The immunological status indices of monkeys inoculated with a meningococcal B vaccine

The method for the determination of bacterial antibodies to group B meningococci was worked out. The method was used for the determination of antibodies to group B meningococcal vaccine produced in the USSR. The dynamic study of antibodies to protein, polysaccharide and lipopolysaccharide antigens of group B meningococci was made by the method of the enzyme immunoassay (EIA), and the safety of the vaccine was studied by the determination of autoantibodies active against brain tissue antigens. The data thus obtained were indicative of the immunological activity of group B protein-polysaccharide vaccines, manifested by the capacity for stimulating bactericidal antibodies whose level increased 8- to 10-fold after the immunization of monkeys in 2 and 3 injections. Similarity in the dynamics of the formation of bacteriolysins and antibodies to protein antigen, as determined in EIA, was noted. The vaccine was found to stimulate no cytotoxic anticerebral antibodies in the glia migration test, which was indicative of the safety of group B meningococcal vaccine.     

Bordetella pertussis fimbriae are effective carrier proteins in Neisseria meningitidis serogroup C conjugate vaccines

Serogroup C meningococcal conjugate vaccines generally use diphtheria or tetanus toxoids as the protein carriers. The use of alternative carrier proteins may allow multivalent conjugate vaccines to be formulated into a single injection and circumvent potential problems of immune suppression in primed individuals. Bordetella pertussis fimbriae were assessed as carrier proteins for Neisseria meningitidis serogroup C polysaccharide. Fimbriae were conjugated to the polysaccharide using modifications of published methods and characterised by size exclusion chromatography; co-elution of protein and polysaccharide moieties confirmed conjugation. The conjugates elicited boostable IgG responses to fimbriae and serogroup C polysaccharide in mice, and IgG:IgM ratios indicated that the responses were thymus-dependent. High bactericidal antibody titres against a serogroup C strain of N. meningitidis were also observed. In a mouse infection model, the conjugate vaccine protected against lethal infection with N. meningitidis. Therefore, B. pertussis fimbriae are effective carrier proteins for meningococcal serogroup C polysaccharide and could produce a vaccine to protect against meningococcal disease and to augment protection against pertussis.     

An OMV Vaccine Derived from a Capsular Group B Meningococcus with Constitutive FetA Expression: Preclinical Evaluation of Immunogenicity and Toxicity

Following the introduction of effective protein-polysaccharide conjugate vaccines against capsular group C meningococcal disease in Europe, meningococci of capsular group B remain a major cause of death and can result in debilitating sequelae. The outer membrane proteins PorA and FetA have previously been shown to induce bactericidal antibodies in humans. Despite considerable antigenic variation among PorA and FetA OMPs in meningococci, systematic molecular epidemiological studies revealed this variation is highly structured so that a limited repertoire of antigenic types is congruent with the hyperinvasive meningococcal lineages that have caused most of the meningococcal disease in Europe in recent decades. Here we describe the development of a prototype vaccine against capsular group B meningococcal infection based on a N. meningitidis isolate genetically engineered to have constitutive expression of the outer membrane protein FetA. Deoxycholate outer membrane vesicles (dOMVs) extracted from cells cultivated in modified Frantz medium contained 21.8% PorA protein, 7.7% FetA protein and 0.03 μg LPS per μg protein (3%). The antibody response to the vaccine was tested in three mouse strains and the toxicological profile of the vaccine was tested in New Zealand white rabbits. Administration of the vaccine, MenPF-1, when given by intramuscular injection on 4 occasions over a 9 week period, was well tolerated in rabbits up to 50 μg/dose, with no evidence of systemic toxicity. These data indicated that the MenPF-1 vaccine had a toxicological profile suitable for testing in a phase I clinical trial.     

Detection of blood group A-like substance in bacterial and viral vaccines by countercurrent immunoelectrophoresis using Helix pomatia lectin

The technique of countercurrent immunoelectrophoresis (CI), using the N-acetyl glucosamine-binding lectin from Helix pomatia, provided a rapid, sensitive, inexpensive, specific and reliable method for assaying blood group A-like substances in both bacterial and viral vaccines. Blood group A-like substance was detected in the pneumococcal polysaccharide vaccine manufactured by Merck Sharp & Dohme up to 1981 and in a staphylococcus vaccine ( Staphage Lysate) manufactured by Delmont Laboratories. Other US licensed vaccines, including diphtheria and tetanus toxoids, pertussis, meningococcal polysaccharide and influenza vaccines, did not contain detectable amounts of this substance. Human anti-A globulins did not provide a satisfactory reagent for the CI assay because they contained precipitating activities to the vaccine components.     

Meningococcal polysaccharide vaccines: A review

Polysaccharides produced by Neisseria meningitidis are pharmaceutically important molecules, and are the active components of vaccines against N. meningitidis serogroups A, C, W135 and Y. Effective vaccines based on capsular polysaccharide, polysaccharide conjugates and outer membrane vesicles have been developed for strains expressing capsular polysaccharides that define the sero groups A, C, Y and W135. However, conventional approaches to develop a vaccine for group B strains have been largely unsuccessful. This review focuses on the various aspects of fermentative production of meningococcal polysaccharide from N. meningitidis, methods of conjugation for improving the immunogenicity of polysaccharide vaccine, and efficient and cost effective methods for the purification of N. meningitidis capsular polysaccharide and outer membrane vesicles. In addition, different analytical techniques for the quantitative determination of polysaccharide vaccine and evaluation of structural integrity of conjugate vaccine have been described.     

Antibody studies in mice of outer membrane antigens for use in an improved meningococcal B and C vaccine

Abstract Since 1988, N. meningitidis , B:4:P1.15, ET-5 complex, has been responsible for an epidemic of meningococcal disease in Greater São Paulo, Brazil. Despite current trials to develop an effective vaccine against group B meningococci, children less than 2 years old have not been protected. It has been suggested that iron-regulated proteins (IRPs) should be considered as potential antigens for meningococcal vaccines. The vaccines under study consisted of outer-membrane vesicles depleted of lipooligosaccharide from three serogroup B strains and one serogroup C strain, IRPs, meningococcal group C polysaccharide and aluminum hydroxide. Four different protein and C polysaccharide concentrations were studied. The ELISA and bactericidal results showed a higher antibody response when 2 injections of 2.0 μg doses were administered. Despite higher IgG reactivity against antigen preparations containing IRPs seen in ELISA, the bactericidal activity was not increased if the target strain was grown in iron-restricted medium. The influence of addition of alkaline-detoxified lipooligosaccharide (dLOS) on immunogenicity of the vaccine was also investigated, and the dLOS provided for a more functionally specific antibody response.     

In vivo human immune response to transferrin-binding protein 2 and other iron-regulated proteins of Neisseria meningitidis

Abstract When grown under iron restriction, Neisseria meningitidis expresses new outer-membrane proteins, some of which are antigenic and potentially useful as vaccine components. This is particularly relevant to N. meningitidis serogroup B, against which neither polysaccharide nor conjugate vaccines are effective. We investigated recognition of N. meningitidis serogroup B outer-membrane antigens by three sera from patients recovered from meningitis. Recognition of antigens from the homologous strain provided information on in vivo expression during infection and immunogenicity, while cross-reactivity with outer membrane proteins from the other two strains and from another five strains in our collection allowed evaluation of antigenic heterogeneity. Our results demonstrate that transferrin-binding protein 2 (TBP2) is immunogenic in humans, to varying degrees depending on the strain, and that TBP2s (like the equivalent proteins of Haemophilus influenzae type b) are among the most important iron-regulated outer membrane antigens expressed during infection. Other immunogenic outer membrane proteins (some iron-regulated) are also expressed during infection; in a previous study in mouse, three of these proteins (with M _r of 50, 70 and 77 kDa) did not induce an immune response. Our cross-reactivity data provide some support for Robki et al.'s two-group classification of N. meningitidis strains, and provide evidence against the possibility that the antigenic domains shared by the TBP2s of all N . meningitidis strains induce immune responses in vivo.     

Prediction and characterization of T-cell epitopes for epitope vaccine design from outer membrane protein of Neisseria meningitidis serogroup B

Neisseria meningitidis serogroup B (MC58) is a leading cause of meningitis and septicaemia, principally infects the infants and adolescents. No vaccine is available for the prevention of these infections because the serogroup B capsular polysaccharide is unable to stimulate an immune response, due to its similarity with polysialic acid. To overcome these obstacles, we proposed to develop a peptide based epitope vaccine from outer membrane protein contained in outer membrane vesicles (OMV) based on our computational analysis. In OMV a total of 236 proteins were identified, only 15 (6.4%) of which were predicted to be located in outer membrane. The major requirement is the identification and selection of T-cell epitopes that act as a vaccine target. We have selected 13 out of 15 outer membrane proteins from OMV proteins. Due to similarity of the fkpA and omp85 with the human FKBP2 and SAMM50 protein, we removed these two sequences from the analysis as their presence in the vaccine is likely to elicit an autoimmune response. ProPred and ProPred1 were used to predict promiscuous helper T Lymphocytes (HTL) and cytotoxic T Lymphocytes (CTL) epitopes and MHCPred for their binding affinity in N. meningitidis serogroup B (MC58), respectively. Binding peptides (epitopes) are distinguished from nonbinding peptides in properties such as amino acid preference on the basis of amino acid composition. By using this dataset, we compared physico-chemical and structural properties at amino acid level through amino acid composition, computed from ProtParam server. Results indicate that porA, porB, opc, rmpM, mtrE and nspA are more suitable vaccine candidates. The predicted peptides are expected to be useful in the design of multi-epitope vaccines without compromising the human population coverage.     

B群脑膜炎球菌疫苗的研制策略

脑膜炎奈瑟菌主要引起儿童细菌性脑脊髓膜炎和败血症,有较高的发病率和病死率。现用疫苗能够控制A、C、W135和Y群脑膜炎球菌引起的感染,而由于B群荚膜多糖免疫原性弱,外膜蛋白变异性高等原因,仍无安全和具有广泛保护性的疫苗用于控制B群脑膜炎球菌的感染。目前,B群脑膜炎球菌大多已成为引起发达国家侵袭性脑膜炎疾病的主要病原体。随着研究的不断深入,B群脑膜炎球菌疫苗的研究已经取得了很大的进展,外膜囊(Out membrane vesicles,OMV)疫苗已经在控制特异性菌株爆发流行中取得了成功。然而,人们对具有广泛保护性的B群脑膜炎球菌疫苗的探索仍在继续。本文对近年来B群脑膜炎球菌基于不同型抗原疫苗的各种研制策略及其存在的问题进行了综述。     

Molecular mimetics of polysaccharide epitopes as vaccine candidates for prevention of Neisseria meningitidis serogroup B disease

Neisseria meningitidis is a major cause of meningitis and sepsis. Despite nearly 25 years of work, there is no promising vaccine candidate for prevention of disease caused by meningococcal B strains. This review summarizes newer approaches for eliciting protective meningococcal B immune responses, including the use of molecular mimetics of group B polysaccharide and conserved membrane proteins as immunogens. The capsular polysaccharide of this organism is conserved and serum antibody to this capsule confers protection against disease. However, the immunogenicity of meningococcal B polysaccharide-based vaccines is poor. Further, a portion of the antibody elicited has autoantibody activity. Recently, our laboratory produced a panel of murine monoclonal antibodies (Mabs) that react specifically with capsular polysaccharide epitopes on meningococcal B that are distinct from host polysialic acid. These Mabs elicit complement-mediated bactericidal activity and confer passive protection in animal models. The anti-capsular Mabs were used to identify molecular mimetics from phage display peptide libraries. The resulting peptides were antigenic mimetics as defined by binding to the Mabs used to select them but, to date, are poor immunogenic mimetics in failing to elicit anti-capsular antibodies.     

Meningococcal lipopolysaccharides: virulence factor and potential vaccine component.

Lipopolysaccharides (LPS) are surface components of the outer membrane of Neisseria meningitidis. Today, 12 different types of meningococcal LPS (immunotypes) are known, of which 3 are prevalent in the western world. The differences between these immunotypes are in the oligosaccharide part of the LPS molecule and consist of small differences in the oligosaccharide structure, the amount and location of phosphoethanolamine groups, and the degree of O acetylation of individual monosaccharides. Although the differences between the various immunotypes are small, they have a profound influence on the immunochemical and immunological properties of these molecules. Furthermore, each individual strain synthesizes a number of different LPS molecules. The expression of the various components (protective epitopes) is influenced by growth conditions and growth phase. Meningococci can endogenously sialyate their LPS, which constitutes one of the mechanisms by which N. meningitidis can evade the response of the human host. Meningococcal LPS play a key role in the induction of septic shock and can probably enhance the invasiveness of meningococcal strains and shield protective epitopes. Therefore, incorporation of (detoxified) LPS or oligosaccharide components derived therefrom might be very beneficial for the efficacy of a vaccine against group B meningococci. An overview of the development of vaccines against group B meningococci is given, and the status and potential of meningococcal LPS-derived (synthetic) oligosaccharide-protein conjugate vaccines are discussed.     

Antigenic cross-reactivity between outer membrane proteins of Neisseria meningitidis and commensal Neisseria species

Two mouse sera against outer membrane proteins from a pathogenic Neisseria meningitidis strain and a commensal N. lactamica strain and two human sera from patients recovering from meningococcal meningitis were used to identify antigens common to pathogenic and commensal Neisseria species. Two major antigens of 55 kDa and 32 kDa, present in all N. meningitidis and N. lactamica strains tested, were demonstrable with all the sera used; the 55-kDa protein was iron-regulated. Demonstration of other common antigens was dependent on the serum used: a 65-kDa antigen was visualised with the human and the mouse anti-N. lactamica sera; a 37-kDa antigen identified as the meningococcal ferric binding protein (FbpA) was only detected with the mouse sera, and two antigens of 83 kDa and 15 kDa were only shown with the mouse anti-N. meningitidis serum. The results demonstrate the existence of several outer membrane antigens common to N. lactamica and N. meningitidis strains, in agreement with the hypothesis that natural immunity against meningitis is partially acquired through colonisation by commensal species, and open new perspectives for the design of vaccine formulations and the development of strategies for vaccination against meningitis.     

Simple and rapid technique for monitoring the quality of meningococcal polysaccharides by high performance size-exclusion chromatography

The molecular size of meningococcal polysaccharides is an important physico-chemical parameter which correlates with immunogenicity. This paper describes the experimental conditions for high-performance size-exclusion chromatography on a PL Aquagel-OH 60 column to follow changes in the size distribution and therefore in the distribution coefficient (K(D)) of the meningococcal polysaccharides of groups A, C, Y and W-135 used to formulate anti-Neisseria meningitidis vaccines. The experimental conditions were also found to be suitable for a rapid monitoring of the quality (no group A polysaccharide depolymerization) of the tetravalent meningococcal polysaccharide vaccine.     

Evaluation of the immunological activity and safety of group B meningococcal vaccine prepared from a natural complex of specific polysaccharide and outer membrane proteins

Immunological activity and safety of group B meningococcal vaccine prepared from a natural complex of specific polysaccharide and outer membrane proteins were under study. The immunological safety of the vaccine was evaluated by the absence of antibodies to denaturated and native DNA (d-DNA and n-DNA). As shown with the use of the enzyme immunoassay (EIA), the administration of the vaccine did not induce antibody formation to d-DNA and n-DNA during the observation period. The titer of bactericidal antibodies in the immune bacteriolysis assay (IBA) to the vaccine strain B:2b:P1.2 after immunization increased four-fold and greater in 80% of the vaccinated persons. The significant increase of bactericidal antibodies to heterologous strains B:2a:P1.2 and B:15:P1.7 was registered in 20-30% of the vaccinees, respectively. A month after the repeated vaccination an increase in specific IgG antibodies to the complex antigen was found to occur according to EIA results. The use of RIB made it possible to evaluate the preventive activity of group B meningococcal vaccine as a whole and to suppose that the vaccine induced mainly type-specific response.     

The antibody response of animals to a corpuscular meningococcal group-B preparation with different methods of immunization]

As revealed in animal experiments, the formation of antibodies to group-B N. meningitidis antigens (group-specific polysaccharide, lipopolysaccharide and outer membrane proteins) in response to administration of meningococcal corpuscular preparations depends on the method of administration, the dose, and the number of administrations. In the sera of rabbits, immunized orally, antibodies to all three antigens in sufficiently high titers have been detected.     

Expression of foreign LpxA acyltransferases in Neisseria meningitidis results in modified lipid A with reduced toxicity and retained adjuvant activity

A major problem in the development of vaccines against Gram-negative bacteria is the endotoxic -activity of lipopolysaccharide (LPS), which is determined by its lipid A moiety. Nevertheless, LPS would be an interesting vaccine component because of its immune-stimulating properties. In the present study, we have changed the fatty acid composition of Neisseria meningitidis LPS by replacing the lpxA gene of strain H44/76 with the Escherichia coli or Pseudomonas aeruginosa homologue. The majority of the O-linked 3-OH C12 in N. meningitidis lipid A was replaced by 3-OH C14 (strain HA01E) and 3-OH C10 (strain HA25P) respectively. Both strains, but most notably strain HA01E, had reduced amounts of LPS compared with the wild-type strain. In addition, growth was severely impaired for HA01E. The major outer membrane proteins were expressed normally. Outer membrane complexes of both strains normalized on their LPS content showed a 10-fold reduction in their ability to induce tumour necrosis factor (TNF)-alpha. Immunogenicity studies in BALB/c mice revealed that the adjuvant activity of the LPS was not affected. Thus, the replacement of the O-linked fatty acids in meningococcal lipid A results in immunogenic outer membranes with reduced endotoxic activity, more suitable for use in outer membrane vesicle vaccines.     

Measurement of opsonophagocytic activity of antibodies specific toNeisseria meningitidis serogroup A capsular polysaccharide-serogroup B outer membrane vesicle conjugate in animal model

Neisseria meningitidis is efficiently phagocytosed by polymorphonuclear leukocytes (PMNS) following opsonization with opsonic antibodies; opsonophagocytosis is the primary mechanism for clearance of meningococci from the host. Thus, in testing meningococcal vaccines, the level of opsonophagocytic antibodies appears to correlate with vaccine-induced protection. Our previous studies demonstrated that the conjugation ofN. meningitidis serogroup A capsular polysaccharide (CPSA) to serogroup B outer membrane vesicle (OMV) could induce a high level of bactericidal antibody response against serogroup A meningococci in animals. The purpose of this study was to evaluate opsonophagocytic activity of the conjugate of CPSA to OMV (CPSA-OMV). In order to evaluate the potential efficacy of CPSA-OMV a flow cytometric opsonophagocytic assay was used. The conjugate and controls were injected intramuscularly into four groups of rabbits with boosters on days 14, 28 and 42 following primary immunization. The rabbits were bled prior to injection and two weeks after each injection. Opsonophagocytic activity of antibodies in hyperimmune sera through rabbit PMNS were measured with flow cytometer, using dihydrorhodamine-123 as a probe. The results indicated that our conjugate could induce a highly significant level of opsonophagocytic activity against serogroup A meningococci after 56 days compared to the control groups (P<0.05). We conclude that this conjugate represents a vaccine candidate against serogroups A and B meningococci after further investigation.     

Analysis of Neisseria lactamica antigens putatively implicated in acquisition of natural immunity to Neisseria meningitidis

Sera from healthy human volunteers, patients convalescent from meningococcal meningitis, and mice immunized with outer membrane proteins from Neisseria meningitidis and Neisseria lactamica strains were used to analyze and identify antigens cross-reactive to both neisserial species. All classes of meningococcal proteins except class 1 (PorA) and class 5 cross-reacted with N. lactamica proteins and two other proteins of 65 and 55 kDa (an iron-regulated protein). Results obtained with the mouse sera demonstrate that cross-reactive antibodies can be elicited by either N. meningitidis or N. lactamica. These results support the suggestion that N. lactamica contributes to the development of natural immunity against N. meningitidis during the first years of life. The use of vaccines containing proteins other than PorA could interfere in colonization of mucosal surfaces by N. lactamica, hampering the natural mechanisms of immunity acquisition in humans. Only convalescent sera reacted with the 55 and 65 kDa proteins, which suggests that they might be relevant for pathogenicity.     

Proteomic analysis of a meningococcal outer membrane vesicle vaccine prepared from the group B strain NZ98/254

In the absence of a suitable carbohydrate-based vaccine, outer membrane vesicle (OMV) vaccines have been used to disrupt outbreaks of serogroup B meningococcal disease for more than 20 years. Proteomic technology provides physical methods with the potential to assess the composition and consistency of these complex vaccines. 2-DE, combined with MS, were used to generate a proteome map of an OMV vaccine, developed to disrupt a long-running outbreak of group B disease in New Zealand. Seventy four spots from the protein map were identified including the outer membrane protein (OMP) antigens: PorA, PorB, RmpM and OpcA. Protein identification indicates that, in addition to OMPs, OMV vaccines contain periplasmic, membrane-associated and cytoplasmic proteins. 2-D-DIGE technology highlighted differences between preclinical development batches of vaccines from two different manufacturers.     

A novel monoclonal antibody to Neisseria meningitidis serogroup X capsular polysaccharide and its potential use in quantitation of meningococcal vaccines

A novel murine hybridoma monoclonal antibody (MAb) was produced against the capsular polysaccharide (CP) of Neisseria meningitidis serogroup X (MenX) in order to develop a sandwich enzyme linked immunosorbent assay (ELISA) for the quantitation of the meningococcal polysaccharide. The MAb only reacted with the CP from MenX and did not react with CPs from N. meningitidis serogroups A, C, Y and W (MenA, MenC, MenY, MenW). The affinity constant (Ka) of the MAb measured by non-competitive ELISA was 7.25 × 10⁷ M⁻¹. The application of this MAb in a sandwich ELISA was demonstrated by its ability to properly quantitate three lots of an experimental meningococcal CP-based vaccine. The MAb obtained in this work could be a valuable reagent for the detection and quantitation of future meningococcal vaccines containing MenX CP.