key: cord-0748371-5zhjn3u2
authors: Walls, Alexandra C.; Sprouse, Kaitlin R.; Bowen, John E.; Joshi, Anshu; Franko, Nicholas; Navarro, Mary Jane; Stewart, Cameron; Cameroni, Elisabetta; McCallum, Matthew; Goecker, Erin A.; Degli-Angeli, Emily J.; Logue, Jenni; Greninger, Alex; Corti, Davide; Chu, Helen; Veesler, David
title: SARS-CoV-2 breakthrough infections elicit potent, broad and durable neutralizing antibody responses
date: 2022-01-20
journal: Cell
DOI: 10.1016/j.cell.2022.01.011
sha: df7aa697e3cd6a40f193ac88e4ec39e948001d16
doc_id: 748371
cord_uid: 5zhjn3u2

Although infections among vaccinated individuals lead to milder COVID-19 symptoms relative to unvaccinated subjects, the specificity and durability of antibody responses elicited by breakthrough cases remain unknown. Here, we demonstrate that breakthrough infections induce serum binding and neutralizing antibody responses that are markedly more potent, durable and resilient to spike mutations observed in variants than those in subjects who received only two doses of vaccine. However, we show that breakthrough cases, subjects who were vaccinated after infection and individuals vaccinated three times have serum neutralizing activity of comparable magnitude and breadth, indicating that increased number of exposures to SARS-CoV-2 antigen(s) enhance the quality of antibody responses. Neutralization of SARS-CoV was moderate, however, underscoring the importance of developing vaccines eliciting broad sarbecovirus immunity for pandemic preparedness.

The SARS-CoV-2 Delta (B.1.617.2) variant of concern emerged at the end of 2020 and became dominant globally by mid-2021. Mutations in the spike (S) glycoprotein Walls et al., 2020a; Wrapp et al., 2020) and in the nucleoprotein (N) have been suggested to account for its enhanced transmissibility, replication kinetics and viral loads in oropharyngeal and nose-throat swabs of infected individuals relative to the ancestral Wuhan-Hu-1 virus and other variants Liu et al., 2021b; Mlcochova et al., 2021; Saito et al., 2021; Syed et al., 2021) . Moreover, multiple S mutations in the N-terminal domain and receptor-binding domain have been shown to promote immune evasion (McCallum et al., 2021a (McCallum et al., , 2021b Mlcochova et al., 2021; Suryadevara et al., 2021; Ying et al., 2021) . These characteristics combined with waning of serum neutralizing antibody titers over time in vaccinated individuals has resulted in breakthrough infections that are usually associated with much milder symptoms than infection of unvaccinated individuals (Levine-Tiefenbrun et al., 2021; Mlcochova et al., 2021) .

Understanding the magnitude and breadth of immune responses following breakthrough infections is key to guiding vaccination policies and pandemic preparedness efforts . Serum neutralizing antibody titers represent the current best correlate of protection against SARS-CoV-2 in animal challenge studies (Arunachalam et al., 2021; Case et al., 2020a; Corbett et al., 2021; Hassan et al., 2021; Khoury et al., 2021; McMahan et al., 2021; Winkler et al., 2020) and multiple clinical trials have shown the benefits of therapeutic administration of monoclonal antibodies in humans . Furthermore, serum neutralizing antibodies are used in ongoing comparative clinical trials as key success metrics for the next generation of vaccines (e.g., NCT05007951 and NCT04864561 comparing GBP510 and VLA2001 to AZD1222, respectively). To understand whether the sequence of infection and/or vaccination as well as repeated exposures alter the specificity, magnitude, and breadth of antibody responses, we followed and compared serum antibodies in individuals who were vaccinated, previously infected and then vaccinated, or vaccinated and then infected predominantly with the SARS-CoV-2 Delta variant.

We compared serum binding titers following infection, vaccination, or both in groups of ~15 individuals enrolled through the HAARVI longitudinal cohort study at the University of Washington in Seattle ( Table S1 ). The breakthrough group (13/16 confirmed Delta infection-see methods) was composed of n=1 Johnson and Johnson Ad26.COV2.S, n=1 Covishield (Oxford-Astrazeneca), n=5 Moderna mRNA-1273 and n=9 Pfizer Cominarty vaccinees. For the infected then vaccinated (infected/vaccinated) cohort, all samples were obtained prior to September 2020, indicating these infections were likely with Washington-1-like isolates (according to outbreaks.info), and comprised n=1 Jansen Ad26.COV2.S, n=3 Moderna mRNA-1273 and n=11 Pfizer Cominarty vaccinees. The vaccinated-only group was made up of n=3 Moderna mRNA-1273 and n=12 Pfizer Cominarty vaccinees ( Table S1 ). Benchmarking of these samples was carried out with human convalescent plasma (HCP) which were collected prior to October 2020 in Washington State (all samples were obtained prior to July 2020 except one which was drawn in September 2020), indicating these infections were likely with Washington-1-like isolates (Table  S1 ). Eight individuals from the infected/vaccinated cohort and eight individuals from the vaccinated-only group received a third vaccine dose. All these samples were compared to plasma from SARS-CoV-2 naive individuals, who had blood drawn prior to vaccination (Table S1) , as J o u r n a l P r e -p r o o f confirmed by the lack of SARS-CoV-2 nucleocapsid (N) reactivity using the Roche anti-N immuno assay (only convalescent samples were positive) (Table S1) .

Serum IgG, IgA, and IgM binding titers were evaluated using ELISAs with the SARS-CoV-2 Hexapro S antigen . The cohorts were followed longitudinally for up to 180 days after initial blood draws to evaluate differences in durability of antibody responses. Responses were highest amongst individuals who were exposed to SARS-CoV-2 S three or four times through vaccination or a combination of infection and vaccination. The magnitude of IgG responses for vaccinated individuals who experienced a breakthrough infection was greatest 30 days post-positive PCR test and reduced ~3 fold by day 60 ( Figure 1A , Table 1, Table S2 , Data S1). Infected/vaccinated individuals had binding GMTs which peaked 10 days after receiving a second vaccine dose, dropped ~2 fold at day 112 and remained stable at day 180 ( Figure 1A , Table 1, Table S2 , Data S1). Peak binding titers were ~1.6 fold lower than the breakthrough samples at 30 days post infection. Following a third vaccination, the infected/vaccinated individuals experienced a 3.6-fold rise of binding GMT relative to day 10 post second shot ( Figure  1A , Table 1, Table S2 , Data S1). Vaccinated-only individuals, who received 2 doses, had binding GMTs 12 and ~7.5 fold lower than breakthrough and infected/vaccinated individuals at peak timepoints (30 days post positive PCR or 10 days post second vaccination, respectively). Binding titers were reduced 1.7 and 3 fold at 112 and 180 days after the second vaccination, respectively. Following administration of a third vaccine dose, we observed a 27 fold binding GMT increase for vaccinated-only individuals relative to 10 days post second vaccination ( Figure 1A , Table 1,  Table S2 , Data S1). Peak binding GMT for infected-only individuals (30 days post positive PCR) was well below all other groups, suggesting infection alone or a single exposure do not induce as robust antibody responses as compared to multiple vaccinations or a mixture of vaccination and infection . SARS-CoV-2 naive individuals had very low responses overall (at the limit of detection of our assay) ( Figure 1A , Table 1, Table S2 , Data S1). These data suggest that repeated exposures through vaccination, infection or a combination of both induce potent polyclonal serum antibody binding titers. Furthermore, we observed a greater durability of binding GMTs over 180 days for the infected/vaccinated relative to the vaccinated-only (two doses) cohorts, which could result from increased number of exposures, different exposure spacing (Parry et al. 2021) , or due to actual infection versus vaccination-only.

Peak S-specific IgA binding GMTs for breakthrough cases, infected/vaccinated individuals, vaccinated-only individuals and for HCP followed a trend roughly similar to IgG binding titers ( Figure 1B , Data S1). Conversely, IgM binding titers were approximately an order of magnitude higher for HCP relative to other cohorts ( Figure 1C , Data S1), presumably as a result of extensive immunoglobulin class switching and affinity maturation occurring for the latter cases, mirroring findings made with memory B cells in vaccinated individuals (Cho et al., 2021) . Collectively, these data indicate that breakthrough infections, vaccination following infection or vaccination only trigger similarly robust anamnestic responses.

To assess how vaccination and infection affect the breadth of binding antibody responses, we performed ELISAs using prefusion diproline-stabilized (2P) SARS-CoV S as antigen (Kirchdoerfer et al., 2018; Pallesen et al., 2017) . We determined binding GMTs for breakthrough cases, infected/vaccinated individuals, and vaccinated-only subjects at 30 days post PCR positive test or 10 days post second vaccination. Infected/vaccinated individuals had higher SARS-CoV S binding GMTs compared to breakthrough and vaccinated-only subjects ( Figure 1D , Data S1). Upon receiving a third immunization, SARS-CoV S-specific responses increased 1.5 fold and 9.5 fold for infected/vaccinated and vaccinated-only subjects, respectively. We observed the lowest SARS-CoV S binding responses for HCP, and for SARS-CoV-2 naive individuals (limit of detection). These results suggest that antibody cross-reactivity within the sarbecovirus subgenus improves with repeated exposures to SARS-CoV-2 S ( Figure 1D , Data S1). We also investigated antibody cross-reactivity beyond the sarbecovirus subgenus using the prefusion-stabilized S trimers of the common-cold causing embecoviruses OC43 (Tortorici et al., 2019) and HKU1 as ELISA antigens. We determined that GMTs were not markedly enhanced with repeated SARS-CoV-2 S exposures relative to SARS-CoV-2 naive individuals ( Figure 1E -F, Data S1). These findings show that exposure to SARS-CoV-2 S does not enhance emebecovirus cross-reactive antibody responses substantially in the cohorts examined, even compared to SARS-CoV-2 naive individuals who might have been previously exposed to seasonal coronaviruses.

To understand the magnitude and durability of neutralizing antibody responses among the different groups, we determined serum neutralizing activity for all samples obtained longitudinally using vesicular stomatitis virus (VSV) pseudotyped with SARS-CoV-2 G614 S using VeroE6-TMPRSS2 target cells (Kaname et al., 2010; Lempp et al., 2021) . Breakthrough infections resulted in potent neutralizing activity for up to 60 days following positive PCR test (Figure 2A Table 2, Table S2 , Data S1). Infected/vaccinated individuals had similarly potent neutralizing activity and a 2-fold neutralizing activity decay after 180 days (Figure 2A Table 2, Table S2 , Data S1). Ten days following a third vaccination, neutralizing GMT for infected/vaccinated individuals increased 2.5 fold relative to ten days following their second dose (Figure 2A Table 2, Table S2 , Data S1). Vaccinated-only (2 doses) individuals had serum neutralizing activity ~10 fold lower than breakthrough or infected/vaccinated individuals at peak timepoints, which decayed 3-fold after 180 days (Figure 2A Table 2, Table S2 , Data S1). Following a third vaccination, neutralizing GMT for vaccinated-only individuals rose 8.4 fold compared to 10 days after receiving the second dose, reaching levels comparable to breakthrough cases 30 days after infection and to infected/vaccinated subjects 10 days post second vaccine dose (Figure 2A Table 2, Table S2 , Data S1). HCP had weak neutralizing potency, in line with their low binding titers (Figure 2A  Table 2, Table S2 , Data S1). These data suggest that the number of exposures to S is more important than the type of exposure (infection vs. vaccination) to determine the magnitude of serum neutralizing activity. These findings are in agreement with studies of individuals infected and subsequently vaccinated with mRNA vaccines or Jansen Ad26.COV2.S (Keeton et al., 2021; Krammer et al., 2021; Saadat et al., 2021; Stamatatos et al., 2021; Tan et al., 2021a) . However, the durability of neutralizing antibody responses for breakthrough and infected/vaccinated cases set these cohorts apart from vaccinated-only individuals who experienced a decay of ~65% over 6 months after the second vaccine dose. It remains to be determined if waning of binding and neutralizing GMTs will occur with similar kinetics after the third immunization of vaccinated-only subjects than after two doses or if they will mirror the durable antibody responses observed in breakthrough and infected/vaccinated cohorts. Hence, multiple types of exposure or increased number of exposures to SARS-CoV-2 antigen(s) enhance the magnitude and durability of serum neutralizing activity possibly due to sustained antibody production by long lived plasma cells, increased affinity maturation yielding antibodies with greater neutralization potency, or a combination of both (Cho et al., 2021; Turner et al., 2021a Turner et al., , 2021b .

We also observed durable neutralizing activity for the breakthrough serum samples against both Delta S and Beta S VSV pseudoviruses ( Figure 2B -C Table 2, Table S2 , Data S1). Neutralizing GMTs increased against Delta and Beta from 30 to 60 days post positive PCR test. The dampening in neutralization potency relative to G614 S VSV for both Delta S (1.2-1.4 fold GMT reduction) and Beta S (2.8-3.0 fold GMT reduction) was smaller than previous findings with vaccinated-only or convalescent cohorts (Cele et al., 2021; Madhi et al., 2021; McCallum et al., 2021a; Planas et al., 2021a; Walls et al., 2021a; Wibmer et al., 2021) . For infected/vaccinated subjects, we determined that neutralizing GMTs against Delta were reduced 1.8-2.0 fold relative to G614 S VSV (Figure 2A -B, F Table 2, Table S2 , Data S1). After receiving a third vaccine dose, these individuals experienced a 2.0 fold increase in neutralizing titers against Delta S VSV, corresponding to a 2.3 fold GMT reduction relative to G614 S VSV (Figure 2A -B, F Table 2,  Table S2 , Data S1). Therefore, although the magnitude of neutralizing GMT increased, the breadth of broadly neutralizing antibody responses remains similar against Delta S VSV ( Figure  2A -B). Beta S VSV neutralizing GMTs for infected/vaccinated individuals were reduced 3.1-3.8fold, compared to G614 S VSV, and were of overall similar magnitude to those of breakthrough cases. Following the third vaccination, these individuals had a 2.0 fold increase in neutralizing titers against Beta S VSV, yielding a 3.9 fold GMT reduction relative to G614 S VSV (Figure 2A -B, F Table 2, Table S2 , Data S1). Prior to boosting, this cohort experienced a ~2 fold reduction of neutralizing GMTs over 180 days against both Delta S and Beta S VSV pseudoviruses, mirroring neutralization of G614 S VSV (Figure 2-C, F Table 2, Table S2 , Data S1). The vaccinated-only cohort had neutralizing GMTs against Delta S VSV that were reduced 1.7-5 fold relative to G614 S VSV, and of an overall magnitude ~20 fold lower than breakthrough or infected/vaccinated subjects ( Figure 2B ,F Table 2, Table S2 , Data S1). Triple vaccinated-only subjects displayed an 18.8 fold increase compared to the same individuals after receiving only two vaccine doses at the same time point, bringing their neutralizing GMTs close to other individuals who had three exposures ( Figure 2B , F Table 2, Table S2 , Data S1). Beta S VSV pseudovirus neutralizing GMTs for vaccinated-only (2 doses) individuals were 4-5.1 fold lower relative to G614 S VSV ( Figure 2C ,F Table 2, Table S2 , Data S1). Triple vaccinated-only subjects displayed a 9.0 fold neutralizing GMT increase compared to the same time point after receiving only two vaccine doses, consistent with the overall enhancement of neutralizing titers against all pseudoviruses tested.

The SARS-CoV-2 Omicron variant was first detected in November 2021 and was shown to evade antibody-mediated immunity to an unprecedented level due to 37 S amino acid mutations (Cameroni et al., 2021; Cao et al., 2021; Liu et al., 2021a; Planas et al., 2021b; VanBlargan et al., 2021) . We therefore set out to evaluate serum neutralizing activity of breakthrough, infected/vaccinated, or triple vaccinated individuals against Omicron. Breakthrough cases experienced a ~7-8 fold decrease in neutralization potency compared to G614 VSV S ( Figure 2D , F Table 2, Table S2 , Data S1). Infected/vaccinated individuals experienced a ~6.6 fold decrease in neutralization capacity after 10 days, 14 fold decrease after 112 days, and an 18 J o u r n a l P r e -p r o o f fold decrease after 180 days compared to G614 VSV S ( Figure 2D , F Table 2, Table S2 , Data S1Vaccinated-only (2 doses) subjects experienced ≥11-22-fold reductions of neutralizing GMTs against Omicron versus G614 S VSV, which are likely underestimates due to the limit of detection of our assay (Figure 2D ,F Table 2, Table S2 , Data S1). Following a third vaccination, we observed enhanced neutralizing activity against Omicron for both infected/vaccinated subjects (1.3 fold GMT rise compared to 10 days post second dose) and vaccinated-only individuals (16 fold GMT rise compared to 10 days post second dose) ( Figure 2D , F Table 2, Table S2 , Data S1), in agreement with recent data (Garcia-Beltran et al., 2021) . The latter group had neutralizing GMT against Omicron S VSV after 3 doses which is comparable to that against G614 S VSV after 2 doses. No Omicron neutralization was detected for HCP ( Figure 2D , F Table 2, Table S2 , Data S1), which concurs with other studies (Cameroni et al., 2021) . These data suggest that repeated exposures, even to a distinct antigen, improves the potency and resilience of serum neutralizing activity to variants, including against Beta S and Omicron S VSV, in line with previous studies of infected/vaccinated individuals (Keeton et al., 2021; Stamatatos et al., 2021) . Finally, we observed a subtle advantage for subjects who experienced both infections and vaccination (irrespective of the order) in terms of neutralization breadth against SARS-CoV-2 variants relative to triple vaccinated-only individuals.

To assess the breadth of serum neutralizing activity among these different cohorts against viruses belonging to a different sarbecovirus clade than SARS-CoV-2, we used a SARS-CoV S VSV pseudotype. Individuals with three mixed exposures had neutralizing GMTs reductions of 24-37 fold (breakthrough) (Figure 2E -F Table 2, Table S2 , Data S1) and 28-46 fold (infected/vaccinated) ( Figure 2E -F Table 2, Table S2 , Data S1) compared to G614 S VSV, which is approximately twice the magnitude of immune evasion observed with Omicron (Cameroni et al., 2021) . After receiving a third vaccine dose, infected/vaccinated individuals had ~2.5 fold enhanced serum neutralizing responses against SARS-CoV S VSV relative to the same subjects at 10 days post second vaccination ( Figure 2E -F Table 2, Table S2 , Data S1). Although it amounts to a 27 fold reduction compared to peak neutralizing G614 S VSV GMT, this neutralizing activity approaches that of vaccinated-only individuals against G614 S VSV after 2 doses (Figures 2A, E-F) . The vaccinated-only subjects had low neutralizing activity until receiving a third vaccine dose yielding GMT matching those of breakthrough cases or of infected/vaccinated individuals (after two vaccinations) (Figure 2E-F Table 2, Table S2 , Data S1), further suggesting that the number of exposures is more important than the type of exposure to determine the overall magnitude and breadth of serum neutralizing activity. No SARS-CoV neutralization was detected for HCP, in line with the weak neutralizing activity of this cohort even against SARS-CoV-2 ( Figure  2E -F Table 2, Table S2 , Data S1).

We show that breakthrough cases or infected/vaccinated subjects are endowed with greater potency, breadth and durability of serum neutralizing activity relative to individuals who received 2 doses of COVID-19 vaccine or those who were infected only by SARS-CoV-2 in 2020. Serum neutralizing GMTs for the breakthrough cases and infected/vaccinated subjects were greater against the Delta and Beta S variants at all time points than that of vaccinated-only individuals (after two doses) against G614 S pseudovirus at peak titers. The latter levels of neutralization potency were associated with ~95% protection from symptomatic COVID-19 disease in phase 3 clinical trials of the Pfizer Cominarty (Polack et al., 2020) and Moderna mRNA-1273 (Baden et al., 2021) vaccines and are reminiscent of observations made with infected/vaccinated samples (Stamatatos et al., 2021) . The marked enhancement of serum neutralizing activity for vaccinated-only subjects after receiving three doses, to levels comparable to breakthrough and infected/vaccinated cases, suggest that the number of exposures and/or delay between exposures to SARS-CoV-2 S, whether through vaccination or infection, correlates with the strength of neutralizing antibody responses as well as resilience to variants. It remains to be determined whether the number of exposures also correlates with durability of antibody responses for vaccinated-only individuals, as shown here with breakthrough cases up to 60 days and infected/vaccinated subjects for 180 days. The reduction of serum neutralizing activity against SARS-CoV presented here following up to 3 SARS-CoV-2 S exposures, along with previous studies (Martinez et al., 2021; Stamatatos et al., 2021; Walls et al., 2021a) , suggest that combinations of COVID-19 disease and vaccination would leave the population more vulnerable to infection by a genetically divergent sarbecovirus, due to the more extensive sequence differences between SARS-CoV-2 S and SARS-CoV S compared to any two SARS-CoV-2 S variants. However, four exposures to SARS-CoV-2 S, through infection followed by three vaccinations, elicit SARS-CoV serum neutralizing titers with a magnitude that is equivalent to protective levels for SARS-CoV-2 based on clinical trial evaluation of COVID-19 vaccines (Baden et al., 2021; Polack et al., 2020) . These findings suggest that repeated exposures may improve elicitation of broadly neutralizing sarbecovirus antibodies, but not broadly reactive betacoronavirus antibodies based on the comparable cross-reactive responses to OC43 and HKU1 S observed for all cohorts. Moreover, a recent study indicated that survivors of SARS-CoV infection who subsequently received a COVID-19 vaccine had broader sarbecovirus neutralizing antibody responses than subjects only exposed to SARS-CoV-2 virus or vaccine (Tan et al., 2021b) . These data lend further support to the ongoing development of several vaccine candidates (Cohen et al., 2021; Martinez et al., 2021; Walls et al., 2021a) designed to specifically elicit broad sarbecovirus immunity and could protect against SARS-CoV-2 variants as well as putative future zoonotic sarbecoviruses.

Although this study uses pseudotyped virus assays, rather than authentic virus, very good correlation was established between the two assays and with protection from challenge (Arunachalam et al., 2021; Case et al., 2020b) . This study is based on cohorts of 15 individuals or fewer and may be limited by sample size. Finally, we emphasize that our study is focused on serum antibody responses and that other aspects of the immune system participate in controlling viral infections, including memory B cells and T cells. Table S1 . Statistical significance was determined by Kriskal Wallis and Dunn's multiple comparisons test and is shown in Table S2 Table S1 . Statistics shown in Table S2 . Normalized curves and fits are shown in Data S1. Figures 1A and 2 Data S1: Raw data fits and curves of ELISA and Neutralization plots, related to Figures 1 and 2 Star Methods:

Lead contact:

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, David Veesler (dveesler@uw.edu).

Materials generated in this study will be made available on request after signing a materials transfer agreement with the University of Washington. This work is licensed under a Creative CommonsAttribution 4.0 International (CC BY 4.0) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/. This license does not apply tofigures/photos/artwork or other content included in the article that is credited to a third party;obtain authorization from the rights holder before using such material.

Data and Code availability: Data generated in this study are available upon request.

Cell lines: Expi293F (derived from 293 cells which are female) cells are derived from the HEK293F cell line (Life Technologies). Expi293F cells were grown in Expi293 Expression Medium (Life Technologies), cultured at 36.5°C with 8% CO2 and shaking at 130 rpm. HEK293T/17 is a female human embryonic kidney cell line (ATCC). The VeroE6-TMPRSS2 cell line is an African Green monkey Kidney cell line expressing TMPRSS2 (Lempp et al. 2021) . All adherent cells were cultured at 37°C with 5% CO2 in flasks with DMEM + 10% FBS (Hyclone) + 1% penicillinstreptomycin. Cell lines were not tested for mycoplasma contamination nor authenticated.

Human Subjects Sera/Plasma: Blood samples were collected from participants as part of the Hospitalized or Ambulatory Adults with Respiratory Viral Infections (HAARVI) study and was approved by the University of Washington Human Subjects Division Institutional Review Board (STUDY00000959). Baseline socio-demographic and clinical data for these individuals are summarized in Data S1. All samples were sera except for the convalescent and 60 day breakthrough samples which were ACD plasma. Naive samples were obtained from INGM, Ospedale Maggio Policlinico of Milan and approved by the local review board Study Polimmune.

Sequencing of Breakthrough Individuals: Participants were enrolled after SARS-CoV-2 positive RT-PCR results that showed S-gene dropout or delay using a primer set that predates the S probe set in the "TaqPath" test kit sold by Thermo Fisher. Specifically, the Delta S 157-158 deletion causes S-gene dropout in our diagnostic RT-qPCR assay. The RT-qPCR assay uses probes for human RNase P and SARS-CoV-2 Orf1b and S gene. S-gene dropout or delay was identified using the following individual sample criteria: 1) internal control RNase P amplification was present, 2) Orf1b amplification was present, and 3) S-gene specific probes showed no amplification or a delay of >5 cycles compared to Orf1b due to the 156-157 deletion in delta. Three samples (278C, 284C, and 286C) included in this analysis were either not sequenced due to low volumes or failed sequencing due to low viral loads, but according to outbreaks.info over 90% of all cases in Washington state at the time were Delta SARS-CoV-2.

SARS-CoV-2 genome sequencing was then conducted using a targeted enrichment approach. SARS-CoV-2 was detected using a laboratory-developed test (LDT) or research assay. For the LDT, SARS-CoV-2 detection was performed using real-time RT-PCR with a probe sets targeting Orf1b and S with FAM fluor (Life Technologies 4332079 assays # APGZJKF and #APXGVC4) multiplexed with an RNaseP probe set with VIC or HEX fluor (Life Technologies A30064 or IDT custom) each in duplicate on a QuantStudio 6 instrument (Applied Biosystems). RNA from positive specimens was converted to cDNA using random hexamers and reverse transcriptase (Superscript IV, Thermo) and a sequencing library was constructed using the Illumina TruSeq RNA Library Prep for Enrichment kit (Illumina). The sequencing library was enriched for SARS-CoV-2 using the Twist Respiratory Virus Research Panel (Twist). Libraries were sequenced on a MiSeq or NextSeq instruments. The resulting reads were assembled against the SARS-CoV-2 reference genome Wuhan-Hu-1/2019 (Genbank accession MN908947) using the bioinformatics pipeline https://github.com/seattleflu/assembly. Consensus sequences were deposited to Genbank and GISAID (Bedford et al., 2020) . Sequence quality was determined using Nextclade version 1.0.0-alpha.8 (https://clades.nextstrain.org/). Lineage was assigned using the Pangolin COVID-19 Lineage Assigner version 3.1.11 (https://pangolin.cog-uk.io/) (Paredes et al., 2021) Roche Anti-N Analysis: 0.5mL serum/plasma samples were tested in CAP/CLIA-accredited clinical laboratory using the FDA-authorized Roche Elecsys Anti-SARS-CoV-2 for anti-nucleocapsid antibodies using manufacturer's established positivity cut-off index of ≥1.0 (Theel et al., 2021) .

The SARS-CoV-2 Hexapro S , SARS-CoV S 2P (Pallesen et al., 2017; Walls et al., 2020b) , HKU1 S2P, and OC43 S (Tortorici et al., 2019) ectodomains were produced in Expi293F Cells (ThermoFisher Scientific) grown in suspension using Expi293 Expression Medium (ThermoFisher Scientific) at 37°C in a humidified 8% CO2 incubator with constant rotation at 130 rpm. Cells grown to a density of 3 million cells per mL were transfected using the ExpiFectamine 293 Transfection Kit (ThermoFisher Scientific) and cultivated for four days prior to supernatent harvest. SARS-CoV-2 Hexapro S, SARS-CoV S2P, and HKU1 S2P ectodomains were purified from clarified supernatants using a HisTrapHP column (Cytiva) and washed with 10 column volumes of 25 mM sodium phosphate pH 8.0 and 150 mM NaCl before elution on a gradient up to 500 mM imidazole. OC43 S, fused to a strep tag, supernatant was clarified by 10 minute centrifugation at 800xg and brought to 100 mM Tris-HCL pH 8, 150 mM NaCl, and 18.1 mL/liter biotin blocking solution (BioLock). After a 20-minute incubation, supernatant was further centrifuged at 10,000xg for 20 minutes. Supernatant was then bound to a 1 mL StrepTrap HP column (Cytiva) and washed with 10 column volumes of 100 mM Tris-HCl, 150 mM NaCl, and 1 mM EDTA pH 8 before elution in wash buffer supplemented with 2.5 mM desthiobiotin. Purified protein was buffer exchanged into 20 mM Tris-HCl pH 8.0 and 100 mM NaCl, concentrated using 100 kDa MWCO centrifugal filters (Amicon Ultra) to 1-2 mg/mL, and flash frozen.

J o u r n a l P r e -p r o o f ELISA: For anti-S ELISA, 50 μL of 2-6 μg/mL S was plated onto 384-well Nunc Maxisorp (ThermoFisher) plates in PBS and sealed overnight at 4°C. The next day plates were washed 4 × in Tris Buffered Saline Tween (TBST-20mM Tris pH 8, 150mM NaCl, 0.1% Tween) using a plate washer (BioTek) and blocked with Casein (ThermoFisher) for 1 h at 37°C. Plates were washed 4 × in TBST and 1:5 serial dilutions of human sera or plasma were made in 50 μL TBST starting at 1:10, 1:50, or 1:250 and incubated at 37°C for 1 h. Plates were washed 4 × in TBST, then anti-human (Invitrogen) horseradish peroxidase-conjugated antibodies were diluted 1:5,000 and 50 μL added to each well and incubated at 37°C for 1 h. Plates were washed 4 × in TBST and 50 μL of TMB (SeraCare) was added to every well for 5 min at room temperature. The reaction was quenched with the addition of 50 μL of 1 N HCl. Plates were immediately read at 450 nm on a VarioSkanLux plate reader (ThermoFisher) and data plotted and fit in Prism (GraphPad) using nonlinear regression sigmoidal, 4PL, X is log(concentration) to determine EC50 values from curve fits. Where the curve did not reach an OD450 of 4, a constraint of OD450 4 was placed on the upper bounds of the fit. VSV Pseudovirus Production: G614 SARS-CoV-2 S (YP 009724390.1)(Crawford et al. 2020), Delta S, Beta S, Omicron S, and SARS-CoV S pseudotyped VSV viruses were prepared as described previously (McCallum et al., 2021a; Walls et al., 2021b) . Briefly, HEK293T cells in DMEM supplemented with 10% FBS, 1% PenStrep seeded in 10-cm dishes were transfected with the plasmid encoding for the corresponding S glycoprotein using lipofectamine 2000 (Life Technologies) following the manufacturer's instructions. One day post-transfection, cells were infected with VSV(G*ΔGluciferase) (Kaname et al., 2010) and after 2 h were washed five times with DMEM before adding medium supplemented with anti-VSV-G antibody (I1-mouse hybridoma supernatant, CRL-2700, ATCC). Virus pseudotypes were harvested 18-24 h post-inoculation, clarified by centrifugation at 2,500 x g for 5 min, filtered through a 0.45 μm cut off membrane, concentrated 10 times with a 30 kDa cut off membrane, aliquoted and stored at -80°C. VSV Pseudovirus Neutralization: VeroE6-TMPRSS2 (Lempp et al., 2021) were cultured in DMEM with 10% FBS (Hyclone), 1% PenStrep and 8 µg/mL puromycin (to ensure retention of TMPRSS2) with 5% CO2 in a 37°C incubator (ThermoFisher). Cells were trypsinized using 0.05% trypsin and plated to 40,000 cells/well. The following day, cells were checked to be at 80% confluence. In an empty half-area 96-well plate, a 1:3 serial dilution of sera was made in DMEM and diluted pseudovirus was then added and incubated at room temperature for 30-60 min before addition of the sera-virus mixture to the cells at 37°C. 2 hours later, 40 μL of a DMEM solution containing 20% FBS and 2% PenStrep was added to each well. After 17-20 hours, 40 μL/well of One-Glo-EX substrate (Promega) was added to the cells and incubated in the dark for 5-10 min prior to reading on a BioTek plate reader. Measurements were done at least in duplicate using distinct batches of pseudoviruses and one representative experiment is shown. Relative luciferase units were plotted and normalized in Prism (GraphPad). Nonlinear regression of log(inhibitor) versus normalized response was used to determine IC50 values from curve fits. Normality was tested using the J o u r n a l P r e -p r o o f D'agostino-Pearson test and in the absence of a normal distribution, kruskal-wallis tests were used to compare two groups to determine whether differences reached statistical significance. Fold changes were determined by comparing individual animal IC50 and then averaging the individual fold changes for reporting.

8-15 subjects were included in each group as detailed in Table S1 . Experiments were all done in at least biologic duplicates. Normality was tested using the D'agostino-Pearson test and in the absence of a normal distribution, Kruskal Wallis tests were used to compare two groups to determine whether differences reached statistical significance. Significance is indicated with stars: * , p < 0.05; * * * * p < 0.0001 and non-significant groups are not shown. Fold changes were determined by comparing individual animal IC50 and then averaging the individual fold changes for reporting.

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SARS-CoV-2 spike conformation determines plasma neutralizing activity

Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift

Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies

Replication-Competent Vesicular Stomatitis Virus Vaccine Vector Protects against SARS-CoV-2-Mediated Pathogenesis in Mice

Neutralizing Antibody and Soluble ACE2 Inhibition

Replication-Competent VSV-SARS-CoV-2 and a Clinical Isolate of SARS-CoV-2

Escape of SARS-CoV-2 501Y.V2 from neutralization by convalescent plasma

Anti-SARS-CoV-2 receptor binding domain antibody evolution after mRNA vaccination

Mosaic nanoparticles elicit cross-reactive immune responses to zoonotic coronaviruses in mice

Immune Responses in Fully Vaccinated Individuals Following Breakthrough Infection with the SARS-CoV-2 Delta Variant in

Immune correlates of protection by mRNA-1273 vaccine against SARS-CoV-2 in nonhuman primates

Tackling COVID-19 with neutralizing monoclonal antibodies

mRNA-based COVID-19 vaccine boosters induce neutralizing immunity against SARS-CoV-2 Omicron variant

A single intranasal dose of chimpanzee adenovirus-vectored vaccine protects against SARS-CoV-2 infection in rhesus macaques

Structure-based design of prefusion-stabilized SARS-CoV-2 spikes

Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis

Acquisition of complement resistance through incorporation of CD55/decay-accelerating factor into viral particles bearing baculovirus GP64

Prior infection with SARS-CoV-2 boosts and broadens Ad26.COV2.S immunogenicity in a variant-dependent manner

Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection

Stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis

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

Lectins enhance SARS-CoV-2 infection and influence neutralizing antibodies

Viral loads of Delta-variant SARS-CoV-2 breakthrough infections after vaccination and booster with BNT162b2

Viral infection and transmission in a large

Striking antibody evasion manifested by the Omicron variant of SARS-CoV-2

Delta spike P681R mutation enhances SARS-CoV-2 fitness over Alpha variant

Efficacy of the ChAdOx1 nCoV-19 Covid-19 Vaccine against the B.1.351 Variant

Chimeric spike mRNA vaccines protect against Sarbecovirus challenge in mice

Molecular basis of immune evasion by the Delta and Kappa SARS-CoV-2 variants

N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV

Correlates of protection against SARS-CoV-2 in rhesus macaques

SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion

Structure-based design of a highly stable, covalently-linked SARS-CoV-2 spike trimer with improved structural properties and immunogenicity

Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen

Associations between SARS-CoV-2 variants and risk of COVID-19 hospitalization among confirmed cases

Sensitivity of infectious SARS-CoV-2 B.1.1.7 and B.1.351 variants to neutralizing antibodies

Considerable escape of SARS-CoV-2 Omicron to antibody neutralization

Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine

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

Enhanced fusogenicity and pathogenicity of SARS-CoV-2 Delta P681R mutation

mRNA vaccination boosts crossvariant neutralizing antibodies elicited by SARS-CoV-2 infection

Neutralizing and protective human monoclonal antibodies recognizing the N-terminal domain of the SARS-CoV-2 spike protein

Rapid assessment of SARS-CoV-2 evolved variants using virus-like particles

Ad26.COV2.S or BNT162b2 Boosting of BNT162b2 Vaccinated Individuals

Pan-Sarbecovirus Neutralizing Antibodies in BNT162b2-Immunized SARS-CoV-1 Survivors

SARS-CoV-2 Serologic Assays Dependent on Dual-Antigen Binding Demonstrate Diverging Kinetics Relative to Other Antibody Detection Methods

Structural basis for human coronavirus attachment to sialic acid receptors

SARS-CoV-2 mRNA vaccines induce persistent human germinal centre responses

SARS-CoV-2 infection induces long-lived bone marrow plasma cells in humans

1.1.529 Omicron virus escapes neutralization by several therapeutic monoclonal antibodies

Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus Fusion

Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines

Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines. bioRxiv

SARS-CoV-2 501Y.V2 escapes neutralization by South African COVID-19 donor plasma

Human neutralizing antibodies against SARS-CoV-2 require intact Fc effector functions and monocytes for optimal therapeutic protection

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Protective activity of mRNA vaccines against ancestral and variant SARS-CoV-2 strains. bioRxiv

We thank Hideki Tani (University of Toyama) for providing the reagents necessary for preparing VSV pseudotyped viruses. We thank Antonio Lanzavecchia for coordination of the SARS-CoV-2 naive blood donors. We thank Christina Lockwood and Lea Starita for their assistance in providing the sequencing data for participant identification. This study was supported by the National Institute of Allergy and Infectious Diseases (DP1AI158186 and HHSN272201700059C to D.V.), a Pew Biomedical Scholars Award (D.V.), an Investigators in the Pathogenesis of Infectious Disease Awards from the Burroughs Wellcome Fund (D.V.),

J o u r n a l P r e -p r o o f

ETOC Blurb: Individuals with breakthrough COVID-19 infection, those previously infected/vaccinated individuals those vaccinated thrice have potent and durable serum binding and neutralizing antibody responses against variants of concern, including Omicron. Neutralization of SARS-CoV, however, was moderate thus urging the need for developing broad vaccines for pandemic preparedness.