Recombinant virus-like particles as a carrier of B- and T-cell epitopes of hepatitis C virus (HCV)

The major aim of the project was the development of virus-like particles (VLP) displaying B- and T-cell epitopes of hepatitis C virus (HCV) proteins. To this end, hepatitis B virus core (HBc) particles were used as a carrier of HCV epitopes. Fragments of HCV genes encoding core (aa 98) and NS3 (aa 155) proteins were fused to the 3' terminus of the truncated HBV core gene. All recombinant plasmids led to relatively high levels of expression of chimeric proteins in E. coli, which resulted in the formation of complete "mature" VLP. Chimeric HBc/HCV VLPs were purified by combination of gel filtration and sucrose gradient centrifugation, and used for immunogenicity studies in mice. All variants of hybrid particles induced high humoral and cellular responses to HBcAg. Immunization with the HBc/HCV core particles led to relatively low antibody and T-cell proliferative responses to HCV core epitopes. The HBc/HCV NS3 particles were able to induce high levels of anti-NS3 antibodies in the absence of proliferative responses to HCV epitopes. Thus, the results of the current study have demonstrated the principal possibility of using VLP on the basis of HBcAg for creation of a new type of HCV-specific immunogen.     

Efficient induction of T helper 1 CD4+ T-cell responses to hepatitis C virus core and E2 by a DNA prime-adenovirus boost

Hepatitis C virus (HCV) is an important causative agent of liver disease, but currently there is no available prophylactic vaccine against HCV infection. Here, we investigated the HCV E2- and core-specific T-cell responses induced by DNA (D) and/or recombinant adenovirus (A) vaccines. In single (D versus A) or double immunizations (D-D versus A-A), the recombinant adenovirus vaccines induced higher levels of IFN-gamma secreting T-cell response and cytotoxic T lymphocytes (CTL) response than the DNA vaccines. However, a heterologous (D-A) regimen elicited the highest level of T helper 1 (Th1) CD4(+) T-cell responses. Furthermore, three E2-specific CTL epitopes were mapped using a peptide pool spanning the E2 protein sequence (a.a. 384-713) in BALB/c mice, and one of these (E2 405-414: SGPSQKIQLV) was shown to be immunodominant. Interestingly, no significant differences were found in the repertoire of E2-specific T-cell responses or in the immunodominance hierarchy of the three epitopes induced by D-D, D-A, A-A, and A-D, indicating that the breadth and hierarchy of T-cell responses is independent of these different vaccination regimens. In conclusion, the heterologous DNA prime-recombinant adenovirus boost regimen described offers an efficient promising strategy for the development of an effective T-cell-based HCV vaccine.     

A recombinant adenovirus prime-virus-like particle boost regimen elicits effective and specific immunities against norovirus in mice

Prime-boost vaccination using recombinant viral vectors and proteins has emerged as a highly effective strategy for protecting against viral pathogens. However, the ability of such regimens to provide immunity against norovirus (NV), an important cause of acute epidemic gastroenteritis worldwide, has never been assessed. In this study, we analyzed NV-specific humoral, mucosal, and cellular immune responses following intranasal immunization with the recombinant adenovirus expressing the NV GGII4 capsid protein (rAd) prime-NV virus-like particle (VLP) boost, VLP prime-rAd boost, or repeated NV VLP regimens. Our results show that mice primed with rAd and boosted with VLP had stronger humoral, mucosal, and interferon-γ responses than those immunized with VLP prime-rAd boost or VLP alone. These results demonstrate that adenovirus prime-VLP boost vaccination is an effective strategy for induction of immune responses against NV and is a promising strategy to improve current VLP-based NV vaccine development.     

Therapeutic vaccine IC41 as late add-on to standard treatment in patients with chronic hepatitis C

We examined the effect of the hepatitis C virus (HCV) peptide vaccine IC41 on HCV-specific T-cell responses and virological relapse rates in patients with chronic HCV genotype 1 infection when added to pegylated interferon plus ribavirin standard therapy. 35 patients received 6 vaccinations with IC41 from weeks 28 to 48 of standard antiviral treatment and were followed-up for another 6 months. IC41 vaccination did not prevent HCV-RNA relapse in patients with ongoing interferon standard treatment but HCV-specific T-cell responses were inducible and were associated with lower relapse rates. An increase of HCV-specific T-cell responses occurred in 73% of patients, responses were more frequent and stronger in patients with sustained virologic response than in patients who relapsed. Optimized vaccine responses may enhance sustained virologic response rates obtained with standard treatment of chronic hepatitis C.     

Recombinant retrovirus-derived virus-like particle-based vaccines induce hepatitis C virus-specific cellular and neutralizing immune responses in mice

While the immunological correlates of hepatitis C virus (HCV)-specific immunity are not well understood, it is now admitted that an effective vaccine against HCV will need to induce both cellular and humoral immune responses and address viral heterogeneity to prevent immune escape. We developed a vaccine platform specifically aimed at inducing such responses against HCV antigens displayed by recombinant retrovirus-based virus-like particles (VLPs) made of Gag of murine leukemia virus. Both ex vivo produced VLPs and plasmid DNA encoding VLPs can be used as vaccines. Here, we report that immunizations with plasmid DNA forming VLPs pseudotyped with HCV E1 and E2 envelope glycoproteins (HCV-specific plasmo-retroVLPs) induce strong T-cell-mediated immune responses that can be optimized by using proper DNA delivery methods and/or genetic adjuvants. Additionally, multigenotype or multi-specific T-cell responses were observed after immunization with plasmids that encode VLPs pseudotyped with E1E2 derived from numerous viral genotypes and/or displaying NS3 antigen in capsid proteins. While homologous prime-boost immunizations with HCV-specific plasmo-retroVLPs or ex vivo produced VLPs induce a low level of specific antibody responses, optimal combination of plasmo-retroVLPs and VLPs was identified for inducing HCV-specific T-cell and B-cell responses as well as neutralizing antibodies. Altogether, these results have important meanings for the development of anti-HCV preventive vaccines and exemplify the flexibility and potential of our retrovirus-based platform in inducing broad cellular and humoral immune responses.     

Immunization of chimpanzees with an envelope protein-based vaccine enhances specific humoral and cellular immune responses that delay hepatitis C virus infection

Two chimpanzees, one na?ve (Ch1601) and one recovered from hepatitis C virus (HCV) acute infection (Ch1587), were vaccinated with recombinant envelope glycoproteins (E1E2) and then challenged with 100 CID50 of HCV. Results of the challenge were compared to infection in a non-vaccinated control animal. Immunization generated high antibody titers to E1E2 including antibody specifically directed to the hypervariable region 1 (HVR1) in addition to strong and specific HVR1 T-cell proliferative responses. Upon challenge with HCV, viremia was delayed 3 weeks in both vaccinated animals compared to the non-immunized (control) animal. Ch1601 HCV RNA titers were maintained below 5 x 10(4) copies/ml, and alanine aminotransferase levels were only minimally elevated. An increase in intrahepatic cytokine mRNA levels coincided with a fall in HCV RNA to non-quantifiable levels. Despite this apparent control of virus replication the animal became persistently infected. Ch1587 had a significantly shorter and milder viremia, compared to the re-infection of the non-vaccinated control animal. This data indicates that a strategy inducing a T-cell immune response combined with antibody responses to E1E2 would make a viable candidate for an HCV vaccine.     

Virus-like particle vaccine protects against H3N2 canine influenza virus in dog

In the present study, virus-like particles (VLPs) were evaluated as a candidate veterinary vaccine against canine influenza virus (CIV) subtype H3N2. Specific pathogen-free (SPF) beagle dogs received a single injection of a VLP vaccine containing hemagglutinin (HA) and M1 protein of CIV H3N2 (H3 HA VLP). The vaccine was tested at 3 different doses with an adjuvant and 1 dose without an adjuvant. To evaluate the immunogenicity and protective efficacy of the H3 HA VLP vaccine, we performed hemagglutination inhibition tests to determine serological immune responses and conducted challenge studies using SPF beagle dogs. The addition of Montanide ISA 25 adjuvant significantly increased the immunogenicity of the H3 HA VLP vaccine. The experimental infection study showed that a single dose of H3 HA VLP vaccine induced protection against wild-type virus challenge in dogs. These results provide support for continued development of the VLP as an animal vaccine against influenza virus.     

携带丙型肝炎病毒非结构蛋白3基因的重组腺病毒构建研究

目的构建表达丙型肝炎病毒(HCV)非结构蛋白3 (NS3)的复制缺陷型重组腺病毒,为防治HCV感染的基因免疫和基因治疗提供实验基础. 方法将HCV NS3基因定向克隆到穿梭质粒pAdTrack-CMV上,经与腺病毒骨架质粒pAdEasy-1在大肠埃希菌BJ5183内同源重组后,转染293细胞进行包装,并反复感染293细胞进行扩增. 结果通过PCR扩增、酶切、核酸测序及Western杂交法检测鉴定,均证实插入穿梭质粒中的片段为HCV NS3基因(基因型1b);所包装出的复制缺陷型重组腺病毒AdEasy-GFP-NS3具有良好的感染能力,并可在293细胞中表达HCV NS3蛋白. 结论 AdEasy-GFP-NS3携带有HCV NS3基因,能有效地感染293细胞并高效表达,从而为丙型肝炎基因疫苗的进一步研究奠定了基础.     

Neutralization effects of antibody elicited by chimeric HBV S antigen viral-like particles presenting HCV neutralization epitopes

Hepatitis C virus (HCV) infection is a major public health problem despite effectual direct-acting antivirals (DAAs) therapy. Development of a prophylactic vaccine is essential to block spread of HCV infection. The HBV small surface antigen (HBsAg-S) can self-assemble into virus-like particles (VLPs), has higher immunogenicity and is used as a vaccine against HBV infections. Chimeric HBsAg-S proteins with foreign epitopes allow VLP formation and induce the specific humoral and cellular immune responses against the foreign proteins. In this study, we investigated the immune responses induced by chimeric VLPs with HCV neutralizing epitopes and HBV S antigen in mice. The chimeric HCV-HBV VLPs expressing neutralizing epitopes were prepared and purified. BALB/c mice were immunized with purified chimeric VLPs and the serum neutralizing antibodies were analyzed. We found that these chimeric VLPs induced neutralizing antibodies against HCV in mice. Additionally, the murine serum neutralized infections with HCV pseudoparticles and cell-cultured viruses derived from different heterologous 1a, 1b and 2a genotypes. We also found that immunization with chimeric VLPs induced anti-HBsAg antibodies. This study provides a novel strategy for development of a HCV prophylactic neutralizing epitope vaccine and a HCV-HBV bivalent prophylactic vaccine.     

Comparative analysis of immune responses and cytokine profiles elicited in rabbits by the combined use of recombinant fowlpox viruses,plasmids and virus-like particles in prime-boost vaccination protocols against SHIV

Three different prime-boost immunization protocols were tested in rabbits and their immune response was evaluated and compared with the final aim of identifying a vaccine strategy that might be able to protect non-human primates from infection with the pathogenic chimera simian/human immunodeficiency virus (SHIV)(89.6P). Protocols were based on priming with two fowlpox (FP) recombinant vectors and two expression plasmids, which express either the simian immunodeficiency virus (SIV)mac(239) gag/pol or the human immunodeficiency virus (HIV-1)env(89.6P) genes, followed by boosting with virus-like particles (VLP). All protocols were effective in eliciting homologous neutralizing Ab and highlighted the efficacy of VLP boosting. The FP vector was less efficient than plasmid DNA in inducing Ab against the gag core proteins. Analysis of cytokine expression 5 months after last immunization indicated that priming with pcDNA3gag/pol(SIV) and FPenv(89.6P) followed by VLP boosting generated a T helper (Th0) profile and a good Ab titer, suggesting a potential protocol to be tested in the SHIV-macaque model of HIV-1 infection.     

Utility of the Sindbis replicon system as an Env-targeted HIV vaccine

Sindbis replicon-based vaccine vectors are designed to combine the immunostimulatory properties of replicating viruses with the superior safety profile of non-replicating systems. In this study we performed a detailed assessment of Sindbis (SIN) replicon vectors expressing HIV-1 envelope protein (Env) for the induction of cell-mediated and humoral immune responses in a small animal model. SIN-derived virus-like particles (VLP) elicited Env-specific antibody responses that were detectable after boosting with recombinant Env protein. This priming effect could be mediated by replicon activity alone but may be enhanced by Env attached to the surface of VLP, offering a potential advantage for this mode of replicon delivery for Env based vaccination strategies. In contrast, the Env-specific CTL responses that were elicited by SIN-VLP were entirely dependent on replicon activity. SIN-VLP priming induced more durable humoral responses than immunization with protein only. This is important from a vaccine perspective, given the intrinsic tendency of Env to induce short-lived antibody responses in the context of vaccination or infection. These results indicate that further efforts to enhance the magnitude and durability of the HIV-1 Env-specific immune responses generated by Sindbis vectors, either alone or as part of prime-boost regimens, are justified.     

The generation of a simian adenoviral vectored HCV vaccine encoding genetically conserved gene segments to target multiple HCV genotypes

BackgroundHepatitis C virus (HCV) genomic variability is a major challenge to the generation of a prophylactic vaccine. We have previously shown that HCV specific T-cell responses induced by a potent T-cell vaccine encoding a single strain subtype-1b immunogen target epitopes dominant in natural infection. However, corresponding viral regions are highly variable at a population level, with a reduction in T-cell reactivity to these variants. We therefore designed and manufactured second generation simian adenovirus vaccines encoding genomic segments, conserved between viral genotypes and assessed these for immunogenicity.MethodsWe developed a computer algorithm to identify HCV genomic regions that were conserved between viral subtypes. Conserved segments below a pre-defined diversity threshold spanning the entire HCV genome were combined to create novel immunogens (1000–1500 amino-acids), covering variation in HCV subtypes 1a and 1b, genotypes 1 and 3, and genotypes 1–6 inclusive. Simian adenoviral vaccine vectors (ChAdOx) encoding HCV conserved immunogens were constructed. Immunogenicity was evaluated in C57BL6 mice using panels of genotype-specific peptide pools in ex-vivo IFN-ϒ ELISpot and intracellular cytokine assays.ResultsChAdOx1 conserved segment HCV vaccines primed high-magnitude, broad, cross-reactive T-cell responses; the mean magnitude of total HCV specific T-cell responses was 1174 SFU/10⁶ splenocytes for ChAdOx1-GT1-6 in C57BL6 mice targeting multiple genomic regions, with mean responses of 935, 1474 and 1112 SFU/10⁶ against genotype 1a, 1b and 3a peptide panels, respectively. Functional assays demonstrated IFNg and TNFa production by vaccine-induced CD4 and CD8 T-cells. In silico analysis shows that conserved immunogens contain multiple epitopes, with many described in natural HCV infection, predicting immunogenicity in humans.ConclusionsSimian adenoviral vectored vaccines encoding genetic segments that are conserved between all major HCV genotypes contain multiple T-cell epitopes and are highly immunogenic in pre-clinical models. These studies pave the way for the assessment of multi-genotypic HCV T-cell vaccines in humans.     

Chapter 12: Prophylactic HPV vaccines: underlying mechanisms

Human papillomavirus virus-like particles (HPV VLP) can be generated by the synthesis and self-assembly in vitro of the major virus capsid protein L1. HPV L1 VLPs are morphologically and antigenically almost identical to native virions, and this technology has been exploited to produce HPV L1 VLP subunit vaccines. The vaccines elicit high titres of anti-L1 VLP antibodies that persist at levels 10 times that of natural infections for at least 48 months. At present the assumption is that the protection achieved by these vaccines against incident HPV infection and HPV-associated ano-genital pathology is mediated via serum neutralising Immunoglobulin G (IgG). However, since there have been very few vaccine failures thus far, immune correlates of protection have not been established. The available evidence is that the immunodominant neutralising antibodies generated by L1 VLPs are type-specific and are not cross-neutralising, although highly homologous HPV pairs share minor cross-neutralisation epitopes. Important issues remaining to be addressed include the duration of protection and genotype replacement.     

Recombinant adenoviral vector expressing HCV NS4 induces protective immune responses in a mouse model of Vaccinia-HCV virus infection: A dose and route conundrum

Hepatitis C virus (HCV) leads to chronic infection in the majority of infected patients presumably due to failure or inefficiency of the immune responses generated. Both antibody and cellular immune responses have been suggested to be important in viral clearance. Non-replicative adenoviral vectors expressing antigens of interest are considered as attractive vaccine vectors for a number of pathogens. In this study, we sought to evaluate cellular and humoral immune responses against HCV NS4 protein using recombinant adenovirus as a vaccine vector expressing NS4 antigen. We have also measured the effect of antigen doses and routes of immunization on the quality and extent of the immune responses, especially their role in viral load reduction, in a recombinant Vaccinia-HCV (Vac-HCV) infection mouse model. Our results show that an optimum dose of adenovirus vector (2 × 10⁷ pfu/mouse) administered intramuscularly (i.m.) induces high T cell proliferation, granzyme B-expressing CD8⁺ T cells, pro-inflammatory cytokines such as IFN-γ, TNF-α, IL-2 and IL-6, and antibody responses that can significantly reduce the Vac-HCV viral load in the ovaries of female C57BL/6 mice. Our results demonstrate that recombinant adenovirus vector can induce both humoral and cellular protective immunity against HCV-NS4 antigen, and that immunity is intricately controlled by route and dose of immunizing vector.     

A virus-like particle vaccine confers protection against enterovirus D68 lethal challenge in mice

Enterovirus D68 (EV-D68) is increasingly associated with severe acute respiratory infection and acute flaccid myelitis (AFM) in children around the world. However, neither vaccines nor therapeutic drugs are available for EV-D68. Here we report the development of a virus-like particle (VLP) based experimental EV-D68 vaccine. We found that EV-D68 VLPs could be successfully generated in insect cells infected with a recombinant baculovirus co-expressing the P1 precursor and 3CD protease of EV-D68. Biochemical and electron microscopic analyses revealed that EV-D68 VLPs were composed of VP0, VP1, and VP3 capsid proteins derived from precursor P1 and were visualized as spherical particles of ∼30 nm in diameter. Immunization of mice with EV-D68 VLPs resulted in the production of serum antibodies that displayed potent serotype-specific neutralizing activities against EV-D68 virus in vitro. Passive transfer of anti-VLP sera completely protected neonatal recipient mice from lethal EV-D68 infection. Moreover, maternal immunization with these VLPs provided full protection against lethal EV-D68 challenge in suckling mice. Together, these results demonstrate that the recombinant EV-D68 VLP is a promising vaccine candidate against EV-D68 infection.     

Evaluation of influenza virus-like particles and Novasome adjuvant as candidate vaccine for avian influenza

The development of safe and effective vaccines for avian influenza viruses is a priority for pandemic preparedness. Adjuvants improve the efficacy of vaccines and may allow antigen sparing during a pandemic. We have previously shown that influenza virus-like particles (VLPs) comprised of HA, NA, and M1 proteins represent a candidate vaccine for avian influenza H9N2 virus [Pushko P, Tumpey TM, Fang Bu, Knell J, Robinson R, Smith G. Influenza virus-like particles comprised of the HA, NA, and M1 proteins of H9N2 influenza virus induce protective immune responses in BALB/c mice. Vaccine 2005;23(50):5751-9]. In this study, an H9N2 VLP vaccine and recombinant HA (rH9) vaccine were evaluated in three animal models. The H9N2 VLP vaccine protected mice and ferrets from challenge with A/Hong Kong/1073/99 (H9N2) virus. Novasome adjuvant improved immunogenicity and protection. Positive effect of the adjuvant was also detected using the rH9 vaccine. The results have implications for the development of safe and effective vaccines for avian influenza viruses with pandemic potential.     

Hepatitis B virus core particles containing multiple epitopes confer protection against enterovirus 71 and coxsackievirus A16 infection in mice

Human enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two major causative agents of hand-foot-and-mouth disease (HFMD). To investigate novel combined vaccines to prevent EV71 and CA16 infection, we constructed chimeric virus-like particles (tHBc/SPA or tHBc/SP VLPs) displaying conserved epitopes of EV71 (aa 208–222 of VP1 and aa 248–263 of VP2) and CA16 (aa271-285 of VP1) using a truncated hepatitis B virus core carrier (tHBc). Immunization with the chimeric VLPs induced epitope- or virus-specific IgG and neutralization antibodies against EV71 and CA16 in the mice. Compared with inactivated EV71, the chimeric VLPs induced significantly increased Th1 cytokine (IFN-γ, IL-2) production and decreased Th2 cytokine (IL-4, IL-10) responses. Neonatal mice born to dams immunized with the recombinant particles were completely protected from lethal EV71 and partially protected from CA16 infection. Co-expression of the conserved human MHC class I CD4+ T cell epitope (aa248-263 of VP2) did not improve the antiviral immunity of the chimeric VLP vaccine in mice. Our results demonstrate that experimental combination vaccines comprised of EV71 and CA16 epitopes induce both humoral and cellular immune responses and therefore support further preclinical and clinical development of a bivalent VLP vaccine targeting both CA16 and EV71.     

Oral administration of genetically modified Bifidobacterium displaying HCV-NS3 multi-epitope fusion protein could induce an HCV-NS3-specific systemic immune response in mice

More than 170 million people worldwide are chronic HCV (Hepatitis C virus) carriers, and about 30% of them will develop progressive liver disease, such as cirrhosis and hepatocellular carcinoma. A combination of pegylated interferon-α with ribavirin, the standard treatment for HCV infection, has been effective in fewer than 50% of patients infected with HCV genotype 1. A strong T cell response against the nonstructural protein 3 (NS3) is important for recovery from acute HCV infection, and an early multi-specific CD4+ helper and CD8+ cytotoxic T cell response is critical for HCV clearance. In the present study, we successfully constructed a genetically modified Bifidobacterium longum (B. longum) displaying recombinant HCV-NS3 peptides containing some CD4 and CD8 epitopes located in the HCV-NS3 region as an oral vaccine against chronic HCV infection. The oral administration of this vaccine could induce NS3-specific immune responses in mice through intestinal mucosal immunity. Our findings suggest that this novel oral vaccine has great potential as a novel oral vaccine against chronic HCV infection.     

Ebola virus glycoprotein Fc fusion protein confers protection against lethal challenge in vaccinated mice

Ebola virus is a Filoviridae that causes hemorrhagic fever in humans and induces high morbidity and mortality rates. Filoviruses are classified as “Category A bioterrorism agents”, and currently there are no licensed therapeutics or vaccines to treat and prevent infection. The Filovirus glycoprotein (GP) is sufficient to protect individuals against infection, and several vaccines based on GP are under development including recombinant adenovirus, parainfluenza virus, Venezuelan equine encephalitis virus, vesicular stomatitis virus (VSV) and virus-like particles. Here we describe the development of a GP Fc fusion protein as a vaccine candidate. We expressed the extracellular domain of the Zaire Ebola virus (ZEBOV) GP fused to the Fc fragment of human IgG1 (ZEBOVGP-Fc) in mammalian cells and showed that GP undergoes the complex furin cleavage and processing observed in the native membrane-bound GP. Mice immunized with ZEBOVGP-Fc developed T-cell immunity against ZEBOV GP and neutralizing antibodies against replication-competent VSV-G deleted recombinant VSV containing ZEBOV GP. The ZEBOVGP-Fc vaccinated mice were protected against challenge with a lethal dose of ZEBOV. These results show that vaccination with the ZEBOVGP-Fc fusion protein alone without the need of a viral vector or assembly into virus-like particles is sufficient to induce protective immunity against ZEBOV in mice. Our data suggested that Filovirus GP Fc fusion proteins could be developed as a simple, safe, efficacious, and cost effective vaccine against Filovirus infection for human use.     

Protection of cynomolgus monkeys against HEV infection by oral administration of recombinant hepatitis E virus-like particles

Hepatitis E virus (HEV) is an important causative agent of enterically-transmitted hepatitis. Successful vaccine development is crucial in controlling global HEV infection. HEV capsid protein, with 111 amino acids truncated at the N-terminus, was efficiently expressed in the baculovirus expression system. Expressed protein spontaneously assembled into virus-like particles (VLPs) and was released into culture medium. When cynomolgus monkeys were orally inoculated with 10mg of purified rHEV VLPs, serum IgM, IgG, and IgA responses were observed. All these antibody responses were obtained without adjuvants. When the monkeys were challenged with native HEV by intravenous injection, they were protected against infection or developing hepatitis. These results suggested that recombinant HEV (rHEV) VLPs can be a candidate for the oral hepatitis E vaccine.