Shailendra Kumar Verma

Bacterially expressed recombinant envelope protein domain III of Japanese encephalitis virus (rJEV-DIII) elicits Th1 type of immune response in BALB/c mice

Japanese encephalitis is a major cause of encephalitis in Asia. Cases occur largely in rural areas of the South and East Asian region resulting in significant morbidity and mortality. Multiple vaccines exist to control Japanese encephalitis, but all suffer from problems. Envelope protein domain III of Japanese encephalitis virus is involved in binding to host receptors and it contains specific epitopes that elicit virus-neutralizing antibodies. Earlier, the protective efficacy of domain III has been evaluated in mice by some researchers, but these studies are lacking in explanation of humoral and cellular immune responses. We have earlier reported cloning, expression, purification and in vitro refolding of Japanese encephalitis virus envelope protein domain III (rJEV-DIII). Ninety percent JEV is neutralized when the serum against refolded rJEV-DIII is used at a dilution of 1:80. In the present study, we have evaluated the immunomodulatory potential of refolded rJEV-DIII protein in BALB/c mice with Freunds complete/incomplete adjuvants. Mice were tested for humoral immune response by ELISA. Cell-mediated immune response was tested by lymphocyte proliferation assay and cytokine profiling. The rJEV-DIII generated high IgG antibody and its isotypes (IgG2a and IgG3) and induced significant expression of INF-gamma and IL-2 cytokines. The rJEV-DIII induced significant lymphoproliferation of splenocytes. In conclusion rJEV-DIII induced Th1 type of immune response which plays an important role in protection for intracellular pathogens.

Antibodies Against Refolded Recombinant Envelope Protein (Domain III) of Japanese Encephalitis Virus Inhibit the JEV Infection to Porcine Stable Kidney Cells

Japanese encephalitis virus (JEV) is a mosquito-borne viral zoonosis of public health importance. Global efforts have been made towards development of vaccine for prevention of Japanese encephalitis. The envelope protein of JEV is associated with viral binding to cellular receptors, membrane fusion, and the induction of protective neutralizing antibody response in hosts. Here we report that the antibodies raised against refolded domain III of envelope protein of JEV neutralize the JE virus and inhibit the JEV infection to Porcine Stable Kidney (PS) cells. A reverse transcriptase-PCR amplified gene encoding domain III of JEV envelope protein was cloned into pET28a+ vector and over expressed in E. coli. The recombinant JEV-DIII protein was purified by affinity chromatography under denaturing conditions. The rJEV-DIII was refolded by oxido-redux shuffle and purified to homogeneity by ion-exchange chromatography. Refolded rJEV-DIII was characterized using biochemical and biophysical methods. The polyclonal antibodies were raised against in vitro refolded rJEV-DIII protein in BALB/c mice with Freunds adjuvant. Ninety percent JEV is neutralized when the serum against refolded rJEV-DIII is used at a dilution of 1:80 as against 60.5% neutralization capacity with the same dilution of serum raised against denatured rJEV-DIII. The method of expression and purification of biologically functional rJEV-DIII protein described in this study may help in better understanding the biology of JE virus and the development of better vaccine candidate. Since the expression system uses E. coli as the heterologous host, the process is easy and amenable to inexpensive scale-up.

Use of a recombinant burkholderia intracellular motility a protein for immunodiagnosis of glanders.

ABSTRACT Glanders, caused by the Gram-negative, nonmotile bacterium Burkholderia mallei, is a contagious and highly fatal disease of equines. During the last decade, the number of glanders outbreaks has increased steadily. The disease also has high zoonotic significance and B. mallei is listed biological warfare agent. The complement fixation test (CFT) is a routinely used and internationally recognized test to screen equine sera for the glanders. However, discrepant results have been observed using the CFT. The low sensitivity and specificity of the CFT and enzyme-linked immunosorbent assay (ELISA) have been linked to the use of crude test antigens. We expressed a novel recombinant Burkholderia intracellular motility A (rBimA) protein in Escherichia coli for the diagnosis of equine glanders. Purified rBimA was used in an indirect ELISA format. All of the 21 true-positive serum samples used in the study tested positive, whereas only 17 of the 1,524 potentially negative sera tested positive by indirect ELISA, thus exhibiting 100% sensitivity and 98.88% specificity. Also, rBimA protein did not react with melioidosis patient and normal healthy human serum samples, showing its high specificity. The developed assay can be used as a simple and rapid tool for diagnosis of glanders in equine serum samples. An Indian patent (1328/DEL/2010) has been filed for the reagent.

Plague Vaccine Development: Current Research and Future Trends

Plague is one of the world’s most lethal human diseases caused by Yersinia pestis, a Gram-negative bacterium. Despite overwhelming studies for many years worldwide, there is no safe and effective vaccine against this fatal disease. Inhalation of Y. pestis bacilli causes pneumonic plague, a fast growing and deadly dangerous disease. F1/LcrV-based vaccines failed to provide adequate protection in African green monkey model in spite of providing protection in mice and cynomolgus macaques. There is still no explanation for this inconsistent efficacy, and scientists leg behind to search reliable correlate assays for immune protection. These paucities are the main barriers to improve the effectiveness of plague vaccine. In the present scenario, one has to pay special attention to elicit strong cellular immune response in developing a next-generation vaccine against plague. Here, we review the scientific contributions and existing progress in developing subunit vaccines, the role of molecular adjuvants; DNA vaccines; live delivery platforms; and attenuated vaccines developed to counteract virulent strains of Y. pestis.

Immunogenicity and protective potential of a bacterially expressed recombinant dihydrolipoamide succinyltransferase (rE2o) of Brucella abortus in BALB/c mice

Brucellosis is one of the world’s major zoonoses. No vaccine is available for the prevention of brucellosis in human. Efforts are needed to develop an effective, safe, stable, vaccine with long lasting immunity against human brucellosis. Here, we cloned and expressed recombinant dihydrolipoamide succinyltransferase (rE2o) of Brucella abortus in Escherichia coli and purified up to homogeneity by metal affinity chromatography. The purified rE2o is immunoreactive with brucellosis positive cattle sera. The immunogenicity and the protective potential of recombinant dihydrolipoamide succinyltransferase (rE2o) were evaluated in BALB/c mice with two different adjuvants i.e., Freund’s and aluminium hydroxide gel. Mice were tested for humoral immune response by ELISA. Cell mediated immune response was tested by lymphocyte proliferation assay and cytokine profiling. The recombinant E2o (rE2o) generated high IgG antibody and its isotypes IgG1, and induced significant production of INF-γ, IL-10 and IL-4 cytokines. The rE2o protein induced significant lymphoproliferation of splenocytes. Altogether, these results suggest that rE2o induces a mixed but a predominant Th2 type of immune response in BALB/c mice and provides partial protection against challenge with pathogenic Brucella abortus.

HSP70 domain II of Mycobacterium tuberculosis modulates immune response and protective potential of F1 and LcrV antigens of Yersinia pestis in a mouse model.

No ideal vaccine exists to control plague, a deadly dangerous disease caused by Yersinia pestis. In this context, we cloned, expressed and purified recombinant F1, LcrV antigens of Y. pestis and heat shock protein70 (HSP70) domain II of M. tuberculosis in E. coli. To evaluate the protective potential of each purified protein alone or in combination, Balb/C mice were immunized. Humoral and cell mediated immune responses were evaluated. Immunized animals were challenged with 100 LD50 of Y. pestis via intra-peritoneal route. Vaccine candidates i.e., F1 and LcrV generated highly significant titres of anti-F1 and anti-LcrV IgG antibodies. A significant difference was noticed in the expression level of IL-2, IFN-γ and TNF-α in splenocytes of immunized animals. Significantly increased percentages of CD4+ and CD8+ T cells producing IFN-γ in spleen of vaccinated animals were observed in comparison to control group by flow cytometric analysis. We investigated whether the F1, LcrV and HSP70(II) antigens alone or in combination can effectively protect immunized animals from any histopathological changes. Signs of histopathological lesions noticed in lung, liver, kidney and spleen of immunized animals on 3rd day post challenge whereas no lesions in animals that survived to day 20 post-infection were observed. Immunohistochemistry showed bacteria in lung, liver, spleen and kidney on 3rd day post-infection whereas no bacteria was observed on day 20 post-infection in surviving animals in LcrV, LcrV+HSP70(II), F1+LcrV, and F1+LcrV+HSP70(II) vaccinated groups. A significant difference was observed in the expression of IL-2, IFN-γ, TNF-α, and CD4+/CD8+ T cells secreting IFN-γ in the F1+LcrV+HSP70(II) vaccinated group in comparison to the F1+LcrV vaccinated group. Three combinations that included LcrV+HSP70(II), F1+LcrV or F1+LcrV+HSP70(II) provided 100% protection, whereas LcrV alone provided only 75% protection. These findings suggest that HSP70(II) of M. tuberculosis can be a potent immunomodulator for F1 and LcrV containing vaccine candidates against plague.

A Recombinant Trivalent Fusion Protein F1–LcrV–HSP70(II) Augments Humoral and Cellular Immune Responses and Imparts Full Protection against Yersinia pestis

Plague is one of the most dangerous infections in humans caused by Yersinia pestis, a Gram-negative bacterium. Despite of an overwhelming research success, no ideal vaccine against plague is available yet. It is well established that F1/LcrV based vaccine requires a strong cellular immune response for complete protection against plague. In our earlier study, we demonstrated that HSP70(II) of Mycobacterium tuberculosis modulates the humoral and cellular immunity of F1/LcrV vaccine candidates individually as well as in combinations in a mouse model. Here, we made two recombinant constructs caf1–lcrV and caf1–lcrV–hsp70(II). The caf1 and lcrV genes of Y. pestis and hsp70 domain II of M. tuberculosis were amplified by polymerase chain reaction. Both the recombinant constructs caf1–lcrV and caf1–lcrV–hsp70(II) were cloned in pET28a vector and expressed in Escherichia coli. The recombinant fusion proteins F1–LcrV and F1–LcrV–HSP70(II) were purified using Ni-NTA columns and formulated with alum to evaluate the humoral and cell mediated immune responses in mice. The protective efficacies of F1–LcrV and F1–LcrV–HSP70(II) were determined following challenge of immunized mice with 100 LD50 of Y. pestis through intraperitoneal route. Significant differences were noticed in the titers of IgG and it’s isotypes, i.e., IgG1, IgG2b, and IgG3 in anti- F1–LcrV–HSP70(II) sera in comparison to anti-F1–LcrV sera. Similarly, significant differences were also noticed in the expression levels of IL-2, IFN-γ and TNF-α in splenocytes of F1–LcrV–HSP(II) immunized mice in comparison to F1–LcrV. Both F1–LcrV and F1–LcrV–HSP70(II) provided 100% protection. Our research findings suggest that F1–LcrV fused with HSP70 domain II of M. tuberculosis significantly enhanced the humoral and cellular immune responses in mouse model.

Characterization of pandemic influenza A (H1N1) virus hemagglutinin specific polyclonal antibodies for biosensor applications.

In this study, recombinant hemagglutinin protein (rH1N1HA) of Pandemic influenza virus and polyclonal antibodies against it for biosensor applications have been characterized. For rapid and high sensitive detection of H1N1 virus or its antibodies, PCR‐free and label free detection method based on a surface plasmon resonance technique has been proposed. The glycosylated H1N1HA protein was expressed in yeast and the authenticity of the expressed protein was confirmed by Western blotting. Rabbit polyclonal antibodies developed against rH1N1HA protein were evaluated for their ability to neutralize H1N1 virus through plaque reduction neutralization test and indirect ELISA. Affinity purified anti‐H1N1HA IgG were characterized further for their specificity, affinity of interaction, the association and dissociation rates at which they interact through surface plasmon resonance technique. The equilibrium constant and maximum binding capacity of analyte was found to be 49.7 nM and 47.28m°, respectively. The assay could detect a lowest IgG of 0.5 ng on a rH1N1HA coated chip. Combined with the high sensitivity of surface plasmon resonance technique and specificity of the reagents, it is possible to develop a rapid detection assay for monitoring influenza infections. J. Med. Virol. 86:363–371, 2014.

Characterization of Immune Responses to Yersinia pestis (Indian Isolate) Infection in Mouse Model

Yersinia pestis, causative agent of plague, is one of the deadliest pathogens around globe. Innate immune response is first line of host defense against pathogens. Here, we have investigated innate and adaptive immune response in plague infected mice, gene expression levels of TLR1-9 and CD14, MyD88, NF-A„¸B, TNF-α, MAPKp38, IL-1β were studied in peritoneal macrophages of plague infected mice in a time dependent manner (0 h, 24 h, 48 h, 72 h, 96 h and 120 h of post infection) by qRT-PCR. We also evaluated the immune response to Yersinia outer proteins (Yops) in Y. pestis infected mice. Selected genes (11 numbers) of Y. pestis encoding virulent factors viz, YpkA, YopH, YopM, V antigen, Pla, YopN, YopJ, YopE, YopK, F1 and pH6 antigen were amplified by PCR, cloned and expressed in Escherichia coli . To study the IgG and its isotypes level, ELISA and immunoblotting were performed using purified recombinant antigens. The major antigens recognized by murine plague infected sera were V antigen, YopH, YopM, YopE, F1 but very weak immunoreaction was observed in case of Pla. We observed a significant difference in IgG isotypes (IgG1, IgG2a, IgG2b and IgG3) to V antigen and F1, whereas in case of YopH and YopE (IgG1 and IgG2b) only. We also observed significant increase in the expression of CD14 at 48 h post infection, TLR4 and MyD88 at 72 h post infection in Y. pestis infected mouse peritoneal macrophages. Our findings suggest that both innate and humoral immune responses are crucial for protection.