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1.
Chemistry ; 26(68): 15953-15968, 2020 Dec 04.
Article in English | MEDLINE | ID: mdl-32578281

ABSTRACT

With the emergence of multidrug resistant Salmonella strains, the development of anti-Salmonella vaccines is an important task. Currently there are no approved vaccines against Salmonella Paratyphi A, the leading cause of paratyphoid fever. To fill this gap, oligosaccharides corresponding to the O-polysaccharide repeating units from the surface of Salmonella Paratyphi A have been synthesized through convergent stereoselective glycosylations. The synthetic glycan antigen was conjugated with a powerful immunogenic carrier system, the bacteriophage Qß. The resulting construct was able to elicit strong and long-lasting anti-glycan IgG antibody responses, which were highly selective toward Salmonella Paratyphi A associated glycans. The availability of well-defined glycan antigen enabled the determination that one repeating unit of the polysaccharide is sufficient to induce protective antibodies, and the paratose residue and/or the O-acetyl modifications on the backbone are important for recognition by antibodies elicited by a Qß-tetrasaccharide conjugate. Immune sera provided excellent protection to mice from lethal challenge with Salmonella Paratyphi A, highlighting the potential of the synthetic glycan-based vaccine.


Subject(s)
Oligosaccharides , Paratyphoid Fever , Salmonella paratyphi A , Typhoid-Paratyphoid Vaccines , Animals , Antibodies, Bacterial/immunology , Antigens, Bacterial/immunology , Mice , Oligosaccharides/immunology , Paratyphoid Fever/prevention & control , Salmonella paratyphi A/immunology , Typhoid-Paratyphoid Vaccines/administration & dosage , Typhoid-Paratyphoid Vaccines/chemistry , Vaccines, Synthetic
2.
Hum Vaccin Immunother ; 15(6): 1310-1316, 2019.
Article in English | MEDLINE | ID: mdl-31021700

ABSTRACT

Typhax is an investigational typhoid fever vaccine candidate that was GMP manufactured applying Protein Capsular Matrix Vaccine (PCMV) technology. It consists of Vi polysaccharide antigen, derived from S. Typhi, non-covalently entrapped in a glutaraldehyde catalyzed cross-linked α-poly-L-lysine and CRM197 protein matrix. Analysis of Typhax determined the average molecular weight of the vaccine particles was approximately 6 x 106 Daltons, corresponding to particles containing 1-2 molecules of Vi polysaccharide and 10-20 molecules of CRM197 protein. The ratio of the concentration of Vi to CRM197 protein in Typhax is 2.4:1. Preclinical immunogenicity studies in mice demonstrated that Typhax was immunogenic and elicited a significant increase in anti-Vi IgG antibody titers following each immunization. The anti-Vi IgG antibody response elicited by Typhax in rabbits increased as the dose increased from 0.1 µg to 2.5 µg. Further, at the 2.5 and 10 µg dose levels, the anti-Vi IgG antibody titers increased after the second and third immunizations. At the 10 µg dose level, 100% of rabbits seroconverted. In the non-human primate (NHP) study, 100% seroconversion was observed at both 2.5 µg and 10 µg dose levels after the first immunization. A murine in vivo immunopotency study demonstrated that Typhax stored at 4°C was stable for at least 30 months. Collectively, the Typhax in vitro profile, preclinical immunogenicity studies, and rabbit toxicology study indicate that Typhax is a viable typhoid fever vaccine candidate for Phase 1 clinical trial evaluation.


Subject(s)
Antibodies, Bacterial/blood , Immunogenicity, Vaccine , Polysaccharides, Bacterial/chemistry , Polysaccharides, Bacterial/immunology , Typhoid-Paratyphoid Vaccines/chemistry , Typhoid-Paratyphoid Vaccines/immunology , Animals , Female , Immunoglobulin G/blood , Macaca mulatta , Mice , Mice, Inbred BALB C , Rabbits , Salmonella typhi , Seroconversion , Typhoid Fever/prevention & control , Vaccines, Conjugate/immunology
3.
PLoS One ; 13(9): e0203631, 2018.
Article in English | MEDLINE | ID: mdl-30216367

ABSTRACT

Salmonella Typhi and Salmonella Paratyphi A are the leading causative agents of enteric fever which cause morbidity and mortality worldwide. Currently, there is no combination vaccine which could protect infection from both the strains. In this paper, we are focusing on the development of a novel bivalent typhoidal Outer Membrane Vesicles (OMVs) based immunogen against enteric fever. We have isolated Salmonella Typhi and Paratyphi A OMVs and also characterized OMVs associated antigens. Then we immunized adult mice with three doses of our newly formulated bivalent immunogen orally (25 µg/200 µl). After three doses of oral immunization, we found our immunogen could significantly induce humoral response. We have also found serum IgG against LPS, Vi-polysaccharide etc. OMV immunization induces CD4, CD8 and CD19 population in immunized mice spleen. It also induces Th1 and Th17-cell mediated immunity. We also found bivalent OMVs immunization can prevent more than lethal dose of heterologous Salmonella strains mediated systemic infection in adult mice model. We determined that, the protective immune responses depend on the humoral and cell-mediated immune response. Furthermore, we have evaluated the mode of protective immune response carried out by anti-OMVs antibody by significantly inhibiting bacterial motility and mucin penetration ability. Taken together, these findings suggest that our bivalent immunogen could be used as a novel candidate vaccine against enteric fever.


Subject(s)
Salmonella typhi/immunology , Typhoid Fever/immunology , Typhoid-Paratyphoid Vaccines/chemistry , Animals , Antibodies, Bacterial/immunology , Female , Mice , Mice, Inbred BALB C , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Typhoid Fever/prevention & control
4.
Molecules ; 23(7)2018 Jul 17.
Article in English | MEDLINE | ID: mdl-30018230

ABSTRACT

Typhoid fever due to Salmonella Typhi and invasive nontyphoidal Salmonella (iNTS) infections caused by serovars Enteritidis (SE) and Typhimurium (STm) are major pediatric health problems in sub-Saharan Africa. Typhoid has high complication rates, and iNTS infections have high case fatality rates; moreover, emerging antimicrobial resistance is diminishing treatment options. Vi capsule-based typhoid conjugate vaccine (Typbar-TCV™), licensed in India and pre-qualified by the World Health Organization, elicits durable immunity when administered to infants, but no iNTS vaccines are licensed or imminent. We have developed monovalent SE and STm glycoconjugate vaccines based on coupling lipopolysaccharide-derived core-O polysaccharide (COPS) to phase 1 flagellin protein (FliC) from the homologous serovar. Herein, we report the immunogenicity of multivalent formulations of iNTS COPS:FliC conjugates with Typbar-TCV™. Rabbits immunized with the trivalent typhoid-iNTS glycoconjugate vaccine generated high titers of serum IgG antibody to all three polysaccharide antigens for which anti-COPS IgG antibodies were directed primarily against serogroup-specific OPS epitopes. Responses to SE and STm FliC were lower relative to anti-COPS titers. Post-vaccination rabbit sera mediated bactericidal activity in-vitro, and protected mice after passive transfer against challenge with virulent SE or STm Malian blood isolates. These results support accelerated progression to clinical trials.


Subject(s)
Antibodies, Bacterial/immunology , Glycoconjugates , Immunogenicity, Vaccine , Salmonella typhi , Typhoid Fever , Typhoid-Paratyphoid Vaccines , Animals , Glycoconjugates/chemistry , Glycoconjugates/immunology , Glycoconjugates/pharmacology , Rabbits , Salmonella typhi/chemistry , Salmonella typhi/immunology , Typhoid Fever/immunology , Typhoid Fever/prevention & control , Typhoid-Paratyphoid Vaccines/chemistry , Typhoid-Paratyphoid Vaccines/immunology , Typhoid-Paratyphoid Vaccines/pharmacology
5.
Biosci Rep ; 38(5)2018 10 31.
Article in English | MEDLINE | ID: mdl-29875175

ABSTRACT

It is established that the immunogenicity of polysaccharides is enhanced by coupling them to carrier proteins. Cross reacting material (CRM197), a nontoxic variant of diphtheria toxin (DT) is widely used carrier protein for polysaccharide conjugate vaccines. Conventionally, CRM197 is isolated by fermentation of Corynebacterium diphtheriae C7 (ß197) cultures, which often suffers from low yield. Recently, several recombinant approaches have been reported with robust processes and higher yields, which will improve the affordability of CRM197-based vaccines. Vaccine manufacturers require detailed analytical information to ensure that the CRM197 meets quality standards and regulatory requirements. In the present manuscript we have described detailed structural characteristics of Escherichia coli based recombinant CRM197 (rCRM197) carrier protein. The crystal structure of the E. coli based rCRM197 was found to be identical with the reported crystal structure of the C7 CRM197 produced in C. diphtheriae C7 strain (Protein Data Bank (PDB) ID: 4EA0). The crystal structure of rCRM197 was determined at 2.3 Å resolution and structure was submitted to the PDB with accession number ID 5I82. This is the first report of a crystal structure of E. coli derived recombinant CRM197 carrier protein. Furthermore, the rCRM197 was conjugated to Vi polysaccharide to generate Typhoid conjugate vaccine (Vi-rCRM197) and its immunogenicity was evaluated in Balb/C Mice. The Vi-rCRM197 conjugate vaccine was found to generate strong primary α-Vi antibody response and also showed a booster response after subsequent vaccination in mice. Overall data suggest that E. coli based recombinant CRM197 exhibits structural and immunological similarity with the C7 CRM197 and can be used as a carrier protein in conjugate vaccine development.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/immunology , Drug Carriers/chemistry , Recombinant Proteins/immunology , Vaccines, Conjugate/pharmacology , Amino Acids/analysis , Animals , Bacterial Proteins/genetics , Blotting, Western/methods , Crystallography, X-Ray , Escherichia coli/genetics , Female , Immunoblotting , Mass Spectrometry , Mice, Inbred BALB C , Molecular Weight , Polysaccharides/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Typhoid-Paratyphoid Vaccines/chemistry , Typhoid-Paratyphoid Vaccines/pharmacology , Vaccines, Conjugate/administration & dosage , Vaccines, Conjugate/genetics
6.
Vaccine ; 35(51): 7121-7126, 2017 12 18.
Article in English | MEDLINE | ID: mdl-29150208

ABSTRACT

Typhoid fever remains a serious public health problem with a high impact on toddlers and young children. Vaccines against the Vi capsular polysaccharide are efficacious against typhoid fever demonstrating that antibodies against Vi confer protection. The currently licensed Vi typhoid vaccines have however limited efficacy and are manufactured by a complex process from wild-type bacteria. Due to these inherent issues with the current vaccines, an alternative vaccine based on an O-acetylated high molecular weight (HMW) polygalacturonic acid (GelSite-OAc™) was generated. The HMW polygalacturonic acid shares the same backbone as the Vi polysaccharide of Salmonella Typhi. The GelSite-OAc™ has a high molecular weight (>1 × 106 Da) and a high degree of O-acetylation (DOAc) (>5 µmole/mg), both exceeding the potency specifications of the current Vi vaccine. Studies in Balb/c mice demonstrated that GelSite-OAc™ was highly immunogenic, inducing a strong antigen-specific antibody response in a DOAc- and dose-dependent manner which was comparable to or higher than those induced by the licensed Vi vaccine. Importantly, the GelSite-OAc™ was shown to be fully protective in mice against lethal challenge with Salmonella Typhi. Furthermore, the GelSite-OAc™ demonstrated a boosting effect or memory response, exhibiting a >2-fold increase in antibody levels upon the second immunization with either GelSite-OAc™ or the Vi vaccine. This novel boosting effect is unique among polysaccharide antigens and potentially makes GelSite-OAc™ effective in people under 2 years old. Together these results suggest that the GelSite-OAc™ could be a highly effective vaccine against Salmonella Typhi.


Subject(s)
Pectins/immunology , Polysaccharides, Bacterial/chemistry , Polysaccharides, Bacterial/immunology , Typhoid Fever/prevention & control , Typhoid-Paratyphoid Vaccines/chemistry , Typhoid-Paratyphoid Vaccines/immunology , Vaccines, Synthetic/immunology , Acetylation , Animals , Antibodies, Bacterial/blood , Antibody Formation/immunology , Disease Models, Animal , Immunization, Secondary , Immunogenicity, Vaccine , Immunoglobulin G/blood , Immunologic Memory , Mice , Pectins/administration & dosage , Pectins/chemistry , Polysaccharides, Bacterial/administration & dosage , Salmonella typhi/immunology , Typhoid Fever/immunology , Typhoid Fever/microbiology , Typhoid-Paratyphoid Vaccines/administration & dosage , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/chemistry
7.
J Immunol Methods ; 450: 27-33, 2017 11.
Article in English | MEDLINE | ID: mdl-28735760

ABSTRACT

Efficacious typhoid vaccines for young children will significantly reduce the disease burden in developing world. The Vi polysaccharide based conjugate vaccines (Vi-rEPA) against Salmonella Typhi Vi positive strains has shown high efficacy but may be ineffective against Vi negative S. Typhi. In this study, for the first time, we report the synthesis and evaluation of polysaccharide-protein conjugates of Vi negative S. Typhi as potential vaccine candidates. Four different conjugates were synthesized using recombinant exoprotein A of Pseudomonas aeruginosa (rEPA) and human serum albumin (HSA) as the carrier proteins, using either direct reductive amination or an intermediate linker molecule, adipic acid dihydrazide (ADH). Upon injection into mice, a significantly higher antibody titer was observed in mice administrated with conjugate-1 (OSP-HSA) (P=0.0001) and conjugate 2 (OSP-rEPA) (P≤0.0001) as compared to OSP alone. In contrast, the antibody titer elicited by conjugate 3 (OSPADH-HSA) and conjugate 4 (OSPADH-rEPA) were insignificant (P=0.1684 and P=0.3794, respectively). We conclude that reductive amination is the superior method to prepare the S. Typhi OSP glycoconjugate. Moreover, rEPA was a better carrier protein than HSA. Thus OSP-rEPA conjugate seems to be efficacious typhoid vaccines candidate, it may be evaluated further and recommended for the clinical trials.


Subject(s)
ADP Ribose Transferases/immunology , Bacterial Toxins/immunology , Exotoxins/immunology , O Antigens/immunology , Polysaccharides, Bacterial/immunology , Salmonella typhi/immunology , Typhoid-Paratyphoid Vaccines/immunology , Virulence Factors/immunology , ADP Ribose Transferases/administration & dosage , ADP Ribose Transferases/chemistry , Amination , Animals , Antibodies, Bacterial/blood , Bacterial Toxins/administration & dosage , Bacterial Toxins/chemistry , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Exotoxins/administration & dosage , Exotoxins/chemistry , Female , Immunization , Immunization Schedule , Injections, Intraperitoneal , Mice, Inbred BALB C , O Antigens/administration & dosage , O Antigens/chemistry , Oxidation-Reduction , Proton Magnetic Resonance Spectroscopy , Recombinant Proteins/immunology , Serum Albumin/immunology , Serum Albumin, Human , Typhoid-Paratyphoid Vaccines/administration & dosage , Typhoid-Paratyphoid Vaccines/chemistry , Vaccines, Conjugate/immunology , Virulence Factors/administration & dosage , Virulence Factors/chemistry , Pseudomonas aeruginosa Exotoxin A
8.
Vaccine ; 34(35): 4263-4269, 2016 07 29.
Article in English | MEDLINE | ID: mdl-27372155

ABSTRACT

Salmonella (S.) enterica infections are an important global health problem with more than 20 million individuals suffering from enteric fever annually and more than 200,000 lethal cases per year. Although enteric fever can be treated appropriately with antibiotics, an increasing number of antibiotic resistant Salmonella strains is detected. While two vaccines against typhoid fever are currently on the market, their availability in subtropical endemic areas is limited because these products need to be kept in uninterrupted cold chains. Hence, the development of a thermally stable vaccine that induces mucosal immune responses would greatly improve human health in endemic areas. Here, we have combined the high structural stability of Salmonella typhi outer membrane proteins (porins) with their microencapsulation into poly(lactic-co-glycolic acid) (PLGA) to generate an orally applicable vaccine. Encapsulated porins were protected from acidic degradation and exhibited enhanced immunogenicity following oral administration. In particular, the vaccine elicited strong S. typhi-specific B cell responses in Peyer's patches and mesenteric lymph nodes. In sum, PLGA microencapsulation substantially improved the efficacy of oral vaccination against S. typhi.


Subject(s)
Bacterial Outer Membrane Proteins/immunology , Lactic Acid/chemistry , Polyglycolic Acid/chemistry , Typhoid-Paratyphoid Vaccines/immunology , Animals , Antibodies, Bacterial/blood , Bacterial Outer Membrane Proteins/chemistry , Lymph Nodes/immunology , Mice, Inbred C57BL , Peyer's Patches/immunology , Polylactic Acid-Polyglycolic Acid Copolymer , Protein Stability , Salmonella typhi , Typhoid-Paratyphoid Vaccines/chemistry
9.
Eur J Pharm Biopharm ; 102: 115-22, 2016 May.
Article in English | MEDLINE | ID: mdl-26969261

ABSTRACT

Gastrointestinal (GI) models that mimic physiological conditions in vitro are important tools for developing and optimizing biopharmaceutical formulations. Oral administration of live attenuated bacterial vaccines (LBV) can safely and effectively promote mucosal immunity but new formulations are required that provide controlled release of optimal numbers of viable bacterial cells, which must survive gastrointestinal transit overcoming various antimicrobial barriers. Here, we use a gastro-small intestine gut model of human GI conditions to study the survival and release kinetics of two oral LBV formulations: the licensed typhoid fever vaccine Vivotif comprising enteric coated capsules; and an experimental formulation of the model vaccine Salmonella Typhimurium SL3261 dried directly onto cast enteric polymer films and laminated to form a polymer film laminate (PFL). Neither formulation released significant numbers of viable cells when tested in the complete gastro-small intestine model. The poor performance in delivering viable cells could be attributed to a combination of acid and bile toxicity plus incomplete release of cells for Vivotif capsules, and to bile toxicity alone for PFL. To achieve effective protection from intestinal bile in addition to effective acid resistance, bile adsorbent resins were incorporated into the PFL to produce a new formulation, termed BR-PFL. Efficient and complete release of 4.4×10(7) live cells per dose was achieved from BR-PFL at distal intestinal pH, with release kinetics controlled by the composition of the enteric polymer film, and no loss in viability observed in any stage of the GI model. Use of this in vitro GI model thereby allowed rational design of an oral LBV formulation to maximize viable cell release.


Subject(s)
Bacterial Vaccines/chemistry , Gastric Mucosa/metabolism , Intestine, Small/metabolism , Typhoid-Paratyphoid Vaccines/chemistry , Vaccines, Attenuated/chemistry , Administration, Oral , Bacterial Vaccines/administration & dosage , Bile/metabolism , Capsules/chemistry , Chemistry, Pharmaceutical/methods , Humans , Hydrogen-Ion Concentration , Models, Biological , Polymers/chemistry , Typhoid-Paratyphoid Vaccines/administration & dosage , Vaccines, Attenuated/administration & dosage
10.
Glycoconj J ; 30(5): 511-22, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23053636

ABSTRACT

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.


Subject(s)
Hexosyltransferases/immunology , Membrane Proteins/immunology , O Antigens/immunology , Polysaccharides, Bacterial/immunology , Salmonella typhi/drug effects , Typhoid Fever/prevention & control , Typhoid-Paratyphoid Vaccines/immunology , Animals , Antibodies, Bacterial/biosynthesis , Campylobacter jejuni/chemistry , Campylobacter jejuni/genetics , Campylobacter jejuni/immunology , Carbohydrate Sequence , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli/immunology , Female , Glycoconjugates/chemistry , Glycoconjugates/immunology , Glycosylation , Hexosyltransferases/chemistry , Hexosyltransferases/genetics , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mice , Molecular Sequence Data , O Antigens/chemistry , O Antigens/genetics , Polysaccharides, Bacterial/chemistry , Polysaccharides, Bacterial/genetics , Protein Engineering , Salmonella typhi/immunology , Salmonella typhi/pathogenicity , Typhoid Fever/immunology , Typhoid Fever/microbiology , Typhoid-Paratyphoid Vaccines/administration & dosage , Typhoid-Paratyphoid Vaccines/chemistry , Typhoid-Paratyphoid Vaccines/genetics , Vaccines, Conjugate
11.
PLoS One ; 7(11): e47039, 2012.
Article in English | MEDLINE | ID: mdl-23144798

ABSTRACT

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 CRM(197), 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 CRM(197) as carrier protein. The newly developed conjugation chemistry yielded immunogenic conjugates with strong serum bactericidal activity against S. Paratyphi A.


Subject(s)
Bacterial Proteins/chemistry , O Antigens/chemistry , Paratyphoid Fever/prevention & control , Salmonella paratyphi A/immunology , Typhoid-Paratyphoid Vaccines/chemistry , Vaccines, Conjugate/chemistry , Animals , Bacterial Proteins/immunology , Bacterial Proteins/therapeutic use , Carbohydrate Sequence , Female , Humans , Mice , Molecular Sequence Data , O Antigens/immunology , O Antigens/therapeutic use , Paratyphoid Fever/blood , Paratyphoid Fever/immunology , Paratyphoid Fever/veterinary , Salmonella paratyphi A/chemistry , Typhoid-Paratyphoid Vaccines/immunology , Typhoid-Paratyphoid Vaccines/therapeutic use , Vaccines, Conjugate/immunology , Vaccines, Conjugate/therapeutic use
12.
Hum Vaccin Immunother ; 8(2): 189-93, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22426380

ABSTRACT

Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), is a major health problem particularly in developing countries. The available vaccines have certain limitations regarding their efficacy, and inability to induce an immune response especially in individuals under 2 years of age. Conjugate vaccines which consist of a bacteria-specific polysaccharide chemically bound to a carrier protein overcome these problems by inducing a T-cell dependent immune response characterized by enhanced immunogenicity in all ages. In this study, O-specific polysaccharides (OSP) of S. Typhi were conjugated to diphtheria toxoid (DT) using adipic acid dihydrazide (ADH) as a linker. These conjugates (OSP-AH-DT) were then evaluated for their immunogenicity using mice as a model and showed significantly higher levels of IgG ELISA titers (P = 0.0241 and 0.0245) than lipopolysaccharides alone. Different immunization  schedules were compared and it was found that schedule-B (three injections with 4-weeks interval) induced higher immune responses than schedule-A (three injections with 2-weeks interval). We showed that diphtheria toxoid can be successfully employed as a carrier protein for conjugation with Salmonella OSP and play an important role in facilitating adequate immune response.


Subject(s)
Antibodies, Bacterial/blood , Diphtheria Toxoid/immunology , O Antigens/immunology , Salmonella typhi/immunology , Typhoid Fever/prevention & control , Typhoid-Paratyphoid Vaccines/immunology , Animals , Antibodies, Bacterial/immunology , Carrier Proteins/immunology , Diphtheria Toxoid/chemistry , Female , Immunization Schedule , Immunoglobulin G/biosynthesis , Mice , Mice, Inbred BALB C , O Antigens/chemistry , Typhoid Fever/immunology , Typhoid-Paratyphoid Vaccines/chemistry , Vaccines, Conjugate/chemistry , Vaccines, Conjugate/immunology
13.
Vaccine ; 30(5): 853-61, 2012 Jan 20.
Article in English | MEDLINE | ID: mdl-22172503

ABSTRACT

A conjugate vaccine for Salmonella enterica serovar Typhi was produced by chemically linking Vi, purified from Citrobacter, to the non-toxic mutant diphtheria toxin CRM(197) via an adipic dihydrazide spacer using N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide coupling chemistry. The polysaccharide purification process was developed based on Vi precipitation from culture supernatant with cetyl trimethylammonium bromide (CTAB), solubilization of the CTA-polysaccharide salt with ethanol followed by exchange of the CTA(+) counter ion with Na(+). The purified Vi polysaccharide was fully O-acetylated and with high purity. The conjugation process was optimized to obtain a scalable process that has been used for GMP production at pilot scale of vaccine currently in clinical trials.


Subject(s)
Citrobacter/immunology , Polysaccharides, Bacterial/isolation & purification , Typhoid-Paratyphoid Vaccines/isolation & purification , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Citrobacter/chemistry , Humans , Polysaccharides, Bacterial/chemistry , Polysaccharides, Bacterial/metabolism , Technology, Pharmaceutical/methods , Typhoid-Paratyphoid Vaccines/chemistry , Typhoid-Paratyphoid Vaccines/metabolism , Vaccines, Conjugate/chemistry , Vaccines, Conjugate/metabolism
14.
Vaccine ; 29(44): 7618-23, 2011 Oct 13.
Article in English | MEDLINE | ID: mdl-21843575

ABSTRACT

In this study it was demonstrated that the immunogenicity of Vi polysaccharide-diphtheria toxoid conjugates was related to the physical and chemical structure of the conjugate. Conjugates were prepared in two steps, firstly binding adipic acid dihydrazide (ADH) spacer molecules to diphtheria toxoid (DT) carrier protein then secondly binding varying amounts of this derivatized DT to a fixed amount of Vi capsular polysaccharide purified from Salmonella enterica Serovar Typhi. As the amount of DT bound to the Vi increased the size of the conjugate increased but also the degree of cross-linking increased. The immunogenicity of the conjugates was tested in mice and measured by ELISA for anti Vi and anti DT IgG responses, and the results revealed a trend that as the amount of DT bound to the Vi increased the anti Vi responses increased. This study establishes a correlation between physico-chemical characteristics of the conjugate and the magnitude of the anti Vi and anti DT responses.


Subject(s)
Diphtheria Toxoid/chemistry , Diphtheria Toxoid/immunology , Polysaccharides, Bacterial/chemistry , Polysaccharides, Bacterial/immunology , Typhoid-Paratyphoid Vaccines/chemistry , Typhoid-Paratyphoid Vaccines/immunology , Adipates/metabolism , Animals , Antibodies, Bacterial/blood , Antitoxins/blood , Enzyme-Linked Immunosorbent Assay , Humans , Immunoglobulin G/blood , Mice , Protein Binding , Vaccines, Combined/chemistry , Vaccines, Combined/immunology , Vaccines, Conjugate/chemistry , Vaccines, Conjugate/immunology
15.
Clin Vaccine Immunol ; 18(3): 460-8, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21248155

ABSTRACT

Typhoid fever remains a major health problem in developing countries. Young children are at high risk, and a vaccine effective for this age group is urgently needed. Purified capsular polysaccharide from Salmonella enterica serovar Typhi (Vi) is licensed as a vaccine, providing 50 to 70% protection in individuals older than 5 years. However, this vaccine is ineffective in infants. Vi conjugated to a carrier protein (i.e., an exoprotein A mutant from Pseudomonas aeruginosa [rEPA]) is highly immunogenic, provides long-term protection, and shows more than 90% protective efficacy in children 2 to 5 years old. Here, we describe an alternative glycoconjugate vaccine for S. Typhi, Vi-CRM(197), where Vi was obtained from Citrobacter freundii WR7011 and CRM(197), the mutant diphtheria toxin protein, was used as the carrier. We investigated the optimization of growth conditions for Vi production from C. freundii WR7011 and the immunogenicity of Vi-CRM(197) conjugates in mice. The optimal saccharide/protein ratio of the glycoconjugates was identified for the best antibody production. We also demonstrated the ability of this new vaccine to protect mice against challenge with Vi-positive Salmonella enterica serovar Typhimurium.


Subject(s)
Adjuvants, Immunologic/administration & dosage , Bacterial Proteins/administration & dosage , Citrobacter freundii/chemistry , Polysaccharides, Bacterial/immunology , Polysaccharides, Bacterial/isolation & purification , Typhoid-Paratyphoid Vaccines/immunology , Adjuvants, Immunologic/chemistry , Animals , Bacterial Proteins/chemistry , Female , Mice , Mice, Inbred BALB C , Polysaccharides, Bacterial/chemistry , Salmonella Infections, Animal/prevention & control , Salmonella typhimurium/immunology , Salmonella typhimurium/pathogenicity , Typhoid-Paratyphoid Vaccines/chemistry , Vaccines, Conjugate/chemistry , Vaccines, Conjugate/immunology
16.
Clin Vaccine Immunol ; 17(1): 73-9, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19889941

ABSTRACT

Typhoid fever remains a serious public health problem in developing countries, especially among young children. Recent studies showed more than 50% of typhoid cases are in children under 5 years old. Licensed vaccines, such as Salmonella enterica serovar Typhi capsular Vi, did not confer protection against typhoid fever for this age group. Vi conjugate, prepared by binding Vi to Pseudomonas aeruginosa recombinant exoprotein A (rEPA), induces protective levels of antibody at as young as 2 years old. Because of the lack of regulatory precedent for rEPA in licensing vaccines, we employed diphtheria toxoid (DT) as the carrier protein to accommodate accessibility in developing countries. Five lots of Vi-DT conjugates were prepared using adipic acid dihydrazide (ADH) as the linker. All 5 lots showed consistency in their physical and chemical characteristics and final yields. These Vi-DT conjugates elicited levels of IgG anti-Vi in young mice significantly higher than those in mice injected with Vi alone and induced a booster response upon reinjection. This booster effect was absent if the Vi replaced one of the two conjugate injections. Vi-DT was stable under repeated freeze-thaw (20 cycles). We plan to perform clinical evaluation of the safety and immunogenicity of Vi-DT when added to the infant combination vaccines.


Subject(s)
Diphtheria Toxoid/chemistry , Diphtheria Toxoid/immunology , Polysaccharides, Bacterial/chemistry , Polysaccharides, Bacterial/immunology , Salmonella typhi/immunology , Typhoid-Paratyphoid Vaccines/chemistry , Typhoid-Paratyphoid Vaccines/immunology , Animals , Antibodies, Bacterial/blood , Drug Stability , Female , Freezing , Immunization, Secondary , Immunoglobulin G/blood , Mice , Salmonella typhi/chemistry
18.
Vaccine ; 15(3): 321-4, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9139493

ABSTRACT

A 13 amino acid peptide corresponding to a potent BALB/c mouse T cell epitope of hen's egg lysozyme (HEL) was substituted singly at five sites in the d flagellin of Salmonella muenchen. The resulting chimeric proteins were unable to expand T cells capable of being stimulated by the HEL epitope and induced T cell populations which either failed to respond or responded at a low level to a normally highly stimulatory flagellin T cell epitope that was present in all chimeras. The results suggested that substitution of a T cell epitope in flagellin may alter the processing of the resulting immunogen.


Subject(s)
Epitopes/immunology , Flagellin/immunology , Lymphocyte Activation/drug effects , Muramidase/immunology , Salmonella/immunology , T-Lymphocytes/immunology , Typhoid-Paratyphoid Vaccines/immunology , Amino Acid Sequence , Animals , Antigen Presentation , Epitopes/pharmacology , Flagellin/genetics , Flagellin/pharmacology , Lymphocyte Activation/genetics , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Muramidase/pharmacology , Typhoid-Paratyphoid Vaccines/chemistry , Typhoid-Paratyphoid Vaccines/genetics , Vaccines, Synthetic/chemistry , Vaccines, Synthetic/immunology
19.
Dev Biol Stand ; 87: 277-81, 1996.
Article in English | MEDLINE | ID: mdl-8854028

ABSTRACT

Live oral attenuated vaccines against typhoid fever (Salmonella typhi Ty21a) and cholera (Vibrio cholerae CVD 103-HgR) have been licensed for human use. Vaccine potency is dependent upon each dose containing a minimum number of viable organisms and galenic parameters. To ensure long-term stability, such vaccines must be stored at 5 degrees C +/- 3 degrees C. However, exposure to ambient temperatures (25 degrees C) for short periods of time (< 7 days) does not compromise vaccine potency. Brief exposures (< or = 24 hours) to temperatures as high as 37 degrees C will also not render the vaccine unsuitable for use. The Ty21a vaccine is available either as enteric-coated capsules or as a "liquid formulation", while CVD 103-HgR is presented only in the latter form. Each galenic formulation presents unique challenges with regard to the production of stable vaccines. Residual moisture, excipients, and processing temperatures during manufacturing were all found to markedly affect vaccine stability.


Subject(s)
Cholera Vaccines/chemistry , Typhoid-Paratyphoid Vaccines/chemistry , Capsules , Child, Preschool , Cholera Vaccines/immunology , Drug Packaging , Drug Stability , Drug Storage , Excipients , Gastric Acid , Humans , Hydrogen-Ion Concentration , Pancreatic Juice , Preservatives, Pharmaceutical , Refrigeration , Solutions , Temperature , Time Factors , Typhoid-Paratyphoid Vaccines/immunology , Vaccines, Attenuated/chemistry , Vaccines, Attenuated/immunology
20.
Arch Intern Med ; 155(21): 2293-9, 1995 Nov 27.
Article in English | MEDLINE | ID: mdl-7487253

ABSTRACT

Typhoid is still prevalent in many parts of the world. We reviewed all published and unpublished studies of a newly licensed vaccine composed of the Vi capsular polysaccharide of Salmonella typhi, the causative agent of the disease, which had been licensed previously outside the United States. These included observational studies and double-blind randomized studies done in the United States, Europe, and the developing world in which children and adults unexposed to typhoid or those living in endemic areas were enrolled. A single dose of 25 micrograms of the purified polysaccharide was given by intramuscular injection. The vaccine was well tolerated, inducing only minor reactions in fewer than 10% of subjects. An antibody response occurred in about 90% of subjects and lasted about 3 years. Seroconversion was shown in children as young as 2 years. Protective efficacy was evaluated in two studies conducted in areas in which typhoid is endemic; the efficacy was 55% and 75%, respectively, in adults and in children older than 5 years. The Vi vaccine compares favorably with other typhoid vaccines in regard to safety, patient compliance, immunogenicity, and efficacy. Vi polysaccharide is a well-standardized antigen that is effective in a single parenteral dose, is safer than whole-cell vaccine, and may be used in children 2 years of age or older.


Subject(s)
Antigens, Bacterial/immunology , Polysaccharides, Bacterial , Typhoid-Paratyphoid Vaccines/immunology , Adult , Child, Preschool , Double-Blind Method , Humans , Immunization, Secondary , Product Surveillance, Postmarketing , Randomized Controlled Trials as Topic , Typhoid-Paratyphoid Vaccines/adverse effects , Typhoid-Paratyphoid Vaccines/chemistry , Typhoid-Paratyphoid Vaccines/supply & distribution
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