Vi antigen of <Emphasis Type="Italic">Salmonella</Emphasis> enetrica serovar Typhi — biosynthesis,regulation and its use as vaccine candidate

Vi capsular polysaccharide (Vi antigen) was first identified as the virulence antigen of Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever in humans. The presence of Vi antigen differentiates S. Typhi from other serovars of Salmonella. Vi antigen is a linear polymer consisting of α-1,4-linked-N-acetyl-galactosaminuronate, whose expression is controlled by three chromosomal loci, namely viaA, viaB and ompB. Both viaA and viaB region are present on Salmonella Pathogenicity Island-7, a large, mosaic, genetic island. The viaA region encodes a positive regulator and the viaB locus is composed of 11 genes designated tviA-tviE (for Vi biosyhthesis), vexA-vexE (for Vi antigen export) and ORF 11. Vi polysaccharide is synthesized from UDP-N-acetyl glucosamine in a series of steps requiring TviB, TviC, and TviE, and regulation of Vi polysaccharide synthesis is controlled by two regulatory systems, rscB-rscC (viaA locus) and ompR-envZ (ompB locus), which respond to changes in osmolarity. This antigen is highly immunogenic and has been used for the formulation of one of the currently available vaccines against typhoid. Despite advancement in the area of vaccinology, its pace of progress needs to be accelerated and effective control programmes will be needed for proper disease management.     

Molecular Characterization of the viaB Locus Encoding the Biosynthetic Machinery for Vi Capsule Formation in Salmonella Typhi

The Vi capsular polysaccharide (CPS) of Salmonella enterica serovar Typhi, the cause of human typhoid, is important for infectivity and virulence. The Vi biosynthetic machinery is encoded within the viaB locus composed of 10 genes involved in regulation of expression (tviA), polymer synthesis (tviB-tviE), and cell surface localization of the CPS (vexA-vexE). We cloned the viaB locus from S. Typhi and transposon insertion mutants of individual viaB genes were characterized in Escherichia coli DH5α. Phenotype analysis of viaB mutants revealed that tviB, tviC, tviD and tviE are involved in Vi polymer synthesis. Furthermore, expression of tviB-tviE in E. coli DH5α directed the synthesis of cytoplasmic Vi antigen. Mutants of the ABC transporter genes vexBC and the polysaccharide copolymerase gene vexD accumulated the Vi polymer within the cytoplasm and productivity in these mutants was greatly reduced. In contrast, de novo synthesis of Vi polymer in the export deficient vexA mutant was comparable to wild-type cells, with drastic effects on cell stability. VexE mutant cells exported the Vi, but the CPS was not retained at the cell surface. The secreted polymer of a vexE mutant had different physical characteristics compared to the wild-type Vi.     

Engineering, conjugation, and immunogenicity assessment of Escherichia coli O121 O antigen for its potential use as a typhoid vaccine component

State-of-the-art production technologies for conjugate vaccines are complex, multi-step processes. An alternative approach to produce glycoconjugates is based on the bacterial N-linked protein glycosylation system first described in Campylobacter jejuni. The C. jejuni N-glycosylation system has been successfully transferred into Escherichia coli, enabling in vivo production of customized recombinant glycoproteins. However, some antigenic bacterial cell surface polysaccharides, like the Vi antigen of Salmonella enterica serovar Typhi, have not been reported to be accessible to the bacterial oligosaccharyltransferase PglB, hence hamper development of novel conjugate vaccines against typhoid fever. In this report, Vi-like polysaccharide structures that can be transferred by PglB were evaluated as typhoid vaccine components. A polysaccharide fulfilling these requirements was found in Escherichia coli serovar O121. Inactivation of the E. coli O121 O antigen cluster encoded gene wbqG resulted in expression of O polysaccharides reactive with antibodies raised against the Vi antigen. The structure of the recombinantly expressed mutant O polysaccharide was elucidated using a novel HPLC and mass spectrometry based method for purified undecaprenyl pyrophosphate (Und-PP) linked glycans, and the presence of epitopes also found in the Vi antigen was confirmed. The mutant O antigen structure was transferred to acceptor proteins using the bacterial N-glycosylation system, and immunogenicity of the resulting conjugates was evaluated in mice. The conjugate-induced antibodies reacted in an enzyme-linked immunosorbent assay with E. coli O121 LPS. One animal developed a significant rise in serum immunoglobulin anti-Vi titer upon immunization.     

Differential Killing of Salmonella enterica Serovar Typhi by Antibodies Targeting Vi and Lipopolysaccharide O:9 Antigen

Salmonella enterica serovar Typhi expresses a capsule of Vi polysaccharide, while most Salmonella serovars, including S. Enteritidis and S. Typhimurium, do not. Both S. Typhi and S. Enteritidis express the lipopolysaccharide O:9 antigen, yet there is little evidence of cross-protection from anti-O:9 antibodies. Vaccines based on Vi polysaccharide have efficacy against typhoid fever, indicating that antibodies against Vi confer protection. Here we investigate the role of Vi capsule and antibodies against Vi and O:9 in antibody-dependent complement- and phagocyte-mediated killing of Salmonella. Using isogenic Vi-expressing and non-Vi-expressing derivatives of S. Typhi and S. Typhimurium, we show that S. Typhi is inherently more sensitive to serum and blood than S. Typhimurium. Vi expression confers increased resistance to both complement- and phagocyte-mediated modalities of antibody-dependent killing in human blood. The Vi capsule is associated with reduced C3 and C5b-9 deposition, and decreased overall antibody binding to S. Typhi. However, purified human anti-Vi antibodies in the presence of complement are able to kill Vi-expressing Salmonella, while killing by anti-O:9 antibodies is inversely related to Vi expression. Human serum depleted of antibodies to antigens other than Vi retains the ability to kill Vi-expressing bacteria. Our findings support a protective role for Vi capsule in preventing complement and phagocyte killing of Salmonella that can be overcome by specific anti-Vi antibodies, but only to a limited extent by anti-O:9 antibodies.     

Antigenic Variation among Bordetella: BORDETELLA BRONCHISEPTICA STRAIN MO149 EXPRESSES A NOVEL O CHAIN THAT IS POORLY IMMUNOGENIC*

The O chain polysaccharide (O PS) of Bordetella bronchiseptica and Bordetella parapertussis lipopolysaccharide is a homopolymer of 2,3-diacetamido-2,3-dideoxygalacturonic acid (GalNAc3NAcA) in which some of the sugars are present as uronamides. The terminal residue contains several unusual modifications. To date, two types of modification have been characterized, and a survey of numerous strains demonstrated that each contained one of these two modification types. Host antibody responses against the O PS are directed against the terminal residue modifications, and there is little cross-reactivity between the two types. This suggests that Bordetella O PS modifications represent a means of antigenic variation. Here we report the characterization of the O PS of B. bronchiseptica strain MO149. It consists of a novel two-sugar repeating unit and a novel terminal residue modification, with the structure Me-4-α-l-GalNAc3NAcA-(4-β-d-GlcNAc3NAcA-4-α-l-GalNAc3NAcA-)_5–6-, which we propose be defined as the B. bronchiseptica O3 PS. We show that the O3 PS is very poorly immunogenic and that the MO149 strain contains a novel wbm (O PS biosynthesis) locus. Thus, there is greater diversity among Bordetella O PSs than previously recognized, which is likely to be a result of selection pressure from host immunity. We also determine experimentally, for the first time, the absolute configuration of the diacetimido-uronic acid sugars in Bordetella O PS.     

The Bordetella Bps polysaccharide is critical for biofilm development in the mouse respiratory tract

Bordetellae are respiratory pathogens that infect both humans and animals. Bordetella bronchiseptica establishes asymptomatic and long-term to life-long infections of animal nasopharynges. While the human pathogen Bordetella pertussis is the etiological agent of the acute disease whooping cough in infants and young children, it is now being increasingly isolated from the nasopharynges of vaccinated adolescents and adults who sometimes show milder symptoms, such as prolonged cough illness. Although it has been shown that Bordetella can form biofilms in vitro, nothing is known about its biofilm mode of existence in mammalian hosts. Using indirect immunofluorescence and scanning electron microscopy, we examined nasal tissues from mice infected with B. bronchiseptica. Our results demonstrate that a wild-type strain formed robust biofilms that were adherent to the nasal epithelium and displayed architectural attributes characteristic of a number of bacterial biofilms formed on inert surfaces. We have previously shown that the Bordetella Bps polysaccharide encoded by the bpsABCD locus is critical for the stability and maintenance of three-dimensional structures of biofilms. We show here that Bps is essential for the formation of efficient nasal biofilms and is required for the colonization of the nose. Our results document a biofilm lifestyle for Bordetella in mammalian respiratory tracts and highlight the essential role of the Bps polysaccharide in this process and in persistence of the nares.     

In-vitro and in-vivo analysis of the production of the Bordetella type three secretion system effector A in Bordetella pertussis,Bordetella parapertussis and Bordetella bronchiseptica

Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica are three closely related pathogens.They all possess the gene coding for the Bordetella type three secretion system effector A (bteA) toxin that became a focus of interest since it was demonstrated that B. pertussis Japanese non-vaccine-type isolates produce BteA unlike vaccine-type isolates. We thus explored the in-vitro production of BteA in B. pertussis isolates collected in France during periods of different vaccine policy as well as in B. parapertussis and B. bronchiseptica isolates. We also analyzed the in-vivo induction of anti-BteA antibodies after infection with different isolates of the three species.We produced a recombinant His6-tagged BteA (rBteA) protein. Specific rBteA polyclonal serum was prepared which enabled us to screen Bordetella isolates for in-vitro BteA production: 99.0% (293/296) of tested B. pertussis isolates, including French vaccine strains, and 97.5% (79/81) of B. bronchiseptica isolates produced BteA in-vitro but only the latter was capable of inducing an in-vivo immune response. No in-vitro or in-vivo production of BteA was detected by any of the B. parapertussis isolates tested.     

The Vi Capsular Polysaccharide Enables Salmonella enterica Serovar Typhi to Evade Microbe-Guided Neutrophil Chemotaxis

Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever, a disseminated infection, while the closely related pathogen S. enterica serovar Typhimurium (S. Typhimurium) is associated with a localized gastroenteritis in humans. Here we investigated whether both pathogens differ in the chemotactic response they induce in neutrophils using a single-cell experimental approach. Surprisingly, neutrophils extended chemotactic pseudopodia toward Escherichia coli and S. Typhimurium, but not toward S. Typhi. Bacterial-guided chemotaxis was dependent on the presence of complement component 5a (C5a) and C5a receptor (C5aR). Deletion of S. Typhi capsule biosynthesis genes markedly enhanced the chemotactic response of neutrophils in vitro. Furthermore, deletion of capsule biosynthesis genes heightened the association of S. Typhi with neutrophils in vivo through a C5aR-dependent mechanism. Collectively, these data suggest that expression of the virulence-associated (Vi) capsular polysaccharide of S. Typhi obstructs bacterial-guided neutrophil chemotaxis.     

Host–Pathogen Interaction in Invasive Salmonellosis

Salmonella enterica infections result in diverse clinical manifestations. Typhoid fever, caused by S. enterica serovar Typhi (S. Typhi) and S. Paratyphi A, is a bacteremic illness but whose clinical features differ from other Gram-negative bacteremias. Non-typhoidal Salmonella (NTS) serovars cause self-limiting diarrhea with occasional secondary bacteremia. Primary NTS bacteremia can occur in the immunocompromised host and infants in sub-Saharan Africa. Recent studies on host–pathogen interactions in Salmonellosis using genome sequencing, murine models, and patient studies have provided new insights. The full genome sequences of numerous S. enterica serovars have been determined. The S. Typhi genome, compared to that of S. Typhimurium, harbors many inactivated or disrupted genes. This can partly explain the different immune responses both serovars induce upon entering their host. Similar genome degradation is also observed in the ST313 S. Typhimurium strain implicated in invasive infection in sub-Saharan Africa. Virulence factors, most notably, type III secretion systems, Vi antigen, lipopolysaccharide and other surface polysaccharides, flagella, and various factors essential for the intracellular life cycle of S. enterica have been characterized. Genes for these factors are commonly carried on Salmonella Pathogenicity Islands (SPIs). Plasmids also carry putative virulence-associated genes as well as those responsible for antimicrobial resistance. The interaction of Salmonella pathogen-associated molecular patterns (PAMPs) with Toll-like receptors (TLRs) and NOD-like receptors (NLRs) leads to inflammasome formation, activation, and recruitment of neutrophils and macrophages and the production of pro-inflammatory cytokines, most notably interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)-α, and interferon-gamma (IFN)-γ. The gut microbiome may be an important modulator of this immune response. S. Typhimurium usually causes a local intestinal immune response, whereas S. Typhi, by preventing neutrophil attraction resulting from activation of TLRs, evades the local response and causes systemic infection. Potential new therapeutic strategies may lead from an increased understanding of infection pathogenesis.     

Head-to-Head Comparison of Humoral Immune Responses to Vi Capsular Polysaccharide and Salmonella Typhi Ty21a Typhoid Vaccines–A Randomized Trial

Background

The two typhoid vaccines, the parenteral Vi capsular polysaccharide and the oral live whole-cell Salmonella Typhi Ty21a vaccine, provide similar levels of protection in field trials. Sharing no antigens, they are thought to confer protection by different mechanisms. This is the first head-to-head study to compare the humoral immune responses to these two vaccines.

Methods

50 age- and gender-matched volunteers were immunized, 25 with the Vi and 25 with the Ty21a vaccine. Circulating plasmablasts reactive with whole-cell Salmonella Typhi or one of the typhoidal antigenic structures, Vi, O-9,12, and H-d antigens, were identified as antibody-secreting cells (ASC) with ELISPOT. Homing receptor (HR) expressions were determined. These results were compared with ASC in four patients with typhoid fever. Antibodies to S. Typhi lipopolysaccharides were assessed in cultures of ALS (antibodies in lymphocyte supernatants) and in serum with ELISA.

Results

In 49 out of 50 vaccinees, no typhoid-specific plasmablasts were seen before vaccination. On day 7, response to Vi antigen was mounted in 24/25 volunteers in the Vi, and none in the Ty21a group; response to S. Typhi and O-9,12 was mounted in 49/50 vaccinees; and to H-d in 3/50. The numbers of typhoid-specific plasmablasts (total of ASC to Vi, O-9,12 and H-d antigens) proved equal in the vaccination groups. The HR expressions indicated a mainly systemic homing in the Vi and intestinal in the Ty21a group, the latter resembling that in natural infection. Plasmablasts proved more sensitive than serum and ALS in assessing the immune response.

Conclusions

The typhoid-specific humoral responses to Vi and Ty21a vaccines are similar in magnitude, but differ in expected localization and antigen-specificity. The unforeseen O antigen-specific response in the Vi group is probably due to lipopolysaccharide contaminating the vaccine preparation. Only the response to Ty21a vaccine was found to imitate that in natural infection.

Trial Registration

Current Controlled Trials Ltd. c/o BioMed Central ISRCTN68125331 http://www.controlled-trials.com/ISRCTN68125331/     

Glycoconjugate vaccines against Salmonella enterica serovars and Shigella species: existing and emerging methods for their analysis

The global spread of enteric disease, the increasingly limited options for antimicrobial treatment and the need for effective eradication programs have resulted in an increased demand for glycoconjugate enteric vaccines, made with carbohydrate-based membrane components of the pathogen, and their precise characterisation. A set of physico-chemical and immunological tests are employed for complete vaccine characterisation and to ensure their consistency, potency, safety and stability, following the relevant World Health Organization and Pharmacopoeia guidelines. Variable requirements for analytical methods are linked to conjugate structure, carrier protein nature and size and O-acetyl content of polysaccharide. We investigated a key stability-indicating method which measures the percent free saccharide of Salmonella enterica subspecies enterica serovar Typhi capsular polysaccharide, by detergent precipitation, depolymerisation and HPAEC-PAD quantitation. Together with modern computational approaches, a more precise design of glycoconjugates is possible, allowing for improvements in solubility, structural conformation and stability, and immunogenicity of antigens, which may be applicable to a broad spectrum of vaccines. More validation experiments are required to establish the most effective and suitable methods for glycoconjugate analysis to bring uniformity to the existing protocols, although the need for product-specific approaches will apply, especially for the more complex vaccines. An overview of current and emerging analytical approaches for the characterisation of vaccines against Salmonella Typhi and Shigella species is described in this paper. This study should aid the development and licensing of new glycoconjugate vaccines aimed at the prevention of enteric diseases.     

High-resolution genotyping of the endemic Salmonella Typhi population during a Vi (typhoid) vaccination trial in Kolkata

Background

Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), is a major health problem especially in developing countries. Vaccines against typhoid are commonly used by travelers but less so by residents of endemic areas.

Methodology

We used single nucleotide polymorphism (SNP) typing to investigate the population structure of 372 S. Typhi isolated during a typhoid disease burden study and Vi vaccine trial in Kolkata, India. Approximately sixty thousand people were enrolled for fever surveillance for 19 months prior to, and 24 months following, Vi vaccination of one third of the study population (May 2003–December 2006, vaccinations given December 2004).

Principal Findings

A diverse S. Typhi population was detected, including 21 haplotypes. The most common were of the H58 haplogroup (69%), which included all multidrug resistant isolates (defined as resistance to chloramphenicol, ampicillin and co-trimoxazole). Quinolone resistance was particularly high among H58-G isolates (97% Nalidixic acid resistant, 30% with reduced susceptibility to ciprofloxacin). Multiple typhoid fever episodes were detected in 22 households, however household clustering was not associated with specific S. Typhi haplotypes.

Conclusions

Typhoid fever in Kolkata is caused by a diverse population of S. Typhi, however H58 haplotypes dominate and are associated with multidrug and quinolone resistance. Vi vaccination did not obviously impact on the haplotype population structure of the S. Typhi circulating during the study period.     

Representational difference analysis identifies a strain-specific LPS biosynthesis locus in Bordetella spp.

Bordetella pertussis and B. bronchiseptica are genetically very closely related but differ significantly in their virulence properties. Using Representational Difference Analysis (RDA), 11 DNA fragments specific for B. pertussis Tohama I or B. bronchiseptica BB7865 were identified. All B. bronchiseptica BB7865-derived fragments also hybridized with chromosomal DNA from B. parapertussis but not from the B. pertussis strains Tohama I and W28, underlining the close phylogenetic relationship between B. bronchiseptica and B. parapertussis. The B. pertussis type strain BP18323 is a special case, as it contains DNA sequences characteristic for both B. pertussis and B. bronchiseptica. As demonstrated by pulsed-field gel electrophoresis, several of the BB7865-derived fragments are present on a single 30-kb XbaI fragment. Based on the sequences of putative coding regions, four of these fragments may code for proteins involved in carbohydrate metabolism or transport. In agreement with this notion, a mutant for one of these loci synthesizes a significantly altered lipopolysaccharide that lacks the O-specific side chains. The analysis of the corresponding genomic region in various Bordetella species showed that this locus is present in B. bronchiseptica and B. parapertussis but not in B. pertussis. This confirms that the RDA approach has identified a novel strain-specific LPS biosynthesis locus which accounts for the differences between the LPS structures elaborated by different Bordetella species.     

A Conserved Acetyl Esterase Domain Targets Diverse Bacteriophages to the Vi Capsular Receptor of Salmonella enterica Serovar Typhi

A number of bacteriophages have been identified that target the Vi capsular antigen of Salmonella enterica serovar Typhi. Here we show that these Vi phages represent a remarkably diverse set of phages belonging to three phage families, including Podoviridae and Myoviridae. Genome analysis facilitated the further classification of these phages and highlighted aspects of their independent evolution. Significantly, a conserved protein domain carrying an acetyl esterase was found to be associated with at least one tail fiber gene for all Vi phages, and the presence of this domain was confirmed in representative phage particles by mass spectrometric analysis. Thus, we provide a simple explanation and paradigm of how a diverse group of phages target a single key virulence antigen associated with this important human-restricted pathogen.Bacteriophages are dependent for their survival on the presence of susceptible host bacteria in their environment. The first stage of recognition of the bacterial host normally involves binding of a specific phage attachment protein to a receptor molecule on the bacterial surface. Bacteria can evade phage infection by various mechanisms, including accumulating escape mutations in the receptor, acquiring phage inhibitory proteins, or directly modifying the receptor, for example, lipopolysaccharide (LPS) (43). In addition, phage can adapt to recognize different receptors through a number of genetic mechanisms involving evolution of their attachment proteins (20) or by tropism switching (21, 22).Phage can exploit capsular exopolysaccharides as receptors, some of which are associated with virulence in pathogens (5, 23, 35). A notable example is the Vi capsule found in Salmonella enterica serovar Typhi (S. Typhi) and some isolates of S. Dublin and Citrobacter freundii (29). The Vi capsule of S. Typhi is an important virulence factor, facilitating the bacteria to escape opsonization and other forms of immune surveillance (14, 30) as well as potentially helping the bacteria to evade phage that would otherwise target the O:9 LPS, which the Vi capsule can, at least in part, mask (27). In the middle of the last century, a set of lytic phages were isolated that utilized the Vi capsule as a receptor (6). These Vi phages were exploited in diagnostic laboratories as a “typing set” to distinguish between different strains of S. Typhi isolated from typhoid patients (8). A secondary typing set was generated from Vi typing phage II by adapting this phage to grow on different S. Typhi hosts (6). At this time, typhoid was still common in many parts of Europe and North America, and clinicians tested some of these Vi phages for their potential in phage therapy experiments with human typhoid patients (11). Although this work showed significant promise, phage therapy gradually disappeared from clinical practice in many countries as antibiotics became readily available.S. Typhi is a monophyletic serovar of the broad enteric species S. enterica (16, 31). Interestingly, S. Typhi is host restricted to humans and has no known zoonotic source. Unlike many other S. enterica serovars, S. Typhi normally causes a systemic infection and does not persist in the intestine efficiently, where high levels of bacteriophage are present. Although it is rare in developed countries, S. Typhi is still a significant cause of mortality in many developing countries (26). Most current clinical isolates are Vi positive when first isolated (2), but it is noteworthy that the Vi capsule biosynthesis and export genes are carried by an operon within a potentially unstable island called Salmonella pathogenicity island 7 (SPI-7) (29).Although some phenotypic characterization of the Vi phage has been undertaken (1), very little has been performed at the molecular level. We previously showed that Vi typing phage II-E1 is related to the S. Typhimurium phage ES18 (4, 28), with synteny in many capsid and tail proteins. We have now further characterized the other members of this S. Typhi Vi phage collection, designated types I, III, IV, V, VI, and VII (abbreviated from here on as Vi phages I, III, IV, etc.) (6, 11), by utilizing electron microscopy and genomic analysis. This analysis shows that this collection of Vi phages represents a diverse group of bacteriophages that have adapted to growth on S. Typhi through convergent evolution within their tail spike protein genes and the acquisition of conserved acetyl esterase domains.     

Physiochemical properties of the Vi antigen before and after mild alkaline hydrolysis

Sedimentation and diffusion constants were determined for electrophoretically purified Vi antigen before and after mild alkaline hydrolysis. The molecular weight of the intact antigen was found to be 1.7 × 10⁶. Mild alkaline hydrolysis completely removed O-acetyl, reduced the molecular weight to 1.0 × 10⁵, and lowered the intrinsic viscosity from 8.0 deciliters/g to approximately 0.5 deciliter/g. Infrared spectra of the two antigen preparations were also compared.     

Identification of novel vaccine candidate against Salmonella enterica serovar Typhi by reverse vaccinology method and evaluation of its immunization

Salmonella enterica serovar Typhi (S. Typhi) is an essential enteric fever causing bacterium worldwide. Due to the emergence of multidrug-resistant strains, urgently attention is needed to prevent the global spread of them. Vaccination is an alternative approach to control these kinds of infections. Currently available commercial vaccines have significant limitations such as non-recommendation for children below six years of age and poor long-term efficacy. Thus, the development of a new vaccine overcoming these limitations is immediately required. Reverse Vaccinology (RV) is one of the most robust approaches for direct screening of genome sequence assemblies to identify new protein-based vaccines. The present study aimed to identify potential vaccine candidates against S. Typhi by using the RV approach. Immunogenicity of the best candidate against S. Typhi was further investigated. The proteome of S. Typhi strain Ty2 was analyzed to identify the most immunogenic, conserved, and protective surface proteins. Among the predicted vaccine candidates, steD (fimbrial subunit) was the best for qualifying all the applied criteria. The synthetic steD gene was expressed in E.coli, and the mice were immunized with purified recombinant steD protein and then challenged with a lethal dose of S. Typhi. Immunized animals generated high protein-specific antibody titers and demonstrated 70% survival following lethal dose challenge with S. Typhi. Pretreatment of the S. Typhi cells with immunized mice antisera significantly decreased their adhesion to Caco-2 cells. Altogether, steD as a protective antigen could induce a robust and long term and protective immunity in immunized mice against S. Typhi challenge.     

O:2-CRM197 Conjugates against Salmonella Paratyphi A

Enteric fevers remain a common and serious disease, affecting mainly children and adolescents in developing countries. Salmonella enterica serovar Typhi was believed to cause most enteric fever episodes, but several recent reports have shown an increasing incidence of S. Paratyphi A, encouraging the development of a bivalent vaccine to protect against both serovars, especially considering that at present there is no vaccine against S. Paratyphi A. The O-specific polysaccharide (O:2) of S. Paratyphi A is a protective antigen and clinical data have previously demonstrated the potential of using O:2 conjugate vaccines. Here we describe a new conjugation chemistry to link O:2 and the carrier protein CRM₁₉₇, using the terminus 3-deoxy-D-manno-octulosonic acid (KDO), thus leaving the O:2 chain unmodified. The new conjugates were tested in mice and compared with other O:2-antigen conjugates, synthesized adopting previously described methods that use CRM₁₉₇ as carrier protein. The newly developed conjugation chemistry yielded immunogenic conjugates with strong serum bactericidal activity against S. Paratyphi A.