Murine Immune Responses to Neisseria meningitidis Group C Capsular Polysaccharide and a Thymus-Dependent Toxoid Conjugate Vaccine

The polysaccharide (PS) capsules of many pathogenic bacteria are poor immunogens in infants and young children as a result of the delayed response to PS antigens during ontogeny. The development of polysaccharide-protein conjugate vaccines for Haemophilus influenzae type b, which have proven to be efficacious in this age group, has led to active development by a number of investigators of conjugate vaccines for other diseases. We describe here the response of several mouse strains to the capsular PS of Neisseria meningitidis group C (MCPS) conjugated to tetanus toxoid (MCPS-TT) and the same response in BALB/c mice as a model of the immune consequences of conjugate vaccine immunization. The use of a conjugate vaccine results in a shift in the isotype elicited in response to the MCPS, from immunoglobulin M (IgM) and IgG3 to primarily IgG1. A response to MCPS-TT is seen even among mouse strains which respond poorly to MCPS itself, emphasizing the importance of a strain survey when choosing a mouse model for a vaccine. The marked increase in IgG1 antibody titer was accompanied by a large increase in bactericidal activity of sera from these animals. Animals primed with the conjugate vaccine demonstrated a booster response after secondary immunization with either the MCPS or the conjugate. The ability to produce a boosted IgG1 anti-MCPS response to the MCPS can be transferred to adoptive recipients by B cells alone from mice primed with MCPS-TT but not mice primed with MCPS alone. These data indicate that in BALB/c mice a single immunization with MCPS-TT is sufficient to induce a shift to IgG1 and generate a memory B-cell population that does not require T cells for boosting.     

Response of Mice to Injection of Ribosomal Fraction from Group B Neisseria meningitidis

Ribosomes of strain NOR-7 of group B Neisseria meningitidis were isolated by a procedure that included treatment of the cells with sodium dodecyl sulfate, disruption in a French pressure cell, and differential centrifugation. These preparations consisted of 66% ribonucleic acid and 24% protein and sedimented as a single component with a constant of approximately 66S. When used in immunodiffusion tests with homologous rabbit antiserum, untreated ribosomes formed two precipitin lines, when treated with ribonuclease three lines, and when Pronase-digested only one distinct line. Qualitatively indistinguishable reactions were obtained with the same antiserum and ribosomes from group A meningococci, but no precipitation occurred with those of Escherichia coli. When injected into mice, group B ribosomes elicited an increase in the number of antibody-producing spleen cells demonstrable by the hemolytic plaque technique using unsensitized sheep erythrocytes. Sensitization of the erythrocytes with increasing amounts of supernatant fluid of meningococcal cultures progressively reduced the number of demonstrable plaque-forming cells. Neuraminidase treatment of the erythrocytes increased immune hemolysis, whereas Pronase digestion reduced it. Injected mice were protected against homologous and heterologous meningococcal challenge. Both hemolysis and protection-inducing activities of the ribosomes were unimpaired by ribonuclease, but were reduced by Pronase. It is concluded that the immunological response elicited by the meningococcal ribosomes does not involve the group-specific carbohydrate antigen. The immunological mechanism by which the mice are protected against meningococcal challenge remains unknown.     

Molecular Mimetics of Neisseria meningitidis Serogroup B Polysaccharide

Strains of Neisseria meningitidis serogroup B (NmB) are an important cause of meningitis and sepsis. Efforts to develop a NmB vaccine have been hampered by poor immunogenicity of the polysaccharide capsule, which cross-reacts with host polysialic acid, and the danger of eliciting autoantibodies. To investigate the potential of molecular mimetics to circumvent these problems, we prepared murine monoclonal antibodies (mAbs) against the N-propionyl derivative (N-Pr) of NmB polysaccharide [10]. Several mAbs were found that reacted with capsular polysaccharide epilopes, which were distinct from host polysialic acid. These mAbs also passively conferred protection against experimental bacteremia. We used these mAbs to screen novel independently folding peptide phage display libraries, and pools of combinatorial small molecules, each consisting of-30 to-700 small molecules of diverse composition. To date, several mimetic candidates have been identified. One is a peptide selected from a library of independently folding αβ peptides, and others are peptoid [23] dimers or trimers selected from the small molecule pools. The peptoids contain an indan-type of ring system, and some of them also contain a large hydrophobic group such as oleyl amine or dehydroabietyl amine, and a positively charged group at the amino-terminus. Both the αβ peptide from the phage library, and the peptoids from the small molecule pools, inhibit binding of the mAbs to N-Pr NmB polysaccharide. Future studies will focus on the structure/activity relationship of these mimetics, and the development of immunogens that may be capable of eliciting anti-capsular antibody without autoantibody activity.     

Physicochemical Properties of Neisseria meningitidis Group X Polysaccharide Antigen

Neisseria meningitidis group X occurs in human carrier populations and is rarely implicated in serious disease. This organism possesses a capsular group antigen which is an acidic polysaccharide. It is composed of the amino sugars, glucosamine, glucosamine-6-phosphate, galactosamine, and the simple hexose, glucose. The group X capsular antigen has an S_20,w⁰ of 3.6, and the acidic nature of the polysaccharide is reflected in an isoelectric point of 3.65. The meningococcal A, B, C, and Y polysaccharide group antigens are also composed primarily of amino sugars. The chemical composition of the group X antigen most closely resembles the capsular antigen of N. meningitidis group Y, which is also predominately a carrier organism.     

Production and Degradation of Serogroup B Neisseria meningitidis Polysaccharide

The antigenicity of experimental Mycoplasma pneumoniae vaccines prepared from antigen grown in a medium buffered with N-2-hydroxyethylpiperazine-N′-2′-ethanesulfonic acid was tested in young, adult males. Formalin-inactivated antigens from a high-passage strain and a low-passage strain at various dilutions (12 to 123 μg of N/ml) were injected intramuscularly in 1-ml doses. Antibody responses were tested by the metabolic inhibition (MI) technique. Sixty-five to 86% of the volunteers in all vaccine groups responded with fourfold or greater MI antibody rises, but only nine (39%) of 23 antibody-free subjects converted compared to 53 (88%) of 60 of those with pre-existing antibody. A booster vaccination did not increase the number of converters or enhance the geometric mean titers. The antigen concentrations of vaccines with 24 or more μg of N/ml appeared to be above the threshold needed for maximal antibody responses in the dose range tested. MI antibody rises could not be detected in sputa and nasal washings obtained from a small group of vaccinees. The results of this study suggest that the new vaccines offer little or no improvement in antigenicity in man over earlier inactivated vaccines.     

Humoral immune responses to Neisseria meningitidis in children

An understanding of the nature of immunity to serogroup B meningococci in childhood is necessary in order to establish the reasons for poor responses to candidate vaccines in infancy. We sought to examine the nature of humoral immune responses following infection in relation to age. Serum bactericidal activity was poor in children under 12 months of age despite recent infection with Neisseria meningitidis. The highest levels of bactericidal activity were seen in children over 10 years of age. However, infants produced levels of total immunoglobulin G (IgG) and IgG subclass antibodies similar to those in older children in a meningococcal enzyme-linked immunosorbent assay. Most antibody was of the IgG1 and IgG3 subclasses. This striking age dependency of bactericidal antibody response following infection is not apparently due to failure of class switching in infants but might be due to qualitative differences in antibody specificity or affinity.     

Neisseria meningitidis Lipopolysaccharide Modulates the Specific Humoral Immune Response to Neisserial Porins but Has No Effect on Porin-Induced Upregulation of Costimulatory Ligand B7-2

The role of lipopolysaccharide (LPS) in the specific humoral response to meningococcal porins was investigated by measuring anti-PorA or -PorB antibody levels in mice immunized with wild-type meningococcal strain H44/76 or with its recently described LPS-negative mutant. Two murine strains were used for these immunizations: C3H/HeJ, which is LPS hyporesponsive, or C3H/HeOuJ, which is LPS responsive. A high level of anti-PorB immunoglobulin G (IgG) response was induced in both strains of mice immunized with either organism. The response induced by the wild-type strain was greater in C3H/HeOuJ mice than in C3H/HeJ mice, while the response induced by the LPS-negative mutant was similar in the two murine strains. Additionally, the anti-PorB response was similar in C3H/HeJ mice immunized with either bacterial strain. In general, the anti-PorA IgG response was lower than the anti-PorB response. These findings indicate that the presence of LPS is not essential for the induction of an antineisserial porin humoral response but can augment such a response. To determine whether LPS has any effect on the B-cell-stimulatory effect of neisserial porins (essential for the adjuvant activity of neisserial porins), B cells from both murine strains were incubated with outer membrane complexes (OMCs) prepared from strain H44/76 and its LPS-negative mutant. OMCs from either meningococcal strain were able to increase the surface expression of the costimulatory ligand B7-2 on B cells from either murine strain. Consistent with previously reported findings, LPS does not significantly affect the ability of neisserial porins to induce the costimulatory ligand B7-2.     

Physicochemical Properties of Neisseria meningitidis Group C and Y Polysaccharide Antigens

Chemical and physical studies of the Neisseria meningitidis group C and Y antigens indicated that these preparations are acidic polysaccharides. The group C antigen contains predominately n-acetyl neuraminic acid, with small amounts of glucose, glucosamine, and mannose, whereas the group Y antigen contains glucose, glucosamine, and mannose. The molecular weights of the group C and Y antigens were found to be 88,000 and 197,000, respectively. The group C antigen was susceptible to neuraminidase digestion. Finally, the immunological reactivity of neither antigen was inhibited in hemagglutination or immunodiffusion systems by neutral or substituted sugars.     

Characterization of Spheroplast Membranes of Neisseria meningitidis Group B

Spheroplast membranes (spheroplast envelopes) of strain 2091 of group B Neisseria meningitidis were prepared by a procedure that included lysozyme treatment of the cells and osmotic lysis of the resulting spheroplasts. Electron microscopy revealed that the membranes consisted of two unit layers, generally parallel to each other. The membrane preparation migrated as a single component in a 40 to 70% sucrose gradient and consisted of 62% protein, 28% lipid, 9% ribonucleic acid, small amounts of carbohydrate, hexosamine, and deoxyribonucleic acid. When 1 or 10 mug (dry weight) was injected intravenously into rabbits, a mild pyrogenic reaction was elicited. In immunodiffusion tests, immune rabbit serum prepared against spheroplast membranes produced three major precipitin lines, with the homologous antigen solubilized with sodium dodecyl sulfate, and a single line with untreated antigen. The immune serum also reacted with a cell wall antigen, and to a lesser extent with some of the cytoplasmic antigens. Succinate dehydrogenase and reduced nicotinamide adenine dinucleotide (NADH) oxidase activities were found to be associated with the spheroplast membranes. NADH dehydrogenase also was associated with the membranes but was gradually released and recovered in other fractions. Glutamate-oxaloacetate transaminase, glutamate, glucose-6-phosphate, and isocitrate dehydrogenase activities were not found in the membrane preparation. About one-third of these enzymatic activities were recovered in the supernatant fluid after the sedimentation of the spheroplasts and two-thirds were recovered in the cytoplasmic fraction. N-acetylneuraminic acid (NAN)-condensing enzyme and cytidine monophosphate-NAN synthesizing enzyme also were identified in this organism. These enzymes were not associated with the membranes and were recovered from extracts from whole cells, spheroplasts, or cells exposed to osmotic shock, as well as from spheroplast supernatant and shock fluids. It is concluded that the spheroplast membranes of the strain of meningococci used in these studies are typical of those recovered from gram-negative bacteria.     

Modulation of the Immune System by Neisseria meningitidis

The immunomodulatory potential of Neisseria meningitidis was investigated. Spleen cells from mice injected intraperitoneally with low to moderate doses of meningococci (10(4)-10(7)) were found to display enhanced responses to the mitogens lipopolysaccharide (LPS), phytohaemagglutinin (PHA), and concanavalin A (Con A). In contrast, high doses of meningococci (10(8)-10(9)) caused a marked decrease in mitogenic reactivity. Meningococci-injected mice also displayed a dose-dependent suppression of a primary anti-sheep red blood cell (SRBC) plaque-forming cell (PFC) response. The timing between the injection of SRBC and of meningococci appeared to play an important role in the induction of suppression by the organisms. Thus, decreased PFC responses were observed only when the bacteria were injected prior to the antigen. When meningococci were injected at the same time or after SRBC, normal or even increased PFC responses developed. Kinetic experiments showed that the onset of suppression of both mitogen and antibody responses by meningococci was very rapid, so that by 6-7 h after injection of the bacteria, mice showed markedly reduced mitogen responses and became essentially unable to mount an antibody response against SRBC. Suppression of mitogen responses was relatively transient, since reactivity returned to normal after 48 h. However, the ability of infected animals to mount a normal anti-SRBC response did not fully return until 12 days after the infection. Spleen cells from meningococci-infected mice also showed markedly depressed PFC responses when stimulated with SRBC in vitro but failed to suppress the response of normal spleen cells in mixed cultures. These observations indicate that putative suppressor cells, if they exist at all, are too insignificant in terms of numbers and/or efficiency to account for the observed immunosuppression. A more likely explanation for the inhibition, which is supported by our data, presented here and elsewhere, is that certain surface components of meningococci are capable of imparting immunosuppressive signals directly onto target lymphocytes.     

Translocation and surface expression of lipidated serogroup B capsular Polysaccharide in Neisseria meningitidis

The capsule of N. meningitidis serogroup B, (alpha2-->8)-linked polysialic acid and the capsules of other meningococcal serogroups and of other gram-negative bacterial pathogens are anchored in the outer membrane through a 1,2-diacylglycerol moiety. Previous work on the meningococcal cps complex in Escherichia coli K-12 indicated that deletion of genes designated lipA and lipB caused intracellular accumulation of hyperelongated capsule polymers lacking the phospholipid substitution. To better understand the role of lip and lipB in capsule expression in a meningococcal background, the location, sequence, and relationship to related bacterial capsule genes were defined and specific mutations in lipA and lipB were generated in the serogroup B meningococcal strain NMB. The lipA and lipB genes are located on the 3' end of the ctr operon and are most likely transcribed independently. Inactivation of lipA, lipB, and both resulted in the same total levels of capsular polymer production as in the parental controls; however, these mutants were as sensitive as an unencapsulated mutant to killing by normal human serum. Immunogold electron microscopy and flow cytometric analyses revealed intracellular inclusions of capsular polymers in lipA, lipB, and lipA lipB mutants. Capsular polymers purified from lipA, lipB, and lipA lipB mutants were lipidated. The phospholipid anchor was shown by gas chromatography-mass spectroscopy analysis to be a phosphodiester-linked 1,2-dipalmitoyl (C16:0) glycerol moiety and was identical in structure to that found on the wild-type meningococcal capsule polymers. Thus, lipA and lipB do not encode proteins responsible for diacylglycerophosphatidic acid substitution of the meningococcal capsule polymer; rather, they are required for proper translocation and surface expression of the lipidated polymer.     

Development and Characterization of Anti-idiotype Based Peptide and DNA Vaccines which Mimic the Capsular Polysaccharide of Neisseria meningitidis Serogroup C

Anti-idiotypic antibodies and peptides which mimic antigens may offer an alternate strategy for converting a thymus-independent (TI) antigen to a thymus dependent (TD) antigen. We have developed an anti-idiotype based peptide mimic of the capsular polysaccharide of N. meningitidis serogroup C (MCPS) which induces a T-dependent protective immune response in mice. Subsequent studies have demonstrated that immunization of severe combined immunodeficient mice reconstituted with human B cells respond to immunization with the MCPS peptide mimic with bactericidal anti-polysaccharide directed antibody response. We hypothesized that administration of a DNA vaccine resulting in endogenous expression of this carbohydrate peptide mimic would induce anti-MCPS antibodies.     

Anti-Pneumococcal Capsular Polysaccharide Antibody Response and CD5 B Lymphocyte Subsets

The role of CD19⁺ CD5⁺ and CD19⁺ CD5⁻ B cell subpopulations in the antibody response to pneumococcal capsular polysaccharides (caps-PSs) is controversial. In the present study, we evaluated the role of human CD19⁺ CD5⁺ and CD19⁺ CD5⁻ cell populations in the serotype-specific antibody response to caps-PS. After vaccination of 5 healthy human adults with Pneumovax (23-valent pneumococcal polysaccharide vaccine [PPV23]), IgG anti-caps-PS serotype 4 antibody-producing cells resided mainly in the CD19⁺ CD5⁻ B cell subset, as assessed by enzyme-linked immunosorbent spot (ELISpot) analysis. Moreover, in a humanized SCID mouse model, CD19⁺ CD5⁻ B cells were more effective than CD19⁺ CD5⁺ cells in producing IgG anti-cap-PS antibodies. Finally, an association was found between the level of IgG anti-caps-PS antibodies and the number of CD19⁺ CD5⁻ B cells in 33 humans vaccinated with PPV23. Taken together, our data suggest that CD5 defines a functionally distinct population of B cells in humans in the anti-caps-PS immune response.     

Human B Lymphocyte In Vitro Response to the Group B Streptococcal Type III Capsular Carbohydrate

Anti-group B streptococcal type III polysaccharide-specific human B cells from the peripheral circulation can be activated and delected in an in vitro culture system. It is possible to detect both IgM- and IgG-producing cells from both seropositive and seronegative donors. The specificity of the response was demonstrated by inhibition with excess liquid phase antigen and the use of related but antigenically distinct control antigens. The response was absent without the addition of T cells, optimal at 10% and 25% T cells respectively for IgM- and IgG-secreting cells, and undetectable using 50% T cells. The optimal antigen concentration for in vitro B-cell activation is 2.5 × 10⁻⁴ μ /ml. Cells from 5 of 6 seropositive donors and 3 of 7 seronegative donors produced specific IgM antibody after culture with antigen. We conclude that the control of the human antibody response to the group B streptococcal type III polysaccharide is influenced by T cells. The response seen in the culture system may be of value in assessing future vaccine candidates designed to prevent neonatal infections.     

Humoral Immune Response to Some Benzylpenicillin Preparations

In this study the humoral immune responses to long-term administration of benzylpenicillin preparations with and without adjuvants were compared. In 44.4% of the patients on long-term treatment with a benzylpenicillin preparation containing oil and alum monostearate, an induction of benzylpenicilloyl (BPO)-specific IgG, IgM and IgE was demonstrated during and after the course. Patients treated with a benzylpenicillin preparation containing no oil and alum monostearate showed only a very weak BPO-specific IgM and IgG response during the course. In patients in whom long-term treatment with a benzylpenicillin preparation without adjuvants was initiated by a benzylpenicillin preparation containing oil and alum monostearate, not only BPO-speific IgM and/or IgG but also IgE were demonstrated in 13.0%. The differences in immunoesponse in the various long-term courses were significant ( P < 0.05). The data suggest that the presence of such adjuvants as oil and alum monostearate has an influence on the synthesis of BPO-specific antibodies. However, it can not be excluded that the difference in immunogenicity had some unknown connection with the differences in penicillin/blood levels.     

Humoral Immune Response to the Antigen Administered as an Immune Complex

Antigen (HSA) bound in immune complexes at equivalence with syngeneic anti-HSA antibodies elicit much stronger humoral immune response then soluble HSA. On the other hand, administration of immune complexes formed with xenogeneic (rabbit) anti-HSA antibodies suppressed humoral immune response against HSA, but not against rabbit IgG in mice. We suggest that immunization with antigen bound in immune complex might represent a powerful tool in enhancing humoral immune responses.     

Fulminant Neisseria meningitidis B to revealing multiple myeloma

We report the case of a 64 years-old woman, presenting in our hospital with purpura fulminans due to invasive meningococcal B infection. Biological exams led to the diagnosis of multiple myeloma. This case illustrates the importance of immunosuppression in this hematologic disease and allows us to insist on the prophylactic measures to be implemented.     

Differences in Surface Expression of NspA among Neisseria meningitidis Group B Strains

NspA is a highly conserved membrane protein that is reported to elicit protective antibody responses against Neisseria meningitidis serogroups A, B and C in mice (D. Martin, N. Cadieux, J. Hanel, and B. R. Brodeur, J. Exp. Med. 185:1173-1183, 1997). To investigate the vaccine potential of NspA, we produced mouse anti-recombinant NspA (rNspA) antisera, which were used to evaluate the accessibility of NspA epitopes on the surface of different serogroup B strains by an immunofluorescence flow cytometric assay and by susceptibility to antibody-dependent, complement-mediated bacteriolysis. Among 17 genetically diverse strains tested, 11 (65%) were positive for NspA cell surface epitopes and 6 (35%) were negative. All six negative strains also were resistant to bactericidal activity induced by the anti-rNspA antiserum. In contrast, of the 11 NspA surface-positive strains, 8 (73%; P < 0.05) were killed by the antiserum and complement. In infant rats challenged with one of these eight strains, the anti-rNspA antiserum conferred protection against bacteremia, whereas the antiserum failed to protect rats challenged by one of the six NspA cell surface-negative strains. Neither NspA expression nor protein sequence accounted for differences in NspA surface accessibility, since all six negative strains expressed NspA in outer membrane preparations and since their predicted NspA amino acid sequences were 99 to 100% identical to those of three representative positive strains. However, the six NspA cell surface-negative strains produced, on average, larger amounts of group B polysaccharide than did the 11 positive strains (reciprocal geometric mean titers, 676 and 224, respectively; P < 0.05), which suggests that the capsule may limit the accessibility of NspA surface epitopes. Given these strain differences in NspA surface accessibility, an rNspA-based meningococcal B vaccine may have to be supplemented by additional antigens.