Competent immune responses to SARS-CoV-2 variants in older adults following two doses of mRNA vaccination

Aging is associated with a reduced magnitude of primary immune responses to vaccination. mRNA-based SARS-CoV-2 vaccines have shown efficacy in older adults but virus variant escape is still unclear. Here we analyze humoral and cellular immunity against an early-pandemic viral isolate and compare that to the P.1 (Gamma) and B.1.617.2 (Delta) variants in two cohorts (<50 and >55 age) of mRNA vaccine recipients. We further measure neutralizing antibody titers for B.1.617.1 (Kappa) and B.1.595, with the latter SARS-CoV-2 isolate bearing the spike mutation E484Q. Robust humoral immunity is measured following second vaccination, and older vaccinees manifest cellular immunity comparable to the adult group against early-pandemic SARS-CoV-2 and more recent variants. More specifically, the older cohort has lower neutralizing capacity at 7-14 days following the second dose but equilibrates with the younger cohort after 2-3 months. While long-term vaccination responses remain to be determined, our results implicate vaccine-induced protection in older adults against SARS-CoV-2 variants and inform thinking about boost vaccination.

R NA viruses have a high mutation rate resulting in diverse viral populations 1 . Since its emergence in the human population in late 2019, SARS-CoV-2 has infected more than 295 million people globally leading to the appearance of multiple new variants. Several variants of concern (VOC) quickly became dominant in their countries of identification, spreading across the globe. B.1.1.7 (Alpha) was identified in the UK in December 2020 and spread to more than 90 other countries 2 . Other identified VOC with broad spread include B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta). These lineages are transmitted more efficiently, conferring evolutionary advantage over ancestral virus 3 . Two mRNA vaccines based on the first published genome sequence of SARS-CoV-2 (Wuhan/Hu-1/2019), BNT162b2 (Pfizer) and mRNA-1273 (Moderna), are being deployed to reduce COVID-19 disease severity and transmission, raising the obvious question of whether they will afford similar protection against variants of concern. Reduced plasma antibody (Ab) neutralization titers against pseudovirus in mRNA vaccine recipients has been reported for multiple variants 4 . Another report showed that B.1.351 (Beta) variant might be more effective in escaping humoral immunity than B.1.1.7 (Alpha) variant 5 , as one may expect from the location of its mutations to Ab-binding regions. Despite reduced humoral immunity, T cell responses to Alpha and Beta variants were preserved in adult mRNA vaccine recipients 6 . Similar results were obtained with adenovirus vaccine recipients, which showed 3-5 fold lower neutralizing antibody titers against Beta and Gamma variants but uncompromised T cell responses 7 .
Efficacy and immunogenicity of many vaccines are known to be decreased in advanced age [8][9][10] . Fortunately, SARS-CoV-2 mRNA based vaccines were shown to be both well tolerated and highly effective in older adults [11][12][13] . It remains unknown whether the breadth of the immune response to these mRNA vaccines will remain sufficient for protection against new viral variants.
Here we find that the magnitude and neutralization capacity of humoral memory to these vaccines is not reduced in our older adult cohort (>55 years) as might have been expected based on historic vaccine studies. We confirm lower neutralizing antibody titers against several variants in both of our cohorts. Importantly, robust cellular immunity against WA1, P.1 (Gamma), and B.1.617.2 (Delta) is preserved in most participants. Overall, the effect of age on the immune response to SARS-CoV-2 variants in mRNA vaccinees was measurable, but minimal. The age-related decrease is most evident in the antibody response at 7-14 days after the second vaccine dose. We conclude that effective immunity in older adults is attainable with mRNA vaccines.

Results and discussion
A total of 40 participants were enrolled in our study before receiving either the BNT162b2 (Pfizer) or mRNA-1273 (Moderna) COVID vaccine. Blood draws were collected prior to initial dose of vaccine, 7-9, and 18-26 days after first vaccine dose, and 7-14 days, 2-3 months, and 6 months after their second dose. These time points are designated as T0, T1, T2, T3, T4, and T5 on the graphs. One participant was excluded from the study because they had a high neutralizing antibody titer and a strong antigen (Ag) specific T cell response before vaccination, indicating prior infection. Our final cohort included 22 participants under 50 years of age (<50) and 17 participants over the age of 55 (>55).
Antibody ELISA assays for the receptor-binding domain (RBD) and S2 region of spike protein on plasma samples at each time point show similar rates and levels of seroconversion between cohorts (Fig. 1a, b). Area under curve (AUC) values confirm that participants over age 55 have robust and comparable antibody responses to the younger cohort with no significant differences measured at any time point (Fig. 1c). Neutralizing antibody test titers utilizing WA1 were also comparable between both cohorts across time points (Fig. 2a). These data taken together are important because they demonstrate that the humoral immune response in older adults is preserved to these mRNA vaccines when tested with the virus they were generated against. We found a diminished capacity of both cohorts to neutralize the P.1(Gamma), AZ-E484Q, and B.1.617.1 (Kappa), and B.1.617.2 (Delta) at the third time point with equivalent neutralization in memory ( Fig. 2a and Table 1). E484Q, a mutation in the RBD of spike protein, is present in both B.1.351 (Beta) and P.1 (Gamma) and has already been demonstrated to impact neutralization capacity 14 . We chose AZ-E484Q to test against vaccine induced antibodies because it has spike E484Q and D614G mutations in common with B.1.617.1 (Kappa) but not L452R. L452R has already been shown by others to impact neutralization in pseudotyped virus systems 15 but it remained an open question how E484Q impacts neutralization capacity 16 . Our use of authentic B.1.595 SARS-CoV-2, which in spike bears only E484Q and D614G, demonstrates that E484Q also affords the virus an opportunity to escape neutralization. Understanding how this particular mutation impacts neutralization may be important during the emergence of future variants. Finally, we compared neutralizing antibody titers between vaccine brands at T3 and T4 and determined that antibody responses to the Pfizer vaccine were lower than for Moderna when tested against WA but were statistically indistinguishable when evaluated against the variant viruses ( Supplementary Fig. 1A). Antibody assays are considered the gold-standard when assessing the quality of immunity after vaccination because antibodies can provide sterilizing immunity. However, the establishment of memory B cell populations is also critical to lasting vaccine efficacy as these are the resources available for response to the virus, or a variant, upon next encounter. To assess whether there is an impact of age on the formation of memory B cell populations we measured the frequency and number of circulating memory B cells specific for SARS-CoV-2 pre-vaccination and one week after booster dose by dual staining with tetramers specific for RBD and S1 using flow cytometry 17 (gating strategy in Fig. 3a). Both groups of participants had a small (0.1-0.3% of all B cells) but detectable population of antigen specific cells double positive for S1 and RBD tetramer (Fig. 3a). This population increased as a percentage of B cells post vaccination in all but two (one from each cohort) participants with no difference between groups (Fig. 3b). By multiplying percentages of tetramer positive B cells with total B cell percentage and lymphocyte counts we have calculated the absolute numbers of SARS-CoV-2 specific B cells in circulation and again observed no difference between cohorts for S1 + RBD + double positive cells (Fig. 3c). Similar results were obtained with S1 single-positive cells again with no difference between adult and older participants post vaccination in percentage of S1 single positive cells (Fig. 3d) or their absolute numbers (Fig. 3e). Next, we examined the phenotype of the SARS-CoV-2 specific B cells (gating strategy in Supplementary  Fig. 1B) with respect to class switching, as it has been previously reported that aging is associated with a decline in the percentage and numbers of switched memory B cells 18 . To investigate this possibility, we examined differentiation and class switching of total tetramer (S1+) positive B cells by flow cytometric staining for CD27, IgM, IgD, CD21 and CD11c (representative flow cytometric gating in Supplementary Fig. 1A). Ag-specific B cells from adult and older participants expressed CD27 at identical levels ( Supplementary Fig. 1C) and equal numbers of both CD27 positive and negative cells were class switched ( Supplementary  Fig. 1D, E). Adult and older participants also displayed no difference in classical memory (CD21+) phenotype among the class switched tetramer-positive cells ( Supplementary Fig. 1F). Thus overall, we conclude that induction and differentiation of SARS-CoV-2 specific B cells through vaccination was not compromised by aging.
Finally, we measured antigen-specific T cells elicited by vaccinated. Given that ELISpot was previously reported to be a highly sensitive method for detection of rare antigen specific T cells 19 , we simultaneously measured the number of T cells expressing costimulatory molecules CD137 and OX-40 by flow cytometry (used in several reports examining SARS-CoV-2 specific T cell immunity -20,21 (representative flow cytometry in Supplementary Fig. 2A) and performed IFN-γ ELISpot on PBMCs from select participants stimulated with peptide pools corresponding to the spike protein of WA1. Confirming prior data, ELISpot proved to be a much more sensitive method for enumeration of Ag-specific T cells. We detected a statistically significant increase in ELISpots per 10 6 PBMC's after vaccination analyzing just 5 samples, whereas parallel flow cytometry samples showed no significant differences (Supplementary Fig. 2B-E). The only limitation of ELISpot, as compared to flow cytometry based enumeration, is that total T cell responses are measured without separate quantification of CD4 and CD8 responses.
We also analyzed Ag-specific T cell responses to stimulation with spike protein peptide pools from WA1 versus two VOC by ELISpot. Participant PBMC's were stimulated with 16-mer overlapping peptide pools corresponding to the spike protein of WA1, P.1 (Gamma) and B.1.617.2 (Delta). In accordance with previously published results 22 , mRNA vaccines induced a robust T cell response to WA1, the ancestral strain of SARS-CoV-2, which did not differ for Gamma and Delta variants, as evidenced by a tenfold increase in ELISpots from post-vaccination samples stimulated by S peptide pools compared to unstimulated wells (Fig. 4a). Of note, the data represented in Fig. 4a shows concatenated time points for each peptide pool to demonstrate the resolution of this assay. Next, we parsed that data to compare T cell responses of both cohorts at different time points postvaccination and subtracted the number of spots in the unstimulated wells for each sample to properly calculate the number of

ARTICLE
Ag-specific ELISpots. Data from old mice [23][24][25] showed that induction of Ag-specific T cell responses becomes delayed and decreased with age. In our data, there was a slightly lower response in the older cohort at day 7 post first dose with all three variants which was statistically significant only with the WA1 peptide pool (Fig. 4b). However, after a second dose both groups had a very robust T cell response against all three viral variants examined (Fig. 4c-e). Therefore, while we acknowledge that the primary Ag-specific T cell response is lower, and likely delayed, in some of our older participants, we conclude that the outcome after booster dose is competent T cell mediated immunity against WA1 and tested VOC in all mRNA vaccine recipients in our study. This is in agreement with our previous studies of vaccination in aged mice which showed that at least two cycles of T0 T3 S1 tetramer RBD tetramer S1 tetramer RBD tetramer Doublets and dead cells excluded and S1 and RBD tetramer positive cells analyzed as a frequency of total CD19 + B cells; b Percentage of tetramer double positive (S1 + RBD + ) B cells were increased after vaccine booster dose (T3) compared to pre-vaccination (T0) and to an equal extent in adult and older adult cohorts. c Absolute numbers of S1 + RBD + B cells were also equally increased in <50 and >55 cohorts. d Percentage of S1+ tetramer single positive B cells were equally increased in <50 and >55 cohorts; e as was their absolute number per ml of blood. n = 22 < 50 cohort and n = 16 > 55 cohort. Line is median; Mann-Whitney U test. in vivo restimulation are required for adequate ag-specific T cell response in aged animals 26 . All of these data taken together demonstrate the rather expected blunted primary response in older adults and the improvement of this response to the levels seen in adults following a second vaccine dose. Since IFN-γ is not the only effector cytokine produced by T cells following antigen stimulation, we have additionally measured polyfunctional responses. Spike peptide pools induce a dramatic number of IFNγ spots in comparison to unstimulated wells, but also an increase in IL-2 and GrB spots (Fig. 5a). We observed no difference between the age groups in IL-2 or GrB spots (Fig. 5b) in response to WA/2020 or Delta peptide pools at post-second dose time points (T3 and T4). Similarly, there was no difference between the number of polyfunctional double positive (Fig. 5c) or triple positive cells (Fig. 5d). We also analyzed FLUORISpot responses in recipients of mRNA vaccines from different manufacturers. We did not measure any difference in IFN-γ, IL-2 or GrB T cell responses between recipients of Moderna vs. Pfizer mRNA vaccine ( Supplementary Fig. 3A, B). It is well established that primary immune responses wane with age and contribute to the increased susceptibility to infection experienced by older adults (reviewed in ref. 27 ). There is also evidence that generation of immune memory in older adults is reduced, but not maintenance of memory. Studies with multiple pathogens have shown decreased T cell receptor repertoire with age 28,29 which could mean easier escape from existing immunity for pathogen variants. All of the above-mentioned findings warrant an extensive and long-term monitoring of immunity in SARS-CoV-2 vaccinees, especially those over age 55.
The emergence of SARS-CoV-2 impacted older adults especially hard with more than 80% of deaths in those over age 65 30 and mortality rates rising sharply above the age of 55 31 . Vaccination reduces these rates dramatically 32 demonstrating that the principles of immunology hold true for this virus and that immune memory is what our species is lacking. Well tolerated and effective, SARS-CoV-2 mRNA vaccines induce potent humoral and cellular immune responses 11,22 . Their deployment also offers an opportunity to establish, in a truly immune naive population, correlates, and contours, of protective immunity. Recent elegant studies in rhesus macaques have shown that even sub-sterilizing neutralizing antibody titers are protective and decrease SARS-CoV-2 severity 33 . The same research study demonstrated a protective role for T cell memory responses and this is supported by human research showing that accumulation of oligoclonal CD8 T cells in bronchoalveolar lavage fluid inversely correlated with disease severity 34 . Recently, Collier et al. 35 analyzed 102 partially and 38 fully vaccinated participants and concluded that participants >80 years of age produced lower primary and secondary antibody neutralizing responses, including those against variants 35 . They did not analyze T cell immunity against variants and, somewhat curiously, did not observe increased T cell responses following the second dose in their older groups (>80). These authors argue that older adults-those over 80-remain vulnerable at least until they receive the second vaccine dose. Our results agree with these conclusions with regard to antibody immunity, and suggest that T cell immunity in response to mRNA vaccines is robust in older adults and against variants (Fig. 6), even though we did not analyze participants in the octogenarian bracket. Clinical efficacy of the mRNA vaccines in protecting older adults has been strong, consistent with both our data and those by ref. 35 . The decrease in antibody titer when challenged with SARS-CoV-2 variants does suggest that the breadth of immunity may be narrower in advanced age; a challenge that can be met with booster doses of heterologous sequence virus. Further studies on the durability and breadth of protection by current and future heterologous vaccines in older adults will be necessary to answer these and other germane questions on their immunity and SARS-CoV-2 protection in older adults.

Methods
Study participants. This study, collection, and use of human blood was approved by the University of Arizona Institutional Review Board (Protocol#2102460536). We collected blood from 23 adults <50 years old and 17 above 55 years old. Demographics are provided in Table 2. Informed consent was obtained for all participants. Participants received a compensation of 25 USD for each blood draw. Samples for all participants were collected prior to vaccination, 1 week after first dose (mRNA vaccine: Pfizer N = 23; Moderna N = 17), day before booster dose and 7-10 days after booster. Many participants were also sampled at 3 or 6-9 months following first dose. The time points are labeled T0, T1, T2, T3, T4, and T5 in all graphs. Blood for complete blood count was collected in BD vacutainer with EDTA and submitted to Sonora Quest Laboratories (Arizona). Blood for peripheral blood mononuclear cells (PBMCs) and plasma was collected in BD Vacutainer with sodium heparin. Plasma was separated by centrifugation at 1000 g for 10 min and PBMC was isolated from the buffy coat by Ficoll-Paque PLUS (GE Healthcare) and cryopreserved in fetal calf serum + 10% DMSO. ELISpot assays. T cell specific immunity to peptide pools corresponding to spike, nucleocapsid, and matrix proteins were measured as previously described 17  Flow cytometry. Cryopreserved PBMC (2-5 × 10 6 /sample) were thawed in prewarmed RPMI-1640 with L-glutamine (Lonza) + 10% FCS. Thawed PBMCS were rested overnight at 37°C in X-VIVO 15 Serum-free Hematopoietic Cell Medium (Lonza) supplemented with 5% human Ab serum. Cells were stained with surface antibodies in PBS (Lonza) + 2% FCS, and then stained with the live dead fixable blue dye (Thermofisher). B cell tetramers were assembled by mixing 100 μg ml −1 of C-terminal AviTagged RBD or S1 (ACROBiosystems) with 100 μg ml −1 of streptavidin-PE (eBiosciences) or streptavidin-BV421 (BioLegend), respectively, at a 5:1 molar ratio in which 1/10 of the final volume of streptavidin was added every 5 min. Samples were stained for 1 h at 4°C. List of antibodies used for flow cytometric staining in Supplementary Table I. Samples were acquired using a Cytek Aurora cytometer (Cytek) and analyzed by FlowJo software (Tree Star).
Statistical analysis. SPSS and Graph Pad Prism were used for statistical analysis. Upon inspection of data distribution by Shapiro-Wilks normality test group differences were calculated as described in each figure legend. Fig. 6 Schematic of blood collection time points and overall depiction of T cell and antibody responses measured in adult and older adult cohorts over a two dose series of COVID mRNA vaccine. Two doses of mRNA vaccine potently stimulate T cell and antibody immune responses, even in older adults, to both ancestral and variant SARS-CoV-2. These data demonstrate that the immune system is quite competent to be leveraged for immune defense and that mRNA vaccines can be an effective strategy.