Vi capsular polysaccharide-protein conjugates for prevention of typhoid fever. Preparation, characterization, and immunogenicity in laboratory animals.

The Vi has proven to be a protective antigen in two double masked, controlled clinical trials in areas with high rates of typhoid fever (approximately 1% per annum). In both studies the protective efficacy of the Vi was approximately 70%. Approximately 75% of subjects in these areas responded with a fourfold or greater rise of serum Vi antibodies. In contrast, the Vi elicited a fourfold or greater rise in 95-100% of young adults in France and the United States. Methods were devised, therefore, to synthesize Vi-protein conjugates in order to both enhance the antibody response and confer T-dependent properties to the Vi (and theoretically increase its protective action in populations at high risk for typhoid fever). We settled on a method that used the heterobifunctional crosslinking reagent, N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP), to bind thiol derivatives of the Vi to proteins. This synthetic scheme was reproducible, provided high yields of Vi-protein conjugates, and was applicable to several medically relevant proteins such as diphtheria and tetanus toxoids. The resultant conjugates were more immunogenic in mice and juvenile Rhesus monkeys than the Vi alone. In contrast to the T-independent properties of the Vi, conjugates of this polysaccharide with several medically relevant proteins induced booster responses in mice and in juvenile Rhesus monkeys. Clinical studies with Vi-protein conjugates are planned. This scheme is also applicable to synthesize protein conjugates with other polysaccharides that have carboxyl functions.

An outbreak of Salmonella Paratyphi A in a boarding school: a community-acquired enteric fever and carriage investigation.

Salmonella Paratyphi A (SPA) is rapidly becoming a common cause of enteric fever in South East Asia. A large outbreak of SPA occurred in a boarding middle school in China in 2004. There were 394 suspected cases; 95·5% were students. The highest incidence was in the youngest children (7th grade). Forty-four of 151 (29%) blood cultures and 4/54 (7·4%) rectal swabs were positive for SPA; three were from kitchen workers. The geometric mean levels of serum IgG anti-lipopolysaccharide (anti-LPS) from patients was higher than from healthy individuals [35·25 vs. 5·20 ELISA units (EU), P<0·001]. A kitchen worker with a positive rectal swab, negative blood culture and a high level of serum IgG anti-LPS (529·65 EU), was identified as a possible SPA carrier. No SPA was isolated from water or food samples. A survey of students' habits indicated drinking unboiled water as being the main reason for contracting the disease. Hand washing was the second most important factor. A food handler with possible SPA carriage could also have been a risk factor. Attention to maintaining a safe water supply, enhancing food-handler hygiene and proper hand washing can help to prevent similar outbreaks in the future.

Prevention of invasive bacterial diseases by immunization with polysaccharide-protein conjugates.

Covalent binding of CPS to T cell-dependent carrier proteins to form conjugates can be done by clinically acceptable methods. As a component of a conjugate, two immunologic properties of CPS are changed: 1) their immunogenicity is increased and; 2) reinjection induces a booster response in the young (T cell-dependence). Serum antibodies induced by the CPS alone, or as a component of a conjugate, are qualitatively similar: the difference between antibodies elicited by the CPS or the conjugate is quantitative. A clinical trial with a Hib-DT conjugate showed that conjugates could confer immunity in an age group not protected by the CPS alone. (table; see text) Induction of serum CPS antibodies confers protection against capsulated bacteria in the bloodstream: their role in the interaction of these pathogens on the mucous membranes has not been characterized. Preliminary in vitro experiments suggest that secretory antibodies to non-capsular structures may also exert protective immunity.

O-acetylation affects the binding properties of the carboxyl groups on the Vi bacterial polysaccharide.

The capsular polysaccharide of Salmonella typhi and Citrobacter freundii (Vi) is a linear homopolymer of (1-->4)-linked 2-acetamido-2-deoxy-alpha-D-galactopyranosyluronic acid partially O-acetylated at the C-3 position. The physico-chemical properties of the carboxyl groups of the Vi polysaccharide, as a function of different degrees of O-acetylation, were studied by potentiometric titration, circular dichroism, and their reaction with the bulky nucleophile 2-nitro-phenylhydrazine (NPH). Potentiometric titrations with K+ and Ca2+ hydroxides showed that the difference in the free energy of binding between the two cations (delta GKCa) was inversely proportional to the degree of O-acetylation. Similar cationic effects were found when measuring circular dichroism. Moreover there was also an inverse relation between the degree of O-acetylation and the extent of binding of NPH to the carboxyl groups. These data all indicate that O-acetyl groups hinder the association of carboxyls with cations and nucleophilic reagents. This provides a possible explanation for the importance of the O-acetyl and the relative unimportance of the carboxyl groups in contributing to the immunologic properties of the Vi.

Characterization of colominic acid by circular dichroism and viscosity analysis.

Conformations of oligo- and poly-(alpha (2-->8)-D-Neu pNAc) (colominic acid) and its derivatives were studied by circular dichroism (CD) spectroscopy and viscometry to understand the molecular basis of their unusual antigenic properties. No temperature-dependent conformational transition between 5 and 70 degrees C or divalent salt effect of Ca2+ or Mg2+ was observed in colominic acid or its N-deacetylated form by CD spectroscopy. However, CD spectroscopy indicated that the distribution of conformers in oligocolominic acid changes continuously from n = 2 to octamer, and there was no further change of the conformer distribution for n > 9. Colominic acid exhibited a much lower intrinsic viscosity compared with the values for other polyelectrolytes of similar linear charge density, such as polynucleic acids. The apparent absence of induced conformational transition by salt or temperature, and the high flexibility indicated that the binding of colominic acid to its antibodies may not contain a significant amount of specific conformationally controlled determinant. Instead, our data suggest that more than nine saccharide units are needed for a cooperative binding process.

Circular dichroism of the O-specific polysaccharide of Vibrio cholerae O1 and some related derivatives.

The O-specific polysaccharide (O-SP) of Vibrio cholerae O1 is a homopolymer of alpha-(1 --> 2)-linked 4-amino-4, 6-dideoxy-D-mannopyranose whose amino group is acylated with 3-deoxy-L-glycero-tetronic acid [N-(3-deoxy-L-glycero- tetronyl)-alpha-D-perosamine]. The circular dichroism (CD) of the O-SP as well as of a number of N-acyl (formyl, acetyl, 4-hydroxybutyl, 3-deoxy-L-and D-glycero-tetronyl) derivatives of methyl alpha-glycosides of 4-amino-4,6-dideoxy-D-mannopyranose (methyl alpha-D-perosaminide) has been studied for solutions in water, acetonitrile and 1,1,1-trifluoroethanol. The strong solvent dependence of the sign and intensity of the CD observed for the monosaccharide amides bearing achiral acyl groups is explained by solvent-mediated change of the orientation of the amido group relative to the proximal hydroxyl group at C-3. A change in the population of the nonplanar conformers with a pyramidal arrangement of bonds at the amido nitrogen has also been considered. The effect of solvents upon the CD spectra of compounds bearing chiral N-acyl substituents is less pronounced than that of their counterparts bearing achiral N-acyl substituents. The sign of the CD for the O-SP was found negative in all solvents used. This result is in agreement with the negative sign of the CD of the n --> pi electron transition observed, independent of the solvent, for the monosaccharide derivative containing the L-glycero-3-deoxytetronamido group, and the positive sign found for its D-glycero-counterpart.

Conformational differences among mono- and oligosaccharide fragments of O-specific polysaccharides of Vibrio cholerae O1 revealed by circular dichroism.

The circular dichroism (CD) of synthetic mono- and oligosaccharides that represent the terminal, non-reducing group of O-antigens of Vibrio cholerae O1 from the subtypes Ogawa and Inaba was measured in various solvents. We found differences in the CD of the monosaccharides of these subtypes that decrease with increasing chain lengths of the oligosaccharides. The differences can be explained by different orientations of the N-acyl side chain of the terminal monosaccharides. The linear relationship of ellipticity versus the number of residues in an oligosaccharide chain follows the principle of optical superposition. This, together with a similar contribution by internal units to the overall ellipticity, suggests an identical, regular conformation of oligosaccharide fragments of both Ogawa and Inaba series.

Ultrasonic irradiation of bacterial polysaccharides. Characterization of the depolymerized products and some applications of the process.

Ultrasonic irradiation (u.i.) has been used to depolymerize biopolymers including DNA, dextran, and the Vi capsular polysaccharide from Citrobacter freundii. Representative bacterial polysaccharides were subjected to u.i. and the effect of this energy upon their molecular weight and chemical structure was characterized. U.i. depolymerized a neutral polysaccharide (dextran) and acidic polysaccharides containing either a phosphoric diester linkage [Haemophilus influenzae type b (Hib) and pneumococcus types 6A and 6B] or a uronic acid moiety (pneumococcus type 9N). Prolonged u.i. depolymerized all the polysaccharides to a finite and similar molecular mass (approximately 50 000 daltons). The rate of depolymerization induced by u.i. depended on the viscosity of the solvent and the concentration of the polysaccharide. 13C-N.m.r. data of the native Hib polysaccharide and its depolymerized products indicated that u.i. did not alter the chemical structure of the repeating units. Determination of the monophosphate terminal residues by 31P-n.m.r. spectroscopy and of the reducing end groups by the Park-Johnson reaction indicated that both the phosphoric diester and the glycosidic linkages were cleaved. The Vi polysaccharide, prepared as an investigational vaccine, could not be analyzed for its chemical structure by 13C-n.m.r. spectroscopy owing to its high viscosity but depolymerization by u.i. permitted this analysis. The finite molecular weight of the products observed after prolonged u.i. is best explained by the postulation that the mechanical torque necessary to rupture the linkages is dependent upon the length of the polysaccharide. The method of u.i. for depolymerization is useful for the preparation of homogeneous, low-molecular-weight polysaccharides without alteration of the chemical structure of the repeating units.

Simultaneous determination of glucosamine and glucosamine 4-phosphate in Lipid A with high-performance anion-exchange chromatography (HPAEC).

A method for simultaneous determination of glucosamine (GlcN) and glucosamine 4-phosphate (GlcN-4-P) in Lipid A with high-performance anion-exchange chromatography (HPAEC) is described. Lipid A is hydrolyzed with 4 M HCl for 16 h at 100 degrees C, and the peaks of glucosamine and glucosamine 4-phosphate were measured. The true GlcN value can be computed from the GlcN value after correction for the incomplete hydrolysis of GlcN-4-P, or by the combined yield of GlcN and GlcN-4-P.

Hypothesis: how licensed vaccines confer protective immunity.

By examining experience with evaluation of licensed vaccines we theorize that a critical level of serum IgG confers protection against infectious diseases by killing or inactivating the inoculum. We found that efficacy is reliably predicted by measurement of serum antibodies elicited by vaccines, that serum IgG antibodies alone account for the protection conferred by passive immunization, that vaccine-induced "herd" immunity is best explained by inactivation of the inoculum on epithelial surfaces by serum antibodies and that serum antibodies induced by active immunization will neither treat disease symptoms nor eliminate the pathogen. If valid, this theory should facilitate research because knowledge of the pathogenesis of the disease symptoms may not be essential for vaccine development.

Application of optical properties of the Vi capsular polysaccharide for quantitation of the Vi antigen in vaccines for typhoid fever.

The capsular polysaccharide of Salmonella typhi and of Citrobacter freundii (Vi) is a linear homopolymer of alpha 1,4-linked N-acetylgalactosaminuronic acid, variably O-acetylated at the C-3 position. Vaccines composed of Vi confer protection against typhoid fever with an efficacy of about 70%; Vi has recently been conjugated to proteins to increase its immunogenicity and effectiveness (I.L. Acharya, R. Tapa, V.L. Gurubacharya, M.B. Shrestha, C.U. Lowe, D.D. Bryla, R. Schneerson, J.B. Robbins, T. Crampton, B. Trollfors, M. Cadoz, D. Schulz, and J. Armand, N. Engl. J. Med. 317:1101-1104, 1987; K.P. Klugman, I. Gilbertson, H.J. Kornhof, J.B. Robbins, R. Schneerson, D. Schulz, M. Cadoz, and J. Armand, Lancet ii:1165-1169, 1987; S.C. Szu, A.L. Stone, J.D. Robbins, R. Schneerson, and J.B. Robbins, J. Exp. Med. 166:1510-1524, 1987). Vi, however, cannot be measured by conventional colorimetric methods. Two optical techniques were adapted to quantitate Vi in vaccines. The first, Fourier-transformed infrared spectroscopy, was performed on salt-free, freeze-dried samples. The intensities of the absorbance peaks of Vi were proportional to the amount of Vi within the range of 0.25 to 2.0 mg. The amount of Vi was determined from integrated absorptions at the 1,235- or 1,417-cm-1 band. The second technique, spectrophotometric titration, was more sensitive than the Fourier-transformed infrared spectroscopy and could be performed on dilute solutions. The metachromatic effect of the reaction between the aromatic cationic dye acridine orange and the carboxyl groups of Vi was quantitative within +/- 2% in the range of 20 to 700 micrograms of Vi per ml. The accuracy of the titration of Vi in the vaccines was within +/- 8%. These two methods may be applicable to measure other capsular polysaccharides in vaccines.

Detection of "neutral" type 7F and type 14 pneumococcal capsular polysaccharides by immunoelectrophoresis.

Type 7F and type 14 pneumococcal capsular polysaccharides, neutral at pH 8.6, were studied by immunoelectrophoresis at pH 5. Three techniques were used: rocket, countercurrent, and reversed immunoelectrophoresis. Our results show that these two capsular polysaccharides types can be detected at pH 5 with high sensitivity.

Evaluation of synthetic schemes to prepare immunogenic conjugates of Vibrio cholerae O139 capsular polysaccharide with chicken serum albumin.

Vibrio cholerae serotype O139 is a new etiologic agent of epidemic cholera. There is no vaccine available against cholera caused by this serotype. V. cholerae O139 is an encapsulated bacterium, and its polysaccharide capsule is an essential virulent factor and likely protective antigen. This study evaluated several synthetic schemes for preparation of conjugates of V. cholerae O139 capsular polysaccharide (CPS) with chicken serum albumin as the carrier protein (CSA) using 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) or 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as activating agents. Four conjugates described here as representative of many experiments were synthesized in 2 steps: 1) preparation of adipic acid hydrazide derivative of CPS (CPS(AH)) or of CSA (CSA(AH)), and 2) binding of CPS(AH) to CSA or of CPS to CSA(AH). Although all conjugates induced CPS antibodies, the conjugate prepared by EDC-mediated binding of CPS and CSA(AH) (EDC:CPS-CSA(AH)) was statistically significantly less immunogenic than the other three conjugates. Representative sera from mice injected with these three conjugates contained antibodies that mediated the lysis of V. cholerae O139 inoculum. Evaluation of the different synthetic schemes and reaction conditions in relation to the immunogenicity of the resultant conjugates provided the basis for the preparation of a V. cholerae O139 conjugate vaccine with a medically useful carrier protein such as diphtheria toxin mutant.

Rabbit antibodies to the cell wall polysaccharide of Streptococcus pneumoniae fail to protect mice from lethal challenge with encapsulated pneumococci.

A conjugate, composed of the cell wall polysaccharide (C polysaccharide) of Streptococcus pneumoniae and bovine serum albumin (BSA), was prepared with the bifunctional agent N-succinimidyl-3-(2-pyridyldithio)-propionate. Analysis with monoclonal antibodies provided evidence that the phosphocholine (PC) moiety of the C polysaccharide was retained during the conjugation procedure. The C polysaccharide-BSA conjugate elicited antibodies to C polysaccharide in rabbits; no PC-specific antibodies were detected in globulins prepared from these hyperimmune sera obtained early and late after a second immunization. Rabbit hyperimmune sera were taken after multiple intravenous injections of the pneumococcus strain SRC-2, which has a capsulelike structure composed of the C polysaccharide. Globulin prepared from these antisera had both C polysaccharide- and PC-specific antibodies. Antibodies to C polysaccharide elicited by the C polysaccharide-BSA conjugate failed to protect mice against intraperitoneal challenge with a strain of type 3 or type 6A pneumococci. The anti-SRC-2 globulin conferred protection against both of these pneumococcal strains. Absorption of the SRC-2 globulin with C polysaccharide, however, failed to change its protective activity. These data provide evidence that antibodies to the C polysaccharide do not confer immunity against infection of mice with encapsulated pneumococci inoculated by the intraperitoneal route.

Protection of mice against Salmonella typhimurium with an O-specific polysaccharide-protein conjugate vaccine.

Serious infections with salmonellae remain a threat in many human populations. Despite extensive study of salmonella infections in animals and clinical experience with killed cellular vaccines, there are no vaccines against serotypes other than Salmonella typhi licensed for human use. Serum antibodies to the O-specific polysaccharide (O-SP) of salmonellae protect mice against invasive infection. In order to render it immunogenic, we have conjugated the O-SP of Salmonella typhimurium to carrier proteins by various schemes. O-SP conjugated to tetanus toxoid (O-SP-TT) elicited antibodies in outbred mice after three subcutaneous injections without adjuvant. The O-SP alone elicited no detectable antibody. The antibody response to O-SP-TT was boosted by successive doses and consisted of immunoglobulin G (IgG) and IgM. Most mice only produced antibodies specific for the abequose (O:4 factor) region of the O-SP. Occasional animals also produced antibodies to the core oligosaccharide. Immunized mice were protected against intraperitoneal challenge with S. typhimurium, demonstrating a 160-fold increase in the 50% lethal dose. Passive immunization with conjugate-induced IgM or IgG also protected against challenge. These results indicate that an O-SP-TT conjugate, when given by a route and formulation acceptable for human use, protects mice against challenge with S. typhimurium.

Perspective: hypothesis: serum IgG antibody is sufficient to confer protection against infectious diseases by inactivating the inoculum.

The theory proposed is that a critical level of specific serum IgG is sufficient to confer protection against infectious diseases by inactivating the inoculum of the pathogen. This theory relies heavily on evaluation of licensed vaccines and includes the following: Measurement of serum antibodies only reliably predicts the efficacy of vaccines, according to regulatory agencies. Serum IgG antibodies alone account for the protection conferred by passive immunization. "Herd" immunity conferred by vaccines on viral and bacterial diseases is best explained by serum antibodies that inactivate the inoculum on mucosal surfaces, thus reducing the pathogen's transmission. Once the disease is manifest, serum antibodies induced by active immunization will neither relieve symptoms nor eliminate the pathogen; specific IgG must be present when the host encounters the pathogen in order to confer protective immunity. Information about the initial pathogen-host contact is vital, whereas knowledge of the symptomatology of the disease may not be essential for vaccine development.

Protection against pneumococcal infection in mice conferred by phosphocholine-binding antibodies: specificity of the phosphocholine binding and relation to several types.

Some anti-phosphocholine antibodies protect mice against challenge with certain, but not all, pneumococcal types. We found that both their isotype and reactivity with the cell wall C-polysaccharide of encapsulated pneumococci, as measured by immunodiffusion, were important in predicting the protective activity of anti-phosphocholine antibodies. We propose that the specificity of the protective antibodies includes the backbone of the phosphocholine-containing structure.

Characterization of the Salmonella paratyphi C Vi polysaccharide.

The Vi capsular polysaccharide (Vi) is both a virulence factor and a protective antigen of Salmonella typhi; its pathogenic role for Salmonella paratyphi C is less well understood. We found no differences between the antigenic and immunogenic properties and the structure of the Vi from representative strains of S. paratyphi C, S. typhi, and Citrobacter freundii. There were, however, differences in both the amount produced per cell and the degree of association with the cell among the Vi from the three species of Enterobacteriaceae. S. paratyphi C produced less Vi than both the wild-type S. typhi and C. freundii did, and it showed the fastest release of Vi into the media. These findings may provide an explanation for the inability of the Vi to inhibit completely the agglutination of S. paratyphi C by anti-O sera. In an outbreak of enteric fever caused by S. paratyphi C, 66 of 78 isolates (85%) were Vi positive.

Covalent linkage between the capsular polysaccharide and the cell wall peptidoglycan of Streptococcus pneumoniae revealed by immunochemical methods.

The attachment of capsular polysaccharide to Streptococcus pneumoniae was examined using monoclonal and polyclonal antibodies. Among the strains examined, the capsular polysaccharide of types 2, 4, 6A, 6B, 7F, 8, 14, 19F and 23F was bound to the pneumococci whereas that of a type 3 strain was not. Sequential treatment with 2% SDS at 100 degrees C, pronase, and EDTA did not dissociate the capsular polysaccharide from the pneumococci. Treatment of the cells with mutanolysin, a muramidase that degrades the cell wall peptidoglycan of pneumococci and other streptococci, released both the capsular and the cell wall C-polysaccharide (C-Ps). Type 6A capsular polysaccharide released from cell walls by mutanolysin treatment, was fractionated by high performance liquid chromatography and examined by immunoelectrophoresis. It was found to be bound to both the C-Ps and the peptidoglycan. The bond between the capsular polysaccharide and the peptidoglycan has not yet been identified but is probably covalent, as the two components could not be dissociated after boiling in SDS. Based on our studies with type 6A, we propose that capsular polysaccharide and C-Ps of the pneumococcus are linked to the peptidoglycan at different sites and, thereby, indirectly to each other. Studies in mice showed that the peptidoglycan enhanced the serum antibody response to C-Ps but not to type 6A polysaccharide.

Comparative immunogenicity of conjugates composed of Escherichia coli O111 O-specific polysaccharide, prepared by treatment with acetic acid or hydrazine, bound to tetanus toxoid by two synthetic schemes.

Escherichia coli O111, of various H types and virulence factors, causes enteritis throughout the world, especially in young children. This O type is found rarely in healthy individuals. Serum antibodies to the O-specific polysaccharide of O111 lipopolysaccharide (LPS) protect mice and dogs against infection with this E. coli serotype. The O111 O-specific polysaccharide is composed of a pentasaccharide repeat unit with two colitoses bound to the C-3 and C-6 of glucose in a trisaccharide backbone; this structure is identical to that of Salmonella adelaide (O35), another enteric pathogen. Nonpyrogenic O111 O-specific polysaccharide was prepared by treatment of its LPS with acetic acid (O-SP) or the organic base hydrazine (DeA-LPS). The O-SP had a reduced concentration of colitose. These products were derivatized with adipic acid dihydrazide (ADH) or thiolated with N-succinimidyl-3(2-pyridyldithio) propionate (SPDP). The four derivatives were covalently bound to tetanus toxoid (TT) by carbodiimide-mediated condensation or with SPDP to form conjugates. Immunization of BALB/c and general-purpose mice by a clinically acceptable route showed that DeA-LPS-TTADH, of the four conjugates, elicited the highest level of LPS antibodies. Possible reasons to explain this differential immunogenicity between the four conjugates are discussed.