Immunogenicity of various presentation forms of PorA outer membrane protein of Neisseria meningitidis in mice.

In this study we compare different vaccine formulations containing meningococcal PorA outer membrane protein; purified PorA, outer membrane vesicles (OMV) and immune-stimulating complexes (iscom). Bactericidal antibodies could be generated by the OMV and iscom formulation but not with purified PorA using either A1PO4 or Quil-A as adjuvant. OMV and iscom formulations revealed similar immunogenicity when tested in a dose response manner, with respect to bactericidal as well as OMV-binding antibodies. The anti-OMV IgG subclass response induced by PorA in OMV formulation was found in all subclasses IgG1, IgG2a, IgG2b, IgG3. OMP-iscoms induced very high IgG1 anti-OMV antibodies but almost no IgG3 response. Also, OMP-iscoms appeared to be a potent inducer of antibodies directed against linear peptides corresponding to surface exposed loops of PorA. In addition, iscoms as well as purified PorA with Quil-A as adjuvant (but not with A1PO4) induced high levels of antibodies against purified PorA. In summary, in addition to the OMV formulation, only iscoms containing PorA are able to generate an anamnestic and bactericidal antibody response.

In vitro cariogenicity of trans-galactosyl-oligosaccharides.

Trans-galactosyl-oligosaccharides (TOS) are a class of oligosaccharides produced by transgalactosylation of lactose. TOS are used as bifidogenic factors in human and animal nutrition. TOS can be present in the oral cavity and form a risk of caries. All oral bacteria tested were able to degrade and ferment both TOS and galactosyllactose (GLL), one of its components. Growth was improved compared with carbohydrate-free media and acid was produced after 24 h incubation of the bacteria with TOS and GLL. Degradation patterns, using HPAEC, showed degradation of most components. GLL was degraded only partially. Rapid acidification was only observed for Streptococcus mutans, resulting in a pH of 5.4 within 30 min. All other strains fermented TOS and GLL only slowly. Plaque formation could not be detected on both substrates. It can be concluded that TOS and GLL present only a small risk of caries formation, unless proven otherwise in animal studies.

Standardization and a multilaboratory comparison of Neisseria meningitidis serogroup A and C serum bactericidal assays. The Multilaboratory Study Group.

A standardized serum bactericidal assay (SBA) is required to evaluate the functional activity of antibody produced in response to Neisseria meningitidis serogroup A and C vaccines. We evaluated assay parameters (assay buffer, target strains, growth of target cells, target cell number, complement source and concentration, and methods for growth of surviving bacteria) which may affect the reproducibility of SBA titers. The various assay parameters and specificity of anticapsular antibody to five serogroup A strains (A1, ATCC 13077, F8238, F9205, and F7485) and four serogroup C strains (C11, G7880, G8050, and 1002-90) were evaluated with Centers for Disease Control and Prevention meningococcal quality control sera. The critical assay parameters for the reproducible measurement of SBA titers were found to include the target strain, assay incubation time, and complement. The resulting standardized SBA was used by 10 laboratories to measure functional anticapsular antibody against serogroup A strains F8238 and serogroup C strain C11. In the multilaboratory study, SBA titers were measured in duplicate for 14 pairs of sera (seven adults and seven children) before and after immunization with a quadrivalent polysaccharide (A, C, Y, and W-135) vaccine. The standardized SBA was reliable in all laboratories regardless of experience in performing SBAs. For most sera, intralaboratory reproducibility was +/- 1 dilution; interlaboratory reproducibility was +/- 2 dilutions. The correlation between median titers (interlaboratory) and enzyme-linked immunosorbent assay total antibody concentrations was high for both serogroup A (r = 0.86; P < 0.001; slope = 0.5) and serogroup C (n = 0.86; P < 0.001; slope = 0.7). The specified assay, which includes the critical parameters of target strain, incubation time, and complement source, will facilitate interlaboratory comparisons of the functional antibody produced in response to current or developing serogroup A and C meningococcal vaccines.

Phase I clinical trial with a hexavalent PorA containing meningococcal outer membrane vesicle vaccine.

A meningococcal outer membrane vesicle (OMV) vaccine was prepared from two production strains designed to express three serosubtype-specific class 1 outer membrane proteins or PorA. The resulting hexavalent PorA OMV vaccine contained the serosubtypes P1.7,16; P1.5,2; P1.19,15; P1.7h,4; P1.5c,10; P1.12,13 and were used to immunize adult volunteers. A single immunization with two dosages, 7.5 and 15 micrograms of the individual PorAs, was studied. The vaccine was considered safe for further use. Approximately half of the volunteers demonstrated a fourfold increase in bactericidal antibody activity against six test strains expressing the specific PorAs when given the higher dosage. This bactericidal activity was found to be directed against PorA.

Interlaboratory study evaluating quantitation of antibodies to Haemophilus influenzae type b polysaccharide by enzyme-linked immunosorbent assay.

An interlaboratory study was conducted to determine whether an enzyme-linked immunosorbent assay (ELISA) with an antigen preparation composed of various-sized fragments of Haemophilus influenzae type b polysaccharide conjugated to human serum albumin could be standardized across laboratories and whether the ELISA-derived results from different laboratories are equivalent to those obtained by the standard radioactive antigen binding assay (RABA) for quantitation of anti-H, influenzae type b polysaccharide antibodies. Twenty coded human serum samples were quantitated by ELISA in 11 laboratories and by RABA in 5 laboratories. The mean RABA-derived values served as the basis for all comparisons. While the overall correspondence of antibody values between the two methods was good, significant differences were found among some of the 11 ELISA data sets and among the mean RABA values. Seven laboratories generated higher ELISA antibody values for low-titered sera. Four laboratories generated antibody concentrations that were not statistically different between the two assay methods. The results therefore indicate that the ELISA can tolerate substantial variations in protocol, such as the use of different plates and different antibody reagents, without affecting the quantitation of serum antibodies. However, attention should be focused on low-titered sera, as some assay conditions may yield spurious results. This ELISA is a serologic assay which can serve as an alternative to the RABA for quantitation of antibodies to H. influenzae type h polysaccharide.

Polysaccharicb-conjugate vaccines.

It was recognized early this century that small molecules, called haptens, can be made immunogenic after conjugation to carrier proteins (1), This principle was thereafter applied successfully to improve the rmmunogenicity of (poly)saccharides (2, 3). We now know that the carrier proteins ensure the involvement of T-helper lymphocytes in the activation of the haptenor polysaccharide-specific antibody producing B lymphocytes (Fig. 1). In contrast to small molecules or haptens, polysaccharides (or other macromolecules with a repeating structure) are able to induce an immune response, most likely by directly activating B lymphocytes. Antigens that are able to induce an immune response without the involvement of T-helper lymphocytes are named TI (thymus independent) antigens (4) (Table 1). TI-2 antigens, such as plain polysaccharides, are not able to activate relatively immature B-cells. This is in contrast to TI-1 antigens, which can activate immature B-cells because of their mitogenic activity. Lipopolysaccharides are examples of TI-1 antigens. T-cells with specificity for saccharide structures that are recognized in association with the major histocompatibility complex (MMC) structures have never been found nor described; binding to MHC and stimulation of T-cells appears to be limited to peptides. The findings of T-cell regulation of the immune response against polysaccharides (5-7) without biochemical demonstration of the specificity of the molecular interactions can best be explained by assuming a role for antiidiotypic antibodies and T-cells.

Conjugates of synthetic cyclic peptides elicit bactericidal antibodies against a conformational epitope on a class 1 outer membrane protein of Neisseria meningitidis.

Bactericidal antibodies directed against surface loops of class 1 outer membrane proteins play a crucial role in protection against meningitis and sepsis caused by Neisseria meningitidis. So far, all efforts to obtain protective antibodies against these apparently conformational epitopes by using linear peptide analogs have been in vain. In this study, conjugates of head-to-tail cyclic peptides encompassing the predicted top of a protective surface loop were used for immunization. A series of 18 cyclic peptides with a ring size ranging from 7 to 17 residues, conjugated to tetanus toxoid, was investigated. Antipeptide and anti-whole-cell immunoglobulin G (IgG) titers elicited by the conjugates were determined. Conjugates of three peptides, containing 14, 15, and 17 amino acid residues (peptides 7, 12, and 13, respectively), induced an anti-whole-cell titer when Quillaja saponin A was used as the adjuvant. When alum was used as the adjuvant, the conjugate of peptide 12 did not elicit an anti-whole-cell response. From the Quillaja saponin A group, some of the sera obtained with conjugates of peptides 7 and 12 and all sera obtained with the peptide 13 conjugate were bactericidal in vitro. None of the sera evoked with alum as the adjuvant showed bactericidal activity. Nonbactericidal sera contained IgG1 primarily, whereas bactericidal sera showed significant titers of IgG2a and IgG2b. Class 1 protein-derived synthetic cyclic peptides which are capable of eliciting bactericidal antibodies, such as peptide 13 derived from meningococcal strain H44/76, represent potential candidates for a (semi)synthetic vaccine against meningococcal disease.

Immunization of mice with pneumolysin toxoid confers a significant degree of protection against at least nine serotypes of Streptococcus pneumoniae.

Pneumolysin is the thiol-activated cytolysin produced by Streptococcus pneumoniae. Mice were immunized with a genetically engineered toxoid version of pneumolysin, which was derived from a serotype 2 pneumococcus. The toxoid carried the mutation Trp-433-->Phe. Alum was used as the adjuvant. Immunized mice had significantly increased levels of anti-pneumolysin antibodies, principally immunoglobulin G1. Mice were challenged intraperitoneally or intranasally with 12 strains covering capsular serotypes 1 to 6, 7F, 8, and 18C. Following challenge, the survival rate and/or the time of death of nonsurvivors (survival time) was significantly greater than that of sham-immunized mice for all nine serotypes. However, differences in the degree of protection were noted between different strains. The route of challenge also appeared to influence the degree of protection. Nevertheless, the significant, albeit in some cases partial, protection provided against all nine pneumococcal serotypes supports the conclusion that pneumolysin toxoids warrant consideration for inclusion in a human vaccine.

Development, characterization, and biological properties of meningococcal immunotype L3,7,(8),9-specific monoclonal antibodies.

In this study, we characterize the properties of nine monoclonal antibodies (MAbs) that recognize meningococcal lipopolysaccharides (LPS). The following three specific MAbs that had not been described previously were elicited in BALB/c mice by using an immunotype L3,7,9 oligosaccharide-tetanus toxoid conjugate in combination with Quil A: 4D1-B3, 3A12-E1, and 4A8-B2. These MAbs reacted with L3,7,9 LPS on immunoblots and in the LPS enzyme-linked immunosorbent assay (ELISA) and recognised strains containing L3, L3,7, L8 (except 3A12-E1), or L9 LPS in the whole-cell ELISA. The six other MAbs have been described in previous studies (K. Saukkonen, M. Leinonen, H. Abdillahi, and J.T. Poolman, Vaccine 7:325-328, 1989; R.J.P.M. Scholten, B. Kuipers, H.A. Valkenburg, J. Danjert, W.D. Zollinger, and J.T. Poolman, J. Med. Microbiol., in press) and were obtained after immunization with outer membrane protein complexes containing LPS: MN15A11, MN15A8-1, MN15A17-1, MN11A11G, MN14F20-11, and MN14F21-11. MN15A11 was specific for L3,7,9 LPS and displayed properties similar to those of 3A12-E1. MN15A17-1, MN14F20-1, and MN11A11G were cross-reactive, and MN14F21-11 was specific for the L1,8 immunotype. Epitope specificities of MAbs reacting with L3,7,(8),9 strains were analyzed. MAbs 4D1-B3, 3A12-E1, and 4A8-B2 recognized phosphoethanolamine group-containing oligosaccharide-specific epitopes. MN15A11 and MN15A17-1 were probably directed against a conformational epitope, although for MN5A11 recognition of an unknown L3,7,9-specific epitope in the 2-keto-3-deoxyoctulosonic acid (KDO)-lipid A region cannot be excluded. MN15A8-1, a strongly cross-reactive MAb, recognized a determinant which included the KDO-lipid A region and the more terminal saccharides.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of nonhuman primate antibodies against Haemophilus influenzae type b polysaccharide with human antibodies in oligoclonality and in vivo protective potency.

Nonhuman primates are often used as a model for studying vaccines for humans. However, it is not always clear how closely the antibody responses in these species mimic human responses. Recent studies have characterized the human antibody response to Haemophilus influenzae type b (Hib) in great detail. In this study, we have compared the antibody response to Hib of humans with those of other primates. Studies of isoelectric points and V kappa subgroup usage show that, like humans, nonhuman primates produce oligoclonal antibodies. Also, monkey antibodies to the Hib polysaccharide are as protective as human antibodies in an in vivo model of Hib infection. Thus, we conclude that nonhuman primates produce antibodies to Hib polysaccharide that are structurally and functionally similar to human antibodies and are a good model for testing human vaccines.

Synthetic trimer and tetramer of 3-beta-D-ribose-(1-1)-D-ribitol-5-phosphate conjugated to protein induce antibody responses to Haemophilus influenzae type b capsular polysaccharide in mice and monkeys.

Synthetic oligosaccharides derived from the capsular polysaccharide (PRP) of Haemophilus influenzae type b were conjugated to carrier proteins via a thioether linkage. Conjugates were made of trimeric and tetrameric ribose-ribitol-phosphate and tetanus toxoid or diphtheria toxin. All conjugates elicited anti-PRP antibody responses with an increasing immunoglobulin G/immunoglobulin M ratio in adult mice and monkeys. Trimer conjugates elicited lower anti-PRP antibody responses compared with tetramer conjugates. Adult monkeys responded equally well to the tetrameric oligosaccharide-tetanus toxoid conjugate as to the oligosaccharide-CRM197 conjugate (HbOC), which elicits protective levels of serum antibodies in human infants after two or three injections.

In vitro activation of human T lymphocytes by Haemophilus influenzae type b capsular polysaccharides.

Polyribosilribitolphosphate (PRP), the capsular polysaccharide from Haemophilus influenzae type b, is a T-cell-independent type 2 antigen. In vitro culture of adult peripheral blood T cells with 15 micrograms/ml PRP leads to induction of interleukin-2 receptor (IL-2R) expression on up to 10% of T cells. These cells are CD4+ and carry the alpha beta T-cell receptor. PRP, at concentrations above 1-5 micrograms/ml, can also induce in vitro proliferation of both adult and neonatal T cells. We conclude that PRP acts as a human T-cell mitogen. The in vitro proliferative response as well as IL-2R expression was studied in T cells derived from adults after vaccination with native PRP, with PRP conjugated to a carrier protein, or with diphtheria toxoid. Vaccination with conjugated PRP decreased the doses of PRP required for in vitro induction of IL-2R expression and T-cell proliferation. This indicates that vaccination with PRP conjugated to a carrier protein improves the in vitro T-cell response to PRP activation.

Immunogenicity of a Streptococcus pneumoniae type 4 polysaccharide--protein conjugate vaccine is decreased by admixture of high doses of free saccharide.

In this study we report that the priming capacity of a Streptococcus pneumoniae type 4 polysaccharide-protein conjugate to booster immunizations with the native capsular polysaccharide is dose dependent. Furthermore, it is shown by admixture experiments that simultaneous administration of high doses of free saccharide (0.5-25 micrograms) of different chain lengths (varying from M(r) 1.6-120 kDa) decreases the anti-polysaccharide antibody response. Presence of low doses of saccharide (up to 10%), which are usually present in conjugates prepared by the carbodiimide coupling procedure, did not influence the anti-polysaccharide antibody response in adult and neonatal mice.

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.

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.

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.

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.