key: cord-0836493-id0qa28t authors: Taborska, Pavla; Lastovicka, Jan; Stakheev, Dmitry; Strizova, Zuzana; Bartunkova, Jirina; Smrz, Daniel title: SARS-CoV-2 spike glycoprotein-reactive T cells can be readily expanded from COVID-19 vaccinated donors date: 2021-05-29 journal: bioRxiv DOI: 10.1101/2021.05.27.446089 sha: 526b409d8f519f169aa3c549f784eeaab786f319 doc_id: 836493 cord_uid: id0qa28t Introduction The COVID-19 vaccine was designed to provide protection against infection by the severe respiratory coronavirus 2 (SARS-CoV-2) and coronavirus disease 2019 (COVID-19). However, the vaccine’s efficacy can be compromised in patients with immunodeficiencies or the vaccine-induced immunoprotection suppressed by other comorbidity treatments, such as chemotherapy or immunotherapy. To enhance the protective role of the COVID-19 vaccine, we have investigated a combination of the COVID-19 vaccination with ex vivo enrichment and large-scale expansion of SARS-CoV-2 spike glycoprotein-reactive CD4+ and CD8+ T cells. Methods SARS-CoV-2-unexposed donors were vaccinated with two doses of the BNT162b2 SARS-CoV-2 vaccine. The peripheral blood mononuclear cells of the vaccinated donors were cell culture-enriched with T cells reactive to peptides derived from SARS-CoV-2 spike glycoprotein. The enriched cell cultures were large-scale expanded using the rapid expansion protocol (REP) and the peptide-reactive T cells evaluated. Results We show that vaccination with the SARS-CoV-2 spike glycoprotein-based mRNA COVID-19 vaccine induced humoral response against SARS-CoV-2 spike glycoprotein in all tested healthy SARS-CoV-2-unexposed donors. This humoral response was found to correlate with the ability of the donors’ PBMCs to become enriched with SARS-CoV-2 spike glycoprotein-reactive CD4+ and CD8+ T cells. Using an 11-day rapid expansion protocol, the enriched cell cultures were expanded nearly a thousand fold, and the proportions of the SARS-CoV-2 spike glycoprotein-reactive T cells increased. Conclusions These findings show for the first time that the combination of the COVID-19 vaccination and ex vivo T cell large-scale expansion of SARS-CoV-2-reactive T cells could be a powerful tool for developing T cell-based adoptive cellular immunotherapy of COVID-19. vivo T cell large-scale expansion of SARS-CoV-2-reactive T cells could be a powerful tool for developing T cell-based adoptive cellular immunotherapy of COVID-19. COVID-19 is transforming into more severe and contagious forms as the severe respiratory coronavirus 2 (SARS-CoV-2) mutates during the pandemic [1] . The recently appeared new mutations of the virus seem to evade the immune system more efficiently, including evasion of the coronavirus disease 2019 (COVID-19) vaccineinduced immunity [2] . This evasion lies in the decreased capability of SARS-CoV-2specific antibodies to neutralize the virus efficiently [3] . Since the antibody-mediated protection depends on the structure of the target antigen, any mutation causing a productive conformational change of the target antigen can decrease the antibody binding and erode its protective role [4, 5] . The current COVID-19 vaccines are nearly exclusively targeting a single protein of the virus, the spike glycoprotein, so chances of evasion could be high. The antiviral immune response also relies on adaptive cellular immunity where the antiviral effectors are, instead of antibodies, the cytotoxic CD8 + T cells which recognize infected cells expressing viral proteins [6] . Unlike antibodies, the viral proteins are recognized in the form of protein fragments (peptides) presented in the context of the major-histocompatibility complexes and T cell receptors (TCR) [6] . Recent studies show that SARS-CoV-2 T cell-based immunity is negligibly impacted by the current mutated variants of SARS-CoV-2 [7] and, therefore, could counteract the debilitating impact these mutations might have on the parallel humoral immunity [8] . However, many immunocompromised patients, patients with immunodeficiencies, or patients with a comorbidity treatment-suppressed immunity, such as patients undergoing chemotherapy or immunotherapy, may not sufficiently mobilize the cellular immunity against SARS-CoV-2 after vaccination. It is, therefore, necessary to find new ways to enhance their cellular immunity against the virus. This study examined the impact of the COVID-19 vaccine on the induction of the humoral and cellular responses in 8 healthy SARS-CoV-2-unexposed donors. We investigated whether the cellular response to the vaccine could be enhanced by the ex vivo enrichment and large-scale expansion and hence represent an avenue for promoting the SARS-CoV-2-specific cellular immunity in patients who could not fully benefit from the COVID-19 vaccines. We first investigated the humoral response of the mRNA SARS-CoV-2 spike glycoprotein-based COVID-19 vaccine, BNT162b2, in 8 healthy donors who tested negative for the presence of antibodies specific to SARS-CoV-2 spike glycoprotein and who reported no previous history of COVID-19 and/or positive tests for SARS-CoV-2. The donors were vaccinated with two doses of the vaccine within a (3-4)-week interval. Samples, the serum and unclotted blood, were collected during the 2 days before each vaccination and 3-4 weeks after the second dose of the vaccine. Using a microblot system, the sera were analyzed for the presence of SARS-CoV-2-specific IgA, IgG, or IgM antibodies against the virus proteins: receptor-binding domain (RBD) and S2 subunits of the spike glycoprotein (S2), nucleocapsid protein (NCP), envelope protein (EP), and papain-like protease (PLP) [9] . As negative controls, the sera were analyzed for the presence of antibodies specific to human ACE-2 protein [10] or proteins from other coronaviruses: S1 subunit of the Middle East respiratory syndrome-related coronavirus spike glycoprotein (MERS-CoV) [11], nucleocapsid protein of SARS-CoV [12] , human coronavirus 229E [13] and NL63 [14] . As shown in Fig. 1A , the COVID-19 vaccination induced no production of SARS-CoV-2 unrelated antibodies. The COVID-19 vaccination also did not induce production of antibodies specific to the SARS-CoV-2 nucleocapsid protein, envelope protein, or papain-like protease (Fig. 1B) , which indicated no previous SARS-CoV-2 infection. On the other hand, the vaccine induced the production of antibodies specific to SARS-CoV-2 glycoprotein. As shown in Fig. 1B (middle panel), the RBD-specific IgG antibodies were already induced in all the tested donors after the first dose of the vaccine and their levels were further enhanced after the second dose of the vaccine. Only one donor after the first dose and two donors after the second dose of the vaccine produced IgG antibodies against the S2 subunit of the virus spike glycoprotein (Fig. 1B , middle panels), indicating stronger immunogenicity of its RBD domain. This stronger immunogenicity was even more pronounced for the IgAspecific antibodies where the vaccine induced the production of only the RBD-specific The cellular immunity against SARS-CoV-2 is increasingly considered to be as important for the effective protection against the virus as the humoral immunity [15] . Since our data showed that the COVID-19 vaccine specifically induced humoral response against SARS-CoV-2 spike glycoprotein, we next investigated whether the COVID-19 vaccination impacted the reactivity of the donors' CD4 + and CD8 + T cells to peptides derived from SARS-CoV-2 spike glycoprotein. We first found that the COVID-19 vaccination did not affect the viability of the isolated donors' peripheral blood mononuclear cells (PBMCs) ( Fig. 2A and 2B ). The vaccination also had a minimal effect on the proportions of T cells and their CD4 + and CD8 + subpopulations ( Fig. 2A and 2B ). To determine the reactivity of the donors' CD4 + and CD8 + T cells to peptides derived from the SARS-CoV-2 spike glycoprotein, the donors' PBMCs were stimulated with a pool of peptides derived from the glycoprotein (Fig. 2C ). The peptide pool-stimulated cells were either analyzed by intracellular cytokine staining (ICS) after a 5 h stimulation or cultured for 12 days in the presence of IL-2 to enrich the cell cultures for the peptidespecific T cells [16] . Following the stimulation of the 12-day-enriched cell cultures with the peptide pool for 5 h, the presence of the peptide-specific T cells was determined by ICS (Fig. 2C ). As shown, the 12-day cell culture enrichment decreased the viability of the cultured cells ( Our data showed that the COVID-19 vaccine promoted both humoral and cellular immunity against SARS-CoV-2 spike glycoprotein. We next analyzed whether the extent of the humoral response correlated with the ability of PBMCs to become enriched with SARS-CoV-2 spike glycoprotein-reactive CD4 + and CD8 + T cells. As shown in Fig. 4 , the extent of the humoral response correlated with the ability of PBMCs to become enriched with SARS-CoV-2 spike glycoprotein-reactive CD4 + and CD8 + T cells. The levels of the RBD-specific IgG antibodies were found to correlate with the extent of the PBMCs' ex vivo enrichment with the SARS-CoV-2 spike glycoprotein-reactive TNFαproducing CD4 + and TNFα-, IFNγ-or TNFα/IFNγ-producing CD8 + T cells (Fig. 4A ). Comparable data were obtained upon the correlations with the levels of RBD-specific IgA antibodies (Fig. 4A ). The only exception was with no correlation found for the TNFαproducing CD4 + T cells (Fig. 4B, left panel) . Overall, the data showed a close association between the COVID-19 vaccination-induced humoral and cellular responses against the SARS-CoV-2 spike glycoprotein. The peptide-enrichment experiments showed that COVID-19 vaccination could significantly enhance or even induce the PBMC's ability to become enriched with SARS-CoV-2 spike glycoprotein-reactive CD4 + and CD8 + T cells. We further investigated whether this enrichment could also have the potential to become an avenue for a T cell- This study showed that COVID-19 vaccines could elicit both a humoral and cellular response against the virus. Using the cell culture techniques and peptides derived from the virus antigen, the vaccine-induced antigen-reactive T cells can be ex vivo-enriched and large-scale-expanded and as such represent a potential therapeutic tool for the enhancement of cellular immunity after COVID-19 vaccination. The previous reports have shown that the BNT162b2 vaccine potentiated both the humoral and cellular responses [18] . Our data confirmed that vaccination of healthy donors with this vaccine indeed induced a humoral immune response that led to the production of SARS-CoV-2 spike glycoprotein-specific IgG and IgA antibodies. This response was highly specific as the vaccination induced no detectable antibodies specific to other SARS-CoV-2 proteins or proteins from other coronaviruses. These data, therefore, confirmed the precision of the vaccine-based prophylactic immunotherapy. The cellular immunity is the important layer of the immune protection against viruses as it prevents the virus amplification after infection [19, 20] . The effector cells of this arm of immunity are primarily the cytotoxic CD8 + T cells [6] . This study showed that no such SARS-CoV-2 spike glycoprotein-specific CD8 + T cells were detected in the peripheral blood of either non-vaccinated or 2-dose-vaccinated donors. These cells were also not detectable in the non-vaccinated donors even after the peptide-mediated enrichment. But, once the donors obtained 2 doses of the COVID-19 vaccine, the peptide-mediated enrichment already produced cell cultures containing the SARS-CoV-2 spike glycoprotein-specific CD8 + T cells. These data showed that the vaccination was important for increasing the frequency of the SARS-CoV-2 spike glycoprotein-specific CD8 + T cells to the levels that allow their peptide-mediated enrichment in the cell culture. These findings corroborate previous reports showing increased frequencies of T cells reactive to peptides derived from the tumor-associated antigens in the peptideenriched cell cultures after the patients' vaccination with ex vivo-produced dendritic cells loaded with whole inactivated tumor cells [21, 22] . Our results showed that humoral and T cell-based immune responses went hand in hand in the tested healthy donors. However, patients with compromised immunity or undergoing therapies that compromise their immunity may not respond well with both arms of the adaptive immunity, and the protective potential of the COVID-19 vaccines can then be undermined in these patients [23] . The large-scale expanded antigenspecific T cells have been utilized for adoptive cellular immunotherapy (ACI) of cancer [24] . Both prophylactic and therapeutic anti-viral ACI has also been utilized after the hematopoietic stem cell (HSC) transplantations, where viral infections are an important cause of morbidity and mortality [25, 26] . The restoration of the viral immunity is often successfully attained by adoptive transfer of the HSC donor's ex vivo expanded virusspecific CD8 + T cells [25, 26] . The expanded SARS-CoV-2-reactive T cells could, therefore, be also implemented in these therapeutic strategies to compensate for insufficiencies of the SARS-CoV-2-specific cellular immunity. The findings of the study show that the combination of the COVID-19 vaccines with the ex vivo peptide-mediated enrichment and large-scale expansion could represent a viable approach for the production of T cells for cellular therapy of COVID-19. The study involved 8 healthy donors who were negative for SARS-CoV The cryopreserved PBMCs were reconstituted at the concentration 2 × 10 6 The cultured cells or isolated PBMCs were stimulated with 0.5 g/ml concentration of the peptide pool. After 1 h of culture (37 °C, 5% CO 2 ), the cells were supplemented with brefeldin A solution (BioLegend, San Diego, CA) and cultured for 4 h. The samples stimulated with the peptide solvent alone (20% DMSO in PBS) were used as unstimulated controls. The cells were transferred to a V-bottom 96-well plate (Nalgene) and stained as described [29] with live/dead fixable stain and the following antibodies: The REP was performed for the large-scale expansion of the peptide-enriched cell cultures [17] . As the feeder cells were used PBMCs isolated from buffycoats as described [30] . The buffy coats were obtained from the Institute of Hematology and The means of values±SEM were calculated from the indicated sample size (n) using GraphPad Prism 6 (GraphPad Software, La Jolla, CA) and the statistical significance (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) between two groups of samples determined by Wilcoxon matched-pair signed-rank tests and between three or more groups the statistical significance was determined by matched-pair 1-way ANOVA with Dunn's post test. The associations between two variables were assessed by the Spearman's rank-order correlation coefficient (r) and the statistical significance of the correlation (p) determined. Graphical images were created with Biorender.com (accessed in March and April, 2021). 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