Prophylactic Evaluation and Immunological Characterization of Recombinant Heavy Chain Myosin of Adult Female Brugia Malayi

Abstract

Lymphatic filariasis (LF) is a major tropical disease caused by mosquito borne parasite worm Wuchereria bancrofti, Brugia malayi or B. timori is widespread in Asia, Africa and Latin America. Of the three species, W. bancrofti is responsible for more than 90% of cases and is found throughout the tropics and in some sub-tropical areas including Asia, East Africa, Latin America, the Middle East, the Pacific Rim and Pacific Island. Brugia malayi is less prominent and constitute the remaining 10% incidence. India alone accounts for 40% of the global prevalence of infection leading to loss of 1 billion US $ (~Rs. 5000 crores) each year in form of secondary treatment costs and loss of working days due to filariasis which highlights the considerable burden this disease places on individuals and on the community. Filariasis is second only to malaria as the most important vector borne disease in India. The disease is endemic in 22 states and union territories. WHO has identified filariasis as one of six potentially eradicable infectious diseases and initiated ‘Global Programme to Eliminate Lymphatic Filariasis’ (GPELF) for its elimination as a public health problem by the year 2020. The current global filariasis control efforts (GPELF) advocate few strategies for the treatment and control including (i) MDA with single-dose albendazole and ivermectin (Mectizan) tablets in areas, where LF is co-endemic with onchocerciasis, annually for 4–6 years, (ii) single dose albendazole and diethylcarbamazine (DEC) in areas, where onchocerciasis is not endemic, annually for 4–6 years, (iii) exclusive use of DEC-fortified tablet or cooking salt for 1–2 years in some settings, (iv) vector control measures, especially the use of insecticide-treated materials, where appropriate and cost-effective, (v) home-based self-management of lymphoedema and elephantiasis for affected individuals and (vi) improved access to surgical intervention for men with hydrocoele. Despite development of MDA programme, newer control strategies, sensitive and specific immunodiagnostic assays, control of lymphatic filariasis still appears a far-fetched dream. Diethylcarbamazine (DEC) or ivermectin principally act on circulating microfilariae resulting in reappearance of microfilaraemia after withdrawal of the drug, moreover both the drug have a little adulticidal effect. Recently the evidence of emergence of resistance against mainstay drugs like albendazole and ivermectin have started to show up which also makes this challenge tougher than past. In absence of adequate chemotherapeutic tools, control of infection by immunological means remains the only alternative. Despite extensive research till date no vaccine has developed for the control of human filariasis. Multicellular parasites (including filaria) have used millions of years of co-adaptation to divert the host’s immune system to ensure their long-term survival. In view of this fact a vaccine against these parasites is not an easy task. A systematic approach necessitates identification, production and purification of protective immunogens, usage of adjuvant, carriers, and mode of presentation/delivery and determination of protective immune responses. Most of the studies in the past have employed the strategy of identifying immunogen from different filarial life stages to be utilized as vaccine candidate. With this perspective, efforts in past were done in our laboratory for the identification of immunogenic antigens from a cDNA library of adult female B. malayi. This identification of clones carrying immuno-reactive antigen was done on the basis of high immune reactivity of clone with hyperimmune serum against adult B. malayi somatic antigens raised in rabbit. Out of numerous immuno-reactive clones identified from this exercise, one coded for B. malayi myosin. Being identified as immunogenic clone, the prospect to use this protein for vaccination studies was feasible. The present study was thus planned to evaluate the immunization potential of recombinant myosin of B. malayi.

Procuring recombinant protein for immunological and vaccination studies The present work was initiated with sub-cloning of the cDNA clone in a prokaryotic vector to facilitate the production of recombinant protein in bulk quantity. Cloning was done in pET28b cloning vector and was transformed in E. coli DH5α cells for maintenance of recombinant ligated vector and in E. coli Bl21DE3 cell for over-expression of recombinant protein. The conditions for over-expression of recombinant protein were optimized through extensive and repeated experimentation. Over-expressed protein in bacterial cells cannot be used for immunological work therefore purification of over-expressed recombinant protein was carried out. Recombinant protein had a His-tag (a short chain of six amino acid-histidine) at N-terminus of the protein encoded by the vector. The presence of this His-tag fused with recombinant protein facilitated its purification to homogeneity by immobilized metal affinity chromatography (IMAC) using commercially available Ni-NTA column (Chelating Sepharose, Fast Flow with Ni+4 metal coupled, Qiagen). The charged Nickel in the column binds with six histidine residues present at N terminal of the expressed protein which can be eluted with buffer containing high concentration of imidazole. Thus single band purified recombinant protein of size ~73 kDa to be used for further studies was obtained in bulk amount i.e. ~20mg/liter of E. coli culture. Positive reaction with anti-his antibody confirmed presence of purified recombinant protein expressed after IPTG induction as sharp single band at ~73 kDa position in the blot

Serological reactivity and prophylactic studies Serological reactivity of recombinant protein was carried with human bancroftian sera belonging to various disease categories. The recombinant myosin reacted strongly with all the categories of human sera exhibiting its broad-spectrum recognition by W. bancrofti infected human sera. Recombinant protein did not react with non endemic normal sera demonstrating lack of any non specific reaction. Recombinant protein also displayed strong positive reaction with filaria-infected mastomys sera. Uniform degree of reaction of purified recombinant protein with both bancroftian as well as brugian sera (from animal) imply that the outcome of vaccination with B. malayi can be extended for W. bancrofti as well. Further, the recombinant protein was utilized for vaccination studies for the determination of its prophylactic potential. The immunization studies were carried out in widely used animal rodent models for filariasis (Mastomys coucha and Meriones unguiculatus) that are well established and maintained in our laboratory. Vaccination was carried out with recombinant myosin emulsified in FCA/FIA by subcutaneous administration of emulsion following standard protocol. Appropriate age and sex matched control animals were included in the study. Experimental animals (control and vaccinated) were challenged with infective stage larvae (L3) on the completion of immunization schedule. L3 were obtained from mosquitoes reared and bred in our insectarium. Since worm establishment and maturation takes upto 12 weeks, observations were started from 12 weeks post challenge till 20th week. Microfilaraemia burden was measured every fortnight from 12-20 weeks following which animals were euthanized for the recovery of adult worms. Adult female worms were observed for their fecundity. Vaccination study produced fruitful outcome in form of significantly reduced microfilarial density in blood and remarkable decrease in adult worm burden in vaccinated group of animals. Vaccination with recombinant myosin led to ~80% reduction in microfilaraemia and ~60% reduction in adult worm burden as compared to controls in mastomys. Furthermore vaccination also increased sterility in adult female worms, which was ~55% higher than controls.

Vaccination studies with other adjuvants and delivery systems Mastomys were used as experimental model and FCA/FIA as adjuvant in the initial experiment. The results of initial vaccination studies were quite encouraging as the recombinant vaccine was able to generate fair degree of protection. Further we tried to use other adjuvants and animal models in order to improve protectively of recombinant myosin. Therefore in the subsequent study two new adjuvants montanide and alum (both approved for use in humans) were included for comparison with FCA/FIA. Apart from mastomys jirds were also included in the study. This implies that vaccination of recombinant protein was done in both animal models with all the three adjuvants separately. Immunization schedule was same as earlier experiment, followed by challenge with equal number of infective stage larvae as used in the previous experiment. Monitoring aspects as observed and recorded in earlier experiment were followed in both the models, omitting microfilaraemia recording in jird model where it is little impractical. Mastomys model generated analogous outcome as achieved in the previous experiment. However both of the human compatible adjuvants used in the study were unable to improve the efficacy over FCA, however positive aspect of the experiment was that the protection efficacy of alum group was nearly same degree as of FCA group. Montanide was unable to impart its adjuvant affect with recombinant myosin and did not impart higher protection in vaccinated animals. There was a slight variation in vaccination results in jird model. Although all vaccinated group of jirds exhibited significant reduction in adult worm burden as compared to control jirds, none of them could generate higher degree of protection in this model when compared to that of mastomys. Interestingly alum group generated higher protection (~54%) as compared to FCA group (~45%) in jird. All these results reflect efficiency of recombinant myosin in eliciting protective immunity against B. malayi infections, although the degree of protection in different models had exhibited slight variation. Since the conventional adjuvants were able to provide similar degree of protection as of FCA the purpose of enhancement of efficacy of recombinant myosin remained stand still. Therefore additional strategy was planned involving utilization of adjuvant properties of biocompatible lipid molecules i.e. liposomes. The fusogenic potential along with the ability to induce CMI response renders liposomes to be excellent delivery agent or adjuvant. These properties were exploited for improving efficacy of recombinant protein. Recombinant myosin was entrapped in egg PC liposome (PC+Myosin) and E. coli lipid liposome-escheriosome (EC+Myosin) to be utilized for vaccination in mastomys model. The antigen dose was reduced to 10 µg /animal instead of 25 µg/animal used in earlier experiment. The animals received two immunization doses before challenge and were euthanized on days 35 post L3 challenge to determine adult worm establishment. This implies that each animal received a total of 20 µg recombinant protein compared to 75 µg in earlier experiments, which was obvious due to use of liposomes. Liposomized myosin was able to increase protection efficiency and upto 70% reduction in adult worm establishment was observed in EC+myosin group. PC+myosin group also generated significant degree of protection which was though lower than the EC+myosin group. This experiment reconfirmed the potential of recombinant myosin as vaccine candidate along with the validation of approach for enhancement of its proficiency through improvement in mode of delivery.

Immunological characterization of recombinant myosin Immunization studies provided adequate evidence of vaccination potential of recombinant myosin, however array of immunological events generated by vaccination of this protein were still unrevealed. A study was therefore designed to determine the in vivo immunological scenario rendered by myosin vaccination, which would enable the elucidate the most probable mode of action of the protein that led to protection against B. malayi. Balb/c mice were selected as standard animal model largely due to its wide use and commercial availability of specific antibodies and reagents which is not in case of either mastomys or jird. Balb/c mice were immunized with recombinant myosin emulsified with all the adjuvants and liposomes in separate groups as per standard schedule. Each animal received 25 µg of recombinant myosin in the adjuvant groups as compared to liposomes vaccinated group which received 10 µg/animal. Appropriate age and sex matched control animals were included in the study for comparison. Blood was collected before vaccination and every week after booster dose for serum collection from all the groups (control and vaccinated) for determination of humoral response by ELISA. Serum was utilized for determination of total IgG, antibody isotypes and antibody dependent cellular adhesion and cytotoxicity. Animals of all the groups were euthanized on day 7 post last booster dose and their macrophages (PEC’s) and splenocytes were used for different immunological assays. PEC’s were utilized to determine nitric oxide (NO) release by Griess method and oxidative burst by DCF-DA dye on flow cytometer. Splenocytes were utilized for the determination of extracellular production of pro-inflammatory (IFNγ, TNF-α and IL-1β) and anti-inflammatory (IL4, IL6 and IL10) cytokine production by commercially available cytokine detection kit. Surface staining of splenocytes was done with different monoclonal antibodies against CD markers for the identification of B cells (CD19), T helper cell (CD4+), T cytotoxic cells (CD8+), natural killer cells (CD94), activated macrophages (CD80/86) and Th2 cell marker (CD30). CD4+ and CD8+ cells were further used for determination of their ability for intracellular production of proinflammatory (IL2, IFN-γ) and anti-inflammatory (IL4, IL10) cytokines. The cell phenotypes and intracellular cytokine production were determined by flow cytometry. The results of immunological assays on analysis presented a clear format of the mechanism generated with response to myosin vaccination which led to protection against B. malayi infection. Vaccination generated high level of humoral response which was evident by significantly high antibody (IgG) titer and B cell population (CD19) in myosin vaccinated group mice. IgG1, IgG2a and IgG2b were major isotypes produced indicating a mixed Th1 and Th2 response. Splenocytes from myosin vaccinated group of mice produced pro-inflammatory cytokines like IFN-γ and TNF-α as detected by extracellular cytokine kit. This is indicative of Th1 response generation by myosin vaccination. Myosin exerted its primary and immediate immunological action against invading L3 by generation of nitric oxide and reactive oxygen intermediates as evident by high production of both NO and ROI by macrophages from vaccinated group. Macrophages and NK cells appear to be also involved in L3 killing by ADCC as the percentage of specific CD markers for activated macrophages (CD80/CD86) and NK cells (CD94) were significantly higher in vaccinated group of mice. Secondary and long lasting immune response of myosin was exerted through T helper (CD4+) and T cytotoxic cells (CD8+) as both these cells were significantly higher in vaccinated group of animals with higher population of CD4+ cells. The helper cells are responsible for the production of proinflammatory cytokines (IL2 and IFN-γ) as determined by intracellular cytokine staining which was significantly higher in vaccinated group. The production of anti-inflammatory cytokines was not detected by either extracellular or intracellular methods which indicate absence of Th2 response due to myosin vaccination. The work embodied in the present thesis is a comprehensive attempt to determine vaccination efficacy and immunological characterization of recombinant B. malayi myosin in animal model of filariasis. Conclusively, the outcome of the present study indicates recombinant myosin to be a good candidate antigen for vaccine against B. malayi infection.