Development Of Recombinant Subunit Vaccines Against Bluetongue Virus Using Baculovirus And E.coli Expression Systems

Bluetongue?BT?is an insect-borne disease of livestock caused by bluetongue virus?BTV?,the prototype member of the genus Orbivirus within the family Reoviridae.The disease is classified as an emerging disease in several countries and can cause major loss of livestock in infected sheep herds.Infection of ruminants occurs throughout much of the temperate and tropical regions of the world,coincident with the distribution of specific species of biting midges belonging to the species Culicoides.The spread of BTV to previously disease-free regions which prohibit the use of the current BTV live-attenuated vaccine has highlighted the need for a new generation of vaccines.Subunit vaccines are one of the attractive alternative strategies.Subunit vaccines against BTV would target the outercapsid protein VP2,the main neutralization-specific antigen.The VP2 can be co-expressed with other capsid and core proteins to form a particle that resembles the intact BTV virus like particle?VLP?.In the current study we discussed in details the production and evaluation of protein-based subunit vaccines against BTV.The work has been dissected into three parts.?1?Expression of full-length BTV proteins in baculovirus expression systemIn this part of work,a baculovirus expression system was successfully utilized to express BTV-16?VP2,VP3,VP7,and VP5?,BTV-1?VP2 and VP5?,and BTV-8?VP2?proteins for later immunization of animals with the purified proteins aiming to produce an effective subunit vaccine against BTV.Baculovirus expression system offers a correct folding of the expressed proteins.The expression conditions?optimum expression time and temperature for protein binding to the Ni-NTA resins,pH,binding and elution buffers?were optimized for each individual protein.All proteins,except for VP5,were successfully expressed and the obtained yields of the purified proteins were sufficient for later use in the proposed subunit vaccine.?2?Prokaryotic expression of full-length BTV-16 VP3,VP5,VP7,NS2 and truncated VP5BTV-16 VP3,VP7,NS2 and VP5-41aa genes were amplified and used for cloning into pSMK,pET32a or p PROEX-HTb plasmids.The confirmed clones of pSMKBTV16VP3 and pSMKBTV16VP7were used to transform E.coli BL21-codon Plus?DE3?-RIL competent cells,while pET32aBTV16VP5-41aa and pPROEX-HTb BTV16NS2 were transformed into BL21?DE3?competent cells,and later induced with IPTG for expression.Expression conditions were optimized in order to get high expression.The lower temperature?16°C?for the expression of the His-sumo tagged proteins was found to enhance the solubility of the expressed proteins,while the His-tagged NS2 protein was expressed as a soluble protein at 37°C.In contrast,VP5-41aa could not yield a soluble protein therefore it has been purified under the hybrid condition that assumed to partially enhances for the refolding of the expressed protein.Furthermore,the SUMO-tagged proteins of VP3 and VP7 were assembled into core-like particles?CLP?in vitro after the cleavage of the His-sumo moiety.?3?Immunogenicity evaluation of protein-based subunit vaccines against bluetongue virus in BALB/c mice and sheepIn this study,the main objective was to develop a subunit vaccine candidate targeting BTV-16 as this strain has been previously isolated from clinically infected sheep in China.Other objective of the study was to examine the possibility of the vaccine candidate to be used for heterologous serotype protection.With this purpose,BTV-1VP2 and BTV-8VP2 were also expressed and used to replace BTV-16VP2.To characterize ovine and murine immune responses to the expressed proteins,both sheep and mice were subcutaneously immunized twice with one of five protein combinations in Montanide^TM ISA201 VG adjuvant or a placebo.?n=5/group?.Utilizing BTV-1 cELISA,significantly higher antibody titers were detected in the immunized animals than the controls.The vaccine induced a protein specific humoral response as well as lymphocyte proliferative responses upon re-stimulation of peripheral blood mononuclear cells?PBMC?from immunized sheep.Serum neutralization test against serotype BTV-1showed a promising results for cross serotypic protection of the vaccine against BTV-8 and BTV-16.The data suggest that the recombinant purified proteins could represent an important part of a novel vaccine against BTV.In conclusion,we began with eight immunologic BTV proteins that were produced in scalable expression systems.We followed systematic steps of protein identification and purification method optimizations.The purified proteins were utilized to ultimately develop an experimental vaccine candidate composed of the stable and purified recombinant BTV-16 proteins VP2,VP3,VP7,NS2 and VP5-41aa,in different combinations with Montanide ^TM ISA201 VG adjuvant.The replacement of BTV-16VP2 with either BTV-1VP2 or BTV-8VP2 was also discussed.Since the evaluated novel vaccine induced both humoral and cell-mediated immune responses and all vaccinated sera were able to neutralize BTV-1 in part,indicating a cross serotype responses induced by the vaccine,we believe that this rational recombinant subunit vaccine can be adapted to additional or multiple BTV serotypes depending on local epidemiology and the disease occurrence and will represent a defense line in case of BTV outbreak or emergence.