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J T Poolman

Publications and source records attributed to J T Poolman.

At least 73 records · Page 4Linked to original sources
PubMed1991

In vitro induction of a Haemophilus influenzae type b polysaccharide-specific antibody response in human peripheral blood lymphocytes of individuals recently vaccinated with an oligosaccharide-protein conjugate.

In this paper an in vitro culture system for the induction of an antibody response to the Haemophilus influenzae type b polysaccharide (PRP) is described. Anti-PRP IgM and IgG antibody-secreting cells (ASC) and anti-diphtheria toxoid (DT) IgG ASC were detected in cultures of blood B and T cells derived from donors 4 to 6 wk after immunization with Haemophilus influenzae type b oligosaccharide-mutant diphtheria toxin (CRM197) conjugate (HbOC) and required in vitro restimulation with HbOC. When lymphocytes from HbOC-vaccinated donors were stimulated with PRP, anti-PRP IgM and IgG ASC could be detected in 50% offGe cases. Lymphocytes from PRP-vaccinated donors or non-vaccinated donors consistently failed to generate anti-PRP antibodies after in vitro stimulation with HbOC. Optimal in vitro responses were observed at concentrations of 0.06 to 0.6 micrograms/ml of Ag. At higher doses of Ag (6 micrograms/ml) anti-PRP and anti-DT antibody responses were suppressed. The in vitro generation of anti-PRP and anti-DT ASC, as detected by a spot-forming cell assay was shown to be T cell dependent, Ag dependent, and Ag specific. This culture system provides a model for the study of human B cell activation and immunoregulation by polysaccharide-protein conjugates and polysaccharides.

Adult↗
PubMed1991

T cell recognition of Neisseria meningitidis class 1 outer membrane proteins. Identification of T cell epitopes with selected synthetic peptides and determination of HLA restriction elements.

No vaccine is yet available against serogroup B meningococci, which are a common cause of bacterial meningitis. Some outer membrane proteins (OMP), LPS, and capsular polysaccharides have been identified as protective Ag. The amino acid sequence of the protective B cell epitopes present within the class 1 OMP has been described recently. Synthetic peptides containing OMP B cell epitopes as well as capsular polysaccharides or LPS protective B cell epitopes have to be presented to the immune system in association with T cell epitopes to achieve an optimal Ir. The use of homologous, i.e., meningococcal, T cell epitopes has many advantages. We therefore investigated recognition sites for human T cells within the meningococcal class 1 OMP. We have synthesized 16 class 1 OMP-derived peptides encompassing predicted T cell epitopes. Peptides corresponding to both surface loops and trans-membrane regions (some of which occur as amphipathic beta-sheets) of the class 1 OMP were found to be recognized by T cells. In addition, 10 of 11 peptides containing predicted amphipathic alpha-helices and four of five peptides containing T cell epitope motifs according to Rothbard and Taylor (Rothbard, J. B., and W. R. Taylor. 1988. EMBO J 7:93) were recognized by lymphocytes from one or more volunteers. Some of the T and B cell epitopes were shown to map to identical regions of the protein. At least six of the peptides that were found to contain T cell epitopes show homology to constant regions of the meningococcal class 3 OMP and the gonococcal porins PIA and PIB. Peptide-specific T cell lines and T cell clones were established to investigate peptide recognition in more detail. The use of a panel of HLA-typed APC revealed clear HLA-DR restriction patterns. It seems possible now to develop a (semi-) synthetic meningococcal vaccine with a limited number of constant T cell epitopes that cover all HLA-DR locus products.

Adult↗
PubMed1991

A comparative study of the immunogenicity of pneumococcal type 4 polysaccharide and oligosaccharide tetanus toxoid conjugates in adult mice.

A number of pneumococcal type 4 poly- and oligosaccharide tetanus toxoid conjugates were prepared using identical conjugation methods. Purified conjugates with similar m.w. were injected in mice; polysaccharide conjugates (PS4TT) were more immunogenic than oligosaccharide conjugates (OS4TT). Polysaccharide conjugates with a PS4:TT ratio less than 1 (w/w) appeared to be the most immunogenic conjugates. This was observed in both NIH (outbred) and BALB/c (inbred) strains of mice. Oligosaccharide tetanus toxoid conjugates required w/w ratios of greater than 1 to acquire optimal immunogenicity. Oligosaccharides (12 repeating units) of pneumococcal type 4, obtained by periodate cleavage, appeared to retain full antigenicity as measured by competition ELISA. Both PS4TT and OS4TT conjugates induced an antibody response with the characteristics of a T cell-dependent type of immune response. An anti-PS4 IgG and IgM booster effect could be demonstrated for all conjugates. The IgG subclass response induced by PS4TT and OS4TT conjugates is primarily IgG1 but IgG3 is also detectable. However, the amounts of anti-PS4 IgG3 differed for the various conjugates. We conclude that the immune response induced by pneumococcal type 4 saccharide tetanus toxoid conjugates can be manipulated by variation of the saccharide:protein ratio and the saccharide chain length, whereas keeping the m.w. of such conjugates at constant values.

Animals↗
PubMed1991

Point mutation in meningococcal por A gene associated with increased endemic disease.

The por A gene, which encodes expression of meningococcal class 1 outer membrane protein, responsible for antigenic subtype specificity, has been cloned and sequenced in an isolate of Neisseria meningitidis (B:15:P1.7,16) from a patient in the Gloucester area with meningococcal meningitis. Comparison of the sequence with that of the equivalent gene from the P1.7,16 reference strain reveals a point mutation which generates a single aminoacid change in the epitope responsible for P1.16 specificity. Monoclonal antibodies with P1.16 specificity do not react with synthetic peptides that correspond to the altered epitope, and do not promote complement-mediated bactericidal killing of the isolate. Analysis of other strains shows widespread distribution of infections due to B:15:P1.7,16 meningococci with the altered epitope (P1.16b) in England and Wales.

Antibodies, Monoclonal↗
PubMed1991

Characterization of Neisseria meningitidis serogroup A strains from an outbreak in France by serotype, serosubtype, multilocus enzyme genotype and outer membrane protein pattern.

In an attempt to determine the epidemiological relationship between cases of infection caused by Neisseria meningitidis serogroup A in France between August 1987 and December 1988, and an outbreak which occurred in Mecca in August 1987, markers such as serotype, serosubtype, multilocus enzyme genotype and outer membrane protein (OMP) pattern were used to characterize the bacterial isolates. From a total of 28 cases, 20 strains were isolated. Sixteen isolates, including one which undoubtedly originated from the Mecca epidemic, were homogenous (serotype 4, serosubtype P1.9, multilocus enzyme genotype of clone III-1, identical OMP pattern). Four isolates had a different OMP pattern with little or no P1 protein, no P4 and no P6. Two of these isolates belonged to other clones (I.1 and group I) and they were considered to be unrelated to the Mecca epidemic strains. The origin of the remaining two isolates was not clear. It is concluded that the majority of the Neisseria meningitidis serogroup A strains recently isolated in France originate from the Mecca epidemic.

Adolescent↗
PubMed1991

The influence of the adjuvant Quil A on the epitope specificity of meningococcal lipopolysaccharide anti-carbohydrate antibodies.

Rabbits were immunized with immunotype L3,7,9 phosphoethanolamine (PEA) group containing oligosaccharide-tetanus toxoid conjugates both with and without the addition of the adjuvant Quil A. The epitope specificity of the antibodies present in these antisera was analysed in an immunotype L2 and L3,7,9 specific inhibition ELISA using the homologous and heterologous lipopolysaccharide, oligosaccharide and partial dephosphorylated oligosaccharide as inhibitors. Two groups of antisera could be identified. In one group of antisera, at least two antibody populations are present, namely directed against the PEA group containing determinants on immunotype L3,7,9 lipopolysaccharide and against immunotype L2 specific epitopes in which no PEA group is present. In the second group of antisera, one but probably more antibody populations are detected with a similar specificity towards the conserved epitopes of both immunotypes. In general, immunization with the conjugates only resulted in the induction of antibodies against the PEA group containing epitopes on the L3,7,9 lipopolysaccharide (80%). Antibodies directed against the conserved epitopes of both immunotypes are mainly evoked with the conjugates in combination with the adjuvant Quil A (80%). Although these results suggest that the epitope specificity of the antibodies induced depends on the use of Quil A, the influence of genetic factors cannot be excluded. At the moment it is not known whether the differences in epitope specificities are reflected in biological function of these antibodies. However, the induction of antibodies with clearly different epitope specificities after immunization of different rabbits with the same antigen stresses the importance of this kind of analysis when developing a vaccine based on oligosaccharide-protein conjugates.

Animals↗
PubMed1991

Pneumococcal conjugate vaccines.

We have prepared conjugates of pneumococcal type 4 polysaccharides (PS4) or oligosaccharides to tetanus toxoid using the carbodiimide method. The use of a spacer, 6-aminohexanoic acid, resulted in higher incorporation of carrier protein. Conjugates contained up to 10% free polysaccharide, but no free protein. In general, polysaccharide conjugates induced higher anti-PS4 IgG antibody titers than oligosaccharide conjugates. Conjugates with the highest amount of incorporated protein were the most immunogenic. The response to conjugated PS4 does show characteristics of a T cell-dependent antibody response, in terms of both isotype distribution and induction of immunological memory. Repeated immunization with high doses of PS4TT conjugate resulted in a virtually negative anti-PS4 IgG response, suggestive of the induction of high dose tolerance.

Animals↗
PubMed1991

Effect of carrier priming on immunogenicity of saccharide-protein conjugate vaccines.

Previous studies with saccharide-protein conjugates have demonstrated that antibody responses to the saccharide can be improved by the preexistence of carrier immunity. Here we report that prior exposure to the carrier protein can either enhance or suppress antibody response to polysaccharides administered in saccharide-protein conjugates. A dose-dependent role for carrier priming in the antisaccharide antibody response to three saccharide-protein conjugate vaccines, i.e., a Streptococcus pneumoniae type 4 polysaccharide-tetanus toxoid (TT) conjugate (PS4TT), a Neisseria meningitidis group C polysaccharide-TT conjugate (MenCTT), and a N. meningitidis group C oligosaccharide-diphtheria mutant toxin conjugate (MenCCRM), was investigated. The results showed that an increase in the antipolysaccharide antibody response could be obtained for both PS4TT and MenCTT but not for MenCCRM with low-dose carrier priming (0.025 to 0.25 microgram). However, suppression of the antipolysaccharide antibody response was observed with the PS4TT and MenCTT vaccines with high-dose (25-micrograms) carrier priming. There was no suppression effect with MenCCRM. The increase in the antipolysaccharide antibody response was shown to be restricted to the immunoglobulin G1 (IgG1) subclass, whereas suppression with high-dose carrier priming affected all antipolysaccharide subclass antibodies induced by PS4TT (IgG1, IgG2b, and IgG3) and only two of the four subclass antibodies induced by MenCTT (IgG2a and IgG2b). The increase in the antipolysaccharide antibody response was also present at the antipolysaccharide IgM antibody level but was not observed at the anti-carrier IgG antibody level.

Animals↗
PubMed1991

Minimal oligosaccharide structures required for induction of immune responses against meningococcal immunotype L1, L2, and L3,7,9 lipopolysaccharides determined by using synthetic oligosaccharide-protein conjugates.

The 12 types of meningococcal lipopolysaccharide (LPS) (immunotypes) contain immunotype-specific and cross-reactive epitopes situated on the oligosaccharide part of the LPS molecules. To identify useful cross-reactive epitopes and to determine minimal oligosaccharide structures required for the induction of an immune response against the most prevalent immunotypes, L1, L2, and L3,7,9, synthetic as well as native LPS-derived oligosaccharides were conjugated with tetanus toxoid. L3,7,9 phosphoethanolamine (PEA) group-containing oligosaccharide-tetanus toxoid conjugates evoked high immunoglobulin G (IgG) antibody levels in rabbits which were detected by an L2-, L3,7,9-, and, depending on the antiserum, L1-specific enzyme-linked immunosorbent assay (ELISA). Inhibition studies revealed that an identical antibody population was detected by L1 and L3,7,9 ELISA, indicating a similar tertiary structure of the inner core oligosaccharide of these two immunotypes. These antibodies recognize PEA group-containing epitopes present on the L1 and L3,7,9 LPS. An L2 PEA group-containing oligosaccharide-tetanus toxoid conjugate elicited L2- and L3,7,9-specific IgG antibodies, but in contrast with the L3,7,9 conjugates, no L1-specific IgG antibodies were evoked. These results indicate that L1 and L2 LPS do not contain cross-reactive epitopes, whereas both L2 and L3,7,9 LPS and L1 and L3,7,9 LPS possess common determinants. Three linear oligosaccharides and one branched oligosaccharide, representing partial structures of the inner core oligosacchardes of meningococcal LPS, were synthesized. Only the branched synthetic oligosaccharide-containing conjugate was able to induce and L1- and L3,7,9-specific immune response, whereas the linear oligosaccharide-protein conjugates evoked L2-specific immune responses. The branched oligosaccharide (beta-D-Glcp(1----4)-[L-alpha-D-Hepp(1----3)]-L-alpha-D-Hepp ) is therefore considered a minimal structure required for the induction of an immune response against L1 and L3,7,9 LPS and part of a cross-reactive epitope between these two immunotypes. For L2-specific immune responses, oligosaccharide structures terminating in beta-D-Glcp(1----4), alpha-D-GlcNAcp(1----2), or L-alpha-D-Hepp(1----5) are needed. The results suggest that it is possible to prepare an oligosaccharide structure with the ability to evoke an immune response against L1, L2, and L3,7,9 LPS. A feasible structure for such a "hybrid" oligosaccharide is discussed.

Animals↗
PubMed1991

Preparation, characterization, and immunogenicity of meningococcal immunotype L2 and L3,7,9 phosphoethanolamine group-containing oligosaccharide-protein conjugates.

A method was developed for the well-defined coupling of phosphoethanolamine group (PEA)- and carboxylic acid group-containing polysaccharides and oligosaccharides to proteins without the need for extensive modification of the carbohydrate antigens. The carboxylic acid group of the terminal 2-keto-3-deoxyoctulosonic acid moiety was utilized to introduce a thiol function in meningococcal immunotype L2 and L3,7,9 lipopolysaccharide-derived oligosaccharides. The thiol group-containing oligosaccharides were subsequently coupled to bromoacetylated proteins. Immunotype L2 and L3,7,9 PEA group-containing oligosaccharide-tetanus toxoid conjugates were prepared, and their immunogenicities were studied in rabbits. Both the immunotype L2 and immunotype L3,7,9 conjugates evoked high immunoglobulin G (IgG) antibody titers after the first booster injection. These conjugates also displayed an ability to induce long-lasting IgG antibody levels which could be detected until 9 months after one booster injection at week 3. The adjuvant Quil A enhanced the immune response to all the conjugates to a minor extent, which is in contrast with reported adjuvant effects of Quil A on these types of antigens in mice. A conjugate prepared from the dephosphorylated L3,7,9 oligosaccharides evoked a significantly lower IgG response than a similar PEA-containing conjugate, and enzyme-linked immunosorbent assay inhibition studies indicated a different epitope specificity. Furthermore, antisera elicited with the complete bacteria contained antibodies directed against PEA-containing epitopes, which stresses the importance of the presence of unmodified PEA groups in meningococcal lipopolysaccharide-derived oligosaccharide-protein conjugates. The procedure developed offers an elegant solution for the specific coupling of meningococcal PEA-containing oligosaccharides to proteins and may therefore be a very useful tool in the development of a vaccine against group B meningococci.

Animals↗
PubMed1991

Topology of outer membrane porins in pathogenic Neisseria spp.

In Escherichia coli, membrane-spanning amphipathic beta-sheet structures are characteristic of many outer membrane proteins. By applying the principles that have been recognized for them to the four classes of neisserial porins, we have constructed a model for the topology of the porins within the outer membrane. This model predicts eight surface-exposed loops, both in the meningococcal class 1 and 2 proteins and in the gonococcal PIA and PIB proteins. The transmembrane sequences are highly conserved among these porins and are able to form an amphipathic beta-sheet structure. The surface-exposed hydrophilic loops show extensive variation in both length and sequence. Experimental evidence in support of this model has been obtained by using antisera against synthetic peptides which correspond to surface-exposed loops in class 1 and 2 proteins. Thus, binding to the cell surface was observed with antibodies against loops 1, 4, and 5 of class 1 and loops 1 and 5 of class 2. In class 1, these loops are the longest ones and show the highest sequence diversity among strains of different subtypes. Mapping of epitopes recognized by monoclonal antibodies with bactericidal activity has also provided strong support for the model. The epitopes are located in loops 1 and 4 of class 1 protein, loop 5 of PIB, and loop 6 of PIA. A nonbactericidal antibody that binds only weakly to whole cells was shown to recognize loop 3 of PIB. These results suggest that the longest loops are immunodominant, provide the binding sites for bactericidal antibodies, and display the greatest variation among different strains.

Amino Acid Sequence↗
PubMed1991

Serotypes and subtypes of Neisseria meningitidis serogroup B strains associated with meningococcal disease in Canada, 1977-1989.

Typing of Neisseria meningitidis serogroup B disease isolates was carried out using a panel of serotype-and subtype-specific monoclonal antibodies (MAbs) in enzyme-linked immunosorbent assays (ELISA). Three hundred and sixty-two strains isolated from 1977 to 1986 were typed using five serotyping and seven subtyping reagents and outer membrane vesicles as antigens. Serotype 2b accounted for 30% of the disease isolates. The most common subtype was P1.2, which occurred on 18.5% of all strains or 48.6% of the serotype 2b strains. Of the 362 strains typed, 135 (37.3%) were serotyped and 122 (33.7%) were subtyped. Overall, 185 (51.1%) of the strains could be assigned a serotype and (or) subtype. Strains (221) isolated during the years 1987-1989 were typed using a panel of 6 serotyping and 12 subtyping reagents by whole-cell ELISA. Strains of serotypes 4 (21.7%) and 15 (20.8%) were the most common and carried a wide variety of subtypes. The most common subtypes were P1.2 (11.8%) and P1.16 (9.5%). Of the 221 strains analyzed, 132 (59.7%) were assigned a serotype and 123 (55.7%) a subtype and with all 18 MAbs, 192 (86.9%) of the strains were serotyped and (or) subtyped. Two different MAbs to the four epitopes 2a, 15, P1.2, and P1.16 gave discordant reactions of 0.3, 6.6, 2.6, and 2.2%, respectively, when used to analyze over 300 strains of N. meningitidis.

Canada↗
PubMed1990

Deduced amino acid sequences of class 1 protein (PorA) from three strains of Neisseria meningitidis. Synthetic peptides define the epitopes responsible for serosubtype specificity.

The previously determined nucleotide sequence of the porA gene, encoding the class 1 outer membrane protein of meningococcal strain MC50, has been used to clone and sequence the porA gene from two further strains with differing serosubtype specificities. Comparison of the predicted amino acid sequences of the three class 1 proteins revealed considerable structural homology with major variation confined to two discrete regions (VR1 and VR2). The high degree of structural homology between the sequences gave predicted secondary structures that were almost identical, with the variable domains located in hydrophilic regions that are likely to be surface located and hence accessible to antibody binding. The predicted amino acid sequences have been used to define the epitopes recognized by mAbs with serosubtype specificity. A series of overlapping decapeptides spanning each of the class 1 protein sequences have been synthesized on solid-phase supports and probed with mAbs. Antibodies with P1.16 and P1.15 subtype specificity reacted with sequences in the VR2 domain, while antibodies with P1.7 subtype specificity reacted with sequences in the VR1 domain. Further peptides have been constructed to define the minimum epitopes recognized by each antibody. Thus we have been able to define linear peptides on each class 1 protein molecule that are responsible for subtype specificity and that represent targets for a protective immune response.

Amino Acid Sequence↗
PubMed1990

Solid-phase synthesis and applications of N-(S-acetylmercaptoacetyl) peptides.

The reagent pentafluorophenyl S-acetylmercaptoacetate was used to modify the N-terminus of resin-bound side-chain-protected peptides. The modification was carried out in an automated cycle in the final stage of fluorenylmethoxycarbonyl (Fmoc)/polyamide-mediated solid-phase synthesis. Side-chain deprotection and cleavage from the resin with aqueous trifluoroacetic acid gave the N-(S-acetylmercaptoacetyl) peptides. The S-acetylmercaptoacetyl peptides were transformed into reactive thiol-containing peptides by incubation with hydroxylamine at neutral pH. The S-deacetylation was performed in the presence of a sulfhydryl-reactive compound (or intramolecular group) to enable immediate capture of the sensitive thiol. Three applications were investigated. An S-acetylmercaptoacetyl peptide, containing a sequence of a meningococcal membrane protein, was incubated with hydroxylamine in the presence of 5-(iodoacetamido)fluorescein to give the corresponding fluorescein-labeled peptide in 62% yield. The same peptide was also S-deacetylated in the presence of bromoacetylated poly-L-lysine to afford a peptide/polylysine conjugate. Finally, a peptide corresponding to a sequence of herpes simplex virus glycoprotein D was prepared. This peptide, containing an N-terminal-S-acetylmercaptoacetyl group and an additional C-terminal S-(3-nitro-2-pyridinesulfenyl)cysteine residue, was converted into a cyclic disulfide peptide (20%).

Autoanalysis↗
PubMed1990

Characterization of outer membrane proteins from Actinobacillus actinomycetemcomitans.

Outer membranes were prepared from whole cells of various strains of Actinobacillus actinomycetemcomitans and analysed by SDS-polyacrylamide gel (12.5%) electrophoresis (SDS-PAGE). In all strains four common major outer membrane proteins (OMPs) with molecular masses of 30, 34, 36 and 39 kDa could be distinguished. Heating the OMP preparation of strain Y4 at 60, 70, 90 and 100 degrees C produced a band of 30 kDa, which gradually lost its intensity from 70 degrees C onwards concomitantly with the development of two new protein bands of 34 and 36 kDa. Furthermore, the 36 kDa OMP appeared susceptible to proteolysis by trypsin; degraded products apparently produced a new electrophoretic band of 27 kDa. Y4-derived OMP fractions were solubilized with a Triton-SDS mixture to investigate the presence of peptidoglycan-associated proteins. The 39 kDa OMP was found to be peptidoglycan-associated.

Actinobacillus↗