MALDI glycotyping of O-antigens from a single colony of gram-negative bacteria.

Polypeptide-targeted MALDI-TOF MS for microbial species identification has revolutionized microbiology. However, no practical MALDI-TOF MS identification method for O-antigen polysaccharides, a major indicator for epidemiological classification within a species of gram-negative bacteria, is available. We describe a simple MALDI glycotyping method for O-antigens that simultaneously identifies the molecular mass of the repeating units and the monosaccharide composition of the O-antigen. We analyzed the Escherichia coli O1, O6, and O157-type strains. Conventional species identification based on polypeptide patterns and O-antigen polysaccharide typing can be performed in parallel from a single colony using our MALDI-TOF MS workflow. Moreover, subtyping within the same O-antigen and parallel colony-specific O-antigen determination from mixed strains, including the simultaneous identification of multiple strains-derived O-antigens within selected colony, were performed. In MALDI glycotyping of two Enterobacteriaceae strains, a Citrobacter freundii strain serologically cross-reactive with E. coli O157 gave a MALDI spectral pattern identical to E. coli O157. On the other hand, an Edwardsiella tarda strain with no reported O-antigen cross-reactivity gave a MALDI spectral pattern of unknown O-antigen repeating units. The method described in this study allows the parallel and rapid identification of microbial genera, species, and serotypes of surface polysaccharides using a single MALDI-TOF MS instrument.

Lipopolysaccharide with long O-antigen is crucial for Salmonella Enteritidis to evade complement activity and to facilitate bacterial survival in vivo in the Galleria mellonella infection model.

Bacterial resistance to serum is a key virulence factor for the development of systemic infections. The amount of lipopolysaccharide (LPS) and the O-antigen chain length distribution on the outer membrane, predispose Salmonella to escape complement-mediated killing. In Salmonella enterica serovar Enteritidis (S. Enteritidis) a modal distribution of the LPS O-antigen length can be observed. It is characterized by the presence of distinct fractions: low molecular weight LPS, long LPS and very long LPS. In the present work, we investigated the effect of the O-antigen modal length composition of LPS molecules on the surface of S. Enteritidis cells on its ability to evade host complement responses. Therefore, we examined systematically, by using specific deletion mutants, roles of different O-antigen fractions in complement evasion. We developed a method to analyze the average LPS lengths and investigated the interaction of the bacteria and isolated LPS molecules with complement components. Additionally, we assessed the aspect of LPS O-antigen chain length distribution in S. Enteritidis virulence in vivo in the Galleria mellonella infection model. The obtained results of the measurements of the average LPS length confirmed that the method is suitable for measuring the average LPS length in bacterial cells as well as isolated LPS molecules and allows the comparison between strains. In contrast to earlier studies we have used much more precise methodology to assess the LPS molecules average length and modal distribution, also conducted more subtle analysis of complement system activation by lipopolysaccharides of various molecular mass. Data obtained in the complement activation assays clearly demonstrated that S. Enteritidis bacteria require LPS with long O-antigen to resist the complement system and to survive in the G. mellonella infection model.

Preclinical validation of an Escherichia coli O-antigen glycoconjugate for the prevention of serotype O1 invasive disease.

A US collection of invasive Escherichia coli serotype O1 bloodstream infection (BSI) isolates were assessed for genotypic and phenotypic diversity as the basis for designing a broadly protective O-antigen vaccine. Eighty percent of the BSI isolate serotype O1 strains were genotypically ST95 O1:K1:H7. The carbohydrate repeat unit structure of the O1a subtype was conserved in the three strains tested representing core genome multi-locus sequence types (MLST) sequence types ST95, ST38, and ST59. A long-chain O1a CRM197 lattice glycoconjugate antigen was generated using oxidized polysaccharide and reductive amination chemistry. Two ST95 strains were investigated for use in opsonophagocytic assays (OPA) with immune sera from vaccinated animals and in murine lethal challenge models. Both strains were susceptible to OPA killing with O1a glycoconjugate post-immune sera. One of these, a neonatal sepsis strain, was found to be highly lethal in the murine challenge model for which virulence was shown to be dependent on the presence of the K1 capsule. Mice immunized with the O1a glycoconjugate were protected from challenges with this strain or a second, genotypically related, and similarly virulent neonatal isolate. This long-chain O1a CRM197 lattice glycoconjugate shows promise as a component of a multi-valent vaccine to prevent invasive E. coli infections. IMPORTANCE: The Escherichia coli serotype O1 O-antigen serogroup is a common cause of invasive bloodstream infections (BSI) in populations at risk such as newborns and the elderly. Sequencing of US BSI isolates and structural analysis of O polysaccharide antigens purified from strains that are representative of genotypic sub-groups confirmed the relevance of the O1a subtype as a vaccine antigen. O polysaccharide was purified from a strain engineered to produce long-chain O1a O-antigen and was chemically conjugated to CRM197 carrier protein. The resulting glycoconjugate elicited functional antibodies and was protective in mice against lethal challenges with virulent K1-encapsulated O1a isolates.

Distribution of serotypes and antibiotic resistance of invasive Pseudomonas aeruginosa in a multi-country collection.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of acute and chronic infections and is frequently associated with healthcare-associated infections. Because of its ability to rapidly acquire resistance to antibiotics, P. aeruginosa infections are difficult to treat. Alternative strategies, such as a vaccine, are needed to prevent infections. We collected a total of 413 P. aeruginosa isolates from the blood and cerebrospinal fluid of patients from 10 countries located on 4 continents during 2005-2017 and characterized these isolates to inform vaccine development efforts. We determined the diversity and distribution of O antigen and flagellin types and antibiotic susceptibility of the invasive P. aeruginosa. We used an antibody-based agglutination assay and PCR for O antigen typing and PCR for flagellin typing. We determined antibiotic susceptibility using the Kirby-Bauer disk diffusion method. RESULTS: Of the 413 isolates, 314 (95%) were typed by an antibody-based agglutination assay or PCR (n = 99). Among the 20 serotypes of P. aeruginosa, the most common serotypes were O1, O2, O3, O4, O5, O6, O8, O9, O10 and O11; a vaccine that targets these 10 serotypes would confer protection against more than 80% of invasive P. aeruginosa infections. The most common flagellin type among 386 isolates was FlaB (41%). Resistance to aztreonam (56%) was most common, followed by levofloxacin (42%). We also found that 22% of strains were non-susceptible to meropenem and piperacillin-tazobactam. Ninety-nine (27%) of our collected isolates were resistant to multiple antibiotics. Isolates with FlaA2 flagellin were more commonly multidrug resistant (p = 0.04). CONCLUSIONS: Vaccines targeting common O antigens and two flagellin antigens, FlaB and FlaA2, would offer an excellent strategy to prevent P. aeruginosa invasive infections.

Lipopolysaccharide O-antigen molecular and supramolecular modifications of plant root microbiota are pivotal for host recognition.

Lipopolysaccharides, the major outer membrane components of Gram-negative bacteria, are crucial actors of the host-microbial dialogue. They can contribute to the establishment of either symbiosis or bacterial virulence, depending on the bacterial lifestyle. Plant microbiota shows great complexity, promotes plant health and growth and assures protection from pathogens. How plants perceive LPS from plant-associated bacteria and discriminate between beneficial and pathogenic microbes is an open and urgent question. Here, we report on the structure, conformation, membrane properties and immune recognition of LPS isolated from the Arabidopsis thaliana root microbiota member Herbaspirillum sp. Root189. The LPS consists of an O-methylated and variously acetylated D-rhamnose containing polysaccharide with a rather hydrophobic surface. Plant immunology studies in A. thaliana demonstrate that the native acetylated O-antigen shields the LPS from immune recognition whereas the O-deacylated one does not. These findings highlight the role of Herbaspirillum LPS within plant-microbial crosstalk, and how O-antigen modifications influence membrane properties and modulate LPS host recognition.

Structural Determination and Genetic Identification of the O-Antigen from an Escherichia coli Strain, LL004, Representing a Novel Serogroup.

The O-antigen is the outermost component of the lipopolysaccharide layer in Gram-negative bacteria, and the variation of O-antigen structure provides the basis for bacterial serological diversity. Here, we determined the O-antigen structure of an Escherichia coli strain, LL004, which is totally different from all of the E. coli serogroups. The tetrasaccharide repeating unit was determined as →4)-ß-d-Galp-(1→3)-ß-d-GlcpNAc6OAc(~70%)-(1→3)-ß-d-GalpA-(1→3)-ß-d-GalpNAc-(1→ with monosaccharide analysis and NMR spectra. We also characterized the O-antigen gene cluster of LL004, and sequence analysis showed that it correlated well with the O-antigen structure. Deletion and complementation testing further confirmed its role in O-antigen biosynthesis, and indicated that the O-antigen of LL004 is assembled via the Wzx/Wzy dependent pathway. Our findings, in combination, suggest that LL004 should represent a novel serogroup of E. coli.

Rationalizing the design of a broad coverage Shigella vaccine based on evaluation of immunological cross-reactivity among S. flexneri serotypes.

No vaccine to protect against an estimated 238,000 shigellosis deaths per year is widely available. S. sonnei is the most prevalent Shigella, and multiple serotypes of S. flexneri, which change regionally and globally, also cause significant disease. The leading Shigella vaccine strategies are based on the delivery of serotype specific O-antigens. A strategy to minimize the complexity of a broadly-protective Shigella vaccine is to combine components from S. sonnei with S. flexneri serotypes that induce antibodies with maximum cross-reactivity between different serotypes. We used the GMMA-technology to immunize animal models and generate antisera against 14 S. flexneri subtypes from 8 different serotypes that were tested for binding to and bactericidal activity against a panel of 11 S. flexneri bacteria lines. Some immunogens induced broadly cross-reactive antibodies that interacted with most of the S. flexneri in the panel, while others induced antibodies with narrower specificity. Most cross-reactivity could not be assigned to modifications of the O-antigen, by glucose, acetate or phosphoethanolamine, common to several of the S. flexneri serotypes. This allowed us to revisit the current dogma of cross-reactivity among S. flexneri serotypes suggesting that a broadly protective vaccine is feasible with limited number of appropriately selected components. Thus, we rationally designed a 4-component vaccine selecting GMMA from S. sonnei and S. flexneri 1b, 2a and 3a. The resulting formulation was broadly cross-reactive in mice and rabbits, inducing antibodies that killed all S. flexneri serotypes tested. This study provides the framework for a broadly-protective Shigella vaccine which needs to be verified in human trials.

The prevailing O serogroups among the serologically differentiated clinical Proteus spp. strains in central Poland.

In the years 2006-2011, 617 Proteus spp. strains isolated mostly from urine and wounds or other clinical sources were collected in Lódz, Poland, to determine the offensive O serotypes frequently occurring among patients. P. mirabilis exhibited the most intensive swarming growth and was dominating species (86.9%), followed by P. genomospecies, P. vulgaris, and P. penneri. Ninety four per cent strains were recognized as S (smooth) forms. Serological studies (involving ELISA-enzyme-linked immunosorbent assay and Western blotting using native and adsorbed rabbit antisera) enabled classification of 80% S isolates into respective Proteus O serogroups among the 83 ones, described so far. The remaining strains seemed to be serologically unique. Despite the observed big serological variety of Proteus spp. isolates, we found the O78 serogroup recently described in Poland as dominating and identified other widespread serotypes: O3, O6, O10, O11, O27, O28, and O30 reported earlier as predominating also in other countries; O77 and O79 detected lately in Poland; O16, O18, O20, and O50. No unique structural feature of the prevalent O serotypes has been indicated. However, the prevalence of some O serogroups indicates that particular serotypes may be in some ways beneficial to the strains producing these kinds of O antigen.

Defining Polysaccharide-Specific Antibody Targets against Vibrio cholerae O139 in Humans following O139 Cholera and following Vaccination with a Commercial Bivalent Oral Cholera Vaccine, and Evaluation of Conjugate Vaccines Targeting O139.

Cholera caused by Vibrio cholerae O139 could reemerge, and proactive development of an effective O139 vaccine would be prudent. To define immunoreactive and potentially immunogenic carbohydrate targets of Vibrio cholerae O139, we assessed immunoreactivities of various O-specific polysaccharide (OSP)-related saccharides with plasma from humans hospitalized with cholera caused by O139, comparing responses to those induced in recipients of a commercial oral whole-cell killed bivalent (O1 and O139) cholera vaccine (WC-O1/O139). We also assessed conjugate vaccines containing selected subsets of these saccharides for their ability to induce protective immunity using a mouse model of cholera. We found that patients with wild-type O139 cholera develop IgM, IgA, and IgG immune responses against O139 OSP and many of its fragments, but we were able to detect only a moderate IgM response to purified O139 OSP-core, and none to its fragments, in immunologically naive recipients of WC-O1/O139. We found that immunoreactivity of O139-specific polysaccharides with antibodies elicited by wild-type infection markedly increase when saccharides contain colitose and phosphate residues, that a synthetic terminal tetrasaccharide fragment of OSP is more immunoreactive and protectively immunogenic than complete OSP, that native OSP-core is a better protective immunogen than the synthetic OSP lacking core, and that functional vibriocidal activity of antibodies predicts in vivo protection in our model but depends on capsule thickness. Our results suggest that O139 OSP-specific responses are not prominent following vaccination with a currently available oral cholera vaccine in immunologically naive humans and that vaccines targeting V. cholerae O139 should be based on native OSP-core or terminal tetrasaccharide. IMPORTANCE Cholera is a severe dehydrating illness of humans caused by Vibrio cholerae serogroup O1 or O139. Protection against cholera is serogroup specific, and serogroup specificity is defined by O-specific polysaccharide (OSP). Little is known about immunity to O139 OSP. In this study, we used synthetic fragments of the O139 OSP to define immune responses to OSP in humans recovering from cholera caused by V. cholerae O139, compared these responses to those induced by the available O139 vaccine, and evaluated O139 fragments in next-generation conjugate vaccines. We found that the terminal tetrasaccharide of O139 is a primary immune target but that the currently available bivalent cholera vaccine poorly induces an anti-O139 OSP response in immunologically naive individuals.

A rationally designed oral vaccine induces immunoglobulin A in the murine gut that directs the evolution of attenuated Salmonella variants.

The ability of gut bacterial pathogens to escape immunity by antigenic variation-particularly via changes to surface-exposed antigens-is a major barrier to immune clearance1. However, not all variants are equally fit in all environments2,3. It should therefore be possible to exploit such immune escape mechanisms to direct an evolutionary trade-off. Here, we demonstrate this phenomenon using Salmonella enterica subspecies enterica serovar Typhimurium (S.Tm). A dominant surface antigen of S.Tm is its O-antigen: a long, repetitive glycan that can be rapidly varied by mutations in biosynthetic pathways or by phase variation4,5. We quantified the selective advantage of O-antigen variants in the presence and absence of O-antigen-specific immunoglobulin A and identified a set of evolutionary trajectories allowing immune escape without an associated fitness cost in naive mice. Through the use of rationally designed oral vaccines, we induced immunoglobulin A responses blocking all of these trajectories. This selected for Salmonella mutants carrying deletions of the O-antigen polymerase gene wzyB. Due to their short O-antigen, these evolved mutants were more susceptible to environmental stressors (detergents or complement) and predation (bacteriophages) and were impaired in gut colonization and virulence in mice. Therefore, a rationally induced cocktail of intestinal antibodies can direct an evolutionary trade-off in S.Tm. This lays the foundations for the exploration of mucosal vaccines capable of setting evolutionary traps as a prophylactic strategy.

Antibodies Elicited by the Shigella sonnei GMMA Vaccine in Adults Trigger Complement-Mediated Serum Bactericidal Activity: Results From a Phase 1 Dose Escalation Trial Followed by a Booster Extension.

Shigella is the second most deadly diarrheal disease among children under five years of age, after rotavirus, with high morbidity and mortality in developing countries. Currently, no vaccine is widely available, and the increasing levels of multidrug resistance make Shigella a high priority for vaccine development. The single-component candidate vaccine against Shigella sonnei (1790GAHB), developed using the GMMA technology, contains the O antigen (OAg) portion of lipopolysaccharide (LPS) as active moiety. The vaccine was well tolerated and immunogenic in early-phase clinical trials. In a phase 1 placebo-controlled dose escalation trial in France (NCT02017899), three doses of five different vaccine formulations (0.06/1, 0.3/5, 1.5/25, 3/50, 6/100 µg of OAg/protein) were administered to healthy adults. In the phase 1 extension trial (NCT03089879), conducted 2-3 years following the parent study, primed individuals who had undetectable antibody levels before the primary series received a 1790GAHB booster dose (1.5/25 µg OAg/protein). Controls were unprimed participants immunized with one 1790GAHB dose. The current analysis assessed the functionality of sera collected from both studies using a high-throughput luminescence-based serum bactericidal activity (SBA) assay optimized for testing human sera. Antibodies with complement-mediated bactericidal activity were detected in vaccinees but not in placebo recipients. SBA titers increased with OAg dose, with a persistent response up to six months after the primary vaccination with at least 1.5/25 µg of OAg/protein. The booster dose induced a strong increase of SBA titers in most primed participants. Correlation between SBA titers and anti-S. sonnei LPS serum immunoglobulin G levels was observed. Results suggest that GMMA is a promising OAg delivery system for the generation of functional antibody responses and persistent immunological memory.

Impact of Immunoglobulin Isotype and Epitope on the Functional Properties of Vibrio cholerae O-Specific Polysaccharide-Specific Monoclonal Antibodies.

Vibrio cholerae causes the severe diarrheal disease cholera. Clinical disease and current oral cholera vaccines generate antibody responses associated with protection. Immunity is thought to be largely mediated by lipopolysaccharide (LPS)-specific antibodies, primarily targeting the O-antigen. However, the properties and protective mechanism of functionally relevant antibodies have not been well defined. We previously reported on the early B cell response to cholera in a cohort of Bangladeshi patients, from which we characterized a panel of human monoclonal antibodies (MAbs) isolated from acutely induced plasmablasts. All antibodies in that previous study were expressed in an IgG1 backbone irrespective of their original isotype. To clearly determine the impact of affinity, immunoglobulin isotype and subclass on the functional properties of these MAbs, we re-engineered a subset of low- and high-affinity antibodies in different isotype and subclass immunoglobulin backbones and characterized the impact of these changes on binding, vibriocidal, agglutination, and motility inhibition activity. While the high-affinity antibodies bound similarly to O-antigen, irrespective of isotype, the low-affinity antibodies displayed significant avidity differences. Interestingly, despite exhibiting lower binding properties, variants derived from the low-affinity MAbs had comparable agglutination and motility inhibition properties to the potently binding antibodies, suggesting that how the MAb binds to the O-antigen may be critical to function. In addition, not only pentameric IgM and dimeric IgA, but also monomeric IgA, was remarkably more potent than their IgG counterparts at inhibiting motility. Finally, analyzing highly purified F(ab) versions of these antibodies, we show that LPS cross-linking is essential for motility inhibition.IMPORTANCE Immunity to the severe diarrheal disease cholera is largely mediated by lipopolysaccharide (LPS)-specific antibodies. However, the properties and protective mechanisms of functionally relevant antibodies have not been well defined. Here, we have engineered low and high-affinity LPS-specific antibodies in different immunoglobulin backbones in order to assess the impact of affinity, immunoglobulin isotype, and subclass on binding, vibriocidal, agglutination, and motility inhibition functional properties. Importantly, we found that affinity did not directly dictate functional potency since variants derived from the low-affinity MAbs had comparable agglutination and motility inhibition properties to the potently binding antibodies. This suggests that how the antibody binds sterically may be critical to function. In addition, not only pentameric IgM and dimeric IgA, but also monomeric IgA, was remarkably more potent than their IgG counterparts at inhibiting motility. Finally, analyzing highly purified F(ab) versions of these antibodies, we show that LPS cross-linking is essential for motility inhibition.

Effect of O-Antigen Chain Length Regulation on the Immunogenicity of Shigella and Salmonella Generalized Modules for Membrane Antigens (GMMA).

Recently, generalized modules for membrane antigens (GMMA) technology has been proposed as an alternative approach to traditional glycoconjugate vaccines for O-antigen delivery. Saccharide length is a well-known parameter that can impact the immune response induced by glycoconjugates both in terms of magnitude and quality. However, the criticality of O-antigen length on the immune response induced by GMMA-based vaccines has not been fully elucidated. Here, Shigella and Salmonella GMMA-producing strains were further mutated in order to display homogeneous polysaccharide populations of different sizes on a GMMA surface. Resulting GMMA were compared in mice immunization studies. Athymic nude mice were also used to investigate the involvement of T-cells in the immune response elicited. In contrast with what has been reported for traditional glycoconjugate vaccines and independent of the pathogen and the sugar structural characteristics, O-antigen length did not result in being a critical parameter for GMMA immunogenicity. This work supports the identification of critical quality attributes to optimize GMMA vaccine design and improve vaccine efficacy and gives insights on the nature of the immune response induced by GMMA.

The Role of ß-Catenin in Th1 Immune Response against Tuberculosis and Profiles of Expression in Patients with Pulmonary Tuberculosis.

ß-Catenin is a key molecule of canonical Wnt/ß-catenin pathway. Its roles and expression profiles in T cells of tuberculosis (TB) remain unclear. The aim of this study was to explore the role of ß-catenin in CD4+ T cells and its expression characteristics in patients with pulmonary tuberculosis (PTB). In this study, CD4+ T cell-specific ß-catenin conditional knockout mice (ß-CAT-cKO mice) were aerosol infected with Mycobacteria tuberculosis (Mtb) H37RV with wild-type mice as controls. Four weeks after infection, the mRNA expression of IFN-γ, TNF-α, and TCF-7 in the lungs of mice was measured. CD4, CD8, ß-catenin, IFN-γ, and TNF-α in mononuclear cells from the lungs and spleens were measured by flow cytometry, and the pathological changes of lungs were also observed. Patients with PTB were enrolled, with blood samples collected and PBMCs isolated. The expressions of ß-catenin, IFN-γ, TNF-α, and PD-1 in CD4+ and CD8+ T cells were measured by flow cytometry. Results showed a decreased frequency of and reduced IFN-γ/TNF-α mRNA expression and secretion by CD4+ T cells in the lungs of infected ß-CAT-cKO mice compared with infected wild-type controls, and only slightly more inflammatory changes were observed in the lungs. ß-catenin expressions in CD4+ and CD8+ T cells were significantly decreased in blood cells of patients with severe PTB compared with those in mild PTB. The stimulation of peripheral blood mononuclear cells (PBMCs) with lithium chloride (LiCl), a stimulant of ß-catenin, resulted in the increase in CD4+ T cell frequency, as well as their secretion of IFN-γ and TNF-α. ß-Catenin demonstrated a moderately positive correlation with PD-1 in CD4+ T cells. ß-Catenin along with PD-1 and IFN-γ in CD4+ T cells had a high correlation with those in CD8+ T cells. In conclusion, ß-catenin may be involved in the regulation of Th1 response and CD4+ T cell frequency in TB.

Shielding Effect of Escherichia coli O-Antigen Polysaccharide on J5-Induced Cross-Reactive Antibodies.

Escherichia coli is the leading cause of severe mastitis in dairy farms. As E. coli mastitis is refractory to the hygienic control measures adapted to contagious mastitis, efficient vaccines are in demand. Existing mastitis vaccines, based on the use of killed rough E. coli J5 as the antigen, aim at inducing phagocytosis by neutrophils. We assessed the binding of J5-induced antibodies to isogenic rough and smooth strains along with a panel of mastitis-associated E. coli Analysis by enzyme-linked immunosorbent assay revealed that antibodies to OmpA or killed J5 bind readily to rough E. coli but poorly to smooth strains. Flow cytometry analysis indicated that immunization with J5 induced antibodies that cross-reacted with rough E. coli strains but with only a small subpopulation of smooth strains. We identified type 1 fimbriae as the target of most antibodies cross-reacting with the smooth strains. These results suggest that the O-polysaccharide of lipopolysaccharide shields the outer membrane antigens and that only fiber antigens protruding at the bacterial surface can elicit antibodies reacting with mastitis-associated E. coli We evaluated J5-induced antibodies in an opsonophagocytic killing assay with bovine neutrophils. J5 immune serum was not more efficient than preimmune serum, showing that immunization did not improve on the already high efficiency of naturally acquired antibodies to E. coli In conclusion, it is unlikely that the efficiency of J5 vaccines is related to the induction of opsonic antibodies. Consequently, other research directions, such as cell-mediated immunity, should be explored to improve E. coli mastitis vaccines.IMPORTANCE Despite intensive research, mastitis remains an important disease in dairy cattle with a significant impact on animal welfare, use of antibiotics, and, in the end, the economy of dairy farms. Although vaccines available so far have shown limited efficacy against coliform mastitis, vaccination is considered one of the measures that could limit the consequences of mastitis. One reason for the lack of efficiency of current vaccines likely stems from the current evaluation of vaccines that relies mostly on measuring antibody production against vaccine antigens. This report clearly shows that vaccine-induced antibodies fail to bind to most mastitis-associated E. coli strains because of the presence of an O-antigen and, thus, do not allow for improved phagocytosis of pathogens. As a consequence, this report calls for revised criteria for the evaluation of vaccines and suggests that cell-mediated immunity should be targeted by new vaccinal strategies. More generally, these results could be extended to other vaccine development strategies targeting coliform bacteria.

Dissecting the contribution of O-Antigen and proteins to the immunogenicity of Shigella sonnei generalized modules for membrane antigens (GMMA).

GMMA are exosomes released from engineered Gram-negative bacteria resembling the composition of outer membranes. We applied the GMMA technology for the development of an O-Antigen (OAg) based vaccine against Shigella sonnei, the most epidemiologically relevant cause of shigellosis. S. sonnei OAg has been identified as a key antigen for protective immunity, and GMMA are able to induce anti-OAg-specific IgG response in animal models and healthy adults. The contribution of protein-specific antibodies induced upon vaccination with GMMA has never been fully elucidated. Anti-protein antibodies are induced in mice upon immunization with either OAg-negative and OAg-positive GMMA. Here we demonstrated that OAg chains shield the bacteria from anti-protein antibody binding and therefore anti-OAg antibodies were the main drivers of bactericidal activity against OAg-positive bacteria. Interestingly, antibodies that are not targeting the OAg are functional against OAg-negative bacteria. The immunodominant protein antigens were identified by proteomic analysis. Our study confirms a critical role of the OAg on the immune response induced by S. sonnei GMMA. However, little is known about OAg length and density regulation during infection and, therefore, protein exposure. Hence, the presence of protein antigens on S. sonnei GMMA represents an added value for GMMA vaccines compared to other OAg-based formulations.

High efficiency biosynthesis of O-polysaccharide-based vaccines against extraintestinal pathogenic Escherichia coli.

Extraintestinal pathogenic Escherichia coli (ExPEC) has presented a major clinical infection emerged in the past decades. O-polysaccharide (OPS)-based glycoconjugate vaccines produced using the bacterial glycosylation machinery can be utilized to confer protection against such infection. However, constructing a low-cost microbial cell factory for high-efficient production of OPS-based glycoconjugate vaccines remains challenging. Here, we engineered a glyco-optimized chassis strain by reprogramming metabolic network. The yield was enhanced to 38.6 mg L-1, the highest level reported so far. MS analysis showed that designed glycosylation sequon was modified by target polysaccharide with high glycosylation efficiency of 90.7 % and 76.7 % for CTB-O5 and CTB-O7, respectively. The glycoconjugate vaccines purified from this biosystem elicited a marked increase in protection against ExPEC infection in mouse model, compared to a non-optimized system. The glyco-optimized platform established here is broadly suitable for polysaccharide-based conjugate production against ExPEC and other surface-polysaccharide-producing pathogens.

Salmonella Paratyphi A Outer Membrane Vesicles Displaying Vi Polysaccharide as a Multivalent Vaccine against Enteric Fever.

Typhoid and paratyphoid fevers have a high incidence worldwide and coexist in many geographical areas, especially in low-middle-income countries (LMIC) in South and Southeast Asia. There is extensive consensus on the urgent need for better and affordable vaccines against systemic Salmonella infections. Generalized modules for membrane antigens (GMMA), outer membrane exosomes shed by Salmonella bacteria genetically manipulated to increase blebbing, resemble the bacterial surface where protective antigens are displayed in their native environment. Here, we engineered S Paratyphi A using the pDC5-viaB plasmid to generate GMMA displaying the heterologous S Typhi Vi antigen together with the homologous O:2 O antigen. The presence of both Vi and O:2 was confirmed by flow cytometry on bacterial cells, and their amount was quantified on the resulting vesicles through a panel of analytical methods. When tested in mice, such GMMA induced a strong antibody response against both Vi and O:2, and these antibodies were functional in a serum bactericidal assay. Our approach yielded a bivalent vaccine candidate able to induce immune responses against different Salmonella serovars, which could benefit LMIC residents and travelers.

O-Antigen Extraction, Purification, and Chemical Conjugation to a Carrier Protein.

A variety of bacterial infections have been tackled by glycoconjugates over the recent years, and more vaccines are either under development at preclinical level or in clinical trials. So far, licensed glycoconjugate vaccines have made use of capsular polysaccharides or derived fragments. Today, many glycoconjugates are making use of other classes of sugars, in particular, the O-antigen portion of lipopolysaccharide molecules. Here, we report a simplified method for O-antigen extraction and purification that avoids the step of lipopolysaccharide isolation. Also, a selective chemistry for terminal linkage of O-antigen chains to a carrier protein is described, together with analytical methods for intermediates and final conjugate characterization.