Middle East respiratory syndrome coronavirus spike protein delivered by modified vaccinia virus Ankara efficiently induces virus-neutralizing antibodies

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) has recently emerged as a causative agent of severe respiratory disease in humans. Here, we constructed recombinant modified vaccinia virus Ankara (MVA) expressing full-length MERS-CoV spike (S) protein (MVA-MERS-S). The genetic stability and growth characteristics of MVA-MERS-S make it a suitable candidate vaccine for clinical testing. Vaccinated mice produced high levels of serum antibodies neutralizing MERS-CoV. Thus, MVA-MERS-S may serve for further development of an emergency vaccine against MERS-CoV.

Fig 1

Generating and characterizing recombinant MVA. (A) Schematic diagram of the MVA genome and the locations of major deletion sites I to IV, with deletion III being the site used to insert the MERS-CoV S gene sequences. Flank-1 and flank-2 refer to MVA DNA sequences adjacent to deletion site III which were originally prepared by PCR and cloned into MVA transfer plasmids targeting deletion site III for insertion of recombinant genes. In MVA vector plasmids pIIIH5red-S and -S_HA, the S coding gene sequences (MERS-S/S_HA) are placed under transcriptional control of the vaccinia virus promoter PmH5 and introduced by homologous recombination between the flanking sequences in the vector and the MVA genome. MVA-MERS-S and MVA-MERS-S_HA were isolated in plaque passages by screening for transient coexpression of the fluorescent marker gene mCherry under transcriptional control of the vaccinia virus late promoter P11. Repetitive sequences (del) are designed to remove the mCherry marker by intragenomic homologous recombination (marker gene deletion). (B) Genetic integrity and genetic stability of MVA-MERS-S and MERS-S_HA. PCR analysis of genomic viral DNA using oligonucleotide primers to confirm the identity (MERS-S) and proper insertion (deletion III) of S gene sequences. (C) Multiple-step growth analysis of recombinant MVA-MERS-S. Recombinant MVA (MVA-S) and wild-type MVA (MVA) can be efficiently amplified in CEF (multiplicity of infection [MOI], 0.1) but fail to productively grow in HeLa and HaCat human cell lines.

Fig 2

Immunostaining of S proteins in recombinant MVA-infected cells. (A) Transient expression of the marker protein mCherry served to localize single virus-infected cells (left panel). Monoclonal antibody directed against the HA tag (anti-HA) (right panel) reveals the presence of S_HA in Vero cells infected (MOI, 0.1) with MVA-MERS-S_HA (MVA-S_HA). (B) Polyclonal antibodies from a MERS-CoV-infected cynomolgous macaque (anti-MERS-CoV) detected S-producing cell foci in CEF infected with MVA-MERS-S (MVA-S; MOI, 0.1) but no foci when CEF were infected with nonrecombinant MVA (MVA).

Fig 3

Synthesis of full-length S glycoprotein in recombinant MVA-infected cells. (A) Western blot analysis of cell lysates from MVA-MERS-S (MVA-S)- or MVA-MERS-S_HA (MVA-S_HA)-infected Vero cells 24 h postinfection. Polypeptides were analyzed by SDS-PAGE and immunoblotting using serum from a MERS-CoV-infected macaque (1:1,000; upper panel) or monoclonal rat anti-HA tag antibody (1:50; lower panel). Lysates from uninfected (Vero) or wild-type MVA-infected (MVA) cells served as controls. (B) Western blot analysis of MVA expressed S_HA following treatment with glycosidases. Vero cells were infected with MVA-MERS-S_HA for 24 h. Cell lysates were incubated with (+) or without (−) glycosidase PNGase F or endo H and analyzed by SDS-PAGE and immunoblotting with monoclonal rat anti-HA tag antibody (1:50). Numbers on the left indicate molecular masses of marker proteins in kilodaltons.

Fig 4

Antibody responses after intramuscular immunization with recombinant MVA-MERS-S. BALB/c mice (n = 10) were vaccinated twice within a 21-day interval with 10⁸ PFU MVA-MERS-S (MVA-S). Groups of mice (n = 4) vaccinated with wild-type MVA (MVA) or saline (PBS) served as controls. MERS-CoV virus-neutralizing titers (VNT) were determined 20 days after primary immunization (A) and 10 days after the second immunization (B and C) using Huh7 (A and B) and Vero cells (C). Sera obtained after the second boost were also tested against SARS-CoV on Vero cells (D). Serum from a rabbit (17) immunized with the MERS-CoV receptor binding domain (RBD) (E) or sera from mice obtained after the second MVA-S boost (F) were preincubated with PBS, the RBD, or a SARS-CoV-derived control protein (CTRL) at 5 μg/ml for 1 h before incubation with MERS-CoV, followed 1 h later by inoculation on Vero cells.