Publications by authors named "Shivakiran Makam"

8 Publications

  • Page 1 of 1

Functional characterization of a broad and potent neutralizing monoclonal antibody directed against outer membrane protein (OMP) of Salmonella typhimurium.

Appl Microbiol Biotechnol 2020 Mar 29;104(6):2651-2661. Epub 2020 Jan 29.

Department of Microbiology, Defence Research & Development Establishment (DRDE - DRDO), Gwalior, Madhya Pradesh, 474 002, India.

In the present study, we have generated a murine monoclonal antibody (mAb) named Sal-06 by using the crude outer membrane protein preparation of Salmonella enteric subsp. enterica serovar Typhimurium ATCC 14028 strain as antigen. Sal-06mAb belonging to IgG1 isotype demonstrated broad cross-reactivity to standard and isolated strains of genus Salmonella and others such as Escherichia coli, Klebsiella pneumonia, and Proteus mirabilis. Cross-reactivity across several bacterial genera indicated that the epitopes reactive to Sal-06mAb are conserved among these members. Neutralizing effects of Sal-06mAb on Salmonella growth and survival was evaluated in vitro using bacteriostatic and bactericidal activity with and without complement and bacterial invasion inhibition assay. Sal-06mAb demonstrated a bacteriostatic effect on the growth of S. typhimurium ATCC 14028 strain which is both time and concentration (of mAb) dependent. It was also found that the bacterial growth inhibition was complement independent. When the bacterial cells were preincubated with Sal-06mAb, it reduced the adherence and invasion of bacterial cells into A549 epithelial cell line. This was confirmed by CFU count analysis, phase contrast, and fluorescence microscopy. Scanning electron microscope (SEM) imaging confirmed the antimicrobial effects of Sal-06mAb on S. typhimurium ATCC 14028. The development of broadly reactive and cross protective Sal-06mAb opens new possibilities for immunotherapy of sepsis caused by Gram-negative Enterobacteriaceae members.
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http://dx.doi.org/10.1007/s00253-020-10394-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7080182PMC
March 2020

A Bivalent Protein r-PAbxpB Comprising PA Domain IV and Exosporium Protein BxpB Confers Protection Against Spores and Toxin.

Front Immunol 2019 19;10:498. Epub 2019 Mar 19.

School of Biotechnology, Jawaharlal Nehru University, New Delhi, India.

Anthrax vaccines primarily relying only on protective antigen (PA), the cell binding component in anthrax toxins provide incomplete protection when challenged with spores of virulent encapsulated strains. Alternatively, formaldehyde inactivated spores (FIS) or recombinant spore components generate anti-spore immune responses that inhibit the early stages of infection and augment the PA protective efficacy. In the present study domain IV of PA was spliced with exosporium antigen BxpB via a flexible G4S linker to generate a single functional antigen r-PAbxpB that was further assessed for its protective efficacy against anthrax toxins and spore infection. Immunization of mice with r-PAbxpB elicited significantly high titer antibodies comprising IgG1:IgG2a isotypes in 1:1 ratio, balanced up-regulation of both Th1 (IL2, IL12, IFN-γ) and Th2 (IL4, IL5, IL10) cytokines and high frequencies of CD4+ and CD8+ T cell subsets. The anti-r-PAbxpB antibodies significantly enhanced spore phagocytosis, and killing within macrophages; inhibited their germination to vegetative cells and completely neutralized the anthrax toxins as evidenced by the 100% protection in passive transfer studies. Active immunization with r-PAbxpB provided 100 and 83.3% protection in mice I.P. challenged with 5 × LD LD of toxins and 5 × 10 cfu/ml Ames spores, respectively while the sham immunized group succumbed to infection in 48 h. Therefore, the ability of r-PAbxpB to generate protective immune responses against both spores and toxin and provide significant protection suggests it as an efficient vaccine candidate against infection.
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http://dx.doi.org/10.3389/fimmu.2019.00498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433990PMC
September 2020

A bivalent protein r-PB, comprising PA and BclA immunodominant regions for comprehensive protection against Bacillus anthracis.

Sci Rep 2018 05 8;8(1):7242. Epub 2018 May 8.

School of Biotechnology, Jawaharlal Nehru University, New Delhi, 110067, India.

Anthrax infection is primarily initiated by B. anthracis endospores that on entry into the host germinate to vegetative cells and cause severe bacteremia and toxaemia employing an array of host colonisation factors and the lethal tripartite toxin. The protective efficacy of conventional protective antigen (PA) based anthrax vaccines is improved by co-administration with inactivated spores or its components. In the present study, using structural vaccinology rationale we synthesized a bivalent protein r-PB encompassing toxin (PAIV) and spore components (BclACTD) and characterized its protective efficacy against B. anthracis infection. Active immunization of mice with r-PB generated high titer circulating antibodies which facilitated the phagocytic uptake of spores, inhibited their germination to vegetative cells and completely neutralized anthrax toxins in vivo resulting in 100 % survival against anthrax toxin challenge. Proliferation of CD4+ T cell subsets with up-regulation of Th1 (IFN-γ, IL-2, and IL-12), Th2 (IL-5, IL-10) cytokines and balanced expression of IgG1:IgG2a antibody isotypes indicated the stimulation of both Th1 and Th2 subsets. The immunized mice exhibited 100 % survival upon challenge with B. anthracis spores or toxin indicating the ability of r-PB to provide comprehensive protection against anthrax. Our results thus demonstrate r-PB an efficient vaccine candidate against anthrax infection.
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http://dx.doi.org/10.1038/s41598-018-25502-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5940697PMC
May 2018

An efficient method for integration of PCR fragments into adjacent or overlapping restriction sites during gene cloning.

3 Biotech 2018 Apr 24;8(4):197. Epub 2018 Mar 24.

Department of Biotechnology, Vignan's Foundation for Science, Technology and Research (VFSTR), Vadlamudi, Guntur District, Andhra Pradesh 522213 India.

In the present work, a simple and straightforward method was developed to clone any PCR-amplified products into restriction sites that are very close, adjacent or overlapping in the expression vector. The novelty of the methodology involves a crucial primer-designing step by adding appropriate overhangs to the 5' ends of primers based on the multiple cloning sites (MCS) (polylinker) region of expression vector. After PCR amplification, actual cloning is performed not in adjacent RE sites, but in sites that are little distant in the MCS. However, the sites lost during this cloning step are maintained intact since they are provided by the cloned PCR product (through the primer overhangs). Gene for green fluorescent protein (GFP) was cloned and expressed employing this strategy to demonstrate its simplicity. This method is highly useful for vector modification without losing the restriction sites present in the MCS.
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http://dx.doi.org/10.1007/s13205-018-1214-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866252PMC
April 2018

A novel IgY-Aptamer hybrid system for cost-effective detection of SEB and its evaluation on food and clinical samples.

Sci Rep 2015 Oct 19;5:15151. Epub 2015 Oct 19.

DRDO-BU-CLS, Bharathiar University Campus, Coimbatore, Tamil Nadu- 641046, India.

In the present study, we introduce a novel hybrid sandwich-ALISA employing chicken IgY and ssDNA aptamers for the detection of staphylococcal enterotoxin B (SEB). Cloning, expression and purification of the full length recombinant SEB was carried out. Anti-SEB IgY antibodies generated by immunizing white leg-horn chickens with purified recombinant SEB protein and were purified from the immunized egg yolk. Simultaneously, ssDNA aptamers specific to the toxin were prepared by SELEX method on microtiter well plates. The sensitivity levels of both probe molecules i.e., IgY and ssDNA aptamers were evaluated. We observed that the aptamer at 250 ngmL(-1) concentration could detect the target antigen at 50 ngmL(-1) and the IgY antibodies at 250 ngmL(-1), could able to detect 100 ngmL(-1) antigen. We further combined both the probes to prepare a hybrid sandwich aptamer linked immune sorbent assay (ALISA) wherein the IgY as capturing molecule and biotinylated aptamer as revealing probe. Limit of detection (LOD) for the developed method was determined as 50 ngmL(-1). Further, developed method was evaluated with artificially SEB spiked milk and natural samples and obtained results were validated with PCR. In conclusion, developed ALISA method may provide cost-effective and robust detection of SEB from food and environmental samples.
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http://dx.doi.org/10.1038/srep15151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4609960PMC
October 2015

Functional characterization and evaluation of in vitro protective efficacy of murine monoclonal antibodies BURK24 and BURK37 against Burkholderia pseudomallei.

PLoS One 2014 10;9(3):e90930. Epub 2014 Mar 10.

Microbiology Division, Defence Food Research Laboratory, Mysore, Karnataka, India.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0090930PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3948747PMC
March 2016

Protective antigen and extractable antigen 1 based chimeric protein confers protection against Bacillus anthracis in mouse model.

Mol Immunol 2014 May 8;59(1):91-9. Epub 2014 Feb 8.

Microbiology Division, Defence Food Research Laboratory, Siddarthanagar, Mysore 570011, Karnataka, India. Electronic address:

Recombinant bivalent chimeric protein was generated comprising of domain 4 of protective antigen (PA4) and carboxy terminal region of extractable antigen 1 (EA1C) by overlap extension PCR. The immunogenicity and protective efficacy of recombinant chimeric protein (PE) and protein mixture (PAEA) along with the individual components, PA4 and EA1C were evaluated in this study. We found that PE and PAEA exhibited higher endpoint titer and elevated IgG1 response. Compared to PA4 and EA1C, the chimeric protein PE and protein mixture PAEA exhibited 1.52 and 1.39 times more proliferative effect on lymphocytes in vitro. The spore uptake by anti-PE and anti-PAEA antibodies was significantly more than the individual components. We further evaluated the effects of antisera on the toxins in vitro and in vivo. Anti-PE and anti-PAEA antibodies displayed nearly 80% protection against crude toxin activity on RAW 264.7 cell lines. We further demonstrated that the anti-PE and anti-PAEA antibodies displayed better protection in controlling the edema induced by crude toxin. Passive immunization with anti-PE and anti-PAEA provided protection against toxin challenge in mice. The present study reveals that the chimeric protein consisting of heterologous regions of PA and EA1 can render better protection than PA4 or EA1C alone against toxins and bacilli.
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http://dx.doi.org/10.1016/j.molimm.2014.01.012DOI Listing
May 2014

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

World J Microbiol Biotechnol 2013 Dec 23;29(12):2379-88. Epub 2013 Jun 23.

Microbiology Division, Defence Food Research Laboratory, Siddartha Nagar, Mysore, 570011, Karnataka, India.

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 100 cfu ml⁻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.
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http://dx.doi.org/10.1007/s11274-013-1406-1DOI Listing
December 2013