Protease inhibitors are among the most powerful antiviral drugs. Nirmatrelvir is the first protease inhibitor specifically developed against the SARS-CoV-2 protease 3CL ^pro that has been licensed for clinical use. To identify mutations that confer resistance to this protease inhibitor, we engineered a chimeric vesicular stomatitis virus (VSV) that expressed a polyprotein composed of the VSV glycoprotein (G), the SARS-CoV-2 3CL ^pro , and the VSV polymerase (L). Viral replication was thus dependent on the autocatalytic processing of this precursor protein by 3CL ^pro and release of the functional viral proteins G and L, and replication of this chimeric VSV was effectively inhibited by nirmatrelvir. Using this system, we applied nirmatrelvir to select for resistance mutations. Resistance was confirmed by retesting nirmatrelvir against the selected mutations in additional VSV-based systems, in an independently developed cellular system, in a biochemical assay, and in a recombinant SARS-CoV-2 system. We demonstrate that some mutants are cross-resistant to ensitrelvir and GC376, whereas others are less resistant to these compounds. Furthermore, we found that most of these resistance mutations already existed in SARS-CoV-2 sequences that have been deposited in the NCBI and GISAID databases, indicating that these mutations were present in circulating SARS-CoV-2 strains.

Since emergence of the initial SARS-CoV-2 BA.1, BA.2 and BA.5 variants, Omicron has diversified substantially. Antigenic characterization of these new variants is important to analyze their potential immune escape from population immunity and implications for future vaccine composition. Here, we describe an antigenic map based on human single-exposure sera and live-virus isolates that includes a broad selection of recently emerged Omicron variants such as BA.2.75, BF.7, BQ, XBB and XBF variants. Recent Omicron variants clustered around BA.1 and BA.5 with some variants further extending the antigenic space. Based on this antigenic map we constructed antibody landscapes to describe neutralization profiles after booster immunization with bivalent mRNA vaccines based on ancestral virus and either BA.1 or BA.4/5. Immune escape of BA.2.75, BQ, XBB and XBF variants was also evident in bivalently boosted individuals, however, cross-neutralization was improved for those with hybrid immunity. Our results indicate that future vaccine updates are needed to induce cross-neutralizing antibodies against currently circulating variants.

In the SARS-CoV-2 pandemic, the so far two most effective approved antivirals are the protease inhibitors nirmatrelvir, in combination with ritonavir (Paxlovid) and ensitrelvir (Xocova). However, antivirals and indeed all antimicrobial drugs are sooner or later challenged by resistance mutations. Studying such mutations is essential for treatment decisions and pandemic preparedness. At the same time, generating resistant viruses to assess mutants is controversial, especially with pathogens of pandemic potential like SARS-CoV-2. To circumvent gain-of-function research with non-attenuated SARS-CoV-2, a previously developed safe system based on a chimeric vesicular stomatitis virus dependent on the SARS-CoV-2 main protease (VSV-M^pro) was used to select mutations against ensitrelvir. Ensitrelvir is clinically especially relevant due to its single-substance formulation, avoiding drug-drug interactions by the co-formulated CYP3A4 inhibitor ritonavir in Paxlovid. By treating VSV-M^pro with ensitrelvir, highly-specific resistant mutants against this inhibitor were selected, while being still fully or largely susceptible to nirmatrelvir. We then confirmed several ensitrelvir-specific mutants in gold standard enzymatic assays and SARS-CoV-2 replicons. These findings indicate that the two inhibitors can have distinct viral resistance profiles, which could determine treatment decisions.

Nirmatrelvir was the first protease inhibitor (PI) specifically developed against the SARS-CoV-2 main protease (3CL ^pro /M ^pro ) and licensed for clinical use. As SARS-CoV-2 continues to spread, variants resistant to nirmatrelvir and other currently available treatments are likely to arise. This study aimed to identify and characterize mutations that confer resistance to nirmatrelvir. To safely generate M ^pro resistance mutations, we passaged a previously developed, chimeric vesicular stomatitis virus (VSV-M ^pro ) with increasing, yet suboptimal concentrations of nirmatrelvir. Using Wuhan-1 and Omicron M ^pro variants, we selected a large set of mutants. Some mutations are frequently present in GISAID, suggesting their relevance in SARS-CoV-2. The resistance phenotype of a subset of mutations was characterized against clinically available PIs (nirmatrelvir and ensitrelvir) with cell-based and biochemical assays. Moreover, we showed the putative molecular mechanism of resistance based on in silico molecular modelling. These findings have implications on the development of future generation M ^pro inhibitors, will help to understand SARS-CoV-2 protease-inhibitor-resistance mechanisms and show the relevance of specific mutations in the clinic, thereby informing treatment decisions. Teaser: Understanding how SARS-CoV-2 could counter the antiviral drug nirmatrelvir and what it means for the future of COVID-19 treatment.

In response to a large outbreak of the SARS-CoV-2 Beta (B.1.351) variant in the district Schwaz, Austria, a rapid mass vaccination campaign with BNT162b2 was carried out in spring 2021, immunizing more than 70% of the adult population within one week. Subsequent analysis revealed that the mass vaccination was associated with a significant reduction in new SARS-CoV-2 infections compared to control districts. Here, we aimed to evaluate both SARS-CoV-2-specific T- and B-cell responses at 35 ± 8 and 215 ± 7 days after the second dose in 600 study subjects who participated at both time points. Overall, a robust antibody and T-cell response was measured at day 35, which waned over time. Nevertheless, all persons preserved seropositivity and T cell response could still be detected in about half of the participants at day 215. Further, antibody response correlated negatively with age; however, in persons who experienced SARS-CoV-2 infection prior to study enrolment, the serum levels of both S- and N-specific antibodies surprisingly increased with age. In contrast, there was no correlation of T cell response with age. We could not detect any sex-related difference in the immune responses. SARS-CoV-2 infections prior to study enrolment or incident infections before day 215 resulted in higher antibody levels and T cell responses at day 215 compared to study participants with no history of infection. Collectively, our data support that vaccination with BNT162b2 against COVID-19 provides a durable immune response and emphasize the usefulness of vaccination even after a natural infection.

Complementing the adult seroprevalence data collected at the time of the rapid SARS-CoV-2 mass vaccination in the district of Schwaz in 2021, we set out to establish the seroprevalence of SARS-CoV-2 among the pediatric population of the district. A total of 369 children, mean age 9.9 (SD 3.4), participated in the study, answering a structured questionnaire on the history of SARS-CoV-2 infection, household contacts, symptoms and history of vaccination. We determined binding and neutralizing antibody levels using plasma samples provided. We estimated the overall prevalence of SARS-CoV-2 infection in the general pediatric population at the time of the study using the census data from Statistik Austria and daily reports of officially confirmed cases. Excluding study participants who reported a history of PCR-confirmed infection, the age-standardized seroprevalence of previously unknown SARS-CoV-2 infection among the general pediatric population of the district was 27% (95% CI: 26.1–27.8). Adding this to the officially documented cases, the true overall prevalence was 32.8% (95% CI: 31.9–33.6) in contrast to the officially documented 8.0% (95% CI: 7.5–8.5) by June 2021. This translated into a proportion of 75.7% (95% CI: 74.4–77.0) of cases being officially undocumented, suggesting a high extent of silent SARS-CoV-2 infections in the pediatric population and possibly silent transmission.