Global prevalence of SARS-CoV-2 3CL protease mutations associated with nirmatrelvir or ensitrelvir resistance.

BACKGROUND: Nirmatrelvir-ritonavir (Paxlovid) and ensitrelvir are 3-chymotrypsin-like cysteine protease (3CLpro) inhibitors which have been approved for the treatment of COVID-19 in 2021 and 2022, respectively. Previous studies have identified 3CLpro mutations that are associated with reduced susceptibility to these antivirals. The aim of the current study was to estimate the global prevalence of 3CLpro inhibitor-resistant SARS-CoV-2 strains. METHODS: We compiled a list of 3CLpro mutations which have been associated with nirmatrelvir or ensitrelvir resistance based on either viral replication or 3CLpro activity assays, and determined their prevalence among 13.4 million sequences deposited in GISAID as of December 14, 2022, about 1 year after the approval of nirmatrelvir-ritonavir. We analyzed the prevalence for different time periods, SARS-CoV-2 lineages and geographical locations. FINDINGS: Overall, 0.5% (67,095/13,446,588) of the sequences contained at least one mutation that was shown to affect the inhibitory activity of nirmatrelvir or ensitrelvir on viral replication or 3CLpro activity. We did not observe any increasing trend of resistance after the widespread clinical use of nirmatrelvir-ritonavir. G15S (2070 per million) and T21I (1386 per million) were the most prevalent mutations, and these mutations were dominant in some SARS-CoV-2 lineages. E166V and S144E, previously shown to affect the inhibitory activity of nirmatrelvir on viral replication or protease activity by > 100-folds, were found in <1 per million sequences. INTERPRETATION: Our data suggest that 3CLpro inhibitor resistance is currently rare. However, continuous global genotypic and phenotypic surveillance would be crucial in the early detection of resistant mutants. FUNDING: Richard and Carol Yu, May Tam Mak Mei Yin, The Shaw Foundation Hong Kong, Michael Tong, Marina Lee, Government Consultancy Service, the Emergency Key Program of Guangzhou Laboratory (See acknowledgements for full list).

Three-dose vaccination-induced immune responses protect against SARS-CoV-2 Omicron BA.2: a population-based study in Hong Kong.

Background: The ongoing outbreak of SARS-CoV-2 Omicron BA.2 infections in Hong Kong, the model city of universal masking of the world, has resulted in a major public health crisis. Although the third vaccination resulted in strong boosting of neutralization antibody, vaccine efficacy and correlate of immune protection against the major circulating Omicron BA.2 remain to be investigated. Methods: We investigated the vaccine efficacy against the Omicron BA.2 breakthrough infection among 470 public servants who had received different SARS-CoV-2 vaccine regimens including two-dose BNT162b2 (2 × BNT, n = 169), three-dose BNT162b2 (3 × BNT, n = 168), two-dose CoronaVac (2 × CorV, n = 34), three-dose CoronaVac (3 × CorV, n = 67) and third-dose BNT162b2 following 2 × CorV (2 × CorV+1BNT, n = 32). Humoral and cellular immune responses after three-dose vaccination were further characterized and correlated with clinical characteristics of BA.2 infection. Findings: During the BA.2 outbreak, 27.7% vaccinees were infected. The timely third-dose vaccination provided significant protection with lower incidence rates of breakthrough infections (2 × BNT 46.2% vs 3 × BNT 13.1%, p < 0.0001; 2 × CorV 44.1% vs 3 × CorV 19.4%, p = 0.003). Investigation of immune responses on blood samples derived from 90 subjects in three-dose vaccination cohorts collected before the BA.2 outbreak revealed that the third-dose vaccination activated spike (S)-specific memory B cells and Omicron cross-reactive T cell responses, which correlated with reduced frequencies of breakthrough infections and disease severity rather than with types of vaccines. Moreover, the frequency of S-specific activated memory B cells was significantly lower in infected vaccinees than uninfected vaccinees before vaccine-breakthrough infection whereas IFN-γ+ CD4 T cells were negatively associated with age and viral clearance time. Critically, BA.2 breakthrough infection boosted cross-reactive memory B cells with enhanced cross-neutralizing antibodies to Omicron sublineages, including BA.2.12.1 and BA.4/5, in all vaccinees tested. Interpretation: Our results imply that the timely third vaccination and immune responses are likely required for vaccine-mediated protection against Omicron BA.2 pandemic. Although BA.2 conferred the highest neutralization resistance compared with variants of concern tested before the emergence of BA.2.12.1 and BA.4/5, the third dose vaccination-activated S-specific memory B cells and Omicron cross-reactive T cell responses contributed to reduced frequencies of breakthrough infection and disease severity. Neutralizing antibody potency enhanced by BA.2 breakthrough infection in vaccinees with prior 3 doses of CoronaVac or BNT162b2 may reduce the risk of infection against ongoing BA.2.12.1 and BA.4/5. Funding: Hong Kong Research Grants Council Collaborative Research Fund, Health and Medical Research Fund, Wellcome Trust, Shenzhen Science and Technology Program, the Health@InnoHK, Innovation and Technology Commission of Hong Kong, China, National Program on Key Research Project, Emergency Key Program of Guangzhou Laboratory, donations from the Friends of Hope Education Fund and the Hong Kong Theme-Based Research Scheme.

Infection of healthcare workers despite a high vaccination rate during the fifth wave of COVID-19 due to Omicron variant in Hong Kong.

Background: No nosocomial infection was recorded in our healthcare workers (HCWs) during the early phase of the coronavirus disease 2019 (COVID-19) pandemic. With the emergence of the Omicron variant of increased transmissibility, infection in HCWs occurred as expected. We aimed to study the epidemiology of infection in HCWs and to describe the infection control measures during the outbreak of the Omicron variant. Methods: With daily rapid antigen testing and molecular confirmation test for COVID-19, infected HCWs were interviewed by infection control nurses (ICNs) to investigate the potential source of infection. The epidemiology of COVID-19 in Hong Kong served as reference. Results: During the fifth wave of COVID-19 (31 December 2021 to 31 May 2022), 1,200,068 cases were reported (incidence 95 times higher than in preceding waves in Hong Kong; 162,103 vs 1,707 per million population respectively, p<0.001). The proportion of infected HCWs was significantly higher than that of the general population (24.9%, 1,607/6,452 vs 16.2%, 12,000,068/7,403,100 respectively; p<0.01). The proportion of infected non-clinical staff was significantly higher than that of clinical staff (31.8%, 536/1,687 vs 22.5%, 1,071/4,765 respectively; p<0.001). Of 82.8% (1,330/1,607) infected HCWs interviewed by ICNs, 99.5% (1,324/1,330) had been fully vaccinated; 49.5% (659/1,330) had no identifiable source; 40.7% (541/1,330) were probably infected from household members; 9.8% (130/1,330) had possible exposure to confirmed patients or HCWs, but no lapse in infection control measures or inappropriate use of personal protective equipment was recalled. Conclusion: Omicron variant is highly transmissible such that breakthrough infection occurred despite high level of vaccination.

Peptidic defective interfering gene nanoparticles against Omicron, Delta SARS-CoV-2 variants and influenza A virus in vivo.

Defective interfering genes (DIGs) are short viral genomes and interfere with wild-type viral replication. Here, we demonstrate that the new designed SARS-CoV-2 DIG (CD3600) can significantly inhibit the replication of SARS-CoV-2 including Alpha, Delta, Kappa and Omicron variants in human HK-2 cells and influenza DIG (PAD4) can significantly inhibit influenza virus replication in human A549 cells. One dose of influenza DIGs prophylactically protects 90% mice from lethal challenge of A(H1N1)pdm09 virus and CD3600 inhibits SARS-CoV-2 replication in hamster lungs when DIGs are administrated to lungs one day before viral challenge. To further investigate the gene delivery vector in the respiratory tract, a peptidic TAT2-P1&LAH4, which can package genes to form small spherical nanoparticles with high endosomal escape ability, is demonstrated to dramatically increase gene expression in the lung airway. TAT2-P1&LAH4, with the dual-functional TAT2-P1 (gene-delivery and antiviral), can deliver CD3600 to significantly inhibit the replication of Delta and Omicron SARS-CoV-2 in hamster lungs. This peptide-based nanoparticle system can effectively transfect genes in lungs and deliver DIGs to inhibit SARS-CoV-2 variants and influenza virus in vivo, which provides the new insight into the drug delivery system for gene therapy against respiratory viruses.

Human Intestinal Organoids Recapitulate Enteric Infections of Enterovirus and Coronavirus.

Enteroviruses, such as EV-A71 and CVA16, mainly infect the human gastrointestinal tract. Human coronaviruses, including SARS-CoV and SARS-CoV-2, have been variably associated with gastrointestinal symptoms. We aimed to optimize the human intestinal organoids and hypothesize that these optimized intestinal organoids can recapitulate enteric infections of enterovirus and coronavirus. We demonstrate that the optimized human intestinal organoids enable better simulation of the native human intestinal epithelium, and that they are significantly more susceptible to EV-A71 than CVA16. Higher replication of EV-A71 than CVA16 in the intestinal organoids triggers a more vigorous cellular response. However, SARS-CoV and SARS-CoV-2 exhibit distinct dynamics of virus-host interaction; more robust propagation of SARS-CoV triggers minimal cellular response, whereas, SARS-CoV-2 exhibits lower replication capacity but elicits a moderate cellular response. Taken together, the disparate profile of the virus-host interaction of enteroviruses and coronaviruses in human intestinal organoids may unravel the cellular basis of the distinct pathogenicity of these viral pathogens.