Sequence Homology Between Pandemic, Endemic, and Seasonal Coronaviruses
To evaluate the potential for cross-reactivity, we first compared the spike protein sequence homology among SARS-CoV-2, MERS-CoV, SARS-CoV, HCoV-OC43, and HCoV-HKU1 (Fig. 1, Supplementary Fig. 1). The greatest homology was between SARS-CoV-2 and SARS-CoV (76% identity, 87% similarity), followed by MERS-CoV (42% identity, 58% similarity) and lastly HCoV-OC43/HCoV-HKU1 (OC43 30% identity, 41% similarity; HKU1 29% identity, 40% similarity). Embecoviruses HCoV-OC43 and HCoV-HKU1 are more similar to each other (64% identity, 75% similarity) than to the two endemic betacoronaviruses. There is a larger fraction of homology towards the C-terminus of the protein in all coronavirus spike proteins, which represents the major structural regions of the protein including the heptad repeat regions responsible for insertion of the fusion peptide into the host cell membrane. Homology is significantly lower in the N-terminal regions of spike, with significant lack of similarity in the regions including the receptor-binding domain, correlating with the difference in receptors and determinants used for host cell entry in the different betacoronaviruses (MERS-CoV: receptor dipeptidyl peptidase-4 (DPP4), SARS-CoV/SAR-CoV-2: ACE2, HCoV-OC43/HCoV-HKU1: the sugar N-acetylneuraminic acid) .
Serologic Reactivity of Anti-Spike IgG, IgM, and IgA Antibodies
Functional cross-reactivity was determined through the use of enzyme-linked immunosorbent assays (ELISAs) measuring IgG, IgM, and IgA subclasses, representing mature, early stage, and mucosal specific serologic responses, respectively. We produced recombinant soluble spike proteins of SARS-CoV-2, MERS-CoV, SARS-CoV, HCoV-OC43, and HCoV-HKU1 using the Expi293 expression system, which yielded pure, intact ectodomain trimers suitable for ELISA . Notably, the yields of all coronavirus spike proteins were significantly different even though four of five were cloned in identical vectors and contained the same modifications to the wild-type sequences (elimination of furin cleavage site, prefusion-stabilizing proline mutations (2P), similar C-terminal tags), none of which was expected to alter serologic recognition due to their internal locations. The HCoV-OC43 construct has all of these features but the wild-type furin cleavage site is present. Using similar expression conditions, SARS-CoV-2 spike was produced at a maximum of 2 mg/L culture, while the other spike proteins were significantly easier to produce with yields of 5, 11, 8, and 6 mg/L respectively for SARS-CoV, MERS-CoV, HCoV-OC43, and HCoV-HKU1.
We next utilized a semi-automated ELISA protocol to detect serum antibodies from pre-2019 archival samples and samples from a community with high SARS-CoV-2 prevalence during the 2020 pandemic (Fig. 2). In serum samples collected from healthy volunteers prior to 2019, there was minimal reactivity with SARS-CoV-2, MERS-CoV and SARS-CoV. The majority of tested samples (n = 114) displayed high IgG reactivity with HCoV-OC43 and HCoV-HKU1 spike proteins, consistent with the extensive spread of seasonal betacoronavirus infections within the USA (Fig. 2a–c). As reported previously, we detected a high proportion of donors who seroconverted and were SARS-CoV-2 IgG+ in a community in New York City, along with a significant number of IgM and IgA seropositive donors, including several donors who were non-symptomatic, of these 68 donors, 62 were seropositive, and 22 had a previous known PCR+ diagnosis (Fig. 2c) . All samples had low levels of IgM reactivity against MERS-CoV, SARS-CoV, HCoV-OC43, and HCoV-HKU1 (Fig. 2d–f). IgA antibodies were present at higher levels than IgM, but still well below levels of IgG, correlating well with biologic prevalence of antibody classes in response to pathogens (Fig. 2g–i).
Minimal Linear Correlation of SARS-CoV-2 Signal Intensity with Other Betacoronaviruses
When comparing the assay absorbance signal (optical density, OD) between SARS-CoV-2 and the other spike proteins in the high-incidence population, we saw a stronger correlation of signal intensity between SARS-CoV-2 and SARS-CoV IgG (Correlation = 0.711, R2 = 0.505) and the lowest correlation with HCoV-HKU1 (Correlation = 0.281, R2 = 0.079) (Fig. 3a, Supplementary Fig. 2). Though there was not a precise linear correlation for IgG, donors who represented signal intensity in the lower 50% of SARS-CoV-2 absorbance readings did have a significantly lower MERS-CoV and SARS-CoV signal intensity when compared to the upper 50% of SARS-CoV-2 intensity (Fig. 4). Overall, these data suggest some cross-reactivity occurs that is more easily detectable at high titers of antibody.
Cross-Reactivity of SARS-CoV-2 IgG Antibodies with Endemic and Seasonal Coronaviruses
Since we observed a difference in the IgG signal intensity of other betacoronaviruses with high levels of SARS-CoV-2 antibodies, we further analyzed the relationship between SARS-CoV-2 seroprevalence and antibody titer with SARS-CoV, MERS-CoV, HCoV-OC43, and HCoV-HKU1 in pre-pandemic (pre-2019), high-prevalence symptomatic donors, and high-prevalence asymptomatic donors (Fig. 5, Supplementary Fig. 3). Overall, archival pre-2019 samples displayed an equivalent low signal intensity of SARS-CoV-2, MERS-CoV, and SARS-CoV spike reactivity (Fig. 5a). One donor from this group with high reactivity for SARS-CoV-2 was negative for both MERS-CoV and SARS-CoV. As previously discussed, the majority of donors were HCoV-OC43 and HCoV-HKU1 seropositive due to the broad circulation of these viruses in humans. In the high SARS-CoV-2 incidence community, for both symptomatic and asymptomatic individuals, there appeared to be a correlation in SARS-CoV-2 signal intensity with MERS-CoV and SARS-CoV. To further analyze this, we directly compared the signal intensity of archival sample controls to the high-incidence pandemic population (Fig. 5b). There was a significant difference in signal intensity of MERS-CoV (p < 0.0001), SARS-CoV (p < 0.0001), HCoV-OC43 (p = 0.0123), and HCoV-HKU1 (p < 0.0001), suggesting potential cross-reactivity of SARS-CoV-2 IgG antibodies with all four spike proteins.