Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Jul 28:6:7712.
doi: 10.1038/ncomms8712.

Evaluation of candidate vaccine approaches for MERS-CoV

Lingshu Wang  1 , Wei Shi  1 , M Gordon Joyce  1 , Kayvon Modjarrad  2 , Yi Zhang  1 , Kwanyee Leung  1 , Christopher R Lees  1 , Tongqing Zhou  1 , Hadi M Yassine  1 , Masaru Kanekiyo  1 , Zhi-yong Yang  3 , Xuejun Chen  1 , Michelle M Becker  4 , Megan Freeman  4 , Leatrice Vogel  5 , Joshua C Johnson  6 , Gene Olinger  6 , John P Todd  1 , Ulas Bagci  7 , Jeffrey Solomon  8 , Daniel J Mollura  8 , Lisa Hensley  6 , Peter Jahrling  6 , Mark R Denison  9 , Srinivas S Rao  1 , Kanta Subbarao  5 , Peter D Kwong  1 , John R Mascola  1 , Wing-Pui Kong  1 , Barney S Graham  1
Affiliations

Evaluation of candidate vaccine approaches for MERS-CoV

Lingshu Wang et al. Nat Commun. .

Abstract

The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) as a cause of severe respiratory disease highlights the need for effective approaches to CoV vaccine development. Efforts focused solely on the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein may not optimize neutralizing antibody (NAb) responses. Here we show that immunogens based on full-length S DNA and S1 subunit protein elicit robust serum-neutralizing activity against several MERS-CoV strains in mice and non-human primates. Serological analysis and isolation of murine monoclonal antibodies revealed that immunization elicits NAbs to RBD and, non-RBD portions of S1 and S2 subunit. Multiple neutralization mechanisms were demonstrated by solving the atomic structure of a NAb-RBD complex, through sequencing of neutralization escape viruses and by constructing MERS-CoV S variants for serological assays. Immunization of rhesus macaques confers protection against MERS-CoV-induced radiographic pneumonia, as assessed using computerized tomography, supporting this strategy as a promising approach for MERS-CoV vaccine development.

PubMed Disclaimer

Conflict of interest statement

L.W., W.S., M.G.J., K.M., P.D.K., J.R.M., W.P.K. and B.S.G. are inventors on a U.S. patent application describing the use of Spike glycoprotein as a vaccine antigen and monoclonal antibodies D12, F11, G2, G4 as potential therapeutics and aids in vaccine design.

Figures

Figure 1
Figure 1. MERS-CoV Spike glycoprotein vaccine design and immunogenicity in mice.
Candidate vaccine immunogens were designed on the basis of the Spike glycoprotein sequence of the England1 MERS coronavirus and elicited high neutralization titres. (a) Schematic representation of MERS-CoV Spike protein cDNAs and recombinant proteins. Five vaccine constructs were made: three DNA and two protein subunits. DNA constructs consisted of full-length Spike or truncated versions that either had the transmembrane domain or the entire S2 subunit deleted. The protein constructs contain either a truncated Spike molecule with the transmembrane domain deleted (S-ΔTM) or the S1 subunit. RBD, receptor-binding domain; SP, signal peptide; TM, transmembrane domain; FTH, foldon (trimerization domain), thrombin (cleavage site) followed by histidine tag; 3CHis, Human rhinovirus 3C protease cleavage site, followed by 6 × histidine tag. (b) Immunogenicity of eight vaccine regimens. Five mice per group were immunized with plasmid DNA only at weeks 0, 3 and 6 (groups 1–3); plasmid DNA at weeks 0 and 3 and protein plus Ribi adjuvant at week 6 (groups 4–6); or protein plus Ribi adjuvant at weeks 0 and 4 (groups 7 and 8). Two weeks after each immunization, sera were collected and neutralizing antibody titres were measured against pseudotyped MERS-CoV England1 virus. Open, grey and black bars, respectively, represent the IC90 neutralization titres (GMT from five mice per group with 95% confidence interval) from the post-prime, first post-boost and second post-boost sera. Each sample was tested in triplicate; all assays were repeated once. A nonparametric two-tailed t-test (Mann–Whitney) was used for statistical analysis, and the relevant P values are indicated. (c) MERS-CoV vaccines induced cross-neutralization to eight MERS-CoV strains. The sera from the mice immunized with MERS-CoV S DNA three times, primed with S DNA and boosted with S1 protein plus Ribi adjuvant, or primed and boosted with S1 protein plus Ribi adjuvant were assayed for neutralization to the eight strains of MERS-CoV and SARS-CoV pseudotyped viruses as indicated. IC90 titre is shown. Data are presented as the mean of triplicates with s.e.’s.
Figure 2
Figure 2. Varied vaccination regimens elicit neutralizing antibodies that target different regions of the MERS-CoV Spike glycoprotein.
Immunization with different vaccine regimens elicited neutralizing antibodies that target the Spike glycoprotein within and outside the RBD. (a) Cell adsorption assay. Sera from mice immunized with MERS-CoV S DNA-only, S DNA prime and S1 protein plus Ribi adjuvant, or S1 protein plus Ribi adjuvant prime and boost were evaluated for neutralization activity against pseudotyped MERS-CoV England1 after adsorption with HEK 293T cell-surface-expressed MERS-CoV Spike proteins: S, RBD, S1, S2. Serum neutralization was tested at a single dilution. Sera adsorbed with untransfected HEK 293T cells served as controls and retained 95% of neutralization activity. Each bar represents the mean of triplicate assays with s.e.’s. The experiment was repeated once to ensure reproducible results; one of the two experiments is shown. (b) Protein competition neutralization assay. Sera at a single dilution from the immunized mice were also assayed for neutralization of MERS-CoV England1 pseudovirus in the presence of soluble MERS-CoV RBD, S1 and S2 proteins at concentrations of 0.016–50 μg ml−1. Each data point represents the mean of triplicate assays with s.e.’s. The experiment was repeated once to ensure reproducible results; one of the two experiments is shown.
Figure 3
Figure 3. Isolated mAbs exhibit high binding affinity against multiple Spike glycoprotein (Spike) domains.
Four mAbs were characterized for their binding specificity. Each of the mAbs was tested, using ELISA, in triplicate, for binding to soluble RBD, S1 and S2 conjugated to Fc (S2–hFc) respectively.
Figure 4
Figure 4. MERS-CoV-neutralizing mAbs target multiple regions of the RBD.
Vaccine-induced mAb D12 binds to the DPP4-interacting region of the viral Spike RBD, blocking receptor binding. (a) (Left) Comparison of RBD binding to D12 and DPP4. RBD (cyan) with receptor-binding motif (residues 484–567, magenta) and D12 are shown in ribbon representation. The D12 heavy chain is light blue and the light chain is light green. The heavy chain complementarity determining regions (CDR) are light blue (CDR H1), blue (CDR H2) and dark blue (CDR H3), while the light chain CDRs are cyan (CDR L2), green (CDR L2) and pale yellow (CDR L3). The main interacting regions are in CDR H2, CDR H3 and CDR L2. (Right) DPP4 is shown in ribbon representation (green) with Asn 229 and the attached N-glycan (yellow) shown as sticks. The RBD is oriented as shown in the left panel. (b) Antibody:RBD and DPP4:RBD crystal structure complexes. RBD in surface representation is shown with the D12 heavy and light chain binding region coloured blue and green, respectively. The CDR loops are shown as ribbons and coloured as in (a; left). The rotated RBD shows the full D12 paratope: D12 CDR H2 interacts with RBD W535 and E536 residues, which predominantly interact with the Asn 229-associated N-glycan on DPP4 (centre). RBD, shown in surface representation with the DPP4-interacting region coloured green. Major interacting regions of DPP4 are shown as ribbon representations with Asn 229 and N-glycan shown as sticks (Supplementary Fig S11; right). (c) D12 and RBD interface. All CDRs are shown in ribbon representation, with interacting residues shown as sticks, and hydrogen bonds represented by dotted lines. (d,e) Crystal structure of MERS England1 RBD and effect of critical RBD mutations on binding. RBD residues 506 and 509, identified by mutagenesis analysis, are highlighted in green. Critical RBD residues 532, 535 and 536, identified by structural definition and viral resistance evolution, reduce or eliminate D12 binding (shown in red). ELISA results show that this set of mutations eliminate F11 or D12 binding.
Figure 5
Figure 5. MERS-CoV Spike immunogens elicit potent, long-lived neutralization in NHPs and protect from severe lung infiltrates.
Selected immunogens from mouse studies were evaluated in NHPs. (a) Schematic representation of full-length MERS-CoV Spike protein cDNA and recombinant S1 protein. The DNA construct consists of full-length Spike and transmembrane domain. The protein construct contains a truncated Spike with the S1 subunit. RBD, receptor-binding domain; SP, signal peptide; TM, transmembrane domain; 3CHis, Human rhinovirus 3C protease cleavage site, followed by 6 × histidine tag. (b) Immunogenicity of three vaccine regimens. Six NHPs, per group, were immunized intramuscularly using plasmid DNA with electroporation at weeks 0, 4 and 8; plasmid DNA with electroporation at weeks 0 and 4; and protein plus AlPO4 at week 8 or protein plus AlPO4 at weeks 0 and 8. Two weeks after immunization and at weeks 12 and 18, neutralizing antibody titres were measured against pseudotyped MERS-CoV England1. Different symbols indicate sera from six NHPs per group collected at indicated time points. IC90 neutralization titres (GMT with 95% confidence interval) from sera were determined. Nonparametric two-tailed t-test (Mann–Whitney) was used for statistical analysis. (c) Spike immunogens protect against pulmonary disease in NHPs. Six unimmunized NHPs and 12 NHPs were immunized with one of two selected candidate vaccine immunogens ((1) full-length S DNA prime/S1 subunit protein boost; (2) S1 subunit protein prime/S1 subunit protein boost) and challenged with MERS-CoV 19 weeks after last vaccine boost. Intratracheal inoculation of 5 × 106 p.f.u. of the JordanN3 strain (GenBank ID: KC776174.1) was performed on each animal. The per cent abnormal lung volume in all NHPs peaked on day 3 post challenge; however, the lung infiltrates were significantly more extensive and prolonged in the unvaccinated compared with vaccinated NHPs. A nonparametric two-tailed t-test (Mann–Whitney) was used. *P value <0.05; ** P value<0.01. (d) Abnormal lung segmental images from selected animals on day 6 post challenge. The images correspond to NHP lung volume data points circled in black in Fig. 5c. The CT images and abnormal lung segments for all 18 animals are shown in Supplementary Fig. 14 a–c.
See this image and copyright information in PMC

References

    1. ECDC. Epidemiological Update: Middle East Respiratory Syndrome Coronavirus (MERS-CoV) European Centre for Disease Prevention and Control (2014).
    1. WHO. Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Summary and Literature Update–as of 11 June 2014 World Health Organization (2014).
    1. Assiri A, et al. Epidemiological, demographic, and clinical characteristics of 47 cases of Middle East respiratory syndrome coronavirus disease from Saudi Arabia: a descriptive study. Lancet Infect. Dis. 2013;13:752–761. doi: 10.1016/S1473-3099(13)70204-4. - DOI - PMC - PubMed
    1. Breban R, Riou J, Fontanet A. Interhuman transmissibility of Middle East respiratory syndrome coronavirus: estimation of pandemic risk. Lancet. 2013;382:694–699. doi: 10.1016/S0140-6736(13)61492-0. - DOI - PMC - PubMed
    1. Cauchemez S, et al. Middle East respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility. Lancet Infect. Dis. 2014;14:50–56. doi: 10.1016/S1473-3099(13)70304-9. - DOI - PMC - PubMed

Publication types