key: cord-0265653-oa6k5y16
authors: Rodda, L. B.; Morawski, P. A.; Pruner, K. B.; Fahning, M. L.; Howard, C. A.; Franko, N.; Logue, J.; Eggenberger, J.; Stokes, C.; Golez, I.; Hale, M.; Gale, M.; Chu, H. Y.; Campbell, D. J.; Pepper, M.
title: Imprinted SARS-CoV-2-specific memory lymphocytes define hybrid immunity
date: 2022-01-13
journal: nan
DOI: 10.1101/2022.01.12.22269192
sha: 7c1b1f7d79158a3cc753fb42008f9a78bbc0df4c
doc_id: 265653
cord_uid: oa6k5y16

Immune memory is tailored by cues that lymphocytes perceive during priming. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic created a situation in which nascent memory could be tracked through additional antigen exposures. Both SARS-CoV-2 infection and vaccination induce multifaceted, functional immune memory, but together they engender improved protection from disease, termed hybrid immunity. We therefore investigated how vaccine-induced memory is shaped by previous infection. We found that following vaccination, previously infected individuals generated more SARS-CoV-2 RBD-specific memory B cells and variant-neutralizing antibodies and a distinct population of IFN- and IL-10-expressing memory SARS-CoV-2 spike-specific CD4+ T cells than previously naive individuals. While additional vaccination could increase humoral memory, it did not recapitulate the distinct CD4+ T cell cytokine profile in previously naive individuals. Thus, imprinted features of SARS-CoV-2-specific memory lymphocytes define hybrid immunity.

The generation of immune memory is influenced by signals that B and T cells of the adaptive immune system perceive during a primary immune response. This system has evolved such that key cues from an invading pathogen or vaccine are relayed to lymphocytes so they can provide specific functional outputs to combat infection and protect from re-infection. The site of antigen encounter, specific inflammatory signals, and the number and frequency of antigen exposures all influence the resulting memory pool, yet the specific rules governing memory formation remain undefined.

Understanding how the confluence of these parameters influences immune memory function and maintenance is critical for optimizing protective vaccines.

In the context of the ongoing SARS-CoV-2 pandemic, so-called "hybrid/super-immunity" induced by a combination of prior SARS-CoV-2 infection and subsequent vaccination, provides greater protection against re-infection and severe COVID-19 disease than either infection or vaccination alone (Abu-Raddad et al., 2021; Crotty 2021; Gazit et al., 2021; Goldberg et al., 2021; Reynolds et al., 2021) . Studies investigating immune correlates of hybrid-immunity have revealed improved breadth and neutralizing ability of circulating antibodies from previously infected individuals (Cho et al., 2021; Schmidt et al., 2021; Stamatatos et al., 2021; Wang et al., 2021) but the specific changes in the cellular immune compartment associated with this immune state remain undefined. Additionally, it is not understood whether further activation of immune memory in vaccinated only individuals could achieve similar qualities, as previously infected individuals have had an additional antigen exposure.

To answer these questions, we tracked circulating SARS-CoV-2-specific antibodies and memory lymphocytes in a cohort of Naive (N) or SARS-CoV2-Previously Infected (PI) subjects over the course of three vaccinations. We focused on visualizing RBD-specific antibodies and B cells and spike (S)-specific CD4+ T cells as they are critical mediators of protection in infected individuals over a two year time period and targets of SARS-CoV-2-directed vaccines (Corbett et al., 2021; Feng et al., 2021; Gilbert et al., 2021; Khoury et al., 2021) .

We find that the immune memory landscape elicited by vaccination of previously infected subjects is distinct from the immune memory of SARS-CoV-2-naive individuals.

Three months after two doses of vaccination, PI individuals maintained a higher quantity of RBD-specific plasma antibodies with superior plasma neutralization of viral variants including Omicron. PI individuals also retained increased numbers of RBD-specific B cells enriched for IgG+ classical and activated MBCs compared to N individuals.

However, these differences were normalized by administration of a third vaccine dose.

Examination of the T cell compartment revealed that although N and PI individuals generated equivalent numbers of S-specific CD4+ T cells after vaccination, there was a profound functional skewing towards a Th1 phenotype in PI subjects. CD4+ T cell functional differences between N and PI subjects persisted following the administration of a vaccine booster. Further, although the third dose of vaccine provided a boost in S1A). PI participants reported a positive SARS-CoV-2 test and mildly symptomatic disease an average of 9 months before their pre-vaccination blood draw.

To determine the distinct humoral features of hybrid immunity, we first compared the circulating SARS-CoV-2 RBD-specific B cells in N and PI participants pre-and post-vaccination. Tetramer enrichment allowed us to identify rare SARS-CoV-2 RBD-specific B cells from PBMCs to track the memory response ( Figure S1B ).

Pre-vaccination, PI individuals had significantly more RBD-specific antigen-experienced B cells (CD21 + CD27 + and CD21 -CD27 +/-)(Cancro and Tamayko, 2021) ( Figures 1B and   1C , S1C) and plasma RBD-specific IgG and IgA than N individuals (Figures 1D and   1E ), reflecting the sustained memory response to their previous SARS-CoV-2 infection ( Figures 1B and 1C , S1C) (Rodda et al., 2021 , Dan et al., 2021 . Two dose vaccination induced a robust humoral response in both N and PI individuals including increased numbers of RBD-specific antigen-experienced B cells (Figure 1C) , an acute burst of RBD-specific plasmablasts ( Figure S1D ) and increased titers of RBD-specific IgG and IgA antibody (Figures 1D and 1E) . The numbers of RBD-specific antigen-experienced B cells and RBD-specific antibodies in both groups contracted to levels higher than those at pre-vaccination. However, PI individuals formed and retained higher numbers of RBD-specific antigen-experienced B cells and RBD-specific IgG and IgA than N individuals at 3 and 6 months post-vaccination (Figures 1C-E) . These findings corroborate recent work and support the likely functional role of the elevated RBD-specific antibody and MBCs in hybrid immunity (Goel et al., 2021) .

6 . CC-BY 4.0 International license It is made available under a 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 January 13, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 To determine if a third vaccine dose could overcome these differences by engaging the memory response in N individuals in additional expansion, we measured the humoral response in both groups 2 weeks after a third dose. While RBD-specific B cells in N individuals responded by increasing RBD-specific IgG and IgA antibody to the levels in PI individuals (Figures 1D-F) , the antibody response in N individuals to a third SARS-CoV-2 antigen exposure did not match the acute response in PI individuals to their third exposure (vaccination 2) (Figures 1C and 1D ). This muted response to a third exposure in N and PI individuals most likely reflects efficient clearance of this smaller antigen dose by the high quantity of antigen-specific memory now established in both groups. However, data from a further memory time point post-3rd vaccination is required to determine if this muted response is sufficient to induce a sustained expansion of RBD-specific MBCs and plasma antibody in N individuals to match that in PI individuals.

CD4+ T cell responses to vaccination were also assessed in N and PI individuals using an activation induced marker (AIM) assay based on expression of CD69 and CD137 to detect SARS-CoV-2 specificity (Tarke et al., 2021) (Figures S2A-S2D) . PBMCs from N or PI donors isolated before and after vaccination were re-stimulated in the presence of peptide pools optimized for the induction of MHC class II-dependent CD4+ T cell responses (15-mers), and containing predicted T cell epitopes across a range of common HLA haplotypes against the viral membrane and nucleocapsid (M/N) or spike (S) proteins (Table S2) .

Pre-vaccination, CD4+ T cells from PI donors showed responses to both M/N and S, consistent with prior SARS-CoV-2 infection and the presence of memory cells (Figures   2A and 2B) , whereas responses in N donors were undetectable above individual donor background (Figures S3A and S3B) . Throughout the course of vaccination, responses to M/N remained negative in N donors, but endured, undiminished in PI donors at all time points, indicating that SARS-CoV-2-specific CD4+ T cells primed during infection, persist at least 18 months following initial antigen exposure ( Figure 2B ). Following initial two-dose vaccination, S-reactive AIM+CD4+ T cells were significantly elevated above pre-vaccination levels in both N and PI individuals, with no significant differences in peak CD4+ T cell response between groups (Figures 2B, S3C, and S3D) . Therefore COVID-19 vaccination induces numerically equivalent populations of S-specific cells in all subjects, regardless of prior SARS-CoV-2 exposure status, that persist for at least three months.

We next examined the relative composition of effector and memory populations within the SARS-CoV-2-reactive T cell compartment in N and PI subjects. Prior to vaccination, there was no difference in the frequency of S-reactive central memory (CM) T cells between N and PI groups, however PI individuals maintained significantly elevated frequencies of effector memory (EM and EMRA) and follicular helper (Tfh) cells ( Figures 2C, 2D) . Two-dose vaccination induced a significant increase in nearly all AIM+ effector and memory populations in both groups, with EM and Tfh populations dominating the response ( Figure S4A ). In addition, two-dose vaccination was sufficient to normalize numerical differences identified in pre-vaccination N and PI donors, while a third dose was not associated with any additional increase in these populations in either N or PI donors (Figure 2D and S4A) . Therefore, vaccination induces numerically equivalent S-specific memory Tfh and EM CD4+ T cell responses in N and PI individuals that do not further increase in response to an additional vaccine dose.

Memory B cells function by activating, proliferating and forming antibody-secreting plasmablasts more rapidly than naive B cells (Cancro and Tamayko, 2021) . Prior to vaccination, RBD-specific B cells in N individuals were predominantly Naive (CD21 + CD27 -) while RBD-specific B cells in PI individuals were predominantly non-Naive antigen-experienced B cells (CD21 + CD27 + and CD21 -CD27 +/-)(Figures S5A and S5B). Two vaccinations induced dominant populations of activated MBCs (CD21 -CD27 +/-CD11c + ) (Cancro and Tomayko, 2021) in both groups at 1 week post-vaccination (Figures 3A and 3B) . By 3 months post-vaccination, RBD-specific antigen experienced cells in both groups were enriched for IgG + Classical MBCs (cMBCs, CD21 + CD27 + ) (Figure 3C) , which can rapidly form IgG-secreting plasmablasts upon antigen exposure (Laidlaw and Ellebedy, 2021) However, a significantly greater population of activated MBCs remained in PI individuals at this memory time point than 9 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 13, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 in N individuals. This may reflect prolonged antigen retention and recent egress of memory B cells from an ongoing germinal center (GC) which would contribute to the increased quantity of RBD-specific antigen-experienced cells including cMBCs and long lived plasma cells producing RBD-specific antibodies in PI individuals with hybrid immunity (Figures 1C-E) (Betts et al., 2017; Kim et al., 2019) . While mucosal infections such as SARS-CoV-2 are associated with IgA + MBCs particularly at the site of infection, PI individuals had only a small proportion of circulating IgA+ RBD-specific antigen-experienced B cells at 9 months post-infection and they were not further induced in N or PI individuals by vaccination ( Figure S5C ).

To assess if infection prior to vaccination impacts the breadth or ability of SARS-CoV-2 specific MBCs and antibodies to recognize and combat SARS-CoV-2 variants of concern (VOCs), we measured the ability of RBD-specific MBCs and antibody to recognize the B.1.351 (β) variant and plasma antibody to neutralize the B.1.617.2 (Δ) and B.1.1.529 (o) variants. SARS-CoV-2(β) and SARS-CoV-2(o) have been found to significantly reduce neutralization by vaccine-induced antibodies as compared to the original SARS-CoV-2(Wu-1) strain which was also used to make the mRNA vaccines relevant to this study (Cameroni et al., 2021 , Collier et al., 2021 . Using SARS-CoV-2 RBD(Wu-1) and SARS-CoV-2 RBD(β) tetramers simultaneously (Figure S1B), we found that the majority of RBD-specific antigen-experienced B cells in all N and PI participants could recognize both variants (Figures S5D and S5E) . This technique does not allow us to distinguish cross-reactive cells that bind epitopes shared by the variant RBDs from cells that bind epitopes with the 3 amino acid differences between the 10 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 13, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 variants. While vaccination induced increased RBD(β)-specific IgG antibody titers in both groups, PI individuals had significantly higher RBD(β)-specific IgG titers than N individuals pre-and 6 months post-vaccination ( Figure S5F) . Additionally, we found that plasma from PI individuals 6 months post-vaccination was significantly better at neutralizing SARS-CoV-2(Wu-1) virus as well as pseudotyped SARS-CoV-2(Δ)-spike and SARS-CoV-2(o)-spike lentiviruses than plasma from N individuals (Figures 3D and   S5G ). SARS-CoV-2(Wu-1) neutralization was highly correlated with RBD(Wu-1)-specific IgG antibody titers as we and others have shown previously and likely contributes to the improved breadth in PI individuals post-vaccination ( Figure S5H ) (Rodda et al., 2021; Goel et al., 2021) . The increased antibody response to a third vaccine dose in N individuals ( Figures 1D, 1E and S5F) correlated with sharply increased neutralization capacity of the plasma antibody against the variants tested reaching the levels in PI individuals pre-and post-3rd vaccination (Figures 3D and S5F) . Thus, vaccination induces robust humoral memory in N and PI participants, but a third dose is needed for N individuals to achieve the breadth and VOC neutralizing efficacy of the plasma antibody formed in hybrid immunity.

During T cell priming, the context of the initial antigen exposure directs CD4+ T cells towards different helper and memory fates with distinct functional capacities. To determine how a primary infection versus vaccination or total number of antigen 11 . CC-BY 4.0 International license It is made available under a 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 January 13, 2022. ; exposures differentially impacted functional outcomes, we compared the CD4+ T helper lineage composition of SARS-CoV-2 AIM+ T cells based on differential chemokine receptor expression in N and PI subjects. Prior to vaccination, we found that S-reactive T cells in PI subjects were primarily Th1 and Th1/17 memory cells (Figure 4A and 4B) .

In response to vaccination, the frequency of nearly all T helper subsets examined were increased in both N and PI individuals (Figure S6A) , consistent with previous reports (Goel et al., 2021; Guerrera et al., 2021; Painter et al., 2021) . However, PI subjects maintained an elevated frequency of both CXCR3+CCR6-Th1 and CXCR3+CCR6+

Th1/17 cells, whereas N donors showed increased numbers of CXCR3-CCR6-CCR4+ Th2 cells by 3 months post-vaccine two ( Figure 4B ). Th1 and Th1/17 AIM+ cells continued to trend higher in PI individuals following a third vaccine dose, but this difference did not reach statistical significance. In addition, at this acute timepoint post-third vaccine, the frequency of Th2 populations were normalized between groups ( Figure 4B ). These data demonstrate that T helper polarization in response to initial vaccination differs according to prior SARS-CoV-2 infection status, and that additional vaccine doses may have a normalizing effect on some aspects of this response.

To complement the phenotypic analysis of CD4+ T cells, we employed a similar assay to directly interrogate cytokine production from SARS-CoV-2-specific CD4+ T cells following stimulation with M/N or S peptide pools. As demonstrated previously, CD154 and CD69 can be used to identify activated, antigen-reactive T cells (Reiss et al., 2017) that represent the primary cytokine producers in response to stimulation (Figures S7A   and S7B) . Importantly, the response measured in CD154+CD69+ SARS-CoV-2 reactive 12 . CC-BY 4.0 International license It is made available under a 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 January 13, 2022. ; https://doi.org/10.1101/2022.01.12.22269192 doi: medRxiv preprint T cells to peptide stimulation mirrors that of AIM+ CD69+CD137+ cells shown in Figure   2 , including an M/N response exclusive to PI subjects and robust induction of S-specific cells following two-dose immunization that was maintained after a third vaccination dose in both groups (Figures S8A-S8C) .

In PI subjects, we measured a strong cytokine response to infection in S-(Figures (Figures 4G and 4H) . However, few spike-reactive T cells co-produced IL-4 and IL-13 ( Figure S9E ), suggesting the absence of bonafide Th2 polarization. Consistent with our phenotypic analysis of T helper polarization based on differential chemokine receptor expression, PI individuals not only exhibited a strong induction of IFN--producing S-reactive CD4+ T cells following vaccination, but this response remained significantly elevated compared with the N group following both 13 . CC-BY 4.0 International license It is made available under a 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 January 13, 2022. ; second and third vaccine doses (Figures 4C and 4D) . PI donor CD4+ T cells also produced higher levels of IL-10 after two-dose vaccination compared with N donors, which also trended higher after a third vaccine dose despite failing to reach statistical significance (Figures 4E and 4F) . Production of IL-21, although not detected following infection, was observed at comparable levels in both N and PI individuals following twoand three-dose vaccination (Figures S9C and S9D) .

The S-reactive CD4+ T cell compartment was further distilled into subpopulations using a dimensionality reduction analysis on pooled CD154+CD69+ cells from all donors.

UMAP followed by FlowSOM analysis identified 15 distinct clusters, of which 13 were expanded following infection or vaccination, and 6 included cells which produced cytokine(s) (Figures S11A-P) . Two of these clusters, CL4 and CL13, were specifically enriched in PI donor T cells following vaccination. CL4 was defined largely by the production of IFN-and low CD127 expression, indicative of terminally differentiated Th1 cells ( Figure S11E ). CL13 was defined by co-production of IFN-and IL-10, while also displaying low CD127 expression, resembling FOXP3-type-1 regulatory (Tr1) cells ( Figure S11N ) (Jankovic et al., 2007; Sun et al., 2009) In contrast, increased vaccine-induced T cells producing IL-4 were specifically enriched in CL15 only in N donors. These cells additionally co-produced IL-2, IFN-, and small amounts of IL-21 ( Figure S11P) , suggesting a Tfh-like phenotype (Olatunde et al., 2021) . Collectively, this unbiased analysis of the S-reactive CD4 T cell compartment identifies the emergence of functionally distinct subsets which differ following vaccination in PI and N participants according to prior SARS-CoV-2 infection history.

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The copyright holder for this preprint this version posted January 13, 2022. ;

To determine how the number of antigen exposures may alter distinct cytokine production profiles seen in N and PI individuals, we compared S-reactive T cells from N individuals after three doses of vaccination (3 antigen exposures) to those from PI individuals after two doses of vaccination (3 antigen exposures). A comparison of AIM+ S-specific CD4+ T cells in triple vaccinated N subjects to double vaccinated PI individuals normalized differences between Th1 and Th1/17 frequencies as assessed by CXCR3 and CCR6 expression (Figures 4A and 4B) . We also found similar frequencies of CM, EM, EMRA and and Tfh cells when normalizing for the number of antigen exposures (Figures 2B and 2D) . However, when cytokine production was assessed between triple vaccinated N donors and double vaccinated PI donors, the PI group maintained a dramatically elevated frequency of IFN--and IL-10-producing S-specific CD4+ T cells compared with the N group (Figures 4C and 4D) . These data indicate that T cell priming by infection or vaccination promotes distinct effector states in which phenotypic outcomes can be normalized by additional antigen exposure, while functional differences persist through multiple vaccine doses.

Protective immunity generated in response to infection or vaccination is characterized by an expanded population of antigen-specific memory cells that rapidly expresses effector molecules upon antigen re-exposure. In order to prevent or control reinfection, memory cells are functionally tailored by the initial priming event, however it is unclear 15 . CC-BY 4.0 International license It is made available under a 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 January 13, 2022. ; https://doi.org/10.1101/2022.01.12.22269192 doi: medRxiv preprint how subsequent antigen exposures alter the quantity and quality of the response. The emergence of a novel coronavirus and expeditious generation of vaccines provided an unprecedented opportunity to examine the formation of a nascent antigen-specific memory pool in human volunteers, and the impact of previous infection on the quantity, quality and durability of vaccine-induced immune memory. This is particularly important as SARS-CoV-2 infection prior to vaccination, referred to as "hybrid immunity", provides a protective advantage over vaccination alone (Abu-Raddad et al., 2021; Crotty 2021; Gazit et al., 2021; Goldberg et al., 2021) .

In this study, we examined the distinguishing features of hybrid immunity to SARS-CoV-2 in comparison to vaccination alone over time and with subsequent antigen exposures. We found that certain features of the humoral response differed in N and PI subjects following vaccination that could contribute to hybrid immunity. These include the enhanced numbers of RBD-specific memory B cells and SARS-CoV-2 variant-neutralizing plasma antibody. These features were normalized following booster vaccination, and therefore could be a function of the number of antigen exposures.

However the cytokine profiles of spike-specific memory CD4+ T cells in previously infected and then vaccinated individuals exhibited a unique IFN-and IL-10 cytokine-producing profile that could not be achieved by multiple vaccinations in the absence of prior infection, and this represents the first immune correlate of hybrid immunity that is stably imprinted on responding cells during initial infection.

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The copyright holder for this preprint this version posted January 13, 2022. ; https://doi.org/10.1101/2022.01.12.22269192 doi: medRxiv preprint Enhanced numbers of memory B cells and breadth of antibody specificity are proposed to be essential to the mechanisms underlying the superior immunity described in PI individuals after vaccination (PV2) (Cho et al., 2021; Schmidt et al., 2021; Schmidt et al., 2021; Stamatatos et al., 2021; Wang et al., 2021) . In accordance with these studies, we found infection prior to vaccination induced higher numbers of RBD-specific MBC, IgG and IgA circulating antibodies, and increased plasma neutralization of multiple VOCs including SARS-CoV-2(o). Furthermore, differences in the number and timing of SARS-CoV-2 antigen exposures impacts memory B cell response maturation (Goel et al., 2021) . This is likely to explain the quantitative differences in vaccine-induced humoral memory and antibody breadth between N and PI individuals described in our study because additional vaccination normalized the differences between groups. We additionally observed however that RBD-specific MBCs in PI individuals were enriched Sustained low-levels of activation may enrich the ongoing germinal center responses that have been shown to last for at least 6 months post-vaccination and support generation of higher affinity and broadly neutralizing antibodies critical for combating variants (Cho et al., 2021; Kim et al., 2021; Turner et al., 2021) and likely contributing to the protective advantage of hybrid immunity. Additional data from memory time points 17 . CC-BY 4.0 International license It is made available under a 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 January 13, 2022. ;

after third vaccination are needed to determine whether this enhanced activation at memory time points can be matched by repeat vaccination. CC-BY 4.0 International license It is made available under a 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 January 13, 2022. ; https://doi.org/10.1101/2022.01.12.22269192 doi: medRxiv preprint (Brinkmann et al., 1993; Lazarevic et al., 2013) . Further, the cytokines IL-6, IL-12, or IL-27 drive the expression of IL-10 in FOXP3-CD4+ helper T cells through the adapter c-maf (Gabryšová et al., 2018; Pot et al., 2009; Yang et al., 2005) . Finally, infection with SARS-CoV-2 recruits effector T cells to the lung tissue, generating bona fide tissue-resident memory (Trm) cells in the human lung (Poon et al., 2021) . Therefore, S-reactive CD4+ T cells from PI individuals may receive additional tissue-restricted signals, before contracting into memory cells (Klicznik et al., 2019; Wijeyesinghe et al., 2021) . Indeed, Trm cells produce the highest levels of IFN-and IL-10 in a mouse-adapted SARS-CoV infection (Zhao et al., 2016) . Therefore, it may also be the case that the increase in cytokine production we see in PI donors is due to the presence of circulating S-reactive T cells which previously received tissue-restricted cues.

The distinct CD4+ T cell cytokine signature we describe likely contributes to the increased protection observed in PI individuals with hybrid-immunity. IFN-is produced rapidly by memory T cells in vivo, driving an antiviral response program which is CC-BY 4.0 International license It is made available under a 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 January 13, 2022. ; https://doi.org/10.1101/2022.01.12.22269192 doi: medRxiv preprint study also reported that concomitant treatment with rTNF exacerbates SARS-CoV-mediated disease in mice. IL-10 can inhibit TNF and other pro-inflammatory cytokines in order to regulate inflammatory responses to infection (De Waal Malefyt et al., 1991; Gazzinelli et al., 1996; Hunter et al., 1997) . Therefore, increased production of IL-10 by S-specific Th1 cells potentiated through viral infection could be a critical element of hybrid immunity that limits future symptomatic SARS-CoV-2 infection. Additional work is needed to explore this crucial role of memory T cell-derived IL-10 in secondary challenge. 20 . CC-BY 4.0 International license It is made available under a 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 January 13, 2022. 24 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. CC-BY 4.0 International license It is made available under a 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 January 13, 2022. ; https://doi.org/10.1101/2022.01.12.22269192 doi: medRxiv preprint post-vaccination two (N: 15, PI: 23), 6 months post-vaccination two (N: 8, PI: 9) and 2 weeks post-vaccination three (N: 8, PI: 10). Not all samples were run for both plasma and PBMC assessments.

Venous blood from study volunteers was collected in EDTA tubes and spun at 700xg for 10 min. Plasma was collected, heat-inactivated at 56 o C for 30 min and stored at -80 o C.

Cellular fraction was resuspended in phosphate buffered saline (PBS) and PBMCs were separated from red blood cells using Sepmate PBMC Isolation Tubes (STEMCELL Technologies) according to manufacturer's instruction or cells were resuspended in HBSS, washed, overlaid with ficoll, spun at 400xg for 30 min with no brake and PBMCs collected. Cells were washed twice in HBSS and frozen at -80 o C before being stored in liquid nitrogen.

Recombinant SARS-CoV-2 RBD (Wuhan-1, Wu-1) was generated by standard transient transfection followed by IMAC purification as described previously (Walls et al., 2020) .

Recombinant SARS-CoV-2 RBD (B.1.1.351, β) was generated by transient transfection from the SARS-CoV-2-Beta-RBD-Avi (K417N-E484K-N501Y) construct synthesized by GenScript into CMVR with an N-terminal mu-phosphatase signal peptide and a C-terminal octa-histidine tag, flexible linker, and avi tag (GHHHHHHHHGGSSGLNDIFEAQKIEWHE) as described previously (Tortorici et al., 2021) . For tetramer generation, RBD proteins were biotinylated with the BirA500 kit 28 . CC-BY 4.0 International license It is made available under a 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 January 13, 2022. quenched with 1M HCl. Sample optical density (OD) was measured by a spectrophotometer at 450nm and 570nm.

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The copyright holder for this preprint this version posted January 13, 2022. Supernatants were harvested the next day, filtered through 0.2uM filters, and concentrated by overnight centrifugation at 4°C. Pellets were then resuspended in PBS at 100X.

PVNT assays were performed as previously described (Crawford et al., 2020) . Briefly, heat inactivated plasma was diluted 1:5 followed by four 3-fold serial dilutions all in duplicate and mixed 1:1 with 10 6 relative luciferase units of SARS-CoV-2(Δ)-spike or SARS-CoV-2(o)-spike pseudotyped lentivirus in DMEM with 10% heat-inactivated FBS, 30 . CC-BY 4.0 International license It is made available under a 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 January 13, 2022. ; 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. After 1 hour incubation at 37 o C, the plasma/virus mixtures were added to 96-well poly-L-lysine-coated plates seeded with human ACE2-expressing 293T cells (BEI Resources: NR-52511) 20 hours prior. Each plate contained wells with no plasma and wells with no plasma and 293T cells as a background control. Human ACE2-Fc (BEI Resources) was used as a positive control with a starting dilution at 10ug/ml. After 

PRNT assays were performed as previously described (Erasmus et al., 2020) . Briefly, heat inactivated plasma was diluted 1:5 followed by four 4-fold serial dilutions and mixed 1:1 with 600 PFU/mL SARS-CoV-2 WA-1 (BEI resources) in PBS+0.3% cold water fish skin gelatin (Sigma). After 30 min of incubation at 37 o C, the plasma/virus mixtures were added to 12 well plates of Vero cells and incubated for 1 h at 37 o C, rocking every 15 min. All dilutions were done in duplicate, along with virus only and no virus controls. Plates were then washed with PBS and overlaid with a 1:1 mixture of 2.4% Avicel RC-591 (FMC) and 2X MEM (ThermoFisher) supplemented with 4% 31 . CC-BY 4.0 International license It is made available under a 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 January 13, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 heat-inactivated FBS and Penicillin/Strep-tomycin (Fisher Scientific.) After a 48h incubation, the overlay was removed, plates were washed with PBS, fixed with 10% formaldehyde (Sigma-Aldrich) in PBS for 30 min at room temp and stained with 1% crystal violet (Sigma-Aldrich) in 20% EtOH. Percent neutralization was calculated as (1 -# sample plaques/# positive control plaques) x 100. Data was analyzed in Prism (GraphPad).

SARS-CoV-2 15-mer peptides, 1mg each (BEI Resources), were provided lyophilized and stored at -80C. Peptides were selected for reactivity against a broad range of class I and class II HLA sub-types for targeted coverage of CD4+ and CD8 T cell epitopes identified previously (Tarke et al., 2021) (Table S2) . Before use, peptides were warmed to room temperature for 1 hour then reconstituted in DMSO to a concentration of 10 mg/mL. Individual peptides were combined in equal ratios to create Membrane/Nucleocapsid (182ug/mL each, 55 peptides) or Spike (200ug/mL each, 49 peptides) megapools, maintaining a total concentration of 10mg/mL.

Approximately 20e6 PBMC from SARS-CoV-2 naive or previously infected individuals were divided in two for full phenotypic or cytokine analysis. For broad surface phenotyping 10e6 PBMC per sample were further divided into four 5mL polystyrene tubes and cells were pelleted at 250xg for 5 minutes. Pellets were resuspended at 5e6/mL in one of the following treatment conditions: DMSO (Sigma-Aldrich, >99.5% cell 32 . CC-BY 4.0 International license It is made available under a 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 January 13, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 culture grade), 1ug/mL CEFX Ultra SuperStim Pool (JPT, PM-CEFX-2), or 5ug/mL SARS-CoV-2 Membrane and Nucleocapsid or Spike peptide megapools. Stimulation was performed for 18 hours in ImmunoCult-XF T cell Expansion Medium (StemCell Technologies). For intracellular cytokine assessment 2e6/mL PBMC were stimulated using 6.6ug/mL SARS-CoV-2 peptide megapools, or an equivalent volume of DMSO (Sigma-Aldrich, >99.5% cell culture grade) for 12 hours in RPMI complete T cell medium containing anti-human CD40 antagonist mAb (Miltenyi, clone HB10), 5uL LAMP-1 BV510 mAb (BioLegend, clone H4A3), 2uM CaCl2, and 1.8uL Monensin (Becton Dickinson) -for the final 8 hours of culture.

PBMCs were thawed at 37 o C and washed twice before first staining with decoy tetramer and then with RBD tetramer prior to incubation with anti-PE magnetic beads and magnetic bead enrichment (Miltenyi Biotec) as previously described (Krishnamurty et al., 2016) . Cells in the positive fraction were stained with surface antibodies for B cell phenotypes (Table S3 ). Subsequent analysis of T cells was performed on unlabeled PBMC flow-thru resulting from B cell RBD-tetramer pulldown.

PBMC flow-thru were stimulated with peptide megapools as described above. For surface phenotyping, cells were washed and barcoded using four different fluorescently labeled CD45 antibodies to create eight unique barcodes as previously described (Becht et al., 2021) . Using this method to limit technical variability, all four stimulation conditions for both pre-and post-vaccination samples were combined and fully stained 33 . CC-BY 4.0 International license It is made available under a 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 January 13, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 simultaneously for 20 minutes at 37 o C. Cells were then fixed for 10 minutes at room temperature using 1% paraformaldehyde (Sigma-Aldrich). For intracellular cytokine staining, cells were first incubated with anti-CXCR5 antibody at room temperature for 40 minutes. Cells were then stained with surface antibodies for 25 minutes at 4 o C and then fixed and permeabilized with Fixation Permeabilization kit (Becton Dickinson) for 15 minutes at room temperature before staining for intracellular cytokines for 20 minutes at room temperature (Table S3) .

Data were acquired on a five-laser Cytek Aurora (T cell surface phenotyping and T cell intracellular cytokine analysis) or BD FACS Symphony A3 or A5 (B cell surface phenotyping). Control PBMCs or UltraComp eBeads (ThermoFisher) were used for compensation. Up to 10 7 live PBMC were acquired per sample for T cells and all enriched PBMCs were acquired for B cells. Data were analyzed using SpectroFlow (Cytek Biosciences) and FlowJo10 (Becton Dickinson) software.

AIM-positive (CD154+CD69+) cells from all data files were concatenated with keywords and subjected to Phenograph clustering algorithm using k=40 nearest neighbors (Levine et al., 2015) and UMAP dimensionality reduction plugins using parameters IL-2, IFN-, IL-10, IL-4, IL-21, CD127, CD25, and CXCR5 in FlowJo 10 (Becton Dickinson). Clusters were then enumerated per 1M non-naive T cells for each sample by multiplying the percentage "cluster X parameter" by percentage "AIM+" and then by 1 million.

. CC-BY 4.0 International license It is made available under a 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 January 13, 2022. 

36 . CC-BY 4.0 International license It is made available under a 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 January 13, 2022. ; https://doi.org/10. 1101 /2022 All data generated or analysed during this study are included in this published article (and its supplementary information files) or available from the corresponding author upon reasonable request with the exception of a few blood draw samples that were used up in this study.

Correspondence and requests for materials should be addressed to M.P.

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The copyright holder for this preprint this version posted January 13, 2022. ; https://doi.org/10.1101/2022.01.12.22269192 doi: medRxiv preprint Statistics for unpaired data determined by 2-tailed Mann-Whitney tests and, for paired data, by 2-tailed Wilcoxon signed-rank tests: not significant (ns), *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001. Error bars represent mean and SD. See also:

Supplementary Table 1 and Figure S1 . CC-BY 4.0 International license It is made available under a 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 January 13, 2022. CC-BY 4.0 International license It is made available under a 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 January 13, 2022. ; https://doi.org/10.1101/2022.01.12.22269192 doi: medRxiv preprint neutralization at 1:270 plasma dilution (right). Dashed line indicates the average % neutralization of plasma from N individuals pre-vaccination. Statistics determined by 2-tailed Mann-Whitney tests for all tests because of the limited pairing with 2w PV3: not significant (ns), *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001. Error bars represent mean and SD. Pre-vaccination (Pre), one week post two-dose COVID-19 mRNA vaccination (1w PV2), three months post two-dose vaccination (3m PV2), two weeks post third vaccination dose (2w PV3). See also: Figure S5 . 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 January 13, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 spike-reactive AIM CD4+ T cells from Naive (blue) and Previously Infected (red) donors comparing pre-vaccine (Pre) and one week post two-dose mRNA vaccination (1w Post-V2). "+": marker was gated, "-": marker was excluded, "+/-": analysis was agnostic Mann-Whitney test for group analyses: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Error bars represent mean and SD. Dashed lines indicate average donor 42 . CC-BY 4.0 International license It is made available under a 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 January 13, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 background level. Pre-vaccination (Pre), one week post two-dose COVID-19 mRNA vaccination (1w PV2), three months post two-dose vaccination (3m PV2), two weeks post third vaccination dose (2w PV3). CC-BY 4.0 International license It is made available under a 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 January 13, 2022. ; https://doi.org/10.1101/2022.01.12.22269192 doi: medRxiv preprint vaccination (1w PV2), three months post two-dose vaccination (3m PV2), two weeks post third vaccination dose (2w PV3). 45 . CC-BY 4.0 International license It is made available under a 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 January 13, 2022. ; 

. CC-BY 4.0 International license It is made available under a 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 January 13, 2022. ; https://doi.org/10.1101/2022.01.12.22269192 doi: medRxiv preprint Enumeration of each cluster is per 1M non-naive (nn) CD4+ T Cells in SARS-CoV-2 Naive (blue) and SARS-CoV-2 Previously Infected (red) donors. Pre-vaccination (Pre), one week post two-dose COVID-19 mRNA vaccination (1w PV2), three months post two-dose vaccination (3m PV2). Significance was determined by multiple unpaired Mann-Whitney test for group analyses and by Wilcoxon matched-paired signed rank test for longitudinal analyses: *P < 0.05, **P < 0.01. Table S1 : Study Cohort CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 13, 2022. ; https://doi.org/10.1101/2022.01.12.22269192 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 13, 2022. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 13, 2022. ; https://doi.org/10.1101/2022.01.12.22269192 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 13, 2022. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

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We thank the participants of the HAARVI Research study, Healthy Adult Specimen Repository study and COVID-19/SARS-CoV-2 Prevalence and Antibody Therapy