HCV envelope protein E1 / E2 encoded as VSV vector for anti-hepatitis c virus vaccine

Abstract

The present invention relates to the field of vaccination, in particular to vaccination against hepatitis C virus (HCV). The present invention provides a composition comprising at least a portion of the HCV core protein, the HCV E1 and HCV E2 proteins, and the VSV-G protein of a particular HCV strain. These proteins can be assembled in rVSV HCV particles. This has been determined to induce broadly neutralizing antibodies that are particularly advantageous. The invention also provides nucleic acids encoding the HCV protein and the VSV protein but not encoding the VSV-G protein. Vaccines comprising the particles, compositions or nucleic acids are disclosed that are particularly suitable for prophylactic vaccination of HCV. A process for preparing the rVSV-HCV particles or compositions of the invention as well as the particles and compositions prepared are also subject matter of the invention.

Description

[0001] The present invention relates to the field of vaccination, in particular vaccination against hepatitis C virus (HCV). The present invention provides a composition comprising at least part of the HCV core protein, HCV E1 and HCV E2 proteins of a specific HCV strain, and VSV-G protein. These proteins can be assembled in rVSV-HCV pseudovirions. This has been determined to induce particularly advantageous broadly neutralizing antibodies. The present invention also provides nucleic acids encoding the HCV proteins and VSV proteins but not encoding the VSV-G protein. Vaccines comprising particles, compositions or nucleic acids are disclosed as being particularly suitable for preventive vaccination against HCV. Methods for preparing the VSV particles or complexes of the present invention and the prepared particles and compositions are also the subject of the present invention.

[0002] HCV is a global health problem with an estimated 71 million people chronically infected worldwide. Among those infected, 50%-80% develop chronic disease and are at risk for developing cirrhosis and hepatocellular carcinoma.

[0003] The advent of highly effective direct-acting antivirals (DAAs) has revolutionized patient care. However, providing DAAs to all patients worldwide remains a major public health challenge. In addition, in rare cases, treatment failure occurs due to resistance-associated mutations, and HCV reinfection may occur after treatment-induced cure.

[0004] Therefore, preventive vaccines are important to control the HCV disease burden. One approach to vaccine development utilizes the induction of broadly neutralizing antibodies (bNAbs) targeting envelope glycoproteins E1 and E2. Several studies support the important role of neutralizing antibodies in spontaneous HCV clearance (Pestka et al. Rapid induction of virus-neutralizing antibodies and viral clearance in a single-source outbreak of hepatitis C. Proc Natl Acad Sci U SA 2007;104:6025-30; Logvinoff et al. Neutralizing antibody response during acute and chronic hepatitis C virus infection. Proc Natl Acad Sci US A 2004;101:10149-54; Osburn et al. Spontaneous control of primary hepatitis C virus infection and immunity against persistent reinfection. Gastroenterology 2010;138:315-24; Raghuraman et al. Spontaneous clearance of chronic hepatitis C virus infection is associated with appearance of neutralizing antibodies and reversal of T-cell exhaustion. J Infect Dis 2012;205:763-71).

[0005] In addition, passive immunoprophylaxis studies in animals have confirmed the importance of antibodies in protecting against HCV infection (Youn et al., Sustained E2 antibody response correlates with reduced peak viremia after hepatitis C virus infection in the chimpanzee. Hepatology 2005;42:1429-36; Law et al., Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge. Nat Med 2008;14:25-7; Vanwolleghem et al., Polyclonal immunoglobulins from a chronic hepatitis C virus patient protect human liver-chimeric mice from infection with a homologous hepatitis C virus strain. Hepatology 2008;47:1846-55).

[0006] However, HCV is a difficult target for vaccine development due to its extraordinary diversity and flexibility as well as HCV's different immune evasion mechanisms. In addition, there are limitations in testing candidate vaccines because there are no robust animal models to directly protect people from HCV infection (Bankwitz et al., für Impfstoffe gegenHepatitis-C-Virus-Infektionen. Bundesgesundheitsbl 2022, doi.org / 10.1007 / s00103-021-03477-9).

[0007] HCV is highly variable, and virus isolates are classified into eight genotypes (GT) and multiple subtypes. The genetic diversity even exceeds that of HIV-1 (Smith et al., Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: updated criteria and genotype assignment web resource. Hepatology 2014; 59: 318-27).

[0008] The envelope protein is the target of neutralizing antibodies and represents the most variable protein in the virus. The hypervariable region (HVR), such as HVR1 in the E2 protein, is highly immunogenic and may lead to the induction of non-protective antibodies. HVR1 can even be deleted without the virus losing infectivity (Bankwitz et al., Hepatitis C virus hypervariable region 1 modulates receptor interactions, conceals the CD81 binding site and protects conserved neutralizing epitopes. J Virol 84: 5751-5763; Prentoe et al., Hypervariable region 1 differentially impacts viability of hepatitis C virus strains of genotypes 1 to 6 and impairs virus neutralisation. J Virol 85: 2224-2234). For successful vaccine design, it is crucial to induce cross-protective antibodies against different HCV variants.

[0009] However, the lack of robust animal models and the use of different experimental systems to quantify antibody responses complicate the comparison of vaccine efficacy. Typically, infection assays based on retroviral HCV pseudoparticles (HCVpp) or cell culture-derived HCV particles (HCVcc) are used to quantify antibody efficacy. In recent years, several HCVpp or HCVcc panels have been developed (Kinchen et al., Defining breadth of hepatitis C virus neutralization. Front Immunol 2018; 9: 9). To some extent, these panels include a large number of different E1-E2 proteins (Tarr et al., Hepatitis C patient-derived glycoproteins exhibit marked differences insusceptibility to serum neutralizing antibodies: genetic subtype defines antigenic but not neutralization serotype. J Virol 2011; 85: 4246-57). However, in some cases, these panels only include GT1-derived glycoproteins (Osburn et al., Clearance of hepatitis Cinfection is associated with the early appearance of broad neutralizing antibody responses. Hepatology 2014; 59: 2140-51; Wasilewski et al., Hepatitis C virus resistance to broadly neutralizing antibodies measured using replication-competent virus and pseudoparticles. J Gen Virol 2016; 97: 2883-93). It is unclear whether these GT-selective panels adequately report the full functional diversity of HCV sampled globally, including GT 1 to 7 strains.In other cases, viruses have E1-E2 genes that carry cell culture adaptations that may affect neutralization by viral antibodies (Gottwein et al., Development and characterization of hepatitis C virus genotype 1-7 cell culture systems: role of CD81 and scavenger receptor class B type I and effect of antiviral drugs. Hepatology 2009; 49: 364-77). In addition, there are well-documented functional differences between the cellular entry of HCVpp and HCVcc, including dependence on entry factors (Sainz et al., Identification of the Niemann-Pick C1-like 1cholesterol absorption receptor as a new hepatitis C virus entry factor. Nat Med 2012;18:281-5) and sensitivity to membrane fusion inhibitors (He et al., Repurposing of the antihistamine chlorcyclizine and related compounds for treatment of hepatitis C virus infection. Sci Transl Med 2015;7; Perin et al., Flunarizine prevents hepatitis C virus membrane fusion in a genotype-dependent manner by targeting the potential fusion peptide within E1. Hepatology 2016;63:49-62).Although studies have demonstrated good concordance between HCVpp and HCVcc neutralization (Kinchen et al., 2008), HCVpp tends to be more sensitive to neutralization than HCVcc (Wasilewski et al., 2016; Urbanowicz et al., A diverse panel of hepatitis C virus glycoproteins for use invaccine research reveals extremes of monoclonal antibody neutralization resistance. J Virol 2015;90:3288-301), and the above differences may hinder the assessment of important determinants and features of virus-antibody interactions.

[0010] Therefore, a reference virus panel was established for rigorous and balanced quantification of the breadth and potency of HCV-specific antibodies across a wide range of HCV strains sampled globally (Bankwitz et al., Hepatitis C reference viruses highlight potent antibody responses and diverse viral functional interactions with neutralising antibodies. Gut 2021;70(9):1734-1745): To this end, the neutralizing potency of polyclonal immunoglobulins from patients infected with HCV genotypes (GT) 1-6 was analyzed in 13 HCV strains representing five viral GTs. HCV neutralization was plotted on neutralization plots using the metric multidimensional scaling. K-means clustering was employed to guide virus clustering and selection of representative strains. Viruses were mapped to six distinct neutralization clusters, composed in part of viruses from different GTs. The neutralization sensitivity of the viruses varied widely, with GT2a.J6 being the most resistant and GT5a.SA13 being the most sensitive. There was no correlation between virus neutralization and genetic distance, indicating that functional neutralization clustering differs from sequence-based clustering. Calibrating reference viruses representing these clusters with purified antibodies from 496 patients infected with viruses from GT1 to GT6 readily identified individuals with exceptionally strong and broadly neutralizing antibodies. It revealed comparable antibody cross-neutralization and diversity between samples from different viral GTs, confirming reports of balanced HCV cross-neutralization in highly diverse human samples.

[0011] Virus strains from this panel were used, for example, to identify HCV-infected subjects with superior antibody responses, and the most potent antibodies were cloned and analyzed (Weber et al., Analysis of antibodies from HCV eliteneutralizers identifies genetic determinants of broad neutralization. Immunity 55(2):341-345.e7).

[0012] For example, Bankwitz et al. described previous vaccination approaches in 2022. The only candidate vaccines tested in humans are based on viral vectors ChAd3 and MVA, each encoding HCV nonstructural proteins NS3, NS4A, NS4B, NS5A, and inactivated NS5B (NSmut) for a heterologous basal-boost immunization strategy. Despite the induction of CD4 and CD8 T cell responses, there was no significant difference in the incidence of chronic HCV infection (Capone et al., Modulation of the immune response induced by gene electrotransfer of a hepatitis C virus DNA vaccine in nonhuman primates. J Immunol 2006; 177: 7462-7471; Swadling et al., A human vaccines strategy based on chimpanzee adenoviral and MVA vectors that primes, boosts and sustains functional HCV-specific T cell memory. Sci Transl Med 2014; 6: 261ra13).

[0013] In contrast, other approaches focus on the induction of antibodies, such as inactivated HCV particles obtained from cell culture, which can induce protective antibodies (Akazawa et al., Neutralizing antibodies induced by cell culture-derived hepatitis C virus protect against infection in mice. Gastroenterology 2013; 145: 447-445.e1-4; Gottwein et al., Viral hepatitis: cell-culture derived HCV-a promising vaccine antigen. Nat Rev Gastroenterol Hepatol 2013; 10: 508-509; Yokokawa et al., Induction of humoral and cellular immunity by immunization with HCV particles vaccine in a non-human primate model. Gut 2018; 372-379). However, these inactivated HCV particles are difficult to concentrate and purify (Wolf et al., Downstream processing of cell-culture-derived virus particles. Expert Rev Vaccines 2011; 1451-1475; Nestola et al., Improved virus purification processes for vaccination and gene therapy. Biotechnl Bioeng 2015; 112: 843-857).

[0014] Vesicular stomatitis virus (VSV)-based vaccine vectors are used as vehicles for the production of HCV virus-like particles (VLPs), which induce humoral and cellular immune responses (Ezelle et al., Generation of Hepatitis C Virus-Like Particles by Use of a Recombinant Vesicular Stomatitis Virus Vector. J Virol 2002; 76: 12325-12334). Similar VSV-based vectors have been successfully used to protect mice against SARS-CoV-2 pathogenesis (Case et al., Replication-Competent Vesicular Stomatitis Virus Vaccine Vector Protects against SARS-CoV-2-Mediated Pathogenesis in Mice. Cell Host & Microbe 2020; 28: 465-474) and Ebola vaccines (VSV-EBOV; Jones et al., Live attenuated recombinant vaccine protects nonhuman primates against Ebola and Marburgviruses. Nature Medicine 2005; 11: 786-790).

[0015] However, tests of VLPs encoding E1 and E2 produced by baculovirus in the insect cell line Sf9 did not induce neutralizing antibodies in three out of four chimpanzees (Elmowalid et al., Immunization with hepatitis C virus-like particles results in control of hepatitis C virus infection in chimpanzees. PNAS 2007; 4: 8427-8432). Other VLPs have targeted dendritic cells with lipopeptides (Chua et al., Hepatitis C VLPs delivered to dendritic cells by a TLR2 targeting lipopeptider results in enhanced antibody and cell-mediated responses PLoS One 2012; 7: e47492), or a mixture of different HCV genotypes used as a quadrivalent vaccine candidate (Christiansen et al., Immunological responses following administration of a genotype 1a / 1b / 2 / 3aquadrivalent HCV VLP vaccine. Sci Rep 2018; 8: 6483). HCV glycoproteins are also contained in chimeric HBV-HCV particles (Beaumont et al., Chimeric hepatitis B virus / hepatitis C virus envelope proteins elicit broadly neutralizing antibodies and constitute a potential bivalent prophylactic vaccine. Hepatology 2013; 57: 1303-1313).

[0016] Recent approaches are based on recombinantly produced HCV proteins, such as HCV-E1E2 (rE1E2), which has been successfully tested in clinical phase I studies for vaccination of chimpanzees and orangutans against hepatitis C virus infection (Choo et al., Vaccination of chimpanzees against infection by the hepatitis C virus. PNAS 1994; 91: 1294-1298; Frey et al., Safety and immunogenicity of HCV E1E2 vaccine adjuvanted with MF59 administered to healthy adults. Vaccine 2010; 28: 6367-6373; Law et al., PLoS One 2013; 8: e59776; Ray et al., Characterization of antibodies induced by vaccination with hepatitis C virus envelope glycoproteins. J Infect Dis 2010; 202: 862-866), or secreted forms of E1E2 heterodimers (Wang et al., Induction of broadly neutralizing antibodies using a secreted form of the hepatitis C virus E1E2heterodimer as a vaccine candidate. PNAS 2022;119(11):e2112008119). However, most methods do not take into account the genetic variability of HCV.

[0017] In view of this, the inventors have solved the problem of providing a vaccine suitable for the preventive vaccination of humans against HCV, overcoming the problems arising from the prior art candidate vaccines, for example, a vaccine suitable for protecting against multiple, preferably all HCV strains. The present invention, in particular the subject matter of the claims, solves this problem.

[0018] The present invention provides a composition comprising

[0019] a) at least the 60 C-terminal amino acids of the HCV core protein,

[0020] b) HCV E1 protein of the first HCV strain

[0021] c) HCV E2 protein of a first HCV strain, and

[0022] d) VSV-G protein,

[0023] The first HCV strain is selected from GT2a.J6, GT2r.2r and GT5a.SA13.

[0024] The proteins can be combined to form particles, in particular rVSV-HCV pseudovirions. Therefore, the present invention also provides rVSV-HCV pseudovirions, which comprise

[0025] a) at least the 60 C-terminal amino acids of the HCV core protein,

[0026] b) HCV E1 protein of the first HCV strain

[0027] c) HCV E2 protein of a first HCV strain, and

[0028] d) VSV-G protein,

[0029] Wherein the first HCV strain is selected from GT2a.J6, GT2r.2r and GT5a.SA13. A composition comprising said particles is also a subject of the present invention.

[0030] GT2a.J6, GT2r.2r and GT5a.SA13 are HCV neutralization clusters or HCV biotypes defined by Bankwitz et al. in 2021. As described therein, there is no correlation between neutralization and genetic distance. It is understood herein that an HCV strain is selected from the neutralization group if it belongs to the neutralization cluster.

[0031] In the present invention, it is preferred that the first HCV strain belongs to cluster GT2a.J6. The cluster includes strain GT2a. As further shown herein, the inventors were able to demonstrate that in the context of the present invention, E1 and E2 proteins from GT2a.J6, for example genotype GT2a (Genebank accession number MT955904, see https: / / www.ncbi.nlm.nih.gov / nuccore / MT955904), induce antibodies with particularly good neutralization properties and high cross-reactivity between strains.

[0032] E1 and E2 proteins from HCV strains belonging to clusters GT2r.2r and GT5a.SA13 have also been shown to neutralize antibodies with reasonable cross-reactivity. Therefore, they can be used instead or in addition. As shown in Bankwitz et al., 2021, cluster GT2r.2r includes strains GT2r and GT2k (Genebank accession numbers MT955910 (https: / / www.ncbi.nlm.nih.gov / nuccore / MT955910) MT955913 (https: / / www.ncbi.nlm.nih.gov / nuccore / MT955913)). Cluster GT5a.SA13 includes strain GT5a.

[0033] Using the methods described in Bankwitz et al., 2021, other HCV strains or genotypes can be characterized as belonging to one of the clusters. This includes artificial HCV viral variants, or HCV variants with modified E1 and E2 proteins. In brief, to this end, the neutralizing potency of polyclonal immunoglobulins from multiple patients infected with HCV genotypes (GT) 1-6 (e.g., at least 50 patients) can be analyzed between different HCV strains representing different viral GTs. Using the metric multidimensional scaling, HCV neutralization is plotted on a neutralization plot, and K-means clustering is employed to guide viral clustering.

[0034] Since this requires a fairly comprehensive analysis, the target HCV strain or genotype is preferably characterized as belonging to one of the defined clusters by a method comprising the following steps:

[0035] a) integrating the target protein sequence into the complete HCV genome;

[0036] b) preparing HCVcc;

[0037] c) neutralization assay using 6 different monoclonal antibodies, HC33.8, AR3C, AP33, HC84.27, HEPC3 and HEPC74; preferably at a concentration of 50 μg antibody / mL, and

[0038] d) determining the percent neutralization efficiency of each of said antibodies, and

[0039] e) comparing the neutralization percentage of each of said antibodies with the neutralization percentage of each of said antibodies against a corresponding strain being HCVcc,

[0040] Wherein if the neutralization percentage of each of the antibodies against the target strain is + / - 10% of the neutralization percentage of each of the antibodies against the corresponding strain as HCVcc, the target HCV strain or genotype is considered to belong to the same neutralization cluster.

[0041] For example, rVSV HCV with E1 / E2 proteins from a specific target strain has not been characterized as belonging to one of the neutralization clusters of Bankwitz et al. 2021. If the neutralization percentage of each of the antibodies against the target strain is + / - 10% of the neutralization percentage of each of the antibodies against the GT2a.J6 strain as HCVcc, it can be characterized as belonging to the neutralization cluster of GT2a.J6. An exemplary preparation of HCVcc is shown in Example 2. The neutralization fingerprint, i.e., the neutralization percentage of each of the antibodies against the GT2a.J6 strain, the GT2r.2r strain, and the GT5a.SA13 strain as HCVcc is as follows Figure 7 Some or all of the steps of the method can be performed as described in the legend, preferably in Example 3.

[0042] The E1 and E2 proteins of the first HCV strain have sequence identity with the E1 and / or E2 proteins of the reference strains of the corresponding cluster (GT2a, GT2r or GT2k and GT5a), preferably, the identity with E1 and E2 can be at least 90%, optionally, at least 95%, at least 99%, or preferably 100%.

[0043] For this comparison, the sequence identity of the hypervariable region of the E2 protein (E2 hypervariable region (HVR1), including the first 27 amino acid residues at the N-terminus of E2) was ignored. The sequence identity of different domains or regions of the protein may be different, and in particular the sequence identity of the hypervariable region may be as low as 0%, such as 10-100%, 20-90%, 30-80%, 40-70% or 50-60%, or one or more regions may be partially or completely missing (e.g., 10%-100%, 20-90%, 30-80%, 40-90% or 50-80%).

[0044] Preferably, the HCV E1 protein of the first strain belongs to cluster GT2a.J6, and has at least 90%, such as at least 95%, amino acid identity with SEQ ID NO:1, and / or the HCV E2 protein has at least 90% (e.g., at least 95%) amino acid identity with SEQ ID NO:2 (optionally, excluding amino acids 1-27, i.e., HVR1). Based on this, the rVSV-HCV pseudovirion is named rVSV-HCV-E1 / E2-J6-particles. In a preferred embodiment, the sequence of the HCV E1 protein comprises SEQ ID NO:1 or is SEQ ID NO:1, and / or the sequence of the HCV E2 protein comprises SEQ ID NO:2 or is SEQ ID NO:2 (optionally, excluding amino acids 1-27, i.e., HVR1).

[0045] If the HCV of the first strain is from GT2r.2r, the HCV E1 protein has at least 90%, for example at least 95% amino acid identity with SEQ ID NO:4, and / or the HCV E2 protein has at least 90%, for example at least 95% amino acid identity with SEQ ID NO:5 (optionally, excluding amino acids 1-27, i.e., HVR1). Based on this, the rVSV-HCV pseudovirion is referred to as rVSV-HCV-E1 / E2-2r-particles. Preferably, in this case, the sequence of the HCV E1 protein is SEQ ID NO:4, and / or the sequence of the HCV E2 protein is SEQ ID NO:5 (optionally, excluding amino acids 1-27, i.e., HVR1).

[0046] If the HCV of the first strain is from GT5a.SA13, the HCV E1 protein has at least 90%, such as at least 95% amino acid identity with SEQ ID NO:7, and / or the HCV E2 protein has at least 90%, such as at least 95% amino acid identity with SEQ ID NO:8 (optionally, excluding amino acids 1-27, i.e. HVR1). Based on this, the rVSV-HCV pseudovirus particles are referred to as rVSV-HCV-E1 / E2-SA13-particles. Preferably, in this case, the sequence of the HCV E1 protein is SEQ ID NO:7, and / or the sequence of the HCV E2 protein is SEQ ID NO:8 (optionally, excluding amino acids 1-27, i.e. HVR1).

[0047] It has been previously discovered that the 60 C-terminal amino acids of the HCV core protein are necessary for the correct expression and / or processing of the E1 and E2 proteins. More amino acids of the core protein may also be included. Typically, in order to obtain optimal expression, the HCV core protein is also the HCV core protein of the first HCV strain. However, the HCV core protein of another HCV strain from the same or another HCV neutralization cluster may also be used. The 60 C-terminal amino acids of the HCV core protein may also have at least 90%, for example at least 95%, sequence identity with the HCV core protein, and the HCV core protein is preferably from the first strain.

[0048] Preferably, only a portion of the HCV core protein is used, in particular a portion that does not allow the production of HCV virus-like particles together with the E1 and E2 proteins. In Ezelle et al., 2002, a similar approach was used, but they found that the E1 / E2-containing particles formed were mostly not incorporated with VSV-G. This is best avoided by using a smaller portion of the core protein, such as the 60 C-terminal amino acids of the HCV core protein.

[0049] Preferably, if the HCV strain is from GT2a.J6, the sequence of the 60 C-terminal amino acids of the HCV core protein is SEQ ID NO:3, the sequence of the HCV E1 protein is SEQ ID NO:1, and the sequence of the HCV E2 protein is SEQ ID NO:2 (optionally, excluding amino acids 1-27, i.e. HVR1).

[0050] Optionally, if the HCV strain is from GT2r.2r, the sequence of the 60 C-terminal amino acids of the HCV core protein is SEQ ID NO:6, the sequence of the HCV E1 protein is SEQ ID NO:4, and the sequence of the HCV E2 protein is SEQ ID NO:5 (optionally, excluding amino acids 1-27, i.e., HVR1). Optionally, if the HCV strain is from GT5a.SA13, the sequence of the 60 C-terminal amino acids of the HCV core protein is SEQ ID NO:9, the sequence of the HCV E1 protein is SEQ ID NO:7, and the sequence of the HCV E2 protein is SEQ ID NO:8 (optionally, excluding amino acids 1-27, i.e., HVR1).

[0051] In addition to the mentioned HCV proteins, the VSV-G protein is also contained in the particles of the present invention. This has the advantage that the pseudovirions can infect a wider selection of cells. VSV-G allows clathrin-mediated endocytosis of viral particles into endosomes, resulting in transduction of almost all cell types. Therefore, the in vivo reproduction rate of the virus is higher and the immune response is enhanced. VSV-G can have the sequence of SEQ ID NO: 16 or at least 80%, such as at least 90% or at least 99% sequence identity thereof.

[0052] In addition to its primary function of mediating fusion of the viral envelope and host cell membrane with a variety of different cells, VSV-G can also stabilize pseudovirions and allow for the preparation of higher titer compositions containing rVSV-HCV pseudovirions.

[0053] Pseudovirions, also called pseudoviruses, are viruses that carry foreign viral envelope proteins. Thus, in this case, the recombinant VSV virus contains the HCV proteins in addition to VSV-G.

[0054] The particles of the present invention preferably do not contain nucleic acids encoding VSV-G proteins. Therefore, infected cells no longer produce VSV-G proteins. On the contrary, a large amount of E1 / E2 proteins are formed, and rVSV-HCV pseudovirions containing the HCV proteins but not containing VSV-G are produced. Therefore, compared with wild-type VSV, pseudoviruses preferably have limited replication ability and attenuation ability. The rVSV-HCV pseudovirions can complete a single round of infection through efficient VSV-G proteins, and the VSV-G proteins are complemented during the in vitro particle production process. The offspring pseudovirions produced by infected cells completely lack VSV-G proteins. Therefore, compared with wild-type VSV, its attenuation includes limited infection titers and severely restricted host tropism.

[0055] The VSV-G gene of VSV may be replaced by sequences encoding HCV proteins (ie, HCV core protein and at least the 60 C-terminal amino acids of E1 and E2 proteins).

[0056] Without intending to be bound by this theory, it is believed that both the administered rVSV-HCV pseudovirions containing the administered VSV-G and the rVSV-HCV pseudovirions produced by infected cells that do not contain VSV-G contribute to the induction of the desired immune response. In human subjects, the rVSV-HCV pseudovirions can further infect hepatocytes expressing the human receptor proteins occludin and CD81. As further described herein, the inventors found in mouse experiments with transgenic mice expressing these receptors that this is not detrimental to the immune response, but also does not appear to be necessary.

[0057] The particles of the invention may also contain VSV nucleoprotein, VSV phosphoprotein, VSV matrix protein and / or VSV polymerase. These enable the pseudovirion of the invention to effectively infect cells. Suitably, variants may also be used.

[0058] The particles of the present invention may also comprise nucleic acids encoding HCV and optionally VSV proteins. Preferably, the nucleic acids encode the 60 C-terminal amino acids of HCV core protein, HCV E1 protein and HCV E2 protein, VSV nucleoprotein, VSV phosphoprotein, VSV matrix protein and VSV polymerase. The coding region is preferably operably linked to a promoter, such as a T7 promoter. Preferably, the nucleic acid does not encode VSV-G. The nucleic acid is typically RNA.

[0059] The rVSV HCV particles may also comprise, for example, at least one other HCV protein selected from P7 (assembly factor), N2, NS3, NS4A, NS4B, NS5A and NS5B, and preferably, the nucleic acid may encode the other HCV protein. Alternatively or additionally, the particles may comprise peptides from at least one of the proteins, for example, comprising at least one, preferably at least 2, at least 5, at least 10 or at least 20 HCV T cell epitopes, for example, T cell epitope counts that can be recognized in the context of multiple HLA alleles, such as HLA alleles (commonly expressed in at least 20%, at least 25%, at least 40% or at least 50% of the population and / or conserved between different HCV strains). Preferably, the combination of T cell epitopes is selected so that at least 80%, preferably at least 90% of human individuals can present at least one of the T cell epitopes on their HLA. The T cell epitope may be an MHC class I restricted MHC class II restricted T cell epitope. Can comprise peptide, described peptide comprises T cell epitope from different HCV strains and / or different proteins.This can further enhance immune response, and can produce additional antibody to these albumen and / or T cell response.The amount of additional protein and / or encoded peptide is subject to the restriction of carrier maximum size.Preferably, the maximum size of carrier is 16-18kb.In optimized version, particle comprises, and nucleic acid encodes, for example full-length HCV core protein E1, E2, P7 and NS2.Also can select other HCV proteins, but at least one HCV protein must be missing, thereby avoid functional complete HCV genome.

[0060] The present invention also provides a composition comprising the rVSV HCV particles of the present invention, and optionally a suitable solvent, such as a buffer and / or other excipients, such as fillers such as trehalose and / or salts (such as NaCl, Tris, citrate or phosphate). The composition can be dry, such as a freeze-dried composition. Such a dry composition may be advantageous for storage, for example at a temperature of up to 40° C. For administration, the composition is typically a solution comprising a buffer, i.e. a dry composition that can be reconstituted. Although the composition may comprise an adjuvant, one of the advantages of the present invention is that an adjuvant is not required. The composition may be a pharmaceutical composition, in particular a vaccine. It should be suitable for administration to humans, for example, it should be free of bacteria and other viruses, and non-toxic.

[0061] In one embodiment of the present invention, the composition comprising the rVSV-HCV particles of the present invention optionally further comprises rVSV-HVC particles comprising

[0062] a) at least the 60 C-terminal amino acids of the HCV core protein,

[0063] b) an HCV E1 protein of a second HCV strain, and

[0064] c) HCV E2 protein of a second HCV strain,

[0065] d) VSV-G protein,

[0066] Wherein the second HCV strain is different from the first HCV strain. Optionally, the second HCV strain is an HCV strain selected from GT2a.J6, GT2r.2r and GT5a.SA13. For example, rVSV-HCV-E1 / E2-J6-particles can be combined with rVSV-HCV-E1 / E2-2r-particles and / or rVSV-HRV-E1 / E2-SA13-particles.

[0067] In particular, the second HCV strain belongs to a neutralization cluster different from the first HCV strain. This combination of envelope proteins from different neutralization clusters may allow induction of antibodies that react more widely to different HCV strains. Of course, the composition may also include other rVSV-HCV particles with HCV E1 and E2 proteins from the third or another strain of still different neutralization clusters (e.g., providing a composition comprising rVSV-HCV-E1 / E2-J6-particles, rVSV-HCV-E1 / E2-2r-particles and rVSV-HCV-E1 / E2-SA13-particles), and / or additional antigens or vaccines, such as rVSV-EBOV. This may be useful for combined vaccination.

[0068] In one embodiment, the present invention also provides a nucleic acid encoding

[0069] a) at least the 60 C-terminal amino acids of the HCV core protein,

[0070] b) HCV E1 protein of the first HCV strain,

[0071] c) HCV E2 protein of a first HCV strain, and

[0072] d) VSV nucleoprotein, VSV phosphoprotein, VSV matrix protein and VSV polymerase,

[0073] Wherein the first HCV strain is an HCV strain selected from GT2a.J6, GT2r.2r and GT5a.SA13. Preferably, the coding sequence can be operably linked to a promoter active in human cells, such as a T7 promoter. The nucleic acid can also include a packaging signal that allows packaging in pseudovirions of the present invention, such as a VSV packaging signal.

[0074] The nucleic acid can be DNA, such as a plasmid or RNA. The nucleic acid does not encode VSV-G. It can be used to produce rVSV-HCV particles of the present invention. The nucleic acid can include, for example, SEQ ID NO:10 encoding the 60 C-terminal amino acids, E1 protein and E2 protein of the HCV core protein of GT2a-J6, SEQ ID NO:11 encoding the 60 C-terminal amino acids, E1 protein and E2 protein of the HCV core protein of GT2r.2r, or SEQ ID ID:12 encoding the 60 C-terminal amino acids, E1 protein and E2 protein of the HCV core protein of GT5a.SA13. SEQ ID NO:10 is preferred. It can be RNA, preferably a recombinant VSV nucleic acid that does not encode VSV-G. Alternatively, it can be a DNA comprising a DNA sequence corresponding to the RNA sequence. A preferred rVSV-based vector construct is provided in SEQ ID NO:13, which encodes the protein of the VSV particles of the present invention based on GT2a-J6. Another rVSV-based vector construct encoding a protein of a VSV particle of the invention based on GT2r.2r is provided in SEQ ID NO: 14. Another rVSV-based vector construct encoding a protein of a VSV particle of the invention based on GT5a.SA13 is provided in SEQ ID NO: 15.

[0075] Another object of the present invention is a method for preparing the rVSV-HCV particles of the present invention, comprising

[0076] a) culturing cells expressing VSV-G protein and transfected with the nucleic acid of the present invention under conditions suitable for producing the rVSV-HCV particles, and

[0077] b) separating the particles, and

[0078] c) optionally, concentrating the particles, and

[0079] d) Optionally, formulating the particles into a formulation suitable for storage and / or administration as a vaccine.

[0080] The cells expressing the VSV-G protein can be, for example, BHK-G43 cells. Mifepristone can induce the expression of VSV-G. Cells can be transfected with nucleic acids by transfecting with a starting virus comprising the nucleic acid.

[0081] The separation of particles includes separation from the particle-producing cells, and separation from at least part, preferably all, of the cell culture medium. The particles can be further concentrated. The separation and / or concentration of particles can be achieved by ultracentrifugation and / or spin column purification systems. For example, as described above, the preparation suitable for storage can be dry, such as lyophilized, or dissolved, such as in a buffer. The storage preparation is suitable for administration to the human body after reconstitution, such as in water for injection or a suitable buffer.

[0082] The present invention also provides rVSV-HCV particles obtained by the method, wherein the particles are preferably the particles of the present invention as described herein.

[0083] The present invention also provides a vaccine comprising the rVSV HCV particles of the present invention, the nucleic acid of the present invention or the composition of the present invention.

[0084] The vaccine is used for prophylactic vaccination of a subject. The subject can be a human subject or a mouse subject, preferably a human subject. Optionally, the subject is a subject who has been successfully treated for chronic HCV infection. Unfortunately, treatment of chronic HCV infection does not prevent reinfection and the risk is high. In this case, the vaccination of the present invention reduces the risk of reinfection or prevents reinfection. However, the subject may have never been infected with HCV. The subject may also be another subject at risk of HCV infection, such as a user of injected drugs, men who have sex with men, a person exposed to an increased risk of hospital infection, or a medical staff member.

[0085] The invention also provides a syringe comprising the vaccine of the invention, for example in dry form or as a solution.

[0086] Advantageously, the vaccines of the invention induce antibodies that can confer protection to a subject against a variety of HCV strains, such as strains belonging to clusters GT3a.S53, GT2r.2r, GT2b.2b4, GT1b.J4, GT5a.SA13 and GT2a.J6.

[0087] Vaccination is usually a preventive vaccination. Therapeutic vaccination is also conceivable.

[0088] The present invention is further described in the following examples with reference to the accompanying drawings and sequences, but is not limited thereto. For the purpose of the present invention, all references cited herein are fully incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1: Clustering of HCV reporter virus biotypes and rVSV-HCV-E1 / E2-J6 constructs. (A) Depiction of 13 HCVcc reference reporter viruses, which were tested for neutralization capacity by antibodies isolated from patients. The so-called clustering enables the classification of reference viruses into 6 neutralization clusters based on their neutralization phenotype (Bankwitz et al., 2021). Each cluster is represented by a specific biotype (labels in the figure). These 13 reference viruses or the subdivision of 6 representative biotypes form the basis for all further methods (Figure from: Bankwitz et al., 2021) (B) Schematic representation of one of the rVSV-HCV pseudovirions used for the production of rVSV-HCV pseudovirions. The glycoprotein (G protein) was deleted from the VSV genome and replaced with either the GFP (control vector) sequence or the 60 C-terminal amino acid sequence of the HCV core protein plus the HCV glycoprotein E1 plus the HCV glycoprotein E2. Six constructs were generated: genotype 1b isolate J4 (representing biotype cluster 4), genotype 2a isolate J6 (representing biotype cluster 6), genotype 2b isolate 2b4 (representing biotype cluster 3), genotype 2r isolate 2r (representing biotype cluster 2), genotype 3a isolate S52 (representing biotype cluster 1), and genotype 5a isolate SA13 (representing biotype cluster 5).

[0090] Figure 2 : Schematic representation of the in vivo immunization schedule and in vitro follow-up test system used to evaluate antibody responses in mice. (A) In vivo immunization studies were performed in C57BL / 6 wild-type mice as well as in recipient transgenic hOC mice (liver-specific expression of the human HCV entry receptor CD81 and occludin). 1 × 10 8 Animals were immunized intramuscularly with rVSV-HCV pseudovirions. Minimal blood was drawn at the 5th and 6th weeks of the experiment, and the final maximum blood volume of each animal was drawn at the 9th week. Serum was separated by centrifugation and used for subsequent determinations. (B) The presence of binding antibodies in serum was tested using an ELISA binding assay for cell lysates infected with 13 reference viruses of HCVcc, and the presence of neutralizing antibodies was tested using a neutralization assay for reference viruses produced by 6 different cell cultures.

[0091] Figure 3 : ELISA binding data of rVSV-HCV-immunized mouse sera with lysates of cells infected with 13 HCV reporter viruses representing six established biotype clusters. All binding data of 74 analyzed mouse serum samples (rows) with cell lysates infected with 13 HCVcc reference viruses determined in ELISA binding assays (columns) are sorted. OD values ​​are shown on a scale between 0 and 1 (black 1 indicates the strongest binding, white 0 indicates the weakest binding).

[0092] Figure 4 : Neutralization data of mouse sera and six representatives of established biotype clusters and description of the dynamic range of the assay. (AF) The ability of all mouse sera from immune BL6 and hOC animals to neutralize 6 HCV biotypes by diluting them 1:20. All data points represent the measurements of a single animal, the bar graphs represent the average of a group of animals, and the error bars represent the standard deviation (SD). The results for BL6 mice and hOC mice are shown separately. Left: Neutralization of the reference virus by mouse sera (one reference virus was neutralized in each figure) is expressed as x-fold neutralization compared to the average value achieved by the control animal sera. Right: Raw data graph of luciferase measurement, with the remaining infectivity of the reference virus represented in logarithm. The dynamic range of the assay (light gray area) is defined by the average remaining luciferase counts after adding control mouse sera as the upper boundary (upper dashed line) and the background of the measurement system as the lower boundary (lower dashed line).

[0093] Figure 5 : Summary of all neutralization data and immune group ranking. (AH) Neutralization indicates that the reference virus of the mouse serum sample (one group of immune animals in each figure) is x times the average neutralization of the control animal serum. (I) (AH) Data summary. The neutralization data of all reference viruses in different mouse groups are summarized and used as the basis for candidate virus ranking. Statistics calculated using Brown-Forsythe and Welch ANOVA tests (ns=not significant; *p<0.05; **p<0.01; ***p<0.001). For (AI), the following applies: All data points represent the measurements of a single animal, the bar graph represents the average of a group of animals, and the error bar graph represents the standard deviation (SD). The results for BL6 mice and hOC mice are shown separately. (J) The final ranking of the neutralization data of a single immune mouse group is shown as the average x-fold neutralization compared to the corresponding control group.

[0094] Figure 6 : Correlation of differences in population-specific binding and neutralizing capacity of all representative reporter viruses tested. (A) Binding data (see Figure 3 ) and the neutralization data determined with sera from immune mice (see Figure 4 and Figure 5). Correlations were calculated based on all 74 animals. The area between the dotted lines represents the 95% confidence interval. (B) Summary of ELISA binding data. One data point represents the average of the ELISA binding assay OD for cell lysates infected with all 13 reference viruses tested. (C) Summary of neutralization data. One data point represents the average neutralization rate for all 6 reference viruses tested. For (BC), all data points represent the measurements of a single animal, the bar graphs represent the average of a group of animals, and the error bars represent the standard deviation. (DF) See description of (AC). Difference: Correlations were calculated using only n=10 animals from the GT2b.2b4 immunization group. (GI) See description of (AC). Difference: Correlations were calculated based only on n=9 animals from the GT2a.J6 immunization group.

[0095] Figure 7 : Unique neutralization fingerprints of rVSV-HCV-E1 / E2 candidate vaccines using six monoclonal antibodies. (AC) Radar charts depict the neutralization sensitivity of the HCVcc versions of the three best rVSV-HCV constructs with six different monoclonal HCV-specific antibodies, resulting in unique neutralization fingerprints. Values ​​are plotted as the percentage of neutralization (0% to 100%) of a specific HCVcc infection at a concentration of 50 μg / ml of monoclonal antibody. The black line represents the average neutralization value of n=3 biological replicates, and the dotted line represents the interval of plus or minus 10%. DETAILED DESCRIPTION

[0096] sequence

[0097] SEQ ID NO:1 HCV E1 protein of GT2a-J6

[0098] SEQ ID NO: 2 HCV E2 protein of GT2a-J6

[0099] SEQ ID NO: 360 C-terminal amino acids of HCV core protein of GT2a-J6

[0100] SEQ ID NO:4GT2r.2r HCV E1 protein

[0101] SEQ ID NO: 5GT2r.2r HCV E2 protein

[0102] SEQ ID NO: 6GT2r.60 C-terminal amino acids of HCV core protein of 2r

[0103] SEQ ID NO:7 HCV E1 protein of GT5a.SA13.

[0104] SEQ ID NO:8 HCV E2 protein of GT5a.SA13.

[0105] SEQ ID NO:9 The 60 C-terminal amino acids of the HCV core protein of GT5a.SA13.

[0106] SEQ ID NO: 10 Nucleic acid encoding the 60 C-terminal amino acids of the HCV core protein, E1 protein and E2 protein of GT2a-J6

[0107] SEQ ID NO: 11 is a nucleic acid encoding the 60 C-terminal amino acids of the HCV core protein of GT2r.2r, the E1 protein, and the E2 protein.

[0108] SEQ ID NO: 12 is a nucleic acid encoding the 60 C-terminal amino acids of the HCV core protein of GT5a.SA13, the E1 protein and the E2 protein.

[0109] SEQ ID NO: 13 rVSV vector construct encoding proteins of the VSV particle of the present invention based on GT2a-J6.

[0110] SEQ ID NO: 14 rVSV vector construct encoding proteins of the VSV particle of the present invention based on GT2r.2r.

[0111] SEQ ID NO: 15 rVSV vector construct encoding proteins of the VSV particle of the present invention based on GT5a.SA13.

[0112] SEQ ID NO:16VSV-G

[0113] Example

[0114] 1. Propagation and Isolation / Concentration of rVSV-HCV-E1 / E2 Particles

[0115] To propagate rVSV-HCV-E1 / E2 particles, transgenic BHK-G43 cells were inoculated 24 h before infection. Mifepristone-dependent expression of VSV-G surface protein in BHK-G43 cells was induced for 6 h before infection. For induction, 5% FCS and 10 -9 M mifepristone-containing DMEM medium and incubate the cells at 37°C for 6 hours. The rVSV-HCV-E1 / E2 virus stock was added to the medium at a final dilution of 1:100 to 1:200. The supernatant was harvested after 24 hours and cell debris was removed by low-speed centrifugation. To remove the spent mifepristone-containing cell culture medium, the particle-containing supernatant was applied to a spin column-based purification system ( Pro Purification System with 100 kDa Ultra-0.5 device). The rVSV pseudoparticles suppressed by the filter unit were eluted with plain DMEM or PBS without additives. Aliquots were prepared and frozen in liquid nitrogen. Titrations were performed on BHK-21 cells (the basic cell line of transgenic BHK-G43 cells).

[0116] 2. Production of infectious cell culture-derived hepatitis C virus stock solution (HCVcc stock solution)

[0117] Up to 20 μg of reporter viral plasmid DNA was linearized using appropriate restriction enzymes. According to the instructions of the supplier, Qiagen Spin Miniprep kit was used to purify plasmid DNA. Subsequently, 2 μg of restricted and purified plasmid DNA was used as a template for in vitro transcription. The reaction was completed in a total volume of 100 μL, containing the following components: 80 mM HEPES (pH 7.5), 12 mM MgCl2, 2 mM spermidine, 40 mM dithiothreitol (DTT), 3.125 mM concentration of each ribonucleoside triphosphate, 1 U RNase inhibitor (Promega), 0.6 U T7 RNA polymerase (Promega) per microliter. After incubating the reaction mixture at 37 ° C for 2 hours, 0.3 U T7 polymerase / μL was added, and the reaction continued for another 2 hours at 37 ° C. Subsequently, transcription was terminated by adding 7.5 U DNase (Promega) and incubating at 37 ° C for 30 minutes. In vitro transcribed RNA was purified using the NucleoSpin RNA cleanup kit (Macherey & Nagel) according to the manufacturer's instructions. To generate HCVcc reporter viral particles, we electroporated Huh7.5.1 cells with 5 μg of in vitro transcribed reporter viral RNA. Briefly, single cell suspensions were prepared by trypsin treatment, and cells were washed with phosphate-buffered saline (PBS), counted, and plated at 10 per ml. 7The cell density of 10 cells was resuspended in Cytomix. Cytomix consisted of 2 mM ATP, 5 mM glutathione, 120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4 / KH2PO4 (pH 7.6), 25 mM Hepes, 2 mM EGTA, 5 mM MgCl2. ATP and glutathione were added before use. 400 μL of the cell suspension in Cytomix was gently mixed with 5 μg of in vitro transcribed RNA, transferred to an electroporation cuvette (gap width 0.4 cm; BioRad), and electroporated with a BioRad Gene-Pluser at 975 μF and 270 V. The electroporated cells were quickly transferred to fresh DMEM and seeded in a cell culture vessel. We collected and pooled the supernatants of the electroporated cells 48, 72, and 96 hours after electroporation, filtered through a 0.45 μm filter, and stored them at -80 °C.

[0118] 3. HCVcc neutralization assay using HCV-specific monoclonal antibodies or mouse serum

[0119] To neutralize HCV infection, 100 μL of Huh-7.5 cell suspension (10 cells / mL) was added to the cells 24 hours before inoculation. 5 Cells) were inoculated into 96-well plates. The HCVcc reporter virus was mixed with 50 μg / mL of monoclonal antibody or 1:20 diluted immune mouse heat-inactivated serum (30 minutes at 56°C) and incubated at 37°C for 45 minutes. The mixture was then used to inoculate cells at 37°C for 4 hours in triplicate. Thereafter, 170 μL of DMEM was added to the cells. Infection was quantified 72 hours after virus inoculation by measuring luciferase activity. To this end, the cells were washed once with PBS and lysed directly on the plate by adding 35 μL Milli Q water. After a freeze-thaw cycle, the lysate was resuspended and relative light units (RLU) were measured in a plate photometer (Lumat LB Centro, Berhold, Germany) after adding luciferase substrate (1 μM coelenterazine in water). Neutralization data were analyzed using GraphPad Prism V9.0.0 (GraphPad Software, La Jolla, California, USA).

[0120] 4. ELISA method to detect HCV binding antibodies

[0121] To evaluate the antibody titers of the vaccinated mouse sera, nunc-Immuno plates (Thermo Scientific) were coated with cell lysates of HCV infected cells. Briefly, Huh-7.5.1 cells were transfected with HCV viral RNA and cell lysates were harvested 96 hours after infection with RIPA buffer. Plates were pretreated with snowdrop agglutinin (GNA) (500 ng / well). The wells were washed twice with TBST (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween-20, Sigma), blocked with 100 μl / well of BLOTTO (5% skim milk powder, 5% normal goat serum in TBST buffer) and left at room temperature for 1 hour. After subsequent washing with TBST, each well of the plate was coated with 50 μl of cell lysate (diluted 1:10 in BLOTTO). Mouse antiserum and AP33 monoclonal antibody were diluted to 1:200 in Blotto. Anti-mouse IgG-HRP antibody (sigma 1:1000) and TMB substrate (sigma) were added for reaction and quenched with 1 M sulfuric acid; the absorbance was measured at 450 nm (nonspecific background was 630 nm) using an ELISA plate reader (BioTech).

[0122] 5. Comparison and analysis of antibodies induced by different rVSV-HCV-E1 / E2 particles

[0123] For each of the six biotypes identified by Bankwitz et al. 2021, rVSV-HCV-E1 / E2 pseudovirions encoding functional E1 and E2 envelope proteins of the biotype were generated. VSV-G was deleted from the VSV genome and replaced by either the GFP (control vector) sequence or the sequence of the 60 C-terminal amino acids of the HCV core protein plus the HCV glycoprotein E1 plus the HCV protein E2, as shown in Figure 1b. Six constructs were prepared: genotype 1b isolate J4 (representing biotype cluster 4), genotype 2a isolate J6 (representing biotype cluster 6, see SEQ ID NO:13), genotype 2b isolate 2b4 (representing biotype cluster 3), genotype 2r isolate 2r (representing biotype cluster 2, see SEQ ID NO:14), genotype 3a isolate S52 (representing biotype cluster 1), and genotype 5a isolate SA13 (representing biotype cluster 5, see SEQ ID:15).

[0124] Prepare particles (as described in Example 1). Since the cells that produce them express VSV-G, the particles also contain the glycoprotein in addition to the HCV envelope protein. Therefore, the particles can also infect cells through VSV-G. After infection, the newly formed pseudoviral particles no longer contain VSV-G because it is not encoded in the nucleic acid. Therefore, a second round of infection is only possible in cells that express at least a minimum set of human HCV entry receptors CD81 and occludin. Therefore, in in vivo immunization studies, in addition to C57BL / 6 wild-type mice (where a second round of infection is excluded), receptor transgenic hOC mice with liver-specific expression of human HCV entry receptors CD81 and occludin were also used.

[0125] according to Figure 2 Particles were titrated according to the protocol shown and mice were infected with the particles. 1 × 10 per ml was injected intramuscularly at the beginning of the experiment, 4 weeks later, and 7 weeks later. 8 Animals were immunized with rVSV HCV pseudovirions, and animals were immunized with an injection volume of 60 μL. Minimum blood was drawn at the 5th and 6th weeks of the experiment, and the final maximum blood volume of each animal was drawn at the 9th week. Serum was separated by centrifugation and used for subsequent determinations. The presence of binding antibodies in serum was tested using an ELISA binding assay for cell lysates infected with HCVcc 13 reference viruses (as described in Example 4), and the presence of neutralizing antibodies was tested using a neutralization assay for reference viruses produced by 6 different cell cultures, as described in Example 3).

[0126] Figure 3-6 The results shown indicate that all vectors are able to induce antibodies in mice that can bind to at least some of the different envelope protein systems tested, but that there are fundamental differences in the quality of these antibodies, particularly with respect to the induction of neutralizing antibodies.

[0127] All 56 immunized mice showed the production of HCV E1E2 binding antibodies, such as Figure 2 B. The reactivity of all immunized animal sera to 13 E1E2 envelope proteins of reference viruses previously characterized as belonging to different neutralizer clusters was quantitatively compared by ELISA assay (Bankwitz et al., 2021) ( Figure 3 ). Positive binding (defined as at least two times the OD compared to the control group) was found to at least one E1E2 complex in 13 representatives of the six biotypes. In total, 28 of the 56 sera contained antibodies capable of binding to 12 or even all 13 of the lysates used, demonstrating the presence of very broadly binding antibodies.

[0128] As a core method for further analyzing the estimated antiviral effects of these antibodies, the inventors used neutralization assays to demonstrate the presence of neutralizing antibodies in serum, as described in Example 3. The internal quality and comparability of the neutralization assays ( Figure 4 ) and the ability to detect neutralizing antibodies ( Figure 5 ) were analyzed. Neutralization assays were performed on the same six biotypes used to prepare the rVSV-HCV constructs for immunization. All biotypes were successfully neutralized by the corresponding mouse sera from both immune mouse strains ( Figure 4 , left). In addition, the specific measurement window for each HCVcc neutralization was individually defined as the dynamic range between the plate background and the control serum neutralization level ( Figure 4 , right column, shows the minimum analysis window. For GT1b.J4, Figure 4 A could detect a 166-fold reduction in infectivity compared to the control, while the largest analysis window (GT2a.J6, Figure 4 B) Infectivity was reduced 13.043-fold compared to the control.

[0129] Figure 5 Figures AH depict the measured neutralization of all viral particles produced in cell culture by each group of immunized animals. Figure IJ summarizes the results of each group of animals against all tested viruses. These tests did not show any neutralizing activity (above background) of sera from naive mice or animals immunized with a GFP expression vector as a control ( Figure 5 In the groups immunized with VSV constructs with envelope proteins of the GT1b.J4, GT2b.2b4 or GT3a.S52 genotypes, low levels of neutralization could be observed only in some animals ( Figure 5 C, E, G). In contrast, in animals immunized with GT2r.2r, GT5a.SA13, and GT2a.J6 ( Figure 5 D, F, and H), significant neutralization of the sera was detected.

[0130] The sera from the group of animals immunized with GT2a.J6 were the most promising group ( Figure 5 D). As an average of all tested viruses, in B16 mice, the reduction in infectivity was 9.8-fold higher and highly significant compared to the sera of control mice. In hOC mice, a 5.4-fold higher and highly significant reduction was observed ( Figure 5 IJ). This rVSV-HCV-GT2a.J6 vaccine vector induced highly potent neutralizing antibodies against all six reference viruses, representing all six different neutralizing clusters / biotypes of HCV ( Figure 5 D).

[0131] Another important parameter is the correlation of the binding characteristics with the neutralizing characteristics of antibodies induced by immunization with the candidate vaccine vector ( Figure 6 On the basis of all animals (n=74), there was a highly significant positive correlation (P<0.0001) between measured binding and measured neutralization in mouse sera (r=0.7173), see Figure 6 A. However, there were clear differences between the candidate vaccines: for animals immunized with rVSV-HCV-E1E2-GT2b.2b4, there was only a low correlation (2 = 0.2263). Figure 6 E, marked) cannot be converted into strong neutralizing properties ( Figure 6 In contrast, animals vaccinated with rVSV-HCV-GT2a.J6 showed the most significant positive correlation between groups (r = 0.8707, Figure 6 G, labeled). Good binding properties of the antibody ( Figure 6 H, labeled) and the strong neutralization properties of the entire assay ( Figure 6 I, labeled) corresponding. Therefore, both vectors discussed here induce antibodies that can bind to different E1E2 variants. However, rVSV-HCV-GT2a.J6 is particularly effective in inducing neutralizing antibodies that can neutralize different biotype HCV variants.

[0132] This confirmed that rVSV-HCV expressed E1 and E2 proteins from HCV strain GT2a.J6 (see Figure 1A ), such as rVSV-HCV-GT2a.J6, is most suitable as a vaccine candidate.

Claims

1. rVSV-HCV pseudovirion, comprising a) at least the 60 C-terminal amino acids of the HCV core protein, b) HCV E1 protein of the first HCV strain c) HCV E2 protein of the first HCV strain, and d) VSV-G protein, The first HCV strain is an HCV strain selected from GT2a.J6, GT2r.2r and GT5a.SA13.

2. The particle according to claim 1, wherein the HCV is HCV strain GT2a.J6.

3. The particle of claim 2, wherein the HCV E1 protein has at least 90% amino acid identity with SEQ ID NO: 1, and / or the HCV E2 protein has at least 90% amino acid identity with SEQ ID NO: 2, optionally excluding amino acids 1-27, in, Preferably, the sequence of the HCV E1 protein is SEQ ID NO: 1, and / or the sequence of the HCV E2 protein is SEQ ID NO: 2, optionally excluding amino acids 1-27.

4. The particle according to any one of the preceding claims, wherein the HCV core protein is also the HCV core protein of the first HCV strain, in, Preferably, the sequence of the 60 C-terminal amino acids of the HCV core protein is SEQ ID NO: 3, wherein the sequence of the HCV E1 protein is SEQ ID NO: 1, and the sequence of the HCV E2 protein is SEQ ID NO: 2, optionally excluding amino acids 1-27.

5. The particle of any of the preceding claims, further comprising VSV nucleoprotein, VSV phosphoprotein, VSV matrix protein and VSV polymerase.

6. The particle according to any of the preceding claims, further comprising nucleic acid encoding the 60 C-terminal amino acids of HCV core protein, HCV E1 protein and HCV E2 protein, VSV nucleoprotein, VSV-phosphoprotein, VSV-matrix protein and VSV polymerase.

7. The particle according to any one of the preceding claims, which does not comprise nucleic acid encoding VSV-G protein.

8. The particle of any preceding claim, comprising at least one additional HCV protein selected from the group consisting of P7, N2, NS3, NS4A, NS4B, NS5A and NS5B.

9. A composition comprising the particles according to any one of the preceding claims, and optionally a suitable solvent and / or excipient.

10. The composition particle according to claim 9, optionally, the composition particle further comprises: a) at least the 60 C-terminal amino acids of the HCV core protein, b) HCV E1 protein of the second HCV strain and c) HCV E2 protein of a second HCV strain, d) VSV-G protein, wherein the second HCV strain is different from the first HCV strain, in, Optionally, the second HCV strain is an HCV strain selected from GT2a.J6, GT2r.2r and GT5a.SA13.

11. A nucleic acid encoding a) at least the 60 C-terminal amino acids of the HCV core protein, b) HCV E1 protein of a first HCV strain, c) HCV E2 protein of the first HCV strain, and d) VSV nucleoprotein, VSV phosphoprotein, VSV matrix protein and VSV polymerase, The first HCV strain is an HCV strain selected from GT2a.J6, GT2r.2r and GT5a.SA13.

12. A method for preparing the particles according to any one of claims 1 to 8, comprising a) culturing cells expressing VSV-G protein and transfected with the nucleic acid of claim 11 under conditions suitable for the production of said particles, and b) separating the particles, and c) concentrating the particles, and d) Optionally, formulating the particles into a formulation suitable for storage and / or administration as a vaccine.

13. rVSV-HCV particles obtainable by the method of claim 12.

14. A vaccine comprising the rVSV-HCV particle of any one of claims 1-8 or 13, the nucleic acid of claim 11, or the composition of any one of claims 9-10.

15. The vaccine according to claim 14, for use in the prophylactic vaccination of a subject, optionally a subject who has been successfully treated for chronic HCV infection.

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