Two doses of SARS-CoV-2 vaccination induce more robust immune responses to emerging SARS-CoV-2 variants of concern than does natural infection.

Both natural infection with SARS-CoV-2 and immunization with vaccines induce protective immunity. However, the extent to which such immune responses protect against emerging variants is of increasing importance. Such variants of concern (VOC) include isolates of lineage B.1.1.7, rst identied in the UK, and B.1.351, rst identied in South Africa. Our data conrm that VOC, particularly those with substitutions at residues 484 and 417, escape neutralization by antibodies directed to the ACE2-binding Class 1 and the adjacent Class 2 epitopes but are susceptible to neutralization by the generally less potent antibodies directed to Class 3 and 4 epitopes on the anks of the receptor-binding domain. To address the potential threat posed by VOC, we sampled a SARS-CoV-2 uninfected UK cohort recently vaccinated with BNT162b2 (Pzer-BioNTech, two doses delivered 18-28 days apart), alongside a cohort sampled in the early convalescent stages after natural infection in the rst wave of the pandemic in Spring 2020. We tested antibody and T cell responses against a reference isolate of the original circulating lineage, B, and the impact of sequence variation in the B.1.1.7 and B.1.351 VOC. Neutralization of the VOC compared to B isolate was reduced, and this was most evident for the B.1.351 isolate. This reduction in antibody neutralization was less marked in post-boost vaccine-induced responses compared to naturally induced immune responses and could be largely explained by the potency of the homotypic antibody response. After a single vaccination, which induced only modestly neutralizing homotypic antibody titres, neutralization against the VOC was completely abrogated in the majority of vaccinees. Importantly, high magnitude T cell responses were generated after two vaccine doses, with the majority of the T cell response directed against epitopes that are conserved between the prototype isolate B and the VOC. These data indicate that VOC may evade protective neutralizing responses induced by prior infection, and to a lesser extent by immunization, particularly after a single vaccine dose, but the impact of the VOC on T cell responses appears less marked. The results emphasize with 2% Triton X-100 and stained for the nucleocapsid (N) antigen or spike (S) antigen using monoclonal (mAbs) After with peroxidase-conjugated and were using logistic (Hill in


Introduction
The emergence of new lineages of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on three continents towards the end of 2020, and their rapid expansion at the expense of the previously dominant lineages, poses signi cant challenges to public health (WHO | SARS-CoV-2 Variants) 1 . In order to address these challenges effectively, there is an urgent need to understand the biological consequences of the mutations found in these lineages, and the consequential impact on their susceptibility to current control measures, including vaccines, drugs and non-pharmaceutical interventions.
Three variants (B.1.1.7, B.1.351 and P1) have been termed variants of concern (VOC). All three variants share the N501Y substitution in the receptor-binding domain (RBD) of spike glycoprotein (S), which increases binding a nity of S with the virus's cellular receptor, angiotensin-converting enzyme 2 (ACE2) 2 (see Figure 1). As of 1 March 2021, N501Y is present globally in 77% of currently sequenced samples (data from GISAID -Initiative). Lineage B.1.1.7, rst identi ed in the UK in September 2020 (0F0F [1]), is characterized by additional mutations in S, such as deletion of residues 69 & 70 and the P681H substitution, for which plausible effects on the virus biology are proposed, as well as ve other mutations in S, a premature stop codon in ORF8, three substitutions and a deletion in ORF1 and two amino acid substitutions in nucleoprotein (N), of as-yet unknown signi cance. Lineage B.1.351 3 was rst identi ed in November 2020 in South Africa and is characterized by two additional substitutions of likely signi cance in RBD, namely, K417N and E484K. The former is predicted to disrupt a salt bridge with D30 of ACE2, a characteristic of SARS-CoV-2 in distinction to severe acute respiratory syndrome coronavirus (SARS-CoV-1), but may not impact on binding, whereas the latter, which might disrupt the interaction of RBD with K31 of human ACE2, may enhance ACE2 binding 24 . On 1 March 2021, this lineage accounted for 5% of all current sequences globally, and 100% of those identi ed in South Africa. The third variant of concern, P.1 (formerly B.1.1.28.1) is characterized by K417T, in addition to E484K and N501Y, and accounted for 80% of all viruses sequenced in Brazil on 1 March 2021. In early 2021, E484K had been detected rst in lineage B.1.1.7 in the United Kingdom (UK) 5 and subsequently in lineages A23.1, B.1 and B.1.177, as well as in imported cases of B.1.51 and P.21F1F [2] . Our data con rm that VOC, particularly those such as B.1.351 with substitutions at residues 484 and 417, escape neutralization by antibodies directed to the ACE2-binding Class 1 and the adjacent Class 2 epitopes, but are susceptible to neutralization by the generally less potent antibodies directed to Class 3 and 4 epitopes on the anks of the RBD The immune correlates of protection against infection and disease caused by SARS-CoV-2 are imperfectly understood (reviewed by 6,7 ). Classically, neutralization by antibody, measured by reduction in plaque or infectious foci by authentic virus in vitro is considered a major component of protection, though indirect effects of antibody, such as complement activation and opsonization may also play a role in vivo. Recent studies have demonstrated that symptomatic re-infection within six months after the rst wave in the UK was very rare in the presence of anti-S or anti-N IgG antibodies 8,9 . Virus-speci c lymphocytes may play an important direct role in protection, in addition to their indirect effect mediated through help to antibody-producing cells. Robust T cell immune responses (with CD4+ T cells dominating) to S, M, N and some ORF antigens are readily detected after infection, correlate with disease severity and are durable for at least several months [10][11][12] . Furthermore, CD8 depletion studies in nonhuman primate (NHP) challenge studies suggest T cells also play a protective role especially when antibody levels are low 13,14 15 . Nevertheless, passive infusion of neutralizing antibody has been shown to be su cient to mediate effective protection against SARS-CoV-2 in these NHP studies 14 . Although studies in NHPs of both adenovirus-26 and DNA-based vaccine candidates found that levels of neutralizing antibodies but not of T cells were signi cantly correlated with viral clearance 13,16 recent reports involving subunit vaccine candidates in NHP found not only neutralizing antibodies, but also Nspeci c CD4+ responses were a statistically signi cant correlate of protection 1713,16 , recent reports involving subunit vaccine candidates in NHP found not only neutralizing antibodies, but also N-speci c CD4+ responses were a statistically signi cant correlate of protection 17 .
Multiple vaccines have been reported to have e cacy against COVID-19 (coronavirus disease 2019) in phase III clinical trials. Of these, three -P zer/BNT162b2, Moderna/mRNA-1273 and Sputnik V -that were reported to have e cacies against symptomatic infection in the mid-90% range, had also induced classical neutralizing antibody titres substantially higher than those found on average in convalescent patients [18][19][20] . In contrast, one -CoronaVac -that showed approximately 50% e cacy, had been reported to induce neutralizing titres several-fold lower than those found in convalescent patients 21 25 and AZD1222 26 . Each of the studies report reduced e cacy in South African populations. Vaccine correlates of protection, and the relative contribution of T cell and humoral immunity, are yet to be precisely de ned since detailed immune analysis in people with vaccine breakthrough infections is lacking.
In pseudotype virus neutralization assays, it appears that convalescent sera from patients exposed to prototype strain of SARS-CoV-2, in distinction to vaccine-elicited responses, may not be effective in neutralizing lineage B.1.351 27,28 . As the lineage-de ning substitutions include changes in previously identi ed antibody epitopes and regions of S associated with its processing and rearrangement during cellular infection, this is a very plausible observation. In order to test whether convalescent sera and sera from vaccine recipients were similarly affected in their ability to neutralize authentic virions, we have undertaken classical neutralization assays against reference isolates of both B.1.1.7 and B.1.351 compared to the early pandemic B isolate. We nd that, while cross-neutralization of B.1.1.7 is only modestly reduced compared to that of the prototype B lineage, cross-neutralization of B.1.351 may be markedly reduced in convalescent sera, and after a single vaccine dose. However, both the neutralization of VOC and the generation of viral speci c T cells, is signi cantly enhanced by a boost vaccination. In addition, vaccination not only induces enhanced reactivity to S from endemic human coronaviruses, but also results in signi cant cross-reactivity to both SARS-CoV-1 and Middle East respiratory syndromerelated coronavirus (MERS-CoV).
Since viral mutations may also affect T cell recognition, we also evaluate the contribution of T cells that target epitopes located at sites of amino acid substitution in the spike glycoproteins of VOC. We show that the majority of T cell responses in recipients of two doses of the BNT162b2 vaccine are generated by epitopes that are invariant between the prototype B lineage virus and VOC.
Whilst the T cell data is encouraging, the loss of neutralizing antibody recognition against VOC suggest that reformulation of vaccines to address new variant lineages ought to be considered and indicates that seasonal re-vaccination might be required for this virus. HCWs not known to be previously infected with SARS-CoV-2, were recruited after vaccination with the COVID-19 mRNA Vaccine BNT162b2 (P zer). 11 participants were recruited post-prime (mean 29 days after a single dose, range 18-41). 25 participants were recruited postboost (mean 8 days after the second dose, range 7-17 days) and assessed again for T cell reactivity 28 days boost. An additional 13 unvaccinated, non-SARS-CoV-2 exposed HCW were recruited and assessed for T cell reactivity. Four unvaccinated participants were recruited under the Observational Biobanking study approvals SthObs (18/YH/0441) and assessed for neutralizing antibodies. Pre-pandemic negative control sera, used for binding assays, were obtained from a prior vaccine study of the National Vaccine Evaluation Consortium performed in 2017. Ethics approval from NHS Heath Research Authority -NRES  human IgG (clone G18-145, Cat. No. 555787, BD). The CD3 neg CD19 pos CD20 neg CD27 hi CD38hi IgG pos plasmablasts were gated as single cells.
Sorted single cells were used to produce human IgG mAbs, as previously described 33 . Brie y, the variable region genes from each single cell were ampli ed in a reverse transcriptase polymerase chain reaction (RT-PCR: QIAGEN, Germany) using a cocktail of sense primers speci c for the leader region and antisense primers to the Cγ constant region for heavy chains and Cκ and Cλ for light chains. The RT-PCR products were ampli ed in separate PCR for the individual heavy and light chain gene families using nested primers to incorporate unique restriction sites at the ends of the variable gene as previously described 33 . Monoclonal antibodies C121 (Class 2) and S309 (Class 3) were derived from the published sequences 34,35 by gene synthesis (GeneArt). These variable genes were then cloned into expression vectors for the heavy and light chains. Plasmids were transfected into the Expi293F cell line for expression of recombinant full-length human IgG mAbs in serum-free transfection medium. The mAbs were then a nity puri ed using a MabSelectSure column (Cytiva, USA) according to the manufacturer's protocol and buffer exchanged into 1xPBS using a 10k MWCO Amicon Ultracentrifugal Unit. and samples were diluted 1:10 -1:100 in diluent buffer. Importantly, an ACE2 calibration curve which consists of a monoclonal antibody with equivalent activity against spike variants was used to interpolate results as arbitrary units. Furthermore, internal controls and the WHO international standard were added to each plate. After 1-hour incubation recombinant human ACE2-SULFO-TAG™ was added to all wells.
After a further 1-hour plates were washed and MSD GOLD™ Read Buffer B was added, plates were then immediately read using a MESO® SECTOR S 600 Reader.
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 (P zer) 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 P zer vaccine showed a non-signi cant increase in binding to SARS-CoV-2 spike and RBD compared to those receiving single dose and a signi cant 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 signi cant 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 exempli ed 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 signi cant 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 signi cantly 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.

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. 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 signi cant deviations in heterotypic neutralization potency against a currently approved reference serum 20/130 (NIBSC, Figure 3B).  Figure 3D), whereas 2/25 individuals showed more modest titres (10 < NT50 < 100). These sera neutralized the B. 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  ( 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 signi cant 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 signi cant correlations were observed between humoral and T cell responses (see Supplementary table 3c).

Discussion
Our results show that both binding and neutralization by antibodies induced by the S protein of prototypic lineage B is diminished to S from recent VOC; B.1.351 to a greater extent than B.1.1.7. This broad trend masks both qualitative and quantitative differences in antibody responses by individuals, whose serum may contain differing proportions of antibodies to neutralizing epitopes that we show here are sometimes conserved between lineages.
Given the cost and di culty of authentic virus neutralization assays, it is encouraging that in our hands, both a high-throughput spike-binding assay and a spike-ACE2 binding-inhibition assay provide a signi cant correlation with the neutralizing potency -both homotypic and heterotypic -of human sera.
It is also reassuring to nd that the majority of T cell responses in recipients of two doses of the BNT162b2 vaccine are generated by epitopes that are invariant between the prototype and two of the Consistent with this, our data show that neutralization of sera and T cell activity are independent 16 .Moreover, in over 90% of the recipients of two vaccine doses, heterotypic neutralizing titres (NT50) remain comfortably above the level associated with immune protection in non-human primate challenge studies 13,16 . However, in a majority of individuals whose homotypic neutralization titres were more modest -including over 50% of convalescent COVID-19 individuals and recipients of a single dose of vaccine -heterotypic neutralization dropped to negligible levels. This loss of cross-neutralization was particularly notable against B.1.351 with potential implications for vaccine effectiveness in populations where this VOC dominates and when only moderate levels of S antibodies are generated after vaccination.
It should be noted that neutralization escape, observed in a well of a micro-titre plate, is not direct evidence of vaccine failure. Non-neutralizing antigen-speci c antibodies, T cells and innate lymphocytes clearly have the potential to contribute to vaccine e cacy 42 . The acceptance that prior infection with in uenza virus results in reduced disease against subsequent infection with heterosubtypic strains, in both human and animal challenge studies, provides further evidence that cellular components and nonneutralizing antibodies make an important contribution to protection [43][44][45] . We also note that the recent Nevertheless, our results re-emphasize the urgent need to deploy the most effective vaccine strategies as widely and rapidly as possible in order to provide population protection against the emerging lineages of concern of SARS-CoV-2. Our ndings show clearly that the weaker responses generated for example by natural infection or single doses of vaccine, do not provide adequate cross-neutralization. The results support the recommendations by P zer, the FDA and EMA for a two-dose vaccine regimen.    Cross-correlation of immune parameters following two vaccine doses. For each serum, pairwise Spearman correlation analyses were undertaken between the value of binding of serum antibody to coronavirus antigens, the ACE2-spike binding-inhibition potency (see Figure 2), and the homotypic and heterotypic neutralizing titre of the same sera (see Figure 3). A. Heatmap of Spearman's r parameter for each comparison in which spike binding data was available (n = 56). Colour mapping is dual gradient from Blue (r = 1.0) through White (r = 0.5) to Red (r = 0). Values outside this range are Black. B. Heatmap

Declarations
Spearman's r parameter for each comparison in which ACE2-spike binding-inhibition data were available (n = 35). Colour mapping as in A.