key: cord-0849026-5ld9m2om authors: Chivu‐Economescu, Mihaela; Bleotu, Coralia; Grancea, Camelia; Chiriac, Daniela; Botezatu, Anca; Iancu, Iulia V.; Pitica, Ioana; Necula, Laura G.; Neagu, Ana; Matei, Lilia; Dragu, Denisa; Sultana, Camelia; Radu, Elena L.; Nastasie, Alina; Voicu, Oana; Ataman, Marius; Nedeianu, Saviana; Mambet, Cristina; Diaconu, Carmen C.; Ruta, Simona Maria title: Kinetics and persistence of cellular and humoral immune responses to SARS‐CoV‐2 vaccine in healthcare workers with or without prior COVID‐19 date: 2022-01-18 journal: J Cell Mol Med DOI: 10.1111/jcmm.17186 sha: c41858eb50fc2a11ccdd5953f51c1e3ec9bed400 doc_id: 849026 cord_uid: 5ld9m2om SARS‐CoV‐2 vaccines are highly efficient against severe forms of the disease, hospitalization and death. Nevertheless, insufficient protection against several circulating viral variants might suggest waning immunity and the need for an additional vaccine dose. We conducted a longitudinal study on the kinetics and persistence of immune responses in healthcare workers vaccinated with two doses of BNT162b2 mRNA vaccine with or without prior SARS‐CoV‐2 infection. No new infections were diagnosed during follow‐up. At 6 months, post‐vaccination or post‐infection, despite a downward trend in the level of anti‐S IgG antibodies, the neutralizing activity does not decrease significantly, remaining higher than 75% (85.14% for subjects with natural infection, 88.82% for vaccinated after prior infection and 78.37% for vaccinated only). In a live‐virus neutralization assay, the highest neutralization titres were present at baseline and at 6 months follow‐up in persons vaccinated after prior infection. Anti‐S IgA levels showed a significant descending trend in vaccinated subjects (p < 0.05) after 14 weeks. Cellular immune responses are present even in vaccinated participants with declining antibody levels (index ratio 1.1–3) or low neutralizing activity (30%–40%) at 6 months, although with lower T‐cell stimulation index (p = 0.046) and IFN‐γ secretion (p = 0.0007) compared to those with preserved humoral responses. Severe acute respiratory syndrome coronavirus-2 (SARS- spread rapidly around the globe infecting by the end of November 2021 over 250 million individuals and causing over 5 million deaths. In December 2020, the first vaccines became available, and until the end of November 2021, more than 8 billion doses were administered worldwide, with about 55% of the world population currently immunized. 1 Immunity gained from natural infection or vaccination provides a significant degree of protection against both reinfection and progression towards a severe form of COVID-19 requiring hospitalization. It is currently estimated that 89% of the individuals recovered after a natural infection are protected for at least 8 months, 2 while vaccinated people have a reduction of 50%-95% in their risk of infection, 3 depending on the type of vaccine, the interval between doses, time elapsed from the completion of the vaccination regimen, age, immuno-competence, prior infection with SARS-CoV-2 and the circulating viral variants. During the clinical trials, mRNA-based vaccines have demonstrated the highest efficacy against symptomatic COVID-19: 95% for BNT162b2 (Pfizer/BioNTech) 4 for mRNA-1273 (Moderna), 5 followed by vector-based vaccines: 92% efficacy against symptomatic infection for Gam-COVID-Vac (Gamaleya), 6 70% for ChAdOx1-nCoV19 (AstraZeneca) 7 and 67% for Ad26.COV2.S (Janssen). 8 After introducing large-scale vaccination, numerous independent studies confirmed the real-world effectiveness of vaccination, with results similar to the clinical trials during the dominance of Alpha variant and a slight decrease, especially against symptomatic infection, after emergence and global spread of Delta variant. Currently, there is a debate on the necessity of additional vaccine doses, with supporters citing waning immunity and decreased efficacy against viral variants and opponents highlighting incomplete scientific evidence, differences according to the individual immuno-competence status, high risk of exposure, as well as ethical reasons related to inequalities in vaccine access. In August 2021, the US CDC published a report showing that mRNA vaccine protection against infection has declined from 91.7% in May to 79.8% in July, while vaccine effectiveness against hospitalization remained relatively stable (91.9%-95.3%). 9 Another CDC study showed a more pronounced decline of mRNA vaccines efficiency against infection among nursing home residents (from 74.7% in March 2021 to 53.1% in July 2021, when B.1.617.2 (Delta) variant became dominant). 10 In the general population, the number of severe SARS-CoV-2 infections after vaccination remains low, with more than 40% of hospitalized breakthrough cases registered in immunocompromised persons, 11, 12 who are at risk for prolonged SARS-CoV-2 shedding and viral evolution. Preliminary unpublished data from Israel suggest a major drop down in vaccine effectiveness against SARS-CoV-2 infection (39%), especially in those vaccinated in the early months of 2021, with protection retained against hospitalization (81%). 13 Similar data were reported in a test negative, case-control study in Qatar with significantly diminished levels of protection against symptomatic and asymptomatic infection 20 weeks after the second dose of BNT162b2 vaccine, but maintenance of protection against severe, critical and fatal forms of (9/21) in those with prior SARS-CoV-2 infection. There were no significant age or comorbidities differences between the two groups (p > 0.05). Based on these data, we divided the participants as follows: 10 ml of venous blood was collected on EDTAK3, centrifuged at 1500 g for 15 min to separate plasma that was stored at −80°C for downstream antibody analysis. For cellular immunity analysis, peripheral blood mononuclear cells (PBMC) were separated onto Ficoll-Paque PLUS density gradient media (1.077 g/ml), washed with RPMI and treated with ACK erythrocyte lysis buffer (Thermo Fisher Scientific) for 5 min. Cells were washed again and used in ex vivo stimulation experiments. Anti-S IgG and IgA isotypes and anti-NCP IgM antibodies were de- In order to assess the antibodies neutralizing capacity, a commercial SARS-CoV-2 surrogate virus neutralization test (sVNT) was used (GenScript cPass™ SARS-CoV-2 Neutralization Antibody Detection Kit, Genscript). This functional assay is based on antibody-mediated blockade of the interaction between ACE2 receptor and the receptor-binding domain (RBD) of the spike protein. The method was performed as previously described. 17 Briefly, plasma samples, positive and negative controls were diluted 1:10 and mixed with an equal volume of enzyme-conjugated RBD, incubated at 37°C for 30 min and transferred to a plate coated with recombinant human ACE2 for 15 min. After plate washing, the enzyme substrate (100 µl tetramethylbenzidine) was added for 15 min at room temperature, and the optical density (OD) was assessed at 450 nm. Each sample's neutralizing capacity was calculated using the following formula: % inhibition = (1 − (OD450 of sample/Average OD450 of negative controls) × 100. A value of >30% inhibition is considered positive for neutralizing activity. The virus was isolated from 500 µl of nasopharyngeal swab incubated on a confluent monolayer of Vero E6 cells for 1 h at 37°C fol- In vitro neutralizing assay was performed using a viral strain isolated from a patient diagnosed with SARS-CoV-2 infection and F I G U R E 1 Schematic representation of the groups investigated for the longitudinal immune response confirmed as an Alpha variant (B.1.408 betacoronavirus hCoV-19/ Romania/IIsolate3247BLVL/2020) by virus whole-genome sequencing using MiSeq NGS system (Illumina) (GSAID accession ID: EPI_ISL_1081959). Patients' plasma samples were decomplemented by heat inactivation (56°C for 1 h), subjected to serial twofold dilution (1:2-1:512) in microtitre plates and incubated with 100 median tissue culture infectious dose (TCID) 50 of SARS-CoV-2 in a 1:1 ratio for 1 h at 37°C. 100 µl antibody-virus mixture was inoculated for 1 h at 37°C, on VERO E6 cells, seeded in 96-well plates at a concentration of 7500 cells per well the day before. The inoculum was removed, and a maintenance medium with 2% FBS was added. The cytopathic effect (CPE) was quantified using IncuCyte Live Cell Imaging System 5 days after inoculation. Neutralizing antibody titre was determined by identifying the highest plasma dilution without observable CPE and expressed as a geometric mean titre (GMT). 18 supplemented with 10% human serum and subsequently stimulated with 1 µg/ml PepTivator. For the positive and negative control, PBMCs were stimulated with 2.5 µg/ml Phytohemagglutinin-L (Invitrogen, Thermo Scientific) or 2 µl of sterile water with 10% DMSO, respectively. Cells were incubated for 20 h at 37°C, 5% CO 2 . After specific stimulation, the cells were labelled with specific antibodies for flow cytometric analysis of lymphocytes and supernatants were harvested for interferon gamma (IFNγ) release detection. Interferon-gamma release assay was used to determine the magnitude of the SARS-CoV-2-specific T-cell response. Supernatants from 1 × 10 6 PBMC stimulated with PepTivator SARS-CoV-2 Prot_S (Miltenyi Biotec) for 20 h, were analysed for IFNγ concentrations by ELISA, according to manufacturer's instructions (R&D Systems). Data were analysed using GraphPad Prism 5 software. Line plots present means with a 95% confidence interval. Antibody responses were compared between groups using 1-way ANOVA Tukey's multiple comparison test. For correlations between two groups, Pearson r correlation coefficient was used. All tests were performed with a nominal significance threshold of p < 0.05. *p < 0.05, **p < 0.01 and ***p < 0.001. We Figure 3C ). Thus, titres ≤4 correspond to an average neutralization capacity of 43.15% ± 5.16%, titres of 8 corresponds to 66.46% ± 10.13%, titres of 16 corresponds to 84.50% ± 5.79%, titres of 32-64 correspond to 90.27% ± 5.08% and titres ≥128 to neutralization capacities of over 94.38% ± 0.67%. A subset of patients, representing 35.48% (11/31) of V group, 11 .11% (1/9) of I + V and 33.33% (4/12) of I group had more (Table 1) . A comparison between the three groups showed that subjects from the I + V category are better IFNγ secretors, a fact correlated with a superior T-cell stimulation index ( Figure 6 ). The key findings of our study on the kinetics of the specific SARS- sponse. 31 The persistence of virus-specific memory B cells has been demonstrated at more than 240 days of COVID-19 symptom onset, even though plasma antibody levels were declining. 32, 33 Additionally, in our study, an important decrease in the anti-S IgG titres seems to be compensated by higher neutralizing capacity and a robust cellular immune response, demonstrated by a high level of IFNγ synthesis by stimulated T cells. Whole-blood cytokine release assays after stimulation with SARS-CoV-2-specific peptides have been already proposed as an additional diagnostic tool. 34 Our study also brings essential information regarding SARS-CoV-2-specific T-cell responses. It is observed that in good responders the CD4 + T-cell index is correlated with a higher secretion of IFNγ. This IFNγ secretion mainly by TCD4 + cells seems to be specific to SARS- 38, 39 For unvaccinated individuals with natural infection, it seems that disease severity may contribute to the heterogeneity of cellular immune response, as those asymptomatic or mildly symptomatic had In conclusion, our study shows the persistence of neutralizing antibodies at 6 months after vaccination with an mRNA vaccine or after SARS-CoV-2 prior infection, with individual variability associated with specific antibodies' level and dynamics. Although there is a downward trend in the titre of IgG and especially in IgA anti-S antibodies, the neutralizing capacity is maintained, and effective cellular immune responses are mounted even in low responders. Nonetheless, there is considerable heterogeneity in both humoral and cellular immunity. Moreover, the decline in antibodies titre did not translate into reduced protection against symptomatic or asymptomatic disease. The strongest cellular immune response was detected in subjects that were vaccinated after prior natural infection, but there were also subjects with good cellular immune response in vaccinated and individuals recovered after natural infection. We have to acknowledge the limitations of our study due to the rather small number and homogeneous age of the enrolled subjects The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Data curation (equal); Formal analysis (equal); Investigation (lead) Writing -original draft (lead) Writing -review & editing (equal). Coralia Bleotu: Conceptualization (equal) Data curation (equal); Formal analysis (equal) Investigation (equal) Writing -review & editing (equal). Camelia Grancea: Data curation (equal) Investigation (equal) Writing -review & editing (equal) Data curation (equal); Formal analysis (equal) Investigation (equal) Anca Botezatu: Data curation (equal) Investigation (equal) Writing -review & editing (equal). Iulia V. Iancu: Data curation (equal) Investigation (equal) Ioana Pitica: Data curation (equal) Investigation (equal) Laura Georgiana Necula: Data curation (equal) Investigation (equal) Writing -review & editing (equal). Ana I. Neagu: Data curation (equal); Formal analysis (equal) Lilia Matei: Data curation (equal); Formal analysis (equal) Denisa Dragu: Data curation (equal); Formal analysis (equal) Writing -review & editing (equal) Camelia Sultana: Data curation (equal); Formal analysis (equal) Investigation (equal) Writing -review & editing (equal). Elena L. 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The data that support the findings of this study are available from the corresponding author upon reasonable request.