Epitope specificities of the group Y and W-135 polysaccharides of Neisseria meningitidis.

Previous studies have identified the length dependency of several polysaccharide (PS) protective epitopes. We have investigated whether meningococcal polysaccharides Y and W-135 possess such epitopes. Oligosaccharides (OSs) consisting of one or more disaccharide repeating units (RU) were derived from the capsular PSs of group Y and W-135 meningococci (GYMP and GWMP, respectively) by mild acid hydrolysis. The relative affinities of anticapsular antibodies binding to derivative OSs of different chain lengths were measured in inhibition enzyme-linked immunosorbent assays. As OS size increased from two to three RU, there was a notable increase in binding inhibition of rabbit anti-group Y antiserum. This pattern of antibody binding inhibition was also observed for rabbit antiserum to group W-135, though the inhibition increase was much more pronounced. In the cases of both OS species, the concentration of inhibiting antigen required to achieve 50% inhibition of rabbit immunoglobulin binding increased progressively as the inhibiting disaccharide chain length increased from 1 RU through greater than 50 RU. These data suggest that antibodies directed against both of these meningococcal PSs recognize conformational epitopes only fully expressed in higher-molecular-weight forms of these antigens.

Protective meningococcal capsular polysaccharide epitopes and the role of O acetylation.

Previous studies with group C meningococcal polysaccharide-tetanus toxoid (GCMP-TT) conjugates had suggested that the GCMP O-acetyl group masked the protective epitope for group C meningococci through steric hindrance or altered conformations. For this report, we confirmed this phenomenon and performed comparative studies with group Y meningococcal polysaccharide (GYMP)-TT to determine whether it might extend to other serogroups. The de-O-acetylated (dOA) polysaccharides (PSs) resulted in higher serum bactericidal activities (SBA) towards the O-acetylated (OA) meningococcal strains from the respective serogroups. High-resolution H-nuclear magnetic resonance spectroscopy at 500 MHz and competitive inhibition serum bactericidal assays were used to characterize the nature of the protective epitope. In head-to-head comparisons with OA PSs as SBA inhibitors, the dOA PSs provided 10 to 1,000 times better inhibition for GCMP in human and mouse antisera and 6 to 13 times better inhibition for GYMP in mouse antisera, using OA strains in all assays. In addition, the SBA for OA strains was highly correlated with dOA PS-specific immunoglobulin G (r=0.72 to 0.98) for both GCMP and GYMP. The results suggest that there may be a generalized role for the O-acetyl group to provide an epitope of misdirected immunogenicity for meningococcal PS capsules, enabling escape from immune surveillance. In addition to greater chemical consistency, the dOA forms of GCMP and GYMP conjugate vaccines endow greater immunologic competence to the PSs, rendering them capable of eliciting higher levels of functional antibodies toward the protective epitopes.

Specificity of the immune response to a modified group B meningococcal polysaccharide conjugate vaccine.

Antibodies to a modified group B meningococcal polysaccharide vaccine were examined for antigenic and functional specificities. Bactericidal determinants were investigated by using immunoaffinity columns and competitive inhibition of bactericidal activity in an in vitro killing assay. We conclude that nearly all of the vaccine-induced bactericidal activity is specific for the native polysaccharide.

Monitoring activation sites on polysaccharides by GC-MS.

A method has been developed to determine the location and order of activation for potential saccharide antigens used in conjugate vaccine development. Saccharides were monitored for activation by sodium periodate oxidation and subsequent analysis by gas chromatography-mass spectrometry (GC-MS). Pneumococcal serotype polysaccharides 7F and 18C were evaluated as polysaccharides containing multiple potential sites for activation. Sialyllactose was used as a model oligosaccharide compound to evaluate oxidation of terminally linked sialic acids and reducing sugar residues. Oxidized saccharides were analyzed by monosaccharide composition and/or linkage analysis to elucidate specific activation of cis versus trans diols, as well as diols containing primary versus secondary alcohols, at specified levels of periodate. Samples (100-500 microg) were sequentially oxidized, reduced, methanolyzed, and derivatized in a single reaction vial for routine analysis by GC-MS.

Group B streptococcal type II and III conjugate vaccines: physicochemical properties that influence immunogenicity.

Recent efforts toward developing vaccines against group B streptococci (GBS) have focused on increasing the immunogenicity of GBS polysaccharides by conjugation to carrier proteins. However, partial depolymerization of GBS polysaccharides for the production of vaccines is a difficult task because of their acid-labile, antigenically critical sialic acids. Here we report a method for the partial depolymerization of type II and III polysaccharides by mild deaminative cleavage to antigenic fragments with reducing-terminal 2,5-anhydro-d-mannose residues. Through the free aldehydes of their newly formed end groups, the fragments were conjugated to tetanus toxoid by reductive amination. The resulting conjugates stimulated the production in animals of high-titer type II- and III-specific antibodies which induced opsonophagocytic killing of type II and III strains of group B streptococci. For the type II conjugates, immunogenicity increased as oligosaccharide size decreased, whereas for type III conjugates, the size of the oligosaccharides did not significantly influence immunogenicity. When oligosaccharides of defined size were conjugated through sialic acid residues, the resulting cross-linkages were shown to affect immunogenicity. When oligosaccharides were conjugated through terminal aldehyde groups generated by deamination, modification of the exocyclic chain of sialic acid did not influence immunogenicity.

Addition of glycerol for improved methylation linkage analysis of polysaccharides.

A presolubilization procedure with the use of glycerol is shown to be applicable for the structural analysis of polysaccharides. Neutral, acidic, high-molecular-weight and low-molecular-weight polysaccharides were solubilized in glycerol prior to methylation and subsequent linkage analysis by GC-MS. All four types of polysaccharides showed significant increases in derivatization following presolubilization as measured by recovery of partially methylated alditol acetates.

Group A streptococcus (GAS) carbohydrate as an immunogen for protection against GAS infection.

Previous studies have shown that human serum containing anti-group A streptococcus carbohydrate (GAS CHO) antibodies were opsonic for different M protein-carrying serotypes. To investigate the role that anti-GAS CHO antibodies play in passive and active protection, mice were immunized subcutaneously or intranasally with GAS CHO conjugated to tetanus toxoid, and mortality and oral colonization were monitored after challenge with live GAS. Compared with control mice, immunized mice were significantly protected against systemic or nasal challenge with GAS. Furthermore, studies of serum samples and throat cultures from Mexican children revealed an inverse relationship between high serum titers of anti-GAS CHO antibodies and the presence of GAS in the throat. Anti-GAS CHO antibodies were also tested for cross-reactivity with human tissues and cytoskeletal proteins. No cross-reactivity was observed in either assay. The present study demonstrates that GAS CHO is both immunogenic and protective against GAS infections.

Doubly branched hexasaccharide epitope on the cell wall polysaccharide of group A streptococci recognized by human and rabbit antisera.

A number of epitope specificities associated with the cell wall polysaccharide antigen of group A streptococci were identified in a polyclonal rabbit antiserum induced in rabbits by whole group A streptococci and in polyclonal convalescent human antisera from children that had recovered from streptococcal A infections. The identification was achieved by using a series of synthetic oligosaccharides, glycoconjugates, and bacterial polysaccharide inhibitors to inhibit the binding of the group A helical polysaccharide to the polyclonal antisera. The exclusively dominant epitope expressed in the convalescent human antisera was the doubly branched extended helical hexasaccharide with the structure alpha-L-Rhap(1-->2)[beta-D-GlcpNAc(1-->3)]alpha-L-Rhap(1-->3)alpha-L-Rhap(1-->2)[beta-D-GlcpNAc(1-->3)]alpha-L-Rhap. The hexasaccharide epitope also bound with the highest immunoreactivity to the rabbit antiserum. In contrast, the human antisera did not show significant binding to the singly branched pentasaccharide with the structure alpha-L-Rhap(1-->2)alpha-L-Rhap(1-->3)alpha-L-Rhap(1-->2)[beta-D-GlcpNAc(1-->3)]alpha-L-Rhap or the branched trisaccharide alpha-L-Rhap(1-->2)[beta-D-GlcpNAc(1-->3)]alpha-l-Rhap, although both these haptens bound significantly to the same rabbit antiserum, albeit with less immunoreactivity than the hexasaccharide. Inhibition studies using streptococcal group A and B rabbit antisera and the inhibitors indicated above also suggested that the group A carbohydrate, unlike the group B streptococcal polysaccharide, does not contain the disaccharide alpha-L-Rhap(1-->2)alpha-L-Rhap motif at its nonreducing chain terminus, stressing the importance of mapping the determinant specificities of these two important streptococcal subcapsular group polysaccharides to fully understand the serological relationships between group A and group B streptococci.

An integrity assay for a meningococcal type B conjugate vaccine.

The development of an analytical procedure for the evaluation of a conjugate vaccine's structural wholeness or integrity is described. The principle component of the vaccine was the N-propionylated group B meningococcal polysaccharide (NPr-GBMP) covalently attached to a carrier protein. The goal of the procedure was to determine whether any whole polysaccharide, oligosaccharide, or monosaccharide, from minute to moderate levels, became detached off the conjugate. Free saccharide was isolated from the formulation, which included an aluminum hydroxide adjuvant for analysis. Due to its linkage, the NPr-GBMP did not release sialic acid efficiently with acid hydrolysis to the extent necessary for accurate quantitation. To accomplish depolymerization, the NPr-GBMP was subjected to methanolysis, 3N hydrochloric acid in methanol for 16h at 80 degrees C. The main product of the methanolysis reaction was a de-N-acylated methyl glycoside of sialic acid. N-acetylneuraminic acid oligomers and colominic acid were used to confirm the methanolysis depolymerization efficiency of the alpha(2 --> 8) saccharides; with the treatment all oligomers produced a common methyl glycoside. For this determination anion exchange chromatography and size exclusion chromatography were both interfaced to an integrated pulsed amperometric detector. Sensitivity and linearity were demonstrated to be sufficient for the application with vaccine dose formulations with low total saccharide concentrations.

Prevención de infecciones por Neisseria miningitidis del serogrupo C, con una nueva vacuna / Prevention of Neisseria meningitidis serogroup C infections with a new vaccine

La incidencia de la enfermedad meningocócica difiere en muchas regiones del mundo y también existen diferencias en los serogrupos de Neisseria meningitidis que provocan la enfermedad. En Africa, prevalecen los serogrupos A W135, y en Europa, el serogrupo B. en los Estados Unidos, predominan los serogrupos C, B e Y, mientras que, en la Argentina, hay un leve predominio del serogrupo B (60 por ciento). También existen marcadas variaciones entre las diferentes provincias de la Argentina. Asimismo, la mortalidad es más alta en individuos con enfermedad meningocócica por serogrupo C. En este artículo, analizamos el desarrollo clínico que condujo a la aprobación de una nueva vacuna conjugada polisacárida C (NeisVac-C), que actualmente se comercializa en 27 países, inclusive la Argentina. La vacuna confiere protección contra la enfermedad meningocócica provocada por elserogrupo C y se diferencia de las vacunas polisacáridas previas utilizadas en que, mediante conjugación a una proteína transportadora (toxoide tetánico), se transforma en un antígeno dependiente de células T. Esto evoca la producción de IgG bactericida y células B de memoria con mayor inmunogenicidad y memoria inmunitaria. Además, se propuso que el patrón específico de de-O-acetilación de NeisVac-C contribuye a la actividad bactericida inducida por la vacuna. Se demostró que ésto es especialmente importante en niños, y NeisVac-C tuvo una inmunogenecidad superior después de la inmunización primaria y de la provocación. Comunicamos los principales hallazgos delprograma de desarrollo clínico y resumimos los datos de inmunogenicidad y seguridad obtenidos durante el período y la vigilancia poscomercialización en el Reina Unido

Prevención de infecciones por Neisseria miningitidis del serogrupo C, con una nueva vacuna / Prevention of Neisseria meningitidis serogroup C infections with a new vaccine

La incidencia de la enfermedad meningocócica difiere en muchas regiones del mundo y también existen diferencias en los serogrupos de Neisseria meningitidis que provocan la enfermedad. En Africa, prevalecen los serogrupos A W135, y en Europa, el serogrupo B. en los Estados Unidos, predominan los serogrupos C, B e Y, mientras que, en la Argentina, hay un leve predominio del serogrupo B (60 por ciento). También existen marcadas variaciones entre las diferentes provincias de la Argentina. Asimismo, la mortalidad es más alta en individuos con enfermedad meningocócica por serogrupo C. En este artículo, analizamos el desarrollo clínico que condujo a la aprobación de una nueva vacuna conjugada polisacárida C (NeisVac-C), que actualmente se comercializa en 27 países, inclusive la Argentina. La vacuna confiere protección contra la enfermedad meningocócica provocada por elserogrupo C y se diferencia de las vacunas polisacáridas previas utilizadas en que, mediante conjugación a una proteína transportadora (toxoide tetánico), se transforma en un antígeno dependiente de células T. Esto evoca la producción de IgG bactericida y células B de memoria con mayor inmunogenicidad y memoria inmunitaria. Además, se propuso que el patrón específico de de-O-acetilación de NeisVac-C contribuye a la actividad bactericida inducida por la vacuna. Se demostró que ésto es especialmente importante en niños, y NeisVac-C tuvo una inmunogenecidad superior después de la inmunización primaria y de la provocación. Comunicamos los principales hallazgos delprograma de desarrollo clínico y resumimos los datos de inmunogenicidad y seguridad obtenidos durante el período y la vigilancia poscomercialización en el Reina Unido(AU)