Short-lived booster effect and stable CD8+ T cell memory after 3rd COVID-19 vaccine dose

doi:10.21203/rs.3.rs-1617057/v1

Download PDF

Biological Sciences - Article

Short-lived booster effect and stable CD8+ T cell memory after 3rd COVID-19 vaccine dose

https://doi.org/10.21203/rs.3.rs-1617057/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 08 Aug, 2022

Read the published version in Nature Communications →

Version 1

posted

You are reading this latest preprint version

Reports of waning immunity after COVID-19 vaccination (1-3) have recently led to large booster vaccination campaigns. Previous studies showed that basic immunization with two mRNA vaccine doses elicits a robust spike-specific CD8+ T cell response (4-6). The effect of mRNA booster vaccination on the spike-specific CD8+ T cell response remains, however, unclear. Indeed, very little is known about the efficacy, duration and effects on long-term immunity and recall responses in breakthrough infections. In this study, we show that spike-specific CD8+ T cells are immediately and vigorously activated and expanded in all tested individuals after the 3rd and 4th mRNA vaccine shots. However, this CD8+ T cell boost response is characterized by a steep contraction and lasts only for about 30-60 days compared to a prolonged contraction after natural infection. Booster vaccination did not affect long-term spike-specific CD8+ T cell immunity reflected by a stable stem cell memory pool that already reached maximum frequencies after basic immunization. Accordingly, rapid and full-fledged recall responses of boosted spike-specific CD8+ T cells were detectable after breakthrough infection with delta and omicron. Thus, in addition to the previously reported cross-reactivity (7-12) also a robust activation and effector response determines the efficacy of the CD8+ T cell response targeting emerging variants of concern. Neutralizing antibody responses displayed hardly any boost effect towards omicron, further highlighting the relevance of spike-specific CD8+ T cell immunity. In sum, these data will inform future vaccination strategies facing the next COVID-19 wave expected for late 2022/early 2023.

CD8+ T cells

neutralizing antibodies

SARS-CoV-2

mRNA vaccination

COVID-19

booster vaccination

antiviral immunity

immunological memory

adaptive immune response

More than two years after the outbreak of the SARS-CoV-2 pandemic, constantly emerging variants of concern (VOCs) still fuel the pandemic situation and vaccination is considered to be an important measure to accelerate the transition from the pandemic into the endemic stage¹³. mRNA vaccines against SARS-CoV-2 have been proven to rapidly induce protection against symptomatic COVID-19 and death by inducing robust antibody and T cell responses^4-6,14. However, reports of waning humoral immunity^1-3 and high SARS-CoV-2 infection incidences with the VOC omicron (B.1.1.529) have recently led to large booster vaccination campaigns. Immediate beneficial effects of these booster immunizations were detectable with respect to the neutralization capacity of the humoral response¹⁵. The effect of mRNA booster vaccination on the spike-specific CD8+ T cell response remains, however, unclear. Indeed, there is little information available about the efficacy, duration and effects on long-term immunity and recall responses in breakthrough infections.

To address these important questions, we longitudinally traced and deeply profiled SARS-CoV-2 mRNA vaccine-boosted spike-specific CD8+ T cell responses on a single epitope level in vaccinees starting at baseline of prime vaccination until 4 months after 3^rd or even 1-2 months after 4^th vaccination. Additionally, we analyzed the spike-specific CD8+ T cell response in breakthrough infections with delta (B.1.617.2) and omicron after booster immunization. Moreover, we compared the kinetics of the spike-specific CD8+ T cell response with the neutralizing capacity of the humoral response. Our data show that after the 3^rd mRNA vaccine dose there is a substantial but short-lived booster effect on spike-specific CD8+ T cells without marked effect on the memory response and a minor boost effect on the neutralizing antibody response against omicron. These observations may advise future booster vaccination campaigns.

Transient spike-specific CD8+ T cell booster responses

To assess the longevity of boosted adaptive effector responses, we longitudinally collected sera and peripheral blood mononuclear cells (PBMCs) from 13 volunteers (Supplementary Table 1) before and after 3^rd mRNA vaccination with Comirnaty/Pfizer-Biontech or Spikevax/Moderna (Extended Data Fig. 1a). All volunteers included in this cohort did not have a history of SARS-CoV-2 infection and received their vaccinations with a three-week interval between 1^st and 2^nd and six-to-nine-months interval between 2^nd and 3^rd dose. First, we analyzed the kinetics of the spike-specific CD8+ T cell response targeting the A*01/S₈₆₅ (n=7) or A*02/S₂₆₉epitope (n=8). A*01/S₈₆₅- and A*02/S₂₆₉-specific CD8+ T cells are dominant within the overall spike-specific CD8+ T cell response after mRNA vaccination^5,10 and target epitopes that are highly conserved in all VOCs including the omicron variants BA.1 and BA.2 (Extended Data Fig. 1b) but not in common cold coronaviruses⁵. To increase the detection sensitivity of spike-specific CD8+ T cells targeting single epitopes, we performed peptide-loaded MHC-class I tetramer-based enrichment^5,16. Spike-specific CD8+ T cell frequencies had already reached their set point approx. 60 days after the 2^nd dose and were rapidly boosted with peak expansion one week after the 3^rd dose in all tested donors (Fig. 1a). Robust boosting of the spike-specific CD8+ T cell response by the 3^rd dose was also reflected by high CD38, Ki-67 and T-BET expression and thus by rapid activation, strong proliferation and substantial induction of an effector cell program (Fig. 1b and Extended Data Fig. 1c). However, within 30-60 days the circulating spike-specific CD8+ T cell frequency, activation, proliferation and effector cell differentiation decreased rapidly to a similar level as before the 3^rd dose (Fig. 1a/b and Extended Data Fig. 1c), thus following the same kinetics as after the 2^nd dose. Similarities between the spike-specific CD8+ T cell response after the 2^nd and 3^rd dose, respectively, are also supported by t-distributed stochastic neighborhood embedding (t-SNE) and pseudotemporal diffusion analysis of T cell phenotypes including CD38, T-BET, BCL-2, PD-1, and TOX expression (Fig. 1c and Ext. Data Fig. 1d/e). Phenotypic differences (e.g., in T-BET and TOX expression) were detectable between the spike-specific CD8+ T cell response after the 1^st dose compared to the 2^nd and 3^rd doses and only minor differences were apparent with respect to the targeted epitopes A*01/S₈₆₅ or A*02/S₂₆₉ (Fig. 1c and Extended Data Fig. 1e/f). With respect to the neutralizing antibody response, boosting efficacy clearly depended on the VOC (Fig. 1d and Extended Data Fig. 1g; p<0.0001). In particular, neutralization was only slightly increased against the omicron variant BA.1 (B.1.1.529) by the 3^rd dose while neutralization of the parental B.1 strain and delta (B.1.617.2) peaked after approx. 3 weeks followed by a decrease to baseline neutralization after 4 months (Fig. 1d). Of note, after the 4^th dose similar kinetics (Fig. 1e) and dynamics (Extended Data Fig. 2a-c) of spike-specific CD8+ T cell and neutralizing antibody responses were detectable. Hence, after booster vaccination, there is a transient mRNA vaccine-associated booster response of spike-specific CD8+ T cells and neutralizing antibodies of about 2 and 4 months, respectively.

Stable memory T cell response irrespective of booster immunization

To investigate the effect of booster immunization on long-term spike-specific CD8+ T cell immunity, we comparatively analyzed the spike-specific CD8+ T cell response after three and nine months post 2^nd dose versus three months after the 3^rd dose. The memory T cell subset distribution (Fig. 2a and Ext. Data Fig. 3a/b) and activation/differentiation footprint (Fig. 2b and Ext. Data Fig. 3c) of spike-specific CD8+ T cells were similar at the tested time points, indicating stable long-term memory features without a clear effect of the 3^rd dose. To more precisely quantify and characterize the T cell memory compartment, we analyzed on the one hand spike-specific CD8+ T cells highly expressing BCL-2 (BCL-2^hi) to map the overall memory pool irrespective of certain subsets and on the other hand focused on stem cell memory T cells (T_SCM, defined by expression of CD45RA, CCR7, CD27, CD28 and CD95; Ext. Data Fig. 3a) that are essential for long-term T cell immunity¹⁷. Spike-specific BCL2^hi CD8+ T cells and CD8+ T_SCM cells were induced after the 1^st dose and their frequencies remained stable after the 2^nd dose throughout the subsequent vaccination (Fig. 2c/d). t-SNE analysis revealed that spike-specific BCL-2^hi CD8+ T cells showed minor phenotypic differences such as higher T-BET expression 3 months post 2^nd and 3^rd dose compared to 9 months after the 2^nd dose (Fig. 2e and Ext. Data Fig. 3d) probably reflecting a more resting state at later time points post immunization. In contrast, phenotypic characteristics of CD8+ T_SCM remained completely stable, indicated by a complete intermingling within the t-SNE analysis (Fig. 2f). Furthermore, the spike-specific CD8+ T cell memory pool was also conserved during and after the 4^th dose (Ext. Data Fig. 4a-c). Thus, these results indicate that booster immunizations give rise to a rapid effector T cell response that is based on a stable memory T cell pool as illustrated by a subset of spike-specific CD8+ T cell expressing high levels of BCL-2 in parallel to T-BET^hi, PD-1^hi, TOX⁺subsets at peak expansion after booster immunization (Ext. Data Fig. 2a).

Conserved spike-specific CD8+ T cell recall capacity

Next, we analyzed the recall capacity of spike-specific CD8+ T cells after the 3^rd dose. For this, we stained for TCF-1 expression, a transcription factor indicative of the proliferative capacity of T cells¹⁸. The frequencies of TCF-1⁺ spike-specific CD8+ T cells were stable suggesting a conserved pool of proliferative competent T cells throughout the 2^nd and 3^rd dose (Fig. 3a). Accordingly, the peptide-specific in vitro expansion capacity of spike-specific CD8+ T cells was also conserved before and after the 3^rd dose (Fig. 3b and Ext. Data Fig. 5a/b). Furthermore, after two weeks of expansion, spike-specific CD8+ T cells produced similar amounts of IFNg and TNF, including co-production irrespective of whether the input cells were obtained before or after the 3^rd dose (Fig. 3c and Ext. Data Fig. 5b-d). Spike-specific production of IFNg and TNF were assessed in relation to the frequency of spike-specific CD8+ T cells after expansion as a measure of the effector function per cell. In order to estimate the cytotoxic potential of spike-specific CD8+ T cells after in vitro expansion, we analyzed degranulation as indicated by CD107a expression in relation to the frequency of spike-specific CD8+ T cells (Fig. 3d and Ext. Data Fig. 5e) and Granzyme B (Fig. 3e) and Perforin (Fig. 3f) expression of spike-specific CD8+ T cells. Similar to cytokine production, the cytotoxic potential was also largely conserved with almost all spike-specific CD8+ T cells expressing Granzyme B and Perforin. Thus, recall capacity tested in vitro comprising expansion capacity, differentiation to cytokine-producing and cytotoxic effector cells is robust and stable before and after 3^rd vaccination.

Recall response in breakthrough infections

To determine the recall capacity in vivo and to compare booster responses after vaccination versus infection, we next analyzed the spike-specific CD8+ T cell response and the neutralizing antibody capacity in breakthrough infections. Specifically, we compared the adaptive effector immune response after the 4^th antigen contact, either by the 4^th vaccine dose (n=5) or by breakthrough infection with omicron (n=12) or delta (n=2) after three mRNA vaccine doses. The spike-specific CD8+ T cell response was rapidly and robustly induced and similar after the 4^th vaccine dose versus an omicron or delta breakthrough infection (Fig. 4a). Spike-specific CD8+ T cells peaked in a classical effector cell response with pronounced expression of CD38, Ki-67 and T-BET (Fig. 4b and Ext. Data Fig. 6a-c). Variations within the proportion of spike-specific CD8+ T cells expressing these markers may be due to differences in infection time-point or time point of symptom onset, which is especially relevant in breakthrough infections with omicron that are often characterized by mild symptoms in vaccinated individuals. However, within one to two months after breakthrough infection and 4^th vaccine dose, a fully functional early T cell memory was present with similar reactivation capacities comprising expansion, cytokine production and degranulation (Fig. 4c and Ext. Data Fig. 6d). Furthermore, phenotypic characteristics of early spike-specific memory CD8+ T cells were similar after breakthrough infections and 4^th vaccine dose as depicted by t-SNE analysis including CD38, CCR7, TCF-1 and BCL-2 (Fig. 4d) and memory subset distribution with transitional and effector memory subsets being dominant (Ext. Data Fig. 6e). Again, the frequencies of the BCL-2^hi memory pool within the spike-specific CD8+ T cells was stable before and after the 4^th antigen contact irrespective of breakthrough infection or 4^th vaccine dose (Fig. 4e). Hence, vaccine-elicited spike-specific CD8+ T cell immunity exhibited a substantial recall capacity in vivo even towards VOCs such as omicron. However, the neutralizing antibody response after the 4^th antigen contact differed from the spike-specific CD8+ T cells in the following aspects (Fig. 4f): First, neutralization capacity depended on the targeted SARS-CoV-2 variant regardless of whether the 4^th antigen exposure was through vaccination or breakthrough infection. Second, the neutralizing antibody response depended on the 4^th antigen contact with differences in vaccination and breakthrough infections. Third, the neutralizing antibody response exhibited a prolonged decay after breakthrough infections compared to vaccination. Thus, in contrast to spike-specific CD8+ T cells, recall of the neutralizing antibody response was less efficient with respect to VOCs.

Here, we mapped the dynamics of mRNA vaccine- versus SARS-CoV-2 infection-boosted spike-specific adaptive effector immunity. By analyzing the dominant A*01/S₈₆₅- and A*02/S₂₆₉-specific CD8+ T cells responses, we observed a rapid and robust expansion of spike-specific CD8+ T cells with a similar slope and amplitude after the 3^rd and 4^th vaccine dose versus breakthrough infections with SARS-CoV-2 delta and omicron variants. Notably, kinetics and magnitude were comparable to the spike-specific CD8+ T cell response after the 2^nd vaccine dose with a remarkably high recruitment of spike-specific CD8+ T cells to the effector pool that is reflected by high expression of activation and proliferation markers, like CD38 and Ki-67. This observation highlights the effectiveness of mRNA vaccination to elicit rapid and functional CD8+ T cell responses. Thus, given the important role of CD8+ T cells to control SARS-CoV-2 infection and protect from severe COVID-19^19-22, mRNA booster vaccination represents a valuable tool to immediately react to high viral burden in the population in order to e.g., protect vulnerable groups and to reduce the risk of overwhelming the public health system.

Importantly, however, the spike-specific CD8+ T cell booster responses after 3^rd and 4^th mRNA vaccine only lasted approximately 1-2 months until it decreased to a similar level as prior booster vaccination. This rapid contraction of the spike-specific CD8+ T cell response has to be taken into account when booster vaccination strategies are planned especially for risk groups but most likely also for broader populations this fall. Furthermore, the steep decline of the spike-specific CD8+ T cell boost response is in stark contrast to a more prolonged contraction evident after SARS-CoV-2 breakthrough infection irrespective of the infecting variant. This prolonged contraction has also been reported for non-spike epitope-specific CD8+ T cells after SARS-CoV-2 infection¹⁶ indicating a more general difference in virus-specific CD8+ T cells induced by SARS-CoV-2 infection versus mRNA vaccination. Possible explanations include differences in antigen half-life, innate immunity, co-stimulation, cytokines, antigen presentation and CD4+ T cell responses that are all required for coordinating CD8+ T cell responses^6,20,21 after mRNA vaccination versus SARS-CoV-2 infection. Hence, a better understanding of the interplay between the different immune components in viral infection versus vaccination is required to adapt mRNA vaccines prolonging antigen-specific CD8+ T cell booster responses in the context of SARS-CoV-2 but also of other targets.

A durable and functionally competent spike-specific memory CD8+ T cell response was already detectable after the 2^nd dose of mRNA vaccine and thus after completed basic immunization^5,6,8. Here, we now show that this well-established durable spike-specific CD8+ T cell memory response is not tremendously affected by the 3^rd dose. In particular, a pool of T_SCM cells that have been reported to maintain long-term CD8+ T cell immunity after yellow fever virus (YFV) vaccination^17,23 and that were reported to be detectable after basic immunization with SARS-CoV-2 mRNA vaccination²⁴ were already induced within spike-specific CD8+ T cells after prime vaccination. The spike-specific CD8+ T_SCM cell pool reached final size after the 2^nd dose and remained constant throughout the 3^rd and 4^th dose. Thus, 3^rd mRNA vaccinations do not amplify long-term CD8+ T cell immunity and also do not drive the CD8+ T cell memory pool into relevant senescence. This is in line with a recent report showing that repeated antigen exposure does also not induce T cell exhaustion of spike-specific CD8+ T cells²⁵. It rather appears that the spike-specific CD8+ T cell booster response is an effector response based on a stable memory pool. Importantly, spike-specific memory CD8+ T cells were capable to efficiently mount recall responses in vitro and in vivo after breakthrough infections with delta or omicron. This recall efficiency is explained by the previously described cross-reactivity of the vaccine-elicited spike-specific CD8+ T cell response^7-12, diversification of the T cell repertoire²⁵ and of the rapid kinetics and effector differentiation dynamics shown in this study. It is, however, important to note that our study is limited to healthy, rather young individuals without higher risk to develop severe COVID-19²¹ and with the capability to establish robust vaccine-elicited immune responses^4-6,8,14. Future studies have to show whether individuals above the age of 60 develop comparable immunity after mRNA vaccination. Still, our observations challenge the discussion about the necessity to frequently apply mRNA booster vaccination to healthy individuals that are not compromised in their immune response in three-to-six-months intervals.

In contrast to spike-specific CD8+ T cells, boosting the neutralizing antibody response by currently applied, non-adapted mRNA vaccines clearly depends on the targeted SARS-CoV-2 variant. In particular, neutralizing capacity against omicron is hardly increased. Of note, neutralizing capacity against omicron is also only moderately increased after breakthrough infections with omicron probably reflecting antibody escape. Similar to the spike-specific CD8+ T cell response, neutralizing capacity declines slower after infection versus vaccination. Yet, it appears that neither after vaccination nor after infection the neutralization capacity of the humoral response is sufficient to protect from omicron infection as indicated by the high numbers of breakthrough infections in vaccinees and convalescents. In sum, our study highlights that mRNA vaccines are potent inducers of a robust, functionally competent and durable spike-specific CD8+ T cell immunity already after completed basic immunization. This contributes to protection from symptomatic disease that is especially relevant for at-risk individuals and has also important implications for vaccine development targeting other CD8+ T cell-controlled infectious agents and cancers.

1 Chemaitelly, H. et al. Waning of BNT162b2 vaccine protection against SARS-CoV-2 infection in Qatar. N Engl J Med 385, e83, doi:10.1056/NEJMoa2114114 (2021).

2 Goldberg, Y. et al. Waning immunity after the BNT162b2 vaccine in Israel. N Engl J Med 385, e85, doi:10.1056/NEJMoa2114228 (2021).

3 Levin, E. G. et al. Waning immune humoral response to BNT162b2 COVID-19 vaccine over 6 months. N Engl J Med 385, e84, doi:10.1056/NEJMoa2114583 (2021).

4 Kalimuddin, S. et al. Early T cell and binding antibody responses are associated with COVID-19 RNA vaccine efficacy onset. Med (N Y) 2, 682-688 e684, doi:10.1016/j.medj.2021.04.003 (2021).

5 Oberhardt, V. et al. Rapid and stable mobilization of CD8+ T cells by SARS-CoV-2 mRNA vaccine. Nature 597, 268-273, doi:10.1038/s41586-021-03841-4 (2021).

6 Painter, M. M. et al. Rapid induction of antigen-specific CD4+ T cells is associated with coordinated humoral and cellular immunity to SARS-CoV-2 mRNA vaccination. Immunity 54, 2133-2142 e2133, doi:10.1016/j.immuni.2021.08.001 (2021).

7 Gao, Y. et al. Ancestral SARS-CoV-2-specific T cells cross-recognize the Omicron variant. Nat Med 28, 472-476, doi:10.1038/s41591-022-01700-x (2022).

8 Goel, R. R. et al. mRNA vaccines induce durable immune memory to SARS-CoV-2 and variants of concern. Science 374, abm0829, doi:10.1126/science.abm0829 (2021).

9 Keeton, R. et al. T cell responses to SARS-CoV-2 spike cross-recognize Omicron. Nature 603, 488-492, doi:10.1038/s41586-022-04460-3 (2022).

10 Lang-Meli, J. et al. SARS-CoV-2-specific T-cell epitope repertoire in convalescent and mRNA-vaccinated individuals. Nat Microbiol 7, 675-679, doi:10.1038/s41564-022-01106-y (2022).

11 Mateus, J. et al. Low-dose mRNA-1273 COVID-19 vaccine generates durable memory enhanced by cross-reactive T cells. Science 374, eabj9853, doi:10.1126/science.abj9853 (2021).

12 Skelly, D. T. et al. Two doses of SARS-CoV-2 vaccination induce robust immune responses to emerging SARS-CoV-2 variants of concern. Nat Commun 12, 5061, doi:10.1038/s41467-021-25167-5 (2021).

13 Krammer, F. SARS-CoV-2 vaccines in development. Nature 586, 516-527, doi:10.1038/s41586-020-2798-3 (2020).

14 Sahin, U. et al. BNT162b2 vaccine induces neutralizing antibodies and poly-specific T cells in humans. Nature 595, 572-577, doi:10.1038/s41586-021-03653-6 (2021).

15 Schmidt, F. et al. Plasma Neutralization of the SARS-CoV-2 Omicron Variant. N Engl J Med 386, 599-601, doi:10.1056/NEJMc2119641 (2022).

16 Schulien, I. et al. Characterization of pre-existing and induced SARS-CoV-2-specific CD8(+) T cells. Nat Med 27, 78-85, doi:10.1038/s41591-020-01143-2 (2021).

17 Akondy, R. S. et al. Origin and differentiation of human memory CD8 T cells after vaccination. Nature 552, 362-367, doi:10.1038/nature24633 (2017).

18 Utzschneider, D. T. et al. T Cell Factor 1-Expressing Memory-like CD8(+) T Cells Sustain the Immune Response to Chronic Viral Infections. Immunity 45, 415-427, doi:10.1016/j.immuni.2016.07.021 (2016).

19 Apostolidis, S. A. et al. Cellular and humoral immune responses following SARS-CoV-2 mRNA vaccination in patients with multiple sclerosis on anti-CD20 therapy. Nat Med 27, 1990-2001, doi:10.1038/s41591-021-01507-2 (2021).

20 Dan, J. et al. Observations and perspectives on adaptive immunity to SARS-CoV-2. Clin Infect Dis, doi:10.1093/cid/ciac310 (2022).

21 Sette, A. & Crotty, S. Adaptive immunity to SARS-CoV-2 and COVID-19. Cell 184, 861-880, doi:10.1016/j.cell.2021.01.007 (2021).

22 Kedzierska, K. & Thomas, P. G. Count on us: T cells in SARS-CoV-2 infection and vaccination. Cell Rep Med 3, 100562, doi:10.1016/j.xcrm.2022.100562 (2022).

23 Fuertes Marraco, S. A. et al. Long-lasting stem cell-like memory CD8+ T cells with a naive-like profile upon yellow fever vaccination. Sci Transl Med 7, 282ra248, doi:10.1126/scitranslmed.aaa3700 (2015).

24 Guerrera, G. et al. BNT162b2 vaccination induces durable SARS-CoV-2-specific T cells with a stem cell memory phenotype. Sci Immunol 6, eabl5344, doi:10.1126/sciimmunol.abl5344 (2021).

25 Minervina, A. A. et al. SARS-CoV-2 antigen exposure history shapes phenotypes and specificity of memory CD8(+) T cells. Nat Immunol, doi:10.1038/s41590-022-01184-4 (2022).

Acknowledgements

We thank all donors for participating in the current study and FREEZE-biobank-Center for biobanking of the Freiburg University Medical Center and the Medical Faculty for support. We thank Dr. Sandra Ciesek and Dr. Marek Widera (Institute of Medical Virology, University Hospital Frankfurt am Main, Germany) for providing the B.1.1.529 BA.1 isolate. We thank Dr. Jonas Fuchs (Institute of Virology, University of Freiburg, Freiburg) for sequence confirmation of the SARS-CoV-2 stocks used in this study. This study was supported by grants from the German Research Foundation (272983813 to B.B., T.B., R.T., C.N.-H. and M.H., and; 256073931 to B.B., M.S., R.T., C.N.-H. and M.H.; 413517907 to H.L. and J.L.M). This work was also supported by the project “Virological and immunological determinants of COVID-19 pathogenesis – lessons to get prepared for future pandemics (KA1-Co-02 “COVIPA”)”, a grant from the Helmholtz Association's Initiative and Networking Fund (to R.T. and M.H.). M.H. is furthermore supported by the Margarete von Wrangell fellowship (State of Baden-Wuerttemberg).

Author contributions

M.R., H.L., V.K., A.G., S.G., K.C. and D.R. planned, performed and analyzed experiments, interpreted data and contributed to writing the manuscript with the help of V.O., K.H., N.G. and C.B.. M.R., H.L., V.K., A.G., S.G., K.C. and D.R. contributed equally to this work. H.L., A.G., N.R., J.L.-M., S.R., C.S., S.B. and D.S. were responsible for donor recruitment. F.E. performed four-digit HLA-typing by next generation sequencing. B.B. and T.B. contributed to data interpretation. M.S. and G.K. provided virological expertise, supervised virological work and interpreted data. R.T., C.N.-H. and M.H. designed and supervised the study, interpreted data, wrote the manuscript and contributed to experimental design and planning. M.H., C.N.-H. and R.T. are shared last authors.

Declaration of interest

The authors have nothing to declare.

Study cohort

In total, 38 individuals receiving SARS-CoV-2 vaccinations were recruited at the Freiburg University Medical Center, Germany. Of those, blood was collected from 31 individuals vaccinated three times with the mRNA vaccines bnt162b/Comirnaty or mRNA-1273/Spikevax, with 5 individuals receiving a fourth vaccination, without a SARS-CoV-2 infection history and 13 individuals with SARS-CoV-2 breakthrough infections after a third mRNA vaccination. Breakthrough infections were confirmed by positive PCR-testing from oropharyngeal swab. All 13 individuals with breakthrough infections included in this study had mild symptoms without respiratory insufficiency (according to WHO guidelines¹). Characteristics of the participants are summarized in Supplementary Table 1 including the results of the HLA-genotyping performed by next-generation sequencing.

Ethics

Written informed consent was obtained from all study participants. The study was conducted in accordance to federal guidelines, local ethics committee regulations (Albert-Ludwigs-Universität, Freiburg, Germany; vote: 322/20, 21-1135 and 315/20) and the Declaration of Helsinki (1975).

PBMC isolation

PBMCs were isolated from venous blood samples collected in EDTA blood collection tubes by density centrifugation with lymphocyte separation medium (Pancoll separation medium, PAN Biotech GmbH). PBMCs were stored at -80°C until further processing. The cells were thawed in pre-warmed RPMI cell culture medium supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, 1.5% 1M HEPES (all purchased from Thermo Scientific) and 50 U/mLBenzonase (Sigma).

Sequence alignment

Sequence homology was analysed in Geneious® 11.0.5 (https://www.geneious.com/) using Clustal Omega 1.2.2 alignment with default settings². Reference genome of human ancestral SARS-CoV-2 (MN908947.3) was obtained from NCBI database. Genome sequences of SARS-CoV-2 variants of concern (VOCs) B.1, B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.1.529 BA.1 and B.1.1.529 BA.2 were identified via CoVariants (https://covariants.org/). Spike epitopes in ancestral strain and all VOCs were aligned according to their homology on an amino acid level.

Peptides and tetramers for T cell analysis

Peptides were manufactured with an unmodified N-terminus and an amidated C-terminus with standard Fmoc chemistry (Genaxxon Bioscience). All peptides showed a purity of >70%. To generate tetramers, SARS-CoV-2 spike peptides (A*01/S₈₆₅: LTDEMIAQY, A*02/S₂₆₉: YLQPRTFLL) were loaded on biotinylated HLA class I (HLA-I) easYmer® (immunAware) according to manufacturer’s instructions. Subsequently, peptide-loaded-HLA class I monomers were tetramerized with phycoerythrin (PE)-conjugated streptavidin according to the manufacturer’s instructions.

In vitro expansion of spike-specific CD8+ T cells and assessment of effector function

1.5 x 10⁶ PBMCs were stimulated with the spike protein-derived peptides A*01/S₈₆₅ or A*02/S₂₆₉ and anti-CD28 monoclonal antibody (0.5µg/mL) for 14 days in RPMI cell culture medium supplemented with rIL-2 (20 IU/ml, StemCell Technologies). At day 4, 7 and 11, 50% of the culture medium was exchanged with freshly prepared medium containing 20 IU/mLrIL-2. After 14 days, PBMCs were stimulated with peptides again, and stained for CD107a for 1 h at 37 °C to analyse degranulation. Subsequently, brefeldin A (GolgiPlug, 0.5 μl/mL) and monensin (GolgiStop, 0.5 μl/mL) (all BD Biosciences) were added and incubation continued for four more hours, followed by surface and intracellular staining with anti-IFNy, anti-TNF and anti-IL-2-specific antibodies. For calculation of the expansion capacity and to assess the cytotoxic capacity of the expanded cells, peptide-loaded HLA class I tetramer staining was performed together with intracellular staining of Granzyme B, Granzyme K, Perforin and Granulysin.

Magnetic bead-based enrichment of spike-specific CD8+ T cells

CD8+ T cells targeting spike epitopes were enriched as described previously³. In brief, 5 x 10⁶ to 20 x 10⁶ PBMCs were stained with PE-coupled peptide-loaded HLA class I tetramers for 30 min at room temperature followed by incubation with magnetic anti-PE microbeads. Subsequent positive selection of magnetically labelled cells was achieved by using MACS technology (Miltenyi Biotec) according to the manufacturer’s protocol. The enriched spike-specific CD8+ T cells were analysed using multicolor flow cytometry. Cell frequencies were calculated as previously described³. Of note, only samples with ≥ 5 virus-specific CD8+ T cells were included in subsequent analyses. Accordingly, the detection limit of virus-specific CD8+ T cells in this study was: 0.25 – 1 x 10^-6, depending on the initial cell input.

Multiparametric flow cytometry for T cell analysis

The following antibodies were used for multiparametric flow cytometry: anti-CCR7-PE-CF594 (150503, 1:50), anti-CCR7-BV786 (G034H7, 1:50), anti-CD4-BV786 (L200, 1:200), anti-CD8-BUV395 (RPA-T8, 1:400), anti-CD8-BV510 (SK1, 1:100), anti-CD8-BV421 (RPA-T8, 1:200), anti-CD11a-BV510 (HI111, 1:25), anti-CD28-BV421 (CD28.2, 1:100), anti-CD38-APC-R700 (HIT2, 1:400), anti-CD39-BV650 (TU66, 33:1), anti-CD45RA-BUV496 (HI100, 1:800), anti-CD45RA-BUV737 (HI100, 1:800), anti-CD107a-APC (H4A3, 1:100), anti-CD127-BUV737 (HIL-7R-M21, 1:50), anti-EOMES-PerCP-eF710 (WD1928, 1:10), anti-Granzyme B-PE-CF594 (GB11, 1:800), anti-Granzyme B-BV510 (GB11, 1:50), anti-IFN-γ-FITC (25723.11, 1:8), anti-PD-1-BV605 (EH12.1, 1:50), anti-PD-1-PE-Cy7 (EH12.2H7, 1:200), anti-TNF-PE-Cy7 (Mab11, 1:400), anti-HLA-DR-BUV395 (G46-6, 1:200), anti-CD137-BV650 (4B4-1, 1:100), anti-CD95-APC (DX2, 1:100) (BD Biosciences), anti-BCL-2-BV421 (100, 1:200), anti-CCR7-BV785 (G043H7, 1:50), anti-CD57-BV605 (QA17A04, 1:200), anti-CXCR3-PerCP-Cy5.5 (G025H7, 1:33), anti-IL-2-PerCP-Cy5.5 (MQ1-17H12, 1:100), anti-Ki-67-BV711 (CD28.2, 1:200), anti-Perforin-PerCP-Cy5.5 (B-D48, 1:200), anti-Granzyme K-PE-Cy7 (GM26E7, 1:25), anti-Granulysin-APC (DH2, 1:100) (BioLegend), anti-TCF-1-AlexaFluor488 (C63D9, 1:100) (Cell Signaling), anti-CD14-APC-eFluor780 (61D3, 1:400), anti-CD19-APC-eFluor780 (HIB19, 1:400), anti-CD27-FITC (O323, 1:100), anti-KLRG1-BV711 (13F12F2, 1:50), anti-T-BET-PE-Cy7 (4B10, 1:200), anti-TOX-eFluor660 (TRX10, 1:100) (Thermo Fisher). To discriminate live cells from dead cells, fixable Viability Dye (APC-eFluor780, 1:400) from Thermo Fisher or 7-AAD from BD Biosciences (ViaProbe, 1:33) was used. To facilitate staining of intranuclear and cytoplasmic targets, FoxP3/Transcription Factor Staining Buffer Set (Thermo Fisher) and Fixation/Permeabilization Solution Kit (BD Biosciences) were used, respectively. Finally, cells were fixed in 2% paraformaldehyde (Sigma) and samples were analysed on FACSCanto II or LSRFortessa with FACSDiva software version 10.6.2 (BD), or CytoFLEX (Beckman Coulter) with CytExpert Software version 2.3.0.84. Further analyses of the data were performed using FlowJo version 10.6.2 (Treestar).

Dimensional reduction of multiparametric flow cytometry data

For dimensionality reduction, flow cytometry data was analysed with R version 4.1.1 and the Bioconductor CATALYST package (release 3.13)⁴. Initially, viable and tetramer-positive CD8+ T cells (or subsets of those) were identified using FlowJo 10 in two separate multiparametric flow cytometry panels (activation panel: HLA-DR, BCL-2, PD-1, CD137, Ki67, TCF-1, EOMES, T-BET, TOX, CD38, CD45RA, CCR7; differentiation panel: CD45RA, CCR7, CD27, CD28, CD127, CD11a, CD57, CXCR3, CD95, CD57, CD39, KLRG1, PD-1). To facilitate visualization of the dimensionality reduction by t-SNE and diffusion map analysis, cell counts were sampled down to at least 20 cells per sample, and marker expression intensities were transformed by arcsinh-transformation with a cofactor of 150.

Serum IgG determination

Detection of anti-SARS-CoV-2 spike-IgG was performed using Anti-SARS-CoV-2-QuantiVac-ELISA (IgG) from Euroimmun according to the manufacturer’s instructions (anti-SARS-CoV-2 S IgG; <35.2 BAU/mL: negative, ≥ 35.2 BAU/mL: positive).

Neutralization assay

Samples of vaccinated individuals and those with breakthrough infections were tested in a plaque reduction neutralization assay as previously described⁵. In brief, VeroE6 cells were seeded in 12-well plates at a density of 4 × 10⁵ cells per well. Serum samples were diluted at ratios of 1:16, 1:32, 1:64, 1:128, 1:256, 1:512 and 1:1024 in a total volume of 50 μl PBS. For each sample, a serum-free negative control was included. Diluted sera and negative controls were subsequently mixed with 90 plaque-forming units (PFU) of authentic SARS-CoV-2 (either B.1, B.1.617.2 (delta) and B.1.1.529 BA.1 (omicron)) in 50 μl PBS (1,600 PFU/mL) resulting in final sera dilution ratios of 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024 and 1:2048. After incubation at room temperature for 1h, 400 μl PBS was added to each sample and the mixture was subsequently used to infect VeroE6 cells 24 h after seeding. After 1.5 h of incubation at room temperature, inoculum was removed and the cells were overlaid with 0.6% Oxoid-agar in DMEM, 20 mM HEPES (pH 7.4), 0.1% NaHCO3, 1% BSA and 0.01% DEAE-Dextran. Cells were fixed 72 h after infection using 4% formaldehyde for 30 min and stained with 1% crystal violet upon removal of the agar overlay. PFU were counted manually. Plaques counted for serum-treated wells were compared to the average number of plaques in the untreated negative controls, which were set to 100%. Calculation of PRNT50 values was performed using a linear regression model in GraphPad Prism 9 (GraphPad Prism Software).

Statistics

GraphPad Prism software version 9.3.1 was used for statistical analysis. Statistical significance was assessed by Kruskal-Wallis test, one-way ANOVA with mixed-effects model, two-way ANOVA with full model and main model. Statistical analysis was performed for A*01/S₈₆₅ (n=7) and A*02/S₂₆₉ (n=8) longitudinally analysed CD8+ T cell responses in Fig. 1A, 1B, 2C, 3A, 3B and Ext. Data. Fig 1C, 5C, 5D, 5E, for n=28 subjects longitudinally followed in Fig. 1D, for A*01/S₈₆₅ (n=2) and A*02/S₂₆₉ (n=3) T cell responses longitudinally followed in Fig. 1E, for n=26 subjects in Ext. Data. Fig 1G, for n=2 subjects in Ext. Data. Fig 2C, for n=7 at 3 months after 2^nd vaccination, n=11 at 9 months after 2^nd vaccination and n=11 at 3 months after 3^rd vaccination in Fig. 2A and Ext. Data. Fig. 3B, for n=4 at 3 months after 2^nd vaccination, n=8 at 9 months after 2^nd vaccination and n=10 at 3 months after 3^rd vaccination in Ext. Data. Fig. 3D, for A*01/S₈₆₅ (n=7) and A*02/S₂₆₉ (n=6) longitudinally analysed CD8+ T cell responses in Fig. 2D, for n=8 at 3 months after 2^nd vaccination, n=12 at 9 months after 2^nd vaccination and n=11 at 3 months after 3^rd vaccination in Fig. 2B, for n=4 in Ext. Data. Fig. 4A, for A*01/S₈₆₅ (n=2) and A*02/S₂₆₉(n=2) longitudinally analysed CD8+ T cell responses in Ext. Data. Fig. 4B, for n=10 at 3 months after 2^nd vaccination, n=12 at 9 months after 2^nd vaccination and n=11 at 3 months after 3^rd vaccination in Fig. 3C, for n=10 at 3 months after 2^nd vaccination, n=11 at 9 months after 2^nd vaccination and n=11 at 3 months after 3^rd vaccination in Fig. 3D, for n=6 at 3 months after 2^nd vaccination, n=12 at 9 months after 2^nd vaccination and n=10 at 3 months after 3^rd vaccination in Fig. 3E, for n=6 at 3 months after 2^nd vaccination, n=12 at 9 months after 2^nd vaccination and n=11 at 3 months after 3^rd vaccination in Fig. 3F, for omicron infection n=12, delta infection n=2 and 4^th vaccination n=5 longitudinally analysed T-cell responses in Fig. 4A, for omicron infection n=11, delta infection n=2 and 4^th vaccination n=4 analysed T cell responses in Fig. 4B and in peak response in Ext. Data. Fig. 6A, for omicron infection n=12, delta infection n=2 and 4^th vaccination n=3 longitudinally analysed T cell responses in Fig. 4E, for omicron infection n=11, delta infection n=1 and 4^th vaccination n=3 in Fig. 4F, for omicron infection n=6, delta infection n=2 and 4^th vaccination n=4 analysed T cell responses after 1 month in Ext. Data. Fig. 6A and Ext. Data Fig. 6E, for omicron infection n=6, delta infection n=2 and 4^th vaccination n=2 analysed T cell responses in Ext. Data. Fig. 6D.

Data availability statement

Source data files will be provided with this manuscript. All requests for additional raw and analyzed data and materials are promptly reviewed by the University of Freiburg Center for Technology Transfer to verify if the request is subject to any intellectual property or confidentiality obligations. Patient-related data not included in the paper were generated as part of clinical examination and may be subject to patient confidentiality. Any data and materials that can be shared will be released via a Material Transfer Agreement.

Code availability statement

R code to reproduce the analyses of multiparametric flow-cytometry data is available at https://github.com/sagar161286/SARSCoV2_specific_CD8_Tcells.

Online references

1 World Health Organization (WHO). Living guidance for clinical management of COVID-19. 23 November 2021

2 Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7, 539, doi:10.1038/msb.2011.75 (2011).

3 Alanio, C., Lemaitre, F., Law, H. K., Hasan, M. & Albert, M. L. Enumeration of human antigen-specific naive CD8+ T cells reveals conserved precursor frequencies. Blood 115, 3718-3725, doi:10.1182/blood-2009-10-251124 (2010).

4 Crowell, H., Zanotelli, V., Chevrier, S. & Robinson, M. CATALYST: Cytometry dATa anALYSis Tools. R package version 1.16.2. (2021).

5 Oberhardt, V. et al. Rapid and stable mobilization of CD8+ T cells by SARS-CoV-2 mRNA vaccine. Nature 597, 268-273, doi:10.1038/s41586-021-03841-4 (2021).

There is NO Competing Interest.

Patientscharacteristics.pdf
Supplementary Table 1
Slide5.jpg
Extended Figure 1
Slide6.jpg
Extended Figure 2
Slide7.jpg
Extended Figure 3
Slide8.jpg
Extended Figure 4
Slide9.jpg
Extended Figure 5
Slide10.jpg
Extended Figure 6

Download PDF

Journal Publication

published 08 Aug, 2022

Read the published version in Nature Communications →

Version 1

posted

You are reading this latest preprint version

Short-lived booster effect and stable CD8+ T cell memory after 3rd COVID-19 vaccine dose

Status:

Journal Publication

Version 1

Abstract

Figures

introduction

Results

Discussion

References

Declarations

online methods

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1