key: cord-0336273-ub9gwib8
authors: Guerrera, Gisella; Picozza, Mario; D’Orso, Silvia; Placido, Roberta; Pirronello, Marta; Verdiani, Alice; Termine, Andrea; Fabrizio, Carlo; Giannessi, Flavia; Sambucci, Manolo; Balice, Maria Pia; Caltagirone, Carlo; Salvia, Antonino; Rossini, Angelo; Battistini, Luca; Borsellino, Giovanna
title: The BNT162b2 mRNA vaccine induces polyfunctional T cell responses with features of longevity
date: 2021-09-28
journal: bioRxiv
DOI: 10.1101/2021.09.27.462006
sha: 60c42f20ec6da8fda6a0cb860e66d54b93cd24d7
doc_id: 336273
cord_uid: ub9gwib8

Vaccination against SARS-CoV-2 infection has shown to be effective in preventing hospitalization for severe COVID-19. However, multiple reports of break-through infections and of waning antibody titers have raised concerns on the durability of the vaccine, and current discussions on vaccination strategies are centered on evaluating the opportunity of a third dose administration. Here, we monitored T cell responses to the Spike protein of SARS-CoV-2 in 71 healthy donors vaccinated with the Pfizer–BioNTech mRNA vaccine (BNT162b2) for up to 6 months after vaccination. We find that vaccination induces the development of a sustained anti-viral memory T cell response which includes both the CD4+ and the CD8+ lymphocyte subsets. These lymphocytes display markers of polyfunctionality, are fit for interaction with cognate cells, show features of memory stemness, and survive in significant numbers the physiological contraction of the immune response. Collectively, this data shows that vaccination with BNT162b2 elicits an immunologically competent and potentially long-lived SARS-CoV-2-specific T cell population. Understanding the immune responses to BNT162b2 provides insights on the immunological basis of the clinical efficacy of the current vaccination campaign and may instruct future vaccination strategies.

provides insights on the immunological basis of the clinical efficacy of the current vaccination campaign and may instruct future vaccination strategies.

Mass-vaccination against COVID-19 has quickly shown to be effective and to confer high levels of protection against COVID-19 in real-world settings (1) . However, one notion that all immunologists learned during the pandemic was that natural infection with coronaviruses induces short-lived immunity (2) , with reinfections occurring frequently. This notion was quickly revised by subsequent studies showing that T cell immunity to SARS is actually long lived (3, 4) . The study of immune responses in COVID-19 patients with various degrees of disease severity have revealed that indeed infection with SARS-CoV-2 elicits a robust immune response, involving both the innate and the adaptive arms of the immune system and theoretically effective in protecting from reinfections (5, 6) . However, this protection is not absolute and impeccable, and cases of reinfection do occur (7) . Humoral immunity provides a shield against reinfection through the generation of neutralizing antibodies, which are easily measurable and are widely used as indicators of protective immunity (8, 9) . Notably, subjects with undetectable or impaired humoral responses can nonetheless clear the infection, suggesting that antigen-specific T cells are themselves effective at containing the virus (10) (11) (12) . Evidence of T cell involvement in the immune response to SARS-CoV-2 came from reports showing that the emergence of activated T cells precedes recovery from COVID-19 (13) (14) (15) (16) , and was later confirmed in in vitro studies which characterized antigen-specific CD4+ and CD8+ lymphocytes reactive with overlapping peptide pools from the SARS-CoV-2 Spike protein (15, (17) (18) (19) . These cells persist in COVID-19 convalescents (4, (20) (21) (22) (23) , and have been shown to reduce viral loads in non-human primate models of infection; crucially, they arise also following vaccination both with mRNA-and adenoviral-vaccines (24) (25) (26) (27) . It is necessary and urgent to identify the cellular immune components induced by vaccination and to measure their persistence, also in the view of reports of waning immunity over time and in recent updates in vaccination strategies which now propose administration of a third dose. Here, we

show the results of a longitudinal study on T cell responses in 71 health-care workers and scientists vaccinated with the BNT162b2 vaccine following the European Medicines Agency (EMA)-approved vaccination schedule, up to 6 months after the first dose. We find robust induction of neutralizing antibodies and of Spike-specific CD4+ and CD8+ lymphocytes which persist in the periphery after the physiological contraction of the initial response. These cells are detectable in most individuals also before vaccination, denoting the presence of a pre-existing pool of cross-reactive cells, and they are expanded significantly after the boost. We show that vaccine-induced T cells are mostly central and effector memory cells, and that they are equipped with overlapping sets of molecules which enable them to perform multiple immune functions: facilitation of B cell differentiation and antibody production, direct cytotoxic activity, and cytokine production, and that they are equally represented in both sexes.

Importantly, we show that vaccination also induces the generation of potentially long-lived memory stem cells, pilasters of durable immunity.

The antibody response to vaccination with BNT162b2 was measured in serum samples obtained at the day of the boost (T1), 14 days later (T2), and 6 months after the first dose (T3). As expected from previous studies, all individuals in our cohort were devoid of neutralizing antibodies (nAbs) at baseline, and significant levels of nAbs were obtained in 100% of individuals only after the second dose ( Fig.1A) , (T1: median 28; T2, median 1786; T3: median 517). Age and gender are key variables in immunity induced by vaccination, whose effectiveness decreases with age and is usually lower in males (28) . Thus, we analyzed the differences in antibody levels in male and female donors and also correlated them with age. We find that antibody titers correlate negatively with age at all time points, confirming previous results (29, 30) (Fig. 1C ). In our cohort antibody levels induced by BNT162b2 are comparable between males and females, as shown also in other studies (30) (Fig. 1B) , although females seem to retain lower levels of nAbs 6 months after vaccination. Thus, this data shows that BNT162b2 aptly induces the production of nAbs which decrease over time but are however maintained at high levels for at least 6 months.

Induction and durability of the Spike-specific T cell response To investigate the cellular immune response induced by vaccination, we exposed freshly obtained peripheral blood mononuclear cells (PBMC) to peptide pools spanning the entire sequence of the Spike (S) protein. Blood collection was performed at baseline (T0), on the day of the boost (day 21, T1), 14 days later (T2), and after 6 months (T3). Several effector functions of CD4+ cells consist in the upregulation of surface molecules for intercellular communication, and these may remain trapped inside the cells if the secretion inhibitors required for intracellular cytokine detection are present during antigenic stimulation. Thus, to fully capture the T cell antigen-specific response and to maximize sensitivity, two separate assays for the detection of the expression of surface Activation-Induced Markers (AIM) and for Intracellular Cytokine Staining (ICS) were set up ( Fig. S1 for gating strategy). Moreover, all assays were performed on freshly isolated PBMCs in order to avoid the inevitable cell loss during the freezing/thawing procedure, and to obtain accurate absolute cell counts. AIM+ CD4+ T cells were defined by upregulation of CD40L and CD69, while CD137 and CD69 expression identified the AIM+ CD8+ subset ( Fig. 2A) (Fig. 2B,C) .The total magnitude of the T cell response (that is, including both CD4+ and CD8+ cells) increased significantly following priming (T1) and rechallenge (T2), and decreased 6 months after the first dose (T3) (Fig. 2D) . Importantly, the Stimulation Index (SI, the ratio of AIM+ T cells in stimulated over unstimulated samples) of AIM+ CD4+ cells increased dramatically after the first dose, remained at high levels after boosting (Fig. 2E) , and increased further after 6 months, reaching a median of 25,7. The SI of AIM+ CD8+ T cells peaked 14 days after the boost, and declined by 1/3 at the latest time-point. Moreover, the fraction of individuals showing CD4+ with SI >3 increased at all time points, denoting the establishment of an antigen-specific T cell population (Fig. 2F ).

The frequency of S-specific CD4+ (and not CD8+) T cells induced by vaccination correlated inversely with age only 21 days after the first dose (T1), although a tendency towards reduced numbers of activated T cells with increasing age was observed at all time points (Fig. S2A) , again with no significant differences between males and females (Fig. S2B ).

The numbers of AIM+ cells correlated with antibody levels only after priming and not at subsequent time points (Fig. S2C) , suggesting that humoral and cellular immune responses follow different kinetics. However, machine learning indicated that the number of AIM+ CD4+ cells 21 days after priming is the best predictor of nAbs levels after 6 months, as expected from a T cell-dependent B cell response (Fig. S2D) .

Thus, vaccination induces clearly detectable and robust antigen-specific T cells in both sexes, arising before the development of high antibody titres, with a major expansion occurring after the first dose of vaccination followed by consolidation of the response after the boost, and persistence for up to 6 months. Cytokine secretion was also measured, at the peak of the response (T2), in the supernatants of cultures stimulated with the peptide pools. Production of high levels of IFNg and IL-2 was confirmed, whereas IL-17 and IL-4 were barely detectable (<5 pg/ml), confirming the Th1 differentiation profile of S-specific cells (Fig. S3B) .

Thus, vaccination induces the emergence in both males and females of a robust CD4+ and CD8+ cytokine response by T cells already after priming, while full effector functions marked by polyfunctionality are acquired only following the boost, and then maintained for at least 6 months. 5 D and F) . This is in contrast to Cluster 1 (Naïve-like cells) which remains stable after a first decrease in frequency at T2, and to Cluster 2 (CCR7+ CD127+ CD45RA-central memory CD8+ T cells) which is not significantly reduced at T2 but increases in size at 6-months. Manual analysis of AIM+ CD8+ T cells confirmed the transient increase at T2 of CD38+, HLA-DR+ and CD25+ cell frequencies, whereas CD39+ and PD-1+ cells steadily decrease or increase, respectively, at T2 and T3 compared to T1 (Fig. S4) .

Thus, antigen-specific T cells acquire phenotypic features of activation and functional capability early after the booster, and most of these attributes are less evident and partially replaced, in the long run, by characteristics distinctive of more quiescent memory cells.

Among the desirable outcomes of vaccination lies the generation of a pool of stem memory cells, which can rapidly and efficiently differentiate in an army of highly effective and polyfunctional lymphocytes in case of re-encounter with their nominal antigen (31) . Stemness includes long-term persistence, a key aspect in this age of pandemics and uncertainties on the durability of the novel vaccines. Thus, we searched for these cells within the antigen-responsive CD4+ and CD8+ subsets.

After the first dose, CD4+AIM+CD45RA+CD27+CCR7 high CD95+ cells, representing CD4+TSCM (T Stem cell memory) cells, were detectable in 88% of individuals (Fig. S5 ); 2 weeks after the second dose, this fraction was still 88%; after 6 months, 91% of individuals showed these cells (Fig.6A ). The number of circulating CD4+ TSCM rose from a median of 0,01 cells/ml at baseline to 14,7 cells/ml on the day of the boost, and further to 15,28 cell/ml two weeks after the second dose; after 6 months these cells were slightly increased in number (median 23,9 cells/ml). CD8+ TSCM followed similar kinetics, with 94%, 87%, and 96% of individuals showing these cells (0.05, 6, 8, and 8 cells/ml at baseline, T1, T2, and T3 respectively). To investigate the possible impact of TSCM on future immune responses, we applied a machine learning approach to test if the number of TSCM induced by vaccination significantly predicts immunological outcomes at distant time points. We find that indeed the number of TSCM induced after priming is a significant predictor of the number of both CD4+ and CD8+ activated T cells at the latest time point (Fig. 6B) .

These findings show that vaccination with BNT162b2 induces the emergence of a population of cells with features of longevity, which remain numerically stable in the peripheral blood for at least 6 months, and which predict the persistence of T cell responses.

Specific viral control is achieved through the action of effector cells of the adaptive immune system: the antibody-producing progeny of B cells, and the functionally diverse population of T cells derived from a small pool of naïve progenitors which expand and differentiate to achieve the ability to provide B cell help, to directly kill virally infected cells, and to sustain the immune response through the production of cytokines, all while maintaining a source of non-terminally differentiated but antigen-experienced cells which can rapidly expand upon antigen re-encounter. In all of this, T cells are indispensable: an optimal antibody response is the consequence of a competent underlying T cell response, and T cell responses alone can successfully clear infection with SARS-CoV-2, as shown in COVID-19 patients lacking B cells (10, 12) , and in seronegative COVID-19-recovered individuals (32) (33) (34) . Here, we investigate the immune response occurring after vaccination with BNT162b2 to understand how the vaccine animates T cells specific for the S-protein, in order to predict whether this response will be durable and if it is similar in individuals of both sexes and at different ages. The timing of our glimpses into the immune system's antigen-specific dynamics was such that we could observe the emergence and the evolution of the effector functions of vaccine-induced T cells, and then record the physiological contraction of the immune response and study the features of the surviving antigen-specific cells. The use of freshly obtained blood cells permitted the detection of fragile markers, avoided the bias introduced by freezing/thawing procedures, and provided the possibility to precisely calculate absolute cell counts, a measure routinely used to guide clinical decisions in infectious diseases, such as HIV infection (35) .

The simultaneous measurement of serum levels of neutralizing antibodies provided insights on the effective induction of the humoral arm of the adaptive response. NAbs were induced by vaccination in all individuals in our cohort. Although titers did decline with time, they were maintained at high levels for the entire period of our observation (6 months), in agreement with previous studies (27, 36) . Importantly, the efficacy of the vaccine in inducing antibodies was equivalent in both sexes, and correlated inversely with age, as expected.

We find that most individuals harbour Spike-specific T cells already at baseline, likely due to the presence of a pool of naïve progenitors and of memory clones which are cross-reactive with other coronaviruses (3, 15-17, 37, 38) . These cells are highly responsive to antigen encounter, and their SI increases with time. CD8+ cells show a less vigorous response compared to the CD4+ subset possibly due to the sub-optimal stimulation of CD8+ cells by 15-mers, as those used in our assays (39) . Moreover, CD4+ T cells have also other abilities, such as those related to cytokine production and to direct cytotoxic function. In agreement with previous results, we find that antigenspecific CD4+ T cells induced by vaccination with BNT162b2 produce cytokines typical of the Th1 profile (IFNg, TNFa), and minimal levels of IL-4 and IL-17. In infants the presence in the peripheral blood of ³100 influenza-specific IFNg-producing cells/ml confers protection against clinical influenza (47) , and a correlation between both magnitude and polyfunctionality of T cell responses and resistance to infection has been described in other vaccine settings (48) (49) (50) (51) (52) (53) (54) . In this study, we show that 6 months after vaccination with BNT162b2 over 300 CD4+ and CD8+ Spike-specific IFNg-producing T cells/ml are clearly detected in the periphery, with diverse functionalities. CD4+ cells acquire both helper and cytotoxic functions which are particularly evident at the peak of the antigen-specific response, 14 days after the boost. At this time point we detect the highest frequencies of cells with a polyfunctional phenotype, in both CD4+ and CD8+ S-specific T cells. Interestingly, the CD4+ subset is more spared from the physiological contraction of the immune response, 6 months after receiving the first dose of BNT162b2, and it shows polyfunctional features of both cytotoxicity and ability to provide Bcell help. At the same time point, the antigen-specific CD8+ T cell subset seems to drop at lower levels, although these cells are still clearly detectable and are significantly expanded compared to the baseline. This data is consistent with studies which have investigated cellular immune responses in individuals recovering from COVID-19 (16, 20, (55) (56) (57) (58) , and with other studies on mRNA vaccination (59, 60) , and suggest that vaccine-induced T cells have the prerequisites to confer protection from subsequent infections. Moreover, the persistence of a numerically consistent pool of antigen-specific T cell, which we find to be still increased 6fold from baseline after 6 months, may be the source for of effector cells which can promptly expand in case of antigen re-encounter (61).

The finding that CD4+ and CD8+ TSCM are present throughout the 6-month period of this study suggests that immunity offered by vaccination should be long-lived, since it induces a reservoir of cells with multipotent capacity which likely are the very cells that provide long-lasting protection, and whose generation is a target of vaccination (62) (63) (64) . Importantly, the number of TSCM was stable during the period of our study, and crucially those induced after priming are highly significant predictors of future T cell responses. This knowledge may inform current vaccination strategies and decisions on third dose vaccine administration, which may be spared for the fragile or immunologically impaired and re-directed to the unvaccinated, globally.

This study has the inevitable limits of human studies, and was performed on circulating lymphocytes, which may be different from those who reside at mucosal barriers and which confer immediate protection against infection. Also, we did not investigate Spike-specific B lymphocytes, and dosage of antibody titers was the only read-out of successful induction of humoral immunity. Similarly, the innate arm of the immune response, which has intriguingly been shown to be activated by vaccination(65), was not included in this study; both of these elements are currently under active investigation the world over. Further time points will also be necessary to measure effective durability of anti-SARS-CoV-2 immune responses.

Moreover, although donors were equally distributed for age and sex, our sample was limited in size.

This notwithstanding, some considerations may be made. On the whole, the results of this study can be visualized as the dynamic and integrated emergence of a theoretically effective Spikespecific adaptive immune response, characterized by a T cell response which precedes in time the development of high levels of anti-Spike neutralizing antibodies (Fig.7) . T Immunologically fragile individuals, however, or those who are unavoidably exposed to high titers of virus may be better protected by a third dose of vaccination which has been shown to increase antibody levels, thus providing an immediate albeit temporary shield against viral entry in the host cell. In an equitable world, and based on the current data on vaccine effectiveness in preventing severe disease, after having secured the fragile from infection the absolute precedence should be given to unvaccinated individuals globally, in the unified endeavor to curb viral circulation and to prevent disease. AIM assay In vitro stimulation of freshly obtained PBMC was performed as described (66) .

In brief, 200 ml of cell suspensions (10 x 10 6 cells/ml) were seeded in U-bottom 96-well plates at a density of (0,2 ml/well) and stimulated for 18 hours with or without PepTivator SARS-CoV-2 protein S, S1 and S+ peptide pools (1 mg/ml each, Miltenyi Biotec). Purified aCD40 (0,5 μg/µl, Miltenyi Biotech) was added at culture start to enhance endpoint CD40L staining by inhibiting its recycling. Supernatants and cells from these culture wells were collected for cytokine measurement and flow cytometry, respectively. (right) cells at the different time points. Timepoints were compared by non parametric Kruskall-Wallis test; lines represent median with interquartile range. ****p < 0.0001; no symbol, not significant. B) The relevance of T1 CD4 T SCM cells/ml in predicting T3 AIM+ CD4 (top) and CD8 (bottom) cells/ml was tested with two General Linear Models with stepwise selection aiming to optimize Akaike Information Criterion (AIC) (Bozdogan, 1987). Model's R 2 were 0.21 and 0.16 for AIM+ CD4 and CD8 cells respectively. The number of CD4 T SCM in T1 cells/ml was deemed as significant in predicting both CD4+ and CD8+ AIM+ cell numbers in T3 (****p < 0.0001; *p < 0.05). Lines represent linear regression. 

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The authors declare that they have no competing interests. Table S3 .