Elicit: Immune Responses to Tozinameran S Antigen

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Immune Responses to Tozinameran S Antigen

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May 5, 2026

What immune responses (neutralizing antibodies, cellular immunity) are induced by Tozinameran's spike (S) antigen expression?

Tozinameran's spike antigen induces robust neutralizing antibody responses peaking at 2-4 weeks that decline over 6 months, alongside persistent spike-specific Th1-type CD4+ and cytotoxic CD8+ T cell responses with long-lived memory populations showing superior variant cross-reactivity.

Abstract

BNT162b2 (Tozinameran) vaccination induces robust spike-specific humoral and cellular immune responses in immunocompetent populations, with 95-98% seroconversion after two doses and peak neutralizing antibody levels at 2-4 weeks post-second dose. Antibody titers decline substantially over 6 months, with 50-90% reductions from peak levels, though this occurs alongside persistent spike-specific memory T and B cells capable of recall responses through 9 months. Cellular immunity emerges early, with T cell responses detectable by day 15, characterized by Th1-type CD4+ cells expressing IL-2, IFN-γ, and TNF-α and cytotoxic CD8+ responses. T stem cell memory populations persist stably through 6 months in 88% of individuals. Prior SARS-CoV-2 infection profoundly amplifies vaccine responses, with recovered individuals achieving peak neutralizing antibody titers after a single dose equivalent to two-dose vaccination in naïve individuals. However, immunocompromised populations show markedly attenuated responses, with dialysis patients achieving only 88.9% seroconversion and 67.7% T cell response rates compared to 93.3% in age-matched controls.

Cellular immunity demonstrates superior cross-reactivity with variants compared to humoral responses. While Beta (B.1.351) variant neutralization shows 2- to 10-fold reductions, CD4+ T cell activation remains unaffected by mutations in B.1.1.7 and B.1.351 variants, with no significant differences in T cell responses against Beta and Delta variants. Age negatively correlates with antibody magnitude and persistence, while T cell responses decline in individuals with increased immunosenescent CD8+ TEMRA cells. Booster doses administered 6-12 months post-vaccination restore waning antibody responses, with greater benefit observed in naïve versus previously infected individuals. The evidence indicates Tozinameran’s spike antigen elicits complementary humoral and cellular immunity, with antibodies providing initial sterilizing protection that wanes over months while durable memory T cell populations persist to enable rapid recall responses upon antigen re-exposure.

Methods

We analyzed 10 sources from an initial pool of 200, using 7 screening criteria. Each paper was reviewed for 8 key aspects that mattered most to the research question. More on methods

Records from Elicit search

n = 200

Papers screened using: Vaccine Type, Immune Response Outcomes, Quantitative Measurements, Study Design, Sample Size, Publication Type, Research Focus

n = 200

Papers screened out

n = 190

Papers included for extraction

n = 10

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Paper search

We performed a semantic search across over 138 million academic papers from the Elicit search engine, which includes all of Semantic Scholar and OpenAlex.

We ran this query: “What immune responses (neutralizing antibodies, cellular immunity) are induced by Tozinameran’s spike (S) antigen expression?”

The search returned 200 total results from Elicit.

We retrieved 200 papers most relevant to the query for screening.

Screening

We screened in sources based on their abstracts that met these criteria:

We considered all screening questions together and made a holistic judgement about whether to screen in each paper.

Data extraction

We asked a large language model to extract each data column below from each paper. We gave the model the extraction instructions shown below for each column.

Extract study design and participant characteristics for Tozinameran immune response studies, including:

Extract details of Tozinameran (BNT162b2) vaccine administration affecting spike antigen immune responses, including:

Extract all neutralizing antibody response data to Tozinameran’s spike antigen, including:

Extract all cellular immunity data to Tozinameran’s spike antigen, including:

Extract timing and kinetics of immune responses to spike antigen after Tozinameran vaccination, including:

Extract data on immune response cross-reactivity against SARS-CoV-2 spike variants after Tozinameran vaccination, including:

Extract factors that influence immune responses to Tozinameran’s spike antigen, including:

Extract the main conclusions about immune responses induced by Tozinameran’s spike antigen expression, including:

Results

Characteristics of Included Studies

All 10 studies evaluated immune responses following BNT162b2 (Tozinameran) vaccination, with full texts available for 7 studies and abstract-only data for 3 studies. Study designs, populations, and follow-up durations varied substantially across the included evidence.

Study

Full text retrieved?

Study type

Sample size

Population characteristics

Prior SARS-CoV-2 infection

Follow-up duration

Dosing schedule

E. Schrezenmeier et al., 2021

Yes

Observational study

43 (36 completed to week 3, 32 to week 10)

Chronic dialysis patients, median age 74 years, 69.4% male

Excluded; 3 developed infection after first dose

Up to 10 weeks post-second dose

Two doses 21 days apart

D. Geers et al., 2021

Yes

Prospective cohort study

121 healthcare workers

Median age 41 years, 68.9% female, healthcare workers

19% (n=23) recovered from mild COVID-19

Samples at 14-21 days post-first dose and 21-28 days post-second dose

Two doses 21 days apart

G. Guerrera et al., 2021

Yes

Cohort study

71 healthy donors

Healthy individuals aged 22-66 years, equal male/female distribution

Not previously infected (implied)

Up to 6 months

Two doses; first dose followed by boost at day 21, assessments at 2 weeks and 6 months post-boost

P. Naaber et al., 2021

No

Longitudinal prospective study

122 volunteers

Age correlation examined; specific demographics not provided

Not mentioned

Up to 6 months

Two doses

R. Payne et al., 2021

No

Cohort study

589 individuals (substudy)

UK healthcare workers

Prior infection amplified response

Extended dosing interval 6-14 weeks

Single dose or extended interval (6-14 weeks vs 3-4 weeks)

B. Pulendran & P. Arunachalam, 2021

Yes

Cohort study

56 healthy volunteers

Healthy volunteers; demographics not detailed

4 volunteers had prior confirmed infection

At least 42 days

Two doses approximately 21 days apart

J. Takeuchi et al., 2021

Yes

Longitudinal cohort study

100 hospital workers

Hospital workers aged ≥20 years, no immunosuppression, no prior COVID-19

None; exclusion criterion

Approximately 8 months post-second dose

Two doses 3 weeks apart

Salomé Desmecht et al., 2022

Yes

Prospective cohort study

208 healthcare workers

Healthcare workers >18 years, Liège University Hospital, Belgium

50 experienced, 158 naïve

Up to 12 months

Two doses 3-4 weeks apart, plus booster at 6-12 months

Youmna El-Orfali et al., 2025

No

Cohort study (inferred)

12 participants

Not specified

Not mentioned

Up to 9 months post-booster

Prime and booster doses

I. Cassaniti et al., 2021

No

Longitudinal observational study

145 healthcare workers

Healthcare workers, both naïve and previously infected

Both naïve and previously infected included

Not specified

Two doses

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The included studies represented diverse populations, with healthy volunteers and healthcare workers comprising the majority of participants, while one study specifically examined immunocompromised dialysis patients. Sample sizes ranged from 12 to 208 participants. Most studies administered two doses 21 days apart, though one examined extended dosing intervals of 6-14 weeks, and two included booster doses. Follow-up durations varied from several weeks to 12 months, with most studies providing data beyond 6 months.

Neutralizing Antibody Responses

Seroconversion Rates and Antibody Titers

Studies consistently demonstrated high seroconversion rates following two-dose vaccination in immunocompetent populations, though with notable variation in timing and magnitude. Among dialysis patients, only 55.6% developed SARS-CoV-2 IgG antibodies at the first sampling (~1 week post-second dose), increasing to 88.9% at 3-4 weeks and declining to 84.4% at 10 weeks. In contrast, 98.4% of immunocompetent naïve healthcare workers developed both neutralizing antibodies and specific T cells after the second dose.

Antibody kinetics showed consistent patterns across studies. Guerrera et al. reported median neutralizing antibody levels of 28 at the day of boost (T1), peaking at 1786 fourteen days later (T2), and declining to 517 at 6 months (T3). Takeuchi et al. found no participants were seropositive on day 15, rising to 96% on day 29, then decreasing to 81% and 58% on days 61 and 82-96 respectively. The magnitude of decline was substantial: IgG-S-RBD titers decreased by 53.1% at day 61, 73.7% at day 82-96, and 96.2% at day 263 compared to peak levels on day 29.

Study

Population

Peak antibody timing

Peak seroconversion rate

Decline at 3-6 months

E. Schrezenmeier et al., 2021

Dialysis patients

3-4 weeks post-second dose

88.9%

84.4% at 10 weeks

G. Guerrera et al., 2021

Healthy donors

14 days post-second dose

100%

Median titer 517 at 6 months (from peak 1786)

J. Takeuchi et al., 2021

Hospital workers

Day 29

96%

81% at day 61, 58% at day 82-96

P. Naaber et al., 2021

Volunteers

Shortly after second dose

Not specified

Similar to one-dose or convalescent levels at 6 months

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The observed decline in antibody levels was consistent with waning immunity over time. Naaber et al. found that antibody levels at 6 months post-vaccination were similar to those in individuals who received only one dose or recovered from COVID-19, indicating substantial reduction from peak levels.

Neutralization Capacity Against Variants

Studies evaluating variant cross-reactivity revealed differential neutralization capacity depending on the specific variant tested. For the B.1.1.7 (Alpha) variant, Geers et al. reported higher neutralizing antibody titers compared to wild-type virus (2.5-fold and 2.2-fold increases). However, the B.1.351 (Beta) variant showed consistent immune escape, with 2.7-fold and 3.3-fold decreases in neutralizing capacity and approximately 10-fold reduction in neutralizing antibody levels compared to the parent strain. Among immunocompetent naïve subjects, 80.9% retained detectable neutralizing antibodies against the Beta variant and 86.2% against the Delta variant.

Temporal decline in variant neutralization was evident. Naaber et al. found that antibodies efficiently blocked ACE2 receptor binding to spike proteins of five variants of concern at one week post-vaccination, but this capacity decreased by three months. The fold-reduction in neutralization against B.1.351 compared to the original strain was consistently 2- to 4-fold across antibody-based assays.

Effects of Prior SARS-CoV-2 Infection

Prior SARS-CoV-2 infection substantially amplified humoral responses. Geers et al. demonstrated that recovered individuals achieved a geometric mean plateau PRNT50 titer of 1:1874 after a single vaccination, with no further increase after the second dose. In contrast, naïve individuals required two doses to achieve comparable levels, with only 7 of 12 developing neutralizing antibodies after one dose, rising to 12 of 12 after the second. Desmecht et al. confirmed higher anti-spike antibody levels and neutralization capacity in experienced versus naïve individuals at all timepoints prior to booster administration. Median neutralization titers in experienced individuals reached 1:640 after the first dose, while naïve individuals showed minimal increase.

Cellular Immune Responses

CD4+ and CD8+ T Cell Activation

T cell responses were detected across multiple studies using various assay methods. Among dialysis patients, 67.7% demonstrated SARS-CoV-2-specific T cell responses 3 weeks after the second vaccination, significantly lower than 93.3% in age-matched controls. Takeuchi et al. found that T cell responses, measured by IFN-γ production using the QuantiFERON SARS-CoV-2 assay, were detected as early as day 15 after the first dose and peaked after the second dose before declining over time. Positive T cell responses were defined as ≥0.20 IU/mL IFN-γ concentration.

Pulendran and Arunachalam characterized the quality of cellular responses, finding that CD4+ T cell responses were primarily Th1-type expressing IL-2, IFN-γ, or TNF-α with low levels of IL-4 induction. CD8+ T cell responses were dominated by IFN-γ and TNF-α production, with 25 of 31 participants showing IFN-γ responses at least 3 times higher than baseline. Importantly, no significant correlation existed between T cell responses and neutralizing antibody levels.

Study

Assay method

CD4+ T cell response

CD8+ T cell response

Response rate

E. Schrezenmeier et al., 2021

IGRA

Detected in dialysis patients

Not separately quantified

67.7% in dialysis patients vs 93.3% in controls

J. Takeuchi et al., 2021

QuantiFERON SARS-CoV-2

IFN-γ production ≥0.20 IU/mL

IFN-γ production ≥0.20 IU/mL

Detected day 15, peaked after second dose

B. Pulendran & P. Arunachalam, 2021

Intracellular cytokine staining

Th1-type (IL-2, IFN-γ, TNF-α)

IFN-γ and TNF-α dominant

25/31 participants >3× baseline IFN-γ

Salomé Desmecht et al., 2022

QuantiFERON Human IFN-γ

Measured by IFN-γ secretion

Not separately quantified

Higher in experienced vs naïve at T4

P. Naaber et al., 2021

Not specified

Memory T cells developed

Lower with immunosenescent TEMRA cells

87% developed spike-specific memory T cells

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Desmecht et al. found experienced individuals had higher cellular immunity compared to naïve individuals at 6 months (T4), though this difference disappeared after the booster dose. The correlation between anti-spike IgG and IFN-γ was more pronounced in experienced individuals.

Memory T Cell Formation and Phenotypes

Guerrera et al. provided detailed characterization of memory T cell phenotypes, demonstrating that BNT162b2 induced CD4+ T stem cell memory (TSCM) detectable in 88% of individuals, with levels increasing from baseline to 6 months. These cells displayed markers of immunological maturity and stem cell memory, surviving the physiological contraction phase and persisting for at least 6 months. El-Orfali et al. confirmed that vaccination resulted in spike-specific memory CD4+ T cells that proliferate upon reactivation, promote a protective Th1 response, and induce IgG/IgA class switching in memory B cells. Memory CD8+ T cells similarly proliferated upon reactivation and upregulated granzyme and perforin to eliminate infected cells.

The temporal emergence of T cell responses was notable. Takeuchi et al. observed that T cell responses were detected on day 15, earlier than the onset of neutralizing antibody activity, suggesting T cells may provide initial protection before robust humoral immunity develops. This early T cell activation preceded peak antibody responses by approximately 2 weeks.

T Cell Cross-Reactivity with Variants

In contrast to the partial escape observed with humoral immunity, cellular immunity demonstrated robust cross-reactivity across variants. Geers et al. found no differences in CD4+ T cell activation when peripheral blood mononuclear cells were stimulated with peptide pools spanning the mutated spike regions of B.1.1.7 and B.1.351, indicating that these variants do not escape T cell-mediated immunity. CD4+ T cells equally recognized wild-type, B.1.1.7, and B.1.351 spike proteins. Cassaniti et al. similarly reported that T cell responses were less affected by SARS-CoV-2 variants, with no significant difference in the frequency of responders when challenged with Beta and Delta variants.

Response Kinetics and Durability

Temporal Dynamics of Immune Responses

The kinetics of antibody and T cell responses followed distinct but complementary trajectories. Guerrera et al. documented that antibody responses appeared before high titers developed, displaying markers of immunological maturity, and survived physiological contraction for at least 6 months. Peak antibody responses occurred 14 days after the second dose (T2), with median levels of 1786, declining to 517 at 6 months. T stem cell memory cells remained stable throughout this period.

Pulendran and Arunachalam measured responses at baseline, day 21, and day 42 post-vaccination, finding peak antibody responses around day 42 with approximately 5-fold increases after the second dose compared to the first. Cellular immunity was more readily detectable 7 days after the second dose. Innate immune responses showed different kinetics, with cytokine levels returning to baseline by day 28.

Study

Antibody peak timing

T cell peak timing

Duration of detectable responses

Key kinetic features

G. Guerrera et al., 2021

14 days post-second dose

14 days post-second dose

Up to 6 months for both

TSCM remained stable for 6 months

J. Takeuchi et al., 2021

Day 29

Day 29

Approximately 6-10 weeks

T cells detected earlier than neutralizing activity

Salomé Desmecht et al., 2022

2 weeks post-second dose

Not separately specified

Up to 12 months

Decline starting 2 weeks after peak

B. Pulendran & P. Arunachalam, 2021

Around day 42

7 days post-second dose

Not specified beyond 42 days

5-fold increase after second dose

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Long-Term Persistence and Waning Immunity

Long-term follow-up revealed progressive decline in humoral immunity with more stable cellular responses. Takeuchi et al. found immune responses sustained for approximately 6 to 10 weeks but not beyond 7 months following the second vaccination. The decline in IgG-S-RBD titers was substantial: 53.1% at day 61, 73.7% at day 82-96, and 96.2% at day 263 compared to peak levels. Similarly, anti-SARS-CoV-2 IgA decreased from 91.67% at 3-4 weeks to 59.38% at 10 weeks in dialysis patients.

Desmecht et al. provided the longest follow-up data, showing immune responses detectable up to 12 months. However, antibody levels declined substantially, particularly in naïve individuals, necessitating booster doses. El-Orfali et al. demonstrated that spike-specific memory T and B cells persisted for up to 9 months post-immunization and could mount recall responses upon antigen reactivation.

The comparison between first and second dose responses revealed amplification effects. Desmecht et al. found higher antibody titers in the experienced group after the first dose, with peak responses occurring two weeks after the second dose in naïve individuals. Payne et al. showed that extended dosing intervals (6-14 weeks vs 3-4 weeks) resulted in higher neutralizing antibody levels and enrichment of IL-2-expressing CD4+ T cells.

Response Modifying Factors

Age and Immunosenescence

Age emerged as a consistent negative predictor of antibody responses across multiple studies. Naaber et al. found antibody responses correlated negatively with age, while Guerrera et al. reported that vaccine effectiveness decreases with age and antibody titers correlate negatively with age. However, the relationship was more nuanced for cellular immunity. Naaber et al. observed that T cell responses were lower in individuals with increased proportions of immunosenescent CD8+ TEMRA cells, a marker of immune aging. Pulendran and Arunachalam documented age-related differences in response patterns: younger subjects had greater changes in monocyte and inflammatory responses, while older subjects showed increased responses in B and T cell modules. Despite these differences, no significant difference in neutralizing antibody responses existed between males and females.

Takeuchi et al. found younger age (<40 years) was associated with higher IgG-S-RBD titers, while Desmecht et al. confirmed a significant negative association between age and persistence of humoral responses. The age effect on antibody responses appeared stronger than the effect on cellular immunity, though both were influenced by advancing age.

Impact of Prior SARS-CoV-2 Infection

Prior infection was the strongest amplifier of vaccine-induced immunity across all studies that examined this factor. Geers et al. found recovered individuals showed a recall response with high levels of antibodies and T cells after a single vaccination, sufficient to match or exceed naïve individuals receiving two doses. Payne et al. confirmed that prior infection amplified and accelerated the immune response, while Cassaniti et al. reported increased immunogenicity in experienced subjects, with sustained neutralizing antibody activity against all tested variants.

Desmecht et al. provided the most comprehensive analysis, showing experienced individuals had higher anti-spike antibodies and neutralization capacity at all timepoints prior to booster administration. The median neutralization titer in experienced individuals increased to 1:640 after the first dose, while naïve individuals showed minimal increase. After the booster dose, experienced individuals who had breakthrough infections demonstrated even higher cellular and humoral responses. The correlation between anti-spike IgG and IFN-γ was more pronounced in experienced versus naïve individuals.

Comorbidities and Immunocompromised States

Schrezenmeier et al.’s study of dialysis patients revealed profound effects of chronic kidney disease on vaccine immunogenicity. Only 55.6% of dialysis patients developed SARS-CoV-2 IgG antibodies at the first sampling, increasing to 88.9% at the second sampling, compared to near-universal seroconversion in immunocompetent populations. T cell responses were similarly impaired, with 67.7% of dialysis patients showing responses compared to 93.3% in age-matched controls. Multiple mechanisms likely contributed to this impairment, including decreased B and T cell counts, impaired clearance of uremic toxins, systemic inflammation, and malnutrition.

The magnitude of antibody responses was also reduced. Schrezenmeier et al. found significantly lower seroconversion rates and antibody titers in dialysis patients compared to age-matched non-dialysis controls. Gender differences were noted but appeared attenuated by advanced age. These findings highlighted the vulnerability of immunocompromised populations and suggested the need for modified vaccination strategies, including additional booster doses.

Sex and Body Mass Index

Sex differences in immune responses showed variable patterns across studies. Guerrera et al. reported comparable antibody levels between males and females, though females retained lower levels of neutralizing antibodies at 6 months. Takeuchi et al. found females tended to have higher IgG-S-RBD titers, though this was a trend rather than a statistically significant difference. Conversely, males showed higher IFN-γ production by CD4+ T cells on day 29, suggesting potential sex-specific differences in cellular versus humoral immunity balance.

Body mass index also influenced responses, with Takeuchi et al. reporting that individuals with normal BMI tended to have higher IgG-S-RBD titers compared to those with higher BMI. However, no significant differences in T cell responses were observed based on BMI.

Booster Dose Effects

Two studies specifically examined the impact of booster doses administered 6-12 months after initial vaccination. Desmecht et al. found the booster dose induced substantial increases in both humoral and cellular immune responses, particularly in naïve individuals. Neutralization capacity, which had decreased in naïve individuals by 6 months (T4), was restored after the booster. The benefit of the booster dose was greater in naïve individuals compared to those with prior infection, suggesting that hybrid immunity (vaccination plus natural infection) provided more durable protection than vaccination alone.

El-Orfali et al. confirmed that blood samples taken at 1, 3, 6, and 9 months post-booster dose showed sustained spike-specific memory T and B cell responses capable of mounting recall responses upon reactivation. Memory B cells were primed to secrete neutralizing antibodies upon reactivation, while memory CD4+ T cells promoted protective Th1 responses and induced IgG/IgA class switching.

Synthesis

The included studies revealed substantial heterogeneity in immune response magnitude and durability, driven by distinct population characteristics, prior infection status, age, and comorbid conditions rather than conflicting findings. Three key patterns emerged that reconcile apparent discrepancies.

First, population-specific immune capacity determined baseline vaccine responsiveness. Studies of healthy adults and healthcare workers consistently reported >95% seroconversion after two doses, while dialysis patients achieved only 88.9% seroconversion with significantly lower antibody titers and T cell responses (67.7% vs 93.3% in controls). This divergence reflects fundamental differences in immune competence rather than vaccine failure. Dialysis patients exhibit decreased B and T cell counts, impaired toxin clearance, systemic inflammation, and malnutrition—mechanistic factors that predict attenuated responses to any vaccine. The appropriate conclusion is not that BNT162b2 has variable efficacy, but that immunocompromised populations require augmented vaccination strategies (additional doses, higher antigen loads, or adjuvants) to achieve protection comparable to healthy cohorts.

Second, prior SARS-CoV-2 infection created a distinct immunological state that fundamentally altered vaccine kinetics and magnitude. Hybrid immunity (infection plus vaccination) consistently produced higher and more durable responses than vaccination alone. Geers et al. showed that recovered individuals achieved peak neutralizing antibody titers (geometric mean 1:1874) after a single vaccine dose with no further increase after the second, while naïve individuals required two doses to reach comparable levels. Desmecht et al. extended this finding across the full vaccination course, demonstrating experienced individuals maintained higher anti-spike antibodies and neutralization capacity at all timepoints. The mechanistic explanation involves memory B cell priming: prior infection establishes antigen-specific memory cells that undergo rapid proliferation and affinity maturation upon vaccine boost, bypassing the slower germinal center reaction required in naïve individuals. This explains why booster doses provided greater benefit to naïve individuals—they were reaching parity with the immune state already achieved through hybrid immunity. Critically, cellular immunity showed even more pronounced advantages in experienced individuals, with stronger T cell responses at 6 months and robust cross-reactivity maintained against variants.

Third, the temporal decline in antibody levels does not indicate vaccine failure but rather normal immune contraction following resolution of acute antigenic stimulation. Multiple studies documented 50-90% reductions in antibody titers between peak (2-4 weeks post-second dose) and 6 months. However, this occurred alongside stable or increasing memory T cell populations, particularly CD4+ T stem cell memory (TSCM) cells that persisted through 6 months. The mechanistic framework explains this pattern: following antigen clearance, short-lived plasma cells die off while long-lived plasma cells migrate to bone marrow niches and memory B cells enter quiescence. The observed antibody decline reflects contraction of the short-lived plasma cell pool, not loss of immunological memory. Supporting this, El-Orfali et al. demonstrated that memory B cells remained primed to secrete neutralizing antibodies upon reactivation through 9 months, while memory T cells proliferated and upregulated effector functions upon antigen re-exposure. The key metric is therefore not circulating antibody titer at arbitrary timepoints but rather memory cell persistence and recall capacity—both of which remained robust through available follow-up periods.

The apparent contradiction between waning antibody titers and claims of sustained immunity is reconciled by distinguishing between correlates of protection at different stages post-vaccination. In the first 3-6 months, high circulating antibody levels provide sterilizing immunity preventing infection. Beyond 6 months, as antibody levels decline, protection shifts toward memory-mediated responses that prevent severe disease. Takeuchi et al.’s finding that T cell responses appeared earlier than neutralizing antibodies (day 15 vs day 29) supports a model where cellular immunity provides a critical protective layer even before peak humoral responses develop and persists after antibody waning. This explains how breakthrough infections can occur in vaccinated individuals while severe disease remains rare—memory cells rapidly expand upon infection, containing viral replication before clinical deterioration.

Age effects operated through both quantitative and qualitative mechanisms. Older individuals showed reduced antibody titers and increased immunosenescence markers (CD8+ TEMRA cells), but Pulendran and Arunachalam revealed distinct age-stratified response patterns: younger subjects mounted stronger innate inflammatory responses while older subjects showed preserved or enhanced B and T cell module responses. This suggests age-related immune decline is not uniform across all compartments. The modest inverse correlation between age and neutralizing antibodies may be partially offset by compensatory increases in other protective mechanisms, explaining why real-world vaccine effectiveness against severe disease remained high across age groups despite lower antibody titers in elderly populations.

For variant cross-reactivity, the divergence between humoral and cellular immunity proved critical. Beta (B.1.351) variant showed 2- to 10-fold reductions in neutralizing antibody responses, raising concerns about immune escape. However, CD4+ T cell responses showed no impairment against Beta or Delta variants, with T cells equally recognizing wild-type and variant spike proteins. The mechanistic basis is epitope breadth: neutralizing antibodies target a limited set of RBD epitopes susceptible to single amino acid substitutions (K417N/T, E484K in Beta), while T cells recognize diverse linear epitopes across the entire spike protein, most of which are conserved across variants. This differential susceptibility explains why breakthrough infections increased with Beta despite maintained protection against severe disease—antibodies failed to prevent infection, but T cells controlled viral replication and pathology.

The studies collectively support a model where BNT162b2 induces robust humoral and cellular immunity in immunocompetent individuals, with response magnitude and durability strongly influenced by prior infection status, age, and immune competence. For healthy adults, two doses produce peak responses at 2-4 weeks with physiological contraction to steady-state memory by 6 months. For individuals with prior infection, a single dose achieves hybrid immunity superior to two-dose naïve vaccination. For immunocompromised populations, standard dosing produces suboptimal responses requiring augmented strategies. In all cases, cellular immunity proves more durable and cross-reactive than humoral immunity, suggesting memory T cells are the primary mediator of long-term protection against severe disease even as antibody-mediated protection against infection wanes.

References

D. Geers, M. Shamier, S. Bogers, G. den Hartog, L. Gommers, and 20 more\ (2021).SARS-CoV-2 variants of concern partially escape humoral but not T cell responses in COVID-19 convalescent donors and vaccine recipients. Science immunology

G. Guerrera, M. Picozza, S. D’Orso, R. Placido, Marta Pirronello, and 11 more\ (2021).BNT162b2 vaccination induces durable SARS-CoV-2–specific T cells with a stem cell memory phenotype. Science immunology

P. Naaber, L. Tserel, K. Kangro, E. Sepp, Virge Jürjenson, and 10 more\ (2021).Dynamics of antibody response to BNT162b2 vaccine after six months: a longitudinal prospective study. The Lancet Regional Health - Europe

R. Payne, S. Longet, J. Austin, D. Skelly, W. Dejnirattisai, and 131 more\ (2021).Immunogenicity of standard and extended dosing intervals of BNT162b2 mRNA vaccine. Cell

B. Pulendran, P. Arunachalam\ (2021).Systems biological assessment of human immunity to BNT162b2 mRNA vaccination. Research Square

J. Takeuchi, A. Fukunaga, S. Yamamoto, A. Tanaka, K. Matsuda, and 10 more\ (2021).SARS-CoV-2 specific T cell and humoral immune responses upon vaccination with BNT162b2: a 9 months longitudinal study. Scientific Reports

Salomé Desmecht, Aleksandr Tashkeev, Nicole Maréchal, Hélène Perée, Y. Tokunaga, and 16 more\ (2022).Kinetics and Persistence of the Cellular and Humoral Immune Responses to BNT162b2 mRNA Vaccine in SARS-CoV-2-Naive and -Experienced Subjects: Impact of Booster Dose and Breakthrough Infections. Frontiers in Immunology

Youmna El-Orfali, Rana H. Mansour, Habib Al-Kalamouni, Ziad C. Jabbour, Antoine G. Abou Fayad, and 3 more\ (2025).Longitudinal T and B cell recall responses following vaccination with the Pfizer-BioNTech mRNA vaccine BNT162b2. Journal of Allergy and Clinical Immunology

I. Cassaniti, F. Bergami, E. Percivalle, E. Gabanti, J. Sammartino, and 15 more\ (2021).Humoral and cell-mediated response against SARS-CoV-2 variants elicited by mRNA vaccine BNT162b2 in healthcare workers: a longitudinal observational study. Clinical Microbiology and Infection

E. Schrezenmeier, L. Bergfeld, D. Hillus, J. Lippert, Ulrike A. Weber, and 14 more\ (2021).Immunogenicity of COVID-19 Tozinameran Vaccination in Patients on Chronic Dialysis. Frontiers in Immunology

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BNT162b2 vaccination induces durable SARS-CoV-2–specific T cells with a stem cell memory phenotype

G. Guerrera, M. Picozza, S. D’Orso, R. Placido, Marta Pirronello, Alice Verdiani, Andrea Termine, Carlo Fabrizio, Flavia Giannessi, M. Sambucci, M. Balice, C. Caltagirone, A. Salvia, A. Rossini, L. Battistini, G. Borsellino

Science immunology·

2021·

214 citations

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Study Design

- Study type: Cohort study - Sample size for immune response analysis: 71 healthy donors - Population characteristics: Healthy individuals aged 22-66 years, equal distribution of males and females - Prior SARS-CoV-2 infection status: Not explicitly mentioned, but likely not previously infected - Inclusion/exclusion criteria: Scheduled for vaccination with BNT162b2, signed informed consent forms - Study setting and duration of follow-up: Santa Lucia Foundation, up to 6 months after vaccination

Tozinameran Administration

- Number of doses administered: Two doses - Dosing schedule and intervals between doses: First dose (T0), second dose 21 days later (T1), and follow-up at 2 weeks after the second dose (T2) and 6 months after the first dose (T3) - Vaccine formulation/batch details: Not mentioned - Any deviations from standard protocol: Not mentioned - Timing relative to prior SARS-CoV-2 infection: Not mentioned

Neutralizing Antibody Responses

- Neutralizing antibody levels/titers at each timepoint: Baseline (T0): 0; Day of the boost (T1): median 28; 14 days later (T2): median 1786; 6 months after the first dose (T3): median 517. - Seroconversion rates and timing: 100% seroconversion after the second dose. - Neutralization capacity against original spike and variants: Not mentioned. - IgG and IgA antibody levels specific to spike protein: Not mentioned. - Comparison to controls or reference groups: Not mentioned. - Assay methods used: Electrochemiluminescence sandwich immunoassay (ECLIA) with a cut-off of 0.8 U/ml. - Geometric mean titers and fold-changes from baseline: T1: median 28; T2: median 1786; T3: median 517.

Cellular Immune Responses

- CD4+ T cell responses: Facilitate B cell differentiation and antibody production, direct cytotoxic activity, cytokine production. - CD8+ T cell responses: Cytotoxic markers and functionality. - Memory T cell formation: Generation of stem cell memory cells persisting for at least 6 months. - Assay methods: Flow cytometry, intracellular cytokine staining. - Quantitative results: CD4+ TSCM detectable in 88% of individuals, increased from baseline to 6 months. - Statistical comparisons: Not explicitly mentioned in the provided text.

Response Timing

- Timepoints: Baseline (T0), day of the boost (T1), 14 days later (T2), 6 months after the first dose (T3) - Peak response timing: Antibodies peaked at T2 (14 days after the second dose), cellular immunity peaked at T2 - Duration of detectable responses: Up to 6 months for both antibodies and cellular immunity - Decline rates: Antibody levels decreased from T2 to T3 (median 1786 to 517) - Comparison between first and second dose responses: First dose induces significant T cell response, consolidated after second dose - Long-term persistence data: T stem cell memory cells remain stable for at least 6 months

Variant Cross-Reactivity

Not mentioned (the paper does not provide specific data on variant cross-reactivity, neutralizing antibody levels against variants, or cellular immunity cross-reactivity with variant spike proteins)

Response Modifying Factors

- Age effects on antibody and cellular responses: Effectiveness decreases with age; antibody titers correlate negatively with age. - Impact of comorbidities: Not mentioned. - Effects of prior SARS-CoV-2 infection: Not mentioned. - Sex/gender differences in immune responses: Comparable antibody levels between males and females; females retain lower levels of neutralizing antibodies at 6 months. - Medication effects: Not mentioned. - Other population-specific factors: Study limited to healthy donors; focus on circulating lymphocytes.

Key Findings

- Overall immunogenicity assessment: The BNT162b2 vaccine induces a sustained CD4+ and CD8+ T cell response and high levels of neutralizing antibodies, indicating strong immunogenicity. - Relative strength of humoral vs. cellular responses: Both humoral and cellular responses are induced, with T cell responses persisting for at least 6 months and antibody levels decreasing over time but remaining high. - Clinical correlates of protection: The vaccine-induced T cell response is associated with protection against severe disease and breakthrough infections. - Comparison to other vaccines or natural infection: Not explicitly compared in the paper. - Authors' conclusions about spike antigen immunogenicity: The spike antigen induces a long-lived and immunologically competent T cell population. - Limitations or concerns about immune response quality: The study focuses on circulating lymphocytes, which may not reflect mucosal immunity.

Description SARS-CoV-2 mRNA vaccination induces stem cell memory T cells specific for the virus that persist up to 6 months. SARS-CoV-2 mRNA vaccination induces long-term T cell responses Vaccinations for SARS-CoV-2 are being administered globally to halt the spread of the infection. Despite the prevalence of these vaccines, the long-term immune responses they induce are still poorly characterized. Here, Guerrera et al. took longitudinal blood samples from 71 BNT162b2 mRNA–vaccinated individuals and looked at the T cell responses to vaccination. They found that the vaccine induced polyfunctional CD4+ and CD8+ T cell memory responses that peaked 2 weeks after the second dose of the vaccine and contracted at 6 months after vaccination. Despite this waning, the mRNA vaccine induced T stem cell memory cells after the first dose that remained stable out to 6 months. Thus, BNT162b2 provides robust, long-term T cell memory responses against SARS-CoV-2. Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is effective in preventing hospitalization from severe COVID-19. However, multiple reports of breakthrough infections and of waning antibody titers have raised concerns on the durability of the vaccine, and current vaccination strategies now propose administration of a third dose. Here, we monitored T cell responses to the Spike protein of SARS-CoV-2 in 71 healthy donors vaccinated with two doses of the Pfizer-BioNTech mRNA vaccine (BNT162b2) for up to 6 months after vaccination. We found that vaccination induced the development of a sustained anti-viral CD4+ and CD8+ T cell response. These cells appeared before the development of high antibody titers, displayed markers of immunological maturity and stem cell memory, survived the physiological contraction of the immune response, and persisted for at least 6 months. Collectively, these data show that vaccination with BNT162b2 elicits an immunologically competent and long-lived SARS-CoV-2–specific T cell population.

Introduction

Mass-vaccination against COVID-19 has quickly shown to be effective and to confer high levels of protection against COVID-19 in real-world settings (1). However, one notion that all immunologists learned during the pandemic was that natural infection with coronaviruses induces short-lived immunity (2), with reinfections occurring frequently. This notion was quickly revised by subsequent studies showing that T cell immunity to SARS is actually long lived (3,4). The study of immune responses in COVID-19 patients with various degrees of disease severity have revealed that indeed infection with SARS-CoV-2 elicits a robust immune response, involving both the innate and the adaptive arms of the immune system and theoretically effective in protecting from reinfections (5,6). However, this protection is not absolute and impeccable, and cases of reinfection do occur (7). Humoral immunity provides a shield against reinfection through the generation of neutralizing antibodies, which are easily measurable and are widely used as indicators of protective immunity (8, 9) . Notably, subjects with undetectable or impaired humoral responses can nonetheless clear the infection, suggesting that antigen-specific T cells are themselves effective at containing the virus (10)(11)(12). Evidence of T cell involvement in the immune response to SARS-CoV-2 came from reports showing that the emergence of activated T cells precedes recovery from COVID-19 (13)(14)(15)(16), and was later confirmed in in vitro studies which characterized antigen-specific CD4+ and CD8+ lymphocytes reactive with overlapping peptide pools from the SARS-CoV-2 Spike protein (15,(17)(18)(19). These cells persist in COVID-19 convalescents (4,(20)(21)(22)(23), and have been shown to reduce viral loads in non-human primate models of infection; crucially, they arise also following vaccination both with mRNA-and adenoviral-vaccines (24)(25)(26)(27). It is necessary and urgent to identify the cellular immune components induced by vaccination and to measure their persistence, also in the view of reports of waning immunity over time and in recent updates in vaccination strategies which now propose administration of a third dose. Here, we show the results of a longitudinal study on T cell responses in 71 health-care workers and scientists vaccinated with the BNT162b2 vaccine following the European Medicines Agency (EMA)-approved vaccination schedule, up to 6 months after the first dose. We find robust induction of neutralizing antibodies and of Spike-specific CD4+ and CD8+ lymphocytes which persist in the periphery after the physiological contraction of the initial response. These cells are detectable in most individuals also before vaccination, denoting the presence of a pre-preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this this version posted September 28, 2021. ; https://doi.org/10.1101/2021.09. 27.462006 doi: bioRxiv preprint 3 existing pool of cross-reactive cells, and they are expanded significantly after the boost. We show that vaccine-induced T cells are mostly central and effector memory cells, and that they are equipped with overlapping sets of molecules which enable them to perform multiple immune functions: facilitation of B cell differentiation and antibody production, direct cytotoxic activity, and cytokine production, and that they are equally represented in both sexes.

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

Induction and persistence of neutralizing antibodies

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

Induction and durability of the Spike-specific T cell response

To investigate the cellular immune response induced by vaccination, we exposed freshly obtained peripheral blood mononuclear cells (PBMC) to peptide pools spanning the entire sequence of the Spike (S) protein. Blood collection was performed at baseline (T0), on the day of the boost (day 21, T1), 14 days later (T2), and after 6 months (T3). Several effector functions of CD4+ cells consist in the upregulation of surface molecules for intercellular communication, and these may remain trapped inside the cells if the secretion inhibitors required for intracellular cytokine detection preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this this version posted September 28, 2021. ; https://doi.org/10.1101/2021.09.27.462006 doi: bioRxiv preprint are present during antigenic stimulation. Thus, to fully capture the T cell antigen-specific response and to maximize sensitivity, two separate assays for the detection of the expression of surface Activation-Induced Markers (AIM) and for Intracellular Cytokine Staining (ICS) were set up (Fig. S1 for gating strategy). Moreover, all assays were performed on freshly isolated PBMCs in order to avoid the inevitable cell loss during the freezing/thawing procedure, and to obtain accurate absolute cell counts. AIM+ CD4+ T cells were defined by upregulation of CD40L and CD69, while CD137 and CD69 expression identified the AIM+ CD8+ subset (Fig. 2A ). After paired background subtraction from parallel unstimulated cultures, 99% of donors (66/67) had detectable numbers of AIM+ CD4+ cells at baseline (median 349 cells/ml, range 63-4639); 21 days after the first dose of vaccine these cells were identified in all donors, and at higher levels (median 2218 cells/ml, range 319-110394, p <0.0001), and further still at 14 days after the boost (median 3608 cells/ml, range 566-101864); 6 months after the first dose AIM+ CD4+ cell numbers were still increased 5-fold compared to baseline (median 1660, range 349-74859). AIM+ CD8+ T lymphocytes were present at baseline in only 63% of the donors (42/67, median 209 cells/ml, range 18-2695), and increased following vaccination (59/71, median 900 cells/ml, range 54-21142, p<0,0001), with further expansion after the second dose (64/69, median 1539 cells/ml, range 46-18,638), and persisted after 6 months in all donors (median 706 cell/ml, range 78-27593) (Fig. 2B, C ).The total magnitude of the T cell response (that is, including both CD4+ and CD8+ cells) increased significantly following priming (T1) and rechallenge (T2), and decreased 6 months after the first dose (T3) (Fig. 2D ). Importantly, the Stimulation Index (SI, the ratio of AIM+ T cells in stimulated over unstimulated samples) of AIM+ CD4+ cells increased dramatically after the first dose, remained at high levels after boosting (Fig. 2E ), and increased further after 6 months, reaching a median of 25,7. The SI of AIM+ CD8+ T cells peaked 14 days after the boost, and declined by 1/3 at the latest time-point. Moreover, the fraction of individuals showing CD4+ with SI >3 increased at all time points, denoting the establishment of an antigen-specific T cell population (Fig. 2F ).

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

The numbers of AIM+ cells correlated with antibody levels only after priming and not at subsequent time points (Fig. S2C ), suggesting that humoral and cellular immune responses follow different kinetics. However, machine learning indicated that the number of AIM+ CD4+ cells 21 days after priming is the best predictor of nAbs levels after 6 months, as expected from a T cell-dependent B cell response (Fig. S2D ). Thus, vaccination induces clearly detectable and robust antigen-specific T cells in both sexes, arising before the development of high antibody titres, with a major expansion occurring after the first dose of vaccination followed by consolidation of the response after the boost, and persistence for up to 6 months.

Cytokine production by S-specific T cells. Cytokine production was assessed by intracellular flow cytometry following stimulation o.n. with the peptide pools in the presence of monensin and brefeldin (Fig. 3A ). Measurement of cytokine production showed that at baseline 62% and 50% of the tested donors showed IFNg+ CD4+ and CD8+ cells, respectively (CD4: median 90 cells/ml, range 1-1536; CD8: median 116 cells/ml, range 9-592) (Fig. 3B, C ). Three weeks after the first dose, 90% of individuals showed IFNg+ CD4+ T cells (median 275 cells/ml, range 5-5799), while CD8+IFNg+ (median 446 cell/ml, range 2-6606) were found in 77% of individuals. Two weeks after the boost, 97% and 69% of individuals showed IFNg+ CD4+ (median 17111 cell/ml, range 200-19595) and CD8+ T cells (median 592 cells/ml,, range 1-36917), respectively; these cells were maintained for 6 months (CD4: median 360 cell/ml, range 51-13028; CD8: median 315, range 13-4586). 53% of donors at baseline showed IL-2+ CD4+ T cells (median 13 cells/ml, range 2-171); this fraction increased to 84% after the first dose (median 139 cells/ml, range 4-2595) then reached 94% after the boost (median 1838 cells/ml, range 140-17302), and remained detectable after 6 months (median 446 cells/ml, range 20-5678). Polyfunctional IFN-g+IL-2+ CD4+ T cells were induced in 99% of the individuals only following the booster dose (median 825 cells/ml, range 43-7088), and persisted for at least 6 months after the first dose (median 169 cells/ml, range 9-1533) (Fig. 3D ). Polyfunctional CD8+ T cells co-expressing IFNg+ and lysosomal associated membrane glycoprotein (LAMP-1, CD107a) increased significantly after the boost (T0: median 33 cells/ml, range 1-1528; T1: median 54 cells/ml, range 2-694; T2: median 108 cells/ml, range 11-11965; T3: median 106 cells/ml, range 1-1854) (Fig. 3D ). Induction of these cytokinesecreting S-specific T cells was equivalent in both sexes at all time points (Fig. S3A ). The number of TNF-a-secreting CD4+ and CD8+ cells was highest at the peak of the secondary response, 14 days after the second dose (Fig. S3B ); at the same time point polyfunctional TNFa+IFNg+ cells decreased significantly, and after 6 months they were found to be at prevaccination levels. Further analysis of CD107a, TNFa and Granzyme B co-expression among CD4+ and CD8+ T cells producing IFNg+ revealed changing patterns between time points (p<0.001 for all comparisons except for IFNg+ CD4+ T cells between T1 and T2, Figure 3E ), with the fraction of polyfunctional CD4+ cells increasing at each time point, and a predominance of CD8+ cells with 2 functions at the latest time point, indicating a dynamic refolding of functional programs in antigen-specific T cells following boost and and contraction.

Cytokine secretion was also measured, at the peak of the response (T2), in the supernatants of cultures stimulated with the peptide pools. Production of high levels of IFNg and IL-2 was confirmed, whereas IL-17 and IL-4 were barely detectable (<5 pg/ml), confirming the Th1 differentiation profile of S-specific cells (Fig. S3B ).

Thus, vaccination induces the emergence in both males and females of a robust CD4+ and CD8+ cytokine response by T cells already after priming, while full effector functions marked by polyfunctionality are acquired only following the boost, and then maintained for at least 6 months.

Features of Spike-specific T cells.

We then characterized the antigen-specific T cells induced by vaccination through the definition of their differentiation status (Fig. 4 ). The study of the composition of naïve, memory, and effector cell fractions within AIM+ cells in the peripheral blood shows a high frequency of cells with a naïve phenotype at baseline which drops after vaccination, matched by a striking increase in the fraction of effector and central memory cells in both CD4+ and CD8+ subsets, suggesting differentiation from a pool of S-specific cell precursors after antigen exposure and successful induction of a memory cell pool (Fig. 4A and B ). The fraction of terminally differentiated CD45RA+CCR7-in both CD4+ and CD8+ subsets drastically decreases following vaccination, suggesting the establishment of a lively and nonterminal immune response. Six months after the first dose, S-specific cells which have survived the physiological contraction of the immune response are mostly central-and effector memory cells, and in the CD8+ subset these cells also include a significant fraction of terminally differentiated effectors. It should be noted that the fraction of AIM+ T cells at baseline necessarily includes also non Spike-specific CD4+ and CD8+ lymphocytes, (as suggested by the low stimulation index and by the similar distribution of the differentiation subsets in AIM+ and in total CD4+ or CD8+ T cells, respectively), and these cannot be excluded from the analysis. After priming and boosting, however, the very high stimulation indices indicate that AIM+ cells are completely represented by S-specific T lymphocytes. For this reason, we limited further and deeper phenotypic analysis of Spike-specific cells to the time points after vaccination.

Thus, although the absolute number of antigen-specific T cells does not significantly increase following the second dose, the composition of this subset differs significantly between priming and recall responses, and indicates the establishment of an efficient population of S-specific cells comprising a significant fraction of non-terminally differentiated and potentially longlived memory cells.

Effector features of Spike-Specific CD4+ and CD8+ cells. We then explored by unbiased analysis the changes in time in the expression of immunologically relevant markers occurring in antigen-specific T cells. We used FlowSOM clustering to identify 4 CD4+ T cell clusters segregated by the expression pattern of CD137, CD39, ICOS, PD-1, HLA-DR, CD25, CXCR5, CD95, CCR7, CD45RA, CD38, and CD127 and superimposed those clusters in a UMAP plot generated by embedding the same set of markers (Fig. 5A ). Since Ki67, a marker of cell proliferation, was never expressed on AIM+ cells (not shown), we excluded it from analysis. AIM+ CD4+ T cells distribute differently in the UMAP plot at the three different time points (Fig. 5B ), highlighting an overall phenotypic shift. Among these cell clusters, cluster 3 and 4 result unchanged in frequency, while clusters 1 and 2 show significant changes over time (Fig. 5C ). Cluster 2, which increases in frequency at each time point, is characterized by high expression of activation markers (CD25, CD38, CD39, HLA-DR, CD137) and displays features typical of T follicular helper (Tfh) cells (i.e. expression of ICOS-L, PD-1, and CXCR5) while cluster 1 is composed by cells which show a non-activated profile, and decreases with time (Fig. 5A and C ). This data indicates that the antigen-specific CD4+ T cell phenotype is shaped and "perfectioned" by the second encounter with the antigen, and the acquired phenotype persists in the peripheral blood for at least 6 months. Data analysis through manual gating and measurement of single marker expression on the antigen-specific T cells, revealed the emergence and persistence in time, albeit at lower levels after 6 months, of CD4+ subsets displaying high levels of PD-1, ICOS, and CXCR5 (Fig. S4 ), denoting their ability to interact with B cells in lymphoid follicles. At the 6-month time point expression of activation markers such as CD25, CD39, CD38, and HLA-DR is greatly reduced, in line with what expected after the physiological contraction of the immune response. Also S-specific CD8+ lymphocytes are remodeled by vaccine doses, as shown by the distinct distribution with time along the UMAP axes (Fig. 5E ). Two clusters, comprising highly (Cluster 4) and slightly less (Cluster 3) activated AIM+ CD8+ T cells, show transient expansion after the booster dose (Fig. 5 D and F ). This is in contrast to Cluster 1 (Naïve-like cells) which remains stable after a first decrease in frequency at T2, and to Cluster 2 (CCR7+ CD127+ CD45RA-central memory CD8+ T cells) which is not significantly reduced at T2 but increases in size at 6-months. Manual analysis of AIM+ CD8+ T cells confirmed the transient increase at T2 of CD38+, HLA-DR+ and CD25+ cell frequencies, whereas CD39+ and PD-1+ cells steadily decrease or increase, respectively, at T2 and T3 compared to T1 (Fig. S4 ).

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

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

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

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

DISCUSSION

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

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

We find that most individuals harbour Spike-specific T cells already at baseline, likely due to the presence of a pool of naïve progenitors and of memory clones which are cross-reactive with other coronaviruses (3, 15-17, 37, 38) . These cells are highly responsive to antigen encounter, and their SI increases with time. CD8+ cells show a less vigorous response compared to the CD4+ subset possibly due to the sub-optimal stimulation of CD8+ cells by 15-mers, as those used in our assays (39).

High antibody titers induced by influenza vaccination have been shown to correlate positively with the frequency of T cells expressing follicular helper molecules, including CXCR5 and ICOS (40,41). Relevant to infection with SARS-CoV-2, an interesting study has shown the absence of germinal centers and a block in the differentiation of Tfh cells in post-mortem tissues from COVID-19 patients (42), with loss of both transitional and follicular B cells in severe disease. Here, we find a consistent population of T cells equipped with the molecules needed for interaction with B cells, which survives the contraction of the immune response and is clearly detectable 6 months after vaccination albeit with a measurable (but not statistically significant) decrease in magnitude. The number of antigen-specific cells expressing CXCR5, crucial for positioning T cells in the germinal center within lymph nodes, was increased 5-fold at the latest time point compared to the initial measurement on the emerging population of antigen-specific cells, three weeks after the first dose. Similarly, T cells expressing ICOS and PD-1 composed a significant fraction of the antigen-specific subset, and were numerically maintained for at least 6 months. These findings show that vaccination with BNT162b2 appropriately induces the differentiation of the T cell subset specialized in providing B cell help and thus in sustaining the generation of high-affinity antibodies in germinal centers, and that these cells persist in the periphery for at least 6 months. Importantly, AIM+ T cell numbers correlate with serum antibody levels only in the primary response, measured 3 weeks after the first dose, while subsequent measurements indicated that T cell responses and antibody levels follow different kinetics: thus, the sole measurement of nAbs does not inform on the whole immunological ensemble induced by vaccination, although high antibody levels seem to correlate with protection from infection both in animal models and in humans (43)(44)(45)(46). Moreover, CD4+ T cells have also other abilities, such as those related to cytokine production and to direct cytotoxic function. In agreement with previous results, we find that antigenspecific CD4+ T cells induced by vaccination with BNT162b2 produce cytokines typical of the Th1 profile (IFNg, TNFa), and minimal levels of IL-4 and IL-17. In infants the presence in the peripheral blood of ³100 influenza-specific IFNg-producing cells/ml confers protection against clinical influenza (47), and a correlation between both magnitude and polyfunctionality of T cell responses and resistance to infection has been described in other vaccine settings (48)(49)(50)(51)(52)(53)(54). In this study, we show that 6 months after vaccination with BNT162b2 over 300 CD4+ and CD8+ Spike-specific IFNg-producing T cells/ml are clearly detected in the periphery, with diverse functionalities. CD4+ cells acquire both helper and cytotoxic functions which are particularly evident at the peak of the antigen-specific response, 14 days after the boost. At this time point we detect the highest frequencies of cells with a polyfunctional phenotype, in both CD4+ and CD8+ S-specific T cells. Interestingly, the CD4+ subset is more spared from the physiological contraction of the immune response, 6 months after receiving the first dose of BNT162b2, and it shows polyfunctional features of both cytotoxicity and ability to provide Bcell help. At the same time point, the antigen-specific CD8+ T cell subset seems to drop at lower levels, although these cells are still clearly detectable and are significantly expanded compared to the baseline. This data is consistent with studies which have investigated cellular immune responses in individuals recovering from COVID-19 (16,20,(55)(56)(57)(58), and with other studies on mRNA vaccination (59,60), and suggest that vaccine-induced T cells have the prerequisites to confer protection from subsequent infections. Moreover, the persistence of a numerically consistent pool of antigen-specific T cell, which we find to be still increased 6fold from baseline after 6 months, may be the source for of effector cells which can promptly expand in case of antigen re-encounter (61).

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

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

Moreover, although donors were equally distributed for age and sex, our sample was limited in size. This notwithstanding, some considerations may be made. On the whole, the results of this study can be visualized as the dynamic and integrated emergence of a theoretically effective Spikespecific adaptive immune response, characterized by a T cell response which precedes in time the development of high levels of anti-Spike neutralizing antibodies (Fig. 7 ). T cells induced after the first encounter with the Spike protein are mostly effector cells, which secrete intermediate levels of cytokines, express the highest levels of Granzyme B, have not acquired polyfunctionality, and a fraction is also competent for the interaction with B cells; notably, also cells with features of stemness and longevity appear. After the boost, the peak of the response shows a fully activated, cytotoxically empowered, multifunctional, and B cell "friendly" T cell population, and this also corresponds to the highest levels of neutralizing antibodies in the serum. What remains after 6 months is a population of T cells with features of polyfunctionality and ability to provide B cell help; at this time point, Spike-specific T cells which have survived the immunological contraction are highly specific and prone to give rise to effective and rapid antiviral responses, both by sustaining the production of neutralizing antibodies and by exerting direct cytotoxicity towards already infected cells. Concomitantly, the pool of TSCM is stably maintained. In our cohort we did not find differences between males and females in any of the investigated immunological measurements.

This model provides the immunological basis of the current clinical data showing the effectiveness of the vaccine in preventing severe disease, of the low frequency of breakthrough infections, and of the reduced time-window of contagiousness of re-infected individuals, all signs of effective immunity. Previous literature on T cell responses following vaccination shows similar immunological patterns in previously validated and approved anti-viral vaccines with outcomes of pluri-decennial immunity. This also suggests that a third booster dose may not be needed in most individuals, since over 90% of vaccinees develop T cells with features of longevity and show high numbers of circulating and appropriately modeled antigen-specific memory cells, ready to effectively and rapidly engage in a possible re-encounter with the virus.

Immunologically fragile individuals, however, or those who are unavoidably exposed to high titers of virus may be better protected by a third dose of vaccination which has been shown to increase antibody levels, thus providing an immediate albeit temporary shield against viral entry in the host cell. In an equitable world, and based on the current data on vaccine effectiveness in preventing severe disease, after having secured the fragile from infection the absolute precedence should be given to unvaccinated individuals globally, in the unified endeavor to curb viral circulation and to prevent disease.

Study Design

This work started to prospectively define cellular immune responses against the SARS-CoV-2 Spike-protein induced by mRNA vaccine administration. To this aim, we enrolled 71 individuals from the sanitary personnel and from scientists operating at the Santa Lucia Foundation which were scheduled for vaccination with Pfizer-BioNTech BNT162b2 between 12 January and 2 February 2021 (Table S1 ). All donors signed informed consent forms approved by the Ethical Committee of the Santa Lucia Foundation. Venous blood was obtained immediately before the first dose (T0), 21 days thereafter, at the time of boosting (T1), and two weeks after the second dose (T2), and 6 months after the first dose (T3). To avoid gender and age biases, an equal number of female and male volunteers, as well as an equal number of subjects 22-45 and 45-66 years old, was included. Not all subjects were analyzed at each time point due to precautionary quarantine of some individuals during the study. All data is presented in table 3 .

Evaluation of anti SARS-CoV-2 Antibodies

The measurement of anti SARS-CoV-2 neutralizing Abs was performed by electrochemiluminescence sandwich immunoassay (ECLIA) through Roche Elecsys Anti-SARS-CoV-2 S (Roche diagnostics, Switzerland). The neutralizing Ab were measured on a Cobas 601 modular analyzer (Roche diagnostics, Switzerland), using a cut-off of 0.8 U/ml to determine Ab levels. In particular, Elecsys Anti-SARS-CoV-2 S U/mL measurements are equivalent to WHO International Standard Binding Arbitrary Units per mL (BAU/mL), according to which higher values than 0.8 BAU/mL are considered positive.

AIM assay

In vitro stimulation of freshly obtained PBMC was performed as described (66).

In brief, 200 ml of cell suspensions (10 x 10 6 cells/ml) were seeded in U-bottom 96-well plates at a density of (0,2 ml/well) and stimulated for 18 hours with or without PepTivator SARS-CoV-2 protein S, S1 and S+ peptide pools (1 mg/ml each, Miltenyi Biotec). Purified aCD40 (0,5 μg/µl, Miltenyi Biotech) was added at culture start to enhance endpoint CD40L staining by inhibiting its recycling. Supernatants and cells from these culture wells were collected for cytokine measurement and flow cytometry, respectively.

Intracellular Cytokine Staining PBMC were incubated with the addition of BV421conjugated antiCD107a (1/200 dilution, BD Biosciences), Monensin and Brefeldin A (5μM and 10 μg/ml, respectively, both from Sigma-Aldrich) after the first hour of incubation to allow accumulation of cytokines into the cytoplasm and avoid endo-lysosome acidification which can quench fluorescence from the aCD107a reinternalized during degranulation. At the end of the culture, cells were harvested and directly stained for flow cytometry.

Flow cytometry staining and acquisition

Post-culture cells were pelleted in V-bottom 96well plates and resuspended in 30 μl of antibodies at pre-optimized concentrations and diluted in Brilliant Stain Buffer (BD Biosciences), then incubated for 15' at RT. After a washing step, cell pellets were fixed either in FoxP3 fixation/permeabilization Buffer (ThermoFisher) for 20' at RT (AIM Assay) or in Formaldehyde 4% in PBS (ICS Assay). ICS was performed by incubating cells in 30 μl of antibodies against cytokines and Granzyme B diluted in a Saponin (Sigma-Aldrich) 0.5% W/V solution. The complete list of antibodies used for surface staining and ICS is shown in Table S2 . Samples were acquired on a fully equipped Cytoflex LX within 6 hours from staining and fixation. Quality Control beads (Beckman Coulter) were used daily to check and standardize instrument performances. Data was analyzed with FlowJo v. 10.7

Cytokine measurements IL4, IL17, TNFa and IFNg were measured in frozen culture supernatants from the AIM assays described above by a bead-based sandwich ELISA (MACSPlex Cytokine Kit, Miltenyi Biotech) according to manufacturer instructions.

AU Neutralizing IgG

Anticorpi T0 T1 T2 T3

Author contributions:

GB, LB, and AR conceptualized the study. AR, AS and CC performed vaccination supervision and donor enrollment. GG and MP set up and supervised all experimental procedures. GG, RP, SD, AV, MPirr, MS, FG performed cell stimulations, stainings, flow cytometry experiments, and cytokine measurements. MPB performed antibody dosage. GB prepared the dataset for analysis. GB and MP performed descriptive statistics. AT and CF did the statistical analysis.

Competing interests:

The authors declare that they have no competing interests.

Data and materials availability:

All data is included in Table S3 .

Acknowledgements

Acknowledgments:We thank the volunteers for donating their blood and time, and the nurses for their assistance.We are grateful to Daniela F. Angelini for critically reading the manuscript.

Funding

Funding: This work was partially supported by the Italian Ministry of Health to L.B. (COVID-2020-12371735).Funding acquisition: LB.Writing -original draft: GB.Writing -review & editing: GB, MP, LB, with input from all authors.

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