Construction And Application Of Virus-like Particles For Middle East Respiratory Syndrome

Middle East respiratory syndrome coronavirus (MERS-CoV) was isolated and identified in 2012 as a novel and fatal human coronavirus. MERS-CoV is the pathogen of Middle East respiratory syndrome, a respiratory disease with symptoms including fever, cough and acute respiratory distress and with a mortality rate of over 35%. Since its emergence it has spread to over 27 countries. Primary human cases have been reported in a number of Middle Eastern countries, but travel-associated cases have been reported from countries in Europe, Africa, and Asia. With the globalization of world economies, the risk of natural infection or imported cases in China is increasing. Licensed vaccines or therapeutics anti-MERS are still lacking, which constitutes a roadblock in controlling disease outbreaks and spread. While there has been only one report of MERS-CoV infection in China-an imported case in Guangzhou in 2015 from Korea-China has a large population density and, to prevent possible viral spread, as happened with SARS-CoV, the development of safe and immunogenic vaccine candidates and specific treatments as strategic stocks are urgently needed.Since handling MERS-CoV presents biosafety risks, the virus must be handled in a BSL-3 laboratory by trained personnel. This poses a barrier to research and large scale production of traditional inactivated or live-attenuated vaccines. Due to the risk of residual virulence in inactivated and attenuated vaccines, exploration of safe and effective immunogens has important practical applications. Virus-like particles (VLPs) are constructed from one or several viral structural proteins without incorporation of genetic material, and therefore VLPs are non-infectious. VLPs can display immunogenic epitopes at a high density, making them highly effective vehicles for vaccine development.A recombinant baculovirus co-expressing the genes of the MERS-CoV spike (S), membrane (M) and envelope (E) proteins has been constructed for generation of MERS-CoV VLPs and characterized by genomic PCR and indirect immuninofluorescence. Grown in army worm Sf9 cells, the recombinant has been purified on a discontinuous sucrose gradient for further studies. Immunoelectron microscopy with gold particles showed that protein S had been incorporated into the VLPs. Western blots, and bands corresponding to viral proteins S, M and E were observed in the MERS-CoV VLPs; i.e., indicating successful assembly. We then confirmed the structural integrity of VLPs based on those results.The immunogenicity of MERS-CoV VLPs as a vaccine candidate was evaluated in BALB/c mice and rhesus macaques. Titers of serum IgG were determined by ELISA, with endpoints reaching 1:384 and 1:1,067, in mice and monkeys respectively. In these nimals inoculation with MERS-CoV VLPs resulted in production of virus-neutralizing antibody titers of up to 1:208 and 1:33 respectively. MERS-CoV VLPs also elicited T-helper 1 cell (Thl)-mediated immunity in macaques and T-helper 2 cell (Th2)-mediated immunity in mice, as measured by ELISpot. Our results showed that MERS-CoV VLPs are immunogenic, able to elicit robust levels of specific humoral and cell-mediated immunity in vaccinated mice and monkeys. These data demonstrate that MERS-CoV VLPs have good immunogenicity and represent a promising vaccine candidate.Passive immunotherapy with anti-serum against viruses and bacteria has been found an effective and economic approach to the prevention and treatment of infectious disease. Healthy horses received multi-point injections of purified MERS-CoV VLPs to produce hyperimmune serum. The protective efficacy of these equine antibodies was assessed by neutralization assay and further assessed in vivo in a transduced mouse model using an adenovirus vector expressing hDPP4. Equine antibody in titers of 1:20,900 cleared the virus from the lungs of challenged mice. Our results indicate that the pre-and postexposure efficacy of such hyperimmune serum preparations can provide technical and material reserves for the control of MERS.An advantage of VLPs is that antigens can be incorporated into their well-characterized structure and foreign epitopes may be presented at the surface. To explore the possibility of utilizing the MERS-CoV receptor-binding domain (RBD) in a subunit vaccine, canine parvovirus VP2 genes were fused with RBD genes and insert into the pFastBacl vector to generate a recombinant baculovirus. The structure of this recombinant was confirmed by genomic PCR and indirect immuninofluorescence. It was grown in Sf9 cells and purified using saturated ammonium sulfate. Empty particles similar to parvoviruses were observed by electron microscopy. Immunoelectron microscopy and western blot analyses of the chimeric VLPs detected specific signals, suggesting that the RBD had been assembled alonf with CPV-VP2 in the chimeric structures.The immunogenicity of the parvovirus-like VLPs candidate was evaluated as a potential MERS-CoV vaccine in BALB/c mice. Titers of serum IgG were determined by ELISA, with endpoints reaching 1:416. Mice inoculated with sVLPs induced virus-neutralizing antibody titers up to 1:72. In our research, mice immunized with the chimeric VLPs induced higher levels of cytokines such as IFN-y, IL-2 and IL-4 than untreated controls. These results indicate that injection of the chimeric VLPs induced specific humoral and cellular immunity in BALB/c mice, showing that presentation of MERS-CoV RBD on the surface of VLPs provides a safe and effective strategy for vaccine design.This study was focused on research into the development of MERS-CoV vaccines and antibodies. Two vaccine candidates were generated, and their stuctural features and immune activity against MERS-CoV were analysed. The production and testing of horse hyperimmune serum has provided a means for technical storage of reagents for the control of MERS and other zoonoses.