Biology subjects

Miyakawa, K.

Publications and source records attributed to Miyakawa, K..

7 recordsLinked to original sources

bioRxiv2025

Combinational adjuvants delivered by ink-jet potentiate naked mRNA vaccines for robust protection against infectious diseases

The use of lipid nanoparticles (LNPs) to enhance delivery and immunogenicity of mRNA vaccines raises safety concerns due to strong reactogenicity. Developing naked mRNA vaccines, which can be delivered through liquid jet-injection to the skin, may offer a safer alternative, but their immunogenicity has been limited. To address this challenge, we screened clinically relevant adjuvants to enhance the potency of naked mRNA jet-injection vaccines in mouse models. Although many adjuvants showed little or inhibitory effects on inducing humoral or cellular immunity--despite their known ability to potentiate conventional non-mRNA vaccine platforms--we identified a combination of aluminum phosphate and CpG oligonucleotide that substantially enhanced both humoral and cellular immune responses with increased IgG2a/IgG1 ratio. This adjuvanted naked mRNA vaccine induced IgG2a and cellular immunity at levels comparable to those achieved with LNPs but with lower local and systemic reactogenicity. Mechanistic investigations revealed a key role for type I interferon signaling during immunization. Notably, this vaccine formulation in mice virtually eliminated infection by both SARS-CoV-2 and influenza A virus (A/California/7/2009 H1N1) in plaque-forming assays. Collectively, the adjuvanted naked mRNA jet-injection vaccine represents a novel, effective, and safe platform against infectious diseases, with potential for clinic-ready translational applications.

bioengineering↗

bioRxiv2025

A compact glutamic acid decarboxylase 67 promoter enables inhibitory neuron-targeted AAV gene therapy for treatment-resistant epilepsy

Epilepsy often becomes treatment-resistant, partly due to impaired inhibitory neurotransmission and reduced {gamma}-aminobutyric acid (GABA) function. Enhancing inhibitory neuron activity via gene therapy may restore excitation-inhibition (E/I) balance. We developed a compact 410-bp glutamic acid decarboxylase 67 promoter (cmGAD67) that enables strong, selective transgene expression in inhibitory neurons while preserving AAV packaging capacity. When delivered systemically, AAV vectors carrying cmGAD67 preferentially targeted parvalbumin interneurons and supported effective circuit manipulation. To evaluate therapeutic potential, we expressed glutamic acid decarboxylase 65 (GAD65) under cmGAD67 (AAV-GAD65) in pentylenetetrazole (PTZ) epilepsy models. Systemic AAV-GAD65 suppressed abnormal delta oscillations, reduced seizure-like events, normalized anxiety-like behavior, and improved survival in a severe PTZ paradigm. Biochemical analyses confirmed increased cortical and hippocampal GABA levels, linking behavioral and electrophysiological improvements to enhanced inhibitory neurotransmitter synthesis. Prior clinical evidence indicates that AAV-GAD65 delivery to the subthalamic nucleus is safe and effective in Parkinsons disease. Building on this foundation, our findings establish the cmGAD67 promoter as a powerful platform for inhibitory neuron-targeted AAV gene therapy and highlight AAV-cmGAD67-GAD65 as a promising approach for treatment-resistant epilepsy and other disorders involving disrupted E/I balance.

neuroscience↗

bioRxiv2024

Neutralizing antibody evasion of SARS-CoV-2 JN.1 derivatives KP.3, KP.3.1.1, LB.1, and XEC

The emergence of SARS-CoV-2 variants poses ongoing challenges to vaccine efficacy. We evaluated neutralizing antibody responses against JN.1 and its derivatives (KP.3, KP.3.1.1, LB.1, and XEC) in healthcare workers who received seven doses of BNT162b2, including XBB.1.5 monovalent vaccine. In COVID-19-naive individuals, KP.3.1.1 and LB.1 showed substantial immune escape, while previously infected individuals maintained neutralization activity against all variants. We also demonstrated that JN.1-based immunization induces robust cross-neutralizing activity against emerging variants. A single amino acid deletion at position 31 in the spike protein significantly impacted immune evasion. These findings support the potential effectiveness of JN.1-based vaccines while highlighting the need for continued surveillance and vaccine optimization.

immunology↗

bioRxiv2024

Rapid neutralizing assay for circulating H5N1 influenza virus in dairy cows

A rapid and safe neutralization assay is required for emerging highly pathogenic avian influenza viruses, including the H5N1 subtype, which was recently found in cows. Herein, we report a novel neutralization assay using HiBiT-tagged virus-like particles (hiVLPs). Our hiVLP-based neutralization test demonstrated a higher quantitative value and shorter assay time than conventional methods. We used this assay to evaluate whether the neutralizing antibodies induced by the candidate vaccine virus (NIID-002) were cross-reactive with cow-derived H5N1. Our results suggest that the circulating H5N1 virus in cows shares antigenic characteristics with NIID-002, providing significant implications for the development and preparation of vaccines.

microbiology↗

bioRxiv2024

Characterization of sediment and granite hosted deep underground research laboratories reveals diverse microbiome functions, limited temporal variation and substantial genomic conservation

Underground research laboratories (URLs) provide a window on the deep biosphere and enable investigation of potential microbial impacts on nuclear waste, CO2 and H2 stored in the subsurface. We carried out the first multi-year study of groundwater microbiomes sampled from defined intervals between 140 and 400 m below the surface of the Horonobe and Mizunami URLs, Japan. We reconstructed draft genomes for >90% of all organisms detected over a four year period. The Horonobe and Mizunami microbiomes are dissimilar, likely because the Mizunami URL is hosted in granitic rock and the Horonobe URL in sedimentary rock. Despite this, hydrogen metabolism, rubisco-based CO2 fixation, reduction of nitrogen compounds and sulfate reduction are well represented functions in microbiomes from both URLs, although methane metabolism is more prevalent at the organic- and CO2-rich Horonobe URL. High fluid flow zones and proximity to subsurface tunnels select for candidate phyla radiation bacteria in the Mizunami URL. We detected near-identical genotypes for approximately one third of all genomically defined organisms at multiple depths within the Horonobe URL. This cannot be explained by inactivity, as in situ growth was detected for some bacteria, albeit at slow rates. Given the current low hydraulic conductivity and groundwater compositional heterogeneity, ongoing inter-site strain dispersal seems unlikely. Alternatively, the Horonobe URL microbiome homogeneity may be explained by higher groundwater mobility during the last glacial period. Genotypically-defined species closely related to those detected in the URLs were identified in three other subsurface environments in the USA. Thus, dispersal rates between widely separated underground sites may be fast enough relative to mutation rates to have precluded substantial divergence in species composition. Species overlaps between subsurface locations on different continents constrain expectations regarding the scale of global subsurface biodiversity. Overall, microbiome and geochemical stability over the study period has important implications for underground storage applications.

microbiology↗

bioRxiv2022

Fe(III)-dependent anaerobic methane-oxidizing activity in a deep underground borehole demonstrated by in-situ pressure groundwater incubation

The family Methanoperedenaceae archaea mediate anaerobic oxidation of methane (AOM) in various terrestrial environments. In this study, we newly developed a high-pressure laboratory incubation system by controlling hydraulic pressure ranging from ambient to 5 MPa. Using the system, we investigated groundwater from 214- and 249-m deep boreholes at Horonobe Underground Research Laboratory, Japan, where the high and low abundances of Methanoperedenaceae archaea have been revealed by genome-resolved metagenomics, respectively. We incubated the groundwater samples amended with or without amorphous Fe(III) as an electron acceptor and 13C-labelled methane at an in-situ pressure of 1.6 MPa. After three to seven-day incubation, AOM activities were not detected from the 249-m deep groundwater but from the 214-m deep groundwater. The AOM rates were 93.7 {+/-} 40.6 and 27.7 {+/-} 37.5 nM/day with and without Fe(III) amendment. To clarify the differences in AOM activity between the 214- and 249-m deep groundwater samples, we characterized Fe(III) contents in suspended particulates collected by filtration. The particulates were not visible in the 249-m deep groundwater on the filter, while they were abundant and contained Fe(III)-bearing phyllosilicates in the 214-m deep groundwater. These results support the in-situ activity of Fe(III)-dependent AOM in the deep subsurface borehole.

microbiology↗

bioRxiv2021

Nanobodies recognizing conserved hidden clefts of all SARS-CoV-2 spike variants

We are in the midst of the historic coronavirus infectious disease 2019 (COVID-19) pandemic caused by severe respiratory syndrome coronavirus 2 (SARS-CoV-2). Although countless efforts to control the pandemic have been attempted--most successfully, vaccination1-3--imbalances in accessibility to vaccines, medicines, and diagnostics among countries, regions, and populations have been problematic. Camelid variable regions of heavy chain-only antibodies (VHHs or nanobodies)4 have unique modalities: they are smaller, more stable, easier to customize, and, importantly, less expensive to produce than conventional antibodies5, 6. We present the sequences of nine alpaca nanobodies that detect the spike proteins of four SARS-CoV-2 variants of concern (VOCs)--namely, the alpha, beta, gamma, and delta variants. We show that they can quantify or detect spike variants via ELISA and lateral flow, kinetic, flow cytometric, microscopy, and Western blotting assays7. The panel of nanobodies broadly neutralized viral infection by pseudotyped SARS-CoV-2 VOCs. Structural analyses showed that a P86 clone targeted epitopes that were conserved yet unclassified on the receptor-binding domain (RBD) and located inside the N-terminal domain (NTD). Human antibodies have hardly accessed both regions; consequently, the clone buries hidden crevasses of SARS-CoV-2 spike proteins undetected by conventional antibodies and maintains activity against spike proteins carrying escape mutations.

molecular biology↗