Gaurav Batra

Dengue vaccines: state of the art

Importance of the field: Dengue is a significant public health problem transcending geographical boundaries to place nearly 50% of the global population at risk. A vaccine to prevent dengue is an unmet need that should be addressed urgently. Areas covered in the review: A brief introduction to dengue and a detailed review of the classical and modern approaches being undertaken currently to develop dengue vaccines described in recent patent literature, highlighting the inherent hurdles and challenges. What the reader will gain: An understanding of the approaches being deployed to develop multiple viral and non-viral vaccine candidates and an appreciation of the complexity of developing dengue vaccines. Take home message: Live viral vaccines, which have advanced to clinical trials, have revealed new challenges, emphasizing the need to pursue non-viral alternatives simultaneously.

Pichia pastoris-expressed dengue virus type 2 envelope domain III elicits virus-neutralizing antibodies.

A tetravalent dengue vaccine that can protect against all four serotypes of dengue viruses is a global priority. The host-receptor binding, multiple neutralizing epitope-containing carboxy-terminal region of the dengue envelope protein, known as domain III (EDIII), has emerged as a promising subunit vaccine antigen. One strategy to develop a tetravalent dengue subunit vaccine envisages mixing recombinant EDIIIs, corresponding to the four dengue virus serotypes. Towards this objective, a recombinant clone of the methylotrophic yeast Pichia pastoris, harboring the EDIII gene of dengue virus type 2 (EDIII-2) for its intracellular expression, was created. Recombinant EDIII-2 protein, expressed by this clone was purified to near homogeneity by affinity chromatography, with final yields of >50mg/l culture. Groups of Balb/c mice were immunized with this protein, separately formulated in two adjuvants, alum and montanide ISA 720. The EDIII-2 antigen, formulated in either adjuvant, elicited high levels of neutralizing antibodies to dengue virus type 2 in mice as analyzed by Plaque Reduction Neutralization Test (PRNT). This study demonstrates the feasibility of using P. pastoris to produce EDIII antigens capable of eliciting potent virus-neutralizing antibodies.

Single Antigen Detects both Immunoglobulin M (IgM) and IgG Antibodies Elicited by All Four Dengue Virus Serotypes

ABSTRACT The resurgence of dengue (DEN) virus infections in the last few decades coupled with the lack of a preventive vaccine and specific antiviral drugs has jointly contributed to making this a significant global public health problem. Currently, symptomatic supportive treatment and fluid replacement therapy are the only means available to minimize DEN-induced mortality. As the clinical symptoms associated with DEN virus infections are indistinguishable from those of many other viral, bacterial, and parasitic infections, specific diagnostic tests assume critical importance in the unequivocal identification of DEN virus infections. We have designed a novel chimeric antigen based on envelope domain III (EDIII), a critical antigenic region of the major structural protein of DEN viruses. We fused EDIIIs corresponding to each of the four DEN virus serotypes using pentaglycyl linkers, overexpressed the resultant tetravalent chimeric protein in Escherichia coli, and affinity purified it in high yields, obtaining ∼30 mg protein of >95% purity per liter of culture. We show that this tetravalent antigen could specifically recognize anti-DEN virus antibodies of both the immunoglobulin M (IgM) and IgG classes. Using a large panel of IgM antibody capture-enzyme-linked immunosorbent assay- and hemagglutination inhibition-confirmed DEN virus-infected and uninfected patient sera (n = 289), we demonstrate that this tetravalent antigen can function as a diagnostic tool of high sensitivity and specificity.

Optimization of conditions for secretion of dengue virus type 2 envelope domain III using Pichia pastoris.

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.

Evaluation of envelope domain III-based single chimeric tetravalent antigen and monovalent antigen mixtures for the detection of anti-dengue antibodies in human sera

BackgroundFlavivirus 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.MethodsA 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.ResultsThe 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.ConclusionsThe 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.

Expression, purification and characterization of in vivo biotinylated dengue virus envelope domain III based tetravalent antigen

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 20mg protein of approximately 95% purity, from 1L of induced E. coli shake flask culture, and the efficiency of biotinylation was estimated to be approximately 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.

Complexity of rice Hsp100 gene family: lessons from rice genome sequence data

Elucidation of genome sequence provides an excellent platform to understand detailed complexity of the various gene families. Hsp100 is an important family of chaperones in diverse living systems. There are eight putative gene loci encoding for Hsp100 proteins in Arabidopsis genome. In rice, two full-length Hsp100 cDNAs have been isolated and sequenced so far. Analysis of rice genomic sequence by in silico approach showed that two isolated rice Hsp100 cDNAs correspond to Os05g44340 and Os02g32520 genes in the rice genome database. There appears to be three additional proteins (encoded by Os03g31300, Os04g32560 and Os04g33210 gene loci) that are variably homologous to Os05g44340 and Os02g32520 throughout the entire amino acid sequence. The above five rice Hsp100 genes show significant similarities in the signature sequences known to be conserved among Hsp100 proteins. While Os05g44340 encodes cytoplasmic Hsp100 protein, those encoded by the other four genes are predicted to have chloroplast transit peptides.

Array-in-well binding assay for multiparameter screening of phage displayed antibodies

Phage display is a well-established and powerful tool for the development of recombinant antibodies. In a standard phage display selection process using a high quality antibody phage library, a large number of unique antibody clones can be generated in short time. However, the pace of the antibody discovery project eventually depends on the methodologies used in the next screening phase to identify the clones with the most promising binding characteristics e.g., in terms of specificity, affinity and epitope. Here, we report an array-in-well binding assay, a miniaturized and multiplexed immunoassay that integrates the epitope mapping to the evaluation of the binding activity of phage displayed antibody fragments in a single well. The array-in-well assay design used here incorporates a set of partially overlapping 15-mer peptides covering the complete primary sequence of the target antigen, the intact antigen itself and appropriate controls printed as an array with 10×10 layout at the bottom of a well of a 96-well microtiter plate. The streptavidin-coated surface of the well facilitates the immobilization of the biotinylated analytes as well-confined spots. Phage displayed antibody fragments bound to the analyte spots are traced using anti-phage antibody labelled with horseradish peroxidase for tyramide signal amplification based highly sensitive detection. In this study, we generated scFv antibodies against HIV-1 p24 protein using a synthetic antibody phage library, evaluated the binders with array-in-well binding assay and further classified them into epitopic families based on their capacity to recognize linear epitopes. The array-in-well assay enables the integration of epitope mapping to the screening assay for early classification of antibodies with simplicity and speed of a standard ELISA procedure to advance the antibody development projects.

Array-In-Well Epitope Mapping of Phage-Displayed Antibodies

Novel affinity reagents, such as single chain (scFv) antibody fragments, can be generated by isolating them from recombinant protein libraries using phage display selection. A successful selection process against a target protein can produce a number of binder candidates among which the desired binders are identified by screening and characterization of individual clones. Obtaining information on the binding properties, such as the binding epitope, already during the screening step helps to choose the most useful candidates for further development at early phase saving time and resources. To this end, we describe here an Array-in-Well-based screening procedure to perform activity testing and epitope mapping for filamentous phage-displayed scFvs in an integrated manner with a single assay.