Group B Streptococcus (GBS) is a life-threatening opportunistic pathogen, particularly in pregnant women, infants, and the elderly. Currently, maternal vaccination is considered the most viable long-term option for preventing GBS mother-to-infant infection, and two polysaccharide conjugate vaccines utilizing CRM197 as a carrier protein have undergone clinical phase II trials. Surface immunogenic protein (Sip), present in all identified serotypes of GBS strains so far, is a protective surface protein of GBS. In this study, the type Ia capsular polysaccharide (CPS) of GBS was utilized as a model to develop candidate antigens for a polysaccharide conjugate vaccine by coupling it with the Sip of GBS and the traditional carrier protein CRM197. Serum analysis from immunized New Zealand rabbits and CD1 mice revealed that there was no significant difference in antibody titers between the Ia-Sip group and Ia-CRM197 group; however, both were significantly higher than those observed in the Ia polysaccharide group. Opsonophagocytosis and passive immune protection results using rabbit serum indicated no significant difference between the Ia-Sip and Ia-CRM197 groups, both outperforming the Ia polysaccharide group. Furthermore, serum from the Ia-Sip group had a cross-protective effect on multiple types of GBS strains. The challenge test results in CD1 mice demonstrated that the Ia-Sip group provided complete protection against lethal doses of bacteria and also showed cross-protection against type III strain. Our study demonstrates for the first time that Ia-Sip is immunogenic and provides serotype-independent protection in glycan conjugate vaccines, which also indicates Sip may serve as an excellent carrier protein for GBS glycan conjugate vaccines and provide cross-protection against multiple GBS strains.

The isolation and characterization of novel probiotics from dairy products, fermented foods, and the gut have gained significant attention. In particular, Streptococcus salivarius shows promise for use in oral probiotic preparations. In this study, we isolated two strains of S. salivarius—S.82.15 and S.82.20—from the oral cavity of a healthy individual. These strains exhibited distinct antimicrobial profiles. We thoroughly assessed the morphology and growth patterns of both strains and confirmed auto-aggregation and hemolytic activity. Through comprehensive genomic analysis, we found notable strain differences within the same bacterial species isolated from the same individual. Notably, the presence or absence of plasmids varied between the two strains. The genome of S.82.15 spans 2,175,688 bps and contains 1994 coding DNA sequences (CDSs), while S.82.20 has a genome size of 2,414,610 bps, a GC content of 40.62%, and 2276 annotated CDSs. Both strains demonstrated antibacterial activity against Group A Streptococcus (GAS), Micrococcus. luteus, and Porphyromonas gingivalis. To investigate the antibacterial properties further, we identified a gene cluster of salivaricin 9 on the plasmid of S.82.20 and a blp gene family on the chromosomes of both S.82.15 and S.82.20. Moreover, the gene expression of the blp family was upregulated when the isolated strains were co-cultured with GAS.

Botulinum neurotoxin serotype A (BoNT/A), the most toxic of the seven serotypes produced by Clostridium botulinum, poses significant public health risks because of its involvement in foodborne outbreaks and potential use in bioterrorism. In this study, we developed high-affinity monoclonal antibodies for BoNT/A detection using single-cell fluorescence-activated cell sorting and nested PCR. The optimized antibody pair demonstrated exceptional sensitivity, detecting recombinant BoNT/A at concentrations as low as 0.02 ng/mL with a linear range of 0.02–10 ng/mL, while maintaining high specificity against BoNT/B, E, and F. Biolayer interferometry confirmed superior binding kinetics, and a time-resolved fluoroimmunoassay (TRFIA) demonstrated consistent performance in complex food matrices, including ham sausage and soybean paste. These rabbit-derived monoclonal antibodies enable ultrasensitive detection of BoNT/A across diverse food matrices, offering a powerful tool for food safety monitoring and biosecurity.

Lassa virus (LASV), a member of the Arenaviridae family, is the causative agent of Lassa fever (LF), an acute zoonotic hemorrhagic disease transmitted by rodents, characterized by high infectivity and mortality rates. Due to the nonspecific nature of early clinical symptoms, the development of rapid, sensitive, and specific diagnostic methods is critical for effective epidemic control. In this study, the Lassa virus glycoprotein complex (LASV-G) was selected as the target antigen. High-affinity rabbit monoclonal antibodies were generated using a single B-cell cloning approach, and an AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay)-based homogeneous, no-wash detection system was established. Sixteen LASV-G-specific monoclonal antibodies were isolated through flow cytometric sorting, and the optimal antibody pair (56–24) was identified by AlphaLISA pairing and performance screening. The established AlphaLISA system exhibited a limit of detection (LOD) of 0.025 ng/mL, representing approximately a 30-fold increase in sensitivity compared with conventional Enzyme Linked Immunosorbent Assay (ELISA), while reducing the total assay time to less than 30 min. The coefficient of variation (CV) was below 8%, and no cross-reactivity was observed with Ebola, dengue, yellow fever, Zika, or influenza virus antigens. These findings demonstrate that the developed AlphaLISA assay possesses high sensitivity, rapid detection, and good tolerance to matrix effects, significantly improving the efficiency of early LASV antigen detection. This work provides a potential platform for the rapid on-site screening and epidemiological surveillance of highly pathogenic viruses.

Background: Mycoplasma pneumoniae is a major cause of respiratory infections in children and adolescents. Increasing macrolide resistance and the absence of a commercially available vaccine highlight the need for effective preventive strategies. Our preliminary antigen-screening work identified MPN162 as a potential vaccine antigen, although its immunogenicity alone was limited. This study evaluated whether fusion with cholera toxin B subunit (CTB) and intratracheal delivery could improve MPN162-induced protection. Methods: A recombinant CTB-MPN162 fusion protein, designated CTB-162, was expressed in Escherichia coli. BALB/c mice were immunized with MPN162 or CTB-162 by intratracheal or subcutaneous administration. Antigen-specific serum IgG and IgA responses were measured by ELISA. Following M. pneumoniae challenge, pulmonary bacterial DNA burden was quantified by digital PCR, and lung inflammatory cytokines were measured. The short-term safety of a single high dose of CTB-162 was assessed using body weight, serum biochemical and inflammatory markers, and histopathology. Results: Intratracheal administration induced significantly higher anti-MPN162 IgG and IgA responses than subcutaneous injection and resulted in a lower pulmonary bacterial DNA burden after challenge. CTB-162 elicited stronger IgG responses than MPN162 alone, while the 5 μg dose induced the highest IgA response among the tested groups. Following challenge, the 5 μg CTB-162 dose reduced the pulmonary M. pneumoniae DNA burden by more than three orders of magnitude compared with the PBS group and significantly decreased pulmonary IL-6, IL-1β, and TNF-α levels. The 1 μg dose also provided significant protection. A single 15 μg dose caused no detectable short-term systemic toxicity. Conclusions: CTB fusion and intratracheal delivery enhanced the immunogenicity and protective efficacy of MPN162 in mice. CTB-162 is therefore a promising mucosal subunit vaccine candidate for further preclinical development.