Omicron BA.1 neutralizing antibody response following Delta breakthrough infection compared with booster vaccination of BNT162b2.

BACKGROUND: Longitudinal data are lacking to compare booster effects of Delta breakthrough infection versus third vaccine dose on neutralizing antibodies (NAb) against Omicron. METHODS: Participants were the staff of a national research and medical institution in Tokyo who attended serological surveys on June 2021 (baseline) and December 2021 (follow-up); in between, the Delta-dominant epidemic occurred. Of 844 participants who were infection-naïve and had received two doses of BNT162b2 at baseline, we identified 11 breakthrough infections during follow-up. One control matched to each case was selected from boosted and unboosted individuals. We compared live-virus NAb against Wild-type, Delta, and Omicron BA.1 across groups. RESULTS: Breakthrough infection cases showed marked increases in NAb titers against Wild-type (4.1-fold) and Delta (5.5-fold), and 64% had detectable NAb against Omicron BA.1 at follow-up, although the NAb against Omicron after breakthrough infection was 6.7- and 5.2-fold lower than Wild-type and Delta, respectively. The increase was apparent only in symptomatic cases and as high as in the third vaccine recipients. CONCLUSIONS: Symptomatic Delta breakthrough infection increased NAb against Wild-type, Delta, and Omicron BA.1, similar to the third vaccine. Given the much lower NAb against Omicron BA.1, infection prevention measures must be continued irrespective of vaccine and infection history while the immune evasive variants are circulating.

Efficacy of convalescent plasma therapy for COVID-19 in Japan: An open-label, randomized, controlled trial.

BACKGROUND: Convalescent plasma is a potential therapeutic option for patients with coronavirus disease 2019 (COVID-19). Despite its use for treating several viral infections, we lack comprehensive data on its efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: We conducted a multicenter, open-label, randomized controlled trial of convalescent plasma therapy with high neutralizing activity against SARS-CoV-2 in high-risk patients within five days after the onset of COVID-19 symptoms. The primary endpoint was the time-weighted average change in the SARS-CoV-2 viral load in nasopharyngeal swabs from days 0-5. RESULTS: Between February 24, 2021, and November 30, 2021, 25 patients were randomly assigned to either convalescent plasma (n = 14) or standard of care (n = 11) groups. Four patients discontinued their allocated convalescent plasma, and 21 were included in the modified intention-to-treat analysis. The median interval between the symptom onset and plasma administration was 4.5 days (interquartile range, 3-5 days). The primary outcome of the time-weighted average change in the SARS-CoV-2 viral load in nasopharyngeal swabs did not significantly differ between days 0-5 (1.2 log10 copies/mL in the convalescent plasma vs. 1.2 log10 copies/mL in the standard of care (effect estimate, 0.0 [95% confidence interval, -0.8-0.7]; P = 0.94)). No deaths were observed in either group. CONCLUSIONS: The early administration of convalescent plasma with high neutralizing activity did not contribute to a decrease in the viral load within five days compared with the standard of care alone.

Immunogenicity and safety of single booster dose of KD-414 inactivated COVID-19 vaccine in adults: An open-label, single-center, non-randomized, controlled study in Japan.

Although vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease 2019 (COVID-19) induce effective immune responses, vaccination with booster doses is necessary because of waning immunity. We conducted an open-label, non-randomized, single-arm study in adults in Japan to assess the immunogenicity and safety of a single booster dose of the KD-414 purified whole-SARS-CoV-2-virion inactivated vaccine candidate after vaccination with a primary series of BNT162b2. The primary endpoint was serum neutralizing activity at 7 days after booster injection compared with the primary series of BNT162b2. The SARS-CoV-2-structural protein-binding antibody level and T cell response against SARS-CoV-2-Spike (S) peptides were also examined as secondary endpoints, and safety profile assessments were conducted. Twenty subjects who participated in a previous study declined an injection of KD-414 (non-KD-414 group) and received a booster dose of BNT162b2 instead. The non-KD-414 group was compared to the KD-414 group as a secondary outcome. A single dose of KD-414 induced lower serum neutralizing activity against the wild-type virus within 7 days compared to after the primary series of BNT162b2 but significantly induced anti-SARS-CoV-2-S1-receptor-binding domain-binding immunoglobulin G (IgG) antibodies and SARS-CoV-2-S peptide-specific CD4+ and CD8+ T cell responses. Local or systemic symptoms were significantly lower in the participants who received KD-414 than in those who received BNT162b2 as the third COVID-19 vaccine dose. The present data indicate that a single booster dose of KD-414 induces a substantial immune response in BNT162b2-primed individuals and has a good safety profile, thereby supporting further clinical trials to identify rational targets.

Identification of SARS-CoV-2 Mpro inhibitors containing P1' 4-fluorobenzothiazole moiety highly active against SARS-CoV-2.

COVID-19 caused by SARS-CoV-2 has continually been serious threat to public health worldwide. While a few anti-SARS-CoV-2 therapeutics are currently available, their antiviral potency is not sufficient. Here, we identify two orally available 4-fluoro-benzothiazole-containing small molecules, TKB245 and TKB248, which specifically inhibit the enzymatic activity of main protease (Mpro) of SARS-CoV-2 and significantly more potently block the infectivity and replication of various SARS-CoV-2 strains than nirmatrelvir, molnupiravir, and ensitrelvir in cell-based assays employing various target cells. Both compounds also block the replication of Delta and Omicron variants in human-ACE2-knocked-in mice. Native mass spectrometric analysis reveals that both compounds bind to dimer Mpro, apparently promoting Mpro dimerization. X-ray crystallographic analysis shows that both compounds bind to Mpro's active-site cavity, forming a covalent bond with the catalytic amino acid Cys-145 with the 4-fluorine of the benzothiazole moiety pointed to solvent. The data suggest that TKB245 and TKB248 might serve as potential therapeutics for COVID-19 and shed light upon further optimization to develop more potent and safer anti-SARS-CoV-2 therapeutics.

Preserved SARS-CoV-2 neutralizing IgG activity of in-house manufactured COVID-19 convalescent plasma.

PURPOSE: In the current study, we aimed to evaluate the neutralizing IgG activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as well as the coagulation factors of convalescent plasmas which we manufactured in-house without a fast-freezing technique. METHODS: We collected plasmas from eligible participants who had confirmed certain titers of neutralizing antibodies. The plasmas were frozen and stored in the ordinary biofreezer without a fast-freezing function. The purified-IgG neutralizing activity of 20 samples from 19 participants and the coagulation factors of 49 samples from 40 participants were evaluated before and after freezing. RESULTS: Purified-IgG maintained its neutralizing activities, with the median 50 % inhibitory concentration (IC50) of 10.11 mg/ml (IQR 6.53-18.19) before freezing and 8.90 m g/ml (IQR 6.92-28.27) after thawing (p = 0.956). On the contrary, fibrinogen and factor Ⅷ decreased significantly after freezing and thawing in our environment. No significant temperature deviation was observed during the storage period. CONCLUSION: Neutralizing IgG activity, which largely contributes to the antiviral activity of convalescent plasma, did not change through our in-house manufacturing, without fastfreezing and storage conditions for more than 200 days. Ordinary freezers without the fast-freezing function are suitable enough to manufacture and store convalescent plasmas. Hospitals or facilities without specified resources could easily collect and store convalescent plasmas in case of upcoming emerging or re-emerging infectious diseases on-demand with appropriate neutralizing antibody levels measurements.

Neutralizing antibodies after three doses of the BNT162b2 vaccine, breakthrough infection, and symptoms during the Omicron-predominant wave.

OBJECTIVES: To investigate the role of immunogenicity after the third vaccine dose against Omicron infection and COVID-19-compatible symptoms of infection. METHODS: First, we examined vaccine effectiveness (VE) of the third dose against the second dose during the Omicron wave among the staff at a tertiary hospital in Tokyo. In a case-control study of third vaccine recipients, we compared the preinfection live-virus neutralizing antibodies (NAb) against Omicron between breakthrough cases and their controls who had close contact with patients with COVID-19. Among these cases, we examined the association between NAb levels and the number of COVID-19-compatible symptoms. RESULTS: Among the 1456 participants for VE analysis, 60 breakthrough infections occurred during the Omicron wave. The third dose VE for infection was 54.6%. Among the third dose recipients, NAb levels against Omicron did not differ between the cases (n = 22) and controls (n = 21). Among the cases, those who experienced COVID-19-compatible symptoms had lower NAb levels against Omicron than those who did not. CONCLUSION: The third vaccine dose was effective in decreasing the risk of SARS-CoV-2 infection during Omicron wave compared with the second dose. Among third dose recipients, higher preinfection NAb levels may not be associated with a lower risk of Omicron infection. Contrarily, they may be associated with fewer symptoms of infection.

Neutralization activity of IgG antibody in COVID­19­convalescent plasma against SARS-CoV-2 variants.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluated the anti-SARS-CoV-2 antibody levels, anti-spike (S)-immunoglobulin G (IgG) and anti-nucleocapsid (N)-IgG, and the neutralization activity of IgG antibody in COVID­19­convalescent plasma against variants of SARS-CoV-2, alpha, beta, gamma, delta, kappa, omicron and R.1 strains. The study included 30 patients with clinically diagnosed COVID-19. The anti-S-IgG and anti-N-IgG levels ranged from 30.0 to 555.1 and from 10.1 to 752.6, respectively. The neutralization activity (50% inhibition concentration: IC50) for the wild-type Wuhan strain ranged from < 6.3 to 81.5 µg/ml. IgG antibodies were > 100 µg/ml in 18 of 30 (60%) subjects infected with the beta variant. The IC50 values for wild-type and beta variants correlated inversely with anti-S-IgG levels (p < 0.05), but no such correlation was noted with anti-N-IgG. IgG antibodies prevented infectivity and cytopathic effects of six different variants of concern in the cell-based assays of wild-type, alpha, gamma, delta, kappa and R.1 strains, but not that of the beta and omicron strains. IgG is considered the main neutralizing activity in the blood, although other factors may be important in other body tissues.

Recent Drug Development and Medicinal Chemistry Approaches for the Treatment of SARS-CoV-2 Infection and COVID-19.

COVID-19, caused by SARS-CoV-2 infection, continues to be a major public health crisis around the globe. Development of vaccines and the first cluster of antiviral drugs has brought promise and hope for prevention and treatment of severe coronavirus disease. However, continued development of newer, safer, and more effective antiviral drugs are critically important to combat COVID-19 and counter the looming pathogenic variants. Studies of the coronavirus life cycle revealed several important biochemical targets for drug development. In the present review, we focus on recent drug design and medicinal chemistry efforts in small molecule drug discovery, including the development of nirmatrelvir that targets viral protein synthesis and remdesivir and molnupiravir that target viral RdRp. These are recent FDA approved drugs for the treatment of COVID-19.

Generation of Angiotensin-Converting Enzyme 2/Transmembrane Protease Serine 2-Double-Positive Human Induced Pluripotent Stem Cell-Derived Spheroids for Anti-Severe Acute Respiratory Syndrome Coronavirus 2 Drug Evaluation.

We newly generated two human induced pluripotent stem cell (hiPSC)-derived spheroid lines, termed Spheroids_4MACE2-TMPRSS2 and Spheroids_15M63ACE2-TMPRSS2, both of which express angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2), which are critical for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Both spheroids were highly susceptible to SARS-CoV-2 infection, and two representative anti-SARS-CoV-2 agents, remdesivir and 5h (an inhibitor of SARS-CoV-2's main protease), inhibited the infectivity and replication of SARS-CoV-2 in a dose-dependent manner, suggesting that these human-derived induced spheroids should serve as valuable target cells for the evaluation of anti-SARS-CoV-2 activity. IMPORTANCE The hiPSC-derived spheroids we generated are more expensive to obtain than the human cell lines currently available for anti-SARS-CoV-2 drug evaluation, such as Calu-3 cells; however, the spheroids have better infection susceptibility than the existing human cell lines. Although we are cognizant that there are human lung (and colonic) organoid models for the study of SARS-CoV-2, the production of those organoids is greatly more costly and time consuming than the generation of human iPSC-derived spheroid cells. Thus, the addition of human iPSC-derived spheroids for anti-SARS-CoV-2 drug evaluation studies could provide the opportunity for more comprehensive interpretation of the antiviral activity of compounds against SARS-CoV-2.

Potent and biostable inhibitors of the main protease of SARS-CoV-2.

Potent and biostable inhibitors of the main protease (Mpro) of SARS-CoV-2 were designed and synthesized based on an active hit compound 5h (2). Our strategy was based not only on the introduction of fluorine atoms into the inhibitor molecule for an increase of binding affinity for the pocket of Mpro and cell membrane permeability but also on the replacement of the digestible amide bond by a surrogate structure to increase the biostability of the compounds. Compound 3 is highly potent and blocks SARS-CoV-2 infection in vitro without a viral breakthrough. The derivatives, which contain a thioamide surrogate in the P2-P1 amide bond of these compounds (2 and 3), showed remarkably preferable pharmacokinetics in mice compared with the corresponding parent compounds. These data show that compounds 3 and its biostable derivative 4 are potential drugs for treating COVID-19 and that replacement of the digestible amide bond by its thioamide surrogate structure is an effective method.

Regulation of Retroviral and SARS-CoV-2 Protease Dimerization and Activity through Reversible Oxidation.

Most viruses encode their own proteases to carry out viral maturation and these often require dimerization for activity. Studies on human immunodeficiency virus type 1 (HIV-1), type 2 (HIV-2) and human T-cell leukemia virus (HTLV-1) proteases have shown that the activity of these proteases can be reversibly regulated by cysteine (Cys) glutathionylation and/or methionine oxidation (for HIV-2). These modifications lead to inhibition of protease dimerization and therefore loss of activity. These changes are reversible with the cellular enzymes, glutaredoxin or methionine sulfoxide reductase. Perhaps more importantly, as a result, the maturation of retroviral particles can also be regulated through reversible oxidation and this has been demonstrated for HIV-1, HIV-2, Mason-Pfizer monkey virus (M-PMV) and murine leukemia virus (MLV). More recently, our group has learned that SARS-CoV-2 main protease (Mpro) dimerization and activity can also be regulated through reversible glutathionylation of Cys300. Overall, these studies reveal a conserved way for viruses to regulate viral polyprotein processing particularly during oxidative stress and reveal novel targets for the development of inhibitors of dimerization and activity of these important viral enzyme targets.

SARS-CoV-2-Neutralizing Humoral IgA Response Occurs Earlier but Is Modest and Diminishes Faster than IgG Response.

Secretory immunoglobulin A (IgA) plays a crucial role in mucosal immunity for preventing the invasion of exogenous antigens; however, little is understood about the neutralizing activity of serum IgA. Here, to examine the role of IgA antibodies against COVID-19 illnesses, we determined the neutralizing activity of serum/plasma IgG and IgA purified from previously SARS-CoV-2-infected and COVID-19 mRNA vaccine-receiving individuals. We found that serum/plasma IgA possesses substantial but rather modest neutralizing activity against SARS-CoV-2 compared to IgG with no significant correlation with the disease severity. Neutralizing IgA and IgG antibodies achieved the greatest activity at approximately 25 and 35 days after symptom onset, respectively. However, neutralizing IgA activity quickly diminished to below the detection limit approximately 70 days after onset, while substantial IgG activity was observed until 200 days after onset. The total neutralizing activity in sera/plasmas of those with COVID-19 largely correlated with those in purified IgG and purified IgA and levels of anti-SARS-CoV-2-S1-binding IgG and anti-SARS-CoV-2-S1-binding IgA. In individuals who were previously infected with SARS-CoV-2 but had no detectable neutralizing IgA activity, a single dose of BNT162b2 or mRNA-1273 elicited potent serum/plasma-neutralizing IgA activity, but the second dose did not further strengthen the neutralization antibody response. The present data show that the systemic immune stimulation with natural infection and COVID-19 mRNA-vaccines elicits both SARS-CoV-2-specific neutralizing IgG and IgA responses in serum, but the IgA response is modest and diminishes faster than the IgG response. IMPORTANCE Secretory dimeric immunoglobulin A (IgA) plays an important role in preventing the invasion of foreign objects by its neutralizing activity on mucosal surfaces, while monomeric serum IgA is thought to relate to the phagocytic immune system activation. Here, we report that individuals with the novel coronavirus disease (COVID-19) developed both systemic neutralizing IgG (nIgG) and IgA (nIgA) active against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although the nIgA response was quick and reached the highest activity earlier than the nIgG response, nIgA activity was modest and diminished faster than nIgG activity. In individuals who recovered from COVID-19 but had no detectable nIgA activity, a single dose of COVID-19 mRNA vaccine elicited potent nIgA activity, but the second dose did not further strengthen the antibody response. Our study provides novel insights into the role and the kinetics of serum nIgA against the pathogen in both naturally infected and COVID-19 mRNA vaccine-receiving COVID-19-convalescent individuals.

Neutralization activity of sera/IgG preparations from fully BNT162b2 vaccinated individuals against SARS-CoV-2 Alpha, Beta, Gamma, Delta, and Kappa variants.

In the present prospective study, 225 individuals in Kumamoto General Hospital, Japan, who received two-doses of BNT162b2 vaccine were enrolled/followed up over 150 days and neutralizing activity (NT50) of their sera and antiviral activity (EC50) of IgG purified from sera on day-60 post-1st-dose were determined against wild-type SARS-CoV-2 (SARS-CoV-2Wuhan) (n = 211) and 9 variants (Alpha, Beta, Gamma, Delta, and Kappa) (n = 45). Time-dependent changes of IgG-activity (n = 25) against SARS-CoV-2Wuhan and variants were also examined. Day-60 sera showed reduced NT50 by more than 50% against all variants examined, and greatest reduction was seen with Beta. IgG fractions of high-responders and moderate-responders showed similar fold-changes in EC50 against each variant compared to SARS-CoV-2Wuhan. Evaluation of EC50 of IgG obtained at different time-points (day-28 to -150) revealed time-dependent reduction of activity against all variants. However, against Delta, relatively long-lasting favorable antiviral activity (at least 150 days) was observed. Our data strongly suggest that the successful antecedent scale-up of mRNA-based vaccine administrations in Japan was the primary contributor to the lessening of the otherwise more devastating SARS-CoV-2 pandemic wave caused by the Delta variant. The present data that the effectiveness of vaccine against the then-dominant SARS-CoV-2 variant was likely associated with the moderation of the COVID-19 pandemic wave suggest that as in the case of influenza vaccines, the development of multivalent mRNA-based vaccines represent a generalizable approach to pre-emptively respond pandemic with mutable pathogens.

SARS-CoV-2 specific T cell and humoral immune responses upon vaccination with BNT162b2: a 9 months longitudinal study.

The humoral and cellular immune responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) upon the coronavirus disease 2019 (COVID-19) vaccination remain to be clarified. Hence, we aimed to investigate the long-term chronological changes in SARS-CoV-2 specific IgG antibody, neutralizing antibody, and T cell responses during and after receiving the BNT162b2 vaccine. We performed serological, neutralization, and T cell assays among 100 hospital workers aged 22-73 years who received the vaccine. We conducted seven surveys up to 8 months after the second vaccination dose. SARS-CoV-2 spike protein-specific IgG (IgG-S) titers and T cell responses increased significantly following the first vaccination dose. The highest titers were observed on day 29 and decreased gradually until the end of the follow-up period. There was no correlation between IgG-S and T cell responses. Notably, T cell responses were detected on day 15, earlier than the onset of neutralizing activity. This study demonstrated that both IgG-S and T cell responses were detected before acquiring sufficient levels of SARS-CoV-2 neutralizing antibodies. These immune responses are sustained for approximately 6 to 10 weeks but not for 7 months or later following the second vaccination, indicating the need for the booster dose (i.e., third vaccination).

Antibody titers and neutralizing activity in cases of COVID-19 after a single dose of vaccination.

Vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have shown high efficacy in preventing the onset of disease. However, the immune response to infection immediately after the first vaccination remains unknown. We examined the anti-SARS-CoV-2-binding-antibody titers and neutralizing activity in patients who developed coronavirus disease 2019 after the first vaccination. The amount of anti-SARS-CoV-2-binding antibodies and neutralizing activity drastically increased from the first to the second collection. Our results may provide important data on the course of immune response following vaccination.

COVID-19 in Japan: An update on national policy, research, clinical practice, and vaccination campaign.

As countries worldwide take steps such as vaccination campaigns to combat the COVID-19 pandemic, academia is actively promoting the timely sharing of scientific information across borders. As an international academic journal, Global Health & Medicine (GHM) has quickly accepted COVID-19-related papers and published results of series of studies since the beginning of 2020. In particular, the "First Special Issue on COVID-19" (April 2020) and the "Second Special Issue on COVID-19" (April 2021) included a wide range of articles presenting frontline data on the COVID-19 response in Japan, China, the United States, Italy, the United Kingdom, West Africa, and other various countries and areas worldwide. This "Third Special Issue on COVID-19" (April 2022) features the practical experiences of front-line clinicians, researchers, and other healthcare professionals from Japan and it presents updated data on i) national policy, ii) research, iii) clinical practice, and iv) the vaccination campaign. Our hope is that the rapid publication and sharing of information will help, in any way possible way, in the global fight against COVID-19.