Evaluation of serologic and antigenic relationships between middle eastern respiratory syndrome coronavirus and other coronaviruses to develop vaccine platforms for the rapid response to emerging coronaviruses

Sudhakar Agnihothram, Robin Gopal, Boyd L Yount Jr, Eric F Donaldson, Vineet D Menachery, Rachel L Graham, Trevor D Scobey, Lisa E Gralinski, Mark R Denison, Maria Zambon, Ralph S Baric, Sudhakar Agnihothram, Robin Gopal, Boyd L Yount Jr, Eric F Donaldson, Vineet D Menachery, Rachel L Graham, Trevor D Scobey, Lisa E Gralinski, Mark R Denison, Maria Zambon, Ralph S Baric

Abstract

Background: Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012, causing severe acute respiratory disease and pneumonia, with 44% mortality among 136 cases to date. Design of vaccines to limit the virus spread or diagnostic tests to track newly emerging strains requires knowledge of antigenic and serologic relationships between MERS-CoV and other CoVs.

Methods: Using synthetic genomics and Venezuelan equine encephalitis virus replicons (VRPs) expressing spike and nucleocapsid proteins from MERS-CoV and other human and bat CoVs, we characterize the antigenic responses (using Western blot and enzyme-linked immunosorbent assay) and serologic responses (using neutralization assays) against 2 MERS-CoV isolates in comparison with those of other human and bat CoVs.

Results: Serologic and neutralization responses against the spike glycoprotein were primarily strain specific, with a very low level of cross-reactivity within or across subgroups. CoV N proteins within but not across subgroups share cross-reactive epitopes with MERS-CoV isolates. Our findings were validated using a convalescent-phase serum specimen from a patient infected with MERS-CoV (NA 01) and human antiserum against SARS-CoV, human CoV NL63, and human CoV OC43.

Conclusions: Vaccine design for emerging CoVs should involve chimeric spike protein containing neutralizing epitopes from multiple virus strains across subgroups to reduce immune pathology, and a diagnostic platform should include a panel of nucleocapsid and spike proteins from phylogenetically distinct CoVs.

Keywords: Diagnostics; MERS-CoV Vaccine Design; Serology; Synthetic Genomics.

Figures

Figure 1.
Figure 1.
Growth of Middle East respiratory syndrome coronavirus (MERS-CoV) Hu isolates at indicated multiplicity of infection (MOI) in Vero cells (A) and Calu-3 cells (B). Infected cultures were sampled in triplicates at times indicated, and viral titers (shown as plaque-forming units [PFU]/mL) were determined by plaque assay on Vero cells. Error bars indicate standard error of the mean. C, Northern blot analysis of RNA harvested 12 hours after infection from Vero cells infected with MERS-CoV Hu isolates at a MOI of 5. D, Western blots of lysates harvested 12 hours after infection from Calu-3 2B4 cells infected with MERS-CoV Hu isolates at a MOI of 5 that were probed with antisera to spike (S) and nucleocapsid (N) proteins. β-actin indicates loading control.
Figure 2.
Figure 2.
A, Serum from each of 4 young and aged mice immunized with Venezuelan equine encephalitis virus replicons (VRPs) expressing spike (S) or nucleocapsid (N) proteins were tested in a plaque reduction neutralization test to neutralize Middle East respiratory syndrome coronavirus (MERS-CoV) Hu isolates. Error bars indicate standard error of the mean. B and C, NA01 patient sera collected at indicated dates after hospitalization were analyzed in an enzyme-linked immunosorbent assay, using cell lysates expressing S and N antigens from VRPs. D, Indicated dilutions of NA01 patient sera collected on November 16, 2012 were screened with 1:800 dilutions of mouse antisera to S, N, bat CoV (BtCoV) HKU 5.5 N, or SARS-CoV S in an in vitro competition assay for binding to MERS-CoV or SARS-CoV. E, The full-length genome sequences of 51 CoVs were downloaded from GenBank or PATRIC, aligned with ClustalX, and phylogenetically compared by maximum likelihood estimation, using 100 bootstraps. The tree shows that CoVs are divided into 3 distinct phylogenetic groups, defined as α, β, and γ. This taxonomic nomenclature replaced the former group 1, 2, and 3 designation, respectively. Classical subgroup clusters are marked as 2a–2d for the β-CoVs and as 1a and 1b for the α-CoVs. The tree was generated using maximum likelihood estimation with the PhyML package. The scale bar represents nucleotide substitutions. Only nodes with bootstrap support of >70% are labeled. Accession numbers and definitions of various CoV strains will be provided upon request.
Figure 3.
Figure 3.
Western Blots showing cross-reactivity between nucleocapsid (N; A and B) and spike (S; C and D) proteins of Middle East respiratory syndrome coronavirus (MERS-CoV) Hu isolates and N and S proteins of bat CoV (BtCoV) HKU 4.2 and HKU 5.5 (E). Plaque reduction neutralization tests showing absence of cross-neutralization of MERS-CoV Hu isolates by antisera to BtCoV HKU 4.2 and 5.5 S proteins. Serum from groups of 4 mice immunized with Venezuelan equine encephalitis virus replicons was tested in this assay. Error bars indicate standard error of the mean. Note the cross-reactivity of antisera to BtCoV HKU 5.5 S protein to S proteins of MERS-CoV Hu isolates (D) but the absence of cross-neutralization.
Figure 4.
Figure 4.
Western Blots showing no cross-reactivity between nucleocapsid (N; A) and spike (S; B) proteins of Middle East respiratory syndrome coronavirus (MERS-CoV) Hu isolates and SARS-CoV. C, Plaque reduction neutralization tests showing the absence of cross-neutralization of MERS-CoV Hu isolates by antisera to SARS-CoV S and of SARS-CoV by antisera to MERS-CoV/SA-1/2012 S protein and BtCoV 279 S protein. Note that antisera to SARS-CoV S neutralize SARS-CoV. Serum from groups of 4 mice immunized with Venezuelan equine encephalitis virus replicons was tested in this assay and error bars indicate standard error of the mean. D, Enzyme-linked immunosorbent assay results showing the absence of reactivity of NA01 patient sera to SARS-CoV S antigen.
Figure 5.
Figure 5.
Western blots showing cross-reactivity between nucleocapsid (N; A and B) and spike (S; C and D) proteins of Middle East respiratory syndrome coronavirus (MERS-CoV) Hu isolates and N and S proteins of BtCoV 279 and HKU 3. E, Plaque reduction neutralization tests showing absence of cross-neutralization of MERS-CoV Hu isolates by antisera to BtCoV 279S and HKU 3 S proteins. Serum from groups of 4 mice immunized with Venezuelan equine encephalitis virus replicons was tested in this assay. Error bars indicate standard error of the mean.
Figure 6.
Figure 6.
A, NA01 patient serum specimens collected at indicated dates were analyzed in an enzyme-linked immunosorbent assay (ELISA), using cell lysates expressing indicated antigens. B, Mouse antisera to the indicated antigens were screened in an ELISA. C, Human antisera to indicated CoVs were screened in an ELISA with cell lysates expressing indicated antigens.
Figure 7.
Figure 7.
Western blots showing the cross-reactivity of spike (S) proteins of bat coronavirus (BtCoV) HKU 2.298 (A) and BtCoV 1A (B) with Middle East respiratory syndrome coronavirus (MERS-CoV) Hu isolates. C, Plaque reduction neutralization tests showing the absence of cross-neutralization of MERS-CoV Hu isolates by antisera. Serum from groups of 4 mice immunized with Venezuelan equine encephalitis virus replicons was tested in this assay. Error bars indicate standard error of the mean.

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