Safety and Humoral Responses to SARS-CoV-2 mRNA Vaccination of Previously Infected and Naive Populations

Since COVID-19 risk of reinfection is of great concern, the safety and e�cacy 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 �rst vaccine dose was 2260 AU/ml, compared to 238 AU/ml 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 su�cient 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.


Background
Vaccines to prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are considered the most promising approach for curbing the pandemic.There are several types of SARS-CoV-2 vaccines and mRNA-based vaccines were the rst to be approved by the FDA with an e cacy of 94-95% for preventing a symptomatic disease [1][2][3].The safety and e cacy of the mRNA-based vaccines were evaluated in prospective clinical trials on SARS-CoV-2 naïve population and the data on the vaccine usability in a previously infected population is gathered from real life case series data [4][5][6][7].
Following a SARS-CoV-2 infection, most patients develop detectable serum antibodies to the receptorbinding domain of the viral spike protein along with associated neutralizing activities [8][9][10][11][12][13][14][15].It was previously reported that some cases of asymptomatic and mildly symptomatic patients failed to mount neutralizing antibodies [8-11, 16, 17].However, other studies indicate that the vast majority do develop detectable levels of IgM, IgG-S and IgG-N that can persist for more than six months after the acute infection [12,14,18,19].The minimal level of antibodies required for infection immunity has yet to be determined.It is also known that in addition to protective antibodies, immunity for recurrent infections includes SARS-CoV-2-speci c memory lymphocytes, including the S-antigen presenter by mRNA-based vaccine, that upon antigen reencounter are activated to generate antibodies and secrete a variety of cytokines [15,20].It is still not clear if seronegative previously infected individuals are also at an advantage upon recurrent infection.
In addition to vaccine availability, the issue of whether to immunize previously infected SARS-CoV-2 patients is still debated since most of the vaccine related side effects have been attributed to overactivation of the immune system [21].
The aim of this study was to evaluate the safety and e cacy of a single injection protocol of SARS-CoV-2 mRNA-based vaccine (BNT162b2) in a previously COVID-19 infected population and compare it to the standard two injection protocol given to the infection-naïve population.

Cohort characteristics
A total of 78 con rmed post-COVID-19 infection patients, who performed a serological test after the rst vaccine dose, were identi ed in Shamir Medical Center's database.Of them, 45 (57.7%) had a prevaccination SARS-CoV-2 serology test as well.Within this subgroup, 9/45 (20%) were seronegative before vaccination.
The infection naive population consisted of 177 cases.Among this cohort, 71 (40.1%) participants had pre-vaccination serology test results, and 24 (33.8%) of them preformed a serology test after the rst 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.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 postinfected 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 rst 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 rst 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 pre-vaccination antibody measurements.A strong correlation was demonstrated 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. 3. Notably, the post-infected participants who were found seronegative prior to their vaccination had signi cantly higher titer levels after a single vaccine dose 1410.0AU/ml [IQR, 1006.2-1607.5](n = 9), compared to the infection-naive group after two vaccination dosages 238 AU/ml [IQR, 186.75-302.25](n = 177).

Discussion
This study evaluates the safety and e cacy 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][2][3].The reported e cacy after the rst dose is 52% [21].Similar to previous case series that evaluated antibody levels after a single mRNA-based vaccine dose in post-infected individuals [5][6][7], our post-infected population developed, approximately 10-fold higher antibody titer levels compared to the antibody levels after a second dose in the infection-naïve cohort, two weeks after vaccination.A linear correlation was found between the pre and post-rst vaccine dose antibody titers in the post-infected population.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.With the risk of reinfection increasing with time, several COVID-19 reinfection cases have already been documented [22][23][24][25][26][27][28][29].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.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 rst 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 rst and the second vaccine injections respectively [1][2][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 nding 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.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 su cient 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 pro le 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.

Study population
A retrospective analysis was conducted on a cohort of individuals, 18 years or older, who received BNT162b2 mRNA COVID-19 Vaccine (P zer, New York, US, and BioNTech, Mainz, Germany), and preformed serological tests, within 7 to 21 days after vaccination, at the Shamir Medical Center, between December 2020, and February 2021.Two population groups were identi ed: individuals previously infected with SARS-CoV-19, and 1:2 ratio matched infection naive population, who received two vaccination injections.Epidemiologic, demographic, pre-existing conditions data, and symptom information after each vaccine dose were collected by telephone interview.The study was approved by Shamir Medical Center institutional review board (IRB) (No. 029-21-ASF), with a waiver for informed consent.Because of the retrospective nature of the study, questionnaire completion was implied as informed consent.All research was performed in accordance with relevant guidelines and regulations.

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 signi cant variables.Local and systemic side effects are presented as numbers, percentages, and associated Clopper-Pearson 95% con dence 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 ve years), gender, and comorbidities (in count of pre-existing conditions as listed in Table 1

Figure 1 Local
Figure 1

Figure 3 Scatterplot
Figure 3 ). Hamming distance was used to compute the similarity metric.A value of p < 0.05 was considered signi cant.Data were statistically analyzed using the Matlab Statistics Toolbox, R2020b (Mathworks, Natick, MA)