Characterization of an EG.5.1 clinical isolate in vitro and in vivo (preprint)

EG.5.1 is a subvariant of the SARS-CoV-2 Omicron XBB variant that is rapidly increasing in prevalence worldwide. EG.5.1 has additional substitutions in its spike protein (namely, Q52H and F456L) compared with XBB.1.5. However, the pathogenicity, transmissibility, and immune evasion properties of clinical isolates of EG.5.1 are largely unknown. In this study, we used wild-type Syrian hamsters to investigate the replicative ability, pathogenicity, and transmissibility of a clinical EG.5.1 isolate. Our data show that there are no obvious differences in growth ability and pathogenicity between EG.5.1 and XBB.1.5, and both EG.5.1 and XBB.1.5 are attenuated compared to a Delta variant isolate. We also found that EG.5.1 is transmitted more efficiently between hamsters compared with XBB.1.5. In addition, unlike XBB.1.5, we detected EG.5.1 virus in the lungs of four of six exposed hamsters, suggesting that the virus tropism of EG.5.1 is different from that of XBB.1.5 after airborne transmission. Finally, we assessed the neutralizing ability of plasma from convalescent individuals and found that the neutralizing activity against EG.5.1 was slightly, but significantly, lower than that against XBB.1.5 or XBB.1.9.2. This suggests that EG.5.1 effectively evades humoral immunity and that the amino acid differences in the S protein of EG.5.1 compared with that of XBB.1.5 or XBB.1.9.2 (i.e., Q52H, R158G, and F456L) alter the antigenicity of EG.5.1. Our data suggest that the increased transmissibility and altered antigenicity of EG.5.1 may be driving its increasing prevalence over XBB.1.5 in the human population.

Severity Predictors of COVID-19 in SARS-CoV-2 Variant, Delta and Omicron Period; Single Center Study (preprint)

Background: The outcomes of coronavirus disease 2019 (COVID-19) treatment have improved due to vaccination and the establishment of better treatment regimens. However, the emergence of variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes COVID-19, and the corresponding changes in the characteristics of the disease present new challenges in patient management. This study aimed to analyze predictors of COVID-19 severity caused by the delta and omicron variants of SARS-CoV-2. Methods: We retrospectively analyzed the data of patients who were admitted for COVID-19 at Yokohama City University Hospital from August 2021 to March 2022. Results: A total of 141 patients were included in this study. Of these, 91 had moderate COVID-19, whereas 50 had severe COVID-19. There were significant differences in sex, vaccination status, dyspnea, sore throat symptoms, and body mass index (BMI) (p <0.0001, p <0.001, p <0.001, p=0.02, p< 0.0001, respectively) between the moderate and severe COVID-19 groups. Regarding comorbidities, smoking habit and renal dysfunction were significantly different between the two groups (p=0.007 and p=0.01, respectively). Regarding laboratory data, only LDH level on the first day of hospitalization was significantly different between the two groups (p<0.001). Multiple logistic regression analysis revealed that time from the onset of COVID-19 to hospitalization, BMI, smoking habit, and LDH level were significantly different between the two groups (p<0.03, p=0.039, p=0.008, p<0.001, respectively). The cut-off value for the time from onset of COVID-19 to hospitalization was four days (sensitivity, 0.73; specificity, 0.70). Conclusions: Time from the onset of COVID-19 to hospitalization is the most important factor in the prevention of the aggravation of COVID-19 caused by the delta and omicron SARS-CoV-2 variants. Appropriate medical management within four days after the onset of COVID-19 is essential for preventing the progression of COVID-19, especially in patients with smoking habits.

Age stratified seroprevalence of antibodies against SARS CoV 2 in the pre and post vaccination era, February 2020 to March 2022, Japan (preprint)

Japan has reported a small number of COVID-19 cases relative to other countries. Because not all infected people receive diagnostic tests for COVID-19, the reported number of COVID-19 cases must be lower than the actual number of infections. Assessments of the presence of antibodies against the spike protein of SARS-CoV-2 can retrospectively determine the history of natural infection and vaccination. In this study, we assessed SARS-CoV-2 seroprevalence by analyzing over 60,000 samples collected in Japan from February 2020 to March 2022. The results showed that about 5% of the Japanese population had been infected with the virus by January 2021. The seroprevalence increased with the administration of vaccinations to adults; however, among the elderly, it was not as high as the vaccination rate, probably due to poor immune responses to the vaccines and waning immunity. The infection was spread during the epidemic waves caused by the SARS-CoV-2 Delta and Omicron variants among children who were not eligible for vaccination. Nevertheless, their seroprevalence was as low as 10% as of March 2022. Our study underscores the low incidence of SARS-CoV-2 infection in Japan and the effects of vaccination on immunity at the population level.

Multi-omics Characterization of Neutrophil Extracellular Trap Formation in Severe and Mild COVID-19 Infections (preprint)

The detailed mechanisms of COVID-19 infection pathology remain poorly understood. To improve our understanding of SARS-CoV-2 pathology, we performed a multi-omics analysis of an immunologically naive SARS-CoV-2 clinical cohort from the plasma of uninfected controls, mild, and severe infections. A comparison of healthy controls and patient samples showed activation of neutrophil degranulation pathways and formation of neutrophil extracellular trap (NET) complexes that were activated in a subset of the mild infections and more prevalent in severe infections (containing multiple NET proteins in individual patient samples). As a potential mechanism to suppress NET formation, multiple redox enzymes were elevated in the mild and severe symptom population. Analysis of metabolites from the same cohort showed a 24- and 60-fold elevation in plasma L-cystine, the oxidized form of cysteine, which is a substrate of the powerful antioxidant glutathione, in mild and severe patients, respectively. Unique to patients with mild infections, the carnosine dipeptidase modifying enzyme (CNDP1) was up-regulated. The strong protein and metabolite oxidation signatures suggest multiple compensatory pathways working to suppress oxidation and NET formation in SARS-CoV-2 infections.

Analysis of humoral immunity against emerging SARS-CoV-2 variants: a population-based prevalence study in Yokohama, Japan (preprint)

Background Little is known about the population prevalence of antibodies against emerging immune escape variants of SARS-CoV-2. Methods A population-based prevalence study was conducted in Yokohama City, the most populous municipality of Japan. Quantitative measurements of immunoglobulin G against SARS-CoV-2 spike protein (SP-IgG) and qualitative measurements of neutralization antibodies against the Omicron BA.1 and BA.2 variants were performed. Results Of 6,000 randomly selected residents aged 20-74, 1,277 participated in the study during a period from January 30 to February 28, 2022. Of them, 3% had prior diagnosis of COVID-19, 96% received at least two-doses of SARS-CoV-2 vaccines, and 94% were positive for SP-IgG. The positive rates of neutralizing antibodies were 28% to Omicron BA.1 and BA.2 variants in a random sample of 10% of participants (n=123) and 100% to BA.1 and BA.2 among participants who received the third vaccination at least 7 days before (n=66). Conclusions In this population-based prevalence study in Japan, most had SP-IgG antibodies but the overall neutralizing antibody positive rate was 28% against the Omicron BA.1 and BA.2 variants. The population-level insufficient humoral immunity against the Omicron variants may explain the outbreak of COVID-19 during this period in Japan.

SARS-CoV-2 delta AY.1 variant cluster in an accommodation facility for COVID-19: Research study (preprint)

Background: This is a case report on a cluster infection of novel severe acute respiratory syndrome coronavirus 2 delta AY.1 variant at an accommodation facility and the subsequent attempts to isolate individuals who tested positive. Methods The background that facilitated this cluster was investigated, and the conditions in which infection was established, the infection route, and the effectiveness of routine measures were evaluated. Ninety-nine staff members had been working at the accommodation facility at the time of infection, and it was estimated that 10 members were infected with the delta AY.1 variant. Results Our results suggest that infection of staff from a patient staying overnight should be excluded. The factors contributing to the cluster infection involved short-distance conversations with individuals wearing non-woven three-layer masks moved out of position (non-woven) and gathering together with individuals wearing non-woven masks in hypoventilated conditions. Our findings also indicate that this variant is possibly airborne and can infect individuals in enclosed spaces with poor ventilation, even when either infected or exposed individuals wear non-woven masks. Conclusions The routine maintenance of systems established for the detection of infections and prompt and appropriate preventive measures following the identification of positive individuals will help prevent further cluster infections.

SARS-CoV-2 Delta AY.1 variant Cluster in an accommodation facility for COVID-19 (preprint)

This is a case report on a cluster infection of novel severe acute respiratory syndrome coronavirus 2 delta AY.1 variant at an accommodation facility and the subsequent attempts to isolate individuals who tested positive. The background that facilitated this cluster was investigated, and the conditions in which infection was established, the infection route, and the effectiveness of routine measures were evaluated. Ninety-nine staff members had been working at the accommodation facility at the time of infection, and it was estimated that 10 members were infected with the delta AY.1 variant. Our results suggest that infection of staff from a patient staying overnight should be excluded. The factors contributing to the cluster infection involved short-distance conversations with individuals wearing non-woven three-layer masks moved out of position (non-woven) and gathering together with individuals wearing non-woven masks in hypoventilated conditions. Our findings also indicate that this variant is possibly airborne and can infect individuals in enclosed spaces with poor ventilation, even when either infected or exposed individuals wear non-woven masks. The routine maintenance of systems established for the detection of infections and prompt and appropriate preventive measures following the identification of positive individuals will help prevent further cluster infections.

Vaccine-induced humoral and cellular immunity against SARS-CoV-2 at 6 months post BNT162b2 vaccination (preprint)

To evaluate vaccine-induced humoral and cell-mediated immunity at 6 months post BNT162b2 vaccination, immunoglobulin G against SARS-CoV-2 spike protein (SP IgG), 50% neutralizing antibody (NT50), and spot-forming cell (SFC) counts were evaluated by interferon-{gamma} releasing ELISpot assay of 98 healthy subjects (median age, 43 years). The geometric mean titers of SP IgG and NT50 decreased from 95.2 (95% confidence interval (CI) 79.8-113.4) to 5.7 (95% CI 4.9-6.7) and from 680.4 (588.0-787.2) to 130.4 (95% CI 104.2-163.1), respectively, at 3 weeks and 6 months after the vaccination. SP IgG titer was negatively correlated with age and alcohol consumption. Spot-forming cell counts at 6 months did not correlate with age, gender, and other parameters of the patients. SP IgG, NT50, and SFC titers were elevated in the breakthrough infected subjects. Although the levels of vaccine-induced antibodies dramatically declined at 6 months after vaccination, a certain degree of cellular immunity was observed irrespective of the age.

Persistence of robust humoral immune response in COVID-19 convalescent individuals over 12 months after infection (preprint)

SARS-CoV-2 infection elicits varying degrees of protective immunity conferred by neutralizing antibodies (nAbs). Here we report the persistence of nAb responses over 12 months after infection despite its decreasing trend noticed from 6 months. The study included sera from 358 individuals who had been infected with SARS-CoV-2 between January and May 2020. Samples were collected at 6 and 12 months after onset. The titers of IgG to the viral nucleocapsid protein (NP) and receptor-binding domain of the spike protein (RBD) were measured by CLEIA. The nAb titer was determined using lentivirus-based pseudovirus or authentic virus. Antibody titers of NP-IgG, RBD-IgG, and nAbs were higher in severe and moderate cases than in mild cases at 12 months after onset. While the nAb levels were likely to confer adequate protection against wild-type viral infection, the neutralization activity to recently circulating variants in some of the mild cases ([~]30%) was undermined, implying the susceptibility of reinfection to the variants of concerns (VOCs). COVID-19 convalescent individuals have robust humoral immunity even at 12 months after infection albeit that the medical history and background of patients could affect the function and dynamics of antibody response to the VOCs.

Antibody titers against the Alpha, Beta, Gamma, and Delta variants of SARS-CoV-2 induced by BNT162b2 vaccination measured using automated chemiluminescent enzyme immunoassay (preprint)

BackgroundLevels of 50% neutralizing titer (NT50) reflect a vaccine-induced humoral immunity after the vaccination against the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Measurements of NT50 are difficult to implement in large quantities. A high-throughput laboratory test is expected for determining the level of herd immunity against SARS-CoV-2. MethodsWe analyzed samples from 168 Japanese healthcare workers who had completed two doses of the BNT162b2 vaccine. We analyzed immunoglobulin G (IgG) index values against spike protein (SP) using automated chemiluminescent enzyme immunoassay system AIA-CL and analyzed the background factors affecting antibody titer. SP IgG index was compared with 50% neutralization titers. ResultsThe median SP IgG index values of the subjects (mean age = 43 years; 75% female) were 0.1, 1.35, 60.80, and 97.35 before and at 2, 4, and 6 weeks after the first dose, respectively. At 4 and 6 weeks after the first dose, SP IgG titers were found to have positive correlation with NT50 titer (r=0.7535 in 4 weeks; r=0.4376 in 6 weeks). Proportions of the SP IgG index values against the Alpha, Beta, Gamma, and Delta variants compared with the original strain were 2.029, 0.544, 1.017, and 0.6096 respectively. Older age was associated with lower SP IgG titer index 6 weeks after the first dose. ConclusionsSP IgG index values were raised at 3 weeks after two doses of BNT162b2 vaccination and have positive correlation with NT50. SP IgG index values were lower in the older individuals and against Beta and Delta strain.

Neutralizing efficacy of vaccines against the SARS-CoV-2 Mu variant (preprint)

The rise of mutant strains of SARS-CoV-2 poses an additional problem to the existing pandemic of COVID-19. There are rising concerns about the Mu variant which can escape humoral immunity acquired from infections from previous strains or vaccines. We examined the neutralizing efficacy of the BNT162b2 mRNA vaccine against the Mu variant and report that the vaccine has 76% neutralizing effectiveness against the Mu compared to 96% with the original strain. We also show that Mu, similar to the Delta variant, causes cell-to-cell fusion which can be an additional factor for the variant to escape vaccine-mediated humoral immunity. Despite the rise in vaccine escape strains, the vaccine still possesses adequate ability to neutralize majority of the mutants.

Machine Learning Supports Clinical Decision in Optimized Treatment for COVID-19 Patients (preprint)

Background: It has been difficult to distinguish between mild, moderate, and severe cases at an early stage for COVID-19 patients, making it challenging to decide an optimized treatment for each patient. This machine learning system could predict the clinical course and be used to develop a novel method to provide optimal treatment based on risk. Method: We applied machine learning techniques to international clinical data from a large cohort of patients with COVID-19 at 15 hospitals in Japan and three hospitals in New York City from January 1, 2020 to March 30, 2021. We analyzed clinical information of over 2000 COVID-19 patients comprising various races and ethnicities and built a severity and mortality prediction model. Furthermore, using a severity index with machine learning allowed early detection of patients most at-risk for developing severe illness to support the decision for the patient to receive optimized therapy. Findings: We developed an international COVID-19 early prediction model for use at the time of hospital admission that predicts disease severity and mortality with high accuracy, 0.88 (AUC). Using the novel method of severity-matched analysis to assess treatment effectiveness, in the high-risk group, the Kaplan–Meier estimates of mortality by Day 30 were 26% in the dexamethasone treatment group and 63% in the non-treatment group. The Kaplan–Meier estimates of mortality were low at 3% with remdesivir and dexamethasone in combination and 49% with no remdesivir and dexamethasone treatment by Day 30. There may be an add-on effect of remdesivir to conventional dexamethasone. Interpretation: The severity prediction index can be calculated, which can assist with an optimized treatment for COVID-19 patients in each risk group. The severity-matched treatment system could support the recommendation of optimized treatments, such as dexamethasone, remdesivir, or heparin, in high-risk groups by calculating the severity index predicted at the time of the first visit.Trial Registration Details: The trial registration number was 2020142NI. Funding Information: K.T., K.I., and Y.D. received funding from the Japan Agency for Medical Research and Development (AMED) (19fk0108153h0001). K.T. received funding from AMED (19jm0610015h0001). K.T. received funding from the Healthy Longevity Global Grand Challenge, Catalyst Award.Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: The study protocol was centrally reviewed by the Institutional Review Board of Tokyo University. The requirement for consent was waived given the retrospective and non-interventional nature of the study.

Rapid detection of neutralizing antibodies to SARS-CoV-2 variants in post-vaccination sera (preprint)

The uncontrolled spread of the COVID-19 pandemic has led to the emergence of different SARS-CoV-2 variants across the globe. The ongoing global vaccination strategy to curtail the COVID-19 juggernaut, is threatened by the rapidly spreading Variants of Concern (VOC) and other regional mutants, which are less responsive to neutralization by infection or vaccine derived antibodies. We have previously developed the hiVNT system which detects SARS-CoV-2 neutralizing antibodies in sera in less than three hours. In this study, we modify the hiVNT for rapid qualitative screening of neutralizing antibodies (nAb) to multiple variants of concern (VOC) of SARS-CoV-2, and assess the neutralizing efficacy of the BNT162b2 mRNA vaccine on seven epidemiologically relevant SARS-CoV-2 variants. Here we show that the BNT162b2 mRNA vaccine can activate humoral immunity against the major SARS-CoV-2 mutants that are currently in circulation. Albeit a small sample size, we observed that one dose of vaccine was sufficient to elicit a protective humoral response in previously infected people. Using a panel of seven SARS-CoV-2 variants and a single prototype virus, our modified hiVNT would be useful for large-scale community wide testing to detect protective immunity that may confer vaccine/immune passport in the ongoing COVID-19 pandemic.

Real-world evaluation of a computed tomography-first triage strategy for suspected coronavirus disease 2019 in emergency outpatients in Japan: an observational cohort study (preprint)

Background: Coronavirus disease 2019 (COVID-19) continues to spread worldwide. Because of the absence of reliable rapid diagnostic systems, patients with COVID-19 symptoms are suspected of disease. Computed tomography (CT) in patients with suspected COVID-19 may be reasonable for triaging, and CT-first triage strategies have been proposed. However, clinical evaluation of a CT-first triage protocol is lacking. The aim of this study is to investigate the real-world efficacy and limitations of a CT-first triage strategy in patients with suspected COVID-19.Methods: This was a single-center cohort study evaluating outpatients with suspected COVID-19 who underwent a medical examination at Yokohama City University Hospital and who were prospectively registered between 9 February and 5 May 2020. We treated patients according to the CT-first triage protocol. CT findings were classified into five categories according to the COVID-19 Reporting and Data System (CO-RADS). With the CT-first triage protocol, patients with a suspicious clinical history, symptoms, or suspicious findings on chest CT were allocated to the COVID-19 suspected group. The primary outcome was efficacy of the CT-first triage protocol for outpatients with suspected COVID-19. We conducted additional analyses of the isolation time of outpatients with suspected COVID-19 and reached final diagnoses.Results: In total, 108 outpatients with suspected COVID-19 were examined at our hospital. Forty-eight patients (44.9%) were categorized as CO-RADS 1, 26 patients (24.3%) as CO-RADS 2, 14 patients (13.1%) as CO-RADS 3, 6 patients (5.6%) as CO-RADS 4, and 13 patients (12.1%) as CO-RADS 5. One patient was excluded because of pregnancy. Using the CT-first triage protocol, 48 (44.9%) patients were suspected of having COVID-19. Nine patients (18.8%) in this group were positive for severe acute respiratory syndrome coronavirus 2 using polymerase chain reaction; no patients in the group not suspected of having COVID-19 were diagnosed with COVID-19 during follow up. The protocol significantly shortened the duration of isolation for the not-suspected versus the suspected group (70.5 vs. 1037.0 minutes, P < .001). Conclusions: Our CT-first triage protocol was acceptable for triaging outpatients with suspected COVID-19. This protocol will be helpful for appropriate triage, especially in areas where polymerase chain reaction is limited.