Activity and cross-reactivity of antibodies induced in mice by immunization with a group B meningococcal conjugate.

The capsular polysaccharide of group B Neisseria meningitidis is composed of a linear homopolymer of alpha(2-8) N-acetyl neuraminic acid or polysialic acid (PSA) that is also carried by isoforms of the mammalian neural cell adhesion molecule (NCAM), which is especially expressed on brain cells during development. Here we analyzed the ability of antibodies induced by the candidate vaccine N-propionyl polysaccharide tetanus toxoid conjugate to recognize PSA-NCAM. We hyperimmunized mice to produce a pool of antisera and a series of immunoglobulin G monoclonal antibodies and evaluated their self-reactivity profile by using a battery of tests (immunoprecipitation, immunoblotting, and immunofluorescence detection on live cells and human tissue sections) chosen for their sensitivity and specificity to detect PSA-NCAM in various environments. We also searched for the effects of the vaccine-induced antibodies in two functional assays involving cell lysis or cell migration. Although they were highly bactericidal, all the antibodies tested showed very low or no recognition of PSA-NCAM, in contrast to PSA-specific monoclonal antibodies used as controls. Different patterns of cross-reactions were revealed by the tests used, likely due to affinity and specificity differences among the populations of induced antibodies. Furthermore, neither cell lysis nor perturbation of migration was observed in the presence of the tested antibodies. Importantly, we showed that whereas enzymatic removal of PSA groups from the surfaces of live cells perturbed their migration, blocking them with PSA-specific antibodies was not functionally detrimental. Taken together, our data indicated that this candidate vaccine induced antibodies that could not demonstrate an immunopathologic effect.

Allelic diversity of the two transferrin binding protein B gene isotypes among a collection of Neisseria meningitidis strains representative of serogroup B disease: implication for the composition of a recombinant TbpB-based vaccine.

The distribution of the two isotypes of tbpB in a collection of 108 serogroup B meningococcal strains belonging to the four major clonal groups associated with epidemic and hyperendemic disease (the ET-37 complex, the ET-5 complex, lineage III, and cluster A4) was determined. Isotype I strains (with a 1.8-kb tbpB gene) was less represented than isotype II strains (19.4 versus 80.6%). Isotype I was restricted to the ET-37 complex strains, while isotype II was found in all four clonal complexes. The extent of the allelic diversity of tbpB in these two groups was studied by PCR restriction analysis and sequencing of 10 new tbpB genes. Four major tbpB gene variants were characterized: B16B6 (representative of isotype I) and M982, BZ83, and 8680 (representative of isotype II). The relevance of these variants was assessed at the antigenic level by the determination of cross-bactericidal activity of purified immunoglobulin G preparations raised to the corresponding recombinant TbpB (rTbpB) protein against a panel of 27 strains (5 of isotype I and 22 of isotype II). The results indicated that rTbpB corresponding to each variant was able to induce cross-bactericidal antibodies. However, the number of strains killed with an anti-rTbpB serum was slightly lower than that obtained with an anti-TbpA(+)B complex. None of the sera tested raised against an isotype I strain was able to kill an isotype II strain and vice versa. None of the specific antisera tested (anti-rTbpB or anti-TbpA(+)B complex) was able to kill all of the 22 isotype II strains tested. Moreover, using sera raised against the C-terminus domain of TbpB M982 (amino acids 352 to 691) or BZ83 (amino acids 329 to 669) fused to the maltose-binding protein, cross-bactericidal activity was detected against 12 and 7 isotype II strains, respectively, of the 22 tested. These results suggest surface accessibility of the C-terminal end of TbpB. Altogether, these results show that although more than one rTbpB will be required in the composition of a TbpB-based vaccine to achieve a fully cross-bactericidal activity, rTbpB and its C terminus were able by themselves to induce cross-bactericidal antibodies.

Comparison of polymorphonuclear cells from healthy donors and differentiated HL-60 cells as phagocytes in an opsonophagocytic assay using antigen-coated fluorescent beads.

Polymorphonuclear cells (PMNs) from healthy donors and differentiated HL-60 cells were compared in an opsonophagocytic assay using fluorescent latex beads coated with Streptococcus pneumoniae polysaccharide conjugates. Serum-specific phagocytosis was efficiently mediated by both sources of cells, as measured by flow cytometry, but the mean number of beads ingested per cell was three- to fivefold higher when PMNs were used than when HL-60 cells were used. Nevertheless, differentiated HL-60 cells could be a convenient and standardized source of cells to evaluate the functionality of specific antibodies to vaccine candidates as a coating on fluorescent beads.

Evaluation of recombinant transferrin-binding protein B variants from Neisseria meningitidis for their ability to induce cross-reactive and bactericidal antibodies against a genetically diverse collection of serogroup B strains.

Transferrin-binding protein B (TbpB) is a surface-exposed protein, variable among strains of Neisseria meningitidis, that has been considered as a vaccine candidate. To define a TbpB molecule that would give rise to broadly cross-reactive antibodies with TbpB of many strains, specific antisera were produced against three recombinant TbpB variants from strain M982: one corresponding to the full-length TbpB; one in which stretches of amino acids located in the central part of the molecule, described as hypervariable, have been deleted; and one corresponding to the N-terminal half of the molecule, described as the human transferrin binding domain. The reactivity of these antisera against 58 serogroup B strains with a 2.1-kb tbpB gene representing different genotypes, serotypes, and subtypes and different geographic origins was tested on intact meningococcal cells. In parallel, the bactericidal activity of the antisera was evaluated against 15 of the 58 strains studied. Of the 58 strains, 56 (98%) reacted with the antiserum specific for the N-terminal half of TbpB M982; this antiserum was bactericidal against 9 of 15 strains (60%). On the other hand, 43 of 58 strains reacted with the antiserum raised to full-length TbpB while 12 of 15 (80%) were killed with this antiserum. The antiserum specific to TbpB deleted of its central domain gave intermediate results, with 53 of 58 strains (91.3%) recognized and 10 of 15 (66.6%) killed. These results indicate that the N-terminal half of TbpB was sufficient to induce cross-reactive antibodies reacting with the protein on meningococcal cells but that the presence of the C-terminal half of the protein is necessary for the induction of cross-bactericidal antibodies.

Comparative safety and immunogenicity of an acellular versus whole-cell pertussis component of diphtheria-tetanus-pertussis vaccines in Senegalese infants.

A diphtheria and tetanus toxoid two-component acellular pertussis vaccine (DTaP), consisting of 25 micrograms glutaraldehyde-detoxified pertussis toxin (PT) and 25 micrograms native filamentous hemagglutinin (FHA), was compared with diphtheria and tetanus toxoid whole-cell pertussis vaccine (DTwP) in a randomized, double-blind manner in 286 Senegalese infants inoculated at two, four, and six months of age. In infants receiving DTaP a significantly lower rate of local reactions, crying and fever was observed than in infants receiving DTwP. One month after the third dose, the geometric mean titres for FHA antibodies were higher in the DTaP group, whereas increases in PT antibody titres were higher in the DTwP group. More than 90% of the infants had a fourfold or more increase in antibodies to both PT and FHA with either vaccine. Diphtheria, tetanus, and polio antibody responses were also measured and found to be comparable between the two groups. The results of this pilot study support the implementation of a field trial to compare the protective efficacy of these vaccines against pertussis in the same setting.

Evaluation of transferrin-binding protein 2 within the transferrin-binding protein complex as a potential antigen for future meningococcal vaccines.

Because the meningococcal transferrin receptor was shown to elicit bactericidal and protective antibodies in laboratory animals, we undertook a study of the protective role of each of the polypeptides within the Tbp1-Tbp2 complex. We developed a procedure to purify from Neisseria meningitidis B16B6 the two proteins in milligram amounts and raised specific antisera in rabbits and mice. Only antisera specific for Tbp2 displayed bactericidal activity against the parent strain. Mice immunized with purified Tbp2 survived a lethal challenge to a similar degree as animals immunized with the Tbp1-Tbp2 complex, demonstrating that Tbp2 played an important role in the protective activity observed with the complex. Both Tbp1- and Tbp2-specific antisera inhibited transferrin binding to the purified receptor in a solid-phase binding assay, suggesting that the antibodies were able to interact with the Tbp1 molecule only when it was removed from its membrane environment. Finally, Tbp2-specific immunoglobulins were able to lower the growth rate of the meningococci when human transferrin was their sole iron source. Therefore, in all four different systems tested, Tbp2 or antibodies specific for Tbp2 displayed biological characteristics close to those of the Tbp1-Tbp2 complex. This suggests that Tbp2 plays an important role in the protective activity of the complex, eliciting antibodies that are not only bactericidal but also inhibitory for meningococcal growth.

Transferrin-binding proteins isolated from Neisseria meningitidis elicit protective and bactericidal antibodies in laboratory animals.

Transferrin-binding proteins (Tbps) were affinity-isolated from group B Neisseria meningitidis strain B16B6 and used to raise specific antisera. Administration of the antisera to mice loaded with human transferrin before bacterial challenge significantly protected the animals from death. In active immunization studies, mice received three 25 micrograms injections of purified Tbps over a period of 28 days, 7 days after which they were challenged with N. meningitidis. The survival rate in immunized mice was much higher than in control groups. In both active and passive immunization experiments mice were protected against at least 100 LD50. A specific Tbp antiserum was highly bactericidal against the parent strain and against approximately half of the strains tested.

Bordetella pertussis and Bordetella parapertussis: two immunologically distinct species.

Bordetella pertussis and Bordetella parapertussis are closely related species. Both are responsible for outbreaks of whooping cough in humans and produce similar virulence factors, with the exception of pertussis toxin, specific to B. pertussis. Current pertussis whole-cell vaccine will soon be replaced by acellular vaccines containing major adhesins (filamentous hemagglutinin and pertactin) and major toxin (pertussis toxin). All of these factors are antigens that stimulate a protective immune response in the murine respiratory model and in clinical assays. In the present study, we examined the protective efficacies of these factors, and that of adenylate cyclase-hemolysin, another B. pertussis toxin, against B. parapertussis infection in a murine respiratory model. As expected, pertussis toxin did not protect against B. parapertussis infection, since this bacterium did not express this protein, but the surprising result was that none of the other factors were protective against B. parapertussis infection. Furthermore, B. parapertussis adenylate cyclase-hemolysin, although it protected against B. parapertussis infection, did not protect against B. pertussis infection. Despite a high degree of homology between both B. pertussis and B. parapertussis species, no cross-protection was observed. Our results outline the fact that, as in other gram-negative bacteria, Bordetella surface proteins vary immunologically.

Characterization of the serum antibody response induced by Haemophilus influenzae type b tetanus protein-conjugate vaccine in infants receiving a DTP-combined vaccine from 2 months of age.

The serum antibody response induced by Haemophilus influenzae type b capsular polysaccharide (CPS)-tetanus protein conjugate vaccine combined to DTP vaccine was characterized in infants receiving three injections from 2 months of age. Sixty-five per cent and 94% of infants had anti-CPS antibody levels > or = 1 micrograms ml-1 after the second and third dose, respectively. The antibody response was mostly made up of IgG with a marked IgG1 predominance. Significant rises in bactericidal and in complement-mediated opsonic activities were observed after immunization. These data clearly show that this vaccine can be successfully administered in one syringe together with DTP vaccine during the regular infant immunization programme.

Acellular pertussis vaccines: evaluation of reversion in a nude mouse model.

An animal model has been developed to assess the safety of acellular pertussis vaccines in terms of reversion to toxicity. Adsorbed pertussis toxoid preparations, alone or combined in a DTP formulation, were administered to nude mice intraperitoneally. In parallel, groups of positive and negative control mice received pertussis toxin and buffer, respectively. The circulating white blood cells of the animals were monitored for 28 days. Mice immunized with glutaraldehyde toxoid preparations did not develop a lymphocytosis during the observation period, whereas mice immunized with an experimental formalin pertussis toxoid vaccine exhibited a high lymphocytosis six days after vaccine administration, demonstrating, in this model, a reversion of the toxoid. The nude mouse model thus appears to reveal the in-vivo reversion of pertussis toxoids and could be included in the quality control panel for the assessment of the safety of acellular pertussis vaccine.

The influence of immunoglobulin on the development of Pseudomonas aeruginosa infection in an immunocompromised mouse model.

Pretreatment with normal immunoglobulin G (normal IgG) isolated from human placental blood, in secondary immune deficiency after anaesthesia, surgery and corticosteroid therapy seems to be an effective protection against bacterial infection in clinical practice. For this purpose, we tried to verify the effectiveness of normal IgG in prevention of bacterial infection in an animal model. The influence of treatment with normal human IgG has been studied in mice exposed to a sublethal infection with Pseudomonas aeruginosa in various states of immunocompetence such as under anaesthesia and corticosteroid therapy. Results, evaluated as percentages of survivors, confirm significant protection against infection with P. aeruginosa despite immunodepression with hydrocortisone and anaesthesia (96.6% of survivors in the group treated with IgG as opposed to 30% for the untreated corresponding group--P less than 0.001).