Impaired Functional T-Cell Response to SARS-CoV-2 After Two Doses of BNT162b2 mRNA Vaccine in Older People

Long-term care facility (LTCF) older residents display physiological alterations of cellular and humoral immunity that affect vaccine responses. Preliminary reports suggested a low early postvaccination antibody response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The aim of this study was to focus on the specific T-cell response. We quantified S1-specific IgG, neutralizing antibody titers, total specific IFNγ-secreting T cells by ELISpot, and functionality of CD4+- and CD8+-specific T cells by flow cytometry, after two doses of the BNT162b2 vaccine in younger and older people, with and without previous COVID-19 infection (hereafter referred to as COVID-19-recovered and COVID-19-naive subjects, respectively). Frailty, nutritional, and immunosenescence parameters were collected at baseline in COVID-19-naive older people. We analyzed the immune response in 129 young adults (median age 44.0 years) and 105 older residents living in a LCTF (median age 86.5 years), 3 months after the first injection. Humoral and cellular memory responses were dramatically impaired in the COVID-19-naive older (n = 54) compared with the COVID-19-naive younger adults (n = 121). Notably, older participants’ neutralizing antibodies were 10 times lower than the younger’s antibody titers (p < 0.0001) and LCTF residents also had an impaired functional T-cell response: the frequencies of IFNγ+ and IFNγ+IL-2+TNFα+ cells among specific CD4+ T cells, and the frequency of specific CD8+ T cells were lower in COVID-19-naive older participants than in COVID-19-naive young adults (p < 0.0001 and p = 0.0018, respectively). However, COVID-19-recovered older participants (n = 51) had greater antibody and T-cell responses, including IFNγ+ and IFNγ+IL-2+TNFα+-specific CD4+ T cells (p < 0.0001), as well as TNFα+-specific CD8+ T cells (p < 0.001), than COVID-19-naive older adults. We also observed that “inflammageing” and particularly high plasma levels of TNFα was associated to poor antibody response in the older participants. In conclusion, our results show that the COVID-19-naive older people had low counts and impaired specific CD4+ and CD8+ T cells, in addition to impaired antibody response, and that specific studies are warranted to assess the efficiency of SARS-CoV-2 mRNA-based vaccines, as in other immunocompromised subjects. Our study also shows that, despite their physiological alterations of immunity, vaccination is highly efficient in boosting the prior natural memory response in COVID-19-recovered older people.

Long-term care facility (LTCF) older residents display physiological alterations of cellular and humoral immunity that affect vaccine responses. Preliminary reports suggested a low early postvaccination antibody response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The aim of this study was to focus on the specific T-cell response. We quantified S1-specific IgG, neutralizing antibody titers, total specific IFNgsecreting T cells by ELISpot, and functionality of CD4 + -and CD8 + -specific T cells by flow cytometry, after two doses of the BNT162b2 vaccine in younger and older people, with and without previous COVID-19 infection (hereafter referred to as COVID-19-recovered and COVID-19-naive subjects, respectively). Frailty, nutritional, and immunosenescence parameters were collected at baseline in COVID-19-naive older people. We analyzed the immune response in 129 young adults (median age 44.0 years) and 105 older residents living in a LCTF (median age 86.5 years), 3 months after the first injection. Humoral and cellular memory responses were dramatically impaired in the COVID-19-naive older (n = 54) compared with the COVID-19-naive younger adults (n = 121). Notably, older participants' neutralizing antibodies were 10 times lower than the younger's antibody titers (p < 0.0001) and LCTF residents also had an impaired functional T-cell response: the frequencies of IFNg + and IFNg + IL-2 + TNFa + cells among specific CD4 + T cells, and the

INTRODUCTION
Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the beginning of the worldwide coronavirus disease 2019 (COVID-19) pandemic, unprecedented efforts have been made to develop vaccines. Considered among the most at risk of developing severe COVID-19, long-term care facility (LTCF) older residents were among the first to be vaccinated. In addition to age, older adults usually cumulate other risk factors for COVID-19 and death, including diabetes, hypertension, cardiovascular disease, and/or malignancy (1). Furthermore, the closed environment and the relative inability of residents to adopt preventive health measures led to numerous outbreaks in LTCFs worldwide (1). For these reasons, there were high hopes for anti-SARS-CoV-2 vaccines, especially among the older and healthcare workers (HCW) in LTCFs. However, the older display physiological alterations of cellular and humoral immunity that affect vaccine responses (2,3), and, due to their age and frailty, they were not included in clinical trials evaluating the BNT162b2 mRNA vaccine (4)(5)(6)(7).
The aims of this study were (i) to assess the specific memory humoral and cellular response after two doses of the BNT162b2 mRNA vaccine in older LTCF residents in comparison with HCWs, with a focus on the functionality of specific T cells, (ii) to evaluate the impact of prevaccine immunization, by comparing the postvaccinal response in older adults without and in older adults with prior COVID-19, and finally (iii) to evaluate the impact of frailty, nutrition, and immunosenescence features on postvaccination immune response.

Study Design and Participants
This was a prospective single-center study conducted at the Lille University Hospital, in the North of France. Participants were consecutively included in the study and were healthcare workers (HCW; hereafter referred to as young adults) aged 18-65 years and LCTF residents (hereafter referred to as older residents or older adults) aged >65 years who consented to be vaccinated with BNT162b2 mRNA vaccine and were willing to comply with the study procedures. None of the enrolled participants had a recent, current, or persistent infectious disease, any neoplasia diagnosis in the last 5 years, or treatment with steroids and/or immunosuppressants. Participant characteristics collected at baseline included confirmation of prior SARS-CoV-2 infection, determined by polymerase chain reaction (PCR) and/or high antibody titer to SARS-CoV-2 spike S1 domain: participants with a history of positive PCR and/or who tested positive for anti-S1 antibodies were considered COVID-19-recovered, and the other participants as "COVID-19-naive." Among older adults, Geriatric Nutritional Risk Index was calculated according to the Lorentz formula: GNRI = (1.489 × albumin, g/l) + (41.7 × present/ideal body weight), with the ideal weight calculated according to the Lorentz formula (8). Frailty was assessed with the Clinical Frailty Scale as proposed by Rockwood et al. (9) and using the Fried frailty phenotype criteria (10). All participants received the two-dose BNT162b2 vaccination at a 3-week dosing interval: the first dose was administered at Day 0 (D0) and the second dose between D21 and D28. Serum samples were collected for all participants at D0, and D90 (± 14 days) after the first dose.

Anti-SARS-CoV-2 Antibodies
Anti-SARS-CoV2 spike S1 domain-specific immunoglobulin G (IgG) was assessed in serum samples using ELISA (Quantivac, Euroimmun Lübeck, Germany), with a sensitivity of 90.3 and a specificity of 99.8% according to the manufacturer's data. The maximum IgG level that could be determined with appropriate precision after dilution was 1,920 relative units per milliliter (RU/ml).

SARS-CoV-2 Neutralization Assay
Neutralizing antibodies were investigated using a live virus neutralization assay (LV-NT). A classical B.1.1.7 lineage (20I/ 501Y.V1) SARS-CoV-2 strain, previously isolated from a clinical specimen and propagated in Vero E6 cells, was used in all experiments. The whole genome sequence of the viral isolate was submitted to GISAID (accession reference EPI_ISL_1653931). In brief, serial twofold dilutions (starting from 1:10) of the heated serum (56°C for 30 min) were incubated for 1 h at 37°C with a viral solution containing 100 TCID50 of SARS-CoV-2 and then added to Vero E6 cell monolayers in a 96-well plate. The cytopathic effect was recorded after 3 days, and the serum virus neutralization titer (V-NT50) was defined as the reciprocal value of the highest dilution that showed at least 50% protection of cells. A sample with a titer ≥20 was defined as positive. Negative signals were set to 0 for statistical analyses.

SARS-CoV-2 Pseudovirus Neutralization Assay
To further assess the neutralizing activity of sera, retroviral pseudoparticles containing the SARS-CoV-2 glycoprotein S (SARS-CoV-2pp) were produced as previously described (11), with a plasmid encoding the human "codon-optimized" sequence of the SARS-CoV-2 glycoprotein spike (accession number: MN908947). The supernatants containing the SARS-CoV-2pp were harvested at 48-h posttransfection and filtered through a 0.45-µm membrane and stored at −80°C. The serum neutralization test was performed as previously described (12). In brief, 20 µl of SARS-CoV-2pp were incubated in the diluted serum at a final volume of 50 µl of DMEM+Glutamax+penicillin-streptomycin+10% fetal calf serum (FCS) for 30 min at room temperature. The mixture was then added to HEK 293TT-ACE2 plated the day before (HEK 2932TT cells stably expressing the hACE2 receptor are seeded at 4,500 cells/well in a volume of 50 µl of DMEM+Glutamax+penicillin-streptomycin+10% FCS mixture) (13). At 48-h postinfection, Luciferase activity was measured using the Luciferase Assay System kit (Charbonnièresles-Bains, Promega FR, Charbonnières-les-Bains, France) as recommended by the manufacturer and expressed as relative luciferase units (RLUs). RLUs were compared and normalized with the wells where pseudoparticles were added in the absence of serum (100%). Serum pseudovirus neutralization titer 50 (PV-NT50) was expressed as the maximal dilution of the sera where the reduction of the signal is greater than 50%. The titer was multiplied by 781, since the initial volume of the sera tested was 8 µl and had to be normalized to 1 ml (14).

Peripheral Blood Mononuclear Cells Preparation
Isolation and numeration of peripheral blood mononuclear cells (PBMCs) were performed from 10 to 15 ml of freshly collected heparinized blood samples. In brief, T-cell Xtend (Oxford Immunotec, Abingdon, UK) at a concentration of 25 µl/ml of blood was added 15 min prior to isolation to remove cell debris and aggregates. SepMate-50 ml (StemCell Technologies, Vancouver, Canada) was then used for density gradient centrifugation. PBMCs were collected and washed twice using RPMI. Isolated cells were suspended in AIM-V medium and counted using flow cytometry with CD45 staining (Beckman Coulter, Brea, CA, USA) and Flow-Count Fluorospheres (Beckman Coulter). Normalization of the cell suspension was performed at a final concentration of 2.5.10 6 cells/ml for T-CoV-Spot assay and 10.10 6 cells/ml for flow cytometry analyses.

IFNg ELISpot Assay-T-CoV-Spot Assay
T-CoV-Spot assay was performed as previously described (8). In brief, overlapping peptide pools covering the N-terminal S1 domain were used (PepTivator_SARS-CoV-2, Miltenyi Biotec, Bergisch Gladbach, Germany). Peptides consisted of 15-mer sequences with 11 amino acids overlap. Microtiter plates coated with anti-IFNg antibodies (T-SPOT.TB, Oxford Immunotec) were used. The cell suspension was normalized at a final concentration of 2.5 × 10 6 cells/ml, and plating with SARS-CoV-2 antigens was manually performed (2.5 × 10 5 PBMCs added per well). Peptide pools were added at a concentration of 0.5 mg/ml. Following an incubation at 37°C for 16-20 h in a humidified atmosphere containing 5% CO 2 , wells were washed and incubated with conjugate reagent for 1 h at 2°C-8°C. After a washing step, wells were developed for 7 min with substrate solution. The reaction was stopped by adding distilled water. Plates were allowed to dry in an oven at 37°C for 1 h. Spot-forming cells (SFCs) were detected using the CTL ImmunoSpot plate reader. Appropriate negative and positive controls were used (15).

Fluorescence-Activated Cell Sorter Data Analysis
Fluorescence-activated cell sorter (FACS) data were analyzed with Kaluza Analysis Software (Beckman Coulter). The gating strategy for analysis of antigen-specific T cells is illustrated in the Supplementary Figure S1. For the activation-induced marker (AIM) T-cell assay, a specific T-cell response was considered positive when the stimulation index was 2 or higher, i.e., when the antigen-stimulated cultures contained at least twofold higher frequencies of CD154 + CD69 + cells among alive (7-AAD−) CD4 + T cells (AIM + CD4 + T cells), or CD107a + CD69 + cells among alive CD8 + T cells (AIM + CD8 + T cells), compared with the unstimulated control sample. No further background subtraction was applied. Coexpression of intracellular cytokines was assessed among AIM + CD4 + and CD8 + T cells using a Boolean gating strategy. Unsupervised analysis was conducted using t-distributed stochastic neighbor embedding (t-SNE) in AIM + CD4 + or CD8 + T cells (Cytobank, Beckman Coulter). All datasets were extracted from the pregating made with Kaluza on AIM + T cells, group concatenations were made, and all data were imported into Cytobank. Unsupervised cell subset identification (clustering) was also performed for analysis of cytokine productions by AIM + CD4 + and CD8 + T cells. Percentages of each main subsets of specific T cells (according to production of 0/1, 2, or 3 cytokines) obtained by the unsupervised FlowSOM analysis (considered the addition of all cluster abundance in the subset) were reported on the subsets (Cytobank, Beckman Coulter).

Statistical Analyses
Categorical variables are expressed as numbers (percentages) and quantitative variables are expressed as median (interquartile range). Normality distribution was assessed graphically and using the Shapiro-Wilk test. Immune parameters were compared within the same group between the baseline and 3-month assessments using the Wilcoxon signed rank test. Comparisons of immune parameters between the four study groups (COVID-19-naive older, COVID-19-recovered older, COVID-19-naive young, and COVID-19-recovered young) were done using the Kruskal-Wallis test followed by post-hoc Dunn's tests for quantitative measures and chi-squared test (or Fisher's exact test in cases of expected cell frequency <5) for responder rates. Comparisons of baseline characteristics in COVID-19-recovered older adults and D90 characteristics in COVID-19-naive older adults (natural post-COVID-19 versus post-BNT162b2 immunization) were done using the Mann-Whitney U test. We assessed the correlation between age, vaccinal response parameters, nutritional, frailty, or immunosenescence parameters by calculating Spearman's rank correlation (r) coefficients, with their 95% confidence intervals based on the Fisher Z-transformation. Statistical tests were done at the two-tailed a level of 0.05. No correction for multiple testing was carried out. Data analyses and graphs were performed using the GraphPad Prism software version 9.1.2 (GraphPad Software, La Jolla, CA, USA).

Ethics
This study was performed in accordance with the Declaration of Helsinki principles for ethical research. The study was approved by the Ile-De-France V (ID-CRB 2021-A00119-32) ethics committee. All participants (and/or their legal representative if required) received detailed information and signed a consent form before participating in the study. The study was registered in ClinicalTrials.gov, with the identifier NCT04760704.  Table 1). Participants were sampled before (D0) and 90 days (D90) after the first dose: 129 young adults and 105 older residents had both samples ( Figure 1). In young and older subjects, with or without prior COVID-19, anti-S1 IgG, neutralizing antibodies, and IFNgsecreting T-cell levels increased from D0 to D90 (Figure 2 and Supplementary Figure S1).

Immunogenicity in COVID-19-Naive Young and Older Subjects
Our primary objective was to compare the specific memory response in COVID-19-naive younger (n = 121/129) and in COVID-19-naive older adults (n = 54/105). At D90, S1 IgG reactivity was detected in almost all participants in both groups (99.2% of younger and 97.2% of older adults), but the median titer of anti-S1 IgG antibodies was two times lower among the older residents (p < 0.001) ( Figure 3A). The difference was greater for neutralizing antibodies, with a geometric mean of 50% serum neutralization titer (NT50)  Figure 3C). The mean NT50 in each group was consistent with the PV-NT50 assay (Supplementary Figure S2). Regarding cellular response, T cells reactive to the S1 subunit detected by ELISpot were less frequent in the older than in the younger group (13.5 [25.0-27.57] versus 29.5 [15.0; 46.5], respectively) (p = 0.002) ( Figure 3D). To confirm our results, all acquired parameters were correlated with age in COVID-19-naive young and older participants. Age negatively correlated with anti-S1 IgG, neutralizing antibody titers, and count of specific IFNg-secreting T cells, which support the differences observed between the two groups ( Figure 3E). There were also strong positive correlations between the immune parameters, which highlights both the conserved links between these different adaptive responses among the older population, and the robustness of the chosen approaches ( Figure 3E and Table 2).
To confirm the significance of our results in specific cellular responses, we used an automatized cluster analysis of T-cell subsets (Supplementary Figure S3). The hierarchical clustering confirmed the lower cytokine production in COVID-19-naive older participants compared with the three other groups of interest. The cluster analysis also showed a higher CD8 + /CD4 + ratio among AIM + T cells in the COVID-19-recovered older group. Similarly, an unsupervised analysis using t-SNE corroborated the reduced cytokine production in COVID-19-naive compared with the COVID-19-recovered older participants (Figure 7).

Immunogenicity of BNT162b Vaccine
According to Frailty, Nutritional, and Immunosenescence Parameters in COVID-19-Naive Older People Finally, we evaluated whether some frailty, nutritional, and immunosenescence parameters could account for the poor  vaccinal response among the COVID-19-naive older adults. As other authors (9), we failed to identify any clear link between vaccinal response and frailty, nutritional state (Supplementary Figure S4), or baseline B cell, total T-cell, and naive T-cell counts (Supplementary Figure S5). We also assessed plasma levels of three major proinflammatory cytokines, IL-1b, IL-6 and TNFa, and IL-10, an anti-inflammatory cytokine, in COVID-19-naive older adults. Plasma IL-1 b levels tended to be negatively correlated with anti-S1 IgG and live virus-neutralizing antibodies. Plasma TNFa levels correlated negatively with both neutralizing titers (Figure 8).

DISCUSSION
Our work demonstrates that COVID-19-naive older adults have a poor memory immune response to BNT162b2 mRNA vaccine compared with the younger adults. Considering the impact of COVID-19 on life expectancy in LCTF residents (16), specific vaccinal strategy may be required in this frail population. Our results are in line with earlier evaluations of antibody response after a first dose (9,10) and between 14 and 28 days after the second one (17)(18)(19)(20), indicating a poorer response in the older people. Even though we did not assess memory-switched B cells, our results obtained 90 days after the first dose and 60 days after the second dose may reflect the memory response established after vaccination rather than a response being initiated and may predict that immunity may wane even more over time.
Our study also brings to light new elements on the function of T cells in the older population after two doses of BNT162b2. As in previous works using FluoroSpot or ELISpot T-cell assays (18,20,21), we also reported here an impaired specific T-cell response after a full vaccination scheme in older people.  Table 2. **p-values <0.01; ***p-values < 0.001; ****p-values < 0.0001; ns, not significant. IFNg SFCs, interferon gamma spot-forming cells; LV-NT50, 50% serum neutralization titer in live virus neutralization assay; pV-NT50, 50% serum neutralization titer in pseudovirus neutralization assay. All correlations are presented in Figure 3E (with Spearman's rank correlation (r) coefficients). NT50 LV-NT assay, 50% serum neutralization titer in live virus neutralization assay; NT50 PV-NT assay, 50% serum neutralization titer in pseudovirus neutralization assay; CI, confidence interval.
Using flow cytometry, we observed that specific IFNg + and triple + CD4 + T cells, the most important subsets in the orchestration of the whole adaptive immune response, were lower in COVID-19-naive older participants than in COVID-19-naive young adults. We also noted that, in the COVID-19naive population, the frequency of AIM + IL-2 + CD8 + T cells (but not IFNg + or TNFa + CD8 + T cells) was greater among the older subjects compared with the young group. This suggests that,  while CD8 + T cells can be highly activated with SARS-CoV-2 antigens in the naive older population, the main effector cytokines required for antiviral response are not produced.
Our study also demonstrated that specific antibody response is greater in COVID-19-recovered older residents (compared with COVID-19-naive), and at a level similar to that of young participants. These results are in agreement with previous reports suggesting that patients with prior COVID-19 infection had a better antibody response, regardless of the age (21)(22)(23). Our work also shows that specific IFNg + and triple + CD4 + T cells, and specific TNFa + CD8 + T cells, the latter being the most important in antiviral defense, were also highly boosted in participants who had a prior COVID-19 infection. Considering the major role of these cell subsets in limiting the disease severity (23,24), a repeated vaccination could be effective in increasing the immune response in the older population.
We also observed that "inflammageing" may play a role in the poor anti-SARS-CoV-2 antibody response in the older, and particularly TNFa. This is in line with previous data in human and mice models, which reported that serum TNFa negatively  correlated with the B-cell response and a vaccine-specific antibody response (24).

Strengths and Limitations
This study is the first one to assess the functionality of specific T cells in older people after two doses of BNT162b: our results obtained by flow cytometry support the results obtained by ELISpot and brings new elements about the quality of the postvaccination T-cell immune response in this at-risk population for severe COVID-19. We are also able to discuss the higher immunogenicity of BNT162b vaccine in older people with previous COVID-19, not only considering the antibody response, but also the T-cell response and its functionality. Our study is, however, limited in that, due to the recommendations applied in France at the time of the study, we were unable to assess whether a single dose of this vaccine after exposure to COVID-19 would have generated a sufficient response in these individuals. Also, the relatively short follow-up period only allowed us to assess the short-term effects of the vaccine. However, these data on the 2-month residual immune memory after the second dose may help anticipate future needs to adapt the vaccination strategy among the older.

CONCLUSION
Our results demonstrate that, with the recommended vaccination scheme (i.e., two doses of BNT162b2), both antibody and cellular responses are impaired in the COVID-19-naive older population compared with the younger group: this definitely confirms that specific studies are necessary to assess the immunogenicity of mRNA vaccines in frail older people (6,7). Recent studies showed only a slightly lower immunogenicity after two doses with the delta variant that is currently responsible of a large majority of COVID-19 cases in many countries. We can suppose that the reduced immunogenicity of the BNT162b2 vaccine among older people may be similar or lower for the delta variant. For this reason, some countries have recently decided to recommend a third dose in older people and not only in immunocompromised patients. Our study illustrates that, even if the ability to respond to neoantigens A B FIGURE 7 | Unsupervised analysis of CD4 + and CD8 + T-cell functionality in older participants using t-distributed stochastic neighbor embedding (t-SNE). AIM + CD4 + (A) and AIM + CD8 + (B) T cells from older participants were concatenated and subjected to unsupervised analysis using t-SNE; highlighted (z-dimension) are areas with IFNg, IL-2, or TNFa cell expression in COVID-19-naive and COVID-19-recovered older adults. To be noted, the higher frequency of IFNg + CD4 + T cells and of TNFa + CD8 + T cells in COVID-19-recovered older adults (arrows).
is impaired in the older, the post-COVID-19 memory immune response is improved by an additional boost. Our work highlights the need of specific studies to assess the efficiency of SARS-CoV-2 mRNA-based vaccines in older people living in LCTFs, as in other immunocompromised subjects, and notably to confirm that a third dose may improve protective immunity.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Ile-De-France V (ID-CRB 2021-A00119-32) ethics committee. The patients/participants provided their written informed consent to participate in this study.