Bacterial infections pose a significant global health threat, exacerbated by antimicrobial resistance. The CDC estimated 3 million antimicrobial-resistant infections in the USA annually in 2019, with resistance rising globally. New strategies, including vaccination, are urgently needed. While vaccines exist for pathogens like *Clostridium tetani*, *Bordetella pertussis*, and *Streptococcus pneumoniae*, there are no approved vaccines against many deadly pathogens, notably *Staphylococcus aureus* (*S. aureus*), responsible for numerous infections and deaths. The prevalence of methicillin-resistant *S. aureus* (MRSA) further underscores this need. A key challenge in vaccine development is antigen selection. Many pathogens, including *S. aureus*, possess poly-β-(1→6)-N-acetylglucosamine (PNAG) in their cell walls. PNAG is a virulence factor aiding immune evasion, but its structural heterogeneity (varying degrees and positions of free amines versus N-acetamides) complicates vaccine design. Previous studies showed that PNAG pentasaccharides and nonasaccharides, with either all amines free or fully acetylated, conjugated to tetanus toxoid (TT), induced varying levels of protective immunity. However, the optimal PNAG structure for maximal protection remains unknown. This study aimed to synthesize a comprehensive library of PNAG pentasaccharides with systematically varied acetylation patterns to identify the most effective vaccine epitopes.

Several studies have explored the immunological properties of PNAG. Previous research synthesized PNAG pentasaccharides and nonasaccharides with fully acetylated or fully deacetylated amine groups, conjugating them with an immunogenic protein carrier, tetanus toxoid (TT). Immunization studies demonstrated that TT conjugates with PNAG bearing all amines induced protective immunity, whereas those with fully acetylated PNAG did not. Despite this, previous PNAG-based vaccines only included glycans with either all amines free or fully acetylated. The optimal PNAG structure and the effect of specific amine/acetylation patterns on vaccine efficacy remained unclear. This lack of structurally defined PNAG sequences highlighted the need for a comprehensive library to elucidate the impact of varied acetylation patterns on epitope recognition and vaccine efficacy.

This study employed a divergent synthetic strategy to create a library of 32 PNAG pentasaccharides encompassing all possible combinations of free amine and N-acetylation locations. The synthesis began with thioglycoside **3**, which was glycosylated with 3-azido-1-propanol **4** to yield compound **5**. After Alloc group removal and N-acetylation, azide reduction and amidation produced compound **6**. Two key linchpin pentasaccharide intermediates (**1** and **2**) were synthesized, bearing different orthogonal protective groups (Boc, Alloc, Troc, and Fmoc) on glucosamine units A, B, C, and D, with the reducing end (E) either N-acetylated or N-trifluoroacetylated. Orthogonal deprotection and acetylation steps allowed for the divergent synthesis of the 32 PNAG pentasaccharides. These were then conjugated to mutant bacteriophage Qβ (mQβ) as a carrier protein for vaccination using sulfhydryl chemistry. A conjugate with tetanus toxoid heavy chain (TTHC) was also produced for comparison. Immunogenicity was evaluated by immunizing mice (C57BL/6 and CD1) and rabbits with the mQβ-PNAG conjugates. Antibody titers were measured by ELISA. Epitope specificity was analyzed using a glycan microarray with an anti-PNAG monoclonal antibody (mAb F598). The protective efficacy of the vaccines was assessed in mouse bacteremia challenge models with *S. aureus* ATCC29213 and MRSA strain 1058. Active immunization involved multiple subcutaneous injections of the vaccine. A passive protection model involved transferring rabbit antisera to mice. Complement deposition and opsonophagocytic killing (OPK) assays evaluated the functional activity of the elicited antibodies. Finally, the impact of vaccination on the gut microbiome was evaluated using 16S rRNA sequencing.

The researchers successfully synthesized a comprehensive library of 32 PNAG pentasaccharides with all possible amine/acetylation patterns. Analysis with mAb F598 revealed that the presence of N-acetylglucosamine (GlcNAc) at position B, and additional GlcNAc at position D, was crucial for optimal antibody binding. This guided the selection of PNAG10 and PNAG26 for vaccine development. Comparative immunogenicity studies showed that the mQβ-PNAG conjugates elicited significantly higher IgG antibody titers against the immunizing antigens (PNAG10 and PNAG26) in mice and rabbits compared to the TTHC-PNAG conjugate. Furthermore, these antibodies showed superior complement deposition and opsonic killing activity *in vitro*. Active immunization with mQβ-PNAG conjugates offered near-complete protection against lethal *S. aureus* challenges in mice, significantly better than the TTHC-PNAG conjugate. The passive immunization model, using diluted rabbit antisera, also demonstrated significant protection against *S. aureus* and MRSA challenges. Importantly, the vaccines were biocompatible, showing no significant adverse effects, and did not significantly alter the gut microbiome.

This study successfully identified PNAG structures that are more immunogenic than previously studied PNAG epitopes leading to the development of improved vaccines. The comprehensive PNAG library allowed for a detailed analysis of epitope specificity, revealing the importance of GlcNAc at specific positions. The use of mQβ as a carrier resulted in superior antibody responses and protective immunity compared to traditional tetanus toxoid. The strong protection observed in both active and passive immunization models confirms the efficacy of the newly designed vaccine. The finding that the vaccines do not significantly disrupt the gut microbiome suggests a good safety profile. These results highlight the potential of this approach for developing next-generation vaccines against *S. aureus*, including drug-resistant strains.

This research demonstrates a powerful strategy for designing effective vaccines against *Staphylococcus aureus*, particularly MRSA. The creation of a comprehensive PNAG pentasaccharide library, coupled with the use of mQβ as a carrier, led to the identification of highly immunogenic and protective epitopes. The resulting vaccines showed exceptional efficacy in both active and passive protection models, while also demonstrating excellent biocompatibility and minimal impact on the gut microbiome. Future research could focus on clinical trials to further validate the efficacy and safety of these vaccines and explore their potential against a wider range of bacterial infections.

While this study demonstrates significant promise, several limitations should be considered. The mouse models, while valuable, might not fully recapitulate human immune responses. The study focused primarily on *S. aureus* ATCC29213 and a specific MRSA strain; further testing is needed to assess efficacy against a broader range of *S. aureus* strains and other PNAG-producing pathogens. The long-term duration of protection afforded by these vaccines still needs to be established. Additional studies examining potential side effects beyond those observed in the current investigation are needed before widespread clinical applications.