Zeliang Chen

Background/Objectives: Brucellosis is a widespread intracellular zoonosis that continues to impose substantial public-health and economic burdens in endemic regions. No human brucellosis vaccine has yet been approved, and most existing multi-epitope candidates rely solely on computational predictions, without population-specific human leukocyte antigen (HLA) optimization or in vivo epitope validation. REC1 is therefore an early-stage, proof-of-concept, human-oriented multi-epitope candidate, with epitopes screened against high-frequency HLA alleles in the Chinese population. This study developed a multi-epitope vaccine candidate, REC1, using an integrated reverse-vaccinology workflow. Methods: We predicted Major Histocompatibility Complex (MHC) I and II epitopes against high-frequency HLA alleles representative of the Chinese population (AFND). Population coverage of the final incorporated epitope set was estimated with the IEDB Population Coverage tool under Hardy–Weinberg equilibrium, while paired HLA-DQ heterodimers, which the tool cannot model, were evaluated independently from Chinese DQA1-DQB1 haplotype frequencies. Epitopes supported by in vivo immunoreactivity were then assembled into the modular REC1 construct, which we characterized in silico for physicochemical properties, structural architecture, and simulated immune responses. Results: IFN-γ ELISpot shortlisted seven MHC-I and five MHC-II candidate immunoreactive epitopes derived from five Brucella antigens, of which five MHC-I and five MHC-II (one per antigen) were incorporated into REC1. The incorporated set was predicted to cover 91.17% of the China Oriental population on a conservative class I+II estimate (98.88% when HLA-DP was included; the HLA-DQ component alone covered 58–79%, 68.17% in Han). Epitope-level BLAST showed that all twelve shortlisted epitopes were 100% conserved across major Brucella species, except that the two Omp31 epitopes are absent from the Omp31-deleted B. abortus. The 35.86 kDa REC1 construct was predicted to be highly stable (instability index = 25.25) and hydrophilic, with a distinct architecture in which the carrier domain is ordered and the epitope region is disordered. Immune simulations suggested that REC1 can induce Th1-polarized humoral and cellular responses and may support long-term immunological memory. Molecular docking predicted that the L7/L12 carrier forms well-defined interfaces with TLR2, CD14, C1q, and DC-SIGN, and a representative peptide–MHC model confirmed canonical groove binding of a CTL epitope. Conclusions: REC1 exhibits favorable in silico properties, and its embedded peptides show IFN-γ immunogenicity in mice (candidate immunoreactive epitopes), lending preliminary support to its value as a multi-epitope vaccine candidate. HLA-matched validation and challenge studies are still needed to establish protective efficacy. Our study also offers a validated reverse vaccinology framework for future brucellosis vaccine development.

Mammarenaviruses cause serious diseases such as Lassa fever and Argentine hemorrhagic fever that threaten global public health; however, vaccines and therapeutic options remain limited. Therefore, developing a broadly effective vaccine against mammarenaviruses is a priority. In this study, we systematically analyzed the main structural protein sequences of ten mammarenaviruses. We combined antigenic epitopes from the Immune Epitope Database (IEDB) and used inverse vaccination and immunoinformatics methods to screen for highly conserved B- and T-cell epitopes with strong immunogenicity. We then added tissue plasminogen activator (tPA), which is the most effective vaccine against these pathogens. To construct a broad-spectrum multi-epitope fusion mRNA vaccine candidate, we included a tPA signal peptide, PADRE adjuvant, and linkers. The mRNA sequences were algorithm-optimized. We assessed the structural stability and immunogenicity of the candidate vaccine using molecular docking, molecular dynamics (MD) simulation, and immunosimulation. The designed vaccine had good antigenicity and structural stability, forming stable complexes with a variety of intrinsic immunoreceptors and triggering a strong, sustained, and comprehensive immune response during immunosimulation. Our study findings posit the designed vaccine as a potential broad-spectrum multi-epitope vaccine candidate against mammarenaviruses. Further real-world studies are required to validate these results.

This study evaluated the potential of slightly acidic electrolyzed water (SAEW) as an efficient and safe biofilm disinfectant for medical devices and implants. The results showed that SAEW rapidly eradicated planktonic bacteria and biofilms, outperforming NaClO. SAEW degraded proteins and eDNA in extracellular polymeric substances, thereby penetrating biofilms and acting on bacteria. SAEW-treated bacteria could not maintain normal morphology, resulting in bacterial lysis and death. SAEW downregulated biofilm-related genes, including cna, pvl and clfA of Staphylococcus aureus, and aap, icaR and sara of Staphylococcus epidermidis. Additionally, SAEW cleared biofilms on surgical devices and implants within 10 min or less. Furthermore, no significant difference in corrosion efficiency was observed between the SAEW group and the negative control group when tested on stainless steel, zinc alloy and brass. In conclusion, SAEW exhibited robust antibacterial and biofilm-eliminating capabilities, showing great potential as a disinfectant for medical devices and implants.

Brucellosis is a zoonotic infectious disease caused by Brucella species, posing serious threats to public health and livestock industries worldwide. According to the World Health Organization (WHO), over one million new cases are reported annually. However, the actual number may be underestimated due to the bacteria’s intracellular persistence and subclinical bacteremia. These features and Brucella’s genetic variability and immune evasion strategies hinder accurate diagnosis and effective treatment. Thus, there is an urgent need for sensitive and specific molecular probes. This study used whole Brucella bacteria as the target antigen for aptamer selection via the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method, combined with a microplate-based screening approach. To enhance specificity, a multi-organism counter-selection strategy was implemented using Rhizobium fischeri, Escherichia coli, and Salmonella. After 13 rounds of selection, a high-affinity aptamer library was obtained. Candidate aptamers were cloned, sequenced using T-vector cloning, and analyzed phylogenetically. Five candidates (WS-6, WS-17, WS-44, WS-26, and WS-32) were selected for further characterization. Binding affinity and specificity assays identified WS-6 as the top-performing aptamer (Kd = 16.23 ± 5.84 nM). WS-6 also demonstrated thermal and pH stability. Its target protein was identified as the outer membrane protein BamA (β-barrel assembly machinery A) using Co-immunoprecipitation (Co-IP), mass spectrometry, and molecular docking. Western blotting and pull-down assays confirmed this interaction. Functional studies showed that WS-6-loaded liposomes selectively recognized Brucella and significantly reduced bacterial invasion in host cells. qPCR analysis demonstrated a notable decrease in bacterial load in WS-6-treated cells (Ct values from 22.34 to 20.64, P < 0.05). Mutational analysis of WS-6 confirmed the importance of GC-rich binding sites. Moreover, a truncated version of BamA (BamAK) retained binding capability, validating it as the key antigenic domain. In summary, this study demonstrates the potential of SELEX-derived aptamers as diagnostic and therapeutic tools for Brucella infection. Identifying BamA as a target provides a molecular basis for the future development of precise detection methods and targeted therapies against brucellosis. Graphical abstract

Background: African swine fever (ASF) is a highly contagious acute febrile disease with a near 100% mortality rate. There are currently no safe and effective vaccines for this disease. Cellular immunity plays an important role in the process of anti-viral, activating an effective cellular immune response is a prerequisite for the effectiveness of the vaccine. Methods: To effectively activate cellular immune responses, 133 immunodominant T cell epitopes (TEPs) were identified and synthesized into ten recombinant multi-epitope proteins (MEPs). These MEPs were subsequently conjugated to porcine ferritin (pFTH1) to generate MEPs-pFTH1 nanoparticles. Animal experiments were conducted to evaluate their immunogenicity and biocompatibility. Results: Animal experiments demonstrated that both MEPs and MEPs-pFTH1 nanoparticles induced significant humoral and cellular immune responses. Compared to MEPs monomers, the MEPs-pFTH1 nanoparticles induced a 10- to 100-fold increase in IgG and IgG2a antibody titers (p < 0.05), as well as a significantly higher number of IFN-γ⁺ cells. Serum from pigs immunized with MEPs-pFTH1 nanoparticles can significantly inhibit ASFV replication. Conclusions: Our novel self-assembled porcine ferritin nanovaccine candidate can induce strong humoral and cellular immune responses in swine and mice that effectively inhibit ASFV replication. Therefore, the nanovaccine is a highly biocompatible and safe candidate vaccine for ASF that warrants further investigation, such as conducting animal challenge experiments to evaluate the effectiveness of the vaccine.

Brucellosis presents notable diagnostic challenges, particularly during the chronic phase of infection, due to the typically low bacterial load in tissues, which may evade detection by conventional Polymerase Chain Reaction (PCR) methods. Traditional PCR techniques are susceptible to cross-reactions and can produce false-positive or false-negative results. Furthermore, bacteriological and serological tests have recognized limitations in sensitivity and specificity, potentially complicating accurate diagnosis. We developed a novel reverse transcription quantitative PCR (RT-qPCR) assay targeting Brucella species’ highly abundant and conserved 23S rRNA gene to address these challenges. The 23S rRNA gene was selected as an optimal molecular target due to the approaches’ number and genetic stability across Brucella strains. Primers were designed based on a conserved region (Sequence ID: NR_103168.2) to ensure broad applicability, and their specificity was verified through BLAST analysis. A key feature of the 23S-RT-qPCR method is incorporating a reverse transcription step to convert RNA into complementary DNA (cDNA), which may enhance detection sensitivity. Using the Brucella S2 vaccine strain as a template, experimental results indicated that the 23S-RT-qPCR method reduced cycle threshold (Ct) values by approximately 2–3 units (14.67–16.74) compared to conventional qPCR. Statistical analysis using a T-test showed that this reduction was significant (P < 0.05). The performance of the 23S-RT-qPCR method was further evaluated using clinical samples and compared with the IS711 detection method. The 23S-RT-qPCR method demonstrated a lower false-positive rate (2.6%) and false-negative rate (7.6%) compared to the IS711 method, which had a false-positive rate of 5.2% and a false-negative rate of 7.6%. These results suggest that the 23S-RT-qPCR method offers improved sensitivity and specificity, reducing false-positive and false-negative rates in clinical sample analysis. Overall, this method may provide a more reliable approach for diagnosing chronic brucellosis and could have broader applicability in detecting other bacterial pathogens.

Background: Antibiotic resistance of many bacteria, including Methicillin-resistant Staphylococcus aureus (MRSA), has become a major threat to global health. Zinc Oxide Quantum dots (ZnO-QDs) show good antibacterial activity, but most of them are insoluble in water, limiting their application range, and there is a lack of research on drug resistance inducement. Methods: The water-soluble zinc oxide quantum dots modified by APTES (ZnO@APTES QDs) were prepared by a microwave assisted synthesis. Then ZnO@APTES QDs were characterized through various methods. After confirmation of synthesized ZnO@APTES QDs, its bactericidal effect on MRSA was detected through in vitro and in vivo experiments, and its mechanism of action was analyzed. Results: Characterization analysis revealed that the ZnO@APTES QDs have a particle size of 5 nm. The minimum inhibitory concentrations (MIC) were determined to be 64 μg mL^{− 1} for Escherichia coli (E. coli) and 32 μg mL^{− 1} for MRSA. The ZnO@APTES QDs showed significant inhibition of MRSA biofilm formation and effectively disrupted mature biofilms. Notably, the ZnO@APTES QDs did not induce tolerance or resistance even after 30 days of repeated exposure, whereas antibiotics led to a rise in bacterial MIC within 3 days and a 60-fold increase after 30 days. Mechanistic analysis indicated that the positively charged quantum dots interact with bacterial surfaces, altering membrane fluidity. Once inside the bacteria, the ZnO@APTES QDs generate reactive oxygen species (ROS), causing DNA damage and bacterial cell death. Moreover, the ZnO@APTES QDs possessed good biocompatibility and demonstrated significant therapeutic efficacy against drug-resistant bacterial infections in both macrophage and mouse wound infection models. Conclusion: In summary, we have synthesized a highly effective water-soluble ZnO@APTES QDs that shows strong antibacterial and therapeutic efficacy against MRSA and other bacteria. The ZnO@APTES QDs holds significant potential for development as a new treatment agent for combating antibiotic-resistant infections.