Safety and dose-sparing effect of Japanese encephalitis vaccine administered by microneedle patch in uninfected, healthy adults (MNA-J): a randomised, partly blinded, active-controlled, phase 1 trial.

BACKGROUND: It is unclear whether microneedle vaccinations of Japanese encephalitis virus can induce sufficient neutralising antibodies and reduce the amount of vaccine needed. We aimed to assess the safety and dose-sparing effect of a microneedle vaccine patch against Japanese encephalitis in healthy individuals who are naive to both the vaccine and natural infection. METHODS: The MNA-J study was a randomised, partly blinded, active-controlled, phase 1 clinical trial at Hokkaido University (Sapporo, Japan) that enrolled healthy adults aged 20-34 years with no history of Japanese encephalitis vaccination nor of infection as confirmed by seronegativity. We excluded individuals who had been infected with or vaccinated against Japanese encephalitis. Eligible participants were randomly assigned (1:1:1) to one of three groups to receive inactivated Japanese encephalitis vaccine administered twice, 3 weeks apart, by either 2·5 µg per injection by subcutaneous injection, 0·63 µg per patch by high-dose microneedle array (MNA-25%), or 0·25 µg per patch by low-dose microneedle array (MNA-10%). The randomisation sequence, using stratification by cohort and blocks of six, was computer-generated by a statistician who was unaware of group assignment. After administration, the remaining amount of unadministered vaccine was measured by ELISA and calculated as the delivered amount of vaccine. The primary outcome was the neutralising antibody titre at day 42 after first immunisation. Successful seroconversion was defined as post-vaccination titres of 1·3 (log10) or higher in individuals whose pre-vaccination titres had been less than 1 (log10). This study is registered with the Japan Registry of Clinical Trials (s011190004). FINDINGS: Between Aug 31 and Sept 2, 2019, 39 participants were enrolled and each was randomly assigned to a group (n=13 per group). No serious adverse events were observed. All participants in the microneedle array groups had a localised erythematous reaction. The amount of vaccine delivered by microneedle array to each participant was 0·63-1·15 µg (50-92%) of the full 1·26 µg for the MNA-25% group and 0·25-0·41 µg (51-84%) of the full 0·50 µg for the MNA-10% group. All participants demonstrated seroconversion at day 42, and the mean titres (log10) were 2·55 for MNA-25%, 2·04 for MNA-10%, and 2·08 for subcutaneous injection. INTERPRETATION: A microneedle patch of the Japanese encephalitis vaccine is safe, well tolerated, and immunogenically effective. The dose-sparing effect suggests a significant potential to reduce the amount of immunogens needed. However, improved delivery is needed to make it more tolerable and user friendly. FUNDING: FUJIFILM.

Isolation of Aluminum-Tolerant and -Absorbing Yeast.

Aluminum ions are toxic to bacteria and are thus frequently used for preservation in the food industry. However, at higher concentrations, aluminum is toxic to animals. The extraction of aluminum from aluminum-contaminated foods would therefore be beneficial. Based on the discovery of yeast strains that can tolerate and absorb toxic metals, we aimed to identify strains that could tolerate and absorb aluminum. In this study, yeast were isolated from soil samples and cultured in medium containing the toxic concentration of aluminum chloride (5 mM) for Saccharomyces cerevisiae BY4741. Among aluminum-tolerant strains, two strains, Alt-OF2 and Alt-OF5, were identified as aluminum-absorbing. D1/D2 sequencing revealed that both strains belonged to the genus Schizoblastosporion (syn. Nadsonia).

Total Synthesis of Brasilicardins A and C.

The first total synthesis of brasilicardins A and C, novel diterpenoid-saccharide-amino acid hybrid metabolites with unique immunosuppressive activity, is described. The key step is a Diels-Alder/reductive angular methylation sequence capitalizing on a trans-fused bicyclic α-cyano-α,ß-enone as its precursor to construct the 8,10-dimethyl-trans/syn/trans-perhydrophenanthrene skeleton. Other notable features include an anti-selective aldol reaction, a stereocontrolled glycosylation of a C2 alcohol, and a one-pot, two-step global deprotection sequence that did not damage these sensitive molecules.

Structural and thermodynamic analyses reveal critical features of glycopeptide recognition by the human PILRα immune cell receptor.

Before entering host cells, herpes simplex virus-1 uses its envelope glycoprotein B to bind paired immunoglobulin-like type 2 receptor α (PILRα) on immune cells. PILRα belongs to the Siglec (sialic acid (SA)-binding immunoglobulin-like lectin)-like family, members of which bind SA. PILRα is the only Siglec member to recognize not only the sialylated O-linked sugar T antigen (sTn) but also its attached peptide region. We previously determined the crystal structure of PILRα complexed with the sTn-linked glycopeptide of glycoprotein B, revealing the simultaneous recognition of sTn and peptide by the receptor. However, the contribution of each glycopeptide component to PILRα binding was largely unclear. Here, we chemically synthesized glycopeptide derivatives and determined the thermodynamic parameters of their interaction with PILRα. We show that glycopeptides with different sugar units linking SA and peptides (i.e. "GlcNAc-type" and "deoxy-GlcNAc-type" glycopeptides) have lower affinity and more enthalpy-driven binding than the wild type (i.e. GalNAc-type glycopeptide). The crystal structures of PILRα complexed with these glycopeptides highlighted the importance of stereochemical positioning of the O4 atom of the sugar moiety. These results provide insights both for understanding the unique O-glycosylated peptide recognition by the PILRα and for the rational design of herpes simplex virus-1 entry inhibitors.

Rapid Screening by Cell-Based Fusion Assay for Identifying Novel Antivirals of Glycoprotein B-Mediated Herpes Simplex Virus Type 1 Infection.

Herpes simplex virus type 1 (HSV-1) is a causative agent for a variety of diseases. Although antiherpetic drugs such as acyclovir have been developed to inhibit virus replication through interaction with DNA kinases, their continuous administration leads to an increase in the frequency of drug-resistant HSV-1, which is an important clinical issue that requires urgent solution. Recently, we reported that the sialylated O-linked sugar T antigen (sTn) and its attached peptide region (O-glycosylated sTn peptide) derived from the HSV-1 glycoprotein B (gB) protein inhibited HSV-1 infection by specifically targeting paired immunoglobulin-like type 2 receptor alpha (PILRα) in vitro. In this study, to further identify novel inhibitors of gB-mediated HSV-1 infection in vitro, we established a cell-based fusion assay for rapid drug screening. Chinese hamster ovary (CHO) cells were transfected with expression plasmids for HSV-1 gB, gD, gH, and gL, and T7 RNA polymerase, and were designated as the effector cells. The CHO-K1 cells stably expressing PILRα were transfected with the expression plasmid for firefly luciferase under the T7 promoter, and were designated as the target cells. The effector and target cells were co-cultured, and luminescence was measured when both cells were successfully fused. Importantly, we found that cell-to-cell fusion was specifically inhibited by O-glycosylated sTn peptide in a dose dependent manner. Our results suggested that this virus-free cell-based fusion assay system could be a useful and promising approach to identify novel inhibitors of gB-mediated HSV-1 infection, and will aid in the development of antiviral therapeutic strategies for HSV-1-associated diseases.

Glycosylation with 2-Acetamido-2-deoxyglycosyl Donors at a Low Temperature: Scope of the Non-Oxazoline Method.

A direct construction of 1,2-trans-ß-linked 2-acetamido-2-deoxyglycosides was investigated. The 3,4,6-tri-O-benzyl- and 3,4,6-tri-O-acetyl-protected glycosyl diethyl phosphites and 4,6-O-benzylidene-protected galactosyl diethyl phosphite each reacted with a variety of acceptor alcohols in the presence of a stoichiometric amount of Tf2NH in CH2Cl2 at -78 °C to afford the corresponding ß-glycosides in good to high yields with complete stereoselectivity. Some experiments provided strong evidence that the corresponding oxazolinium ions are not responsible for the reaction. We demonstrated that glycosylations with the corresponding glycosyl imidate and thioglycoside also proceeded at a low temperature, indicating the possibility of these donors being attractive alternatives to the phosphite. A plausible reaction mechanism, which features glycosyl triflimide and contact ion pair as reactive intermediates, is proposed on the basis of the results obtained with 2-acetamido-2-deoxymannosyl donors.

Synthesis of the Tetrasaccharide Repeating Unit from Acinetobacter baumannii Serogroup O18 Capitalizing on Phosphorus-Containing Leaving Groups.

The first convergent synthesis of the tetrasaccharide repeating unit of the polymeric O antigen isolated from Acinetobacter baumannii serogroup O18 has been achieved. The ManNAcß1→4Gal and GalNAcß1→3Gal units were successfully obtained through ß-selective glycosylation with 2-azido-4,6-O-benzylidene-2-deoxymannosyl diphenyl phosphate and Tf2NH-promoted glycosylation with 2-acetamido-2-deoxygalactosyl diethyl phosphite, respectively. The disaccharide units could be coupled with the aid of TMSClO4 as an activator of the diphenyl phosphate leaving group, and global deprotection completed the synthesis of the tetrasaccharide.