Influence of glycan structure on the colonization of Streptococcus pneumoniae on human respiratory epithelial cells.

Virtually all living cells are encased in glycans. They perform key cellular functions such as immunomodulation and cell-cell recognition. Yet, how their composition and configuration affect their functions remains enigmatic. Here, we constructed isogenic capsule-switch mutants harboring 84 types of capsular polysaccharides (CPSs) in Streptococcus pneumoniae. This collection enables us to systematically measure the affinity of structurally related CPSs to primary human nasal and bronchial epithelial cells. Contrary to the paradigm, the surface charge does not appreciably affect epithelial cell binding. Factors that affect adhesion to respiratory cells include the number of rhamnose residues and the presence of human-like glycomotifs in CPS. Besides, pneumococcal colonization stimulated the production of interleukin 6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), and monocyte chemoattractantprotein-1 (MCP-1) in nasal epithelial cells, which also appears to be dependent on the serotype. Together, our results reveal glycomotifs of surface polysaccharides that are likely to be important for colonization and survival in the human airway.

Assembly of Bacterial Capsular Polysaccharides and Exopolysaccharides.

Polysaccharides are dominant features of most bacterial surfaces and are displayed in different formats. Many bacteria produce abundant long-chain capsular polysaccharides, which can maintain a strong association and form a capsule structure enveloping the cell and/or take the form of exopolysaccharides that are mostly secreted into the immediate environment. These polymers afford the producing bacteria protection from a wide range of physical, chemical, and biological stresses, support biofilms, and play critical roles in interactions between bacteria and their immediate environments. Their biological and physical properties also drive a variety of industrial and biomedical applications. Despite the immense variation in capsular polysaccharide and exopolysaccharide structures, patterns are evident in strategies used for their assembly and export. This review describes recent advances in understanding those strategies, based on a wealth of biochemical investigations of select prototypes, supported by complementary insight from expanding structural biology initiatives. This provides a framework to identify and distinguish new systems emanating from genomic studies.

The GE296_RS03820 and GE296_RS03830 genes are involved in capsular polysaccharide biosynthesis in Riemerella anatipestifer.

Riemerella anatipestifer is a pathogenic bacterium that causes duck serositis and meningitis, leading to significant harm to the duck industry. To escape from the host immune system, the meningitis-causing bacteria must survive and multiply in the bloodstream, relying on specific virulence factors such as capsules. Therefore, it is essential to study the genes involved in capsule biosynthesis in R. anatipestifer. In this study, we successfully constructed gene deletion mutants Δ3820 and Δ3830, targeting the GE296_RS03820 and GE296_RS03830 genes, respectively, using the RA-LZ01 strain as the parental strain. The growth kinetics analysis revealed that these two genes contribute to bacterial growth. Transmission and scanning electron microscopy (TEM and SEM) and silver staining showed that Δ3820 and Δ3830 produced the altered capsules and compounds of capsular polysaccharides (CPSs). Serum resistance test showed the mutants also exhibited reduced C3b deposition and decreased resistance serum killing. In vivo, Δ3820 and Δ3830 exhibited markedly declining capacity to cross the blood-brain barrier, compared to RA-LZ01. These findings indicate that the GE296_RS03820 and GE296_RS03830 genes are involved in CPSs biosynthesis and play a key role in the pathogenicity of R. anatipestifer. Furthermore, Δ3820 and Δ3830 mutants presented a tendency toward higher survival rates from RA-LZ01 challenge in vivo. Additionally, sera from ducklings immunized with the mutants showed cross-immunoreactivity with different serotypes of R. anatipestifer, including 1, 2, 7 and 10. Western blot and SDS-PAGE assays revealed that the altered CPSs of Δ3820 and Δ3830 resulted in the exposure of some conserved proteins playing the key role in the cross-immunoreactivity. Our study clearly demonstrated that the GE296_RS03820 and GE296_RS03830 genes are involved in CPS biosynthesis in R. anatipestifer and the capsule is a target for attenuation in vaccine development.

Heptavalent O-Antigen Bioconjugate Vaccine Exhibiting Differential Functional Antibody Responses Against Diverse Klebsiella pneumoniae Isolates.

Klebsiella pneumoniae is the leading cause of neonatal sepsis and is increasingly difficult to treat owing to antibiotic resistance. Vaccination represents a tractable approach to combat this resistant bacterium; however, there is currently not a licensed vaccine. Surface polysaccharides, including O-antigens of lipopolysaccharide, have long been attractive candidates for vaccine inclusion. Herein we describe the generation of a bioconjugate vaccine targeting 7 predominant O-antigen subtypes in K. pneumoniae. Each bioconjugate was immunogenic in isolation, with limited cross-reactivity among subtypes. Vaccine-induced antibodies demonstrated varying degrees of binding to a wide variety of K. pneumoniae strains. Furthermore, serum from vaccinated mice induced complement-mediated killing of many of these strains. Finally, increased capsule interfered with the ability of O-antigen antibodies to bind and mediate killing of some K. pneumoniae strains. Taken together, these data indicate that this novel heptavalent O-antigen bioconjugate vaccine formulation exhibits limited efficacy against some, but not all, K. pneumoniae isolates.

Trapping and retaining intermediates in glycosyltransferases.

Glycosyltransferases (GTs) attach sugar molecules to a broad range of acceptors, generating a remarkable amount of structural diversity in biological systems. GTs are classified as either "retaining" or "inverting" enzymes. Most retaining GTs typically use an SNi mechanism. In a recent article in the JBC, Doyle et al. demonstrate a covalent intermediate in the dual-module KpsC GT (GT107) supporting a double displacement mechanism.

Mechanism and linkage specificities of the dual retaining ß-Kdo glycosyltransferase modules of KpsC from bacterial capsule biosynthesis.

KpsC is a dual-module glycosyltransferase (GT) essential for "group 2" capsular polysaccharide biosynthesis in Escherichia coli and other Gram-negative pathogens. Capsules are vital virulence determinants in high-profile pathogens, making KpsC a viable target for intervention with small-molecule therapeutic inhibitors. Inhibitor development can be facilitated by understanding the mechanism of the target enzyme. Two separate GT modules in KpsC transfer 3-deoxy-ß-d-manno-oct-2-ulosonic acid (ß-Kdo) from cytidine-5'-monophospho-ß-Kdo donor to a glycolipid acceptor. The N-terminal and C-terminal modules add alternating Kdo residues with ß-(2→4) and ß-(2→7) linkages, respectively, generating a conserved oligosaccharide core that is further glycosylated to produce diverse capsule structures. KpsC is a retaining GT, which retains the donor anomeric carbon stereochemistry. Retaining GTs typically use an SNi (substitution nucleophilic internal return) mechanism, but recent studies with WbbB, a retaining ß-Kdo GT distantly related to KpsC, strongly suggest that this enzyme uses an alternative double-displacement mechanism. Based on the formation of covalent adducts with Kdo identified here by mass spectrometry and X-ray crystallography, we determined that catalytically important active site residues are conserved in WbbB and KpsC, suggesting a shared double-displacement mechanism. Additional crystal structures and biochemical experiments revealed the acceptor binding mode of the ß-(2→4)-Kdo transferase module and demonstrated that acceptor recognition (and therefore linkage specificity) is conferred solely by the N-terminal α/ß domain of each GT module. Finally, an Alphafold model provided insight into organization of the modules and a C-terminal membrane-anchoring region. Altogether, we identified key structural and mechanistic elements providing a foundation for targeting KpsC.

Quorum sensing positively regulates CPS-dependent Autographiviridae phage infection in Vibrio alginolyticus.

Quorum sensing (QS) orchestrates many bacterial behaviors, including virulence and biofilm formation, across bacterial populations. Nevertheless, the underlying mechanism by which QS regulates capsular polysaccharide (CPS)-dependent phage-bacterium interactions remains unclear. In this study, we report that QS upregulates the expression of CPS-dependent phage receptors, thus increasing phage adsorption and infection rates in Vibrio alginolyticus. We found that QS upregulated the expression of the ugd gene, leading to increased synthesis of Autographiviridae phage receptor CPS synthesis in V. alginolyticus. The signal molecule autoinducer-2 released by Vibrio from different sources can potentially enhance CPS-dependent phage infections. Therefore, our data suggest that inhibiting QS may reduce, rather than improve, the therapeutic efficacy of CPS-specific phages. IMPORTANCE: Phage resistance is a direct threat to phage therapy, and understanding phage-host interactions, especially how bacteria block phage infection, is essential for developing successful phage therapy. In the present study, we demonstrate for the first time that Vibrio alginolyticus uses quorum sensing (QS) to promote capsular polysaccharide (CPS)-specific phage infection by upregulating ugd expression, which is necessary for the synthesis of Autographiviridae phage receptor CPS. Although increased CPS-specific phage susceptibility is a novel trade-off mediated by QS, it results in the upregulation of virulence factors, promoting biofilm development and enhanced capsular polysaccharide production in V. alginolyticus. This suggests that inhibiting QS may improve the effectiveness of antibiotic treatment, but it may also reduce the efficacy of phage therapy.

Inhibition of porcine origin Klebsiella pneumoniae capsular polysaccharide and immune escape by BY3 compounded traditional Chinese medicine residue fermentation broth.

Klebsiella pneumoniae (K. pneumoniae) is a gram-negative conditionally pathogenic bacterium that causes disease primarily in immunocompromised individuals. Recently, highly virulent K. pneumoniae strains have caused severe disease in healthy individuals, posing significant challenges to global infection control. Capsular polysaccharide (CPS), a major virulence determinant of K. pneumoniae, protects the bacteria from being killed by the host immune system, suggesting an urgent need for the development of drugs to prevent or treat K. pneumoniae infections. In this study, BY3 compounded traditional Chinese medicine residue (TCMR) was carried out using Lactobacillus rhamnosus as a fermentation strain, and BY3 compounded TCMR fermentation broth (BY3 fermentation broth) was obtained. The transcription of K. pneumoniae CPS-related biosynthesis genes after treatment with BY3 fermentation broth was detected using quantitative real-time polymerase chain reaction. The effects of BY3 fermentation broth on K. pneumoniae serum killing, macrophage phagocytosis, complement deposition and human ß-defensin transcription were investigated. The therapeutic effect of BY3 fermentation broth on K. pneumoniae-infected mice was also observed, and the major active components of BY3 fermentation broth were analysed via LC‒MS analysis, network pharmacology, and molecular docking. The results showed that BY3 fermentation broth inhibited K. pneumoniae CPS production and downregulated transcription of CPS-related biosynthesis genes, which weakened bacterial resistance to serum killing and phagocytosis, while promoting bacterial surface complement C3 deposition and human ß-defensin expression. BY3 fermentation broth demonstrated safety and therapeutic effects in vivo and in vitro, restoring body weight and visceral indices, significantly reducing the organ bacterial load and serum cytokine levels, and alleviating pathological organ damage in mice. In addition, three natural compounds-oleanolic acid, quercetin, and palmitoleic acid-were identified as the major active components in the BY3 fermentation broth. Therefore, BY3 fermentation broth may be a promising strategy for the prevention or treatment of K. pneumoniae infections.

Epoxide-Mediated Trans-Thioglycosylation and Application to the Synthesis of Oligosaccharides Related to the Capsular Polysaccharides of C. jejuni HS:4.

The enzyme-resistant thioglycosides are highly valuable immunogens because of their enhanced metabolic stability. We report the first synthesis of a family of thiooligosaccharides related to the capsular polysaccharides (CPS) of Campylobacter jejuni HS:4 for potential use in conjugate vaccines. The native CPS structures of the pathogen consist of a challenging repeating disaccharide formed with ß(1→4)-linked 6-deoxy-ß-D-ido-heptopyranoside and N-acetyl-D-glucosamine; the rare 6-deoxy-ido-heptopyranosyl backbone and ß-anomeric configuration of the former monosaccharide makes the synthesis of this family of antigens very challenging. So far, no synthesis of the thioanalogs of the CPS antigens have been reported. The unprecedented synthesis presented in this work is built on an elegant approach by using ß-glycosylthiolate as a glycosyl donor to open the 2,3-epoxide functionality of pre-designed 6-deoxy-ß-D-talo-heptopyranosides. Our results illustrated that this key trans-thioglycosylation can be designed in a modular and regio and stereo-selective manner. Built on the success of this novel approach, we succeeded the synthesis of a family of thiooligosaccharides including a thiohexasaccharide which is considered to be the desired antigen length and complexity for immunizations. We also report the first direct conversion of base-stable but acid-labile 2-trimethylsilylethyl glycosides to glycosyl-1-thioacetates in a one-pot manner.

AI-2 quorum sensing-induced galactose metabolism activation in Streptococcus suis enhances capsular polysaccharide-associated virulence.

Bacteria utilize intercellular communication to orchestrate essential cellular processes, adapt to environmental changes, develop antibiotic tolerance, and enhance virulence. This communication, known as quorum sensing (QS), is mediated by the exchange of small signalling molecules called autoinducers. AI-2 QS, regulated by the metabolic enzyme LuxS (S-ribosylhomocysteine lyase), acts as a universal intercellular communication mechanism across gram-positive and gram-negative bacteria and is crucial for diverse bacterial processes. In this study, we demonstrated that in Streptococcus suis (S. suis), a notable zoonotic pathogen, AI-2 QS enhances galactose utilization, upregulates the Leloir pathway for capsular polysaccharide (CPS) precursor production, and boosts CPS synthesis, leading to increased resistance to macrophage phagocytosis. Additionally, our molecular docking and dynamics simulations suggest that, similar to S. pneumoniae, FruA, a fructose-specific phosphoenolpyruvate phosphotransferase system prevalent in gram-positive pathogens, may also function as an AI-2 membrane surface receptor in S. suis. In conclusion, our study demonstrated the significance of AI-2 in the synthesis of galactose metabolism-dependent CPS in S. suis. Additionally, we conducted a preliminary analysis of the potential role of FruA as a membrane surface receptor for S. suis AI-2.

A challenging case of carbapenem resistant Klebsiella pneumoniae-related pyogenic liver abscess with capsular polysaccharide hyperproduction: a case report.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections are a major public health problem, necessitating the administration of polymyxin E (colistin) as a last-line antibiotic. Meanwhile, the mortality rate associated with colistin-resistant K. pneumoniae infections is seriously increasing. On the other hand, importance of administration of carbapenems in promoting colistin resistance in K. pneumoniae is unknown. CASE PRESENTATION: We report a case of K. pneumoniae-related pyogenic liver abscess in which susceptible K. pneumoniae transformed into carbapenem- and colistin-resistant K. pneumoniae during treatment with imipenem. The case of pyogenic liver abscess was a 50-year-old man with diabetes and liver transplant who was admitted to Abu Ali Sina Hospital in Shiraz. The K. pneumoniae isolate responsible for community-acquired pyogenic liver abscess was isolated and identified. The K. pneumoniae isolate was sensitive to all tested antibiotics except ampicillin in the antimicrobial susceptibility test and was identified as a non-K1/K2 classical K. pneumoniae (cKp) strain. Multilocus sequence typing (MLST) identified the isolate as sequence type 54 (ST54). Based on the patient's request, he was discharged to continue treatment at another center. After two months, he was readmitted due to fever and progressive constitutional symptoms. During treatment with imipenem, the strain acquired blaOXA-48 and showed resistance to carbapenems and was identified as a multidrug resistant (MDR) strain. The minimum inhibitory concentration (MIC) test for colistin was performed by broth microdilution method and the strain was sensitive to colistin (MIC < 2 µg/mL). Meanwhile, on blood agar, the colonies had a sticky consistency and adhered to the culture medium (sticky mucoviscous colonies). Quantitative real-time PCR and biofilm formation assay revealed that the CRKP strain increased capsule wzi gene expression and produced slime in response to imipenem. Finally, K. pneumoniae-related pyogenic liver abscess with resistance to a wide range of antibiotics, including the last-line antibiotics colistin and tigecycline, led to sepsis and death. CONCLUSIONS: Based on this information, can we have a theoretical hypothesis that imipenem is a promoter of resistance to carbapenems and colistin in K. pneumoniae? This needs more attention.

The bacterial tyrosine kinase system CpsBCD governs the length of capsule polymers.

Many pathogenic bacteria are encased in a layer of capsular polysaccharide (CPS). This layer is important for virulence by masking surface antigens, preventing opsonophagocytosis, and avoiding mucus entrapment. The bacterial tyrosine kinase (BY-kinase) regulates capsule synthesis and helps bacterial pathogens to survive different host niches. BY-kinases autophosphorylate at the C-terminal tyrosine residues upon external stimuli, but the role of phosphorylation is still unclear. Here, we report that the BY-kinase CpsCD is required for growth in Streptococcus pneumoniae Cells lacking a functional cpsC or cpsD accumulated low molecular weight CPS and lysed because of the lethal sequestration of the lipid carrier undecaprenyl phosphate, resulting in inhibition of peptidoglycan (PG) synthesis. CpsC interacts with CpsD and the polymerase CpsH. CpsD phosphorylation reduces the length of CPS polymers presumably by controlling the activity of CpsC. Finally, pulse-chase experiments reveal the spatiotemporal coordination between CPS and PG synthesis. This coordination is dependent on CpsC and CpsD. Together, our study provides evidence that BY-kinases regulate capsule polymer length by fine-tuning CpsC activity through autophosphorylation.

Capsular polysaccharide correlates with immune response to the human gut microbe Ruminococcus gnavus.

Active inflammatory bowel disease (IBD) often coincides with increases of Ruminococcus gnavus, a gut microbe found in nearly everyone. It was not known how, or if, this correlation contributed to disease. We investigated clinical isolates of R. gnavus to identify molecular mechanisms that would link R. gnavus to inflammation. Here, we show that only some isolates of R. gnavus produce a capsular polysaccharide that promotes a tolerogenic immune response, whereas isolates lacking functional capsule biosynthetic genes elicit robust proinflammatory responses in vitro. Germ-free mice colonized with an isolate of R. gnavus lacking a capsule show increased measures of gut inflammation compared to those colonized with an encapsulated isolate in vivo. These observations in the context of our earlier identification of an inflammatory cell-wall polysaccharide reveal how some strains of R. gnavus could drive the inflammatory responses that characterize IBD.

Diagnostic Performances of an in-House Immunochromatography Test Based on the Monoclonal Antibody 18B7 to Glucuronoxylomannan for Clinical Suspected Cryptococcosis: a Large-Scale Prototype Evaluation in Northern Thailand.

OBJECTIVE: Cryptococcosis predominantly presents as a meningoencephalitis in Thailand. Early and expeditious diagnosis is essential for reducing both mortality and morbidity associated with cryptococcal meningitis. We aim to define and establish the diagnostic performances between the benchmark commercially available diagnostic kit (CrAg® LFA) and the large-scale prototype of an inexpensive in-house immunochromatographic test (ICT) based on monoclonal antibody (MAb) 18B7. METHODS: We have developed the large-scale prototype for the rapid detection of cryptococcal polysaccharide antigens by utilizing a single antibody sandwich ICT format employing MAb 18B7, which is highly specific to Cryptococcus neoformans glucuronoxylomannan (GXM) antigens. An in-house MAb18B7 ICT was manufactured in accordance with industry standards under the control of the International Organization for Standardization (ISO) 13485. RESULTS: The diagnostic sensitivity, specificity, and accuracy for the in-house MAb 18B7 ICT were 99.10%, 97.61%, and 97.83%, respectively. The agreement kappa (κ) coefficient was 0.968 based on the retrospective evaluation of 580 specimens from patients living in northern Thailand with clinically suspected cryptococcosis. CONCLUSION: The data suggest that this in-house MAb 18B7 ICT will be highly beneficial for addressing the issue of cryptococcal infection in Thailand. Moreover, it is anticipated that this inexpensive ICT can play a pivotal role in various global strategies aimed at eradicating cryptococcal meningitis among individuals living with HIV by 2030.

New Obolenskvirus Phages Brutus and Scipio: Biology, Evolution, and Phage-Host Interaction.

Two novel virulent phages of the genus Obolenskvirus infecting Acinetobacter baumannii, a significant nosocomial pathogen, have been isolated and studied. Phages Brutus and Scipio were able to infect A. baumannii strains belonging to the K116 and K82 capsular types, respectively. The biological properties and genomic organization of the phages were characterized. Comparative genomic, phylogenetic, and pangenomic analyses were performed to investigate the relationship of Brutus and Scipio to other bacterial viruses and to trace the possible origin and evolutionary history of these phages and other representatives of the genus Obolenskvirus. The investigation of enzymatic activity of the tailspike depolymerase encoded in the genome of phage Scipio, the first reported virus infecting A. baumannii of the K82 capsular type, was performed. The study of new representatives of the genus Obolenskvirus and mechanisms of action of depolymerases encoded in their genomes expands knowledge about the diversity of viruses within this taxonomic group and strategies of Obolenskvirus-host bacteria interaction.

Expanding polysaccharide-protein coupling of glycoconjugate vaccines.

For the preparation of glycoconjugate vaccines, polysaccharide antigens can usually be chemically modified to generate reactive functional groups (e.g., the formation of aldehyde groups by periodate oxidation of adjacent diols) for covalent coupling with proteins. In a recent issue of JBC, Duke et al. showed that an alternative agent, galactose oxidase (GOase) isolated from the fungus Fusarium sp. can generate aldehyde groups in a unique chemoenzymatic approach to prepare a conjugate vaccine against Streptococcus pneumoniae. These findings introduce a new strategy for the design and development of glycoconjugate vaccines.

Harnessing galactose oxidase in the development of a chemoenzymatic platform for glycoconjugate vaccine design.

In the preparation of commercial conjugate vaccines, capsular polysaccharides (CPSs) must undergo chemical modification to generate the reactive groups necessary for covalent attachment to a protein carrier. One of the most common approaches employed for this derivatization is sodium periodate (NaIO4) oxidation of vicinal diols found within CPS structures. This procedure is largely random and structurally damaging, potentially resulting in significant changes in the CPS structure and therefore its antigenicity. Additionally, periodate activation of CPS often gives rise to heterogeneous conjugate vaccine products with variable efficacy. Here, we explore the use of an alternative agent, galactose oxidase (GOase) isolated from Fusarium sp. in a chemoenzymatic approach to generate a conjugate vaccine against Streptococcus pneumoniae. Using a colorimetric assay and NMR spectroscopy, we found that GOase generated aldehyde motifs on the CPS of S. pneumoniae serotype 14 (Pn14p) in a site-specific and reversible fashion. Direct comparison of Pn14p derivatized by either GOase or NaIO4 illustrates the functionally deleterious role chemical oxidation can have on CPS structures. Immunization with the conjugate synthesized using GOase provided a markedly improved humoral response over the traditional periodate-oxidized group. Further, functional protection was validated in vitro by measure of opsonophagocytic killing and in vivo through a lethality challenge in mice. Overall, this work introduces a strategy for glycoconjugate development that overcomes limitations previously known to play a role in the current approach of vaccine design.

Investigation of core machinery for biosynthesis of Vi antigen capsular polysaccharides in Gram-negative bacteria.

Salmonella enterica serovar Typhi causes typhoid fever. It possesses a Vi antigen capsular polysaccharide coat that is important for virulence and is the basis of a current glycoconjugate vaccine. Vi antigen is also produced by environmental Bordetella isolates, while mammal-adapted Bordetella species (such as Bordetella bronchiseptica) produce a capsule of undetermined structure that cross-reacts with antibodies recognizing Vi antigen. The Vi antigen backbone is composed of poly-α-(1→4)-linked N-acetylgalactosaminuronic acid, modified with O-acetyl residues that are necessary for vaccine efficacy. Despite its biological and biotechnological importance, some central aspects of Vi antigen production are poorly understood. Here we demonstrate that TviE and TviD, two proteins encoded in the viaB (Vi antigen production) locus, interact and are the Vi antigen polymerase and O-acetyltransferase, respectively. Structural modeling and site-directed mutagenesis reveal that TviE is a GT4-family glycosyltransferase. While TviD has no identifiable homologs beyond Vi antigen systems in other bacteria, structural modeling suggests that it belongs to the large SGNH hydrolase family, which contains other O-acetyltransferases. Although TviD possesses an atypical catalytic triad, its O-acetyltransferase function was verified by antibody reactivity and 13C NMR data for tviD-mutant polysaccharide. The B. bronchiseptica genetic locus predicts a mode of synthesis distinct from classical S. enterica Vi antigen production, but which still involves TviD and TviE homologs that are both active in a reconstituted S. Typhi system. These findings provide new insight into Vi antigen production and foundational information for the glycoengineering of Vi antigen production in heterologous bacteria.

Conformational comparisons of Pasteurella multocida types B and E and structurally related capsular polysaccharides.

Pasteurella multocida, an encapsulated gram-negative bacterium, is a significant veterinary pathogen. The P. multocida is classified into 5 serogroups (A, B, D, E, and F) based on the bacterial capsular polysaccharide (CPS), which is important for virulence. Serogroups B and E are the primary causative agents of bovine hemorrhagic septicemia that is associated with significant yearly losses of livestock worldwide, primarily in low- and middle-income countries. The P. multocida disease is currently managed by whole-cell vaccination, albeit with limited efficacy. CPS is an attractive antigen target for an improved vaccine: CPS-based vaccines have proven highly effective against human bacterial diseases and could provide longer-term protection against P. multocida. The recently elucidated CPS repeat units of serogroups B and E both comprise a N-acetyl-ß-D-mannosaminuronic acid/N-acetyl-ß-D-glucosamine disaccharide backbone with ß-D-fructofuranose (Fruf) side chain, but differ in their glycosidic linkages, and a glycine (Gly) side chain in serogroup B. Interestingly, the Haemophilus influenzae types e and d CPS have the same backbone residues. Here, comparative modeling of P. multocida serogroups B and E and H. influenzae types e and d CPS identifies a significant impact of small structural differences on both the chain conformation and the exposed potential antibody-binding epitopes (Ep). Further, Fruf and/or Gly side chains shield the immunogenic amino-sugar CPS backbone-a possible common strategy for immune evasion in both P. multocida and H. influenzae. As the lack of common epitopes suggests limited potential for cross-reactivity, a bivalent CPS-based vaccine may be necessary to provide adequate protection against P. multocida types B and E.

Characterization of pneumococcal serotype 7F in vaccine conjugation.

Streptococcus pneumoniae is a highly invasive bacterial pathogen that can cause a range of illnesses. Pneumococcal capsular polysaccharides (CPS) are the main virulence factors that causes invasive pneumococcal disease (IPD). Pneumococcal CPS serotype 7F along with a few other serotypes is more invasive and likely to cause IPD. Therefore, 7F is a target for pneumococcal vaccine development, and is included in the two recently approved multi-valent pneumococcal conjugated vaccines, i.e. VAXNEUVANCE and PREVNAR 20.To support process and development of our 15-valent pneumococcal conjugated vaccine (PCV15), chromatographic methods have been developed for 7F polysaccharide and conjugate characterization. A size-exclusion chromatography (SEC) method with UV, light scattering and refractive index detections was employed for concentration, size and conformation analysis. A reversed-phase ultra-performance liquid chromatography (RP-UPLC) method was used for analysis of conjugate monosaccharide composition and degree of conjugation. The collective information obtained by these chromatographic analysis provided insights into the pneumococcal conjugate and conjugation process.