Epitope mapping of Brugia malayi ALT-2 and the development of a multi-epitope vaccine for lymphatic filariasis.

Human lymphatic filariasis is a neglected tropical disease, causing permanent and long-term disability with severe immunopathology. Abundant larval transcript (ALT) plays a crucial role in parasite establishment in the host, due to its multi-faceted ability in host immune regulation. Although ALT protein is a key filarial target, its exact function is yet to be explored. Here, we report epitope mapping and a structural model of Brugia malayi ALT-2, leading to development of a multi-epitope vaccine. Structural analysis revealed that ALT represents unique parasitic defence proteins belonging to a toxin family that carries a 'knottin' fold. ALT-2 has been a favourite vaccine antigen and was protective in filarial models. Due to the immunological significance of ALT-2, we mapped B-cell epitopes systematically and identified two epitope clusters, 1-30 and 89-128. To explore the prophylactic potential of epitope clusters, a recombinant multi-epitopic gene comprising the epitopic domains was engineered and the protective efficacy of recombinant ALT epitope protein (AEP) was tested in the permissive model, Mastomys coucha. AEP elicited potent antibody responses with predominant IgG1 isotype and conferred significantly high protection (74.59%) compared to ALT-2 (61.95%). This proved that these epitopic domains are responsible for the protective efficacy of ALT-2 and engineering protective epitopes as a multi-epitope protein may be a novel vaccine strategy for complex parasitic infections.

Chimeric Epitope Vaccine from Multistage Antigens for Lymphatic Filariasis.

Lymphatic filariasis, a mosquito-borne parasitic disease, affects more than 120 million people worldwide. Vaccination for filariasis by targeting different stages of the parasite will be a boon to the existing MDA efforts of WHO which required repeated administration of the drug to reduce the infection level and sustained transmission. Onset of a filaria-specific immune response achieved through antigen vaccines can act synergistically with these drugs to enhance the parasite killing. Multi-epitope vaccine approach has been proved to be successful against several parasitic diseases as it overcomes the limitations associated with the whole antigen vaccines. Earlier results from our group suggested the protective efficacy of multi-epitope vaccine comprising two immunodominant epitopes from Brugia malayi antioxidant thioredoxin (TRX), several epitopes from transglutaminase (TGA) and abundant larval transcript-2 (ALT-2). In this study, the prophylactic efficacy of the filarial epitope protein (FEP), a chimera of selective epitopes identified from our earlier study, was tested in a murine model (jird) of filariasis with L3 larvae. FEP conferred a significantly (P < 0.0001) high protection (69.5%) over the control in jirds. We also observed that the multi-epitope recombinant construct (FEP) induces multiple types of protective immune responses, thus ensuring the successful elimination of the parasite; this poses FEP as a potential vaccine candidate.

Tandem antioxidant enzymes confer synergistic protective responses in experimental filariasis.

Helminth parasites use antioxidant defence strategies for survival during oxidative stress due to free radicals in the host. Accordingly, tissue-dwelling filarial parasites counteract host responses by releasing a number of antioxidants. Targeting these redox regulation proteins together, would facilitate effective parasite clearance. Here, we report the combined effect of protective immune responses trigged by recombinant Wuchereria bancrofti thioredoxin (WbTRX) and thioredoxin peroxidase (WbTPX) in an experimental filarial model. The expression of WbTRX and WbTPX in different stages of the parasite and their cross-reactivity were analysed by enzyme-linked immunosorbent assay (ELISA). The immunogenicity of recombinant proteins and their protective efficacy were studied in animal models when immunized in single or cocktail mode. The antigens showed cross-reactive epitopes and induced high humoral and cellular immune responses in mice. Further, parasite challenge against Brugia malayi L3 larvae in Mastomys coucha conferred significant protection of 57% and 62% against WbTRX and WbTPX respectively. The efficacy of L3 clearance was significantly higher (71%) (P <  0.001) when the antigens were immunized together, showing a synergistic effect in multiple-mode vaccination. Hence, the study suggests WbTRX and WbTPX to be attractive vaccine candidates when immunized together and provides a tandem block for parasite elimination in the control of lymphatic filariasis.

Dominant T-cell epitopes of filarial BmALT-2 and their cytokine profile in BALB/c mice.

Filarial nematodes down-regulate the host immune response to establish infection by inducing IL-10-mediated T-cell suppression.Abundant larval transcript (ALT) proteins are of major interest as they are expressed abundantly in the L3 stage, implicated in protective immunity and may play a role in immune evasion. The T-cell epitopes of BmALT-2 and the cytokine responses induced by these peptides were investigated in a mouse model using synthetic peptides, which could be exploited in the design of a potent epitope-based vaccine. Five regions were found to carry T-cell epitopes inducing high levels of cellular proliferation. The regions 55­68 and 73­91 of ALT-2 induced very high levels of IL-10 secretion and hence could be involved in immunomodulation in the host.

Crucial epitopes of Wuchereria bancrofti abundant larval transcript recognized in natural infection.

Lymphatic filariasis is a tropical disease, with over 40 million people seriously incapacitated due to lymphangitis and elephantiasis. Over 99% of infections are caused by the nematode Wuchereria bancrofti. Expressed sequence tag (EST) analysis of filarial genome identified novel infective larval (L3) stage-specific antigen, abundant larval transcript (ALT-2), which was shown to be highly essential for parasite establishment and survival in the host. The unique structural features and immunological characteristics of ALT-2 indicate the presence of critical motifs that needs to be explored in natural human infection for understanding and management of the disease. In order to dissect the epitopes of ALT protein recognized in humans, eight peptides spanning the entire protein sequence were selected based on in-silico epitope prediction and synthesized. Analysis of the reactivity of W. bancrofti ALT-2 synthetic peptides with clinical sera (n = 40) from endemic areas identified epitopes recognized by putatively immune sera, of which two comprise the highly variable acidic domain (21-60). Interestingly, our study also revealed crucial epitopes recognized by microfilaremic (MF) sera with significantly high IgG4 isotype (p < 0.001), implicated in immunomodulation. The epitope peptides identified were highly specific for MF sera and, thus, have the potential to be exploited as diagnostic markers.

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.

Loss of Werner syndrome protein function promotes aberrant mitotic recombination.

The chromosome 8p11-12 Werner syndrome (WRN ) locus encodes a RecQ helicase protein of unknown function that possesses both 3' --> 5' helicase and 3' --> 5' exonuclease activities. We show that WRN cell lines display a marked reduction in cell proliferation following mitotic recombination, and generate few viable gene conversion-type recombinants. These findings indicate that WRN plays a role in mitotic recombination, and that a loss of WRN function may promote genetic instability and disease via recombination-initiated mitotic arrest, cell death, or gene rearrangement.

Cell fusion corrects the 4-nitroquinoline 1-oxide sensitivity of Werner syndrome fibroblast cell lines.

We have shown that Werner syndrome (WRN) fibroblast cell lines are unusually sensitive to the DNA-damaging agent 4-nitroquinoline 1-oxide (4NQO), though not to gamma radiation or to hydrogen peroxide. The fusion of 4NQO-sensitive WRN and 4NQO-resistant control fibroblast cell lines generated proliferating WRN x control cell hybrids that expressed WRN protein and were 4NQO-resistant. These results establish the recessive nature of 4NQO sensitivity in WRN cell lines and provide a cellular assay for WRN protein function.