key: cord-0912033-m59rkufo
authors: Vicenti, Ilaria; Gatti, Francesca; Scaggiante, Renzo; Boccuto, Adele; Zago, Daniela; Basso, Monica; Dragoni, Filippo; Zazzi, Maurizio; Parisi, Saverio Giuseppe
title: Single-dose BNT162b2 mRNA COVID-19 vaccine significantly boosts neutralizing antibody response in health care workers recovering from asymptomatic or mild natural SARS-CoV-2 infection
date: 2021-05-19
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2021.05.033
sha: 752cccc901f020d7d5a7708e6d4b641acf76372c
doc_id: 912033
cord_uid: m59rkufo

Objectives To measure SARS-CoV-2 neutralizing antibody (NtAb) titres in previously infected or uninfected healthcare workers (HCW) who received one or two doses of BNT162b2 mRNA COVID-19 vaccine. Methods NtAbs were titrated as dose inhibiting 50% virus replication (ID50) by live virus microneutralization. Forty-one HCW recovering from mild or asymptomatic infection were evaluated on the day of first-dose vaccination (T1_inf) and 21 days later (T2_inf). Sixteen uninfected HCW were evaluated 20 days after the first dose (T2_uninf) and 20 days after the second dose of vaccine (T3_inf). Results At T2_inf, but not at T1_inf, there was a significant correlation between days from diagnosis (median [IQR] 313 [285-322]) and NtAb levels (p = 0.011). NtAb titres increased at T2_inf with respect to T1_inf (1544 [732-2232] vs. 26 [10-88]; p < 0.001). Likewise, there was a significant increase in NtAb titres at T3_uninf with respect to T2_uninf (183 [111-301] vs. 5 [5-15]; p < 0001). However, NtAb levels at T2_inf were significantly higher than those at T2_uninf and T3_uninf (p < 0.0001 for both analyses). Conclusions A single vaccination in people with mild or asymptomatic previous infection further boosts SARS-CoV-2 humoral immunity to levels higher than those obtained by complete two-vaccination in uninfected subjects.

While there is obvious consensus that mass vaccination is the key strategy to keep the current SARS-CoV-2 pandemic under control, the need to vaccinate those who have recovered from natural infection is still under debate. As a matter of fact, several countries have been vaccinating previously infected people and this category has not been excluded from the main vaccine field trials (Krammer et al., 2021; Saadat et al., 2021; Manisty et al., 2021) , although placing them lower on the vaccination priority list was suggested [www has-sante.fr accessed on March 31, 2021].

Further, a short-lasting humoral protection after infection was described (Marot et al., 2021 ) but a robust T-cell response after a single dose of vaccine was reported (Prendecki et al., 2021) .

To investigate the role of post-infection vaccination (BNT162b2 mRNA COVID-19 vaccine), we enrolled 57 Italian healthcare workers (HCW) including 41 SARS-CoV-2 infected individuals (median age 45 [34-51] years, male 34.1%) recovering from asymptomatic infection or mild disease and 16 uninfected controls (median age 49 years , male 31.2%). A written informed consent was obtained from all the HCW to be enrolled in a neutralizing antibody (NtAb) follow-up study, as approved by the local Ethics Committee. The previously infected HCW were tested on the day of first-dose vaccination (T1_inf) and 21±4 days thereafter (T2_inf). The uninfected controls were tested on the day of first-dose vaccination (T1_uninf, all confirmed to be negative), 20±4 days thereafter (T2_uninf) and then 20±4 days following the second-dose vaccination (T3_uninf) ( Figure   1 ). For SARS-CoV-2 virus neutralization, two-fold serial dilutions (starting at 1:10 dilution) of heat-inactivated sera were incubated with 100 TCID 50 of SARS-CoV-2 virus (lineage B.1) at 37°C, 5% CO2 for 1 h in 96-well plates. At the end of incubation, 10,000 pre-seeded Vero E6 cells per J o u r n a l P r e -p r o o f well (ATCC catalog no. CRL-1586) were treated with serum-virus mixtures and incubated at 37°C, 5% CO 2 . Each run included an uninfected control, a virus back titration to confirm the virus inoculum and a known SARS-CoV-2 neutralizing serum yielding a median (IQR) titre of 69 (59.3-69.9) in 5 independent runs. After 72h, cell viability was determined through the commercial kit Cell-titer Glo2.0 (Promega) following manufacturer's instructions. The serum neutralization titre (ID 50 ) was defined as the reciprocal value of the sample dilution that showed a 50% protection of virus cytopathic effect. Antibodies with ID 50 titres ≥10 were defined as SARS-CoV-2 seropositive and neutralizing; sera with ID 50 <10 were defined as negative and scored as 5 for statistical analysis. NtAb titers were expressed as median (IQR) and the non-parametric Wilcoxon Signed Rank Sum test and Mann-Whitney test were used to analyze changes in paired and unpaired data, respectively. Pearson analysis was used to measure the correlation between NtAb titers before vaccination and the time from diagnosis. Analyses were run by IBM SPSS Statistics, version 20, and all p values were 2-sided.

Previously infected HCW were vaccinated after a median (IQR) of 313.0 (285.5-322.5) days since diagnosis. Seventeen subjects had mild disease: they experienced symptoms as fever (15, 

NtAb titres in the uninfected group, when measured both at T2_uninf and at T3_uninf (p <0.0001 for both analyses) (Figure 2 ).

This data supports vaccination of people with mild or asymptomatic previous infection to further boost SARS-CoV-2 immunity, however it remains to be proved whether this translates into increased protection from reinfection with respect to immunity driven by natural infection only.

Response to vaccination appeared to be variable and correlate with time from diagnosis in this selected population, indicating that different patterns of response are obtained following vaccination and suggesting that maturation of immunity improves the efficacy of the vaccination booster. This data, together with the wide range of NtAb levels following natural infection, could suggest reserving vaccination to those with lower response and/or more remote infection, although there is currently limited evidence that protection from reinfection correlates with NtAb titres (Cohen and Burbelo, 2020; Selhorst et al., 2020; Harvey et al., 2021) . It must be noted that variability in prevaccination NtAb levels could be partially explained by additional, asymptomatic exposure to SARS-CoV-2 following recovery from primary infection, further complicating our understanding of the kinetics of NtAb response. Finally, the relatively low NtAb titres detected in the uninfected, vaccinated group is apparently of concern but, importantly, the observation that SARS-CoV-2 infections and morbidity are consistently declining in countries with high vaccination rates (Dagan et al., 2021) is reassuring. 

This work was supported by University of Padova, grant number DOR-2019 and DOR-2020 to SGP and MB.

A written informed consent was obtained from all the HCW to be enrolled in a neutralizing antibody (NtAb) follow-up study, as approved by the local Ethics Committee.

The authors declare that they have no conflict of interest. 

Reinfection with SARS-CoV-2: Implications for Vaccines

Vaccine in a Nationwide Mass Vaccination Setting

Association of SARS-CoV-2 Seropositive Antibody Test With Risk of Future Infection

Antibody Responses in Seropositive Persons after a Single Dose of SARS-CoV-2 mRNA Vaccine

Antibody response to first BNT162b2 dose in previously SARS-CoV-2-infected individuals

Rapid decline of neutralizing antibodies against SARS-CoV-2 among infected healthcare workers

Effect of previous SARS-CoV-2 infection on humoral and T-cell responses to single-dose BNT162b2 vaccine

Binding and Neutralization Antibody Titers After a Single Vaccine Dose in Health Care Workers Previously Infected With SARS-CoV-2

We would like to thank Alessia Lai for making the SARS-CoV-2 lineage B strain available for this study.Preliminary data from this study were presented as Science Spotlight™ presentation at virtual CROI 2021.J o u r n a l P r e -p r o o f