Infection Genetics and Evolution - INFECT GENET EVOL, 2008
Recently we had prepared a recombinant antigen (Ld-rKE16) from a newly isolated Indian strain of ... more Recently we had prepared a recombinant antigen (Ld-rKE16) from a newly isolated Indian strain of Leishmania donovani (MHOM/IN/KE16/1998) with high sensitivity and specificity and the same has been commercialized. While comparing the sequence data of kinesin gene of this (KE16) strain and its expressed protein with another commercially available recombinant antigen (Lc-rK39) from kinesin gene of L. chagasi we found significant genetic and amino acid variations. This prompted us to undertake the present study to unravel whether the kinesin gene and its expressed protein from another old but Indian isolate of L. donovani (MHOM/IN/DD8/1968) had any genetic and amino acid heterogeneity. Sequencing of the kinesin gene revealed that the kinesin gene of DD8 strain is 3016bp long and has immunodominant region consisting of 4.8 tandem repeats, 117 base pairs each. Further blast analysis of the immunodominant regions of 5 strains of L. donovani revealed that it has only 79% homology with L. ch...
Tuberculosis is a major health problem in India, and the emergence of multidrug resistant (MDR) a... more Tuberculosis is a major health problem in India, and the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis (Mtb) has further complicated the situation. Though several studies characterizing drug sensitive and drug resistant strains are available in literature, almost all studies are done on unrelated strains. Therefore, the objective of this study was to compare the proteomic data of four sequential isolates of Mtb from a single patient who developed MDR-TB during the course of anti-tuberculosis therapy (ATT). In this study, using two-dimensional (2D) gel electrophoresis and MALDI-TOF mass spectrometry, we compared and analyzed the cell lysate proteins of Mtb sequential clinical isolates from a patient undergoing anti-TB treatment. The mRNA expression levels of selected identified proteins were determined by quantitative real-time polymerase chain reaction (qRT-PCR). The genotypes of all four isolates remained homologou...
Nontuberculous mycobacteria are often underdiagnosed due to lack of proper diagnostic facilities.... more Nontuberculous mycobacteria are often underdiagnosed due to lack of proper diagnostic facilities. To overcome this, we created a rapid PCR method for the species-specific diagnosis of Mycobacterium tuberculosis and its differentiation from other mycobacteria. A set of PCR primers targeting the gene encoding for early-secreted antigen-6 (ESAT-6) of the M. tuberculosis complex was designed and standardized on mycobacterial standard strains and on 75 recent isolates from AIDS patients and 70 isolates from HIV-negative patients seen at the hospital of the All India Institute of Medical Sciences, New Delhi, India. All 145 fresh mycobacterial isolates were identified using phenotypic methods and 16S rRNA PCR followed by sequencing of hypervariable region A. The ESAT-6 PCR detected all of the M. tuberculosis strains correctly (100% sensitivity), but none of the nontuberculous Mycobacterium spp. gave positive results (100% specific). Most nontuberculous mycobacteria were identified in patie...
Background & objectives: Tuberculosis is a major health problem in India, and the emergence of mu... more Background & objectives: Tuberculosis is a major health problem in India, and the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis (Mtb) has further complicated the situation. Though several studies characterizing drug sensitive and drug resistant strains are available in literature, almost all studies are done on unrelated strains. Therefore, the objective of this study was to compare the proteomic data of four sequential isolates of Mtb from a single patient who developed MDR-TB during the course of anti-tuberculosis therapy (ATT). Methods: In this study, using two-dimensional (2D) gel electrophoresis and MALDI-TOF mass spectrometry, we compared and analyzed the cell lysate proteins of Mtb sequential clinical isolates from a patient undergoing anti-TB treatment. The mRNA expression levels of selected identified proteins were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Results: The genotypes of all four isolates remained homologous, indicating no re-infection. The initial isolate (before treatment) was sensitive to all first-line drugs, but the consecutive isolates were found to be resistant to isoniazid (INH) and rifampicin (RIF) and developed mutations in the katG, inhA and rpoB. the intensities of 27 protein spots were found to be consistently overexpressed in INH and RIF resistant isolates. The most prominent and overexpressed proteins found during the development of drug resistance were GarA (Rv1827), wag31 (Rv2145c), Rv1437 and Rv2970c. Interpretation & conclusions: This preliminary proteomic study provides an insight about the proteins that are upregulated during drug resistance development. These upregulated proteins, identified here, could prove useful as immunodiagnostic and possibly drug resistant markers in future. However, more studies are required to confirm these findings.
International Journal of Peptide and Protein Research, 2009
Using solid phase methodology, we have synthesized five peptides (16-18 residues long) correspond... more Using solid phase methodology, we have synthesized five peptides (16-18 residues long) corresponding to repeat sequences of four antigens of a human malarial parasite, Plasmodium falciparum. Three of these antigens (RESA, FIRA, and ABRA) are found in the asexual blood-stages of the parasite, while the remaining one (CSP) is found in the sporozoites. The synthetic peptides, conjugated to bovine serum albumin, elicited high levels of antibodies in rabbits, and these antibodies were found to cross-react with the heterologous peptides. The degree of cross-reactivity, as estimated in an ELISA, was quite remarkable among all the peptides. The peptide corresponding to the RESA tetrapeptide repeat was found to be the most immunogenic and highly cross-reactive. For this reason this tetrapeptide repeat unit, peptide 1, may be a suitable candidate for inclusion in a multiple epitope polypeptide vaccine design. Conformational studies using circular dichroism spectroscopy show that these peptides have similar conformational characteristics with a common feature OF ∼30% and ∼50% helical content in water and TFE respectively. Theoretical predictions regarding conformation using the Chou-Fasman method have also been presented.
Immunisation with two chemically synthesised, linear, multiple epitope peptides (MEP) containing ... more Immunisation with two chemically synthesised, linear, multiple epitope peptides (MEP) containing B and T cell epitopes from two conserved blood-stage antigens of the human malaria parasite, Plasmodium falciparum, induced high levels of circulating antibodies without the use òf a carrier protein. Immunisation of mice with MEP constructs (P1 and P2) induced antibodies against the various epitope sequences included in their structures, although the immune response was focused more towards the N terminal and the middle portion of the peptides. In vitro T cell proliferation assays indicated that only one of the two Th epitopes included in P1 and P2 are functional. Both P1 and P2, based on P. falciparum sequences, cross-reacted with sera from P. yoelii-infected mice. Immunisation with P1 in CFA, but not with P2, provided partial protection to mice against P. yoelii challenge infection. Peptide P1 was highly immunogenic in alum also, and a somewhat higher level of protection was observed as compared to CFA immunisation. We found that immunisation with P1 induced antibody responses in different strains of mice, although to different extents. These results suggest that linear, multiple epitope peptides may offer attractive alternatives as subunit vaccine candidate molecules, but at the same time highlight the fact that the design principles are far from being clear and have yet to be worked out.
Infection Genetics and Evolution - INFECT GENET EVOL, 2008
Recently we had prepared a recombinant antigen (Ld-rKE16) from a newly isolated Indian strain of ... more Recently we had prepared a recombinant antigen (Ld-rKE16) from a newly isolated Indian strain of Leishmania donovani (MHOM/IN/KE16/1998) with high sensitivity and specificity and the same has been commercialized. While comparing the sequence data of kinesin gene of this (KE16) strain and its expressed protein with another commercially available recombinant antigen (Lc-rK39) from kinesin gene of L. chagasi we found significant genetic and amino acid variations. This prompted us to undertake the present study to unravel whether the kinesin gene and its expressed protein from another old but Indian isolate of L. donovani (MHOM/IN/DD8/1968) had any genetic and amino acid heterogeneity. Sequencing of the kinesin gene revealed that the kinesin gene of DD8 strain is 3016bp long and has immunodominant region consisting of 4.8 tandem repeats, 117 base pairs each. Further blast analysis of the immunodominant regions of 5 strains of L. donovani revealed that it has only 79% homology with L. ch...
Tuberculosis is a major health problem in India, and the emergence of multidrug resistant (MDR) a... more Tuberculosis is a major health problem in India, and the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis (Mtb) has further complicated the situation. Though several studies characterizing drug sensitive and drug resistant strains are available in literature, almost all studies are done on unrelated strains. Therefore, the objective of this study was to compare the proteomic data of four sequential isolates of Mtb from a single patient who developed MDR-TB during the course of anti-tuberculosis therapy (ATT). In this study, using two-dimensional (2D) gel electrophoresis and MALDI-TOF mass spectrometry, we compared and analyzed the cell lysate proteins of Mtb sequential clinical isolates from a patient undergoing anti-TB treatment. The mRNA expression levels of selected identified proteins were determined by quantitative real-time polymerase chain reaction (qRT-PCR). The genotypes of all four isolates remained homologou...
Nontuberculous mycobacteria are often underdiagnosed due to lack of proper diagnostic facilities.... more Nontuberculous mycobacteria are often underdiagnosed due to lack of proper diagnostic facilities. To overcome this, we created a rapid PCR method for the species-specific diagnosis of Mycobacterium tuberculosis and its differentiation from other mycobacteria. A set of PCR primers targeting the gene encoding for early-secreted antigen-6 (ESAT-6) of the M. tuberculosis complex was designed and standardized on mycobacterial standard strains and on 75 recent isolates from AIDS patients and 70 isolates from HIV-negative patients seen at the hospital of the All India Institute of Medical Sciences, New Delhi, India. All 145 fresh mycobacterial isolates were identified using phenotypic methods and 16S rRNA PCR followed by sequencing of hypervariable region A. The ESAT-6 PCR detected all of the M. tuberculosis strains correctly (100% sensitivity), but none of the nontuberculous Mycobacterium spp. gave positive results (100% specific). Most nontuberculous mycobacteria were identified in patie...
Background & objectives: Tuberculosis is a major health problem in India, and the emergence of mu... more Background & objectives: Tuberculosis is a major health problem in India, and the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis (Mtb) has further complicated the situation. Though several studies characterizing drug sensitive and drug resistant strains are available in literature, almost all studies are done on unrelated strains. Therefore, the objective of this study was to compare the proteomic data of four sequential isolates of Mtb from a single patient who developed MDR-TB during the course of anti-tuberculosis therapy (ATT). Methods: In this study, using two-dimensional (2D) gel electrophoresis and MALDI-TOF mass spectrometry, we compared and analyzed the cell lysate proteins of Mtb sequential clinical isolates from a patient undergoing anti-TB treatment. The mRNA expression levels of selected identified proteins were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Results: The genotypes of all four isolates remained homologous, indicating no re-infection. The initial isolate (before treatment) was sensitive to all first-line drugs, but the consecutive isolates were found to be resistant to isoniazid (INH) and rifampicin (RIF) and developed mutations in the katG, inhA and rpoB. the intensities of 27 protein spots were found to be consistently overexpressed in INH and RIF resistant isolates. The most prominent and overexpressed proteins found during the development of drug resistance were GarA (Rv1827), wag31 (Rv2145c), Rv1437 and Rv2970c. Interpretation & conclusions: This preliminary proteomic study provides an insight about the proteins that are upregulated during drug resistance development. These upregulated proteins, identified here, could prove useful as immunodiagnostic and possibly drug resistant markers in future. However, more studies are required to confirm these findings.
International Journal of Peptide and Protein Research, 2009
Using solid phase methodology, we have synthesized five peptides (16-18 residues long) correspond... more Using solid phase methodology, we have synthesized five peptides (16-18 residues long) corresponding to repeat sequences of four antigens of a human malarial parasite, Plasmodium falciparum. Three of these antigens (RESA, FIRA, and ABRA) are found in the asexual blood-stages of the parasite, while the remaining one (CSP) is found in the sporozoites. The synthetic peptides, conjugated to bovine serum albumin, elicited high levels of antibodies in rabbits, and these antibodies were found to cross-react with the heterologous peptides. The degree of cross-reactivity, as estimated in an ELISA, was quite remarkable among all the peptides. The peptide corresponding to the RESA tetrapeptide repeat was found to be the most immunogenic and highly cross-reactive. For this reason this tetrapeptide repeat unit, peptide 1, may be a suitable candidate for inclusion in a multiple epitope polypeptide vaccine design. Conformational studies using circular dichroism spectroscopy show that these peptides have similar conformational characteristics with a common feature OF ∼30% and ∼50% helical content in water and TFE respectively. Theoretical predictions regarding conformation using the Chou-Fasman method have also been presented.
Immunisation with two chemically synthesised, linear, multiple epitope peptides (MEP) containing ... more Immunisation with two chemically synthesised, linear, multiple epitope peptides (MEP) containing B and T cell epitopes from two conserved blood-stage antigens of the human malaria parasite, Plasmodium falciparum, induced high levels of circulating antibodies without the use òf a carrier protein. Immunisation of mice with MEP constructs (P1 and P2) induced antibodies against the various epitope sequences included in their structures, although the immune response was focused more towards the N terminal and the middle portion of the peptides. In vitro T cell proliferation assays indicated that only one of the two Th epitopes included in P1 and P2 are functional. Both P1 and P2, based on P. falciparum sequences, cross-reacted with sera from P. yoelii-infected mice. Immunisation with P1 in CFA, but not with P2, provided partial protection to mice against P. yoelii challenge infection. Peptide P1 was highly immunogenic in alum also, and a somewhat higher level of protection was observed as compared to CFA immunisation. We found that immunisation with P1 induced antibody responses in different strains of mice, although to different extents. These results suggest that linear, multiple epitope peptides may offer attractive alternatives as subunit vaccine candidate molecules, but at the same time highlight the fact that the design principles are far from being clear and have yet to be worked out.
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Papers by Pawan Sharma
resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis (Mtb)
has further complicated the situation. Though several studies characterizing drug sensitive and drug
resistant strains are available in literature, almost all studies are done on unrelated strains. Therefore,
the objective of this study was to compare the proteomic data of four sequential isolates of Mtb from a
single patient who developed MDR-TB during the course of anti-tuberculosis therapy (ATT).
Methods: In this study, using two-dimensional (2D) gel electrophoresis and MALDI-TOF mass
spectrometry, we compared and analyzed the cell lysate proteins of Mtb sequential clinical isolates from
a patient undergoing anti-TB treatment. The mRNA expression levels of selected identified proteins were
determined by quantitative real-time polymerase chain reaction (qRT-PCR).
Results: The genotypes of all four isolates remained homologous, indicating no re-infection. The initial
isolate (before treatment) was sensitive to all first-line drugs, but the consecutive isolates were found
to be resistant to isoniazid (INH) and rifampicin (RIF) and developed mutations in the katG, inhA and
rpoB. the intensities of 27 protein spots were found to be consistently overexpressed in INH and RIF
resistant isolates. The most prominent and overexpressed proteins found during the development of drug
resistance were GarA (Rv1827), wag31 (Rv2145c), Rv1437 and Rv2970c.
Interpretation & conclusions: This preliminary proteomic study provides an insight about the proteins
that are upregulated during drug resistance development. These upregulated proteins, identified here,
could prove useful as immunodiagnostic and possibly drug resistant markers in future. However, more
studies are required to confirm these findings.
resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis (Mtb)
has further complicated the situation. Though several studies characterizing drug sensitive and drug
resistant strains are available in literature, almost all studies are done on unrelated strains. Therefore,
the objective of this study was to compare the proteomic data of four sequential isolates of Mtb from a
single patient who developed MDR-TB during the course of anti-tuberculosis therapy (ATT).
Methods: In this study, using two-dimensional (2D) gel electrophoresis and MALDI-TOF mass
spectrometry, we compared and analyzed the cell lysate proteins of Mtb sequential clinical isolates from
a patient undergoing anti-TB treatment. The mRNA expression levels of selected identified proteins were
determined by quantitative real-time polymerase chain reaction (qRT-PCR).
Results: The genotypes of all four isolates remained homologous, indicating no re-infection. The initial
isolate (before treatment) was sensitive to all first-line drugs, but the consecutive isolates were found
to be resistant to isoniazid (INH) and rifampicin (RIF) and developed mutations in the katG, inhA and
rpoB. the intensities of 27 protein spots were found to be consistently overexpressed in INH and RIF
resistant isolates. The most prominent and overexpressed proteins found during the development of drug
resistance were GarA (Rv1827), wag31 (Rv2145c), Rv1437 and Rv2970c.
Interpretation & conclusions: This preliminary proteomic study provides an insight about the proteins
that are upregulated during drug resistance development. These upregulated proteins, identified here,
could prove useful as immunodiagnostic and possibly drug resistant markers in future. However, more
studies are required to confirm these findings.