Testing meningococcal vaccines for mitogenicity and superantigenicity.

Proteins with intrinsic mitogenic properties are widely represented in prokaryotes, such as in different Streptococcus species (1-3), Candida albicans (4), and Eikenella corrodens (5). Specifically, several bacterial porins of Escherichia coli, Shigella dysenteriae, Salmonella typhimurium, Fusobacterium nucleatum, and pathogenic Neisseria species have been shown to induce nonspecific proliferation of lymphocytes (6-12).

CD4 T-cell epitope mapping.

The majority of T cells recognize peptide epitopes bound to major histocompatibility complex (MHC)-encoded glycoproteins on the surface of professional antigen-presenting cells (APC), principally dendritic cells, macrophages, and B cells (1-3). Most T cells are specific for peptide epitopes in association with either classical MHC class Ia molecules (HLA-A, B, and C in humans and H2-K, D, and L in mice) in the case of CD8(+) T cells, or class II molecules (HLA-DR, DP, and DQ in humans and H2-A and E in mice) for CD4(+) T cells. However, a significant proportion of T cells recognize peptide antigens bound to nonclassical MHC class Ib molecules such as the human HLA-E (mouse analog Qa1) (4) and mouse H2-M3 (5). In addition, some T cells recognize not peptides but lipid or glycolipid antigens bound to nonclassical MHC class Ib molecules such as CD1 in both humans and mice (6).

The role of calcium homeostasis and flux during bacterial antigen processing in murine macrophages.

We report that MHC class II (MHC-II)-restricted antigen processing of two CD4(+) T cell epitopes from the surface M protein of Streptococcus pyogenes in murine macrophages is dependent on intact calcium homeostasis and flux. We have previously shown that the CD4(+) T cell epitope 308-319 of the type 5 M protein is presented by newly synthesized MHC-II molecules via the classical pathway, while 17-31 is loaded on recycling MHC-II molecules via the recycling pathway. In this report we show that processing of viable bacteria for 308-319 presentation depended on the availability of intra- and extra cellular calcium, intact gadolinium-sensitive and/or T-type calcium channels, as well as on thapsigargin-sensitive homeostasis of intracellular calcium. In contrast, processing of 17-31 was independent of both intracellular calcium and gadolinium-sensitive calcium channels. The data suggest that alternative antigen processing pathways have different requirements for intracellular calcium homeostasis.

Functional activities and epitope specificity of human and murine antibodies against the class 4 outer membrane protein (Rmp) of Neisseria meningitidis.

Antibodies against the class 4 outer membrane protein (OMP) from Neisseria meningitidis have been purified from sera from vaccinees immunized with the Norwegian meningococcal group B outer membrane vesicle vaccine. The human sera and purified antibodies reacted strongly with the class 4 OMP in immunoblots, whereas experiments with whole bacteria showed only weak reactions, indicating that the antibodies mainly reacted with parts of the class 4 molecule that were not exposed. The purified human anti-class 4 OMP antibodies and the monoclonal antibodies (MAbs) were neither bactericidal nor opsonic against live meningococci. Three new MAbs against the class 4 OMP were generated and compared with other, previously described MAbs. Three linear epitopes in different regions of the class 4 OMP were identified by the reaction of MAbs with synthetic peptides. The MAbs showed no blocking effect on bactericidal activity of MAbs against other OMPs. However, one of the eight purified human anti-class 4 OMP antibody preparations, selected from immunoblot reactions among sera from 27 vaccinees, inhibited at high concentrations the bactericidal effect of a MAb against the class 1 OMP. However, these antibodies were not vaccine induced, as they were present also before vaccination. Therefore, this study gave no evidence that vaccination with a meningococcal outer membrane vesicle vaccine containing the class 4 OMP induces blocking antibodies. Our data indicated that the structure of class 4 OMP does not correspond to standard beta-barrel structures of integral OMPs and that no substantial portion of the OmpA-like C-terminal region of this protein is located at the surface of the outer membrane.

Two T cell epitopes from the M5 protein of viable Streptococcus pyogenes engage different pathways of bacterial antigen processing in mouse macrophages.

We studied the mechanisms of MHC class II-restricted bacterial Ag processing of the surface fibrillar M5 protein from viable Streptococcus pyogenes in murine macrophages. Two previously defined T cell epitopes were studied using T cell hybridomas specific for 308-319/Ad, associated with the cell wall on the surface of streptococci, and 17-31/Ed, located at the protruding amino terminus of M5. Studies with metabolic inhibitors showed that slow (1 h) processing of M5 308-319 occurred in late endosomes and was dependent on newly synthesized MHC class II molecules and microtubules and on communications between early and late endosomes, consistent with engagement of the classical MHC class II processing pathway. In contrast, fast (15 min) bacterial Ag processing of 17-31 occurred in early endosomes independently of newly synthesized MHC class II molecules and microtubules and of trafficking between early and late endosomes, consistent with the recycling MHC class II processing pathway. Finally, bacterial Ag processing of the epitopes exhibited differential sensitivity to blocking with anti-MHC class II Abs. Thus, two T cell epitopes of a single protective Ag from the surface of whole bacteria are routed to distinct MHC class II processing pathways.

Different endosomal proteolysis requirements for antigen processing of two T-cell epitopes of the M5 protein from viable Streptococcus pyogenes.

We studied endosomal proteolysis of the surface fibrillar M5 protein from viable Streptococcus pyogenes as an essential step involved in major histocompatibility complex class II-restricted antigen processing of two immunodominant CD4(+) T-cell epitopes (17-31/Ed and 308-319/Ad). Intracellular proteolysis of viable streptococci for presentation of 17-31, bound by serine proteinase cleavage sites, was mediated by serine proteinases, whereas processing of soluble recombinant M5 protein required in addition cysteine proteinases. Furthermore, processing of 17-31 was resistant to ammonium chloride and thus was not dependent on endosome acidification. Cysteine and serine proteinase cleavage sites were located adjacent to 308-319, and its processing was dependent on serine, cysteine, and aspartic proteinases, as well as on endosomal acidification. The data suggest that antigen processing of two major T-cell epitopes on streptococcal M5 protein occurred in different endosomal compartments by different classes of intracellular proteinases.

T-Cell epitope mapping the PorB protein of serogroup B Neisseria meningitidis in B10 congenic strains of mice.

T-cell epitope mapping the meningococcal serotype 15 PorB protein performed in this study in three congenic strains of mice with B10 genetic background revealed at least three murine T-cell epitopes (55-72, 163-180, and 226-261), located in the highly conserved putative transmembrane regions of Neisserial porins. Proliferation assays with popliteal lymph node cells derived from mice immunized with the PorB protein or with synthetic 18-mer peptides showed that epitope 163-180 immunized only in the H-2d haplotype, epitope 55-72 could be presented by both H-2f and H-2s molecules, while the 226-261 region covered by three overlapping peptides could be efficiently recognized in context of all three MHC class II haplotypes studied. Inhibition experiments with blocking I-Aalpha- and I-Ealpha-specific mAb showed that peptide 163-180 was presented by I-Ad and peptide 244-261 was presented by both I-Af and I-As. In addition, evidence was obtained that peptide 226-243 was presented in context of H-2d or I-As haplotypes and peptide 55-72 was presented in context of I-Af and I-As loci. Finally, the Norwegian outer membrane vesicle vaccine, but not the purified PorB protein, could recall responses in mice immunized with synthetic peptides corresponding to the 226-261 region. Altogether, these results suggest that T-cell epitopes identified on the serotype 15 PorB protein, particularly those presented by several MHC class II molecules (e.g., 226-261), could have important implications for the development of meningococcal vaccines.

Vaccine-induced IgG antibodies to the linear epitope on the PorB outer membrane protein promote opsonophagocytosis of Neisseria meningitidis by human neutrophils.

The serotype 15 PorB protein of Neisseria meningitidis contains an N-terminal linear immunodominant B-cell epitope located on the putative loop 1 (VR1) region. This epitope has previously been shown to stimulate antibody formation in 74% of the vaccinees after three doses of the Norwegian group B outer-membrane vesicle (OMV) vaccine. In the present study, the purified PorB protein and the 23mer synthetic peptide D63b2 covering VR1 region were immobilized onto N-hydroxysuccinimide-activated matrix and used for affinity purification of the specific IgG antibodies from sera of three selected vaccinees. PorB- and peptide D63b2-specific IgG preparations bound to the PorB protein on immunoblots and reacted with strain 44/76 and OMV complexes expressing the serotype 15 PorB protein, but not with the PorB-deficient mutant, suggesting high specificity for the PorB protein. Both PorB- and peptide D63b2-specific IgG were marginally bactericidal, but enabled strong opsonophagocytosis measured as respiratory burst response of human neutrophils and internalization of opsonized FTTC-labeled meningococci. The data indicate that about 30-57% of the bulk serum opsonic activity for the 44/76 bacteria could be ascribed to linear epitope-specific IgG1, thus contributing to vaccine-induced protection against systemic meningococcal disease via the opsonophagocytic route of pathogen clearance.

Immune responses to linear epitopes on the PorB protein of Neisseria meningitidis in patients with systemic meningococcal disease.

Neisserial porins, the major protein constituents of the outer membrane capable of inducing antibody responses in humans, are considered to be meningococcal vaccine candidates, so it is important to map the relevant B-cell epitopes. For B-cell epitope analyses of the serotype 15 PorB protein in Neisseria meningitidis, paired sera from selected patients with systemic meningococcal disease (SMD) were screened with synthetic 12mer peptides spanning the PorB protein sequence, and/or its variable region 1 (VR1). A 'SMD-related' linear B-cell epitope was found within the VR1 region consisting of 14 residues (17svFHQNGQVTEvtt30). A 23mer soluble peptide (D63b2) that covered the VR1 region, including the complete 17svFHQNGQVTEvtt30 sequence, was recognized, whereas no detectable binding was observed to a 16mer peptide (D63a1) containing most of the essential sequence (19FHQNGQVTEvtt30). A low frequency of IgG responses specific for the PorB linear epitopes was found in convalescent-phase sera from 132 SMD patients studied, as judged from both immunoblotting studies (24/132; 18.2%) and reactivity with peptide D63b2 (18/132; 13.6%). Peptide D63b2 significantly inhibited IgG binding to the denatured PorB protein on immunoblots, suggesting that this B-cell epitope was one of the main linear epitopes on the PorB protein recognized by sera from some SMD patients.

T-cell responses to outer membrane proteins of Neisseria meningitidis: comparative study of the Opa, Opc, and PorA proteins.

Former studies have shown that the class 5 outer membranes proteins (Opa and Opc proteins) of Neisseria meningitidis are at least as immunogenic as meningococcal porin proteins. High antibody titers to class 5 proteins have been observed in sera obtained during convalescence after meningococcal infection. A strong increase in anti-class 5 antibodies has also been observed in vaccinees who received a meningococcal outer membrane vesicle preparation. The enhanced B-cell response to class 5 proteins may be due to the presence of immunodominant helper T-cell epitopes in these proteins. In order to investigate this hypothesis, we tested purified Opa, Opc, and class 1 proteins for recognition by human T cells. a hierarchy of T-cell immunogenicity was observed among the outer membrane proteins, the Opa protein being more immunogenic than the other proteins. In most cases, the proliferative responses elicited by Opc were higher than the responses observed for the class 1 protein. The epitopes recognized by the immune T cells were identified by using overlapping synthetic peptides spanning the protein sequences of OpaB, Opa5d, and Opc.

A linear B-cell epitope on the class 3 outer-membrane protein of Neisseria meningitidis recognized after vaccination with the Norwegian group B outer-membrane vesicle vaccine.

The class 3 outer-membrane protein (OMP) of Neisseria meningitidis is a potential target for bactericidal and opsonic antibodies in humans. Synthetic peptides spanning the class 3 OMP from the vaccine strain 44/76 (B:15:P1.7,16:L3,7) were synthesized on pins and screened with serum obtained from Norwegian adolescents immunized with a meningococcal serogroup B outer-membrane vesicle (OMV) vaccine. A strong IgG response to a single peptide (19FHQNGQVTEVTT30) located within loop 1 (VR1) was stimulated after three doses of OMV vaccine in three vaccinees selected on the basis of their antibody response to class 3 OMP. No clear linear B-cell epitopes were recognized by four different murine serotype 15-specific mAbs. A 23mer peptide (D63b2) containing loop 1 of the class 3 OMP was synthesized, and the IgG responses were measured in pre- and post-vaccination serum from 27 vaccinees. Specific IgG rose significantly in 37% of vaccinees 6 weeks after the second dose and in 74% of the vaccinees 6 weeks after the third dose of the OMV vaccine. Most immune sera reacted distinctly on immunoblots with denatured class 3 OMP, and the immunoblotting reactivity correlated strongly with concentration of the IgG antibodies specific for peptide D63b2. When added to a post-vaccination serum from one vaccinee, peptide D63b2 competed efficiently with the class 3 OMP for specific antibody binding on immunoblots and in pin ELISA. The results show that the significant part of the humoral response to the meningococcal class 3 OMP elicited by vaccination with the Norwegian OMV vaccine was directed against a single continuous epitope.

Emergence of a new virulent clone within the electrophoretic type 5 complex of serogroup B meningococci in Norway.

An increase in B:15:P1.12 meningococci among isolates from patients with Neisseria meningitidis infection in Norway in recent years led to further characterization of such strains. Between 1987 and 1992, B:15:P1.12 strains constituted 9.8% (24 strains) of B:15 isolates. The B:15:P1.12 strains belonged to the electrophoretic type 5 (ET-5) complex, but 17 (71%) strains were a new clone (ET-5c) not found elsewhere in the world. All but one strain of ET-5c were responsible for a localized outbreak of systemic meningococcal disease in western Norway. A novel monoclonal antibody (202,G-12), developed against the unknown variable region 2 on the class 1 protein of one of these strains, bound to 19 of the 15:P1.12 strains, 4 strains bound the subtype P1.13 reference monoclonal antibody MN24H10.75, and the remaining strain showed no reaction. Sequencing of porA genes demonstrated a series of nine threonine residues in the deduced variable region 2 of the latter strain, while four and five threonine residues were found in the corresponding regions of strains reacting with the monoclonal antibodies 202,G-12 and MN24H10.75, respectively. Epitope mapping with synthetic peptides showed that 202,G-12 bound to a sequence of 11 amino acids which included the four threonine residues specific for subtype P1.13a. Immunoglobulin G antibodies against the P1.7,16 subtype protein, induced in volunteers after vaccination with the Norwegian meningococcal vaccine, did not cross-react on immunoblots with the subtype protein of clone ET-5c. Thus, postvaccination class 1 protein antibodies, assumed to be protective, may not be effective against infection with the new clone.

Comparison of three human-murine heteromyeloma cell lines for formation of human hybridomas after electrofusion with human peripheral blood lymphocytes from meningococcal cases and carriers.

Peripheral blood lymphocytes from meningitis patients and healthy meningococcal carriers were fused by electrofusion with the three human-murine heteromyeloma cell lines CB-F7, K6H6B5 and H7NS. 934 hybridomas producing human immunoglobulins were obtained in 30 fusions. Heteromyeloma K6H6B5 yielded a significantly higher proportion of hybridomas producing IgG antibodies than did the two other cell lines. CB-F7 and K6H6B5 yielded comparable numbers of hybridomas whose supernatants reacted with homologous bacteria, whereas the cell line H7NS was less efficient.

A comparison of human and murine monoclonal IgGs specific for the P1.7 PorA protein of Neisseria meningitidis.

Monoclonal human IgG SS269 reacts with Neisseria meningitidis expressing the P1.7 PorA protein and with linear peptides containing NGGAS, which accounts for the P1.7 specificity. Murine monoclonal antibody to P1.7 reacts with peptides containing the overlapping epitope, ASGQ. The human and murine antibodies have similar affinities. The low avidity human antibody is very inefficient at stimulating complement-mediated bactericidal killing while the high avidity murine antibody efficiently kills bacteria. However, efficient opsonophagocytosis was mediated even at low concentrations of the human antibody and in the absence of complement, suggesting that low avidity antibodies might be protective against disease.

Down's syndrome, Edwards' syndrome, Patau's syndrome--synthesis of glycosaminoglycans.

Synthesis of glycosaminoglycans (GAGs) by fibroblasts derived from seven patients with Down's syndrome, five patients with Edwards' syndrome, and two patients with Patau's syndrome were studied in cell culture. The aneuploid strains were compared with diploid fibroblasts from age-matched controls. In terms of hyaluronic acid and sulfated GAG synthesis, the amount of synthesized hyaluronic acid was not significantly different between postnatal aneuploid strains and controls.

Anti-bacterial specificities in the human fetal B cell repertoire.

To study the human fetal B cell repertoire, liver and spleen lymphocytes were fused with the human x mouse heteromyeloma line CB-F7. Initially, 2310 IgM and 181 IgG producing hybridoma lines were established. Culture supernatants were analysed for binding activity to antigens from both the exogeneous and the endogeneous environments. For IgG secreting cell lines no antigenetic specificity has been detected. However, independently from the gestational age, monoclonal IgM antibodies binding to bacterial antigens (tetanus toxoid, lipid A, N. meningitidis antigens) were found. In particular, nearly 10% of the hybridomas obtained from fetal liver produced antibodies binding to lipid A (endotoxin). Among the IgM antibodies with anti-bacterial specificities approximately 30% were found to be polyspecific, i.e. these antibodies recognized auto-antigens of different molecular origin as well (ssDNA, keratin, myosin, actin). We conclude that polyreactive natural IgM antibodies may be generated during fetal life, which may take part in the formation of a humoral anti-infectious first line defense barrier in the neonate.

Synthesis of glycosaminoglycans in fibroblasts from abortuses with trisomy, triploidy, and from children with Down's syndrome.

A comparative study has been made of glycosaminoglycan (GAG) accumulation in human fibroblasts with trisomy 7 and triploidy from spontaneous abortuses, fibroblasts with triploidy from induced abortuses, fibroblasts from patients with Down's syndrome and diploid fibroblasts from age-matched controls. The study demonstrated that the incorporation of [3H] glucosamine into hyaluronic acid by fibroblasts with trisomy 7 and triploidy, established from spontaneous abortuses, and from two out of three induced abortuses with triploidy, was 2.6-5.3 times lower than control incorporation. One strain of fibroblasts from an induced abortus with triploidy (IMG-1062) did not show any differences in GAG production when compared with diploid fibroblasts. However, the strains from children with Down's syndrome revealed normal or even increased levels of hyaluronic acid production. The data support the contention that the decreased hyaluronic acid synthesis in fibroblasts with an abnormal karyotype is related to spontaneous abortion.

Characterization of costal cartilage collagen in funnel chest.

Parallel measurements of pyridinoline content, the ratio of soluble:insoluble collagen, and the percentage of endogenous collagenolysis were made in samples of costal cartilage from forty-five children with funnel chest (FC) and from twenty-two control children. From an analysis of the influence of different factors, such as FC type (isolated or syndromic), a diagnosed syndrome, extent of FC depression (second or third), and age, two biochemical variants of FC-variants a and b, which were not related to the presence or absence of a known concurrent syndrome, were distinguished. These variants differed from each other in all the parameters under study. Variant a occurred about five times less frequently than b, and was characterized by a pyridinoline content of about 50% of that of b, an elevated soluble:insoluble collagen ratio, and an increased percentage of endogenous collagenolysis compared to controls. For variant b, the pyridinoline content fell within normal limits, but the soluble:insoluble collagen ratio, and the percentage of endogenous collagenolysis were below normal. The data suggest that the formation of variant a may be related to defect(s) in collagen crosslinking, whereas the formation of variant b may result from other unknown factor(s) involved in the formation and maturation of costal cartilage.

Collagen and fibronectin synthesis by trisomic and triploid fibroblasts from human spontaneous abortuses.

Collagen and fibronectin synthesis by trisomic and triploid fibroblasts derived from human spontaneous abortuses was studied. It was demonstrated that the level of fibronectin and collagen production in fibroblasts with trisomy 7, trisomy 9, and triploidy was reduced as compared with diploid cells. A correlation between this observation and an increased rate of intracellular 14C-procollagen degradation was also established for the anomalous strains. No difference in hydroxylation of 14C-proline residues in alpha 1(I) and alpha 2(I) collagen chains and no fluctuation in the collagen type (I): type III ratio was found in the strains with the abnormal karyotypes. It was concluded that differentiation of the abnormal fibroblasts was impaired. The data also favour the hypothesis that the deficiency of the fibroblasts in producing proteins may account for a variety of anatomic abnormalities of embryos.