Mohan Lal Dubey

Immunogenicity and efficacy of single antigen Gp63, polytope and polytopeHSP70 DNA vaccines against visceral Leishmaniasis in experimental mouse model.

Polytope approach of genetic immunization is a promising strategy for the prevention of infectious disease as it is capable of generating effective cell mediated immunity by delivering the T cell epitopes assembled in series. Leishmaniasis is a significant world wide health problem for which no vaccine exists. In this study we have compared immunogenicity and efficacy of three types of DNA vaccines: single antigen Gp63 (Gp63/pcDNA), polytope (Poly/pcDNA) and Polytope fused with hsp70 (Poly/hsp/pcDNA) against visceral leishmaniasis in susceptible BALB/c mice. Mice vaccinated with these plasmids generated strong Th1 immune response as seen by dominating IFN-γ over IL-10 cytokine. Interestingly, cytotoxic responses generated by polytope DNA plasmid fused with hsp70 of Leishmania donovani were significantly higher when compared to polytope and single antigen Gp63 vaccine. Challenge studies revealed that the parasite load in liver and spleen was significantly lower with Poly/hsp/pcDNA vaccination compared to other vaccines. Therefore, our study indicates that polytope DNA vaccine is a feasible, practical and effective approach for visceral leishmaniasis.

Plasmodium falciparum induced perturbations of the erythrocyte antioxidant system

Erythrocyte antioxidants catalase, superoxide dismutase, reduced glutathione and glutathione peroxidase were studied in cells harbouring different growth stages of Plasmodium falciparum. Catalase and superoxide dismutase showed significant decrease during parasite maturation indicating hampered metabolism of hydrogen peroxide and superoxide anions. Glutathione peroxidase also exhibited a downward trend during the growth of P. falciparum, while there was a moderate accumulation of reduced glutathione. These findings suggest decreased utilization of the reduction potential in detoxification of reactive oxygen species. The fall in all three antioxidant enzymes studied was highly significant (P less than 0.001) in erythrocytes with mature stages of the parasite (trophozoites, schizonts). The increased vulnerability of erythrocytes to damage, which parallels the growth phases of the parasite emphasizes the need for early treatment of P. falciparum malaria to minimise red cell destruction and the resulting anaemia.

Cysteine proteinase 30 (CP30) and antibody response to CP30 in serum and vaginal washes of symptomatic and asymptomatic Trichomonas vaginalis-infected women

Infection with Trichomonas vaginalis may be asymptomatic or with symptoms suggestive of vaginitis. Because cysteine proteinase 30 (CP30) of T. vaginalis is known to be a virulence marker that plays a role in cytoadherence, the aim of this study was to analyse the presence of CP30 and antibody to CP30 in clinical samples of symptomatic and asymptomatic infected women. CP30 was detected in all the serum and vaginal washes (VWs) of symptomatic women and in 65% of the serum and 80% of the VWs of asymptomatic women. This suggested that the majority of asymptomatic women also exhibit CP30 in the serum and VWs. Antibody to CP30 was detected in all the serum samples of symptomatic and asymptomatic women and in the VWs of only 54·5% of the symptomatic and 35% of the asymptomatic women. Antibody to CP30 was also detected in 3/20 of the serum samples and in none of the VWs from uninfected women. Significantly higher amounts of antibody (mean OD values) were observed in serum and VWs of symptomatic as compared to asymptomatic and healthy women (P < 0·001). These results indicate that besides CP30, other factors may also be playing a role in leading to symptomatic infection, because CP30 was detected in clinical samples from all the symptomatic and the majority of the asymptomatic women. Although anti‐CP30 antibodies do not appear to be protective, detection of antibody to CP30 antigen in serum samples may be used as a diagnostic tool.

Correlation of Molecular Markers, Pfmdr1-N86Y and Pfcrt-K76T, with In Vitro Chloroquine Resistant Plasmodium falciparum, Isolated in the Malaria Endemic States of Assam and Arunachal Pradesh, Northeast India

The mechanism of chloroquine (CQ) resistance in Plasmodium falciparum is not clearly understood. However, CQ resistance has been shown to be associated with point mutations in Pfcrt and Pfmdr1. These genes encode for digestive vacuole transmembrane proteins Pfcrt and Pgh1, respectively. The present study was carried out to analyze the association of Pfcrt-K76T and Pfmdr1-N86Y mutations with CQ resistance in Northeast Indian P. falciparum isolates. 115 P. falciparum isolates were subjected to in vitro CQ sensitivity testing and PCR-RFLP analysis for the Pfmdr1-N86Y and Pfcrt-K76T mutations. 100 isolates of P. falciparum were found to be resistant to CQ by the in vitro susceptibility test (geometric mean EC50 2.21 µM/L blood) while 15 were found to be CQ sensitive (geometric mean EC50 0.32 µM/L blood). All the CQ resistant isolates showed the presence of Pfmdr1 and Pfcrt mutations. CQ sensitive isolates were negative for these mutations. Strong linkage disequilibrium was observed between the alleles at these two loci (Pfmdr1-N86Y and Pfcrt-K76T). The results indicate that Pfmdr1-N86Y and Pfcrt-K76T mutations can be used as molecular markers to identify CQ resistance in P. falciparum. The result necessitates the evaluation of CQ in vivo therapeutic efficacy in endemic areas for more effective malaria control strategies.

Utilization of Monoclonal Antibodies for Detection of Plasmodium falciparum Antigen in Cerebrospinal Fluid of Cerebral Malaria Patients

A uniform protein profile of bands at 34, 43, and 52 kDa was obtained with all the cerebrospinal fluid (CSF) samples of malaria (10 in number) and non-malaria patients (31 in number) by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). An immunoreactive band was observed at 43 kDa in CSF samples of cerebral malaria patients but not in non-malaria cases when tested with rabbit anti-Plasmodium falciparum antibodies by Western blot analysis. Eleven reactive monoclonal antibodies against P. falciparum were stabilized and expanded. Nine monoclonal antibodies were reactive to CSF samples of cerebral malaria and non-malaria and P. falciparum antigen by dot-ELISA and a common immunoreactive band at 43 kDa by Western blot. One clone Cl-2 was reactive at 43 kDa with CSF of the cerebral malaria patients and also in P. falciparum antigen but at 66 kDa with non-malarial CSF samples in Western blot. The other two clones (Cl-6 and 14) reacted with 3/31 (90% specific) and 8/31 (74%) CSF samples of non-malaria patients, respectively. The monoclonal antibody based ELISA reported in the present study using clone-6 can therefore offer another possibility for developing rapid, easy-to-perform, low-cost tests for diagnosis of cerebral malaria in CSF samples. Western blot using clone-2 might be useful for the detection of cerebral malaria antigen in CSF.