{'fraction_non_alphanumeric': 0.04453550077248606, 'fraction_numerical': 0.0327802780949822, 'mean_word_length': 4.686783804430863, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 2, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Differential effects of the second SARS-CoV-2 mRNA vaccine dose on T cell immunity in naive and COVID-19 recovered individuals

The rapid development of mRNA-based vaccines against the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) led to the design of accelerated vaccination schedules that have been extremely effective in naive individuals. While a two-dose immunization regimen with the BNT162b2 vaccine has been demonstrated to provide a 95% efficacy in naive individuals, the effects of the second vaccine dose in individuals who have previously recovered from natural SARS-CoV-2 infection has not been investigated in detail. In this study, we characterize SARS-CoV-2 spike-specific humoral and cellular immunity in naive and previously infected individuals during and after two doses of BNT162b2 vaccination. Our results demonstrate that, while the second dose increases both the humoral and cellular immunity in naive individuals, COVID-19 recovered individuals reach their peak of immunity after the first dose. These results suggests that a second dose, according to the current standard regimen of vaccination, may be not necessary in individuals previously infected with SARS-CoV-2.


In brief
Scarce SARS-CoV-2 vaccine supplies are influencing vaccination policies in some countries. Lozano-Ojalvo et al. report that subjects with previous exposure to SARS-CoV-2 mount powerful immune responses after the first BNT162b2 vaccine dose, suggesting single vaccination regimens in COVID-19 recovered individuals.

INTRODUCTION
The BNT162b2 mRNA coronavirus disease 2019  vaccine was authorized for emergency use by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) in December 2020 (Krammer, 2020). The vaccination strategy for the BNT162b2 vaccine involves an accelerated two-dose vaccination regimen administered 21 days apart, which has been demonstrated to induce a spike-specific humoral and cellular immunity associated with a 95% efficacy in naive individuals (Polack et al., 2020). However, in individuals with prior exposure to SARS-CoV-2, the utility of the second dose has been challenged. While robust spike-specific antibodies and T cells are induced by a single dose vaccination in SARS-CoV-2 seropositive individuals (Krammer et al., 2021;Prendecki et al., 2021), the second vaccination dose does not appear to further increase the spike-specific humoral response in COVID-19 recovered individuals (Samanovic et al., 2021) .
The effects of the second dose of mRNA vaccine on the spikespecific T cell response has just started to be investigated in both naive and in SARS-CoV-2 pre-exposed individuals. Understanding the spike-specific T cell response is critical, as protection from disease severity and infection are likely to be dependent on the coordinated activation of both the humoral and cellular arms of adaptive immunity (McMahan et al., 2021). However, the complexity of monitoring the T cell magnitude and function has so far prevented the measurement of the immune cellular parameters in a robust number of vaccinated individuals during the full vaccination schedule. To address this problem, we investigated spike-SARS-CoV-2 antigen-specific T cell responses in naive and COVID-19 recovered individuals during full BNT162b2 mRNA vaccination.

RESULTS
We first investigated the spike-specific T cell response by a spectral flow cytometric analysis using peripheral blood mononuclear cells (PBMCs) stimulated in vitro with DMSO or SARS-CoV-2 spike peptide pool (Le  in naive and COVID-19 recovered individuals before and after vaccination (20 days after the second dose). We observed that spike-specific (CD154 + ) memory (CD45RA À CCR7 À ) CD4 + T cells producing both interferon (IFN)-g and interleukin (IL)-2 were present in COVID-19 recovered individuals, as well as in naive subjects 20 days after vaccination (Figures S1A and S1B). Functional phenotyping of these cells revealed that spike-specific IFN-g-and IL-2-secreting CD4 + T cells are mainly represented by T helper (Th)1 (CCR6 À CCR4 À CXCR3 + ) cells ( Figure S1C). We next analyzed the dynamic changes of functional spikespecific T cell response by measuring the quantity of IFN-g and IL-2 present in whole blood stimulated with a spike peptide pool (Le . In contrast to more complex and detailed flow cytometry-based analyses that require larger amounts of blood, this approach requires only 1 mL of blood, which facilitates multiple longitudinal tests in a single individual and reliable quantified SARS-CoV-2-specific T cells in SARS-CoV-2-infected individuals (Le  and vaccine recipients (Kalimuddin et al., 2021). Figure 1 shows the results obtained in 92 individuals with and without previous documented SARS-CoV-2 infection (47 naive; mean age 39.9 years [range, 20-62]; female 78%) and in 45 PCR/antigen test-confirmed SARS-CoV-2 recovered individuals (39% at 6-9 months after infection, 26% at 3-6 months, 35% at 1-3 months) (mean age 44.3 years [range, 20-76]; female 76%) (Table 1), tested at multiple time points (i.e., prior to and 10 and 20 days after the first and second dose of vaccine).
Prior to vaccination, the spike peptide pool induced in whole blood a higher IFN-g production in COVID-19 recovered individuals than in naive subjects (median pre-vaccination in naive: 1.0 pg/mL [n = 20] and in COVID-19 recovered: 46.9 pg/mL [n = 21]) ( Figure 1A). We observed a similar trend in spike-specific IL-2 production (median pre-vaccination in naive: 0.5 pg/mL [n = 20] and COVID-19 recovered: 56.9 pg/mL [n = 21]) ( Figure 1B; Table 1). Evaluation of the spike-specific T cell response 10 days after the first dose indicates that COVID-19 recovered individuals mount a stronger IFN-g and IL-2 response in comparison with naive subjects (median day 10 after first vaccine dose in naive: 109.5 pg/mL [n = 20] and in COVID-19 recovered: 547.8 pg/mL [n = 21] for IFN-g; naive: 15.85 pg/mL [n = 16] and COVID-19 recovered: 76.9 pg/mL [n = 21] for IL-2) ( Figures 1A  and 1B). COVID-19 recovered individuals maintain their T cell immunity on day 20 after the first vaccine dose, while IFN-g response in naive individuals decreases, although it is not statistically significant (median day 20 after first vaccine dose in naive: 52.9 pg/mL [n = 23] and in COVID-19 recovered: 351.3 pg/mL [n = 21]). A similar trend in spike-specific IL-2 production was observed, although no decrease was seen in naive individuals (median day 20 after first vaccine dose in naive: 127.0 pg/mL [n = 22] and in COVID-19 recovered: 223.5 pg/mL [n = 20]). These results indicate that individuals with pre-existing immunity exert a more potent and sustained T cell response to SARS-CoV-2 spike after the first dose of the vaccine, consistent with recent investigations (Tauzin et al., 2021;Painter et al., 2021).
We next studied the effects of the second dose of the vaccine. Sampling on day 10 after the second dose confirmed the beneficial effects of the recall vaccine in naive individuals who increase their IFN-g and IL-2 (median day 10 after the second vaccine dose in naive: 211.8 pg/mL [n = 20] for IFN-g and 187.0 pg/ mL [n = 16] for IL-2). On the contrary, COVID-19 recovered individuals did not increase their IFN-g or IL-2 production on day 10 after the second vaccine dose (median day 10 after the second vaccine dose in COVID-19 recovered: 212.0 pg/mL [n = 21] for IFN-g and 148.5 pg/mL [n = 18] for IL-2). These findings indicate that, while naive subjects significantly increase their immunity against SARS-CoV-2 spike protein after the second dose of the vaccine, COVID-19 recovered individuals do not seem to benefit from the standard regimen for COVID-19 vaccination. Interestingly, we observed that, while IFN-g secretion in COVID-19 recovered individuals decreases on day 20 after the second dose, IL-2 levels are not decreased (median day 20 after the first vaccine dose in naive: 87.0 pg/mL [n = 23] and in COVID-19 recovered: 136.1 pg/mL [n = 23] for IFN-g; naive: 121.5 pg/mL [n = 24] and COVID-19 recovered: 86.4 pg/mL [n = 23] for IL-2), which suggests that IL-2 secretion by spike-specific Th1 cells might represent a better biomarker to measure T cell immunity in vaccinated individuals (Figures 1A and 1B; Table 1).
Our analysis of the humoral response depicts a similar trend when measuring SARS-CoV-2 spike-specific immunoglobulin (Ig)G levels, which indicates that naive individuals achieve significant concentrations of IgG antibodies after the second vaccine dose ( Figure 1C). This suggests that protection is achieved following the standard two-dose regimen for COVID-19 vaccination in naive individuals, consistent with a recent report (Ebinger et al., 2021). On the contrary, COVID-19 recovered individuals reach the peak of the response on day 10 after the first dose, which is consistent with the cellular response shown in Figures 1A and 1B. We also observed a better correlation between IL-2 and IgG in comparison to IFN-g and IgG ( Figure S2), which supports the potential use of IL-2 as biomarker for cellular immunity.  (pre-vaccination [pre], 10 and 20 days after the first vaccine dose [10 and 20, respectively], and 10 and 20 days after the second vaccine dose [30 and 40, respectively]) after overnight stimulation of whole blood with SARS-CoV-2 peptide pools. IFN-g and IL-2 concentration levels were determined using ELLA single plex cartridges (n = 92 individuals; 47 naive and 45 COVID-19 recovered). (C) Kinetics of SARS-CoV-2 spike-specific IgG serum levels in naive and COVID-19 recovered individuals measured by ACCESS SARS-CoV-2 CLIA. For IgG, IFN-g, and IL-2 determination, the group means method was used to compare pre-treatment versus post-treatment time points and also among posttreatment time points. All individual time points were measured using technical triplicates. A two-sample t test assuming unpaired populations for pre-and posttreatment was performed for each of the markers, separately for the COVID-19 recovered and naive cohorts. The p value for the test statistic was set to the 0.05 level of significance, and the Benjamini-Hochberg (BH) method was used for multiple testing correction.

DISCUSSION
From our studies of the humoral and cellular spike-specific immunity in recipients of the BNT162b2 mRNA COVID-19 vaccine, we conclude that a single dose of the vaccine elicits the generation of specific antibodies and a T cell immune response in naive and COVID-19 recovered individuals, consistent with recently published data (Prendecki et al., 2021;Tauzin et al., 2021). In naive subjects, the T cell response is boosted in conjunction with spike-specific IgG levels after the second dose. On the contrary, the second vaccine dose not increase the spike-specific T cell response in individuals with a pre-existing immunity against SARS-CoV-2 and further evidences that a single-dose vaccine is sufficient to induce protective immunity in Covid-19 recovered individuals (Reynolds et al., 2021).
Overall, our data support the vaccination scheme determined in clinical trials with prompt administration of the second dose in individuals without previous SARS-CoV-2 exposure (Kadire et al., 2021), but they suggest that in individuals with pre-existing immunity against SARS-CoV-2 the second dose of the vaccine may not be necessary. Longitudinal evaluation of the humoral and cellular immunity, especially in naive individuals with a reduced response to mRNA SARS-CoV-2 BNT162b2 (Grupper et al., 2021), may also justify the need for a third vaccination dose to boost the immune response in immunosuppressed individuals (Kamar et al., 2021). At present, there is a shortage of doses to vaccinate a large proportion of the population, and public health authorities are designing priority vaccination strategies according to age, comorbidities, and risk assessment criteria. The present data support a single vaccine dose in COVID-19 recovered individuals.

DECLARATION OF INTERESTS
A.B. declares the filing of a patent application relating to the use of peptide pools in whole blood for detection of SARS-CoV-2 T cells (pending). The remaining authors declare no competing interests.

INCLUSION AND DIVERSITY
One or more of the authors of this paper self-identifies as a member of the LGBTQ+ community.

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Jordi Ochando (jordi.ochando@mssm.edu).

Materials availability
Further information and requests for resources and reagents should be directed to Jordi Ochando (jordi.ochando@mssm.edu). This study did not generate new unique reagents.
Data and code availability d All data reported in this paper will be shared by the lead contact upon request. d This paper does not report original code. d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS
Peripheral blood samples were obtained from healthy naive and COVID-19 recovered donors. Peripheral blood samples for humoral and cellular immunity antibody isolation were obtained from male and female donors, and the information of individuals are in Table 1. Age of these patients are undisclosed due to privacy policy. The study protocols for the collection of clinical specimens from individuals with and without SARS-CoV-2 infection were reviewed and approved by Hospital La Paz, Hospital 12 de Octubre, Hospital Gregorio Marañ ó n, IIS-Fundació n Jimenez Díaz, Hospital Universitario Marqué s de Valdecilla-IDIVAL and Hospital Puerta de Hierro Clinical Research Ethics Committee (CEIm), and Mount Sinai Hospital Institutional Review Board (IRB). All the study participants provided their written informed consent for the collection of the samples and their subsequent analysis.

METHOD DETAILS
SARS-CoV-2 Peptide pools SARS-CoV-2 peptide pools of 15-mers (55 peptides) covering 40.5% of the spike (S) protein contain most of the SARS-CoV2 spike epitope published to date (Grifoni et al., 2020) or pools of Prot S/S1/S+ from Miltenyi, were used as reported in (Le . Whole-blood culture with SARS-CoV-2 peptide pools 320 mL of whole blood drawn on the same day were mixed with 80 mL RPMI and stimulated with pools of SARS-CoV-2 peptides (S; 2 mg/ml) or DMSO control. After 15 hours of culture, the supernatant (plasma) was collected and stored at À80 C until quantification of cytokines.

Cytokine quantification and analysis
Cytokine concentrations in the plasma were quantified using Ella with microfluidic multiplex cartridges measuring IFN-g and IL-2 following the manufacturer's instructions (ProteinSimple, San Jose, California). The level of cytokines present in the plasma of DMSO controls was subtracted from the corresponding peptide pool stimulated samples.