Carla C. A. M. Peeters

Production, characterization and control of a Neisseria meningitidis hexavalent class 1 outer membrane protein containing vesicle vaccine.

An experimental serogroup B meningococcal vaccine was prepared from two genetically engineered strains; each expressing three different class 1 outer membrane proteins (OMPs) (PorA). The two strains expressed the subtypes P1.7,16;P1.5,2;P1.19,15 and P1.5c,10;P1.12,13;P1.7h,4, respectively. Outer membrane vesicles (OMV) were prepared from these strains by deoxycholate extraction, mixed with aluminiumphosphate as adjuvant and formulated to final vaccines. The class 1 OMPs represent ca 90% of the protein in the vaccine. The vaccine was found safe for human use and induced a bactericidal immune response in mice against five of the six wild type strains, which served as donors for the various por A genes.

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.

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.

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.

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.

The history of pneumococcal conjugate vaccine development: dose selection

Pneumococcal conjugate vaccines (PCVs) differ in polysaccharide (PS) dose, carrier protein and conjugation method. PCV development proceeded initially upon principles successfully proven in Haemophilus influenzae type b (Hib) conjugate vaccine development. However, the need to successfully incorporate multiple serotypes while minimizing the total PS dose and total carrier protein load saw some early vaccine candidates fail. Dose–range studies of individual serotypes indicated that much lower PS doses were needed compared with Hib conjugate vaccines, although subsequent studies confirmed that lower Hib PS doses were possible. Furthermore, the immune response to individual serotype doses was carrier protein dependent. A ‘one-size fits most’ approach has characterized PS dose selection, but peculiarities of individual serotypes are increasingly apparent, raising the question whether re-formulation of PCVs to maximize individual serotype performance is needed.

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.

Induction of anti-pneumococcal cell wall polysaccharide antibodies by type 4 pneumococcal polysaccharide-protein conjugates

We have prepared polysaccharide-protein conjugates consisting of type 4 pneumococcal capsular polysaccharide (PS4) coupled to tetanus toxoid. The PS4 preparation used contained 2.5% pneumococcal cell wall polysaccharide (CPs). During the conjugation process, in addition to PS4-protein conjugates, CPs-protein conjugates were also formed. After immunization with PS4-protein conjugates, CPs-protein conjugates that are present as a contaminant induce IgG anti-CPs antibodies in mice. Pneumococcal oligosaccharides, prepared by periodate oxidation of PS4, did not contain detectable amounts of CPs; hence, oligosaccharide-protein conjugates did not induce anti-CPs antibodies.