Immune responses against hepatitis C virus genotype 3a virus-like particles in mice: A novel VLP prime-adenovirus boost strategy.

Chronic hepatitis C virus (HCV) infection represents a major health threat to global population. In India, approximately 15-20% of cases of chronic liver diseases are caused by HCV infection. Although, new drug treatments hold great promise for HCV eradication in infected individuals, the treatments are highly expensive. A vaccine for preventing or treating HCV infection would be of great value, particularly in developing countries. Several preclinical trials of virus-like particle (VLP) based vaccine strategies are in progress throughout the world. Previously, using baculovirus based system, we have reported the production of hepatitis C virus-like particles (HCV-LPs) encoding structural proteins for genotype 3a, which is prevalent in India. In the present study, we have generated HCV-LPs using adenovirus based system and tried different immunization strategies by using combinations of both kinds of HCV-LPs with other genotype 3a-based immunogens. HCV-LPs and peptides based ELISAs were used to evaluate antibody responses generated by these combinations. Cell-mediated immune responses were measured by using T-cell proliferation assay and intracellular cytokine staining. We observed that administration of recombinant adenoviruses expressing HCV structural proteins as final booster enhances both antibody as well as T-cell responses. Additionally, reduction of binding of VLP and JFH1 virus to human hepatocellular carcinoma cells demonstrated the presence of neutralizing antibodies in immunized sera. Taken together, our results suggest that the combined regimen of VLP followed by recombinant adenovirus could more effectively inhibit HCV infection, endorsing the novel vaccine strategy.

The stability of secreted, acid-labile H77/JFH-1 hepatitis C virus (HCV) particles is altered by patient isolate genotype 1a p7 sequences.

Secreted infectious particles generated by the genotype 2a JFH-1 hepatitis C virus infectious clone are resistant to acidic pH, whereas intracellular virions remain acid-labile. Thus, JFH-1 particles are thought to undergo pH maturation as they are secreted from the cell. Here, we demonstrate that both infectious intracellular and secreted genotype 1a (H77)/JFH-1 chimaeric particles display enhanced acid sensitivity compared with JFH-1, although pH maturation still occurs upon release. Introduction of p7 sequences from genotype 1a infected HCV patients into the H77/JFH-1 background yielded variable effects on infectious particle production and sensitivity to small molecule inhibitors. However, two selected patient p7 sequences increased the acid stability of secreted, but not intracellular H77/JFH-1 particles, suggesting that p7 directly influences particle pH maturation via an as yet undefined mechanism. We propose that HCV particles vary in acid stability, and that this may be dictated by variations in both canonical structural proteins and p7.

Efficient dendrimer-DNA complexation and gene delivery vector properties of nitrogen-core poly(propyl ether imine) dendrimer in mammalian cells.

Dendrimers as vectors for gene delivery were established, primarily by utilizing few prominent dendrimer types so far. We report herein studies of DNA complexation efficacies and gene delivery vector properties of a nitrogen-core poly(propyl ether imine) (PETIM) dendrimer, constituted with 22 tertiary amine internal branches and 24 primary amines at the periphery. The interaction of the dendrimer with pEGFPDNA was evaluated through UV-vis, circular dichroism (CD) spectral studies, ethidium bromide fluorescence emission quenching, thermal melting, and gel retardation assays, from which most changes to DNA structure during complexation was found to occur at a weight ratio of dendrimer:DNA ∼ 2:1. The zeta potential measurements further confirmed this stoichiometry at electroneutrality. The structure of a DNA oligomer upon dendrimer complexation was simulated through molecular modeling and the simulation showed that the dendrimer enfolded DNA oligomer along both major and minor grooves, without causing DNA deformation, in 1:1 and 2:1 dendrimer-to-DNA complexes. Atomic force microscopy (AFM) studies on dendrimer-pEGFP DNA complex showed an increase in the average z-height as a result of dendrimers decorating the DNA, without causing a distortion of the DNA structure. Cytotoxicity studies involving five different mammalian cell lines, using [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] (MTT) assay, reveal the dendrimer toxicity profile (IC50) values of ∼400-1000 µg mL(-1), depending on the cell line tested. Quantitative estimation, using luciferase assay, showed that the gene transfection was at least 100 times higher when compared to poly(ethylene imine) branched polymer, having similar number of cationic sites as the dendrimer. The present study establishes the physicochemical behavior of new nitrogen-core PETIM dendrimer-DNA complexes, their lower toxicities, and efficient gene delivery vector properties.

Monoclonal antibodies against Hepatitis C genotype 3a virus like particle inhibit virus entry in cell culture system.

The envelope protein (E1-E2) of Hepatitis C virus (HCV) is a major component of the viral structure. The glycosylated envelope protein is considered to be important for initiation of infection by binding to cellular receptor(s) and also known as one of the major antigenic targets to host immune response. The present study was aimed at identifying mouse monoclonal antibodies which inhibit binding of virus like particles of HCV to target cells. The first step in this direction was to generate recombinant HCV-like particles (HCV-LPs) specific for genotypes 3a of HCV (prevalent in India) using the genes encoding core, E1 and E2 envelop proteins in a baculovirus expression system. The purified HCV-LPs were characterized by ELISA and electron microscopy and were used to generate monoclonal antibodies (mAbs) in mice. Two monoclonal antibodies (E8G9 and H1H10) specific for the E2 region of envelope protein of HCV genotype 3a, were found to reduce the virus binding to Huh7 cells. However, the mAbs generated against HCV genotype 1b (D2H3, G2C7, E1B11) were not so effective. More importantly, mAb E8G9 showed significant inhibition of the virus entry in HCV JFH1 cell culture system. Finally, the epitopic regions on E2 protein which bind to the mAbs have also been identified. Results suggest a new therapeutic strategy and provide the proof of concept that mAb against HCV-LP could be effective in preventing virus entry into liver cells to block HCV replication.