2018 Enhanced protection in mice induced by immunization with inactivated whole viruses compare to spike protein of midd

Deng et al. Emerging Microbes 1234567890():,; protein mediates coronavirus entry into host cells by fi rst binding to a receptor on the host-cell surface via its receptor-binding domain (RBD)12. Although both the S and N proteins can induce T-cell responses, neutralizing antibodies are almost solely directed against the S protein, which is the major immunodominant factor. Thus, cur- rent MERS-CoV vaccine candidates primarily use the S protein or (parts of) the gene coding for this glycopro- tein4, 5. Vaccines against MERS-CoV infection have been developed using purifi ed coronavirus S protein, as well as DNA or viral vector-based vaccines expressing the full- length MERS-CoV S protein or part of the S protein1318. These vaccines have been tested for their ability to induce virus-neutralizing antibodies in mice or large animals, such as monkeys or camels7, 17. Several MERS vaccines have been developed among vaccine platforms but have been shown to confer variable degrees of immunogenicity, which necessitates the adjustment of the dose, adjuvant, and site of administration to induce optimal protective responses4, 5, 19. Furthermore, ongoing efforts to develop MERS-CoV vaccines should consider their immunity profi les against different antigens and correlates of protection. An ideal MERS vaccine should induce a potent neu- tralizing antibody response without inducing harmful immune effects, such as virus-enhanced antibodies or immunopathology. Several previous reports relative to inactivated SARS-CoV or MERS-CoV vaccines have led to safety concerns in humans2026, which are reminiscent of those reported in mice given a formalin-inactivated, whole-virus respiratory syncytial virus (RSV) vaccine and challenged with infectious RSV27, 28. However, pre- clinical evaluations of a subunit or inactivated whole-virus vaccine and Th1-type adjuvant for SARS-CoV have shown induction of serum neutralizing antibodies and protection against infection in mice challenged with an infectious virus21. Therefore, an appropriate adjuvant or even an adjuvant combination is required for an effective and safe vaccine formulation. CpG oligodeoxynucleotides (namely, CpG), which are shortsyntheticDNAsequencesconsistingof unmethylated CG dinucleotides, are currently being developed as vaccine adjuvants that promote Th1-type immune responses27. Our previous data demonstrated the advantages of combination of two adjuvants, CpG and alum, for the induction of both Th1 and Th2 immunity in mice15, 16, 29, 30. The current study determined the effects of a inactivated whole MERS-CoV(IV) or S protein vaccine with a combined (alum+CpG) adjuvant on pro- tection against MERS-CoV and the risk of lung immu- nopathology in mice. Furthermore, vaccination with a IV formulationcontainingotherstructuralproteins (N, M, and E) than the S protein enhanced protection against MERS-CoV, as well as led to reduced viral antigenexpressionandpathologicaldamageand almost no virus isolation from the lungs of mice post- challenge. Results S protein and IV formulations induced similar levels of the anti-S IgG response Immunogens of the IV and S proteins were fi rst char- acterized by Western blotting using anti-S (Fig. 1a) and anti-N monoclonal (Fig. 1b) antibodies produced by our laboratory31, 32. The S protein migrates as three poly- peptides that are specifi cally recognized by antibodies in Fig. 1 Vaccine candidates and immunization schedule. Western blot analyses of Middle East respiratory syndrome coronavirus (MERS-CoV) S and inactivated whole MERS-CoV(IV) vaccines using mouse anti-S (a) and anti-NP monoclonal antibodies (mAbs) (b). Schematic of the study (c) Deng et al. Emerging Microbes 110-kDa band, which represents S glycoprotein cleavage into an amino- terminal domain (S1); and 210-kDa band, which might represent a dimer of the S1 protein8, 18, 33. IV migrates as two polypeptides, and the upper band represents an N- glycosylated full-length S protein and the lower band represents S18, 18, 33. In addition, IV was loaded onto SDSPAGE gels and characterized by Western blotting using anti-N mouse monoclonal antibodies that were produced by our laboratory (Fig. 1b). The results showed a 46-kDa band, which was the predicted molecular mass of IV in a previous report32. To assess the immunity induced by vaccines developed from the S protein and IV and a combined adjuvant, adult female BALB/c mice (six per group) were given three i.m. immunizations at 4-week intervals of combined adjuvant alone or a formulation with either the S protein or IV at a dosage of 1g of S protein (Fig. 1c). After the fi rst priming (at 2 weeks), a robust S protein-specifi c immunoglobulin response was detected in both the S and IV vaccine groups; the titers in mice immunized with IV were sig- nifi cantly higher than the titers in those immunized with the S antigen (Fig. 2a). After the second immunization, S the protein-specifi c IgG titers were 105at 6 weeks and did not signifi cantly differ