The oral protease inhibitor nirmatrelvir is of key importance for prevention of severe coronavirus disease 2019 (COVID-19). To facilitate resistance monitoring, we studied severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) escape from nirmatrelvir in cell culture. Resistant variants harbored combinations of substitutions in the SARS-CoV-2 main protease (Mpro). Reverse genetics revealed that E166V and L50F + E166V conferred high resistance in infectious culture, replicon, and Mpro systems. While L50F, E166V, and L50F + E166V decreased replication and Mpro activity, L50F and L50F + E166V variants had high fitness in the infectious system. Naturally occurring L50F compensated for fitness cost of E166V and promoted viral escape. Molecular dynamics simulations revealed that E166V and L50F + E166V weakened nirmatrelvir-Mpro binding. Polymerase inhibitor remdesivir and monoclonal antibody bebtelovimab retained activity against nirmatrelvir-resistant variants, and combination with nirmatrelvir enhanced treatment efficacy compared to individual compounds. These findings have implications for monitoring and ensuring treatments with efficacy against SARS-CoV-2 and emerging sarbecoviruses.

Capsid virus-like particles (cVLP) have proven safe and immunogenic and can be a versatile platform to counter pandemics. We aimed to clinically test a modular cVLP COVID-19 vaccine in individuals who were naive to SARS-CoV-2. In this phase 1, single-centre, dose-escalation, adjuvant-selection, open-label clinical trial, we recruited participants at the Radboud University Medical Center in Nijmegen, Netherlands, and sequentially assigned them to seven groups. Eligible participants were healthy, aged 18-55 years, and tested negative for SARS-CoV-2 and anti-SARS-CoV-2 antibodies. Participants were vaccinated intramuscularly on days 0 and 28 with 6 μg, 12 μg, 25 μg, 50 μg, or 70 μg of the cVLP-based COVID-19 vaccine (ABNCoV2). A subgroup received MF59-adjuvanted ABNCoV2. Follow-up was for 24 weeks after second vaccination. The primary objectives were to assess the safety and tolerability of ABNCoV2 and to identify a dose that optimises the tolerability-immunogenicity ratio 14 days after the first vaccination. The primary safety endpoint was the number of related grade 3 adverse events and serious adverse events in the intention-to-treat population. The primary immunogenicity endpoint was the concentration of ABNCoV2-specific antibodies. The trial is registered with ClinicalTrials.gov, NCT04839146. 45 participants (six to nine per group) were enrolled between March 15 and July 15, 2021. Participants had a total of 249 at least possibly related solicited adverse events (185 grade 1, 63 grade 2, and one grade 3) within a week after vaccination. Two serious adverse events occurred; one was classified as a possible adverse reaction. Antibody titres were dose-dependent with levels plateauing at a vaccination dose of 25-70 μg ABNCoV2. After second vaccination, live virus neutralisation activity against major SARS-CoV-2 variants was high but was lower with an omicron (BA.1) variant. Vaccine-specific IFNγ⁺ CD4⁺ T cells were induced. Immunisation with ABNCoV2 was well tolerated, safe, and resulted in a functional immune response. The data support the need for additional clinical development of ABNCoV2 as a second-generation SARS-CoV-2 vaccine. The modular cVLP platform will accelerate vaccine development, beyond SARS-CoV-2. EU, Carlsberg Foundation, and the Novo Nordisk Foundation.

Nirmatrelvir, which targets the SARS-CoV-2 main protease (Mpro), is the first-in-line drug for prevention and treatment of severe COVID-19, and additional Mpro inhibitors are in development. However, the risk of resistance development threatens the future efficacy of such direct-acting antivirals. To gain knowledge on viral correlates of resistance to Mpro inhibitors, we selected resistant SARS-CoV-2 under treatment with the nirmatrelvir-related protease inhibitor boceprevir. SARS-CoV-2 selected during five escape experiments in VeroE6 cells showed cross-resistance to nirmatrelvir with up to 7.3-fold increased half-maximal effective concentration compared to original SARS-CoV-2, determined in concentration–response experiments. Sequence analysis revealed that escape viruses harbored Mpro substitutions L50F and A173V. For reverse genetic studies, these substitutions were introduced into a cell-culture-infectious SARS-CoV-2 clone. Infectivity titration and analysis of genetic stability of cell-culture-derived engineered SARS-CoV-2 mutants showed that L50F rescued the fitness cost conferred by A173V. In the concentration–response experiments, A173V was the main driver of resistance to boceprevir and nirmatrelvir. Structural analysis of Mpro suggested that A173V can cause resistance by making boceprevir and nirmatrelvir binding less favorable. This study contributes to a comprehensive overview of the resistance profile of the first-in-line COVID-19 treatment nirmatrelvir and can thus inform population monitoring and contribute to pandemic preparedness.

The continuous emergence of SARS-CoV-2 variants of concern has rendered many therapeutic monoclonal antibodies (mAbs) ineffective. To date, there are no clinically authorized therapeutic antibodies effective against the recently circulating Omicron sub-lineages BA.2.86 and JN.1. Here, we report the isolation of broad and potent neutralizing human mAbs (HuMabs) from a healthcare worker infected with SARS-CoV-2 early in the pandemic. These include a genetically unique HuMab, named K501SP6, which can neutralize different Omicron sub-lineages, including BQ.1, XBB.1, BA.2.86 and JN.1, by targeting a highly conserved epitope on the N terminal domain, as well as an RBD-specific HuMab (K501SP3) with high potency towards earlier circulating variants that was escaped by the more recent Omicron sub-lineages through spike F486 and E484 substitutions. Characterizing SARS-CoV-2 spike-specific HuMabs, including broadly reactive non-RBD-specific HuMabs, can give insight into the immune mechanisms involved in neutralization and immune evasion, which can be a valuable addition to already existing SARS-CoV-2 therapies.

The continuous re-emergence of yellow fever virus (YFV) poses a serious public health threat, with no approved specific antiviral drugs for treatment. Sofosbuvir and uprifosbuvir, developed for treatment of chronic hepatitis C, are nucleotide analogs with anti-YFV activity in vitro, and sofosbuvir has been used off-label in YFV-infected individuals. To study the barrier to resistance of these drugs, escape experiments were performed with the YFV 17D vaccine strain in human hepatoma Huh7.5 cells. Compared to the original virus, sofosbuvir and uprifosbuvir escape variants exhibited 8.6- to 10.7-fold increased 50% effective concentration (EC₅₀) values. Escape viruses developed putative resistance-associated-substitutions (RAS) M478K and A482G in the NS5 RNA-dependent RNA polymerase domain. Reverse-engineered YFV 17D mutants demonstrated that A482G was the primary determinant of drug resistance, resulting in 5.3- to 5.7-fold increased EC₅₀; however, the substitutions acted synergistically when combined, leading to 12.3- and 11.1-fold increased EC₅₀. A482G conferred slight cross-resistance to bemnifosbuvir, while no cross-resistance was observed for remdesivir, galidesivir, or NITD008. Escape and mutant viruses had slight delays in growth kinetics in Huh7.5 cells and were viable in C6/36 and Aag2-AF5 mosquito cells. Structural analysis of a predicted AlphaFold3 YFV NS5-RNA structure suggests that M478K and A482G induce conformational changes in RdRp motif F, which may reduce sofosbuvir and uprifosbuvir affinities. Sofosbuvir RAS described for other orthoflaviviruses did not substantially affect drug susceptibility in reverse-engineered YFV 17D. This study demonstrates that YFV can develop resistance to sofosbuvir and uprifosbuvir with only two NS5 substitutions that have slight impact on viral fitness.

The continuous emergence of SARS-CoV-2 variants of concern with mutated spike receptor binding domains has rendered many therapeutic mAbs ineffective. To date, there are no clinically authorized therapeutic antibodies effective against the predominant circulating sub-lineages BQ and XBB. Here, we report the isolation of broad and potent neutralizing HuMabs from a Danish healthcare worker infected with SARS-CoV-2 early in the pandemic. These HuMabs include a novel and genetically unique non-RBD-specific HuMab (K501SP6) which can neutralize Omicron sub-lineages BQ and XBB, and an RBD-specific HuMab (K501SP3) with high potency towards earlier circulating variants but was escaped by Omicron sub-lineages BA.5, BQ and XBB through F486 and E484 substitutions. Characterizing SARS-CoV-2 spike-specific HuMabs, including broadly reactive non-RBD-specific HuMabs, can give insight into the immune mechanisms involved in neutralization and immune evasion, which can be a valuable addition to already existing SARS-CoV-2 therapies.