Dr. Y. NARASIMHA REDDYJ. SHIVA JYOTHI2019-12-092019-12-092019-09-09http://krishikosh.egranth.ac.in/handle/1/5810137161D-650Bluetongue (BT) disease, caused by bluetongue virus (BTV) is an acute hemorrhagic fever of domesticated and wild ruminants. India is endemic to BTV, and twenty-nine distinct serotypes have been identified worldwide so far. Although live attenuated and inactivated vaccines are being developed, and used in response to endemic serotypes or a particular outbreak, they are not without their share of drawbacks. Insufficient inactivation or attenuation of the virus in the development of the vaccine can cause possible teratological effects, severe clinical disease with outbreaks, and emergence of novel virus strains due to gene reassortment with wild type viruses. Shortcomings as mentioned above warrant rigorous quality control, which is tricky in developing countries like India. Moreover, the threat posed by the range of different BTV serotypes has highlighted the need for more widely cross-protective BTV vaccines. Hence the current study is proposed with the central objectives to develop a recombinant subunit protein based vaccine that is cross-protective against all the known serotypes of BTV. Data sets of complete sequences for segments 2 (encoding VP2), segment 6 (encoding VP5) and segment 5 (encoding NS1) of the BTV genome were compiled from GenBank. In the Seg-2 alignment, a number of gaps due to insertions and deletions among the different serotypes were observed where as VP5, NS1 nucleotide and protein sequences were rather conserved. The amino acid sequence alignment showed that, although VP2 is very variable, several regions are relatively more conserved between serotypes. According to the present study analysis, most conserved region of VP2 is evident from 338-383 aa. Interestingly, 3D homology modeling of VP2 protein of different serotypes of BTV revealed different structures whereas conserved VP2 (cVP2) region of those serotypes revealed similar protein structure. Being rather conserved, VP5 of different BTV serotypes revealed similar protein structure on homology modelling. To better understand the evolutionary dynamics, selection pressures acting on individual codons of VP2 and VP5 was next determined. The global estimate of dN/dS was 0.16 for VP2 and 0.058 for VP5 indicating strong purifying selection. Moreover, high number of negatively selected codons was identified in both (850/988 for VP2 and 506/528 for VP5). Even more interesting was the observation that all the codons in the putative conserved region of VP2 (cVP2) are under strong negative selection. Two major histocompatability complex (MHC) class I, two MHC class II binding, and three B cell epitopes were predicted with significant percentile ranks suggesting a strong immunogenicity potential of cVP2. Primers were designed for the amplification of sequences of BTV-16 (that was isolated from 2016 field outbreaks of BT in Andhra Pradesh) cVP2, and codon optimized sequence of VP5 and NS1. Following amplification of the cVP2 by RT-PCR the amplified 265 bp cVP2 and codon optimized 1610 bp VP5, 1671 bp NS1 were subjected to restriction enzyme digestion before cloning into pRSET-B (cVP2) and pET-28b (+) for VP5 and NS1 vectors, respectively. Recombinant plasmids (cVP2, VP5 and NS1) were then transformed into the E. coli BL21 (DE3) expression hosts. Protein expression was isopropyl β-D-1-thiogalactopyranoside (IPTG) dependent for cVP2 and autoinduction was used for VP5 and NS1 recombinant proteins. The expressed proteins were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. All the three recombinant proteins were found to be in inclusion bodies. Hence, optimized conditions were used for the purification of the recombinant proteins cVP2, VP5 and NS1 by Ni-NTA column. The purity of the eluted His-Tag fusion protein was checked by SDS-PAGE that revealed a band with molecular weights of expected size of 9.35 KDa for cVP2, 59.8 KDa for VP5 and 61.2 KDa for NS1. Purified proteins were then desalted by dialysis against desired buffer and were quantified by Bradford’s method of protein estimation with BSA as standard. Immunization studies were carried with 36 BALB/c female mice of 4 weeks age. Mice were divided into 6 groups (G1 to G6) of 6 mice in each group. Immunization was done subcutaneously in the back of neck on day 0, day14 and day 28 with recombinant proteins cVP2, VP5 and NS1. Group 1 received 25 μg of cVP2, Group 2 received 25 μg of VP5 and Group 3 received 25 μg of NS1, Group 4 received a mixture of all the three recombinant proteins at a concentration of 25 μg each. Group 5 received the commercial pentavalent vaccine (Raksha Blu, Indian Immunologicals). All the above groups also received equal volume of Montanide as adjuvant. The control group (Group 6) received only Montanide. Blood samples were obtained on day 41 by cardiac puncture, serum was separated and stored at -80oC till use. Detection of protein-specific antibodies was done by standardizing the indirect-enzyme linked immuno sorbent assay (I-ELISA) by checker-board titration method. Results showed that the animals developed antibodies pertaining to the given recombinant proteins (protein-specific antibodies). Furthermore, the ability of these antibodies to neutralize the BTV serotypes was checked by serum neutralization test. Neutralization assay was carried out with eleven available BTV serotypes (BTV-1, 2, 4, 5, 9, 10, 12, 16, 21, 23, and 24). These serotypes represent 8 of the 10 BTV nucleotype groups. Varying neutralizing titres were observed for different BTV serotypes against different groups. Recombinant proteins elicited higher neutralizing antibody (Nab) titres as compared to inactivated vaccine group, except for neutralization of BTV-1 where the vaccine group elicited higher Nab titres. Interestingly, the cocktail of recombinant proteins did not elicit higher Nab titres than individual protein alone as was expected; Nab titre in the mixed group was either similar to or less than the titre of individual protein groups. BTV-12 could only be neutralized by serum raised against inactivated vaccine. Moreover, BTV-5 and 24 could not be neutralized by any of the groups tested. Consistent absence of neutralization with all the serotypes was seen with the control group serum. NS1 recombinant protein developed for use in subunit vaccine in the current study was also found to be a tool in the detection of BTV antibodies from field samples by I-ELISA. Since, the developed subunit vaccine was shown to induce specific immune response to the administered proteins and that additionally could neutralize several BTV serotypes, it is tempting to consider that the conserved recombinant proteins of cVP2, VP5 and NS1 are of significance as recombinant vaccine. Additional investigations in the determinants of cellular immune response of these proteins and immunogenicity studies in the natural host will shed more light into the immune response generated by these vaccines to be used as successful recombinant broad-spectrum vaccines.ennullThesis