key: cord-0429986-3i4jefut authors: Echaide, M.; Labiano, I.; Delgado, M.; Fernandez de Lascoiti, A.; Ochoa, P.; Garnica, M.; Ramos, P.; Chocarro de Erauso, L.; Fernandez, L.; Arasanz, H.; Bocanegra, A.; Blanco, E.; Pineiro, S.; Zuazo, M.; Morente, P.; Vera, R.; Alsina, M.; Escors, D.; Kochan, G. title: Profiling of immune responses to COVID-19 and vaccination uncovers potent adjuvant capacities of SARS CoV-2 infection to vaccination leading to memory T cell responses with a Th17 signature in cancer patients date: 2022-05-27 journal: nan DOI: 10.1101/2022.05.27.22275672 sha: 226fb442828d15139a82711fb5058fc84d7eb962 doc_id: 429986 cord_uid: 3i4jefut Previous studies have shown differing immune responses in cancer patients towards natural infection and vaccination compared to healthy individuals. Therefore, it is yet unclear whether cancer patients show differential responses to SARS CoV-2 natural infection and vaccination with current mRNA vaccines. Immune profiling was performed in three cohorts of healthy donors and oncologic patients: infected with SARS CoV-2, BNT162b2-vaccinated, and vaccinated with previous SARS CoV-2 infection. Vaccine was found to be a poor inductor of S-specific T cell responses compared to natural infection, which acted as a potent adjuvant for vaccination in antibody and T cell responses. Antibodies towards the M protein were a biomarker of disease severity, while the major targets for T cell responses in natural infection were the M and S protein, but not the N protein. T cell responses quickly decayed after 6 months post-vaccination. T cell profiling showed that vaccination expands effector T cells rather than memory T cell subsets unless the subjects had previous COVID-19 disease. Cancer patients with previous COVID-19 and subsequently vaccinated exhibited exacerbated CD8 responses, with elevated IL-17 CD4 and CD8 T cell subsets, and neutrophils. Concluding, a previous COVID-19 infection has potent adjuvant effects for vaccination leading to memory T cell differentiation, but with enhanced inflammatory responses in cancer patients. Severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) caused a new outbreak of pneumonia in Wuhan, China, in December 2019 [1] . Since then, it has caused the COVID-19 pandemic [2] . Patients with cancer are thought to be at higher risk of contracting a severe disease leading to death [3] . Patients with cancer often present comorbidities and risk factors associated with COVID-19 severity, including older age, chronic inflammation and genetic alterations associated with severe disease [2] [3] [4] [5] . Patients with cancer are usually immunocompromised by the disease and antineoplastic treatments [3, [6] [7] [8] . Another frequent feature in cancer patients is T cell senescence. During aging, T cells proceed towards terminal differentiation by a sequential loss of CD27 and CD28 co-receptor surface expression [9, 10] . T cell senescence is characterized loss of effector functions and impaired anti-viral immunity. Senescent T cells are enriched in effector phenotypes such as effector-memory (CD62L-CD45RA-) and effector T cells (CD62L-CD45RA+), with a loss of central memory (CD62L+ CD45RA-) and naïve (CD62L+ CD45RA+) phenotypes [10] . On top of this, cancers can exacerbate chronic inflammation, which may favour pro-inflammatory cytokine release that may contribute to COVID-19 clinical syndrome [11, 12] . It is yet unclear who these alterations impact immunity against SARS-CoV-2 and responses to vaccination [3, 13, 14] . Immune responses to SARS CoV-2 infection in healthy subjects are diverse and complex [15, 16] , and only few studies have addressed this in cancer patients. In general, oncologic patients have shown comparable antibody responses [13, [17] [18] [19] . However, T cell responses were strongly reduced in oncologic patients [17] . Although regarded as a risk population, cancer patients were underrepresented in clinical trials assessing vaccine safety and efficacy [20, 21] . Overall, high seroconversion rates were shown with comparable or slightly lower antibody titres in patients with solid tumours compared to healthy donors [19, 20, [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] . However, a meta-analysis of 35 studies suggested lower protection by vaccination in oncologic patients [35] . T cell activities towards the S protein [22, 23, 29, 33] showed from insufficient responses [22] , lower activation rates [23, 33] or comparable to healthy donors [29] . So far, detailed information on SARS CoV-2 immunity and responses to vaccination in patients with cancer is still lacking [14, 35] . For instance, three of the structural proteins (S, M and N) are the main components of the coronavirion [36, 37] , but only the S protein is included in most vaccine formulations in Europe. Therefore, immune responses towards other structural proteins remain poorly studied [38, 39] . Finally, it is still far from clear whether previous infection affects the responses to vaccination in cancer patients, particularly in T cell immunity. This study was conducted according to the principles of the Declaration of Helsinki. ). The total sample size of the study was established a priori to achieve a minimum power of 0.8 considering a large effect size (f=0.4) using Gpower 3.1 [40] . General clinical characteristics and SARS CoV-2-related parameters of the study cohort are summarized in Table 1 and Table 2 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Blood collection, PBMC, myeloid cells and T cell purification, activation and flow cytometry were carried out as previously described [41] . The following fluorochrome- For ELISA and stimulation studies M, S and N proteins were produced using Bac-to-Bac baculovirus expression. Briefly, synthetic genes encoding S1 (1-303 amino acid), full length N and the cytoplasmic domain of the M protein (1-100 amino acid) were fused to histidine tags and cloned. Protein production and purification by Ni-NTA affinity and size exclusion chromatographies were performed following standard protocols (Bac-to-Bac Thermofisher). Donor sera were obtained from peripheral blood, centrifuged and frozen at -20ºC. For detection of S and N specific antibodies, a 96-well plate was coated with 5 µg/mL of the corresponding protein, followed by blocking with 1X PBS-2% BSA. 1:800, 1:250 and 1:80 sera dilutions were used for detection of anti-S antibodies, anti-N antibodies and anti-M antibodies, respectively. Anti-human IgGs HRP-labelled antibody (ThermoFisher) was used as secondary antibody. ELISAS were developed with 100 µL TMB substrate (Sigma) and read at 450 nm. Statistical analyses were performed with GraphPad 8. Variables under study were tested for normality (Kruskal-Wallis test), homogeneity of variances (F test), and homogeneity (Spearman´s coefficient of variation). Antibody titres and percentages of cell types as quantified by flow cytometry were either not normally distributed or showed high variability. For multi-group comparisons of these variables, non-parametric Kruskal-Wallis tests were performed followed by pair-wise comparisons with Dunn's test. For experiments involving only two independent groups, the non-parametric U of Mann Whitney was used. The percentages of T cell phenotypes were normally distributed, homogeneous and with comparable variances. In this case, one-way ANOVAs were carried out followed by a posterori pair-wise comparisons with Tukey's test. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Clinical and SARS-CoV-2-related characteristics of the cohorts are summarized in Table 1 and Table 2 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Neumo., neumopathies; NT, non-treated; TKI, tyrosin-kinase inhibitors; Q1, quartile 1; Q3, quartile 3; vac, vaccinated. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. CoV, SARS CoV-2 infection; vac, vaccinated; CT, chemotherapy; IT, immunotherapy; NT, non-treated; SD, standard deviation; TKI, tyrosin-kinase inhibitors; Q1, quartile 1; Q3, quartile 3; vac, vaccinated. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. IgG antibody responses were evaluated towards SARS CoV-2 infection and vaccination. IgG antibody titres towards the viral proteins S, M and N were quantified, and sera from pre-pandemic donors served as technical negative controls (TC). Infected healthy and oncologic individuals (H-CoV and O-CoV) had low S-specific IgG titres [42] . No differences were observed as well between H-N/N (healthy no COVID, no All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 27, 2022. Dunn's test for selected pair-wise comparisons. *, **, *** and **** indicate a p value <0.05, <0.01, <0.001 and <0.0001, respectively. Specific T cell responses towards the three main structural proteins were evaluated in peripheral blood mononuclear cells (PBMCs). PBMCs were incubated with viral proteinspecific peptivators and upregulation of the early activation markers CD154 and CD137 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Most oncologic patients have dysfunctional T cell immunity with altered T cell phenotypes [42] . To investigate if this was the case after infection or vaccination, CD62L and CD45RA expression profiles were characterized in S-specific CD4 T cells All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The strongest inducer of CD8 T cell responses was having a previous infection with COVID-19 both for O and H donors but not vaccination alone ( figure 3a-3c and supplementary figure S7 ). There was a tendency to increased percentages of S-specific CD8 T cells in O patients which could be caused by the differences in COVID-19 severity All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. As severe COVID-19 is associated with exacerbated inflammatory responses, IFNγ and IL-17 expression was evaluated first within S and M-specific CD4 T cells following stimulation (supplementary figure S9) . Overall, the proportion of inflammatory CD4 T In H donors, the CoV-V group showed the strongest responses ( figure 4a, 4b) . Similar results were observed for M-specific IFNg-CD4 T cells (figure 4d-4f) . (figure 4g, 4h) . O patients showed a non-significant trend towards increased IL-17 CD4 T cells compared to H donors (figure 4i). Equivalent results were obtained for the M protein ( figure 4j-4l) . Overall, these results indicated a stronger inflammatory response in O patients compared to H donors that could be associated to disease severity or cancer (Supplementary figure S2b). As expected from our previous results, inflammatory CD4 T cell subsets decayed 6 months after vaccination (figure 4m, 4n). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. test. NST, technical control of non-stimulated PMBCs. *, **, *** indicate significant (P<0.05), very significant (P<0.01) and highly significant (P<0.001) differences. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Inflammatory S and M-specific CD8 T cell subsets were quantified (supplementary figure S9b). Infection but not vaccination was the strongest inducer of S-specific IFNγ- (figure 5a-5c) . Similar results were obtained for Mspecific T cells (figure 5d, 5e), without differences between H donors and O patients (figure 5f). There were however marked differences for for IL17-CD8 T cells, which were increased in subjects with previous COVID -19 following vaccination (figure 5g, 5h) . Although there was high variability, we observed a tendency towards increased IL17- H donors (figure 5i) . Similar results were obtained for M-specific CD8 T cells (figure 5j, 5k) , which were comparable between H donors and O patients (figure 5l). Although responses were in general low, inflammatory S-and M-specific CD8 T cells decayed 6 months after vaccination (figure 5m, 5n) All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. H and O groups. j, k, l) All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. test. NST, technical control of non-stimulated PMBCs. *, **, *** indicate significant (P<0.05), very significant (P<0.01) and highly significant (P<0.001) differences. The percentages of monocytes, granulocytes and neutrophils were quantified in peripheral blood and no differences were found in H donors (figure 6a-c) (figure 6b-6f) . Hence, COVID-19 could perturb systemic immunity in cancer patients towards responses mediated by granulocytes rather than monocytes. Baseline percentages of circulating CD19+ CD14-B cells were also quantified, without finding significant differences (supplementary figure S10a-S10c). Nevertheless, there was a tendency in O patients to have decreased percentages of circulating B cells (supplementary figure S10d). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Oncologic patients usually have a compromised immunity from cancer progression and treatments [10, 43] which may impact on responses to COVID-19 and vaccination. In this study we included S-, M-and N-specific T cell profiling and myeloid cell signatures. Most of donors had been vaccinated with mRNA BNT162b2. This vaccine is a potent inducer of S-specific antibodies [44-46] and we found that antibody responses were not impaired in cancer patients in agreement with others [47] . In this study, we confirmed All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 27, 2022. ; https://doi.org/10.1101/2022.05.27.22275672 doi: medRxiv preprint that antibody titres decreased over time which would limit serological protection to 6 months [48] . Importantly, T cell responses after vaccination also decayed after 6 months. Indeed, vaccination did not preferentially expand memory T cell subsets, unless the subjects had previous COVID-19. Hence resolution of COVID-19 disease followed by vaccination may confer longer protection for both healthy donors and oncologic patients. Vaccination mainly induced CD4 T cells, in contrast to SARS CoV-2 infection, which could explain its potency of raising antibody responses possibly through activated CD4 T helper cells [49] . T cell responses towards M and S proteins were found previously [50, 51] , but we extended this observation to O patients. In general, M protein was found to be a potent target for CD8 T cell responses even when compared to the S protein. Therefore, the M-protein could be key for developing novel vaccines. O patients showed differences in T cell immunity compared to healthy donors. Their T cell repertoire was skewed towards differentiated phenotypes expressing IFNγ as shown before [23, 52] , but also IL17 as assessed here in H donors and O patients [53] . Vaccination induced IFNγ and elevated IL-17 in CD4 T cells, a marker of Th17 responses [23, 52] . Indeed, SARS-CoV-2 infection also induced a Th17 signature, which could be important for disease severity. Finally, the profiles of circulating myeloid subsets was in agreement with oncologic patients having more inflammatory profile, as expected in cancer patients [43] . This could be detrimental for vaccine efficacy. Elevated neutrophil counts are frequent in O patients [54, 55] , which were even higher in subjects with previous COVID-19. A relationship between COVID-19 severity and higher monocyte and granulocyte content was found in early studies [56, 57] . Concluding, cancer patients showed antibody, T cell and myeloid responses to infection and vaccination. Previous SARS CoV-2 infection had potent adjuvant effects for subsequent vaccination. However, cancer patients showed baseline inflammation, which could be exacerbated upon infection followed by vaccination. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. figure S5. Sequence alignment. a, b, c) Conserved region of the spike protein (a), membrane protein (b) and nucleocapsid protein (c) of SARS-CoV-2 (in red) with the indicated human coronavirus counterparts: 229E (in green), NL-63 (in orange), OC-43 (in blue) and HKU-1 (in purple). Conserved regions are highlighted in grey. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 27, 2022. ; https://doi.org/10.1101/2022.05.27.22275672 doi: medRxiv preprint k) Statistical significance was tested by ANOVA followed by Tukey´s pair-wise comparisons. *, ** and *** indicate, significant (P<0.05), very significant (P<0.01) and highly significant (P<0.001) differences. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. subjects. Statistical significance was tested with Krustal-Wallis followed by Dunn´s pair-wise comparisons. *, **, and *** indicate significant (P<0.05), very significant (P<0.01) and highly significant (P<0.001) differences. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Feng Z. 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The OncoImmunology group is funded by the Spanish Association against Cancer All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.