Distinctive Dynamics and Functions of the CD4+CD25+FOXP3+ Regulatory T Cell Population in Patients with Severe and Mild COVID-19.

Although CD4+CD25+FOXP3+ regulatory T (TREG) cells have been studied in patients with COVID-19, changes in the TREG cell population have not been longitudinally examined during the course of COVID-19. In this study, we longitudinally investigated the quantitative and qualitative changes in the TREG cell population in patients with COVID-19. We found that the frequencies of total TREG cells and CD45RA-FOXP3hi activated TREG cells were significantly increased 15-28 d postsymptom onset in severe patients, but not in mild patients. TREG cells from severe patients exhibited not only increased proliferation but also enhanced apoptosis, suggesting functional derangement of the TREG cell population during severe COVID-19. The suppressive functions of the TREG cell population did not differ between patients with severe versus mild COVID-19. The frequency of TREG cells inversely correlated with SARS-CoV-2-specific cytokine production by CD4+ T cells and their polyfunctionality in patients with mild disease, suggesting that TREG cells are major regulators of virus-specific CD4+ T cell responses during mild COVID-19. However, such correlations were not observed in patients with severe disease. Thus, in this study, we describe distinctive changes in the TREG cell population in patients with severe and mild COVID-19. Our study provides a deep understanding of host immune responses upon SARS-CoV-2 infection in regard to TREG cells.

A heterologous AZD1222 priming and BNT162b2 boosting regimen more efficiently elicits neutralizing antibodies, but not memory T cells, than the homologous BNT162b2 regimen.

BACKGROUND: Comparative analyses of SARS-CoV-2-specific immune responses elicited by diverse prime-boost regimens are required to establish efficient regimens for the control of COVID-19. METHOD: In this prospective observational cohort study, spike-specific immunoglobulin G (IgG) and neutralizing antibodies (nAbs) alongside spike-specific T-cell responses in age-matched groups of homologous BNT162b2/BNT162b2 or AZD1222/AZD1222 vaccination, heterologous AZD1222/BNT162b2 vaccination, and prior wild-type SARS-CoV-2 infection/vaccination were evaluated. RESULTS: Peak immune responses were achieved after the second vaccine dose in the naïve vaccinated groups and after the first dose in the prior infection/vaccination group. Peak titers of anti-spike IgG and nAb were significantly higher in the AZD1222/BNT162b2 vaccination and prior infection/vaccination groups than in the BNT162b2/BNT162b2 or AZD1222/AZD1222 groups. However, the frequency of interferon-γ-producing CD4+ T cells was highest in the BNT162b2/BNT162b2 vaccination group. Similar results were observed in the analysis of polyfunctional T cells. When nAb and CD4+T-cell responses against the Delta variant were analyzed, the prior infection/vaccination group exhibited higher responses than the groups of other homologous or heterologous vaccination regimens. CONCLUSION: nAbs are efficiently elicited by heterologous AZD1222/BNT162b2 vaccination, as well as prior infection/vaccination, whereas spike-specific CD4+T-cell responses are efficiently elicited by homologous BNT162b2 vaccination. Variant-recognizing immunity is more efficiently generated by prior infection/vaccination than the other homologous or heterologous vaccination regimens.

Estimation of SARS-CoV-2 Neutralizing Activity and Protective Immunity in Different Vaccine Types Using Three Surrogate Virus Neutralization Test Assays and Two Semiquantitative Binding Assays Targeting the Receptor-Binding Domain.

Estimating neutralizing activity in vaccinees is crucial for predicting the protective effect against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As the plaque reduction neutralization test (PRNT) requires a biosafety level 3 facility, it would be advantageous if surrogate virus neutralization test (sVNT) assays and binding assays could predict neutralizing activity. Here, five different assays were evaluated with respect to the PRNT in vaccinees: three sVNT assays from GenScript, Boditech Med, and SD Biosensor and two semiquantitative binding assays from Roche and Abbott. The vaccinees were subjected to three vaccination protocols: homologous ChAdOx1, homologous BNT162b2, and heterologous administration. The ability to predict a 50% neutralizing dose (ND50) of ≥20 largely varied among the assays, with the binding assays showing substantial agreement (kappa, ~0.90) and the sVNT assays showing relatively poor performance, especially in the ChAdOx1 group (kappa, 0.33 to 0.97). The ability to predict an ND50 value of ≥118.25, indicating a protective effect, was comparable among different assays. Applying optimal cutoffs based on Youden's index, the kappa agreements were greater than 0.60 for all assays in the total group. Overall, relatively poor performance was demonstrated in the ChAdOx1 group, owing to low antibody titers. Although there were intra-assay differences related to the vaccination protocols, as well as interassay differences, all assays demonstrated fair performance in predicting the protective effect using the new cutoffs. This study demonstrates the need for a different cutoff for each assay to appropriately determine a higher neutralizing titer and suggests the clinical feasibility of using various assays for estimation of the protective effect. IMPORTANCE The coronavirus disease 2019 (COVID-19) pandemic continues to last, despite high COVID-19 vaccination rates. As many people experience breakthrough infection after prior infection and/or vaccination, estimating the neutralization activity and predicting the protective effect are major issues of concern. However, since standard neutralization tests are not available in most clinical laboratories, it would be beneficial if commercial assays could predict these aspects. In this study, we evaluated the performance of three sVNT assays and two semiquantitative binding assays targeting the receptor-binding domain with respect to the PRNT. Our results suggest that these assays could be used for predicting the protective effect by adjusting the cutoffs.

Kinetics of vaccine-induced neutralizing antibody titers and estimated protective immunity against wild-type SARS-CoV-2 and the Delta variant: A prospective nationwide cohort study comparing three COVID-19 vaccination protocols in South Korea.

Introduction: Despite vaccine development, the COVID-19 pandemic is ongoing due to immunity-escaping variants of concern (VOCs). Estimations of vaccine-induced protective immunity against VOCs are essential for setting proper COVID-19 vaccination policy. Methods: We performed plaque-reduction neutralizing tests (PRNTs) using sera from healthcare workers (HCWs) collected from baseline to six months after COVID-19 vaccination and from convalescent COVID-19 patients. The 20.2% of the mean PRNT titer of convalescent sera was used as 50% protective value, and the percentage of HCWs with protective immunity for each week (percent-week) was compared among vaccination groups. A correlation equation was deduced between a PRNT 50% neutralizing dose (ND50) against wild type (WT) SARS-CoV-2 and that of the Delta variant. Results: We conducted PRNTs on 1,287 serum samples from 297 HCWs (99 HCWs who received homologous ChAdOx1 vaccination (ChAd), 99 from HCWs who received homologous BNT162b2 (BNT), and 99 from HCWs who received heterologous ChAd followed by BNT (ChAd-BNT)). Using 365 serum samples from 116 convalescent COVID-19 patients, PRNT ND50 of 118.25 was derived as 50% protective value. The 6-month cumulative percentage of HCWs with protective immunity against WT SARS-CoV-2 was highest in the BNT group (2297.0 percent-week), followed by the ChAd-BNT (1576.8) and ChAd (1403.0) groups. In the inter-group comparison, protective percentage of the BNT group (median 96.0%, IQR 91.2-99.2%) was comparable to the ChAd-BNT group (median 85.4%, IQR 15.7-100%; P =0.117) and significantly higher than the ChAd group (median 60.1%, IQR 20.0-87.1%; P <0.001). When Delta PRNT was estimated using the correlation equation, protective immunity at the 6-month waning point was markedly decreased (28.3% for ChAd group, 52.5% for BNT, and 66.7% for ChAd-BNT). Conclusion: Decreased vaccine-induced protective immunity at the 6-month waning point and lesser response against the Delta variant may explain the Delta-dominated outbreak of late 2021. Follow-up studies for newly-emerging VOCs would also be needed.

Enhanced antibody responses in fully vaccinated individuals against pan-SARS-CoV-2 variants following Omicron breakthrough infection.

Omicron has become the globally dominant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant, creating additional challenges due to its ability to evade neutralization. Here, we report that neutralizing antibodies against Omicron variants are undetected following COVID-19 infection with ancestral or past SARS-CoV-2 variant viruses or after two-dose mRNA vaccination. Compared with two-dose vaccination, a three-dose vaccination course induces broad neutralizing antibody responses with improved durability against different SARS-CoV-2 variants, although neutralizing antibody titers against Omicron remain low. Intriguingly, among individuals with three-dose vaccination, Omicron breakthrough infection substantially augments serum neutralizing activity against a broad spectrum of SARS-CoV-2 variants, including Omicron variants BA.1, BA.2, and BA.5. Additionally, after Omicron breakthrough infection, memory T cells respond to the spike proteins of both ancestral and Omicron SARS-CoV-2 by producing cytokines with polyfunctionality. These results suggest that Omicron breakthrough infection following three-dose mRNA vaccination induces pan-SARS-CoV-2 immunity that may protect against emerging SARS-CoV-2 variants of concern.

Heterologous ChAdOx1 and Bnt162b2 vaccination induces strong neutralizing antibody responses against SARS-CoV-2 including delta variant with tolerable reactogenicity.

OBJECTIVES: We assessed humoral responses and reactogenicity following the heterologous vaccination compared to the homologous vaccination groups. METHODS: We enrolled healthcare workers (HCWs) who were either vaccinated with ChAdOx1 followed by BNT162b2 (heterologous group) or 2 doses of ChAdOx1 (ChAdOx1 group) or BNT162b2 (BNT162b2 group). Immunogenicity was assessed by measuring antibody titers against receptor-binding domain (RBD) of SARS-CoV-2 spike protein in all participants and neutralizing antibody titer in 100 participants per group. Reactogenicity was evaluated by a questionnaire-based survey. RESULTS: We enrolled 499 HCWs (ChAdOx1, n = 199; BNT162b2, n = 200; heterologous ChAdOx1/BNT162b2, n = 100). The geometric mean titer of anti-receptor-binding domain antibody at 14 days after the booster dose was significantly higher in the heterologous group (11 780.55 binding antibody unit (BAU)/mL [95% CI, 10 891.52-12 742.14]) than in the ChAdOx1 (1561.51 [95% CI, 1415.03-1723.15]) or BNT162b2 (2895.90 [95% CI, 2664.01-3147.98]) groups (both p < 0.001). The neutralizing antibody titer of the heterologous group (geometric mean ND50, 2367.74 [95% CI, 1970.03-2845.74]) was comparable to that of the BNT162b2 group (2118.63 [95% CI, 1755.88-2556.32]; p > 0.05) but higher than that of the ChAdOx1 group (391.77 [95% CI, 326.16-470.59]; p < 0.001). Compared with those against wild-type SARS-CoV-2, the geometric mean neutralizing antibody titers against the Delta variant at 14 days after the boosting were reduced by 3.0-fold in the heterologous group (geometric mean ND50, 872.01 [95% CI, 685.33-1109.54]), 4.0-fold in the BNT162b2 group (337.93 [95% CI, 262.78-434.57]), and 3.2-fold in the ChAdOx1 group (206.61 [95% CI, 144.05-296.34]). The local or systemic reactogenicity after the booster dose in the heterologous group was higher than that of the ChAdOx1 group but comparable to that of the BNT162b2 group. DISCUSSION: Heterologous ChAdOx1 followed by BNT162b2 vaccination with a 12-week interval induced a robust humoral immune response against SARS-CoV-2, including the Delta variant, that was comparable to the homologous BNT162b2 vaccination and stronger than the homologous ChAdOx1 vaccination, with a tolerable reactogenicity profile.

Antiviral Efficacies of FDA-Approved Drugs against SARS-CoV-2 Infection in Ferrets.

Due to the urgent need of a therapeutic treatment for coronavirus (CoV) disease 2019 (COVID-19) patients, a number of FDA-approved/repurposed drugs have been suggested as antiviral candidates at clinics, without sufficient information. Furthermore, there have been extensive debates over antiviral candidates for their effectiveness and safety against severe acute respiratory syndrome CoV 2 (SARS-CoV-2), suggesting that rapid preclinical animal studies are required to identify potential antiviral candidates for human trials. To this end, the antiviral efficacies of lopinavir-ritonavir, hydroxychloroquine sulfate, and emtricitabine-tenofovir for SARS-CoV-2 infection were assessed in the ferret infection model. While the lopinavir-ritonavir-, hydroxychloroquine sulfate-, or emtricitabine-tenofovir-treated group exhibited lower overall clinical scores than the phosphate-buffered saline (PBS)-treated control group, the virus titers in nasal washes, stool specimens, and respiratory tissues were similar between all three antiviral-candidate-treated groups and the PBS-treated control group. Only the emtricitabine-tenofovir-treated group showed lower virus titers in nasal washes at 8 days postinfection (dpi) than the PBS-treated control group. To further explore the effect of immune suppression on viral infection and clinical outcome, ferrets were treated with azathioprine, an immunosuppressive drug. Compared to the PBS-treated control group, azathioprine-immunosuppressed ferrets exhibited a longer period of clinical illness, higher virus titers in nasal turbinate, delayed virus clearance, and significantly lower serum neutralization (SN) antibody titers. Taken together, all antiviral drugs tested marginally reduced the overall clinical scores of infected ferrets but did not significantly affect in vivo virus titers. Despite the potential discrepancy of drug efficacies between animals and humans, these preclinical ferret data should be highly informative to future therapeutic treatment of COVID-19 patients.IMPORTANCE The SARS-CoV-2 pandemic continues to spread worldwide, with rapidly increasing numbers of mortalities, placing increasing strain on health care systems. Despite serious public health concerns, no effective vaccines or therapeutics have been approved by regulatory agencies. In this study, we tested the FDA-approved drugs lopinavir-ritonavir, hydroxychloroquine sulfate, and emtricitabine-tenofovir against SARS-CoV-2 infection in a highly susceptible ferret infection model. While most of the drug treatments marginally reduced clinical symptoms, they did not reduce virus titers, with the exception of emtricitabine-tenofovir treatment, which led to diminished virus titers in nasal washes at 8 dpi. Further, the azathioprine-treated immunosuppressed ferrets showed delayed virus clearance and low SN titers, resulting in a prolonged infection. As several FDA-approved or repurposed drugs are being tested as antiviral candidates at clinics without sufficient information, rapid preclinical animal studies should proceed to identify therapeutic drug candidates with strong antiviral potential and high safety prior to a human efficacy trial.

Age-dependent pathogenic characteristics of SARS-CoV-2 infection in ferrets.

While the seroprevalence of SARS-CoV-2 in healthy people does not differ significantly among age groups, those aged 65 years or older exhibit strikingly higher COVID-19 mortality compared to younger individuals. To further understand differing COVID-19 manifestations in patients of different ages, three age groups of ferrets are infected with SARS-CoV-2. Although SARS-CoV-2 is isolated from all ferrets regardless of age, aged ferrets (≥3 years old) show higher viral loads, longer nasal virus shedding, and more severe lung inflammatory cell infiltration, and clinical symptoms compared to juvenile (≤6 months) and young adult (1-2 years) groups. Furthermore, direct contact ferrets co-housed with the virus-infected aged group shed more virus than direct-contact ferrets co-housed with virus-infected juvenile or young adult ferrets. Transcriptome analysis of aged ferret lungs reveals strong enrichment of gene sets related to type I interferon, activated T cells, and M1 macrophage responses, mimicking the gene expression profile of severe COVID-19 patients. Thus, SARS-CoV-2-infected aged ferrets highly recapitulate COVID-19 patients with severe symptoms and are useful for understanding age-associated infection, transmission, and pathogenesis of SARS-CoV-2.

Development and Evaluation of a Preceptor Education Program Based on the One-Minute Preceptor Model: Participatory Action Research.

This participatory action research study was conducted to confirm the implementation process and effect of developing and applying a preceptor education program based on the One-Minute Preceptor Model to foster the competence of preceptor clinical nurses. The study was conducted for eight weeks from March 2020 on 30 preceptor nurses in South Korea. Nursing standards were developed for two weeks and six cycles (comprising four stages) were performed. Data collection was integrated using both quantitative and qualitative approaches. For quantitative data, the Clinical Core Competency of Preceptor (CCCP) and General Communication Competence Scale (GICC-15) results were collected from preceptor nurses through questionnaires. Reflection journals of nurses' experiences were also analyzed through content analysis and frequency of keywords using WordClouds. There was no significant change in CCCP or GICC-15 results among preceptor nurses. However, nurses' experiences were associated with the growth and development of competencies such as evidence-based practice, quality feedback, and self-reflection. The program was effective in developing nurse competencies. Therefore, it is necessary to encourage One-Minute Preceptor Model activities among preceptors through an action research approach and to actively support research and practice in clinical settings, as well as to provide organizational and systematic support.

Abnormality in the NK-cell population is prolonged in severe COVID-19 patients.

BACKGROUND: Our understanding of adaptive immune responses in patients with coronavirus disease 2019 (COVID-19) is rapidly evolving, but information on the innate immune responses by natural killer (NK) cells is still insufficient. OBJECTIVE: We aimed to examine the phenotypic and functional status of NK cells and their changes during the course of mild and severe COVID-19. METHODS: We performed RNA sequencing and flow cytometric analysis of NK cells from patients with mild and severe COVID-19 at multiple time points in the course of the disease using cryopreserved PBMCs. RESULTS: In RNA-sequencing analysis, the NK cells exhibited distinctive features compared with healthy donors, with significant enrichment of proinflammatory cytokine-mediated signaling pathways. Intriguingly, we found that the unconventional CD56dimCD16neg NK-cell population expanded in cryopreserved PBMCs from patients with COVID-19 regardless of disease severity, accompanied by decreased NK-cell cytotoxicity. The NK-cell population was rapidly normalized alongside the disappearance of unconventional CD56dimCD16neg NK cells and the recovery of NK-cell cytotoxicity in patients with mild COVID-19, but this occurred slowly in patients with severe COVID-19. CONCLUSIONS: The current longitudinal study provides a deep understanding of the NK-cell biology in COVID-19.

SARS-CoV-2-specific T cell memory is sustained in COVID-19 convalescent patients for 10 months with successful development of stem cell-like memory T cells.

Memory T cells contribute to rapid viral clearance during re-infection, but the longevity and differentiation of SARS-CoV-2-specific memory T cells remain unclear. Here we conduct ex vivo assays to evaluate SARS-CoV-2-specific CD4+ and CD8+ T cell responses in COVID-19 convalescent patients up to 317 days post-symptom onset (DPSO), and find that memory T cell responses are maintained during the study period regardless of the severity of COVID-19. In particular, we observe sustained polyfunctionality and proliferation capacity of SARS-CoV-2-specific T cells. Among SARS-CoV-2-specific CD4+ and CD8+ T cells detected by activation-induced markers, the proportion of stem cell-like memory T (TSCM) cells is increased, peaking at approximately 120 DPSO. Development of TSCM cells is confirmed by SARS-CoV-2-specific MHC-I multimer staining. Considering the self-renewal capacity and multipotency of TSCM cells, our data suggest that SARS-CoV-2-specific T cells are long-lasting after recovery from COVID-19, thus support the feasibility of effective vaccination programs as a measure for COVID-19 control.

Differences in seroprevalence between epicenter and non-epicenter areas of the COVID-19 outbreak in South Korea.

To compare the standardized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroprevalence of high epicenter region with non-epicenter region, serological studies were performed with a total of 3,268 sera from Daegu City and 3,981 sera from Chungbuk Province. Indirect immunofluorescence assay (IFA) for SARS-CoV-2 IgG results showed a high seroprevalence rate in the Daegu City (epicenter) compared with a non-epicenter area (Chungbuk Province) (1.27% vs. 0.91%, P = 0.0358). It is noteworthy that the highest seroprevalence in Daegu City was found in elderly patients (70's) whereas young adult patients (20's) in Chungbuk Province showed the highest seroprevalence. Neutralizing antibody (NAb) titers were found in three samples from Daegu City (3/3, 268, 0.09%) while none of the samples from Chungbuk Province were NAb positive. These results demonstrated that even following the large outbreak, the seropositive rate of SARS-CoV-2 in the general population remained low in South Korea.

PD-1-Expressing SARS-CoV-2-Specific CD8+ T Cells Are Not Exhausted, but Functional in Patients with COVID-19.

Memory T cell responses have been demonstrated in COVID-19 convalescents, but ex vivo phenotypes of SARS-CoV-2-specific T cells have been unclear. We detected SARS-CoV-2-specific CD8+ T cells by MHC class I multimer staining and examined their phenotypes and functions in acute and convalescent COVID-19. Multimer+ cells exhibited early differentiated effector-memory phenotypes in the early convalescent phase. The frequency of stem-like memory cells was increased among multimer+ cells in the late convalescent phase. Cytokine secretion assays combined with MHC class I multimer staining revealed that the proportion of interferon-γ (IFN-γ)-producing cells was significantly lower among SARS-CoV-2-specific CD8+ T cells than those specific to influenza A virus. Importantly, the proportion of IFN-γ-producing cells was higher in PD-1+ cells than PD-1- cells among multimer+ cells, indicating that PD-1-expressing, SARS-CoV-2-specific CD8+ T cells are not exhausted, but functional. Our current findings provide information for understanding of SARS-CoV-2-specific CD8+ T cells elicited by infection or vaccination.

Critical role of neutralizing antibody for SARS-CoV-2 reinfection and transmission.

Cases of laboratory-confirmed SARS-CoV-2 reinfection have been reported in a number of countries. Further, the level of natural immunity induced by SARS-CoV-2 infection is not fully clear, nor is it clear if a primary infection is protective against reinfection. To investigate the potential association between serum antibody titres and reinfection of SARS-CoV-2, ferrets with different levels of NAb titres after primary SARS-CoV-2 infection were subjected to reinfection with a heterologous SARS-CoV-2 strain. All heterologous SARS-CoV-2 reinfected ferrets showed active virus replication in the upper respiratory and gastro-intestinal tracts. However, the high NAb titre group showed attenuated viral replication and rapid viral clearance. In addition, direct-contact transmission was observed only from reinfected ferrets with low NAb titres (<20), and not from other groups. Further, lung histopathology demonstrated the presence of limited inflammatory regions in the high NAb titre groups compared with control and low NAb groups. This study demonstrates a close correlation between a low NAb titre and SARS-CoV-2 reinfection in a recovered ferret reinfection model.