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.

DNA vaccination with VP2 gene fragment confers protection against Infectious Bursal Disease Virus in chickens.

Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens by destruction of antibody producing B cells in the Bursa of Fabricius and poses a potential threat to the poultry industry. We have examined the protective efficacy of a subunit DNA vaccine against IBDV infection in chickens in this study. An immunodominant VP2 gene fragment (VP252-417) was cloned into CMV promoter based DNA vaccine vector pVAX1 and in vitro expression of the DNA encoded antigens was confirmed by transfection of CHO cells with vaccine constructs followed by RT-PCR and western blot analysis using IBDV-antiserum. Two weeks old chickens were immunized intramuscularly with pVAXVP252-417 and the in vivo transcription of the plasmid DNA was confirmed by RT-PCR analysis of DNA injected muscle tissue at different intervals of post immunization. Tissue distribution analysis revealed that the plasmid DNA was extensively distributed in muscle, spleen, kidney, liver, and bursa tissues. Chickens immunized with pVAXVP252-417 developed high titer (1:12,000) of anti-VP252-417 antibodies. Further, chicken splenocytes from pVAXVP252-417 immunized group showed a significantly high proliferation to the whole viral and recombinant antigen (P<0.01) compared to control groups, which implies that pVAXVP252-417 codes for immunogenic fragment which has epitopes capable of eliciting both B and T cell responses. This is evident by the fact that, pVAXVP252-417 immunized chicken conferred 75% protection against virulent IBDV (vIBDV) challenge compared to the control group. Thus, the present study confirms that the immunodominant VP2 fragment can be used as a potential DNA vaccine against IBDV infection in chickens.

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.