key: cord-0268991-gyax5oi0 authors: Sievers, B. L.; Chakraborty, S.; Xue, Y.; Gelbart, T.; Gonzalez, J. C.; Cassidy, A. G.; Golan, Y.; Prahl, M.; Gaw, S. L.; Arunachalam, P. S.; Blish, C. A.; Boyd, S. D.; Davis, M. M.; Jagannathan, P.; Nadeau, K. C.; Pulendran, B.; Singh, U.; Scheuermann, R. H.; Frieman, M. H.; Vashee, S.; Wang, T. T.; Tan, G. S. title: Magnitude and breadth of neutralizing antibody responses elicited by SARS-CoV-2 infection or vaccination date: 2022-01-01 journal: nan DOI: 10.1101/2021.12.30.21268540 sha: 32477431aa0602aeb3af7372b2f36208fbe0bd4a doc_id: 268991 cord_uid: gyax5oi0 Multiple SARS-CoV-2 variants that possess mutations associated with increased transmission and antibody escape have arisen over the course of the current pandemic. While the current vaccines have largely been effective against past variants, the number of mutations found on the Omicron (B.1.529) spike appear to diminish the efficacy of pre-existing immunity. Using pseudoparticles expressing the spike of several SARS-CoV-2 variants, we evaluated the magnitude and breadth of the neutralizing antibody response over time in naturally infected and in mRNA-vaccinated individuals. We observed that while boosting increases the magnitude of the antibody response to wildtype (D614), Beta, Delta and Omicron variants, the Omicron variant was the most resistant to neutralization. We further observed that vaccinated healthy adults had robust and broad antibody responses while responses were relatively reduced in vaccinated pregnant women, underscoring the importance of learning how to maximize mRNA vaccine responses in pregnant populations. Findings from this study show substantial heterogeneity in the magnitude and breadth of responses after infection and mRNA vaccination and may support the addition of more conserved viral antigens to existing SARS-CoV-2 vaccines. First identified in Botswana in November 2021, the SARS-CoV-2 Omicron variant (B.1.1.529) is rapidly becoming the dominant circulating variant of concern (VOC)(1, 2). The Omicron virus harbors a striking 59 amino acid substitutions throughout its genome relative to the ancestral Wuhan-hu-1 SARS-CoV-2 virus, referred to as D614 here. Thirty-seven of these mutations are within the spike protein, the target of neutralizing antibody responses against this virus. As neutralizing antibodies are the major correlate of protection against coronavirus disease 2019 (COVID-19) (3, 4) , this degree of mutational change raises questions about the effectiveness of neutralizing antibodies that were elicited by infection with SARS-CoV-2 D614 infection or by current mRNA vaccines which encode the wildtype (WT) spike. To define the extent of escape by Omicron from neutralizing antibodies in the population, we evaluated the magnitude and breadth of the response against the D614 WT virus along with three VOCs, Beta (B.1.351), Delta (B.1.617.2) and Omicron (B.1.1.529). Understanding these neutralizing antibody responses will enable us to assess the state of pre-existing immunity elicited by the WT virus and can inform the design of the next generation of COVID-19 vaccines. The spike glycoprotein of SARS-CoV-2 has two major antigenic domains; mutations in these regions can contribute to antigenic escape and reduced immunity against infection (5) . The receptor binding domain (RBD) interacts directly with the receptor for SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2), and amino acid changes in RBD can impact the affinity of spike for ACE2 and thus transmissibility and virulence of viral variants. The Beta variant has notable mutations (L18F, D80A, D215G, ∆ 242-244 and R246L) in the amino terminal domain (NTD) and RBD (K417N, E484K and N501Y) (6) of the spike protein that is associated with antibody escape as previously reported (7) (8) (9) . The emergence of the Delta with changes in the RBD resulted in higher transmissibility and ultimately has become the predominant circulating strain of SARS-CoV-2 currently. Unlike Beta, Delta has only two mutations in the RBD (L452R, E484Q) relative to the WT virus that are associated with antibody escape (4) . Based on these different mutational profiles of the Beta and Delta spike proteins, we chose to include these VOCs in the present study along with Omicron. Omicron harbors a relative abundance of mutations with 37 non-synonymous changes in the spike alone, 11 in the NTD and 15 in the RBD. Based on the structural features of the Omicron spike and recent findings by other groups (10) (11) (12) (13) , we anticipated that Omicron would be at least as resistant to current neutralizing antibodies in the population as the Beta variant and likely far more resistant compared to the WT and Delta viruses. To study the relative susceptibility of the spikes of SARS-CoV-2 VOC to neutralizing antibodies in the population, we studied activity in sera or plasma from three cohorts of naturally infected or mRNA-vaccinated individuals against D614, Beta, Delta and Omicron VOCs. In a natural infection cohort, we tested plasma collected during the peak phase of mild COVID-19 (day 28 post study enrollment) and two time points during the convalescent period -at days 210 and 300 post study enrollment. To understand the breadth of neutralizing antibodies elicited by mRNA vaccination, we studied a cohort of pregnant individuals who received two doses of the Pfizer or Moderna vaccines during pregnancy; pregnancy is a risk factor for poor outcomes in COVID-19, thus this cohort provides important insights into immunity in this vulnerable population. A second cohort of healthcare workers received three doses of the Pfizer vaccine. These approved SARS-CoV-2 mRNA vaccines code for the original D614 SARS-CoV-2 spike protein. While mRNA vaccines have been extremely successful at inducing potent neutralizing antibody responses, their effectiveness will depend in large part to the degree of antigenic drift in circulating SARS-CoV-2 variants. Using these three distinct cohorts, we evaluated the magnitude and breath of neutralizing antibody titers over time after natural infection and mRNA vaccination. We first evaluated a total of 54 plasma samples from three study time-points for neutralizing antibody responses in a group of individuals from a longitudinal cohort of mild SARS-CoV-2 patients enrolled in an outpatient study at Stanford Hospital Center (14) . This study was a trial evaluating the efficacy of interferon lambda in mild COVID-19 but only subjects from the placebo arm have been studied here. Subjects in this study were infected with SARS-CoV-2 during the first half of 2020, a time when the D614 virus was the dominant circulating SARS-CoV-2 strain. Three time points were chosen for this analysis: day 28 post enrollment, the previously characterized peak antibody response, and days 210 and 300 post enrollment ( Figure 1A ) (15) . Neutralizing antibody titers were highest on day 28 for all VOCs and waned over time in the convalescent period on days 210 and 300 ( Figure 1B) . Notably, both Beta and Omicron VOC were much more resistant to neutralization even on day 28 compared to WT and Delta, perhaps reflecting the key mutations found in both the Beta and Omicron spike that have been previously described to contribute to antibody escape (8) . While neutralizing activity against D614 and Delta was measurable in many subjects at day 300, activity against the Beta and Omicron variants was largely absent by this later timepoint (Figures 1B, C, D) . Magnitude and breadth of the antibody response following vaccination All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. To understand the neutralizing antibody responses elicited by SARS-CoV-2 mRNA vaccines, we studied two separate cohorts of vaccinated individuals. One cohort comprised pregnant subjects that were enrolled in a vaccine study in the University of California San Francisco Health system (n=9 at baseline before vaccination and n=33 following 2 doses of mRNA vaccine)(16, 17). These subjects received either the Pfizer (n=10) or Moderna (n=23) mRNA vaccines during pregnancy. We tested paired neutralizing antibody responses in a subset of subjects (n=9) from serum taken before vaccination (baseline) and in serum collected after the second immunization (post-dose 2 (PD2)) ( Figure 2A) . As expected, the PD2 neutralizing titers were substantial against D614 (the homologous spike) after two vaccine doses, with a mean pNT50 of ~128 fold over baseline. Response at PD2 in all subjects were progressively lower than D614, with neutralizing titers against Delta (91x)>Beta (40x)>Omicron (10.2x) ( Figure 2B ). The magnitude of neutralizing titers against Delta, Beta and Omicron were all significantly reduced relative to D614 ( Figure 2C ). Substantial heterogeneity was present among vaccinees in the breadth of neutralizing antibody responses. This was evidenced in the wide range of ratios displayed by individuals in neutralizing titers against Delta, Beta and Omicron variants relative to D614 ( Figure 2D ). Finally, we evaluated the neutralizing antibody responses from a cohort of healthcare workers previously vaccinated with the Pfizer/BNT162b2 mRNA vaccine at the Stanford Hospital Center (n=137 samples from 4 study timepoints) (18, 19) . Four time points were chosen for this analysis. An early timepoint following the second dose (seven days post dose 2 or study day 28), followed by a late timepoint at study day 210 prior to dose 3 enabled us to study the durability of neutralizing responses after 2 doses of vaccine. In addition, we defined neutralizing responses on day 7 or between day 21-28 post dose 3 ( Figure 3A ). Neutralizing antibody titers were generally highest at the timepoints 7 days after dose 2 and dose 3 against the homologous D614 and all variants. As expected, titers had waned substantially against all variants by day 210 after dose 2. The third vaccine dose increased the titers against all variants substantially, to a level that matched, and in most cases surpassed, titers observed at 7 days post dose 2 ( Figure 3B ). By the day 21-28 post dose 3 timepoint, some individuals already had reduced neutralizing antibody levels while others maintained durable levels All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 January 1, 2022. ; approximating those observed on 7 days post dose 3. The antibody titers against Omicron were measurable in all subjects after dose 3 but were significantly lower than other variants (Figures 3C and D) . As in the pregnancy cohort, substantial heterogeneity was observed in the breadth of the neutralizing antibody responses, evidenced by the ratio of titers against variants to D614 (Figures 3C and D) . Using three longitudinal cohorts, we measured the magnitude and breath of the antibody response against WT, Delta, Beta and Omicron SARS-CoV-2 viruses following natural infection or mRNA vaccination. Infection during the early pandemic when D614 was the dominant circulating SARS-CoV-2 virus elicited a relatively low neutralizing antibody response against Beta and Omicron variants while immunity against Delta was not significantly reduced over D614. There was significant heterogeneity in this cohort with respect to both the magnitude and breadth of neutralizing responses against all variants tested. In two vaccine cohorts, we observed robust responses elicited by mRNA SARS-CoV-2 vaccines. In all three cohorts, responses against Beta and Omicron were reduced relative to the homologous D614 and Delta, as has been previously described (5) (6) (7) . In a cohort of healthcare workers where we were able to evaluate responses after 3 vaccine doses, there was clearly a benefit to both magnitude and breadth of the response conferred by the 3 rd dose. Most importantly, titers against Omicron were robust after dose 3 relative to dose 2. There was substantial heterogeneity in responses, particularly with respect to breadth, after 2 and 3 doses of mRNA vaccines. Determinants of broadly neutralizing antibody responses after homologous vaccine boosts are not well understood at this time and are an important topic to address in future studies. Because COVID-19 is a risk factor for adverse outcomes in pregnancy, it is critical to understand the response to mRNA vaccines in this population. Here, we show a relatively consistent response in the magnitude of response after two vaccine doses in this population but note that, while timepoints were not exactly matched with those in the healthy vaccine cohort, the titers were generally lower in pregnancy. Understanding optimal timing during pregnancy for booster doses will be important for protecting this population(20). Most approved vaccines rely solely on eliciting immune responses against a single immunogen, the spike. While clinical trials have demonstrated excellent effectiveness of mRNA vaccines up to now, it is evident that they will likely have somewhat reduced effectiveness against variants that are substantially antigenically drifted such as Omicron. As SARS-CoV-2 evolves, the efficacy of vaccines that relies on eliciting responses against a highly mutable antigen will always come into question. While it is noted that T-cell epitopes are generally not highly impacted by changes seen in the variants (21) as B cell epitopes we expect that supplementing spikebased vaccines with other more conserved viral antigens would elicit more potent and broad immunity against SARS-CoV-2 infections and COVID-19 (22). The role of non-neutralizing antibodies and T cells in immunity against VOCs warrants further study to better define all immune mechanisms, in addition to neutralizing antibodies, that can control viral replication (23-26) Characterization of these samples at Stanford was performed under a protocol approved by the Institutional Review Board of Stanford University (protocol #55718). Stanford Lambda cohort: 120 participants were enrolled in a phase 2 randomized controlled trial of Peginterferon Lambda-1a (Lambda, NCT04331899) Inclusion/exclusion criteria and the study protocol for the trial have been published (12) . Briefly, adults aged 18-75 years with an FDA emergency use authorized reverse transcription-polymerase chain reaction (RT-PCR) positive for SARS-CoV-2 within 72 hours prior to enrollment were eligible for study participation. Exclusion criteria included hospitalization, respiratory rate >20 breaths per minute, room air oxygen saturation <94%, pregnancy or breastfeeding, decompensated liver disease, recent use of investigational and/or immunomodulatory agents for treatment of COVID-19, and prespecified lab abnormalities. All participants gave written informed consent, and all study procedures were approved by the Institutional Review Board of Stanford University (IRB-55619). Participants were randomized to receive a single subcutaneous injection of Lambda or saline placebo. Peripheral blood was collected at enrollment, day 5, and day 28 post enrollment. A subset of participants (n=80) returned for long-term follow-up visits 4-, 7-, and 10-months post enrollment, with peripheral blood obtained. Longitudinal samples from the 56 SARS-CoV-2infected outpatients who were in the placebo arm of the broader Lambda study were obtained and assessed here. (Table 1) were introduced by primers to each amplicon which has 30-35 bp homologous sequences at each end to the adjacent fragments. These amplicons were digested with DpnI (NEB) to remove template DNA and purified by Qiagen PCR purification kit. 50 fmol of each amplicon and 15 fmol of YCP/BAC vector were covalently joined using standard Gibson assembly reaction (NEB), transformed into E.coli DH10B competent cells (Thermo Fisher), and plated on LB medium All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 January 1, 2022. ; https://doi.org/10.1101/2021.12.30.21268540 doi: medRxiv preprint with 12.5 mg/ml chloramphenicol. E.coli transformants were verified to contain correct mutations using PCR and Sanger sequencing (GeneWiz). Plasmids were isolated from E. coli by the Purelink HiPure Plasmid Midiprep Kit (Thermo Fisher). Primers used for spike gene construction and verification are listed in Table 2 . Lastly, the spike genes lacking the cytoplasmic domain by deleting the last 18 amino acides were then cloned into the pCAGGS expression vector. To generate VSV pseudotyped with SARS-CoV-2 S, we first Twenty-five microliters containing ~500 fluorescent forming units (FFUs) of a VSV encoding eGFP gene All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 January 1, 2022. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 January 1, 2022. ; https://doi.org/10.1101/2021.12.30.21268540 doi: medRxiv preprint (which was not certified by peer review) 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 January 1, 2022. ; (which was not certified by peer review) 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 January 1, 2022. Ratio of pNT50 of variants over D614 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 January 1, 2022. ; https://doi.org/10.1101/2021.12.30.21268540 doi: medRxiv preprint (which was not certified by peer review) 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 January 1, 2022. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 January 1, 2022. ; https://doi.org/10.1101/2021.12.30.21268540 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 January 1, 2022. ; https://doi.org/10.1101/2021.12.30.21268540 doi: medRxiv preprint . 3 5 1 L 1 8 F , D 8 0 A , D 2 1 5 G , R 2 4 6 I , K 4 1 7 N , E 4 8 4 K , N 5 0 1 Y , D 6 1 4 G , A 7 0 1 V B . 1 . 6 1 7 . 2 T 1 9 R , G 1 4 2 D , E 1 5 6 -, F 1 5 7 -, R 1 5 8 G , L 4 5 2 R , T 4 7 8 K , D 6 1 4 G , P 6 8 1 R , D 9 5 0 N B . 1 . 5 2 9 A 6 7 V , H 6 9 -, V 7 0 -, T 9 5 I , G 1 4 2 D , V 1 4 3 -, Y 1 4 4 -, Y 1 4 5 -, N 2 1 1 -, L 2 1 2 I , i n s 2 1 4 E P E , G 3 3 9 D , S 3 7 1 L , S 3 7 3 P , S 3 7 5 F , K 4 1 7 N , N 4 4 0 K , G 4 4 6 S , S 4 7 7 N , T 4 7 8 K , E 4 8 4 A , Q 4 9 3 R , G 4 9 6 S , Q 4 9 8 R , N 5 0 1 Y , Y 5 0 5 H , T 5 4 7 K , D 6 1 4 G , H 6 5 5 Y , N 6 7 9 K , P 6 8 1 H , N 7 6 4 K , D 7 9 6 Y , N 8 5 6 K , Q 9 5 4 H , N 9 6 9 K , L 9 8 1 F , T T G A G T T C T G G T T C T A A G A T T A A C A C A C T I n _ T 1 9 R _ F A G T G T G T T A A T C T T A G A A C C A G A A C T C A A T T I n _ G 1 4 2 D _ R C A C T T T C C A T C C A A C T T T T G T T G T T T T T G T G G T A A T T T T A T T C T A G T G C G I n _ L 4 5 2 R _ R G A T A G A T T T C A G T T G A A A T A T C T C T C T C A A A A G G T T T G A G A T T A G A C T T C C T A A A C A A T C T A T A C C G G T A A T T A T A A T T A C I n _ T 4 7 8 K _ F T T T T G A G A G A G A T A T T T C A A C T G A A A T C T A T T T T G 3 3 9 D _ R G C G T T A A A A A C T T C A T C A A A A G G G C A C A A G T G 3 3 9 D _ F A C T T G T G C C C T T T T G A T G A A G T T T T T A A C G C S 3 7 1 L S 3 7 3 P S 3 7 5 F _ R T A A C A C T T A A A A G T G A A A A A T G G T G C G A G A T T A T A T A G G A C A G A S 3 7 1 L S 3 7 3 P S 3 7 5 F _ F T C T G T C C T A T A T A A T C T C G C A C C A T T T T T C A C T T T T A A G T G T T A S A _ G 2 2 8 1 T T T G T T A C C G G C C T G A T A G A S 4 7 7 N -Y 5 0 5 H _ F A C A A A C C T T G T A A T G G T G T T G C A G G T T T T A A T T G T T A C T T T C C T T T A C G A T C A T A T A G T T T C C G A C C C A C T T A T G G T G T T G G T C A C C A A C C A T A C A G A G T T 5 4 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 January 1, 2022. S A _ A 2 3 4 0 3 G _ R T C T G T G C A G T T A A C A C C C T G A T A A A G A A C A G S A _ A 2 3 4 0 3 G _ F C T G T T C T T T A T C A G G G T G T T A A C T G C A C A G A H 6 5 5 Y _ R A T G A G T T G T T G A C A T A T T C A G C C C C T A T T A A H 6 5 5 Y _ F T T A A T A G G G G C T G A A T A T G T C A A C A A C T C A T N 6 7 9 K P 6 8 1 H _ R C T A C G T G C C C G C C G A T G A G A C T T A G T C T G A G T C T G A N 6 7 9 K P 6 8 1 H _ F T C A G A C T C A G A C T A A G T C T C A T C G G C G G G C A C G T A G N 7 6 4 K _ R T C C A G T T A A A G C A C G T T T T A A T T G T G T A C A A N 7 6 4 K _ F T T G T A C A C A A T T A A A A C G T G C T T T A A C T G G A D 7 9 6 Y _ R T A A A A C C A C C A A A A T A T T T A A T T G G T G G T G T D 7 9 6 Y _ F A C A C C A C C A A T T A A A T A T T T T G G T G G T T T T A N 8 5 T C T G T A A T G G T T C C A T T T T C H u 1 -2 T T T T T G T A C A C A A T T H u 1 -2 6 -R G A A A G T G T G C T T T T C C A T C A H u 1 -2 7 -F G C A T G T G A C T T A T G T C C C T G R C O 4 9 3 G T C T C A C C T A A A T A G C T T G G All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 January 1, 2022. ; https://doi.org/10.1101/2021.12.30.21268540 doi: medRxiv preprint Covid-19: Omicron may be more transmissible than other variants and partly resistant to existing vaccines, scientists fear Omicron SARS-CoV-2 variant: a new chapter in the COVID-19 pandemic Correlates of protection against symptomatic and asymptomatic SARS-CoV-2 infection Delayed production of neutralizing antibodies correlates with fatal COVID-19 Defining variant-resistant epitopes targeted by SARS-CoV-2 antibodies: A global consortium study The impact of spike mutated variants of SARS-CoV2 [Alpha, Beta, Gamma, Delta, and Lambda] on the efficacy of subunit recombinant vaccines The effect of spike mutations on SARS-CoV-2 neutralization Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7 Evidence of escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift. bioRxiv SARS-CoV-2 Omicron has extensive but incomplete escape of Pfizer BNT162b2 elicited neutralization and requires ACE2 for infection. medRxiv Booster of mRNA-1273 Vaccine Reduces SARS-CoV-2 Omicron Escape from Neutralizing Antibodies. medRxiv Plasma neutralization properties of the SARS-CoV-2 Omicron variant. medRxiv Peginterferon Lambda-1a for treatment of outpatients with uncomplicated COVID-19: a randomized placebo-controlled trial Divergent early antibody responses define COVID-19 disease trajectories. bioRxiv Correspondence and requests for materials should be addressed to T.T.W and G.S.T