Broad SARS-CoV-2 spike-specific CD4+ T-cell response preserves recognition of variants of concern

doi:10.21203/rs.3.rs-1501801/v1

CD4 + T-cells are essential for protection against viruses including SARS-CoV-2. Mutations in SARS-CoV-2 variants of concern (VOC) can enhance infectivity and reduce antibody recognition. CD4 + T-cell sensitivity to mutations is less well understood because few epitopes have been mapped. Characterising > 100 SARS-CoV-2-specific CD4 + T-cell clones from convalescent healthcare workers, we mapped and HLAII restricted 21 epitopes across three viral proteins. Responses to the same spike epitopes were also present after vaccination of uninfected individuals. Lack of CD4 + T-cell cross-reactivity with endemic beta-coronaviruses suggests these responses arose from naïve T-cells rather than pre-existing cross-reactive coronavirus-specific T-cell responses. 10/17 spike epitopes were mutated in VOCs and CD4 + T-cell recognition of 7 was impaired, including 3 of 4 epitopes mutated in Omicron. Broad CD4 + T-cell targeting of epitopes likely limits evasion by current VOCs. However, continued genomic surveillance is vital to identify new emerging mutations able to evade CD4 + T-cell immunity.

Coordinated adaptive immunity is essential for protection and clearance of viral infections including severe acute respiratory syndrome coronavirus-2 (SARS-COV-2)¹. Virus-specific neutralising antibodies are considered the main correlate of protection against SARS-CoV-2 infection but wane over time^{2, 3}. T-cell responses are more durable^{2, 4, 5} and increasing evidence supports their role in restricting SARS-CoV-2 infection and limiting the severity of COVID-19^{6, 7}. Worldwide efforts have rapidly delivered SARS-CoV-2 vaccines, mostly designed against the spike (S) protein that mediates host cell entry. Studies enumerating T-cell responses to whole SARS-CoV-2 S protein using pepmixes (pools of overlapping peptides covering the entire protein sequence) show that memory T-cell responses to S in previously infected or vaccinated individuals are dominated by CD4 + T-cells^{4, 8–10}; essential mediators of functional anti-viral memory CD8 + T-cells and antibody responses¹¹.

Several SARS-CoV-2 CD4 + T-cell epitopes have been identified, but mostly in assays that use high concentrations of peptides, and HLA restrictions have largely been inferred based on in silico HLA binding algorithms^{12, 13}. As such, detailed knowledge of the specificity of CD4 + T-cell responses at the epitope level is currently lacking. Furthermore, whether the epitopes are naturally generated via the MHC-II antigen processing pathway, the identity of the HLA-restricting alleles and whether epitopes can be presented by closely related subtypes, are largely unknown¹⁴. Crucially, this has limited investigation of two key questions at the epitope level. Firstly, the extent to which SARS-CoV-2-specific CD4 + T-cells cross-react with other human coronaviruses (HCoVs). In some unexposed individuals, low frequency CD4 + T-cell reactivity to SARS-CoV-2 pepmixes has been observed^15–19, which can expand following infection or vaccination^{19, 20}. This is presumed to represent cross-reactive T-cells primed by prior exposure to other HCoVs (HKU1, OC43, NL63, 229E, SARS or MERS), however the clinical relevance of such responses during SARS-CoV-2 infection is yet unclear⁶. Cross-reactivity of in vitro expanded S-specific CD4 + T-cell cultures has been reported^{15, 16, 19, 21} but understanding of whether this phenomenon reflects broad cross-recognition across S or reactivity against particularly conserved regions requires detailed knowledge of the epitopes targeted by CD4 + T-cells.

Secondly, the extent to which T-cells induced by ancestral SARS-CoV-2 proteins can protect against variants of concern (VOCs) is a critical question. In particular, the highly transmissible Omicron VOC contains several mutations within the receptor binding domain (RBD) of S, the main target of neutralising antibodies^{22, 23}. Accordingly, these reduce neutralisation by S-specific antibodies induced by ancestral SARS-CoV-2 variants or vaccines designed against the original S protein reference sequence^24–26. This includes most currently approved vaccines including adenovirus vector-based ChAdOx-1 S (Astrazeneca) and Ad26.COV2.S (Janssen), and mRNA-based BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna). Whilst neutralising antibody titres can be partially restored by booster vaccination^{27, 28}, Omicron has caused a high prevalence of secondary and vaccine breakthrough infections²⁹. Ex vivo studies of previously infected or vaccinated individuals using pepmixes have shown minimal reductions in the overall frequency of CD4 + T-cell responses against the Alpha (B.1.1.7), Beta (B1.351), Gamma (P1) and Delta (B.1.717.2)^{26, 30–32} or Omicron (B.1.1.529) VOCs^{25, 33, 34}. However, biologically relevant differences in epitope-specific recognition efficiency may have been missed³⁵. Little information exists at the epitope level to understand the extent of CD4 + T-cell epitopes evaded by current VOCs or to predict CD4 + T-cell epitope loss in future SARS-CoV-2 variants.

Here, we performed a detailed analysis of CD4 + T-cell immunity against SARS-CoV-2. Examining over 100 CD4 + T-cell clones, we identified and characterised 21 HLA class II (HLAII)-restricted T-cell epitopes. Responses to most epitopes located in the S protein were also present in vaccinated individuals of appropriate HLAII genotype. Cross-reactivity was only observed against the closely related S2 region of the SARS virus with no cross-reactive responses observed for any other b-HCoV. Mutations were present in 10 of the 17 S epitopes within one or more SARS-CoV-2 VOC. Minor amino acid (aa) changes in 7 epitope sequences, including those within the RBD region of Omicron, were sufficient to reduce or evade recognition by S-specific CD4 + T-cells. However, the breadth of responses to multiple CD4 + T-cell epitopes seen in each individual suggests that current VOC mutations confer only limited evasion from CD4 + T-cell surveillance.

Breadth of SARS-CoV-2 specific CD4 + T-cell immunity following natural infection

Peripheral blood mononuclear cells (PBMCs) were collected from 20 healthcare workers post infection (HCW-PI) in June-September 2020 following infection during the first wave of SARS-CoV-2 WT (D614G) infection in the UK. All individuals had detectable antibodies to S and nucleoprotein (N) 3–6 months post infection (Supplementary Fig. 1). To characterise T-cell immunity against whole antigens, PBMCs and CD8-depleted PBMCs (in which CD4 + T-cells comprise an increased percentage) were tested against SARS-CoV-2 pepmixes in ex vivo interferon-gamma (IFNg) Elispot assays. As expected, responses to a control pepmix of HLA class I (HLAI)-restricted epitopes decreased significantly in CD8-depleted PBMCs. In contrast, responses to S (tested as two pools, S1 and S2), Membrane (M) and N were relatively increased in CD8-depleted PBMCs (Fig. 1a and b) indicating a predominance of CD4 + T-cell memory (Fig. 1c), consistent with other studies^{4, 8}. Also as previously reported, we detected occasional weak responses to individual SARS-CoV-2 proteins in CD8-depleted PBMCs from 14 uninfected healthy volunteers (Fig. 1c) who had no detectable S or N-specific antibody responses (Supplementary Fig. 1)¹⁵. These responses may represent cross-reactive T cells primed by prior exposure to b-HCoVs. However, they were significantly lower than responses detected in HCW-PI, and not present in every individual, despite equivalent reactivity to the control HLAII pepmix (Fig. 1c).

We next examined the CD4 + T-cell response to S, M and N proteins following natural SARS-CoV-2 infection at the epitope level. Here, polyclonal CD4 + T-cell lines established from HCW-PI using an initial pepmix stimulation were tested with individual 20mer peptides (overlapping by 10aa ensuring complete coverage of these proteins). For each individual, the S-specific polyclonal CD4 + T-cell line was seen to contain multiple responses against peptides distributed throughout the protein, including the RBD, S1 and S2 regions (Fig. 1d). Similarly broad distributions of responses to M and N were also seen in the same individuals, indicating that SARS-CoV-2 induces a broad CD4 + T-cell response^{12, 15}.

Characterisation of CD4 + T-cell epitopes

Detailed knowledge of the identity, HLAII restriction and frequency of individuals responding to individual SARS-CoV-2 epitopes is lacking. We therefore isolated CD4 + T-cell clones specific for 21 epitopes (17 S, 2 M, and 2 N epitopes) from 6 individuals of varying HLAII type. Example characterisations of S-specific T-cell clones SSAN c3 and FNCY c42 are depicted in Fig. 2, and for M and N-specific clones in Supplementary Fig. 2. Using standard approaches³⁶, we identified the epitope recognised by each T-cell clone by titrating four 20mer peptides (overlapping by 15aa) covering the relevant region of T-cell reactivity (Fig. 2a). This defined the SSAN (aa 161–180) and FNCY (aa 486–505) peptides as epitopes for c3 and c42, respectively. Functional assays showed T-cell clone avidities against the epitope peptides were similar to CD4 + T-cells against other viruses^36–38. Importantly, T-cell clones specific for all 17 epitopes within S recognised autologous LCL pre-exposed to low concentrations of purified S protein (1ng/ml) demonstrating that all epitopes are efficiently generated via the exogenous MHCII processing pathway (Fig. 2b).

We next identified the relevant HLAII-restricting alleles by testing each T-cell clone against (i) peptide-loaded autologous LCL in the presence of anti-HLA-DR, -DP and -DQ blocking antibodies (Fig. 2c), and (ii) allogeneic LCLs with partially matched HLA-DR, -DP or -DQ types, as appropriate (Fig. 2d). This revealed the HLAII restriction of SSAN c3 as HLA-DPB1*04:01 and of FNCY c42 as HLA-DRB1*01:01. Table 1 summarises the optimal peptide and HLAII restriction for all 21 S, M and N epitopes identified. All epitopes identified were presented by HLA-DR or HLA-DP alleles, with no HLA-DQ-restricted T-cells found. Several HLA-DRB1*04 restricted epitopes were identified, which discriminated between HLA subtypes. Those presented by HLA-DRB1*04:01 could also be presented by *04:04 and often by *04:03, whereas HLA-DRB1*04:02-restricted epitopes were only presented by this subtype.

Mapping such a broad range of epitopes allowed us to investigate if natural infection and vaccination elicited similar CD4 + T-cell immunity. To address this important question, we first repeated the ex vivo IFNg Elispots on CD8-depleted PBMCs from the same HCW-PI, now using epitope peptides appropriate to each individual’s HLAII type (Table 1). Responses to all 17 epitopes were detected ex vivo in at least one donor. Of the 11 epitopes presented by HLA alleles shared by multiple donors, responses to nine were detected in more than one individual. Next, we examined relative immunogenicity following SARS-CoV-2 vaccination by collecting post-vaccine blood samples from the uninfected donors we had previously analysed in Fig. 1. All nine donors now had S-specific antibodies but lacked N-specific antibodies, confirming lack of prior natural infection (Supplementary Fig. 3). The HLA genotypes of these donors allowed testing with 14 of the S epitopes and responses to 13 were seen in ex vivo assays, many of which were recognised by more than one individual (Table 1). These findings demonstrated that SARS-CoV-2 infection and vaccination both induce broad CD4 + T-cell responses to shared epitopes and that in both contexts HLAII genotype is a key determinant of the SARS-CoV-2 S-specific CD4 + T-cell response.

Cross-reactivity of SARS-CoV-2 S-specific CD4 + T-cells with other human b-HCoVs

PBMCs from SARS-CoV-2 uninfected individuals react with high concentrations of SARS-CoV-2 antigen pepmixes demonstrating cross-reactive T-cells exist (Fig. 1)^15–18. However, the extent to which pre-existing cross-reactive T-cell immunity, primed by prior exposure to b-HCoVs, contributes to controlling SARS-CoV-2 infection remains unclear. Significantly higher levels of SARS and HKU1 S-specific IgG antibodies were seen in HCW-PI compared to uninfected counterparts (Supplementary Fig. 1c), in agreement with prior work demonstrating that SARS-CoV-2 infection boosts antibody titres against other HCoVs⁸. Our panel of S-specific T-cell clones allowed us to investigate the less well studied question of whether epitope-specific CD4 + T-cells elicited by natural SARS-COV-2 infection can similarly cross-react with other closely related HCoVs.

Figure 3a shows the evolutionary relationship of S proteins from all b-HCoVs known to infect humans (SARS, MERS, HKU1 and OC43) compared to SARS-CoV-2. Within the N-terminal S1 region, the aa sequence of SARS is most similar to SARS-CoV-2 (64.7%) whilst the aa similarity of all other b-HCoV S1 regions is low, < 32% (Fig. 3b). The C-terminal S2 regions exhibit greater overall similarity, particularly SARS-CoV-2 and SARS which share 90.0% aa identity. However, the similarity of the SARS-CoV-2 S2 region with the other b-HCoV is much lower, below 45%³⁹.

To assess T-cell cross-reactivity, SARS-CoV-2 S-specific CD4 + T-cell clones were tested against SARS, MERS, HKU1 and OC43 S1 or S2 pepmixes (Fig. 3c and 3d). Where possible, multiple T-cell clones per epitope were tested. As expected, all clones showed similar levels of recognition against S1 or S2 SARS-CoV-2 pepmix and the clone’s cognate epitope peptide. However, within the more divergent S1 region no cross-reactivity was detected against any other b-HCoV. Representative results for the S1-specific SSAN, VVLS and FNCY CD4 + T-cell clones are shown in Fig. 3c with results for clones against all 8 SARS-CoV-2 S1 epitopes summarised in Fig. 3e. Notably, each SARS-CoV-2 epitope had at least two and as many as 16 aa differences compared to the corresponding regions of SARS, the virus with greatest overall sequence similarity. S1 epitope sequences within the other b-HCoVs were even more divergent (Fig. 3c and Supplementary Fig. 4).

Within the more similar S2 region, CD4 + T-cells specific for the NFSQ epitope also did not cross-recognise any other b-HCoV (Fig. 3d). The corresponding epitope in SARS contains 2 central mutations and has high sequence diversity among the other b-HCoVs. Cross-reactivity was observed for T-cells against the other 8/9 S2 epitopes but, as illustrated for STEC and SFIE-specific clones, this was limited to the highly homologous S2 region of SARS (Fig. 3d and 3e). All SARS-CoV-2 S2 epitopes sequences were either identical in SARS or contained a maximum of two aa differences (Fig. 3d and Supplementary Fig. 4). However, again no cross-reactivity was observed for any S2 epitope-specific T-cell clone against the other more divergent b-HCoVs (Fig. 3e).

Collectively, these data show S epitope-specific CD4 + T-cells isolated following SARS-CoV-2 infection can recognise highly homologous epitopes within the S2 region of SARS but do not cross-react with MERS or extant b-HCoVs (HKU1 and OC43). Of note, all HCW did have antibody immunity against endemic b-HCoVs, consistent with prior infection with these viruses (Supplementary Fig. 1). We therefore infer that the T-cell clones isolated were primed by SARS-CoV-2 infection.

Spike-specific CD4 + T-cell recognition of SARS-CoV-2 variants of concern

Having shown that CD4 + T-cell recognition is sensitive to modest sequence differences between the b-HCoVs, we next examined recognition of SARS-CoV-2 VOCs at the epitope level. The VOCs examined included Alpha (B.1.1.7), Beta (B.1.351), Gamma (P1), Delta (B.1.617.2) and Omicron (B.1.1.529). Amino acid substitutions or deletions within VOCs were present in 10 of 17 S epitope sequences. Some were common to multiple VOC, such as the N501Y FNCY mutation present in Alpha, Beta and Gamma variants, whilst others were unique to particular viral isolates, such as the N764K STEC mutation in Omicron (Fig. 4a and b). To study the impact of these mutations on CD4 + T-cell recognition, we employed the S-specific T-cell clones from HCW-PI who had been infected during the WT (D614G) SARS-CoV-2 wave. Peptide titrations were used to detect the effects of mutation at biologically relevant low concentrations that may be missed with higher concentrations.

VOC mutations in certain epitopes did not affect epitope-specific CD4 + T-cell recognition while others reduced or eliminated recognition (Fig. 4a and 4b). Single central aa substitutions in the TLVK (N969K, Omicron) and SGTN (D80Y, P2) epitopes eliminated T cell recognition, except at supra-physiological peptide concentrations for the latter. Two separate point mutations in QLIR, A1022S (Beta) and T1027I (Gamma), both impaired T-cell recognition at equivalent peptide concentrations. However, several epitope peptides containing point mutations, LSET (T307A, P3), STEC (N764K, Omicron) and TYVT (A1022S, Beta), were recognised equally to the WT epitope. The double aa deletion in P3 (D43-44) had no effect on LVDL and TRFQ recognition, however the triple aa deletion (D42-44) in the Beta VOC reduced T-cell stimulation compared to the WT peptide.

The epitopes with most mutations were GGNY and FNCY, both present within the RBD region, a frequent target of mutation in the VOC^{22, 23}. Although single point mutations arising in earlier VOCs in GGNY (L452R, Delta and B.1.324) and FNCY (N501Y, Alpha, Beta and Gamma) did not affect epitope-specific CD4 + T-cell recognition, the multiple mutations in Omicron, GGNY (G446S, L452R, R457N) and FNCY (Q493K, G496S, Q498R, N501Y, Y505H), eliminated recognition of both epitopes. Overall, recognition of 7 of the 17 epitopes was affected by mutations present in one or more VOCs. Importantly, the affected epitopes were restricted through a range of HLAII alleles, and in every case where recognition of an epitope was lost the same person also possessed responses against epitopes that were not impacted by mutation.

Although powerful tools, T-cell clones only allow a small number of TCRs to be studied. In vivo, virus epitope-specific CD4 + T-cell responses comprise multiple different TCRs⁴⁰ and it is possible that other TCRs specific for the same epitopes may be unaffected by these mutations. To examine the generalisability of our findings, we repeated the titrations using CD8-depleted PBMCs from HCW-PI or uninfected individuals post-vaccine in ex vivo IFNg Elispot assays (Fig. 5). We tested 5 epitopes, 4 that had shown decreased recognition against variant peptides (SGTN, LVDL, GGNY and QLIR) and one that had not (STEC). For each epitope tested, the pattern of results was strikingly similar to those obtained with corresponding T-cell clones (Fig. 4), indicating that our findings are representative of the circulating epitope-specific CD4 + T-cell responses (Fig. 5). The frequency of CD4 + T-cells responding to SGTN was severely reduced by point mutation in P2 (D80Y) compared to the WT peptide and was lower against the QLIR variant peptides at the same concentrations (A1022S, Beta and T1027I, Gamma). In contrast, similar magnitudes of responses were detected against the STEC Omicron variant (N764K) and WT peptides, as seen using T-cell clones (Fig. 5). For LVDL, the Beta peptide containing the triple aa deletion (D42-44) was poorly recognised compared to the WT and double aa deleted (D43-44) peptides. Most strikingly, no response was detected to the mutated GGNY sequence from Omicron (G446S, L452R, R457N), as seen with the T-cell clones, confirming loss of recognition of this epitope in Omicron.

Together, these data highlight the acute sensitivity of SARS-CoV-2 S-specific CD4 + T-cells to small aa changes in epitope sequence and the potential for epitope evasion through modest virus mutations. However, the 7 affected epitopes were not all present in every VOC and CD4 + T-cells induced against ancestral SARS-CoV-2 S protein were able to recognise at least 14 of the 17 studied S epitopes in all current VOCs.

CD4 + T-cells are central organisers of anti-viral immune responses^{11, 41} and dominate memory T-cell responses against the major vaccine target for SARS-CoV-2, S^{4, 8–10}. Studies of SARS-CoV-2-specific CD4 + T-cells have largely examined responses to whole antigens using pools of overlapping peptides. Although S-derived epitopes have been reported^{12–14, 43, 44}, for most, epitope generation from native antigen has not been demonstrated and HLAII restrictions have not been defined. Critically, the current lack of fundamental understanding of SARS-CoV-2-specific CD4 T-cell immunity has prevented important questions being investigated at the epitope level. These include the extent of epitope-specific CD4 + T-cell cross-reactivity with other b-HCoVs and the ability of epitope-specific T-cells induced by ancestral SARS-CoV-2 infection or vaccination to protect against VOCs.

To enable detailed investigation of SARS-CoV-2-specific CD4 + T-cells, we studied HCW infected during the first wave of infection in the UK. In line with previous ex vivo studies assessing whole antigens^{4, 8–10}, we detected robust CD4 + T-cell memory against S, M and N peptide pools in HCW-PI. These varied in magnitude between individuals but were consistently higher in CD8-depleted than matched whole PBMCs, reflecting the numerical dominance of CD4 + T-cell memory^{4, 8–10}. Mapping of responses in HCW-PI following in vitro expansion, showed CD4 + T-cell reactivity to multiple peptides spread across S, M and N in every individual. This sensitive method allows detection of low frequency cells or central memory cells not detectable in ex vivo IFNg assays^{28, 42}. Thus, the total number of SARS-CoV-2 epitope-specific responses induced by infection in each individual may be larger than previously estimated^{12, 15}.

Through isolation and characterisation of over 100 CD4 + T-cell clones we identified 21 HLAII-restricted epitopes; 17 in S, two in M and two in N. This greatly expands the number of known CD4 + T-cell epitopes and provides experimental confirmation and HLAII restriction for some previously reported^{12–14, 43, 44}. Furthermore, all 17 S-derived epitopes we describe were processed and presented from low quantities of protein demonstrating their biological relevance⁴⁵. Interestingly, the epitopes we characterised were skewed towards HLA-DP and HLA-DR restriction alleles, a pattern also noted for SARS-CoV-2 by others^{15, 28} and distinct from other human viruses investigated using T-cell clones^{38, 46, 47}. Responses to the same S epitopes were detected in individuals of appropriate HLAII genotype after either SARS-CoV-2 infection or vaccination, indicating that HLAII genotype is a key determinant of the SARS-CoV-2 S-specific CD4 + T-cell response regardless of the route of antigen exposure. We also observed epitopes restricted through prevalent HLAII alleles such as the HLA-DRB1*04 subtypes, DRB1*01:01, DRB1*15:01 and DPB1*04:01⁴⁸. Thus, it is likely that many of the responses will be widespread in the population.

In addition to SARS-CoV-2, several endemic HCoVs infect humans causing mild disease. If T-cells elicited by prior infection with these viruses cross-reacted with SARS-CoV-2 they could modulate the course of disease. Consistent with several groups, we observed low-frequency CD4 + T-cell responses to SARS-CoV-2 S, M and N in people uninfected by SARS-CoV-2 using high peptide concentrations in ex vivo assays^15–18. However, in this study, all the S-specific CD4 + T-cell clones derived from SARS-CoV-2-infected individuals efficiently recognised SARS-CoV-2 S pepmix but never recognised S pepmixes from the endemic OC43 and HKU1 b-HCoV viruses. This suggests strongly that these responses arose from naïve T-cells within the repertoire rather than pre-existing memory. Similar results were recently observed for CD8 + T-cells against the immunodominant HLA-B7:02-restricted N epitope⁴⁹. Indeed, the only cross-reactivity we observed was within the highly conserved S2 region of SARS (90% aa similarity) to which the UK HCWs studied had never been exposed. Collectively these data argue for limited expansion of pre-existing cross-reactive S-specific CD4 + T-cells during SARS-CoV-2 infection.

This view is consistent with prior reports of S cross-reactivity that suggest cross-reactive CD4 + T-cells are rare components within a limited number of S epitope-specific responses^{15, 16, 21}. At the epitope level, spike-specific CD4 + T cell cross-reactivity has mainly been observed within the S2 809–830 linker region (encompassing the HLA-DRB1*08:01 SFIE epitope studied here); in polyclonal T-cell lines^{16, 19}, T-cell clones^{15, 21} and using HLA-DP*04-restricted MHCII tetramers¹⁴. Compared to SARS-CoV-2 primed T-cell responses, the HCoV primed CD4 + T-cells were present at low frequency in the blood¹⁴, had 5-fold lower functional avidity against SARS-CoV-2 peptides²¹ and exhibited less expansion following SARS-CoV-2 infection¹⁴.

In our study we only investigated cross-reactivity of CD4 + T cells specific for S, a protein highly targeted by mutation. In comparison, other viral proteins are more conserved across HCoVs and T-cell cross-reactivity is therefore more likely. Accordingly, in SARS-CoV-2 unexposed individuals, pre-existing T cell immunity has been reported against the more highly conserved N and ORF1ab-encoded NSP proteins^{16, 17, 50}. HCoV cross-reactive T cells in SARS-CoV-2-seronegative HCWs were frequently directed against epitopes within the early expressed replication transcription complex (encompassing Nsp7, Nsp12 and NSp13)⁵⁰. Interestingly, these expanded following putative abortive SARS-CoV-2 infection in exposed HCW who remained seronegative, consistent with a role in protection from overt infection. Ultimately, the extent to which pre-existing cross-reactive T-cell immunity contributes to controlling SARS-CoV-2 infection in an individual will be determined by a complex combination of factors including the conservation of the epitopes presented by their HLA genotype as well as their TCR repertoire and previous history of HCoV exposure¹⁴.

The SARS-CoV-2 pandemic has been exacerbated by emergence of VOCs with mutations in S that reduce the neutralisation capacity of antibodies induced against ancestral S sequences^24–26. Because spike is the only viral antigen in the most widely used vaccines, understanding how sequence mutations affect T-cell recognition is vital for vaccine development efforts and understanding the potential impact of emerging VOCs for vaccinated individuals. Studies of total S-specific T-cells have shown minimal reductions in the overall frequency of response to Alpha, Beta, Gamma, Delta VOCs^{26, 31, 51} and Omicron^{33, 34, 52} in previously infected or vaccinated individuals. However, the use of high concentrations of peptide pools in these studies masks biologically important differences in epitope-recognition efficiency³⁵.

We identified aa substitutions or deletions in 10 of 17 S CD4 + T-cell epitopes; some were common to multiple VOCs while others were unique to particular viral isolates. Combining data from T-cell clones and experiments using ex vivo PBMCs we found variable effects of intra-epitope mutations on CD4 + T-cell recognition. As expected, triple aa deletion and multiple aa substitutions within individual epitopes had the greatest impact on CD4 + T-cell recognition⁵³. Of note, the GGNY and FNCY epitopes within the frequently mutated RBD region were eliminated in Omicron, despite point mutations in earlier VOCs being recognised. Overall, three of the four epitopes mutated in Omicron were no longer recognised by epitope-specific CD4 + T cells. However, the effect of point mutation is complex. A single mutation dramatically decreased T-cell recognition (e.g. SGTN D80Y, P2; TLVK N969K, Omicron) or had no effect (LSET T307A, P3; STEC N764K, Omicron; TYVT A1022S, Beta). The epitope mapping we present therefore provides a rational basis for VOC risk stratification. It highlights the need for further careful experimental definition of immunodominant CD4 + T-cell epitopes, identification of the essential aa required for HLAII binding and TCR engagement, and the need to consider each epitope individually.

In conclusion, our study demonstrates the fine sensitivity of SARS-CoV-2 S-specific CD4 + T-cells to aa differences in epitope sequence and the potential for SARS-CoV-2 evasion of ancestral virus or vaccine-elicited CD4 + T-cells through viral evolution. The breadth of SARS-CoV-2 S epitopes targeted in every individual shows current VOC mutations are likely to have only limited impact on overall CD4 + T-cell surveillance. However, continued mutation of SARS-CoV-2 could lead to further epitope loss highlighting the importance of continued virus sequence monitoring of emerging VOCs.

Donor characteristics and ethical statement

The UPH CIA ethics for the study was approved by the North West – Preston Research Ethics Committee, UK (20/NW/0240) and all participants gave written informed consent. Blood was collected from 20 healthcare workers (HCW), infected in spring 2020 during the first wave of SARS-CoV-2 in the UK, WT (D614G), between 1.5 and 6 months following primary infection. Control samples were collected from SARS-CoV-2 uninfected individuals confirmed to be seronegative for SARS-CoV-2 (Fig. S1), or prior to the pandemic as part of an ethically approved study (South Birmingham Research Ethics Committee 14/WM/1254). Subsequent samples were collected from uninfected individuals following double vaccination with Pfizer BioNtech BNT162b2 or Astrazeneca ChAdOx-1.

Sample Preparation

Plasma and PBMCs were isolated from heparinised blood using standard Ficoll-Hypaque centrifugation. The resulting PBMC layer was washed twice with RPMI and either used directly or cryopreserved prior to use. Where stated, PBMCs were depleted of CD8 + T-cells using anti-CD8 Dynabeads (Invitrogen) to > 94%, as measured by flow cytometry. Autologous Lymphoblastoid cell lines (LCLs) were generated by transformation with B95.8 EBV as previously described⁵⁴ and HLAII sequencing was performed at Anthony Nolan, UK.

Synthetic peptides and protein

Pepmixes containing 15mer peptides overlapping by 11aa covering the full length of WT (D614G) SARS-CoV-2 S1 and S2 (PM-WCPV-S-2), M (PM-WCPV-VME-2) and N (PM-WCPV-NCAP-2), the S1 and S2 regions of SARS (PM-CVHSA-S-1), MERS (PM-MERS-CoV-1), HKU1 (PM-HKU1-S-1) and OC43 (PM-OC43-S-1), and control HLAII CEFX pepmix (PM-CEFX-3) were purchased from JPT. Individual 20-mer peptides overlapping by 15aa covering the full sequences of SARS-CoV-2 S, M and N were purchased from Alta Biosciences, UK. Purified epitope peptides (> 85% purity) and corresponding peptides (20mers or 15mers) from SARS-CoV-2 VOCs were synthesised by Alta Biosciences, UK, and Genscript, Netherlands. All peptides were resuspended in dimethylsulphoxide (DMSO) at a concentration of 5mg/ml. The concentration of all purified epitope peptides was confirmed using a Pierce Quantitative Colorimetric Peptide assay (Thermo Scientific) that binds to the peptide amide backbone, and peptides were adjusted to equivalent concentrations. The SARS-CoV-2 S protein is a soluble prefusion stabilised form, containing proline substitutions at positions F817P, A892P, A899P, A942P, K986P, V987P⁵⁵. Epitope sequences recognised by the isolated CD4 + T-cell clones are free of these stabilising mutations.

Interferon-g Elispot assay

Whole or CD8-depleted PBMCs (0.2-0.4x10⁶ cells) were resuspended in standard media (RPMI supplemented with 8% batch-tested FCS, 100 IU/mL penicillin and 100 µg/mL streptomycin) and added to duplicate or triplicate wells of IFNg Elispot Pro Kit (Mabtech) plates containing 2mg/ml pepmix, 5mg/ml purified epitope peptide or titrated concentration as stated, DMSO (negative control) and PHA or anti-CD3 (positive controls). Samples were incubated at 37°C for 16-18hr. Plates were developed in accordance with the manufacturer’s instructions and read on a Bioreader 5000 Fg (Bio-sys GmbH). To quantify antigen-specific responses, mean DMSO values were deducted from all test wells and the results were expressed as spot forming cells (sfc) per 10⁶ cells.

Polyclonal T-cell generation and analysis

Polyclonal CD4 + T-cell lines, generated by stimulation of CD8-depleted PBMCs with 2mg/ml S, M or N pepmix, were cultured in RPMI supplemented with 5% batch-tested human serum (Gibco) and 50IU/ml IL-2. Following 2–4 bi-weekly repeat stimulations with pepmix, 50,000 polyclonal T-cells were incubated for 16-18hr in V-bottom microtest plate wells with 1ug/ml individual 20-mer peptides overlapping by 10aa covering S, M or N or DMSO (negative control). IFNg release into the supernatant was tested by ELISA (Invitrogen) according to the manufacturer’s protocol. Responses were considered positive if greater than twice the mean of the control wells.

T-cell clone isolation and assays

CD4 + T-cell clones were isolated from polyclonal cultures as previously described⁵⁶. Briefly, polyclonal CD4 + T-cells were selected on rechallenge with 2mg/ml appropriate pepmix using an IFNg cell enrichment kit (Miltenyi Biotech) followed by MACS separation. Enriched cells were cloned by limiting dilution seeding, to establish T-cell clones originating from single cells. Growing microcultures were screened for reactivity against individual 20mer peptides and selected clones were expanded using standard methods³⁶.

Standard numbers of CD4 + T-cell clones (2000 or 5000 cells per well) were incubated in V-bottom microtest plate wells with standard numbers (5x10⁴ cells per well) of autologous or partially HLAII-matched LCLs. In various assays, LCLs were either pre-exposed for 1hr to 5mM epitope peptide, 2mg/ml pepmix, 1ng/ml soluble prefusion-stabilised SARS-CoV-2 S protein⁵⁵ or equivalent volumes of DMSO (negative control) before washing and addition to the wells, or added with a titrated concentration of peptide. In blocking assays, peptide-loaded or DMSO-exposed LCLs were incubated for 1hr with purified monoclonal antibodies against HLA-DR (L243, Biolegend), HLA-DQ (SPV-L3, Biotium) and HLA-DP (B7/21, Life Technologies) before addition of T-cells. In all other assays, T-cells were added immediately. The supernatant medium harvested after 16-18hrs was assayed for IFNg by ELISA.

Serological analysis

Quantitative IgG antibody titres against SARS-CoV-2 S and N and HCoV family S proteins were measured using a multiplex serology assays (V-PLEX COVID-19 Coronavirus Panel 2 [IgG] kit, catalogue number K15369U), according to the manufacturer’s instructions. In brief, 96-well plates were blocked and washed. Samples were pre-diluted 1:4000 in provided sample diluent and added to the wells in duplicate alongside the reference standard and assay kit controls. Following incubation, washing and addition of anti-IgG detection antibodies, read buffer was added to all wells and plates were immediately measured using a MESO Quickplex SQ 120 System (Meso Scale Discovery). Data were generated by Methodological Mind software (version 1.0.36), adjusted for sample dilutions and analysed using MSD Discovery Workbench (version 4.0).

Bioinformatic and statistical analysis

Spike amino acid alignments were performed using the MUSCLE algorithm⁵⁷ with default settings. Protein sequence identity was calculated using Expasy⁵⁸. Correlation plots were prepared in R version 4.0.3 using corrplot (v0.84). All Statistical tests, as stated in each figure, were performed in GraphPad Prism (v. 9.3.1). Statistically significant results were presented as *p < 0.05, **p < 0.01.

HCoV, human coronavirus; VOC, variant of concern; LCL, lymphoblastoid cell line; HCW, healthcare workers; PI, post-infection; PV, post vaccine

ACKNOWLEDGEMENTS

This work was supported by the UK Coronavirus Immunology Consortium (UK-CIC) funded by NIHR/MRC MR/V028448/1. MC is supported by the International AIDS Vaccine Initiative (IAVI) through Grant INV-008352/OPP1153692 funded by the Bill and Melinda Gates Foundation and the University of Southampton Coronavirus Response Fund.

AUTHOR CONTRIBUTIONS

HL designed and supervised the study. ET, EJ, TH, BK, NK, SD and HL performed in vitro assays. ET, EJ, TH, BK, NK, SD, PM, GT and HL perfomed data analysis. PP and HP conducted sample collection. MN and MC generated and provided essential reagents. GT and HL led on data interpretation and wrote the paper. All authors commented on the manuscript.

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Table I – SARS-CoV-2 CD4+ T-cell epitopes and responses

Protein	aa coordinates	Epitope	HLA II restriction	HCW-PI	PV
Spike	71-90	SGTNGTKRFDNPVLPFNDGV	DPB1*05:01	2/2	NT
Spike	161-180	SSANNCTFEYVSQPFLMDLE	DPB1*04:01	2/2	1/2
Spike	226-245	LVDLPIGINITRFQTLLALH	DRB104:01, 04:03, *04:04	1/1	1/2
Spike	236-255	TRFQTLLALHRSYLTPGDSS	DRB1*04:02	1/1	0/1
Spike	296-315	LSETKCTLKSFTVEKGIYQT	DRB104:01, 04:04	1/2	2/2
Spike	446-465	GGNYNYLYRLFRKSNLKPFE	DPB1*02:01	3/3	1/2
Spike	486-505	FNCYFPLQSYGFQPTNGVGY	DRB1*01:01	2/4	1/2
Spike	511-530	VVLSFELLHAPATVCGPKKS	DRB1*01:01	3/4	2/3
Spike	746-765	STECSNLLLQYGSFCTQLNR	DRB1*15:01	2/2	5/5
Spike	801-820	NFSQILPDPSKPSKRSFIED	DRB104:01, 04:03, *04:04	2/2	2/2
Spike	816-835	SFIEDLLFNKVTLADAGFIK	DRB1*08:01	1/1	2/2
Spike	891-910	GAALQIPFAMQMAYRFNGIG	DRB1*07:01	3/5	2/3
Spike	956-975	AQALNTLVKQLSSNFGAISS	DRB1*01:01	2/4	2/3
Spike	961-980	TLVKQLSSNFGAISSVLNDI	DRB104:01, 04:04	1/1	2/2
Spike	1006-1025	TYVTQQLIRAAEIRASANLA	DRB1*04:02	1/1	NT
Spike	1011-1030	QLIRAAEIRASANLAATKMS	DRB104:01, 04:04	1/2	1/2
Spike	1061-1080	VFLHVTYVPAQEKNFTTAPA	DRB1*04:02	1/1	NT

Membrane	146-165	RGHLRIAGHHLGRCDIKDLP	DRB4*01:03	NT	N/A
Membrane	161-180	IKDLPKEITVATSRTLSYYK	DRB1*04:02	NT	N/A

Nucleoprotein	196-215	RNSSRNSTPGSSRGTSPARM	DPB1*09:01	NT	N/A
Nucleoprotein	281-300	QTQGNFGDQELIRQGTDYKH	DRB4*01:03	NT	N/A

HCW-PI – Healthcare workers Post infection; PV – uninfected individuals post vaccine.

There is NO Competing Interest.

SupplFig1.tif
Supplementary Fig. 1. IgG antibody titres against SARS-CoV-2 S and N proteins and S protein from all b-HCoVs in HCW-PI and uninfected individuals. Significance was determined by Mann-Whitney U test, **p<0.001, *p<0.05.
SupplFig2.tif
Supplementary Fig. 2. Characterisation of novel membrane and nucleoprotein CD4+ T-cell epitopes. (a) Mapping of M and N-specific T-cell epitopes. 1000 T-cells/well were cocultured overnight with autologous LCLs (5x10⁴ cells/well) loaded with 20mer peptides overlapping by 15aa (10^-5 to 10^-11M) covering the region of T-cell reactivity. (b) T-cell clone recognition of unmanipulated and peptide-pulsed autologous LCLs either alone or in the presence of mAbs against HLA-DP (B7.21), HLA-DQ (SPV-L3) or HLA-DR (L243). (c) T-cell clones cocultured with autologous LCL and partially HLAII-matched LCLs either unmanipulated or pre-pulsed with epitope peptide. (a-c) Results show mean IFNg release ±SD measured by ELISA, representative of 3 experiments.
SupplFig3.tif
Supplementary Fig. 3. IgG S and N antibody titres in uninfected individuals pre and post SARS-CoV-2 double vaccination. Significance was determined by Wilcoxon test **p<0.001.
SupplFig4.tif
Supplementary Fig. 4. Alignment of S-derived T-cell epitopes with the corresponding b-HCoV sequences.

Broad SARS-CoV-2 spike-specific CD4+ T-cell response preserves recognition of variants of concern

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Results

Breadth of SARS-CoV-2 specific CD4 + T-cell immunity following natural infection

Characterisation of CD4 + T-cell epitopes

Cross-reactivity of SARS-CoV-2 S-specific CD4 + T-cells with other human b-HCoVs

Spike-specific CD4 + T-cell recognition of SARS-CoV-2 variants of concern

Discussion

Methods

Donor characteristics and ethical statement

Sample Preparation

Synthetic peptides and protein

Interferon-g Elispot assay

Polyclonal T-cell generation and analysis

T-cell clone isolation and assays

Serological analysis

Bioinformatic and statistical analysis

Non-standard Abbreviations

Declarations

References

table

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1