Development Of Spike Based Subunit And Chimeric Virus Like Particle Vaccines Against Middle East Respiratory Syndrome Cornonavirus (MERS-CoV) Infection

In June of 2012 a novel human coronavirus, later named Middle East respiratory syndrome coronavirus (MERS-CoV), was isolated from a sixty-year old man died from acute respiratory failure and renal failure. Now MERS-CoV is considered a threat to global public health. As of April 26 2016, the World Health Organization (WHO) reported 1728 confirmed cases of MERS-CoV infection, including 624 deaths (a case fatality rate of 36.1%). Now, studies show that camels are a likely primary source of the MERS-CoV that is infecting humans. But the route of transmission between camels and people which is the key point to stop transmission of the MERS-CoV, is far from clearly understood. The continued threat of MERS-CoV necessitates the development of an effective vaccine.MERS-CoV is an enveloped, single-stranded, positive-sense RNA virus. The spike-surface glycoprotein (S), the small-envelope (E) protein, the matrix (M) protein, and the nucleocapsid (N) protein construct the structural proteins of MERS-CoV. The S protein is cleavaged into the N-terminal receptor-binding S1 and C-terminal membrane fusion S2 subunits. The S1 subunit consists of a C domain which contains the receptor binding domain (RBD), and an N domain named N terminal domain (NTD). The RBD of MERS-CoV enriches neutralizing epitopes. So lots of vaccines against MERS-CoV infection target the recombinant RBD protein. However, highly purified proteins are typically not inherently immunogenic and need adjuvants to stimulate the innate immune system.In view of these, our study firstly evaluated the immunogenicity of RBD-based subunit vaccine using different adjuvant formulations and delivery routes in mice. Different vaccination regimens were compared in BALB/c mice immunized intramuscularly with a vaccine containing recombinant RBD protein in combination with either aluminium hydroxide (alum) alone, alum and polyriboinosinic acid (poly I:C), alum and cysteine-phosphate-guanine (CpG). The immune responses of mice vaccinated with RBD, incomplete Freund's adjuvant (IFA) and CpG by a subcutaneous route were also investigated. The results showed that the combination of alum and CpG by intramuscular injection optimized the development of RBD-specific humoral and cellular immunity following subunit vaccination.Secondly, we evaluated the immunological responses of the recombinant RBD protein vaccine in a rhesus macaques challenge model of MERS-CoV. The results indicated that the recombinant RBD vaccination induced robust and sustained IgG and neutralization antibodies in the rhesus macaque model, as well as significant T-cell response was observed among the 200?g of recombinant RBD vaccination group. In addition, the recombinant RBD vaccination alleviated the respiratory disease and decreased the viral loads in the lungs and oropharyngeal swabs after MERS-CoV infection. The vaccination also lightened the impairment of the tissues in the rhesus macaque model.Thirdly, we exploited the recombinant NTD protein vaccine in a mice model against MERS-CoV infection. After three times of immunization, the recombinant NTD protein vaccine induced powerful humoral and cellular immunity response in BALB/c mice. Then, the recombinant NTD protein vaccine was evaluated in a MERS-CoV challenge model of mouse prior transfection with an adenoviral vector expressing human Dipeptidyl peptidase-4 (DPP4). The results showed the recombinant NTD protein vaccine decreased the impairment of lung species in immunized mice and partially protected the mice against the MERS-CoV challenge.Finally, we acquired chimeric virus like particles (VLPs) of MERS-CoV consisting of influenza virus H5N1 matrix (M1) protein core and S protein of MERS-CoV on the surface by a baculorvirus expression vector system. We immunized mice with the VLPs vaccine adjuvated with alum and CpG by intramuscular injection. The results showd the chimeric VLPs induced high titers of S specific IgG and neutralization antibodies in mice after the third immunization. But the VLPs failed in inducing effective cellular immune responses. The protection effects of the chimeric vaccine need further study in a suitable animal model such as mice or non human primates.Above all, the study evaluated recombinant protein vaccine such as RBD, NTD and chimeric VLPs of MERS-CoV in mice or rhesus macaque model. The results showed that all of these three kinds of vaccine could induce effective immune responses in immunized animal. Among them, some kinds of vaccine could partially protect animal from MERS-CoV challenge. Effective vaccine against MERS-CoV infection needs further study.