Prediction models for neutralization activity against emerging SARS-CoV-2 variants: A cross-sectional study.

Objective: Despite extensive vaccination campaigns to combat the coronavirus disease (COVID-19) pandemic, variants of concern, particularly the Omicron variant (B.1.1.529 or BA.1), may escape the antibodies elicited by vaccination against SARS-CoV-2. Therefore, this study aimed to evaluate 50% neutralizing activity (NT50) against SARS-CoV-2 D614G, Delta, Omicron BA.1, and Omicron BA.2 and to develop prediction models to predict the risk of infection in a general population in Japan. Methods: We used a random 10% of samples from 1,277 participants in a population-based cross-sectional survey conducted in January and February 2022 in Yokohama City, the most populous municipality in Japan. We measured NT50 against D614G as a reference and three variants (Delta, Omicron BA.1, and BA.2) and immunoglobulin G against SARS-CoV-2 spike protein (SP-IgG). Results: Among 123 participants aged 20-74, 93% had received two doses of SARS-CoV-2 vaccine. The geometric means (95% confidence intervals) of NT50 were 65.5 (51.8-82.8) for D614G, 34.3 (27.1-43.4) for Delta, 14.9 (12.2-18.0) for Omicron BA.1, and 12.9 (11.3-14.7) for Omicron BA.2. The prediction model with SP-IgG titers for Omicron BA.1 performed better than the model for Omicron BA.2 (bias-corrected R 2 with bootstrapping: 0.721 vs. 0.588). The models also performed better for BA.1 than for BA.2 (R 2 = 0.850 vs. 0.150) in a validation study with 20 independent samples. Conclusion: In a general Japanese population with 93% of the population vaccinated with two doses of SARS-CoV-2 vaccine, neutralizing activity against Omicron BA.1 and BA.2 were substantially lower than those against D614G or the Delta variant. The prediction models for Omicron BA.1 and BA.2 showed moderate predictive ability and the model for BA.1 performed well in validation data.

Enhancement of humoral and cellular immunity against SARS-CoV-2 by a third dose of BNT162b2 vaccine in Japanese healthcare workers.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has raised concerns regarding vaccine effectiveness. We investigated humoral and cellular immune responses against SARS-CoV-2 in healthcare workers before and after a third (booster) dose of the BNT162b2 mRNA vaccine. It significantly enhanced both humoral and cellular immunity in previously uninfected individuals. However, cellular immunity was not enhanced in previously infected persons, suggesting that three antigenic stimuli by vaccination or natural infection reached a plateau of cellular immunity. Even with reinforced immunity to SARS-CoV-2, we confirmed several post-booster breakthrough cases caused by the Omicron variant.

Intratracheal trimerized nanobody cocktail administration suppresses weight loss and prolongs survival of SARS-CoV-2 infected mice.

BACKGROUND: SARS-CoV-2 Omicron variants are highly resistant to vaccine-induced immunity and human monoclonal antibodies. METHODS: We previously reported that two nanobodies, P17 and P86, potently neutralize SARS-CoV-2 VOCs. In this study, we modified these nanobodies into trimers, called TP17 and TP86 and tested their neutralization activities against Omicron BA.1 and subvariant BA.2 using pseudovirus assays. Next, we used TP17 and TP86 nanobody cocktail to treat ACE2 transgenic mice infected with lethal dose of SARS-CoV-2 strains, original, Delta and Omicron BA.1. RESULTS: Here, we demonstrate that a novel nanobody TP86 potently neutralizes both BA.1 and BA.2 Omicron variants, and that the TP17 and TP86 nanobody cocktail broadly neutralizes in vitro all VOCs as well as original strain. Furthermore, intratracheal administration of this nanobody cocktail suppresses weight loss and prolongs survival of human ACE2 transgenic mice infected with SARS-CoV-2 strains, original, Delta and Omicron BA.1. CONCLUSIONS: Intratracheal trimerized nanobody cocktail administration suppresses weight loss and prolongs survival of SARS-CoV-2 infected mice.

Antibodies are made by the immune system to identify and inactivate infectious agents such as viruses. Alpacas produce a simple type of antibodies called nanobodies. We previously developed two nanobodies named P17 and P86 that inactivate SARS-CoV-2. In this study, we modified these nanobodies to create two nanobodies named TP17 and TP86. The cocktail of these nanobodies inactivated different types of SARS-CoV-2 viruses including Omicron BA.1 and BA.2. The cocktail also prolonged survival of mice infected with lethal doses of SARS-CoV-2.

Prospective Clinical Evaluation of the Diagnostic Accuracy of a Highly Sensitive Rapid Antigen Test Using Silver Amplification Technology for Emerging SARS-CoV-2 Variants.

The COVID-19 pandemic caused by SARS-CoV-2 remains a serious health concern worldwide due to outbreaks of SARS-CoV-2 variants that can escape vaccine-acquired immunity and infect and transmit more efficiently. Therefore, an appropriate testing method for COVID-19 is essential for effective infection control and the prevention of local outbreaks. Compared to reverse-transcription polymerase chain reaction (RT-PCR) tests, antigen tests are used for simple point-of-care testing, enabling the identification of viral infections. In this study, we tested the clinical usefulness of the FUJIFILM COVID-19 Ag test, an antigen test based on silver amplification and immunochromatographic technology. The FUJIFILM COVID-19 Ag test was shown to detect a lower viral concentration as compared to other conventional kits without significant performance loss in detecting prevalent SARS-CoV-2 variants. We tested nasopharyngeal and nasal swabs from a single patient during two different epidemic periods dominated by various SARS-CoV-2 variants. We observed that the sensitivity of the FUJIFILM COVID-19 Ag test was 95.7% and 85.7% in nasopharyngeal and nasal swabs, respectively. These results suggest that the FUJIFILM COVID-19 Ag test is highly sensitive and applicable when RT-PCR testing is unavailable. Furthermore, these results indicate that high-frequency testing using nasal swab specimens may be a valuable screening strategy.

Development of a Fully Automated Desktop Analyzer and Ultrahigh Sensitivity Digital Immunoassay for SARS-CoV-2 Nucleocapsid Antigen Detection.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has had a significant impact on public health and the global economy. Several diagnostic tools are available for the detection of infectious diseases, with reverse transcription-polymerase chain reaction (RT-PCR) testing specifically recommended for viral RNA detection. However, this diagnostic method is costly, complex, and time-consuming. Although it does not have sufficient sensitivity, antigen detection by an immunoassay is an inexpensive and simpler alternative to RT-PCR. Here, we developed an ultrahigh sensitivity digital immunoassay (d-IA) for detecting SARS-CoV-2 nucleocapsid (N) protein as antigens using a fully automated desktop analyzer based on a digital enzyme-linked immunosorbent assay. METHODS: We developed a fully automated d-IA desktop analyzer and measured the viral N protein as an antigen in nasopharyngeal (NP) swabs from patients with coronavirus disease. We studied nasopharyngeal swabs of 159 and 88 patients who were RT-PCR-negative and RT-PCR-positive, respectively. RESULTS: The limit of detection of SARS-CoV-2 d-IA was 0.0043 pg/mL of N protein. The cutoff value was 0.029 pg/mL, with a negative RT-PCR distribution. The sensitivity of RT-PCR-positive specimens was estimated to be 94.3% (83/88). The assay time was 28 min. CONCLUSIONS: Our d-IA system, which includes a novel fully automated desktop analyzer, enabled detection of the SARS-CoV-2 N-protein with a comparable sensitivity to RT-PCR within 30 min. Thus, d-IA shows potential for SARS-CoV-2 detection across multiple diagnostic centers including small clinics, hospitals, airport quarantines, and clinical laboratories.

Antimicrobial strategy for targeted elimination of different microbes, including bacterial, fungal and viral pathogens.

The continuous emergence of microbial pathogens for which there are no effective antimicrobials threatens global health, necessitating novel antimicrobial approaches. Here, we present a targeted antimicrobial strategy that can be applied to various microbial pathogens. A photoimmuno-conjugate composed of an antibody against the target pathogen and a photoplastic phthalocyanine-derivative probe that generates photo-induced mechanical stress was developed based on photoimmuno-technology. This strategy, named as photoimmuno-antimicrobial strategy (PIAS), eliminates targeted pathogens, regardless of the target species or drug-resistance status. Specifically, PIAS acts on a broad range of microbes, including the bacterial pathogen Staphylococcus aureus, fungal pathogen Candida albicans, including their drug-resistant strains, and viral pathogen SARS-CoV-2, the causative agent of COVID-19. Furthermore, PIAS protects mice from fatal infections without damaging the non-targeted host microbiota and tissues. This study may contribute to the development of next-generation anti-infective therapies.

A panel of nanobodies recognizing conserved hidden clefts of all SARS-CoV-2 spike variants including Omicron.

We are amid the historic coronavirus infectious disease 2019 (COVID-19) pandemic. Imbalances in the accessibility of vaccines, medicines, and diagnostics among countries, regions, and populations, and those in war crises, have been problematic. Nanobodies are small, stable, customizable, and inexpensive to produce. Herein, we present a panel of nanobodies that can detect the spike proteins of five SARS-CoV-2 variants of concern (VOCs) including Omicron. Here we show via ELISA, lateral flow, kinetic, flow cytometric, microscopy, and Western blotting assays that our nanobodies can quantify the spike variants. This panel of nanobodies broadly neutralizes viral infection caused by pseudotyped and authentic SARS-CoV-2 VOCs. Structural analyses show that the P86 clone targets epitopes that are conserved yet unclassified on the receptor-binding domain (RBD) and contacts the N-terminal domain (NTD). Human antibodies rarely access both regions; consequently, the clone buries hidden crevasses of SARS-CoV-2 spike proteins that go undetected by conventional antibodies.

Characterization and Utilization of Disulfide-Bonded SARS-CoV-2 Receptor Binding Domain of Spike Protein Synthesized by Wheat Germ Cell-Free Production System.

The spike protein (SP) of SARS-CoV-2 is an important target for COVID-19 therapeutics and vaccines as it binds to the ACE2 receptor and enables viral infection. Rapid production and functional characterization of properly folded SP is of the utmost importance for studying the immunogenicity and receptor-binding activity of this protein considering the emergence of highly infectious viral variants. In this study, we attempted to express the receptor-binding region (RBD) of SARS-CoV-2 SP containing disulfide bonds using the wheat germ cell-free protein synthesis system. By adding protein disulfide isomerase (PDI) and endoplasmic reticulum oxidase (ERO1α) to the translational reaction mixture, we succeeded in synthesizing a functionally intact RBD protein that can interact with ACE2. Using this RBD protein, we have developed a high-throughput AlphaScreen assay to evaluate the RBD-ACE2 interaction, which can be applied for drug screening and mutation analysis. Thus, our method sheds new light on the structural and functional properties of SARS-CoV-2 SP and has the potential to contribute to the development of new COVID-19 therapeutics.

Evasion of vaccine-induced humoral immunity by emerging sub-variants of SARS-CoV-2.

Background: Emergence of vaccine-escaping SARS-CoV-2 variants is a serious problem for global public health. The currently rampant Omicron has been shown to possess remarkable vaccine escape; however, the selection pressure exerted by vaccines might pave the way for other escape mutants in the near future. Materials & methods: For detection of neutralizing antibodies, the authors used the recently developed HiBiT-based virus-like particle neutralization test system. Sera after vaccination (two doses of Pfizer/BioNTech mRNA vaccine) were used to evaluate the neutralizing activity against various strains of SARS-CoV-2. Results: Beta+R346K, which was identified in the Philippines in August 2021, exhibited the highest vaccine resistance among the tested mutants. Surprisingly, Mu+K417N mutant exhibited almost no decrease in neutralization. Imdevimab retained efficacy against these strains. Conclusions: Mutations outside the receptor-binding domain contributed to vaccine escape. Both genomic surveillance and phenotypic analysis synergistically accelerate identifications of vaccine-escaping strains.

Prior to the Omicron variant, the SARS-CoV-2 Beta sub-variant found in the Philippines in August 2021 exhibited remarkable vaccine-escaping capacity. Although Omicron is, at the time of writing, causing most of the infections globally, both genomic surveillance and phenotypic analysis should be reinforced to accelerate the identification of newly emerging vaccine-escaping SARS-CoV-2 variants.

Detecting SARS-CoV-2 neutralizing immunity: highlighting the potential of split nanoluciferase technology.

The coronavirus disease 2019 (COVID-19) pandemic has progressed over 2 years since its onset causing significant health concerns all over the world and is currently curtailed by mass vaccination. Immunity acquired against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be following either infection or vaccination. However, one can never be sure whether the acquired immunity is adequate to protect the individual from subsequent infection because of three important factors: individual variations in humoral response dynamics, waning of protective antibodies over time, and the emergence of immune escape mutants. Therefore, a test that can accurately differentiate the protected from the vulnerable is the need of the hour. The plaque reduction neutralization assay is the conventional gold standard test for estimating the titers of neutralizing antibodies that confer protection. However, it has got several drawbacks, which hinder the practical application of this test for wide-scale usage. Hence, various tests have been developed to detect protective immunity against SARS-CoV-2 that directly or indirectly assess the presence of neutralizing antibodies to SARS-CoV-2 in a lower biosafety setting. In this review, the pros and cons of the currently available assays are elaborated in detail and special focus is put on the scope of the novel split nanoluciferase technology for detecting SARS-CoV-2 neutralizing antibodies.

Vaccine-induced humoral response against SARS-CoV-2 dramatically declined but cellular immunity possibly remained at 6 months post BNT162b2 vaccination.

To evaluate vaccine-induced humoral and cell-mediated immunity at 6 months after completion of two doses of 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-γ 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.

Molecular and Epidemiological Characterization of Emerging Immune-Escape Variants of SARS-CoV-2.

The successive emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has presented a major challenge in the management of the coronavirus disease (COVID-19) pandemic. There are growing concerns regarding the emerging variants escaping vaccines or therapeutic neutralizing antibodies. In this study, we conducted an epidemiological survey to identify SARS-CoV-2 variants that are sporadically proliferating in vaccine-advanced countries. Subsequently, we created HiBiT-tagged virus-like particles displaying spike proteins derived from the variants to analyze the neutralizing efficacy of the BNT162b2 mRNA vaccine and several therapeutic antibodies. We found that the Mu variant and a derivative of the Delta strain with E484K and N501Y mutations significantly evaded vaccine-elicited neutralizing antibodies. This trend was also observed in the Beta and Gamma variants, although they are currently not prevalent. Although 95.2% of the vaccinees exhibited prominent neutralizing activity against the prototype strain, only 73.8 and 78.6% of the vaccinees exhibited neutralizing activity against the Mu and the Delta derivative variants, respectively. A long-term analysis showed that 88.8% of the vaccinees initially exhibited strong neutralizing activity against the currently circulating Delta strain; the number decreased to 31.6% for the individuals at 6 months after vaccination. Notably, these variants were shown to be resistant to several therapeutic antibodies. Our findings demonstrate the differential neutralization efficacy of the COVID-19 vaccine and monoclonal antibodies against circulating variants, suggesting the need for pandemic alerts and booster vaccinations against the currently prevalent variants.

Phosphopeptide enrichment using Phos-tag technology reveals functional phosphorylation of the nucleocapsid protein of SARS-CoV-2.

Phosphorylation of viral proteins serves as a regulatory mechanism during the intracellular life cycle of infected viruses. There is therefore a pressing need to develop a method to efficiently purify and enrich phosphopeptides derived from viral particles in biological samples. In this study, we utilized Phos-tag technology to analyze the functional phosphorylation of the nucleocapsid protein (N protein; NP) of severe respiratory syndrome coronavirus 2 (SARS-CoV-2). Viral particles were collected from culture supernatants of SARS-CoV-2-infected VeroE6/TMPRSS2 cells by ultracentrifugation, and phosphopeptides were purified by Phos-tag magnetic beads for LC-MS/MS analysis. Analysis revealed that NP was reproducibly phosphorylated at serine 79 (Ser79). Multiple sequence alignment and phylogenetic analysis showed that the Ser79 was a distinct phospho-acceptor site in SARS-CoV-2 but not in other beta-coronaviruses. We also found that the prolyl-isomerase Pin1 bound to the phosphorylated Ser79 in NP and positively regulated the production of viral particles. These results suggest that SARS-CoV-2 may have acquired the potent virus-host interaction during its evolution mediated by viral protein phosphorylation. Moreover, Phos-tag technology can provide a useful means for analyzing the functional phosphorylation of viral proteins. SIGNIFICANCE: In this study, we aimed to investigate the functional phosphorylation of SARS-CoV-2 NP. For this purpose, we used Phos-tag technology to purify and enrich virus-derived phosphopeptides with high selectivity and reproducibility. This method can be particularly useful in analyzing viral phosphopeptides from cell culture supernatants that often contain high concentrations of fetal bovine serum and supplements. We newly identified an NP phosphorylation site at Ser79, which is important for Pin1 binding. Furthermore, we showed that the interaction between Pin1 and phosphorylated NP could enhance viral replication in a cell culture model.

Persistence of Robust Humoral Immune Response in Coronavirus Disease 2019 Convalescent Individuals Over 12 Months After Infection.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection elicits varying degrees of protective immunity conferred by neutralizing antibodies (nAbs). In this study, we report the persistence of nAb responses over 12 months after infection despite their decreasing trend noticed from 6 months. METHODS: The study included sera from 497 individuals who had been infected with SARS-CoV-2 between January and August 2020. Samples were collected at 6 and 12 months after onset. The titers of immunoglobulin (Ig)G to the viral nucleocapsid protein (NP) and receptor-binding domain (RBD) of the spike protein were measured by chemiluminescence enzyme immunoassay. The nAb titer was determined using lentivirus-based pseudovirus or authentic virus. RESULTS: 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. Although 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 to reinfection with the variants of concerns (VOCs). CONCLUSIONS: Coronavirus disease 2019 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.

BACKGROUND: Levels of 50% neutralizing titer (NT50) reflect the 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. METHODS: We 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. RESULTS: The 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. CONCLUSIONS: SP IgG index values were rised 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.

All-Trans Retinoic Acid Exhibits Antiviral Effect against SARS-CoV-2 by Inhibiting 3CLpro Activity.

The pandemic of COVID-19 caused by SARS-CoV-2 continues to spread despite the global efforts taken to control it. The 3C-like protease (3CLpro), the major protease of SARS-CoV-2, is one of the most interesting targets for antiviral drug development because it is highly conserved among SARS-CoVs and plays an important role in viral replication. Herein, we developed high throughput screening for SARS-CoV-2 3CLpro inhibitor based on AlphaScreen. We screened 91 natural product compounds and found that all-trans retinoic acid (ATRA), an FDA-approved drug, inhibited 3CLpro activity. The 3CLpro inhibitory effect of ATRA was confirmed in vitro by both immunoblotting and AlphaScreen with a 50% inhibition concentration (IC50) of 24.7 ± 1.65 µM. ATRA inhibited the replication of SARS-CoV-2 in VeroE6/TMPRSS2 and Calu-3 cells, with IC50 = 2.69 ± 0.09 µM in the former and 0.82 ± 0.01 µM in the latter. Further, we showed the anti-SARS-CoV-2 effect of ATRA on the currently circulating variants of concern (VOC); alpha, beta, gamma, and delta. These results suggest that ATRA may be considered as a potential therapeutic agent against SARS-CoV-2.