key: cord-0752654-iqyl8uq1
authors: Edelstein, Michael; Beiruti, Karine Wiegler; Ben-Amram, Hila; Bar-Zeev, Naor; Sussan, Christian; Asulin, Hani; Strauss, David; Bathish, Younes; Zarka, Salman; Abu Jabal, Kamal
title: Antibody-mediated Immunogenicity against SARS-CoV-2 following priming, boosting and hybrid immunity: insights from 11 months of follow-up of a healthcare worker cohort in Israel, December 2020-October 2021
date: 2022-03-12
journal: Clin Infect Dis
DOI: 10.1093/cid/ciac212
sha: 881a5c10636de1c8cf00e6b1e63cdf41694f220a
doc_id: 752654
cord_uid: iqyl8uq1

BACKGROUND: We determined circulating anti-S SARS-CoV-2 IgG antibody titres in a vaccinated healthcare workers (HCWs) cohort from Northern Israel in the 11 months following primary vaccination according to age, ethnicity and previous infection status. METHODS: All consenting HCWs were invited to have their IgG levels measured before vaccination and at 6 subsequent timepoints using a quantitative S1/S2 IgG assay. All HCWs with suspected COVID-19 were PCR tested. We described trends in circulating IgG geometric mean concentration (GMC) by age, ethnicity, timing of boosting and previous infection status and compared strata using Kruskall-Wallis tests. RESULTS: Among 985 vaccinated HCWs, IgG titres between 1 month post 2 (nd) dose to pre-boosting gradually decreased in all age groups. Younger or previously infected individuals had higher initial post-vaccination IgG levels (p<0.001 in both cases); differences substantially decreased or disappeared at 7-9 months, before boosting. The proportion of individuals infected prior to initiating vaccination and re-infected after dose 1 was comparable to the proportion of breakthrough infection post-dose 2 in those not previously infected (4.2 vs 4.7%). Pre-infection IgG levels in the 40 participants with breakthrough infection after dose 2 were similar to levels measured at the same timepoint in vaccinated HCWs who remained uninfected (p>0.3). Post-dose3 IgG levels were more than 10-fold those 1month post-dose 2. CONCLUSIONS: Immunity waned in all age groups and previously infected individuals, reversed by boosting. IgG titres decrease and reinfections in individuals with hybrid immunity (infection+vaccination) suggests they may also require further doses. Our study also highlights the difficulty in determining protective IgG levels.

M a n u s c r i p t 3 Background Ten months after SARS-CoV-2 was declared a pandemic, mass vaccination campaigns commenced with vaccines showing trial efficacy of over 90% against symptomatic illness [ 1, 2, 3] . Postintroduction empirical observational studies confirmed vaccine effectiveness against severe disease and death [ 4] , and initially apparent effectiveness against infection [ 4] raised hopes of control and perhaps elimination. However, bottlenecks in production, supply and delivery and challenges in regulatory capacity meant many low-and middle-income countries remain at very low vaccination coverage [ 5] , and vaccine hesitancy led to gaps in coverage even in countries with ready access to vaccine doses. In addition, viral variants emerged with relative immune evasion (e.g. Beta) or increased transmissibility (e.g. Delta) [ 6, 7] that together with waning of humoral immunity [ 8, 9] left 2-dose recipients sub-optimally protected.

In Israel, mass vaccination started in December 2020 using two doses of BNT162b2 mRNA vaccine scheduled 21 days apart as per manufacturer recommendation. By November 2021 two-dose population coverage was 80% among persons aged 30 years and over and 75% for those aged between 16-29 [ 10] . In June 2021 COVID-19 community transmission ceased briefly, following which importation of the Delta variant caused the largest epidemic yet experienced in the country.

Israel rapidly initiated booster doses. Experimental and observational data comparing 3 vs 2 doses, demonstrated the effectiveness of boosters against symptomatic infection with the Delta variant [ 11, 12] . However, given the low rates of severe disease outcomes among 2 dose recipients, the absolute risk reduction in severe disease is more modest, and inversely the number needed to vaccinate to avert one severe outcome is high. Thus, the appetite to introduce boosters was initially variable, and by November 2021 no other country offered universal boosting. In September 2021 the World Health Organization called for a moratorium on boosting until the end of 2021 [ 13] . In the UK, in September 2021, the Joint Committee for Vaccination and Immunization, the independent body advising the government on vaccine policy, recommended boosting to vulnerable individuals only [ 14] . The duration of clinical protection conferred by the booster remains unknown, nor do we yet have a clear-cut humoral correlate of protection.

A c c e p t e d M a n u s c r i p t 4 Ziv Medical Center (ZMC) is a 300-bed government regional referral hospital located in Safed, Northern Israel. Like all hospitals in the country it started offering vaccination to its healthcare workers (HCW) in December 2020, achieving over 90% coverage by late January 2021, followed by boosting from July 2021. We conducted prospective serosurveillance of HCWs to evaluate trends over time in SARS-CoV-2 humoral immunity by age, vaccination, infection status and time elapsed between priming and boosting, and other predictors. Using the same cohort, we have previously published findings of vigorous anamnestic responses among previously infected single-dose recipients, and the need for second dose among individuals experiencing breakthrough primary SARS-CoV-2 infection shortly after their first dose. [ 15, 16] Here we describe trends in antibody-mediated immunity over 11 months following vaccination by age, ethnicity, infection status and time elapsed between priming and boosting, and compare anamnestic responses resulting from 3 rd dose receipt to those resulting from breakthrough infection.

All ZMC employees were invited to participate. We verified prior infection status among consenting participants by measuring the presence of anti-Nucleocapsid (N) IgG antibodies using a highly sensitive and specific SARS-CoV-2 IgG qualitative assay (Abbott, Abbot Park, US) [ 17] . Workers with detectable anti-N IgG antibodies and/or documented past positive SARS-CoV-2 PCR were considered previously infected. Thereafter quantitative anti-SARS-CoV-2 Spike (S) IgG levels were measured using the LIAISON Diasorin SARS-CoV-2 S1/S2 IgG assay [ 17] at six time points from dose 1; t 1 : 21 days (range 15-35 days), t 2 : 51 days (range 41-65 days), t 3 : 100-150 days, t 4 : 151-210 days, t 5 : 211-270 days and t 6 : 271-310 days. Where the IgG level reading reached the maximum, serial dilutions were performed in order to obtain a precise quantitative value. HCWs were asked to report any arising symptoms. Those whose symptoms were consistent with the standard clinical case definition of COVID-19 were tested by reverse transcription polymerase chain reaction (rt-PCR).

Individuals with a positive PCR test were classified as infected post-vaccination (breakthrough A c c e p t e d M a n u s c r i p t 5 infection). Antibody levels were reported using geometric mean concentration (GMC) in arbitrary units/ml (AU/ml) alongside 95% confidence intervals (95% CI). We used log-GMC when reporting trends graphically. anti-S IgG GMCs were reported by strata defined by number of vaccine doses received, infection status (never infected, infected prior to vaccination, infected after full vaccination), age (according to age at recruitment), ethnicity and timing of boosting. We tested to reject the null hypothesis of no difference in GMC across strata using Kruskall Wallis tests. In order to determine any differences in immunogenicity by age and ethnicity we restricted analysis to never infected individuals who had received at least 2 doses of vaccine. We restricted the ethnicity analysis to 

Of 1500 employees, 985 consented to take part in the study, received at least one dose of vaccine and had at least one serological test post vaccination. Of these, 86 received only a single dose, 141 received 2 doses and 758 received 3 doses (Table 1 ). The median time between doses 1 and 2 was 21 days, and 223 days between doses 2 and 3. HCWs who received a single priming dose (generally because of previous infection) and a second dose more than 6 months after the first were considered boosted. One hundred and eighteen HCWs were infected prior to vaccination, of which 5 (4.2%)

were re-infected after vaccination. Of the 856 participants who received at least two doses and were seronegative at the initiation of vaccination, 82 (9.6%) were infected after initiating their vaccine course of which 40 (4.7%) 30 days or more after receipt of dose 2. The proportion of individuals not infected prior to vaccination initiation who had a breakthrough infection following the beginning of their vaccination course ranged from 7.1% in the 55+ (16/208) to 12.7% in the under 35 (26/178).

There was no statistically significant association between age group and the incidence of (Table 1) .

We observed a decrease in circulating IgG levels in all subgroups from after receipt of the second dose (t2) until boosting or infection (t4, 5 or 6 depending on the subgroup, table 2). At t 1 , compared with never infected individuals, those previously infected (referred to in the literature as having hybrid immunity or "superimmunity" [ 18] ) had 13-fold higher GMC (876.6 vs 63.9 AU/ml, p<0.0001).

Among the same individuals the fold-difference at t 4 (5-7 months post dose 1) was 1.9 (268.4 vs 139.1 AU/ml, p<0.001). At t 5 there was no statistically significant difference in GMC between the two groups although the number of previously infected individuals with available data at this time point was very small (n=4). Among never-infected participants, younger age was associated with higher GMC post dose 1(t 1 ) (table 2, p<0.001) but the difference in GMC was barely significant by t 5 (7-9 months post dose 1 but prior to dose 3, table 2 and Fig 1, p=0.05 ). There was no association between GMC and ethnicity among never infected, fully vaccinated individuals at any time point.

Of the 899 HCW who received ≥2 doses (including those infected prior to vaccination), 44 (4.9%)

were confirmed positive on PCR between 30 days after dose 2 and before dose 3, including 4 reinfections among those infected prior to vaccination. Of those infected for the first time, 4 tested PCR positive prior to t4, 20 had a positive PCR test between t 4 and t 5 and 16 between t 5 and t 6 . Among those first infected after vaccination, IgG GMC just prior to infection was not different than among those who remained uninfected at the same time point (184 vs 139 AU/ml, p=0.3 for those infected between tests 4 and 5, 165 vs114 AU/ml, p=0.9 for those infected between tests 5 and 6). The 40 previously uninfected individuals experiencing breakthrough infections were younger than never infected HCWs (mean age 39 vs 45 years old, p<0.002).

Of the 302 never infected HCWs who received dose 3 and were tested 1-2 months afterwards, t 6 GMC (1-2 months post dose 3) was 2618 (95%CI 2411-2843 AU/ml), while among the 21 non-boosted individuals infected after dose 2 for whom data was available, GMC was significantly higher (4213 A c c e p t e d M a n u s c r i p t 7 AU/ml, p< 0.001, Fig 2) . Among those never-infected, all age groups saw an increase in IgG levels 18-fold or more post boosting. Post-boosting GMC in the 36 individuals boosted 6-7 months after dose 2 was lower compared with those 266 boosted 8-9 months after dose 2 (2012-vs 2713, p=0.03).

However, individuals boosted earlier were older (mean age 50 vs 45, p<0.01)

Our convenience cohort provided a well representative setting in which to monitor serologic responses over time. Consistent with other observational data [ 8] , we found that 6 months post dose 2

IgG titres had waned in all age groups and initial differences in IgG levels had reduced. This phenomenon occurs irrespective of previous infection status. We also found that despite initially higher GMCs in younger individuals, after 6-7 months differences were much smaller or no longer apparent, suggesting that all age groups might require boosting to achieve optimal protection.

Previously infected individuals, who had IgG levels one order of magnitude higher than those never infected after one dose [ 15] also saw their circulating IgG levels drop at 6-7 months, with levels less than twice as high as those never infected. The proportion of reinfections among individuals infected pre-vaccination was comparable to the proportion of breakthrough infections among never infected individuals who received two doses. These findings suggest that, in line with other observational studies [ 19] , hybrid or super-immunity (natural immunity boosted by vaccination), wanes and may eventually need boosting as well, at least if decision making is based on circulating IgG levels.

Observational data has shown a high effectiveness of boosting against infection and severe disease [ 11, 12] including against the recently emerged Omicron variant [ 20] . The IgG levels achieved after boosting were one order of magnitude higher than after the priming course and close to levels achieved in those infected after the priming course. Our data do not allow to estimate duration of protection and no robust real world or modelling studies that estimate duration of protection are available yet. The large fold-increase in circulating IgG following infection among vaccinated A c c e p t e d M a n u s c r i p t 8 individuals may also have diagnostic value where it is not possible or practical to swab individuals for PCR tests during the narrow window of opportunity that the PCR modality offers.

We have demonstrated previously no difference in GMC by ethnicity following a single dose of vaccine. In the present study we found that this remains consistent after subsequent doses. This findings matter because risk of infection and disease was indeed associated with ethnicity in Israel and elsewhere, both before and after national introduction of COVID-19 vaccine [ 21, 22, 23] .

Our study also highlights the limits of using circulating IgG to determine immunogenicity. Anti-S IgG The decrease in IgG levels in the cohort described in this study occurred during a time of increase in the incidence of reported COVID-19 infection in Israel [ 8] but also at a time of a shift in the dominant circulating strain in Israel from Alpha to Delta. It is therefore a challenge to distinguish the effects of declining immunity from those of higher infectivity attributable to novel variants. In addition, while waning immunity has caused vaccine effectiveness against infection to decrease from over 90% to 50-60% [ 27] , the decrease in effectiveness against severe outcomes such hospitalization and death is much less pronounced [ 28] . While our study supports widespread boosting in all age groups from the immunogenicity perspective, the public health benefit of boosting should be balanced against priming previously unvaccinated individuals, both at the national and global levels, when formulating boosting policies.

Repeated blood sampling in the cohort was challenging. The number of latter tests was small, particularly within strata. We caution against drawing inference from later subgroup comparisons.

Secondly, PCR testing only occurred upon report of symptoms which likely underascertained true infection incidence with potential misclassification of infected asymptomatic participants as never infected. Though we did not observe increases in titres unexplained by either vaccination or reported symptoms. It is also possible that individuals who were infected early during the pandemic were not detected and classified as such at the beginning of the study due to decreasing sensitivity 4-5 months post infection of the anti-N IgG assay used in this study [ 29] . Finally though we compared titres, we did not measure neutralizing ability.

Our study demonstrates antibody waning and high post-boosting IgG levels in all age groups, suggesting widespread boosting policies may be beneficial, although this needs to be substantiated by effectiveness studies going forward. The need for such policy becomes more urgent with the emergence of strains such as Omicron that likely requires much higher antibody titres for neutralisation in order to achieve protection 30, 31] . Our data suggest that immunological waning occurs in vaccinated, naturally infected, and infected-then-vaccinated groups, regardless of age and ethnicity. Ongoing detailed large observational cohorts that measure antibody function and have sufficient clinical outcome incidence will help clarify to what extent, after how long and in terms of M a n u s c r i p t which variants, these individuals are again at risk. We continue to monitor anti-S titres in order to determine the durability of boosted immune responses by age, infection history and interval between priming and boosting. 

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Funding: This work was supported by internal funds from Ziv medical Centre. No external funds were received.

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