key: cord-0704264-gret56j9 authors: Vicenti, Ilaria; Basso, Monica; Gatti, Francesca; Scaggiante, Renzo; Boccuto, Adele; Zago, Daniela; Modolo, Eliana; Dragoni, Filippo; Parisi, Saverio Giuseppe; Zazzi, Maurizio title: Faster decay of neutralizing antibodies in never infected than previously infected healthcare workers three months after the second BNT162b2 mRNA COVID-19 vaccine dose date: 2021-09-02 journal: Int J Infect Dis DOI: 10.1016/j.ijid.2021.08.052 sha: e0c957f285f17e9f0ac24292058688f6cfa4813e doc_id: 704264 cord_uid: gret56j9 Objectives : Our aim was to describe the longitudinal evolution of neutralizing antibody titres (NtAb) in three different cohorts of healthcare workers including vaccinated individuals with and without a previous SARS-CoV-2 infection and previously infected unvaccinated subjects. COVID-19 was mild or asymptomatic in those experiencing infection. Methods : NtAb was tested before BNT162b2 mRNA COVID-19 vaccine (V0), 20±2 days after the first dose (V1_20), 20±3 days (V2_20) and 90±2 days (V2_90) after the second dose in vaccinated HCW and after about 2 months (N_60), 10 months (N_300) and 13 months (N_390) from natural infection in unvaccinated HCW. NtAb was measured by authentic virus neutralization with a SARS-CoV-2 B.1 isolate circulating in Italy at HCW enrolment. Results : Sixty-two HCW were enrolled. NtAb were comparable in infected HCW with no or mild disease at all the study points. NtAb of uninfected HCW were significantly lower with respect to those of previously infected subjects at V1_20, V2_20 and V2_90. The median NtAb fold decrease from V2_20 to V2_90 was higher in the uninfected subjects with respect to subjects with mild infection (6.26 vs 2.58, p=0.03) and to asymptomatic HCW (6.26 vs 3.67, p=0.022). The median Nabt at N_390 was significantly lower with respect to N_60, p=0.007). Conclusions : In uninfected subjects completing the two-dose vaccine schedule, a third mRNA vaccine dose is a reasonable option to counteract the substantial NtAb decline occurring at a significantly higher rate compared to previously infected, vaccinated subjects. Although at low level Nabt are still at detectable level after 13 months in two third of previously infected and not vaccinated subjects. BNT162b2 COVID-19 vaccine is known to induce a rapid production of neutralizing antibodies (Lustig et al., 2021; Vicenti et al., 2021b) , however very limited data on their long-term kinetics has been reported. Favresse et al. (2021b) described a robust humoral response 90 days after the first dose vaccine both in previously seropositive and seronegative subjects but a significant antibody decrease respect to the higher level reached occurred within this period. Interestingly, the administration of a third dose of the BNT162b2 vaccine, about two months from the second dose, to solid-organ transplant recipients significantly improved the immunogenicity of the vaccine (Kamar et al., 2021) . Taken together, this data suggests that the three-month period following the first dose vaccine may be crucial to identify subjects requiring dedicated vaccine schedules. The main aim of this study was to analyse early (about 3 weeks) and late (about 12 weeks) changes in neutralizing antibody titres (NtAb) after the second BNT162b2 SARS-CoV-2 vaccination dose in healthcare workers (HCW) with a previous mild or asymptomatic COVID infection with respect to uninfected HCW. Moreover, we described long-term humoral response of previously infected HCW who were not vaccinated. The study population included HCW who were vaccinated following asymptomatic or mild infection according to WHO classification (World Health Organization, 2020), a control group of vaccinated HCW never been positive in all the test performed for hospital surveillance and a small cohort of previously infected, unvaccinated HCW. SARS-CoV-2 infection was diagnosed in the Veneto area in the period March 1, 2020-May 30, 2020 and had negative molecular test since the end of the prescribed quarantine for surveillance hospital program. All the vaccinated subjects received the BNT162b2 mRNA vaccine and the second dose was administered three weeks after the first dose. In vaccinated subjects, NtAb were tested at four time points: V0 (before receiving the first dose), V1_20 (20±2 days after the first dose), V2_20 (20±3 days after the second dose) and V2_90 (90±2 days after the second dose). In the unvaccinated subjects who acquired natural SARS-CoV-2 infection, the study points analysed included N_60 (baseline, after about 2 months from diagnosis), N_300 after a median of 291 days and N_390 after a median of 394 days. The study was approved by the local Ethics Committee and all the subjects gave written informed consent. For SARS-CoV-2 virus neutralization, two-fold serial dilutions (starting at 1:10 dilution) of heatinactivated 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 well (ATCC catalog no. CRL-1586) were treated with serum-virus mixtures and incubated at 37 °C, 5% CO2. 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 (interquartile range [IQR]) titre of 69 (59.3-69.9) in 5 independent runs. After 72 h, cell viability was determined through the commercial kit Cell-titer Glo2.0 (Promega, Wisconsin, USA) following the 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. Sera with ID 50 titres ≥10 were defined as SARS-CoV-2 positive and neutralizing; sera with ID 50 <10 were defined as negative and scored as 5 for statistical analysis (Vicenti et al., 2021a) . Continuous variables were expressed as the median (IQR) whereas categorical variables were indicated as absolute number and frequency. The Mann-Whitney U test, the Wilcoxon signed rank sum test, the chi-squared test and the Fisher's exact test were applied as appropriate. Statistical analyses were performed using MedCalc® Statistical Software version 20.009 (MedCalc Software Ltd, Ostend, Belgium; https://www.medcalc.org; 2021) and the limit of significance for all analyses was established at p<0.05. Sixty-two HCW were enrolled. A complete set of data was available for 23 previously infected and vaccinated HCW (14 with asymptomatic infection and 9 with mild symptoms), for 13 uninfected, vaccinated HCW and for 9 previously infected unvaccinated HCW. A description of the study population including vaccinated subjects is reported in Table 1 and in Figure 1 and Figure 2 . NtAb values were comparable in HCW with asymptomatic infection and with mild disease at all the four study points. Nabt values of the uninfected HCW were significantly lower with respect to those of the previously infected subjects at V1_20, V2_20 and V2_90. At the last control (V2_90), median NtAb was lower than at V2_20 in subject with mild disease (p=0.007), asymptomatic (p=0.001) and uninfected individuals (p=0.001). Likewise, V2_90 NtAb were lower than V1_20 Nabt in subjects with mild disease and in asymptomatic (p=0.028 and p=0.003 respectively): conversely, V2_90 was higher than V1_20 in uninfected individuals (p=0.002).However, median NtAb at V2_90 was still significantly higher than median NtAb at V_0 both in HCW with past mild disease (p=0.01) and in those experiencing asymptomatic infection (p=0.001). Among HCW with previous infection, 5 patients had no detectable NtAb at V_0 after a median interval from infection of 263 In the uninfected vaccinated group, 7 out of 13 patients had undetectable NtAb at V1_20 but all patients but one had detectable NtAb at V2_90. The median NtAb fold change decrease from V2_20 to V2_90 was higher in uninfected patients with respect to subjects with mild infection (6.26 vs 2.58, p=0.03) and to asymptomatic HCW (6.26 NtAb titres in previously infected and uninfected vaccinated HCW and in previously infected not vaccinated HCW are summarized in Table 2 . Real world data have demonstrated that SARS-CoV-2 vaccines prevent infection in more than 90% of cases (Butt et al., 2021; Dagan et al., 2021) . Accordingly, 74% reduction in the proportion of Neutralizing antibody responses are a correlate of protection and their measurement through live virus assays is the reference method to investigate the magnitude and duration of immunity following vaccination or natural infection.This study evaluated the long-term neutralizing response to SARS-CoV-2 in different HCW groups representative of the possible scenarios: (i) mild or asymptomatic infection followed by 2-dose vaccination about 10 months after diagnosis, (ii) absence of infection as confirmed by regular monitoring, followed by 2-dose vaccination and (iii) past mild or asymptomatic infection not followed by vaccine for a very long time period. This last condition is now no longer allowed in Italy since previously infected HCW get vaccination and therefore constitutes a unicum. In agreement with published data (Vicenti et al., 2021b) , previously infected subjects vaccinated with BNT162b2 demonstrated a strong humoral response after the first dose, with a modest further increase 20 days after the second dose. Our data provided additional support on the long-term efficacy of the vaccine boost after a very long time from infection, a median interval of ten months. The kinetics of NAbTs decay in these patients compared to that following single vaccine dose needs further studies: recently a significant antibody decline 3 months after the first dose in both seronegative and seropositive individuals who received two doses was described (Favresse et al., 2021b) . However, our results were obtained 3 months after the second vaccination, in a well characterized population, with a much longer very long term humoral memory: the interval between infection and first vaccine dose (median of 292 days, with IQR 267-300) with respect to that of reported by Favresse et al (2021a) (mean 99 days with range 34-337 days). Of note, 5 (21.7%) out of 23 previously infected individuals had no detectable NAbT before vaccination; nevertheless these subjects experienced a strong response at V2_90, comparable to HCW with detectable NabT at V0, supporting the persistence of long lasting B memory after natural infection (Wang et al, 2021) . Further, our results support the observation that infected vaccinated patients experience a significantly slower NtAb decay than uninfected vaccinated subjects after 3 months from second dose, indicating the ability of the immune system to mount very high titres of NtAb after repeated antigen stimulation. Although natural infection and vaccination may lead to different responses, and residual and recall immune response may impact differently immune protection, the role of humoral immunity in the first phase of viral infection is well established as a major early obstacle to viral spread, counteracting viral replication and evolution as well as immune escape. Thus, measuring circulating antibody levels, their durability, specificity and recall kinetics is crucial for understanding and predicting the durability of protection . As recently reported, among vaccinated HCW, the breakthrough infections were correlated with NAbT detected within the week immediately before (Bergwerk et al., 2021) .The advantage of high NtAbs against the infection by viral variants was reported too . Based on data reported here, a third mRNA vaccine dose is reasonable in uninfected subjects, to further boost immunity and recapitulate the effect observed in previously infected vaccinated subjects. However, the identification of specific target groups and the definition of the best timing for vaccination need further analysis with a more prolonged follow up on a larger cohort. We included a group of previously infected unvaccinated individuals in the study, who were tested thrice after SARS-CoV-2 infection: about 2 months later, after a median of 291 days (defined as corresponding to V0 in vaccinated HCW) and after a median of 394 days (defined as corresponding to the last time point of vaccinated HCW). We regret for the lack of a study time corresponding to N_60 for vaccinated HCW: however the main aim of the study was the long-term humoral response and N_390 and V2_90 were definitely corresponding. We believe that the description of a control group with a protracted follow-up was an useful contribution to the understanding of the durability of NtAb in this particular category of subjects: middle age, previously healthy and without comorbidities and with mild or asymptomatic infection. These individuals are now almost all vaccinated. Strenghts of the study are the availability of authentic virus neutralization with a SARS-CoV-2 isolate, the homogeneity of study population with previous asymptomatic or mild disease, the long time interval from infection to vaccination, the long follow-up after the second dose of vaccination and the different HCW cohorts tested. The main limitation is the number of the subjects evaluated, but we tried to contribute with data immediately after their availability. In conclusion, data obtained by analysis of response to SARS-CoV-2 infection of this multifaceted population may help to suggest a clinical use of NtAb to inform health policy, i.e. to evaluate the decision to administer a third dose of vaccine and how to monitor the unvaccinated personnel. IV: performed the laboratory experiments, helped to interpret the findings and to write the paper. MB: helped to interpret the findings. SGP: designed and coordinated the study, collected and managed the samples and the data, interpreted the findings, and wrote the paper. MZ: supervised the laboratory experiments, helped to interpret the findings, performed the statistical analysis and wrote the paper. This work was supported by University of Padova, grant numbers DOR-2019 and DOR-2020 to SGP The study was approved by the local Ethics Committee: all the subjects gave written informed consent to the inclusion. The authors declare that they have no conflict of interest. Table 1 . Description of the main characteristics and of the neutralizing antibodies titres at V0, V1_20, V2_20 and V2_90 in the overall study population and in subjects with no symptoms or with mild disease: p refers to differences between HCW with asymptomatic or mild disease and to the differences between control group and HCW with asymptomatic or mild disease. In bold significant p values. Covid-19 Breakthrough Infections in Vaccinated Health Care Workers SARS-CoV-2 Vaccine Effectiveness in a High-Risk National Population in a Real-World Setting Longitudinal analysis shows durable and broad immune memory after SARS-CoV-2 infection with persisting antibody responses and memory B and T cells Prospects for durable immune control of SARS-CoV-2 and prevention of reinfection Vaccine in a Nationwide Mass Vaccination Setting Early antibody response in health-care professionals after two doses of SARS-CoV-2 mRNA vaccine (BNT162b2) Microbiol Infect 2021 Antibody titers decline 3-month post-vaccination with BNT162b2 Three Doses of an mRNA Covid-19 Vaccine in Solid-Organ Transplant Recipients Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection vaccine and correlates of humoral immune responses and dynamics: a prospective, single-centre, longitudinal cohort study in health-care workers Initial impact of SARS-Cov-2 vaccination on healthcare workers in Italy-Update on the 28th of linical management of severe acute respiratory infection when novel coronavirus 201 -n o ) infection is suspected: interim guidance Time Course of Neutralizing Antibody in Health Care Workers With Mild or Asymptomatic COVID-19 Infection 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 Naturally enhanced neutralizing breadth against SARS-CoV-2 one year after infection We would like to thank Alessia Lai for making the SARS-CoV-2 lineage B strain available for this study.