key: cord-0257833-vprne05p authors: Komissarov, A.; Dolzhikova, I.; Efimov, G.; Logunov, D.; Mityaeva, O.; Molodtsov, I.; Naigovzina, N.; Peshkova, I.; Shcheblyakov, D.; Volchkov, P.; Vasilieva, E. title: Boosting of the SARS-CoV-2-specific immune response after vaccination with single-dose Sputnik Light vaccine date: 2021-10-27 journal: nan DOI: 10.1101/2021.10.26.21265531 sha: 0fb233bac8f038d26233329f361872b8ec4991b4 doc_id: 257833 cord_uid: vprne05p Despite the measures taken worldwide, COVID-19 pandemic still progresses. While efficient antiviral drugs are not yet widely available, vaccination is the best option to control the infection rate. Although this option is obvious in case of COVID-19-naive individuals, it is still unclear when individuals who have recovered from a previous SARS-CoV-2 infection should be vaccinated and whether the vaccination raises immune responses against the coronavirus and its novel variants. Here we measured the dynamics of the antibody and T-cell responses, as well as virus neutralizing activity (VNA) in serum against two SARS-CoV-2 variants, B.1.1.1 and B.1.617.2, among 84 individuals with different COVID-19 status who were vaccinated with Sputnik Light vaccine. We showed that vaccination of individuals previously exposed to the virus considerably boosts the existing immune response. In these individuals, RBD-specific IgG titers and VNA in serum were already elevated on the 7th day after vaccination, while COVID-19-naive individuals developed the antibody response and VNA mainly 21 days post-vaccination. Additionally, we found a strong correlation between RBD-specific IgG titers and VNA in serum, and according to these data vaccination may be recommended if the RBD-specific IgG titers drop to 142.7 BAU/mL or below. In summary, the results of the study demonstrate that vaccination is beneficial both for COVID-19-naive and recovered individuals, especially since it raises serum VNA against the B.1.617.2 variant - one of four the SARS-CoV-2 variants of concern. Titers of the immunoglobulins G (IgGs) specific to the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein were analyzed in serum using the automated ARCHITECT i1000SR analyzer with compatible reagent kit (Abbott, USA) according to the manufacturer's standard protocol. Values obtained were recalculated in BAU/mL in accordance with the WHO International Standard [9] ; the IgG value equal to 7.2 BAU/mL was used as a seropositivity cutoff according to the manufacturer's instructions. We evaluated the virus neutralizing activity (VNA) in serum from a microneutralization assay using B.1.1.1 (PMVL-1 (GISAID EPI_ISL_421275)) and B.1.617.2 (T19R G142D E156G F157del R158del L452R T478K D614G P681R D950N) SARS-CoV-2 variants in a 96-well plate and a 50% tissue culture infective dose (TCID50) of 100 as described in [10] , with serum dilutions of 10, 20, 40, 80, 160, 320, 640, 1280, 2560, 5120, and 10,240 times. We performed an IFNγ ELISpot assay using the Human IFNγ Single-Color ELISPOT kit (CTL; USA) with a 96-well nitrocellulose plate pre-coated with human IFNγ capture antibody according to the manufacturer's protocol. Briefly, 3×10 5 freshly isolated PBMC in serum-free CTL-test medium (CTL, USA), supplemented with Glutamax (ThermoFisher Scientific, USA) and penicillin/streptomycin (ThermoFisher Scientific, USA), were plated per well and incubated with SARS-CoV-2 PepTivator N or M or a mixture of S, S1, and S+ peptide pools (Miltenyi Biotec, Germany) at a final concentration of 1 μg/mL each at a final volume of 150 µL/well. Additionally, cells were incubated with media only (negative control) or phytohaemagglutinin (Paneco, Russia) at a final concentration of 10 µg/mL (positive control). Plates were incubated for 16-18 h at 37°C in 5% CO2 atmosphere. The plates were washed twice with PBS, then washed twice with PBS containing 0.05% Tween-20, and incubated with biotinylated anti-human IFNγ detection antibody for 2 h at RT. Plates were washed three times with PBS containing 0.05% Tween-20 followed by incubation with streptavidin-AP for 30 min at RT. We visualized spots by incubation with the substrate solution for 15 min at RT. The reaction was stopped by a gentle rinse with tap water. We airdried plates overnight at RT and then counted spots using the automated spot counter CTL ImmunoSpot Analyzer and ImmunoSpot software (CTL; USA). Samples in which the negative control was greater than 10 spots and/or the positive control was less than 20 spots were considered as invalid. Positivity criteria for ELISpot were developed previously [11] . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Statistical analysis was performed with the Python3 programming language with numpy, scipy and pandas packages. The Mann-Whitney U test (two-sided) was used for comparing distributions of quantitative parameters between independent groups of individuals. The Wilcoxon signed-rank test (two-sided, including zero-differences in the ranking process and splitting the zero rank between positive and negative ones) was performed to assess the changes in the quantitative parameters between different time points for the same subject. To control for type I error, we calculated false discovery rate q-values using the Benjamin-Hochberg (BH) procedure and set a threshold of 0.05 to keep the positive false discovery rate below 5%. In all figures, for simplicity, we ranked p-values by significance levels using the following labels: 5.00e-02 < p-values are marked with 'ns'; 1.00e-02 < p-values ≤ 5.00e-02 are marked with '*'; 1.00e-03 < p-values ≤ 1.00e-02 with '**'; 1.00e-04 < p-values ≤ 1.00e-03 with '***'; and p-values ≤ 1.00e-04 with '****'. For the assessment of the different groups of subjects, a hierarchical cluster analysis using Ward variance minimization algorithm on z-normalized values for RBD-specific IgG levels at three time points for each subject was performed. To select the optimal RBD-specific IgG titers for selection of VNA-positive patients, the binary classifier separating patients into groups with VNA >=20 and <20 for either B.1.1.1 or B.1.617.2 using RBD-specific IgG titers as a single input parameter was built and corresponding ROC curves were used for selection of optimal thresholds. A total of 84 initially non-vaccinated Moscow residents were included in the study ( Table 1 ). In the course of the study, participants were vaccinated with Sputnik Light vaccine and their blood was collected prior to the vaccination, as well as on days 7 and 21 after vaccine administration. Serological testing of the participants on the day of their inclusion in the study revealed that 44 (52.4%) individuals were seropositive for the virus-specific immunoglobulins G (IgGs) ( Figure 1A) . Among them, 36 (81.8%) individuals also demonstrated the presence of SARS-CoV-2-specific T cells in peripheral blood ( Figure 1B) . These data, taken together with the . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 27, 2021. ; https://doi.org/10.1101/2021.10.26.21265531 doi: medRxiv preprint self-reported COVID-19 cases in this group, indicated that these individuals had been previously exposed to SARS-CoV-2. Within the observational period among the cohort, there was a constant increase in the titers of IgGs specific to the receptor binding domain (RBD) of the coronavirus spike (S) protein ( Figure 1A) . Accordingly, the fraction of seropositive individuals also increased from 52.4% to 57.1% on the 7th day, and to 100% on the 21st day post-vaccination. The results of the IFNγ ELISpot demonstrated that the T-cell response developed faster than the antibody one. To find the main patterns of the response to the vaccination, all participants were clusterized according to the observed dynamics in RBD-specific IgG titers. For this purpose, we used the Ward variance minimization algorithm in order to discriminate these patterns in an unbiased way. Three clusters were identified ( is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 27, 2021. ; https://doi.org/10.1101/2021.10.26.21265531 doi: medRxiv preprint frequencies of S-protein-specific T cells. However, in the case of cluster 1, IgG titers had already increased considerably on the 7th day post-vaccination but were only slightly elevated on the 21st day. Frequencies of S-protein-specific T cells in peripheral blood also increased considerably 7 days post vaccination, but then dropped on the 21st day, reaching the same value as for cluster 3 (composed of the SARS-CoV-2-naïve individuals). In contrast to cluster 1, individuals comprising cluster 2 demonstrated no changes in either IgG titers or frequencies of S-protein-specific T cells throughout the observation period. On the 21st day post-vaccination, both parameters were significantly lower than those for individuals without previous SARS-CoV-2 exposure (cluster 3) (Figure 2A, B and Supplementary Figure S3A, B) . (Figure 3B) . For each of the tested SARS-CoV-2 variants, we found a strong correlation between RBDspecific IgG titers and VNA in serum (Figure 3C, D) . Accordingly, these IgG titers can be is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 27, 2021. specific T cells in these seronegative individuals might be explained by previously asymptomatic COVID-19, which has been shown to be associated with lack of antibody response or a rapidly decreasing one [12, 13] , or these T cells might have developed as a result of a previous infection with the "common cold" coronaviruses and are cross-reactive to SARS-CoV-2 [14, 15] . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint However, among seropositive individuals only four persons (11% of the seropositive group) did not respond to the vaccination with Sputnik Light, as evidenced by the lack of increase in anti-SARS-CoV-2 IgG titers, peripheral blood T cells, and VNA in serum. While the reasons for this lack have yet to be understood, the fraction of non-responders is rather small and doesn't compromise the general efficacy of vaccination among COVID-19-recovered individuals. Recent studies have shown that IgGs specific to the coronavirus S-protein, particularly to its RBD portion, also demonstrate neutralizing activity against the virus [18-20]. Similar results were obtained in our study: we found a strong correlation between RBD-specific IgG titers and VNA in serum. This correlation was especially pronounced in the case of COVID-19- is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Taken together, our results showed that vaccination with Sputnik Light in cases of individuals previously exposed to the virus considerably boosts the existing immune response against the virus. In these individuals, RBD-specific IgG titers, S-protein-specific T cells, and VNA in serum were already elevated on the 7th day after vaccination, in contrast to the COVID-19naïve individuals, who developed the antibody response and VNA in serum mainly 21 days post-vaccination. We found a strong correlation between RBD-specific IgG titers and VNA in serum, and according to these data vaccination may be recommended if the RBD-specific IgG titers drop to 142.7 BAU/mL or below. In summary, the results of the study demonstrate that vaccination is beneficial both for COVID-19-naïve and recovered individuals, especially since it raises serum VNA against the B.1.617.2 variant, and Sputnik Light can be efficiently used for this purpose. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Gastritis 6 (7.1%) BMI, body mass index. . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 27, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 27, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 27, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 27, 2021. ; https://doi.org/10.1101/2021.10.26.21265531 doi: medRxiv preprint Figure S2 . Clusterization of the participants by the dynamics of the antibody response. RBD-specific IgG titers were estimated as described in the Materials and Methods section and were represented as a heatmap. Next, participants were clusterized by the changes in the IgG titers with the use of the Ward variance minimization algorithm. Clusters found are indicated as C1, C2, and C3. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 27, 2021. Assessment of protection against reinfection with SARS-CoV-2 among 4 million PCR-tested individuals in Denmark in 2020: a populationlevel observational study Re-infection with SARS-CoV-2 in Patients Safety and immunogenicity of an rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine in two formulations: two open, non-randomised phase 1/2 studies from Russia SARS-CoV-2 specific T cells and antibodies in COVID-19 protection: a prospective study Clinical and immunological assessment of asymptomatic SARS-CoV-2 infections Intrafamilial Exposure to SARS-CoV-2 Associated with Cellular Immune Response without Seroconversion Cross-reactive CD4(+) T cells enhance SARS-CoV-2 immune responses upon infection and vaccination HCoV-and SARS-CoV-2 Cross-Reactive T Cells in CVID Patients Rapid induction of antigen-specific CD4(+) T cells is associated with coordinated humoral and cellular immunity to SARS-CoV-2 mRNA vaccination Shcheblyakov 2 , Pavel Volchkov 4 , and Elena Vasilieva 2 Federal State Budget Institution "National Research Centre for Epidemiology and Microbiology named after Honorary Academician N F Gamaleya" of the Ministry of Health of the Russian Federation, 123098, 18 Gamaleya str The authors would like to thank Dr. Leonid Margolis (Eunice Kennedy Shriver National The authors declare no competing interests.