Cloning and molecular characterization of functional protein(s) of human lymphatic filariid Brugia malayi and their evaluation as drug/vaccine target

Abstract

In recent years, control tools and strategies have become available for lymphatic filariasis. Still there is an unequivocal call for the development of much improved complementary approaches for long-lasting reduction in adult worms. New molecules affecting novel molecular targets are required to discover agents that may kill adult parasites and replace current drugs threatened with development of resistance. The introduction of molecular biology including genomics, proteomics and advance computational approaches has revolutionized filarial research for the identification and production of potential drug or vaccine targets. Discovery of antifilarial drug targets, their validation and development of specific assay is foremost for screening compounds in high-throughput mode to facilitate antifilarial drug discovery. Immunoprophylaxis/vaccination offers a viable approach for interrupting transmission by countering the parasite at the very entry point. Identification of novel protein/s that may act as vaccine candidate against filariasis needs to be pursued more vigorously. The work embodied in the present thesis primarily involves molecular and immunological characterisation of recombinant trehalose-6-phosphate phosphatase of human filariid B. malayi as a possible vaccine candidate. In view of the its vital role in filarial biology and absence of tpp gene in mammals, this enzyme was also exploited as antifilarial drug target by RNA interference studies that validated B. malayi TPP as a promising antifilarial drug target too. The findings derived from the present study endow with following insights:  Bm-TPP was successfully PCR amplified, cloned, expressed and purified to homogeneity as ~60 kDa protein.  Purified protein was found to be in native form as demonstrated by the fluorescence spectrometry analysis.  Bm-TPP gene was found to be expressed in all the major life-stages (Mf, L3 and adult) of B. malayi.  The enzyme belonged to HAD hydrolase super family II protein where the highly conserved residues in HAD hydrolase catalytic domains (I, II, and III) were also found to be completely conserved in Bm-TPP. Bm-TPP showed robust and specific specificity towards its substrate trehalose-6- phosphate and the Km value was 0.42 mM.  Bm-TPP showed absolute requirement of Mg2+ ion as a cofactor whose absence or EDTA treatment renders enzyme inactive.  Small sized siRNA mediated knockdown of Bm-tpp gene in adult worms by in vitro soaking caused reduction in the motility of both male and female worms, however, no such effect was noticed in the worms exposed to non target siRNA or siRNA free medium. MTT reduction assay for adult worm viability also corroborated with the motility findings demonstrating profound adverse effect of target gene silencing on B. malayi viability and survival.  Considerable reduction in the number of Mf released by female B. malayi in vitro post tpp gene silencing was noticed within 24 h reaching ~75% within 48 h demonstrating potent adverse effect of gene silencing on Mf release. Majority of the released Mf were found dead and those which were alive had acute signs of phenotypic deformities.  tpp gene silencing had profound adverse effect on female worm embryogenesis. Female worms soaked in the tpp specific siRNA had all degenerated eggs/embryos in the uteri indicating developmental arrest at early embryonic stage.  tpp gene silencing in L3 brought about lethality to majority of the larvae within 48 h. In vivo inoculation of siRNA treated L3 (which escaped death) in to the peritoneal cavity of susceptible host jird, led to 84.9% reduced establishment of adult worms at day 90 demonstrating that knocking down of tpp gene in L3 adversely affected their survival and further development in the vertebrate host. Significant proportion of female worms (54.5%) which reached adulthood in the jird peritoneal cavity demonstrated defective embryogenesis.  In silico prediction of antigenicity of Bm-TPP demonstrated the presence of 20 antigenic determinants in the recombinant protein. The average propensity of the protein was found to be 1.0233 that reveals high antigenic nature of this enzyme. The recombinant enzyme also showed reactivity with the antibodies present in the sera of various categories of human W. bancrofti patients residing in filaria-endemic area including the putatively immune ‘endemic normal’ demonstrating the usefulness as of Bm-TPP as an immunoprophylactic agent.  Administration of Bm-TPP to naïve BALB/c mice and mastomys along with FCA/FIA triggered the production of high levels of Bm-TPP specific antibodies in the sera. The two rodent species generated predominantly elevated IgG1, IgG2a, IgG2b to Bm-TPP. IgG2a/IgG1 ratio was slightly more than 1, indicating a mixed type of Th1/Th2 immune response with marginal bias towards Th1.  Anti Bm-TPP antibody mediated the in vitro adherence of peritoneal cells on the surface of both Mf and L3 causing cytotoxicity and death to >50% of the parasites. This phenomenon was significantly reduced after depletion of anti Bm-TPP antibodies from the serum.  Flow cytometry data from BALB/c and mastomys demonstrated that Bm-TPP brings about considerable oxidative burst in peritoneal macrophages in the immunized animals indicating triggering of an inflammatory protective response.  Bm-TPP administration in the BALB/c promotes expansion of CD4+, CD8+ and CD 19+ T and B cell population. The recombinant protein up-regulated the production of both pro-inflammatory (IL-2, IFN-γ) and anti-inflammatory (IL-4, IL-10) cytokines in CD4+ T cells in BALB/c thus demonstrating the generation of mixed Th1/Th2 immune response by the recombinant.  Immunization of BALB/c with Bm-TPP resulted into profound reduction (67%) in the transformation of challenged L3 to L4 stage. In susceptible host mastomys, immunization adversely affected the challenged larvae to reach adult stage and animals demonstrated 67.8% reduced adult worm establishment. In addition, the established female worms also released reduced number of microfilariae in to the peripheral circulation (72.4%). The female worms recovered from vaccinated mastomys had reduced fecundity (70%).  Splenocytes isolated from Bm-TPP immunized mastomys that were subsequently challenged with L3 demonstrated heightened lymphoproliferation in presence of nonspecific mitogen or the recombinant protein suggesting development of strong cellular immune response.  Immunogenicity and protective efficacy of Bm-TPP was evaluated with three other adjuvants; Alum, Montanide ISA 720 and Titermax Gold. Among these, significant reduction in microfilarial burden was noticed in TMG formulation (63.4%) followed by MON (50.24%) and Alum (31.23%) on day 180 post L3 challenge.  As regards to adult worm establishment, highest protection was induced with Bm- TPP+TMG (52%) while Bm-TPP+MON provided comparatively lesser protection (36%). ALM revealed most inferior protection (21%).  A considerable proportion of recovered female worms had embryostatic effect when ALM (38.06%), TMG (55.02%) and MON (51.78%) were used as adjuvants with recombinant enzyme.  All the three adjuvants induced noticeable differential level of antibody production to Bm-TPP in mastomys. Highest antibody titer was induced by ALM, TMG and lowest by Montanide. Significant increase in both IgG1 and IgG2a antibody isotype was noticed in all three formulations; however the higher ratio of IgG1 to IgG2a in Alum formulation showed a Th2 biased immune response. TMG and MON skewed the immune response toward Th1 phenotype as illustrated by low IgG1/IgG2a ratio (<1).  Significant expansion of CD4+ T cells was observed by all the adjuvant formulations. CD8+ T cell population was significantly up-regulated in MON and TMG group in contrast to Alum. CD19+ B cell population was up-regulated by all the adjuvant formulations.  Administration of Bm-TPP with MON and TMG in BALB/c elicited a Th1 polarized mixed Th1/Th2 immune response as revealed by increased pro-inflammatory (IL-2, IFN-γ) and anti-inflammatory (IL-4, IL-10) cytokines while Alum generated a predominant Th2 immunity displayed by higher production of IL-4 and IL-10 cytokines.  In conclusion, our findings clearly demonstrate the essential nature of B. malayi trehalose-6-phosphate phosphatase which plays a crucial role in filarial parasite fertility, development and survival. Bm-TPP also possesses the characteristics of a good vaccine candidate that offers profound degree of protection against B. malayi larval challenge. Its absence in the mammals and suitability as a drug /vaccine candidate paves the way for design and synthesis of novel inhibitors against Bm-TPP.