NS1 DNA vaccination protects against Zika infection through T cell-mediated immunity in immunocompetent mice.

The causal association of Zika virus (ZIKV) with microcephaly, congenital malformations in infants, and Guillain-Barré syndrome in adults highlights the need for effective vaccines. Thus far, efforts to develop ZIKV vaccines have focused on the viral envelope. ZIKV NS1 as a vaccine immunogen has not been fully explored, although it can circumvent the risk of antibody-dependent enhancement of ZIKV infection, associated with envelope antibodies. Here, we describe a novel DNA vaccine encoding a secreted ZIKV NS1, that confers rapid protection from systemic ZIKV infection in immunocompetent mice. We identify novel NS1 T cell epitopes in vivo and show that functional NS1-specific T cell responses are critical for protection against ZIKV infection. We demonstrate that vaccine-induced anti-NS1 antibodies fail to confer protection in the absence of a functional T cell response. This highlights the importance of using NS1 as a target for T cell-based ZIKV vaccines.

Pre-clinical evaluation of a quadrivalent HCV VLP vaccine in pigs following microneedle delivery.

The introduction of directly acting antiviral agents (DAAs) has produced significant improvements in the ability to cure chronic hepatitis C infection. However, with over 2% of the world's population infected with HCV, complications arising from the development of cirrhosis of the liver, chronic hepatitis C infection remains the leading indication for liver transplantation. Several modelling studies have indicated that DAAs alone will not be sufficient to eliminate HCV, but if combined with an effective vaccine this regimen would provide a significant advance towards achieving this critical World Health Organisation goal. We have previously generated a genotype 1a, 1b, 2a, 3a HCV virus like particle (VLP) quadrivalent vaccine. The HCV VLPs contain the core and envelope proteins (E1 and E2) of HCV and the vaccine has been shown to produce broad humoral and T cell immune responses following vaccination of mice. In this report we further advanced this work by investigating vaccine responses in a large animal model. We demonstrate that intradermal microneedle vaccination of pigs with our quadrivalent HCV VLP based vaccine produces long-lived multi-genotype specific and neutralizing antibody (NAb) responses together with strong T cell and granzyme B responses and normal Th1 and Th2 cytokine responses. These responses were achieved without the addition of adjuvant. Our study demonstrates that our vaccine is able to produce broad immune responses in a large animal that, next to primates, is the closest animal model to humans. Our results are important as they show that the vaccine can produce robust immune responses in a large animal model before progressing the vaccine to human trials.

Viral vector and route of administration determine the ILC and DC profiles responsible for downstream vaccine-specific immune outcomes.

This study demonstrates that route and viral vector can significantly influence the innate lymphoid cells (ILC) and dendritic cells (DC) recruited to the vaccination site, 24 h post delivery. Intranasal (i.n.) vaccination induced ST2/IL-33R+ ILC2, whilst intramuscular (i.m.) induced IL-25R+ and TSLPR+ (Thymic stromal lymphopoietin protein receptor) ILC2 subsets. However, in muscle a novel ILC subset devoid of the known ILC2 markers (IL-25R- IL-33R- TSLPR-) were found to express IL-13, unlike in lung. Different viral vectors also influenced the ILC-derived cytokines and the DC profiles at the respective vaccination sites. Both i.n. and i.m. recombinant fowlpox virus (rFPV) priming, which has been associated with induction of high avidity T cells and effective antibody differentiation exhibited low ILC2-derived IL-13, high NKp46+ ILC1/ILC3 derived IFN-γ and low IL-17A, together with enhanced CD11b+ CD103- conventional DCs (cDC). In contrast, recombinant Modified Vaccinia Ankara (rMVA) and Influenza A vector priming, which has been linked to low avidity T cells, induced opposing ILC derived-cytokine profiles and enhanced cross-presenting DCs. These observations suggested that the former ILC/DC profiles could be a predictor of a balanced cellular and humoral immune outcome. In addition, following i.n. delivery Rhinovirus (RV) and Adenovius type 5 (Ad5) vectors that induced elevated ILC2-derived IL-13, NKp46+ ILC1/ILC3-derived-IFN-γ and no IL-17A, predominantly recruited CD11b- B220+ plasmacytoid DCs (pDC). Knowing that pDC are involved in antibody differentiation, we postulate that i.n. priming with these vectors may favour induction of effective humoral immunity. Our data also revealed that vector-specific replication status and/or presence or absence of immune evasive genes can significantly alter the ILC and DC activity. Collectively, our findings suggest that understanding the route- and vector-specific ILC and DC profiles at the vaccination site may help tailor/design more efficacious viral vector-based vaccines, according to the pathogen of interest.

Immunological responses following administration of a genotype 1a/1b/2/3a quadrivalent HCV VLP vaccine.

The significant public health problem of Hepatitis C virus (HCV) has been partially addressed with the advent of directly acting antiviral agents (DAAs). However, the development of an effective preventative vaccine would have a significant impact on HCV incidence and would represent a major advance towards controlling and possibly eradicating HCV globally. We previously reported a genotype 1a HCV viral-like particle (VLP) vaccine that produced neutralizing antibodies (NAb) and T cell responses to HCV. To advance this approach, we produced a quadrivalent genotype 1a/1b/2a/3a HCV VLP vaccine to produce broader immune responses. We show that this quadrivalent vaccine produces antibody and NAb responses together with strong T and B cell responses in vaccinated mice. Moreover, selective neutralizing human monoclonal antibodies (HuMAbs) targeting conserved antigenic domain B and D epitopes of the E2 protein bound strongly to the HCV VLPs, suggesting that these critical epitopes are expressed on the surface of the particles. Our findings demonstrate that a quadrivalent HCV VLP based vaccine induces broad humoral and cellular immune responses that will be necessary for protection against HCV. Such a vaccine could provide a substantial addition to highly active antiviral drugs in eliminating HCV.

Delivery methods to increase cellular uptake and immunogenicity of DNA vaccines.

DNA vaccines are ideal candidates for global vaccination purposes because they are inexpensive and easy to manufacture on a large scale such that even people living in low-income countries can benefit from vaccination. However, the potential of DNA vaccines has not been realized owing mainly to the poor cellular uptake of DNA in vivo resulting in the poor immunogenicity of DNA vaccines. In this review, we discuss the benefits and shortcomings of several promising and innovative non-biological methods of DNA delivery that can be used to increase cellular delivery and efficacy of DNA vaccines.

Large scale production of a mammalian cell derived quadrivalent hepatitis C virus like particle vaccine.

A method for the large-scale production of a quadrivalent mammalian cell derived hepatitis C virus-like particles (HCV VLPs) is described. The HCV core E1 and E2 coding sequences of genotype 1a, 1b, 2a or 3a were co-expressed in Huh7 cell factories using a recombinant adenoviral expression system. The structural proteins self-assembled into VLPs that were purified from Huh7 cell lysates by iodixanol ultracentrifugation and Stirred cell ultrafiltration. Electron microscopy, revealed VLPs of the different genotypes that are morphologically similar. Our results show that it is possible to produce large quantities of individual HCV genotype VLPs with relative ease thus making this approach an alternative for the manufacture of a quadrivalent mammalian cell derived HCV VLP vaccine.

Characterization of a hepatitis C virus-like particle vaccine produced in a human hepatocyte-derived cell line.

An effective immune response against hepatitis C virus (HCV) requires the early development of multi-specific class 1 CD8+ and class II CD4+ T-cells together with broad neutralizing antibody responses. We have produced mammalian-cell-derived HCV virus-like particles (VLPs) incorporating core, E1 and E2 of HCV genotype 1a to produce such immune responses. Here we describe the biochemical and morphological characterization of the HCV VLPs and study HCV core-specific T-cell responses to the particles. The E1 and E2 glycoproteins in HCV VLPs formed non-covalent heterodimers and together with core protein assembled into VLPs with a buoyant density of 1.22 to 1.28 g cm-3. The HCV VLPs could be immunoprecipited with anti-ApoE and anti-ApoC. On electron microscopy, the VLPs had a heterogeneous morphology and ranged in size from 40 to 80 nm. The HCV VLPs demonstrated dose-dependent binding to murine-derived dendritic cells and the entry of HCV VLPs into Huh7 cells was blocked by anti-CD81 antibody. Vaccination of BALB/c mice with HCV VLPs purified from iodixanol gradients resulted in the production of neutralizing antibody responses while vaccination of humanized MHC class I transgenic mice resulted in the prodution of HCV core-specific CD8+ T-cell responses. Furthermore, IgG purified from the sera of patients chronically infected with HCV genotypes 1a and 3a blocked the binding and entry of the HCV VLPs into Huh7 cells. These results show that our mammalian-cell-derived HCV VLPs induce humoral and HCV-specific CD8+ T-cell responses and will have important implications for the development of a preventative vaccine for HCV.

Intradermal delivery of DNA encoding HCV NS3 and perforin elicits robust cell-mediated immunity in mice and pigs.

Currently, no vaccine is available against hepatitis C virus (HCV), and although DNA vaccines have considerable potential, this has not been realised. Previously, the efficacy of DNA vaccines for human immunodeficiency virus (HIV) and HCV was shown to be enhanced by including the gene for a cytolytic protein, viz. perforin. In this study, we examined the mechanism of cell death by this bicistronic DNA vaccine, which encoded the HCV non-structural protein 3 (NS3) under the control of the CMV promoter and perforin is controlled by the SV40 promoter. Compared with a canonical DNA vaccine and a bicistronic DNA vaccine encoding NS3 and the proapoptotic gene NSP4, the perforin-containing vaccine elicited enhanced cell-mediated immune responses against the NS3 protein in vaccinated mice and pigs, as determined by ELISpot and intracellular cytokine staining, whereas a mouse challenge model suggested that the immunity was CD8(+) T-cell-dependent. The results of the study showed that the inclusion of perforin in the DNA vaccine altered the fate of NS3-positive cells from apoptosis to necrosis, and this resulted in more robust immune responses in mice and pigs, the latter of which represents an accepted large animal model in which to test vaccine efficacy.

Identification of specific regions in hepatitis C virus core, NS2 and NS5A that genetically interact with p7 and co-ordinate infectious virus production.

The p7 protein of hepatitis C virus (HCV) is a small, integral membrane protein that plays a critical role in virus replication. Recently, we reported two intergenotypic JFH1 chimeric viruses encoding the partial or full-length p7 protein of the HCV-A strain of genotype 1b (GT1b; Virology; 2007; 360:134). In this study, we determined the consensus sequences of the entire polyprotein coding regions of the wild-type JFH1 and the revertant chimeric viruses and identified predominant amino acid substitutions in core (K74M), NS2 (T23N, H99P) and NS5A (D251G). Forward genetic analysis demonstrated that all single mutations restored the infectivity of the defective chimeric genomes suggesting that the infectious virus production involves the association of p7 with specific regions in core, NS2 and NS5A. In addition, it was demonstrated that the NS2 T23N facilitated the generation of infectious intergenotypic chimeric virus encoding p7 from GT6 of HCV.

Identification of residues involved in NS2 homodimerization and elucidation of their impact on the HCV life cycle.

The NS2 protein of hepatitis C virus (HCV) plays a critical role in virus morphogenesis and infectivity. The crystal structure of the C-terminus of the NS2 protein (NS2(Pro)) from the H77 strain indicates that NS2(Pro) forms a homodimer. In this study, using computational modelling, we identified residues at the NS2(Pro) dimer interface that have a role in dimerization and confirmed their capacity to influence dimerization by expression studies. Our modelling analysis identified 22 residues at the NS2(Pro) dimer interface that may be important for dimer formation. Based on the free binding energy, we selected the top five ranked mutations (V162A, M170A, I175A, D186A and I201A) for further study. Western blot analysis revealed that M170A, I175A, I201A, D186A and V162A resulted in a 4.0-, 3.2-, 3.0-, 2.8- and 1.5-fold increase, respectively, in the monomer/dimer ratio compared to wild type, confirming a role in homodimer formation or stability. Japanese Fulminant Hepatitis type 1 mutants expressing M170A, I175A, D186A and I201A demonstrated increasing defects in both RNA replication and the production of infectious virus compared to wild type. This study identified residues at the NS2(Pro) dimer interface that modulate NS2(Pro) homodimerization and demonstrated that abrogation of NS2(Pro) homodimerization results in defects in HCV replication and release of infectious virus.

Reverse genetic analysis of a putative, influenza virus M2 HXXXW-like motif in the p7 protein of hepatitis C virus.

SUMMARY: The p7 protein of hepatitis C virus (HCV) has been classified into a family of viral proteins, designated viroporins that form ion channels. The M2 protein of influenza virus is the prototype viroporin and encodes a HXXXW motif that constitutes the main functional element of the M2 channels. Alignment of different p7 proteins revealed that a HXXXW sequence (positions 17-21) is also highly conserved among some HCV genotypes. To study the putative HXXXW motif in p7, five mutants of the Japanese fulminant hepatitis 1 strain of HCV that encoded H17A, H17G, H17E, Y21A and Y21W were generated. After transfection of human hepatoma cells with the mutant transcripts, unlike H17A and H17G that produced up to 1 log lower viral titres than wild type, H17E and Y21W showed slightly higher infectivity. In conclusion, this study demonstrated that the HXXXW sequence exists in the p7 proteins of some HCV genotypes and that H17 plays an important role in virus replication.

Human dendritic cells pulsed with specific lipopeptides stimulate autologous antigen-specific T cells without the addition of exogenous maturation factors.

SUMMARY: Serum-free culture conditions to generate immature human monocyte-derived DC (Mo-DC) were optimized, and the parameters that influence their maturation after exposure to lipopeptides containing CD4(+) and CD8(+) T-cell epitopes were examined. The lipopeptides contained a single CD4(+) helper T-cell epitopes, one of a number of human leucocyte antigen (HLA)-A2-restricted cytotoxic T-cell epitope and the lipid Pam2Cys. To ensure complete maturation of the Mo-DC, we examined (i) the optimal lipopeptide concentration, (ii) the optimal Mo-DC density and (iii) the appropriate period of exposure of the Mo-DC to the lipopeptides. The results showed that a high dose of lipopeptide (30 microm) was no more efficient at upregulating maturation markers on Mo-DC than a low dose (6 microm). There was an inverse relationship between Mo-DC concentration and the mean fluorescence intensity of maturation markers. In addition, at the higher cell concentrations, the chemotactic capacity of the Mo-DC towards a cognate ligand, CCL21, was reduced. Thus, high cell concentrations during lipopeptide exposure were detrimental to Mo-DC maturation and function. The duration of exposure of Mo-DC to the lipopeptides had little effect on phenotype, although Mo-DC exposed to lipopeptides for 48 rather than 4 h showed an increased ability to stimulate autologous peripheral blood mononuclear cells to release interferon-gamma in the absence of exogenous maturation factors. These findings reveal conditions for generating mature antigen-loaded DC suitable for targeted immunotherapy.

A chimeric GB virus B encoding the hepatitis C virus hypervariable region 1 is infectious in vivo.

Two GB virus B (GBV-B) chimeric genomes, GBV-HVR and GBV-HVRh (with a hinge), containing the coding region of the immunodominant hypervariable region 1 (HVR1) of the E2 envelope protein of Hepatitis C virus (HCV) were constructed. Immunoblot analysis confirmed that HVR1 was anchored to the GBV-B E2 protein. To investigate the replication competence and in vivo stability of in vitro-generated chimeric RNA transcripts, two naïve marmosets were inoculated intrahepatically with the transcripts. The GBV-HVR chimeric genome was detectable for 2 weeks post-inoculation (p.i.), whereas GBV-HVRh reverted to wild type 1 week p.i. Sequencing analysis of the HVR1 and flanking regions from GBV-HVR RNA isolated from marmoset serum demonstrated that the HVR1 insert remained unaltered in the GBV-HVR chimera for 2 weeks. Inoculation of a naïve marmoset with serum collected at 1 week p.i. also resulted in viraemia and confirmed that the serum contained infectious particles. All animals cleared the infection by 3 weeks p.i. and remained negative for the remaining weeks. The chimera may prove useful for the in vivo examination of any HCV HVR1-based vaccine candidates.

Transient and stable expression of the HCV envelope glycoproteins in cell lines and primary hepatocytes transduced with a recombinant baculovirus.

A recombinant baculovirus, RecBV-E, encoding the hepatitis C virus (HCV) envelope proteins, E1 and E2, controlled by the cytomegalovirus promoter was constructed. RecBVs can infect mammalian cells, but fail to express proteins or replicate because the viral DNA promoters are not recognised. The RecBV-E transduced 86% of Huh7 cells and 22% of primary marmoset hepatocytes compared with 35% and 0.4%, respectively, after DNA transfection. Several stable cell lines were generated that constitutively expressed E1/E2 in every cell. No evidence of E1/E2-related apoptosis was noted, and the doubling times of cells were similar to that of the parental cells. A proportion of the E1/E2 was expressed on the surface of the stable cells as determined by flow cytometry and was detected by a conformation-dependent monoclonal antibody. It is likely that the continued expression of E1/E2 in the stable cells resulted from integration of the RecBV DNA. Infection of Huh7 cells, in the absence of G418 selection, failed to result in expression of the foreign gene (in this case, eGFP) beyond 14-18 days. RecBVs that express HCV genes from a CMV promoter represent an effective means by which to transduce primary hepatocytes for expression and replication studies.

A 2a/1b full-length p7 inter-genotypic chimeric genome of hepatitis C virus is infectious in vitro.

The p7 protein of hepatitis C virus (HCV) functions as an ion channel in planar lipid bilayers, and its function is vital for the virus life cycle. In this study, we replaced either the entire or partial p7 of genotype 2a (strain JFH1), an HCV strain that replicates and produces virus progeny in vitro, with the corresponding regions of the p7 protein from genotype 1b (Australian isolate, HCV-A). Compared to wild type, the chimeric viruses reached their peak of infectivity with a delay but they produced a comparable titer to the wild type virus and the progeny viruses were able to infect naive permissive cells. Amantadine treatment of wild type and chimeric viruses reduced the virus titers by about 50% and 45%, respectively. Therefore, in this study, for the first time, we demonstrated that genotype 2a (JFH1 strain) genome encoding a full-length genotype 1b p7 gene produces infectious particles in vitro. These chimeric viruses are valuable instruments for comparative studies of the p7 proteins.

Hepatitis C virus p7 protein is localized in the endoplasmic reticulum when it is encoded by a replication-competent genome.

p7 protein is a small protein encoded by Hepatitis C virus (HCV) that functions as an ion channel in planar lipid bilayers. The function of p7 is vital for the virus life cycle. In this study, the p7 protein of genotype 2a (strain JFH1; the only strain that replicates and produces virus progeny in vitro) was tagged with either an enhanced green fluorescent protein (eGFP) or a haemagglutinin (HA) epitope to facilitate tracking of the protein in the intracellular environment. The tagged viral polyprotein was expressed transiently in the cells after transfection with the recombinant RNA transcripts. Confocal microscopy revealed that the tagged p7 protein was localized in the endoplasmic reticulum (ER) but not associated with mitochondria. Immunoelectron microscopy confirmed the p7 localization data and, moreover, showed that intracellular virus-like particles formed in the cells transfected with the wild-type, but not the recombinant, transcripts. Following a few passages of the transfected cells, the recombinant genome with the HA tag reverted to wild-type and the entire tag was deleted. Therefore, in this study, it has been demonstrated that the p7 protein in the context of the full-length polyprotein encoded by a replication competent genome is only localized to the ER and has a possible role in HCV particle formation.

Expression of the hepatitis C virus structural proteins in mammalian cells induces morphology similar to that in natural infection.

Like many positive-strand RNA viruses, replication of the hepatitis C virus (HCV) is associated with cytoplasmic membrane rearrangements. However, it is unclear which HCV proteins induce these ultrastructural features. This work examined the morphological changes induced by expression of the HCV structural proteins, core, E1 and E2, expressed from a Semliki Forest Virus (SFV) recombinant RNA replicon. Electron microscopy of cells expressing these proteins showed cytoplasmic vacuoles containing membranous and electron-dense material that were distinct from the type I cytoplasmic vacuoles induced during SFV replicon replication. Immunogold labelling showed that the core and E2 proteins localized to the external and internal membranes of these vacuoles, but at times were also associated with some of the internal amorphous material. Dual immunogold labelling with antibodies raised against the core protein and against an endoplasmic reticulum (ER)-resident protein (protein disulphide isomerase) showed that the HCV-induced vacuoles were associated with ER-labelled membranes. This report has identified an association between the HCV core and E2 proteins with induced cytoplasmic vacuoles which are morphologically similar to those observed in HCV-infected liver tissue, suggesting that the HCV structural proteins may be responsible for the induction of these vacuoles during HCV replication in vivo.

Development and characterisation of recombinant hepatitis delta virus-like particles.

Injection of particulate hepatitis B virus surface antigen (HBsAg) in mice leads to the induction of a HBsAg-specific class-I-restricted cytotoxic T lymphocyte (CTL) response. It is proposed that any protein internal to HBsAg will also be able to elicit a specific CTL response. In this study, several carboxy-terminal truncations of hepatitis C virus (HCV) core protein were fused to varying lengths of amino-terminal truncated large hepatitis delta antigen (L-HDAg). These constructs were analysed for their ability to be expressed and the particles secreted in the presence of HBsAg after transfection into HuH-7 cells. The secretion efficiency of the various HCV core-HDAg chimeric proteins was generally poor. Constructs containing full length HDAg appeared to be more stable than truncated versions and the length of the inserted protein was restricted to around 40 amino acids. Thus, the use of L-HDAg as a chimera to package foreign proteins is limited. Consequently, a polyepitope (polytope) containing a B-cell epitope from human papillomavirus (HPV 16) and multiple T-cell epitopes from the HCV polyprotein was used to create the construct, L-HDAg-polyB. This chimeric protein was shown to be reliant on the co-expression of HBsAg for secretion into the cell culture fluid and was secreted more efficiently than the previous HCV core-HDAg constructs. These L-HDAg-polyB virus-like particles (VLPs) had a buoyant density of approximately 1.2 g/cm3 in caesium chloride and approximately 1.15 g/cm3 in sucrose. The VLPs were also immunoprecipitated using an anti-HBs but not an anti-HD antibody. Thus, these recombinant VLPs have similar biophysical properties to L-HDAg VLPs.

Vitamin B12 and hepatitis C: molecular biology and human pathology.

Cobalamins are stored in high concentrations in the human liver and thus are available to participate in the regulation of hepatotropic virus functions. We show that cyanocobalamin (vitamin B12) inhibited the HCV internal ribosome entry site (IRES)-dependent translation of a reporter gene in vitro in a dose-dependent manner without significantly affecting the cap-dependent mechanism. Vitamin B12 failed to inhibit translation by IRES elements from encephalomyocarditis virus (EMCV) or classical swine fever virus (CSFV). We also demonstrate a relationship between the total cobalamin concentration in human sera and HCV viral load (a measure of viral replication in the host). The mean viral load was two orders of magnitude greater when the serum cobalamin concentration was above 200 pM (P < 0.003), suggesting that the total cobalamin concentration in an HCV-infected liver is biologically significant in HCV replication.

Antigenicity and immunogenicity of novel chimeric hepatitis B surface antigen particles with exposed hepatitis C virus epitopes.

The small envelope protein of hepatitis B virus (HBsAg-S) can self-assemble into highly organized virus like particles (VLPs) and induce an effective immune response. In this study, a restriction enzyme site was engineered into the cDNA of HBsAg-S at a position corresponding to the exposed site within the hydrophilic a determinant region (amino acid [aa] 127-128) to create a novel HBsAg vaccine vector allowing surface orientation of the inserted sequence. We inserted sequences of various lengths from hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) E2 protein containing immunodominant epitopes and demonstrated secretion of the recombinant HBsAg VLPs from transfected mammalian cells. A number of different recombinant proteins were synthesized, and HBsAg VLPs containing inserts up to 36 aa were secreted with an efficiency similar to that of wild-type HBsAg. The HVR1 region exposed on the particles retained an antigenic structure similar to that recognized immunologically during natural infection. VLPs containing epitopes from either HCV-1a or -1b strains were produced that induced strain-specific antibody responses in immunized mice. Injection of a combination of these VLPs induced antibodies against both HVR1 epitopes that resulted in higher titers than were achieved by vaccination with the individual VLPs, suggesting a synergistic effect. This may lead to the development of recombinant particles which are able to induce a broad anti-HCV immune response against the HCV quasispecies or other quasispecies-like infectious agents.