparing a yeast lysate vaccine as disclosed herein. The antibodies would thus be part of the vaccine and would be expected to partially or fully deplete.

[0097] IL-6 or IL-lb in the local tissue environment produced by resident and infiltrating immune cells. The antibodies are predicted to minimize Thl7 polarization and may be particularly beneficial for SARS-CoV-2 infection since Thl7 responses are known to contribute to immunopathology in severe COVID-19 (Wu et al J Microbiol Immunol Infect. 2020 Jun; 53(3): 368-370).

[0098] Along this line of immune modulation, the administration of Type 1 interferon (e.g., Interferon alpha or beta) by expression inside the yeast prior to lysate production (or co administered) or co-administered with vaccine may have a pronounced benefit for COVID-19 patients as it may work against the TIIFN-defeating mechanisms possessed by SARS-CoV-2. Restoring TIIFN activity in this fashion may recapitulate the beneficial effects for COVID patients that were observed in recent clinical trials (e.g., Scientific Reports volume 11, Article number: 8059 (2021)). Further, by delivering the TIIFN with a vaccine before or soon after infection in asymptomatic or mildly symptomatic patients rather than as a therapeutic for severely infected subjects, the benefit may be better realized because SARS-CoV-2 titers are low or absent and the immunopathologic environment has not yet been established. In this setting TIIFN: i) shuts down the Thl7 pathway (Martinez, G.J., et al. Ann NY Acad Sci. 2008 1143:188-211) alleviating the negative effects of Thl7 polarization on lung inflammation, and; ii) promotes better immunity against SARS-CoV-2 through the action of antiviral effector molecules encoded by IFN-stimulated genes (McNab, F., et al. Nature Reviews Immunology 2015; 15:87-103).

[0099] In another embodiment, an antigen and/or other agent is physically attached to the yeast vehicle. Physical attachment of the antigen and/or other agent to the yeast vehicle can be accomplished by any method suitable in the art, including covalent and non-covalent association methods which include, but are not limited to, chemically crosslinking the antigen and/or other agent to the outer surface of the yeast vehicle or biologically linking the antigen and/or other agent to the outer surface of the yeast vehicle, such as by using an antibody or other binding partner. Chemical cross-linking can be achieved, for example, by methods including glutaraldehyde linkage, photoaffmity labeling, treatment with carbodiimides, treatment with chemicals capable of linking di-sulfide bonds, and treatment with other cross- linking chemicals standard in the art. Alternatively, a chemical can be contacted with the yeast vehicle that alters the charge of the lipid bilayer of yeast membrane or the composition of the cell wall so that the outer surface of the yeast is more likely to fuse or bind to antigens and/or other agent having particular charge characteristics. Targeting agents such as antibodies, binding peptides, soluble receptors, and other ligands may also be incorporated into an antigen as a fusion protein or otherwise associated with an antigen for binding of the antigen to the yeast vehicle.

[00100] When the antigen or other protein is expressed on or physically attached to the surface of the yeast, spacer arms may, in one aspect, be carefully selected to optimize antigen or other protein expression or content on the surface. The size of the spacer arm(s) can affect how much of the antigen or other protein is exposed for binding on the surface of the yeast. Thus, depending on which antigen(s) or other protein(s) are being used, one of skill in the art will select a spacer arm that effectuates appropriate spacing for the antigen or other protein on the yeast surface. In one embodiment, the spacer arm is a yeast protein of at least 450 amino acids. Spacer arms have been discussed in detail above.

[00101] Another consideration for optimizing antigen surface expression is whether the antigen and spacer arm combination should be expressed as a monomer or as dimer or as a trimer, or even more units connected together. This use of monomers, dimers, trimers, etc. allows for appropriate spacing or folding of the antigen such that some part, if not all, of the antigen is displayed on the surface of the yeast vehicle in a manner that makes it more immunogenic.

[00102] In yet another embodiment, the yeast vehicle and the antigen or other protein are associated with each other by a more passive, non-specific or non-covalent binding mechanism, such as by gently mixing the yeast vehicle and the antigen or other protein together in a buffer or other suitable formulation (e.g., admixture).

[00103] In one embodiment, the yeast vehicle and the antigen or other protein are both loaded intracellularly into a carrier such as a dendritic cell or macrophage to form the therapeutic composition or vaccine of the present invention. Alternatively, an antigen or other protein can be loaded into a dendritic cell in the absence of the yeast vehicle.

[00104] In one embodiment, yeast vehicles useful in the invention include yeast vehicles that have been killed or inactivated. Killing or inactivating of yeast can be accomplished by any of a variety of suitable methods known in the art. For example, heat inactivation of yeast is a standard way of inactivating yeast, and one of skill in the art can monitor the structural changes of the target antigen, if desired, by standard methods known in the art. Making yeast lysates as described herein in another way of inactivating the yeast. Alternatively, other methods of inactivating the yeast can be used, such as chemical, electrical, radioactive or UV methods. See, for example, the methodology disclosed in standard yeast culturing textbooks such as Methods of Enzymology, Vol. 194, Cold Spring Harbor Publishing (1990). Any of the inactivation strategies used should take the secondary, tertiary or quaternary structure of the target antigen into consideration and preserve such structure as to optimize its immunogenicity. [00105] Yeast lysate and yeast vehicles can be formulated into yeast-based immunotherapy compositions or products of the present invention, including preparations to be administered to a subject directly or first loaded into a carrier such as a dendritic cell, using a number of techniques known to those skilled in the art. For example, yeast vehicles can be dried by lyophilization. Formulations comprising yeast vehicles can also be prepared by packing yeast in a cake or a tablet, such as is done for yeast used in baking or brewing operations. In one aspect, the yeast lysates and yeast vehicles can be frozen. In addition, yeast vehicles can be mixed with a pharmaceutically acceptable excipient, such as an isotonic buffer that is tolerated by a host or host cell. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include suspensions containing viscosity-enhancing agents, such as sodium carboxymethylcellulose, sorbitol, glycerol or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol. Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise, for example, dextrose, human serum albumin, and/or preservatives to which sterile water or saline can be added prior to administration.

[00106] In one embodiment of the present invention, a composition can include biological response modifier compounds, or the ability to produce such modifiers (i.e., by transfection of the yeast vehicle with nucleic acid molecules encoding such modifiers. Biological response modifiers have been described above.

[00107] Compositions of the invention can further include any other compounds that are useful for protecting a subject from a particular disease or condition, including SAR-CoV-2, or any compounds that treat or ameliorate any symptom of such an infection.

[00108] The invention also includes a kit comprising any of the compositions described herein, or any of the individual components of the compositions described herein. Reagents may be present in free form or immobilized to a substrate such as a plastic dish, microarray plate, a test tube, a test rod and so on. The kit can also include suitable reagents for the detection of the reagent and/or for the labeling of positive or negative controls, wash solutions, dilution buffers and the like. The kit can also include a set of written instructions for using the kit and interpreting the results. In one embodiment, the kit is formulated to be a high-throughput assay. Kits may be prepared and used for any clinical, research or diagnostic method of the invention. [00109] With respect to agents useful in the invention, a protein or antibody is administered, in one aspect, in an amount that is between about 50 U/kg and about 15,000 U/kg body weight of the subject. In another embodiment, a protein or antibody is administered in an amount that is between about 0.01 pg and about 10 mg per kg body weight of the patient, and more preferably, between about 0.1 pg and about 100 pg per kg body weight of the patient. When the compound to be delivered is a nucleic acid molecule, an appropriate single dose results in at least about 1 pg of protein expressed per mg of total tissue protein per pg of nucleic acid delivered. Small molecules are delivered according to the preferred dosage specified for the given small molecule and can be determined by those of skill in the art.

[00110] In one aspect of the invention, an agent is administered concurrently with the yeast- based immunotherapy composition. In one aspect of the invention, an agent is administered sequentially with the yeast-based immunotherapy composition. In another embodiment, an agent is administered before the yeast-based immunotherapy composition is administered. In another embodiment, an agent is administered after the yeast-based immunotherapy composition is administered. In one embodiment, an agent is administered in alternating doses with the yeast-based immunotherapy composition, or in a protocol in which the yeast-based composition is administered at prescribed intervals in between or with one or more consecutive doses of an agent, or vice versa. In one embodiment, the yeast-based immunotherapy composition is administered in one or more doses over a period of time prior to commencing the administration of an agent. In other words, the yeast-based immunotherapeutic composition is administered as a monotherapy for a period of time, and then the agent administration is added, either concurrently with new doses of yeast-based immunotherapy, or in an alternating fashion with yeast-based immunotherapy. Alternatively, an agent may be administered for a period of time prior to beginning administration of the yeast-based immunotherapy composition. In one aspect, the yeast is engineered to express or carry an agent, or a different yeast is engineered or produced to express or carry an agent.

[00111] A virus-based immunotherapy composition typically comprises a viral vector comprising a virus genome or portions thereof ( e.g ., a recombinant virus) and a nucleic acid sequence encoding at least one antigen(s) from a disease-causing agent or disease state (e.g., a cancer antigen(s), infectious disease antigen(s), and/or at least one immunogenic domain thereof). In some embodiments, a virus-based immunotherapy composition further includes at least one viral vector comprising one or more nucleic acid sequences encoding one or more immunostimulatory molecule(s). In some embodiments, the genes encoding immunostimulatory molecules and antigens are inserted into the same viral vector (the same recombinant virus). The virus-based immunotherapy composition can comprise a recombinant adenoviral 5 virus (Ad5) adenovirus. An example is an E2b deleted adenovirus vector, such as those described in US 6,063,622; US 6,451,596; US 6,057,158: and US 6,083,750, may be used in the practice of the methods and compositions disclosed herein.

[00112] As used herein with respect to administration of a composition, the term “concurrently” means to administer each of the compositions and particularly, the first dose of such compositions, essentially at the same time or within the same dosing period, or within a time period during which the initial effects of priming of the immune system by the immunotherapy composition occurs (e.g., within 1-2 days or less). For clarity, concurrent administration does not require administration of all of the compositions at precisely the same moment, but rather, the administration of all compositions should occur within one scheduled dosing of the patient in order to prime the immune system and achieve the effect of the agent concurrently (e.g., one composition may be administered first, followed immediately or closely by the administration of the second composition, and so on). In some circumstances, such as when the compositions are administered to the same site, the compositions may be provided in admixture, although even when administered at the same site, sequential administration of each composition during the same dosing period may be used. In one aspect, the compositions are administered within the same 1-2 days, and in another aspect on the same day, and in another aspect within the same 12 hour period, and in another aspect within the same 8 hour period, and in another aspect within the same 4 hour period, and in another aspect within the same 1, 2 or 3 hour period, and in another aspect, within the same 1, 2, 3, 4, 6, 7, 8, 9, or 10 minutes. [00113] In one embodiment of the invention, the yeast-based immunotherapy composition and the agent(s) are administered concurrently, but to different physical sites in the patient. For example, one composition or agent can be administered to one or more sites of the individual’s body and the other composition or agent can be administered to one or more different sites of the individual’s body, e.g., on different sides of the body or near different draining lymph nodes. In another embodiment, the immunotherapy composition and the agent are administered concurrently and to the same or substantially adjacent sites in the patient. A substantially adjacent site is a site that is not precisely the same injection site to which the first composition or agent is administered, but that is in close proximity (is next to) the first inj ection site. In one embodiment, the immunotherapy composition and agent are administered in admixture. Some embodiments may include combinations of administration approaches. [00114] In the method of the present invention, compositions and therapeutic compositions can be administered to animal, including any vertebrate, and particularly to any member of the Vertebrate class, Mammalia, including, without limitation, primates, rodents, livestock and domestic pets. Livestock include mammals to be consumed or that produce useful products (e.g., sheep for wool production). Mammals to protect include humans, dogs, cats, mice, rats, goats, sheep, cattle, horses and pigs.

[00115] An “individual” is a vertebrate, such as a mammal, including without limitation a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, mice and rats. The term “individual” can be used interchangeably with the term “animal”, “subject” or “patient”.

[00116] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as, Methods of Enzymology, Vol. 194, Guthrie et ah, eds., Cold Spring Harbor Laboratory Press (1990); Biology and activities of yeasts, Skinner, et ah, eds., Academic Press (1980); Methods in yeast genetics : a laboratory course manual, Rose et ah, Cold Spring Harbor Laboratory Press (1990); The Yeast Saccharomyces: Cell Cycle and Cell Biology, Pringle et al., eds., Cold Spring Harbor Laboratory Press (1997); The Yeast Saccharomyces: Gene Expression, Jones et al., eds., Cold Spring Harbor Laboratory Press (1993); The Yeast Saccharomyces: Genome Dynamics, Protein Synthesis, and Energetics, Broach et al., eds., Cold Spring Harbor Laboratory Press (1992); Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) and Molecular Cloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001), (_jointly referred to herein as “Sambrook”); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987, including supplements through 2001); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York; Harlow and Lane (1999) Using Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (jointly referred to herein as “Harlow and Lane”), Beaucage et al. eds., Current Protocols in Nucleic Acid Chemistry John Wiley & Sons, Inc., New York, 2000); Casarett and DoulTs Toxicology The Basic Science of Poisons, C. Klaassen, ed., 6th edition (2001), and Vaccines, S. Plotkin and W. Orenstein, eds., 3rd edition (1999). General Definitions

[00117] A “TARMOGEN^®” (Globelmmune, Inc., Louisville, Colorado) generally refers to a yeast vehicle expressing one or more heterologous antigens extracellularly (on its surface), intracellularly (internally or cytosolically) or both extracellularly and intracellularly. TARMOGEN^®s have been generally described (see, e.g., U.S. Patent No. 5,830,463). Certain yeast-based immunotherapy compositions, and methods of making and generally using the same, are also described in detail, for example, in U.S. Patent No. 5,830,463, U.S. Patent No. 7,083,787, U.S. Patent No. 7,736,642, Stubbs et al., Nat. Med. 7:625-629 (2001), Lu et al., Cancer Research 64:5084-5088 (2004), and in Bernstein et al., Vaccine 2008 Jan 24;26(4):509- 21, each of which is incorporated herein by reference in its entirety.

[00118] As used herein, the term "analog" refers to a chemical compound that is structurally similar to another compound but differs slightly in composition (as in the replacement of one atom by an atom of a different element or in the presence of a particular functional group, or the replacement of one functional group by another functional group). Thus, an analog is a compound that is similar or comparable in function and appearance but has a different structure or origin with respect to the reference compound.

[00119] The terms "substituted", "substituted derivative" and "derivative", when used to describe a compound, means that at least one hydrogen bound to the unsubstituted compound is replaced with a different atom or a chemical moiety. [00120] A “cell-mediated” immune response (which may be used interchangeably anywhere herein with the term "cellular" immune response) refers generally to the response to an antigen of immune cells including T lymphocytes (including cytotoxic T lymphocytes (CTL)), dendritic cells, macrophages, and natural killer cells, and to all of the processes that accompany such responses, including, but not limited to, activation and proliferation of these cells, CTL effector functions, cytokine production that influences the function of other cells involved in adaptive immune responses and innate immune responses, memory T cell generation, and stem cell-like memory cells .

[00121] “Vaccination” or “immunization” refers to the elicitation (induction) of an immune response against an antigen or immunogenic portion thereof, as a result of administration of the antigen, alone or together with an adjuvant. Vaccination results in a protective or therapeutic effect, wherein subsequent exposure to the antigen (or a source of the antigen) elicits an immune response against the antigen (or source) that reduces or prevents a disease or condition in the animal. The concept of vaccination is well known in the art. The immune response that is elicited by administration of an immunotherapeutic composition (vaccine) can be any detectable change in any facet of the immune response (e.g., cell-mediated response, humoral response, cytokine production), as compared to in the absence of the administration of the composition.

[00122] According to the present invention, “heterologous amino acids” are a sequence of amino acids that are not naturally found (i.e., not found in nature, in vivo) flanking the specified amino acid sequence, or that are not related to the function of the specified amino acid sequence, or that would not be encoded by the nucleotides that flank the naturally occurring nucleic acid sequence encoding the specified amino acid sequence as it occurs in the gene, if such nucleotides in the naturally occurring sequence were translated using standard codon usage for the organism from which the given amino acid sequence is derived. Therefore, at least two amino acid residues that are heterologous to the antigen are any two amino acid residues that are not naturally found flanking the antigen.

[00123] According to the present invention, reference to a "heterologous" protein or "heterologous" antigen, including a heterologous fusion protein, in connection with a yeast vehicle of the invention means that the protein or antigen is not a protein or antigen that is naturally expressed by the yeast, although a fusion protein may include yeast sequences or proteins or portions thereof that are naturally expressed by yeast (e.g., an Aga protein as described herein). For example, a fusion protein of an influenza hemagglutinin protein and a yeast Aga protein is considered to be a heterologous protein with respect to the yeast vehicle for the purposes of the present invention, since such a fusion protein is not naturally expressed by a yeast.

[00124] According to the present invention, the phrase "selectively binds to" refers to the ability of an antibody, antigen-binding fragment or binding partner of the present invention to preferentially bind to specified proteins. More specifically, the phrase "selectively binds" refers to the specific binding of one protein to another (e.g., an antibody, fragment thereof, or binding partner to an antigen), wherein the level of binding, as measured by any standard assay (e.g., an immunoassay), is statistically significantly higher than the background control for the assay. For example, when performing an immunoassay, controls typically include a reaction well/tube that contain antibody or antigen binding fragment alone (i.e., in the absence of antigen), wherein an amount of reactivity (e.g., non-specific binding to the well) by the antibody or antigen-binding fragment thereof in the absence of the antigen is considered to be background. Binding can be measured using a variety of methods standard in the art including enzyme immunoassays (e.g., ELISA), immunoblot assays, etc.).

[00125] Reference to a protein or polypeptide in the present invention includes full-length proteins, fusion proteins, or any fragment, domain, conformational epitope, or homologue of such proteins. More specifically, an isolated protein, according to the present invention, is a protein (including a polypeptide or peptide) that has been removed from its natural milieu (i.e., that has been subject to human manipulation) and can include purified proteins, partially purified proteins, recombinantly produced proteins, and synthetically produced proteins, for example. As such, "isolated" does not reflect the extent to which the protein has been purified. In one aspect of the invention, an isolated protein of the present invention is produced recombinantly. According to the present invention, the terms "modification" and "mutation" can be used interchangeably, particularly with regard to modifications/mutations to the amino acid sequence of proteins or portions thereof.

[00126] As used herein, the term "homologue" is used to refer to a protein or peptide which differs from a naturally occurring protein or peptide (i.e., the "prototype" or "wild-type" protein) by minor modifications to the naturally occurring protein or peptide, but which maintains the basic protein and side chain structure of the naturally occurring form. Such changes include, but are not limited to: changes in one or a few amino acid side chains; changes one or a few amino acids, including deletions (e.g., a truncated version of the protein or peptide) insertions and/or substitutions; changes in stereochemistry of one or a few atoms; and/or minor derivatizations, including but not limited to: methylation, glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol. A homologue can have either enhanced, decreased, or substantially similar properties as compared to the naturally occurring protein or peptide. A homologue can include an agonist of a protein or an antagonist of a protein. Homologues can be produced using techniques known in the art for the production of proteins including, but not limited to, direct modifications to the isolated, naturally occurring protein, direct protein synthesis, or modifications to the nucleic acid sequence encoding the protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis. [00127] A homologue of a given protein may comprise, consist essentially of, or consist of, an amino acid sequence that is at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% identical, or at least about 95% identical, or at least about 96% identical, or at least about 97% identical, or at least about 98% identical, or at least about 99% identical (or any percent identity between 45% and 99%, in whole integer increments), to the amino acid sequence of the reference protein. In one embodiment, the homologue comprises, consists essentially of, or consists of, an amino acid sequence that is less than 100% identical, less than about 99% identical, less than about 98% identical, less than about 97% identical, less than about 96% identical, less than about 95% identical, and so on, in increments of 1%, to less than about 70% identical to the naturally occurring amino acid sequence of the reference protein.

[00128] As used herein, unless otherwise specified, reference to a percent (%) identity refers to an evaluation of homology which is performed using: (1) a BLAST 2.0 Basic BLAST homology search using blastp for amino acid searches and blastn for nucleic acid searches with standard default parameters, wherein the query sequence is filtered for low complexity regions by default (described in Altschul, S.F., Madden, T.L., Schaaffer, A.A., Zhang, L, Zhang, Z., Miller, W. & Lipman, D.J. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs." Nucleic Acids Res. 25:3389-3402, incorporated herein by reference in its entirety); (2) a BLAST 2 alignment (using the parameters described below); (3) and/or PSI-BLAST with the standard default parameters (Position-Specific Iterated BLAST. It is noted that due to some differences in the standard parameters between BLAST 2.0 Basic BLAST and BLAST 2, two specific sequences might be recognized as having significant homology using the BLAST 2 program, whereas a search performed in BLAST 2.0 Basic BLAST using one of the sequences as the query sequence may not identify the second sequence in the top matches. In addition, PSI-BLAST provides an automated, easy-to-use version of a "profile" search, which is a sensitive way to look for sequence homologues. The program first performs a gapped BLAST database search. The PSI-BLAST program uses the information from any significant alignments returned to construct a position-specific score matrix, which replaces the query sequence for the next round of database searching. Therefore, it is to be understood that percent identity can be determined by using any one of these programs. [00129] Two specific sequences can be aligned to one another using BLAST 2 sequence as described in Tatusova and Madden, (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250, incorporated herein by reference in its entirety. BLAST 2 sequence alignment is performed in blastp or blastn using the BLAST 2.0 algorithm to perform a Gapped BLAST search (BLAST 2.0) between the two sequences allowing for the introduction of gaps (deletions and insertions) in the resulting alignment. For purposes of clarity herein, a BLAST 2 sequence alignment is performed using the standard default parameters as follows.

[00130] For blastn, using 0 BLOSUM62 matrix:

[00131] Reward for match = 1

[00132] Penalty for mismatch = -2

[00133] Open gap (5) and extension gap (2) penalties

[00134] gap x dropoff (50) expect (10) word size (11) filter (on)

[00135] For blastp, using 0 BLOSUM62 matrix:

[00136] Open gap (11) and extension gap (1) penalties [00137] gap x dropoff (50) expect (10) word size (3) filter (on).

[00138] An isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subject to human manipulation), its natural milieu being the genome or chromosome in which the nucleic acid molecule is found in nature. As such, "isolated" does not necessarily reflect the extent to which the nucleic acid molecule has been purified, but indicates that the molecule does not include an entire genome or an entire chromosome in which the nucleic acid molecule is found in nature. An isolated nucleic acid molecule can include a gene. An isolated nucleic acid molecule that includes a gene is not a fragment of a chromosome that includes such gene, but rather includes the coding region and regulatory regions associated with the gene, but no additional genes that are naturally found on the same chromosome. An isolated nucleic acid molecule can also include a specified nucleic acid sequence flanked by (i.e., at the 5' and/or the 3' end of the sequence) additional nucleic acids that do not normally flank the specified nucleic acid sequence in nature (i.e., heterologous sequences). Isolated nucleic acid molecule can include DNA, RNA (e.g., mRNA), or derivatives of either DNA or RNA (e.g., cDNA). Although the phrase "nucleic acid molecule" primarily refers to the physical nucleic acid molecule and the phrase "nucleic acid sequence" primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a protein or domain of a protein.

[00139] A recombinant nucleic acid molecule is a molecule that can include at least one of any nucleic acid sequence encoding any one or more proteins described herein operatively linked to at least one of any transcription control sequence capable of effectively regulating expression of the nucleic acid molecule(s) in the cell to be transfected. Although the phrase "nucleic acid molecule" primarily refers to the physical nucleic acid molecule and the phrase "nucleic acid sequence" primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a protein. In addition, the phrase "recombinant molecule" primarily refers to a nucleic acid molecule operatively linked to a transcription control sequence, but can be used interchangeably with the phrase "nucleic acid molecule" which is administered to an animal.

[00140] A recombinant nucleic acid molecule includes a recombinant vector, which is any nucleic acid sequence, typically a heterologous sequence, which is operatively linked to the isolated nucleic acid molecule encoding a fusion protein of the present invention, which is capable of enabling recombinant production of the fusion protein, and which is capable of delivering the nucleic acid molecule into a host cell according to the present invention. Such a vector can contain nucleic acid sequences that are not naturally found adjacent to the isolated nucleic acid molecules to be inserted into the vector. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and in one aspect of the present invention, is a virus or a plasmid. Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulating of nucleic acid molecules, and can be used in delivery of such molecules (e.g., as in a DNA vaccine or a viral vector-based vaccine). Recombinant vectors may be used in the expression of nucleic acid molecules, and can also be referred to as expression vectors. Some recombinant vectors are capable of being expressed in a transfected host cell.

[00141] In a recombinant molecule of the present invention, nucleic acid molecules are operatively linked to expression vectors containing regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the host cell and that control the expression of nucleic acid molecules of the present invention. In particular, recombinant molecules of the present invention include nucleic acid molecules that are operatively linked to one or more expression control sequences. The phrase "operatively linked" refers to linking a nucleic acid molecule to an expression control sequence in a manner such that the molecule is expressed when transfected (i.e., transformed, transduced or transfected) into a host cell.

[00142] According to the present invention, the term “transfection” is used to refer to any method by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can be inserted into a cell. The term "transformation" can be used interchangeably with the term "transfection" when such term is used to refer to the introduction of nucleic acid molecules into microbial cells, such as algae, bacteria and yeast. In microbial systems, the term "transformation" is used to describe an inherited change due to the acquisition of exogenous nucleic acids by the microorganism and is essentially synonymous with the term "transfection." Therefore, transfection techniques include, but are not limited to, transformation, chemical treatment of cells, particle bombardment, electroporation, microinjection, lipofection, adsorption, infection and protoplast fusion.

[00143] The following experimental results are provided for purposes of illustration and are not intended to limit the scope of the invention.

EXAMPLES

[00144] Example 1

[00145] This example provides the general methods used for the examples described below. [00146] Intranasal vaccination: Yeast lysates (pL) were passed through a 26Gx3/8 TB needle 7x and filtered using a 70um filter prior to inoculation of mice. Mice are anaesthetized using isofluorane, and a maximum volume of 20uL is dispensed equally between the right (R) and left (L) nostrils.

[00147] Subcutaneous vaccination: Yeast-lysates were passed through a 26Gx3/8 TB needle. Mice are anaesthetized using isofluorane, placed supine, and 50-150uL total volume is injected s.c. split equally between the left and right hip pocket.

[00148] Serum collection and organ harvest and processing: Peripheral blood was obtained by 1 of 2 methods: terminal cardiac puncture or retroorbital (RO) bleed. For terminal cardiac puncture, mice were euthanized using CO2 and immediately used for whole blood collection. Up to 1 mL of whole blood was taken from the heart ventricle using a 25G needle through the left side of the chest. The syringe was withdrawn slowly to prevent the heart collapsing (terminal procedure). For RO blood collection, mice were anaesthetized using isofluorane. Non-heparinized capillary tubes were inserted into the eye cavity and blood collected by droplet into microtainer tubes. [00149] Spleens were macerated in complete RPMI plus 10% FBS and cells were filtered into a 50 mL falcon tube using a 70 pm mesh filter, then centrifuged at 400 x g for 10 minutes. Red blood cells were lysed for 5 minutes in 2 mL Ammonium Chloride-Potassium (ACK) lysing solution per spleen (RT). 15 mL of complete RPMI media with 10% FBS (cRPMI-10) was added to stop the reaction. Cells were centrifuged as above and resuspended in 10 mL PBS. Viable cells were counted by trypan blue staining on a hemocytometer. Each 50 mL Falcon tube contained no more than 100 x 10⁶ cells.

[00150] In vitro angiotensin II converting enzyme (ACE2) binding assay: To evaluate the effect of various lysate processing methods on protein binding, yeast pL were tested as un processed (Fig. 8 far left 2 groups), following passage through a 26Gx3/8 TB needle (Fig. 8 middle 2 groups), or following sonication for 15 seconds (Fig. 8 right 2 groups). Nunc MaxiSorp ELISA plates were coated with lOOuL/well of 0.5ug/mL human ACE2-hFC protein (IB reagent) diluted in lx Coating buffer (BIOLEGEND®) overnight at 4°C. Wells are washed 3x with 200uL/well DPBS and blocked with 2% NF Milk/DPBS (Blocking buffer) for 2 hours at 37°C. Wells are washed 3x 200uL with 0.05% Tween20/DPBS (Wash buffer) and then lOOuL yeast pL sample (undiluted or 1 : 10, 1 : 50, or 1 :250 diluted in Blocking buffer) was added per well and incubated for 1 hr at 37°C. Wells were washed 4x with 200uL/well Wash buffer and lOOuL/well diluted HRP-conjugated secondary antibody (anti-HIS or anti-V5) diluted 1:5000 in Blocking buffer was added perwell an incubated for 1 hr at 37°C. Wells were washed 4x with 200uL/well Wash buffer and lOOuL of TMB substrate (PIERCE®) was added per well and incubated up to 30 min at RT in the dark after which lOOuL of Stop solution (THERMO®) was added. The optical density of the plate was measured on the BIOTEK® SYNERGY™ HTX plate reader at 450 nm within 30 minutes of stopping the reaction.

[00151] Copper induction and Heat inactivation of yeast. Three-hundred nanograms (ng) of CoV-2 expression plasmid were transfected into 50 pL of competent parental yeast W303a or EBY100* ) that were prepared per the Zymo Research frozen EZ yeast II transfection kit protocol. Transfectants were selected on complete yeast media agar plates lacking uracil (UDA) or lacking both uracil and tryptophan UTDA (RBD#4) at 30°C for 3 days. Three single colony isolates of each construct were re-streaked onto ULDA or UTDA (RBD#4) plates to generate pure clones for analysis of growth and antigen expression. Clonal isolates of each yeast strain were inoculated into 50 mL of UL2 or UT2 medium in a 125 mL sterile vented Erlenmeyer flask and the starter cultures were agitated at 250 rpm for 16h (30°C). Sonicated samples of each culture were counted by hemocytometer then used to seed final 1L cultures of UL2 to a density of 0.05 YU/mL. Final cultures were agitated at 250 rpm for 15-18h (30°C) and cell densities were monitored to prevent growth beyond 4 YU/mL. At a cell density of between 2 and 4 YU/mL, copper sulfate was added to 0.375 mM final concentration to cultures harboring the CUP1 promoter and shaking was continued for an additional 3 hours. All cultures were harvested by centrifugation at 2600 x g for 7 minutes at 20°C. Supernatants were discarded and pellets were resuspended by vortexing. Cells were washed IX with 500 mL of PBS per liter of original culture and again centrifuged as above. Cells were heat-inactivated in pre-heated PBS at 20 to 50 YU/mL final for lh at 56°C, then thrice washed with PBS as above, and the cells were again counted by hemocytometer.

[00152] *Strain information: EBY100 is MAT a, ura3-52, trpl, Ieu2-delta200, his3- delta200, pep4HIS3, prbdl.6R, canl, GAL. The strain has a genomic insertion of AGA1 regulated by a GAL promoter with a URA3 selectable marker.

[00153] Pressure homogenization of yeast cells: Yeast cells were homogenized by passing the cells 20 times through a laboratory homogenizer set to 1500 bar and 4 degrees C. Lysates were stored frozen in single use aliquots at -20 degrees C..

[00154] ELISA: NUNC MAXISORP™ ELISA 96 well plates were coated with 0.5 pg/mL of recombinant Spike protein SI domain (Creative BioMart Cat # 191V), recombinant sheep Fc-Sl domain fusion protein (Native antigen company cat # REC31806), recombinant active, trimerized, His-tagged S protein (R683A, R685A, AcroBiosystems cat # SPN-C52H8), recombinant Spike RBD domain (AcroBiosystems cat # A010-214), or recombinant His- tagged N protein (SinoBiologicals cat # 40588-V08B) that were expressed and purified from human cells or insect cells. The coating buffer (BioLegend Cat # 421701) containing the antigens was incubated on the plate overnight at 4°C in a volume of 100 pL per well. The coating solution was removed and the plate was washed with PBS. Wells were blocked with block 2% non-fat dry milk in PBS (blocking buffer, BB) for 2h at 37°C. Plates were washed with 0.05% Tween20 in PBS (Wash buffer, WB). lOOpL of serum or controls diluted in BB were added to each well and the plate was incubated for lh at 37°C. The plate was washed with WB, then 100 pL of 0.16pg/mL of HRP-conjugated goat-anti-mouse total IgG antibody (Jackson ImmunoRe search Cat # 115-035-003) diluted in blocking buffer was added per well and then incubated for lh at 37°C. For Ig-subtyping assays, HRP-conjugated anti-mouse Ig- subclass specific conjugates were used at a 1/5000 dilution in BB. Following WB washes, 100 pL of TMB substrate (3,3',5,5'-tetramethylbenzidine; Pierce cat # 34021) was added per well and incubated up to 30 minutes at RT. The reaction was stopped by adding an equal volume (lOOpL) of stop solution (Thermo Stop solution, #N600). The optical density of the plate was measured on the BIOTEK® SYNERGY™ HTX plate reader at 450 nm within 30 minutes of stopping the reaction.

[00155] ELISpot: At the indicated day post-vaccination, spleens and lungs were harvested, macerated, and red blood cells were lysed by treatment with ACK and washed in cRPMI-10. 200,000 splenocytes or lung-resident cells per well were stimulated with peptide pools (~luM each peptide) for 48 hours with the indicated peptide pool (JPT, ~luM each peptide) and IFNg production assessed using the R&D systems Mouse IFNg ELISpot plate (Cat # EL485) according to kit instructions.

[00156] ICS: At the indicated day post-vaccination, spleens were harvested and red blood cells were lysed by treatment with ACK and washed in cRPMI-10. Splenocytes were stimulated with peptide pools (JPT, ~1mM each peptide) and Brefeldin A for a maximum of 6 hours at 37°C, then stored at 4°C overnight. Cells were then stained for surface expression for CD3, CD4, and CD8 followed by intracellular cytokine staining for IFNy and TNFa using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience). Flow cytometry was performed using the BD FACSVERSE, with 2xl0⁶ singlets collected per sample. Flow cytometry plots are shown for IFNy and TNFa expression within CD3⁺CD8⁺ or CD3⁺CD44⁺CD4⁺ T cells. [00157] In Vivo CTL: On day 7 post-immunization, splenocytes from naive syngeneic mice were harvested and prepared as targets for killing as follows. Spleens were macerated using a 70 mM mesh filter and red blood cells were lysed by treatment with ACK and washed in cRPMI-10. PBS washed splenocytes were labeled with PKH26 dye and divided equally into two samples. One sample was stained with high concentration (1 pM) CFSE and then pulsed with 100 pM cognate peptides; the other sample was stained with low concentration (0.1 pM) CFSE and mock pulsed with medium only. Both CFSE high and low samples were washed and counted and mixed together at lxlO⁶ cells each. The mixed target cells were inject into mice by intravenously or by retro-orbitally using a U-100 insulin syringe, 28G1/2 (0.36 mm x 13 mm). 18-20 hours after target transfer, mice were euthanized and splenocytes were isolated for flow cytometry analysis by analyzing the percent of CFSE high and CFSE low population from PKH positive cells. The CTL killing was calculated as in formula 1 :

[00158] Example 2

[00159] This example shows that vaccination of mice with a combination of full length SARS-CoV-2 Spike protein (S1-S2) with SARS-CoV-2 Nucleoprotein (N) as well as the combination of the SARS-CoV-2 Spike SI subunit (SI) with SARS-CoV-2 N, induced production of SARS-CoV-2 Spike-specific IgG in serum of mice vaccinated 2-3 times. Vaccination with the SARS-CoV-2 Spike proteins (S1-S2) in the absence of the SARS-CoV-2 N protein did not result in production of SARS-CoV-2 Spike-specific antibodies, indicating collaboration between the SARS-CoV-2 Spike and N protein in driving a SARS-CoV-2 Spike- specific IgG response. Furthermore, vaccination with 5 Y.U. of yeast -lysate expressing full- length SARS-CoV-2 Spike (S1-S2) or 10 Y.U. of yeast -lysate expressing only the SARS- CoV-2 Spike SI subunit induced elevated frequencies of Spike SI subunit-specific CD8 T cells co-producing IFNg and TNFa relative to mice vaccinated with negative control W303a yeast lysate. See Figs. 2A-2F and Fig. 3.

[00160] Example 3

[00161] This example shows that vaccination of mice with COVID S1-S2 yeast lysate produced in vivo cytotoxic killing against cellular targets coated with Spike peptides, while vaccination with null yeast (strain W303) did not (see Figs. 5A-5C). Interestingly, prime-boost immunization with S1-S2 lysate did not generate greater killing efficiency than single immunization. This could due to overloading with yeast protein at the relatively high dose used (80 YU) or that the timing of spleen harvest needs further optimization. These results are particularly meaningful because they demonstrate in vivo CTL-mediated killing of spike- peptide-displaying targets.

[00162] Example 4

[00163] This example shows that vaccination of mice with increasing doses of Aga2-RBD yeast lysate induced dose-dependent SARS-CoV-2 Spike-specific IgG in serum and also elicited a neutralizing antibody response by CPASS™ assay. The results indicate that the Aga2- RBD antigen structure is relevant, and in the presence of the yeast lysate adjuvant can elicit antibodies that should protect against SARS-CoV-2 infection. See Figs. 6 and 7A-7B.

[00164] Example 5

[00165] This example shows that both the N-RBD and Aga2-RBD (RBD#4) recombinant protein products expressed in yeast lysatebind to human ACE2 in vitro , with the N-RBD fusion protein binding ACE2 to a significantly higher extent. Among the various yeast lysate processing methods, needle passage does not significantly impact ACE2 binding capacity of the recombinant proteins, but sonication appears to increase the binding of the N-RBD protein in yeastlystate. This may be due to the effect of sonication on protein solubility or availability (through further break-up of organelle or large protein/lipid complexes) within the yeast lysate. See Fig. 8. [00166] Example 6

[00167] This example shows that intranasal (i.n.) vaccination (see Fig. 9) with yeast pL expressing N-RBD results in successful activation of IFNg production by Nucleoprotein- specific T cells in both the spleen (see Fig. 10A) and the lung (see Fig. 10B) of multiple mice. Interestingly, subcutaneous vaccination of the same N-RBD yeastlysate, even at over 3 times higher concentration, failed to induce antigen-specific T cell IFNg production. Furthermore, i.n. vaccination with N-RBD yeast lysate also induced elevated, although not significant, levels of Spike SI -specific T cell production of IFNg in the lung. These data demonstrate that i.n. deliver of N-RBD yeast lysate is more robust and is capable of eliciting both a local (lung) and systemic (spleen) antigen-specific Thl responses.

[00168] Example 7

[00169] This example shows that intranasal vaccination (see Fig. 11) with N-RBD yeast lysate successfully elicited production of anti-Nucleoprotein IgG in the serum and lung of vaccinated mice. There was a dose-dependent response from 0.5 to 2 Y.U. N-RBD yeast lysate in the lung for total IgG, IgGl, and IgG2a, and a similar trend in the serum with the exception that responses increased from 0.5 to 2 Y.U. but then decreased again in mice that received a 6YU dose. Interestingly, i.n. vaccination with all N-RBD yeast lysate doses elicited N-specific IgGl antibodies, but anti-N IgG2a was only present in the serum of mice that received the two highest N-RBD yeast lysate doses (2 and 6 YU). None of the vaccines resulted in production of Spike-neutralizing antibodies that target the RBD domain as detected using the GENSCRIPT® CPASS™ assay. With regards to cellular immunity, all three doses of N-RBD yeast lysate elicited N-specific T cell production of IFNg in the spleen and lung as detected by ELISpot. There was no significant difference in the number of N-specific IFNg-producing T cells in the spleen or lung between the mice that received different doses of N-RBD yeast lysate. These results indicate that i.n. vaccination with yeast lysate results in a dose-dependent antigen- specific response in the serum and antigen-specific Thl T cell responses in both the lung and spleen. (See Fig.s 12A-12C, 13A-13B and 14A-14B.

[00170] Example 8

[00171] This example shows that subcutaneous prime boost of C57B1/6 mice with N lysate elicits CD8 T cells that can be specifically re-activated in vitro with N peptides. This result combined with the product's ability to enhance S specific antibody responses and to generate N specific IgG are consistent with favorable profile for inclusion in a S+N vaccine against SARS-CoV-2 (see Fig. 15A-15B). [00172] While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in the following claims.

Claims (27)

What is claimed is:

1. A yeast immunotherapeutic composition comprising: a) a yeast vehicle or a yeast lysate prepared from a yeast, wherein the lysate lacks yeast membranes and yeast cell wall; and b) at least one viral antigen, wherein the antigen is: i) expressed by the yeast vehicle, ii) expressed by the yeast and retained in the yeast lysate, or iii) added to the yeast vehicle or yeast lysate; wherein the at least one viral antigen is a SARS-CoV-2 antigen.

2. The immunotherapeutic composition of claim 1, wherein the SARS-CoV-2 antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:2,

SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO:8 or combinations thereof

3. The immunotherapeutic composition of claim 1, wherein the SARS-CoV-2 antigen is a fusion protein.

4. The immunotherapeutic composition of claim 3, wherein the fusion protein has an amino acid sequence that is at least 95% identical to SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16.

5. The immunotherapeutic composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable excipient suitable for administration to a human.

6. The immunotherapeutic composition of claim 1, wherein the yeast vehicle or yeast lysate prepared from a yeast is from a genus selected from the group consisting of: Saccharomyces, Candida , Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces and Yarrowia.

7. The immunotherapeutic composition of claim 6, where the yeast vehicle or yeast lysate prepared from a yeast is from Saccharomyces.

8. The immunotherapeutic composition of claim 7, wherein the yeast vehicle or yeast lysate prepared from a yeast is from Saccharomyces cerevisiae.

9. The immunotherapeutic composition of claim 1, wherein the yeast vehicle is a whole yeast.

10. The immunotherapeutic composition of claim 9, wherein the whole yeast is heat- inactivated.

11. A method to stimulate an immune response to SARS-CoV-2 in an individual comprising administering to the individual a yeast immunotherapeutic composition comprising: a) a yeast vehicle or a yeast lysate prepared from a yeast, wherein the lysate lacks yeast membranes and yeast cell wall; and b) at least one viral antigen, wherein the antigen is: i) expressed by the yeast vehicle, ii) expressed by the yeast and retained in the yeast lysate, or iii) added to the yeast vehicle or yeast lysate; wherein the at least one viral antigen is a SARS-CoV-2 antigen; and c) a pharmaceutically acceptable excipient suitable for administration to the individual.

12. The method of claim 11, wherein the composition is formulated in a pharmaceutically acceptable excipient suitable for administration to the individual by an administration route selected from the group consisting of injection, intranasal, inhalation, oral, and combinations thereof.

13. The method of claim 11, wherein the individual is administered the immunotherapeutic composition in a dose from about 0.1 Y.U. to about 100 Y.U.

14. The method of claim 11, further comprising administering to the individual at least one additional dose of the immunotherapeutic composition.

15. The method of claim 14, wherein the additional dose of the immunotherapeutic composition is administered to the individual from 10 days to 52 days after the initial administration of the immunotherapeutic composition.

16. The method of claim 11, wherein the SARS-CoV-2 antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, or combinations thereof.

17. The method of claim 16, wherein the SARS-CoV-2 antigen is a fusion protein.

18. The method of claim 17, wherein the fusion protein has an amino acid sequence that is at least 95% identical to SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16.

19. The method of claim 11, wherein the yeast vehicle or yeast lysate prepare from a yeast is from a genus selected from the group consisting of: Saccharomyces,

Candida , Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces and Yarrowia.

20. The method of claim 19, wherein the yeast vehicle or yeast lysate prepared from a yeast is from Saccharomyce s.

21. The method of claim 20, wherein the yeast vehicle or yeast lysate prepared from a yeast is from Saccharomyces cerevisiae.

22. The method of claim 11, wherein the yeast vehicle is a whole yeast.

23. The method of claim 22, wherein the whole yeast is heat-inactivated.

24. Use of an immunotherapeutic composition comprising a yeast vehicle or yeast lysate prepared from a yeast and a SARS-CoV-2 antigen comprising at least one SARS-CoV- 2 antigen to stimulate an immune response to SARS-CoV-2.

25. The use of claim 24 wherein the SARS-CoV-2 antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, or combinations thereof.

26. The use of claim 24 wherein the SARS-CoV-2 antigen is a fusion protein.

27. The use of claim 26, wherein the fusion protein has an amino acid sequence that is at least 95% identical to SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO:16.