key: cord-0878440-p41gz6st
authors: García‐Jiménez, Álvaro Fernando; Cáceres‐Martell, Yaiza; Fernández‐Soto, Daniel; Martínez Fleta, Pedro; Casasnovas, José M.; Sánchez‐Madrid, Francisco; Frade, José Miguel Rodríguez; Valés‐Gómez, Mar; Reyburn, Hugh T.
title: Cross‐reactive cellular, but not humoral, immunity is detected between OC43 and SARS‐CoV‐2 NPs in people not infected with SARS‐CoV‐2: Possible role of cT(FH) cells
date: 2022-04-05
journal: J Leukoc Biol
DOI: 10.1002/jlb.4covcra0721-356rrr
sha: a231951a9dccb98dc5db86097e33424eb4a13c44
doc_id: 878440
cord_uid: p41gz6st

Multiple questions about SARS‐CoV‐2 humoral and cellular immunity remain unanswered. One key question is whether preexisting memory T or B cells, specific for related coronaviruses in SARS‐CoV‐2‐unexposed individuals, can recognize and suppress COVID‐19, but this issue remains unclear. Here, we demonstrate that antibody responses to SARS‐CoV‐2 antigens are restricted to serum samples from COVID‐19 convalescent individuals. In contrast, cross‐reactive T cell proliferation and IFN‐γ production responses were detected in PBMCs of around 30% of donor samples collected prepandemic, although we found that these prepandemic T cell responses only elicited weak cT(FH) activation upon stimulation with either HCoV‐OC43 or SARS‐CoV‐2 NP protein. Overall, these observations confirm that T cell cross‐reactive with SARS‐CoV‐2 antigens are present in unexposed people, but suggest that the T cell response to HCoV‐OC43 could be deficient in some important aspects, like T(FH) expansion, that might compromise the generation of cross‐reactive T(FH) cells and antibodies. Understanding these differences in cellular responses may be of critical importance to advance in our knowledge of immunity against SARS‐CoV‐2.

Coronavirus disease 2019 , caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprises a set of clinical features varying from asymptomatic to severe symptoms and even death. 1 

and the endemic human coronaviruses (HCoVs) HKU1, 229E, NL63, and OC43 have been suggested to influence SARS-CoV-2 immunity and in vitro studies have shown notable T-cell reactivity to SARS-CoV-2 antigens in 25-30% of healthy, noninfected individuals. [2] [3] [4] [5] In contrast, cross-reactive humoral immunity against SARS-CoV-2 antigens, principally directed to the S2 domain of the spike glycoprotein, is present in few healthy donor samples. 6, 7 The seasonal HCoVs belong to the Alpha-(229E, NL63) and Beta-(HKU1, OC43) coronavirus genus and are around 30% homologous to SARS-CoV-2 spike or nucleocapsid proteins. Infection with these HCoVs typically causes mild respiratory symptoms and generates short-lived antibody responses that cannot block reinfection only 12 months after infection. 8, 9 It has been suggested, but is not known for certain, that the presence of cross-reactive cellular or humoral responses might modulate the development of COVID-19. 10, 11 The vast majority of published studies assessing specific SARS-CoV-2 T-cell immune recognition use sets of overlapping 10 amino-acid peptides that span part, or the whole, proteome of SARS-CoV-2. In this study, we directly assessed the SARS-CoV-2 T and B cell responses using full-length recombinant protein antigens in sets of sera and PBMCs collected prepandemic as well as from convalescent COVID-19 patients.

Thus, the present work extends knowledge about humoral and cellular immunity to SARS-CoV-2 and investigates immunologic cross-reactivity between coronaviruses from a different point of view.

We first determined the serum antibody responses to SARS-CoV-2 and HCoV-OC43 nucleocapsid protein antigens in a cohort of 24 serum samples collected from healthy blood donors between 2010 and 2014. Serum samples from 7 convalescent COVID-19 blood samples were included in these experiments as positive controls. We used OC43 nucleoprotein (NP) as a representative betacoronavirus antigen that was phylogenetically closer to SARS-CoV-2 and demonstrated the highest degree of amino acid sequence overlap among the common endemic coronaviruses ( Figure S1 ). In general, we could detect a wide variety of immunoglobulin responses to OC43 NP antigen in healthy and convalescent samples, ranging from undetectable HCoV-OC43 antibodies to those with medium or high titer antibody responses, perhaps dependent on the B cell capacity and the period elapsed after infection. However, SARS-CoV-2 NP-specific antibodies were only detected in sera from convalescent COVID-19 patients ( Figure 1 ). These serologic observations are consistent with other reports showing little antibody cross-reactivity between SARS-CoV-2 and endemic coronaviruses in healthy prepandemic donors 6, 12 and potentially suggest that antibody responses to these viral proteins are independent.

To assess T-cell cross-reactivity between coronaviruses, viral antigeninduced T-cell proliferation was determined in PBMCs collected from the prepandemic healthy donors (B1-B14) available for study. PBMCs from COVID-19 convalescent individuals (C1-C6) were used as positive controls. OC43 and SARS-CoV-2 NPs were used to stimulate T-cells. Additionally, we also evaluated the potential of the SARS-CoV-2 spike (S) protein, directly involved in viral entry, 13 and the SARS-CoV-2 nonstructural protein 3CL pro , that potently stimulates humoral immunity. 14 CD4 + , CD8 + , and CD3 + specific T-cell proliferation was assessed by cytometry analysis of CTV dilution ( Figure 2 Thus, antigen-specific T-cell proliferation assays support the observation that preexisting SARS-CoV-2 T-cell immunity is present in some unexposed people. Preexisting T-cells responding to SARS-CoV-2 spike and NPs were mainly found in the CD8 + compartment, contrary to what is expected using soluble exogenous proteins as antigens. This observation is interesting as, if assuming cross-reactivity between HCoVs, there is no clear enrichment for presentation through HLA-I alleles within these antigens ( Figure S2 ). In line with this, convalescent individuals also showed similar but stronger trends in T-cell subset proliferation against these antigens, clearly showing the expansion capacity of SARS-CoV-2-specific CD8 + T-cells when stimulated in vitro. 15 

Cytokine production after antigen-specific or nonspecific restimulation was also measured to gain insight into which CD4 + T cell subsets were involved in the immunity to SARS-CoV-2. To this end, PBMCs from the proliferation assays were rested in IL-2 and then (Figure 3(B) ). 13 These analyses showed that SARS-CoV-2 responding T-cells produce much more IFN-γ than IL-17A or IL-10 and also demonstrated clear evidence for bidirectional T-cell cross-reactivity between HCoV-OC43 and SARS-CoV-2 NPs in pandemic and prepandemic samples.

Thus, our work is consistent with those articles that found T-cell crossreactivity against SARS-CoV-2 peptides with homology to human endemic coronaviruses. 2, 3 These data also argue against the idea that coronavirus CD4 + T-cell cross-responses are practically absent and of low avidity, 16 although our observations do suggest a major role for CD8 + subset that, strikingly, was overexpanded in COVID-19 patients in response to OC43 NP (Figure 2(A) ). Consistent with previous work, examples of healthy donor T-cell IFN-γ production against RBD were also detected. 3 Conversely, the prepandemic samples with the greatest expansion and IFN-γ production against OC43 NP (donors B3, B7, and B8) also demonstrated reactivity against all SARS-CoV-2 antigens tested (Figure 3(C) ), probably indicating a strong and diverse epitope immunity versus HCoV-OC43 that consequently triggers a wider SARS-CoV-2 antigen spectrum. F I G U R E 2 T cell proliferation assays. PBMCs were labeled with the cytoplasmic dye Cell Trace Violet (CTV-5 μM) and pulsed with a specific recombinant protein antigen (10 μg/ml) to evaluate the specific proliferation after culture for 6 days. (A) CD4 + , CD8 + , and CD3 + T cell proliferation were assessed by cytometry analysis for CTV dilution within the total CD3 + population. Samples with proliferation values above 0.5% are labeled. Two-sided Mann-Whitney test (*p < 0.05, **p < 0.01). (B) Expression of CCR7 and CD45RA markers within the CD3 + CTV low events detected in healthy samples responding to both OC43 and SARS-CoV-2 NP. (C) Percentage of convalescent or healthy donors with detectable CD4 + , CD8 + or CD3 + proliferation against the different antigens

The previous experiments indicated that SARS-CoV-2 reactive T cells could be found in PBMCs collected prepandemic, including specific T cell cross-reactivity between SARS-CoV-2 and HCoV-OC43 NPs antigens. Nevertheless, antibodies able to bind SARS-CoV-2 NP antigen were not detected in serum samples from unexposed donors.

and humoral immunity, 17 (Figures 4(A) and 4(B) ). The proportion of cT FH cells in PBMCs upon stimulation was highly increased in these COVID-19 convalescent donors, and marked differences in cT FH responses were observed between COVID-19 samples suffering from mild or severe disease ( Figure S3 ), consistent with data reporting that SARS-CoV-2-specific antibody titers are higher in patients that have experienced severe disease. 18, 19 Moreover, for the majority of COVID-19 F I G U R E 3 T cell cytokine production assays. Proliferating PBMCs were rested in culture with IL-2 (5 U/ml) and then restimulated by exposure to freshly thawed autologous PBMCs pre-pulsed with different coronavirus antigens (10 μg/ml). (A) IFN-γ, IL17-A, and IL-10 T cell production were analyzed after antigen reexposure and (B) after restimulation with different antigens. Samples with positive events above 0.2% were labeled. Bar plots showing mean values ± SEM. (C) Heatmaps showing tendencies of CD3 + proliferation and IFN-γ production after restimulation with the same or different antigens for B1-B8 healthy samples convalescent patients, levels of SARS-CoV-2 NP-specific antibodies and cT FH cells correlated (Figure 4(C) ), supporting the key role of these cells in humoral immunity.

It is conceivable that the presence of cT FH migrating from secondary lymphoid tissue after recent infection might contribute to the higher frequencies of SARS-CoV-2 cT FH cells detected in the COVID-19 convalescent patients; however, humoral immunity to seasonal coronaviruses like OC43 is short lived and reinfections are common, suggesting that cT FH would likely also be frequent in our panel of healthy donors, many of whom have considerable titers of anti-OC43 NP antibodies (Figure 1 ). To try and assay antigenspecific cT FH reactivity when the subject is not undergoing an active immune response, we compared HCoV cT FH reactivity and cross-reactivity to OC43 and SARS-CoV-2 NPs with cT FH responses made by healthy donors against the M1 antigen of another seasonal respiratory virus, influenza. In these experiments, the NP-specific cT FH responses observed in COVID-19 convalescent donors were markedly higher than the responses of prepandemic donors to either OC43 or SARS-CoV-2 NPs (Figure 4(D) ), supporting the idea that CXCR5 + CD4 + from COVID-19 patients are enriched in migrating cT FH . Nevertheless, in healthy samples, the frequency of SARS-CoV-2 and OC43 NP-responsive cT FH among CD4 + T cells responding to those antigens was significantly lower than the frequency of cT FH detected after stimulation with the Influenza A antigen (Figure 4(D) ), consistent with the suggestion that cT FH responses to the coronavirus NP antigens are comparatively weak. Figure S4 ). These data suggest that SARS-CoV-2 antigen-specific T FH cells, that aid B cell responses, were generated efficiently in these patients, indeed the magnitude of the T FH response made by SARS-CoV-2 patients is comparable to that seen in vaccination trials for HIV or Ebola. 20, 21 Moreover, strikingly, recent studies showed that SARS-CoV-2 mRNA vaccination induces cT FH in blood and lymph nodes that are maintained at constant levels for at least 6 months. 22 In contrast, the levels of OC43 NP-specific CD4 + cT FH activation noted in PBMCs from OC43 NP responding prepandemic donors were lower than that induced by the M1 antigen from another seasonal virus like Influenza, and consequently, potential cT FH cells cross-reactive with SARS-CoV-2 NP were rather insignificant. Interestingly, Meckiff et al. 23 also described a marked in vitro expansion of noncytotoxic T FH cells in convalescence that was not found in T-cells from healthy donors that displayed cross-reactivity between SARS-CoV-2 and other coronaviruses. These differences in T FH responses may contribute to explain, at least in part, why antibody cross-reactivity is generally absent in SARS-CoV-2 unexposed samples. An interesting idea that could be explored in future experiments is whether this paucity of circulating T FH cells reflects polarization of the T-cell response after frequent, recurrent seasonal coronavirus infections that may also underlie the typical antibody waning observed. 8 

The present report is clearly limited in sample size and number of antigens studied. Moreover, for obvious reasons, T cell subpopulations are only assessed in peripheral blood through the detection of general or classical markers and not in secondary lymphoid tissues.

However, the use of full-length antigens in prolonged assays may represent a more realistic approach even though clonotype specificities are not determined, and in this sense, the results are clear in highlighting that T FH cells can be a key intermediary population that can influence the development of cross-reactive antibodies in SARS-CoV-2 unexposed people. It is possible that COVID-19 cT FH cells may be more frequent in peripheral blood because of recent infection, while detecting cT FH in healthy human donor peripheral blood tends to be more difficult unless they have undergone a recent response to HCoV. Further studies, using more antigens from other coronaviruses, would help to confirm that T FH cells are underrepresented in the immune memory against common endemic coronaviruses, and importantly, would contribute to determine if this trend can also be present in reinfections with SARS-CoV-2.

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Cross-reactive cellular, but not humoral, immunity is detected between OC43 and SARS-CoV-2 NPs in people not infected with SARS-CoV-2: Possible role of cT FH cells

The authors wish to thank the blood donors, and the Biobank Hospital Universitario Puerta de Hierro Majadahonda (HUPHM)/Instituto de Investigación Sanitaria Puerta de Hierro-Segovia de Arana (IDIPHISA)

(PT17/0015/0020 in the Spanish National Biobanks Network) for the human specimens used in this study.