Urmil Tuteja

Expression and purification of the 26 kDa periplasmic protein of Brucella abortus : a reagent for the diagnosis of bovine brucellosis

Development of a single diagnostic test for brucellosis in animals is the top priority of present‐day research in the field. There is currently a battery of serological tests relying mainly on the use of LPS (lipopolysaccharide) as an antigen, culminating in false positives due to serological cross‐reactivity. Other problems include difficulties in antigen production and the associated biohazard risk. This has prompted the need to develop an alternative antigen to replace LPS. In the present study, we cloned and expressed a BP26 (26 kDa periplasmic protein) antigen gene (bp26) of Brucella abortus. The recombinant periplasmic protein [rBP26 (recombinant BP26)] was expressed to high levels in Escherichia coli and purified in a single step. The purified rBP26 was examined for its binding activity with antibodies in a serum derived from a rabbit immunized intramuscularly with whole‐cell lysate of B. abortus, as well as with commercial Brucella antibody (Difco). The purified rBP26 was used to develop an in‐house plate ELISA and was further tested with a panel of 75 bovine brucellosis sera samples characterized previously by conventional serological tests. The results of both were in excellent agreement. The results show that rBP26 has potential use in the diagnosis of brucellosis, both in the laboratory and in field‐based conditions with high levels of sensitivity and specificity.

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.

Immuno capture PCR for rapid and sensitive identification of pathogenic Bacillus anthracis

Immuno capture PCR (IPCR) is a technique capable of detecting the pathogens with high specificity and sensitivity. Rapid and accurate detection of Bacillus anthracis was achieved using anti-EA1 antibodies to capture the cells and two primer sets targeting the virulence factors of the pathogen i.e., protective antigen (pag) and capsule (cap) in an IPCR format. Monoclonal antibodies specific to B. anthracis were generated against extractable antigen 1 protein and used as capture antibody onto 96 well polystyrene plates. Following the binding of the pathogen, the DNA extraction was carried out in the well itself and further processed for PCR assay. We compared IPCR described here with conventional duplex PCR using the same primers and sandwich ELISA using the monoclonal antibodies developed in the present study. IPCR was capable of detecting as few as 10 and 100xa0cfuxa0ml−1 of bacterial cells and spores, respectively. IPCR was found to be 2–3 logs more sensitive than conventional duplex PCR and the sandwich ELISA. The effect of other bacteria and any organic materials on IPCR was also analyzed and found that this method was robust with little change in the sensitivity in the presence of interfering agents. Moreover, we could demonstrate a simple process of microwave treatment for spore disruption which otherwise are resistant to chemical treatments. Also, the IPCR could clearly distinguish the pathogenic and nonpathogenic strains of B. anthracis in the same assay. This can help in saving resources on unnecessary decontamination procedures during false alarms.

Functional Characterization and Evaluation of In Vitro Protective Efficacy of Murine Monoclonal Antibodies BURK24 and BURK37 against Burkholderia pseudomallei

Burkholderia pseudomallei, the causative agent of melioidosis has been recognized by CDC as a category B select agent. Although substantial efforts have been made for development of vaccine molecules against the pathogen, significant hurdles still remain. With no licensed vaccines available and high relapse rate of the disease, there is a pressing need for development of alternate protection strategies. Antibody-mediated passive protection is promising in this regard and our primary interest was to unravel this frontier of specific mAbs against Burkholderia pseudomallei infections, as functional characterization of antibodies is a pre-requisite to demonstrate them as protective molecules. To achieve this, we designed our study on in vitro-based approach and assessed two mAbs, namely BURK24 and BURK37, reactive with outer membrane proteins and lipopolysaccharide of the pathogen respectively, for their ability to manifest inhibitory effects on the pathogenesis mechanisms of B. pseudomallei including biofilm formation, invasion and induction of apoptosis. The experiments were performed using B. pseudomallei standard strain NCTC 10274 and a clinical isolate, B. pseudomallei 621 recovered from a septicemia patient with diabetic ailment. The growth kinetic studies of the pathogen in presence of various concentrations of each individual mAb revealed their anti-bacterial properties. Minimal inhibitory concentration and minimal bactericidal concentration of both the mAbs were determined by using standards of Clinical and Laboratory Standards Institute (CLSI) and experiments were performed using individual mAbs at their respective bacteriostatic concentration. As an outcome, both mAbs exhibited significant anti-Burkholderia pseudomallei properties. They limited the formation of biofilm by the bacterium and completely crippled its invasion into human alveolar adenocarcinoma epithelial cells. Also, the mAbs were appreciably successful in preventing the bacterium to induce apoptosis in A549 cells. The present study design revealed the protection attributes possessed by BURK24 and BURK37 that has to be further substantiated by additional in vivo studies.

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.

Draft Genome Sequences of Yersinia pestis Strains from the 1994 Plague Epidemic of Surat and 2002 Shimla Outbreak in India.

We report the first draft genome sequences of the strains of plague-causing bacteria, Yersinia pestis, from India. These include two strains from the Surat epidemic (1994), one strain from the Shimla outbreak (2002) and one strain from the plague surveillance activity in the Deccan plateau region (1998). Genome size for all four strains is ~4.49 million bp with 139–147 contigs. Average sequencing depth for all four genomes was 21x.

Reverse Line Blot Macroarray for Simultaneous Detection and Characterization of Four Biological Warfare Agents

The need for a rapid detection and characterization of biowarfare (BW) agents cannot be over emphasized. With diverse array of potential BW pathogen available presently, rapid identification of the pathogen is crucial, so that specific therapy and control measures can be initiated. We have developed a multiplex polymerase chain reaction based reverse line blot macroarray to simultaneously detect four pathogens of BW importance viz. Bacillus anthracis, Yersinia pestis, Brucella melitensis and Burkholderia pseudomallei. The multiplex PCR utilizes 14 pairs of primers targeting 18 specific markers. These markers include genes which are genus specific, species-specific chromosomal sequences and virulence markers of plasmid origin. The assay was evaluated on various human, environment and animal isolates. The assay w successful in simultaneous detection and characterization of isolates of the four pathogens on as a single platform with sensitivity ranging from 0.3xa0pg to 0.3xa0ng of genomic DNA. The assay was able to detect 5xa0×xa0102xa0cfu/ml for B. anthracis, 8xa0×xa0102xa0cfu/ml for Yersinia sp., 1.4xa0×xa0102xa0cfu/ml for B. melitensis and 4xa0×xa0102xa0cfu/ml for B. pseudomallei.

Antimicrobial susceptibility and molecular characterization of Vibrio cholerae from cholera outbreaks in Chennai

The genotype and antibiotic resistance pattern of the toxigenic Vibrio cholerae strains associated with cholera outbreaks vary frequently. Fifty-one V. cholerae strains isolated from cholera outbreaks in Chennai (2002–2005) were screened for the presence of virulence and regulatory genes by multiplex polymerase chain reaction (PCR) assay. Genotyping of the isolates was done by VC1 primers derived from enterobacterial repetitive intergenic consensus (ERIC)-related sequence in V. cholerae. All the isolates possessed toxigenic genes, such as ctxA, ctxB, tcpA, ace, ompU, toxR and zot. Two different El Tor genotypes and one O139 genotype could be delineated by VC1-PCR. One of the El Tor genotypes was similar to the El Tor strains isolated from Bhind district and Delhi during 2004. Antibiotic susceptibility testing revealed greater variability among the isolates tested. All the isolates were found to be susceptible to norfloxacin, ciprofloxacin and tetracycline. Thiry-three per cent of the isolates were found to be resistant to more than 4 antibiotics and could be termed as multiple antibiotic resistant. Coexistence of O139 serogroup along with the El Tor biotype could be identified among the strains recovered during the period 2002–2004. The O139 isolates were found to be more susceptible to the antibiotics tested when compared to the El Tor isolates.

Soluble Expression and Characterization of Biologically Active Bacillus anthracis Protective Antigen in Escherichia coli

Bacillus anthracis secretory protein protective antigen (PA) is primary candidate for subunit vaccine against anthrax. Attempts to obtain large quantity of PA from Escherichia coli expression system often result in the formation of insoluble inclusion bodies. Therefore, it is always better to produce recombinant proteins in a soluble form. In the present study, we have obtained biologically active recombinant PA in small scale E. coli shake culture system using three different expression constructs. The PA gene was cloned in expression vectors bearing trc, T5, and T7 promoters and transformed into their respective E. coli hosts. The growth conditions were optimized to obtain maximum expression of PA in soluble form. The expression construct PA-pET32c in DE3-pLysS E. coli host resulted in a maximum production of soluble PA (15u2009mgu2009L−1) compared to other combinations. Purified PA was subjected to trypsin digestion and binding assay with lethal factor to confirm the proteins functionality. Biological activity was confirmed by cytotoxicity assay on J774.1 cells. Balb/c mice were immunized with PA and the immunogenicity was tested by ELISA and toxin neutralization assay. This study highlights the expression of soluble and biologically active recombinant PA in larger quantity using simpler E. coli production platform.