Biology subjects

Mitsuya, H.

Publications and source records attributed to Mitsuya, H..

7 recordsLinked to original sources

bioRxiv2025

Molecular signature of COVID-19 prior to its exacerbation by multi-omics survey

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a global pandemic due to its high transmissibility and ability to evade innate immune responses. Comprehensive characterization of the disease is essential for elucidating its pathophysiology and clinical progression. In this study, we performed multi-omics analyses of plasma samples collected from SARS-CoV-2-positive patients prior to clinical deterioration of coronavirus disease 2019 (COVID-19). These samples revealed the potential of previously reported clinical parameters, including CRP and neutrophil level, to predict COVID-19 exacerbation in the early stage. Our analysis identified a novel panel of molecules that precede the clinical manifestations associated with COVID-19 progression. These candidate biomarkers exhibited strong correlations with previously reported clinical and immunological parameters. Notably, several inflammation-related markers showed inverse associations with specific interferon subtypes, including IFN-6 and IFN-8, potentially reflecting mechanisms of SARS-CoV-2-mediated immune evasion. Our findings contribute to the understanding of virus-induced acute exacerbation and offer a valuable foundation for future pandemic research.

microbiology↗

bioRxiv2023

HIV-1 resistance to islatravir/tenofovir combination therapy in wild-type or NRTI resistant strains of diverse HIV-1 subtypes

Tenofovir disoproxil fumarate (TDF) and islatravir (ISL, 4-ethynyl-2-fluoro-2-deoxyadensine, or MK-8591) are highly potent nucleoside reverse transcriptase inhibitors. Resistance to TDF and ISL is conferred by K65R and M184V, respectively. Furthermore, K65R and M184V increase sensitivity to ISL and TDF, respectively. Therefore, these two nucleoside analogs have opposing resistance profiles and could present a high genetic barrier to resistance. To explore resistance to TDF and ISL in combination, we performed passaging experiments with HIV-1 WT, K65R, or M184V in the presence of ISL and TDF. We identified K65R, M184V, and S68G/N mutations. The mutant most resistant to ISL was S68N/M184V, yet it remained susceptible to TDF. To further confirm our cellular findings, we implemented an endogenous reverse transcriptase assay to verify in vitro potency. To better understand the impact of these resistance mutations in the context of global infection, we determined potency of ISL and TDF against HIV subtypes A, B, C, D, and circulating recombinant forms (CRF) 01_AE and 02_AG with and without resistance mutations. In all isolates studied, we found K65R imparted hypersensitivity to ISL whereas M184V conferred resistance. We demonstrated that the S68G polymorphism can enhance fitness of drug-resistant mutants in some genetic backgrounds. Collectively, the data suggest that the opposing resistance profiles of ISL and TDF suggest that a combination of the two drugs could be a promising drug regimen for the treatment of patients infected with any HIV-1 subtype, including those who have failed 3TC/FTC-based therapies.

microbiology↗

bioRxiv2022

SARS-CoV-2-neutralizing humoral IgA response occurs earlier but modest and diminishes faster compared to IgG response.

Secretory immunoglobulin A (IgA) plays a crucial role in the mucosal immunity for preventing the invasion of the exogenous antigens, however, little has been 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 and went down below the detection limit approximately 70 days after onset, while substantial IgG activity was observed till 200 days after onset. The total neutralizing activity in sera/plasmas of those with COVID-19 largely correlated with that 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 elicit both SARS-CoV-2-specific neutralizing IgG and IgA response in serum, but the IgA response is modest and diminishes faster compared to IgG response. Author SummaryImmunoglobulin A (IgA) is the most abundant type of antibody in the body mostly located on mucosal surfaces as a dimeric secretory IgA. Such secretory IgA plays an important role in preventing the adherence and invasions of foreign objects by its neutralizing activity, 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 and IgA active against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although the neutralizing IgA response was quick and reached the highest activity 25 days post-symptom-onset, compared to 35 days for IgG response, neutralizing IgA activity was modest and diminished faster than neutralizing IgG response. In individuals, who recovered from COVID-19 but had no detectable neutralizing IgA activity, a single dose of COVID-19 mRNA-vaccine elicited potent neutralizing IgA 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 IgA against the viral pathogen both in naturally-infected and COVID-19 mRNA-vaccine-receiving COVID-19-convalescent individuals.

microbiology↗

bioRxiv2021

Highly-Neutralizing COVID-19-Convalescent-Plasmas Potently Block SARS-CoV-2 Replication and Pneumonia in Syrian Hamsters.

Despite various attempts to treat SARS-CoV-2-infected patients with COVID-19-convalescent plasmas, neither appropriate approach nor clinical utility has been established. We examined the efficacy of administration of highly-neutralizing COVID-19-convalescent plasma (hn-plasmas) and such plasma-derived IgG administration using the Syrian hamster COVID-19 model. Two hn-plasmas, which were in the best 1% of 340 neutralizing-activity-determined convalescent plasma samples, were intraperitoneally administered to SARS-CoV-2-infected hamsters, resulting in significant reduction of viral titers in lungs by up to 32-fold as compared to the viral titers in hamsters receiving control non-neutralizing plasma, while with two moderately neutralizing plasmas (mn-plasmas) administered, viral titer reduction was by up to 6-fold. IgG fractions purified from the two hn-plasmas also reduced viral titers in lungs than those from the two mn-plasmas. The severity of lung lesions seen in hamsters receiving hn-plasmas was minimal to moderate as assessed using micro-computerized tomography, which histological examination confirmed. Western blotting revealed that all four COVID-19-convalescent-plasmas variably contained antibodies against SARS-CoV-2 components including the receptor-binding domain and S1 domain. The present data strongly suggest that administering potent-neutralizing-activity-confirmed COVID-19-convalescent plasmas would be efficacious in treating patients with COVID-19. ImportanceConvalescent plasmas obtained from patients, who recovered from a specific infection, have been used as agents to treat other patients infected with the very pathogen. To treat using convalescent plasmas, despite that more than 10 randomized-controlled-clinical-trials have been conducted and more than 100 studies are currently ongoing, the effects of convalescent plasma against COVID-19 remained uncertain. On the other hand, certain COVID-19 vaccines have been shown to reduce the clinical COVID-19 onset by 94-95%, for which the elicited SARS-CoV-2-neutralizing antibodies are apparently directly responsible. Here, we demonstrate that highly-neutralizing-effect-confirmed convalescent plasmas significantly reduce the viral titers in the lung of SARS-CoV-2-infected Syrian hamsters and block the development of virally-induced lung lesions. The present data provide a proof-of-concept that the presence of highly-neutralizing antibody in COVID-19-convalescent plasmas is directly responsible for the reduction of viral replication and support the use of highly-neutralizing antibody-containing plasmas in COVID-19 therapy with convalescent plasmas.

microbiology↗

bioRxiv2021

Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300

SARS-CoV-2 encodes main protease (Mpro), an attractive target for therapeutic interventions. We show Mpro is susceptible to glutathionylation leading to inhibition of dimerization and activity. Activity of glutathionylated Mpro could be restored with reducing agents or glutaredoxin. Analytical studies demonstrated that glutathionylated Mpro primarily exists as a monomer and that a single modification with glutathione is sufficient to block dimerization and loss of activity. Proteolytic digestions of Mpro revealed Cys300 as a primary target of glutathionylation, and experiments using a C300S Mpro mutant revealed that Cys300 is required for inhibition of activity upon Mpro glutathionylation. These findings indicate that Mpro dimerization and activity can be regulated through reversible glutathionylation of Cys300 and provides a novel target for the development of agents to block Mpro dimerization and activity. This feature of Mpro may have relevance to human disease and the pathophysiology of SARS-CoV-2 in bats, which develop oxidative stress during flight.

microbiology↗

bioRxiv2020

Development of Resistance to 4'-Ethynyl-2-Fluoro-2'-Deoxyadenosine (EFdA) by Wild-Type and Nucleoside Reverse Transcriptase Inhibitor Resistant Human Immunodeficiency Virus Type 1

4-ethynyl-2-fluoro-2-deoxyadenosine (EFdA, MK-8591, islatravir) is a nucleoside reverse transcriptase translocation inhibitor (NRTTI) with exceptional potency against WT and drug-resistant HIV strains. However, HIV resistance to EFdA is not well characterized. We therefore developed resistance to EFdA by serial passages using progressively increasing concentrations of EFdA. The starting virus was either WT or clinically relevant NRTI-resistant viruses K65R, M184V, and D67N/K70R/T215F/K219Q). In all cases, the selected mutations included M184V. Additional mutations in the RT connection domain (R358K and E399K) and one mutation in the RNase H domain (A502V) were noted. Site-specific mutagenesis validated the role for M184V as the primary determinant for resistance to EFdA; none of the connection domain mutations contributed significantly to phenotypic resistance to EFdA. A novel EFdA resistance mutation was also observed in the background of M184V. The A114S/M184V combination of mutations imparted higher resistance to EFdA (~24-fold) than M184V (-8-fold) or A114S (~2-fold) alone. Virus fitness data suggested that A114S affects HIV fitness by itself and in the presence of M184V. This is consistent with biochemical experiments that showed decreases in the enzymatic efficiency (kcat/Km) of WT RT vs. A114S (2.1-fold) and A114S/M184V/502V (6.5-fold), whereas there was no significant effect of A502V on RT or virus fitness. The observed EFdA resistance of M184V by itself and in combination with A114S combined with the strong published in vitro and in vivo data, confirm that EFdA is an excellent candidate as a potential HIV therapeutic.

microbiology↗

bioRxiv2019

The WIPE assay for selection and elimination of HIV-1 provirus in vitro using latency-reversing agents

Persistence of HIV-1 latent reservoir cells during antiretroviral therapy is a major obstacle for curing HIV-1. Latency-reversing agents (LRAs) are under development to reactivate and eradicate latently infected cells; however, there are few useful models for evaluating LRA activity in vitro. Here, we established a long-term cell culture system harboring thousands of different HIV-1-infected cell clones with a wide distribution of HIV-1 provirus similar to that observed in vivo. A combination of an LRA and antiretroviral therapy (ART) significantly reduced viral rebound upon treatment interruption. Experimental investigation and mathematical modeling demonstrated that addition of LRA to ART induced latency-reversing effect and contributed to the eradication of replication competent HIV-1. The widely distributed intact provirus elimination (WIPE) assay will be useful for optimizing therapeutics against HIV-1 latency and investigating mechanistic insights into the clonal selection of heterogeneous HIV-1-infected cells.

microbiology↗