Cholera, caused by Vibrio cholerae, remains a significant global health threat, with the O1 serotype being the predominant cause of recent global outbreaks. Although conjugate vaccines against V. cholerae have been developed via chemical methods, the feasibility of applying novel biosynthetic approaches remains uncertain due to the special structure of its O-polysaccharide, which should be recognized by oligosaccharyltransferases. Here, we successfully established an oligosaccharyltransferase PglL-based glycosylation system in an engineered V. cholerae strain to biosynthesize a nanoscale conjugate vaccine, NP-OPSVc(I). The carrier NP was designed by fusing the cholera toxin B subunit (CTB) with a trimer-forming protein (Tri), which could self‑assemble into a nanoparticle during expression. The resulting glycoprotein exhibited high purity, stability, and retained GM1-binding capacity. We found that the size of NP-OPSVc(I) and its GM1 receptor targeting both contribute to a potent humoral immune response, while its efficient delivery is additionally associated with the induction of intestinal mucosal immunity. In mouse models, the NP-OPSVc(I) demonstrated a favorable safety profile and elicited substantially stronger systemic and mucosal immune responses than conventional conjugates, providing great protection against lethal V. cholerae challenge even at a 10‑fold lower dose without requiring an adjuvant. Furthermore, the immune responses were confirmed in non-human primates, supporting its potential for clinical translation. This work not only developed a novel highly effective candidate vaccine against V. cholerae but also significantly expanded the application scope of the PglL-based platform technology, establishing it as a versatile and broadly applicable enzymatic toolkit for vaccine development.

Klebsiella pneumoniae poses a severe global health threat due to its extensive antibiotic resistance. However, to date, no vaccine against this pathogen has been approved for clinical use worldwide. Although self-assembling nanocarriers present distinct advantages for vaccine design, their ability to effectively load polysaccharide antigens and further elicit mucosal immunity remains unclear. Here, we developed a modular, self-assembling nanovaccine (CNP-OPSKpO1) against K. pneumoniae by loading of K. pneumoniae O1 polysaccharide antigen onto a cholera toxin B subunit (CTB)-based nanoparticle (CNP). After determining the safety of the vaccine via intranasal immunization, we further evaluated its immune efficacy. CNP-OPSKpO1 elicited stronger systemic IgG and mucosal sIgA responses than non-nanoparticulate controls. In a non-lethal pulmonary infection model, CNP-OPSKpO1 vaccination reduced lung bacterial burden by over 5 logs compared to controls, achieving near-complete bacterial clearance. Histopathological analysis further confirmed minimal lung damage in vaccinated animals. In addition, in a lethal pulmonary challenge model, it conferred 90% survival, whereas all mice in the antigen-alone control group died within 4 days. Our work not only provides a safe, effective, and adjuvant-free candidate vaccine against K. pneumoniae but also advances a versatile platform for developing broad-spectrum mucosal vaccines against other pathogens.

Neonatal extraintestinal pathogenic Escherichia coli (ExPEC), which can cause severe long-term sequelae by systemic infections, is gradually becoming the primary pathogen threatening neonatal health. The lack of large-scale genomic epidemiological investigation hinders further understanding of neonatal ExPEC. We conducted this nationwide multicenter study to support further strategies for improving neonatal ExPEC management. The neonatal ExPEC strains and clinical information, including antimicrobial resistance phenotype, were collected from nine centers within 7 provinces across China between 2018 and 2023. Whole-genome sequencing was performed. Sequence types (ST) and serotypes were acquired to characterize the strains. Phylogenetic analysis and pan-genomic analysis were conducted to identify the population structure. Bioinformatics analysis associated with virulence factors, antimicrobial resistance genes, and mobile genetic elements were conducted. To characterize the situation of horizontal gene transfer, we developed a computational tool for identifying horizontal evolutionary patterns from large-scale genomic draft assemblies. Co-occurrence and co-localization metrics were used to describe the synergistic effects and transmission mechanism of genes. A total of 411 neonatal ExPEC strains were included. ST1193 (18·0

A thorough understanding of the functions of virulence regulators in Bacillus anthracis evolution and host adaptation, particularly the systematic host responses they trigger, requires an efficient infection model capable of resolving subtle mechanisms. This study constructed a high-resolution host immune response decoder based on Galleria mellonella to analyze the specific response profiles elicited by different virulence regulators in a capsule-deficient background. By integrating transcriptomic, histopathological, and bacterial colonization analyses, the research delineated distinct host immune stress profiles regulated by AtxA and PlcR. The results showed that the AtxA-deficient strain failed to elicit significant host responses; wild-type infection activated broad systemic immune recognition pathways, while the PlcR-activated strain induced a unique response profile characterized by perturbations in oxidative stress pathways. Its enhanced virulence was associated with the expression of downstream hydrolases and occurred without strong systemic immune activation. This work successfully advanced the G. mellonella model from a phenotypic screener to a mechanistic resolver, providing a new methodological framework for distinguishing B. anthracis virulence regulatory mechanisms at the host response level. This approach not only deciphers pathogen-specific immune signatures but also offers a practical platform for rapid anti-virulence compound screening and guides the design of targeted validation in mammalian systems, thereby accelerating therapeutic strategy development against anthrax.

Background Bacillus anthracis forms dormant spores that constitute the primary infectious agent of anthrax. BA_3317, a membrane protein harboring a quorum-sensing (QS)-related AgrB domain, is essential for sporulation in B. anthracis . Methods We constructed an in-frame deletion mutant of BA_3317 in B. anthracis vaccine strain A16R. Sporulation efficiency was quantified, and mutant morphology was observed via confocal microscopy. To investigate the role of BA_3317 in spore germination, we performed secretion exchange experiments between A16R and ΔBA_3317 at T 0.5 , analyzed the transcriptional activity of spoIIE , and determined lecithinase activity after activating the plcR-papR QS system. Additionally, we identified the upstream regulators of BA_3317 using in vitro promoter pull-down assays. Results Deletion of BA_3317 severely reduced sporulation efficiency and ΔBA_3317 mutant was partially arrested at the asymmetric cell division stage. Cells secretion exchange experiments and spatial reporter assays revealed that BA_3317 exports a signal molecule required for sporulation, and its loss downregulated key sporulation gene spoIIE . The mutant also lacked lecithinase activity via the ectopically activated the plcR-papR QS system, which was restored by adding the PapR heptapeptide, indicating BA_3317 mediates peptide signal transport. BA_3317 expression is repressed by SpoVG prior to asymmetric division and positively regulated by GerE during late sporulation. Conclusion Our findings identify BA_3317 as a critical regulator of B. anthracis sporulation that functions as an exporter of sporulation signaling molecules. This study advances understanding of species-specific sporulation mechanisms in B. anthracis and provides a potential target for anthrax prevention and control.

Bacillus anthracis displays susceptibility to penicillin despite harboring a β-lactamase gene, a phenotype governed by the anti-sigma factor RsiP. While RsiP represses σP-dependent β-lactamase expression, its broader roles in physiology and virulence remain unclear. This study aimed to define the global regulatory functions of RsiP beyond antibiotic resistance. Deletion of rsiP significantly upregulated the nprR gene, which is an important quorum-sensing (QS) system regulator and enhanced protease secretion. The ΔrsiP mutant caused higher mortality in cellular and Galleria mellonella models and triggered elevated inflammatory cytokines (IL-6, IL-1β, TNF-α, MIP-2) in macrophages models. Surprisingly, in DBA/2 mice models, ΔrsiP was attenuated, with increased host survival and reduced bacterial loads. Competitive indices (CI) confirmed fitness defects in mice (spleen CI = 0.39; liver CI = 0.42). These defects were not due to altered oxidative stress tolerance but were attributed to impaired macrophage internalization of ΔrsiP spores, reducing early colonization. Our findings indicate that RsiP not only modulates β-lactam resistance but also influences extracellular protease activity and host adaptation.

Background: Extra-intestinal pathogenic Escherichia coli (ExPEC) represents a major global public health challenge due to its ability to cause diverse clinical infections, including urinary tract infections, bacteremia, neonatal meningitis, and sepsis. The growing prevalence of multidrug-resistant (MDR) ExPEC strains, which rapidly erode antibiotic efficacy, underscores vaccine development as a critical priority. Bioconjugate vaccines have emerged as a promising approach to mitigate ExPEC-associated infections. Methods and Results: In this study, we utilized protein glycan coupling technology (PGCT) based on oligosaccharyltransferase (OST) PglL to engineer a tetravalent bioconjugate vaccine targeting four predominant ExPEC serotypes (O1, O2, O6, and O25). We conducted a series of experiments to demonstrate the efficacy of the conjugate vaccine in eliciting humoral immune responses and inducing the production of specific antibodies against Escherichia coli O1, O2, O6, or O25 serotypes. Conclusions: This work establishes the first application of the O-linked PGCT system for engineering bioconjugate vaccines against ExPEC infections.

Escherichia coli and Klebsiella pneumoniae are major contributors to the global challenge of antimicrobial resistance, posing serious threats to public health. Among current preventive strategies, conjugate vaccines that utilize bacterial surface polysaccharides have emerged as a promising and effective approach to counter multidrug-resistant strains. In this study, both the Wzy/Wzx-dependent and ABC transporter-dependent biosynthetic pathways for antigenic polysaccharides were introduced into E. coli W3110 cells. This dual-pathway engineering enabled the simultaneous biosynthesis of two structurally distinct polysaccharides within a single host, offering a streamlined and potentially scalable strategy for vaccine development. Experimental findings confirmed that both polysaccharide types were successfully produced in the engineered strains, although co-expression levels were moderately reduced. A weak competitive interaction was noted during the initial phase of induction, which may be attributed to competition for membrane space or the shared use of activated monosaccharide precursors. Interestingly, despite a reduction in plasmid copy number and transcriptional activity of the biosynthetic gene clusters over time, the overall polysaccharide yield remained stable with prolonged induction. This suggests that extended induction does not adversely affect final product output. Additionally, two glycoproteins were efficiently generated through in vivo bioconjugation of the synthesized polysaccharides with carrier proteins, all within the same cellular environment. This one-cell production system simplifies the workflow and enhances the feasibility of generating complex glycoprotein vaccines. Whole-cell proteomic profiling followed by MFUZZ clustering and Gene Ontology analysis revealed that core biosynthetic genes were grouped into two functional clusters. These genes were predominantly localized to the cytoplasm and were enriched in pathways related to translation and protein binding. Such insights not only validate the engineered biosynthetic routes but also provide a molecular basis for optimizing future constructs. Collectively, this study presents a robust synthetic biology platform for the co-expression of multiple polysaccharides in a single bacterial host. The approach holds significant promise for the rational design and production of multivalent conjugate vaccines targeting drug-resistant pathogens.

Bacillus anthracis (B. anthracis), Yersinia pestis (Y. pestis), and Brucella spp. are zoonotic bacteria that cause anthrax, plague, and brucellosis, respectively. Outbreaks typically occur in remote regions with poor transportation and limited laboratory testing. Therefore, a simple, sensitive, multiplex nucleic acid detection method is essential for effective disease management and control. Primers and probes for the three pathogens were designed to reduce interference from related strains. Three recombinase polymerase amplification (RPA) reactions were conducted at 39 °C for 10 min to produce species-specific fluorescence signals for the three pathogens. These were integrated, and conditions were optimized for rapid, sensitive triplex-RPA assays without cross-reactivity. A triplex-RPA reaction with lateral flow dipsticks (LFDs) was developed and applied to blood samples, newly isolated strains, and simulated samples. Highly sensitive and specific primers and probes were developed, achieving a maximum sensitivity of 1 copy/µL in single-reaction RPA. The optimized triplex RPA detection technique, combined with fluorescence, effectively identified B. anthracis, Y. pestis, and Brucella spp. within 20 min, whereas LFDs achieved detection in 10 min. The assay also performed comparably to conventional polymerase chain reaction techniques when tested on blood samples, newly isolated strains, and simulated samples. This study offers reliable methods for detecting B. anthracis, Y. pestis, and Brucella spp. in rural hospitals and public health initiatives.

Multidrug-resistant Klebsiella pneumoniae constitutes a significant threat as a nosocomial pathogen, and no licensed vaccines are currently available. Generalized modules for membrane antigens (GMMA) have recently been recognized as a promising platform for developing outer membrane vesicle (OMV) vaccines against numerous infectious diseases. The study was carried out in use of the W3110 ΔwbbH-L ΔlpxM::lpxE in which E. coli was treated in order to eliminate the endogenous polysaccharide and use two new ones (polysaccharides from Klebsiella). The exogenous polysaccharides were accurately displayed on the surface of spontaneously released OMVs. The immune responses evoked by subcutaneous administration of these vaccines were evaluated, and the protective effects were assessed using a mouse intraperitoneal challenge model. Interference in the biosynthesis of endogenous polysaccharides (such as deleting related gene clusters) is a viable approach to increasing the yield of glycoengineered GMMA vaccines (geGMMA). The geGMMA platform, which is conducive to safer large-scale production, lays the foundations for the development of GMMA vaccines decorated with exogenous glycan antigens derived from pathogenic bacteria.

Brucellosis, a global zoonosis caused by Brucella species, currently lacks safe vaccines for human use, while existing veterinary live-attenuated vaccines pose infection risks. Although ferritin nanoparticle materials have shown significant advantages in delivery, there is not much research on loading complex polysaccharide antigens. Here, we engineered a bioconjugate nanovaccine (Fn-OPS) through a bacterial glycosylation-driven synthetic strategy, which couples Yersinia enterocolitica O:9 (YeO9) O-specific polysaccharide (OPS)─a structural analogue of Brucella antigens─to a self-assembled ferritin nanoparticle (Fn). This nanocarrier platform combines the symmetrical architecture and antigen-presenting advantages of Fn with the precision of enzymatic glycosylation, addressing the limitations of complex chemical synthesis methods. After confirming the presence of monodisperse nanoparticles with stability at room temperature and great in vivo safety, we performed murine immunization studies and demonstrated a robust activation of T follicular helper (Tfh) cells and germinal center B cells, leading to the production of high-titer IgG antibodies that are cross-reactive with Brucella lipopolysaccharide. This immune response provides strong protection against Brucella infection. This work establishes a ferritin-based nanoconjugate platform for targeted delivery of complex polysaccharide antigens. It advances a scalable, biosafe strategy for Brucella vaccines, expanding the toolkit for targeted antigen delivery in conjugate vaccine design and broadening applications in infectious disease prophylaxis.

Tetrodotoxin (TTX) is a potent neurotoxin found in nature, the possible presence of TTX in pufferfish ovaries, liver, and other processing waste poses a serious environmental and safety hazard. Lactic acid bacteria (LAB) was found to be able to remove a variety of harmful substances, however, its elimination action and mechanism on TTX is not clear. In this study, the removal effects of different states (activated, inactivated, and fragmented LABs) of three LAB strains on TTX amount and toxicity were tested and compared. It was found that thermal inactivated LABs had the best effect, which could reduce the TTX amount by 49.0%–60.33% and decrease the toxicity of TTX by 57.0%–83.67%. In addition, peptidoglycan (PG) of three strains of LABs was found to be a key component in the removal of TTX, which could decrease the toxicity of TTX by more than 87.0 %. By analyzing the changes of functional groups of PGs, zeta-potential and binding effect between PG and TTX before and after the masking of carboxyl or amino groups of PGs, it was found that the removal effect of PG to TTX was significantly decreased (>55.0%) when the carboxyl or amino groups of PGs were masked. At the same time, the zeta-potential of PG combined with TTX was also decreased. These results suggested that the amino and carboxyl groups of PGs were important action sites for the binding interaction between LAB and TTX, and electrostatic interaction might be one of the ways of binding between PG and TTX. Our study could provide a scientific supporting on the removal action mechanism research of PG to TTX.

Modular self-assembling nanoparticle vaccines, represent a cutting-edge approach in immunology with the potential to revolutionize vaccine design and efficacy. Although many innovative efficient modular self-assembling nanoparticles have been designed for vaccination, the immune activation characteristics underlying such strong protection remain poorly understood, limiting the further expansion of such nanocarrier. Here, we prepared a novel modular nanovaccine, which self-assembled via a pentamer cholera toxin B subunit (CTB) domain and an unnatural trimer domain, presenting S. Paratyphi A O-polysaccharide antigen, and investigated its rapid immune activation mechanism. The nanovaccine efficiently targets draining lymph nodes and antigen-presenting cells, facilitating co-localization with Golgi and endoplasmic reticulum. In addition, dendritic cells, macrophages, B cells, and neutrophils potentially participate in antigen presentation, unveiling a dynamic change of the vaccines in lymph nodes. Single-cell RNA sequencing at early stage and iN vivo/iN vitro experiments reveal its potent humoral immune response capabilities and protection effects. This nanoparticle outperforms traditional CTB carriers in eliciting robust prophylactic effects in various infection models. This work not only provides a promising and efficient candidate vaccine, but also promotes the design and application of the new type of self-assembled nanoparticle, offering a safe and promising vaccination strategy for infection diseases.

The antimicrobial-resistant (AMR) Klebsiella pneumoniae (Kp) poses an enormous threat to human health, with O2 serotypes accounting for up to 35-59% of infections. Although the O-polysaccharide (OPS) of the Kp O2 serotype can be used as an antigen target for vaccine preparation, its simple structure (only galactose repeats) makes it difficult to generate effective antibody responses and protection. Here, we prepared a novel Kp O2 OPS bioconjugate nanovaccine using protein glycan coupling technology (PGCT) and a SpyCatcher/SpyTag (SC/ST) orthogonal assembly system. The hepatitis B virus core antigen (HBc), which can assemble into nanoparticles, was used as a carrier to display OPS on its surface, allowing the bioconjugate to reach the nanoscale. The HBc-OPS exhibited attractive stability without aggregation or degradation for up to 10 months. A series of mouse experiments revealed the OPS-specific antibody activation ability of HBc-OPS and its protective effect against different infection doses. In particular, when coadministered with the AS03 adjuvant, all the mice were protected from higher doses of lethal attacks. Through in vitro and in vivo experiments, we found that the addition of AS03 further promoted the humoral immune response by stimulating increased levels of cytokines and T follicular helper (Tfh), germinal center B (GC B), and antigen-specific memory B cells. Moreover, we found that the use of AS03 as an adjuvant can provide a better protective effect than commonly used CpG-based adjuvants. Therefore, we have developed an attractive, stable, and effective bioconjugate nanovaccine against the Klebsiella pneumoniae O2 serotype. This bioconjugate nanovaccine design greatly potentiated the immunogenicity of polysaccharides, and the orthogonal modular assembly strategy reduced the technical difficulty of bioconjugate nanovaccine preparation, both of which could be applicable to the development of OPS conjugate vaccines for serotypes with low immunogenicity.

Human brucellosis caused by Brucella is a widespread zoonosis that is prevalent in many countries globally. The high homology between members of the Brucella genus and Ochrobactrum spp. often complicates the determination of disease etiology in patients. The efficient and reliable identification and distinction of Brucella are of primary interest for both medical surveillance and outbreak purposes. A large amount of genomic data for the Brucella genus was analyzed to uncover novel probes containing single-nucleotide polymorphisms (SNPs). GAMOSCE v1.0 software was developed based on the above novel eProbes. In conjunction with clinical requirements, an RPA-Cas12a detection method was developed for the on-site determination of B. abortus and B. melitensis by fluorescence and lateral flow dipsticks (LFDs). We demonstrated the potential of these probes for rapid and accurate detection of the Brucella genus and five significant Brucella species in silico using GAMOSCE. GAMOSCE was validated on different Brucella datasets and correctly identified all Brucella strains, demonstrating a strong discrimination ability. The RPA-Cas12a detection method showed good performance in detection in clinical blood samples and veterinary isolates. We provide both in silico and on-site methods that are convenient and reliable for use in local hospitals and public health programs for the detection of brucellosis.

The Toll-like receptor 9 (TLR9) stimulator, CpG oligodeoxynucleotide, has emerged as a potent enhancer of protein subunit vaccines. Incorporating the protein antigen directly with the CpG adjuvant presents a novel strategy to significantly reduce the required dosage of CpG compared to traditional methods that use separate components. In contrast to existing chemical conjugation methods, this study introduces an enzymatic approach for antigen-adjuvant coupling using a recombinant endonuclease DCV fused with SpyTag. This fusion protein catalyzes the covalent linkage between itself and the CpG adjuvant under mild conditions. These conjugates can be further linked with target protein antigens containing the SpyCatcher sequence, yielding stable, covalently-linked antigen-adjuvant complexes. The corresponding complex utilizing the receptor-binding domain (RBD) of SARS-CoV-2 spike protein as the model antigen, elicits high-titer, specific antibody production in mice via both subcutaneous administration and intratracheal inoculation. Notably, the tumor vaccine candidate fabricated by this method has also shown significant inhibition of cancer progression after intratracheal administration. The technique ensures precise, site-specific coupling and preserves the antigen's structural integrity due to the post-purification coupling strategy that simplifies manufacturing and aids in developing inhalable vaccines. In this study, an enzymatic method is introduced that employs a recombinant endonuclease DCV fused with SpyTag to conjugate target protein antigens containing the SpyCatcher sequence with the CpG adjuvant under mild conditions. The target covalently-linked antigen-adjuvant complexes could induce strong and specific antibody production when administered via subcutaneous and intratracheal routes. image

In this study, the pyrroloquinoline quinone (PQQ) high-yield mutant J1-1 of Methylobacterium glucophilum MP688 was used to do bioinformatics analysis to extract the two methanol dehydrogenase (MDH) genes from the genome, mpq0771 and mpq2496. The effects of Ca2+ and La3+ on bacterial growth, PQQ synthesis, MDH expression, and enzyme activity were explored, as well as the roles of the two enzymes in methanol metabolism. The results showed that Ca2+ and La3+ both increased J1-1 bacteriophage growth and decreased PQQ synthesis in the context of fast viral development. In the presence of Ca2+, mpq0771 expression increased in strain J1-1. La3+ inhibited mpq0771 expression while promoting mpq2496 expression and increasing total bacterial MDH viability. The knockout bacteria J1-1Δmpq0771 could not grow in methanol, but with the addition of La3+, it could grow in methanol and synthesize PQQ, and MDH activity was identified. The product encoded by mpq0771 was a Ca2+-dependent MDH, and the product encoded by mpq2496 was a La3+-dependent MDH, and La3+ could increase the production of mpq2496 and replace mpq0771 as a methanol dehydrogenase.

Nanoparticles (NPs) have been widely utilized in vaccine design. Although numerous NPs have been explored, NPs with adjuvant effects on their own have rarely been reported. We produce a promising self-assembled NP by integrating the pentameric Escherichia coli heat-labile enterotoxin B subunit (LTB) (studied as a vaccine adjuvant) with a trimer-forming peptide. This fusion protein can self-assemble into the NP during expression, and polysaccharide antigens (OPS) are then loaded in vivo using glycosylation. We initially produced two Salmonella paratyphi A conjugate nanovaccines using two LTB subfamilies (LTIB and LTIIbB). After confirming their biosafety in mice, the data showed that both nanovaccines (NP(LTIB)-OPSSPA and NP(LTIIbB)-OPSSPA) elicited strong polysaccharide-specific antibody responses, and NP(LTIB)-OPS resulted in better protection. Furthermore, polysaccharides derived from Shigella or Klebsiella pneumoniae were loaded onto NP(LTIB) and NP(LTIIbB). The animal experimental results indicated that LTIB, as a pentamer module, exhibited excellent protection against lethal infections. This effect was also consistent with that of the reported cholera toxin B subunit (CTB) modular NP in all three models. For the first time, we prepared a novel promising self-assembled NP based on LTIB. In summary, these results indicated that the LTB-based nanocarriers have the potential for broad applications, further expanding the library of self-assembled nanocarriers.

Nanoparticles (NPs) have been surfacing as a pivotal platform for vaccine development. In our previous work, we developed a cholera toxin B subunit (CTB)-based self-assembled nanoparticle (CNP) and produced highly promising bioconjugate nanovaccines by loading bacterial polysaccharide (OPS) in vivo. In particular, the Klebsiella pneumoniae O2 serotype vaccine showcased a potent immune response and protection against infection. However, extremely low yields limited its further application. In this study, we prepared an efficient Klebsiella pneumoniae bioconjugate nanovaccine in Escherichia coli with a very high yield. By modifying the 33rd glycine (G) in the CNP to aspartate (D), we were able to observe a dramatically increased expression of glycoprotein. Subsequently, through a series of mutations, we determined that G33D was essential to increasing production. In addition, this increase only occurred in engineered E. coli but not in the natural host K. pneumoniae strain 355 (Kp355) expressing OPSKpO2. Next, T-cell epitopes were fused at the end of the CNP(G33D), and animal experiments showed that fusion of the M51 peptide induced high antibody titers, consistent with the levels of the original nanovaccine, CNP-OPSKpO2. Hence, we provide an effective approach for the high-yield production of K. pneumoniae bioconjugate nanovaccines and guidance for uncovering glycosylation mechanisms and refining glycosylation systems.