Induction and subsequent decline of S1-specific T cell reactivity after COVID-19 vaccination

We analyzed magnitude and duration of SARS-CoV-2-specific T cell responses in healthy, infection-naïve subjects receiving COVID-19 vaccines. Overlapping peptides spanning the N-terminal spike 1 (S1) domain of the spike protein triggered secretion of the T cell-derived cytokine interleukin-2 ex vivo in 94/94 whole blood samples from vaccinated subjects at levels exceeding those recorded in all 45 pre-vaccination samples. S1-specific T cell reactivity was stronger in vaccinated subjects compared with subjects recovering from natural COVID-19 and decayed with an estimated half-life of 134 days in the first six months after the 2nd vaccination. We conclude that COVID-19 vaccination induces robust T cell immunity that subsequently declines. EudraCT 2021–000349-42. https://www.clinicaltrialsregister.eu/ctr-search/search?query=2021-000349-42


Introduction
Most currently employed COVID-19 vaccines contain mRNA or double-stranded DNA that encode the spike protein of SARS-CoV-2. While these vaccines are estimated to have spared tens of millions of lives during the COVID-19 pandemic [1], there remains uncertainty regarding the duration of protection after vaccination. Several studies thus show temporal decline of IgG antibodies against the spike protein four to six months after vaccination [2]. COVID-19 vaccination also entails evolvement of spike-specific T cells [3][4][5] but few studies have analyzed duration of vaccine-induced T cell memory against SARS-CoV-2.
Antibodies and T cells are complementary and partially independent effector arms of immunity. A T cell assay may thus reflect immunological memory that is not captured by serum anti-spike 1 (S1)-IgG. Simple and rapid tests that detect functional SARS-CoV-2 specific T cells in whole blood samples were recently developed [6]. For this study, we used a cytokine release assay to determine S1-specific induction of the T cell-derived cytokine interleukin-2 (IL-2) in whole blood samples from healthy volunteers after COVID-19 vaccination. Our results imply that vaccinated subjects invariably acquire S1-specific CD4 + T cell reactivity in blood, but also that vaccine-induced T cell responses significantly decline in the first six months after vaccination.

Study population
This study included 148 whole blood samples retrieved from 55 healthy hospital and university staff members on up to five occasions between December 2020 and December 2021 at the Sahlgrenska University Hospital and University of Gothenburg. The DurIRVac study was approved by the Swedish Ethical Review Authority (permits no. 2020-03276, 2021-00374 and 2021-00539) and by the Swedish Medical Products Agency (EudraCT no. 2021-000349-42). All donors gave written informed consent prior to enrollment. The median age of study subjects was 43 years (range, 19-70) and 39/55 were female. Forty-five control samples were collected from 45 infection-naïve donors who had not been vaccinated. Nine samples were collected from 9 unvaccinated donors with PCR-confirmed COVID-19 infection approximately one month (median 28 days, range 25-50 days) prior to sampling. Thirty samples were collected one month after the first vaccine dose from 30 infection naïve participants. Sixty-four samples from 32 study participants devoid of confirmed COVID-19 infection were collected one, three and/or six months after two doses of COVID-19 vaccination (BNT162b2 [Pfizer] n = 24, mRNA-1273 [Moderna] n = 4, ChAdOx1 [AstraZeneca] n = 3, ChAdOx1 + mRNA-1273 n = 1). Table S1 accounts for detailed information on the type of samples contributed by each participant.

IgG serology
Chemiluminescent microparticle immunoassays were performed on parallel serum samples using the Alinity system for quantitation of IgG antibodies against the spike receptor-binding domain (RBD) (SARS-CoV-2 IgG II Quant, Abbott, Illinois, USA) and the nucleocapsid (SARS-CoV-2 IgG, Abbott) protein of SARS-CoV-2. IgG levels against the spike RBD (anti-S1-IgG) are reported as WHO international standard binding antibody units (BAU) per ml [7]. The lower limit of detection (LOD) for anti-S1-IgG was 14 BAU/ml, and all values below this limit were set to 50% of LOD.

Assay of peptide-induced IL-2 release in whole blood
Peripheral venous blood was collected in BD vacutainer lithiumheparin tubes (BD, Plymouth, UK). One ml of whole-blood was added to 10 ml-tubes (Sarstedt AB, Helsingborg, Sweden) and was left unstimulated or stimulated with 1 μg/ml/peptide of 15-mer peptides with 11amino acid overlap spanning the complete sequence of the N-terminal S1 domain of the Wuhan wild-type SARS-CoV-2 surface glycoprotein (aa 1-692, total 170 peptides S1; 130-127-041, Miltenyi Biotec). These peptides are designed to preferentially activate CD4 + T cells. Samples were incubated for two days at 37 • C and 5% CO 2 and were then centrifuged after which supernatant plasma was recovered and stored at -80 • C until analysis of IL-2 content.
Levels of IL-2 in whole blood supernatants were determined by the FirePlex®-96 Key Cytokines Immunoassay (ab243549 and ab285173, Abcam) according to the manufacturer's instructions. Samples were acquired on a BD LSRFortessa (BD) and analyzed with Fireplex Analysis Workbench (Abcam). S1-specific responses were determined by subtracting levels of IL-2 in unstimulated control samples from those obtained in S1-peptide-stimulated samples. The background levels of IL-2 in unstimulated control samples were consistently below 5 pg/ml. Values that were zero or negative after subtraction were set to the lowest non-zero value recorded in an S1-stimulated sample.

Isolation of peripheral blood mononuclear cells
In addition to whole blood stimulation, venous blood was used for isolation of peripheral blood mononuclear cells (PBMCs). Blood was diluted 1:2 in PBS and layered onto Ficoll-Paque (Lymphoprep, Stemcell Technologies). After gradient centrifugation, the PBMC layer was recovered, and cells were subsequently cryopreserved in Recovery Cell Culture freezing medium (Life Technologies). Samples were stored at − 140 • C until analysis.

Statistics
Pairwise group comparisons of S1-specific serology and T cell responses in controls, infected and vaccinated subjects were performed using linear mixed-effects models to account for subjects with measures in both groups. The infected group shared however no participants with the two vaccinated groups, so an ordinary Student's t-test was used for these comparisons. Waning of S1-specific antibodies and T cell responses was analyzed by linear mixed-effects models. The impact of age and sex on anti-S1 IgG and S1-induced IL-2 was determined using linear regression in each group separately. All statistical analyses were done on logarithmic values of IL-2 and anti-S1-IgG concentrations to reduce skewness. Statistical analyses were performed using IBM SPSS statistics 28.0 and R (version 4 or later), and figures were prepared using GraphPad Prism software (version 9 or later). P-values were designated as follows: *P < 0.05, **P < 0.01, ***P < 0.001 and **** P < 0.0001. All indicated P-values are two-sided.

COVID-19 vaccination triggers spike 1-specific T cell reactivity in blood
Whole blood samples from uninfected subjects were collected one month after the first or second vaccine dose. Blood samples were stimulated with multimeric S1 peptides followed by analysis of IL-2 in plasma supernatants. We similarly analyzed samples obtained from unvaccinated subjects approximately one month after verified COVID-19 and from uninfected, unvaccinated controls along with estimation of anti-S1-IgG in parallel serum samples in all study groups. S1 peptides triggered antigen-specific secretion of IL-2 in all vaccinated samples retrieved one month after vaccine dose one or two (n = 48) but not in samples from uninfected controls (Fig. 1A). T cells, in particular CD4 + T cells, were the dominant contributors of IL-2 in response to S1-peptide stimulation as >80% of S1 peptide-specific IL-2 + cells were CD3 + and approximately 80% of IL-2 + cells within the CD3 + population were CD4 + (Supplementary Fig. 1). Similarly, S1 peptide stimulation of unvaccinated subjects with previously verified SARS-CoV-2 infection resulted in robust secretion of IL-2 (Fig. 1A). The magnitude of S1-specific T cell reactivity after the first and second vaccination was higher than after natural infection but did not significantly increase after the second immunization compared with the first (Fig. 1A). S1-IgG in serum attained comparable levels after infection and after one vaccine dose, but antibody titers were significantly higher after two immunizations as previously reported [2] (Fig. 1B).

Longevity of S1-specific T cell reactivity after vaccination
To determine the duration of S1-specific T cell responses, additional blood samples were collected approximately three and six months after the second vaccination. No subject had verified breakthrough infection post vaccination as supported by absence of nucleocapsid IgG in serum. Levels of S1-induced IL-2 in whole blood declined with an approximated half-life of 134 days (14% per month) until six months from vaccination ( Fig. 2A). The waning of humoral immunity was more pronounced (halflife of anti-RBD-IgG of approximately 47 days corresponding to 36% reduction per month) in agreement with previous studies [2] (Fig. 2B). Despite the observed waning of immunity post vaccination, all subjects harbored S1-induced IL-2 and anti-S1-IgG levels above those recorded in unvaccinated, uninfected controls six months after vaccination ( Fig. 2A,  B). In this cohort, anti-S1 IgG and S1-induced IL-2 levels were not significantly affected by age or sex at any post-vaccination time-point.

Discussion
We studied the induction and longevity of S1-specific IL-2-producing T cells after the second COVID-19 vaccine dose in otherwise healthy volunteers. Earlier studies report decline of S1-IgG in twice vaccinated subjects beginning within months after the 2nd immunization [2], which partly coincides with reduced protection against SARS-CoV-2 infection. However, few twice vaccinated subjects experience severe COVID-19 [8,9] implying that T cell-mediated protection against severe infection might persist despite waning of S1-IgG.
Recent studies have assessed the longevity of antigen-specific T cell responses after COVID-19 vaccination [3][4][5]. The partially divergent results obtained in these reports may be explained by the readout of activation-induced markers expressed by T cells, which may not capture T cell reactivity with sufficient sensitivity, and by the analysis of S1induced formation of interferon-γ that may be contributed by leukocytes other than T cells, in particular early after infection or vaccination. The added value of the present study was thus to provide a simple and apparently reliable test to reflect S1-specific T cell immunity after vaccination. Our study also included comparison of S1-specific T cell responses between vaccinated subjects and convalescents from natural COVID-19 along with a parallel longitudinal assessment of S1-specific T cell reactivity vs. anti-S1-IgG in serum. S1-IgG antibodies induced by previous non-omicron COVID-19 vaccines reportedly do not efficiently neutralize omicron SARS-CoV-2 variants [10][11][12]. By contrast, T cell responses triggered by natural infection or by vaccination were mostly cross-reactive with omicron  Longevity of S1-specific T cell reactivity and S1-IgG after vaccination. (A, 61 samples from 32 individuals) S1 peptide-induced formation of IL-2 in whole blood and (B, 64 samples from 32 individuals) serum levels of anti-RBD S1 IgG in samples obtained from uninfected individuals one, three and six months after receiving their second COVID-19 vaccine dose. Connecting lines indicate samples from the same individual. Estimates of waning of (A) IL-2 and (B) anti-RBD S1 IgG after two vaccine doses are represented by black regression lines generated by linear mixed-effects models. Dotted lines indicate the highest level of S1 peptideinduced IL-2 (A) and the highest anti-RBD S1 IgG (B) in samples from uninfected, unvaccinated subjects. Statistics by linear mixed-effects models.
virus [10,11,13]. It may thus be assumed that the T cell reactivity observed in this study is relevant to protection against severe omicron infection.
A main finding was that two vaccine doses triggered S1-specific T cell reactivity in all subjects across several vaccine platforms, although the shortage of samples from subjects receiving mRNA-1273 and ChAdOx1 precluded meaningful comparison between vaccines. The initial T cell response after vaccination was significantly stronger than that achieved by natural infection at comparable follow-up. However, six months after the second vaccine dose, levels of S1-induced IL-2 had contracted to approximately 38% of peak levels, which emphasizes that the assessment of the long-term stability of T cell memory after vaccination requires longer follow-up. Notably, all vaccinees showed significant T cell reactivity at six months, exceeding that of all uninfected controls, and the S1-specific IL-2 response at six months after two vaccinations was comparable to that reported in naturally infected subjects at a similar time point after COVID-19 [14]. Additionally, our results suggest that S1 peptide-induced T cell reactivity declines more slowly than anti-S1-RBD IgG after vaccination.
Limitations of this study include a small study population and an unavoidably short follow-up. Also, we cannot exclude undiagnosed infections; however, PCR testing was conducted if symptoms occurred, and at all time points after vaccination samples were negative for nucleocapsid-IgG arguing against this possibility. Further studies are required to determine whether vaccination entails long-term T cellmediated immunity against COVID-19.

Funding
This work was supported by The Swedish Research Council [grant no. 2021-04779 and 2020-01437] and the Sahlgrenska Academy at University of Gothenburg.

Significance statement
• IL-2 release induced by multimeric peptides captures S1-specific T cell reactivity. • The S1-specific T cell response is stronger after vaccination than natural COVID-19. • T cell reactivity declines more slowly than anti-S1 IgG after COVID-19 vaccination.

Declaration of Competing Interest
None.

Data availability
Data will be made available on request.