Genomic evolution of SARS-CoV-2 variants of concern under in vitro neutralising selection pressure following two doses of the Pfizer-BioNTech BNT162b2 COVID-19 vaccine

Basile, K.; Agius, J. E.; Fong, W.; McPhie, K.; Fennel, M.; Ko, D.; Hueston, L.; Lam, C.; Arnott, A.; Chen, S. C.-A.; Maddocks, S.; O Sullivan, M. V. N.; Dwyer, D.; Sintchenko, V.; Kok, J.; Rockett, R. J.; CIDMLS COVID-19 Study Group,

doi:10.1101/2023.09.24.558921

bioRxiv · 10.1101/2023.09.24.558921

Genomic evolution of SARS-CoV-2 variants of concern under in vitro neutralising selection pressure following two doses of the Pfizer-BioNTech BNT162b2 COVID-19 vaccine

Basile, K.·Agius, J. E.·Fong, W.·McPhie, K.·Fennel, M.·Ko, D.·Hueston, L.·Lam, C.·Arnott, A.·Chen, S. C.-A.·Maddocks, S.·O Sullivan, M. V. N.·Dwyer, D.·Sintchenko, V.·Kok, J.·Rockett, R. J.·CIDMLS COVID-19 Study Group,

View original source ↗Export metadata ↓

Abstract

AimsTo explore viral evolution during in vitro neutralisation using next generation sequencing, and to determine whether sera from individuals immunised with two doses of the Pfizer-BioNTech vaccine (BNT162b2) are as effective at neutralising the SARS-CoV-2 variant of concern (VOC) Delta (B 1.617.2) compared to the earlier lineages Beta (B.1.351) and wild-type (A.2.2) virus. MethodsUsing a live-virus SARS-CoV-2 neutralisation assay in Vero E6 cells we determined neutralising antibody titres (nAbT) in 14 participants (vaccine-naive (n=2) and post-second dose of BNT162b2 vaccination (n=12), median age 45 years [IQR 29-65], median time after second dose = 21 days [IQR 19-28] against three SARS-CoV-2 strains: wild-type, Beta and Delta. The determination of nAbT was performed by visual inspection of cytopathic effect (CPE) and in-house quantitative reverse transcriptase real time quantitative polymerase chain reaction (RT-qPCR) to confirm SARS-CoV-2 replication. A total of 110 representative samples including inoculum, neutralisation breakpoints at 72 hrs, negative and positive controls underwent genome sequencing using the Respiratory Viral Oligo Panel version 2 (RVOP) (Illumina Inc. (San Diego, United States of America)) viral enrichment and short read sequencing using (Illumina Inc.San Diego, United States of America)(Figure 1). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=35 SRC="FIGDIR/small/558921v1_fig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@1f04650org.highwire.dtl.DTLVardef@1986fceorg.highwire.dtl.DTLVardef@d47ab9org.highwire.dtl.DTLVardef@1c524d5_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Outline of virus neutralisation assays of emerging SARS-CoV-2 variants of concern C_FIG ResultsThere was a significant reduction in nAbT observed against the Delta and Beta VOC compared with wild-type, 4.4-fold (p = >0.0006) and 2.3-fold (p = 0.0140), respectively (Figure 2). Neutralizing antibodies were not detected in one vaccinated immunosuppressed participant nor the vaccine-naive participants (n=2). The highest nAbT against the SARS-CoV-2 variants investigated was obtained from a participant who was vaccinated following SARS-CoV-2 infection 12 months prior (Table S1). Limited consensus level mutations occurred in the SARS-CoV-2 genome of any lineage during in vitro neutralisation, however, consistent minority allele frequency variants (MFV) were detected in the SARS-CoV-2 polypeptide, spike (S) and membrane protein. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/558921v1_fig2.gif" ALT="Figure 2"> View larger version (18K): org.highwire.dtl.DTLVardef@1f51ee8org.highwire.dtl.DTLVardef@1c03b61org.highwire.dtl.DTLVardef@17a77d2org.highwire.dtl.DTLVardef@15084f4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 2:C_FLOATNO Neutralising Antibody Titres Figure description: Differences in neutralising antibody titres (nAbT) against the Wildtype, Beta and Delta SARS-CoV-2 lineages median of 21 days (IQR 19-28) after receiving the second dose of Pfizer-BioNTech (BNT162b2) vaccine measured by visual inspection of cytopathic effect (CPE) with confirmation of viral replication by SARS-CoV-2 by inhouse reverse transcriptase real time quantitative polymerase chain reaction (RT-qPCR). Results are reported in the box-whiskers plots as medians and upper and lower quartiles. There was a significant fold reduction in nAbT observed between both the Delta (M = 4.4, SD = 2), t(11) =-4.9, p = .00059, and Beta (M=2.3, SD=2) t(11) =-3 p = .01397 compared with wild-type. There was also a significant fold reduction in nAbT between Beta (M = 2.6, SD = 1.4), t(11) =-2.5, p = .02897 and Delta. Key: Delta - Delta (B.1.617.2) lineage; Beta - Beta (B.1.351) lineage; Wildtype - Wildtype (A.2.2) lineage; C_FIG DiscussionSignificant reductions in nAbT post-vaccination were identified, with Delta demonstrating a 4.4-fold reduction. The reduction in nAbT for the VOC Beta has been previously documented, however, limited data is available on vaccine evasion for the Delta VOC, the predominant strain currently circulating worldwide at the time. Studies in high incidence countries may not be applicable to low incidence settings such as Australia as nAbT may be significantly higher in vaccine recipients previously infected with SARS-CoV-2, as seen in our cohort. Monitoring viral evolution is critical to evaluate the impact of novel SARS-CoV-2 variants on vaccine effectiveness as mutational profiles in the sub-consensus genome could indicate increases in transmissibility, virulence or allow the development of antiviral resistance.

Explore related subjects

microbiology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Basile, K., Agius, J. E., Fong, W., McPhie, K., Fennel, M., Ko, D., Hueston, L., Lam, C., Arnott, A., Chen, S. C.-A., Maddocks, S., O Sullivan, M. V. N., Dwyer, D., Sintchenko, V., Kok, J., Rockett, R. J., CIDMLS COVID-19 Study Group,. 2023-09-27. Genomic evolution of SARS-CoV-2 variants of concern under in vitro neutralising selection pressure following two doses of the Pfizer-BioNTech BNT162b2 COVID-19 vaccine. https://doi.org/10.1101/2023.09.24.558921

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Characterization of Lactobacillus rhamnosus-Derived Extracellular Vesicles and Their Cellular Cytoprotective Effects Against Trimethylamine-Induced Oxidative Stress in Caco-2 Cells

Dietary trimethylamine (TMA) underwent gut microbiota-mediated catabolism and subsequent hepatic oxidation to generate trimethylamine N-oxide (TMAO), a key metabolic factor linked to vascular and colorectal diseases. While the systemic effects of circulating TMAO were heavily investigated, the direct localized cellular interactions of its precursor, TMA, at the intestinal epithelial level required further elucidation. This study characterized extracellular vesicles isolated from Lactobacillus rhamnosus (Lr-EVs) and evaluated their cytoprotective potential in an in vitro human intestinal Caco-2 cell model under TMA-induced stress. Exposure to a high concentration of TMA (20 mM) significantly reduced Caco-2 viability to 78.8%, triggered intracellular reactive oxygen species (ROS) accumulation, and compromised tight junction integrity as observed via the disruption of Zonula Occludens-1 (ZO-1) alignment. Co-treatment with Lr-EVs (100-300 g/mL) counteracted this cellular toxicity, enhancing cell survival up to 172%. Quantitative PCR analysis revealed that Lr-EVs suppressed the transcriptional upregulation of the pro-inflammatory gene IL-1{beta}, restored the expression of the metabolic regulator SIRT1, and stabilized transcript levels of key mitochondrial-encoded electron transport chain genes (ND6, CYTB, CO1, and ATP6). Additionally, Lr-EVs exhibited intrinsic radical scavenging activities in cell-free ABTS and DPPH assays. These preliminary in vitro findings demonstrated that Lr-EVs mitigated TMA-induced cellular injury, oxidative stress, and junctional disruption at the transcriptomic level, supporting further investigation of probiotic-derived EVs as prospective cell-free functional ingredients.

microbiology↗

bioRxiv2026

Metagenomic Insights into the Fecal Microbiome, Resistome, and Mobilome of Beef Cattle in Cow-Calf Operations in Florida, United States

Food-producing animals are a focus of One Health approaches to antimicrobial resistance (AMR) as reservoirs of antimicrobial-resistant bacteria (ARB) and antimicrobial resistance genes (ARGs). We hypothesized that microbial composition, ARGs profiles, and mobile genetic element (MGE) profiles would differ by operation size and animal age group. Fecal samples from 162 cows and calves were combined into 54 composite samples, each comprising three animals of the same age group. Across operation sizes, Bacillota (51.4-64.9%), Bacteroidota (12.0-17.3%), Pseudomonadota (10.3-16.5%), and Actinomycetota (3.3-10.9%) were predominant phyla. Alpha diversity did not differ among operation sizes; however, cows exhibited greater observed richness, Shannon diversity, and Simpson diversity than calves (all P < 0.001). Microbial composition did not differ significantly among operation sizes (PERMANOVA, R{superscript 2} = 0.065, P = 0.609) but differed significantly between cows and calves (R{superscript 2} = 0.200, P < 0.001). Seventy-five ARGs representing 14 resistance classes were identified. Normalized ARG hit rates were higher in small operations (0.444) than in medium (0.278) and large operations (0.222; P = 0.088), whereas calves exhibited a higher estimated marginal mean normalized ARG hit rate than cows (0.383 vs. 0.247 hits/million reads; P < 0.001). Of 1,054 curated ARG hits, 23.9% occurred on predicted plasmid contigs, 0.9% on predicted viral contigs, and 7.1% on contigs containing insertion sequences. Overall findings demonstrate differences in fecal microbiome diversity and ARG abundance between cows and calves and indicate that predicted plasmids are associated with a notable proportion of ARG hits in Florida beef cow-calf operations.

microbiology↗

bioRxiv2026

A global post-transcriptional regulator directs Acinetobacter baumannii carbon preference and gut colonization

Acinetobacter baumannii is a multidrug-resistant pathogen that colonizes the gut, increasing the risk of subsequent infection and transmission. Recent work showed that A. baumannii catabolizes ornithine, a non-preferred carbon source, to colonize the gut. However, the mechanisms of carbon preference were unknown in A. baumannii. Here, experimental evolution identified the conserved RNA-binding protein CsrA as a regulator of carbon preference in A. baumannii, a previously unreported CsrA function. RNA immunoprecipitation sequencing (RIP-Seq) revealed hundreds of CsrA-associated transcripts. A. baumannii CsrA directly bound and repressed expression of the ast arginine/ornithine catabolic operons. The Pseudomonas aeruginosa CsrA homolog RsmA repressed arginine/ornithine catabolic operon expression, suggesting this CsrA function extends to other bacterial taxa. Finally, CsrA was required for competitive fitness during A. baumannii gut colonization. These findings uncover a previously unrecognized role for a global regulator in A. baumannii carbon preference and persistence in the gut, a critical reservoir for multidrug-resistant pathogens.

microbiology↗