Biology subjects

Maenaka, K.

Publications and source records attributed to Maenaka, K..

10 recordsLinked to original sources

bioRxiv2024

Thermostability and binding properties of single-chained Fv fragments derived from therapeutic antibodies

Small antibody fragments have recently been used as alternatives to full-length monoclonal antibodies in therapeutic applications. One of the most popular fragment antibodies is single-chain fragment variables (scFvs), consisting of variable heavy (VH) and variable light (VL) domains linked by a flexible peptide linker. scFvs have small molecular sizes, which enables good tissue penetration and low immunogenicity. Despite these advantages, the use of scFvs, especially for therapeutic purpose, is still limited because of the difficulty to regulate the binding activity and conformational stability. In this study, we constructed and analyzed 10 scFv fragments derived from 10 representatives of FDA-approved mAbs to evaluate their physicochemical properties. Differential scanning calorimetry analysis showed that scFvs exhibited relatively high but varied thermostability, from 50 to 70 {degrees}C of melting temperatures, and different unfolding cooperativity. Surface plasmon resonance analysis revealed that scFvs fragments that exhibit high stability and cooperative unfolding likely tend to maintain antigen binding. This study demonstrated the comprehensive physicochemical properties of scFvs derived from FDA-approved antibodies, providing insights into antibody design and development.

bioengineering↗

bioRxiv2023

Virological characteristics of the SARS-CoV-2 Omicron EG.5.1 variant

In middle-late 2023, a sublineage of SARS-CoV-2 Omicron XBB, EG.5.1 (a progeny of XBB.1.9.2), is spreading rapidly around the world. Here, we performed multiscale investigations to reveal virological features of newly emerging EG.5.1 variant. Our phylogenetic-epidemic dynamics modeling suggested that two hallmark substitutions of EG.5.1, S:F456L and ORF9b:I5T, are critical to the increased viral fitness. Experimental investigations addressing the growth kinetics, sensitivity to clinically available antivirals, fusogenicity and pathogenicity of EG.5.1 suggested that the virological features of EG.5.1 is comparable to that of XBB.1.5. However, the cryo-electron microscopy reveals the structural difference between the spike proteins of EG.5.1 and XBB.1.5. We further assessed the impact of ORF9b:I5T on viral features, but it was almost negligible at least in our experimental setup. Our multiscale investigations provide the knowledge for understanding of the evolution trait of newly emerging pathogenic viruses in the human population.

microbiology↗

bioRxiv2023

Regulatory Mimicry of Cyclin-Dependent Kinases by Conserved Herpesvirus Protein Kinases

Herpesviruses encode conserved protein kinases (CHPKs) that target cellular cyclin-dependent kinase (CDK) phosphorylation sites; thus, they are termed viral CDK-like kinases. Tyrosine 15 in the GxGxxG motifs of CDK1 and CDK2, whose phosphorylation down-regulates their catalytic activities, is conserved in the corresponding motifs of CHPKs. We found that herpes simplex virus 2 (HSV-2) CHPK UL13 mimicked the regulatory mechanism of CDKs. This regulatory mimicry was conserved in CHPKs encoded by herpesviruses subclassified into subfamilies other than HSV-2, suggesting CHPKs have regulatory and functional mimicry with CDKs. Phosphorylation of the corresponding Tyr in HSV-2 UL13 was required for the down-regulation of viral replication and pathogenicity, specifically in the central nervous system of mice, and for efficient viral recurrence in guinea pigs. These data highlight the dual impact of the regulatory mimicry of CDKs by CHPK on the fine-tuned regulation of lytic and latent HSV-2 infections in vivo.

microbiology↗

bioRxiv2023

The human immune checkpoint molecule, HLA-G2, induces tolerance in monocytes and dendritic cells via upregulation of PD-L1

Human leukocyte antigen (HLA)-G is a non-classical HLA class I immunomodulatory molecule with restricted expression in the placenta, thymus and regulatory T cells. The spliced isoforms of HLA-G include an 2 domain-deleted isoform, HLA-G2, which specifically binds to the immune checkpoint leukocyte immunoglobulin-like receptor B2 (LILRB2), to suppress immune responses in myelomonocytic cells. We previously reported the structural and receptor binding characteristics of recombinant HLA-G2 protein and its immunosuppressive effects on inflammation in mouse models. However, the function and the mechanism of action of HLA-G2 on human immune cells have not been elucidated. In the present study, we demonstrate the immunosuppressive effect of HLA-G2 on human CD14-positive monocytic cells. HLA-G2 induced the production of the immunosuppressive cytokine, IL-10, and stimulated IL-6/STAT3/indoleamine-2,3-dioxygenase signaling by binding to LILRB2. HLA-G2 binding to LILRB2 also down-regulated cell surface expression of HLA-DR and CD86. Unexpectedly, HLA-G2 up-regulated cell surface expression of PD-L1 in both CD14-positive monocytic cells and interferon-induced dendritic cells (IFN-DCs). This observation suggests HLA-G2/LILRB2 signaling promotes PD-L1 expression. Furthermore, HLA-G2 treatment of IFN-DCs suppressed T cell proliferation in mixed lymphocyte reactions. These findings provide novel insights into the modulation of human immune responses of tolerogenic myelomonocytic cells induced by HLA-G2 binding to LILRB2, and suggest that targeting the HLA-G2-LILRB2 interaction could be a novel approach for immune checkpoint therapy. Significance statementDuring pregnancy, HLA-G isoforms are expressed by fetal trophoblasts to suppress maternal immune responses. Among various HLA-G isoforms, the HLA-G2 homodimer has been expected as an immunosuppressive biologic targeting myelomonocytic antigen-presenting cells via leukocyte immunoglobulin-like receptor B2. We previously reported significant immunosuppressive effects of HLA-G2 in autoimmune mouse models. Here, we first demonstrate that HLA-G2 isoform induces tolerogenic phenotypes of human peripheral immune cells by significantly upregulating an immune checkpoint molecule, PD-L1. Monocyte-derived dendritic cells stimulated by HLA-G2 suppressed T cell proliferation in mixed lymphocyte reactions. These results suggest that HLA-G2 can be a novel candidate for immune checkpoint therapy.

immunology↗

bioRxiv2023

Snapshots from Cryo-ET of active SARS-CoV-2 virions

Understanding the molecular properties of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for tackling future outbreaks. Current structural knowledge of the trimeric spike protein relies on truncated recombinant proteins and/or inactivated full-length forms, which may suffer from overstabilization. Here, we apply cryo-electron tomography (cryo-ET) at a Biosafety Level 3 facility to study the virus structure in its native, active state. The virus particles exhibit variable shapes and sizes with diffusible spikes, with the majority in typical prefusion conformations. Notably, we identified unprecedented, a transient open-trimer prefusion states, revealing a hidden flexibility with opened S1 conformation. Subtomogram averaging of the prefusion spikes indicates a loosely packed trimeric architecture that may facilitate the formation of open-trimer state. A cryo-EM map of recombinant Omicron BA.2.75 spike protein further confirms the presence of this loosely packed trimer as a minor conformational state. The observed dynamics uncover conserved cryptic regions that can be targeted for broadly effective vaccines. Structural analysis of active viruses profoundly impacts our understanding of the overlooked fusion mechanism and vaccine, antibody/drug design.

microbiology↗

bioRxiv2023

Virological characteristics of the SARS-CoV-2 XBB.1.5 variant

Circulation of SARS-CoV-2 Omicron XBB has resulted in the emergence of XBB.1.5, a new Variant of Interest. Our phylogenetic analysis suggests that XBB.1.5 evolved from XBB.1 by acquiring the F486P spike (S) mutation, subsequent to the acquisition of a nonsense mutation in ORF8. Neutralization assays showed similar abilities of immune escape between XBB.1.5 and XBB.1. We determined the structural basis for the interaction between human ACE2 and the S protein of XBB.1.5, showing similar overall structures between the S proteins of XBB.1 and XBB.1.5. The intrinsic pathogenicity of XBB.1.5 in hamsters is lower than that of XBB.1. Importantly, we found that the ORF8 nonsense mutation of XBB.1.5 resulted in impairment of MHC expression. In vivo experiments using recombinant viruses revealed that the XBB.1.5 mutations are involved with reduced virulence of XBB.1.5. Together, these data suggest that the mutations in ORF8 and S could enhance spreading of XBB.1.5 in humans.

microbiology↗

bioRxiv2022

2-Thiouridine is a broad-spectrum antiviral nucleoside analogue against positive-strand RNA viruses

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes significant morbidity and mortality worldwide, seriously impacting not only human health but also the global economy. Furthermore, over 1 million cases of newly emerging or re-emerging viral infections, specifically dengue virus (DENV), are known to occur annually. Because no virus-specific and fully effective treatments against these and many other viruses have been approved, they continue to be responsible for large-scale epidemics and global pandemics. Thus, there is an urgent need for novel, effective therapeutic agents. Here, we identified 2-thiouridine (s2U) as a broad-spectrum antiviral nucleoside analogue that exhibited antiviral activity against SARS-CoV-2 and its variants of concern, including the Delta and Omicron variants, as well as a number of other positive-sense single-stranded RNA (ssRNA+) viruses, including DENV. s2U inhibits RNA synthesis catalyzed by viral RNA-dependent RNA polymerase, thereby reducing viral RNA replication, which improved the survival rate of mice infected with SARS-CoV-2 or DENV in our animal models. Our findings demonstrate that s2U is a potential broad-spectrum antiviral agent not only against SARS-CoV-2 and DENV but other ssRNA+ viruses.

microbiology↗

bioRxiv2020

SARS-CoV-2 variants with mutations at the S1/S2 cleavage site are generated in vitro during propagation in TMPRSS2-deficient cells

The spike (S) protein of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) binds to a host cell receptor which facilitates viral entry. A polybasic motif detected at the cleavage site of the S protein has been shown to broaden the cell tropism and transmissibility of the virus. Here we examine the properties of SARS-CoV-2 variants with mutations at the S protein cleavage site that undergo inefficient proteolytic cleavage. Virus variants with S gene mutations generated smaller plaques and exhibited a more limited range of cell tropism compared to the wild-type strain. These alterations were shown to result from their inability to utilize the entry pathway involving direct fusion mediated by the host type II transmembrane serine protease, TMPRSS2. Notably, viruses with S gene mutations emerged rapidly and became the dominant SARS-CoV-2 variants in TMPRSS2-deficient cells including Vero cells. Our study demonstrated that the S protein polybasic cleavage motif is a critical factor underlying SARS-CoV-2 entry and cell tropism. As such, researchers should be alert to the possibility of de novo S gene mutations emerging in tissue-culture propagated virus strains.

microbiology↗

bioRxiv2020

Multidrug treatment with nelfinavir and cepharanthine against COVID-19

Antiviral treatments targeting the emerging coronavirus disease 2019 (COVID-19) are urgently required. We screened a panel of already-approved drugs in a cell culture model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and identified two new antiviral agents: the HIV protease inhibitor Nelfinavir and the anti-inflammatory drug Cepharanthine. In silico modeling shows Nelfinavir binds the SARS-CoV-2 main protease consistent with its inhibition of viral replication, whilst Cepharanthine inhibits viral attachment and entry into cells. Consistent with their different modes of action, in vitro assays highlight a synergistic effect of this combined treatment to limit SARS-CoV-2 proliferation. Mathematical modeling in vitro antiviral activity coupled with the known pharmacokinetics for these drugs predicts that Nelfinavir will facilitate viral clearance. Combining Nelfinavir/Cepharanthine enhanced their predicted efficacy to control viral proliferation, to ameliorate both the progression of disease and risk of transmission. In summary, this study identifies a new multidrug combination treatment for COVID-19.

microbiology↗

bioRxiv2019

Structure of HIV-2 Nef reveals unique features distinct from HIV-1 involved in immune regulation

The HIV accessory protein Nef plays a major role in establishing and maintaining infection, particularly through immune evasion. Many HIV-2 infected people experience long-term viral control and survival, resembling HIV-1 elite control. HIV-2 Nef has overlapping but also distinct functions from HIV-1 Nef. Here we report the crystal structure of HIV-2 Nef core. The dileucine sorting motif forms a helix bound to neighboring molecules, and moreover, isothermal titration calorimetry demonstrated that the CD3 endocytosis motif can directly bind to HIV-2 Nef, ensuring AP-2 mediated endocytosis for CD3. The highly-conserved C-terminal region forms a -helix, absent from HIV-1. We further determined the structure of SIV Nef harboring this region, demonstrating similar C-terminal -helix, which may contribute to AP-1 binding for MHC-I downregulation. These results provide new insights into the distinct pathogenesis of HIV-2 infection.

biophysics↗