Multifunctional virus-like vesicle (VLV) platforms for infectious disease and cancer immunotherapy applications

Abstract

The present invention provides compositions and methods for therapeutic immunization for the treatment of infectious diseases and / or cancer. The methods of the invention include methods of generating high titer infectious agents and cancer vectors, methods of treating and / or preventing cancer or infection by infectious diseases, and methods of inducing memory T and B cell immune responses against infectious agents and cancer in subjects administered the VLV compositions thus produced. In addition, the present invention encompasses pharmaceutical compositions for vaccinating a subject to protect the subject from cancer or infectious agents.

Description

[0001] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No. 2R44DK113858 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 within the National Institutes of Health (NIH). The government has certain rights in this invention.

[0003] Statement regarding sequence listing

[0004] The sequence listing associated with this application is provided electronically in .xml format and is hereby incorporated by reference into the specification. The sequence listing is provided as a file entitled 25133-100410_sl.xml, created on February 21, 2023, and is approximately 83kB in size.

[0005] CROSS-REFERENCE TO RELATED APPLICATIONS

[0006] This international patent application claims priority to U.S. Provisional Patent Application No. 63 / 312,816, filed on February 22, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0007] The present invention relates to the discovery of compositions and methods of therapeutic platforms for the treatment of infectious diseases and cancers. The methods of the present invention include methods of generating high-titer hybrid VLV vectors, methods of treating and / or preventing infectious diseases and cancers; and methods of inducing memory T and B cell immune responses against infectious agents or cancers in subjects administered with the VLV compositions produced thereby. In addition, the present invention includes pharmaceutical compositions for vaccinating a subject to protect the subject from infection with an infectious disease or cancer. Background Art

[0008] Vaccination is one of the greatest public health success stories. Today, vaccines that prevent many viruses that once caused fatal childhood diseases are routinely used around the world. However, traditional vaccine development methods that use inactivated or attenuated viruses have failed for some of the most deadly human pathogens, so new approaches are needed. Advances in molecular virology have made genetic manipulation of viruses possible, which has opened up new opportunities for vaccine development. Genetic modification of viruses not only allows them to be attenuated, but also allows the incorporation of sequences from other viruses, transforming one pathogen into a vaccine vector for another pathogen. Viral vectors have been studied as potential tools for delivering vaccines because they show advantages over traditional vaccines in that they stimulate a wide range of immune responses, including antibodies (B cells), T helper cells (CD4+T cells), and cytotoxic T lymphocytes (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 against viral vectors that may inactivate the vaccine has been problematic, as has the limited cloning capacity of the transgene of interest. Many vaccine studies are in progress to improve the efficiency of this technology and overcome its limitations.

[0009] Enveloped RNA viruses have a highly organized structure. One or more nucleocapsid proteins encapsidate their RNA, a matrix protein is usually located between the capsid and the membrane, and one or more transmembrane glycoproteins interact with the matrix or nucleocapsid proteins 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 to allow the virus to enter new cells.

[0010] Alphaviruses such as Semliki Forest virus (SFV) are positive-stranded membrane enveloped RNA viruses that encode four nonstructural proteins, called nsP 1-4, and three structural proteins: capsid and E1 and E2 transmembrane glycoproteins. The nsp 1-4 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 the antigenomic RNA, which is then replicated to form full-length positive-strand RNA and subgenomic mRNA encoding structural proteins. The capsid protein encapsidates the genomic RNA in the cytoplasm to generate a nucleocapsid that buds from the cell surface in a membrane containing SFV glycoproteins. Alphavirus RNA replication occurs in a bulb-shaped membrane-bound compartment called a microsphere, which is initially formed on the cell surface and then endocytosed to form a cytopathic vacuole containing multiple microspheres. The replicase protein is localized to the cytoplasmic side of the microsphere. It is unclear how the RNA replicated in the microsphere is packaged into the nucleocapsid before SFV buds. Alphavirus RNA replicons lacking any structural protein genes can still replicate efficiently inside cells, but they cannot multiply outside cells.

[0011] Vesicular stomatitis virus (VSV) is a negative-strand RNA virus that encodes a single membrane glycoprotein (G), matrix protein and nucleocapsid protein and two proteins that form a viral polymerase. It is noteworthy that when cells are transfected with SFV RNA replicons that only encode SFV nonstructural proteins and VSV G proteins, infectious membrane envelope vesicles containing VSV G proteins are generated. These infectious virus-like vesicles (VLV) grow to low titers, but can be passaged in tissue culture cells like viruses. The particles contain replicon RNA and VSV G proteins, but unlike enveloped RNA viruses, they have very low density because they do not contain tightly packed nucleocapsid proteins around RNA. The mechanism for generating these VLVs has not yet been determined. These VLVs expressing other proteins have been shown to be useful as experimental vaccines. However, the VLV system has two obvious limitations as a vaccine platform: the relatively low titer of VLV and the rapid loss of foreign gene expression after passage. Obviously, it is necessary to produce a method for a more efficient viral vector vaccine system that supports stable foreign gene expression while generating high vaccine titers that induce a powerful immune response. Such vaccine systems may be used for cancer and / or infectious disease indications. The present invention satisfies this need.

[0012] Taking HBV immunotherapy as an example

[0013] Although current antiviral therapies for chronic hepatitis B virus (CHB) infection effectively reduce viremia, they rarely eliminate the virus. Therefore, new treatment options remain urgently needed for this serious disease. Because the human immune system is able to control HBV but often fails to do so, immunotherapy, including therapeutic vaccination, represents a promising approach to cure chronic CHB. However, although current HBV vaccine platforms generate potent antibody responses to prevent infection, they often do not generate the broad CD8 T cell responses required to eliminate the virus after infection. Therefore, novel vaccine delivery systems that can generate effective therapeutic immune responses to HBV are urgently needed. We have developed an immunotherapy based on our virus-like vesicle (VLV) platform for the treatment of patients with CHB infection.

[0014] We have determined that RNA replicon-based vectors or VLVs carrying RNA encoding one or more of the major HBV antigens [middle surface envelope glycoproteins (MHBs), hepatitis B core antigen (HBcAg), or polymerase] in a single open reading frame (CARG-101) in a polycistronic unit drive a broad, multispecific immune response that produces significant HBV clearance in the mouse liver. Treatment of mice chronically infected with adeno-associated virus (AAV)-HBV significantly reduced and in some animals eliminated serum HBV surface antigen (HBsAg), a surrogate biomarker for viral persistence in the liver. We significantly enhanced overall gene expression, which led to our next-generation clinical candidate, CARG-201, which induced both T cell responses and antibodies compared to CARG-101. In mice with low antigen load (HBsAg 低 In an AAV mouse model of CHB infection (100%), CARG-201 expressing MHBs and HBcAg under separate subgenomic promoters completely eliminated serum HBsAg in 100% of mice and reduced HBV RNA in the liver to undetectable levels. However, in a more stringent AAV-HBV model (HbsAg 高 ), CARG-201 only reduced HBsAg levels by 80%. Since high antigen load 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.

[0015] Modification of CARG-201 in any, or one or more of three complementary approaches improved efficacy and resulted in complete clearance of serum HBsAg levels in animals:

[0016] First, we incorporated the polymerase (Pol) antigen into CARG-201 to generate CARG-301 (expressing MHBs, HBcAg, and Pol). Vaccines that generate multi-antigen-specific T cells are better positioned to provide the desired therapeutic effect compared to one or two antigens. In addition, Pol is a highly immunogenic CD4 and CD8 T cell target, and due to its high sequence conservation, it prevents the generation of escape mutants in T cell epitopes.

[0017] Second, we engineered CARG-201 to incorporate the human IgK signal sequence of the polymerase (pol) gene and the VSV G glycoprotein signal sequence of the HBc gene. It is known that secretory proteins often lead to the activation of dendritic cells, enhanced HBV antigen presentation, and the generation of new cytotoxic T cell responses through epitope spreading. In this way, the quality and quantity of T cell responses against HBV antigens can be further enhanced compared to soluble and non-secreted counterparts. Secretory proteins also contribute to adaptive immune responses by being taken up by antigen-presenting cells and processed through the major histocompatibility complex (MHC) class II pathway.

[0018] Third, we also aimed to disrupt the programmed 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 in patients with other chronic infections. We predicted that disruption of the PD-1 / PD-L1 pathway would reactivate otherwise exhausted T cell function.

[0019] The combined effects of shRNA-mediated PD-L1 inhibition and improved HBV antigen secretion due to ER targeting endow these modified multivalent constructs with a superior therapeutic index necessary to clear the virus and prevent disease progression and mortality in CHB patients. Summary of the invention

[0020] A high titer VLV vector comprising a DNA sequence containing a promoter sequence, the promoter sequence being operably linked to a DNA sequence encoding a Semliki Forest virus (SFV) nonstructural protein nucleotide sequence, being operably linked to a SFV subgenomic RNA promoter, being operably linked to a DNA encoding an infectious disease or cancer-associated antigen or a fragment thereof, being operably linked to a 2A DNA encoding a 2A peptide, which in turn is operably linked to a vesicular stomatitis virus (VSV) G encoding a VSV G protein. DNA, wherein the SFV nonstructural protein nucleotide sequence comprises at least two 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, wherein the vector lacks the nucleotide sequence encoding the SFV structural protein, and further wherein when the vector is propagated in cell culture, at least 10 virus-like particles (VLV) per ml are obtained. 7 The titer is plaque forming units (pfu).

[0021] In an embodiment, the present disclosure provides a high titer hybrid viral vector for treating, preventing or controlling infectious diseases or cancer, comprising the following operably linked sequence elements:

[0022] a) a first DNA sequence comprising a DNA promoter sequence,

[0023] b) a second DNA sequence encoding a polynucleotide sequence of an alphavirus nonstructural protein,

[0024] c) a third DNA sequence encoding at least two alphavirus subgenomic promoters,

[0025] d) a fourth DNA sequence comprising at least two sequence domains, wherein each of the at least two sequence domains independently comprises:

[0026] i) a sequence domain encoding an antigen associated with an infectious disease, or

[0027] ii) a sequence domain encoding a cancer-associated antigen selected from a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA), including combinations thereof, and

[0028] e) A fifth DNA sequence encoding a glycoprotein of the Vesicular Virus.

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

[0030] In embodiments, the cancer associated antigen is one or more of a TSA or a TAA.

[0031] In an embodiment, at least 1×10 virus-like vesicles (VLV) per mL are obtained. 9 The titer is plaque forming units (pfu).

[0032] In embodiments, VLVs are obtained by purification and concentration using centrifugal ultrafiltration, tangential flow filtration, high-speed centrifugation, chromatography, or other methods.

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

[0034] In an embodiment, each of the at least two sequence domains included in the fourth DNA sequence is a sequence domain encoding an antigen associated with cancer.

[0035] In embodiments, 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 TAA.

[0036] In an 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.

[0037] In an embodiment, the fourth DNA sequence comprises one or more polynucleotide sequences, each of which independently has 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 with the sequence according to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:6 or SEQ ID NO:8.

[0038] In an embodiment, one or more sequence domains of the human short hairpin RNA (shRNA) of the fifth DNA sequence each independently include 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 the sequence according to SEQ ID NO: 13, SEQ ID NO: 22 or SEQ ID NO: 27.

[0039] In an embodiment, the present disclosure provides a high titer hybrid viral vector for producing a virus-like vesicle (VLV) for treating, preventing or combating an infectious disease or cancer, comprising the following operably linked sequence elements:

[0040] a) a first DNA sequence comprising a DNA promoter sequence,

[0041] b) a second DNA sequence encoding a polynucleotide sequence of an alphavirus nonstructural protein,

[0042] c) a third DNA sequence encoding at least two alphavirus subgenomic promoters,

[0043] d) a fourth DNA sequence comprising at least two sequence domains, wherein each of the at least two sequence domains independently comprises:

[0044] i) a sequence domain encoding an antigen associated with an infectious disease, or

[0045] ii) a sequence domain encoding a cancer-associated antigen, wherein the cancer-associated antigen sequence domain is selected from a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA), including combinations thereof,

[0046] e) a fifth DNA sequence comprising one or more heterologous secretion signal sequences; and one or more sequence domains encoding human short hairpin RNA (shRNA); and

[0047] f) The sixth DNA sequence encoding the glycoprotein of Vesicular Virus.

[0048] In an embodiment, the antigen associated with an infectious disease is one or more HBV antigens selected from the group consisting of core (HBcAg) antigen, middle (M) surface HB antigen, large (L) surface HB antigen, small (S) surface HB antigen, HBeAg antigen and HBx antigen.

[0049] In an embodiment, at least 1×10 virus-like vesicles (VLV) per mL are obtained. 9 The titer is plaque forming units (pfu).

[0050] In embodiments, VLVs are obtained by purification and concentration using centrifugal ultrafiltration, tangential flow filtration, high-speed centrifugation, chromatography, or other methods.

[0051] In an embodiment, each of the at least two sequence domains included in the fourth DNA sequence is a sequence domain encoding an antigen associated with an infectious disease.

[0052] In an embodiment, each of the at least two sequence domains included in the fourth DNA sequence is a sequence domain encoding an antigen associated with cancer.

[0053] In embodiments, 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 TAA.

[0054] In an embodiment, the one or more sequence domains of the human short hairpin RNA (shRNA) encoding the fifth DNA sequence each independently include 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.

[0055] In an embodiment, one or more sequence domains of the human short hairpin RNA (shRNA) of the fifth DNA sequence each independently include 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 the sequence according to SEQ ID NO: 13, SEQ ID NO: 22 or SEQ ID NO: 27.

[0056] In an 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.

[0057] In an embodiment, the fourth DNA sequence comprises one or more polynucleotide sequences, each of which independently has 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 with the sequence according to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:6 or SEQ ID NO:8.

[0058] In an embodiment, the fourth DNA sequence comprises a polynucleotide sequence encoding one or more of IL-12 or IL-17.

[0059] In an 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.

[0060] In an embodiment, the fourth DNA sequence comprises a polynucleotide sequence encoding one or more cytokine agonist or antagonist polypeptides targeting IL-2, IL-7, IL-15, IL-18, IL-19, IL-35, IL-21, GM-CSF, IL-17 or Flt3L.

[0061] In an embodiment, the fourth DNA sequence comprises a polynucleotide sequence encoding one or more polypeptide modulators of a target associated with a malignancy selected from PD-L1, CXCL1 or CXCL2.

[0062] In an embodiment, the fifth DNA sequence comprises a polynucleotide sequence encoding one or more shRNA modulators of a target associated with a malignancy selected from PD-L1, CXCL1 or CXCL2.

[0063] In embodiments, the present disclosure provides virus-like vesicles (VLVs) containing replicon RNA produced by the high-titer hybrid viral vectors described herein.

[0064] In embodiments, the present disclosure provides compositions comprising virus-like vesicles (VLVs) produced by the high-titer hybrid viral vectors described herein.

[0065] In embodiments, the present disclosure provides methods of treating and / or preventing an infectious disease or cancer in a mammalian subject, the method comprising administering to a mammalian subject in need thereof a therapeutically effective amount of a VLV composition.

[0066] In embodiments, the disclosure provides methods of immunizing a mammalian subject against an infectious disease or cancer, the method comprising administering to a mammalian subject in need thereof a therapeutically effective amount of a VLV composition.

[0067] In embodiments, the disclosure provides methods of down-regulating a gene associated with an infectious disease or cancer, the method comprising administering to a mammalian subject in need thereof a therapeutically effective amount of a VLV composition.

[0068] In embodiments, the mammalian subject is a human or an animal.

[0069] In an embodiment, the infectious disease is hepatitis B virus infection.

[0070] In an embodiment, the disclosure provides for use of a VLV composition as described herein in the preparation of a medicament for treating, preventing, or prophylaxis of an infectious disease or cancer in a mammalian subject in need thereof.

[0071] In embodiments, the mammalian subject is a human or an animal.

[0072] In an embodiment, the infectious disease is hepatitis B virus infection.

[0073] In an embodiment, the present disclosure provides a method for producing a virus-like vesicle (VLV) for treating, preventing or combating an infectious disease or cancer, the method comprising the steps of:

[0074] a) generating a high titer viral vector comprising at least two alphavirus subgenomic promoters; at least two sequence domains, each sequence domain independently comprising a sequence domain encoding the group consisting of: an antigen associated with an infectious disease, a polymerase antigen (Pol), an antigen associated with cancer selected from a tumor-specific antigen (TSA) and a tumor-associated antigen (TAA), including combinations thereof; one or more heterologous secretion signal sequences; and one or more sequence domains encoding a human short hairpin RNA (shRNA),

[0075] b) transfecting BHK-21 or HEK293 T cells with the high titer viral vector of step (a),

[0076] c) incubating the transfected BHK-21 or HEK293 T cells of step (b) in a buffer solution at a suitable temperature and for a suitable time to propagate VLV; and

[0077] d) separating VLV from BHK-21 or HEK293 T cells and a buffer solution by a technique selected from ultrafiltration, centrifugation, tangential flow filtration, affinity purification, ion exchange chromatography, and combinations thereof;

[0078] Wherein the separation in step (d) produces a high titer of VLV.

[0079] In an embodiment, the high titer viral vector further comprises a DNA promoter sequence and a DNA sequence encoding an alphavirus nonstructural protein polynucleotide sequence,

[0080] In an embodiment, the antigen associated with an infectious disease is one or more HBV antigens selected from the group consisting of core (HBcAg) antigen, middle (M) surface HB antigen, large (L) surface HB antigen, small (S) surface HB antigen, HBeAg antigen and HBx antigen.

[0081] In embodiments, the cancer associated antigen is one or more of a TSA or a TAA.

[0082] In an embodiment, step b) comprises the following sub-steps:

[0083] b1) combining the vector with a transfection reagent; and

[0084] b2) transfecting BHK-21 or HEK293 T cells with the combined vector and reagent of step (b1).

[0085] In embodiments, the transfection reagent is lipofectamine or PEIpro.

[0086] In embodiments, the transfection reagent is lipofectamine.

[0087] In embodiments, the transfection reagent is PEIpro.

[0088] In embodiments, high titer is at least 1×10 8 PFU / mL.

[0089] In embodiments, high titer is at least about 1 x 10 9 PFU / mL.

[0090] In an embodiment, 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, pertussis, 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 aureus enterotoxin B infection.

[0091] These and other aspects of the invention will become apparent from the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Various aspects and advantages of the present disclosure will become apparent from the following exemplary embodiments taken in conjunction with the accompanying drawings, in which:

[0093] Figures 1A to 1E Depicted are the effects of dp-HBc.MHs (CARG-201) and dp-MHs on HBsAg levels in the chronic AAV-HBV model. Figure 1A Depicted are exemplary schematics of single antigen (dp-MHs) and dual antigen (CARG-201) vectors, Figure 1B ELISA analysis of HBsAg (ng / mL) is depicted. Figure 1CDepicted is the qRT-PCR analysis of hepatic HBV RNA. Figure 1D Depicts intracellular staining of HBV-specific CD8 with IFNγ after stimulation with HBsAg or HBcAg peptide pools. + Flow cytometry of T cells, and Figure 1E Characterization of HBV-specific CD8 + ELISPOT of T lymphocytes.

[0094] Figure 2 Depicted is the control of HBV by the therapeutic vaccine candidate CARG-201 in prime-boost immunizations in mice with elevated pre-existing HBV antigen levels.

[0095] Figure 3A-3B The construction and expression of a VLV-based recombinant multivalent HBV vaccine is described. Figure 3A Exemplary regimens for CARG-201 and CARG-301 candidates are depicted, and Figure 3B Depicted is the expression of HBV genes as determined by Western blotting in BHK21 cell lysates.

[0096] Figures 4A-4C The therapeutic vaccine candidates CARG-201 and CARG-301 are depicted to control HBV in a prime-boost immunization in mice with high pre-existing HBV antigen levels. Figure 4A Mean (left panel) and individual (right panel; with mean bars) values ​​of HBsAg levels are plotted as a function of time. Figure 4B Serum anti-HBS at week 17 is depicted, and Figure 4C Serum alanine aminotransferase (ALT) levels are depicted as a function of time.

[0097] Figure 5 Depicted is an exemplary schematic of modified CARG-201 vaccine constructs with enhanced immunogenicity and efficacy by incorporating secretion signals and shRNA for PD-L1.

[0098] Figures 6A to 6B Depicted in childish Comparison of immunogenicity of modified CARG-201 variants in CB6F1 mice. Fig. 6A Splenic cellularity at day 7 after immunization is depicted; Figure 6B The frequencies of cytokine-producing T cells after polyclonal stimulation are depicted; Figure 6C The HBS peptide library is shown, and Fig.6D The HBC peptide library is shown.

[0099] Figure 7A-7BDepicted is the expression and secretion of a VLV-based recombinant modified CARG-301 multivalent HBV vaccine. Fig. 7A An exemplary scheme of a CARG-301 candidate construct is depicted, and Figure 7B Depicted is the expression of HBV genes as determined by western blotting in BHK21 cell lysates.

[0100] Fig. 8A –8C depicts inhibition of PD-L1 expression by shRNA in stably transfected BHK21 cells in vitro. Fig. 8A An exemplary scheme for a VLV therapeutic vaccine is depicted; Figure 8B The mouse cDNA clone of PD-L1 is depicted; Figure 8C Depicted are VLV produced by transfecting BHK21 cells with three versions of the shRNA 3XT2A construct and VLV-3XT2A without shRNA.

[0101] Figures 9A-9C Downregulation of PD-L1 with shRNA-VLV constructs is depicted. Fig. 9A Depicted are exemplary empty VLV constructs in which shRNAs are driven by one or two subgenomic promoters, Fig. 9B Western blot analysis of stable BHK21 cells constitutively expressing PD-L1 is depicted, and Fig. 9C Densitometric quantification of the blots after normalization relative to actin is depicted.

[0102] Figures 10A to 10B CARG-201 demonstrated efficacy in a more stringent AAV-HBV model for HBsAg 高 It significantly reduced serum HBsAg levels and induced core-specific T cells. Fig. 10A Figure 3 Serum HBsAg levels in mice transduced with AAV-HBV1.2-mer and fully established chronicity at week 8 (wk8) after which mice were divided into high antigen (HBsAg high) and low antigen (HBsAg low) groups. Fig. 10B Core-specific T cells and PD1 cells are depicted.

[0103] Figures 11A-11F Depicts that in vivo blockade of the PD-1 / PD-L1 pathway by shRNA significantly inhibited the expression of immune checkpoints and inhibitory receptors in MC38 tumor-bearing mice. CTLA 4 ( Fig.11A )、PD-L1( Fig. 11B )、Tigit( Fig. 11C )、PD-1( Fig.11D )、PD-L2( Fig.11E ) and Lag3( Fig.11F) gene expression.

[0104] Fig.12 Exemplary strategies to improve the efficacy of therapeutic HBV vaccines in animals and humans are described.

[0105] Fig.13 Depicted is an exemplary rationale for the development of optimized VLV candidates: a paradigm for therapeutic vaccines (immunotherapy) against HBV.

[0106] Fig.14 Productivity values ​​(PFU / cm2 in various tissue culture flasks) of CARG-201 are shown. DETAILED DESCRIPTION OF THE INVENTION

[0108] Platform Technology and Methods

[0109] The present disclosure relates to a general platform based on virus-like vesicles for infectious diseases and cancer immunotherapy applications. The VLV platform can generally be used to treat, prevent and / or prevent infectious diseases or cancer. In an embodiment, an exemplary 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, recurrent diseases and other infectious diseases. In an exemplary embodiment, 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 an exemplary embodiment, recurrent diseases may include Plasmodium (malaria), tuberculosis, cholera, pertussis, influenza, pneumococcal disease and gonorrhea. In an exemplary embodiment, 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 aureus enterotoxin B.

[0110] As described herein, hepatitis B virus (HBV) is exemplified as an infectious disease, however the full scope of the present disclosure is intended to cover each of new, recurrent and other infectious diseases. In further embodiments, the full scope of the present disclosure is intended to cover any infectious disease known to those skilled in the art.

[0111] The present disclosure also relates to cancer immunotherapy applications. For example, VLV can encode or produce one or more cytokine agonists, cytokine antagonists, short hairpin RNAs and / or other polynucleotides or polypeptides for treating, preventing and / or protecting malignant tumors or cancers. For example, VLV of the present disclosure can encode IL-12, IL-17, shRNA for one or more of PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, TIGIT, CD90, BTLA, CD160, PD-1, and / or cytokine agonists or antagonist polypeptides selected from one or more of IL-2, IL-7, IL-15, IL-18, IL-19, IL-35, IL-21, GM-CSF and IL-17, Flt3L. Platform VLV can alternatively or additionally downregulate genes associated with malignant tumors, such as PD-L1, CXCL1 and / or CXCL2, and other genes known to those skilled in the art.

[0112] As used herein, the term "target" can be used in conjunction with any biological target that can be used in the vectors, plasmids, pharmaceutical compositions and methods of the present disclosure. "Targeting" includes upregulating, downregulating, modulating, functionally acting as an agonist or antagonist, or interacting with a biological target in any manner that can cause a biological effect. Examples of agents encoded by the vectors described herein that "target" certain biological targets include polypeptides, polynucleotides, shRNAs, siRNAs, antibodies or antigen-binding fragments thereof, or any other useful agent that can be encoded by a vector and / or produced in a VLV.

[0113] Other embodiments disclosed herein will be apparent to those skilled in the art. U.S. Patent No. 9,987,353 to Robek et al., issued on June 5, 2018, U.S. Patent No. 10,435,712 to Rose et al., issued on October 8, 2019, and PCT / US2021 / 012834 filed on 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.

[0114] Background and Significance

[0115] HBV as an example: significance of the problem. HBV infection is a major global public health problem. Approximately 2 billion people worldwide have been infected with hepatitis B virus (HBV) during their lifetime, more than 240 million people are currently infected with HBV, and approximately 600,000 people die each year from HBV-related liver disease. Patients with chronic HVB (CHB) infection, including inactive HBV carriers, are at increased risk of developing cirrhosis, liver failure, and hepatocellular carcinoma (HCC). Although the majority of these patients will not develop HBV-related complications, 15-40% will develop severe complications during their lifetime. CHB has different clinical stages defined by HBV DNA titers, the presence of hepatitis B e antigen (HBeAg, a secreted form of the core protein), and the presence or absence of liver inflammation measured by liver transaminase levels. CHB infection occurs due to a continuous interaction between viral replication and the immune response. T cells are exhausted by continued antigen exposure, which contributes to the persistence of HBV infection. When T cells encounter HBV antigens presented by intrahepatic antigen-presenting cells (APCs) such as dendritic cells (DCs) and Kupffer cells, the co-stimulatory signals received by T cells are very weak. This leads to immune tolerance rather than functional activation. In addition, an immunosuppressive microenvironment with a high proportion of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) is formed in the liver of CHB patients. These provide inhibitory signals to T cells and interfere with T cell-mediated anti-HBV function.

[0116] Limitations of Current HBV Vaccines. Despite its success in preventing HBV infection, the current HBV vaccine (recombinant HBsAg adsorbed on alum) has a number of suboptimal properties. First, it does not induce protective antibody responses in all immunized individuals. Second, two to four doses of the vaccine are recommended to induce durable immunity. This requirement for repeated immunizations makes the vaccine somewhat challenging to administer in many parts of the world, especially those that lack adequate healthcare infrastructure. Third, protective antibody responses wane after immunization, falling below protective levels (>10 IU / L) in up to 60% of vaccinated individuals. Fourth, escape mutations in surface protein genes can produce virus that is resistant to the antibody response generated by the vaccine. Finally, as mentioned above, although it elicits a protective antibody response that prevents infection, the current vaccine does not generate a strong CD8 T cell response and, in clinical trials, has not been effective in controlling viral replication in people already infected with HBV.

[0117] Current treatments for CHB. The current standard of care for CHB includes antiviral and immune-boosting drugs, such as tenofovir, entecavir, and pegylated interferon, which are highly effective in slowing the disease, with only 8-12% of treated patients being cured. Discontinuation of antiviral therapy is often accompanied by a rebound in viral load; therefore, lifelong treatment is required. Although existing antiviral drugs can effectively suppress serum HBV viral load to undetectable levels, they generally fail to achieve seroclearance of hepatitis B surface antigen (HBsAg), which indicates eradication of HBV infection and is the ultimate goal of antiviral treatment for CHB. Failure to achieve HBsAg seroclearance may be due to the emergence of new drug-resistant HBV variants and covalently closed circular DNA (cccDNA) in residual infected hepatocytes. Since none of these clinical therapies can achieve long-term viral control in the majority of CHB patients, there is an urgent need to develop new therapies to improve HBsAg clearance and virological cure.

[0118] New approaches to HBV treatment. Failure of HBsAg seroclearance requires the development of new therapeutic strategies to achieve durable viral remission. One strategy is to directly target the virus by targeting viral entry, viral assembly / encapsidation, preS1 or hepatitis B surface antigen (HBsAg) secretion, envelope formation, and cccDNA. Another strategy is to interfere with host mechanisms by using Toll-like receptor (TLR) agonists, cytokines, and blockade of PD-1 / PD-L1. In addition, therapeutic vaccines based on recombinant HBV proteins or HBV envelope subviral particles, DNA, and T cell peptide epitopes provide another promising strategy for HBV eradication. Therapeutic vaccination aims to eliminate persistent viral infection by enhancing the patient's immune response. Individuals who are acutely infected but eventually clear the virus have relatively strong multispecific T cell responses to HBV. However, in those who are chronically infected, the magnitude of the T cell response is much weaker and directed to fewer viral antigens. This suboptimal immune response persists in chronically infected individuals despite the persistent presence of viral antigens in the liver and blood. Although current HBV vaccines induce potent antibody responses that prevent infection, they do not elicit virus-specific T cells required to control established infection. New technologies to generate effective T cell-dependent HBV immune responses are urgently needed. A promising approach to treat CHB is a therapeutic vaccine that can induce virus-specific CD8 T cells to clear HBV infection.

[0119] Functional cure of HBV. The ultimate goal of HBV treatment is a “functional cure”. According to the AASLD and EASL conference, functional cure is defined as the sustained loss of HBsAg in serum. In this case, although HBV cccDNA remains at low levels, functional adaptive immune responses ensure suppression of viral replication in the absence of treatment, similar to what occurs during acute HBV clearance. In acute infection, clearance of HBV requires a strong HBV-specific CD8 T-cell response, but in CHB, T-cell responses are dysfunctional and cannot be fully restored by NUC. As current treatments rarely achieve a functional cure, alternative treatments that can be given in shorter and limited courses are urgently needed. Chronic HBV infection is the result of a complex interaction between HBV and the host, and an impaired immune response to viral antigens is considered a key factor associated with the chronic HBV carrier state. If this state of immune tolerance can be overcome, the loss of HBeAg or HBsAg from the serum (seroclearance) and sustained control of HBV infection will be achieved.

[0120] Scientific Premise. Current standard-of-care therapies rarely result in functional cure, which is characterized by sustained HBsAg loss (with or without HBsAg antibody seroconversion). The goal of next-generation CHB therapies is to achieve higher functional cure rates within a limited treatment duration. To address this urgent need, we developed CHB-targeted shRNA therapeutics based on a VLV delivery platform. shRNAs can be developed as stand-alone treatments or can be co-developed with therapeutic vaccines to achieve functional cure. Since the human immune system can control HBV but often fails to do so, immunotherapy, including therapeutic vaccines, is a promising approach for treating CHB. However, therapeutic immune responses generated in persistent HBV infection are often weak due to CD8 T cell exhaustion. Virus-specific T cell exhaustion may play an important role in HBV persistence. The interaction between programmed death receptor-1 (PD-1) and its ligand PD-L1 on lymphocytes plays a key role in T cell exhaustion by inducing T cell inactivation, suggesting that the PD-1 / PD-L1 pathway is a good therapeutic candidate for chronic HBV infection. Woodchucks infected with woodchuck hepatitis virus (WHV) 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. Others have shown that in these animals, combination therapy with αPD-L1 and entecavir (ETV) improved control of viremia and antigenemia compared with ETV treatment alone. In addition, others have shown that PD-L1 blockade synergistically enhances HBV-specific CD4 T cells. In addition, there is growing evidence that immune checkpoint inhibitors can enhance ex vivo effector T cell responses in 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 inhibitory pathways, can restore the function of these impaired T cells and lead to functional cure.

[0121] Platform immunotherapy technology. We 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. VLV encodes the Semliki Forest virus (SFV) replicon and an additional structural protein, the vesicular stomatitis virus glycoprotein (VSV-G). After 50 generations of in vitro evolution in BHK-21 cells, which resulted in 10 amino acid changes in SFV nsP1-4, the evolved SFV non-structural proteins promoted high-titer VLV replication in cell culture by improving VLV release efficiency. The expression of VSV-G allows robust and pantropic infection because infectious vesicles composed of SFV replication spheres derived from spherical plasma membrane invaginations are coated with VSV-G proteins and bud from infected cells, spread to uninfected cells, and undergo multiple rounds of infection. VLV is non-pathogenic in mice and rhesus monkeys, has a low risk of genomic integration or reversion to pathogenesis, and is immunogenic in the absence of adjuvants. Recent improvements to the system allow the production of high-titer VLV particles and high gene expression, up to multiple subgenomic promoters. Although VLV mimics the immunostimulatory properties of viral vectors, they are safe and non-pathogenic when administered to mice or rhesus monkeys, and they do not exhibit neurotoxicity when injected directly into the mouse brain. These vectors are meaningful because of their efficacy, ease of producing high-titer particles, and predicted safety due to the lack of viral structural proteins. In addition, VLV has a proven strong ability to deliver nucleic acids to express several antigens, thereby inducing T cell and antibody responses to multiple epitopes of multiple antigens, and thus helps to maximize the potential efficacy of the proposed immunotherapy in patients.

[0122] Over the past 20 years, multiple studies have evaluated therapeutic vaccine candidates for CHB treatment using lipopeptide epitope-based vaccines, DNA-based vaccines, and adenoviral vector vaccines. To date, HBV therapeutic vaccination has been ineffective in reliably inducing functional cure in patients with CHB. The persistence of HBV-specific T-cell hyporesponsiveness and high baseline HBsAg loads in participants, as well as the limited T-cell immunogenicity of the vaccine candidates themselves, are possible reasons that may have hampered the success of these vaccine candidates. Other possibilities for failure include the use of HBsAg, which in healthy settings induces HBs antibodies that block viral entry and prevent infection, which may have minimal impact on T-cell induction and immune recovery in the chronic setting with protein vaccines alone. A large proportion of patients have HBeAg + Disease, which is associated with high HBV antigen levels. High antigen load has been proposed as a barrier to successful rescue of tolerized T cells by therapeutic vaccination.

[0123] The vector of the present invention can generally be a plasmid or other vector encoding a VLV. Therefore, the term "vector" includes plasmids. Plasmids can generally contain any elements required for VLV production. A vector or plasmid can be defined by one or more sequence domains or components, or by one or more sequences. Generally, unless the context is clear, a plasmid can contain additional sequence domains or components as necessary or desired. A sequence can be defined as a polynucleotide sequence or a corresponding amino acid sequence. Some sequence components (such as shRNA) may not have a corresponding amino acid sequence. Exemplary sequence domains are provided in Table 1 below:

[0124] Table 1: SEQ ID NOs. 1–30

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143] 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%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98% 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 the vector or plasmid comprises a sequence consisting of one or more of SEQ ID NOs: 1-15 or encoding one or more of SEQ ID NOs: 1-30, it is intended that the vector or plasmid may comprise additional sequence domains.

[0144] In an exemplary embodiment, the plasmid may have a polynucleotide sequence corresponding to SEQ ID NO: 16 or SEQ ID NO: 17. In an exemplary embodiment, the plasmid may have a polynucleotide sequence having greater than about 70%, or greater than 75%, or greater than 80%, or greater than 85%, or greater than 90%, or greater than 95%, or greater than 96%, or greater than 97%, or greater than 98%, or greater than 99% homology to SEQ ID NO: 16 or SEQ ID NO: 17. 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 the plasmid has a polynucleotide sequence consisting essentially of SEQ ID NO: 16 or SEQ ID NO: 17, it is expected that 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.

[0145] Where sequence "homology" or "identity" is considered, for a DNA sequence or an amino acid sequence, the same percentage "similarity" of the amino acid sequence or amino acid sequence corresponding to the DNA sequence is also considered. The term "similarity" differs from the term "identity" in that it allows for conservative substitutions of amino acid residues with similar physicochemical properties over a defined length of a given alignment. In general, any known reasonable similarity scoring matrix can be used to determine similarity.

[0146] When determining the sequence homology or identity of a first sequence compared to a second sequence, various identity calculations can be performed, such as those implemented in the Basic Local Alignment Algorithm 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 on the number of alignment positions.

[0147] VLV can generally be produced by transfecting any suitable cell line with a suitable plasmid or vector. In one embodiment, VLV is produced as follows: BHK-21 or HEK293T cells are transfected with a vector or plasmid, and the transfected BHK-21 or HEK293 T cells are incubated in a buffer solution at a suitable time and a suitable temperature to propagate VLV; and VLV is separated from BHK-21 or HEK293 T cells and buffer solution by a technique selected from ultrafiltration, centrifugation, tangential flow filtration, affinity purification, ion exchange chromatography, and combinations thereof. In various embodiments, VLV can be produced by any appropriate transduction, incubation, and separation methods.

[0148] The VLV produced can generally be used in therapeutic methods. The VLV produced can be formulated with one or more diluents, excipients or other ingredients into a vaccine composition for treating HBV. The composition can generally be administered by any suitable route, such as oral, parenteral, intravenous or other routes.

[0149] With reference to the embodiments presented herein, the drawings are described in more detail below.

[0150] FIG1 depicts the effects of dp-HBc.MHs (CARG-201) and dp-MHs on HBsAg levels in a chronic AAV-HBV model. Figure 1A Schematic diagrams of single antigen (dp-MHs) and dual antigen (CARG-201) vectors are depicted. Chronic HBV was established in C57BL / 6 mice using AAV2 / AAV8 delivery of the HBV genome. Six weeks after AAV-HBV transduction, HBsAg was balanced in each group before primary immunization with a control vector (expressing eGFP, n=6), dp-MHs (expressing HBV middle protein S antigen, n=8), or CARG-201 (expressing HBV core and middle protein S antigens (n=9)). Four weeks later, animals were boosted with a serotype-switched VLV vector. Figure 1B ELISA analysis of HBsAg (ng / mL) is depicted. Figure 1C qRT-PCR of hepatic HBV RNA is depicted. Figure 1D Figure 3. Intracellular staining of HBV-specific CD8 with IFNγ after stimulation with HBsAg or HBcAg peptide pools. +Flow cytometric analysis of T cells. Figure 1E Characterization of HBV-specific CD8 + ELISPOT analysis of T cells. Data are mean ± SEM. Asterisks indicate significant differences (p < 0.01) between the control group and VLV expressing HBV antigens. HBc = HBcAg (core); MHs = MHB (surface); dp = dual subgenomic promoter; SGP1 / 2, subgenomic promoter 1 or 2.

[0151] Figure 2 The therapeutic vaccine candidate CARG-201 controls HBV in mice during prime-boost immunization in mice with elevated 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 (100-500 ng / mL) than previously used. We used 1×10 11 Mice were transduced with AAV-HBV 1.2-mer containing 100 genome copies, and the group of animals with moderate to high (mean HBsAg ~3,000 ng / mL) HBsAg levels were selected as the immunized group. Sustained HBV replication was determined by measuring serum HBsAg levels 8 weeks after transduction. 8 Two groups of 12 mice were primed im with CARG-201 and VLV-GFP at 100 PFU / mouse and boosted 4 weeks later (Pre). CARG-201 induced a significant reduction in HBsAg, but not in GFP or PBS controls.

[0152] FIG. 3 depicts the construction and expression of a VLV-based recombinant multivalent HBV vaccine. Figure 3A Exemplary schematics of CARG-201 and CARG-301 candidates are depicted. The four nonstructural proteins of the Semliki Forest virus (SFV) replicase are named (nsp1-4). The terminal protein of the HBV polymerase (Pol) is deleted from the four domains that comprise the enzyme. Expression of Pol and core antigen is fused to the downstream gene via the T. cerevisiae virus 2A (T2A) ribosomal skipping site. Figure 3B Depicted is the expression of HBV genes as determined by western blot in BHK21 cell lysates. The expression of both 2 and 3 antigens was compared.

[0153] Figure 4 depicts the control of HBV by the therapeutic vaccine candidates CARG-201 and CARG-301 in prime-boost immunizations in mice with high pre-existing HBV antigen levels. CARG-201 contains 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 (mean HBsAg approximately 3000 ng / mL) of HBsAg were selected for the immunization groups. Sustained HBV replication was determined by measuring serum HBsAg levels 8 weeks after transduction. 8 Three groups of 10 mice were primed with CARG-201, CARG-301, and GFP at 100 PFU / mouse and boosted 4 weeks later (boost 1) and 6 weeks after the first boost (boost 2). Significant HBsAg reduction was observed in both CARG-201 and CARG-301, but neither the mean (left panel) nor the individual values ​​(right panel) were significant in the GFP control. NJ RNA replicon of serotype; boost 1, VSV G IN Serotype; boost 2, VSV G CH Serotype; NJ = New Jersey; IN = Indiana; CH = Chandipura.

[0154] Figure 5 An exemplary schematic diagram of a modified CARG-201 vaccine construct for enhanced immunogenicity and efficacy by incorporating a secretion signal and shRNA against PD-L1 is depicted. The nonstructural proteins of the Semliki Forest Virus (SFV) replicase are designated (nsp1-4). The secretion signal (ss) is derived from the VSV G glycoprotein. The secretory terminal protein is derived from the four domains comprising the enzyme.

[0155] FIG6 depicts a comparison of the immunogenicity of modified CARG-201 variants in naive CB6F1 mice,

[0156] FIG. 7 depicts the expression and secretion of the VLV-based recombinant modified CARG-301 multivalent HBV vaccine. Fig. 7AAn exemplary scheme of the CARG-301 candidate construct is depicted. The four nonstructural proteins of the Semliki Forest virus (SFV) replicase are named (nsp1-4). The terminal protein of the HBV polymerase (Pol) is deleted from the four domains comprising the enzyme. The human IgK signal sequence is fused to its N-terminus. The middle surface antigen (MHBs) and core antigen (HBcAg) have the native and heterologous VSV G signal sequence (ss) fused to their amino termini, respectively. The expression of Pol and core antigen is fused to the downstream gene through the ribosomal skipping site of the 2A (T2A) ribosomal skipping site of the HBV polymerase. Figure 7B The expression of HBV genes as determined by western blot in BHK21 cell lysates is depicted. The expression of the three antigens was compared. The culture supernatants were collected and the secretion of HBV antigens was determined. The secreted polymerase appeared to be rapidly degraded in the culture medium, as detected by only a faint band (indicated by a white asterisk *) detected after the culture supernatant was concentrated. Actin protein was detected in the cell lysate (lane 10), but not in the cell supernatant (lanes 1-9). Adding a mixture of protease inhibitors can inhibit degradation and enhance its detection ability. 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.

[0157] FIG. 8 depicts inhibition of PD-L1 expression by shRNA in stably transfected BHK21 cells in vitro. Fig. 8A Depicted is an exemplary scheme of a VLV therapeutic vaccine (VLV-3xT2A) carrying multiple PD-L1-specific shRNAs 5' and 3' to the VSV G glycoprotein. Expression of HBV major antigens (MHBs, HBcAg, and polymerase) in VLV is linked to VSV G via the picornavirus T2A ribosomal skipping site. Figure 8B Depicted is the cloning of the mouse cDNA clone CD274 / B7-H1 / PD-L1 ORF (Sino-biological Inc) of PD-L1 into the mammalian expression vector pCMV3-Flag-mCD274. BHK21 cells were transfected, and hygromycin-resistant clones were selected and expanded. The expression of PD-L1 in stable cells was analyzed by western blotting using anti-PDL1 antibody. Figure 8CDepicted are VLVs produced by transfecting BHK21 cells with three versions of shRNA3XT2A constructs. VLVs were produced and titers were determined using 0.1 MOI to infect BHK21:PD-L1 stable cell lines. Lysates were prepared by collecting cells after 8, 24, and 32 hours. Downregulation of PDL1 was analyzed by western blot and compared with uninfected cell lysates. Band intensities were standardized and quantified relative to β-actin by GelQuant.Net software (BiochemLabSolutions.com).

[0158] FIG. 9 depicts downregulation of PDL1 using shRNA-VLV constructs. Fig. 9A Depicted are exemplary schematics of empty VLV constructs in which shRNAs are driven by one or two subgenomic promoters. Fig. 9B Depicted is a western blot analysis of stable BHK21 cells constitutively expressing PD-L1. Cell lysates were prepared and analyzed 6 hours after infection. Fig. 9C Depicted are densitometry quantification of blots after normalization to actin. Scr; scrambled shRNA; sh373 and sh486; Position of shRNA relative to PD-L1 sequence (bp).

[0159] Figure 10 depicts the efficacy of CARG-201 in a more stringent AAV-HBV model (HBsAg 高 ) significantly reduced serum HBsAg levels and induced core-specific T cells. Fig. 10A The mice were transduced with AAV-HBV1.2-mer and chronicity was fully established at week 8 (wk8). The mice were then divided into high antigen (HBsAg 高 ) and low antigen (HBeAg 低 ). The mice were then immunized (prime) with the construct shown (prime-wk1). One month after the prime, the mice were boosted with the same construct in which the VSV G glycoprotein from a different serotype was switched. Fig. 10B Core-specific T cells and PD1 cells are shown. Single antigen (dp-GS) was as effective as CARG-201 (dp-CGS) in inducing HBV-specific T cells as measured by ELISPOT. Increasing HBV viral load increased the induction of core-specific T cells after immunization with dp-CGS as measured by intracellular cytokine staining of IFNγ+ in CD8+ T cells (left panel).

[0160] Figure 11 depicts that shRNA blocking the PD-1 / PD-L1 pathway in vivo significantly inhibited the expression of immune checkpoints and inhibitory receptors in MC38 tumor-bearing mice. CARG-2020 is a replicon vector carrying three immunomodulators (IL-12, DIL-17RA, and shRNA), while IL-12 and GFP are replicon vectors expressing rIL-12 (p35 and p40 subunits) and GFP, respectively. MC38 cells were implanted into C57BL / 6 mice (n=10) and 5×10 7 PFU of indicated vectors were injected 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 (Cat#MGK121) from ScienCell (Carlsbad, CA). Inhibitory receptors are depicted in A and D, receptor ligands are depicted in B and E, and other immune checkpoint genes are depicted in C and F.

[0161] Fig.12 Multiple strategies have been explored to improve the efficacy of therapeutic HBV vaccines in animals and humans. Strategies include (i) combining vaccine platforms in heterogeneous prime-boost regimens, (ii) optimizing vaccine immunogens by including multiple HBV antigens incorporating signal sequences, (iii) reversing T cell dysfunction using concomitant inhibition, inhibition of immune checkpoints by shRNA, and (iv) immunizing individuals with lower HBV antigen load by reducing antigen load prior to NUC treatment, siRNA inhibition, nucleic acid polymers (NAPs), or VLV vaccination. Example

[0162] The present invention will now be described with reference to the following examples. These examples are provided for illustrative purposes only, and the present invention should in no way be construed as being limited to these examples, but rather should be construed to encompass any and all variations that become evident as a result of the teaching provided herein.

[0163] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods.

[0164] Key points include:

[0165] Dual promoter (DP) or triple promoter (TP) VLV platforms promote higher gene expression and enhance immunogenicity

[0166] Construct CARG-201 drives complete clearance of HBsAg in mice with low antigenemia

[0167] CARG-201 significantly reduced serum biomarker levels and induced T cells in a stringent mouse model

[0168] CARG-201 and CARG-301 were able to break tolerance in hyperantigenemic mice and drive significant reductions in HBsAg in mice

[0169] Modification of CARG-201 with secreted core antigen or shRNA targeting PD-L1 increases immunogenicity in naive mice

[0170] · shRNA inhibits cells constitutively expressing PD-L1 in vitro. In vivo, CARG-2020 constructs expressing both IL-12 and PD-L1 shRNA downregulate PD-L1 + Expression of multiple immune checkpoints in a tumor cell line (MC38).

[0171] ER-targeted secretion signal sequence enhances secretion of HBV antigens in CARG-301

[0172] - Modified CARG-201 and CARG-301 constructs can be scaled up and produced.

[0173] We have determined that treatment of mice chronically infected with AAV-HBV with CARG-101 (a VLV carrying MHBs with only one HBV antigen) significantly reduced, and in some animals eliminated, serum HBsAg, a surrogate biomarker of viral persistence in the liver. Clearance of HBsAg in 40% of AAV-HBV mice with only a single CARG-101 immunization is a superior outcome compared to other HBV immunotherapies developed in the same animal model.

[0174] Notably, we have now achieved reductions in serum biomarker levels in >80% of hyperantigenemic AAV-HBV mice (n=10), suggesting that CARG-201 (HBcAg and MHBs: two HBV antigens) can break tolerance in a highly tolerant model. To achieve this reduction, we combined an enhanced gene expression strategy with a vigorous prime-boost regimen to achieve complete clearance of HBsAg in mice. Therefore, we selected CARG-201 for advancement to the clinic 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 HBV RNA in the liver to undetectable levels, and (iii) induction of multispecific HBV T cells and antibodies. The reduction of intrahepatic HBV RNA is likely the result of strong immune control under high levels of CD8+ and CD4+ T cell responses, as observed in patients with resolution of acute HBV infection (Figures 1 and 2).

[0175] CARG-201 delivers two HBV antigens (MHBs and HBc) transgenes :

[0176] Achieve robust expression and secretion of HBV S protein and core antigen in vitro

[0177] Induce a broad immune response

[0178] · Resulted in a 2-log reduction in HBV marker surface antigen in the AAV model

[0179] Eliminate the virus as monotherapy or in combination with standard antiviral therapy

[0180] We reasoned that the incorporation of a third antigen, such as polymerase (Pol), combined with prime-boost immunization, might generate a more robust, multispecific, and multifunctional T cell response that ultimately controlled 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 cleared the virus with similar efficacy and kinetics to CARG-201 (Figure 4). In some embodiments, the MHBs can be any known MHBs. In some embodiments, HBcAg can be SEQ ID NO: 10 (DNA), SEQ ID NO: 9 (amino acids). In some embodiments, the polymerase can be SEQ ID NO: 12 (DNA), SEQ ID NO: 10 (amino acids).

[0181] Fig.13is an exemplary understanding of how HBV immunotherapy can achieve a functional cure using CARG-201 or CARG-301. The genome of this partially double-stranded DNA hepatotropic virus uses four overlapping open reading frames to encode seven proteins - core (HBcAg), surface [large (L), medium (M) and small (S) HBs], HBeAg, polymerase and HBx. It is generally believed that a broad multi-specific immune response will be most beneficial. Therefore, in addition to evaluating the efficacy of a single antigen, we also tested the efficacy of multiple antigens delivered on a single vector. As described herein, the multiple antigen approach disclosed herein has several advantages over a single antigen construct.

[0182] Fig.13 An exemplary rationale for developing optimized VLV candidates is depicted: a paradigm for therapeutic vaccines (immunotherapy) against HBV. Therapeutic vaccines are currently being developed for multiple chronic viral infections such as HIV, HCV, HPV, and HSV. As an alternative to antiviral therapy or to support only partially effective standard HBV therapy, VLVs are designed to activate the patient's immune system to fight and ultimately control or even eliminate the virus. Although the success of preventive vaccination is based on the rapid neutralization of invading pathogens by antibodies, viral control and elimination of infected cells requires T cells. Therefore, in addition to activating humoral immune responses to limit viral spread, inducing multi-antigen-specific and multifunctional 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 effectively reducing viremia. HBV surface antigens (MHBs), core antigens (HBcAg), or polymerase (Pol) antigens in the primary vaccination stimulate HBV-specific CD4 T cells, which in turn leads to antibody production by HBV-specific B cells. This leads to the production of antigen-neutralizing antibodies, ideally resulting in seroconversion from HBsAg to anti-HBs. Vaccination also induces CD8 CTLs, which are able to kill infected hepatocytes, ultimately leading to viral clearance. HBsAg, hepatitis B surface antigen; anti-HBs, anti-HBsAg antibodies.

[0183] These data we generated establish that: (i) we can enhance antigen load with concomitant increases in immunogenicity by modifying VLV to carry two or more subgenomic promoters and (ii) VSV G serotype switching is an effective prime-boost strategy. Optimized single-antigen (MHBs) or dual-antigen (MHB and HBc) vectors can drive complete clearance of HBsAg in mice (Figure 1). The data establish that reductions in intrahepatic HBV RNA may be due to strong immune control in the presence of high-level CD8+ and CD4+ T-cell responses, as in patients who resolve acute HBV infection.

[0184] CARG-201 drives HBsAg clearance in hyperantigenemic mice Figure 2 ). The data established that CARG-201 can reduce serum biomarker levels in AAV-HBV mice with high antigenemia by >80%. Optimized two booster immunizations can drive complete clearance in highly antigenic mice (Figure 3). The data also established that serotype conversion is a highly effective prime-boost regimen to significantly reduce (>1log) serum biomarker levels in AAV-HBV mice with high antigenemia by >80%. The RNA replicon-based HBV therapeutic vaccine under development can induce CD8+ T cells against multiple antigenic epitopes in the tolerogenic environment of HCB infection, thereby meeting the needs of HBV immunotherapy. Further genetic manipulation of CARG-201 or CARG-301 will further reduce biomarker levels to >2-3 logs.

[0185] Modifications to VLV and antigen design ( Figure 5 and 7) :

[0186] Adding a secretion signal to the core antigen in CARG-201 to enhance antigen expression. In some embodiments, the secretion signal can be a VSV G secretion signal (e.g., SEQ ID NO: 6 (DNA), SEQ ID NO: 5 (amino acid)) or a human IgK secretion signal (e.g., SEQ ID NO: 8 (DNA), SEQ ID NO: 7 (amino acid)).

[0187] Adding a secretion signal to the core antigen and polymerase antigen in CARG-301. In some embodiments, the secretion signal can be a VSV G secretion signal (e.g., SEQ ID NO: 6 (DNA), SEQ ID NO: 5 (amino acid)) or a human IgK secretion signal (e.g., SEQ ID NO: 8 (DNA), SEQ ID NO: 7 (amino acid)).

[0188] Incorporation of the 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 tolerance. In some embodiments, the shRNA may correspond to SEQ ID NO: 13).

[0189] As shown in Figure 6, modification of CARG-201 with shRNAs for secreted core antigen or 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 and express these variant constructs in vitro enables the use of these constructs for immunogenicity and efficacy in animal models (Figure 7).

[0190] shRNA inhibits PD-L1 expression in stably transfected BHK21 cells in vitro (Figure 8). Incomplete inhibition of PD-L1 by shRNA in BHK-21 cells may be due to steric hindrance caused by the close juxtaposition of three RNA loop structures. These structures simultaneously interfere with transcription and inhibit the correct processing of shRNA transcripts into functional siRNAs by Dicer. To overcome this problem, we introduced one or two copies of shRNA separated and driven by two subgenomic promoters (Figure 9). A single copy of PD-L1-specific shRNA was more effective in eliminating expression than two or more copies. In summary, these data show that VLVs can deliver shRNA to inhibit PD-L1 expression in vitro, suggesting that it may be possible to block the PD-1 / PD-L1 interaction in vivo. Delivery of shRNA carried on VLV-CARG-101 (3×T2A) to block PD-L1 expression raises the exciting possibility that anti-PD-L1 shRNA combined with immunotherapy is an excellent therapeutic strategy for treating CHB infection.

[0191] CARG-201-mediated reductions in serum biomarkers correlate with PD-1+ / The decrease in CD8+ population is associated with (Figure 10). We have shown that shRNA delivered by CARG-201 can inhibit PD-L1 expression in vitro, suggesting that blocking the PD-1 / PD-L1 interaction in vivo is possible. The 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 an excellent therapeutic strategy for treating CHB infection. Fusion of PD-L1 shRNA to CARG-301 may increase CARG-301 efficacy to 100% in a high-stringency AAV-HBV efficacy model and activate HBV-specific exhausted T cells (Figure 7).

[0192] CARG-2020 construct expressing both rIL-12 and PD-L1 shRNA downregulates expression of multiple immune checkpoints (Figure 11). 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 inhibit T cell effector function through multiple inhibitory signaling pathways. Therefore, HBV immunotherapy that simultaneously targets PD-1 ligands as well as other redundant signaling pathways (such as CTLA4 and LAG-3) may provide clinical benefit by improving the therapeutic effect. Work in mouse models and other mechanistic studies has shown that these approaches can work complementary to improve the therapeutic effect. Therefore, it is very likely that the incorporation of shRNA into CARG-301 and the ability to secrete antigens will significantly improve the immunogenicity and efficacy of CARG-301 in vivo.

[0193] CARG-201 delivers a transgenic version of two HBV antigens (MHBs and HBc) :

[0194] Achieve robust expression and secretion of HBV S protein and core antigen in vitro

[0195] Induce a broad immune response

[0196] · Resulted in a 2-log reduction in HBV marker surface antigen in the AAV model

[0197] Eliminate the virus as monotherapy or in combination with standard antiviral therapy

[0198] Modifications in antigen design for CARG-201 and CARG-301:

[0199] Adding secretion signals to the core antigen in CARG-201 to enhance antigen expression

[0200] Adding secretion signals to the core and polymerase antigens in CARG-301

[0201] Incorporation of the PD-L1 shRNA cassette from CARG-2020 into CARG-201 and CARG-301 to improve immunogenicity and overcome immune exhaustion and / or tolerance

[0202] Modification of CARG-201 with secreted core antigen or shRNA against PD-L1 appeared to increase immunogenicity in naive mice, as evidenced by an increase in the frequency of HBV-specific T cells.

[0203] Modifications in CARG-201 have no apparent additive or synergistic effects

[0204] As shown in Table 2, we have completed the production of CARG-301 that secretes all three antigens (secCARG-301). We have also engineered CARG-301 to incorporate a single shRNA (CARG-301.sh) or both a secretion signal and shRNA (secCARG-301.shNA). We will now test the immunogenicity of these constructs and prioritize their use in efficacy studies in chronic mouse models of HBV infection. The availability of constructs in both serotypes will enable us to use a prime-boost regimen when necessary.

[0205] Table 2

[0206]

[0207] To demonstrate the utility of the VLV platform for manufacturing at a larger scale, we compared CARG-201 titers obtained using a protocol of BHK-21 cells transfected with Lipofectamine with a modified protocol of BHK-21 cells transfected with PEIPro transfection reagent (suitable for manufacturing according to 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 industrially for the production of viral vectors. The table is divided into three sections showing titers and productivity at harvest, after filtration, and after concentration by ultrafiltration. Fig.14 The productivity values ​​obtained for CARG-201 are shown (y-axis has PFU / cm in various tissue culture flasks). 2 units).

[0208] In embodiments, lipofectamine or PEIPro can be superior to other transfection agents. Lipofectamine is a transfection reagent containing a lipid subunit, which associates with a transfection construct such as a DNA plasmid to form a liposome. Nucleic acid molecules are negatively charged, and the formed liposomes have a net positive surface charge, which overcomes the electrostatic repulsion of negatively charged cell membranes. PEIPro is another transfection reagent that is a polymeric reagent (containing polyethyleneimine) that condenses DNA into positively charged particles.

[0209] As can be seen from the results in Table 3, transfection with PEIPro can be optimized to produce improved titers, yields and productivity compared to the lipofectamine protocol. Therefore, in some embodiments, PEIPro as a transfection reagent may be particularly advantageous in methods for producing VLVs such as CARG-201.

[0210] Table 3. Process development study. VLV quantification at different conditions for CARG-201-1 VLV production

[0211]

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[0287] Incorporated by Reference

[0288] The entire disclosure of each patent document, including certificates of correction, patent application documents, scientific articles, government reports, websites, and other references mentioned herein, is incorporated herein by reference in its entirety for all purposes. In the event of a conflict in terminology, the present specification controls.

[0289] Equivalent

[0290] Without departing from the spirit or essential features of the present invention, the present invention may be implemented in other specific forms. The foregoing embodiments are considered to be illustrative in all respects and are not intended to limit the invention described herein. In various embodiments of the compositions and methods of the present invention, when the term "comprising" is used for the steps of the compositions or the methods, it is also contemplated that the compositions and methods are substantially composed of or consist of the compositions or steps or components. In addition, it should be understood that the order of the steps or the order for performing certain actions is insignificant as long as the present invention remains operable. Moreover, two or more steps or actions may be performed simultaneously.

[0291] In this specification, singular forms also include plural forms, unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by ordinary technicians in the field to which the present invention belongs. In the event of a conflict, the present specification shall prevail.

[0292] Additionally, it will be appreciated that in some cases a composition may be described as consisting of components prior to mixing or prior to further processing steps such as drying, binder removal, heating, sintering, etc. It will be appreciated that certain components may be further reacted or converted into new materials.

[0293] All percentages and ratios used herein are on a volume (v / v) or weight (w / w) basis as indicated or otherwise indicated.

Claims

1. A high titer hybrid viral vector for treating, preventing or controlling infectious diseases 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 a polynucleotide sequence of an alphavirus nonstructural protein, c) a third DNA sequence encoding at least two alphavirus subgenomic promoters, d) a fourth DNA sequence comprising at least two sequence domains, wherein each of the at least two sequence domains independently comprises: i) a sequence domain encoding an antigen associated with an infectious disease, or ii) a sequence domain encoding a cancer-associated antigen selected from a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA), including combinations thereof, and e) A fifth DNA sequence encoding a glycoprotein of the Vesicular Virus.

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

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

4. The vector of claim 1, wherein at least 1 x 10 9 Titer of plaque forming units (pfu).

5. The vector of claim 4, wherein the VLV is obtained by purification and concentration using centrifugal ultrafiltration, tangential flow filtration, high-speed centrifugation, chromatography or other methods. The vector of claim 1 , wherein each of the at least two sequence domains included 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 included in the fourth DNA sequence is a sequence domain encoding a cancer-associated antigen.

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

9. 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. The vector of claim 9, wherein the fourth DNA sequence comprises one or more polynucleotide sequences, each of which independently has 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 the sequence according to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO:

8.

11. The vector of claim 9, wherein one or more sequence domains of the human short hairpin RNA (shRNA) encoding the fifth DNA sequence each independently comprise 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 the 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 a virus-like vesicle (VLV) for treating, preventing or controlling infectious diseases 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 a polynucleotide sequence of an alphavirus nonstructural protein, c) a third DNA sequence encoding at least two alphavirus subgenomic promoters, d) a fourth DNA sequence comprising at least two sequence domains, wherein each of the at least two sequence domains independently comprises: i) a sequence domain encoding an antigen associated with an infectious disease, or ii) a sequence domain encoding a cancer-associated antigen selected from a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA), including combinations thereof, e) a fifth DNA sequence comprising one or more heterologous secretion signal sequences; and one or more sequence domains encoding human short hairpin RNA (shRNA); and f) The sixth DNA sequence encoding the glycoprotein of Vesicular Virus.

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

14. The vector of claim 12, wherein at least 1 x 10 9 Titer of plaque forming units (pfu).

15. The vector of claim 14, wherein the VLV is obtained by purification and concentration using centrifugal 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 included 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 included in the fourth DNA sequence is a sequence domain encoding a cancer-associated antigen.

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

19. The vector of claim 12, wherein the one or more sequence domains encoding the human short hairpin RNA (shRNA) of the fifth DNA sequence each independently comprise 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. The vector of claim 12, wherein one or more sequence domains of the human short hairpin RNA (shRNA) encoding the fifth DNA sequence each independently comprise 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 the sequence according to SEQ ID NO: 13, SEQ ID NO: 22, or SEQ ID NO:

27.

21. 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. The vector of claim 21, wherein the fourth DNA sequence comprises one or more polynucleotide sequences, each of which independently has 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 the sequence according to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO:

8.

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

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

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

28. Virus-like vesicles (VLV) containing replicon RNA produced by the high-titer hybrid viral vector of any one of claims 12-27.

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

30. A method for treating and / or preventing an infectious disease or cancer in a mammalian subject, the method comprising administering to a mammalian subject in need thereof a therapeutically effective amount of the composition of claim 29.

31. A method of immunizing a mammalian subject against an infectious disease or cancer, the method comprising administering to a mammalian subject in need thereof a therapeutically effective amount of the composition of claim 29.

32. A method for down-regulating a gene associated with an infectious disease or cancer, the method comprising administering to a mammalian subject in need thereof a therapeutically effective amount of the composition of claim 29.

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

34. The method of any one of claims 30-32, wherein the infectious disease is hepatitis B virus infection.

35. Use of the composition of claim 29 in the preparation of a medicament for treating, preventing or prophylacticing an infectious disease or cancer in a mammalian subject in need thereof.

36. The use of claim 34, wherein the mammalian subject is a human or an animal.

37. The use of claim 34, wherein the infectious disease is hepatitis B virus infection.

38. A method for producing a virus-like vesicle (VLV) for treating, preventing or treating an infectious disease or cancer, the method comprising the steps of: a) generating a high titer viral vector comprising at least two alphavirus subgenomic promoters; at least two sequence domains, each sequence domain independently comprising a sequence domain encoding the group consisting of: an antigen associated with an infectious disease, a polymerase antigen (Pol), an antigen associated with cancer selected from a tumor-specific antigen (TSA) and a tumor-associated antigen (TAA), including combinations thereof; one or more heterologous secretion signal sequences; and one or more sequence domains encoding a human short hairpin RNA (shRNA), b) transfecting BHK-21 or HEK293 T cells with the high titer viral vector described in step (a), c) incubating the transfected BHK-21 or HEK293 T cells of step (b) in a buffer solution at a suitable temperature and for a suitable time to propagate VLV; and d) separating VLV from BHK-21 or HEK293 T cells and a buffer solution by a technique selected from ultrafiltration, centrifugation, tangential flow filtration, affinity purification, ion exchange chromatography, and combinations thereof; Wherein the separation in step (d) produces a high titer of VLV.

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

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

41. The method of claim 37, wherein the cancer-associated antigen is one or more of TSA or TAA.

42. The method of claim 37, wherein step b) comprises the following sub-steps: b1) combining the vector with a transfection reagent; and b2) transfecting BHK-21 or HEK293 T cells with the combined vector and reagent of step (b1).

43. The method of claim 41, wherein the transfection reagent is lipofectamine or PEIpro.

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

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

46. ​​The method of claim 43, wherein the high titer is at least 1 x 10 8 PFU / mL.

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

48. 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, pertussis, 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 aureus enterotoxin B infection.

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