Generation of a lethal mouse model expressing human ACE2 and TMPRSS2 for SARS-CoV-2 infection and pathogenesis.

Mouse models expressing human ACE2 for coronavirus disease 2019 have been frequently used to understand its pathogenesis and develop therapeutic strategies against SARS-CoV-2. Given that human TMPRSS2 supports viral entry, replication, and pathogenesis, we established a double-transgenic mouse model expressing both human ACE2 and TMPRSS2 for SARS-CoV-2 infection. Co-overexpression of both genes increased viral infectivity in vitro and in vivo. Double-transgenic mice showed significant body weight loss, clinical disease symptoms, acute lung injury, lung inflammation, and lethality in response to viral infection, indicating that they were highly susceptible to SARS-CoV-2. Pretreatment with the TMPRSS2 inhibitor, nafamostat, effectively reduced virus-induced weight loss, viral replication, and mortality in the double-transgenic mice. Moreover, the susceptibility and differential pathogenesis of SARS-CoV-2 variants were demonstrated in this animal model. Together, our results demonstrate that double-transgenic mice could provide a highly susceptible mouse model for viral infection to understand SARS-CoV-2 pathogenesis and evaluate antiviral therapeutics against coronavirus disease 2019.

Differential beta-coronavirus infection dynamics in human bronchial epithelial organoids.

The lower respiratory system serves as the target and barrier for beta-coronavirus (beta-CoV) infections. In this study, we explored beta-CoV infection dynamics in human bronchial epithelial (HBE) organoids, focusing on HCoV-OC43, SARS-CoV, MERS-CoV, and SARS-CoV-2. Utilizing advanced organoid culture techniques, we observed robust replication for all beta-CoVs, particularly noting that SARS-CoV-2 reached peak viral RNA levels at 72 h postinfection. Through comprehensive transcriptomic analysis, we identified significant shifts in cell population dynamics, marked by an increase in goblet cells and a concurrent decrease in ciliated cells. Furthermore, our cell tropism analysis unveiled distinct preferences in viral targeting: HCoV-OC43 predominantly infected club cells, while SARS-CoV had a dual tropism for goblet and ciliated cells. In contrast, SARS-CoV-2 primarily infected ciliated cells, and MERS-CoV showed a marked affinity for goblet cells. Host factor analysis revealed the upregulation of genes encoding viral receptors and proteases. Notably, HCoV-OC43 induced the unfolded protein response pathway, which may facilitate viral replication. Our study also reveals a complex interplay between inflammatory pathways and the suppression of interferon responses during beta-CoV infections. These findings provide insights into host-virus interactions and antiviral defense mechanisms, contributing to our understanding of beta-CoV infections in the respiratory tract.

Rise in broadly cross-reactive adaptive immunity against human ß-coronaviruses in MERS-recovered patients during the COVID-19 pandemic.

To develop a universal coronavirus (CoV) vaccine, long-term immunity against multiple CoVs, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, Middle East respiratory syndrome (MERS)-CoV, and future CoV strains, is crucial. Following the 2015 Korean MERS outbreak, we conducted a long-term follow-up study and found that although neutralizing antibodies and memory T cells against MERS-CoV declined over 5 years, some recovered patients exhibited increased antibody levels during the COVID-19 pandemic. This likely resulted from cross-reactive immunity induced by SARS-CoV-2 vaccines or infections. A significant correlation in antibody responses across various CoVs indicates shared immunogenic epitopes. Two epitopes-the spike protein's stem helix and intracellular domain-were highly immunogenic after MERS-CoV infection and after SARS-CoV-2 vaccination or infection. In addition, memory T cell responses, especially polyfunctional CD4+ T cells, were enhanced during the pandemic, correlating significantly with MERS-CoV spike-specific antibodies and neutralizing activity. Therefore, incorporating these cross-reactive and immunogenic epitopes into pan-CoV vaccine formulations may facilitate effective vaccine development.

The Association Between Antibody Responses and Prolonged Viable Severe Acute Respiratory Syndrome Coronavirus 2 Shedding in Immunocompromised Patients: A Prospective Cohort Study.

Immunocompromised patients with coronavirus disease 2019 were prospectively enrolled from March to November 2022 to understand the association between antibody responses and severe acute respiratory syndrome coronavirus 2 shedding. A total of 62 patients were analyzed, and the results indicated a faster decline in genomic and subgenomic viral RNA in patients with higher neutralizing and S1-specific immunoglobulin G (IgG) antibodies (both P < .001). Notably, high neutralizing antibody levels were associated with a significantly faster decrease in viable virus cultures (P = .04). Our observations suggest the role of neutralizing antibodies in prolonged virus shedding in immunocompromised patients, highlighting the potential benefits of enhancing their humoral immune response through vaccination or monoclonal antibody treatments.

Virological characteristics and the rapid antigen test as deisolation criteria in immunocompromised patients with COVID-19: A prospective cohort study.

There are limited data supporting current Centers for Disease Control and Prevention guidelines for the isolation period in moderate to severely immunocompromised patients with coronavirus disease 2019 (COVID-19). Adult COVID-19 patients who underwent solid organ transplantation (SOT) or received active chemotherapy against hematologic malignancy were enrolled and weekly respiratory samples were collected. Samples with positive genomic real-time polymerase chain reaction results underwent virus culture and rapid antigen testing (RAT). A total of 65 patients (40 with hematologic malignancy and 25 SOT) were enrolled. The median duration of viable virus shedding was 4 weeks (interquartile range: 3-7). Multivariable analysis revealed that B-cell depletion (hazard ratio [HR]: 4.76) was associated with prolonged viral shedding, and COVID-19 vaccination (≥3 doses) was negatively associated with prolonged viral shedding (HR: 0.22). The sensitivity, specificity, positive predictive value, and negative predictive value of RAT for viable virus shedding were 79%, 76%, 74%, and 81%, respectively. The negative predictive value of RAT was only 48% (95% confidence interval [CI]: 33-65) in the samples from those with symptom onset ≤20 days, but it was as high as 92% (95% CI: 85-96) in the samples from those with symptom onset >20 days. About half of immunocompromised COVID-19 patients shed viable virus for ≥4 weeks from the diagnosis, and virus shedding was prolonged especially in unvaccinated patients with B-cell-depleting therapy treatment. RAT beyond 20 days in immunocompromised patients had a relatively high negative predictive value for viable virus shedding.

Molecular evolution and targeted recombination of SARS-CoV-2 in South Korea.

SARS-CoV-2 variants have continuously emerged globally, including in South Korea. To characterize the molecular evolution of SARS-CoV-2 in South Korea, we performed phylogenetic and genomic recombination analyses using more than 12,000 complete genome sequences collected until October 2022. The variants in South Korea originated from globally identified variants of concern and harbored genetic clade-common and clade-specific amino acid mutations mainly around the N-terminal domain (NTD) or receptor binding domain (RBD) in the spike protein. Several point mutation residues in key antigenic sites were under positive selection persistently with changing genetic clades of SARS-CoV-2. Furthermore, we detected 17 potential genomic recombinants and 76.4% (13/17) retained the mosaic NTD or RBD genome. Our results suggest that point mutations and genomic recombination in the spike contributed to the molecular evolution of SARS-CoV-2 in South Korea, which will form an integral part of global prevention and control measures against SARS-CoV-2.

Evaluation of antiviral drugs against newly emerged SARS-CoV-2 Omicron subvariants.

Ongoing emergence of SARS-CoV-2 Omicron subvariants and their rapid worldwide spread pose a threat to public health. From November 2022 to February 2023, newly emerged Omicron subvariants, including BQ.1.1, BF.7, BA.5.2, XBB.1, XBB.1.5, and BN.1.9, became prevalent global strains (>5% global prevalence). These Omicron subvariants are resistant to several therapeutic antibodies. Thus, the antiviral activity of current drugs such as remdesivir, molnupiravir, and nirmatrelvir, which target highly conserved regions of SARS-CoV-2, against newly emerged Omicron subvariants need to be evaluated. We assessed the antiviral efficacy of the drugs using the half-maximal inhibitory concentration (IC50) against human isolates of 23 Omicron subvariants and four former SARS-CoV-2 variants of concern (VOCs) and compared it with the antiviral efficacy of these drugs against the SARS-CoV-2 reference strain (hCoV/Korea/KCDC03/2020). Maximal IC50-fold changes of remdesivir, molnupiravir, and nirmatrelvir were 1.9 (BA.2.75.2), 1.2 (B.1.627.2), and 1.4 (BA.2.3), respectively, compared to median IC50 values of the reference strain. Moreover, median IC50-fold changes of remdesivir, molnupiravir, and nirmatrelvir against the Omicron variants were 0.96, 0.4, and 0.62, respectively, similar to the 1.02, 0.88, and 0.67, respectively, median IC50-fold changes for previous VOCs. Although K90R and P132H in Nsp 5, and P323L, A529V, G671S, V405F, and ins823D in Nsp 12 mutations were identified, these amino acid substitutions did not affect drug antiviral activity. These results indicate that current antivirals retain antiviral efficacy against newly emerged Omicron subvariants. It is important to continue active surveillance and testing of new variants for drug resistance to enable early identification of drug-resistant strains.

Experimental Infection and Transmission of SARS-CoV-2 Delta and Omicron Variants among Beagle Dogs.

We assessed susceptibility of dogs to SARS-COV-2 Delta and Omicron variants by experimentally inoculating beagle dogs. Moreover, we investigated transmissibility of the variants from infected to naive dogs. The dogs were susceptible to infection without clinical signs and transmitted both strains to other dogs through direct contact.

Short-Term Effectiveness of Oral Nirmatrelvir/Ritonavir Against the SARS-CoV-2 Omicron Variant and Culture-Positive Viral Shedding.

BACKGROUND: Information on the effectiveness of nirmatrelvir/ritonavir against the omicron is limited. The clinical response and viral kinetics to therapy in the real world need to be evaluated. METHODS: Mild to moderate coronavirus disease 2019 (COVID-19) patients with risk factors for severe illness were prospectively enrolled as a treatment group with nirmatrelvir/ritonavir therapy versus a control group with supportive care. Serial viral load and culture from the upper respiratory tract were evaluated for seven days, and clinical responses and adverse reactions were evaluated for 28 days. RESULTS: A total of 51 patients were analyzed including 40 in the treatment group and 11 in the control group. Faster symptom resolution during hospitalization (P = 0.048) was observed in the treatment group. Only minor adverse reactions were reported in 27.5% of patients. The viral load on Day 7 was lower in the treatment group (P = 0.002). The viral culture showed a positivity of 67.6% (25/37) vs. 100% (6/6) on Day 1, 0% (0/37) vs. 16.7 (1/6) on Day 5, and 0% (0/16) vs. 50.0% (2/4) on Day 7 in the treatment and control groups, respectively. CONCLUSIONS: Nirmatrelvir/ritonavir against the omicron was safe and resulted in negative viral culture conversion after Day 5 of treatment with better symptomatic resolution.

Comparing the Immunogenicity and Protective Effects of Three MERS-CoV Inactivation Methods in Mice.

The Middle East respiratory syndrome (MERS) is a fatal acute viral respiratory disease caused by MERS-coronavirus (MERS-CoV) infection. To date, no vaccine has been approved for MERS-CoV despite continuing outbreaks. Inactivated vaccines are a viable option when developed using the appropriate inactivation methods and adjuvants. In this study, we evaluated the immunogenicity and protective effects of MERS-CoV vaccine candidates inactivated by three different chemical agents. MERS-CoV was effectively inactivated by formaldehyde, hydrogen peroxide, and binary ethylene imine and induced humoral and cellular immunity in mice. Although inflammatory cell infiltration was observed in the lungs four days after the challenge, the immunized hDPP4-transgenic mouse group showed 100% protection against a challenge with MERS-CoV (100 LD50). In particular, the immune response was highly stimulated by MERS-CoV inactivated with formaldehyde, and all mice survived a challenge with the minimum dose. In the adjuvant comparison test, the group immunized with inactivated MERS-CoV and AddaVax had a higher immune response than the group immunized with aluminum potassium sulfate (alum). In conclusion, our study indicates that the three methods of MERS-CoV inactivation are highly immunogenic and protective in mice and show strong potential as vaccine candidates when used with an appropriate adjuvant.

Relative infectivity of the SARS-CoV-2 Omicron variant in human alveolar cells.

With the continuous emergence of highly transmissible SARS-CoV-2 variants, the comparison of their infectivity has become a critical issue for public health. However, a direct assessment of the viral characteristic has been challenging because of the lack of appropriate experimental models and efficient methods. Here, we integrated human alveolar organoids and single-cell transcriptome sequencing to facilitate the evaluation. In a proof-of-concept study with four highly transmissible SARS-CoV-2 variants, including GR (B.1.1.119), Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (BA.1), a rapid evaluation of the relative infectivity was possible. Our system demonstrates that the Omicron variant is 5- to 7-fold more infectious to human alveolar cells than the other SARS-CoV-2 variants at the initial stage of infection. To our knowledge, for the first time, this study measures the relative infectivity of the Omicron variant under multiple virus co-infection and provides new experimental procedures that can be applied to monitor emerging viral variants.

Can nirmatrelvir/ritonavir treatment shorten the duration of COVID-19 isolation?

Background: The impact of nirmatrelvir/ritonavir treatment on shedding of viable virus in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unclear. Methods: A prospective cohort study evaluating mildly ill COVID-19 patients was conducted. Virologic responses were compared between nirmatrelvir/ritonavir-treatment and supportive care groups. Risk factors and relevant clinical factors for shedding of viable virus were investigated. Results: A total of 80 COVID-19 patients were enrolled and 222 sputum specimens were collected. Ten patients were dropped during follow-up, and 33 patients in the nirmatrelvir/ritonavir and 37 in the supportive care groups were compared. The median age was 67 years, and 67% were male. Clinical characteristics were similar between groups. Viral loads decreased significantly faster in the nirmatrelvir/ritonavir group compared with the supportive care group (P < 0.001), and the slope was significantly steeper (-2.99 ± 1.54 vs. -1.44 ± 1.52; P < 0.001). The duration of viable virus shedding was not statistically different between groups. In the multivariable analyses evaluating all collected specimens, male gender (OR 2.51, 95% CI 1.25-5.03, P = 0.010), symptom score (OR 1.41, 95% CI 1.07-1.87, P = 0.015), days from symptom onset (OR 0.72, 95% CI 0.59-0.88, P = 0.002), complete vaccination (OR 0.09, 95% CI 0.01-0.87, P = 0.038), and BA.2 subtype (OR 0.49, 95% CI 0.26-0.91, P = 0.025) were independently associated with viable viral shedding, while nirmatrelvir/ritonavir treatment was not. Conclusion: Nirmatrelvir/ritonavir treatment effectively reduced viral loads of SARS-CoV-2 Omicron variants but did not decrease the duration of viable virus shedding.

Preclinical assessment and randomized Phase I study of CT-P63, a broadly neutralizing antibody targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in significant morbidity and mortality worldwide. Despite a successful vaccination programme, the emergence of mutated variants that can escape current levels of immunity mean infections continue. Herein, we report the development of CT-P63, a broad-spectrum neutralizing monoclonal antibody. In vitro studies demonstrated potent neutralizing activity against the most prevalent variants, including Delta and the BA.1 and BA.2 sub-lineages of Omicron. In a transgenic mouse model, prophylactic CT-P63 significantly reduced wild-type viral titres in the respiratory tract and CT-P63 treatment proved efficacious against infection with Beta, Delta, and Omicron variants of SARS-CoV-2 with no detectable infectious virus in the lungs of treated animals. A randomized, double-blind, parallel-group, placebo-controlled, Phase I, single ascending dose study in healthy volunteers (NCT05017168) confirmed the safety, tolerability, and pharmacokinetics of CT-P63. Twenty-four participants were randomized and received the planned dose of CT-P63 or placebo. The safety and tolerability of CT-P63 were evaluated as primary objectives. Eight participants (33.3%) experienced a treatment-emergent adverse event (TEAE), including one grade ≥3 (blood creatine phosphokinase increased). There were no deaths, treatment-emergent serious adverse events, TEAEs of special interest, or TEAEs leading to study drug discontinuation in the CT-P63 groups. Serum CT-P63 concentrations rapidly peaked before declining in a biphasic manner and systemic exposure was dose proportional. Overall, CT-P63 was clinically safe and showed broad-spectrum neutralizing activity against SARS-CoV-2 variants in vitro and in vivo.

Duration of Humoral Immunity and Cross-Neutralizing Activity Against the Alpha, Beta, and Delta Variants After Wild-Type Severe Acute Respiratory Syndrome Coronavirus 2 Infection: A Prospective Cohort Study.

A prospective cohort study was conducted for adults with a diagnosis of with coronavirus disease 2019 (COVID-19). Convalescent blood samples were obtained 4, 6, and 11 months after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The seropositivity of anti-spike antibody was maintained in all patients (100%) until 11 months after COVID-19 diagnosis. Neutralizing antibody levels against wild-type SARS-CoV-2 gradually decreased but remained positive in >50% of patients 11 months after diagnosis: in 98.5% (67 of 68) at 4 months, 86.8% (46 of 53) at 6 months, and 58.8% (40 of 68) at 11 months. However, cross-neutralizing activity against the Beta and Delta variants was attenuated 2.53-fold and 2.93-fold, respectively, compared with the wild-type strain.

Engineered small extracellular vesicles displaying ACE2 variants on the surface protect against SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry is mediated by the interaction of the viral spike (S) protein with angiotensin-converting enzyme 2 (ACE2) on the host cell surface. Although a clinical trial testing soluble ACE2 (sACE2) for COVID-19 is currently ongoing, our understanding of the delivery of sACE2 via small extracellular vesicles (sEVs) is still rudimentary. With excellent biocompatibility allowing for the effective delivery of molecular cargos, sEVs are broadly studied as nanoscale protein carriers. In order to exploit the potential of sEVs, we design truncated CD9 scaffolds to display sACE2 on the sEV surface as a decoy receptor for the S protein of SARS-CoV-2. Moreover, to enhance the sACE2-S binding interaction, we employ sACE2 variants. sACE2-loaded sEVs exhibit typical sEVs characteristics and bind to the S protein. Furthermore, engineered sEVs inhibit the entry of wild-type (WT), the globally dominant D614G variant, Beta (K417N-E484K-N501Y) variant, and Delta (L452R-T478K-D614G) variant SARS-CoV-2 pseudovirus, and protect against authentic SARS-CoV-2 and Delta variant infection. Of note, sACE2 variants harbouring sEVs show superior antiviral efficacy than WT sACE2 loaded sEVs. Therapeutic efficacy of the engineered sEVs against SARS-CoV-2 challenge was confirmed using K18-hACE2 mice. The current findings provide opportunities for the development of new sEVs-based antiviral therapeutics.

Longitudinal Analysis of Memory T-Cell Responses in Survivors of Middle East Respiratory Syndrome.

BACKGROUND: Middle East respiratory syndrome (MERS) is a highly lethal respiratory disease caused by a zoonotic betacoronavirus. The development of effective vaccines and control measures requires a thorough understanding of the immune response to this viral infection. METHODS: We investigated cellular immune responses up to 5 years after infection in a cohort of 59 MERS survivors by performing enzyme-linked immunospot assay and intracellular cytokine staining after stimulation of peripheral blood mononuclear cells with synthetic viral peptides. RESULTS: Memory T-cell responses were detected in 82%, 75%, 69%, 64%, and 64% of MERS survivors from 1-5 years post-infection, respectively. Although the frequency of virus-specific interferon gamma (IFN-γ)-secreting T cells tended to be higher in moderately/severely ill patients than in mildly ill patients during the early period of follow-up, there was no significant difference among the different clinical severity groups across all time points. While both CD4+ and CD8+ T cells were involved in memory T-cell responses, CD4+ T cells persisted slightly longer than CD8+ T cells. Both memory CD4+ and CD8+ T cells recognized the E/M/N proteins better than the S protein and maintained their polyfunctionality throughout the period examined. Memory T-cell responses correlated positively with antibody responses during the initial 3-4 years but not with maximum viral loads at any time point. CONCLUSIONS: These findings advance our understanding of the dynamics of virus-specific memory T-cell immunity after MERS-coronavirus infection, which is relevant to the development of effective T cell-based vaccines.

Persistence of the neutralizing antibody response after SARS-CoV-2 infection.

OBJECTIVE: Neutralizing antibodies are among the factors used to measure an individual's immune status for the control of infectious diseases. We aimed to confirm the persistence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibody levels in patients who had recovered from coronavirus disease 2019 (COVID-19). METHODS: Plasma donors in South Korea who had completely recovered from SARS-CoV-2 infection had follow-up testing to determine the persistence of neutralizing antibodies using a plaque-reduction neutralization test and ELISA. RESULTS: Of the 111 participants-aged 20-29 years, 37/111 (33.3%); 30-39 years, 17/111 (15.3%); 40-49 years, 23/111 (20.7%); 50-59 years, 21/111 (18.9%); 60-65 years, 13/111 (11.7%); male, 43/111 (38.7%); female, 68/111 (61.3%)-66.1% still had neutralizing antibodies approximately 9 months (range 255-302 days) after confirmation of the diagnosis. CONCLUSIONS: In this study we analysed the titre of neutralizing antibodies associated with predicting immune status in individuals with natural infection. Information about the persistence and change in levels of neutralizing antibodies against SARS-CoV-2 can be utilized to provide evidence for developing vaccination schedules for individuals with previous infection.