Quantification of each serogroup polysaccharide of Neisseria meningitidis in A/C/Y/W-135-DT conjugate vaccine by high-performance anion-exchange chromatography-pulsed amperometric detection analysis.

Invasive bacterial meningitis caused by Neisseria meningitidis can be prevented by active immunization with meningococcal polysaccharide or polysaccharide-protein conjugate vaccines. In a tetravalent A/C/Y/W-135-DT meningococcal conjugate vaccine vial, or in a final formulated bulk, accurate identification and quantification of each polysaccharide are critical in product release. Determination of sialic acid serogroups (C, W-135, and Y) unambiguously is complex since all these serogroups contribute to the sialic acid monosaccharide peaks that overlap in the high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD). We report a quantification method that involves generation of monosaccharide standard plots for respective sugars mannosamine-6-phosphate, sialic acid, galactose- and glucose-derived from hydrolysis of mixtures of the four serogroups A, C, W, and Y reference polysaccharides. These plots were then used to obtain the unknown polysaccharide concentrations of A/C/Y/W-135 in vialed vaccine or from formulated final bulks. We also present our results of the HPAEC-PAD profiles on groups C, W-135, and Y polysaccharides when hydrolyzed individually and/or in mixtures to discuss the individual sialic acid peak contributions.

Four monoclonal antibodies against capsular polysaccharides of Neisseria meningitidis serogroups A, C, Y and W135: its application in identity tests.

Murine hybridoma monoclonal antibodies (MAbs) were produced against the capsular polysaccharide (CPs) of serogroups A, C, W135 and Y meningococci (MenA, MenC, MenW, MenY) in order to develop immunological reagents for the identification of meningococcal polysaccharides. Each serogroup-specific MAb reacted with the CPs from its homologous serogroup only and did not react with CPs from the other three serogroups. The affinity constant (Ka) of the four MAbs measured by non-competitive ELISA was 6.62 × 10(9), 2.76 × 10(9), 1.48 × 10(9) and 3.8 × 10(9) M(-1) for MenA, MenC, MenW and MenY MAbs respectively. The application of these MAbs for identity tests was demonstrated by their abilities to correctly identify the CPs from serogroups A, C, W135 and Y in meningococcal CPs-based vaccines through ELISA. The MAbs obtained in this work are a very valuable set of tools for study meningococcal polysaccharides vaccines.

AFCo1 as nasal adjuvant of capsular polysaccharide from Neisseria meningitidis serogroup C induces systemic and mucosal immune responses.

Increasing emphasis is being placed on the mucosal administration of vaccines in order to stimulate mucosal as well as systemic responses. Findings from our group suggest that proteoliposome-derived cochleate (AFCo1) acts as a potent mucosal adjuvant. As an alternative to chemical conjugation, the current study aimed to determine the benefit of using AFCo1 to improve the mucosal and systemic immune responses to capsular polysaccharide of Neisseria meningitidis serogroup C (PsC), a model of a thymus-independent (TI) antigen. Therefore, intranasal (i.n.) immunization of 3 doses 1 week apart with AFCo1 plus PsC in mice was conducted. Highly specific anti-PsC IgA responses and an anti-PsC IgG response were obtained. The subclass pattern induced against PsC was similar to that induced with the meningococcal vaccine. In summary, AFCo1 as nasal adjuvant was demonstrated to be capable of eliciting mucosal and systemic specific responses against a TI antigen.

Preclinical evidence for the potential of a bivalent fHBP vaccine to prevent Neisseria meningitidis Serogroup C Disease.

A bivalent factor H binding protein (fHBP) vaccine for the prevention of disease caused by Neisseria meningitidis serogroup B is currently in clinical development. Since fHBP is also expressed by other meningococcal serogroups, anti-fHBP antibodies may have bactericidal activity against meningococci independent of serogroup. To begin examining the susceptibility of other meningococcal serogroups to anti-fHBP antibodies, meningococcal serogroup C invasive isolates (n = 116) were collected from the Centers for Disease Control and Prevention's Active Bacterial Core surveillance (ABCs) sites during 2000-2001. These isolates were analyzed for the presence of the fhbp gene. All serogroup C isolates contained the gene, and sequence analysis grouped the proteins into two subfamilies, A and B. Flow cytometry analysis demonstrated that fHBP was expressed on the surface of ~70% of isolates in vitro with varying levels of expression. fHBP was accessible to antibodies on the cell surface even in the presence of the polysaccharide capsule. Nine isolates from different geographic regions were identified which harboured an identical single nucleotide deletion that could result in a truncated subfamily B fHBP. Analysis by flow cytometry using a polyclonal fHBP antibody preparation revealed that a subpopulation of each of these isolates expressed fHBP. Rabbit and non-human primate immune sera generated with bivalent fHBP vaccine were tested for bactericidal activity against a panel of diverse serogroup C clinical isolates using human complement. Sera from both species demonstrated serum bactericidal antibody activity against the serogroup C isolates tested. These promising findings suggest that a bivalent fHBP vaccine may be capable of providing protection against meningococcal disease caused by both serogroup C and B.