Construction And Evaluation Of Marburg Virus Vaccine Based On Recombinant Adenoviral Vector

Marburg virus(MARV)is the earlist found filovirus,could infect human and nonhuman primates leading to acute hamorrahgic fever with extra high transmissibility and lethality.The need to prepare a vaccine for MARV is urgent for its potential as bioweapon.MARV glycoprotein is the principal viral component and plays a critical role in infecting cells,which was validated as protective antigen for MARV.Here,we developed a MARV vaccine based on adenovirus serotype 5 and evaluated its immunogenicity on mouse and guinea pig models.We chose the most virulent MARV Angola strain GP antigen and synthesized its genome with and without codon-optimization,then cloned them into shuttle plasmid pDC316 respectively.The recombinant plasmids were co-transfected into HEK293cells with the backbone plasmid pBHGlox-E1,3Cre.After site-specific recombination,the construct expressing MARV-GP vectored by adenovirus was packaged.The immunological evaluation in animals was carried out after preparation of the vaccine candidate.Cellular immune response is the primary part in protecting organism against pathogens and cytotoxin T lymphocyte(CTL)epitope is the basis to evaluate it.We pr edicted several H-2~d-specific T cell epitopes in MARV Angola strain suited for BALB/c mice in several websites.Sevaral CTL epitopes from prediction were synthesized and validated using enzyme-linked immunospot assays(ELISPOT)and intracellular cytokine staining(ICS).Peptide LI-9 could stimulate splenocytes of immunized mice to produce abundant IFN-?and could be used for further evaluation of the vaccine candidate.BALB/c mice and guinea pigs were used to evaluate the candidate MARV vaccine's immuno responses.The antibody titer could maintain quite high level over a year in the immunized mice and over 20 weeks in the inoculated guinea pigs;The candidate MARV vaccine also could induce quick and strong cellular response in immunized mice through ELISPOT and ICS assays.Meanwhile,we found that intramuscular injection could induce mice immunized by Ad5-MAGPopt to produce abundant cytokines and high-level GP-specific antibodies,however,the humoral response was weaker than that stimulated by intranasal route;besides producing massive IgG,a considerable amout of Ig A could be detected though intranasal administration which indicates a mucosal protection.Moreover,we failed to detect any cellular responses in intranasal immunized mice.In addition,cross-reaction studies between the EBOV vaccine and the MARV vaccine showed that the MARV vaccine Ad5-MAGPopt prepared in this study was capable of producing a certain level of anti-EBOV antibody.Protection assay conducted by National laboratory of Canada.shows that low-dose(1.0×10~6 ifu)Ad5-MAGPopt can completely protect mice from MA-MARV challenge,providing 100%protection.Above all,the recombinant adenovirus-vectored MARV vaccine is able to induce rapid robust and durable humoral and cellular immune response in mice and guinea pigs,thus is a potential effective MARV vaccine candidate.