Compositions and methods for preventing or treating hepatitis c viral infections

Abstract

Disclosed herein are method of vaccinating or treating a subject for a hepatitis C virus. In certain embodiments, methods comprise administering to a subject a DNA plasmid encoding hepatitis C virus core, E1, E2, and / or p7 proteins. In certain embodiments, methods further comprise administering a recombinant attenuated vaccinia vector encoding hepatitis C virus non-structural NS3, NS4a, NS4b, NS5a and / or NS5b proteins. In certain embodiments, the subject is a human subject.

Description

[0001] COMPOSITIONS AND METHODS FOR PREVENTING OR TREATING HEPATITIS C VIRAL INFECTIONS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 542,014 filed October 2, 2023. The entirety of this application is hereby incorporated by reference for all purposes.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under All 59819 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0006] INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS AN XML FILE VIA THE OFFICE ELECTRONIC FILING SYSTEM

[0007] The Sequence Listing associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing is 24014PCT.xml. The XML file is 6,732 bytes, was created on September 24, 2024, and is being submitted electronically via the USPTO patent electronic filing system.

[0008] BACKGROUND

[0009] Hepatitis C virus (HCV) infection remains a global public health problem. HCV is often asymptomatic until it is retrospectively identified as causing liver cirrhosis and cancer. Certain antiviral agents are clinically approved. However, treatment of chronic HCV infection with antivirals does not prevent reinfection which is a recurrent problem. Numerous HCV vaccine candidates have been explored showing varying immunogenicity. Identifying an effective human HCV vaccine remains elusive. Thus, there is a need to identify a safe and effective HCV vaccine strategy.

[0010] Page et al., report a randomized trial using a recombinant chimpanzee adenovirus 3 vector priming vaccination followed by a recombinant modified vaccinia Ankara boost to prevent chronic HCV infection. N Engl J Med, 2021, 384, 541-549. Swadling et al. report a human vaccine strategy based on chimpanzee adenoviral and MVA vectors providing T cell responses. Sci Transl Med, 2014, 6(261): 261ral53.

[0011] Chea & Amara report immunogenicity of DNA / MVA HIV vaccines in rhesus macaque models. Expert Rev Vaccines, 2017, 16(10): 973-985.

[0012] Sepulveda-Crespo et al. report a hepatitis C virus vaccine design with a focus on the humoral immune response. J Biomed Sc, 2020, 27:78.

[0013] References cited herein are not an admission of prior art.

[0014] SUMMARY

[0015] Disclosed herein are method of vaccinating or treating a subject for a hepatitis C virus. In certain embodiments, methods comprise administering to a subject a DNA plasmid encoding hepatitis C virus core, El, E2, and / or p7 proteins. In certain embodiments, methods further comprise administering a recombinant attenuated vaccinia vector encoding hepatitis C virus non- structural NS3, NS4a, NS4b, NS5a and / or NS5b proteins. In certain embodiments, methods further comprise administering RNA encoding hepatitis C virus non-structural NS3, NS4a, NS4b, NS5a and / or NS5b proteins. In certain embodiments, the subject is a human subject.

[0016] In certain embodiments, this disclosure relates to methods of vaccinating or treating a subject for a hepatitis C virus comprising administering to a subject a DNA plasmid encoding hepatitis C virus core, El, and / or E2 proteins; and administering a recombinant attenuated vaccinia vector encoding hepatitis C virus non-structural NS3, NS4a, NS4b, NS5a and / or NS5b proteins.

[0017] In certain embodiments, this disclosure relates to methods of vaccinating or treating a subject for a hepatitis C virus comprising administering to a subject a DNA plasmid encoding hepatitis C virus core, El, and / or E2 proteins; and administering a RNA encoding hepatitis C virus non-structural NS3, NS4a, NS4b, NS5a and / or NS5b proteins.

[0018] In certain embodiments, this disclosure relates to methods of vaccinating or treating a subject for a hepatitis C virus comprising administering to a subject a DNA plasmid encoding hepatitis C virus core, El, and / or E2 proteins; and administering a recombinant attenuated vaccinia vector encoding hepatitis C virus non-structural NS3, NS4a, NS4b, NS5a and / or NS5b proteins; and administering a RNA encoding hepatitis C virus non-structural NS3, NS4a, NS4b, NS5a and / or NS5b proteins. In certain embodiments, the NS3 protein has mutations such that the protease activity is reduced or inactivated. In certain embodiments, the mutations are H57A, D81A, and / or S391A.

[0019] In certain embodiments, the NS5b protein has mutations such that the polymerase activity is reduced or inactivated. In certain embodiments, the mutations are G317A, D318A and / or D319A.

[0020] In certain embodiments, methods further comprise administering RNA encoding hepatitis C virus core, El, E2, and / or p7 proteins to the subject.

[0021] In certain embodiments, methods further comprise administering RNA encoding hepatitis C virus non-structural NS3, NS4a, NS4b, NS5a and / or NS5b proteins to the subject.

[0022] In certain embodiments, the NS3 protein has mutations such that the protease activity is reduced or inactivated. In certain embodiments, the mutations are H57A, D81A, and / or S391A.

[0023] In certain embodiments, the NS5b protein has mutations such that the polymerase activity is reduced or inactivated. In certain embodiments, the mutations are G317A, D318A and / or D319A.

[0024] In certain embodiments, methods further comprise isolating B cells from the subject and culturing the B cells such that the B cells produce antibodies and wherein the B cells or isolated antibodies produced from the B cells, or sequences in the antibody are optionally cloned, inserted into a nucleic acid or vector, or the antibody are expressed and isolated, and are administered to the subject.

[0025] In certain embodiments, this disclosure relates to compositions comprising a DNA plasmid, RNA, or recombinant attenuated vaccinia vector encoding hepatitis C virus non-structural NS3, NS4a, NS4b, NS5a and / or NS5b proteins. In certain embodiments, the NS3 protein has mutations such that the protease activity is reduced or inactivated. In certain embodiments, the mutations are H57A, D81A, and / or S391A. In certain embodiments, the NS5b protein has mutations such that the polymerase activity is reduced or inactivated. In certain embodiments, the mutations are G317A, D318 A and / or D319A.

[0026] In certain embodiments, this disclosure relates to a DNA plasmid, RNA, or recombinant attenuated vaccinia vector encoding hepatitis C virus core, El, and E2 proteins and / or hepatitis C virus non-structural NS3, NS4a, NS4b, NS5a and / or NS5b proteins.

[0027] In certain embodiments, this disclosure relates to nucleic acids, mRNA, RNA, DNA, DNA plasmids, and vectors encoding proteins and mutants disclosed herein. In certain embodiments, this disclosure relates to cells (e.g., somatic cells) or other expression systems comprising nucleic acids, mRNA, RNA, DNA, DNA plasmids, and vectors encoding proteins and mutants disclosed herein.

[0028] In certain embodiments, this disclosure contemplates kits comprising a vaccine composition or components disclosed herein, e.g., in a container with a suitable diluent.

[0029] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0030] Figure 1A illustrates three constructs NS3-NS4-NS5(APol), NS3(APro)-NS4-NS5(APol), and VLP DNA immunogen designs used in initial DNA and MVA HCV vaccine evaluations. Inactivating mutations are indicated for polymerase and protease in NS5 and NS3, respectively.

[0031] Figure IB illustrates the NS3(APro)-NS4-NS5(APol), and VLP rMVA constructs with the p7 protein of HCV. Inactivating mutations are indicated for polymerase and protease in NS5 and NS3, respectively.

[0032] Figure 1C illustrated various VLP constructs.

[0033] Figure ID illustrates experiments including P7 in the VLP construct enhanced T cell responses.

[0034] Figure 2A shows a schematic of the BLACK / 6 mouse study. Group 1 received mock immunization; group 2 received NS3-NS4-NS5(APol) DNA / MVA vaccinations; group 3 ( received NS3(APro)-NS4-NS5(APol) DNA / MVA vaccinations; and group 4 received NS3(APro)- NS4-NS5(APol) MVA immunization only.

[0035] Figure 2B shows analysis of peptide specific CD4 T cell cytokine responses with APol showing the frequency of CD4+IFNy+ T cells at peak response post 1st MVA boost.

[0036] Figure 2C shows analysis of peptide specific CD4 T cell cytokine responses with APro and APol. Polyfunctional CD4 T cell response is shown by the frequency of CD4 T cells co-expressing IFNy, TNFa, and IL2 in response to indicated NS proteins.

[0037] Figure 2D shows analysis of peptide specific CD8 T cell cytokine responses with APol. Provided is the frequency of CD8+IFNg+ T cells at peak response post 1st MVA boost.

[0038] Figure 2E shows analysis of peptide specific CD8 T cell cytokine responses with the APro and APol construct. Poly functional CD8 T cell response is shown by the frequency of CD8 T cells co-expressing IFNy and TNFa. Figure 2F shows quantification of CD4 or CD8 IFNy+IL-2+TNFa+ cells in the liver and spleen.

[0039] Figure 3A shows a schematic of immunization studies in rhesus macaques. Group 1 received NS3(APro)-NS4-NS5(APol) DNA / MVA vaccination; and group 2 received tPA-C-El- E2-p7 VLP expressing DNA / MVA vaccinations; and group 3 received both immunizations.

[0040] Figure 3B shows data showing the T cell responses to indicated HCV NS proteins at peak response post 1st MVA boost.

[0041] Figure 4A shows co-immunization with DNA / MVA vaccinations. HCV glycoprotein E2- binding IgG in sera of Balb / c mice was determined at indicated time points.

[0042] Figure 4B shows data on IgG binding, H77 neutralization, and neutralization breath. The magnitude of E2-binding IgG for individual mice is shown at indicated time points for DNA / MVA and MVA-only immunized groups. Provides is percent neutralization of HCV virus-like particles pseudotyped (HCVpp) with H77 (genotype la) glycoprotein.

[0043] Figure 4C shows data on HCV glycoprotein E2-binding IgG in sera of rhesus macaques at indicated time points and longitudinal IgG response showing individual data points and geomean.

[0044] Figure 4D shows percent HCVpp neutralization in the sera of rhesus macaques at indicated time points and line graph showing individual data points with geomean.

[0045] Figure 5A shows a schematic of an immunization study in rhesus macaques adding an RNA boost.

[0046] Figure 5B shows a schematic of VLP and NS immunogens.

[0047] Figure 5C shows a comparison of the total magnitude of CD4 cell response in the VLP vs NS-VLP groups.

[0048] Figure 5D shows a comparison of the total magnitude of CD8 cell response in the VLP vs NS-VLP groups.

[0049] Figure 5E shows a schematic of the HCVla VLP and NSdP mRNA vaccine. Lipid nanoparticles were prepared using a mixture of pegylated lipids, cationic lipids, phospholipids, cholesterol, and the RNA.

[0050] DETAILED DISCUSSION

[0051] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims or as amended during prosecution.

[0052] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0053] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0054] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

[0055] As used in this disclosure and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") have the meaning ascribed to them in U.S. Patent law in that they are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0056] "Consisting essentially of' or "consists of' or the like, have the meaning ascribed to them in U.S. Patent law in that when applied to methods and compositions encompassed by the present disclosure refers to the idea of excluding certain prior art element(s) as an inventive feature of a claim, but which may contain additional composition components or method steps, etc., that do not materially affect the basic and novel characterise c(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein.

[0057] The term “comprising” in reference to a peptide having an amino acid sequence refers a peptide that may contain additional N-terminal (amine end) or C-terminal (carboxylic acid end) amino acids, i.e., the term is intended to include the amino acid sequence within a larger peptide. The term “consisting of’ in reference to a peptide having an amino acid sequence refers a peptide having the exact number of amino acids in the sequence and not more or having not more than a range of amino acids expressly specified in the claim. In certain embodiments, the disclosure contemplates that the “N-terminus of a peptide consists of an amino acid sequence,” which refers to the N-terminus of the peptide having the exact number of amino acids in the sequence and not more or having not more than a range of amino acids specified in the claim; however the C- terminus may be connected to additional amino acids, e g., as part of a larger peptide. Similarly, the disclosure contemplates that the “C-terminus of a peptide consists of an amino acid sequence,” which refers to the C-terminus of the peptide having the exact number of amino acids in the sequence and not more or having not more than a range of amino acids specified in the claim; however, the N-terminus may be connected to additional amino acids, e g., as part of a larger peptide.

[0058] A "subject" refers to any animal, preferably a human patient, livestock, rodent, monkey or domestic pet. The term is used herein to encompasses apparently healthy, non-infected individuals or a patient who is known or unknown to be infected with, diagnosed with, a pathogen.

[0059] As used herein, the term "combination with" when used to describe administration with an additional treatment means that the agent may be administered prior to, together with, or after the additional treatment, or a combination thereof.

[0060] The terms "protein" and "peptide" refer to polymers comprising amino acids joined via peptide bonds and are used interchangeably. Amino acids may be naturally or non-naturally occurring. A "chimeric protein" or "fusion protein" is a molecule in which different portions of the protein are derived from different origins such that the entire molecule is not naturally occurring. A chimeric protein may contain amino acid sequences from the same species of different species as long as they are not arranged together in the same way that they exist in a natural state. Examples of a chimeric protein include sequences disclosed herein that are contain one, two or more amino acids attached to the C-terminal or N-terminal end that are not identical to any naturally occurring protein, such as in the case of adding an amino acid containing an amine side chain group, e.g., lysine, an amino acid containing a carboxylic acid side chain group such as aspartic acid or glutamic acid, a polyhistidine tag, e.g. typically four or more histidine amino acids. Contemplated chimeric proteins include those with self-cleaving peptides such as P2A-GSG. See Wang. Scientific Reports 5, Article number: 16273 (2015).

[0061] In certain embodiments, the disclosure relates to recombinant polypeptides comprising sequences disclosed herein or variants or fusions thereof wherein the amino terminal end or the carbon terminal end of the amino acid sequence are optionally attached to a heterologous amino acid sequence, label, or reporter molecule.

[0062] A "label" refers to a detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes. A label includes the incorporation of a radiolabeled amino acid or the covalent attachment of biotinyl moieties to a polypeptide that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionucleotides (such as18F,35S or131I) fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0063] In certain embodiments, this disclosure contemplates that chimeric proteins disclosed herein may be variants. Variants may include 1 or 2 amino acid substitutions or conserved substitutions. Variants may include 3 or 4 amino acid substitutions or conserved substitutions. Variants may include 5 or 6 or more amino acid substitutions or conserved substitutions. Variants include those with not more than 1% or 2% of the amino acids are substituted. Variants include those with not more than 3% or 4% of the amino acids are substituted. Variants include proteins with greater than 80%, 89%, 90%, 95%, 98%, or 99% identity or similarity.

[0064] Variant peptides can be produced by mutating a vector to produce appropriate codon alternatives for polypeptide translation. Active variants and fragments can be identified with a high probability using computer modeling. Shihab et al. report an online genome tolerance browser. BMC Bioinformatics, 2017, 18(1 ):20. Ng et al. report methods of predicting the effects of amino acid substitutions on protein function. Annu Rev Genomics Hum Genet, 2006, 7:61-80. Teng et al. Approaches and resources for prediction of the effects of non-synonymous single nucleotide polymorphism on protein function and interactions. Curr Pharm Biotechnol, 2008, 9(2): 123-33.

[0065] Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, RaptorX, ESyPred3D, HHpred, Homology Modeling Professional for HyperChem, DNAStar, SPARKS-X, EVfold, Phyre, and Phyre2 software. See also Saldano et al. Evolutionary Conserved Positions Define Protein Conformational Diversity, PLoS Comput Biol. 2016, 12(3):el004775; Marks et al. Protein structure from sequence variation, Nat Biotechnol. 2012, 30(11): 1072-80; Mackenzie et al. Curr Opin Struct Biol, 2017, 44: 161-167 Mackenzie et al. Proc Natl Acad Sci U S A. 113(47):E7438-E7447 (2016); Joseph et al. J R Soc Interface, 2014, 11(95):20131147, Wei et al. Int. J. Mol. Sci. 2016, 17(12), 2118. Variants can be tested in functional assays. Certain variants have less than 10%, and preferably less than 5%, and still more preferably less than 2% changes (whether substitutions, deletions, and so on).

[0066] Sequence "identity" refers to the number of exactly matching amino acids (expressed as a percentage) in a sequence alignment between two sequences of the alignment calculated using the number of identical positions divided by the greater of the shortest sequence or the number of equivalent positions excluding overhangs wherein internal gaps are counted as an equivalent position. In certain embodiments, any recitation of sequence identity expressed herein may be substituted for sequence similarity. Percent “similarity” is used to quantify the similarity between two sequences of the alignment. This method is identical to determining the identity except that certain amino acids do not have to be identical to have a match. Amino acids are classified as matches if they are among a group with similar properties according to the following amino acid groups: Aromatic - F Y W; hydrophobic-A V I L; Charged positive: R K H; Charged negative - D E; Polar - S T N Q. The amino acid groups are also considered conserved substitutions.

[0067] Percent identity can be determined, for example, by comparing sequence information using the default settings of the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (J Mol Biol 197048:443), as revised by Smith and Waterman (Adv Appl Math 1981 2:482). Briefly, the GAP program defines identity as the number of aligned symbols (i.e., nucleotides or amino acids) which are identical, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program include: (1) a unitary comparison matrix (containing a value of 1 for identities and 0 for nonidentities) and the weighted comparison matrix of Gribskov and Burgess (Nucl Acids Res 1986 14:6745), as described by Schwartz and Dayhoff (eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington, D.C. 1979, pp. 353-358); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

[0068] The term "recombinant vector" when made in reference to vectors and nucleic acids refers to a nucleic acid molecule that is comprised of segments of nucleic acid joined together by means of molecular biological techniques. The term recombinant nucleic acid is distinguished from the natural recombinants that result from crossing-over between homologous chromosomes. Recombinant nucleic acids as used herein are an unnatural union of nucleic acids from nonhomologous sources, usually from different organisms.

[0069] The terms "expression vector " refer to a recombinant nucleic acid containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism or expression system, e.g., cellular or cell-free. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

[0070] In certain embodiments, a vector optionally comprises a gene vector element (nucleic acid) such as a selectable marker region, lac operon, a CMV promoter, a hybrid chicken B-actin / CMV enhancer (CAG) promoter, tac promoter, T7 RNA polymerase promoter, SP6 RNA polymerase promoter, SV40 promoter, internal ribosome entry site (IRES) sequence, cis-acting woodchuck post regulatory element (WPRE), scaffold-attachment region (SAR), inverted terminal repeats (ITR), FLAG tag coding region, c-myc tag coding region, metal affinity tag coding region, streptavidin binding peptide tag coding region, polyHis tag coding region, HA tag coding region, MBP tag coding region, GST tag coding region, polyadenylation coding region, SV40 polyadenylation signal, SV40 origin of replication, Col El origin of replication, fl origin, pBR322 origin, or pUC origin, TEV protease recognition site, loxP site, Cre recombinase coding region, or a multiple cloning site such as having 5, 6, or 7 or more restriction sites within a continuous segment of less than 50 or 60 nucleotides or having 3 or 4 or more restriction sites with a continuous segment of less than 20 or 30 nucleotides.

[0071] Protein “expression systems” refer to in vivo and in vitro (cell free) systems. Systems for recombinant protein expression typically utilize somatic cells transfected with a DNA expression vector that contains the template. The cells are cultured under conditions such that they translate the desired protein. Expressed proteins are extracted for subsequent purification. In vivo protein expression systems using prokaryotic and eukaryotic cells are well known. Also, some proteins are recovered using denaturants and protein-refolding procedures. In vitro (cell-free) protein expression systems typically use translation-compatible extracts of whole cells or compositions that contain components sufficient for transcription, translation, and optionally post-translational modifications such as RNA polymerase, regulatory protein factors, transcription factors, ribosomes, tRNA cofactors, amino acids and nucleotides. In the presence of an expression vector, these extracts and components can synthesize proteins of interest. Cell-free systems typically do not contain proteases and enable labelling of the protein with modified amino acids. Some cell free systems incorporated encoded components for translation into the expression vector. See, e.g., Shimizu et al., Cell-free translation reconstituted with purified components, 2001, Nat. Biotechnol, 19, 751-755 and Asahara & Chong, Nucleic Acids Research, 2010, 38(13): el41, both hereby incorporated by reference in their entirety.

[0072] A “selectable marker” is a nucleic acid introduced into a vector that encodes a polypeptide that confers a trait suitable for artificial selection or identification (report gene), e.g., betalactamase confers antibiotic resistance, which allows an organism expressing beta-lactamase to survive in the presence antibiotic in a growth medium. Another example is thymidine kinase, which makes the host sensitive to ganciclovir selection. It may be a screenable marker that allows one to distinguish between wanted and unwanted cells based on the presence or absence of an expected color. For example, the lac-z-gene produces a beta-galactosidase enzyme which confers a blue color in the presence of X-gal (5-bromo-4-chloro-3-indolyl-P-D-galactoside). If recombinant insertion inactivates the lac-z-gene, then the resulting colonies are colorless. There may be one or more selectable markers, e.g., an enzyme that can complement to the inability of an expression organism to synthesize a particular compound required for its growth (auxotrophic) and one able to convert a compound to another that is toxic for growth. URA3, an orotidine-5' phosphate decarboxylase, is necessary for uracil biosynthesis and can complement ura3 mutants that are auxotrophic for uracil. URA3 also converts 5-fluoroorotic acid into the toxic compound 5 -fluorouracil. Additional contemplated selectable markers include any genes that impart antibacterial resistance or express a fluorescent protein. Examples include, but are not limited to, the following genes: ampr, camr, tetr, neor, hygr, abxr, neomycin phosphotransferase type II gene (nptll), p-glucuronidase (gus), green fluorescent protein (gfp), egfp, yfp, mCherry, p-galactosidase (lacZ), lacZa, lacZAM15, chloramphenicol acetyltransferase (cat), alkaline phosphatase (phoA), bacterial luciferase (luxAB), bialaphos resistance gene (bar), phosphomannose isomerase (pmi), xylose isomerase (xylA), arabitol dehydrogenase (atlD), UDP-glucose:galactose-l -phosphate uridyltransferasel (galT), feedback-insensitive a subunit of anthranilate synthase (0ASA1D), 2- deoxy lucose (2-DOGR), benzyladenine-N-3 -glucuronide, E. coli threonine deaminase, glutamate 1 -semialdehyde aminotransferase (GSA-AT), D-amino acidoxidase (DAAO), salt-tolerance gene (rstB), ferredoxin-like protein (pflp), trehalose-6-P synthase gene (AtTPSl), lysine racemase (lyr), dihydrodipicolinate synthase (dapA), tryptophan synthase beta 1 (AtTSBl), dehalogenase (dhlA), mannose-6-phosphate reductase gene (M6PR), hygromycin phosphotransferase (HPT), and D- serine ammonialyase (dsdA).

[0073] Recombinant nuclei acids and viral vectors

[0074] In certain embodiments, the disclosure relates to recombinant viral vectors, recombinant vectors, and recombinant plasmids comprising nucleic acids or other DNA or RNA encoding HCV peptides disclosed herein. In certain embodiments, this disclosure relates to expression systems comprising nucleic acids and vectors disclosed herein. In certain embodiments, the protein is expressed in operable combination with a promoter or heterologous promoter or other transcriptional regulatory sequence.

[0075] In certain embodiments, the disclosure relates to compositions comprising a DNA plasmid, RNA, or recombinant attenuated vaccinia vector encoding hepatitis C virus core, El, E2, and p7 proteins and / or hepatitis C virus non- structural NS3, NS4a, NS4b, NS5a and / or NS5b proteins.

[0076] In certain embodiments, the NS3 protein has mutations such that the protease activity is reduced or inactivated. In certain embodiments, the the mutations are H57A, D81 A, and / or S391 A.

[0077] In certain embodiments, the the NS5b protein has mutations such that the polymerase activity is reduced or inactivated. In certain embodiments, the the mutations are G317A, D318A and / or D319A.

[0078] In certain embodiments, the disclosure relates to a DNA plasmid, RNA, or recombinant attenuated vaccinia vector encoding hepatitis C virus core, El, E2, and p7 proteins and / or hepatitis C virus non- structural NS3, NS4a, NS4b, NS5a and / or NS5b proteins.

[0079] Nucleic acids, vectors, and expression constructs can be introduced in vivo via lipofection (DNA transfection via liposomes prepared from synthetic cationic lipids). Synthetic cationic lipids can be used to prepare liposomes to encapsulate a nucleic acid, vector, or expression construct of the disclosure. A nucleic acid, vector, or expression construct can also be introduced as naked DNA or RNA using methods known in the art, such as transfection, microinjection, electroporation, calcium phosphate precipitation, and by biolistic methods.

[0080] In certain embodiments, this disclosure contemplates that methods disclosed herein are used with recombinant virus, preferably recombinant modified vaccinia virus Ankara (MV A). MVA is an attenuated strain of vaccinia virus originally developed as a vaccine for smallpox. The ability of MVA to infect mammalian, including human host cells, is restricted due to known deletions in the virus genome. In addition to the safe use in human vaccinations, Wyatt et al. report mice with severe combined immunodeficiency disease remained healthy when inoculated with MVA. Proc Natl Acad Sci U S A. 2004, 101 (13):4590-5. See also Rahman et al., PD-1 blockade and vaccination provide therapeutic benefit against SIV by inducing broad and functional CD8(+) T cells in lymphoid tissue. Sci Immunol 6, eabh3034 (2021) and Routhu et al., A modified vaccinia Ankara vector-based vaccine protects macaques from SARS-CoV-2 infection, immune pathology, and dysfunction in the lungs. Immunity 54, 542-556 e549 (2021). MVA can be engineered in deleted regions to express heterologous genes to induce protective immunity to other viruses. As a result of extensive passage in cell culture, the MVA virus genome contains six major deletions, referred to as Del I, II, III, IV, V and VI. Historically, the region around Del II and Del III has been used for insertion of heterologous nucleic acid sequences.

[0081] As used herein, the term heterologous is a comparative term, and refers to a molecule that is from an organism different from that to which it is being referenced and / or that is made synthetically such that the entire sequence does not exist in nature. The molecule can be a protein or a nucleic acid sequence (i.e., RNA or DNA). For example, a heterologous nucleic acid sequence in a recombinant virus vector refers to the fact that the heterologous nucleic acid sequence is or may be from an organism other than the base virus used to construct the recombinant virus vector. As a further example, a heterologous nucleic acid sequence in a recombinant vaccinia virus vector refers to the fact that the heterologous nucleic acid sequence is from an organism other than vaccinia virus or that was made synthetically such that the entire sequence is not naturally occurring.

[0082] A heterologous nucleic acid sequence can be inserted at any location in a recombinant virus vector genome, as long as such insertion does not unintentionally alter the functioning of the resulting recombinant virus vector. For example, a nucleic acid sequence can be inserted into a non-essential region. Such non-essential regions include, but are not limited to, naturally occurring deletions within the viral genome (e.g., Del I, II, III, etc. of modified vaccinia virus Ankara (MVA)), intergenic regions or non-essential genes. A non-essential region is a genomic region, the alteration of which has no, or almost no, discernible effect on viral replication and the production of progeny virus. One example of a non-essential region is a non-essential gene such as, for example, the vaccinia virus hemagglutinin gene.

[0083] Alternatively, a nucleic acid sequences can be inserted into an essential region of the genome (e.g., an essential gene). It will be appreciated that interruption of an essential region will result in a recombinant virus vector unable to complete the virus life cycle and produce progeny virus. However, such recombinant virus vectors can produce progeny virus when grown in cells that provide the missing function. Such a cell can be referred to as a complementing cell because it provides the function usually provided by the essential gene. That is, it "complements" the recombinant virus vector. Conversely, a cell that is unable to provide the missing viral function can be referred to as a non-commenting cell. Such culture systems are contemplated herein. At least one heterologous nucleic acid sequence may be inserted into the gene required for expression of post-replicative viral genes.

[0084] The base HCV NS3 sequence is

[0085] APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATCINGVCWTV YHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVI PVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVEN LETTMRSP VFTDNS SPP AVPQ SFQVAHLHAPTGSGK STKVPAA YAAQGYK VLVLNP SVA ATLGFGAYMSKAHGVDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECH STDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVSHPNIEEVALSTTGEIPFYGKAIP LEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVSTDAL MTGFTGDFD SVIDCNTC VTQTVDF SLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYR FVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFW EGVFTGLTHIDAHFLSQTKQSGENFPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKP TLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVT (SEQ ID NO: 1).

[0086] Protease triad mutants (Position 57, 81, 139)

[0087] APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATCINGVCWTV YAGAGTRTIASPKGPVIQMYTNVDQALVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVI PVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVE NLETTMRSP VFTDNS SPP A VPQSFQVAHLHAPTGSGKSTKVPAA YAAQGYK VLVLNPSV AATLGFGAYMSKAHGVDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDEC HSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVSHPNIEEVALSTTGEIPFYGKA IPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVSTDA LMTGFTGDFDS VIDCNTC VTQTVDF SLDPTFTIETTTLPQD AV SRTQRRGRTGRGKPGIY RFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEF WEGVFTGLTHIDAHFLSQTKQSGENFPYLVAYQATVCARAQAPPPSWDQMWKCLIRLK PTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVT (SEQ ID NO: 2). The base NS5b sequence is

[0088] SMSYSWTGALVTPCAAEEQKLPINALSNSLLRHHNLVYSTTSRSACQRQKKVTF DRLQVLDSHYQDVLKEVKAAASKVKANLLSVEEACSLTPPHSAKSKFGYGAKDVRCH ARKAVAHINSVWKDLLEDSVTPIDTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVC EKMALYDVVSKLPLAVMGSSYGFQYSPGQRVEFLVQAWKSKKTPMGFSYDTRCFDST VTESDIRTEEAIYQCCDLDPQARVAIKSLTERLYVGGPLTNSRGENCGYRRCRASGVLTT SCGNTLTCYIKARAACRAAGLQDCTMLVCGDDLVVICESAGVQEDAASLRAFTEAMTR YS APPGDPPQPE YDLELITSC S SNVSVAHDGAGKRVYYLTRDPTTPLARAAWET ARHTP VNSWLGNIIMFAPTLWARMILMTHFFSVLIARDQLEQALNCEIYGACYSIEPLDLPPIIQR LHGLSAFSLHSYSPGEINRVAACLRKLGVPPLRAWRHRARSVRARLLSRGGRAAICGKY LFNWAVRTKLKLTPIAAAGRLDLSGWFTAGYSGGDIYHSVSHARPRWFWFCLLLLAAG VGIYLLPNR (SEQ ID NO: 3).

[0089] Polymerase catalytic site mutant (Position 317, 318, 319)

[0090] SMSYSWTGALVTPCAAEEQKLPINALSNSLLRHHNLVYSTTSRSACQRQKKVTF DRLQVLDSHYQDVLKEVKAAASKVKANLLSVEEACSLTPPHSAKSKFGYGAKDVRCH ARKAVAHINSVWKDLLEDSVTPIDTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVC EKMALYDVVSKLPLAVMGSSYGFQYSPGQRVEFLVQAWKSKKTPMGFSYDTRCFDST VTESDIRTEEAIYQCCDLDPQARVAIKSLTERLYVGGPLTNSRGENCGYRRCRASGVLTT SCGNTLTCYIKARAACRAAGLQDCTMLVCAAALVVICESAGVQEDAASLRAFTEAMTR YS APPGDPPQPE YDLELITSC S SNVSVAHDGAGKRVYYLTRDPTTPLARAAWETARHTP VNSWLGNIIMFAPTLWARMILMTHFFSVLIARDQLEQALNCEIYGACYSIEPLDLPPIIQR LHGLSAFSLHSYSPGEINRVAACLRKLGVPPLRAWRHRARSVRARLLSRGGRAAICGKY LFNWAVRTKLKLTPIAAAGRLDLSGWFTAGYSGGDIYHSVSHARPRWFWFCLLLLAAG VGIYLLPNR (SEQ ID NO: 4)

[0091] HCV Vaccination methods

[0092] Disclosed herein are method of vaccinating or treating a subject for a hepatitis C virus. In certain embodiments, methods comprise administering to a subject a DNA plasmid encoding hepatitis C virus core, El, and E2 proteins; and administering a recombinant attenuated vaccinia vector encoding hepatitis C virus non-structural NS3, NS4a, NS4b, NS5a and / or NS5b proteins. In certain embodiments, this disclosure relates to methods of vaccinating or treating a subject for a hepatitis C virus comprising administering to a subject a DNA plasmid encoding hepatitis C virus core, El, and / or E2 proteins; and administering a recombinant attenuated vaccinia vector encoding hepatitis C virus non-structural NS3, NS4a, NS4b, NS5a and / or NS5b proteins.

[0093] In certain embodiments, this disclosure relates to methods of vaccinating or treating a subject for a hepatitis C virus comprising administering to a subject a DNA plasmid encoding hepatitis C virus core, El, and / or E2 proteins; and administering a RNA encoding hepatitis C virus non-structural NS3, NS4a, NS4b, NS5a and / or NS5b proteins.

[0094] In certain embodiments, this disclosure relates to methods of vaccinating or treating a subject for a hepatitis C virus comprising administering to a subject a DNA plasmid encoding hepatitis C virus core, El, and / or E2 proteins; and administering a recombinant attenuated vaccinia vector encoding hepatitis C virus non-structural NS3, NS4a, NS4b, NS5a and / or NS5b proteins; and administering a RNA encoding hepatitis C virus non-structural NS3, NS4a, NS4b, NS5a and / or NS5b proteins.

[0095] In certain embodiments, the NS3 protein has mutations such that the protease activity is reduced or inactivated. In certain embodiments, the mutations are H57A, D81A, and / or S391A.

[0096] In certain embodiments, the NS5b protein has mutations such that the polymerase activity is reduced or inactivated. In certain embodiments, the mutations are G317A, D318A and / or D319A.

[0097] In certain embodiments, methods further comprise administering RNA encoding hepatitis C virus core , El, and / or E2 proteins to the subject.

[0098] In certain embodiments, methods further comprise administering RNA encoding hepatitis C virus non-structural NS3, NS4a, NS4b, NS5a and / or NS5b proteins to the subject.

[0099] In certain embodiments, the NS3 protein has mutations such that the protease activity is reduced or inactivated. In certain embodiments, the mutations are H57A, D81A, and / or S391A.

[0100] In certain embodiments, the NS5b protein has mutations such that the polymerase activity is reduced or inactivated. In certain embodiments, the mutations are G317A, D318A and / or D319A.

[0101] In certain embodiments, methods further comprise isolating B cells from the subject and culturing the B cells such that the B cells produce antibodies and wherein the B cells or isolated antibodies produced from the B cells are administered to the subject. In certain embodiments, the chimeric HCV / DNA plasmids and / or chimeric MVA / HCV vectors and / or RNA disclosed herein may be administered in a pharmaceutical composition having a pharmaceutically acceptable excipient.

[0102] Pharmaceutical compositions and Kits

[0103] In certain embodiments, this disclosure contemplates pharmaceutical composition or kits comprising a vaccine composition disclosed herein such as chimeric HCV / DNA plasmids and / or chimeric MVA / HCV vectors and / or RNA disclosed herein with a suitable excipient, diluent and / or adjuvant.

[0104] Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous, pH buffered aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), suitable mixtures thereof, vegetable (such as olive oil, sesame oil) and injectable organic esters such as ethyl oleate.

[0105] In certain embodiments, the pharmaceutically acceptable excipient selected from lactose, sucrose, mannitol, triethyl citrate, dextrose, cellulose, methyl cellulose, ethyl cellulose, hydroxyl propyl cellulose, hydroxypropyl methylcellulose, carboxymethylcellulose, croscarmellose sodium, polyvinyl N-pyrrolidone, crospovidone, ethyl cellulose, povidone, methyl and ethyl acrylate copolymer, polyethylene glycol, fatty acid esters of sorbitol, lauryl sulfate, gelatin, glycerin, glyceryl monooleate, silicon dioxide, titanium dioxide, talc, corn starch, carnauba wax, stearic acid, sorbic acid, magnesium stearate, calcium stearate, castor oil, mineral oil, calcium phosphate, starch, carboxymethyl ether of starch, iron oxide, triacetin, acacia gum, esters, or salts thereof.

[0106] In certain embodiments, the pharmaceutical composition is in the form of a sterilized pH buffered aqueous salt solution, isotonic, or a saline phosphate buffer between a pH of 6 to 8, optionally comprising a saccharide or polysaccharide.

[0107] In certain embodiment, the pharmaceutically acceptable excipient is a cationic or polycationic compound and / or with a polymeric carrier.

[0108] These compositions may also contain adjuvants such as preserving, emulsifying, and dispensing agents. Prevention of the action of microorganisms may be controlled by addition of any of various antibacterial and antifungal agents, example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like.

[0109] In certain embodiments, the disclosure relates to compositions comprising chimeric HCV / DNA plasmids and / or chimeric MVA / HCV vectors and / or RNA disclosed herein and an adjuvant. Contemplated adjuvants suitable for depot and delivery are cationic or polycationic compounds, liposomes, chitosan, alum solution, aluminium hydroxide, aluminium salts aluminium phosphate gel, aluminium hydroxide gel (alum), polyphosphazene, squalene, squalene water emulsion, CpG oligonucleotides (nucleic acids with unmethylated CpG motifs), 1 -alpha, 25-dihydroxy -vitamin D3, calcium phosphate gel, dimethyl dioctadecyl ammonium bromide, dehydroepiandrosterone, dimyristoylphosphatidylcholine, myristoyl phosphatidylglycerol, deoxycholic acid sodium salt, imiquimod, interferon gamma, interleukin-1 beta, interleukin-2, interleukin-7, interleukin- 12, 7- allyl-8-oxoguanosine, acetylmuramyl-alanyl-isoglutamine, N-acetyl muramyl-L-threonyl-D- isoglutamine, NAc-Mur-L-Ala-D-Gln-OCHs, QS-21, Quil-A (Quil-A saponin), sorbitan trioleate, 2,6,10,15,19,23-hexamethyltetracosan, stearyl tyrosine, lipid A, 4 '-monophosphoryl lipid A, 3-0- desacyl-4'-monophosphoryl lipid A (MPL™), liposomes containing lipid A, lipid A adsorbed on aluminium hydroxide.

[0110] In certain embodiments, the chimeric HCV / DNA plasmids and / or chimeric MVA / HCV vectors and / or RNA disclosed herein may be administered naked without being associated with any further vehicle.

[0111] In certain embodiments, kits the chimeric HCV / DNA plasmids and / or chimeric MVA / HCV vectors and / or RNA disclosed herein may include instructions for use, administration means, such as syringes, catheters, brushes, etc. (if the compositions are not already provided in the administration means) or other components necessary for use in medical practice, such as substitute needles or catheters, extra vials or further wound cover means.

[0112] In certain embodiments, the kit comprises a syringe housing the dry and stable hemostatic composition and a syringe containing the diluent (or provided to take up the diluent from another diluent container).

[0113] In certain embodiments, the kit comprises lipofectamine, labeling reagents, primers that bind vaccine components, linearization, precipitation, and / or transcription reagents (for PCR), such as a polymerase, trinucleotides, buffer and DNA. T and B cell targeting DNA / MVA HCV vaccines induce robust immunological responses in mice and rhesus macaques

[0114] It is contemplated that a successful HCV vaccine should induce robust HCV-specific CD4 and CD8 T cell responses and neutralizing antibodies. It is contemplated that an effective and safe NS-based vaccine candidate warrants testing protease inactivated NS3 immunogen. Given constant evolution and escape mutants development in HCV, it is desirable to target multiple viral epitopes to establish a broader anti-viral immunity. Thus, a vaccine regimen combining multiple HCV epitopes with the potential to induce cellular and humoral arms of anti-HCV immunity holds promise for an effective prophylactic vaccine against HCV. In this regard, a DNA prime followed by modified vaccinia Ankara (MVA) boost (DNA / MVA) approach is contemplated to induce a potent CD4 and CD8 T cells and antibody response against HCV.

[0115] A DNA / MVA vaccine strategy for HCV was designed to induce a potent and broad CD4 and CD8 T cell response and neutralizing antibody response. The DNA and MVA vaccines expressed HCV (genotype la) nonstructural (NS) proteins NS3-NS4-NS 5 and structural proteins Core-El -E2 to produce virus-like particles (VLPs). In some constructs, mutations inactivate polymerase and protease activities of NS5 and NS3, respectively to enhance safety. These vaccines were tested in mice and rhesus macaques.

[0116] DNA and MVA vaccine immunogens covering the HCV genotype la proteome

[0117] HCV genotype la (H77) sequence-based DNA and MVA vaccines were designed and constructed expressing either the non-structural (NS) or structural proteins (which form virus-like particles, VLPs). The NS construct expressed NS3, NS4a, NS4b, NS5A, and NS5b proteins. To enhance safety, mutations were introduced in the catalytic sites of NS3 protease (H57A / D81A / S139A mutations) and NS5b polymerase (G317A / S318A / D319A mutations). With DNA, two NS constructs were made, one with mutations in both NS3 and NS5 (APro APol), and the other with mutations only in NS5 (APol) (Figure 1 A). The expression of NS immunogens was evaluated using an anti-NS5a antibody as a surrogate to validate the expression of NS polyprotein after transfecting 293T cells with the DNA constructs. Clear expression of NS5a was detected in cells transfected with either NS(APol) or NS(APro APol) constructs by flow cytometry, and the expression was comparable. The western blot analysis further validated the expression of NS5a (56kDa band) in the NS(APol) construct. This suggested the expression of NS polyprotein and proper cleavage. However, the unprocessed NS polyprotein was not detected in the double mutant by western blotting, although NS5a expression was detected by flow cytometry. The reasons for this failure to detect expression by western blotting are unknown. It is possible that the NS polyprotein assumed a structure that was not detected by the anti-NS5a antibody.

[0118] The DNA VLP construct expressed HCV core, El, E2, and P7 proteins. The expression of El and E2 glycoproteins was confirmed in 293T cells using anti -El and anti-E2 antibodies by flow cytometry. The western blot analysis further validated the expression of El and E2 glycoproteins and their sizes, as indicated by the presence of an about 35kDa band of El and an about 60kDa band of E2 (Figure IE). The transmission electron microscopy images of transfected cells confirmed the formation of HCV VLP like structures.

[0119] Multiple MVA vaccines were made, one with NS(APro APol) dual mutant sequence and the other with VLP sequence. (Figure IB). The NS antigens were inserted in Deletion II of MVA using the pLAS2 vector, and the VLP antigens were inserted in the essential region of MVA using the pLW73 vector. As with the DNA vaccines, a clear expression of NS5a, El and E2 glycoproteins were detected in cells infected with the respective MVAs by flow cytometry. The western blot further validated the expression of MVA-derived El, E2, and core as indicated by a clear detection of an about 35kDa band of El, an about 60kDa band of E2, and an about 20kDa band of C. As with the DNA vaccines, NS5 protein expression by western blot analysis was not detected.

[0120] NS vaccine with or without protease inactivating mutation induced robust T cell responses against multiple NS domains in mice

[0121] Next, the effects of protease and polymerase double mutation in NS immunogen (NSAPro+APol) were compared with the polymerase single mutation (NSAPol) on elicitation of T cell response in a DNA prime / MVA boost (DNA / MVA) approach. Mice were immunized twice with DNA either expressing NSAPol or NSAPro+APol on weeks 0 and 4 and boosted with MVA expressing NSAPro+APol at week 12 (Figure 2A). A group of mice received a single dose of MVA NSAPro+APol at week 12 as a control for DNA prime. At week 13, we analyzed IFNy, TNFa, and IL2 cytokine production in CD4 and CD8 T cell subsets in splenocytes stimulated with individual peptide pools, each covering the HCV H77 proteins NS3, NS4a, NS4b, NS5a, and NS5b (Figure 2B-D).

[0122] The DNA / MVA vaccination induced strong NS-specific IFNy+ CD4 T cells in both NSAPol and NSAPro+APol groups, and the responses were comparable between the two DNA / MVA groups. Encouragingly, protease mutation did not diminish the CD4 T cell cytokine responses to NS antigens. The frequencies of IFNy, TNFa , and IL-2 co-expressing cells were also strong, highlighting the polyfunctional nature of CD4 T cells. The CD4 response was directed against all NS antigens except NS4a. The rank order for IFNy+ CD4 T response was NS5a (geomeans of 0.77% and 0.9%, respectively in NSAPol and NSAPro+APol groups) followed by NS3 (0.29%, 0.34%), NS4b (0.14%, 0.08%) and NS5b (0.1%, 0.17%). A similar pattern was also observed for IFNy, TNFa, and IL-2 co-expressing cells. Overall, CD4 T cell showed highly polyfunctional profde induced by NSAPro+APol vaccine. The CD4 T cell response in the naive and MVA-only groups was generally at or below our detection limit (0.01%).

[0123] The DNA / MVA vaccination also induced a robust IFNy+ CD8 T cell response in both groups, and the response was directed primarily to NS3 protein (geomeans of 5% and 4%, respectively in NSAPol and NSAPro+APol groups). The dominant CD8 response to a specific protein / epitope is frequently seen in mice due to the same MHC I background across groups, and a strong CD8 T cell response to an immunodominant epitope can diminish response against other epitopes. The vaccination also induced strong IFNy, TNFa , and IL-2 co-expressing cells. Overall, these results demonstrated a robust induction of CD4 and CD8 T cell responses by NS-based DNA / MVA vaccinations. These data also established that protease and polymerase dual mutations in NS proteins do not impair immunogenicity.

[0124] Having established NSAPro+APol is equally immunogenic, a set of mice were immunized with NSAPro+APol expressing DNA / MVA vaccines to evaluate T cell response in liver, which is the primary site of HCV infection. A strong CD4 T cell response was observed in liver coexpressing IFNy, TNFa , and IL-2 in response to NS proteins (Figure 2E). The response was higher compared with the response in spleen. Similarly, significantly higher CD8 T cell responses were observed in liver co-expressing IFNy, and TNFa in response to NS antigens (Figure 2F). Encouragingly, significantly higher T cell breadth was observed in liver recognizing multiple NS proteins. These data suggest strong potential of DNA / MVA vaccination to induce potent and broad T cell responses in liver. NS and VLP vaccines induced potent and broad T cell responses in rhesus macaques

[0125] The immunogenicity of NS and VLP DNA / MVA vaccines were evaluated in rhesus macaques (RMs). One advantage of RMs is that, like humans, their class I and class II MHCs are diverse between animals, allowing us to study the breadth of T cell response better than in mice. Another objective of the RM study was to test if co-immunization of NS and VLP vaccines will interfere with the magnitude and breadth of T cell response induced by the individual vaccines. With these goals in mind, three groups of RMs, with five animals in each group, were immunized (Figure 3A). Animals received two DNA vaccines at weeks 0 and 4, and two MVA vaccines at weeks 12 and 20. Group 1 received only NS vaccines, Group 2 received only VLP vaccines, and Group 3 received both NS and VLP vaccines. PBMCs were analyzed for the production of IFNy, TNFot, and IL2 cytokines in CD4 and CD8 T cells following stimulation with peptide pools specific to each of the proteins in the vaccines throughout the study.

[0126] Two doses of DNA vaccines induced low to undetectable HCV-specific CD4 and CD8 T cells. However, the 1st MVA dose strongly boosted CD4 and CD8 T cell responses. In the NS- only group, CD4 T cells targeted NS3, NS5a, and NS5b proteins; however, CD8 T cells targeted primarily NS3 protein. The CD4 response was dominant against NS3 and NS5b, whereas the CD8 T cell response was dominant only against NS3. A similar pattern was also observed in the NS+VLP group, but overall, the responses tended to be lower than in the NS-only group. In the VLP-only group, the CD4 T cell response was primarily targeted to El and E2 proteins, and the CD8 T cell response was targeted to core, El, and E2 proteins. Interestingly, in the NS+VLP group, the frequency of El and E2-specific CD4 T cells was comparable to that observed in the VLP- only group, but the frequency of CD8 T cells tended to be lower. At one week after the second MVA boost, the frequency of CD4 T cells was lower, and the frequency of CD8 T cells was similar compared to one week after the first MVA boost suggesting that the second MVA boost did not significantly boost the T cell response.

[0127] The total number of structural and / or nonstructural proteins targeted by CD4 and CD8 T cells upon immunizations was analyzed in mice and rhesus macaques. In mice, two doses of DNA (prime) and one MVA (boost) expressing NS immunogens induced CD4 T cell breadth recognizing on an average four out of five NS proteins compared to about 1 NS protein recognized by CD4 T cells in mice immunized with a single MVA vaccine without DNA prime. Similarly, DNA / MVA immunized mice induced CD8 T cells that recognized about 3 NS proteins compared to only about 1 NS protein recognized by a single MVA immunization.

[0128] In rhesus macaques two doses of DNA (prime) and two MVA (boost) induced CD4 T cell breadth recognizing on an average three out of five NS proteins (in NS only vaccine group), two VLP proteins out of four (in VLP only vaccine group), and four out of nine combined HCV proteins (in NS + VLP vaccine group). These responses were significantly higher compared to the baseline. Similarly, vaccinations induced CD8 T cell breadth recognizing on an average two out of five NS proteins in NS only vaccine group, two out of four VLP proteins in VLP-only vaccine group, and five out of nine HCV proteins in NS + VLP vaccine group. These responses were significantly higher compared to the baseline. Encouragingly, the genotype la vaccine induced cross-reactive CD8 T cells recognizing genotype lb NS proteins. Similarly, cross-reactive CD4 and CD8 T cells were observed in VLP alone or NS+VLP combined vaccine groups. Overall, these data demonstrated the induction of a broad and cross-reactive T cell response by DNA / MVA vaccinations in mice and rhesus macaques.

[0129] HCV VLP vaccines induced strong anti-E2 antibody response with neutralization potential in mice and macaques

[0130] To determine the humoral response induced by the HCV VLP DNA / MVA vaccine, antibody responses were first analyzed in BALB / c mice. Two groups of mice were immunized either with three doses of DNA (prime) followed by two doses of MVA (boost) or with two doses of MVA only (Figure 4A). One group of naive mice served as unvaccinated control. Following the three DNA immunizations, the binding antibody titer was below the detection limit. However, following the 1st MVA boost, induction of significantly higher antibody response (geomean of 1,812) was observed which further increased upon 2nd MVA boost (geomean of 3,039). However, all except one mouse in the MVA-only group failed to generate a detectable antibody response. Further, the neutralizing potential of the DNA / MVA vaccine-induced antibody was evaluated using HCV H77 pseudoparticles (HCVpp). Neutralizing potential significantly increased post 2nd MVA boost. Encouragingly, pooled sera showed cross-clade neutralizing activity.

[0131] Humoral response induced by the VLP and VLP+NS DNA / MVA vaccines were evaluated in rhesus macaques. The HCV H77 E2-specific IgG in sera were analyzed at week 0 (baseline), week 14 (1 st MVA boost + wk2), and week 22 (2nd MVA boost + wk2). Following the two DNA immunizations, the binding antibody titer was below our detection limit. However, following the 1st MVA boost, a strong and comparable antibody response was observed in both VLP-only (geomean of 29,755) and NS+VLP (geomean of 39,712) groups. Upon 2nd MVA boost, the VLP group showed a marginal increase in the E2-binding IgG (geomean 58,334). However, the NS+VLP group did not show a further boost (geomean 32,543). In general, animals that showed a strong E2-specific Ab response post the 1st MVA did not show a boost following the 2nd MVA, while the animals that showed a weak titer following the 1 st MVA showed a strong boost following the 2nd MVA. These data showed strong induction of anti-HCV E2-binding antibodies and no interference by co-immunization with NS-vaccine.

[0132] The neutralizing potential of the vaccine-induced antibody using HCV H77 pseudoparticles (HCVpp) was analyzed. Encouragingly, most animals in the VLP-only and NS+VLPs groups showed neutralizing activity two weeks after the 1st MVA boost (Figure 4D). Two (2) out of 5 animals in each group showed activity higher than 50% neutralization. Upon the 2nd MVA boost, animals with weak activity post the 1 st MVA showed a boost, while animals with strong activity did not. To investigate possible anti-MVA vector antibody responses potentially interfering with the boost upon 2nd MVA administration, anti-MVA binding Ab titer at two weeks post 1st MVA boost was determined and correlated with HCVpp neutralizing activity post the 2nd MVA. A strong negative correlation between anti-MVA Ab response with HCVpp neutralizing activity was observed. These data indicate that strong anti -vector immunity following the 1 st MVA boost limits the anti-E2 response following the 2nd MVA boost. They also suggest that replacing the second MVA boost with a non-MVA boost further boosts the neutralizing Ab response. E2- binding IgG responses strongly correlated with the magnitude of neutralization, antigen-specific CD4, and CD8 T cells, generating an overall strong and coherent cellular and humoral responses against multiple HCV antigens.

[0133] DNA / MVA / mRNA vaccination with VLP immunogens (Fig. 5A) induced a robust E2- specific antibody response. The vaccine-induced antibody response showed strong autologous tier- 2 neutralizing activity, and low levels of heterologous tier-3 neutralizing activity. The NS vaccine did not interfere with the ability of VLP vaccine to induce a strong antibody response. The homologous prime / boost vaccination with mRNA induced a low level of CD4 and CD8 T cell response and a low level of E2-specific antibody response. The heterologous prime / boost vaccination induced robust CD4 and CD8 T cells in tissues, including the liver. The heterologous prime / boost vaccination induced robust E2-specific plasma cells in BM. The heterologous prime / boost vaccination was far superior to homologous prime / boost vaccination in inducing strong T cells and antibodies in blood and tissues.

[0134] Construction and characterization of DNA and MVA vaccines.

[0135] The DNA and MVA vaccines expressing HCV immunogens were constructed using standard procedures. HCV sequence (GenBank: NC 038882.1; HCV isolate H77; genotype la) for core, El, E2, p7, NS3, NS4a, NS4b, NS5a, and NS5b open reading frames were codon optimized and synthesized using GenScript™ services and cloned into pGA8 vector between Nhel and Clal restriction sites under a CMV promoter. To promote expression, the tissue plasminogen activator element was added at the 5’ end of the coding sequence following the Kozak consensus sequence to generate the DNA vaccines. To generate the single mutant NS3-NS4-NS5(APol) immunogen, three-point mutations (G317A / D318A / D319A) were introduced to the polymerase catalytic site in the NS5b domain of NS3-NS4-NS5 polyprotein sequence. Similarly, to generate the double mutant NS3(APro)-NS4-NS5(APol) immunogen, an additional H57A / D81A / S139A mutations were introduced in the protease catalytic domain of the NS3 protein. The HCV-VLP immunogen was generated using the core, El, and E2 glycoproteins coding sequences, in addition to the p7 domain.

[0136] To generate rMVA vaccines, the coding DNA sequences from the VLP and NS3(APro)- NS4-NS5(APol) constructs were codon optimized for MVA mediated expression and used to generate MVA / HCV-VLP and MVA-HCV / NS3(APro)-NS4-NS5(APol) immunogens, respectively. HCV-NS and VLP gene sequences were cloned into deletion II and essential regions (between genes I8R and GIL) using pLAS-2 and pLW-73 vectors, respectively under the mH5 promoter. These inserts were subsequently recombined into MVA using BHK21 cells to develop recombinant viruses. Recombinant plaques were picked for seven rounds to obtain GFP-negative recombinants and sequenced to confirm the presence of inserts. Viral stock for immunizations was purified from rMVA infected BHK21 cell lysates using 36% sucrose cushion and titrated using DF-1 cells. Absence of wildtype MVA was confirmed by PCR using recombinant specific primers flanking the inserts. Transfection and antigen expression characterization

[0137] To characterize the expression of immunogens 2xl06HEK 293T cells per well were seeded in a 6-well plate and incubated at 37°C overnight in a humidified incubator with 5% CO2. On the following day, lug plasmid of either tPA-NS3-NS4-NS5(APol), tPA-NS3(APro)-NS4-NS5(APol), or tPA-C-El-E2-p7 (VLP) DNA constructs were transfected with lipofectamine. To analyze the expression, HEK293T cells were harvested 36h post-transfection, stained for live and dead, anti- NS5a (Clone #H26), anti-El (Clone #A4), and anti-E2 (Clone #8A6), and analyzed using flow cytometry and western blots. Expression of rMVA vaccines were characterized by infecting BHK21 cells with MVA-HCV / NS or MVA / HCV-VLP at an MOI of 1, and the expression was analyzed 24h post-infection using flowcytometry and western blotting.

[0138] Binding IgG responses using ELISA

[0139] Anti-E2 antibody responses were measured using an enzyme-linked immunosorbent assay (ELISA) using E2 protein immunogen, captured on Nunc MaxiSorp™ microtiter plates. Plates were coated with the E2 protein at Ipg / ml in PBS and incubated overnight at 4°C. The following day, plates were washed, blocked for Ih with blocking buffer (5% milk powder) dissolved in 4% whey buffer prepared in PBS containing 0.05% tween-20) and incubated for 2 h with 3-fold dilutions of either mice or rhesus serum samples. Bound Rhesus IgG was detected using peroxidase-conjugated anti-monkey IgG at 1: 10,000 dilution. Bound mouse IgG was detected using Goat Anti Mouse IgG at 1 :6000 and the tetramethylbenzidine. The reaction was stopped by adding 100 pl of IN H3PO4. End point titers were calculated using titer at 0.1 as cut off (2 times of mean control).

[0140] HCVpp neutralization assay

[0141] Neutralization assays were performed with HCV pseudoparticles (HCVpp) generated via transfection of HEK293T-CD81KO cells. Cells were cotransfected the day after seeding with pNL4.3.Luc.R-E plasmid, pAdvantage™ plasmid, and an HCV E1E2 expression plasmid with Lipofectamine 3000 per manufacturer instructions, thereby producing lentiviral particles bearing HCV E1E2 that are capable of single-round entry into hepatocytes for transduction and reporter expression. Filtered supernatants containing HCVpp were coincubated with heat-inactivated test serum from individual animals for 1 hour at 37 C prior to overlay of this mixture onto Huh7.5 cells. After 6 hours of incubation, media was changed and the cells were incubated for 72 hours prior to cell lysis and addition of luciferase substrate for immediate quantification of relative light units (RLU) on a microplate reader. % Neutralization was calculated based on individual animal baseline serum as % Neutralization = (1 - [Test serumRLU / Baseline serumRLU])*100. mRNA after the DNA / MVA

[0142] Experiments were performed using an mRNA boost after the DNA / MVA vaccination. Experiments indicate that an mRNA booster after DNA / MVA was surprisingly superior (See Figures 1C and ID). Contemplated are methods of administering DNA / MVA followed with an mRNA boost and are methods of administering DNA followed with an mRNA boost as a vaccination modality.

Claims

CLAIMSWhat is claimed is:

1. A method of vaccinating or treating a subject for a hepatitis C virus comprising administering to a subject a DNA plasmid encoding hepatitis C virus core, El, E2, and p7 proteins; and administering a recombinant attenuated vaccinia vector encoding hepatitis C virus non- structural NS3, NS4a, NS4b, NS5a and / or NS5b proteins.

2. The method of claim 1, wherein the NS3 protein has mutations such that the protease activity is reduced or inactivated.

3. The method of claim 2, wherein the mutations are H57A, D81A, and / or S391A.

4. The method of claim 1, wherein the NS5b protein has mutations such that the polymerase activity is reduced or inactivated.

5. The method of claim 4, wherein the mutations are G317A, D318 A and / or D319A.

6. The method of claim 1 further comprising administering RNA encoding hepatitis C virus core , El, and / or E2 proteins to the subject.

7. The method of claim 1 further comprising administering RNA encoding hepatitis C virus non-structural NS3, NS4a, NS4b, NS5a, and / or NS5b proteins to the subject.

8. The method of claim 7, wherein the NS3 protein has mutations such that the protease activity is reduced or inactivated.

9. The method of claim 7, wherein the mutations are H57A, D81A, and / or S391A.

10. The method of claim 7, wherein the NS5b protein has mutations such that the polymerase activity is reduced or inactivated.

11. The method of claim 10, wherein the mutations are G317A, D318A and / or D319A.

12. The method of any of claims 1-11, further comprising administering RNA encoding hepatitis C virus core, El, and / or E2 proteins13. The method of any of claims 1-11, further comprising administering RNA encoding hepatitis C virus non- structural NS3, NS4a, NS4b, NS5a and / or NS5b proteins.

14. A composition comprising a DNA plasmid, RNA, or recombinant attenuated vaccinia vector encoding hepatitis C virus core, El, E2, and p7 proteins and / or hepatitis C virus non- structural NS3, NS4a, NS4b, NS5a and / or NS5b proteins15. The composition of claim 14, wherein the NS3 protein has mutations such that the protease activity is reduced or inactivated.

16. The composition of claim 15, wherein the mutations are H57A, D81A, and / or S391A.

17. The composition of claim 14, wherein the NS5b protein has mutations such that the polymerase activity is reduced or inactivated.

18. The composition of claim 17, wherein the mutations are G317A, D318A and / or D319A.

19. A DNA plasmid, RNA, or recombinant attenuated vaccinia vector encoding hepatitis C virus core, El, E2, and p7 proteins and / or hepatitis C virus non- structural NS3, NS4a, NS4b, NS5a and / or NS5b proteins.

20. A kit comprising a DNA plasmid, RNA, and / or recombinant attenuated vaccinia vector as in claims 14-19.

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