Production of hepatitis E virus-like particles presenting multiple foreign epitopes by co-infection of recombinant baculoviruses.
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ABSTRACT:
SUBMITTER: Shima R
PROVIDER: S-EPMC4764844 | biostudies-other | 2016 Feb
REPOSITORIES: biostudies-other
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Project description:UnlabelledWe previously reported a proof-of-concept study for curing chronic hepatitis B virus (HBV) infection using a foreign-antigen recombinant HBV (rHBV) as a gene therapy vector. Targeted elimination of wild-type HBV (wtHBV)-infected cells could be achieved by functionally activating an in situ T-cell response against the foreign antigen. However, as chronic HBV infection spreads to all hepatocytes, specific targeting of virus-infected cells is thought to be less critical. It is also feared that rHBV may not induce active immunization in a setting resembling natural infection. For this immunotherapeutic approach to be practically viable, in the present study, we used a recombinant adenovirus (rAd) vector for rHBV delivery. The rAd vector allowed efficient transduction of wtHBV-producing HepG2 cells, with transferred rHBV undergoing dominant viral replication. Progeny rHBV virions proved to be infectious, as demonstrated in primary tupaia hepatocytes. These results greatly expanded the antiviral capacity of the replication-defective rAd/rHBV in wtHBV-infected liver tissue. With prior priming in the periphery, transduction with rAd/rHBV attracted a substantial influx of the foreign-antigen-specific T-effector cells into the liver. Despite the fully activated T-cell response, active expression of rHBV was observed for a prolonged time, which is essential for rHBV to achieve sustained expansion. In a mouse model of HBV persistence established by infection with a recombinant adeno-associated virus carrying the wtHBV genome, rAd/rHBV-based immunotherapy elicited a foreign-antigen-specific T-cell response that triggered effective viral clearance and subsequent seroconversion to HBV. It therefore represents an efficient strategy to overcome immune tolerance, thereby eliminating chronic HBV infection.ImportanceAdenovirus-delivered rHBV activated a foreign-antigen-specific T-cell response that abrogated HBV persistence in a mouse model. Our study provides further evidence of the potential of foreign-antigen-based immunotherapy for the treatment of chronic HBV infection.
| S-EPMC3958094 | biostudies-literature
Project description:The yellow fever (YF) 17D vaccine is a live attenuated virus. Three-dimensional (3D) homology modeling of the E protein structure from YF 17D virus and its comparison with that from tick-borne encephalitis virus revealed that it is possible to accommodate inserts of different sizes and amino acid compositions in the flavivirus E protein fg loop. This is consistent with the 3D structures of both the dimeric and trimeric forms in which the fg loop lies exposed to solvents. We demonstrate here that YF 17D viruses bearing foreign humoral (17D/8) and T-cell (17D/13) epitopes, which vary in sequence and length, displayed growth restriction. It is hypothesized that interference with the dimer-trimer transition and with the formation of a ring of such trimers in order to allow fusion compromises the capability of the E protein to induce fusion of viral and endosomal membranes, and a slower rate of fusion may delay the extent of virus production. This would account for the lower levels of replication in cultured cells and of viremia in monkeys, as well as for the more attenuated phenotype of the recombinant viruses in monkeys. Testing of both recombinant viruses (17D/8 and 17D/13) for monkey neurovirulence also suggests that insertion at the 17D E protein fg loop does not compromise the attenuated phenotype of YF 17D virus, further confirming the potential use of this site for the development of new live attenuated 17D virus-based vaccines.
| S-EPMC1143750 | biostudies-literature
Project description:BACKGROUND:Schistosomiasis is a significant health problem in more than 70 countries distributed between Africa, Asia and South America, with an infection rate of one in 30 individuals. Data on Schistosomiasis, Hepatitis B virus (HBV) and Hepatitis C virus (HCV) co-infection are scarce; however, there is a high prevalence in countries where schistosomiasis is endemic. METHODS:A systematic search was performed on published data from 1980-2014. Published papers in the databases Google, Medline, PubMed, and MiPc library were searched using the keywords epidemiology, pathogenesis and outcomes of HBV, HCV and schistosomiasis and data were extracted from the relevant studies. RESULTS:The prevalence of HBV/schistosomiasis co-infection in countries where schistosomiasis is endemic was high, ranging between 9.6 to approximately 64% in Egypt, and a maximum of 15.8% among hospitalized patients in Brazil. Concurrent infection between HBV and schistosomiasis is often associated with countries where schistosomiasis is endemic and may lead to chronic liver inflammation. Similarly, HCV infection rates in schistosomiasis populations range from 1% in Ethiopia reaching up to 50% in Egypt. CONCLUSION:There is controversy regarding the effects of HBV and HCV on schistosomiasis and vice versa. Vaccination might be a solution to the era of schistosomiasis and co-infection with HBV and HCV.
| S-EPMC4323254 | biostudies-other
Project description:The most advanced malaria vaccine, RTS,S, includes the central repeat and C-terminal domains of the Plasmodium falciparum circumsporozoite protein (PfCSP). We have recently isolated human antibodies that target the junctional region between the N-terminal and repeat domains that are not included in RTS,S. Due to the fact that these antibodies protect against malaria challenge in mice, their epitopes could be effective vaccine targets. Here, we developed immunogens displaying PfCSP junctional epitopes by genetic fusion to either the N-terminus or B domain loop of the E2 protein from chikungunya (CHIK) alphavirus and produced CHIK virus-like particles (CHIK-VLPs). The structural integrity of these junctional-epitope-CHIK-VLP immunogens was confirmed by negative-stain electron microscopy. Immunization of these CHIK-VLP immunogens reduced parasite liver load by up to 95% in a mouse model of malaria infection and elicited better protection than when displayed on keyhole limpet hemocyanin, a commonly used immunogenic carrier. Protection correlated with PfCSP serum titer. Of note, different junctional sequences elicited qualitatively different reactivities to overlapping PfCSP peptides. Overall, these results show that the junctional epitopes of PfCSP can induce protective responses when displayed on CHIK-VLP immunogens and provide a basis for the development of a next generation malaria vaccine to expand the breadth of anti-PfCSP immunity.
| S-EPMC8003078 | biostudies-literature
Project description:In order to improve the presentation and immunogenicity of single epitopes, virus-like particles (VLPs) are being used as platforms for the display of foreing epitopes on their surface. The rotavirus major capsid protein VP6 has the ability to self-assemble into empty non-infectious VLPs. In the present study, we analyzed the use of double layered VLPs (made up of VP2 and VP6 rotavirus proteins) as carriers to display a 14 amino acid epitope fused to three different aminoacidic regions of VP6 exposed on the surface of VLPs. Although all chimeric protein were correctly expressed in insect cells, only one of them resulted in spontaneous assembly of VLPs displaying the heterologous epitope on their surface, confirmed by sandwich ELISA and electron microscopy. Furthermore, the injection of chimeric VLPs into mice elicited higher antibody titers than the monomeric chimeric protein. Our results identify an specific amino acid region of VP6 which allows the insertion of at least a 14 amino acid heterolgous epitope and demonstrate its potential as immunogenic carrier.
| S-EPMC2777876 | biostudies-literature
Project description:Virus-like particles (VLPs) are hollow nanoparticles composed of recombinant viral surface proteins without a virus genome. In the present study, we investigated the production of influenza VLPs using recombinant insect cells. DNA fragments encoding influenza A virus hemagglutinin (HA) and matrix protein 1 (M1) were cloned with the Drosophila BiP signal sequence in plasmid vectors containing a blasticidin and a neomycin resistance gene, respectively. After Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with a pair of constructed plasmid vectors, stably transformed cells were established via incubation with blasticidin and G418. Western blot analyses showed that recombinant High Five cells secreted HA and M1 proteins into the culture supernatant. Immunoprecipitation of the culture supernatant with an anti-HA antibody and transmission electron microscopy suggested that secreted HA and M1 proteins were in a particulate structure with a morphology similar to that of an influenza virus. Hemagglutination assay indicated that expressed HA molecules retained hemagglutination activity. In a shake-flask culture, recombinant cells achieved a high HA yield (≈ 10 μg/ml) comparable to the yields obtained using the baculovirus-insect cell system. Recombinant insect cells may serve as excellent platforms for the efficient production of influenza VLPs for use as safe and effective vaccines and diagnostic antigens.
| S-EPMC7427601 | biostudies-literature
Project description:Although effective hepatitis C virus (HCV) antivirals are on the horizon, a global prophylactic vaccine for HCV remains elusive. The diversity of the virus is a major concern for vaccine development; there are 7 major genotypes of HCV found globally. Therefore, a successful vaccine will need to protect against HCV infection by all genotypes. Despite the diversity, many monoclonal antibodies (MAbs) with broadly cross-neutralizing activity have been described, suggesting the presence of conserved epitopes that can be targeted to prevent infection. Similarly, a vaccine comprising recombinant envelope glycoproteins (rE1E2) derived from the genotype 1a HCV-1 strain has been shown to be capable of eliciting cross-neutralizing antibodies in guinea pigs, chimpanzees, and healthy human volunteers. In order to investigate the basis for this cross-neutralization, epitope mapping of anti-E1E2 antibodies present within antisera from goats and humans immunized with HCV-1 rE1E2 was conducted through peptide mapping and competition studies with a panel of cross-neutralizing MAbs targeting various epitopes within E1E2. The immunized-goat antiserum was shown to compete with the binding of all MAbs tested (AP33, HC33.4, HC84.26, 1:7, AR3B, AR4A, AR5A, IGH526, and A4). Antisera showed the best competition against HC84.26 and AR3B and the weakest competition against AR4A. Furthermore, antisera from five immunized human vaccinees were shown to compete with five preselected MAbs (AP33, AR3B, AR4A, AR5A, and IGH526). These data show that immunization with HCV-1 rE1E2 elicits antibodies targeting multiple cross-neutralizing epitopes. Our results further support the use of such a vaccine antigen to induce cross-genotype neutralization.An effective prophylactic vaccine for HCV is needed for optimal control of the disease burden. The high diversity of HCV has posed a challenge for developing vaccines that elicit neutralizing antibodies for protection against infection. Despite this, we have previously shown that a vaccine comprising recombinant envelope glycoproteins derived from a single genotype 1a strain was capable of eliciting a cross-neutralizing antibody response in human volunteers. Here, we have used competition binding assays and peptide binding assays to show that antibodies present in the antisera from vaccinated goats and humans bind epitopes overlapping with those of a variety of well-characterized cross-neutralizing monoclonal antibodies. This provides a mechanism for the cross-neutralizing human antisera: antibodies present in the antisera bind to conserved regions associated with cross-neutralization. Importantly, this work provides further support for a vaccine comprising recombinant envelope glycoproteins, perhaps in a formulation with a vaccine component eliciting strong anti-HCV CD4(+) and CD8(+) T cell responses.
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Project description:Botulinum neurotoxins are one of the most potent toxins found in nature, with broad medical applications from cosmetics to the treatment of various neuropathies. Additionally, these toxins are classified as Category A-Tier 1 agents, with human lethal doses calculated at as little as 90 ng depending upon the route of administration. Of the eight distinct botulinum neurotoxin serotypes, the most common causes of human illness are from serotypes /A, /B, and /E. Protection can be achieved by eliciting antibody responses against the receptor-binding domain of the neurotoxin. Our previous research has shown that recombinant rabies virus-based particles can effectively present heterologous antigens. Here, we describe a novel strategy using recombinant rabies virus particles that elicits a durable humoral immune response against the botulinum neurotoxin receptor binding domains from serotypes /A, /B, and /E. Following intramuscular administration of β-propiolactone-inactivated rabies virus particles, mice elicited specific immune responses against the cognate antigen. Administration of a combination of these vectors also demonstrated antibody responses against all three serotypes based on enzyme-linked immunosorbent assay (ELISA) measurements, with minimal decay within the study timeline. Complete protection was achieved against toxin challenge from the serotypes /A and /B and partial protection for /E, indicating that a multivalent approach is feasible.
| S-EPMC4362357 | biostudies-literature
Project description:Hepatitis C virus (HCV) and hepatitis B virus (HBV) co-infection can be encountered in either virus endemic countries. Co-infection can also be found in populations at risk of parenteral transmission. Previous studies demonstrated a high risk of liver disease progression in patients with HCV/HBV co-infection; thus, they should be treated aggressively. Previous evidence recommended therapy combining peginterferon (pegIFN) alfa and ribavirin for co-infected patients with positive HCV RNA. Recent trials further advise using direct-acting antivirals (DAAs) for the clearance of HCV in the co-infected patients. Reactivation of HBV has been observed in patients post-intervention, with higher risks and earlier onset in those having had HCV cured by DAA- versus pegIFN-based therapy. The mechanism of HBV reactivation is an interesting but unsolved puzzle. Our recent study revealed that in vitro HBV replication was suppressed by HCV co-infection; HBV suppression was attenuated when interferon signaling was blocked. In vivo, the HBV viremia, initially suppressed by the presence of HCV super-infection, rebounded following HCV clearance by DAA treatment and was accompanied by a reduced hepatic interferon response. In summary, major achievements in the treatment of HCV/HBV co-infection have been accomplished over the past 20 years. Future clinical trials should address measures to reduce or prevent HBV reactivation post HCV cure.
| S-EPMC7412310 | biostudies-literature
Project description:To enhance the production efficiency of foreign protein in baculovirus expression systems, the effects of polyhedrin fragments were investigated by fusion expressing them with the enhanced green fluorescent protein (EGFP). Recombinant viruses were generated to express EGFP fused with polyhedrin fragments based on the previously reported minimal region for self-assembly and the KRKK nuclear localization signal (NLS). Fusion expressions with polyhedrin amino acids 19 to 110 and 32 to 110 lead to localization of recombinant protein into the nucleus and mediate its assembly. The marked increase of EGFP by these fusion expressions was confirmed through protein and fluorescence intensity analyses. The importance of nuclear localization for enhanced production was shown by the mutation of the NLS within the fused polyhedrin fragment. In addition, when the polyhedrin fragment fused with EGFP was not localized in the nucleus, some fragments increased the production of protein. Among these fragments, some degradation of only the fused polyhedrin was observed in the fusion of amino acids 19 to 85 and 32 to 85. The fusion of amino acids 32 to 85 may be more useful for the enhanced and intact production of recombinant protein. The production of E2 protein, which is a major antigen of classical swine fever virus, was dramatically increased by fusion expression with polyhedrin amino acids 19 to 110, and its preliminary immunogenicity was verified using experimental guinea pigs. This study suggests a new option for higher expression of useful foreign recombinant protein by using the partial polyhedrin in baculovirus.
| S-EPMC3621880 | biostudies-literature