Recombinant Gene Products Group

Delhi, India

Recombinant Gene Products Group

Delhi, India
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Pilankatta R.,Recombinant Gene Products Group | Pilankatta R.,California Pacific Medical Center | Chawla T.,Recombinant Gene Products Group | Chawla T.,National University of Singapore | And 2 more authors.
Journal of Medical Virology | Year: 2010

In vivo gene delivery using human adenovirus serotype 5 (AdV5) vectors is being explored for vaccination purposes. The presence of anti-AdV5 antibodies in human serum arising from natural exposure to AdV5 can interfere potentially with and compromise the efficacy of rAdV5-based vaccine vectors. In this report, a collection of 114 sera from healthy adult Indian blood donors was analyzed for the presence of anti-AdV5 antibodies, using an AdV5 vector encoding the green fluorescent protein (GFP) to monitor the presence of anti-AdV5 neutralizing antibodies in human sera based on their ability to block virus entry into HeLa cells which express the Coxsackievirus-and-Adenovirus Receptor (CAR). In this assay all samples tested were positive for anti-AdV5 antibodies, with titers varying over a very wide range. It was also observed that these antibodies facilitated the uptake of the reporter AdV5 vector into the monocytic cell line U937 which does not express CAR, but expresses Fc receptors (FcRs) instead. These observations have implications for rAdV5-based vaccine development. © 2010 Wiley-Liss, Inc.

Korrapati A.B.,Recombinant Gene Products Group | Korrapati A.B.,Acharya Nagarjuna University | Vijayalakshmi M.,Acharya Nagarjuna University
Current Trends in Biotechnology and Pharmacy | Year: 2016

RNA interference (RNAi) is a highly conserved, specific and selective biological response of gene regulation mechanism and has been widely exploited for designing novel drug targets to treat infections caused by pathogens including viruses. In the previous study, one of the promising sh-5b RNA (short hairpin RNA, sh- 5b) construct was targeted to the conserved sequence within the 51-NTRs (non-translated regions) of all Dengue serotypes. The current study report the stability (up to 24 months) of recombinant adenovirus (rAd) engineered to express sh-5b RNA for DENV-2 replication inhibition. Stability study was done by gene specific PCR, where as the effect of sh-5b RNA in attenuating DENV-2 replication was further validated by One step RT-PCR by TITAN. The rAds harboring sh-5b RNA to target dengue serotypes might serve as an efficient alternative to conventional drugs for therapeutic intervention to treat DENV infections in humans. © 2017 Association of Biotechnology and Pharmacy. All Rights Reserved.

Batra G.,Recombinant Gene Products Group | Nemani S.K.,Recombinant Gene Products Group | Tyagi P.,Recombinant Gene Products Group | Swaminathan S.,Recombinant Gene Products Group | Khanna N.,Recombinant Gene Products Group
BMC Infectious Diseases | Year: 2011

Background: Flavivirus cross-reactive antibodies in human sera interfere with the definitive identification of dengue virus (DENV) infections especially in areas with multiple co-circulating flaviviruses. Use of DENV envelope domain-III (EDIII) can partially resolve the problem. This study has examined the effect of (i) incorporating the EDIIIs of four DENV serotypes into a single chimeric antigen, and (ii) immobilizing the antigen through specific interaction on the sensitivity and specificity of anti-DENV antibody detection.Methods: A sera panel (n = 164) was assembled and characterized using commercial kits for infection by DENV and a host of other pathogens. Anti-DENV antibodies of both IgM and IgG classes in this panel were detected in indirect ELISAs using a mixture of monovalent EDIIIs, a chimeric EDIII-based tetravalent antigen, EDIII-T, and a biotinylated version of the latter as coating antigens. The sensitivity and specificity of these assays were compared to those obtained using the PanBio Dengue IgG/IgM ELISAs.Results: The performance of dengue IgG and IgM indirect ELISAs, using either a physical mixture of four EDIIIs or the single chimeric EDIII-T antigen, were comparable. Coating of a biotinylated version of the tetravalent antigen on streptavidin plates enhanced sensitivity without compromising specificity.Conclusions: The incorporation of the EDIIIs of the four DENV serotypes into a single chimeric antigen did not adversely affect assay outcome in indirect ELISAs. Oriented, rather than random, immobilization of the tetravalent antigen enhanced sensitivity of detection of anti-DENV antibodies with retention of 100% specificity. © 2011 Batra et al; licensee BioMed Central Ltd.

Arora U.,Recombinant Gene Products Group | Tyagi P.,Recombinant Gene Products Group | Swaminathan S.,Recombinant Gene Products Group | Khanna N.,Recombinant Gene Products Group
Journal of Nanobiotechnology | Year: 2012

Background: Dengue is a global public health problem for which no drug or vaccine is available. Currently, there is increasing interest in developing non-replicating dengue vaccines based on a discrete antigenic domain of the major structural protein of dengue viruses (DENVs), known as envelope domain III (EDIII). The use of bio-nanoparticles consisting of recombinant viral structural polypeptides, better known as virus-like particles (VLPs), has emerged as a potential platform technology for vaccine development. This work explores the feasibility of developing nanoparticles based on E. coli-expressed recombinant Hepatitis B virus core antigen (HBcAg) designed to display EDIII moiety of DENV on the surface.Findings: We designed a synthetic gene construct encoding HBcAg containing an EDIII insert in its c/e1 loop. The fusion antigen HBcAg-EDIII-2 was expressed in E. coli, purified to near homogeneity using Ni+2 affinity chromatography and demonstrated to assemble into discrete 35-40 nm VLPs by electron microscopy. Competitive ELISA analyses showed that the EDIII-2 moieties of the VLPs are accessible to anti-EDIII-2-specific monoclonal and polyclonal antibodies, suggesting that they are surface-displayed. The VLPs were highly immunogenic eliciting high titer anti-EDIII-2 antibodies that were able to recognize, bind and neutralize infectious DENV based on ELISA, immunofluorescence and virus-neutralization assays.Conclusion: This work demonstrates that HBcAg-derived nanoparticles can serve as a useful platform for the display of DENV EDIII. The EDIII-displaying nanoparticles may have potential applications in diagnostics/vaccines for dengue. © 2012 Arora et al.; licensee BioMed Central Ltd.

Patil A.,Recombinant Gene Products Group | Khanna N.,Recombinant Gene Products Group
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2012

The recombinant hepatitis B surface antigen (HBsAg) vaccine provides excellent protection against hepatitis B virus (HBV). However, high costs of its production prevents many underdeveloped and developing nations from implementing HBsAg vaccination. This in turn increases the risk of contracting HBV related diseases. Majority of the commercial HBV vaccines are derived from purified HBsAg expressed in recombinant yeasts. Most of the cost in production of the vaccine is incurred during the downstream processing. The costs associated with HBsAg purification can be decreased by optimizing the pre-chromatography steps and by reducing the impurity burden on chromatography operations. Here in this work we present a novel strategy for the enriched extraction of recombinant HBsAg from Pichia pastoris membranes. We have also developed a simple, easy to operate process for the purification of HBsAg VLPs from the membranes of P. pastoris. This novel strategy, while utilizing a single column chromatographic step in the purification scheme results in the highest recovery of HBsAg VLPs reported in the literature. The yield of HBsAg at the end of purification was nearly 5% (85 μg/g of induced wet cell biomass). The HBsAg purified from this process has shown the presence of VLPs. The immunization of these VLPs in BALB/c mice with alhydrogel adjuvant has shown good titers of neutralizing antibodies. © 2012 Elsevier B.V.

Beesetti H.,Birla Institute of Technology and Science | Khanna N.,Recombinant Gene Products Group | Swaminathan S.,Birla Institute of Technology and Science
Expert Opinion on Investigational Drugs | Year: 2016

Introduction: Dengue has emerged as the most significant arboviral disease of the current century. A drug for dengue is an urgent unmet need. As conventional drug discovery efforts have not produced any promising clinical candidates, there is a shift toward re-positioning pre-existing drugs for dengue to fast-track dengue drug development. Areas covered: This article provides an update on the current status of recently completed and ongoing dengue drug trials. All dengue drug trials described in this article were identified from a list of >230 trials that were returned upon searching the World Health Organization’s International Clinical Trials Registry Platform web portal using the search term ‘dengue’ on December 31st, 2015. Expert opinion: None of the handful of drugs tested so far has yielded encouraging results. Early trial experience has served to emphasize the challenge of drug testing in the short therapeutic time window available, the need for tools to predict ‘high-risk’ patients early on and the limitations of the existing pre-clinical model systems. Significant investment of efforts and resources is a must before the availability of a safe, effective and inexpensive dengue drug becomes a reality. Currently, supportive fluid therapy remains the only option available for dengue treatment. © 2016 Informa UK Limited, trading as Taylor & Francis Group.

Batra G.,Recombinant Gene Products Group | Talha S.M.,Recombinant Gene Products Group | Nemani S.K.,Recombinant Gene Products Group | Dhar N.,Recombinant Gene Products Group | And 2 more authors.
Protein Expression and Purification | Year: 2010

Dengue is a rapidly spreading mosquito-borne viral disease prevalent in over a hundred countries around the world. A definitive identification of dengue infection depends on reliable dengue diagnostic tests. This study describes the design, expression and purification of an in vivo biotinylated chimeric dengue antigen to exploit the high affinity of biotin-streptavidin interaction to detect anti-dengue antibodies. This chimeric antigen incorporates the envelope domain III (EDIII) of the four dengue virus serotypes. A biotin acceptor peptide was fused with the chimeric dengue antigen for in vivo biotinylation in Escherichia coli through simultaneous co-expression of the biotin ligase, BirA. Despite the localization of the chimeric dengue antigen to the insoluble fraction of induced E. coli cells, it was found to be biotinylated in vivo. It was purified to near homogeneity using affinity chromatography with final yields of 20 mg protein of ∼95% purity, from 1 L of induced E. coli shake flask culture, and the efficiency of biotinylation was estimated to be ∼85%. Mouse antibodies specific to recombinant EDIII of each of the four dengue serotypes, captured on microtiter wells sensitized with anti-mouse immunoglobulin antibodies, were recognized specifically and with high efficiency by the biotinylated antigen in conjunction with streptavidin-enzyme conjugate. An evaluation of the biotinylated antigen against a panel of pre-characterized dengue-positive and dengue-negative human sera (n = 164), in an antibody capture ELISA format, showed that it manifested 100% specificity, but also suggested that additional epitopes may need to be included in its design to enhance sensitivity. © 2010 Elsevier Inc. All rights reserved.

Korrapati A.B.,Recombinant Gene Products Group | Swaminathan G.,Recombinant Gene Products Group | Singh A.,Recombinant Gene Products Group | Khanna N.,Recombinant Gene Products Group | Swaminathan S.,Recombinant Gene Products Group
PLoS Neglected Tropical Diseases | Year: 2012

Background: Dengue is a mosquito-borne viral disease caused by four closely related serotypes of Dengue viruses (DENVs). This disease whose symptoms range from mild fever to potentially fatal haemorrhagic fever and hypovolemic shock, threatens nearly half the global population. There is neither a preventive vaccine nor an effective antiviral therapy against dengue disease. The difference between severe and mild disease appears to be dependent on the viral load. Early diagnosis may enable timely therapeutic intervention to blunt disease severity by reducing the viral load. Harnessing the therapeutic potential of RNA interference (RNAi) to attenuate DENV replication may offer one approach to dengue therapy. Methodology/Principal Findings: We screened the non-translated regions (NTRs) of the RNA genomes of representative members of the four DENV serotypes for putative siRNA targets mapping to known transcription/translation regulatory elements. We identified a target site in the 5′ NTR that maps to the 5′ upstream AUG region, a highly conserved cis-acting element essential for viral replication. We used a replication-defective human adenovirus type 5 (AdV5) vector to deliver a short-hairpin RNA (shRNA) targeting this site into cells. We show that this shRNA matures to the cognate siRNA and is able to inhibit effectively antigen secretion, viral RNA replication and infectious virus production by all four DENV serotypes. Conclusion/Significance: The data demonstrate the feasibility of using AdV5-mediated delivery of shRNAs targeting conserved sites in the viral genome to achieve inhibition of all four DENV serotypes. This paves the way towards exploration of RNAi as a possible therapeutic strategy to curtail DENV infection. © 2012 Korrapati et al.

Arora U.,Recombinant Gene Products Group | Tyagi P.,Recombinant Gene Products Group | Swaminathan S.,Recombinant Gene Products Group | Khanna N.,Recombinant Gene Products Group
Vaccine | Year: 2013

Objective: Currently, dengue represents one of the most significant arboviral disease worldwide, for which a vaccine is not yet available. Persistent challenges in live viral dengue vaccines have sparked a keen interest in exploring non-replicating dengue vaccines. We have examined the feasibility of using the methylotrophic yeast Pichia pastoris to develop a chimeric vaccine candidate displaying the dengue virus type-2 (DENV-2) envelope domain III (EDIII), implicated in host receptor binding and in the induction of virus-neutralizing antibodies, on the surface of non-infectious virus-like particles (VLP)-based on the Hepatitis B virus core antigen (HBcAg). Methods: We designed a fusion antigen by inserting DENV-2 EDIII into c/e1 loop of HBcAg. A codon-optimized gene encoding this fusion antigen was integrated into the genome of P. pastoris, under the control of the Alcohol Oxidase 1 promoter. The antigen was expressed by methanol induction and purified to near homogeneity by Ni2+ affinity chromatography. The purified antigen was characterized physically and functionally to evaluate its ability to assemble into VLPs, and elicit DENV-2-specific antibodies in mice. Results: This fusion antigen was expressed successfully to high yields and purified to near homogeneity. Electron microscopy and competitive ELISA analyses showed that it formed VLPs in which the EDIII moiety was accessible to different EDIII-specific antibodies. These VLPs were immunogenic in mice, stimulating the production of antibodies that could specifically recognize DENV-2 and neutralize its infectivity. However, virus-neutralizing antibody titers were modest. Conclusions: Our data show: (i) insertion of EDIII into the c/e1 loop of HBcAg does not compromise particle assembly; and (ii) the chimeric VLPs elicit a specific humoral response against DENV-2. The strategy of displaying dengue virus EDIII using a VLP platform will need further optimization before it may be developed into a viable alternative option. © 2012 Elsevier Ltd.

Tharad M.,Recombinant Gene Products Group | Samuchiwal S.K.,Recombinant Gene Products Group | Bhalla K.,Recombinant Gene Products Group | Ghosh A.,Recombinant Gene Products Group | And 3 more authors.
PLoS ONE | Year: 2011

Background: Protein-protein interactions play a crucial role in enabling a pathogen to survive within a host. In many cases the interactions involve a complex of proteins rather than just two given proteins. This is especially true for pathogens like M. tuberculosis that are able to successfully survive the inhospitable environment of the macrophage. Studying such interactions in detail may help in developing small molecules that either disrupt or augment the interactions. Here, we describe the development of an E. coli based bacterial three-hybrid system that can be used effectively to study ternary protein complexes. Methodology/Principal Findings: The protein-protein interactions involved in M. tuberculosis pathogenesis have been used as a model for the validation of the three-hybrid system. Using the M. tuberculosis RD1 encoded proteins CFP10, ESAT6 and Rv3871 for our proof-of-concept studies, we show that the interaction between the proteins CFP10 and Rv3871 is strengthened and stabilized in the presence of ESAT6, the known heterodimeric partner of CFP10. Isolating peptide candidates that can disrupt crucial protein-protein interactions is another application that the system offers. We demonstrate this by using CFP10 protein as a disruptor of a previously established interaction between ESAT6 and a small peptide HCL1; at the same time we also show that CFP10 is not able to disrupt the strong interaction between ESAT6 and another peptide SL3. Conclusions/Significance: The validation of the three-hybrid system paves the way for finding new peptides that are stronger binders of ESAT6 compared even to its natural partner CFP10. Additionally, we believe that the system offers an opportunity to study tri-protein complexes and also perform a screening of protein/peptide binders to known interacting proteins so as to elucidate novel tri-protein complexes. © 2011 Tharad et al.

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