Emerging SARS-CoV-2 variants of concern evade humoral immune responses from infection and
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vaccination
2
3
4
Tom G. Caniels
1†
, Ilja Bontjer
1†
, Karlijn van der Straten
1†
, Meliawati Poniman
1
, Judith A. Burger
1
, Brent
5
Appelman
2
, Ayesha H.A. Lavell
3
, Melissa Oomen
1
, Gert-Jan Godeke
4
, Coralie Valle
4
, Ramona Mögling
4
,
6
Hugo D.G. van Willigen
1
, Elke Wynberg
5
, Michiel Schinkel
2
, Lonneke A. van Vught
2
, Denise Guerra
1
,
7
Jonne L. Snitselaar
1
, Devidas N. Chaturbhuj
6
, Isabel Cuella Martin
1
, Amsterdam UMC COVID-19 S3/HCW
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study group, John P. Moore
6
, Menno D. de Jong
1
, Chantal Reusken
4
, Jonne J. Sikkens
3
, Marije K.
9
Bomers
3
, Godelieve J. de Bree
7
, Marit J. van Gils
1
*, Dirk Eggink
1,4
*, Rogier W. Sanders
1,6
*
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11
1
Department of Medical Microbiology, Amsterdam UMC, University of Amsterdam, Amsterdam Institute for
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Infection and Immunity, Amsterdam, the Netherlands.
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2
Center for Experimental and Molecular Medicine, Amsterdam UMC, University of Amsterdam,
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Amsterdam Institute for Infection and Immunity, Amsterdam, the Netherlands.
15
3
Department of Internal Medicine, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam Institute
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for Infection and Immunity, Amsterdam, the Netherlands.
17
4
Centre for Infectious Disease Control, National Institute for Public Health and the Environment, Bilthoven,
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the Netherlands.
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5
Department of Infectious Diseases, Public Health Service of Amsterdam, GGD, Amsterdam, the
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Netherlands.
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6
Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York,
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USA.
23
7
Department of Internal Medicine, Amsterdam UMC, University of Amsterdam, Amsterdam Institute for
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Infection and Immunity, Amsterdam, the Netherlands.
25
26
† These authors contributed equally to this work.
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* Correspondence to: r.w.sanders@amsterdamumc.nl, dirk.eggink@rivm.nl,
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m.j.vangils@amsterdamumc.nl
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NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.
Abstract
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Emerging SARS-CoV-2 variants pose a threat to human immunity induced by natural infection and
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vaccination. We assessed the recognition of three variants of concern (B.1.1.7, B.1.351 and P.1) in
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cohorts of COVID-19 patients ranging in disease severity (n = 69) and recipients of the Pfizer/BioNTech
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vaccine (n = 50). Spike binding and neutralization against all three VOC was substantially reduced in the
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majority of samples, with the largest 4-7-fold reduction in neutralization being observed against B.1.351.
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While hospitalized COVID-19 patients and vaccinees maintained sufficient neutralizing titers against all
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three VOC, 39% of non-hospitalized patients did not neutralize B.1.351. Moreover, monoclonal neutralizing
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antibodies (NAbs) show sharp reductions in their binding kinetics and neutralizing potential to B.1.351 and
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P.1, but not to B.1.1.7. These data have implications for the degree to which pre-existing immunity can
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protect against subsequent infection with VOC and informs policy makers of susceptibility to globally
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circulating SARS-CoV-2 VOC.
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Introduction
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With over 140 million confirmed infections and over three million deaths as of April 2021, the coronavirus
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disease 2019 (COVID-19) pandemic shows few signs of abating (1). While severe acute respiratory
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syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, remained relatively stable
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genetically and antigenically during the first stage of the pandemic, higher levels of genetic variation have
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been observed during the second wave of the pandemic with considerable genetic changes compared to
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the original Wuhan Hu-1 strain of SARS-CoV-2. These genetic changes include substitutions within the
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functional domains of the SARS-CoV-2 spike (S) protein resulting in altered phenotypes of the virus. The
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WHO has currently defined three VOC based on possible increase in transmissibility or change in COVID-
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19 epidemiology, increase in virulence or change in clinical disease presentation, or decrease in
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effectiveness of available diagnostics, vaccines and therapeutics. These VOC include B.1.1.7
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(20I/N501Y.V1, first detected in the United Kingdom), B.1.351 (20H/N501Y.V2, first detected in South
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Africa) and B.1.1.28.P1 (P.1, 20J/N501Y.V3, first detected in Brazil), which have since spread globally and
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as of April 2021, cases with these variants have been reported in 132, 82 and 52 countries, respectively
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(2–5). The emergence of these variants has raised concerns as to whether immunity, be it natural
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immunity from prior infection or immunity from vaccination, can protect against these different variants.
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Moreover, it is unclear whether therapeutic monoclonal NAbs isolated from convalescent donors retain
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therapeutic efficacy against these new variants.
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The S protein of coronaviruses is the main target of NAbs and consists of a membrane-proximal
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S2 domain containing the fusion peptide and a membrane-distal S1 domain containing the receptor
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binding domain (RBD) and the N-terminal domain (NTD) (Fig. 1A). The S protein mediates viral entry
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through interaction with the angiotensin-converting enzyme 2 (ACE2) receptor on host cells (6, 7).
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Therefore, substitutions present in the RBD of these emerging VOC are particularly worrisome as they
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might improve binding to the human receptor and thus increase viral fitness and transmissibility. Indeed,
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B.1.1.7, B.1.351 and P.1 all share the N501Y RBD mutation, which contributes to an enhanced interaction
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with the human ACE2 receptor resulting in increased infectivity and transmissibility (Fig. 1A) (8, 9). In
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addition, both B.1.351 and P.1 contain the E484K substitution and a substitution at position 417 (K417N in
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B.1.351 and K417T in P.1) both of which have been implicated in escape from NAbs by several studies
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(Fig. 1A) (10, 11). More recently, E484K has also been observed in B.1.1.7 variants that have adopted this
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mutation independently, leading to a more substantial loss of neutralizing titers in vaccinated individuals
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than for B.1.1.7 alone (12). Additional substitutions mostly arise in the NTD region, located apically on the
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S1 domain. Although the exact functional implication of substitutions within the NTD is not clear, it has
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been shown that this domain is an important target for NAbs (13, 14). VOC B.1.351 and P.1 both carry five
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mutations in this region of which they share one (L18F). Early reports on the B.1.351 lineage reported
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variants with a substitution at position 242 and 246 (L242H and R246I), while other sublineages had a
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three amino acid deletion (Δ242-244) (4). B.1.1.7 does not have substitutions in its NTD but has deletions
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in this region (Δ69-70, Δ144) (Fig. 1A). Other mutations are identified in the S2 domain of the S protein
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and their impact on antibody recognition and infectivity is not yet well understood, partly because the vast
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majority of NAbs target the RBD or NTD. Finally, all these variants have the D614G mutation that defines
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the B.1 lineage and became dominant throughout 2020, now being present in the large majority of
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sequenced SARS-CoV-2 variants (> 99%) (Fig. 1A) (15).
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All three VOC have been linked to increased infectivity and transmissibility, and the first reports
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about specific substitutions in B.1.351 and P.1 associated with escape from immunity by infection or
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mRNA vaccines have become available (10, 16–18). These first reports suggest that pre-existing immunity
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was generally sufficient to neutralize B.1.1.7 to similar levels as WT in mRNA vaccine recipients and in
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convalescent individuals up to nine months after infection (17, 19). In case of B.1.351, the impact is more
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substantial with neutralizing titers from convalescent patients being reduced by ~6 to 13-fold while vaccine
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recipients are reported to have a ~10 to 14-fold reduction (10, 17, 18). Moreover, it is reported that ~40%
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of convalescent patients do not have any neutralizing activity against B.1.351 nine months after primary
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infection (17). A recent study showed that although B.1.351 and P.1 have similar mutations in their RBD,
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sera from convalescent patients as well as mRNA vaccine recipients showed a neutralization reduction of
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~3-fold against P.1 while this was 7-13-fold for B.1.351 (20, 21).
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Although these studies have provided valuable initial insights in altered antigenic properties of
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these VOC, few studies have compared all three VOC side-by-side. Furthermore, comparing immune
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responses from vaccine recipients and individuals who have suffered from mild or severe COVID-19 as
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well as reactivity of NAbs to all three VOC within the same study will provide useful insights that can be
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used for additional surveillance of SARS-CoV-2 variants, the use of monoclonal antibodies for treatment
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and modifications of vaccines in order to increase immune coverage of immunity to include yet unknown
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emerging SARS-CoV-2 variants.
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Results
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Recognition of SARS-CoV-2 VOC by convalescent and vaccine sera is reduced
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Here, we assessed the impact of VOC B.1.1.7, B.1.351 and P.1 on humoral immunity elicited either by
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mRNA vaccination or by natural infection with SARS-CoV-2. To this end, we studied two cohorts of
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individuals: the COSCA cohort that included convalescent COVID-19 patients (n = 69) and the S3 cohort
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that included health care workers (HCW) who were vaccinated twice with the Pfizer/BioNTech COVID-19
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vaccine (n = 50) (Table 1). The COSCA cohort included hospitalized and non-hospitalized patients in the
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Netherlands who were enrolled and sampled four to six weeks after symptom onset (i.e. close to the
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expected peak of humoral immunity) between March 2020 and January 2021 (Table 1). Based on the
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rarity of B.1.351 and P.1 in the Netherlands, no individuals are expected to have been infected with either
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VOC. However, infections with B.1.1.7 became more prevalent towards the end of 2020, with the
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estimated prevalence of B.1.1.7 reaching 24% in the Netherlands in the week of the last inclusion
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(January 2021) (22). As genomic data was not collected as part of this study, we cannot exclude that a
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few patients were infected with B.1.1.7. The S3 cohort consists of health care workers without prior SARS-
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CoV-2 infection who received the approved Pfizer/BioNTech COVID-19 mRNA vaccine twice with a three
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week interval and sampled four weeks after the second vaccination (Table 1).
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We first assessed S protein binding titers of convalescent and vaccinee sera in a custom multiplex
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protein microarray that has been extensively validated for clinical use (table S1) (23). We generated S
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proteins, using previously described stabilization approaches, from all three VOC that had been identified
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at the time of assessment as well as a control wild-type (WT) S protein from the Wuhan Hu-1 virus
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(GenBank: MN908947.3) isolated in December 2019 (6, 24, 25). Overall, the antibody responses against
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each S protein were heterogeneous and differed up to ~1200-fold between the strongest and weakest
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responders. The recognition of the three VOC by convalescent patients was significantly reduced
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compared to WT by an average of 2.4-fold, 3-fold and 4-fold for B.1.1.7, B.1.351 and P.1, respectively (p <
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0.0001 for all, Fig. 1, B and C). Binding titers elicited by the mRNA vaccine were more homogeneous than
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those elicited by natural infection and differed ~10-fold between responders, with all participants having
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half-maximal binding titers (ED
50
s) exceeding 10
3
, except for one poor responder (Fig. 1B). Since the
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variability of binding titers in convalescent sera is considerable, we examined whether this variability was
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related to severity of disease (i.e. hospital admission, Fig. 1D). We observed a highly significant ~8-fold
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difference in ED
50
s between non-hospitalized patients and hospitalized patients, which is in line with
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previous reports of WT SARS-CoV-2 binding titers correlating with severity of disease (p < 0.0001) (26,
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27). This difference in binding titers between non-hospitalized patients and hospitalized patients was
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consistent for all three SARS-CoV-2 VOC studied (Fig. 1D). When comparing immune responses of
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vaccine recipients with convalescent sera, we observed similar S protein binding titers between vaccinee
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and hospitalized patients, which are an average ~4 to 11-fold higher compared to non-hospitalized
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patients for all VOC (Fig. 1D). Taken together, these data indicate that vaccine recipients as well as
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COVID-19 patients exhibit reduced binding to S proteins of the currently circulating VOC, with hospitalized
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patients and vaccine recipients exhibiting higher binding titers overall compared to non-hospitalized
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patients.
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SARS-CoV-2 VOC are substantially less sensitive to serum NAbs
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We next tested the neutralizing activity of convalescent and vaccinee sera (Fig. 2, table S1). To this end,
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we generated lentiviral-based pseudoviruses of the currently widespread SARS-CoV-2 D614G (WT)
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variant as well as B.1.1.7, B.1.351 and P.1. We detected substantial neutralizing activity against the WT
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virus (half maximal neutralization titer, ID
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> 100) in 96% of convalescent patients irrespective of
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hospitalization, and in all vaccine recipients, except the one poor responder (Fig. 2A). Indeed, the overall
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binding titers correlated well with the neutralization titers for all VOC (fig. S1C). Non-hospitalized patients
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had the most heterogeneous responses, with WT neutralizing titers differing by up to ~150-fold, while titers
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against WT were much more homogeneous in hospitalized patients (up to ~20-fold difference) and
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vaccine recipients (up to ~12-fold difference) (Fig. 2A). Consistent with the binding results, we observed a
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