key: cord-1002416-2qpngkqp
authors: Basile, Kerri; Rockett, Rebecca J; McPhie, Kenneth; Fennell, Michael; Johnson-Mackinnon, Jessica; Agius, Jessica E; Fong, Winkie; Rahman, Hossinur; Ko, Danny; Donavan, Linda; Hueston, Linda; Lam, Connie; Arnott, Alicia; Chen, Sharon C-A; Maddocks, Susan; O’Sullivan, Matthew V; Dwyer, Dominic E; Sintchenko, Vitali; Kok, Jen
title: Improved neutralization of the SARS-CoV-2 Omicron variant after Pfizer-BioNTech BNT162b2 COVID-19 vaccine boosting
date: 2021-12-13
journal: bioRxiv
DOI: 10.1101/2021.12.12.472252
sha: 8065e97609a7f4f31e7147189e408f3f344d4468
doc_id: 1002416
cord_uid: 2qpngkqp

In late November 2021, the World Health Organization declared the SARS-CoV-2 lineage B.1.1.529 the fifth variant of concern, Omicron. This variant has acquired 15 mutations in the receptor binding domain of the spike protein, raising concerns that Omicron could evade naturally acquired and vaccine-derived immunity. We utilized an authentic virus, multicycle neutralisation assay to demonstrate that sera collected 1, 3, and 6 months post-two doses of Pfizer-BioNTech BNT162b2 has a limited ability to neutralise SARS-CoV-2. However, four weeks after a third dose, neutralizing antibody titres are boosted. Despite this increase, neutralising antibody titres are reduced 4-fold for Omicron compared to lineage A.2.2 SARS-CoV-2.

In November 2021, the SARS-CoV-2 Omicron (B.1.1.529) variant of concern (VOC) emerged in Gauteng province, South Africa, coinciding with a rapid rise in COVID-19 cases. The World Health Organization designated Omicron a VOC two days following its identification. 1,2 There has been subsequent spread worldwide with Omicron cases now reported in over 50 countries. Early epidemiological reports from South Africa suggest that Omicron has an increased ability to evade prior infection-induced immunity compared with the Delta and Beta VOC. 1 Questions regarding Omicron's ability to evade vaccine-derived immunity were also raised following transmission between two vaccinated individuals whilst in hotel quarantine. 2 Omicron is characterized by over 30 non-synonymous mutations in the spike protein (e.g. E484A, K417N, P681H, N501Y, T478K), many of which are within key epitopes that provide SARS-CoV-2 an advantage over host immune responses, but these are yet to be fully quantified in vitro. Here we present data outlining the reduced ability of post-two and three dose vaccination sera to neutralize Omicron compared to Delta and wildtype lineages, but with improvement after a third vaccine dose.

where SARS-CoV-2 RNA was detected by reverse transcriptase real-time polymerase chain reaction (RT-PCR) were used to inoculate VeroE6 expressing transmembrane serine protease 2 (TMPRSS2) [VeroE6/TMPRSS2; JCRB1819] cells as previously outlined. 3 In brief, cells were seeded at 1-3x10 4 cells/cm 2 whilst in the log phase of replication with Dulbecco's minimal essential medium (DMEM) (Lonza) supplemented with 9% foetal bovine serum (FBS) (HyClone Cytiva) and Geniticin (1mg/mL, Gibco) in 25cm 2 cell culture flasks (Corning Inc). The media was changed within 12 hrs for inoculation media containing 1% FBS and 1% antimicrobials (amphotericin B deoxycholate 25 µg/mL, penicillin 10,000 U/mL and streptomycin 10,000 µg/mL) (Lonza) to prevent microbial overgrowth and then inoculated with 500μL of clinical specimen into 25cm 2 cell culture flasks. Routine mycoplasma testing was performed to exclude mycoplasma contamination of the cell lines and all manipulation of SARS-CoV-2 cultures were performed under biosafety level 3 (BSL3) conditions.

Cultures were inspected daily for cytopathic effect (CPE); the inoculum and supernatant were sampled at 96 hrs for SARS-CoV-2 in-house quantitative reverse transcriptase real time polymerase chain reaction (RT-qPCR) targeting the N-gene as previously described. 4 A decrease in the cycle threshold 

A previously described RT-PCR 5 , targeting the nucleocapsid gene was employed to estimate the viral load of the viral inoculum and post-neutralisation viral culture. Serial (10-fold) dilutions starting at 20,000 copies/µL to 2 copies/µL of a commercially available synthetic RNA control (Wuhan-1 strain, TWIST Biosciences NCBI GenBank accession MN908947.3) were used to generate a standard curve and quantify the viral load of each culture extract. The mean cycle threshold of biological replicates was used to calculate the viral load. A positive change in the viral load between the viral inoculum (0 hours) and 72 hours post-neutralisation was used to indicate positive viral replication.

Tiling PCR was used to amplify the entire SARS-CoV-2 genome from RNA extracts of clinical specimens using primers outlined in the Midnight sequencing protocol, the viral respiratory oligo panel (RVOP, Illumina) (Omicron), Artic v3 primers (Delta), or a previously described long amplicon methodology (Wild-type). 5,6 Each PCR included 12.5µL Q5 High Fidelity 2x Master Mix (New England Biolabs), 1.1µL of either pool 1 or pool 2 10µM primer master mix, 2.5µL of template RNA and molecular grade water was added to generate a total volume of 25µL. Cycling conditions were initial denaturation at 95°C for 2 min, then 35 cycles of: 95°C for 30s, 65°C for 2 min 45s, and a final extension step of 75°C for 10 min. Pool 1 and pool 2 amplicons were combined and purified with a 1:1 ratio of AMPureXP beads (Beckman Coulter) and eluted in 30µL of RNAase free water. Purified products were quantified using Qubit™ 1x dsDNA HS Assay Kit (Thermo Fisher Scientific) and diluted to the desired input concentration for library preparation. Sequencing libraries were prepared using Nextera XT (Illumina) according to the manufacturer's respective instructions and pooled with the aim of producing 1x10 6 reads per library. Sequencing libraries were then sequenced with paired end 76 bp chemistry on the iSeq or MiniSeq (Illumina) platforms.

Raw sequence data were processed using an in-house quality control procedure prior to further analysis as described previously. 5, 7 De-multiplexed reads were quality trimmed using Trimmomatic v0.36 (sliding window of 4, minimum read quality score of 20, leading/trailing quality of 5 and minimum length of 36 after trimming). 8 Briefly, reads were mapped to the reference SARS-CoV-2 genome (NCBI GenBank accession MN908947.3)

using Burrows-Wheeler Aligner (BWA)-mem version 0.7.17 9 , with unmapped reads discarded. Average genome coverage was estimated by determining the number of missing bases (Ns) in each sequenced genome. Variant calling and the generation of consensus sequences was conducted using iVar 10 

Neutralizing antibody titres (nAbT) in sera examined four weeks after a third dose of BNT162b2 were higher than nAbT measured at 1, 3, and 6 months after two doses of BNT162b2 ( Figure 1 , Table S1 ).

However, there was a 4-fold reduction in median nAbT against Omicron in contrast to the wild-type and 1.5-fold decrease of nAbT against Delta VOC following the third dose. Median nAbT after 1, 3, 6 months of BNT162b2 vaccination for all variants were documented with titers of <10 and <20 ( Figure   1 , Table S1 ). Trends in decreasing nAbT in sera collected 1, 3, and 6 month post-two doses of BNT162b2 were observed, comparable to decreasing trends in SARS-CoV-2-specific IgG levels determined by immunofluorescence antibody assays (IFA; Table S1, Figure S1 ). Increases in nAbT were noted four weeks after BNT162b2 boosters, with median titers of 240, 160 and 60 for wild-type, Delta and Omicron respectively. Although median responses were lower for Delta (p=0.28) and

Omicron (p=0.55), this reduction did not reach statistical significance.

Three lineages demonstrated comparable 50% tissue culture infective dose (TCID50) and viral load at inoculation ( Figure S2 ). Nevertheless, Omicron had a slower propagation rate with increases in viral load not detected in culture supernatant until 96 hours post-infection ( Figure S2 ). In contrast, wild-type and Delta cultures showed 4-5 log10 increase in viral load 72 hours post-inoculation ( Figure S3 ).

Omicron has rapidly evolved to encode over 30 protein substitutions in the SARS-CoV-2 spike protein, 

Network for Genomic Surveillance in South Africa (NGS-SA) SARS-CoV-2 Sequencing Update

SARS-CoV-2 Variant of Concern

Cell-based culture of SARS-CoV-2 informs infectivity and safe de-isolation assessments during COVID-19

mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants 2 3

SARS-CoV-2 Genome Sequencing Methods Differ in Their Abilities To Detect Variants from Low-Viral-Load Samples

Rapid and inexpensive wholegenome sequencing of SARS-CoV-2 using 1200 bp tiled amplicons and Oxford Nanopore Rapid Barcoding

Revealing COVID-19 transmission in Australia by SARS-CoV-2 genome sequencing and agent-based modeling

Trimmomatic: A flexible trimmer for Illumina sequence data

Fast and accurate short read alignment with Burrows-Wheeler transform

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

Stability of SARS-CoV-2 phylogenies

Assignment of epidemiological lineages in an emerging pandemic using the pangolin tool

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

The Antibody Response to SARS-CoV-2 Infection

SARS-CoV-2 inactivation testing: interim report

SARS-CoV-2 Omicron has extensive but incomplete escape of Pfizer BNT162b2 elicited neutralization and requires ACE2 for infection

BNT162b2 Vaccine Booster and Mortality Due to Covid-19

The authors acknowledge the technical assistance provided by the Sydney Informatics Hub, a Core Research Facility of the University of Sydney, the COVID Heroes (A serosurvey of healthcare workers