Structure activity studies on group C meningococcal polysaccharide-protein conjugate vaccines: effect of O-acetylation on the nature of the protective epitope.

A series of group C meningococcal polysaccharide-tetanus toxoid (GCMP-TT) conjugates were prepared as vaccines with varying percentages of O-acetylation at the C-7 and C-8 positions of sialic acid residues in the polysaccharide (PS). The immune response in mice was highly dependent on the degree of O-acetylation. Less O-acetylation resulted in higher serum bactericidal activity (SBA) towards the O-acetylated (OA) meningococcal strain, C11. In addition, since an unconjugated de-O-acetylated (dOA) GCMP vaccine was previously shown to be highly immunogenic in humans, we had chosen this dOA form to couple with TT by reductive amination for clinical evaluation. This conjugate vaccine was shown to be well-tolerated and highly immunogenic in adults, children, and infants in the UK. To understand the nature of the GCMP protective epitope, a series of spectroscopic and serological studies were conducted, using high resolution H-NMR spectroscopy at 500 MHz and competitive inhibition SBA assays. The dOA GCMP was 10-1000 times better at inhibiting the SBA for an OA strain than the OA GCMP, suggesting that the GCMP-based protective epitope on the bacterium exists in a dOA form. In addition, SBA for an OA strain is highly correlated with dOA GCMP-specific IgG. NMR data on freshly isolated GCMP indicated that, on the surface of the organism, most of the O-acetylation exists at position C-8, with some regions containing dOA or OA C-7 sialic acid. After extraction of PS and storage in solution, most of the O-acetyl groups migrate to C-7, leaving an epitope that is conformationally related, but not quite identical (due to the presence of the O-acetyl group), to the one contained in the dOA PS. We speculate that the role of the O-acetyl group at the C-8 position of the PS on the organism is to form less immunogenic epitopes, or mask the protective epitope, and thus escape immune surveillance. The dOA form of the vaccine may therefore provide better protection against group C meningococcal disease than the OA form by eliciting a greater proportion of functional antibodies that are directly aimed at the protective epitope.

Safety and immunogenicity of a new Neisseria meningitidis serogroup C-tetanus toxoid conjugate vaccine in healthy adults.

UNLABELLED: We evaluated the safety and immunogenicity of a single dose of a new serogroup C O-deacetylated meningococcal polysaccharide-tetanus toxoid conjugate vaccine in 30 healthy adult volunteers. The vaccine was well tolerated with no serious adverse events and minimal local reactions and systemic symptoms. All subjects developed a fourfold or greater increase in serum bactericidal antibody (SBA) to serogroup C meningococcus. SBA geometric mean titre increased from 11 to 3649 (p<0.001). Serogroup C-specific IgG levels increased postvaccination from 0.65 to 17.02 microg/ml (p<0.001). Bactericidal titres pre- and postimmunisation showed significant correlation with serogroup C-specific IgG (r(2)=0.693). Antibody levels fell by 6 months postvaccination, however, meningococcal C IgG avidity increased indicating the successful induction of a T-cell-dependent antibody response. CONCLUSION: meningococcal C-tetanus toxoid conjugate vaccine is immunogenic and well tolerated in healthy adults.

Preclinical studies on a recombinant group B meningococcal porin as a carrier for a novel Haemophilus influenzae type b conjugate vaccine.

In anticipation of future combination vaccines, a recombinant class 3 porin (rPorB) of group B meningococci was evaluated as an alternative carrier protein for a Haemophilus influenzae type b (Hib) polyribosylribotol phosphate (PRP) conjugate vaccine. The use of rPorB may avoid undesirable immunologic interactions among vaccine components, including epitopic suppression from conventional carriers (e.g. tetanus toxoid [TT]), as well as provide desirable immunomodulatory effects. Rats were found to be more reliable and consistent than mice or guinea pigs for studying antibody responses to the Hib conjugates. Different Hib conjugates, Hib-TT and Hib-rPorB, consisting of PRP conjugated by reductive amination to TT or rPorB, were compared in rats. Commercially available, licensed vaccines, HbOC (HibTITER) and PRP-T (OmniHib), were used as reference controls. Maximum geometric mean ELISA IgG titers were obtained in rats after only two doses, showing booster effects for all. However, Hib-rPorB immunization consistently resulted in responses that were 1-2 orders of magnitude greater than those for the other conjugates, including the licensed control vaccines. A maximum 4600-fold rise was observed for Hib-rPorB after two doses, and, unlike the other conjugates, a 100% response rate was always achieved without adjuvant. These results warrant further investigation of Hib-rPorB in combination with DTaP.

Multivalent pneumococcal capsular polysaccharide conjugate vaccines employing genetically detoxified pneumolysin as a carrier protein.

A genetically detoxified pneumolysin, pneumolysoid (PLD), was investigated as a carrier protein for pneumococcal capsular polysaccharide (CPS). Such a CPS-PLD conjugate might provide additional protection against pneumococcal infections and resultant tissue damage. A single point mutant of pneumolysin was selected, which lacked measurable haemolytic activity, but exhibited the overall structural and immunological properties of the wild type. PLD conjugates were prepared from CPS serotypes 6B, 14, 19F, and 23F by reductive amination. The structural features of free PLD, as well as the corresponding CPS-PLD, as assessed by circular dichroism spectroscopy, were virtually indistinguishable from the wild type counterpart. Each of the CPS monovalent and tetravalent conjugate formulations were examined for immunogenicity in mice at both 0.5 and 2.0 micrograms CPS per dose. Tetanus toxoid (TT) conjugates were similarly created and used for comparison. The resultant conjugate vaccines elicited high levels of CPS-specific IgG that was opsonophagocytic for all serotypes tested. Opsonophagocytic titres, expressed as reciprocal dilutions resulting in 50% killing using HL-60 cells, ranged from 100 to 30,000, depending on the serotype and formulation. In general, the lower dose and tetravalent formulations yielded the best responses for all serotypes (i.e., either equivalent or better than the higher dose and monovalent formulations). The PLD conjugates were also generally equivalent to or better in CPS-specific responses than the TT conjugates. In particular, both the PLD conjugate and the tetravalent formulations induced responses for type 23F CPS that were approximately an order of magnitude greater than that of the corresponding TT conjugate and monovalent formulations. In addition, all the PLD conjugates elicited high levels of pneumolysin-specific IgG which were shown to neutralize pneumolysin-induced haemolytic activity in vitro. As a result of these findings, PLD appears to provide an advantageous alternative to conventional carrier proteins for pneumococcal multivalent CPS conjugate vaccines.

Meningococcal vaccine development: a novel approach.

Neisseria meningitidis is a major world-wide cause of meningitis. Effective capsular polysaccharide (CPS) vaccines that elicit CPS-specific bactericidal (BC) antibodies were previously developed and licensed to protect against meningococcal disease. However, due to their T-cell independent character, CPS vaccines are useless in infants and do not provide immunological memory or long-lasting protection in adults. CPS-protein conjugate vaccines are being developed to improve and broaden vaccine efficacy by creating T-cell dependent antigens. However, group B meningococci (GBM) are responsible for nearly half of meningococcal disease and possess a CPS, composed of polysialic acid, that is poorly immunogenic. N-propionyl (NPr) modification of the GBM polysaccharide (GBMP) has enhanced its immunogenicity, but BC antibodies are not induced at high levels, even when conjugated to conventional protein carriers, unless adjuvants stronger than aluminium hydroxide are used. We have chosen to couple the NPr-GBMP by reductive amination to a recombinant GBM class 3 porin (rPorB), which we have shown to modulate the immune response in animals towards the production of CPS-specific BC antibodies. We have also combined this conjugate with similar CPS-rPorB conjugates for groups A and C meningococci to form a trivalent A/B/C conjugate vaccine. This trivalent meningococcal vaccine has been shown to be safe and highly immunogenic in mice and non human primates, generating CPS-specific BC antibodies for each of the 3 major serogroups, which should provide world-wide protection against meningococcal disease.

Preclinical evaluation of a novel group B meningococcal conjugate vaccine that elicits bactericidal activity in both mice and nonhuman primates.

Group B meningococcal (GBM) conjugate vaccines were prepared using chemically modified N-propionylated polysialic acid, from Escherichia coli K1 polysaccharide capsule, coupled by reductive amination to tetanus toxoid and purified recombinant GBM porin (rPorB). All conjugates elicited high antibody levels in mice with good booster responses. However, only rPorB conjugates elicited bactericidal activity specific against a broad spectrum of five different GBM serotypes. Bactericial activity was completely inhibited by free N-propionylated polysaccharide. In baboons and rhesus monkeys, rPorB conjugates elicited high antibody titers, with IgG booster responses 9- to 15-fold higher than primary responses. Bactericial activity increased 19- to 39-fold over preimmune values, using rabbit complement; increased bactericial activity was also confirmed with human and monkey complement. IgG cross-reactivity for unmodified N-acetyl polysaccharide was <5% for 79% of mice and <10% for 80% of primates. These studies strongly suggest that the N-propionylated polysialic acid-rPorB conjugate is an excellent vaccine candidate for human use.

In vitro adhesion of Escherichia coli to porcine small intestinal epithelial cells: pili as adhesive factors.

Escherichia coli strains with pili (K99 or 987P) known to facilitate intestinal colonization adhered in vitro to porcine intestinal epithelial cells. These strains adhered equally to both ileal and jejunal epithelial cells. A laboratory E. coli strain that has type 1 pili also adhered to porcine intestinal epithelial cells. When nonpiliated cells derived from 987P+, K99+, or type 1 pilus+ strains were used for in vitro adhesion assays, they failed to adhere. The attachment of piliated bacteria to epithelial cells was a saturable process that plateaued at 30 to 40 bacterial cells attached per epithelial cell. Competitive inhibition of bacterial cell attachment to epithelial cells with purified pili showed that only purified 987P competed against the 987P+ strain and only purified type 1 pili competed against the type 1 pilus+ strain. Competition between a K99+ strain and K99 was not consistently achieved. K99+, 987P+, and type 1 pilus+ bacteria could be prevented from adhering to epithelial cells by Fab fragments specific for K99, 987P, or type 1 pili, respectively. Fab fragments specific for non-K99 bacterial surface antigens did not inhibit adhesion of the K99+ strain. It is concluded that adhesion of E. coli to porcine intestinal epithelial cells in vitro is mediated by pili and that the epithelial cells used apparently had different receptors for different pili.