Rapidly Adaptable Multiplexed Yeast Surface Display Serological Assay for Immune Escape Screening of SARS-CoV-2 Variants (preprint)

With numerous variations in the Spike protein, including concentrated mutations in the receptor-binding domain (RBD), the SARS-CoV-2 Omicron variant significantly shifted in the trajectory of the COVID-19 pandemic. To understand individual patient risk profiles in the face of rapidly emerging variants, there is an interest in sensitive serological tests capable of analyzing patient IgG response to multiple variants in parallel. Here, we present a serological test based on yeast surface display and serum biopanning that characterizes immune profiles against SARS-CoV-2 RBD variants. We used this yeast-based multi-variant serology method to examine IgG titers from 30 serum samples derived from COVID-19-convalescent and vaccinated individuals in Switzerland and assessed the relative affinity of polyclonal serum IgG for Wuhan (B lineage), Delta (B.1.617.2 lineage), and Omicron (B.1.1.529 lineage) RBD domains. We validated and benchmarked our system against a commercial lateral flow assay and showed strong concordance. Our assay demonstrates that serum IgGs from patients recovered from severe COVID-19 between March-June 2021 bound tightly to both original Wuhan and Delta RBD variants, but became indistinguishable from background when assayed against Omicron, representing an affinity loss of >10-20 fold. Our yeast immunoassay is easily tailored and parallelized with newly emerging RBD variants.

Global surveillance of potential antiviral drug resistance in SARS-CoV-2: proof of concept focussing on the RNA-dependent RNA polymerase (preprint)

Antiviral treatments for COVID-19 have involved many repurposed drugs. Currently, SARS-CoV-2 RNA-dependent RNA polymerase (RdRp, encoded by nsp12-nsp7-nsp8) has been targeted by numerous inhibitors with debated clinical impact. Among these, remdesivir has been conditionally approved for the treatment of COVID-19 patients. Although the emergence of antiviral resistance, an indirect proxy for antiviral efficacy, poses a considerable healthcare threat, an evolutionary perspective on emerging resistant mutants is still lacking. Here we show that SARS-CoV-2 RdRp is under purifying selection, that potential escape mutations are rare, and unlikely to lead to viral fitness loss. In more than 56,000 viral genomes from 105 countries dating from December 2019 to July 2020 we found negative selective pressure affecting nsp12 (Tajimas D = -2.62), with potential antiviral escape mutations in only 0.3% of sequenced genomes. Those affected known key residues, such as Nsp12:Val473 and Nsp12:Arg555. Of the potential escape mutations found globally, in silico structural models show that this rarely implies loss of stability in RdRp. No potential escape mutation were found in our local cohort of remdesivir treated patients from the first wave (n=8). Our results indicate that RdRp is a suitable drug target, and that remdesivir does not seem to exert high selective pressure. Our study could be the starting point of a larger monitoring effort of drug resistance throughout the COVID-19 pandemic. We recommend the application of repetitive genome sequencing of SARS-CoV-2 from patients treated with antivirals to provide early insights into the evolution or antiviral resistance.

Epidemiology and precision of SARS-CoV-2 detection following lockdown and relaxation measures (preprint)

Introduction: SARS-CoV-2-detection is critical for clinical and epidemiological assessment of the ongoing CoVID-19 pandemic. Aim: To cross-validate manual and automated high-throughput (Roche-cobas6800-Target1/Target2) testing for SARS-CoV-2-RNA, to describe detection rates following lockdown and relaxation, and to evaluate SARS-CoV-2-loads in different specimens. Method: The validation cohort prospectively compared Basel-S-gene, Roche-E-gene, and Roche-cobas6800-Target1/Target2 in 1344 naso-oropharyngeal swabs (NOPS) taken in calendar week 13 using Basel-ORF8-gene-assay for confirmation. Follow-up-cohort-1 and -2 comprised 12363 and 10207 NOPS taken over 10 weeks until calendar week 24 and 34, respectively. SARS-CoV-2-loads were compared in follow-up NOPS, lower respiratory fluids, and plasma. Results: Concordant results were obtained in 1308 cases (97%) including 97 (9%) SARS-CoV-2-positives showing high quantitative correlations (Spearman r>0.95; p<0.001) for all assays. Discordant samples (N=36) had significantly lower SARS-CoV-2-loads (p<0.001). Following lockdown, weekly detection rates declined to <1% reducing single-test positive predictive values from 99.3% to 85.1%. Following relaxation, rates flared up to 4% with similarly high SARS-CoV-2-loads, but patients were significantly younger than during lockdown (34 vs 52 years, p<0.001). SARS-CoV-2-loads in follow-up NOPS declined by 3log10 copies/mL within 10 days post-diagnosis (p<0.001). SARS-CoV-2-loads in NOPS correlated weakly with those in time-matched lower respiratory fluids and plasma, but remained detectable in 14 and 7 cases of NOPS with undetectable SARS-CoV-2, respectively. Conclusion: Evaluated manual and automated assays are highly concordant and correlate quantitatively. Following successful lockdown, declining positive predictive values require dual-target-assays for clinical and epidemiologic assessment. Confirmatory and quantitative follow-up testing should be considered within <5 days, using lower respiratory fluids in symptomatic patients with SARS-CoV-2-negative NOPS.

SARS-CoV-2 phylogeny during the early outbreak in the Basel area, Switzerland: import and spread dominated by a single B.1 lineage variant (C15324T) (preprint)

Background: The first local case of SARS-CoV-2 in Basel, Switzerland, was detected on February 26th 2020. We present a phylogenetic cross-sectional study and explore viral introduction and evolution during the exponential early phase of the local COVID-19 outbreak from February 26th until March 23rd. Methods: We sequenced SARS-CoV-2 samples from naso-oropharyngeal swabs and generated 468 high quality genomes and called variants with our COVID-19 Genome Analysis Pipeline (COVGAP). We analysed viral genetic diversity using PANGOLIN taxonomic lineages. For identification of introduction and dissemination events across the Basel area a time-calibrated phylogeny was inferred including global SARS-CoV-2 genomes. Findings: Our samples exhibit low lineage diversity compared to neighbouring countries. Lineage B.1 (82.7%), detected from March 2nd, dominated infections in Basel. A large clade within B.1 contains 69.1% of our samples, all of which carry the SNP C15324T, suggesting local transmission in spreading events. We have located the geographic origin of this mutation in our tri-national region. The remaining genomes map broadly over the global phylogenetic tree, evidencing several events of introduction from and/or dissemination to other regions of the world. Further, we have identified several transmission events within families. Interpretation: Molecular surveillance of SARS-CoV-2 by phylogenetic reconstruction in the Basel area provides important insights into local transmission (spreading events and family transmission). This phylogenetic analysis enriches epidemiological and contact tracing data, allowing connection of seemingly unconnected events and drawing conclusions, which can be used to inform public health interventions. Funding: No dedicated funding was used for this work.

Effect of Systemic Inflammatory Response to SARS-CoV-2 on Lopinavir and Hydroxychloroquine Plasma Concentrations (preprint)

Background: Coronavirus disease 2019 (COVID-19) leads to inflammatory cytokine release, which can downregulate the expression of metabolizing enzymes. This cascade affects drug concentrations in the plasma. We investigated the association between lopinavir (LPV) and hydroxychloroquine (HCQ) plasma concentrations and the values of acute phase inflammation marker C-reactive protein (CRP). Methods: LPV plasma concentrations were prospectively collected in 92 patients hospitalized at our institution. Lopinavir/ritonavir was administered 12 hourly, 800/200 mg on day 1, and 400/100 mg on day 2 until day 5 or 7. HCQ was given at 800 mg, followed by 400 mg after 6, 24 and 48 hours. Hematological, liver, kidney, and inflammation laboratory values were analyzed on the day of drug level determination. Results: The median age of study participants was 59 (range 24 up to 85) years, and 71% were male. The median duration from symptom onset to hospitalization and treatment initiation was 7 days (IQR 4;10) and 8 days (IQR 5;10), respectively. The median LPV trough concentration on day 3 of treatment was 26.5 ug/mL (IQR 18.9;31.5). LPV plasma concentrations positively correlated with CRP values (r=0.37, p<0.001), and were significantly lower when tocilizumab was preadministrated. No correlation was found between HCQ concentrations and CRP values. Conclusions: High LPV plasma concentrations were observed in COVID-19 patients. The ratio of calculated unbound drug fraction to published SARS-CoV2 EC50 values indicated insufficient LPV concentrations in the lung. CRP values significantly correlated with LPV but not HCQ plasma concentrations, implying inhibition of cytochrome P450 3A4 (CYP3A4) metabolism by inflammation.