Novel α-tocopherol-ferrocene conjugates for the specific delivery of transgenes in liver cancer cells under high serum conditions.

The delivery of therapeutic genes to a specific organ has drawn significant research attention. Among the pool of various delivery vectors, cationic liposomes (non-viral) are potential candidates for delivering therapeutic genes due to their low immunogenic response. Here, we have developed novel ferrocene-conjugated cationic tocopheryl aggregates as non-viral vectors. These formulations can transfer a reporter gene (pGL3; encoded for luciferase protein) specifically to liver cancer cells (HepG2 and Huh7) instead of non-hepatic cancer cells, such as Caco-2 (human colon carcinoma) and HeLa (cervical cancer) cells. The transfection efficiency (TE) of the optimum liposomal formulation is more significant than commercially available Lipofectamine 2000 (L2K). Notably, it retains its TE under high serum conditions (up to 50% FBS). A coupled effect from conjugated ferrocene and tocopherol in the cationic liposomal formulation might be responsible for the cell-specific delivery and higher serum compatibility. Therefore, the present proposed delivery system may provide a platform for further progress in terms of developing hepatotropic gene delivery systems.

Structural basis for neutralization of cytotoxic abrin by monoclonal antibody D6F10.

Abrin, an extremely cytotoxic Type II ribosome-inactivating protein (RIP), is a potential bio-warfare agent. Abrin A-chain (ABA) depurinates an adenosine of sarcin-ricin loop (SRL) from eukaryotic 28S rRNA, thereby arresting protein synthesis and leading to cell death. Monoclonal antibody (mAb) D6F10 is the only known antibody that neutralizes ABA's activity in cell-free systems as well as abrin's toxicity in vitro and in vivo. However, how binding of mAb D6F10 to abrin interferes with abrin's catalytic activity at ribosome is still poorly understood. To provide structural basis for mAb D6F10-mediated rescue of ribosome inactivation by abrin, we determined crystal structures of ABA with and without substrate analogs. The structures of ABA-substrate analogs and ribosome were used in an experiment-guided computational protocol, to construct the ABA-Ribosome complex. A homology model of the variable region (Fv ) of mAb D6F10 was generated and docked with the apo-ABA structure to construct the ABA-D6F10 Fv complex. Structural superposition of ABA common to ABA-D6F10 Fv and ABA-Ribosome complexes reveals steric hindrance as the primary mechanism by which mAb D6F10 neutralizes abrin. In contrast to ABA alone, ABA bound to mAb D6F10 is unable to access the SRL on the ribosome owing to steric clashes of mAb D6F10 with the ribosome. Crystal structures of ABA also reveal a catalytic water molecule implicated in hydrolyzing N-glycosidic bond of the susceptible adenosine by RIPs. Furthermore, our strategy provides structural details of steric hindrance important for neutralization of ricin, another RIP, by mAb 6C2 and hence is of wide applicability. ENZYME: EC3.2.2.22. DATABASE: Structural data have been deposited in the Protein Data Bank (PDB) under the accession numbers 5Z37, 5Z3I, and 5Z3J.

Identification of a novel epitope in the C terminus of hepatitis C virus-E2 protein that induces potent and cross-reactive neutralizing antibodies.

Hepatitis C virus (HCV) is a leading cause of chronic viral hepatitis, but an effective vaccine is still not available to prevent infection. Use of neutralizing antibodies could be a potential therapeutic option. In this study, the presence of anti-HCV antibodies in HCV-infected patients was assessed from 50 patients and the presence of neutralizing antibodies was examined using 'hepatitis C virus-like particles'. Antibodies from two samples exhibited significant inhibitory activity, suggesting that these may neutralize viral infection. Antigenic determinants generating the neutralizing antibodies from these two samples were delineated by epitope mapping using the core, E1 and E2 regions and a stretch of 45 amino acid peptide (E2C45) derived from the C-terminal region of HCV-E2 protein (aa 634-679) was designed. Results suggest that this hitherto uncharacterized region has the potential to generate neutralizing antibodies against HCV and thus be effective in preventing virus entry into liver cells. Computational analysis of the structure of the modelled peptide (E2C45) suggested high conformational entropy for this region. Furthermore, E2C45 peptide-generated antibodies could block virus entry and monoclonal antibodies generated against this peptide could also significantly reduce virus replication in a cell culture system. It is possible that the inhibition could be partly due to a conformational alteration of the CD81-binding region, preventing virus attachment to liver cells. In conclusion, this work focused on the discovery of a novel epitope at the C terminus of E2 that induces potent neutralizing antibodies in HCV-infected patients.

Evaluation of immuno diagnostic assay for the exposure of stage specific filarial infection.

Lymphatic filariasis is a debilitating diseases caused by filarial parasitic nematodes. The infection may be acquired in childhood but the symptoms become apparent only in later life. To evaluate the success of any intervention, sensitive diagnostics were used to identify infection among endemic normals that are likely to develop microfilaremia in due course of time. Capture assay was standardized using the recombinant protein Brugia malayi Abundant Larval Transcript-2 (ALT-2) specific monoclonal and poly-clonal antibodies and evaluated with serum samples of clinical groups from high and low filarial infection area individuals (HIA/LIA), Endemic Normal (EN, n = 478), microfilaeremics (MF, n = 77), chronic pathology (CP, n = 57) and non endemic normal (NEN, n = 20). In order to assess stage-specific infection, ALT-2 capture assay was compared with the early reported Venom allergen homologue (VAH) and microfilariae specific SXP-1 capture assays. Of the 632 serum samples tested, ALT-2 and VAH capture assays detected circulating filarial antigen (CFA) in 57% and 52% of HIA-EN individuals, respectively. As expected, the VAH and SXP-1 capture assays were positive for 100 % of MF individuals. The described capture assays can be useful for the detection of early and stage-specific filarial infections in endemic regions of developing countries.

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.

Evaluation of Brugia malayi sheath protein (Shp-1) as a diagnostic antigen for human lymphatic filariasis.

Lymphatic filariasis is the second leading cause of permanent long-term disability globally and control of this disease needs efficient diagnostic methods. In this study, abundantly expressing microfilarial sheath protein (Shp-1) from Brugia malayi was characterized as a filarial diagnostic candidate using samples from different clinical population. Monoclonal antibodies were developed against E. coli expressed recombinant Shp-1 in order to assess its efficiency in filarial antigen detection assay system. Endemic Normal (EN, n = 170), asymptomatic microfilaeremics (MF, n = 65), symptomatic chronic pathology (CP, n = 45) and non endemic normal (NEN, n = 10) sera were analyzed by antigen capture enzyme-linked immunosorbent assay. Of the 290 individuals, all MF individuals (both brugian and bancroftian) were positive in this assay followed by CP and EN. When compared with SXP-1 and Og4C3 antigen assays, all assays detected Wb MF correctly, Bm MF was detected by Shp-1 and SXP-1 assays, and only Shp-1 was able to detect EN (12%) and CP (29%). Results showed that this assay may be useful for monitoring prior to mass drug administration.

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.