key: cord-0846925-83lrtcds
authors: Banerjee, Arinjay; Lew, Jocelyne; Kroeker, Andrea; Baid, Kaushal; Aftanas, Patryk; Nirmalarajah, Kuganya; Maguire, Finlay; Kozak, Robert; McDonald, Ryan; Lang, Amanda; Gerdts, Volker; Straus, Sharon E.; Gilbert, Lois; Li, Angel Xinliu; Mozafarihashjin, Mohammad; Walmsley, Sharon; Gingras, Anne-Claude; Wrana, Jeffrey L.; Mazzulli, Tony; Colwill, Karen; McGeer, Allison J.; Mubareka, Samira; Falzarano, Darryl
title: Immunogenicity of convalescent and vaccinated sera against clinical isolates of ancestral SARS-CoV-2, beta, delta, and omicron variants
date: 2022-04-14
journal: Med (N Y)
DOI: 10.1016/j.medj.2022.04.002
sha: ba62d6316b8b8e83123cea3093a009cf99937842
doc_id: 846925
cord_uid: 83lrtcds

Background SARS-CoV-2 omicron variant of concern (VOC) has evolved multiple mutations within the spike protein, raising concerns of increased antibody evasion. In this study, we assessed the neutralization potential of COVID-19 convalescent sera and sera from vaccinated individuals against ancestral SARS-CoV-2 and VOCs. Methods The neutralizing activity of sera from 65 coronavirus disease (COVID-19) vaccine recipients and convalescent individuals against clinical isolates of ancestral SARS-CoV-2, beta, delta, and omicron VOCs was assessed using a microneutralization assay. Findings. Convalescent sera from unvaccinated individuals infected by the ancestral virus demonstrated reduced neutralization against beta and omicron VOCs. Sera from individuals that received three doses of the Pfizer or Moderna vaccines demonstrated reduced neutralization of the omicron variant relative to ancestral SARS-CoV-2. Sera from individuals that were naturally infected with ancestral SARS-CoV-2 and subsequently received two doses of the Pfizer vaccine induced significantly higher neutralizing antibody levels against ancestral virus and all VOCs. Infection alone, either with ancestral SARS-CoV-2 or the delta variant was not sufficient to induce high neutralizing antibody titers against omicron. Conclusions In summary, we demonstrate that convalescent and vaccinated sera display varying levels of SARS-CoV-2 VOC neutralization. Data from this data will inform booster vaccination strategies against SARS-CoV-2 VOCs. Funding This research was funded by the Canadian Institutes of Health Research (CIHR). VIDO receives operational funding from the Government of Saskatchewan through Innovation Saskatchewan and the Ministry of Agriculture and from the Canada Foundation for Innovation through the Major Science Initiatives for its CL3 facility.

demonstrated reduced neutralization against beta and omicron VOCs. Sera from individuals that 49 received three doses of the Pfizer or Moderna vaccines demonstrated reduced neutralization of 50 the omicron variant relative to ancestral SARS-CoV-2. Sera from individuals that were naturally 51 infected with ancestral SARS-CoV-2 and subsequently received two doses of the Pfizer vaccine 52 induced significantly higher neutralizing antibody levels against ancestral virus and all VOCs. 53

Infection alone, either with ancestral SARS-CoV-2 or the delta variant was not sufficient to 54 induce high neutralizing antibody titers against omicron. 55

Conclusions. In summary, we demonstrate that convalescent and vaccinated sera display varying 56 levels of SARS-CoV-2 VOC neutralization. Data from this data will inform booster vaccination 57 strategies against SARS-CoV-2 VOCs. 58 SARS-CoV-2 has continued to evolve since its emergence in December 2019 1,2 . Variants of 65 SARS-CoV-2 that demonstrate potential for interference with diagnostics, therapies, and vaccine 66 efficacy, along with evidence for increased transmissibility or disease severity are termed 67 variants of concern (VOCs). The most recent VOC, omicron was first reported in November 68 2021 in Botswana and South Africa 3,4 . The omicron variant has evolved multiple mutations 69 within the spike protein and the receptor binding domain (RBD) that raise concerns regarding a 70 possible increased ability to evade pre-existing antibodies, both from prior infection and from 71 vaccination 5 . The omicron variant has demonstrated increased transmission and a higher level of 72 resistance to antibody-mediated neutralization 4,5 . However, little is known about its 73 pathogenicity and whether disease severity is altered in convalescent, vaccinated or unvaccinated 74 individuals. In Canada, long-term care (LTC) residents were prioritized for third vaccine doses 75 against SARS-CoV-2 based on the observation that antibody titers in older adults waned within 76 six months of their second vaccine dose 6,7 . The neutralizing potential of antibodies generated in 77 LTC residents against VOCs, such as delta and omicron after three doses of mRNA vaccines 78 remain unknown. Thus, to better assess the efficacy of antibody-mediated neutralization against 79 ancestral SARS-CoV-2 and VOCs (beta, delta, and omicron) in naturally infected and vaccinated 80 individuals, we collected sera from multiple cohorts and tested their neutralization ability against 81 clinical isolates of ancestral SARS-CoV-2 and VOCs. 82

J o u r n a l P r e -p r o o f determined (see supplementary Table S3 ). Serum samples were collected 1-2 months after the 109 date of onset of COVID-19 (see supplementary Table S3 ). Convalescent delta sera contained 110 lower levels of neutralizing antibodies against both beta and omicron variants, relative to the 111 delta variant ( Figure 2B ), while titers against ancestral SARS-CoV-2 and delta were comparable 112 ( Figure 2B ). These data suggest that infection with either ancestral SARS-CoV-2 or the delta 113 variant induces cross-neutralizing antibodies with comparable titers against both viruses. 114

However, natural infection with either ancestral SARS-CoV-2 or the delta variant induces lower 115 levels of neutralizing antibodies against both beta and omicron variants. 116 117

We determined levels of neutralizing antibodies in sera (n=10) from individuals who had 120 received two doses of the Pfizer BNT162b2 vaccine after being naturally infected with ancestral 121 SARS-CoV-2 (see supplementary Table S3 ). Two doses of the Pfizer BNT162b2 vaccine after 122 natural infection led to higher levels of neutralizing antibodies against ancestral SARS-CoV-2 123 that were significantly higher than levels against the beta (p=0.0265), delta (p=0.0334) and 124 omicron (p=0.0017) variants ( Figure 2C ). Indeed, infection and subsequent two dose vaccination 125 with Pfizer BNT162b2 induced higher levels of neutralizing antibodies against ancestral virus 126 and all VOCs ( Figure 2D ). 127 128 Neutralization of omicron by sera from individuals that received one dose of the Pfizer 129

To determine neutralizing antibody titers in sera from individuals that received one dose of the 131 Pfizer BNT162b2 mRNA vaccine, we collected sera (n=10) one month after the first dose of the 132 vaccine and tested neutralizing antibody titers against ancestral SARS-CoV-2, beta, delta and 133 omicron variants (see supplementary Table S3 ). Low neutralizing antibody titers against the 134 ancestral virus were detected in some samples; however, no neutralization of the three VOCs 135 was observed, except for one serum sample that had detectable levels of neutralizing antibodies 136 against all VOCs ( Figure 3A) . 137 138

Additional booster vaccinations have been deemed critical in protecting us from VOCs in part by 140 inducing higher levels of neutralizing antibodies. Thus, we tested the levels of neutralizing 141 antibodies in sera collected from long-term care residents that received three doses of the Pfizer 142 BNT162b2 (n=10) 9 or the Moderna mRNA-1273 (n=10) 10 vaccines (see supplementary Table  143 S3). For both vaccine recipients, doses 1 and 2 were received 3-4 weeks apart. The third vaccine 144 dose was received ~7 months after dose 2, and serum samples were collected 1 month after the 145 third dose. Sera from individuals that received three doses of the Pfizer BNT162b2 vaccine 146 induced high neutralizing titers against ancestral SARS-CoV-2, but levels of neutralizing 147 antibodies were significantly lower against beta (p=0.0030), delta (p=0.0142) and omicron 148 (p<0.0001) variants, compared to ancestral SARS-CoV-2 ( Figure 3B ). Sera from individuals that 149 received three doses of the Moderna mRNA-1273 vaccine induced high neutralizing titers 150 against ancestral SARS-CoV-2, but levels of neutralizing antibodies were significantly lower 151 against the beta (p=0.0326) and omicron (p=0.0033) variants, relative to ancestral SARS-CoV-2 152 ( Figure 3C ). Neutralizing antibody titers against ancestral SARS-CoV-2 were not significantly 153 different from the delta variant. Serum samples from individuals that received three doses of the 154 Pfizer BNT162b2 vaccine contained 2.89x, 2.25x and 10.9x lower mean neutralizing antibody 155 titers against beta, delta, and omicron VOCs, respectively, relative to ancestral SARS-CoV-2 156 ( Figure 4 ). Serum samples from individuals that received three doses of the Moderna mRNA-157 1273 vaccine contained 2.83x, 1.72x and 6.44x lower mean neutralizing antibody titers against 158 beta, delta, and omicron VOCs, respectively, relative to ancestral SARS-CoV-2 ( Figure 4) . 159

The emergence of yet another SARS-CoV-2 VOC, omicron has led to increasing speculation 162 about the ability of this variant to escape vaccine and natural infection-mediated immunity. The 163 current generation of COVID-19 mRNA vaccines are designed using the spike gene sequence of 164 ancestral SARS-CoV-2 9,10 . The omicron variant has accumulated 29 amino acid substitutions, 3 165 amino-acid deletions and a 3-residue insertion within the spike protein compared to the ancestral 166 SARS-CoV-2 Wuhan isolate 5 . Accumulating data suggest that the omicron variant is at least 167 partially resistant to neutralization by antibodies in vaccinated individuals, along with partial or 168 complete resistance to neutralization by therapeutic monoclonal antibodies 5 . Emerging data 169 demonstrate that T-cell-mediated immunity generated upon infection or vaccination likely 170 remain effective against the omicron variant 11 , and an additional booster vaccine dose results in 171 higher levels of antibodies against the omicron variant when tested using pseudotyped viruses 12 . 172

Despite these recent advances, considerable gaps currently exist in our knowledge regarding the 173 ability of omicron to cause severe COVID-19 and whether partial or complete escape of vaccine 174 or natural infection-mediated immunity occurs and if escape is age dependent. In addition, it is 175 not known if omicron has altered host range, or if transmissibility is increased and whether there 176 J o u r n a l P r e -p r o o f are changes in cellular tropism. Furthermore, data on neutralizing antibody titers against clinical 177 isolates of omicron are limited. Thus, as part of this study, we determined the levels of 178 neutralizing antibodies in individuals that were naturally infected, infected and subsequently 179 vaccinated, or vaccinated with three doses of mRNA vaccines using clinical isolates of ancestral 180 SARS-CoV-2, beta, delta and omicron variants. 181

When omicron was first detected, multiple laboratories reported difficulties in isolating 182 and generating laboratory stocks of this variant. In this study, we used Vero'76 cells to isolate 183 the omicron variant from a clinical specimen (nasopharyngeal swab) that was collected from a 184

Canadian patient. We also confirmed the whole genome sequences of the omicron variant, along 185 with beta and delta variants ( Figure 1 and Table 1 ). Thus, we report that Vero'76 cells are 186 sufficient to facilitate the isolation and propagation of the omicron variant. 187

Next, we tested the levels of neutralizing antibody titers in convalescent sera against the 188 ancestral virus, beta, delta, and omicron variants ( In summary, our data demonstrate that infection alone, either with ancestral SARS-CoV-206 2 or the delta variant is not sufficient to induce high levels of neutralizing antibodies against 207 omicron, a finding which was also recently reported by Rossler et al. 13 to assess both antibody-and cell-mediated immunity. We did not categorize data by sex, race, 231 age, or ethnicity. In the future, it will be important to identify whether sex, race, age, and 232 ethnicity play a significant role in natural and vaccine-mediated immunity against SARS-CoV-2 233 and VOCs. titer is reported as the average of the three replicates per sample. As a control, each virus isolate 438 that was diluted for our micro-neutralization assay was also back-titered using 2-fold serial 439 dilutions. This microneutralization assay was qualified for intra-assay reproducibility, 440 intermediate precision, accuracy, and robustness, including performance by multiple operators. 441

The assay reports lower overall titers than other 50% microneutralization, plaque-reduction 442 neutralization 50 and reporter pseudovirus assays, however, the assay may not be sensitive for 443 samples with very low neutralizing activity. 

A pneumonia outbreak associated with a new coronavirus 474 of probable bat origin

Evolutionary trajectory of SARS-CoV-2 and emerging variants

Omicron SARS-CoV-2 variant: a new chapter in 479 the COVID-19 pandemic

Rapid epidemic expansion of the 482 SARS-CoV-2 Omicron variant in southern Africa

Considerable escape of 485 SARS-CoV-2 Omicron to antibody neutralization

Neutralizing antibody 488 responses to SARS-CoV-2 variants in vaccinated Ontario long-term care home residents 489 and workers

Antibody Titers Before and After a Third Dose of the 492 SARS-CoV-2 BNT162b2 Vaccine in Adults Aged >/=60 Years

Isolation, Sequence, Infectivity, and 496 Replication Kinetics of Severe Acute Respiratory Syndrome Coronavirus 2. Emerging 497 infectious diseases 26

Safety and Efficacy of the 500 BNT162b2 mRNA Covid-19 Vaccine

Efficacy and Safety of the 504 mRNA-1273 SARS-CoV-2 Vaccine

SARS-CoV-2 spike T cell responses 508 induced upon vaccination or infection remain robust against Omicron

mRNA-based 512 COVID-19 vaccine boosters induce neutralizing immunity against SARS-CoV-2 513 Omicron variant

Omicron Variant Neutralization in Serum from Vaccinated and Convalescent Persons

Neutralizing 519 antibody levels are highly predictive of immune protection from symptomatic SARS-520

CoV-2 infection

CoV-2 require intact Fc effector functions for optimal therapeutic protection

Beta and Delta variants trigger Fc effector function with increased cross-reactivity

A single dose of the 531 SARS-CoV-2 vaccine BNT162b2 elicits Fc-mediated antibody effector functions and T 532 cell responses

mRNA-1273 vaccine-induced 535 antibodies maintain Fc effector functions across SARS-CoV-2 variants of concern

CoV-2 variants of concern partially escape humoral but not T-cell responses in COVID-540 19 convalescent donors and vaccinees

High Potency of a Bivalent Human VH 543 Domain in SARS-CoV-2 Animal Models

Genome Sequencing of SARS-CoV-2 Using Amplicon-Based Sequencing, Random 548 Hexamers, and Bait Capture

Persistence of serum and saliva antibody responses to 551 SARS-CoV-2 spike antigens in COVID-19 patients

Aligning sequence reads, clone sequences and assembly contigs with 556 BWA-MEM

Fast and accurate short read alignment with Burrows-558

Project Data Processing

An amplicon-based 565 sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq 566 and iVar

Haplotype-based variant detection from short-read 568 sequencing

A program for annotating and predicting the effects of single 571 nucleotide polymorphisms

Assignment of Epidemiological 574 Lineages in an Emerging Pandemic Using the Pangolin Tool

A dynamic nomenclature proposal for SARS-CoV-2 lineages to 578 assist genomic epidemiology

Data Structures for Statistical Computing in Python

Augur: a bioinformatics toolkit for phylogenetic 583 analyses of human pathogens

IQ-TREE 2: New Models and Efficient 586 Methods for Phylogenetic Inference in the Genomic Era

ETE 3: Reconstruction, Analysis, and 589 Visualization of Phylogenomic Data

Sera from SARS-CoV-2 convalescents have low neutralizing activity against omicron VOC 2. Two doses of mRNA vaccine following infection boosts neutralizing titers against all VOCs 3. Long-term care residents with three doses of mRNA vaccine neutralize all VOCs eTOC blurb

demonstrate that sera from convalescent individuals following ancestral SARS-CoV-2 or delta infection have low neutralizing activity against omicron. Three doses of mRNA vaccines induce detectable levels of neutralizing antibodies against all VOCs in long-term care residents. Omicron is more resistant to antibody-mediated neutralization compared to other VOCs

In this study, we quantified neutralizing antibody levels in convalescent and vaccinated sera against ancestral SARS-CoV-2 and VOCs. Convalescent sera had lower neutralizing antibody levels against the omicron VOC. Two doses of an mRNA vaccine following infection induced high levels of neutralizing antibodies against all VOCs, including omicron. Three doses of authorized mRNA vaccines induced detectable but lower levels of neutralizing antibodies against VOCs in long-term care residents. Data from our study, along with other published studies

race, ethnicity, and socioeconomic status was not collected. Cohort participants provided 384 informed consent for sharing of serum, and studies were approved by the Sunnybrook Research 385Institute (REB# 149-1994) and/or the Mount Sinai Hospital (REB# 02-0118-U, 20-0339-E, and 386 21-0069-E) Research Ethics Board 21 . For samples from each cohort, samples were selected to 387 have a representative range of anti-spike trimer and anti-RBD antibodies as measured by 388 enzyme-linked immunosorbent assay 22 . 389 390

Sequencing and bioinformatic analyses. cDNA was synthesized from extracted RNA. In brief, 392 4 µL LunaScript RT SuperMix 5X (New England Biolabs, NEB, USA) and 8 µL nuclease free 393 water, were added to 8 µL extracted RNA. cDNA synthesis was performed using the following 394 conditions: 25 °C for 2 min, 55 °C for 20 min, 95 °C for 1 min, and holding at 4 °C. 395Amplicons were generated from cDNA using ARTIC V4 primer pools 396 (https://github.com/artic-network/artic-ncov2019). Two multiplex PCR tiling reactions were 397 prepared by combining 2.5 µL cDNA with 12.5 µL Q5 High-Fidelity 2X Master Mix (NEB, 398 USA), 6µL nuclease free water, and 4 µL of respective 10 µM ARTIC v4 primer pool 399