Spike protein sequence differences in SARS-CoV-2 lineages.
The primary structure of the spike glycoprotein (S), and the characteristic sequence variants of the current three lineages of concern are illustrated in Figure 1. In this study, we analysed the homotypic neutralization of the prototypic, PANGO lineage B isolate, VIC001 (hereafter referred to simply as “B”), by mAbs, sera from convalescent individuals following SARS-CoV-2 infection, and recipients of the BNT162b2 (Pfizer) vaccine, which are each induced by prototypic S antigen. We then assessed heterotypic neutralization of two new VOC (B.1.1.7 and B.1.351). In Figure 1, we indicate the residues of S at which the respective lineage – as well as a third lineage of concern, P.1 – differ from lineage B.
Binding of antibodies to coronavirus proteins, and inhibition of ACE2-spike binding
We probed the antibody-binding properties of sera from vaccinated, convalescent and pre-pandemic control sera using a customised MSD coronavirus antigen immunoassay (Figure 2). We observed that sera from individuals receiving two doses of the Pfizer vaccine showed a non-significant increase in binding to SARS-CoV-2 spike and RBD compared to those receiving single dose and a significant difference from sera of convalescent individuals one month after infection (Figures 2A, and 2B, respectively, p<0.0001 in all cases). The absence of N binding in vaccinees (Figure 2C) supports the designation of these individuals as SARS-CoV-2 unexposed, although it does not prove absence of previous infection.
There was significant antibody binding to both SARS-CoV-1 and MERS-CoV spike protein in vaccinated and COVID-19 convalescent individuals compared to the pre-pandemic control sera (Figures 2D & 2E, respectively). This was particularly marked for SARS-CoV-1 reactivity in fully vaccinated individuals, suggesting that the vaccine can induce a broad response to widely shared epitopes, such as those exemplified by EY 6A 32 and CR3022 36.
We also screened for antibody binding to the spike antigen of the four common circulating coronaviruses (Figure 2 F-I). There is a significant increase in binding to the Betacoronavirus clade A isolates, HCoV-HKU1 and HCoV-OC43, in vaccinated and COVID-19 convalescent sera (p<0.0001) compared to unvaccinated naïve sera. Binding to the Alphacoronavirus isolates, HCoV-229E and, to a lesser extent, HCoV-NL63S was also greater in the vaccinees, but not in convalescent sera.
As a surrogate to neutralization, we assessed the ability of sera to inhibit ACE2-spike binding using MSD plates printed with spike proteins representing the prior circulating B lineage, and the more recently evolved VOC (B.1, B1.1.7, B1.351 and P1). Figure 2J indicates that serum from vaccinated individuals receiving either single or double vaccination was able to inhibit ACE2 binding of SARS-CoV-2 spike. The inhibitory effect was significantly higher (>30-fold, p < 0.001 by Mann Whitney comparison) in those sera derived from individuals receiving the boost vaccination compared to prime. Furthermore, sera from boosted individuals had a >3-fold and 10-fold lower mean inhibitory activity for B.1.351 and B.1.1.7 respectively compared to the heterosubtypic B lineage spike. Following vaccine boost, the mean inhibitory activity of B differs significantly from B.1.351 and P.1 but not B.1.1.7 (Friedman test, p <0.0001).
Neutralization by mAbs to the four epitopes of RBD and by reference serum
We made use of a panel of six, epitope-mapped neutralizing monoclonal antibodies (NmAbs, Figure 3A,) 34,35,37,38 in order to map the neutralization sensitivity of VOC to changes in RBD epitopes. We have devised a “squirrel” diagram to help visualise the binding sites of the various mAbs on the RBD (Figure 3A). One NmAb, FI 3A, a Class 1 RBD monoclonal antibody (binds to the left side of the head of the squirrel), whose homotypic IC50 is of the order of 1 nM, is largely unaffected by the changes in B.1.1.7 (IC50 = 1.365 nM) but does not neutralize B.1.351. Two other NmAbs, GR 12C and C121, that are Class 2 RBD binding mAbs (binding to the right side of the head of the squirrel), and which have homotypic IC50 ~ 0.1 nM, show some reduced effectiveness in neutralizing B.1.1.7 and have lost almost all potency against B.1.351. This might be expected, as class 2 antibodies bind to an epitope that includes residue 484 (reviewed by Barnes et al and 39). In contrast, NmAb FD 11A and S309, which are Class 3 RBD mAbs, that bind to the right haunch of the squirrel, and EY 6A, Class 4 monoclonal antibody, that binds to the left haunch of the squirrel, appears to be unaffected by the mutations in the VOC.
Polyclonal responses generated by different individuals to natural infection or in response to vaccination may include a varying proportion of antibodies to these and other neutralization epitopes. We also noted significant deviations in heterotypic neutralization potency against a currently approved reference serum 20/130 (NIBSC, Figure 3B). While homotypic NT50 was 918.2 (95% CL 729.6 – 1,165), close to the result of 1,280 quoted on the 20/130 data sheet, neutralization of B.1.1.7 was decreased to 125 (86 – 164), and of B.1.351 to 14 (0.1 – 51).
Neutralization by sera from convalescent COVID-19 individuals
Sera from convalescent individuals neutralized prototype B virus with highly variable potency (NT50 range <5 to 1,140, Figures 3C and 3E), though sera from those with mild symptoms were significantly more potent on average than those with asymptomatic infection (NT50 438.4 and 38.5, respectively, P=0.002).
Neutralization titres against B.1.1.7 were below the limit of detection in 9/12 asymptomatic convalescent individuals but were detectable in all those with mild symptoms. The neutralizing potency of mild convalescent sera against B.1.1.7 was significantly greater than that of asymptomatic sera (NT50 133 and 9.3, respectively; Kolmogorov-Smirnov test, P = 0.0005).
The decline in neutralization potency was more marked against the B.1.351 isolate, with convalescent sera from 12/12 asymptomatic and 7/12 mild having undetectably low neutralizing potency. Although there was no significant difference between the mean NT50 of mild versus asymptomatic sera against B.1.351 (119 and <5 respectively, P = 0.25), the reduction in potency overall in relation to prototype B virus was very significant (P = 0.000003)
Neutralization by sera from vaccine recipients.
After a single dose of BNT162b2 vaccine, homotypic neutralization potency was on average comparable to that of an asymptomatically infected cohort (NT50 53.8 and 38.5, respectively, P=0.36), but lower than sera from those who had recovered from mild infection (NT50 438.3, P=.003; see Figures 3D and 3E). Neutralization after one dose was undetectable against B.1.1.7 in 7/11 samples, and in all 11 sera tested against B.1.351.
Sera drawn between 7 and 17 days after a second dose of BNT162b2 vaccine - administered 18 to 28 days after the first - neutralized lineage B virus with high potency (average NT50 = 768) and 23/25 individuals had NT50 >> 1/100, Figure 3D), whereas 2/25 individuals showed more modest titres (10 < NT50 < 100). These sera neutralized the B.1.1.7 isolate with a significantly lower potency (average NT50 = 320; p< 0.0001, Kolmogorov-Smirnov test); the same 23/25 had NT50 titres > 100 and 2/25 NT50 titres 10-100. The decline in neutralization potency against the B.1.351 isolate was further significantly reduced (NT50 = 171; P= 0.000001), but 12/25 retained NT50 titres>100, 11/25 NT50 10-100 with only the 2/25 with modest homotypic neutralization potency having undetectable heterotypic neutralizing potency.
The relationship of the neutralizing titre of each individual’s serum to B to the corresponding titre against each variant apparent in Figure 3D is significant. Spearman correlation coefficients (r) are: 0.76 (B to B.1.1.7, CL 0.52 – 0.98; P = 0.0000092); 0.74 (B to B.1.351, CL 0.48 – 0.88. P = 0.00002); and 0.79 (B.1.1.7 to B.1.351, CL 0.57 – 0.91, P = 0.000002).
T cell responses to spike antigens in prototypic B strain and VOC
Following two doses of BNT162b2, spike-specific T cells were detected in all individuals against spike antigens covering the prototypic B strain, assessed in IFN-γ ELISpot assays peaking 7 days after the second vaccine (mean magnitude 561, range 110-1717 SFC/106 PBMC) (Figure 4A and S4). Spike specific T cells could not be detected in unvaccinated SARS-CoV-2 unexposed HCW (S3). T cells Assessing the contribution of T cells that target epitopes located at the site of B.1.1.7, B.1.351 and P.1 spike mutation sites, we find that T cells target epitopes spanning mutation sites in 18/24 individuals (Figure 4B). In each individual, T cells targeted 0-19 (mean 6) epitopes located at mutation sites (Supplementary Table S2) with a total of 8, 9 and 10 epitopes targeted in lineage B.1.1.7, B.1.351 and P.1 respectively. The overall contribution of T cells targeting mutation regions to the total spike specific response is (mean and range) 13% (0-67%) for B.1.1.7, 14% (0-44%) for B.1. 351 and 10% (0-29%) for P1 (Figure 4C). Although the overall contribution of T cell responses to mutational regions/total spike responses was low, in general multiple individuals had T cells that targeted each of the mutational regions, spanning all spike domains (Figure 4D and Supplementary Table S2). T cell responses to total spike and mutation sites were further assessed in a small number of vaccinees after only a single vaccine; here low magnitude T cell responses were detected (Figure S5A), with T cells targeting mutational regions in 3/5 vaccinees (Figure S5B). Similar to post boost responses, the relative contribution of these to total spike was low (% mean contribution and range; 24% (2-34%) for B.1.1.7, 11% (0-20%) for B.1. 351 and 7% (0-23%) for P1) (Figure S5C).
Prediction of heterotypic neutralization by immunoassay
Authentic virus neutralization assays require specialist staff and facilities that are not widely available, and access to reference isolates of virus that are laborious to distribute. Accordingly, we asked whether high throughput ELISA-style immunoassays could provide a degree of predictive value for heterotypic neutralization following two vaccine doses. We performed Spearman non-parametric correlation analysis between the neutralization results, the spike-binding, and ACE2-spike binding-inhibition results obtained from the same sera, and the degree of T cell response to whole S protein determined by ELISPOT analysis from the same donors, as detailed in the foregoing sections.
The results (summary heatmap in Figures 5A and 5B, in table form in Supplementary Table 3) show that there is a consistently highly significant correlation (P<<0.0001) between both spike-binding and ACE2-spike binding-inhibition activity and authentic virus neutralization. For example, the Spearman r between neutralization by serum of lineage B virus and the binding activity to lineage B RBD is 0.68 (95% CI 0.5 to 0.8, n = 56, P = 1 e-10), and the r between neutralization of lineage B.1.351 and binding to B RBD is 0.71 (0.5 to 0.8, n=56, P = 8 e-10). The correlation between neutralization and ACE2-spike binding-inhibition is, if anything, slightly stronger, with r = 0.71 (0.5 to 0.9, n = 35, P = 4 e-6) for lineage B, and r = 0.79 (0.6 to 0.9, n = 35, P = 2 e-8) for lineage B.1.351. (NB in this assay, the spike sequences correspond to the virus lineage in the neutralization assay.)
Interestingly, binding activity to SARS-CoV-2 S predicted binding to both SARS-CoV-1 S and MERS-CoV S very well (r = 0.92 (0.86 to 0.95), n = 56, P = 2 e-23; and r = 0.55 (0.3 to 0.7), n=56, P = 9 e-6). Moderate correlations (r of the order of 0.5) were seen with binding to the spike of endemic human betacoronaviruses and to the spike of alphacoronavirus OC43, but not to that of alphacoronavirus HCoV-HKU1. No significant correlations were observed between humoral and T cell responses (see Supplementary table 3c).