Rabies is a lethal zoonotic disease that threatens human health. As the only viral surface protein, the rabies virus (RABV) glycoprotein (G) induces main neutralizing antibody (Nab) responses; however, Nab titre is closely correlated with the conformation of G. Virus-like particles (VLP) formed by the co-expression of RABV G and matrix protein (M) improve retention and antigen presentation, inducing broad, durable immune responses. RABV nucleoprotein (N) can elicit humoral and cellular immune responses. Hence, we developed a series of nucleoside-modified RABV mRNA vaccines encoding wild-type G, soluble trimeric RABV G formed by an artificial trimer motif (tG-MTQ), membrane-anchored prefusion-stabilized G (preG). Furthermore, we also developed RABV VLP mRNA vaccine co-expressing preG and M to generate VLPs, and VLP/N mRNA vaccine co-expressing preG, M, and N. The RABV mRNA vaccines induced higher humoral and cellular responses than inactivated rabies vaccine, and completely protected mice against intracerebral challenge. Additionally, the IgG and Nab titres in RABV preG, VLP and VLP/N mRNA groups were significantly higher than those in G and tG-MTQ groups. A single administration of VLP or VLP/N mRNA vaccines elicited protective Nab responses, the Nab titres were significantly higher than that in inactivated rabies vaccine group at day 7. Moreover, RABV VLP and VLP/N mRNA vaccines showed superior capacities to elicit potent germinal centre, long-lived plasma cell and memory B cell responses, which linked to high titre and durable Nab responses. In summary, our data demonstrated that RABV VLP and VLP/N mRNA vaccines could be promising candidates against rabies.

Human programmed cell death protein 1 (hPD-1) is an essential receptor in the immune checkpoint pathway. It has played an important role in cancer therapy. However, not all patients respond positively to the PD-1 antibody treatment, and the underlying mechanism remains unknown. PD-1 is a transmembrane glycoprotein, and its extracellular domain (ECD) is reported to be responsible for interactions and signal transduction. This domain contains 4 N-glycosylation sites and 25 potential O-glycosylation sites, which implicates the importance of glycosylation. The structure of hPD-1 has been intensively studied, but the glycosylation of this protein, especially the glycan on each glycosylation site, has not been comprehensively illustrated. In this study, hPD-1 ECD expressed by human embryonic kidney 293 (HEK 293) and Chinese hamster ovary (CHO) cells was analyzed; not only N- and O-glycosylation sites but also the glycans on these sites were comprehensively analyzed using mass spectrometry. In addition, hPD-1 ECD binding to different anti-hPD-1 antibodies was tested, and N-glycans were found functioned differently. All of this glycan information will be beneficial for future PD-1 studies.

Severe fever with thrombocytopenia syndrome virus (SFTSV) or Dabie bandavirus is an emerging pathogen responsible for SFTS. It is considered a novel threat to human health, given the high associated fatality. SFTSV is a segmented negative-strand RNA virus containing three single-stranded RNAs, with the M segment encoding the glycoproteins Gn and Gc. Gc is vital for viral entry into the host cell surface, along with the Gn protein. As the Gc is the surface-exposable antigen from virions, it is a critical diagnostic marker of infection. Although various SFTSV Gn or N protein-based sero-diagnostic methods have been developed, there are no commercially available sero-diagnostic kits. Therefore, we generated monoclonal antibodies (mAbs) against SFTSV Gc and explored their application in serum diagnostic tests to develop sensitive serodiagnostic tools covering broad-range genotypes (A to F). First, 10 SFTSV Gc antibody-binding fragments (Fabs) were isolated using a phage display system and converted into human IgGs. Enzyme-linked immunosorbent assays (ELISA) of the SFTSV and Rift Valley fever virus (RVFV: same genus as SFTSV) Gc antigens showed that all antibodies attached to the SFTSV Gc protein had high affinity. An immunofluorescence assay (IFA), to verify the cross-reactivity of seven antibodies with high affinities for various SFTSV genotypes (A, B2, B3, D, and F) and detect mAb binding with intact Gc proteins, revealed that five IgG type mAbs were bound to intact Gc proteins of various genotypes. Six high-affinity antibodies were selected using ELISA and IFA. The binding capacity of the six antibodies against the SFTSV Gc antigen was measured using surface plasmon resonance. All antibodies had high binding capacity. Consequently, these antibodies serve as valuable markers in the serological diagnosis of SFTSV.

BACKGROUND: This immunoinformatic study identified potential epitopes from the envelopment polyprotein (Gn/Gc) of Rift Valley fever virus (RVFV), a pathogenic virus causing severe fever in humans and livestock. Effective vaccination is crucial for controlling RVFV outbreaks. The identification of suitable epitopes is crucial for the development of safe and effective vaccines.
METHODS: Protein sequences were obtained from the UniProt database, and evaluated through VaxiJen v2.0 to predict the B and T-cell epitopes within the RVFV glycoprotein. Gn/Gc protein sequences were analyzed with bioinformatics tools and algorithms. The predicted T-cell and B-cell epitopes were evaluated for antigenicity, allergenicity, and toxicity by the VaxiJen v2.0 system, AllerTop v2.0, and ToxinPred server, respectively.
RESULTS: We employed computational methods to screen the RVFV envelopment polyprotein encompassing N-terminal and C-terminal glycoprotein segments, to discover antigenic T- and B-cell epitopes. Our analysis unveiled multiple potential epitopes within the RVFV glycoprotein, specifically within the Gn/Gc protein sequences. Subsequently, we selected eleven cytotoxic T-lymphocytes (CTL) and four helper T-lymphocytes (HTL) for population coverage analysis, which collectively extended to cover 97.04% of the world\'s population, representing diverse ethnicities and regions. Notably, the CTL epitope VQADLTLMF exhibited binding affinity to numerous human leukocyte antigen (HLA) alleles. The identification of glycoprotein (Gn/Gc) epitopes through this immunoinformatic study bears significant implications for advancing the development of an effective RVFV vaccine.
CONCLUSIONS: These findings provide valuable insights into the immunological aspects of the disease and may contribute towards the development of broad-spectrum antiviral therapies targeting other RNA viruses with similar polymerase enzymes.

Both Old World and New World hantaviruses are transmitted through rodents and can lead to hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome in humans without the availability of specific therapeutics. The square-shaped surface spikes of hantaviruses consist of four Gn-Gc heterodimers that are pivotal for viral entry into host cells and serve as targets for the immune system. Previously, a human-derived neutralizing monoclonal antibody, AH100, demonstrated specific neutralization against the Old World hantavirus, Hantaan virus. However, the precise mode binding of this neutralizing monoclonal antibody remains unclear. In the present study, we determined the structure of the Hantaan virus Gn-AH100 antigen-binding fragment complex and identified its epitope. Crystallography revealed that AH100 targeted the epitopes on domain A and b-ribbon and E3-like domain. Epitope mapping onto a model of the higher order (Gn-Gc)4 spike revealed its localization between neighboring Gn protomers, distinguishing this epitope as a unique site compared to the previously reported monoclonal antibodies. This study provides crucial insights into the structural basis of hantavirus neutralizing antibody epitopes, thereby facilitating the development of therapeutic antibodies.IMPORTANCEHantaan virus (HTNV) poses a significant threat to humans by causing hemorrhagic fever with renal syndrome with high mortality rates. In the absence of FDA-approved drugs or vaccines, it is urgent to develop specific therapeutics. Here, we elucidated the epitope of a human-derived neutralizing antibody, AH100, by determining the HTNV glycoprotein Gn-AH100 antigen-binding fragment (Fab) complex structure. Our findings revealed that the epitopes situated on the domain A and b-ribbon and E3-like domain of the HTNV Gn head. By modeling the complex structure in the viral lattice, we propose that AH100 neutralizes the virus by impeding conformational changes of Gn protomer, which is crucial for viral entry. Additionally, sequence analysis of all reported natural isolates indicated the absence of mutations in epitope residues, suggesting the potential neutralization ability of AH100 in diverse isolates. Therefore, our results provide novel insights into the epitope and the molecular basis of AH100 neutralization.

Abrin and ricin, both type II ribosome-inactivating proteins, are toxins of significant concern and are under international restriction by the Chemical Weapons Convention and the Biological and Toxin Weapons Convention. The development of a rapid and sensitive detection method for these toxins is of the utmost importance for the first emergency response. Emerging rapid detection techniques, such as surface-enhanced Raman spectroscopy (SERS) and lateral flow assay (LFA), have garnered attention due to their high sensitivity, good selectivity, ease of operation, low cost, and disposability. In this work, we generated stable and high-affinity nanotags, via an efficient freezing method, to serve as the capture module for SERS-LFA. We then constructed a sandwich-style lateral flow test strip using a pair of glycoproteins, asialofetuin and concanavalin A, as the core affinity recognition molecules, capable of trace measurement for both abrin and ricin. The limit of detection for abrin and ricin was 0.1 and 0.3 ng/mL, respectively. This method was applied to analyze eight spiked white powder samples, one juice sample, and three actual botanic samples, aligning well with cytotoxicity assay outcomes. It demonstrated good inter-batch and intra-batch reproducibility among the test strips, and the detection could be completed within 15 min, indicating the suitability of this SERS-LFA method for the on-site rapid detection of abrin and ricin toxins.

A globally implemented unified phylogenetic classification for human respiratory syncytial virus (HRSV) below the subgroup level remains elusive. We formulated global consensus of HRSV classification on the basis of the challenges and limitations of our previous proposals and the future of genomic surveillance. From a high-quality curated dataset of 1,480 HRSV-A and 1,385 HRSV-B genomes submitted to GenBank and GISAID (https://www.gisaid.org) public sequence databases through March 2023, we categorized HRSV-A/B sequences into lineages based on phylogenetic clades and amino acid markers. We defined 24 lineages within HRSV-A and 16 within HRSV-B and provided guidelines for defining prospective lineages. Our classification demonstrated robustness in its applicability to both complete and partial genomes. We envision that this unified HRSV classification proposal will strengthen HRSV molecular epidemiology on a global scale.

An overview of the role of glycosylation in prostate cancer (PCa) development and progression is presented, focusing on recent advancements in defining the N-glycome through glycomic profiling and glycoproteomic methodologies. Glycosylation is a common post-translational modification typified by oligosaccharides attached N-linked to asparagine or O-linked to serine or threonine on carrier proteins. These attached sugars have crucial roles in protein folding and cellular recognition processes, such that altered glycosylation is a hallmark of cancer pathogenesis and progression. In the past decade, advancements in N-glycan profiling workflows using Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) technology have been applied to define the spatial distribution of glycans in PCa tissues. Multiple studies applying N-glycan MALDI-MSI to pathology-defined PCa tissues have identified significant alterations in N-glycan profiles associated with PCa progression. N-glycan compositions progressively increase in number, and structural complexity due to increased fucosylation and sialylation. Additionally, significant progress has been made in defining the glycan and glycopeptide compositions of prostatic-derived glycoproteins like prostate-specific antigen in tissues and biofluids. The glycosyltransferases involved in these changes are potential drug targets for PCa, and new approaches in this area are summarized. These advancements will be discussed in the context of the further development of clinical diagnostics and therapeutics targeting glycans and glycoproteins associated with PCa progression. Integration of large scale spatial glycomic data for PCa with other spatial-omic methodologies is now feasible at the tissue and single-cell levels.

Glycoproteins are difficult to be detected by imprinting strategy due to their low natural abundance, high flexible conformation and large size. Herein, a high-density boric acid modified metal-organic framework (MOF) surface molecularly imprinted polymer (SMIP) resonant light scattering sensor was constructed for the high-sensitivity detection of target glycoproteins. A MOF with large specific surface area was selected as the substrate material to support the boric acid group with high loading density (4.66 %). The introduction of the boric acid group in the SMIP provided a high-affinity binding site for the recognition and binding of glycoproteins. Shallow surface cavities with rapid mass transfer (equilibrium time 20 min) were thus formed by surface imprinting. Furthermore, high sensitivity (limit of detection 15 pM) was achieved at physiological pH (7.4), which was conducive to the detection of glycoproteins with low natural abundance in complex biological samples and maintaining physiological activity.

Aberrant glycosylation has gained significant interest for biomarker discovery. However, low detectability, complex glycan structures, and heterogeneity present challenges in glycoprotein assay development. Using haptoglobin (Hp) as a model, we developed an integrated platform combining functionalized magnetic nanoparticles and zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) for highly specific glycopeptide enrichment, followed by a data-independent acquisition (DIA) strategy to establish a deep cancer-specific Hp-glycosylation profile in hepatitis B virus (HBV, n = 5) and hepatocellular carcinoma (HCC, n = 5) patients. The DIA strategy established one of the deepest Hp-glycosylation landscapes (1029 glycopeptides, 130 glycans) across serum samples, including 54 glycopeptides exclusively detected in HCC patients. Additionally, single-shot DIA searches against a DIA-based spectral library outperformed the DDA approach by 2-3-fold glycopeptide coverage across patients. Among the four N-glycan sites on Hp (N-184, N-207, N-211, N-241), the total glycan type distribution revealed significantly enhanced detection of combined fucosylated-sialylated glycans, which were the most dominant glycoforms identified in HCC patients. Quantitation analysis revealed 48 glycopeptides significantly enriched in HCC (p < 0.05), including a hybrid monosialylated triantennary glycopeptide on the N-184 site with nearly none-to-all elevation to differentiate HCC from the HBV group (HCC/HBV ratio: 2462 ± 766, p < 0.05). In summary, DIA-MS presents an unbiased and comprehensive alternative for targeted glycoproteomics to guide discovery and validation of glyco-biomarkers.