Continuous mutation of viruses enables evasion of established immune defenses and therapeutics. Here we report a broad-spectrum therapeutic design, termed HRBD, that mimics viral invasion-associated molecular patterns. HRBD demonstrated potent and broad-spectrum inhibition against Pangolin-CoV, SARS-CoV, SARS-CoV-2, and its variants, lowering the half-maximal inhibitory concentration (IC50) by approximately 1000-fold compared to the monomeric RBD. Furthermore, HRBD effectively suppressed syncytium formation induced by the spike proteins of sarbecoviruses, an effect not observed with the monomeric RBD. In hACE2-transgenic mice challenged with SARS-CoV-2, intranasal administration of HRBD reduced viral loads in lung and tracheal tissues by ∼106-fold, with no detectable immunogenicity. Binding analyses revealed that HRBD achieved approximately 1000-fold stronger avidity for hACE2 compared to the RBD monomer, superior to the high-affinity RBD-62 mutant generated by directed evolution, without affecting hACE2 enzymatic activity or subcellular localization. Oligomerization characterization confirmed that HRBD predominantly formed heptamers, visualized as ∼10 nm diameter rings via transmission electron microscopy. This mimicking strategy offers a viable approach for developing broad-spectrum therapeutics against current and future antigenically variable viruses.

Objectives: Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) merges multidrug resistance with hypervirulence, posing unprecedented therapeutic challenges. This study aimed to evaluate the efficacy of a recombinant fusion protein vaccine, KPC-Pal, designed to target both the carbapenemase KPC-2 and the virulence-associated peptidoglycan-associated lipoprotein Pal. Methods: The KPC-Pal fusion protein was constructed, expressed, and purified. Its protective efficacy was systematically assessed in a murine pneumonia model by measuring antigen-specific antibodies, cytokine profiles, and memory cell populations. The synergistic effect with the antibiotic meropenem was evaluated both in vitro and in vivo. Furthermore, the interaction with innate immune signaling via TLR2 was investigated. Results: Immunization with KPC-Pal conferred superior protection, resulting in significantly higher survival rates and reduced bacterial burdens in the lungs compared to immunization with either KPC-2 or Pal alone. It induced a robust Th2-biased humoral response and a mixed Th1/Th2/Th17 cellular immune profile, along with enhanced formation of tissue-resident memory T cells. Antibodies generated against KPC-Pal enhanced the efficacy of meropenem in vitro and in animal models, demonstrating a synergistic effect. While Pal alone strongly activated TLR2-driven inflammatory pathways, the KPC-Pal fusion selectively modulated MAPK signaling, mitigating excessive cytokine production. Additionally, KPC-Pal vaccination elicited cross-reactive antibodies against KPC-3 and KPC-33 variants. Conclusions: KPC-Pal functions as both an antigen and a self-adjuvant, offering a promising dual-target strategy for combating K. pneumoniae infections.

The increasing threat of infections caused by multidrug-resistant Gram-positive pathogens such as MRSA and VRE has driven the structural remodeling and repurposing of traditional antibiotics as a key strategy to combat bacterial resistance. Herein, using bacitracin A as a lead template, we established a site-selective semisynthetic modification strategy via reductive amination of its N-terminal primary amine. 32 derivatives were designed and synthesized, and their structure-activity relationships were systematically evaluated. Optimized derivatives exhibited potent activity against MRSA and VRE, with 4-32-fold enhanced antibacterial potency compared with bacitracin A, together with improved bactericidal properties, safety profiles, and pharmacokinetic characteristics. Mechanistically, hydrophobic N-terminal modification endows bacitracin A with multiple antibacterial modes, including enhanced inhibition of cell wall peptidoglycan biosynthesis, obvious membrane depolarization, and disruption of the purine metabolic pathway. These findings provide a rational strategy for bacitracin optimization and highlight the potential of N-terminal modification for developing improved antibacterial agents.

Background/Objectives: Klebsiella pneumoniae (K. pneumoniae) is a leading cause of serious hospital-acquired and community-acquired infections, with limited treatment options, especially for immunocompromised and critically ill patients. No licensed vaccine is currently available. The FimA antigen, a key fimbrial subunit essential for bacterial adhesion and invasion, represents a promising vaccine target. However, little is known about the immunodominant antibody responses against invasive K. pneumoniae. This study aimed to evaluate the immunogenicity and protective efficacy of recombinant FimA protein, to fine-map its immunodominant linear B-cell epitopes, and to assess the individual and combined protective capacity of these epitopes against both standard and clinically isolated K. pneumoniae strains. Methods: A murine model of lethal K. pneumoniae challenge was used. Recombinant FimA protein was administered to evaluate immunogenicity and protective efficacy. Immunodominant linear B-cell epitopes were identified by overlapping peptide ELISA using immune antisera. The identified epitopes were synthesized and conjugated to keyhole limpet hemocyanin (KLH). Mice were immunized with individual epitope-KLH conjugates or a mixture of all four, then challenged with the standard strain ATCC700721 or with multiple clinical isolates of distinct multilocus sequence types (MLST). Epitope-specific antibody responses (total IgG and IgG subclasses) and survival rates were measured. Results: Immunization with full-length recombinant FimA conferred 90% protection against lethal challenge with the standard strain ATCC700721 and induced robust IgG1-dominant antibody responses. Four novel immunodominant linear B-cell epitopes were identified: FimA97–114, FimA103–120, FimA109–126, and FimA145–160. Structural mapping revealed that the first three epitopes reside within the α-helical region, while FimA145–160 is located in the β-sheet domain. These epitopes are highly conserved, exhibiting 100% sequence identity across 36 diverse K. pneumoniae strains. Among individual epitope-KLH conjugates, FimA109–126-KLH induced the highest epitope-specific antibody titers, followed by FimA103–120-KLH. Immunization with a mixture of all four epitope-KLH conjugates elicited significant cross-protection against multiple clinical isolates, achieving survival rates of 60%, 50%, 50%, and 40% against strains 10CYZ, 13LGY, 19ZXQ, and 22CZY, respectively. Protective immunity was primarily associated with IgG1 subtype responses. Conclusions: This study provides the first fine-mapping and protective evaluation of immunodominant linear B-cell epitopes within K. pneumoniae FimA. The identification of highly conserved, functionally relevant B-cell epitopes and the demonstration of cross-protection conferred by a multi-epitope formulation underscore the potential of FimA-based epitope-driven vaccines. These findings offer a promising strategy for the development of broadly protective vaccines against K. pneumoniae infections.

ABSTRACT Staphylococcus aureus is a leading cause of severe invasive infections and is associated with high mortality. The pore‐forming α‐toxin (Hla) is a key virulence determinant that drives disease severity. We identified two high‐affinity anti‐Hla monoclonal antibodies, Hm0399 and Hm0411, derived from the sera of volunteers vaccinated with the S. aureus vaccine rFSAV. Mechanistic analyses—including toxin neutralization assays, structure solution, and effector‐function characterization—revealed that although both antibodies recognize overlapping epitopes on Hla, they confer protection through distinct and complementary mechanisms. Hm0411 potently neutralizes Hla toxicity by blocking receptor engagement through a defined salt‐bridge network, whereas Hm0399 engages a hydrogen‐bond interface that enhances Fc‐mediated effector functions. A dual‐antibody cocktail comprising Hm0399 and Hm0411 (Hm3‐4) demonstrated robust therapeutic efficacy in the USA300 S. aureus sepsis model and pneumonia model. Notably, protection was not compromised by pre‐existing immune imprinting. Transcriptomic profiling indicated enhanced Fcγ receptor–mediated phagocytosis and suppression of pro‐inflammatory Ca2+ signaling pathways. In addition, combining Hm3‐4 with ultra‐low doses of vancomycin or linezolid synergistically improved therapeutic outcomes in experimental sepsis. Collectively, these findings establish a dual‐antibody strategy targeting Hla that integrates virulence neutralization with augmentation of host immune defense, providing a framework for next‐generation immunotherapies against invasive S. aureus infection.

Hypervirulent Klebsiella pneumoniae (hvKP) variants represent urgent multidrug-resistant threats against which no licensed vaccine exists. FimA is indispensable for KP mucosal colonization and is thus a promising vaccine target; however, its protective immunodominant B-cell epitopes in vaccines with different adjuvants against KP remain undefined. Here, we revealed how adjuvants shape different hierarchies of the immunodominant B-cell epitopes of FimA and studied the humoral anti-FimA response and protective efficacy in a murine model of acute pneumonia. BALB/c mice were intramuscularly immunized with recombinant FimA formulated in three clinically trialed adjuvants: AddaVax, AddaS03, and AlPO4, whereafter they were challenged with lethal doses of the hvKP YBQ strain. Under the experimental conditions used in this immunization-challenge model, AddaS03-immunized mice exhibited 100% survival and reduced pulmonary bacterial load, whereas AddaVax-immunized mice showed only 40% survival. Furthermore, FimA-immunized groups with different adjuvants exhibited different opsonophagocytic killing activities and inflammatory cytokine levels, likely explaining the variability in protective immunity. ELISA-based linear B-cell epitope mapping of FimA with different adjuvants revealed three novel immunodominant epitopes—their hierarchies were altered by different adjuvants. Further, a causal link might exist between FimA epitope hierarchy and protective efficacy in hvKP-infected mice. Immunization with a mixture of these FimA immunodominant epitopes plus AlPO4 achieved an 80% protection rate, where it exerted a potent therapeutic effect in combination with low-dose meropenem. Our findings revealed that adjuvants enhance anti-FimA immunity and modulate FimA epitope hierarchies across different vaccinated groups. Therefore, rational adjuvant selection may elicit effective FimA-induced responses in KP vaccines, wherein different FimA immunodominant epitopes induce protective humoral immune responses.

Intracellular persistence caused by Staphylococcus aureus (S. aureus) is among the primary reasons for recurrence and difficulty in eradicating S. aureus infections. In this study, we identify the secreted protein Hla (α-hemolysin) by S. aureus as a key factor enabling its intracellular retention. We demonstrate that intracellular Hla secreted by S. aureus inhibits lysosome degradation via disrupting lysosomal function, which sustains the survival and proliferation of S. aureus within autophagosomes. Furthermore, we identify the interaction between Hla and intracellular LGALS3 (galectin 3) as crucial for sustaining intracellular survival of S. aureus, resolve the structure of the Hla-LGALS3 complex, and identify the Loop 68-75 region of Hla as the key binding domain with LGALS3. Moreover, the interaction between Hla and LGALS3 influences the recruitment of PDCD6IP/ALIX (programmed cell death 6 interacting protein) to the damaged lysosomal surface, resulting in disruption of lysosomal degradative function. Our results highlight an unknown role of Hla in the intracellular survival of S. aureus and suggest that interrupting the interaction between Hla and LGALS3 May be a potential therapeutic strategy for treating S. aureus infections.Abbreviations: 3 MA: 3-methyladenine; AECII: alveolar epithelial cells II; Agr: accessory gene regulator; ATG13: autophagy related 13; Baf A1: bafilomycin A1; BLI: biolayer interferometry; CFU: colony-forming units; ClfA: clumping factor A; Co-IP: co-immunoprecipitation; CRD: carbohydrate recognition domain; ER: endoplasmic reticulum; ESCRT: endosomal sorting complex required for transport; FnbA: fibronectin-binding protein A; FnbB: fibronectin-binding protein B; Hla: α-hemolysin; IP-MS: immunoprecipitation-mass spectrometry; LGALS3: galectin 3; LLoMe: L-leucyl-L-leucine methyl ester hydrobromide; LMP: lysosomal-membrane permeabilization; MOI: multiplicity of infection; PDCD6IP/ALIX: programmed cell death 6 interacting protein; S. aureus: Staphylococcus aureus; SPA: staphylococcal protein A; SSPA: staphylococcal surface protein A; TEM: transmission electron microscopy; TRAF3: TNF receptor associated factor 3; ULK1: unc-51 like autophagy activating kinase 1.

The past decade has witnessed the recognition of antibiotic resistance as an emerging threat to public health. Polymyxin B (PMB) is a last-resort antibiotic against multidrug-resistant (MDR) Gram-negative bacteria, but it has nephrotoxicity. A cocktail with molecules to sensitize microbes to PMB is an effective approach to rescue and expand its clinical application range. Tuspetinib (TUS) is a selective FMS-like tyrosine kinase 3 (FLT3) inhibitor with activity against acute myeloid leukemia (AML) and a favorable safety profile. In this study, we found that TUS can enhance the antibacterial activity of PMB against Klebsiella pneumoniae and its clinical PMB-resistant isolates. We also revealed the mechanism by which TUS inhibits the activity of GlcNAc6P deacetylase (NagA) and clarified the association between NagA and PMB resistance. Finally, we demonstrated the efficacy of TUS plus PMB therapy in a mouse model of pulmonary infection with a clinical PMB-resistant K. pneumoniae isolate. In all, this work discovers a promising drug combination strategy based on PMB and TUS and underlies the special mode of action that involves inhibiting the activity of NagA.

Lipid II, referred to as the bacterial "Achilles' heel" and serving as the target of vancomycin, remains inaccessible to vancomycin in Gram-negative bacteria due to their outer membrane (OM) barrier, rendering it intrinsically ineffective against these pathogens. Herein, we serendipitously discovered that a simple, single quinoline moiety modification on vancomycin yields quinovancins, which not only restore vancomycin susceptibility against acquired vancomycin-resistant strains but also unexpectedly expand its antibacterial spectrum to encompass certain Gram-negative Enterobacteriaceae (e.g., Escherichia coli, Salmonella) in vitro and in vivo. Intriguingly, substitution with a naphthalene group failed to confer such activity. Mechanistically, quinovancins overcome acquired resistance through augmented "cell wall-membrane" dual inhibitions in Gram-positive bacteria. In Gram-negative bacteria, the quinoline moiety functions as a shuttle, facilitating vancomycin's transit across the OM to the Lipid II site without compromising OM integrity─distinct from conventional OM-disrupting pathways. Besides, the best quinovancin candidate 3d exhibits a synergistic sensitization effect to potentiate meropenem, particularly against carbapenem-resistant E. coli. Furthermore, 3d demonstrates favorable druggability, including rapid and scalable synthesis, great solubility, outperformed in vivo pharmacokinetic profiles, and good safety. This work provides an unanticipated but promising strategy that enables the secondary development of vancomycin to combat bacterial multidrug resistance.

Objective Klebsiella pneumoniae(Kp)is a clinically prevalent opportunistic pathogen,with escalating antimicrobial resistance posing substantial challenges.Current vaccine strategies are constrained by the complexity of serotype diversity and the weak immunogenicity of candidate antigens,hindering effective protection.The construction of a multiepitope vaccine against K.pneumoniae,designated HBc-KpEpi,based on hepatitis B core antigen(HBc)virus-like particles,aims to overcome the weak immunogenicity of epitope peptides and to validate its ability to induce specific immune responses and protective efficacy.Methods After the multi-epitope vaccine HBc-KpEpi was expressed and purified,its purity and self-assembly status were characterized by SDS-PAGE,gel filtration chromatography,and transmission electron microscopy.Its safety was evaluated using cytotoxicity assay,hemolysis assay,and detection of serum biochemical indicators.Female BALB/c mice(6 to 8 weeks old,weighing 18 to 20 g)were randomly divided into the following groups:PBS control group,KpEpi group,and HBc-KpEpi group(n=10).Each group received intranasal immunization with PBS,KpEpi,or HBc-KpEpi(30 μg/mouse)on days 0,14,and 21,respectively.In 7 d after the final immunization,the mice were challenged with a lethal dose(6×106 CFU)or a sublethal dose(5×105 CFU)of the clinical strain YBQ via intratracheal instillation.Detection indicators included survival rate and changes in body weight,bacterial colonization in lung tissue,serum inflammatory cytokines(IL-6,TNF-α,and IL-1β),lung histopathological changes,titers of serum specific antibodies(IgG,IgG1,IgG2a),and proportions of splenic CD4 ⁺ T cell subsets(Th1,Th2,Th17).Meanwhile,the ability of the vaccine to induce the differentiation and maturation of mouse bone marrow-derived dendritic cells(BMDCs)was evaluated through in vitro experiments.Results The HBc-KpEpi protein was successfully prepared with high purity and self-assembled into regular particles.The survival rates in the HBc-KpEpi group were significantly higher than those in the PBS group(P<0.05).The bacterial colonization in lung tissue was significantly lower in the HBc-KpEpi group than that of the PBS and KpEpi groups(P<0.01).The HBc-KpEpi group exhibited significantly lower IL-6 level than the PBS group(P<0.000 1),with the levels of TNF-α and IL-1β showing downward trends.Lung histopathology showed that the alveolar structure remained relatively intact with markedly reduced inflammatory cell infiltration in the HBc-KpEpi group.After immunization,high titers of specific IgG antibodies were induced in the HBc-KpEpi group,with IgG1 as predominant subtype.Flow cytometry detection showed that the proportions of Th2 and Th17 cells in CD4 ⁺ T cells were significantly higher in the HBc-KpEpi group than in the KpEpi group(P<0.05).In vitro experiments indicated that HBc-KpEpi significantly promoted the expression of MHC-Ⅱ,CD40,CD80,and CD86 on the surface of BMDCs(P<0.000 1)Conclusion The HBc-KpEpi vaccine significantly enhances the immunogenicity of epitope peptides,inducing cellular immune responses dominated by Th2 and Th17 as well as high titers of specific antibodies,and effectively reduce bacterial load,alleviate inflammatory damage,and improve the survival rate of mice.

Antimicrobial resistance threatens global public health, urgently requiring new antibiotics. Herein, we report a novel antibacterial scaffold with a 1,2,4-oxadiazole teraryl moiety and γ-lactam fragment via bacteria-based phenotypic screening. Diversified modifications of its four sites yielded 42 derivatives, and comprehensive SAR analysis against multidrug-resistant "superbugs" identified lead compound 8c. 8c exhibits excellent activity against drug-resistant Gram-positive bacteria (e.g., MRSA, VISA, VRE), 4-32-fold more potent than ciprofloxacin and meropenem. 8c shows rapid bactericidal activity, a low propensity to induce resistance, and a moderate safety profile. Mechanistically, 8c inhibits peptidoglycan biosynthesis by targeting the Lipid II cycle and disrupts membrane homeostasis via enhanced permeability and induced hyperpolarization, with this dual action validated by proteomic and lipidomic analyses. It also has excellent liver microsomal stability, favorable in vivo pharmacokinetics, and potent in vivo anti-MRSA efficacy. This novel scaffold is a promising addition to the antibiotic arsenal and warrants further development.

Alpha hemolysin, a pore-forming toxin from Staphylococcus aureus, is a critical virulence factor for bacteria. Previous studies have demonstrated that the Hla mutant H35A (HlaH35A) serves as a potent carrier protein for subunit vaccines, yet its immunomodulatory mechanisms remain incompletely understood. Here, we demonstrate that the HlaH35A fusion enhances vaccine efficacy by targeting A Disintegrin and Metalloproteinase 10 (ADAM10) on dendritic cells (DCs), thereby activating the ADAM10-Notch signaling axis. Using the candidate antigen PA0833 from Pseudomonas aeruginosa as a model, we show that the HlaH35A-PA0833 fusion protein (HPF) significantly augments antigen uptake, DC maturation, and Notch-dependent transcriptional programs, particularly in conventional DCs (cDCs). The HlaH35A fusion drives the differentiation of Notch2-dependent cDC2s, which is marked by ESAM expression and IL-23 secretion. This process promotes Th17 and T follicular helper (Tfh) cell responses in draining lymph nodes, leading to elevated antigen-specific IgG1 titers and robust protection against acute Pseudomonas aeruginosa lung infection. Notably, ADAM10 or Notch inhibition abrogates these effects. Similarly, human monocyte-derived DCs exhibit enhanced maturation and Notch activation via the HlaH35A-ADAM10 interaction. Our findings reveal that HlaH35A is a novel carrier protein that shapes adaptive immunity by modulating cDC2 differentiation via ADAM10-Notch2 signaling, suggesting a promising strategy for Th17/Tfh-oriented vaccine design.

The escalating threat posed by multidrug-resistant bacteria underscores the urgent need for novel antibiotics. Bacitracin, with its unique undecaprenyl pyrophosphate-targeting mechanism, serves as an ideal template for structural optimization. Herein, we developed a site-selective modification strategy targeting the 7-ornithine amino group, a critical yet underexplored residue in bacitracin. This approach revitalized bacitracin's therapeutic potential against multidrug-resistant pathogens, achieving even up to 256-fold improved activity against methicillin-, vancomycin-, and daptomycin-resistant strains while systematically establishing previously unreported SAR at this locus. The lead compound, Bac-51, incorporating an optimized trifluoromethyl biphenyl moiety, demonstrated superior in vitro potency, favorable safety/pharmacokinetic profiles, and single-dose efficacy in a lethal MRSA sepsis murine model. Mechanistic studies revealed it has a dual mode of action: enhanced peptidoglycan biosynthesis inhibition and membrane-disrupting activity, which minimized resistance development and exhibited continuous efficacy against acquired bacitracin-resistant strains. Collectively, Bac-51 represents a next-generation bacitracin analog as a promising candidate for combating multidrug-resistant Gram-positive infections.

Multidrug-resistant Gram-positive superbugs pose a significant menace to global public health, urgently demanding the advent of novel antibiotics. In this study, three biphenyl sulfonium lipoglycopeptides derived from vancomycin were rationally designed and synthesized to combat such resistance. Among them, the most promising derivative, BD-V-2, exhibited outstanding in vitro activity against a diverse array of refractory strains. Notably, in two highly challenging lethal sepsis models induced by MRSA and VREm (vanA), BD-V-2 achieved complete protection of the infected mice with remarkably low single-dose administrations of merely 7 and 2.5 mg/kg, respectively, vividly demonstrating its potent in vivo efficacy. Furthermore, its in vivo pharmacokinetic profile and toxicity assessment indicated favorable druggability. Interestingly, BD-V-2 was found to impart a novel self-assembly property into micelles. In addition, independent and synergistic mechanisms of action targeting the bacterial membrane, via phosphatidylglycerol (PG) interaction, and cell wall, via two more binding sites on lipid II, respectively, interpeptide bridge and pyrophosphate motif, were elucidated. Astonishingly, BD-V-2 was capable of significantly downregulating the expression of the type VII secretion system proteins, uncovering an unprecedented antivirulence mechanism for glycopeptide antibiotics. Collectively, these findings unraveled the hitherto unknown roles of the sulfonium strategy and established BD-V-2 as a highly prospective candidate for future pharmaceutical development.

Pseudomonas aeruginosa (P. aeruginosa), a major cause of severe respiratory infections, particularly ventilator-associated pneumonia (VAP), employs various virulence mechanisms that remain incompletely characterized. In this study, we report that PA0833, an OmpA family outer membrane protein, acts as a novel virulence factor that is involved in the regulation of bacterial pathogenicity. We demonstrate that intranasal administration of recombinant PA0833 alone induces significant pulmonary inflammation in mice, characterized by alveolar disruption, neutrophil infiltration, and elevated pro-inflammatory cytokine levels in BALF, independent of live bacteria. Deletion of PA0833 attenuated virulence, reducing mortality, weight loss, lung inflammation, and tissue damage in P. aeruginosa infected mice, without affecting bacterial growth or colonization. Mechanistically, PA0833 triggered pro-inflammatory cytokine secretion in macrophages via TLR2-dependent activation of the NF-κB pathway, confirmed by TLR2 inhibition and co-immunoprecipitation. Furthermore, PA0833 deletion significantly impaired biofilm formation and reduced extracellular polysaccharide (EPS) content. This defect correlated with downregulation of key genes (mucA, mucB, algB, algD, algU) in the MucA-AlgU EPS biosynthesis pathway. Scanning electron microscopy revealed compromised cell wall integrity in the ΔPA0833 mutant. Collectively, our result indicates that PA0833 contributes to the virulence of P. aeruginosa, and targeting PA0833 offers a promising strategy for novel therapeutics against P. aeruginosa lung infections.

The rapid emergence and spread of antimicrobial resistance have become critical global health issues, leading to significant morbidity and mortality worldwide. With the increase in resistance to multiple drugs, especially frontline clinical antibiotics, there is an urgent need for novel and effective alternative strategies. Herein, we developed a vaccine targeting the antimicrobial resistance enzyme NDM-1, which was first identified in Klebsiella pneumoniae and has quickly spread to other gram-negative bacteria. Our results demonstrate that NDM-1 primarily triggers a humoral immune response and effectively protects mice from lethal Klebsiella pneumoniae infection, as evidenced by increased survival rates, reduced bacterial loads, and decreased lung inflammation in mice. The specific antibodies generated were able to inhibit the enzymatic activity of NDM-1, bacterial growth, and exhibit opsonophagocytic activity against Klebsiella pneumoniae in vitro. Both active and passive immunization with NDM-1 showed an additive effect when combined with meropenem therapy. Furthermore, NDM-1 immunization induced cross-reactivity with NDM-1 variants, potentially providing broad protection against bacteria carrying different NDM genes. Additionally, heptamerization of NDM-1 improved its immunogenicity and protective efficacy in mice. These results highlight the potential of vaccine development based on antibiotic resistance candidates for broadly combatting antimicrobial resistance.

Tissue-resident memory T cells (TRMs) are integral to localized immune defense, characterized by their capacity for long-term tissue residency and rapid effector responses in infectious diseases. Beyond their established roles in antimicrobial immunity, TRMs exhibit unique therapeutic potential in tumor immunology and vaccine design. This review summarizes the advances in TRM biology, emphasizing molecular determinants of tissue residency, functional specialization across tissue niches, and mechanistic insights into pathogen-specific immunity. We highlight the roles of TRMs in cancer and their critical function in combating infections, including viral, bacterial, and parasitic challenges. Furthermore, we critically evaluate vaccine strategies leveraging TRM biology, such as mucosal delivery, adjuvant modulation, and antigen persistence engineering, to enhance protective immunity. Addressing challenges like interspecies discrepancies in TRM longevity and exhaustion dynamics will be pivotal for translating these insights into durable vaccine platforms against infectious diseases and cancer.

Current diagnostic and preventive strategies against Staphylococcus aureus methicillin-resistant strains (MRSA) remain inadequate. Hence, we aimed to identify candidate epitopes as potential therapeutic targets and diagnostic biomarkers. We focused on clinically validated targets and investigated four antigens (Hla, SEB, MntC, and IsdB) currently incorporated into phase III clinical trials of a recombinant five-antigen vaccine (termed rFSAV) and the recently identified leukocidin LukG. Using convalescent serum samples from patients with clinically confirmed MRSA, we identified 10 immunodominant epitopes through ELISA screening of overlapping 18-mer peptides, seven of which named MntC55-72, MntC121-138, MntC271-285, SEB37-54, LukG30-47, LukG235-252, and LukG246-263 have not been previously reported. Immunoprotection trials showed that five epitopes Hla168-185, IsdB384-401, MntC55-72, SEB37-54, and LukG235-252 elicited effective protection in a BALB/c murine sepsis model infected with MRSA252. The combination of these protective epitopes exhibited broad-spectrum efficacy against both the MRSA252 strain and phylogenetically distinct clinical isolates. Diagnostically, the performance of the epitope panel was superior to that of conventional culture methods with a sensitivity of 0.839 and specificity of 0.826 in a 3-h detection window, thus offering rapid and cost-effective advantages. Notably, bioinformatic analysis showed that all identified B-cell epitopes contained predicted CD4+ T-cell epitope sequences, which suggests the potential to elicit combined T-B cell immune responses through MHC-II presentation. Thus, these immunodominant epitopes with dual functions that integrate both diagnostic and immunoprotective capabilities could function as a novel immunodiagnostic toolkit that enables rapid MRSA detection and aid in establishing a multi-epitope vaccine platform. These findings present an integrated strategy that bridges diagnostic development and vaccine design for MRSA management.

Humans, as natural carriers of Staphylococcus aureus (SA), have developed nonprotective immune imprints that can be reactivated by SA antigen vaccination and that contribute to the failure of SA vaccine trials. To test whether an epitope-focused vaccine strategy can overcome this issue, we explored the protective epitope of the notable SA antigen MntC. A surface loop of MntC (Loop101) was found to be essential for SA to absorb manganese(II) ion and survive oxidative stress. Our Loop101-deficient versus -competent MntC-based differential screening identified a Loop101-specific human monoclonal antibody (Hm0686). Hm0686 blocked SA from absorbing manganese(II) ion and exhibited a strong opsonophagocytic activity, suggesting that Hm0686-targeted Loop101 may be a protective epitope. A Loop101 epitope vaccine but not the whole MntC antigen protected against SA infection in mice with prior exposure-induced nonprotective imprints. Thus, this effective protective epitope-based vaccine strategy may be explored to overcome nonprotective immune imprints in humans.