key: cord-0916158-mnbxqs1a
authors: van Gils, M. J.; Willegen, H. D. G.; Wynberg, E.; Han, A. X.; van der Straten, K.; Verveen, A.; Lebbink, R.; Dijkstra, M.; Burger, J. A.; Oomen, M.; Tejjani, K.; Bouhuijs, J. H.; Appelman, B.; Lavell, A. H. A.; Poniman, M.; Caniels, T. G.; Bontjer, I.; van Vught, L. A.; Vlaar, A. P. J.; Sikkens, J. J.; Bomers, M. K.; Sanders, R. W.; Kootstra, N. A.; Russell, C. A.; Prins, M.; de Bree, G. J.; de Jong, M. D.; Group, RECoVERED Study
title: Single-dose SARS-CoV-2 vaccine in a prospective cohort of COVID-19 patients
date: 2021-05-25
journal: nan
DOI: 10.1101/2021.05.25.21257797
sha: 8c04d8d411c4bf730d6bbbe2d24b06f02a3b7ae8
doc_id: 916158
cord_uid: mnbxqs1a

Background The urgent need for, but limited availability of, SARS-CoV-2 vaccines worldwide has led to widespread consideration of dose sparing strategies, particularly single vaccine dosing of individuals with prior SARS-CoV-2 infection. Methods We evaluated SARS-CoV-2 specific antibody responses following a single-dose of BNT162b2 (Pfizer-BioNTech) mRNA vaccine in 155 previously SARS-CoV-2-infected individuals participating in a population-based prospective cohort study of COVID-19 patients. Participants varied widely in age, comorbidities, COVID-19 severity and time since infection, ranging from 1 to 15 months. Serum antibody titers were determined at time of vaccination and one week after vaccination. Responses were compared to those in SARS-CoV-2-naive health care workers after two BNT162b2 mRNA vaccine doses. Results Within one week of vaccination, IgG antibody levels to virus spike and RBD proteins increased 27 to 29-fold and neutralizing antibody titers increased 12-fold, exceeding titers of fully vaccinated SARS-CoV-2-naive controls (95% credible interval (CrI): 0.56 to 0.67 v. control 95% CrI: -0.16 to -0.02). Pre-vaccination neutralizing antibody titers had the largest positive mean effect size on titers following vaccination (95% CrI (0.16 to 0.45)). COVID-19 severity, the presence of comorbidities and the time interval between infection and vaccination had no discernible impact on vaccine response. Conclusion A single dose of BNT162b2 mRNA vaccine up to 15 months after SARS-CoV-2 infection provides neutralizing titers exceeding two vaccine doses in previously uninfected individuals. These findings support wide implementation of a single-dose mRNA vaccine strategy after prior SARS-CoV-2 infection.

The urgent need for, but limited availability of, SARS-CoV-2 vaccines worldwide has led to widespread consideration of dose sparing strategies, particularly single vaccine dosing of individuals with prior SARS-CoV-2 infection.

We evaluated SARS-CoV-2 specific antibody responses following a single-dose of BNT162b2 These findings support wide implementation of a single-dose mRNA vaccine strategy after prior SARS-CoV-2 infection.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The unprecedented rapid development and emergency use authorization of several vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) allows for optimism in the global fight against the coronavirus disease 2019 (COVID- 19) pandemic. 1 However, in many regions, vaccination campaigns are hampered by limited supply or resources, hence vaccine sparing strategies are desirable. Making use of immunological memory after prior natural SARS-CoV-2 infection, 2,3 single dosing represents one such strategy for vaccines requiring two doses for optimal efficacy. Indeed, a number of recent small studies in health care workers (HCW) have shown similar or higher antibody responses and higher vaccine efficacy to a single dose SARS-CoV-2 mRNA vaccine after prior infection, compared to two doses in SARS-CoV-2-naive individuals. [4] [5] [6] [7] [8] [9] [10] [11] However, these studies were performed in relatively young and healthy individuals and provided limited information on the possible influence of COVID-19 severity and the duration since infection on vaccine responses. To inform potential wide implementation of a single dose strategy following natural infection, we evaluated the titers and breadth of antibody responses to a single SARS-CoV-2 mRNA vaccine in an ongoing population-based prospective cohort study of COVID-19 patients, representing a range in age, the presence of comorbidities, COVID-19 severity and time since infection. Antibody responses were compared to those observed after two doses in a cohort of SARS-CoV-2 naive HCW.

The current vaccine study was embedded in the RECoVERED project, an ongoing prospective cohort study of individuals with laboratory-confirmed SARS-CoV-2 infection in Amsterdam, the Netherlands. The RECoVERED cohort was initiated in May 2020 and as of April 2021 enrolled 328 participants, including both home-cared patients with mild infections and hospitalized patients with moderate to severe or critical illness. RECoVERED participants are followed at 1-3-month intervals from illness onset whereby biological specimens and questionnaires are collected at each follow-up visit to address RECoVERED's primary objectives relating to immunology and long-term sequelae of COVID-19. Clinical severity groups were defined in line with the WHO COVID-19 disease severity criteria: 12 Mild disease was defined as having a respiratory rate (RR) <20/min and oxygen saturation (SpO2) on room air >94% during acute illness; moderate disease as having a RR of 20-30/min, SpO2 90-94% and/or receiving oxygen therapy; severe disease as having a RR>30/min or SpO2 <90%; and critical disease as requiring ICU admission.

Cohort participants, invited to receive vaccination according to the Dutch national vaccination campaign before 12 April 2021, were asked to participate in the present vaccine . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Vaccinated HCW without longitudinal serological evidence of prior SARS-CoV-2 infection, participating in a HCW cohort study at the Amsterdam University Medical Centers (S3 study), served as a control group. 13 In this cohort, antibody responses were measured four weeks after the second dose of the BNT162b2 mRNA vaccine. The RECoVERED study, including the vaccine substudy, and the S3 study were approved by the medical ethical review board of the Amsterdam University Medical Centers (NL73759.018.20 and NL73478.029.20, respectively). All participants provided written informed consent.

Levels of Immunoglobulin G (IgG) binding to SARS-CoV-2 receptor-binding domain (RBD), nucleocapsid (N) and spike (S) proteins of wild type virus (Wu-1) and variants of concern (B.1.1.7, B.1.351 and P.1), as well as to control proteins tetanus toxoid, respiratory syncytial virus glycoprotein (RSV-G) and influenza A/H1N1pdm09 virus HA protein, were determined using a custom luminex assay as described previously. 14 In short, proteins were produced in HEK293F cells (Invitrogen) and purified from the cell culture supernatant using affinity chromatography with NiNTA agarose beads (Qiagen). Proteins were covalently coupled to luminex magplex beads using a two-step carbodiimide reaction. Beads were incubated overnight with 1:100,000 diluted serum followed by detection with goat-anti-human IgG-PE (Southern Biotech) on a Magpix (Luminex) as the mean fluorescent intensity (MFI).

Pseudovirus neutralization assay was performed as previously described (Brouwer et al. Cell 2021). Briefly, HEK293T/ACE2 cells 15 were seeded in poly-L-lysine pre-coated 96-well plates. The next day, heat-inactivated 1:100 diluted sera were 3-fold serially diluted and mixed in a 1:1 ratio with pseudovirus Wu-1 D614G (WT) 15 After 1-hour incubation at 37°C the mixtures were added to the cells and incubated for 48 hours at 37°C. The luciferase activity in cell lysates was measured using the Nano-Glo Luciferase Assay System (Promega) and GloMax system (Turner BioSystems). The 50%

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The copyright holder for this preprint this version posted May 25, 2021. ; https://doi.org/10.1101/2021.05.25.21257797 doi: medRxiv preprint inhibitory dilution (ID50) titers were determined as the serum dilution at which infectivity was inhibited by 50% using a non-linear regression curve fit (GraphPad Prism software version 8.3).

A Bayesian hierarchical generalization of the one-way ANOVA model was used to compare control, pre-and post-vaccination neutralizing and IgG antibody binding titers.

Differences between groups are reported as differences in effect sizes. A difference in effect size is non-trivial if it is non-zero, and substantial if greater/lower than 1/-1. This model was also used to estimate the individual effect size of age group, clinical severity, and time since symptom onset group, on the observed vaccine responses.

Furthermore, a Bayesian multilevel model that partially pooled effect size estimates across all study participants was used to estimate the effect size of the predictor variables individually and in combination on post-vaccination serum neutralization levels (Table S1) . We investigated if, and the degree to which, participants' age, sex, presence of comorbidities (i.e. history of cancer, cardiovascular disease, chronic respiratory disease, diabetes mellitus and obesity, separately), COVID-19 severity, time since COVID-19 symptom onset, prevaccination neutralization titers, and infection by a B.1.1.7 lineage variant were correlated with vaccine response. Condition indices were computed to ensure that there was no collinearity among the predictor variables (i.e. condition index <10). A distribution of normalized effect sizes (analogous to regression coefficients) was estimated for each predictor variable as a measure of their relative contributions to vaccine response. Similar to the Bayesian ANOVA model, an effect size is non-trivial if it is non-zero, and substantial if greater/lower than 1.

All Bayesian models were fitted using Markov Chain Monte Carlo (MCMC) with pymc3, 16 implementing a no-u-turn sampler. Four MCMC chains were run with at least 4000 burn-in steps and 2000 saved posterior samples. Convergence for all parameters were verified by checking trace plots, ensuring their ̂ values were <1.05 with sufficient effective sample size (>200).

A total of 155 participants of the RECoVERED cohort received a single-dose of the BNT162b2 mRNA vaccine after a median of nine months following SARS-CoV-2 infection (interquartile range (IQR) 5-12 months). The median age of participants was 55 years (IQR 33-61), 37% were female, 44% had one or more comorbidities and their SARS-CoV-2 infections were classified as mild, moderate, or severe/critical COVID-19 in 33%, 45% and 22% of participants, respectively (Table 1) . Overall, the vaccine was well tolerated with only . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 25, 2021. ; https://doi.org/10.1101/2021.05.25.21257797 doi: medRxiv preprint mild and self-limiting adverse events (Table S2) . A total of 130 of the 155 participants (84%) reported one or more side effects within 48 hours of vaccination, with pain at the injection-site (84%) and fatigue (48%) reported most frequently. The control group consisted of 49 healthy HCW (62% female, median age 44 years (IQR 33-53)) without evidence of previous SARS-CoV-2 infection who received two doses of the BNT162b2 mRNA vaccine.

Prior to vaccination, levels of IgG antibodies binding to S, RBD and N proteins exhibited a wide range, with overall higher levels observed in participants with previous severe/critical COVID-19 ( Figure 1A , S1A). Sharp increases in anti-S and anti-RBD IgG were Table S3 ). Achieved levels were similar or higher to those observed four weeks after two vaccinations in the SARS-CoV-2-naive HCW control group ( Figure S1B ; Table S3 ). When looking at anti-S IgG to three variants of concern (VOC; B.1.1.7, B.1.351 and P.1), levels were comparable to wild-type (WT) S protein both pre-and post-vaccination, with discernible increases for all VOC S proteins after vaccination (Figures 1B, S2A ; Table S3 ).

Neutralizing antibodies were detected (ID50 >100) prior to vaccination in 134 of 155 (86%) participants, correlating with COVID-19 severity and time since infection. Similar to SARS-CoV-2-specific IgG, neutralization titers increased sharply after vaccination (median fold increase 12.5 (IQR 5.2-39.6)), achieving higher titers than observed in the control group ( Figure 2A , S1A-B, Table S4 ). Bayesian multilevel regression analysis showed that anti-S IgG (95% CrI: 0.29, 0.88) correlated more strongly to neutralization levels than anti-RBD IgG (CrI: 0.08, 0.66) before vaccination. However, after vaccination anti-RBD IgG correlated strongly with neutralization (95% CrI: 0.55, 1.41), while no discernable effects of anti-S IgG were observed (CrI: -0.55, 0.28); Figure S3 ). This indicates that neutralization is dominated by IgG binding to RBD after vaccination.

Neutralization of VOC was evaluated in a random selection of 20 participants with detectable WT neutralization titers. While 11 of these 20 participants had undetectable neutralizing activity against one or more VOC before vaccination, neutralization titers rose sharply and were measurable in all participants after vaccination to all three VOC (median fold Figure   . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 25, 2021. ; https://doi.org/10.1101/2021.05.25.21257797 doi: medRxiv preprint 2B, S2B; Table S4 ). The differences between WT and VOC neutralization titers were trivial, except for B.1.351 post-vaccination (95% CrI: -0.90, -0.08), with the ratio between WT and VOC neutralization diminished after vaccination ( Figure S2C ; Table S4 ).

We used a Bayesian multilevel regression model to estimate the effect size of variables potentially affecting post-vaccination neutralization levels in 139 participants from whom complete metadata were available (Table S5) . Pre-vaccination neutralization levels showed the largest positive mean effect with clear posterior support of non-trivial effect size (95% CrI: 0.16, 0.45). In addition, age (95% CrI: -0.29, -0.05) and sex (CrI: 0.04, 0.32) exhibited modest non-trivial effects ( Figure 3A ). Further analyses using the Bayesian ANOVA model showed that mean post-vaccination neutralization levels were discernible for sex (95% CrI: male: -0.15, 0.00; female: 0.00, 0.15) but not between different age groups (95% CrI: ≤45y: -0.05, 0.16; 46-65y: -0.08, 0.12; >65y: -0.24, 0.06; Figure S4A ). This indicates that, while younger individuals were expected to achieve higher neutralizing responses, neutralization levels (Table S6) .

This study demonstrates that higher levels of neutralizing antibodies are achieved within a week after a single dose of a SARS-CoV-2 mRNA vaccine in previously infected individuals compared to those observed in fully vaccinated SARS-CoV-2 naive HCW, irrespective of time since infection. This implies that a single-dose in prior-infected individuals administered up to more than one year after SARS-CoV-2 infection provides antibody responses associated with the vaccine efficacy observed in the phase 3 study of the BNT162b2 mRNA vaccine. 17 Similar favorable vaccine responses after natural infection have been reported but these studies were fairly small, restricted to relatively young and healthy . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 25, 2021. ; https://doi.org/10.1101/2021.05.25.21257797 doi: medRxiv preprint HCW with mostly mild disease who were vaccinated up to nine months after infection. [4] [5] [6] [7] [8] [9] [10] [11] Our prospective COVID-19 cohort allowed extension of these findings to a broader population at risk and showed that these responses were not affected by the presence of underlying comorbidities, COVID-19 disease severity or timing of vaccination since infection. Hence, our study supports wide implementation of single dosing strategies for previously infected individuals.

The Bayesian multilevel regression model showed that pre-vaccination neutralization titers were associated with higher neutralization titers after vaccination, independent of disease severity or time since infection. This may suggest that pre-existing antibodies potentially augment immune responses perhaps through the formation of immune complexes by antibodies binding the vaccine antigen. 18, 19 In keeping with other studies, 6,20,21 age and male sex were inversely correlated with vaccine responses. Even though variation in the neutralization titers was large and effect sizes overlapped between the age groups, younger individuals, especially females, are expected to have higher neutralization responses, which have also been observed with other vaccines (e.g. influenza). 22, 23 The emergence of SARS-CoV-2 variants may pose risks of infection despite immunity Overall, these results suggest that neutralization breadth was not improved after vaccination, most likely because neutralization after vaccination is overwhelmingly dominated by RBD responses, which are shown to be more sensitive to the mutations in the VOC. 27 Nevertheless, a degree of cross-neutralization of these three VOCs was observed in all participants already after a single dose in previously infected individuals.

There are several limitations of our study. As only symptomatic COVID-19 patients were followed we were unable to study vaccine responses after previous asymptomatic SARS-CoV-2 infection. However, an earlier study in HCW has observed no differences in antibody responses to a mRNA vaccine between individuals with prior asymptomatic and symptomatic SARS-CoV-2 infections. 10 Furthermore, the SARS-CoV-2 naive HCW controls were not matched with the previously infected cohort participants for potentially relevant factors such as age, sex or the presence of comorbidities. However, given that antibody responses in the healthier and younger HCW controls were lower, combined with our finding that age is inversely correlated with antibody vaccine response, the observed difference in vaccine response might even have been more pronounced if controls were matched. Finally, participants with severe COVID-19 were overrepresented in the subgroup with >12 months . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

Data supporting the findings in this manuscript are available from the corresponding author upon request.

All authors declare no competing interests. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 25, 2021. ; https://doi.org/10.1101/2021.05.25.21257797 doi: medRxiv preprint no. 21-14 to M.P.). The funders had no role in study design, data collection, data analysis, data interpretation or data reporting. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (Table S3) . Area of binding values below detection limit are shaded in gray.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (Table S4 ). Area of neutralization titers below detection limit are shaded in gray.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 25, 2021. ; https://doi.org/10.1101/2021.05.25.21257797 doi: medRxiv preprint

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The authors wish to thank all RECoVERED and S3 study participants; Mathieu A.