Pneumococcal conjugate vaccines overcome splenic dependency of antibody response to pneumococcal polysaccharides.

Protection against infections with Streptococcus pneumoniae depends on the presence of antibodies against capsular polysaccharides that facilitate phagocytosis. Asplenic patients are at increased risk for pneumococcal infections, since both phagocytosis and the initiation of the antibody response to polysaccharides take place in the spleen. Therefore, vaccination with pneumococcal polysaccharide vaccines is recommended prior to splenectomy, which, as in the case of trauma, is not always feasible. We show that in rats, vaccination with a pneumococcal conjugate vaccine can induce good antibody responses even after splenectomy, particularly after a second dose. The spleen remains necessary for a fast, primary response to (blood-borne) polysaccharides, even when they are presented in a conjugated form. Coadministration of a conjugate vaccine with additional nonconjugated polysaccharides of other serotypes did not improve the response to the nonconjugated polysaccharides. We conclude that pneumococcal conjugate vaccines can be of value in protecting asplenic or hyposplenic patients against pneumococcal infections.

Immunogenicity of Streptococcus pneumoniae type 6B and 14 polysaccharide-tetanus toxoid conjugates and the effect of uncoupled polysaccharide on the antigen-specific immune response.

The immunogenicity of two types of Streptococcus pneumoniae capsular polysaccharide-tetanus toxoid conjugates (PS6BTT and PS14TT) was evaluated in mice. Both conjugates induced high titres of high avidity type-specific anti-PS IgG, which include all IgG isotypes except IgG2a. Repeated immunization resulted in booster responses in both cases. The antibodies induced exhibited opsonic activity, as measured in an in vitro opsonophagocytosis assay, using the mouse macrophage cell line RAW-264. Furthermore, the influence of spiking PS6BTT with free PS6B of either 1000 kDa (native) or 37 kDa was investigated. The results indicate that not only the amount but also the molecular weight of the free PS6B present in the conjugate vaccine affect the anti-PS6B immune response. Large amounts of free PS6B of both molecular weights decrease each anti-PS6B IgG isotype response. However, unlike admixture of the low molecular weight PS6B, addition of the high molecular weight PS6B leads to a rather persistent state of unresponsiveness.

Application of cystamine and N,N'-Bis(glycyl)cystamine as linkers in polysaccharide-protein conjugation.

Pneumococcal polysaccharide type 6B, 14, or 23F (35-70 kDa) was activated with cyanogen bromide and modified with cystamine. After reduction of the spacer, the thiol-containing (i.e. cysteamine-modified) polysaccharide obtained was added in a 5-10-fold molar excess to bromoacetylated tetanus toxoid to give thioether-linked polysaccharide-protein conjugates in a yield of 10-20%. This approach failed for preparing a type 19F polysaccharide-protein conjugate, possibly due to intramolecular elimination of cysteamine from the reduced 19F polysaccharide. When N,N'-bis(glycyl)cystamine was introduced as a spacer molecule, the elimination of the reduced spacer was suppressed, thus allowing preparation of a 19F polysaccharide-tetanus toxoid conjugate (15%).

Polysaccharicb-conjugate vaccines.

It was recognized early this century that small molecules, called haptens, can be made immunogenic after conjugation to carrier proteins (1), This principle was thereafter applied successfully to improve the rmmunogenicity of (poly)saccharides (2, 3). We now know that the carrier proteins ensure the involvement of T-helper lymphocytes in the activation of the haptenor polysaccharide-specific antibody producing B lymphocytes (Fig. 1). In contrast to small molecules or haptens, polysaccharides (or other macromolecules with a repeating structure) are able to induce an immune response, most likely by directly activating B lymphocytes. Antigens that are able to induce an immune response without the involvement of T-helper lymphocytes are named TI (thymus independent) antigens (4) (Table 1). TI-2 antigens, such as plain polysaccharides, are not able to activate relatively immature B-cells. This is in contrast to TI-1 antigens, which can activate immature B-cells because of their mitogenic activity. Lipopolysaccharides are examples of TI-1 antigens. T-cells with specificity for saccharide structures that are recognized in association with the major histocompatibility complex (MMC) structures have never been found nor described; binding to MHC and stimulation of T-cells appears to be limited to peptides. The findings of T-cell regulation of the immune response against polysaccharides (5-7) without biochemical demonstration of the specificity of the molecular interactions can best be explained by assuming a role for antiidiotypic antibodies and T-cells.

Structure of the L2 lipopolysaccharide core oligosaccharides of Neisseria meningitidis.

Three different oligosaccharides were identified following mild acid hydrolysis of the lipopolysaccharide obtained from Neisseria meningitidis serotype 2 and their structures elucidated by combined chemical and physical techniques. The use of 500 MHz 1H nmr in both one- and two-dimensional modes as well as nuclear Overhauser effect experiments were employed. To assist in the structural assignments the oligosaccharides were also degraded by chemical and enzymatic procedures to smaller fragments. The oligosaccharides were all triantennary nonasaccharides in which the longest antenna terminates in lacto-N-neotetraose. Two of the nonasaccharides (major components), not separable by column chromatography, were distinguishable only by their different patterns of phosphorylethanolamine substitution and the third minor component by the absence of this substituent.

Effect of carrier priming on immunogenicity of saccharide-protein conjugate vaccines.

Previous studies with saccharide-protein conjugates have demonstrated that antibody responses to the saccharide can be improved by the preexistence of carrier immunity. Here we report that prior exposure to the carrier protein can either enhance or suppress antibody response to polysaccharides administered in saccharide-protein conjugates. A dose-dependent role for carrier priming in the antisaccharide antibody response to three saccharide-protein conjugate vaccines, i.e., a Streptococcus pneumoniae type 4 polysaccharide-tetanus toxoid (TT) conjugate (PS4TT), a Neisseria meningitidis group C polysaccharide-TT conjugate (MenCTT), and a N. meningitidis group C oligosaccharide-diphtheria mutant toxin conjugate (MenCCRM), was investigated. The results showed that an increase in the antipolysaccharide antibody response could be obtained for both PS4TT and MenCTT but not for MenCCRM with low-dose carrier priming (0.025 to 0.25 microgram). However, suppression of the antipolysaccharide antibody response was observed with the PS4TT and MenCTT vaccines with high-dose (25-micrograms) carrier priming. There was no suppression effect with MenCCRM. The increase in the antipolysaccharide antibody response was shown to be restricted to the immunoglobulin G1 (IgG1) subclass, whereas suppression with high-dose carrier priming affected all antipolysaccharide subclass antibodies induced by PS4TT (IgG1, IgG2b, and IgG3) and only two of the four subclass antibodies induced by MenCTT (IgG2a and IgG2b). The increase in the antipolysaccharide antibody response was also present at the antipolysaccharide IgM antibody level but was not observed at the anti-carrier IgG antibody level.

Structure of the L5 lipopolysaccharide core oligosaccharides of Neisseria meningitidis.

Three different oligosaccharides were isolated by mild acid hydrolysis of the lipopolysaccharides, obtained from Neisseria meningitidis serotype 5, and their structures were elucidated by combined chemical and physical techniques. The use of 500-MHz 1H NMR in both one-dimensional and two-dimensional modes as well as nuclear Overhauser effect experiments were employed. To assist in the structural assignments the purified oligosaccharides were also degraded by chemical and enzymatic procedures to smaller fragments. The largest of the three original oligosaccharides is a triantennary partially O-acetylated decasaccharide in which the largest antenna terminates in a lacto-N-neotetraose unit. The smaller oligosaccharides (heptasaccharide and octasaccharide) except for terminal glycose deletions from the longest antenna are structural replicas of the larger.

Analysis of oligosaccharide epitopes of meningococcal lipopolysaccharides by fast-atom-bombardment mass spectrometry.

A mass-spectrometric approach is presented for the analysis of the structures of lipopolysaccharide-derived oligosaccharides, which are frequently difficult to define by classical methods since they contain chemically labile components. The method involves f.a.b.-m.s. of the oligosaccharides, their peracetylated and permethylated derivatives, their deuterioacetylated and methylated analogues, and the fragments obtained during graded methanolysis of the methylated analogues. Data obtained from two representative meningococcal LPS oligosaccharides define the sequence, patterns of branching, and the extent and location of the phosphorylethanolamine and O-acetyl substituents.

Proof of the occurrence of 5,6-O-(1-carboxyethylidene)-D-galactofuranose units in the capsular polysaccharide of Klebsiella K12.

The 13C-n.m.r. spectra of the capsular polysaccharide of Klebsiella K41 and phage-derived oligosaccharides K41-P1 and K41-P2 were compared with spectra from the structurally similar polysaccharide of Klebsiella K12 and oligosaccharides K12-P1 and K12-P2. This led to the conclusion that K41 and K12 contain one and two galactofuranose residues per repeating unit, respectively, and that the terminal, lateral residue in K12 has the 5,6-O-(1-carboxyethylidene)-D-galactofuranose structure rather than that of a 4,6-acetal of D-galactopyranose as originally stated. This is the first reported occurrence in Nature of such a structural unit.

Homologous and heterologous reactions of bacteriophages phi 41 and phi 12 on the capsular polysaccharides from Klebsiella K41 and K12.

The structures of the capsular polysaccharides from Klebsiella K41 and K12 are very similar and differ only in the lateral, terminal group of their respective repeating units. The bacteriophages phi 41 and phi 12 are shown to hydrolyze the same alpha-galactopyranosyl bond in each of the polysaccharides, giving rise to an oligosaccharide characteristic of the starting polysaccharide, irrespective of the phage employed. The presence of the uronic acid function is essential for the phages to be active, but the carboxyl group of the pyruvic acetal in K12 does not appear to play a role in the recognition process.

Structure and immunochemistry of meningococcal lipopolysaccharides.

The structures of the largest dephosphorylated oligosaccharides (OS) obtained by mild acid hydrolyses of the L2, L3 and L5 serotype lipopolysaccharides (LPS) of Neisseria meningitidis have been elucidated. The OS have extensive regions of structural similarity of which some are responsible for cross-reactivity among the meningococcal LPS. However, the fact that the LPS are predominantly serotype-specific antigens implies that the terminal lacto-N-neotetraose unit, common to all the above OS, is not immunodominant, and that the major LPS serotype specificity originates in the inner core region of the OS.

Structural investigation of the capsular polysaccharide from Klebsiella K19 by chemical and N.M.R. analyses.

Sugar analysis of the capsular antigen K19 from Klebsiella and of the carboxyl-reduced derivative confirmed its classification into the chemotype containing rhamnose, galactose, glucose, and glucuronic acid residues. Partial acid hydrolysis and phage depolymerization of K19 provided respectively a modified, linear form of the polysaccharide and oligosaccharides of the repeating unit, these were used for the structural elucidation of the original polymer. Methylation analysis, Smith degradation, and 1H- and 13C-n.m.r. spectroscopy of the polysaccharide and derivatives permitted formulation of the following structure for K19: (formula; see text)

Depolymerization of the capsular polysaccharide from Klebsiella K19 by the glycanase associated with particles of Klebsiella bacteriophage luminal diameter 19.

The site of cleavage of the capsular polysaccharide from Klebsiella K19 by the endoglycanase associated with particles of Klebsiella bacteriophage luminal diameter 19 was determined. The specific cleavage of the bond Rhap-(1----2)-Rhap provided a series of oligosaccharides having rhamnose at the reducing end. The enzyme is thus an alpha-rhamnosidase. Structural studies on the oligomers confirmed the sequence of the repeating unit of the polysaccharide from K19. The 1H- and 13C-n.m.r. spectra of the homologous series of oligosaccharides corresponding to one, two, three, and four repeat-units exhibit important differences that denot variation of conformation with chain length. The bacteriophage acted on modified forms of K19 polysaccharide to provide a series of linear oligomers, and emphasized the essential role of the negative charge on the uronic acid in the action of the glycanase.