Safety and humoral responses to BNT162b2 mRNA vaccination of SARS-CoV-2 previously infected and naive populations

Since COVID-19 risk of reinfection is of great concern, the safety and efficacy of the mRNA-based vaccines in previously infected populations should be assessed. We studied 78 individuals previously infected with SARS-CoV-19, who received a single dose of BNT162b2 mRNA COVID-19 vaccine, and 1:2 ratio matched infection-naïve cohort who received two injections. The evaluation procedure included symptom monitoring, and serological tests. Among the post-infected population, the median IgG-S response after the first vaccine dose was 3.35 AU, compared to 2.38 AU after the second vaccine injection in the infection naive group. A strong correlation was demonstrated between IgG-S level before vaccination, and the corresponding responses after a single vaccine dose (r = 0.8, p < 0.001) in the post infected population. Short-term severe symptoms that required medical attention were found in 6.8% among the post-infected individuals, while none were found in the infection naïve population. Our data suggest that a single vaccine dose is sufficient to induce an intense immune response in post-infected population regardless of seropositivity. Although some short-term safety issues were observed compared to the infection naïve population, a single dose regimen can be considered safe in post-infected populations.

The infection naive population consisted of 177 cases. Among this cohort, 71 (40.1%) participants had prevaccination serology test results, and 24 (33.8%) of them preformed a serology test after the first dose as well. Cohort baseline characteristics, demographics, and high-risk comorbidities data are provided in Table 1.
Safety and side effects. We assessed the frequency of local and systemic side effects after vaccination in the post-infected and in the infection-naive cohorts. Local and systemic symptom severity was determined based on 3 . Briefly, Mild, does not interferes with daily activity; Moderate, some interference with activity, or temperature > 38.5 °C; Severe, prevents daily activity, temperature > 39 °C, or emergency department visit or hospitalization. Figure 1A shows that similar percentages of participants reported having side effects, by severity, in both cohorts. A total of 55/78 (70.5%) reported having a side effect in the post-infected cohort. Similarly 117/177 (66.1%), and 127/177 (71.8%) reported having any type of side effect in the infection-naive cohort after the first and the second injection respectively (Fig. 1A). Most common side effects were local injection-site symptoms (mild pain, redness and swelling), which occurred a few hours after the injection (Fig. 1B, Table 2). Short term allergic reactions were more pronounced in the post-infected population 4.1% compared to 1.1% in the infection-naïve cohort, as well as the overall severe symptoms that required medical attention (emergency department visit or hospitalization), 6.8% in the post-infected population, and 0.6% after the first vaccine dose, and none after the second in the infection naïve population.
Repeated sampling analysis was performed in a subgroup of 45 post-infected participants who had also prevaccination antibody measurements. Among this group, the median pre-vaccination IgG-S response was 1 www.nature.com/scientificreports/ between IgG-S levels before vaccination, and the corresponding responses after a single vaccination dose (r = 0.8, p < 0.001) as shown in Fig. 4. Notably, the post-infected participants who were found seronegative prior to their vaccination had significantly higher titer levels after a single vaccine dose 3.15 AU [IQR 3.00-3.21] (n = 9, highlighted in Fig. 4

Discussion
This study evaluates the safety and efficacy of a single mRNA-based vaccine dose in post-SARS-CoV-2 infected individuals. We demonstrate that a single dose induces a strong humoral response regardless of seropositivity in previously infected subjects. Although they had higher rates of adverse events compared to the infection-naïve population, a single vaccine dose was generally safe, and this regimen can be considered for the post-infected population. The BNT162b2 mRNA vaccine, administered in a two-dose regimen, provides 95% protection against COVID-19 at least seven days after the second dose 1-3 . The reported efficacy after the first dose is 52% 3 . Similar to previous case series that evaluated antibody levels after a single mRNA-based vaccine dose in post-infected individuals 5-7 , our post-infected population developed, approximately tenfold higher antibody titer levels compared to the antibody levels after a second dose in the infection-naïve cohort, 2 weeks after vaccination. A linear correlation was found between the pre and post-first vaccine dose antibody titers in the post-infected population.  www.nature.com/scientificreports/ Importantly, even among all seronegative participants who had evidence of a previous SARS-CoV-2 infection, a single vaccine dose induced higher titer levels compared to the infection naïve cohort, suggesting immune memory persistence. Specifically, due to the decay of immune memory over time, a single vaccine dose serves as a very potent booster dose for seronegative individuals who were previously infected. With the risk of reinfection increasing with time, several COVID-19 reinfection cases have already been documented [21][22][23][24][25][26][27][28] . The fact that most reinfected patients are asymptomatic or mildly symptomatic, raises the possibility that immunity is maintained and reduces the disease symptom severity even in cases of undetectable antibody levels at the time of reinfection.
Here, we studied post-infected individuals vaccinated 3-6 months after the infection. Although there was no correlation between the time from the infection and antibody titer levels, a single vaccine dose induced a strong humoral response even in seronegative individuals. The higher antibody response after a single vaccination in post infected patients is related to the acquired immune system, developed after the viral exposure. Upon the first infection, B-cells can differentiate into the plasma cells, and produce a first wave of virus neutralizing antibodies, that diminish gradually once the virus was eliminated, or develop into memory B-cells and long-live plasma cells. Memory B-cells preserve specific B-cell receptors (BCRs) of the pathogen that allows them to recognize antigen and mount a specific antibody response. When memory B-cells reencounter their specific antigen, they can proliferate and differentiate into plasma cells faster and of greater magnitude compared with the primary antibody response, and produce a more efficient secondary immune response 29 . Taken together, in post-infected populations, a single dose vaccine along with a serological test may provide an effective alternative regimen.
Local and systemic side effects were reported among BNT162b2 vaccine recipients. Reactogenicity after the first dose was characterized mostly with mild or moderate local reactions, while after the second dose, also systemic side effects were more common and severe. The frequency of severe systemic events was less than 0.9% and 2% after the first and the second vaccine injections respectively 1-3 . In this study, the reported frequency of local and moderate systemic side effect was in agreement with previous studies. However, 6.8% of the post-infection cases required emergency department visits or hospitalization due to over activation of the immune system or allergic reactions that needed medical supervision and care, while none of the infection naïve participants needed medical care. Severe vaccine related side effects were also reported in other studies of post-infected population 4,5 . This finding should be taken into consideration when vaccinating this population.
Although this study provides clear evidence regarding immune memory persistence in post infected individuals, the study has several limitations. The relatively small sample size of the post-infected population affected the infection-naïve cohort selection, due to the matching procedure, that may not represent the overall population. In addition, we studied COVID-19 post-infected individuals who were mildly symptomatic, with relatively low pre-vaccination titer levels. Finally, our study does not include information on cell mediated immunity (CMI) responses, which would provide further insight regarding the immune response, especially in post-infected seronegative individuals. Whether our safety conclusions could be generalized to previously moderate and severe COVID-19 infected patients 30 , has yet to be determined.
In conclusion, this study demonstrates that in previously SARS-CoV-2 infected populations, a single dose of an mRNA-based vaccine is sufficient to induce an intense immune response regardless of seropositivity. This vaccine-induced response correlates with the pre-vaccination IgG-S antibody concentration. The overall safety profile of the post-infected cohort is similar to the infection naive population, supporting the notion that a single dose vaccination approach can be considered in this population.  SARS-CoV-2 serology evaluation. COVID-19 serological tests were performed using the Liaison SARS-CoV-2 S1/S2 IgG (311450, DiaSorin, Saluggia, Italy): A chemiluminescent immunoassay (CLIA) for quantitative determination of anti-S1 and anti-S2 specific IgG antibodies using magnetic beads coated with S1 and S2 antigens. The analyzer automatically calculates SARS-CoV-2 S1/S2 IgG antibody concentrations expressed as arbitrary units (AU/ml), with a positive minimal cutoff level of 15.0 AU/ml.

Statistical methods.
We compared antibody levels and symptom responses between the two study populations of vaccine recipients. Continuous data are expressed as median and interquartile range (IQR). Independent t-tests with two-tail distribution were performed to compare variables between groups, when a normality assumption holds according to Kolmogorov-Smirnov tests. Categorical data were expressed in numbers and percentages and compared by chi-square tests. Univariate analyses were performed using Fisher's exact test to identify significant variables. Local and systemic side effects are presented as numbers, percentages, and associated Clopper-Pearson 95% confidence intervals. Continuous parameters correlations were performed using the Pearson correlation analysis. Boxplot analysis was used to present data distribution, and to detect outliers. Matching criteria included age (in bins of 5 years), gender, and comorbidities (in count of pre-existing conditions as listed in Table 1). Hamming distance was used to compute the similarity metric. A value of p < 0.05 was considered significant. Data were statistically analyzed using the Matlab Statistics Toolbox, R2020b (Mathworks, Natick, MA).