Dengue vaccine development: Global and Indian scenarios

India is home to nearly a third of the global population at risk of dengue, a viral disease caused by four antigenically and genetically distinct dengue viruses. Clinical illness following dengue virus infection can either be mild and self-limiting dengue fever or severe dengue hemorrhagic fever/dengue shock syndrome, with potentially fatal consequences. A live attenuated vaccine known as Dengvaxia, developed by Sanofi, was licensed in 2015. Following this, long-term follow-up of the Sanofi phase III efficacy trial participants has revealed potential safety concerns. This vaccine, which appears to predispose dengue-naïve recipients to an increased risk of hospitalization in the future, is recommended by the World Health Organization only for adults with a history of prior dengue virus infection. A safe and efficacious dengue vaccine continues to be sought globally. India has joined these efforts in recent years, and is poised to initiate the clinical development of two candidates in the near future, one licensed from abroad and the other developed indigenously. This article provides a glimpse of India's efforts to develop dengue vaccines in the context of the global dengue vaccine development and evaluation landscape and highlights key issues and questions confronting the dengue vaccine community. Keywords: Dengue, DSV4, Pichia pastoris, ADE, Dengvaxi

An adenovirus prime/plasmid boost strategy for induction of equipotent immune responses to two dengue virus serotypes

Background: Dengue is a public health problem of global significance for which there is neither an effective antiviral therapy nor a preventive vaccine. It is a mosquito-borne viral disease, caused by Dengue (DEN) viruses, which are members of the Flaviviridae family. There are four closely related serotypes, DEN-1, DEN-2, DEN-3 and DEN-4, each of which is capable of causing disease. As immunity to any one serotype can potentially sensitize an individual to severe disease during exposure to a heterologous serotype, the general consensus is that an effective vaccine should be tetravalent, that is, it must be capable of affording protection against all four serotypes. The current strategy of creating tetravalent vaccine formulations by mixing together four monovalent live attenuated vaccine viruses has revealed the phenomenon of viral interference leading to the manifestation of immune responses biased towards a single serotype. Results: This work stems from the emergence of (i) the DEN virus envelope (E) domain III (EDIII) as the most important region of the molecule from a vaccine perspective and (ii) the adenovirus (Ad) as a promising vaccine vector platform. We describe the construction of a recombinant, replication-defective Ad (rAd) vector encoding a chimeric antigen made of in-frame linked EDIIIs of DEN virus serotypes 2 and 4. Using this rAd vector, in conjunction with a plasmid vector encoding the same chimeric bivalent antigen, in a prime-boost strategy, we show that it is possible to elicit equipotent neutralizing and T cell responses specific to both DEN serotypes 2 and 4. Conclusion: Our data support the hypothesis that a DEN vaccine targeting more than one serotype may be based on a single DNA-based vector to circumvent viral interference. This work lays the foundation for developing a single Ad vector encoding EDIIIs of all four DEN serotypes to evoke a balanced immune response against each one of them. Thus, this work has implications for the development of safe and effective tetravalent dengue vaccines

Simultaneous detection of Human Immunodeficiency Virus 1 and Hepatitis B virus infections using a dual-label time-resolved fluorometric assay

A highly specific and novel dual-label time-resolved immunofluorometric assay was developed exploiting the unique emission wavelengths of the intrinsically fluorescent terbium (Tb3+) and europium (Eu3+) tracers for the simultaneous detection of human immunodeficiency virus 1 (HIV-1) and hepatitis B virus (HBV) infections, respectively. HIV-1 infection was detected using a double antigen sandwich format wherein anti-HIV-1 antibodies were captured using an in vivo biotinylated version of a chimeric HIV-1 antigen and revealed using the same antigen labeled with Tb3+ chelate. Hepatitis B surface antigen (HBsAg), which served as the marker of HBV infection, was detected in a double antibody sandwich using two monoclonal antibodies (mAbs), one chemically biotinylated to capture, and the other labeled with Eu3+ nanoparticles, to reveal. The performance of the assay was evaluated using a collection (n = 60) of in-house and commercially available human sera panels. This evaluation showed the dual-label assay to possess high degrees of specificity and sensitivity, comparable to those of commercially available, single analyte-specific kits for the detection of HBsAg antigen and anti-HIV antibodies. This work demonstrates the feasibility of developing a potentially time- and resource-saving multiplex assay for screening serum samples for multiple infections in a blood bank setting

Escherichia coli–expressed near full length HIV-1 envelope glycoprotein is a highly sensitive and specific diagnostic antigen

Background: The Human Immunodeficiency Virus type 1 (HIV-1) envelope glycoprotein gp160, useful in detecting anti-HIV-1 antibodies, is difficult to express in heterologous hosts. The major hurdles are its signal sequence, strong hydrophobic regions and heavy glycosylation. While it has not been possible to express full length recombinant (r)-gp160 in E. coli, it can be expressed in insect and mammalian cells, but at relatively higher cost. In this work, we report E. coli-based over-expression of r-gp160 variant and evaluate its performance in diagnostic immunoassays for the detection of anti-HIV-1 antibodies. Methods: A deletion variant of r-gp160 lacking hydrophobic regions of the parent full length molecule was expressed in E. coli and purified to near homogeneity using single-step Ni (II)-affinity chromatography. Biotinylated and europium (III) chelate-labeled versions of this antigen were used to set up one- and two-step time-resolved fluorometric double antigen sandwich assays. The performance of these assays was evaluated against a collection of well-characterized human sera (n = 131), that included an in-house panel and four commercially procured panels. Results: In-frame deletion of three hydrophobic regions, spanning amino acid residues 1–43, 519–538 and 676–706, of full length HIV-1 gp160 resulted in its expression in E. coli. Both the one- and two-step assays manifested high sensitivity unambiguously identifying 75/77 and 77/77 HIV-1 positive sera, respectively. Both assays also identified all 52 HIV-seronegative sera correctly. Between the two assays, the mean signal-to-cutoff value of the two-step assay was an order of magnitude greater than that of the one-step assay. Both assays were highly specific manifesting no cross-reactivity towards antibodies specific to other viruses like hepatitis B, C and human T cell leukemia viruses. Conclusions: This study has demonstrated the expression of r-gp160 variant in E. coli, by deletion of hydrophobic regions, and its purification in reasonable yields. This underscores the potential for cost saving in antigen production. Evaluation of this antigen in a double antigen sandwich two-step assay showed it to be a highly sensitive and specific HIV-1 diagnostic reagent. The amenability of this assay to the one-step format suggests its potential utility in developing a rapid point-of-care HIV-1 diagnostic test

Virus-like particles derived from Pichia pastoris-expressed dengue virus type 1 glycoprotein elicit homotypic virus-neutralizing envelope domain III-directed antibodies

Background: Four antigenically distinct serotypes (1–4) of Dengue Viruses (DENVs) cause dengue disease. Antibodies to any one DENV serotype have the potential to predispose an individual to more severe disease upon infection with a different DENV serotype. A dengue vaccine must elicit homotypic neutralizing antibodies to all four DENV serotypes to avoid the risk of such antibody-dependent enhancement in the vaccine recipient. This is a formidable challenge as evident from the lack of protective efficacy against DENV-2 by a tetravalent live attenuated dengue vaccine that has completed phase III trials recently. These trial data underscore the need to explore non-replicating subunit vaccine alternatives. Recently, using the methylotrophic yeast Pichia pastoris, we showed that DENV-2 and DENV-3 envelope (E) glycoproteins, expressed in absence of prM, implicated in causing severe dengue disease, self-assemble into Virus-like Particles (VLPs), which elicit predominantly virus-neutralizing antibodies and confer significant protection against lethal DENV challenge in an animal model. The current study extends this work to a third DENV serotype. Results: We cloned and expressed DENV-1 E antigen in P. pastoris and purified it to near homogeneity. Recombinant DENV-1 E underwent post-translational processing, namely, signal peptide cleavage and glycosylation. Purified DENV-1 E self-assembled into stable VLPs, based on electron microscopy and dynamic light scattering analysis. Epitope mapping with monoclonal antibodies revealed that the VLPs retained the overall antigenic integrity of the virion particles despite the absence of prM. Subtle changes accompanied the efficient display of E domain III (EDIII), which contains type-specific neutralizing epitopes. These VLPs were immunogenic, eliciting predominantly homotypic EDIII-directed DENV-1-specific neutralizing antibodies. Conclusions: This work demonstrates the inherent potential of P. pastoris-expressed DENV-1 E glycoprotein to self-assemble into VLPs eliciting predominantly homotypic neutralizing antibodies. This work justifies an investigation of the last remaining serotype, namely, DENV-4, to assess if it also shares the desirable vaccine potential manifested by the remaining three DENV serotypes. Such efforts could make it possible to envisage the development of a tetravalent dengue vaccine based on VLPs of P. pastoris-expressed E glycoproteins of the four DENV serotypes

Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin

<p>Abstract</p> <p>Background</p> <p>The prevalence of diabetes is predicted to rise significantly in the coming decades. A recent analysis projects that by the year 2030 there will be ~366 million diabetics around the world, leading to an increased demand for inexpensive insulin to make this life-saving drug also affordable for resource poor countries.</p> <p>Results</p> <p>A synthetic insulin precursor (IP)-encoding gene, codon-optimized for expression in <it>P. pastoris</it>, was cloned in frame with the <it>Saccharomyces cerevisiae </it>α-factor secretory signal and integrated into the genome of <it>P. pastoris </it>strain X-33. The strain was grown to high-cell density in a batch procedure using a defined medium with low salt and high glycerol concentrations. Following batch growth, production of IP was carried out at methanol concentrations of 2 g L^-1, which were kept constant throughout the remaining production phase. This robust feeding strategy led to the secretion of ~3 gram IP per liter of culture broth (corresponding to almost 4 gram IP per liter of cell-free culture supernatant). Using immobilized metal ion affinity chromatography (IMAC) as a novel approach for IP purification, 95% of the secreted product was recovered with a purity of 96% from the clarified culture supernatant. Finally, the purified IP was trypsin digested, transpeptidated, deprotected and further purified leading to ~1.5 g of 99% pure recombinant human insulin per liter of culture broth.</p> <p>Conclusions</p> <p>A simple two-phase cultivation process composed of a glycerol batch and a constant methanol fed-batch phase recently developed for the intracellular production of the Hepatitis B surface antigen was adapted to secretory IP production. Compared to the highest previously reported value, this approach resulted in an ~2 fold enhancement of IP production using <it>Pichia </it>based expression systems, thus significantly increasing the efficiency of insulin manufacture.</p

Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of Hepatitis B surface antigen

<p>Abstract</p> <p>Background</p> <p>Hepatitis B is a serious global public health concern. Though a safe and efficacious recombinant vaccine is available, its use in several resource-poor countries is limited by cost. We have investigated the production of Hepatitis B virus surface antigen (HBsAg) using the yeast <it>Pichia pastoris </it>GS115 by inserting the <it>HBsAg </it>gene into the alcohol oxidase 1 locus.</p> <p>Results</p> <p>Large-scale production was optimized by developing a simple fed-batch process leading to enhanced product titers. Cells were first grown rapidly to high-cell density in a batch process using a simple defined medium with low salt and high glycerol concentrations. Induction of recombinant product synthesis was carried out using rather drastic conditions, namely through the addition of methanol to a final concentration of 6 g L^-1. This methanol concentration was kept constant for the remainder of the cultivation through continuous methanol feeding based on the <it>on-line </it>signal of a flame ionization detector employed as methanol analyzer in the off-gas stream. Using this robust feeding protocol, maximum concentrations of ~7 grams HBsAg per liter culture broth were obtained. The amount of soluble HBsAg, competent for assembly into characteristic virus-like particles (VLPs), an attribute critical to its immunogenicity and efficacy as a hepatitis B vaccine, reached 2.3 grams per liter of culture broth.</p> <p>Conclusion</p> <p>In comparison to the highest yields reported so far, our simple cultivation process resulted in an ~7 fold enhancement in total HBsAg production with more than 30% of soluble protein competent for assembly into VLPs. This work opens up the possibility of significantly reducing the cost of vaccine production with implications for expanding hepatitis B vaccination in resource-poor countries.</p

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Dengue: recent advances in biology and current status of translational research

Dengue is a very rapidly growing public health problem being currently faced by &#8764;40% of the global population living in more than a hundred tropical and sub-tropical countries. It is a viral disease caused by four types of dengue viruses, transmitted by mosquitoes, to an estimated 50 million people each year. Vector control methods to contain transmission have not been successful and there is currently no useful diagnostic test, drug or vaccine to combat dengue disease. However, as a result of the heightened awareness of its magnitude and its potential to spread beyond the tropical world, dengue has begun to emerge out of the list of neglected diseases in recent years. New interest in this disease has drawn scientists from multiple disciplines into the dengue arena. This has resulted in novel insights into several aspects of dengue virus biology and identified potential drug targets. Several tetravalent vaccines are being developed. Newer animal models that mirror some of the salient features of dengue disease are becoming available to investigate the mechanism of pathogenesis and to aid in drug and vaccine discovery efforts. The realization that therapeutic and prophylactic intervention can be cost-effective has resulted in vigorous industry-driven translational initiatives to develop drugs and vaccines. Dengue research is at a critical juncture and the implementation of existing knowledge supplemented by a better understanding of pathogenesis promises to make a tangible impact in the combat against dengue in the coming years