Subodh Kumar

Evaluation of immunogenicity and protective efficacy of a plasmid DNA vaccine encoding ribosomal protein L9 of Brucella abortus in BALB/c mice

Brucellosis is a worldwide zoonotic disease. No Brucella vaccine is available for use in humans and existing animal vaccines have limitations. We have previously described the ribosomal protein L9 to have the vaccine potential. In this study, L9 based DNA vaccine (pVaxL9) was generated and evaluated in mouse model. Intramuscular immunisation of pVaxL9 was able to elicit the anti-L9 IgG antibody response of both IgG1 and IgG2a isotypes when compared with PBS and pVax immunised control animals. Heightened antibody response was observed in mice groups immunised with pVaxL9 priming and recombinant L9 boosting (PB) and where pDNA immunisation was carried out by in vivo electroporation (EP). The vaccine groups proliferated splenocytes and released Th1 type cytokines e.g. IFN-γ, TNF-α, IL-2. Further, flow cytometric analysis revealed that IFN-γ was released by both by CD4+ and CD8+ T cells particularly in PB and EP groups when compared with mice immunised with empty control vector. The L9 based pDNA vaccine was able to confer significant protection in mice against challenge with virulent B. abortus with PB and EP groups offering better protection. Taken together, it can be concluded that L9 based DNA vaccine is immunogenic and confer protection in mouse model.

Recombinant Shiga toxin B subunit elicits protection against Shiga toxin via mixed Th type immune response in mice

Shigella dysenteriae is the causative agent of the third commonest bacterial disease for childhood diarrhoea and responsible for millions of deaths per year. It produces potent toxin termed Shiga toxin which is listed in category B biological warfare agent of CDC, USA. Earlier we have reported production of recombinant Shiga toxin B subunit that produced antibodies which neutralized Shiga toxin toxicity in HeLa cells. In the present study, we have evaluated the immunomodulatory potential of rStxB protein in Balb/c mice using Freunds adjuvants. Animal protection with recombinant StxB was conferred through both humoral and cellular immune responses as indicated by an increased antibody titre with predominance of IgG2a and IgG2b isotypes along with elevated levels of IgG1 subtype. Cytokine profile of the mice antiserum and splenocyte also indicates Th2 and Th1 type of immune responses where former dominates in early stage of immunization. Histopathology study of kidneys of vaccinated mice showed remarkable differences when compared to the animals infected with Shigella dysenteriae type1. The present study indicates that recombinant StxB is a promising vaccine candidate and can be used for production of therapeutic antibodies to counter Shiga intoxication.

Gene expression and phosphoprotein profile of certain key neuronal signaling proteins following soman intoxication

Nerve agents irreversibly inhibit acetylcholinesterase (AChE), leading to cholinergic crisis and death at acute exposure levels. The complexity, delayed onset, and persistent nature of nerve agent induced CNS effects need to be elucidated to block their multiple effects. In the present study gene expression and phosphoprotein profile of certain key neuronal proteins were studied after soman exposure. Quantitative real time PCR analysis of c-Fos, Bax, CREB and caspase 3 genes in the hippocampus, cortex and cerebellum showed that only c-Fos and Bax mRNA expression was increased significantly. Western blot analysis also confirmed the induction of c-Fos at early time points both at 0.5 and 1.0 LD(50) dose of soman exposure. Acute soman exposure caused perturbations in the phosphorylation status of ERK, JNK, p38 MAPK, CREB, c-Jun and NF-κB in all the three brain regions. The primary target for soman toxicity, AChE was inhibited in blood and brain up to 90%. Therapeutic treatment comprising of HI-6, atropine and diazepam has completely protected animals from death and reactivated soman inhibited AChE up to 40% in the plasma and RBC. This therapeutic regime also reduced soman induced Bax expression to near control levels, but could not reverse the soman induced changes in c-Fos expression and phosphorylation levels completely. Results suggest that exposure to soman caused persistent changes in these key brain proteins, which could lead to the development of complex neurotoxic effects and there is an urgent need for development of better drugs to stop multiple effects of nerve agents poisoning.

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.

Evaluation of Recombinant Proteins of Burkholderia mallei for Serodiagnosis of Glanders

ABSTRACT Glanders is a contagious disease caused by the Gram-negative bacillus Burkholderia mallei. The number of equine glanders outbreaks has increased steadily during the last decade. The disease must be reported to the Office International des Epizooties, Paris, France. Glanders serodiagnosis is hampered by the considerable number of false positives and negatives of the internationally prescribed tests. The major problem leading to the low sensitivity and specificity of the complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA) has been linked to the test antigens currently used, i.e., crude preparations of whole cells. False-positive results obtained from other diagnostic tests utilizing crude antigens lead to financial losses to animal owners, and false-negative results can turn a risk into a possible threat. In this study, we report on the identification of diagnostic targets using bioinformatics tools for serodiagnosis of glanders. The identified gene sequences were cloned and expressed as recombinant proteins. The purified recombinant proteins of B. mallei were used in an indirect ELISA format for serodiagnosis of glanders. Two recombinant proteins, 0375H and 0375TH, exhibited 100% sensitivity and specificity for glanders diagnosis. The proteins also did not cross-react with sera from patients with the closely related disease melioidosis. The results of this investigation highlight the potential of recombinant 0375H and 0375TH proteins in specific and sensitive diagnosis of glanders.

Unravelling the Molecular Epidemiology and Genetic Diversity among Burkholderia pseudomallei Isolates from South India Using Multi-Locus Sequence Typing.

There is a slow but steady rise in the case detection rates of melioidosis from various parts of the Indian sub-continent in the past two decades. However, the epidemiology of the disease in India and the surrounding South Asian countries remains far from well elucidated. Multi-locus sequence typing (MLST) is a useful epidemiological tool to study the genetic relatedness of bacterial isolates both with-in and across the countries. With this background, we studied the molecular epidemiology of 32 Burkholderia pseudomallei isolates (31 clinical and 1 soil isolate) obtained during 2006–2015 from various parts of south India using multi-locus sequencing typing and analysis. Of the 32 isolates included in the analysis, 30 (93.7%) had novel allelic profiles that were not reported previously. Sequence type (ST) 1368 (n = 15, 46.8%) with allelic profile (1, 4, 6, 4, 1, 1, 3) was the most common genotype observed. We did not observe a genotypic association of STs with geographical location, type of infection and year of isolation in the present study. Measure of genetic differentiation (FST) between Indian and the rest of world isolates was 0.14413. Occurrence of the same ST across three adjacent states of south India suggest the dispersion of B.pseudomallei across the south western coastal part of India with limited geographical clustering. However, majority of the STs reported from the present study remained as “outliers” on the eBURST “Population snapshot”, suggesting the genetic diversity of Indian isolates from the Australasian and Southeast Asian isolates.

Prediction of T cell epitopes of Brucella abortus and evaluation of their protective role in mice

Brucellae are Gram-negative intracellular bacteria that cause an important zoonotic disease called brucellosis. The animal vaccines are available but have disadvantage of causing abortions in a proportion of pregnant animals. The animal vaccines are also pathogenic to humans. Recent trend in vaccine design has shifted to epitope-based vaccines that are safe and specific. In this study, efforts were made to identify MHC-I- and MHC-II-restricted T cell epitopes of Brucella abortus and evaluate their vaccine potential in mice. The peptides were designed using online available immunoinformatics tools, and five MHC-I- and one MHC-II-restricted T cell peptides were selected on the basis of their ability to produce interferon gamma (IFN-γ) in in vivo studies. The selected peptides were co-administered with poly dl-lactide-co-glycolide (PLG) microparticles and evaluated for immunogenicity and protection in BALB/c mice. Mice immunized with peptides either entrapped in PLG microparticles (EPLG-Pep) or adsorbed on PLG particles (APLG-Pep) showed significantly higher splenocyte proliferation and IFN-γ generation to all selected peptides than the mice immunized with corresponding irrelevant peptides formulated PLG microparticles or phosphate-buffered saline (PBS). A significant protection compared to PBS control was also observed in EPLG-Pep and APLG-Pep groups. A plasmid DNA vaccine construct (pVaxPep) for peptides encoding DNA sequences was generated and injected to mice by in vivo electroporation. Significant protection was observed (1.66 protection units) when compared with PBS and empty vector control group animals. Overall, the MHC-I and MHC-II peptides identified in this study are immunogenic and protective in mouse model and support the feasibility of peptide-based vaccine for brucellosis.

Identification of a new diagnostic antigen for glanders using immunoproteome analysis

Glanders is a disease of horses, donkeys and mules. The causative agent Burkholderia mallei, is a biorisk group 3 pathogen and is also a biothreat agent. Simple and rapid diagnostic tool is essential for control of glanders. Using a proteomic approach and immunoblotting with equine sera, we identified 12 protein antigens that may have diagnostic potential. Various immunoreactive proteins e.g. GroEL, translation elongation factor Tu, elongation factor Ts, arginine deiminase, malate dehydrogenase, DNA directed RNA polymerase subunit alpha were identified on 2-dimentional immunoblots. One of these proteins, GroEL, was cloned and expressed in E. coli and purified using Ni-NTA affinity chromatography. The recombinant GroEL protein was evaluated in ELISA format on a panel of glanders positive (n=49) and negative (n=79) equine serum samples to determine its diagnostic potential. The developed ELISA had a sensitivity and specificity of 96 and 98.7% respectively. The results of this study highlight the potential of GroEL in serodiagnosis of glanders.

Evaluation of PCR, DNA hybridization and immunomagnetic separation — PCR for detection of Burkholderia mallei in artificially inoculated environmental samples

Glanders is highly contagious disease of equines, caused by Burkholderia mallei. The disease though rare, can be transmitted to humans. Here, we report a strategy for rapid detection of B. mallei from environmental samples. Different bacteriological media were evaluated and brain heart infusion broth medium with selective supplements (BHIB-SS) of penicillin (200 U/ml) and crystal violet (1:10,00000) was found to support the maximum growth of B. mallei even in the presence of other bacteria like Escherichia coli and Staphylococcus aureus. A polymerase chain reaction (PCR) and a DNA hybridization method was standardized for 823 bp specific dNA sequence of B. mallei. To enable the quicker and direct enrichment of B. mallei bacteria from environmental samples, an immunomagnetic separation (IMS) method was also standardized. Water, husk, grass and gram samples were artificially contaminated by B. mallei bacteria and after enrichment of B. mallei in BHIB-SS, detection was carried out by PCR and DNA hybridization. PCR was found to be a better method of the two with a detection limit of 104–106 CFU/ml (6 h enrichment in BHIB-SS) in water and other particulate matrices. Detection by PCR in the above samples without enrichment in BHIBSS was carried out following IMS where the detection limit was about 1–2 log higher than PCR following enrichment in BHIB-SS. We recommend PCR for 823 bp for detection of B. mallei from environmental samples either following enrichment in BHIB-SS or IMS. IMS-PCR method may be preferred in situations where numbers of B. mallei bacteria are expected to be high and results are required in short time.

Improvement of recombinant-truncated Burkholderia motility protein A (BimA)-based indirect ELISA for equine glanders

ABSTRACT Glanders is a contagious and highly fatal disease of equines with zoonotic potential. It is caused by a Gram-negative, nonmotile bacterium Burkholderia mallei. Complement fixation test (CFT) is one of the most commonly used tests for diagnosis of glanders; however, it has some limitations. A recombinant-truncated Burkholderia intracellular motility A (BimA) protein-based indirect enzyme-linked immunosorbent assay (iELISA) was previously reported by us for glanders diagnosis, which has been re-optimized in this study using a panel of glanders positive (n = 75) and glanders negative (n = 227) serum samples. The improved iELISA exhibited 96% sensitivity and 90.75% specificity. The assay had 98.56% negative predictive value. In the improved iELISA, background for negative samples was reduced and a rational assay cut-off based on ROC curves was introduced. Intra laboratory repeatability of the iELISA was tested by 3 different operators with 100% correlation. The BimA-coated ELISA plates could be used without significant decrease in diagnostic efficacy even after their storage at room temperature or 37°C for 90 days. Overall, the improved iELISA is a sensitive, specific, reproducible, and easy-to-use assay that has potential in serodiagnosis of glanders, more suitably to demonstrate freedom from B. mallei infection in a population.