Homotypic and Heterotypic neutralization of key SARS-CoV-2 lineages by antibody.
The primary structure of the Spike glycoprotein, 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 prototype strain, Victoria/01/2020 (PANGO lineage A), by monoclonal antibodies (mAbs), sera from convalescent individuals following COVID-19, and recipients of the BNT162b2 (Pfizer) vaccine, which are each induced by prototypic S antigen, and heterotypic neutralization by these same antibodies of two new lineages of concern (B1.1.7 and B1.351). 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 prototype.
Neutralization by sera from convalescent COVID-19 individuals
Sera from convalescent individuals neutralized prototype virus with highly variable potency (see Figure 2a). The neutralization potency against Lentivirus (SARS-CoV-2 S) pseudotypes broadly – but imperfectly – corresponded with homotypic neutralization of authentic virus. Sera neutralized the B1.1.7 isolate with a lower potency (2-fold; 95% CL: 1.5 – 3-fold), and those with the lowest homotypic neutralizing potency had undetectable heterotypic potency. The decline in heterotypic neutralization potency against the B1.351 isolate was more marked with 5/9 having undetectably low neutralizing potency.
Neutralization by sera from vaccine recipients.
After a single dose of BNT162b2 vaccine, 10/11 sera showed homotypic neutralization potency equivalent to that of the more modest half of convalescent sera (NT50 ~ 1/100, see Figure 2b). Heterotypic neutralization was undetectable in all 11 sera against B1.351.
Sera from 23/25 individuals drawn between 7 and 17 days after two doses of BNT162b2 vaccine administered 18 to 28 days apart neutralized prototype virus with high potency (NT50 >> 1/100, Figure 2c), whereas 2/25 individuals showed more modest titres (1/10 < NT50 < 1/100). Sera neutralized the B1.1.7 isolate with a lower potency (2-fold; 95% CL: 1.5 – 3-fold), but in no case was neutralization undetectable. The decline in neutralization potency against the B1.351 isolate was somewhat greater on average but only the 2/25 with modest homotypic neutralization potency had undetectable heterotypic neutralizing potency.
The data from convalescent individuals and those having received vaccine are summarized in Fig2D, showing the trend to lower neutralization potency across the three genotypes of virus, which is least marked in those receiving two doses of vaccine. The relationship of the neutralizing titre of each individual’s serum to B VIC01 to their neutralizing titre against each VOC is displayed in Fig 2E and shows clear pairwise correlation. Correlation coefficients are: 0.95 (B VIC01 to B.1.1.7 HMPP1); 0.81 (B VIC01 to B1.351 HV001); and 0.80 (B1.1.7 HMPP1 to B1.351 HV001).
Neutralization by monoclonal, neutralizing mAbs to the four epitopes of RBD and by reference serum
In order to understand better the reasons for differences between the homotypic and heterotypic neutralization potency of polyclonal human sera seen above, we made use of a panel of six, epitope-mapped neutralizing monoclonal antibodies (NmAbs, Figure 2F, and Fig S1) 25–28. We have devised a “squirrel” diagram to help visualise the binding sites of the various mAbs on the RBD (Figure 2d). One NmAb, FI 3A, a Class 1 RBD mAb (binds to the left side of the head of the squirrel) whose homotypic NT50 is of the order of 1 nM, is largely unaffected by the changes in B1.1.7 HMPP1 (NT50 = 1.365) but does not neutralize B1.351 HV001. Two other NmAbs, GR 12C and C121 that are Class 2 RBD binding mAb (that bind to the right side of the head of the squirrel), and which have homotypic NT50 ~ 0.1 nM, show some reduced effectiveness in neutralizing B1.1.7 HMPP1 and have lost all potency against B1.351 HW001. This might be expected, as class 2 antibodies bind to an epitope that includes residue 484 (reviewed by Barnes et al and 29). In contrast, NmAb FD 11A and S309, which are Class 3 RBD mAb, that bind to the right haunch of the squirrel, and EY 6A, Class 4 mAb, that binds to the left haunch of the squirrel, appears to be unaffected by the mutations in the VOCs.
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, Fig 2G). While homotypic NT50 was 1/918.2 (95% CL 1/729.6 – 1/1,165), close to the result of 1:1280 quoted on the 20/130 data sheet, neutralization of B1.1.7 HMPP1 was enhanced by approximately 10-fold, but of B1.351 HV01 was reduced by >10-fold.
Binding of antibodies to Alpha and Beta Coronavirus proteins
We probed the antibody-binding properties of sera from vaccinated, convalescent and control sera using a customised MSD coronavirus antigen array ELISA (Figure 3). We observed that sera from individuals receiving two doses of the Pfizer vaccine showed significantly higher binding to both SARS-CoV-2 spike and RBD compared to those receiving single dose and to convalescent individuals one month after infection (Fig 3 A, and B, respectively). The absence of antibody binding to N (Fig 3C) confirms that all vaccinated individuals are naïve for SARS-CoV-2 infection.
There was significant antibody binding to both SARS-CoV-1 and MERS spike protein in vaccinated and COVID-19 convalescent individuals compared to the negative control sera (Fig 3D & E, respectively). This was particularly marked for SARS-CoV-1 reactivity in fully vaccinated individuals, suggesting that vaccine can induce a broad response to widely shared epitopes, such as those exemplified by EY 6A (see above) and CR302230.
We also screened for antibody binding to the spike antigen of the four common coronaviruses circulating in the UK (Fig 3 F-I). There is a significant increase in binding to all four, particularly to the Betacoronavirus clade A isolates, HKU1 and OC43, in vaccinated and COVID-19 convalescent sera (p<0.0001). Binding to the Alphacoronavirus isolates, 229E and, to a lesser extent, NL63S was also greater in the post-boost vaccinees, but not in convalescent sera.
T cell responses to Spike antigens in prototype strain Victoria and VOCs
Following two doses of BNT162b2, spike-specific T cells were detected in all individuals (mean magnitude 561, range 110-1717 SFC/106 PBMC) against spike antigens covering the Victoria strain, assessed in IFN-γ ELISpot assays (Fig 4A). Assessing the contribution of T cells that target epitopes located at the site of B1.1.7, B1.351 and P.1 specific mutation sites we find that T cells targeted epitopes spanning all spike mutation sites (Fig 4B) (8, 9 and 10 epitopes in lineage B1.1.7, B1.351 and P.1 respectively). In each individual, T cells targeted 0-19, mean 6) epitopes located at mutation sites. The overall contribution of these, to the total spike specific response (mean magnitude and range) is 13% (0-67%), 14% (0-44%) and 10% (0-29%) respectively (Fig 4C).
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. Using the Mesoscale discovery (MSD) assay, we investigated the relationship between the binding activity of serum to homotypic SARS-CoV-2 S and its heterotypic neutralization potency against both SARS-CoV-2 VOCs, and its ability to bind to other pandemic and endemic human coronaviruses. The correlation coefficients are summarized and the individual data are shown in Figure 5. Unsurprisingly, binding to SARS-CoV-2 S correlates very strongly with binding to SARS-CoV-2 S in convalescent sera, though surprisingly less well in post-boost vaccinees, who, reassuringly, showed no reactivity to SARS-CoV-2 N. S binding in vaccinees predicted homotypic and heterotypic neutralization moderately (0.5 >Spearman r > 0.4, *). S binding offered no predictive value for either homotypic or heterotypic neutralization in convalescent sera. Interestingly, binding activity in post-vaccine sera to SARS-CoV-2 R predicted binding to MERS-CoV S very well (r = 0.69, ***) but not to SARS-CoV-1 S. While SARS-CoV-2 S binding activity in vaccinee sera predicted binding to the S of one endemic beta coronavirus (OC43 S r = 0.57, **; HKU1 S, r = 0.44, *) it did not predict binding to the other two endemic coronaviruses.
Conversely, RBD binding predicted neutralization of VIC01 very well (Spearman r = 0.57, **), but neutralization of B1.1.7 less well (r=0.48, *) and of B1.351 not at all (r=0.35, ns), and showed an inverted relationship in binding to other epidemic coronavirus S proteins (SARS-CoV-1 S, r = 0.67, ***; MERS-CoV S, r = 0.52, *; data not shown).