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.

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.

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.

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.

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.

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.

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.

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.

J2N-k hamster model simulates severe infection caused by severe acute respiratory syndrome coronavirus 2 in patients with cardiovascular diseases.

Considering the global impact of the coronavirus disease 2019 (COVID-19) pandemic, generating suitable experimental models is imperative. For pre-clinical studies, researchers require animal models displaying pathological features similar to those observed in patients; therefore, establishing animal models for COVID-19 is crucial. The golden Syrian hamster model mimics conditions observed in humans with mild severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, a golden Syrian hamster model of severe infection has not been reported. J2N-k hamsters are utilized as a cardiomyopathy model; therefore, we used cardiomyopathic J2N-k hamsters showing conditions similar to those of severe COVID-19 complicated with cardiovascular diseases, as patients with cardiovascular diseases exhibit a higher risk of morbidity and mortality due to COVID-19 than patients without cardiovascular diseases. Unlike that in golden Syrian hamsters, SARS-CoV-2 infection was lethal in J2N-k hamsters, with a median lethal dose of 104.75 plaque-forming units for the S clade of SARS-CoV-2 (A, GenBank: MW466791.1). High viral titers and viral genomes were detected in the lungs of J2N-k and golden Syrian hamster models harvested 3 days after infection. Pathological features of SARS-CoV-2-associated lung injury were observed in both models. The J2N-k hamster model can aid in developing vaccines or therapeutics against COVID-19.

Comparison of SARS-CoV-2 variant lethality in human angiotensin-converting enzyme 2 transgenic mice.

This study compared the lethality of severe acute respiratory syndrome coronavirus 2 variants belonging to the S, V, L, G, GH, and GR clades using K18-human angiotensin-converting enzyme 2 heterozygous mice. To estimate the 50% lethal dose (LD50) of each variant, increasing viral loads (100-104 plaque-forming units [PFU]) were administered intranasally. Mouse weight and survival were monitored for 14 days. The LD50 of the GH and GR clades was significantly lower than that of other clades at 50 PFU. These findings suggest that the GH and GR clades, which are prevalent worldwide, are more virulent than the other clades.

Longitudinal Assessment of Antisevere Acute Respiratory Syndrome Coronavirus 2 Immune Responses for Six Months Based on the Clinical Severity of Coronavirus Disease 2019.

BACKGROUND: There is insufficient data on the longevity of immunity acquired after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: We aimed to evaluate the duration of SARS-CoV-2-specific humoral and cellular immunity according to the clinical severity of coronavirus disease 2019 (COVID-19). The study population comprised asymptomatic (n = 14), symptomatic/nonpneumonic (n = 42), and pneumonic (n = 41) patients. RESULTS: The anti-SARS-CoV-2 immunoglobulin class G and neutralizing antibody (NAb) titers lasted until 6 months after diagnosis, with positivity rates of 66.7% and 86.9%, respectively. Older age, prolonged viral shedding, and accompanying pneumonia were more frequently found in patients with sustained humoral immunity. Severe acute respiratory syndrome coronavirus 2-specific T-cell response was strongly observed in pneumonic patients and prominent in individuals with sustained humoral immunity. CONCLUSIONS: In conclusion, most (>85%) patients carry NAb until 6 months after diagnosis of SARS-CoV-2 infection, providing insights for establishing vaccination strategies against COVID-19.

Sustained Responses of Neutralizing Antibodies Against Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in Recovered Patients and Their Therapeutic Applicability.

BACKGROUND: Zoonotic coronaviruses have emerged as a global threat by causing fatal respiratory infections. Given the lack of specific antiviral therapies, application of human convalescent plasma retaining neutralizing activity could be a viable therapeutic option that can bridges this gap. METHODS: We traced antibody responses and memory B cells in peripheral blood collected from 70 recovered Middle East respiratory syndrome coronavirus (MERS-CoV) patients for 3 years after the 2015 outbreak in South Korea. We also used a mouse infection model to examine whether the neutralizing activity of collected sera could provide therapeutic benefit in vivo upon lethal MERS-CoV challenge. RESULTS: Anti-spike-specific IgG responses, including neutralizing activity and antibody-secreting memory B cells, persisted for up to 3 years, especially in MERS patients who suffered from severe pneumonia. Mean antibody titers gradually decreased annually by less than 2-fold. Levels of antibody responses were significantly correlated with fever duration, viral shedding periods, and maximum viral loads observed during infection periods. In a transgenic mice model challenged with lethal doses of MERS-CoV, a significant reduction in viral loads and enhanced survival was observed when therapeutically treated with human plasma retaining a high neutralizing titer (> 1/5000). However, this failed to reduce pulmonary pathogenesis, as revealed by pathological changes in lungs and initial weight loss. CONCLUSIONS: High titers of neutralizing activity are required for suppressive effect on the viral replication but may not be sufficient to reduce inflammatory lesions upon fatal infection. Therefore, immune sera with high neutralizing activity must be carefully selected for plasma therapy of zoonotic coronavirus infection.

Longevity of seropositivity and neutralizing antibodies in recovered MERS patients: a 5-year follow-up study.

OBJECTIVES: We aimed to assess the longevity of spike-specific antibody responses and neutralizing activity in the plasma of recovered Middle East respiratory syndrome (MERS) patients. METHODS: We traced the antibody responses and neutralizing activity against MERS coronavirus (MERS-CoV) in peripheral blood samples collected from 70 recovered MERS patients for 5 years after the 2015 MERS outbreak in South Korea. We also measured the half-life of neutralizing antibody titres in the longitudinal specimens. RESULTS: The seropositivity rate persisted for up to 4 years (50.7-56.1%), especially in MERS patients who suffered from severe pneumonia, and then decreased (35.9%) in the fifth year. Although the spike-specific antibody responses decreased gradually, the neutralizing antibody titres decreased more rapidly (half-life: 20 months) in 19 participants without showing negative seroconversion during the study period. Only five (26.3%) participants had neutralizing antibody titres greater than 1/1000 of PRNT50, and a high neutralizing antibody titre over 1/5000 was not detected in the participants at five years after infection. DISCUSSION: The seropositivity rate of the recovered MERS patients persisted up to 4 years after infection and significantly dropped in the fifth year, whereas the neutralizing antibody titres against MERS-CoV decreased more rapidly and were significantly reduced at 4 years after infection.

A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein.

Vaccines and therapeutics are urgently needed for the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we screen human monoclonal antibodies (mAb) targeting the receptor binding domain (RBD) of the viral spike protein via antibody library constructed from peripheral blood mononuclear cells of a convalescent patient. The CT-P59 mAb potently neutralizes SARS-CoV-2 isolates including the D614G variant without antibody-dependent enhancement effect. Complex crystal structure of CT-P59 Fab/RBD shows that CT-P59 blocks interaction regions of RBD for angiotensin converting enzyme 2 (ACE2) receptor with an orientation that is notably different from previously reported RBD-targeting mAbs. Furthermore, therapeutic effects of CT-P59 are evaluated in three animal models (ferret, hamster, and rhesus monkey), demonstrating a substantial reduction in viral titer along with alleviation of clinical symptoms. Therefore, CT-P59 may be a promising therapeutic candidate for COVID-19.

An infectious cDNA clone of a growth attenuated Korean isolate of MERS coronavirus KNIH002 in clade B.

The MERS-CoV isolated during the 2015 nosocomial outbreak in Korea showed distinctive differences in mortality and transmission patterns compared to the prototype MERS-CoV EMC strain belonging to clade A. We established a BAC-based reverse genetics system for a Korean isolate of MERS-CoV KNIH002 in the clade B phylogenetically far from the EMC strain, and generated a recombinant MERS-CoV expressing red fluorescent protein. The virus rescued from the infectious clone and KNIH002 strain displayed growth attenuation compared to the EMC strain. Consecutive passages of the rescued virus rapidly generated various ORF5 variants, highlighting its genetic instability and calling for caution in the use of repeatedly passaged virus in pathogenesis studies and for evaluation of control measures against MERS-CoV. The infectious clone for the KNIH002 in contemporary epidemic clade B would be useful for better understanding of a functional link between molecular evolution and pathophysiology of MERS-CoV by comparative studies with EMC strain.