Polyosides (encapsulated bacteria)

The polysaccharide capsule which surrounds bacterial species such as Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis and Salmonella typhi is a potent virulence factor by protecting the bacteria from phagocytosis. The host responds with antibody production and specific antibodies plus complement binding to the capsule facilitate opsonization of the micro-organism, which is phagocytized and eliminated. Purified capsular polysaccharides elicit T-independent antibody responses without a memory function, but are often poorly immunogenic in infants where much of the invasive H. influenzae type b (Hib) and pneumococcal infections is seen. Therefore purified polysaccharides have found limited use as vaccines. However, covalent linkage of the capsular polysaccharide, or fractions thereof, to immunogenic carrier proteins creates glycoconjugates which are T-dependent antigens and which elicit antibodies also in infants and which prime for boosting either with the glycoconjugate or the capsular polysaccharide. In the last decade Hib glycoconjugate vaccines have been successfully introduced and in countries with very high immunization coverage the disease has been virtually eliminated and a decline of over 95% has been seen in countries with slightly lower vaccine rates. World-wide use of Hib glycoconjugate vaccines offers the possibility of elimination of invasive Hib disease. Pneumococcal (11 serotypes with coverage of approximately 85% of invasive disease), meningococcal (A, C, W 135, Y but not B) and S. typhi glycoconjugates are in advanced development and offer the prospect of being as successful as the Hib glycoconjugates.     

Control of meningococcal meningitis with meningococcal vaccines.

The development of effective meinigococcal vaccines was based upon the finding that immunity to the meningococcus was directly correlated with serum bactericidal antibodies. Purified high molecular weight capsular polysaccharides of serogroups A and C meningococci stimulated the production of humoral antibodies which had group specific bactericidal activity. In controlled field trials in Army recruits, group C polysaccharide vaccines were highly effective in preventing group C disease. Following its use as a routine immunization in recruits in October 1971 group C meningococcal disease has been almost completely eliminated from Army training centers. Group A vaccine has been field tested in Egyptian school children with great success. Group B polysaccharide has failed to induce bactericidal antibodies in humans and, therefore, new research is underway to attempt to develop a cell wall protein antigen as a vaccine against group B disease.     

Memory immune response generated in Cercopithecus aethiops against meningococcal polysaccharide serogroup C conjugate vaccine

The chemical conjugation of bacterial polysaccharide to carrier proteins has proved to be an efficient tool to improve the immunological response against these bacterial antigens. In this study, we characterized the antibody response generated in a non-human primate model against the meningococcal capsular polysaccharide serogroup C (CCPS) conjugated to the P64k protein. Similar to licensed vaccines the CCPS conjugate is able to generate a good memory immune response with antibody titers threefold higher than the free CCPS. Three different ELISA protocols were used to measure the antibody response and the importance of the coating antigen was demonstrated. The ELISA using the derivatized CCPS showed the best results and had a high correlation with the bactericidal activity. The antibodies elicited showed a high protective capacity when assayed in the infant rat protection model.     

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.     

Polysaccharides, mimotopes and vaccines for fungal and encapsulated pathogens.

Vaccination is a rational alternative to treatment for Cryptococcus neoformans infections, as these infections are currently intractable in immunocompromised (including HIV-infected) individuals. Vaccines composed of the cryptococcal capsular polysaccharide glucuronoxylomannan (GXM), the key C. neoformans virulence factor, elicit protective antibodies in mice, although deleterious antibodies can also be induced. By contrast, polysaccharides are poor immunogens in HIV-infected humans and others with B-cell defects. Peptide mimotopes of GXM can induce protective immunity to C. neoformans in mice, however, our knowledge of the mechanisms of mimotope-induced protection is incomplete and further work is needed if polysaccharide- or mimotope-based vaccines are to be used to manage C. neoformans infection.     

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.     

Vaccine development against Neisseria meningitidis

Meningococcal disease is communicable by close contact or droplet aerosols. Striking features are high case fatality rates and peak incidences of invasive disease in infants, toddlers and adolescents. Vaccine development is hampered by bacterial immune evasion strategies including molecular mimicry. As for Haemophilus influenzae and Streptococcus pneumoniae, no vaccine has therefore been developed that targets all serogroups of Neisseria meningitidis. Polysaccharide vaccines available both in protein conjugated and non‐conjugated form, have been introduced against capsular serogroups A, C, W‐135 and Y, but are ineffective against serogroup B meningococci, which cause a significant burden of disease in many parts of the world. Detoxified outer membrane vesicles are used since decades to elicit protection against epidemic serogroup B disease. Genome mining and biochemical approaches have provided astounding progress recently in the identification of immunogenic, yet reasonably conserved outer membrane proteins. As subcapsular proteins nevertheless are unlikely to immunize against all serogroup B variants, thorough investigation by surrogate assays and molecular epidemiology approaches are needed prior to introduction and post‐licensure of protein vaccines. Research currently addresses the analysis of life vaccines, meningococcus B polysaccharide modifications and mimotopes, as well as the use of N. lactamica outer membrane vesicles.     

Development and evaluation of a rapid multianalyte particle-based flow cytometric assay for the quantification of meningococcal serogroup B-specific IgM antibodies in sera for nonculture case confirmation

Serogroup-specific antibody has been shown to be present in the sera of patients recovering from meningococcal disease, and thus the detection of such antibodies may aid in the confirmation of disease. There are currently no standard methods for measuring meningococcal serogroup B-specific antibody in sera. Here, we report the development of a microsphere-based immunoassay which utilizes colominic acid from Escherichia coli 07:K1 (L):NM to detect immunoglobulin M directed against serogroup B polysaccharide. The serogroup B assay was incorporated into a multiplex assay which also detects serogroup-specific immunoglobulin M for meningococcal serogroups A, C, Y and W-135. Using the method of cross-standardization, serogroup B-specific immunoglobulin M concentrations were assigned to the standard serum CDC 1992. The assay is able to detect increases in specific immunoglobulin M concentrations from acute to convalescent phase serum from serogroup B cases, and can be utilized in conjunction with the previously developed tetraplex immunoglobulin G detection assay for serogroups A, C, Y and W-135.     

Bacterial genome variability and its impact on vaccine design

The majority of currently available successful vaccines induce host responses against antigens that are highly conserved in the targeted pathogens. The diphtheria, tetanus, and pertussis vaccines confer protection by inducing neutralizing antibodies to the conserved bacterial toxins that are the major virulence factors. The Hemophilus influenzae B vaccine induces responses to conserved epitopes in the sugar structure of the bacterial capsular polysaccharide. However, the efficacy of more recently developed vaccines is limited by antigen variation, which also presents a challenge for future vaccine development. This review will explore bacterial genome variability and its impact on vaccine development.     

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.     

Capsule phase variation in Neisseria meningitidis serogroup B by slipped-strand mispairing in the polysialyltransferase gene (siaD): correlation with bacterial invasion and the outbreak of meningococcal disease

A mechanism of capsular polysaccharide phase variation in Neisseria meningitidis is described. Meningococcal cells of an encapsulated serogroup B strain were used in invasion assays. Only unencapsulated variants were found to enter epithelial cells. Analysis of one group of capsule-deficient variants indicated that the capsular polysaccharide was re-expressed at a frequency of 10^?3. Measurement of enzymatic activities involved in the biosynthesis of the α-2,8 polysialic acid capsule showed that polysialyltransferase (PST) activity was absent in these capsule-negative variants. Nucleotide sequence analysis of siaD revealed an insertion or a deletion of one cytidine residue within a run of (dC)₇ residues at position 89, resulting in a frameshift and premature termination of translation. We analysed unencapsulated isolates from carriers and encapsulated case isolates collected during an outbreak of meningococcal disease. Further paired blood-culture isolates and unencapsulated nasopharyngeal isolates from patients with meningococcal meningitis were examined. In all unencapsulated strains analysed we found an insertion or deletion within the oligo-(dC) stretch within siaD, resulting in a frameshift and loss of capsule formation. All encapsulated isolates, however, had seven dC residues at this position, indicating a correlation between capsule phase variation and bacterial invasion and the out-break of meningococcal disease.     

Induction of group 17 specific antibodies by pneumococcal type 17F and 17A polysaccharide vaccines.

Pneumococcal capsular polysaccharide group 17 contains two distinct serotypes, 17F and 17A. Pneumococcal group 17 is present in the licensed 23 valent polysaccharide vaccines. One such vaccine contains type 17A, while the other vaccine contains type 17F. The purpose of these studies was to determine the extent of cross-protection that could be expected, as both type 17F and 17A cause disease. The antibody responses of one group of adults to a vaccine containing type 17F was compared to that of another group that received a type 17A containing vaccine. By ELISA the 17A vaccine induced more cross-reactive antibodies. Opsonophagocytic antibodies are a good predictor of protection and both vaccines induced antibodies opsonic for both 17F and 17A. We conclude that either 17F or 17A will provide similar protection against group 17 disease.     

Induction of meningococcal group B polysaccharide-specific IgG antibodies in mice by using an N-propionylated B polysaccharide-tetanus toxoid conjugate vaccine

Conjugation of the group B meningococcal polysaccharide to tetanus toxoid failed to substantially enhance its immunogenicity in mice. Therefore, additional chemical manipulation of the basic structure of the group B meningococcal polysaccharide was attempted, on the premise that a synthetically derived artificial antigen might be capable of modulating the immune response in mice to produce elevated levels of cross-reactive group B meningococcal polysaccharide-specific antibodies. To achieve this, the antigenicity of the modified polysaccharide to group B meningococcal polysaccharide-specific antibodies had to be preserved, and this criterion could only be satisfied in modifications in which the carboxylate and N-carbonyl groups of the sialic acid residues of polysaccharide remained intact. Therefore, the most successful modifications were accomplished by N-deacetylation of the group B meningococcal polysaccharide with strong base to yield a precursor that could then be N-acetylated or N-arylated with different substituents. For example, the introduction of N-propionyl groups, followed by conjugation of the resultant N-propionylated group B meningococcal polysaccharide to tetanus toxoid, yielded an antigen that when injected in mice induced in them high levels of cross-reactive group B meningococcal polysaccharide-specific IgG antibodies. The T cell dependency of this antigen was established when it was demonstrated that the levels of these B polysaccharide-specific antibodies could be significantly boosted by using both the N-propionylated- and native N-acetylated-group B meningococcal polysaccharide-tetanus toxoid conjugates.     

Elaboration of both the group W135 and group Y capsular polysccharides by a single strain of Neisseria meningitidis

A single strain (8021) of Neisseria meningitidis, isolated from a child with disseminated meningococcal disease, was found to elaborate two serogroup-specific capsular polysaccharides-Y and W135. The original isolate as well as the progeny of ten single colony sub-isolates each agglutinated with both group Y and group W135 serogrouping antisera. The capsular polysccharide of strain 8021 contained the chemical constituents of both the W135 and Y capsular polysaccharides in a ratio of about 2.5:1. The patient responded immunologically to both capsular polysaccharides with haemagglutinating antibodies. Analysis by double diffusion in agar revealed that the capsular polysaccharide of strain 8021 contained individual molecules of group W135 and group Y capsular polysaccharides as well as a mosaic molecule containing both antigenic determinants.     

Fusarium verticillioides (Saccardo) Nirenberg Associated with Hardlock of Cotton

The development of new immune potentiators for human vaccines is an important and expanding field of research. In the present study, the ability of the capsular polysaccharide from Neisseria meningitidis serogroup A (CPS-A), a mannose-containing carbohydrate, to enhance the antibody production against a co-administered model vaccine antigen, is examined. A protein-meningococcal serogroup C capsular polysaccharide (CPS-C) conjugate was selected as the model antigen for this study. After subcutaneous immunization of Balb/C mice, the conjugate mixed with CPS-A induced higher anti-CPS-C IgG and IgG_2a antibody levels and higher anti-meningococcal serogroup C bactericidal titers than the conjugate alone or mixed with CPS-C. The immuno-stimulatory properties exhibited by CPS-A and the fact that vaccines based on purified CPS-A has been safely used during decades to fight the serogroup A meningococcal disease, support the proposal to use CPS-A as immune potentiator for human vaccination studies.     

Difficulties in Establishing a Serological Correlate of Protection After Immunization with Haemophilus Influenzae Conjugate Vaccines

Abstract. in several studies the protective concentration of anti-Haemophilus infiuenzae type b (Hib) capsular polysaccharide (PS) antibodies has been concluded to be around 0·04 to 0·20 μg/ml. After the Finnish Hib polysaccharide vaccine trial it was estimated that 1 μg/ml has to be achieved to predict long term protection after vaccination. These estimates of protective anti-Hib PS antibody concentrations were based on the assumption that protection from invasive Hib disease is mediated by antibodies and the role of cell-mediated immunity is negligible. This assumption was justified since the Hib PS is a T cell-independent antigen. The matter becomes quite different when the character of the PS vaccine is altered by conjugating it to a protein carrier, so that it acquires the ability to stimulate T cells, and the immunological memory plays a role in the protection. The immunized infants are thought to be able to respond with a rapid and high antibody response after exposure to the organism. After immunization with conjugate vaccines, protection can be seen at a lower serum antibody concentration than after polysaccharide vaccine. In addition, higher avidity of anti-Hib PS antibodies is associated with the response to conjugate than PS vaccine, and there are differences between the conjugates. This might have an influence on the functional activity of the antibodies. Hib conjugate vaccines are also able to reduce the carriage rate of Hib. This should be kept in mind when estimating what is needed from protective immune response after immunization with Hib conjugate vaccines.     

Unique intermolecular bactericidal epitope involving the homosialopolysaccharide capsule on the cell surface of group B Neisseria meningitidis and Escherichia coli K1

The N-propionylated group B meningococcal polysaccharide mimics a unique bactericidal epitope on the surface of group B meningococci and Escherichia coli K1. This was confirmed when both the above organisms were able to absorb the bactericidal antibodies from a mouse-anti-N-propionylated group B meningococcal polysaccharide-tetanus toxoid conjugate serum. By using affinity columns it was possible to divide the conjugate antiserum into three distinct populations of both group B polysaccharide cross-reactive and non-cross-reactive antibodies, one of which contained most of the bactericidal activity. The cross-reactive (IgG1) antibodies were absorbed by an affinity column in which the group B polysaccharide was linked to the solid support by a long spacer arm, thereby isolating a population of non-cross-reactive (IgG1) antibodies. Surprisingly the above column also retained another population of non-cross-reactive (IgG2a) and (IgG2b) antibodies which contained most of the bactericidal activity. These latter antibodies were not absorbed by a similar group B polysaccharide-affinity column in which a short spacer arm was employed. Thus the above experiments not only effected a separation of highly bactericidal antibodies but also provided evidence that the long spacer arm is functional in the binding of the bactericidal antibodies to the affinity column. This indicates that the bactericidal epitope is mimicked by the group B polysaccharide in the presence of the long spacer arm, which supports the hypothesis that the epitope is polysaccharide-associated and is probably intermolecular in nature.     

Generation of capsule-deficient Neisseria meningitidis strains by homologous recombination

Capsule-deficient mutants of Neisseria meningitidis serogroup B strain B1940 were constructed by allelic replacement using the plasmids pMF120 and pMF121, which contain the flanking regions of the gene locus for the biosynthesis pathway of the group B meningococcal capsular polysaccharide. Southern blot analysis of chromosomal DNA of the capsule-deficient meningococcal strains confirmed the generation of large deletions in the chromosomal cps gene complex. The same strategy proved useful in constructing meningococcal strains with capsular types A, C, W135, Y and Z.     

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.     

Detection of pneumococcal antibodies in children with acute pneumonia and pleurisy by an immunoenzyme method

The samples of sera and pleural fluid from sick children have been analyzed by means of EIA techniques. To detect the time course of antibody production, the antigenic preparations of pneumococci (monovalent capsular polysaccharides, polyvalent polysaccharide vaccine and complex pneumococcal antigen) have been used. Antibody response observed in the forms of pneumococcal infection, studied in this investigation, has proved to be highly variable. It is expedient to determine antibodies to polysaccharide antigens not earlier than on days 10-12 from the beginning of the disease. But, besides the positive dynamics of antibodies, their unchanged level is sometimes observed in the patients at the beginning of the disease. As a rule, there is a coincidence between the dynamics of antibody formation in response to polysaccharide antigens and to complex pneumococcal antigen.