Analysis of the ontogeny of the murine humoral response to Neisseria meningitidis B capsular polysaccharide reveals levels of complexity relevant to vaccine development.

Although purified capsular polysaccharide of Neisseria meningitidis group B (CpsB) is not immunogenic at any age, CpsB on the bacterial surface elicits antibody responses late in ontogeny. Therefore, a detailed analysis of the ontogeny of the murine anti-CpsB response to N. meningitidis could determine key parameters regarding the poor immunogenicity of CpsB. The effects of bacterial dose, hyperimmunization, age, and sex on the induction of primary and secondary anti-CpsB immunoglobulin isotype profiles were studied. It was demonstrated that the timing and repetition of immunization and of the bacterial dose have a marked differential effect on the primary induction of anti-CpsB immunoglobulin isotypes and on the ability to induce anti-CpsB antibody responses after subsequent rechallenge. It is noteworthy that the ontogeny of the response is related to the appearance of natural anti-CpsB antibodies, but this is not associated with the presence of CpsB cross-reactive antigens in the microflora.

Distinct types of T-cell help for the induction of a humoral immune response to Streptococcus pneumoniae.

Studies have indicated that purified soluble polysaccharide antigens can elicit T cell-independent Ig responses in vivo, although these responses can be modulated by T cells in a noncognate manner. Relatively little is known, however, concerning the parameters that regulate polysaccharide-specific, as well as protein-specific, Ig isotype responses to an intact extracellular bacterium. Using the murine in vivo humoral response to intact Streptococcus pneumoniae as a model it can be shown that CD4+ T-cell receptor alphabeta+ T cells deliver help for both polysaccharide- and protein-specific Ig responses. However, these responses differ fundamentally in their mechanism of action.

The form variation of the capsular polysaccharide K1 is not a critical virulence factor of Escherichia coli in a neonatal mouse model of infection.

Escherichia coli K1 is a prevalent cause of Gram-negative neonatal bacteraemia and meningitis in humans. Its capsular polysaccharide K1 (CpsK1) has been identified as an important virulence factor. Nevertheless, the biological and pathogenic implications of its O-acetylated and non-O-acetylated forms are poorly understood. In an attempt to address this, we monitored the expression of both CpsK1 form variants in a neonatal mouse infection model. In the absence of anti-CpsK1 antibodies, no CpsK1 form variant selection was observed during the course of infection. The administration of monoclonal antibodies specific for CpsK1 provided a high level of protection. The monoclonal antibodies that recognized both CpsK1 forms (MGB12) provided protection from up to 850 LD(50). By contrast, the administration of the monoclonal antibodies (MGB15) specific for non-O-acetylated CpsK1 cleared only bacteria expressing this CpsK1 form; a few mouse pups remained bacteraemic, and the bacteria in the blood had O-acetylated CpsK1. In those pups, the infection progressed in a similar fashion to that in mice not treated with monoclonal antibody. Moreover, when the number of bacteria expressing the O-acetylated CpsK1 in the inoculated dose is considered independently, the LD(50)was similar to that for the original strain in pups that had not been treated with monoclonal antibodies (35 CFU). These results suggest that whereas variation in acetylation form per se does not reinforce virulence, it could enable E. coli to avoid immune defenses. This highlights the importance of using highly specific monoclonal antibodies in immunotherapeutic approaches to E. coli K1 neonatal meningitis.

Dynamics of the murine humoral immune response to Neisseria meningitis group B capsular polysaccharide.

Immunization with Neisseria meningitidis group B capsular polysaccharide (CpsB) elicited responses in adult mice that showed the typical dynamic characteristics of the response to a thymus-independent antigen, in contrast to the thymus-dependent behavior of antibody responses to CpsC. The former had a short latent period and showed a rapid increase in serum antibodies that peaked at day 5, and immunoglobulin M (IgM) was the major isotype even though IgG (mainly IgG2a and IgG2b) was also detectable. This response was of short duration, and the specific antibodies were rapidly cleared from the circulation. The secondary responses were similar in magnitude, kinetics, IgM predominance, and IgG distribution. Nevertheless, a threefold IgG increase, a correlation between IgM and IgG levels, and dose-dependent secondary responses were observed. Hyperimmunization considerably reinforced these responses: 10-fold for IgM and 300-fold for IgG. This favored isotype switch was accompanied by a progressive change in the subclass distribution to IgG3 (62%) and IgG1 (28%), along with the possible generation of B-cell memory. The results indicate that CpsB is being strictly thymus independent and suggest that unresponsiveness to purified CpsB is due to tolerance.

A quantitative ELISA for antigen-specific IgG subclasses using equivalence dilutions of anti-kappa and anti-subclass specific secondary reagents. Application to the study of the murine immune response against the capsular polysaccharide of Neisseria meningitidis serogroup B.

We have developed an enzyme-linked immunosorbent assay (ELISA) to measure murine antigen-specific IgG antibodies of defined subclass using precalibrated equivalence dilutions of anti-kappa (in the standard) and each anti-IgG subclass-specific polyclonal secondary antibody (in the test sample). The calibration of secondary reagents could be carried out easily with a set of monoclonal antibodies (MoAbs) specific for all IgG subclasses. These MoAbs do not require purification or standardization. In addition the MoAbs can be of different antigenic specificity. Once the equivalence dilutions have been determined, they can be applied in a quantitative ELISA using the same antigen in the standard and sample, and using only one IgG subclass standard for the determination of all the IgG subclasses. The method is easy to standardize for many antigenic systems. It is particularly useful when the only standard available is one standardized MoAb of the appropriate specificity, and it could be adapted to use with standard polyclonal antibodies having a known content of total antigen-specific IgG bearing kappa chains but unknown IgG subclass composition. The use of this method to quantitate IgG specific for the capsular polysaccharide of Neisseria meningitidis serogroup B (CpsB) gave highly reproducible measures with an interbatch CV of 5-6% similar for all IgG subclasses and low detection limits ranging from 0.3 ng/well for IgG3 to 0.8 ng/well for IgG2a. The IgG subclass response observed after immunization with live meningococci was mainly IgG2a (74%) and IgG2b (18%). Hyperimmunization modified this IgG distribution to one of mainly IgG3 (62%) and IgG1 (28%) which was maintained in the response to a single immunization 4 weeks later, possibly indicating the generation of resting B cells during continuous stimulation.