key: cord-1055760-z4lxwfeg
authors: Verhagen, Johan; van der Meijden, Edith D.; Lang, Vanessa; Kremer, Andreas E.; Völkl, Simon; Mackensen, Andreas; Aigner, Michael; Kremer, Anita N.
title: Human CD4(+) T cells specific for dominant epitopes of SARS‐CoV‐2 Spike and Nucleocapsid proteins with therapeutic potential
date: 2021-06-01
journal: Clin Exp Immunol
DOI: 10.1111/cei.13627
sha: f9de2a7bcb2e337b53a5555b86e11485e4204fa8
doc_id: 1055760
cord_uid: z4lxwfeg

Since December 2019, Coronavirus disease‐19 (COVID‐19) has spread rapidly across the world, leading to a global effort to develop vaccines and treatments. Despite extensive progress, there remains a need for treatments to bolster the immune responses in infected immunocompromised individuals, such as cancer patients who recently underwent a haematopoietic stem cell transplantation. Immunological protection against COVID‐19 is mediated by both short‐lived neutralising antibodies and long‐lasting virus‐reactive T cells. Therefore, we propose that T cell therapy may augment efficacy of current treatments. For the greatest efficacy with minimal adverse effects, it is important that any cellular therapy is designed to be as specific and directed as possible. Here, we identify T cells from COVID‐19 patients with a potentially protective response to two major antigens of the SARS‐CoV‐2 virus, Spike and Nucleocapsid protein. By generating clones of highly virus‐reactive CD4(+) T cells, we were able to confirm a set of 9 immunodominant epitopes and characterise T cell responses against these. Accordingly, the sensitivity of T cell clones for their specific epitope, as well as the extent and focus of their cytokine response was examined. Moreover, by using an advanced T cell receptor (TCR) sequencing approach, we determined the paired TCRαβ sequences of clones of interest. While these data on a limited population require further expansion for universal application, the results presented here form a crucial first step towards TCR‐transgenic CD4(+) T cell therapy of COVID‐19.

COVID-19 has presented as an unprecedented global health emergency, with 100 million cases worldwide confirmed in little more than 1 year, according to the World Health Organisation COVID-19 dashboard at https://covid19.who.int. A huge effort to develop effective vaccines has led to these being introduced at record pace. Despite this success and assuming that the current vaccines will provide high levels of protection against current and future variants of the virus in healthy individuals, there will remain a substantial group of individuals who may experience reduced efficacy of a vaccine. From our own perspective, patients with haematological malignancies who have undergone haematopoietic stem cell transplantation remain highly vulnerable to severe complications of infection until 6-9 months posttransplant, when they can be vaccinated successfully. A similar reduction in protection may be true for other immunocompromised individuals. The development of new treatments to boost the immune response to SARS-CoV-2 therefore remains relevant.

Treatment of established COVID-19 remains challenging. One method of adoptive immunity to the SARS-CoV-2 virus that has been trialled extensively so far, is the transfer of convalescent plasma. This approach, which first came to prominence during the outbreak of the Spanish Flu in 1918, relies on the transfer of neutralising antibodies. Although various degrees of success have been achieved with this approach in COVID-19, the approach seems mostly successful when plasma with very high titres of antibody is used in mild to moderate disease [1, 2] . In more severe disease, where patients require mechanical ventilation, this approach is much less successful. Moreover, a recent report warned that convalescent plasma treatment of severe disease may promote the evolution of the virus to avoid interaction with neutralising antibodies [3] . This, of course, would be a very worrying development for the wider population as this may also compromise the efficacy of the current generation of vaccines.

In addition to the production of neutralising antibodies, T cell immunity against a number of SARS-CoV-2 proteins has been found to represent a major component of the healthy immune response [4, 5] . This, therefore, opens up another potential avenue of immunotherapy to augment the effect of current approaches. In fact, Cooper et al [6] previously published a method of expanding SARS-CoV-2-specific T cells for allogeneic T cell therapy. However, to limit the risk of adverse effects of cell therapy, it is crucial to direct the T cell response as much as possible. To this end, several groups have previously published immunodominant peptides from the different proteins of the virus [5, [7] [8] [9] , emphasising the involvement of both spike and non-spike antigens in the T cell response. In addition to the extent to which particular peptide regions are recognised, Bacher et al [10] stressed that the nature of the T cell response is crucial

This article is protected by copyright. All rights reserved for successful clearance of the virus. In particular, a focussed, high avidity interaction of Th1 cells rather than diversity seems crucial to limit disease severity.

With this in mind, we characterised T cells derived from the peripheral blood of COVID-19 patients with strong responses to SARS-CoV-2, by generating hundreds of T cells clones reactive to Spike and Nucleocapsid protein. Immunodominant epitopes were determined and the responses of 81 antigenspecific clones assessed for sensitivity and diversity of the cytokine response. This, combined with fulllength analysis of paired TCR and  chains allows for the selection of a small pool of T cells with highly desirable characteristics that together target multiple epitopes. The information provided here facilitates a significant step towards the generation of a T cell therapy, which will bolster the portfolio of immunotherapies available against COVID-19.

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CoV-2 infection were obtained at the University Hospital Erlangen. The criteria for COVID-19 grades were as follows: Severe, intensive care treatment or death; Moderate, no criteria for severe disease fulfilled but requiring supplemental oxygen; Mild, no criteria for moderate or severe disease fulfilled. All participants gave their informed, written consent. HLA typing was performed by Illumina sequencing at the European Federation for Immunogenetics (EFI)-accredited Laboratory for Immunogenetics at the University hospital Erlangen. The study has been performed according to the declaration of Helsinki and was approved and monitored by the ethical committee of the Friedrich-Alexander-Universität Erlangen-Nürnberg (protocol 118_20B and 174_20B). 

PBMCs from COVID-19 patients and healthy volunteers were separated by density gradient centrifugation using Pancoll (PAN Biotech, Aidenbach, Germany) and stored in liquid nitrogen until further processing.

Cells were thawed and stimulated with 1 g/ml of total Spike and Nucleocapsid antigen for 24 hours.

Subsequently, cells were stained with anti-CD3-FITC, anti-CD137-PE, anti-CD8-BV421, anti-CD4-BV510 and 7AAD (all from BD Biosciences). Additionally, for COVID-19 patients only, PBMC were stained ex vivo without prior peptide stimulation with anti-HLA-DR-PE, anti-CD3-BV510, anti-CD4-BV421, anti-CD8-APC-

This article is protected by copyright. All rights reserved Cy7, anti-CD38-APC and 7AAD (all from BD biosciences). Fluorescence intensity was measured on a BD FACSCanto II flow cytometer and analysed using FlowJo (v.10) software. 

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Statistical analysis was performed using GraphPad Prism 8 software, using the appropriate test as indicated in figure legends. p < 0.05 was considered statistically significant.

In order to investigate the T cell response to SARS-CoV-2, we opted to study patients hospitalised due to COVID-19. These patients were reasoned to have a strong and enduring immune response. 18 patients (7 ♀ and 11 ♂, average age of 69 ± 14.4 years) hospitalised between April and November 2020 were selected at random. The patients varied in disease severity and the overall HLA background of our patient group was characteristic for the German population. Details are listed in Table 1 . First, T cells from the peripheral blood of these patients were analysed for the activation markers CD38 and HLA-DR (Figure 1a) , ex vivo. As described previously [11] , a considerable number of CD4 + and, in particular, CD8 + T cells 

To study the antigen-specific T cell response of the three highly reactive patients in more detail, we generated clones from CD4 + cells that were either CD38 + HLA-DR + ex vivo or expressed CD137 after 24hour in vitro stimulation with a pool of peptides that together span the lengths of both Spike and Nucleocapsid protein. After expansion, these clones were restimulated with Spike or Nucleocapsid peptide pools for 24 hours in the presence of exogenous rhIL-2 to determine specificity for either protein by means of IFN- secretion. In this manner, we selected a total of 179 CD4 + T cell clones reactive to Spike (42 activated ex vivo, 137 in vitro stimulated), 53 reactive to Nucleocapsid (11 activated ex vivo, 42 in vitro stimulated) and one that seemed to recognise both. Very few CD8 + T cell clones were generated, most likely due to the methodology applied, and these were not analysed further. The CD4 + T cell clones

were each restimulated with overlapping matrix peptides from the relevant protein and rhIL-2 to determine specificity down to a 15-mer sequence. Specific T cell clone responses were again detected by IFN- ELISA on cell culture supernatant. Although the CD4 + T cell clones recognised peptides throughout the lengths of Spike ( Figure 2a ) and Nucleocapsid (Figure 2b ), a handful of regions were targeted by multiple clones, from T cells activated ex vivo or in vitro, often in two or more of the three donors. We thus classified these regions as immunodominant.

In the next step, we selected 9 dominant peptides or peptide regions, 2 from Nucleocapsid and 7 from

Spike that were each recognised by multiple CD4 + T cell clones from the two most reactive patients, 54

and 91 ( where CD4 + T cell clones recognised two adjacent, overlapping peptides, both peptides were included in further studies and considered as one condition. The 9 peptides selected were all similar or identical to epitopes that have previously been described as immunodominant by at least one other group (Table 2) .

Additionally, 8 out of 9 peptides (89%) were predicted to have high affinity for the HLA haplotypes in our study population. These 9 peptides were recognised by a total of 81 CD4 + T cell clones from COVID-19

patients. The sequence of peptide condition S33-4 is altered near the C terminus by deletion mutation Y144 found in the B.1.1.7 variant of SARS-CoV-2. This, however, did not appear to impair the response of specific T cell clones (Supplementary figure 1), so further analysis was performed with the original sequence only. Two clones, namely clone 172 from patient 54 and clone 158 from patient 91, recognised 2 different peptides within this panel (N72-3 + S42 and S112-3 + S165-6, respectively). TCR analysis, discussed later in this article, however, suggested that clone 172 was not in fact a true clonal expansion.

Further analyses were performed to detail the functional characteristics of the CD4 + T cell clones selected.

The CD4 + T cells most effective at fighting viral infection are considered those that recognise specific peptide at high affinity and have a highly differentiated Th1 cytokine profile. To test our 81 CD4 + T cell clones, each was stimulated with titrated doses of specific peptide ranging from 1 ng/ml to 1 g/ml. In

these experiments, rhIL-2 was omitted to be able to judge cytokine-producing potential outside a proinflammatory environment. Without the addition of exogenous IL-2, not all clones were able to produce IFN- (Figure 3a -i). Moreover, responses to certain peptides, namely N72-3, S42, S112-3 and S201 were recognised more frequently at lower concentrations than others. We did not detect a consistent difference in pattern between clones generated from T cells activated ex vivo and those activated after stimulation in vitro. Although the responses of the two donors to most peptides seemed largely distinct, responses to S42 and S276 were broadly similar in both.

All clones were selected initially for IFN- production in response to antigen, in the presence of exogenous IL-2. However, to be effective in fighting viral infections and to minimise adverse effects, it is important that the T cells demonstrate a highly differentiated Th1 cytokine profile. The avidity of the TCR:pMHC II interaction, which can be affected by the nature of the presented peptide or the characteristics of specific TCRs, might affect T cell polarisation [12, 13] . Therefore, we examined if some CD4 + T cells specific for immunodominant peptides were more likely than others to demonstrate a skewed cytokine response.

Supernatant from cultures stimulated with specific peptide for 48 hour without exogenous IL-2 was therefore analysed for a panel of 12 T helper cell cytokines by a cytometric assay. Of the 12 cytokines investigated, IL17A, IL-17F and IL-22 never exceeded the detection threshold (not shown). As concluded earlier from the ELISA after 24-hour activation, most clones were able to produce IFN- even in the absence of exogenous IL-2 ( Figure 4 ). However, a substantial number of clones seemed poorly differentiated and additionally produced the Th2 cytokines IL-5 and IL-13, and to a lesser extent IL-4.

Although most peptides were detected by T cell clones with various levels of differentiation, T cells specific for some peptides were more likely to demonstrate a mixed Th1/Th2 phenotype than others. T cells specific for peptides S42, S201 and S276, in particular, frequently produced IL-5 and IL-13 ( Figure 4 and supplementary figure 2). Additionally, the incidence of IL-10 production varied depending on antigenspecificity, albeit that this cytokine was secreted mostly at relatively low levels. Nonetheless, this suggests that some CD4 + T cell clones may have an immunosuppressive Tr1-like phenotype that is not desirable for fighting infections. The levels of IL-2 in the supernatants were remarkably low but it is not clear if this is due to limited production or high consumption. There were no clear differences in polarisation between clones from different COVID-19 patients nor was there a general difference between clones derived from cells activated ex vivo or post in vitro activation. Although we cannot conclude for certain that the cytokine patterns directly reflect either the nature of the antigen or the characteristics of the specific TCR rather than merely the differentiation status of individual T cells, we would select TCRs from highly differentiated Th1 cells for potential transgenic T cell therapy, to err on the side of caution.

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The final step in this study was to determine the TCR sequence of the 81 T cell clones. This was done for two reasons. First, to confirm that the samples are indeed clones and not contaminated with other cells. Second, to further identify the cells based on their TCR sequence in order to open up the possibility of CD4 + T cell therapy with clearly defined populations. By using populations of T cell clones rather than single-sorted cells for TCR sequencing, we increased the probability of obtaining paired TCR and TCR chains and also improved reliability. As a result, we obtained reliable paired sequences for all our samples (Table 3 and supplementary data set 1). The sequences confirmed that 4 samples were not indeed clonal T cells. Nevertheless, these were left in the previous sections of the study as they did still respond to SARS-CoV-2 and the sequences of very limited diversity found within these samples may still corroborate information from true clones. Sample P54-172 was not clonal and thus does not represent a dual-specific T cell as earlier data suggested. Non-clonal cells were not used for the next analyses. Interestingly, despite the relatively small number of clones generated, 7 paired TCR and TCR sequences were shared between clones (Table 3 ). Some T cell clones had two in-frame TCR expressed at comparable levels, which seemed to coincide with certain epitopes. The variation in sequences overall seemed highly dependent on the specific epitope ( Figure 5a ). For example, whereas 15 out of 16 (94%) TRAV sequences from clones generated against peptide S42 were identical in TRAV35, clones specific for peptide S276 had 12 different TRAV among a total of 20. Notably, among clones specific for peptide S42, 4 out of 5 (80%) clones from patient 91 expressed the same TRAV35 also found in all 11 clones from patient 54. Two of the clones of patient 91 shared the TRAV35:TRAJ42 combination seen in all clones from patient 54, while 1 clone from patient 91 even shared an identical CDR3 sequence with 4 clones from patient 54. This is remarkable considering the differences in HLA haplotype between these two individuals but does demonstrate that some TCR sequences are not only dominant but also promiscuous over various HLA backgrounds. It should be noted, though, that despite the similarity in  chain sequences, this consistency was not found for the  chain, which may explain the differences in the cytokine responses of individual clones. Finally, we note that, in line with the use of variable and junctional regions, the length of TCR CDR3 sequences varied depending on the cognate antigen, with those specific for S112-3 in particular being notably short (Figure 5b ). TCR CDR3 length was highly variable in all groups (Figure 5c ).

In this limited study, we define a population of CD4 + T cells with potential for immunotherapy of COVID-19. Although vaccines are rolled out at high speed currently, there remains a notable part of the population that will remain dependent on treatment. T cell therapy may bolster the success of current antibody and antiviral treatments. The adequate and rapid treatment of COVID-19 is essential not only to overcome the initial respiratory disease, but concerns are also growing regarding longer term effects of SARS-CoV-2 infiltration of organs including the brain and pancreas [14, 15] .

Here, we focused on people hospitalised with COVID-19 at a range of severity. As also shown previously by others, these patients have a high level of HLA-DR and CD38 expression on both CD4 + and CD8 + T cells [11, [16] [17] [18] , thus suggesting an ongoing anti-viral T cell response. Because only a limited proportion of these in vivo activated T cells were expected to be SARS-CoV-2-reactive, we also looked for cells activated by in vitro stimulation. Although Spike is a major immunological target, in particular for antibody responses, others have previously demonstrated that other viral proteins are also targeted by T cells [4] .

We therefore examined responses to both Spike and Nucleocapsid proteins. In our study, only 3 out of 18 patients showed a clear response to the viral antigens. Other studies that have looked for immunodominant epitopes have mostly found responders in a much larger fraction of patients [7, 8, 10] .

In part, this may be explained by the fact that most other studies used convalescent patients whose immune responses have developed further. More importantly, studies such as Bacher et al [10] . have adopted elaborate techniques to detect as many responders as possible. This approach is much more sensitive than the analysis of CD137 after 24 hours of in vitro stimulation that we adopted. However, the advantage of our approach is that only interactions of relatively high avidity or cells with a low activation threshold are detected. Therefore, although we did not cover the full potential spectrum of the immune responses, we could focus on the strongest responding T cells that likely are most effective in anti-viral protection. Low-avidity interactions with SARS-CoV-2 antigens, most likely due to cross-reactivity, have been described in both healthy individuals and COVID-19 patients, but these may not provide robust protection [10, 17, 19 ]. Indeed, cross-reactive T cells did not demonstrate an overlap with TCR sequences identified in our study [19] . . This suggests that in humans too, the main boon of CD4 + T cells may be to accelerate recovery rather than provide primary protection.

Additionally, a patient with X-linked agammaglobulinaemia (XLA) who demonstrated a seemingly robust T cell response still required antiviral treatment with Remdesivir for a full recovery [22] . Therefore, CD4 + T cell therapy is a promising novel approach for immunotherapy following SARS-CoV-2 infection but may best be applied in combination with antibody treatment and/or antiviral drugs rather than as a standalone treatment.

We define and study 9 immunodominant epitopes, which all share a sequence of 9 or more amino acids with SARS-CoV-2 epitopes described previously [5, [7] [8] [9] . These studies, including ours, all use peptide pools to identify immunodominant epitopes. A very recent publication by Sallusto and colleagues identifies naturally processed epitopes with a focus on the RBD [23]. The two immunodominant, naturally processed and presented epitopes they identify, S346-365 and S446-485, overlap largely with our two peptides from the same region, S89-90 (353-371) and S112-3 (445-463). This confirms the physiological relevance of at least two of our targets. It is debatable how many epitopes need to be targeted for T cellmediated immunity to be successful. In the case of our patients, a large number of epitopes were targeted, but only a limited number of epitopes were targeted by multiple clones. In the case of a potential T cell therapy, therefore, generating substantial numbers of high quality clones against a limited number of dominant antigens may suffice.

One current concern is that the level of mutations in the Spike protein could render the neutralising effect of antibodies induced by the current generation of vaccines less effective. From the point of T cell therapy, this risk can be mitigated by assembling a pool of T cells that targets multiple antigens, including Spike and Nucleocapsid. Spike variants that cause particular concern currently because they spread more quickly and neutralising antibodies induced by existing vaccines may be less effective against them, -147) , but we found that this did not affect the IFN- response of specific CD4 + T cell clones. It has been suggested that mutations affect CD4 + T cell immunity less than neutralising antibodies [5, 24, 25] .

Each patient is thought to harbour T cells recognising at least 30-40 SARS-Cov-2 epitopes, with significant variability from person to person [5] . Moreover, CD4 + T cell epitopes had minimal overlap with antibody epitopes. It is therefore less likely for any particular mutation that avoids T cell recognition to provide a selective advantage and spread throughout the population. Pre-published studies appear to confirm that

This article is protected by copyright. All rights reserved while mutations in variants-of-concern of SARS-CoV-2 proteins impair the antibody protection provided by vaccines, the T cell response remains unaffected [24, 25] . This corroborates our own findings that the responses to immunodominant epitopes we identified were not affected by common Spike mutations.

In a very recent study of people vaccinated with either Pfizer-BioNTech (BNT162b2) or Moderna (mRNA-1273) mRNA-based COVID-19 vaccine, the authors identify 23 dominant peptides of the Spike protein, 5

of which overlap significantly with the 7 Spike peptides we identified as immunodominant in our patients [26] . Only S201 (801-815) and S276 (1101-1115) were not detected as immunodominant after vaccination. Overall, this implies that the T cell responses we identified are thus also relevant in a healthy, younger population exposed to much-used vaccines rather than the virus. This corroborates the potential of transgenic T cells expressing T cell receptors based on the sequences we identified for T cell immunotherapy. Because the SARS-CoV-2 Nucleocapsid protein is not currently targeted by any licenced vaccine, a T cell therapy based on our data could offer additional benefits.

We confirm here that when studying immune responses to viral antigen, it is important not to focus solely on IFN- production to identify cells with a desirable phenotype. Many of our clones that produce large amounts of IFN- in response to even low concentrations of peptide seemed not fully differentiated Th1 cells but also produced Th2 cytokines such as IL-5 and IL-13. Moreover, some co-produced IL-10, suggesting that they may have immunoregulatory, Tr1-like properties [27] . Certain peptides seem to be more likely to be targeted by pluripotent Th1/Th2 cells than others. In cases where we found two T cell clones with identical TCR and  sequences, these did not necessarily have identical cytokine responses to the same cognate peptide. For example, while clones 55 and 166 from patient 54 have identical TCRs and both produce ample IFN- in response to cognate peptide, the former co-produced IL-13. . It may thus be disputed to what extent the characteristics of the TCR contribute to Th1/Th2 differentiation [12, 13] .

Other factors such as, for example, the differentiation status or length of expansion of a clone could also be hypothesised to affect this. This should be kept in mind when translating findings for (transgenic) T cell therapy.

Finally, by using a technique developed for single cell analysis to investigate the TCR repertoire of T cell clones, we managed to reliably obtain paired sequences for all our samples with enough information to clone the exact TCR. This approach of identifying full-length, paired sequences is to our knowledge unique when it comes to characterising SARS-CoV-2-specific T cells. Unlike studies where only one TCR chain or only CDR3 regions were sequenced, we can now select the exact TCR sequences from T cell clones with the most desirable properties. By applying techniques established within our facilities we can generate

This article is protected by copyright. All rights reserved SARS-CoV-2 antigen-specific TCR-transgenic T cells using either a patient's own endogenous T cells or, in the case of haematopoietic stem cell transfer, an HLA-matched donor's. The similarity in TCR sequences that we found between different donors is encouraging for the creation of T cell therapy products that may be applicable over a range of HLA backgrounds, thus augmenting the feasibility of this approach. It should be noted, though, that although our donors demonstrated a range of HLA haplotypes characteristic for the German population, wider investigation of antigen-specific T cell responses with donors from more diverse backgrounds will be required for a global approach. Nevertheless, based on the data generated in this study, we will proceed towards the development of a therapeutic approach that can be tested clinically.

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Bernd Spriewald and colleagues of the laboratory for immunogenetics at the University Hospital Erlangen are acknowledged for all HLA haplotyping. We thank Alina Kämpf, Lina Meretuk and Alina Bauer for excellent technical assistance.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Accepted Article NFSQILPDPSKPSKR (Peng et al [8] and Tarke et al [5] ),

276 HWFVTQRNFYEPQII (S1101-1115) HLA-DRB1*04:05 HLA-DQA1*01:01/DQB1*05:01

Sequential, overlapping peptides recognised by the same T cell clones were combined. Predicted HLA affinity results with percentile ranking of < 10. Overlap of 9 or more amino acids with published sequences is marked in bold, Italic font. Table 3 . TCR  and  chain identification in CD4 + T cell clones grouped by specific epitope. Sequences from patient 91 are in italic font to distinguish them from patient 54 sequences. Four samples had more than one  chain that could not be explained by technical errors and are listed as "not a clone".

Figure Legends concentration (pg/ml) S112-3 P 9 1 -1 5 5 P 9 1 -1 5 8 P 9 1 -4 9 1 P 9 1 -4 9 2 P 9 1 -5 0 2 P 9 1 -5 3 0 P 9 1 -5 3 3 P 9 1 -5 3 6 P 9 1 -5 5 0 P 9 1 -5 7 2 P 9 1 -5 7 9 P 9 1 -4 5 9 cei_13627_f5.pdf This article is protected by copyright. All rights reserved

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Accepted Article This article is protected by copyright. All rights reserved

The authors thank all participants included in this study. The Core Unit Cell Sorting and Immunomonitoring Erlangen (Florentine Schonath and Uwe Applet) supported this work. Prof. dr. med.