Development and Application of a High-Throughput Method for the Purification and Analysis of Surface Carbohydrates from Klebsiella pneumoniae.

Klebsiella pneumoniae (Kp) is a Gram-negative bacterium, and a leading cause of neonatal sepsis in low- and middle-income countries, often associated with anti-microbial resistance. Two types of polysaccharides are expressed on the Kp cell surface and have been proposed as key antigens for vaccine design: capsular polysaccharides (known as K-antigens, K-Ags) and O-antigens (O-Ags). Historically, Kp has been classified using capsule serotyping and although 186 distinct genotypes have been predicted so far based on sequence analysis, many structures are still unknown. In contrast, only 11 distinct OAg serotypes have been described. The characterization of emerging strains requires the development of a high-throughput purification method to obtain sufficient K- and O-Ag material to characterize the large collection of serotypes and gain insight on structural features and potential cross-reactivity that could allow vaccine simplification. Here, this was achieved by adapting our established method for the simple purification of O-Ags, using mild acetic acid hydrolysis performed directly on bacterial cells, followed by filtration and precipitation steps. The method was successfully applied to purify the surface carbohydrates from different Kp strains, thereby demonstrating the robustness and general applicability of the purification method developed. Further, antigen characterization showed that the purification method had no impact on the structural integrity of the polysaccharides and preserved labile substituents such as O-acetyl and pyruvyl groups. This method can be further optimized for scaling up and manufacturing to support the development of high-valency saccharide-based vaccines against Kp.

O-Antigen decorations in Salmonella enterica play a key role in eliciting functional immune responses against heterologous serovars in animal models.

Introduction: Different serovars of Salmonella enterica cause systemic diseases in humans including enteric fever, caused by S. Typhi and S. Paratyphi A, and invasive nontyphoidal salmonellosis (iNTS), caused mainly by S. Typhimurium and S. Enteritidis. No vaccines are yet available against paratyphoid fever and iNTS but different strategies, based on the immunodominant O-Antigen component of the lipopolysaccharide, are currently being tested. The O-Antigens of S. enterica serovars share structural features including the backbone comprising mannose, rhamnose and galactose as well as further modifications such as O-acetylation and glucosylation. The importance of these O-Antigen decorations for the induced immunogenicity and cross-reactivity has been poorly characterized. Methods: These immunological aspects were investigated in this study using Generalized Modules for Membrane Antigens (GMMA) as delivery systems for the different O-Antigen variants. This platform allowed the rapid generation and in vivo testing of defined and controlled polysaccharide structures through genetic manipulation of the O-Antigen biosynthetic genes. Results: Results from mice and rabbit immunization experiments highlighted the important role played by secondary O-Antigen decorations in the induced immunogenicity. Moreover, molecular modeling of O-Antigen conformations corroborated the likelihood of cross-protection between S. enterica serovars. Discussion: Such results, if confirmed in humans, could have a great impact on the design of a simplified vaccine composition able to maximize functional immune responses against clinically relevant Salmonella enterica serovars.

Streptococcus pneumoniae serotype 33G: genetic, serological, and structural analysis of a new capsule type.

IMPORTANCE: Streptococcus pneumoniae (the pneumococcus) is a bacterial pathogen with the greatest burden of disease in Asia and Africa. The pneumococcal capsular polysaccharide has biological relevance as a major virulence factor as well as public health importance as it is the target for currently licensed vaccines. These vaccines have limited valency, covering up to 23 of the >100 known capsular types (serotypes) with higher valency vaccines in development. Here, we have characterized a new pneumococcal serotype, which we have named 33G. We detected serotype 33G in nasopharyngeal swabs (n = 20) from children and adults hospitalized with pneumonia, as well as healthy children in Mongolia. We show that the genetic, serological, and biochemical properties of 33G differ from existing serotypes, satisfying the criteria to be designated as a new serotype. Future studies should focus on the geographical distribution of 33G and any changes in prevalence following vaccine introduction.

Acid hydrolysis conditions for quantification of meningococcal X polysaccharide in a pentavalent vaccine using HPAEC-PAD/ESI-MS.

A selective and sensitive method was evaluated for quantitation of meningococcal X (Men X) polysaccharide in pentavalent meningococcal A, C, W, Y and X conjugate vaccine using different acid hydrolysis conditions like HCl, TFA, HF, HF-TFA, and HF-HCl. High-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using CarboPac PA10 column was used to identify the hydrolyzed products based on retention time and its comparison with monosaccharide standards. Complete release of glucosamine (GlcN) from Men X in monovalent bulk and pentavalent vaccine samples was achieved using HF hydrolysis at 80 °C for 2 h. The Men X HF-hydrolyzed polysaccharide to glucosamine along with the reference standard was identified using collision-induced dissociation (CID) electrospray mass spectroscopy and the MS/MS fragments of m/z 162, m/z 144 and m/z 84. Meningococcal polysaccharide concentration was determined with a correlation coefficient r2 >0.99 using polysaccharide reference standard. The serogroups A, W, and Y were converted to their monosaccharides units and quantified using this method however, milder acid hydrolysis 0.1 M HCl 80 °C 2 h for release of sialic acid for Men C polysaccharide was found to be more suitable. These methods will provide necessary tools and prove to be beneficial to laboratories developing new saccharide-based vaccine combinations.

Effect of trifluoroacetic acid on the antigenicity of capsular polysaccharides obtained from various Streptococcus pneumoniae serotypes.

Determining the safety, antigenicity, and immunogenicity by in vitro and in vivo studies is a prerequisite for the development of new vaccines. And this study investigated it for a vaccine made from Streptococcus pneumoniae serotypes 2, 5, 12F, 18C, and 22F. The crude CPS was purified and partially depolymerized by conventional and trifluoroacetic acid methods. 1H NMR analysis confirmed the identity of the depolymerized CPS which gave similar profiles to reference polysaccharides, except for serotype 18C which was de-O-acetylated during TFA treatment. The antigenicity of the depolymerized CPS prepared by either method was comparable to that of the native CPS for serotypes 2, 5, 18C, and 22F based on multiplex bead based competitive inhibition assay. This study demonstrated a relationship between antigenicity and immunogenicity, which offers more suitable candidates for conjugation. It was found that after partial depolymerization process, the CPS with optimal molecular size resulted in higher antigenicity. The immunogenicity of S. pneumoniae serotype 2 conjugates in mice was evaluated by opsonophagocytic assay and a multiplex bead-based assay, wherein on day 42 after immunization, the total and functional IgG titer was found to be increased by 32-fold.

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.

Metabolomics reveals the response of hydroprimed maize to mitigate the impact of soil salinization.

Soil salinization is a major environmental stressor hindering global crop production. Hydropriming has emerged as a promising approach to reduce salt stress and enhance crop yields on salinized land. However, a better mechanisitic understanding is required to improve salt stress tolerance. We used a biochemical and metabolomics approach to study the effect of salt stress of hydroprimed maize to identify the types and variation of differentially accumulated metabolites. Here we show that hydropriming significantly increased catalase (CAT) activity, soluble sugar and proline content, decreased superoxide dismutase (SOD) activity and peroxide (H2O2) content. Conversely, hydropriming had no significant effect on POD activity, soluble protein and MDA content under salt stress. The Metabolite analysis indicated that salt stress significantly increased the content of 1278 metabolites and decreased the content of 1044 metabolites. Ethisterone (progesterone) was the most important metabolite produced in the roots of unprimed samples in response to salt s tress. Pathway enrichment analysis indicated that flavone and flavonol biosynthesis, which relate to scavenging reactive oxygen species (ROS), was the most significant metabolic pathway related to salt stress. Hydropriming significantly increased the content of 873 metabolites and significantly decreased the content of 1313 metabolites. 5-Methyltetrahydrofolate, a methyl donor for methionine, was the most important metabolite produced in the roots of hydroprimed samples in response to salt stress. Plant growth regulator, such as melatonin, gibberellin A8, estrone, abscisic acid and brassinolide involved in both treatment. Our results not only verify the roles of key metabolites in resisting salt stress, but also further evidence that flavone and flavonol biosynthesis and plant growth regulator relate to salt tolerance.

Modeling of pneumococcal serogroup 10 capsular polysaccharide molecular conformations provides insight into epitopes and observed cross-reactivity.

Streptococcus pneumoniae is an encapsulated gram-negative bacterium and a significant human pathogen. The capsular polysaccharide (CPS) is essential for virulence and a target antigen for vaccines. Although widespread introduction of pneumococcal conjugate vaccines (PCVs) has significantly reduced disease, the prevalence of non-vaccine serotypes has increased. On the basis of the CPS, S. pneumoniae serogroup 10 comprises four main serotypes 10A, 10B, 10C, and 10F; as well as the recently identified 10D. As it is the most prevalent, serotype 10A CPS has been included as a vaccine antigen in the next generation PCVs. Here we use molecular modeling to provide conformational rationales for the complex cross-reactivity reported between serotypes 10A, 10B, 10C, and 10F anti-sera. Although the highly mobile phosphodiester linkages produce very flexible CPS, shorter segments are conformationally defined, with exposed ß -D-galactofuranose ( ß DGalf) side chains that are potential antibody binding sites. We identify four distinct conformational epitopes for the immunodominant ß DGalf that assist in rationalizing the complex asymmetric cross-reactivity relationships. In particular, we find that strongly cross-reactive serotypes share common epitopes. Further, we show that human intelectin-1 has the potential to bind the exposed exocyclic 1,2-diol of the terminal ß DGalf in each serotype; the relative accessibility of three- or six-linked ß DGalf may play a role in the strength of the innate immune response and hence serotype disease prevalence. In conclusion, our modeling study and relevant serological studies support the inclusion of serotype 10A in a vaccine to best protect against serogroup 10 disease.

Purification of capsular polysaccharides isolated from S. pneumoniae serotype 2 by hydrogen peroxide and endonuclease.

A high-quality and cost-effective purification procedure is one of the most important requirements for manufacturing glycoconjugate vaccines. The goal of the present work was to devise a method for removing impurities such as protein and nucleic acid from Streptococcus pneumoniae serotype 2 capsular polysaccharides (CPS). The use of hydrogen peroxide for the reduction of impurities of crude CPS was investigated. Centrifugation followed by filtration decreased protein contaminant of the hydrogen peroxide-treated CPS to meet the limit specified by WHO. The nucleic acid impurity remaining was removed by a further step of endonuclease treatment to yield the purified CPS. Characterization of purified CPS was evaluated by various analytical techniques including 1H NMR and antigenicity by competitive inhibition assay. Various hydrogen peroxide concentrations have significant impact on the antigenic property of CPS. Whereas, optimum process conditions can preserve the native characteristics of CPS.

Genetic and Structural Variation in the O-Antigen of Salmonella enterica Serovar Typhimurium Isolates Causing Bloodstream Infections in the Democratic Republic of the Congo.

Salmonella enterica serovar Typhimurium causes a devastating burden of invasive disease in sub-Saharan Africa with high levels of antimicrobial resistance. No licensed vaccine is available, but O-antigen-based candidates are in development, as the O-antigen moiety of lipopolysaccharides is the principal target of protective immunity. The vaccines under development are designed based on isolates with O-antigen O-acetylated at position C-2 of abequose, giving the O:5 antigen. Serotyping data on recent Salmonella Typhimurium clinical isolates from the Democratic Republic of the Congo (DRC), however, indicate increasing levels of isolates without O:5. The importance and distribution of this loss of O:5 antigen in the population as well as the genetic mechanism responsible for the loss and chemical characteristics of the O-antigen are poorly understood. In this study, we Illumina whole-genome sequenced 354 Salmonella Typhimurium isolates from the DRC, which were isolated between 2002 and 2017. We used genomics and phylogenetics combined with chemical approaches (1H nuclear magnetic resonance [NMR], high-performance anion-exchange chromatography with pulsed amperometric detection [HPAEC-PAD], high-performance liquid chromatography-PAD [HPLC-PAD], and HPLC-size exclusion chromatography [HPLC-SEC]) to characterize the O-antigen features within the bacterial population. We observed convergent evolution toward the loss of the O:5 epitope predominantly caused by recombination events in a single gene, the O-acetyltransferase gene oafA. In addition, we observe further O-antigen variations, including O-acetylation of the rhamnose residue, different levels of glucosylation, and the absence of O-antigen repeating units. Large recombination events underlying O-antigen variation were resolved using long-read MinION sequencing. Our study suggests evolutionary pressure toward O-antigen variants in a region where invasive disease by Salmonella Typhimurium is highly endemic. This needs to be taken into account when developing O-antigen-based vaccines, as it might impact the breadth of coverage in such regions. IMPORTANCE The bacterium Salmonella Typhimurium forms a devastating burden in sub-Saharan Africa by causing invasive bloodstream infections. Additionally, Salmonella Typhimurium presents high levels of antimicrobial resistance, jeopardizing treatment. No licensed vaccine is available, but candidates are in development, with lipopolysaccharides being the principal target of protective immunity. The vaccines under development are designed based on the O:5 antigen variant of bacterial lipopolysaccharides. Data on recent Salmonella Typhimurium clinical isolates from the Democratic Republic of the Congo (DRC), however, indicate increasing levels of isolates without this O:5 antigen. We studied this loss of O:5 antigen in the population at the genetic and chemical levels. We genome sequenced 354 isolates from the DRC and used advanced bioinformatics and chemical methods to characterize the lipopolysaccharide features within the bacterial population. Our results suggest evolutionary pressure toward O-antigen variants. This needs to be taken into account when developing vaccines, as it might impact vaccine coverage.

Streptococcus pneumoniae serotype 15B polysaccharide conjugate elicits a cross-functional immune response against serotype 15C but not 15A.

Protection conferred by pneumococcal polysaccharide conjugate vaccines (PCVs) is associated with PCV-induced antibodies against vaccine-covered serotypes that exhibit functional opsonophagocytic activity (OPA). Structural similarity between capsular polysaccharides of closely related serotypes may result in induction of cross-reactive antibodies with or without a cross-functional activity against a serotype not covered by a PCV, with the former providing an additional protective clinical benefit. Serotypes 15B, 15A, and 15C, in the serogroup 15, are among the most prevalent Streptococcus pneumoniae serotypes associated with invasive pneumococcal disease following the implementation of a 13-valent PCV; in addition, 15B contributes significantly to acute otitis media. Serological discrimination between closely related serotypes such as 15B and 15C is complicated; here, we implemented an algorithm to quickly differentiate 15B from its closely related serotypes 15C and 15A directly from whole-genome sequencing data. In addition, molecular dynamics simulations of serotypes 15A, 15B, and 15C polysaccharides demonstrated that while 15B and 15C polysaccharides assume rigid branched conformation, 15A polysaccharide assumes a flexible linear conformation. A serotype 15B conjugate, included in a 20-valent PCV (PCV20), induced cross-functional OPA serum antibody responses against the structurally similar serotype 15C but not against serotype 15A, both not included in PCV20. In PCV20-vaccinated adults (18-49 years), robust OPA antibody titers were detected against both serotypes 15B (the geometric mean titer [GMT] of 19,334) and 15C (GMTs of 1692 and 2747 for strains PFE344340 and PFE1160, respectively), but were negligible against serotype 15A (GMTs of 10 and 30 for strains PFE593551 and PFE647449, respectively). Cross-functional 15B/C responses were also confirmed using sera from a larger group of older adults (60-64 years).

Rapid generation of Shigella flexneri GMMA displaying natural or new and cross-reactive O-Antigens.

Generalized modules for membrane antigens (GMMA) are exosomes released from engineered Gram-negative bacteria and represent an attractive vaccine platform for the delivery of the O-Antigen (OAg), recognized as the key target for protective immunity against several pathogens such as Shigella. Shigella is a major cause of disease in Low- and Middle-Income countries and the development of a vaccine needs to deal with its large serotypic diversity. All S. flexneri serotypes, except serotype 6, share a conserved OAg backbone, corresponding to serotype Y. Here, a GMMA-producing S. flexneri scaffold strain displaying the OAg backbone was engineered with different OAg-modifying enzymes, either individually or in combinations. This strategy rapidly yielded GMMA displaying 12 natural serotypes and 16 novel serotypes expressing multiple epitopes combinations that do not occur in nature. Importantly, a candidate GMMA displaying a hybrid OAg elicited broadly cross-bactericidal antibodies against a large panel of S. flexneri serotypes.

Deciphering the Mechanism of Binding Selectivity of Chlorofluoroacetamide-Based Covalent Inhibitors toward L858R/T790M Resistance Mutation.

Covalent modification of the oncogenic mutant epidermal growth factor receptor (EGFR) by small molecules is an efficient strategy for achieving an enhanced and sustained pharmacological effect in the treatment of non-small-cell lung cancer. NSP-037 (18), an irreversible inhibitor of the L858R/T790M double-mutant EGFR (EGFRDM) using α-chlorofluoroacetamide (CFA) as a novel warhead, has seven times the inhibition selectivity for EGFRDM over the wild type (EGFRWT), as compared to clinically approved osimertinib (7). Here, we employ multiple computational approaches to elucidate the mechanism underlining this improved selectivity, as well as the effect of CFA on the selectivity enhancement of inhibitor 18 over 7. We find that EGFRDM undergoes significantly larger conformational changes than EGFRWT upon binding to 18. The conformational stability of the diamine side chain and the CFA motif of 18 in the orthosteric site of EGFRDM is identified as key for the disparate binding mechanism and inhibitory prowess of 18 with respect to EGFRWT and EGFRDM and 18's higher selectivity than 7. The binding free energy of the 18-bound complexes is -6.38 kcal/mol greater than that of the 7-bound complexes, explaining the difference in selectivity of these inhibitors. Further, free energy decomposition analysis indicates that the electrostatic contribution of key residues plays an important role in the 18-bound complexes. QM/MM calculations show that the most favored mechanism for the Cys797 alkylation reaction is the direct displacement mechanism through a CFA-based inhibitor, producing a reaction with the lowest energy barrier and most stable product.

Carbohydrate Force Fields: The Role of Small Partial Atomic Charges in Preventing Conformational Collapse.

Although the quality of current additive all-atom force fields for carbohydrates has been demonstrated in many applications, occasional significant differences reported for the hydrodynamic behavior of specific polysaccharides modeled with different force fields is a cause for concern. In particular, irreversible conformational collapse has been noted for some polysaccharide simulations with the GLYCAM06j force field. Here, we investigate the cause of this phenomenon through comparative simulations of a range of saccharides with both the GLYCAM06j and the CHARMM36 carbohydrate force fields. We find that conformational collapse in GLYCAM06j occurs for saccharide chains containing the deoxy sugar α-l-rhamnose after relatively long simulation intervals. Further, we explore the mechanism of conformational collapse and show that this phenomenon arises because of the anomalous low energy in GLYCAM06j (as compared to quantum mechanical calculations) of a specific orientation of α-l-Rha to α-l-Rha glycosidic linkages, which are subsequently sustained by intramolecular interactions in the saccharide chain. We identify the lack of partial charges on aliphatic hydrogens in GLYCAM as the source of this anomaly, demonstrating that addition of small partial atomic charges on the aliphatic protons in rhamnose removes the conformational collapse phenomenon. This work reveals the large cumulative impact that small partial charges may have on the dynamic behavior of polysaccharides and indicates that future reparameterization of the GLYCAM06j force field should investigate the addition of partial charges on all aliphatic hydrogens.

Characterisation of a new cell wall teichoic acid produced by Listeria innocua ZM39 and analysis of its biosynthesis genes.

Listeria innocua is genetically closely related to the foodborne human pathogen Listeria monocytogenes. However, as most L. innocua strains are non-pathogenic, it has been proposed as a surrogate organism for determining the efficacy of antimicrobial strategies against L. monocytogenes. Teichoic acids are one of the three major cell wall components of Listeria, along with the peptidoglycan backbone and cell wall-associated proteins. The polymeric teichoic acids make up the majority of cell wall carbohydrates; the type of teichoic acids directly attached to the peptidoglycan are termed wall teichoic acids (WTAs). WTAs play vital physiological roles, are important virulence factors, antigenic determinants, and phage-binding ligands. The structures of the various WTAs of L. monocytogenes are well known, whereas those of L. innocua are not. In the present study, the WTA structure of L. innocua ZM39 was determined mainly by 1D and 2D NMR spectroscopy and it was found to be the following: [→4)-[α-D-GlcpNAc-(1→3)]-ß-D-GlcpNAc-(1→4)-D-Rbo-(1P→]n This structure is new with respect to all currently known Listeria WTAs and it shares structural similarities with type II WTA serovar 6a. In addition, the genome of strain L. innocua ZM39 was sequenced and the majority of putative WTA synthesis genes were identified.

Partial depolymerization of capsular polysaccharides isolated from Streptococcus pneumoniae serotype 2 by various methods.

Partial depolymerization of bacterial capsular polysaccharides (CPS) is an essential process carried out before its use as an antigenic preparation in a vaccine industry. Choice of CPS depolymerization methods depends on the process robustness, reproducibility, yield, retention of CPS bioactivity, etc. Partial depolymerization methods based on chemicals, enzymes, mechanical, thermal, etc. have been subject of many investigations before. Partial depolymerization of Streptococcus pneumoniae serotype 2 purified CPS was conducted by methods such as acid hydrolysis, microfluidization, ultrasonication, thermal and microwave. Partial depolymerization of the CPS was evaluated by size exclusion high performance liquid chromatography, whereas structural identity and conformity of CPS was ensured by 1H NMR spectroscopy. The antigenicity of CPS was assessed by bead based competitive inhibition assay. Microwave and thermal methods effectively depolymerized CPS and reduced the concentration of cell wall polysaccharide (CWPS) impurity, but both methods have a negative impact on the antigenicity of CPS. Whereas the trifluoroacetic acid treatment not only depolymerized the CPS but completely removed the CWPS while retaining the antigenicity of 92 ± 4% and this method is advantageous over other methods.

Composition and inhibitory properties of endogenous urinary GAGS are different in subjects from two race groups with different occurrence rates of kidney stones: Pilot studies provide unique evidence in support of an inhibitory role for this group of compounds.

BACKGROUND: Calcium oxalate (CaOx) kidney stone disease is common in South African whites (W) but is rare in the black population (B). The possible role of endogenous urinary glycosaminoglycans (GAGs) has not been previously investigated in this context. AIM: To determine concentration, composition, structure and CaOx crystal-inhibiting properties of this group of compounds in ultrafiltered urine of healthy subjects from both groups. MATERIALS AND METHODS: GAGS were isolated from 24 h urine samples and were quantified and characterized by sequential precipitation, Bradford protein assay, high performance liquid chromatography, and anion exchange high performance chromatography. CaOx crystal inhibition was determined in ultrafiltered urinary fractions to which purified GAGS (PG) from each group (PGB and PGW) had been added. Nucleation, growth and aggregation were measured by Coulter particle counting, spectrophotometric assay and [14C]-oxalate deposition. RESULTS: Higher concentrations of chondroitin sulfate (CS) were found in PGB than in PGW. PGB inhibited crystallization to a greater extent than PGW. CONCLUSIONS: We attribute the stronger inhibitory effect of PGB to its higher content of CS and suggest that the superior inhibition of CaOx crystallization by PGB relative to PGW might be a contributory factor in accounting for the lower stone occurrence rate in B.

Cross-reactivity of Haemophilus influenzae type a and b polysaccharides: molecular modeling and conjugate immunogenicity studies.

Haemophilus influenzae is a leading cause of meningitis disease and mortality, particularly in young children. Since the introduction of a licensed conjugate vaccine (targeting the outer capsular polysaccharide) against the most prevalent serotype, Haemophilus influenzae serotype b, the epidemiology of the disease has changed and Haemophilus influenzae serotype a is on the rise, especially in Indigenous North American populations. Here we apply molecular modeling to explore the preferred conformations of the serotype a and b capsular polysaccharides as well as a modified hydrolysis resistant serotype b polysaccharide. Although both serotype b and the modified serotype b have similar random coil behavior, our simulations reveal some differences in the polysaccharide conformations and surfaces which may impact antibody cross-reactivity between these two antigens. Importantly, we find significant conformational differences between the serotype a and b polysaccharides, indicating a potential lack of cross-reactivity that is corroborated by immunological data showing little recognition or killing between heterologous serotypes. These findings support the current development of a serotype a conjugate vaccine.

Evaluation of Critical Quality Attributes of a Pentavalent (A, C, Y, W, X) Meningococcal Conjugate Vaccine for Global Use.

Towards achieving the goal of eliminating epidemic outbreaks of meningococcal disease in the African meningitis belt, a pentavalent glycoconjugate vaccine (NmCV-5) has been developed to protect against Neisseria meningitidis serogroups A, C, Y, W and X. MenA and X polysaccharides are conjugated to tetanus toxoid (TT) while MenC, Y and W polysaccharides are conjugated to recombinant cross reactive material 197 (rCRM197), a non-toxic genetic variant of diphtheria toxin. This study describes quality control testing performed by the manufacturer, Serum Institute of India Private Limited (SIIPL), and the independent control laboratory of the U.K. (NIBSC) on seven clinical lots of the vaccine to ensure its potency, purity, safety and consistency of its manufacturing. In addition to monitoring upstream-manufactured components, samples of drug substance, final drug product and stability samples were evaluated. This paper focuses on the comparison of the vaccine's critical quality attributes and reviews key indicators of its stability and immunogenicity. Comparable results were obtained by the two laboratories demonstrating sufficient levels of polysaccharide O-acetylation, consistency in size of the bulk conjugate molecules, integrity of the conjugated saccharides in the drug substance and drug product, and acceptable endotoxin content in the final drug product. The freeze-dried vaccine in 5-dose vials was stable based on molecular sizing and free saccharide assays. Lot-to-lot manufacturing consistency was also demonstrated in preclinical studies for polysaccharide-specific IgG and complement-dependent serum bactericidal activity for each serogroup. This study demonstrates the high quality and stability of NmCV-5, which is now undergoing Phase 3 clinical trials in Africa and India.

Conformational and Immunogenicity Studies of the Shigella flexneri Serogroup 6 O-Antigen: The Effect of O-Acetylation.

The pathogenic bacterium Shigella is a leading cause of diarrheal disease and mortality, disproportionately affecting young children in low-income countries. The increasing prevalence of antibiotic resistance in Shigella necessitates an effective vaccine, for which the bacterial lipopolysaccharide O-antigen is the primary target. S. flexneri serotype 6 has been proposed as a multivalent vaccine component to ensure broad protection against Shigella. We have previously explored the conformations of S. flexneri O-antigens from serogroups Y, 2, 3, and 5 that share a common saccharide backbone (serotype Y). Here we consider serogroup 6, which is of particular interest because of an altered backbone repeat unit with non-stoichiometric O-acetylation, the antigenic and immunogenic importance of which have yet to be established. Our simulations show significant conformational changes in serogroup 6 relative to the serotype Y backbone. We further find that O-acetylation has little effect on conformation and hence may not be essential for the antigenicity of serotype 6. This is corroborated by an in vivo study in mice, using Generalized Modules for Membrane Antigens (GMMA) as O-antigen delivery systems, that shows that O-acetylation does not have an impact on the immune response elicited by the S. flexneri serotype 6 O-antigen.