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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. Source

Batra G.,Recombinant Gene Products Group | Gurramkonda C.,Recombinant Gene Products Group | Nemani S.K.,Recombinant Gene Products Group | Jain S.K.,Jamia Hamdard University | And 2 more authors.
Journal of Bioscience and Bioengineering | Year: 2010

We have developed a recombinant clone of the methylotrophic yeast Pichia pastoris capable of secreting dengue virus type 2 envelope domain III (sEDIII-2). We explored various induction parameters including media composition, temperature, pH, and methanol concentration, to optimize conditions for sEDIII-2 expression in shake flask culture. Induction at 20°C in the presence of 2% (v/v) methanol in a medium buffered to pH 5.8 resulted in highest secretion of sEDIII-2. This yield could be further enhanced up to 70% by repeated induction of the same initial biomass. Using a fed-batch cultivation strategy, we observed that shake-flask yields can be scaled up ~. 8-fold in a bioreactor. We obtained ~. 94% purity with >70% recovery after purification. This study, which demonstrates for the first time the feasibility of secreting envelope domain III using the P. pastoris host, will be relevant to sub-unit approaches to dengue vaccine development. © 2010 The Society for Biotechnology, Japan. Source

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. Source

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. Source

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. Source

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