A versatile virus-like vesicle (VLV) platform for infectious disease and cancer immunotherapy applications

Abstract

The present invention provides compositions and methods for therapeutic immunity for the treatment of infectious diseases and / or cancer. The methods of the present invention include methods for producing high titer infectious agent and cancer vectors, methods for treating and / or preventing infection by cancer or infectious diseases, and methods for inducing memory T and B cell immune responses against infectious agents and cancer in subjects administered the VLV compositions produced thereby. Additionally, the present invention includes pharmaceutical compositions for vaccinating a subject to protect the subject against cancer or infectious agents.

Description

[Technical field]

[0001] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Grant No. 2R44 DK113858 awarded by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and Grant No. 1R43AI149798-01A1 awarded by the National Institute of Allergy and Infectious Diseases (NIAID), each of which is an institute within the National Institutes of Health (NIH). The U.S. Government has certain rights in this invention.

[0002] (Sequence Listing Statement) The Sequence Listing associated with this application has been provided electronically in .xml format and is hereby incorporated by reference in its entirety in a file named 25133-100410_sl.xml, created on Feb. 21, 2023, and which is approximately 83 kB in size.

[0003] (CROSS-REFERENCE TO RELATED APPLICATIONS) This international patent application claims priority to U.S. Provisional Patent Application No. 63 / 312,816, filed February 22, 2022, which is incorporated by reference in its entirety.

[0004] (Technical field) The present invention relates to the development of compositions and methods for therapeutic platforms for immunity to treat infectious diseases and cancer. The methods of the present invention include methods for producing high titer hybrid VLV vectors, methods for treating and / or preventing infectious diseases and cancer, and methods for inducing memory T and B cell immune responses against infectious agents or cancer in subjects administered the VLV compositions produced thereby. Furthermore, the present invention includes pharmaceutical compositions for vaccinating a subject to protect the subject against infection by infectious diseases or cancer. [Background technology]

[0005] (background) Vaccination is one of the greatest public health success stories. Today, vaccines that protect against many of the viruses that once caused fatal childhood diseases are routinely used worldwide. However, traditional vaccine development methods that use inactivation or attenuation of viruses have failed for some of the most deadly human pathogens, necessitating novel approaches. Advances in molecular virology have enabled genetic engineering of viruses, which has provided new opportunities for vaccine development. Genetic engineering of viruses not only allows for their attenuation, but also allows for the incorporation of sequences from other viruses, turning one pathogen into a vaccine carrier for another. Viral vectors have been investigated as a possible tool to deliver vaccines. This is because viral vectors present an advantage over traditional vaccines in that they stimulate a broad range of immune responses, including antibody (B cells), T helper cells (CD4+ T cells), and cytotoxic T lymphocyte (CTL, CD8+ T cells)-mediated immunity. These viral vector vaccines can be used against a variety of infectious and malignant diseases. However, there are limitations to the use of viral vector-based vaccines.For example, pre-existing anti-vector immunity to the viral vector, which may inactivate the vaccine, poses a problem, as does the limited cloning ability of the transgene of interest.A number of vaccine studies are underway to improve the efficiency of this technology and overcome its limitations.

[0006] Enveloped RNA viruses have a highly organized structure: one or more nucleocapsid proteins encapsidate the RNA, a matrix protein is often present between the capsid and the membrane, and one or more transmembrane glycoproteins interact with the matrix protein or nucleocapsid protein to direct efficient particle assembly. Once the particles are released from the cell, one or more glycoproteins bind to cellular receptors and catalyze membrane fusion, allowing the virus to enter new cells.

[0007] Alphaviruses, such as Semliki Forest virus (SFV), are positive-stranded membrane-enveloped RNA viruses that encode four nonstructural proteins (called nsP1-nsP4) and three structural proteins (capsid, and E1 and E2 transmembrane glycoproteins). The nsp1-nsp4 proteins are translated from the first two-thirds of the genomic RNA. These proteins form a complex that directs the replication of the genomic RNA to form an antigenomic RNA, which is then copied to form a full-length positive-stranded RNA and a subgenomic mRNA that encodes the structural proteins. The capsid proteins package the genomic RNA in the cytoplasm to produce a nucleocapsid that buds from the cell surface at a membrane containing the SFV glycoproteins. Alphavirus RNA replication occurs inside bulb-shaped membrane-bound compartments called spherules, which first form at the cell surface and are then endocytosed to form cytopathic vacuoles containing multiple spherules. Replicase proteins are localized to the cytoplasmic side of the spherules. It is not known how the replicated RNA in the spherules is packaged into nucleocapsids prior to SFV budding. Alphavirus RNA replicons lacking any structural protein genes can still replicate efficiently inside the cell, but they are unable to propagate beyond the cell.

[0008] Vesicular stomatitis virus (VSV) is a negative-stranded RNA virus that encodes a single membrane glycoprotein (G), a matrix protein, a nucleocapsid protein, and two proteins that form the viral polymerase. Notably, when cells are transfected with an SFV RNA replicon that encodes only the SFV nonstructural proteins and the VSV G protein, infectious membrane-enclosed vesicles containing the VSV G protein are generated. These infectious virus-like vesicles (VLVs) grow to low titers but can be passaged like viruses in tissue culture cells. The particles contain the replicon RNA and the VSV G protein, but unlike enveloped RNA viruses, they have a very low density because they do not contain the nucleocapsid protein tightly packed around the RNA. The mechanism by which these VLVs are generated has not been determined. These VLVs expressing other proteins have proven useful as experimental vaccines. However, there are two notable limitations of the VLV system as a vaccine platform: the relatively low titer of the VLV, and the rapid loss of expression of foreign genes during passage. Clearly, there is a need for a method to create a more efficient viral vector vaccine system that supports stable foreign gene expression while at the same time producing high vaccine titers that induce strong immune responses. Such vaccine systems are useful in cancer and / or infectious disease indications. The present invention meets this need.

[0009] (HBV immunotherapy as an example) Current antiviral therapies for chronic hepatitis B (CHB) infection effectively reduce viremia, but they rarely eliminate the virus. Thus, there remains a critical need for novel treatment options for this serious disease. Because the human immune system can control HBV but often fails to do so, immunotherapy, including therapeutic vaccination, represents a promising approach to cure chronic CHB. However, while current HBV vaccine platforms generate strong antibody responses that prevent infection, they typically do not generate the broad CD8 T cell responses required to eliminate the virus after infection. Thus, novel vaccine delivery systems that can generate effective therapeutic immune responses against HBV are urgently needed. The present inventors have developed an immunotherapy based on a virus-like vesicle (VLV) platform for the treatment of patients with CHB infection.

[0010] The inventors have demonstrated that RNA replicon-based vectors or VLVs (CARG-101), carrying RNAs encoding one or more of the HBV major antigens [middle surface envelope glycoprotein (MHBs), hepatitis B core antigen (HBcAg), or polymerase] in a single open reading frame in a polycistronic unit, drive a broad polyspecific immune response that results in substantial clearance of HBV in mouse liver. Treatment of mice chronically infected with adeno-associated virus (AAV)-HBV substantially reduces serum HBV surface antigen (HBsAg), a surrogate biomarker for viral persistence in the liver, and in some animals, eliminates serum HBV surface antigen (HBsAg). The inventors have significantly enhanced global gene expression, which has led to our next-generation clinical candidate, CARG-201, which induces both T cell responses and antibodies compared to CARG-101. CARG-201 expressing MHBs and HBcAg under separate subgenomic promoters successfully inhibited the expression of HBsAg in an AAV mouse model of CHB infection with low antigen load (HBsAg 低), completely removes serum HBsAg in 100% of mice and reduces HBV RNA in the liver to undetectable levels. However, the more rigorous AAV-HBV model (HBsAg 高 ), CARG-201 reduces HBsAg levels by only 80%. Because high antigen burden is observed in many CHB patients and is associated with T cell exhaustion or tolerance, successful immunotherapy should improve immunogenicity and overcome T cell exhaustion and / or tolerance.

[0011] Modification of CARG-201 in any one or more of the three complementary approaches can enhance efficacy and result in complete clearance of serum HBsAg levels in animals.

[0012] First, we incorporated the polymerase (Pol) antigen into CARG-201 to generate CARG-301 (expressing MHBs, HBcAg, plus Pol). A vaccine that generates multi-antigen specific T cells is in a better position to provide the desired therapeutic effect compared to one or two antigens. Furthermore, Pol is a highly immunogenic target for CD4 and CD8 T cells, and due to its high sequence conservation, it may prevent the generation of escape mutants in its T cell epitopes.

[0013] Secondly, we engineered CARG-201 to incorporate a human IgK signal sequence for its polymerase (pol) gene and a VSV G glycoprotein signal sequence for its HBc gene. It is known that secreted proteins generally lead to the activation of dendritic cells, enhanced HBV antigen presentation, and the generation of novel cytotoxic T cell responses through epitope spreading. In this way, the quality and quantity of T cell responses to HBV antigens can be further enhanced compared to their soluble and non-secreted counterparts. Secreted proteins also contribute to adaptive immune responses by being taken up by antigen-presenting cells and processed by their major histocompatibility complex (MHC) class II pathway.

[0014] Thirdly, we also targeted the disruption of the programmed cell death ligand 1 (PD-L1) immune checkpoint by short hairpin RNA (shRNA) to achieve sustained long-term viral suppression or complete elimination of the virus in the liver. Checkpoint inhibition can enhance ex vivo effector T cell responses from patients with other chronic infections. We predicted that disruption of the PD-1 / PD-L1 pathway would reinvigorate other exhausted T cell functions.

[0015] The combined effect of shRNA-mediated PD-L1 inhibition and improved secretion of HBV antigens as a result of ER targeting gives these modified multivalent constructs the superior therapeutic index needed to clear the virus and halt disease progression and mortality in CHB patients. Summary of the Invention [Means for solving the problem]

[0016] (Abstract) A high titer VLV vector comprising a DNA sequence comprising a promoter sequence operably linked to a DNA sequence encoding a Semliki Forest Virus (SFV) nonstructural protein nucleotide sequence, the DNA sequence encoding the Semliki Forest Virus (SFV) nonstructural protein nucleotide sequence being operably linked to a SFV subgenomic RNA promoter, the SFV subgenomic RNA promoter being operably linked to a DNA encoding an antigen associated with an infectious disease or a DNA encoding an antigen associated with a cancer or a fragment thereof, the DNA encoding an antigen associated with an infectious disease or a DNA encoding an antigen associated with a cancer or a fragment thereof being operably linked to a 2A DNA encoding a 2A peptide, the 2A DNA in turn being linked to a vesicular stomatitis virus (VSV) G protein encoding a VSV G protein. and wherein the SFV nonstructural protein nucleotide sequence comprises at least two of the mutations selected from the group consisting of G-4700-A, A-5424-G, G-5434-A, T-5825-C, T-5930-C, A-6047-G, G-6783-A, G-6963-A, G-7834-A, T-8859-A, T-8864-C, G-9211-A, A-10427-G, G-11560-A, A-11871-G and T-11978-C, the vector lacks a nucleotide sequence encoding an SFV structural protein, and further wherein the vector produces at least 10 virus-like vesicles (VLVs) per ml when propagated in cell culture. 7 Titers of plaque forming units (pfu) are obtained.

[0017] In one embodiment, the present disclosure provides a high titer hybrid viral vector for the treatment, prophylaxis or prevention of an infectious disease or cancer, the high titer hybrid viral vector comprising the following operably linked sequence elements: a) a first DNA sequence comprising a DNA promoter sequence; b) a second DNA sequence encoding an alphavirus nonstructural protein polynucleotide sequence; c) a third DNA sequence encoding at least two alphavirus subgenomic promoters; d) a fourth DNA sequence comprising at least two sequence domains, each of the at least two sequence domains comprising: i) a sequence domain that encodes an antigen associated with an infectious disease, or ii) a sequence domain encoding an antigen associated with cancer, selected from a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA), including combinations thereof; a fourth DNA sequence, independently comprising: e) a fifth DNA sequence encoding a vesiculovirus glycoprotein; Includes.

[0018] In one embodiment, the antigen associated with the infection is one or more of HBV antigens selected from the group consisting of core (HBcAg) antigen, middle (M) surface HBs antigen, large (L) surface HBs antigen, small (S) surface HBs antigen, HBeAg antigen, and HBx antigen.

[0019] In one embodiment, the cancer associated antigen is one or more of a TSA or a TAA.

[0020] In one embodiment, at least 1 x 10 per mL of virus-like vesicles (VLVs) 9 Titers of plaque forming units (pfu) are obtained.

[0021] In one embodiment, the VLVs are obtained by purification and concentration using centrifugation, centrifugation ultrafiltration, tangential flow filtration, high speed centrifugation, chromatography, or other methods.

[0022] In one embodiment, each of the at least two sequence domains contained in the fourth DNA sequence is a sequence domain encoding an antigen associated with an infectious disease.

[0023] In one embodiment, each of the at least two sequence domains contained in the fourth DNA sequence is a sequence domain encoding an antigen associated with cancer.

[0024] In one embodiment, the TSA or TAA is one or more of alpha-fetoprotein, melanoma-associated antigen, CD44 glycoprotein, aspartate beta-hydroxylase, carcinoembryonic antigen, and a cancer-specific TSA or cancer-specific TAA.

[0025] In one embodiment, the fourth DNA sequence comprises one or more polynucleotide sequences each independently having at least about 80% homology to a sequence according to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:6, or SEQ ID NO:8.

[0026] In one embodiment, the fourth DNA sequence comprises one or more polynucleotide sequences each independently having at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% homology to a sequence according to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:6, or SEQ ID NO:8.

[0027] In one embodiment, each of the one or more sequence domains encoding a human short hairpin RNA (shRNA) of the fifth DNA sequence independently comprises a polynucleotide sequence having at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% homology to a sequence according to SEQ ID NO:13, SEQ ID NO:22, or SEQ ID NO:27.

[0028] In one embodiment, the present disclosure provides a high titer hybrid viral vector for generating virus-like vesicles (VLVs) for the treatment, prophylaxis or prevention of an infectious disease or cancer, the high titer hybrid viral vector comprising the following operably linked sequence elements: a) a first DNA sequence comprising a DNA promoter sequence; b) a second DNA sequence encoding an alphavirus nonstructural protein polynucleotide sequence; c) a third DNA sequence encoding at least two alphavirus subgenomic promoters; d) a fourth DNA sequence comprising at least two sequence domains, each of the at least two sequence domains comprising: i) a sequence domain that encodes an antigen associated with an infectious disease, or ii) a sequence domain encoding an antigen associated with cancer, selected from a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA), including combinations thereof; a fourth DNA sequence independently comprising: e) a fifth DNA sequence comprising one or more heterologous secretory signal sequences and one or more sequence domains encoding a human short hairpin RNA (shRNA); and f) a sixth DNA sequence encoding a vesiculovirus glycoprotein; Includes.

[0029] In one embodiment, the antigen associated with the infection is one or more of HBV antigens selected from the group consisting of core (HBcAg) antigen, middle (M) surface HBs antigen, large (L) surface HBs antigen, small (S) surface HBs antigen, HBeAg antigen, and HBx antigen.

[0030] In one embodiment, at least 1 x 10 per mL of virus-like vesicles (VLVs) 9 Titers of plaque forming units (pfu) are obtained.

[0031] In one embodiment, the VLVs are obtained by purification and concentration using centrifugation, centrifugation ultrafiltration, tangential flow filtration, high speed centrifugation, chromatography, or other methods.

[0032] In one embodiment, each of the at least two sequence domains contained in the fourth DNA sequence is a sequence domain encoding an antigen associated with an infectious disease.

[0033] In one embodiment, each of the at least two sequence domains contained in the fourth DNA sequence is a sequence domain encoding an antigen associated with cancer.

[0034] In one embodiment, the TSA or TAA is one or more of alpha-fetoprotein, melanoma-associated antigen, CD44 glycoprotein, aspartate beta-hydroxylase, carcinoembryonic antigen, and a cancer-specific TSA or cancer-specific TAA.

[0035] In one embodiment, each of the one or more sequence domains encoding a human short hairpin RNA (shRNA) of the fifth DNA sequence independently comprises a polynucleotide sequence having at least about 80% homology to a sequence according to SEQ ID NO:13, SEQ ID NO:22, or SEQ ID NO:27.

[0036] In one embodiment, each of the one or more sequence domains encoding a human short hairpin RNA (shRNA) of the fifth DNA sequence independently comprises a polynucleotide sequence having at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% homology to a sequence according to SEQ ID NO:13, SEQ ID NO:22, or SEQ ID NO:27.

[0037] In one embodiment, the fourth DNA sequence comprises one or more polynucleotide sequences each independently having at least about 80% homology to a sequence according to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:6, or SEQ ID NO:8.

[0038] In one embodiment, the fourth DNA sequence comprises one or more polynucleotide sequences each independently having at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% homology to a sequence according to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:6, or SEQ ID NO:8.

[0039] In one embodiment, the fourth DNA sequence comprises a polynucleotide sequence encoding one or more of IL-12 or IL-17.

[0040] In one embodiment, the fifth DNA sequence comprises a polynucleotide sequence encoding one or more shRNA sequences targeting PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, TIGIT, CD90, BTLA, CD160, or PD-1.

[0041] In one embodiment, the fourth DNA sequence comprises a polynucleotide sequence encoding one or more cytokine agonist or cytokine antagonist polypeptides targeting IL-2, IL-7, IL-15, IL-18, IL-19, IL-35, IL-21, GM-CSF, IL-17, or Flt3L.

[0042] In one embodiment, the fourth DNA sequence comprises a polynucleotide sequence encoding one or more polypeptide modulators of a malignancy-associated target selected from PD-L1, CXCL1, or CXCL2.

[0043] In one embodiment, the fifth DNA sequence comprises a polynucleotide sequence encoding one or more shRNA modulators of a malignancy-associated target selected from PD-L1, CXCL1, or CXCL2.

[0044] In one embodiment, the present disclosure provides virus-like vesicles (VLVs) containing replicon RNA produced by the high-titer hybrid viral vectors described herein.

[0045] In one embodiment, the disclosure provides a composition comprising virus-like vesicles (VLVs) produced by the high titer hybrid viral vectors described herein.

[0046] In one embodiment, the disclosure provides a method for treating and / or preventing an infection or cancer in a mammalian subject, the method comprising administering a therapeutically effective amount of a VLV composition to a mammalian subject in need thereof.

[0047] In one embodiment, the disclosure provides a method of immunizing a mammalian subject against an infectious disease or cancer, the method comprising administering a therapeutically effective amount of a VLV composition to a mammalian subject in need thereof.

[0048] In one embodiment, the disclosure provides a method for downregulating a gene associated with an infectious disease or cancer, the method comprising administering a therapeutically effective amount of a VLV composition to a mammalian subject in need thereof.

[0049] In one embodiment, the mammalian subject is a human or animal.

[0050] In one embodiment, the infection is a Hepatitis B virus infection.

[0051] In one embodiment, the disclosure provides for the use of a VLV composition described herein in the manufacture of a medicament for the treatment, prevention, or prophylaxis of an infection or cancer in a mammalian subject in need thereof.

[0052] In one embodiment, the mammalian subject is a human or animal.

[0053] In one embodiment, the infection is a Hepatitis B virus infection.

[0054] In one embodiment, the disclosure provides a method of making virus-like vesicles (VLVs) for the treatment, prevention, or prophylaxis of an infectious disease or cancer, the method comprising: a) generating a high titer viral vector comprising at least two alphavirus subgenomic promoters, at least two sequence domains, one or more heterologous secretory signal sequences, and one or more sequence domains encoding human short hairpin RNAs (shRNAs), each of the at least two sequence domains independently comprising a sequence domain encoding an antigen associated with an infectious disease, a sequence domain encoding a polymerase antigen (Pol), a sequence domain encoding an antigen associated with a cancer selected from a tumor specific antigen (TSA) and a tumor associated antigen (TAA), including combinations thereof; b) transfecting BHK-21 cells or HEK293T cells with the high titer viral vector of step (a); c) incubating the transfected BHK-21 or HEK293T cells of step (b) in a buffer solution for an appropriate time and at an appropriate temperature to allow the VLVs to grow; and d) isolating the VLVs from the BHK-21 or HEK293T cells and buffer solution by a technique selected from the group consisting of ultrafiltration, centrifugation, tangential flow filtration, affinity purification, ion exchange chromatography, and combinations thereof; wherein the isolating step (d) produces high titer VLVs.

[0055] In one embodiment, the high titer viral vector further comprises a DNA promoter sequence and a DNA sequence encoding an alphavirus nonstructural protein polynucleotide sequence.

[0056] In one embodiment, the antigen associated with the infection is one or more of HBV antigens selected from the group consisting of core (HBcAg) antigen, middle (M) surface HBs antigen, large (L) surface HBs antigen, small (S) surface HBs antigen, HBeAg antigen, and HBx antigen.

[0057] In one embodiment, the cancer associated antigen is one or more of a TSA or a TAA.

[0058] In one embodiment, step b) comprises the following substeps: b1) combining the vector with a transfection reagent; and b2) transfecting BHK-21 cells or HEK293T cells with the combined vector and reagent of step (b1). Includes.

[0059] In one embodiment, the transfection reagent is lipofectamine or PEIpro.

[0060] In one embodiment, the transfection agent is lipofectamine.

[0061] In one embodiment, the transfection reagent is PEIpro.

[0062] In one embodiment, the high titer is at least 1×10 8 PFU / mL.

[0063] In one embodiment, the high titer is at least about 1×10 9 PFU / mL.

[0064] In one embodiment, the infection is HIV, Severe Acute Respiratory Syndrome-associated coronavirus (SARS-CoV), SARS-CoV-1, SARS-CoV-2, Lyme disease, Escherichia coli O157:H7 (E. coli) infection, Hantavirus infection, Dengue fever, West Nile virus infection, Zika virus infection, Plasmodium infection (malaria), tuberculosis, cholera, whooping cough, influenza, pneumococcal disease, gonorrhea, HSV, HPV, RSV, Hepatitis B virus infection, Hepatitis C virus infection, Tick-borne encephalitis virus infection, Chikungunya virus infection, Yellow fever, Clostridium difficile infection, or Staphylococcus enterotoxin B infection.

[0065] BRIEF DESCRIPTION OF THE DRAWINGS Aspects and advantages of the present disclosure will become apparent from the following illustrative embodiments taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0066] [Figure 1] Figures 1A-1E show the effect of dp-HBc.MHs (CARG-201) and dp-MHs on HBsAg levels in a chronic AAV-HBV model. Figure 1A shows an exemplary schematic of single antigen (dp-MHs) and dual antigen (CARG-201) vectors, Figure 1B shows ELISA analysis of HBsAg (ng / mL), Figure 1C shows qRT-PCR of liver HBV RNA, Figure 1D shows flow cytometry of HBV-specific CD8+ T cells using intracellular staining for IFNγ after stimulation with HBsAg or HBcAg peptide pools, and Figure 1E shows ELISPOT of HBV-specific CD8+ T cells using HBsAg peptide pools.

[0067] [Diagram 2]FIG. 2 shows that therapeutic vaccine candidate CARG-201 in prime-boost immunization controls HBV in mice with higher pre-existing HBV antigen levels.

[0068] [Diagram 3] Figures 3A-B show the construction and expression of a VLV-based recombinant multivalent HBV vaccine. Figure 3A shows an exemplary schematic of CARG-201 and CARG-301 candidates, and Figure 3B shows the expression of HBV genes assayed by Western blot in BHK21 cell lysates.

[0069] [Figure 4] Figures 4A-C show that therapeutic vaccine candidates CARG-201 and CARG-301 in prime-boost immunization control HBV in mice with high pre-existing HBV antigen levels. Figure 4A shows the mean and individual (with mean bar) values ​​of HBsAg levels as a function of time, Figure 4B shows serum anti-HBs at week 17, and Figure 4C shows serum alanine transaminase (ALT) levels as a function of time.

[0070] [Diagram 5] FIG. 5 is an exemplary schematic representation of a modified CARG-201 vaccine construct for enhanced immunogenicity and efficacy by incorporating a secretory signal and shRNA for PD-L1.

[0071] [Figure 6-1] Figures 6A-6B show a comparison of the immunogenicity of modified CARG-201 variants in naive CB6F1 mice. Figure 6A shows splenic cellularity 7 days after immunization; Figure 6B shows the frequency of cytokine-producing T cells after polyclonal stimulation; Figure 6C shows the HBS peptide pool, and Figure 6D shows the HBC peptide pool. [Figure 6-2] Same as above.

[0072] [Figure 7-1]Figures 7A-7B show the expression and secretion of a VLV-based recombinant modified CARG-301 multivalent HBV vaccine. Figure 7A shows an exemplary schematic of the CARG-301 candidate construct, and Figure 7B shows the expression of HBV genes assayed by Western blot in BHK21 cell lysates. [Figure 7-2] Same as above.

[0073] [Figure 8-1] Figures 8A-C show in vitro shRNA inhibition of PD-L1 expression in stably transfected BHK21 cells. Figure 8A shows an exemplary schematic of a VLV therapeutic vaccine; Figure 8B shows a mouse cDNA clone of PD-L1; Figure 8C shows VLVs produced by transfecting BHK21 cells with three versions of the shRNA 3XT2A construct and shRNA-free VLV-3xT2A. [Figure 8-2] Same as above.

[0074] [Figure 9] Figures 9A-C show downregulation of PD-L1 by shRNA VLV constructs. Figure 9A shows exemplary empty VLV constructs in which shRNA is driven by one or two subgenomic promoters, Figure 9B shows western blot analysis of stable BHK21 cells constitutively expressing PD-L1, and Figure 9C shows densitometric quantification of the blots after normalization to actin.

[0075] [Figure 10] Figure 10A-B show that CARG-201 dramatically reduces serum HBsAg levels and induces core-specific T cells in the more stringent AAV-HBV model HBsAg high. Figure 10A shows serum HBsAg levels for mice transduced with AAV-HBV1.2-mer, showing that chronicity was fully established by week 8 (wk8), after which mice separated into high antigen (HBsAg high) and low antigen (HBsAg low). Figure 10B shows core-specific T cells and PD1 cells.

[0076] [Figure 11] Figures 11A-11F show that in vivo shRNA-mediated blockade of the PD-1 / PD-L1 pathway significantly inhibits immune checkpoint and inhibitory receptor expression in MC38 tumor-bearing mice. Gene expression for CTLA4 (Figure 11A), PD-L1 (Figure 11B), Tigit (Figure 11C), PD-1 (Figure 11D), PD-L2 (Figure 11E), and Lag3 (Figure 11F) is shown.

[0077] [Figure 12] FIG. 12 shows exemplary strategies for improving the efficacy of therapeutic HBV vaccines in animals and humans.

[0078] [Figure 13] FIG. 13 shows an exemplary rationale for the development of optimized VLV candidates, which is a paradigm for therapeutic vaccines (immunotherapy) against HBV.

[0079] [Figure 14] FIG. 14 shows productivity values ​​(PFU per cm2 of different tissue culture flasks) for CARG-201. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0080] (Detailed Description) (Platform Technology and Methods) The present disclosure relates to a versatile virus-like vesicle-based platform for immunotherapy applications in infectious diseases and cancer. The VLV platform is generally useful for the treatment, prophylaxis, and / or prevention of infectious diseases or cancer. In one embodiment, a representative infectious disease is Hepatitis B virus infection (HBV). The platform technology and treatment methods disclosed herein are applicable to several infectious diseases, including but not limited to emerging diseases, re-emerging diseases, and other infectious diseases. In a representative embodiment, the emerging diseases may include HIV, severe acute respiratory syndrome-related coronavirus (SARS-CoV), SARS-CoV-1, SARS-CoV-2, Lyme disease, Escherichia coli O157:H7 (E. coli), Hantavirus, Dengue fever, West Nile virus, and Zika virus. In representative embodiments, re-emerging diseases may include Plasmodium (malaria), tuberculosis, cholera, whooping cough, influenza, pneumococcal disease, and gonorrhea. In representative embodiments, other infectious diseases may include HSV, HPV, RSV, Hepatitis B virus, Hepatitis C virus, tick-borne encephalitis virus, Chikungunya virus, yellow fever, Clostridium difficile, Staphylococcus enterotoxin B.

[0081] As disclosed herein, Hepatitis B Virus (HBV) is exemplified as an infectious disease, however, the full scope of the disclosure is intended to include each of the above emerging, re-emerging, and other infectious diseases. In further embodiments, the full scope of the disclosure is intended to include any infectious disease known to one of skill in the art.

[0082] The present disclosure also relates to cancer immunotherapy applications. For example, the VLVs can encode one or more cytokine agonists, cytokine antagonists, small hairpin RNAs, and / or other polynucleotides or polypeptides useful for treating, preventing, and / or preventing malignant tumors or cancer. For example, the VLVs of the present disclosure can encode or produce one or more cytokine agonist or cytokine antagonist polypeptides selected from IL-12; IL-17; shRNA against one or more of PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, TIGIT, CD90, BTLA, CD160, PD-1; and / or one or more of IL-2, IL-7, IL-15, IL-18, IL-19, IL-35, IL-21, GM-CSF, and IL-17, Flt3L. The platform VLVs may alternatively or additionally downregulate genes associated with malignancies (e.g., PD-L1, CXCL1, and / or CXCL2, among others known to those of skill in the art).

[0083] As used herein, the term "target" can be used in relation to any biological target that is useful in the vectors, plasmids, pharmaceutical compositions and methods of the present disclosure. "Targeting" includes upregulating the biological target, downregulating the biological target, regulating the biological target, functionally acting on the biological target as an agonist or antagonist, or interacting with the biological target in any manner that can cause a biological effect. Examples of factors encoded by the vectors described herein that "target" a particular biological target include polypeptides, polynucleotides, shRNAs, siRNAs, antibodies or antigen-binding fragments thereof, or any other useful factors that can be encoded by vectors and / or produced in VLVs.

[0084] Other embodiments disclosed herein will be readily apparent to those skilled in the art. U.S. Patent No. 9,987,353 to Robek et al., issued June 5, 2018, U.S. Patent No. 10,435,712 to Rose et al., issued October 8, 2019, and PCT / US2021 / 012834, filed January 8, 2021 (published as WO / 2021 / 142366 on July 15, 2021), each generally relate to VLV technology and are each incorporated herein by reference in their entirety.

[0085] (Background and Importance) (HBV as an example: importance of the challenge). HBV infection is a major global public health challenge. Globally, approximately 2 billion people will be infected with hepatitis B virus (HBV) during their lifetime, over 240 million people currently have HBV infection, and approximately 600,000 people die from HBV-related liver disease each year. Patients with chronic HBV (CHB) infection (including inactive carriers of HBV) are at increased risk of developing cirrhosis, liver failure, and hepatocellular carcinoma (HCC). Most of these patients will not develop HBV-related complications, but 15%-40% will develop severe complications during their lifetime. CHB has a variety of clinical conditions defined by the presence or absence of liver inflammation measured by HBV DNA titer, the presence of hepatitis B e antigen (HBeAg, a secreted form of the core protein), and hepatic transaminase levels. CHB infection results from a continuous interplay between viral replication and immune response. T cells are exhausted by their continuous antigen exposure, which is responsible for the persistence of HBV infection. When T cells encounter HBV antigens presented by intrahepatic antigen-presenting cells (APCs) (e.g., dendritic cells (DCs) and Kupffer cells), the costimulatory signal received by the T cells is very weak. This results in immune tolerance rather than functional activation. Furthermore, a high proportion of regulatory T cells (T reg An immunosuppressive microenvironment is formed that contains immune-suppressing T cells (IL-1, IL-2, and myeloid-derived immunosuppressive cells (MDSCs), which provide inhibitory signals to T cells and interfere with T cell-mediated anti-HBV function.

[0086] Limitations of the Current HBV Vaccine: Despite its success in preventing HBV infection, the current HBV vaccine (recombinant HBsAg adsorbed to alum) has a number of suboptimal characteristics. First, it does not induce a protective antibody response in all immunized individuals. Second, between two and four doses of the vaccine are recommended to induce long-lasting immunity. This requirement for repeated immunization makes the vaccine somewhat difficult to administer in many parts of the world, especially in areas lacking adequate medical infrastructure. Third, the protective antibody response fades after immunization, decreasing to below protective levels (>10 IU / L) in up to 60% of vaccinated individuals. Fourth, escape mutations in the surface protein genes can result in the virus being resistant to the antibody response generated by the vaccine. Finally, as noted above, although it elicits a protective antibody response that prevents infection, the current vaccine does not generate a strong CD8 T cell response, and it has not been effective in clinical trials to control viral replication in people already infected with HBV.

[0087] (Current Treatments for CHB). The current standard of care for CHB includes antiviral and immune-enhancing drugs (e.g., tenofovir, entecavir, and pegylated IFN), which are highly effective in delaying the disease and are curative in only 8%-12% of treated patients. Cessation of antiviral therapy is often accompanied by a rebound in the viral load, thus necessitating lifelong treatment. Available antiviral drugs can efficiently result in a reduction of serum HBV viral load to undetectable levels, but they usually fail to achieve seroclearance of hepatitis B surface antigen (HBsAg), which indicates eradication of HBV infection, the ultimate goal of antiviral treatment for CHB. The failure to achieve HBsAg seroclearance may be due to emerging drug-resistant HBV mutants and residual covalently closed circular DNA (cccDNA) in infected hepatocytes. None of these clinical treatments achieve long-term virologic control in the majority of patients with CHB, therefore, there is an urgent need to develop novel therapies to improve HBsAg clearance and virologic cure.

[0088] (Novel modalities for HBV treatment). The failure of HBsAg seroclearance necessitates the development of novel therapeutic strategies to achieve sustained viral remission. One of the strategies is to directly target the virus by targeting viral entry, viral assembly / encapsidation, preS1 or Hepatitis B surface antigen (HBsAg) secretion, envelopment and cccDNA. Another strategy is to interfere with the host mechanisms by using Toll-like receptor (TLR) agonists, cytokines and blockade of PD-1 / PD-L1. Furthermore, therapeutic vaccines based on recombinant HBV proteins or HBV-enveloped subviral particles, DNA and T-cell peptide epitopes resend another promising strategy for HBV eradication. Therapeutic vaccination aims to eliminate persistent viral infection by enhancing the patient's immune response. Individuals who become acutely infected but eventually clear the virus have relatively strong multispecific T-cell responses against HBV. However, in chronically infected individuals, the T cell response is rather weak in magnitude and directed against only a few viral antigens. This suboptimal immune response persists in chronically infected individuals despite the continued presence of viral antigens in the liver and blood. Although current HBV vaccines induce strong antibody responses that prevent infection, they do not elicit the virus-specific T cells required to control established infection. Novel technologies that generate effective T cell-dependent immune responses against HBV are urgently needed. One promising approach to treat CHB is a therapeutic vaccine that can induce virus-specific CD8 T cells to clear HBV infection.

[0089] (Functional cure of HBV). The ultimate goal of HBV treatment is "functional cure". According to the AASLD and EASL meetings, functional cure is defined as a sustained loss of HBsAg in serum. In this scenario, HBV cccDNA remains at low levels, but a functional adaptive immune response ensures suppression of viral replication without treatment, similar to what occurs in the clearance of acute HBV. A strong HBV-specific CD8 T cell response is required for HBV clearance in acute infection, but in CHB, the T cell response is dysfunctional and is not restored to improvement by NUC. Since functional cure is rarely achieved with current therapies, alternative treatments that can be given in a shorter, finite course are urgently needed. CHB infection is the result of a complex interaction between HBV and the host, and impaired immune response to viral antigens is thought to be a key factor associated with the CHB carrier state. If this state of immune tolerance can be overcome, loss of HBeAg or HBsAg from serum (seroclearance) and sustained control of HBV infection is achieved.

[0090] (Scientific premise). Current standard treatments rarely result in functional cure, characterized by sustained loss of HBsAg (with or without HBsAg antibody seroconversion). The goal of next-generation CHB treatments is to achieve a higher percentage of functional cures with a finite treatment period. To address this urgent need, we developed a targeted shRNA therapeutic for CHB based on a VLV delivery platform. This shRNA can be developed as a stand-alone treatment or in combination with a therapeutic vaccine to achieve functional cure. Since the human immune system can control HBV but often fails to do so, immunotherapy, including therapeutic vaccines, represents a promising approach to cure CHB. However, therapeutic immune responses generated in persistent HBV infections are often weak due to CD8 T cell exhaustion. Exhaustion of virus-specific T cells may play an important role in HBV persistence. The interaction between the programmed cell death 1 (PD-1) receptor and its ligand PD-L1 on lymphocytes plays an important role in T cell exhaustion by inducing T cell inactivation, indicating that the PD-1 / PD-L1 pathway is a good therapeutic candidate for chronic HBV infection. Woodchucks infected with woodchuck hepatitis virus (WHV) may have increased hepatic expression of PD-1-ligand 1 (PD-L1), increased PD-1 on CD8+ T cells, and limited numbers of virus-specific T cells. In these animals, others have shown that combination therapy with αPD-L1 and entecavir (ETV) improved the control of viremia and antigenemia compared to ETV treatment alone. Furthermore, others have shown that PD-L1 blockade synergistically enhances HBV-specific CD4 T cells. Furthermore, there is accumulating evidence that immune checkpoint inhibitors can enhance ex vivo effector T cell responses from patients with other chronic viral, bacterial, or parasitic infections, including HIV, tuberculosis, and malaria.[64-66] We found that therapeutic shRNA interventions targeting exhausted T cells by blocking these suppressive pathways can restore the function of these impaired T cells, resulting in a functional cure.

[0091] (Platform immunotherapy technology) The inventors have found that alphavirus replicons are excellent vaccine vectors because they are highly immunogenic and target dendritic cells. The virus-like vesicle (VLV) vaccine platform is a capsid-free, self-replicating antigen expression system that represents an attractive alternative to other virus-based vectors. VLVs encode the Semliki Forest virus (SFV) replicon and an additional structural protein, the vesicular stomatitis virus glycoprotein (VSV-G). After in vitro evolution through 50 passages in BHK-21 cells that resulted in 10 amino acid changes in SFV nsP1 to SFV nsP4, the evolved SFV nonstructural proteins promote high-titer VLV replication in cell culture through increased VLV release efficiency. VSV-G expression enables robust pantropic infectivity. The reason is that infectious vesicles composed of SFV replication globules derived from spherical cell membrane invaginations are coated with VSV-G protein, bud off from infected cells, and spread to non-infected cells, causing multiple rounds of infection. VLVs are non-pathogenic in mice and rhesus macaques, pose little risk of genomic integration or reversion to pathogenicity, and are immunogenic in the absence of adjuvants. Recent improvements to this system allow for the production of high titer VLV particles as well as high gene expression up to multiple subgenomic promoters. Although VLVs mimic the viral stimulatory properties of viral vectors, these VLVs are safe and non-pathogenic when administered to mice or rhesus macaques, and they do not exhibit neurotoxicity when injected directly into the mouse brain. These vectors are important because of their potency, ease of producing high titer particles, and predicted safety due to the lack of viral structural proteins. Furthermore, VLVs have a demonstrated large capacity to deliver nucleic acids for expression of several antigens, resulting in the induction of T cell and antibody responses against multiple epitopes of multiple antigens, thus helping to maximize the potential efficacy of proposed immunotherapies in patients.

[0092] In the last two decades, multiple studies have evaluated therapeutic vaccine candidates for CHB treatment using lipopeptide epitope-based vaccines; DNA-based vaccines; and adenovirus vector vaccines. Thus far, attempts at therapeutic vaccination for HBV have not been effective in reliably inducing functional cure in individuals with CHB. The persistent low responsiveness of HBV-specific T cells and the high baseline HBsAg load of participants, as well as the limited T cell immunogenicity of the vaccine candidates themselves, are possible reasons that may have hindered the success of these vaccine candidates. Another possible failure is the use of HBsAg, which in healthy individuals induces HBs antibodies that block viral entry and prevent infection. The effect of using protein vaccines alone on T cell induction and immune regeneration in the chronic setting may be minimal. Most patients develop HBeAg associated with high HBV antigen levels. + High antigen load has been proposed as an obstacle to successful rescue of tolerized T cells by therapeutic vaccination.

[0093] The vector of the present invention may generally be a plasmid or other vector encoding a VLV. Thus, the term "vector" includes plasmids. These plasmids may generally include any element required for VLV production. These vectors or plasmids may be defined by one or more sequence domains or sequence components, or by one or more sequences. Generally, unless otherwise clear from the context, plasmids may include additional sequence domains or sequence components as necessary or desired. A sequence may be defined as a polynucleotide sequence or a corresponding amino acid sequence. Some sequence components (e.g., shRNA) may not have a corresponding amino acid sequence. Exemplary sequence domains are presented in Table 1 below. Table 1: SEQ ID NOs: 1-30 [Table 1-1]

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

Table 1-27

Table 1-28

Table 1-29

Table 1-30

Table 1-31

Table 1-32

Table 1-33

Table 1-34

Table 1-35

Table 1-36

Table 1-37

Table 1-38

Table 1-39

Table 1-40

Table 1-41

[0094] In various embodiments, the vector or plasmid may comprise and / or encode one or more of SEQ ID NOs: 1-30. In various embodiments, the vector or plasmid may comprise and / or encode a sequence or sequence portion having greater than 70% homology, or greater than 75% homology, or greater than 80% homology, or greater than 85% homology, or greater than 90% homology, or greater than 95% homology, or greater than 96% homology, or greater than 97% homology, or greater than 98% homology, or greater than 99% homology to one or more of SEQ ID NOs: 1-30. In some embodiments, the vector or plasmid may comprise a sequence consisting of one or more of SEQ ID NOs: 1-30. When a vector or plasmid comprises a sequence consisting of one or more of SEQ ID NOs: 1-30 or a sequence encoding one or more of SEQ ID NOs: 1-30, it is contemplated that the vector or plasmid may comprise additional sequence domains.

[0095] In exemplary embodiments, the plasmid may have a polynucleotide sequence corresponding to SEQ ID NO: 16 or SEQ ID NO: 17. In exemplary embodiments, the plasmid may have a polynucleotide sequence having greater than about 70% homology to SEQ ID NO: 16 or SEQ ID NO: 17, or greater than 75% homology, or greater than 80% homology, or greater than 85% homology, or greater than 90% homology, or greater than 95% homology, or greater than 96% homology, or greater than 97% homology, or greater than 98% homology, or greater than 99% homology. In some embodiments, the plasmid may have a polynucleotide sequence consisting of SEQ ID NO: 16 or SEQ ID NO: 17. In some embodiments, the plasmid may have a polynucleotide sequence consisting essentially of SEQ ID NO: 16 or SEQ ID NO: 17. When a plasmid has a polynucleotide sequence consisting essentially of SEQ ID NO: 16 or SEQ ID NO: 17, it is contemplated that the plasmids having the same general sequence domains may contain one or more nucleotide and / or amino acid substitutions, additions, or deletions in or between those domains that do not significantly affect the function of the plasmid.

[0096] When sequence "homology" or sequence "identity" is contemplated for a DNA sequence or an amino acid sequence, the same percentage of "similarity" is also contemplated for that amino acid sequence or the amino acid sequence corresponding to that DNA sequence. The term "similarity" differs from the term identity because it allows for conservative substitution of amino acid residues with similar physicochemical properties over a given alignment of a defined length. In general, any known and reasonable similarity scoring matrix can be used to determine similarity.

[0097] When determining the sequence homology or sequence identity of a first sequence compared to a second sequence, various identity calculations can be performed, such as the calculation carried out by the Basic Local Alignment Search Tool (BLAST) of the National Institutes of Health.In some embodiments, standard BLAST settings can be used.For example, BLAST identity can be defined as the number of matching bases relative to the number of aligned positions.

[0098] VLVs can generally be produced by transfecting any suitable cell line with a suitable plasmid or vector. In one embodiment, VLVs are produced by transfecting BHK-21 cells or HEK293T cells with a vector or plasmid, incubating the transfected BHK-21 cells or HEK293T cells in a buffer solution for a suitable time and at a suitable temperature to allow VLVs to grow; and isolating the VLVs from the BHK-21 cells or HEK293T cells and the buffer solution by a technique selected from the group consisting of ultrafiltration, centrifugation, tangential flow filtration, affinity purification, ion exchange chromatography, and combinations thereof. In various embodiments, VLVs are produced by any suitable transduction, incubation, and isolation methods.

[0099] The generated VLVs are generally useful for therapeutic purposes. The generated VLVs can be formulated with one or more diluents, excipients, or other components into a vaccine composition for treating HBV. The composition can generally be administered by any suitable route, such as orally, parenterally, intravenously, or other routes.

[0100] The figures are described in more detail below with reference to the examples presented herein.

[0101] Figure 1 shows the effect of dp-HBc.MHs (CARG-201) and dp-MHs on HBsAg levels in the chronic AAV-HBV model. Figure 1A shows a schematic diagram of single antigen (dp-MHs) and dual antigen (CARG-201) vectors. Chronic HBV was established in C57BL / 6 mice using HBV genome delivery by AAV2 / AAV8. Groups were matched for HBsAg before prime immunization with control vector (expressing eGFP, n=6), dp-MHs (expressing HBV middle S antigen, n=8), or CARG-201 (expressing HBV core antigen and HBV middle S antigen, n=9) 6 weeks after AAV-HBV transduction. Animals were boosted 4 weeks later with serotype-converted VLV vectors. Figure 1B shows ELISA analysis of HBsAg (ng / mL). Figure 1C shows qRT-PCR of hepatic HBV RNA. Figure 1D shows HBV-specific CD8+ cells using intracellular staining for IFNγ after stimulation with HBsAg or HBcAg peptide pools. + Flow cytometry of T cells. Figure 1E shows HBV-specific CD8 + ELISPOT of T cells is shown. Data are means ± standard error of the mean (SEM). Asterisks indicate significant differences between control and VLVs expressing HBV antigens (p<0.01). HBc=HBcAg (core); MHs=MHBs (surface); dp=dual subgenomic promoter; SGP1 / 2, subgenomic promoter 1 or 2.

[0102] Figure 2 shows that therapeutic vaccine candidate CARG-201 in prime-boost immunization controls HBV in mice with higher pre-existing HBV antigen levels. To further understand the impact of antigenemia on CARG-201-mediated HBV control, we evaluated anti-HBV efficacy in mice with even higher antigen levels than previously used (100 ng / mL to 500 ng / mL). We administered 1 × 10 11Groups of animals were transduced with 10 genome copies of AAV-HBV 1.2-mer and had moderate to high (mean HBsAg ≈3,000 ng / mL) levels of HBsAg were selected for immunization. Persistent HBV replication was determined by measuring serum HBsAg levels 8 weeks after transduction. Two groups of 12 mice were immunized with 10 8 PFU / mouse were primed intramuscularly (im) with CARG-201 and VLV-GFP and boosted 4 weeks later (Pre). A significant HBsA reduction occurs for CARG-201, but is not evident in either the GFP or PBS controls.

[0103] Figure 3 shows the construction and expression of a VLV-based recombinant multivalent HBV vaccine. Figure 3A shows an exemplary schematic of the CARG-201 and CARG-301 candidates. The four nonstructural proteins of the Semliki Forest Virus (SFV) replicase are named (nsp1 to nsp4). The HBV polymerase (Pol) lacks its terminal protein from the four structural domains that make up the enzyme. Expression of the Pol and core antigens is fused to downstream genes by the 2A (T2A) ribosomal skip site of the picornavirus Thosea asigna virus. Figure 3B shows the expression of HBV genes assayed by Western blot in BHK21 cell lysates. Both two-antigen and three-antigen expression are compared.

[0104] Figure 4 shows that therapeutic vaccine candidates CARG-201 and CARG-301 in prime-boost immunization control HBV in mice with high pre-existing HBV antigen levels. CARG-201 carries two antigens (HBcAg and MHBs) and CARG-301 (HBcAg, MHBs, polymerase). To further understand the impact of antigenemia on CARG-mediated HBV control, we evaluated anti-HBV efficacy in mice with higher antigen levels, as described in Figure 3. High levels of HBsAg (average HBsAg approx. 3,000ng / mL) were selected for the immunization group. Persistent HBV replication was determined by measuring serum HBsAg levels 8 weeks after transduction. Three groups of 10 mice were injected with 10 8 PFU / mouse were primed intramuscularly (im) with CARG-201, CARG-301 and GFP and boosted 4 weeks later (boost 1) and 6 weeks after the first boost (boost 2). Significant HBsAg reduction occurs for both CARG-201 and CARG-301, but is not evident in the GFP control, both in mean values ​​(left panel) and individual values ​​(right panel). Prime, VSV G NJ RNA replicons encoding serotypes; Boost 1, VSV G IN Serotype; Boost 2, VSV G CH Serotype; NJ=New Jersey; IN=India; CH=Chandipura.

[0105] Figure 5 shows an exemplary schematic representation of modified CARG-201 vaccine constructs for enhanced immunogenicity and efficacy by incorporating secretory signals and shRNA for PD-L1. The nonstructural proteins of the Semliki Forest Virus (SFV) replicase are named (nsp1 to nsp4). The secretory signal (ss) is derived from the VSV G glycoprotein. The secretory terminal protein is derived from the four structural domains that make up the enzyme. FIG. 6 shows a comparison of the immunogenicity of modified CARG-201 variants in naive CB6F1 mice. Figure 7 shows the expression and secretion of the VLV-based recombinant modified CARG-301 multivalent HBV vaccine. Figure 7A shows an exemplary schematic of the CARG-301 candidate construct. The four nonstructural proteins of the Semliki Forest Virus (SFV) replicase are named (nsp1 to nsp4). The HBV polymerase (Pol) lacks its terminal proteins from the four structural domains that make up the enzyme. At its N-terminus, a human IgK signal sequence is fused. Its middle surface antigen (MHBs) and core antigen (HBcAg) have native and heterologous VSV G signal sequences (ss) fused to their amino termini, respectively. Expression of the Pol antigen and core antigen are fused to downstream genes by the 2A (T2A) ribosomal skip site of the picornavirus Thosea asigna virus. Figure 7B shows the expression of HBV genes assayed by Western blot in BHK21 cell lysates. Expression of the three antigens is compared. Culture supernatants were collected and assayed for secretion of HBV antigens. Secreted polymerase was identified as a faint band (white asterisk) that was only detected after concentration of the culture supernatants. * Actin appears to undergo rapid degradation in the culture medium as detected by ELISA (indicated by ELISA). Actin protein is detected in the cell lysate (lane 10) but not in the cell supernatant (lanes 1-9). Addition of a cocktail of protease inhibitors can inhibit the degradation and enhance its detection. HBc, HBcAg; C=control; 301=CARG-301; s301=secCARG-301; 301.sh=CARG-301.shRNA; s301.sh=secCARG-301.shRNA. IN=New Jersey serotype; NJ=New Jersey serotype.

[0106] Figure 8 shows that shRNA inhibits PD-L1 expression in stably transfected BHK21 cells in vitro. Figure 8A shows an exemplary schematic of a VLV therapeutic vaccine (VLV-3xT2A) carrying multiple PD-L1 specific shRNAs at the 5' and 3' ends of the VSV G glycoprotein. Expression of the major antigens of HBV (MHBs, HBcAg and polymerase) in VLVs is linked to VSV G by the 2A (T2A) ribosomal skip site of the picornavirus Thosea asigna virus. Figure 8B shows that the mouse cDNA clone of PD-L1, CD274 / B7-H1 / PD-L1 ORF (Sino biological Inc.), was cloned into the mammalian expression vector pCMV3-Flag-mCD274. BHK21 cells were transfected and hygromycin-resistant clones were selected and expanded. PD-L1 expression in stable cells was analyzed by Western blot using anti-PDL1 antibody. Figure 8C shows VLVs produced by transfecting BHK21 cells with three versions of shRNA 3XT2A construct. VLVs were produced and titered, and infected into stable BHK21:PD-L1 cell lines using 0.1 MOI. Lysates were prepared by harvesting cells after 8, 24, and 32 hours. PDL1 downregulation was analyzed by Western blot and compared to non-infected cell lysates. Band intensities were quantified by GelQuant.NET software (BiochemLabSolutions.com) and normalized to β-actin.

[0107] Figure 9 shows downregulation of PDL1 by shRNA VLV constructs. Figure 9A shows an exemplary schematic representation of empty VLV constructs in which shRNA is driven by one or two subgenomic promoters. Figure 9B shows Western blot analysis of stable BHK21 cells constitutively expressing PD-L1. Six hours after infection, cell lysates were prepared and analyzed. Figure 9C shows densitometric quantification of the blots after normalization to actin. Scr; scrambled shRNA; sh373 and sh486; shRNA at position (bp) relative to the PD-L1 sequence.

[0108] Figure 10 shows that CARG-201 inhibits the more rigorous AAV-HBV model HBsAg 高 Figure 10A shows that AAV-HBV1.2-mer dramatically reduced serum HBsAg levels and induced core-specific T cells in mice. 高 ) and low antigen (HBsAg 低 ) into the CD8+ / CD9+ / CD1 ... + Increased induction of core-specific T cells following immunization with dp-CGS as measured by intracellular cytokine staining for IFNγ+ in T cells (left panel).

[0109] Figure 11 shows that blocking the PD-1 / PD-L1 pathway with shRNA in vivo significantly inhibits the expression of immune checkpoints and inhibitory receptors in MC38 tumor mice. CARG-2020 is a replicon vector carrying three immune modulators (IL-12, DIL-17RA and shRNA), while IL-12 and GFP are replicon vectors expressing rIL-12 (p35 and p40 subunits) and GFP, respectively. C57BL / 6 mice (n=10) were implanted with MC38 cells and their tumors were inoculated with 5x10 6 cells on days 0, 3 and 6. 7 PFU of the indicated vectors were infected intratumorally. When tumors regressed significantly in the CARG-2020 and IL-12 groups but not in the control GFP group, tumors were harvested, RNA was prepared, and analyzed by qPCR using the GeneQuery kit (catalog no. MGK121) from ScienCell (Carlsbad, CA). Inhibitory receptors are shown in A and D, receptor ligands in B and E, and other immune checkpoint genes in C and F.

[0110] Figure 12 explores multiple strategies to improve the efficacy of therapeutic HBV vaccines in animals and humans. Strategies include: (i) combining vaccine platforms in a heterologous prime-boost regimen; (ii) optimizing vaccine immunogens by including multiple HBV antigens incorporating signal sequences; (iii) reversing T cell dysfunction by simultaneous inhibition of immune checkpoints with shRNA; (iv) immunizing individuals with lower HBV antigen loads by reducing antigen load with NUC therapy, siRNA inhibition, nucleic acid polymers (NAPs) or VLVs prior to vaccination. EXAMPLES

[0111] (Example) The invention will now be described with reference to the following examples, which are provided for illustrative purposes only, and the invention should in no way be construed as being limited to these examples, but rather as including any and all variations that become evident as a result of the teachings provided herein.

[0112] Without further description, it is believed that one of ordinary skill in the art can, using the above description and the following illustrative examples, make and use the present compounds and practice the present methods.

[0113] Key points include: Dual promoter (DP) or triple promoter (TP) VLV platforms promote higher gene expression and enhance immunogenicity · Construct CARG-201 drives complete clearance of HBsAg in mice with hypoantigenemia CARG-201 dramatically reduces serum biomarker levels and induces T cells in a stringent mouse model Both CARG-201 and CARG-301 break tolerance in hyperantigenemic mice and drive a dramatic reduction in HBsAg in mice Modification of CARG-201 with shRNA for secreted core antigen or PD-L1 increases immunogenicity in naive mice shRNA inhibits cells that constitutively express PD-L1 in vitro. In vivo, the CARG-2020 construct expressing both IL-12 and PD-L1 shRNA inhibits PD-L1 + Downregulates the expression of multiple immune checkpoints in tumor cell line (MC38) · Endoplasmic reticulum (ER)-targeted secretory signal sequence enhances secreted HBV antigen in CARG-301 The modified CARG-201 and modified CARG-301 constructs can be scaled up and produced.

[0114] We demonstrated that treatment of mice chronically infected with AAV-HBV with CARG-101 (a VLV carrying only one HBV antigen, MHBs) significantly reduced serum HBsAg, a surrogate biomarker for viral persistence in the liver, and in some animals, cleared the serum HBsAg. Clearance of HBsAg in 40% of the AAV-HBV mice by a single immunization with CARG-101 is an excellent result compared to other HBV immunotherapies being developed in the same animal model.

[0115] Remarkably, we have now achieved a reduction in serum biomarker levels in over 80% of AAV-HBV mice (n=10) with hyperantigenemia. This indicates that CARG-201 (two HBV antigens, HBcAg and MHBs) can break tolerance in a highly tolerant model. To achieve this reduction, we have achieved complete clearance of HBsAg in mice in combination with a gene expression enhancement strategy and a robust prime-boost regimen. Thus, we selected CARG-201 to advance to the clinic stage based on the following results: (i) complete reduction of HBsAg in most, but not all, treated animals in a mouse model of persistent HBV replication, (ii) reduction of intrahepatic HBV RNA to undetectable levels, and (iii) induction of multispecific HBV T cells and antibodies. The reduction of intrahepatic HBV RNA was associated with high levels of CD8 T cells, as observed in patients during recovery from acute HBV infection (Figures 1 and 2). + T cell responses and CD4 + This may be the result of strong immune control under T cell responses.

[0116] (CARG-201 delivers transgenes for two HBV antigens (MHBs and HBc): Enables robust expression and secretion of HBV middle S antigen and HBV core antigen in vitro Induce a broad immune response - Causes a >2-log reduction in HBV marker surface antigens in the AAV model Eliminate the virus as a monotherapy or in combination with standard antiviral therapy.

[0117] We reasoned that the incorporation of a third antigen (e.g., polymerase (Pol)) in combination with prime-boost immunization may generate a stronger multi-specific and polyfunctional T cell response that ultimately controls the virus in 100% of infected mice (Figure 3). Therefore, we designed CARG-301, which carries MHBs, HBcAg, plus polymerase (three HBV antigens), and showed that it clears the virus with similar efficacy and kinetics as CARG-201 (Figure 4). In some embodiments, the MHBs can be any known MHBs. In some embodiments, the HBcAg can be SEQ ID NO: 10 (DNA), SEQ ID NO: 9 (amino acid). In some embodiments, the polymerase can be SEQ ID NO: 12 (DNA), SEQ ID NO: 10 (amino acid).

[0118] Figure 13 shows an exemplary understanding of how HBV immunotherapy works to achieve a functional cure using either CARG-201 or CARG-301. The genome of this partially double-stranded DNA hepatotropic virus uses four overlapping open reading frames to code for seven proteins: core (HBcAg), surface [large (L)HBs, middle (M)HBs, and small (S)HBs], HBeAg, polymerase, and HBx. It is generally believed that a broad, multi-specific immune response is most beneficial. Therefore, in addition to evaluating the efficacy of a single antigen, we tested the efficacy of multiple antigens delivered on a single vector. The multiple antigen approach disclosed herein has several advantages over single antigen constructs, as described herein.

[0119] FIG. 13 shows an exemplary rationale for the development of optimized VLV candidates, which is a paradigm for therapeutic vaccines (immunotherapy) against HBV. For multiple chronic viral infections (e.g., HIV, HCV, HPV and HSV), therapeutic vaccines are currently under development. As an alternative to antiviral treatments or to support standard HBV therapies that are only partially effective, the VLVs are designed to activate the patient's immune system to fight the virus and eventually control or even ideally eliminate it. Successful prophylactic vaccination is based on rapid neutralization of invading pathogens by antibodies, whereas viral control and elimination of infected cells require T cells. Thus, in addition to activation of humoral immune responses to limit viral spread, induction of multi-antigen specific and polyfunctional T cell responses against key viral antigens is a paradigm for therapeutic vaccination. Cell-mediated immunity (CARG-201 or CARG-301) inhibits HBV replication, thereby efficiently reducing viremia. Prime vaccination with HBV surface antigen (MHBs), core antigen (HBcAg) or polymerase (Pol) antigen stimulates the help of HBV-specific CD4 T cells, resulting in antibody production by HBV-specific B cells. This results in the production of antigen-neutralizing antibodies, and ideally, seroconversion of HBsAg to anti-HBs. The vaccination also induces CD8 CTLs capable of killing infected hepatocytes, ultimately resulting in viral clearance. HBsAg, hepatitis B surface antigen; anti-HBs, antibodies against HBsAg.

[0120] These data we have generated demonstrate that (i) we can enhance antigen load with concomitant increase in immunogenicity by modifying VLVs to carry two or more subgenomic promoters, and (ii) VSV G serotype conversion is an effective prime-boost strategy. Optimized single antigen (MHBs) or optimized dual antigen (MHBs and HBc) vectors can drive complete clearance of HBsAg in mice (Figure 1). The data show that the reduction of intrahepatic HBV RNA is associated with high levels of CD8, as in patients recovering from acute HBV infection. + T cell responses and CD4 + This may be due to the strong immune regulation under T cell responses.

[0121] CARG-201 drives HBsAg clearance in highly antigenemic mice (Figure 2). The data demonstrate that CARG-201 can reduce serum biomarker levels in over 80% of AAV-HBV mice with hyperantigenemia. Optimized double boost can drive complete clearance in highly antigenemic mice (Figure 3). The data also demonstrate that serotype conversion is a highly effective prime-boost regimen to significantly (greater than 1 log) reduce serum biomarker levels in over 80% of AAV-HBV mice with hyperantigenemia. The RNA replicon-based HBV therapeutic vaccine in development can induce CD8+ T cells against multiple antigen epitopes in the tolerogenic environment of CHB infection, addressing the need for HBV immunotherapy. Further genetic engineering of either CARG-201 or CARG-301 further reduces the biomarker levels by over 2-3 logs.

[0122] Modifications to VLV and antigen design (Figures 5 and 7): Addition of a secretory signal to the core antigen in CARG-201 to enhance antigen expression. In some embodiments, the secretory signal can be a VSV G secretory signal (e.g., SEQ ID NO:6 (DNA), SEQ ID NO:5 (amino acid)), or a human IgK secretory signal (e.g., SEQ ID NO:8 (DNA), SEQ ID NO:7 (amino acid)). Addition of a secretory signal to the core antigen and polymerase antigen in CARG-301. In some embodiments, the secretory signal can be a VSV G secretory signal (e.g., SEQ ID NO:6 (DNA), SEQ ID NO:5 (amino acid)), or a human IgK secretory signal (e.g., SEQ ID NO:8 (DNA), SEQ ID NO:7 (amino acid)). Incorporation of a PD-L1 shRNA cassette from CARG-2020 (VLV carrying IL-12+IL-17R+PD-L1 shRNA) into CARG-201 and CARG-301 to increase immunogenicity and overcome immune exhaustion and / or immune tolerance. In some embodiments, the shRNA may correspond to SEQ ID NO: 13.

[0123] As seen in Figure 6, modification of CARG-201 with either secreted core antigen or shRNA for PD-L1 or both generally increased immunogenicity in naive mice, suggesting that the efficacy of these vaccines may be enhanced in chronic models of HBV. The ability to modify CARG-301 to express these variant constructs in vitro greatly enables the exploitation of these constructs for immunogenicity and efficacy in animal models (Figure 7).

[0124] (shRNA inhibits PD-L1 expression in stably transfected BHK21 cells in vitro (Figure 8)). The incomplete inhibition of PD-L1 by shRNA in BHK-21 cells may be due to steric hindrance caused by the proximal juxtaposition of three RNA loop structures. These structures interfere with transcription and at the same time inhibit the shRNA transcript from being properly processed into functional siRNA by Dicer. To overcome this problem, we incorporated either one copy of shRNA or two copies of shRNA separated and driven by two subgenomic promoters (Figure 9). A single copy of PD-L1-specific shRNA is more effective at suppressing expression than two or more copies. Taken together, these data indicate that shRNA delivery by VLV can inhibit PD-L1 expression in vitro, suggesting that it is possible to block PD-1 / PD-L1 interaction in vivo. Delivery of shRNA carried on VLV-CARG-101(3xT2A) to block PD-L1 expression raises the exciting possibility that anti-PD-L1 shRNA combined with immunotherapy may be a superior therapeutic strategy for the treatment of CHB infection.

[0125] CARG-201-mediated reduction of serum biomarkers enhances PD-1 expression in a chronic HBsAg AAV-HBV model + / CD8 + The PD-L1 expression correlates with a decrease in the HBV-specific T cell population (Figure 10). We have shown that shRNA delivery by CARG-201 can inhibit PD-L1 expression in vitro, suggesting that it is possible to block PD-1 / PD-L1 interaction in vivo. Delivery of shRNA carried on CARG-301 to block PD-L1 expression raises the exciting possibility that anti-PD-L1 shRNA combined with immunotherapy is a good therapeutic strategy for the treatment of CHB infection. Fusion of PD-L1 shRNA to CARG-301 could improve the efficacy of CARG-301 to 100% in a high stringency AAV-HBV efficacy model to activate exhausted HBV-specific T cells (Figure 7).

[0126] (CARG-2020 construct expressing both rIL-12 and PD-L1 shRNA downregulates the expression of multiple immune checkpoints (Figure 11). The shRNA not only inhibits the expression of PD-1 ligands (PD-L1 and PD-L2), but also blocks the T cell co-inhibitory receptors PD-1, CTLA-4, LAG-3 and TIGIT. These immune receptors expressed on activated or exhausted T cells attenuate T cell effector function by diverse inhibitory signaling pathways. Therefore, HBV immunotherapy that simultaneously targets both PD-1 ligands as well as other redundant signaling pathways (e.g., CTLA4 and LAG-3) may provide clinical utility by increasing therapeutic efficacy. Studies in mouse models and other mechanistic studies indicate that these approaches may act in a complementary manner and thus increase therapeutic efficacy. Therefore, incorporation of shRNAs into CARG-301 and their ability to secrete antigens is likely to dramatically improve the immunogenicity and efficacy of CARG-301 in vivo.

[0127] (CARG-201 delivers transgenes for two HBV antigens (MHBs and HBc): Enables robust expression and secretion of HBV middle S antigen and HBV core antigen in vitro Induce a broad immune response ->2-log reduction in HBV marker surface antigens in AAV models Eliminate the virus as a monotherapy or in combination with standard antiviral therapy.

[0128] (Modification of antigen design of CARG-201 and CARG-301): Addition of a secretory signal to the core antigen in CARG-201 to enhance antigen expression Addition of secretory signals to the core and polymerase antigens in CARG-301 Incorporation of a PD-L1 shRNA cassette from CARG-2020 into CARG-201 and CARG-301 to increase immunogenicity and overcome immune exhaustion and / or immune tolerance Modification of CARG-201 with shRNA for secreted core antigen or PD-L1 appears to increase immunogenicity in naive mice, as evidenced by increased frequencies of HBV-specific T cells. · There are no apparent additive or synergistic effects of the modifications in CARG-201.

[0129] As seen in Table 2, we have completed the generation of CARG-301 (secCARG-301) that secretes all three antigens. We have also engineered CARG-301 to incorporate shRNA alone (CARG-301.sh) or both secretion signal and shRNA (secCARG-301.shNA). We are currently testing the immunogenicity of these constructs and prioritizing them for efficacy studies in chronic mouse models of HBV infection. The availability of constructs in both serotypes allows us to use prime-boost regimes, if necessary. (Table 2) [Table 2-1] [Table 2-2]

[0130] To demonstrate the utility of the VLV platform for larger scale production, we compared CARG-201 titers obtained with a protocol using BHK-21 cells transfected with Lipofectamine to those obtained with a modified protocol using BHK-21 cells transfected with PEIPro transfection reagent (suitable for production under cGMP guidelines) and HEK293T cells transfected with either reagent. The primary objective was to demonstrate that CARG-201 can be produced in 293T cells, a substrate cell line used by industry for the production of viral vectors. The table is divided into three parts and shows the titers and productivity at harvest, after filtration, and after concentration by ultrafiltration. Figure 14 shows the productivity values ​​obtained for CARG-201 (y-axis is in units of PFU / cm in different tissue culture flasks). 2 (having).

[0131] In embodiments, lipofectamine or PEIPro may be advantageous over other transfection agents. Lipofectamine is a transfection reagent that includes lipid subunits that assemble with transfection constructs (e.g., DNA plasmids) to form liposomes. Nucleic acid molecules have negative charges, and the liposomes that form have a positive net surface charge that overcomes electrostatic repulsion by negatively charged cell membranes. PEIPro is another transfection reagent that is a polymeric reagent (containing polyethyleneimine) that condenses DNA into positively charged particles.

[0132] From the results in Table 3, it can be seen that transfection with PEIPro can be optimized to produce improved titers, yields, and productivity compared to the Lipofectamine protocol. Thus, in some embodiments, PEIPro as a transfection reagent can be particularly advantageous in methods for producing VLVs (e.g., CARG-201). Table 3. Process Development Studies. VLV Quantification for Various CARG-201-1 VLV Production Conditions [Table 3]

[0133] (References) [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0134] (Incorporated by reference) The entire disclosures of each of the patent documents, patent application documents, scientific papers, government reports, websites, and other references mentioned herein, including certificates of correction, are incorporated herein by reference in their entirety for all purposes. In the event of a conflict in terminology, the present specification will control.

[0135] (Equivalent) The present invention may be embodied in other specific forms without departing from the spirit or essential characteristics of the present invention. The above-described embodiments should be considered in all respects as illustrative rather than limiting on the invention described herein. In various embodiments of the compositions and methods of the present invention, when the term comprising is used in reference to the composition or a described step of the method, it is also intended that the composition and method consist essentially of or consist of the described composition or step or component. Furthermore, it should be understood that the order of steps or the order of performing certain operations is not important so long as the invention remains operable. Moreover, two or more steps or operations may be performed simultaneously.

[0136] As used herein, the singular forms also include the plural forms unless the context clearly indicates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.

[0137] Moreover, it should be recognized that in certain cases the composition may be described as being composed of its components prior to mixing or further processing steps, such as drying, debinding, heating, sintering, etc. It is recognized that certain components may be further reacted or transformed into new materials.

[0138] All percentages and ratios used herein are by volume (vol / vol) or weight (weight / weight) as shown or otherwise indicated.

Claims

1. A high titer hybrid viral vector for the treatment, prophylaxis or prevention of an infectious disease or cancer, comprising the following operably linked sequence elements: a) a first DNA sequence comprising a DNA promoter sequence; b) a second DNA sequence encoding an alphavirus nonstructural protein polynucleotide sequence; c) a third DNA sequence encoding at least two alphavirus subgenomic promoters; d) a fourth DNA sequence comprising at least two sequence domains, each of said at least two sequence domains comprising: i) a sequence domain that encodes an antigen associated with an infectious disease, or ii) a sequence domain encoding an antigen associated with cancer, selected from a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA), including combinations thereof; a fourth DNA sequence independently comprising: e) a fifth DNA sequence encoding a vesiculovirus glycoprotein; A vector comprising:

2. 2. The vector of claim 1, wherein the antigen associated with an infectious disease is one or more of HBV antigens selected from the group consisting of core (HBcAg) antigen, middle (M) surface HBs antigen, large (L) surface HBs antigen, small (S) surface HBs antigen, HBeAg antigen, and HBx antigen.

3. 2. The vector of claim 1, wherein the antigen associated with cancer is one or more of a TSA or a TAA.

4. At least 1 x 10 per mL of virus-like vesicles (VLVs) ９ The vector of claim 1 , which provides a titer of plaque forming units (pfu).

5. The vector of claim 4, wherein the VLV is obtained by purification and concentration using centrifugation, ultrafiltration, tangential flow filtration, high speed centrifugation, chromatography, or other methods.

6. The vector of claim 1 , wherein each of the at least two sequence domains contained in the fourth DNA sequence is a sequence domain encoding an antigen associated with an infectious disease.

7. The vector of claim 1 , wherein each of the at least two sequence domains contained in the fourth DNA sequence is a sequence domain encoding an antigen associated with cancer.

8. 2. The vector of claim 1, wherein the TSA or TAA is one or more of alpha-fetoprotein, melanoma-associated antigen, CD44 glycoprotein, aspartate beta-hydroxylase, carcinoembryonic antigen, and a cancer-specific TSA or cancer-specific TAA.

9. 2. The vector of claim 1, wherein the fourth DNA sequence comprises one or more polynucleotide sequences each independently having at least about 80% homology to a sequence according to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:6, or SEQ ID NO:

8.

10. 10. The vector of claim 9, wherein the fourth DNA sequence comprises one or more polynucleotide sequences each independently having at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% homology to a sequence according to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:6, or SEQ ID NO:

8.

11. 10. The vector of claim 9, wherein each of the one or more sequence domains encoding a human short hairpin RNA (shRNA) of the fifth DNA sequence independently comprises a polynucleotide sequence having at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% homology to a sequence according to SEQ ID NO:13, SEQ ID NO:22, or SEQ ID NO:

27.

12. A high titer hybrid viral vector for producing virus-like vesicles (VLV) for the treatment, prophylaxis or prevention of an infectious disease or cancer, comprising the following operably linked sequence elements: a) a first DNA sequence comprising a DNA promoter sequence; b) a second DNA sequence encoding an alphavirus nonstructural protein polynucleotide sequence; c) a third DNA sequence encoding at least two alphavirus subgenomic promoters; d) a fourth DNA sequence comprising at least two sequence domains, each of said at least two sequence domains comprising: i) a sequence domain that encodes an antigen associated with an infectious disease, or ii) a sequence domain encoding an antigen associated with cancer, selected from a tumor specific antigen (TSA) or a tumor associated antigen (TAA), including combinations thereof; a fourth DNA sequence independently comprising: e) a fifth DNA sequence comprising one or more heterologous secretory signal sequences and one or more sequence domains encoding human short hairpin RNAs (shRNAs); and f) a sixth DNA sequence encoding a vesiculovirus glycoprotein; A vector comprising:

13. 13. The vector of claim 12, wherein the antigen associated with an infectious disease is one or more of HBV antigens selected from the group consisting of core (HBcAg) antigen, middle (M) surface HBs antigen, large (L) surface HBs antigen, small (S) surface HBs antigen, HBeAg antigen, and HBx antigen.

14. At least 1 x 10 per mL of virus-like vesicles (VLVs) ９ The vector of claim 12, which provides a titer of plaque forming units (pfu).

15. The vector of claim 14, wherein the VLV is obtained by purification and concentration using centrifugation, ultrafiltration, tangential flow filtration, high speed centrifugation, chromatography, or other methods.

16. The vector of claim 12, wherein each of the at least two sequence domains contained in the fourth DNA sequence is a sequence domain encoding an antigen associated with an infectious disease.

17. The vector of claim 12, wherein each of the at least two sequence domains contained in the fourth DNA sequence is a sequence domain encoding an antigen associated with cancer.

18. 13. The vector of claim 12, wherein the TSA or TAA is one or more of alpha-fetoprotein, melanoma associated antigen, CD44 glycoprotein, aspartate beta-hydroxylase, carcinoembryonic antigen, and a cancer-specific TSA or cancer-specific TAA.

19. 13. The vector of claim 12, wherein each of the one or more sequence domains encoding a human short hairpin RNA (shRNA) of the fifth DNA sequence independently comprises a polynucleotide sequence having at least about 80% homology to a sequence according to SEQ ID NO: 13, SEQ ID NO: 22, or SEQ ID NO:

27.

20. 13. The vector of claim 12, wherein each of the one or more sequence domains encoding a human short hairpin RNA (shRNA) of the fifth DNA sequence independently comprises a polynucleotide sequence having at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% homology to a sequence according to SEQ ID NO: 13, SEQ ID NO: 22, or SEQ ID NO:

27.

21. 13. The vector of claim 12, wherein the fourth DNA sequence comprises one or more polynucleotide sequences each independently having at least about 80% homology to a sequence according to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:6, or SEQ ID NO:

8.

22. 22. The vector of claim 21 , wherein the fourth DNA sequence comprises one or more polynucleotide sequences each independently having at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% homology to a sequence according to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:6, or SEQ ID NO:

8.

23. 13. The vector of claim 12, wherein the fourth DNA sequence comprises a polynucleotide sequence encoding one or more of IL-12 or IL-17.

24. 13. The vector of claim 12, wherein the fifth DNA sequence comprises a polynucleotide sequence encoding one or more shRNA sequences targeting PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, TIGIT, CD90, BTLA, CD160, or PD-1.

25. 13. The vector of claim 12, wherein the fourth DNA sequence comprises a polynucleotide sequence encoding one or more cytokine agonist or cytokine antagonist polypeptides that target IL-2, IL-7, IL-15, IL-18, IL-19, IL-35, IL-21, GM-CSF, IL-17, or Flt3L.

26. 13. The vector of claim 12, wherein the fourth DNA sequence comprises a polynucleotide sequence encoding one or more polypeptide modulators of a malignancy-associated target selected from PD-L1, CXCL1, or CXCL2.

27. 13. The vector of claim 12, wherein the fifth DNA sequence comprises a polynucleotide sequence encoding one or more shRNA modulators of a malignancy-associated target selected from PD-L1, CXCL1, or CXCL2.

28. A virus-like vesicle (VLV) containing a replicon RNA produced by the high-titer hybrid viral vector according to any one of claims 12 to 27.

29. A composition comprising virus-like vesicles (VLVs) produced by the high-titer hybrid viral vector of any one of claims 12 to 27.

30. 1. A method of treating and / or preventing an infectious disease or cancer in a mammalian subject, comprising:

30. Administering a therapeutically effective amount of the composition of claim 29 to a mammalian subject in need thereof. A method comprising:

31. 1. A method of immunizing a mammalian subject against an infectious disease or cancer, comprising:

30. Administering a therapeutically effective amount of the composition of claim 29 to a mammalian subject in need thereof. A method comprising:

32. 1. A method for downregulating a gene associated with an infectious disease or cancer, comprising:

30. Administering a therapeutically effective amount of the composition of claim 29 to a mammalian subject in need thereof. A method comprising:

32. The method of any one of claims 30 to 32, wherein the mammalian subject is a human or an animal.

33. The method according to any one of claims 30 to 32, wherein the infection is a Hepatitis B virus infection.

34. 30. Use of the composition of claim 29 in the manufacture of a medicament for the treatment, prophylaxis, or prevention of an infection or cancer in a mammalian subject in need thereof.

35. 35. The use according to claim 34, wherein the mammalian subject is a human or an animal.

36. 35. The use according to claim 34, wherein the infection is a Hepatitis B virus infection.

37. 1. A method of making virus-like vesicles (VLVs) for the treatment, prevention, or prophylaxis of an infectious disease or cancer, comprising: a) generating a high titer viral vector comprising at least two alphavirus subgenomic promoters, at least two sequence domains, one or more heterologous secretory signal sequences, and one or more sequence domains encoding human short hairpin RNAs (shRNAs), each of said at least two sequence domains independently comprising a sequence domain encoding an antigen associated with an infectious disease, a sequence domain encoding a polymerase antigen (Pol), a sequence domain encoding an antigen associated with a cancer selected from a tumor specific antigen (TSA) and a tumor associated antigen (TAA), including combinations thereof; b) transfecting BHK-21 cells or HEK293T cells with the high titer viral vector of step (a). c) incubating the transfected BHK-21 or HEK293T cells of step (b) in a buffer solution for a suitable time and at a suitable temperature to allow VLVs to propagate; and d) isolating the VLVs from the BHK-21 or HEK293T cells and buffer solution by a technique selected from the group consisting of ultrafiltration, centrifugation, tangential flow filtration, affinity purification, ion exchange chromatography, and combinations thereof; Including, The method of claim 1, wherein said isolating step of step (d) produces high titer VLV.

38. 38. The method of claim 37, wherein said high titer viral vector further comprises a DNA promoter sequence and a DNA sequence encoding an alphavirus nonstructural protein polynucleotide sequence.

39. 38. The method of claim 37, wherein the antigen associated with the infection is one or more of HBV antigens selected from the group consisting of core (HBcAg) antigen, middle (M) surface HBs antigen, large (L) surface HBs antigen, small (S) surface HBs antigen, HBeAg antigen, and HBx antigen.

40. 38. The method of claim 37, wherein the cancer associated antigen is one or more of a TSA or a TAA.

41. Step b) comprises the following substeps: b1) combining the vector with a transfection reagent; and b2) transfecting BHK-21 cells or HEK293T cells with the combined vector and reagent of step (b1).

38. The method of claim 37, comprising:

42. 42. The method of claim 41, wherein the transfection reagent is pofectamine or PEIpro.

43. 42. The method of claim 41, wherein the transfection agent is lipofectamine.

44. 43. The method of claim 42, wherein the transfection reagent is PEIpro.

45. The high titer is at least 1×10 ８ 44. The method of claim 43, wherein the PFU / mL.

46. The high titer is at least about 1×10 ９ 45. The method of claim 44, wherein the PFU / mL.

47. 13. The vector of claim 1 or claim 12, wherein the infectious disease is HIV, Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV), SARS-CoV-1, SARS-CoV-2, Lyme disease, Escherichia coli O157:H7 (E. coli) infection, Hantavirus infection, Dengue fever, West Nile virus infection, Zika virus infection, Plasmodium infection (malaria), tuberculosis, cholera, whooping cough, influenza, pneumococcal infection, gonorrhea, HSV, HPV, RSV, Hepatitis B virus infection, Hepatitis C virus infection, Tick-borne encephalitis virus infection, Chikungunya virus infection, Yellow fever, Clostridium difficile infection, or Staphylococcal enterotoxin B infection.

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