Immunological characterization of recombinant Wuchereria bancrofti cuticular collagen (COL-4) as putative vaccine candidate for human lymphatic filariasis.

OBJECTIVE: To elucidate immunoprophylactic potential of recombinant Wuchereria bancrofti (W. bancrofti) cuticular collagen (COL-4) in BALB/c mice and filarial clinical samples. METHODS: col-4 gene was PCR amplified from W. bancrofti L3 cDNA library and cloned in pRSET B vector. Recombinant COL-4 was over expressed in salt inducible system and was purified by nickel affinity chromatography. Humoral and cellular responses were measured by ELISA and peripheral blood mononuclear cells (PBMC) of various filarial clinical samples respectively using purified recombinant COL-4 antigen. Then the protective immune responses of COL-4 immunized BALB/c mice were characterized. RESULTS: Sequence analysis of COL-4 with human host proteins reveals lack of homology. The recombinant COL-4 was found to be at 15 kDa fusion protein. The affinity purified COL-4 showed significant reactivity with putatively immune sera and in a similar fashion it demonstrated marked proliferation in PBMC samples. Immunization studies in experimental filarial host (mice) elicited significant titers with protective antibody isotype profile (IgM and IgG). Cellular immune responses were also significant in terms of splenocytes proliferation assay on mice samples. CONCLUSIONS: Our immunological findings in experimental host suggest Th2 mediated immune response. Hence, we propose that W. bancrofti COL-4 could be an efficacious vaccine candidate against lymphatic filariasis.

Evaluation of synthetic peptides of WbSXP-1 for the diagnosis of human lymphatic filariasis.

Parasitic nematodes infect nearly half of the world's human population, resulting in significant morbidity and mortality. Though filariasis is not fatal, it is the second leading cause of permanent and long-term disability worldwide. Filariasis has a spectrum of disease manifestation and infectivity found among the infected individuals and also goes unnoticed for years. Furthermore, there are ample reports emerging on the genetic variation among the parasites population. Hence, it is necessary to develop diagnostics for early detection of the disease. Synthetic peptides that mimic the immunogenic regions and a conserved region similar to that of recombinant antigen will be more useful in developing diagnostics, vaccines, or therapeutics. WbSXP-1 was earlier proven as a good diagnostic antigen; B-cell epitopic analysis showed 4 potent immunodominant regions spanning the whole antigen. These synthetic peptides (N, N1, N2, and N3) were produced and used as a diagnostic candidate to detect anti-SXP antibody and conversely to detect the infected individuals. The monomeric peptides showed good reactivity against microfilareamic (MF) sera. Among them, the peptides N, N1, and N2 were found to be more reactive. Furthermore, multiple chimeric peptides in linear combinations of 2 peptides were tested for its efficacy to detect anti-SXP antibody in infected MF sera. The peptides N:N1 and N1:N2 were synthesized and tested against human clinical sera. This chimeric peptides constructed based on WbSXP-1 were found to be reactive, specifically with MF sera by ELISA. These peptide-based diagnostic method can serve as a standard better tool without cross-reactivity in lymphatic filariasis elimination program.

Molecular characterization of a truncated antigen (Wb14) of SXP-1 of Wuchereria bancrofti from four endemic regions in India.

Wb14 of Wuchereria bancrofti, an orthologue of Brugia malayi SXP-1 and W. bancrofti SXP-1, was amplified from genomic DNA of W. bancrofti microfilaria collected from four distant geographical locations in India viz., Vellore, Bhubaneshwar, Pondicherry and Sevagram. The gene was sub-cloned in a prokaryotic vector pRSET and expressed in Escherichia coli as a truncated protein (approximately 23kDa). The nucleotide sequence of the gene is 98% similar to that of WbSXP-1 and is found to be intron-less. However, the analysis and comparison of the derived amino acid sequence with WbSXP-1 showed that Wb14 is truncated at amino acid position 153. The distribution of the two genes in the studied four geographical locations indicated that WbSXP-1 is prevalent only in parasite samples from Sevagram while Wb14 is present in parasites from all the other locations. Only a limited polymorphism was observed in both the genes among the parasites from different geographical locations.

Brugia malayi: comparison of protective immune responses induced by Bm-alt-2 DNA, recombinant Bm-ALT-2 protein and prime-boost vaccine regimens in a jird model.

Immunization of jirds with Bm-alt-2 elicited partial protection against challenge infection with the filarial parasite Brugia malayi. In this study, we initially compared the protective immune responses elicited following immunization with recombinant Bm-ALT-2 protein regimen and Bm-alt-2 DNA regimen. These studies showed that protein vaccination conferred approximately 75% protection compared to DNA vaccination that conferred only 57% protection. Analysis of the protective immune responses showed that the protein immunization promoted a Th2-biased response with an increase in IL-4, IL-5 and IgG1 responses, whereas, the DNA vaccine promoted a Th1-biased response with profound IFN-gamma and IgG2a responses. Since protein vaccination gave better results than DNA vaccination, we then wanted to evaluate whether a prime-boost vaccination that combined DNA prime and protein boost will significantly increase the protective responses induced by the protein vaccine. Our results suggest that prime-boost vaccination had no added advantage and was comparatively less effective (64% protection) than the Bm-ALT-2 protein alone vaccination. Prime boost vaccination generated mixed Th1/Th2 responses with a slightly diminished Th2 responses compared to protein vaccination. Thus, our results suggest that Bm-ALT-2 protein vaccination regimen may be slightly better than prime-boost vaccine regimen and the mechanism of protection appears to be largely mediated by a Th2-biased response.