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ABSTRACTThe most common infections in primary immune deficiency disease (PIDD) patients involve encapsulated bacteria, mainly Haemophilus influenzaetype b (Hib) and Streptococcus pneumoniae(pneumococcus). Thus, it is important to know the titers of Hib- and pneumococcus-specific antibodies that are present in immune globulin (Ig) intravenous (IGIV) preparations used to treat PIDD. In this study, seven IGIV preparations were tested by enzyme-linked immunosorbent assay and opsonophagocytic activity for antibody titers to the capsular polysaccharides of Hib and five pneumococcal serotypes. Differences in Hib- and pneumococcus-specific antibody titer were observed among various IGIV preparations, with some products having higher- or lower-than-average titers. Opsonic activity also varied among preparations. As expected, IgG2 was the most active subclass of both binding and opsonic activity except against pneumococcal serotype 6B where IgG3 was the most active. This study determines antibody titers against capsular polysaccharides of Hib and pneumococcus in seven IGIV products that have been shown to be effective in reducing infections in PIDD patients. As donor antibody levels and manufacturing methods continue to change, it may prove useful from a regulatory point of view to reassess IGIV products periodically, to ensure that products maintain antibody levels that are important for the health of IGIV recipients.

ABSTRACTThe importance of O-acetyl groups to the immunogenicity of Neisseria meningitidisserogroup A polysaccharide (PS) was examined in studies using human sera and mouse immunization. In 17 of 18 postimmunization human sera, inhibition enzyme-linked immunosorbent assay indicated that the majority of antibodies binding to serogroup A PS were specific for epitopes involving O-acetyl groups. Studies with mice also showed an essential role for O-acetyl groups, where serum bactericidal titers following immunization with de-O-acetylated (de-O-Ac) conjugate vaccine were at least 32-fold lower than those following immunization with O-Ac PS-conjugate vaccine and 4-fold lower than those following immunization with native capsular PS. Inhibition studies using native and de-O-Ac PS confirmed the specificity of murine antibodies to native PS. The dramatic reduction in immunogenicity associated with removal of O-acetyl groups indicates that O acetylation is essential to the immunogenic epitopes of serogroup A PS. Since levels of bactericidal antibodies are correlated with protection against disease, O-acetyl groups appear to be important in protection.

Haemophilus influenzaetype b (Hib) conjugate vaccines represent a new technology wherein an immunogen is targeted to a specific immune response mechanism. Covalent attachment of the Hib polysaccharide to a protein carrier converts the T cell-independent polysaccharide antigen into a protein-like T cell-dependent antigen. The polysaccharide alone is poorly immunogenic in infants (≤12 months old), and conjugation to a protein carrier results in a protein-like antibody response to the Hib polysaccharide in infants. Conjugate vaccines induce mostly IgG antibodies and immunological memory. Later vaccination or natural exposure then induces a booster response to the Hib polysaccharide.

Immunocompromise caused by HIV-1 infection increases the importance of receipt of routine childhood vaccines to prevent infections such as invasive Haemophilus influenzaetype B (Hib) disease. The objectives of the study were to evaluate the immunogenicity of Hib conjugate vaccines among HIV-infected children according to clinical and immunologic disease progression as well as viral load.

Meningococcal groups B and C have been subdivided into a series of serotypes based upon the antigenic specificity of protein serotype antigens (STA). The purpose of these studies was to obtain the STA by gentle methods and determine its anatomic location in the meningococcal cell. The STA was extracted from group B meningococcal strains by either 0.2 M LiCl or 0.2 M CaCl2 and isolated from the extracts by gel filtration on Sepharose 6B or by pelleting the STA by centrifugation at 100,000 g. The isolated STA was a lipoprotein-lipopolysaccharide complex with a mol wt of approximately 4 x 106 daltons. Antisera prepared against the type 2 STA were bactericidal only for homologous serotype strains. The STA proved to be a constituent of the outer membrane of the cell envelope. This was shown by SDS-polyacrylamide gel electrophoresis (PAGE) of the isolated outer membrane and of the purified STA. The type 2 STA complex contains three principal proteins, one of which is predominant with a mol wt of 41,000 daltons. The type 2 STA was dissociated by Triton X-100 and separated by sucrose gradient isodensity centrifugation into two peaks. The denser peak (ρ = 1.26 g/cm3) contained the majority of the 41,000 dalton major outer membrane protein as shown by SDS-PAGE. This peak also contained the type 2 antigenic determinant. Thus the major outer membrane protein, extracted as part of a lipoprotein-lipopolysaccharide complex, contains the type 2 STA determinant.

Most of the 15 protein serotypes found in group B Neisseria meningitidishave distinct major outer membrane protein patterns when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by the Weber-Osborn system. Both serotypes 2 and 11 contain major outer membrane proteins with apparent molecular weights of 41,000 and 28,000 (41K and 28K). The 41K and 28K proteins were purified from the prototype strains of these two serotypes (M986 type 2 and M136 type 11) by preparative slab SDS-PAGE and were chemically characterized. No hexosamine was found in the purified 41K and 28K proteins. Although the two 41K proteins had similar amino acid compositions, their mobilities in Laemmli SDS-PAGE and their fragmentation patterns on SDS-PAGE after cyanogen bromide cleavage were different. The two 28K proteins differed in their amino acid composition, mobilities in Laemmli SDS-PAGE, and cyanogen bromide cleavage products. Peptide maps following chymotrypsin digestion of radioiodinated 41K and 28K proteins revealed distinct peptide maps for all four proteins. Comparison of the peptide maps of two 41K or two 28K proteins indicated that most of the unique peptides were hydrophilic, whereas most of the common peptides were hydrophobic. These results indicated that both of the 41K proteins and the 28K proteins from serotypes 2 and 11 were chemically different, although the proteins having the same molecular weights appeared to share common peptides.

Group B Neisseria meningitidishas been subdivided into 11 distinct serotypes by a sensitive bactericidal inhibition technique. The antigens responsible for induction of bactericidal type-specific antibodies were found to be extractable from the group B cells with heating at 100 C either by 0.017 nHCl in saline or by normal saline. These extracted serotype antigens were detected by a capillary precipitin test. The development of methods for extraction and assay of the serotype antigens permitted studies on their immunochemistry. The serotype antigens were distinct from the group-specific substance. Acid extracts contained abundant serotype antigen, but were devoid of group-specific substance. The identity of serotype antigens as proteins was confirmed by their sensitivity to Pronase and trypsin. The molecular weight of these antigens as estimated by G-200 Sephadex chromatography and by electrophoresis in polyacrylamide gels is in excess of 200,000 daltons. Saline extracts containing the serotype antigen could be fractionated into three distinct fractions with acetic acid: pH 4.5 and pH 3.5 precipitated fractions and a pH 3.5 supernatant fraction. The pH 4.5 precipitated fraction contained the serotype antigen.

Over 10 distinct serotypes of group B Neisseria meningitidishave been found to date by using a sensitive microbactericidal assay developed by the authors. The serotype antigens have now been extracted by hot acid or saline extraction procedures. It was found that these extracted serotype antigens may be used in a simple capillary precipitin method. This method uses unadsorbed, undiluted rabbit antisera. In the capillary precipitin method a 3+ to 4+ reaction was considered significant. The microbactericidal assay and capillary precipitin methods for serotyping group B meningococci show excellent agreement. Group B meningococci were also serotyped by an agar gel double diffusion technique. The latter technique conserves reagents and has the further advantage that it does not show the minor cross-reactions observed in the capillary precipitin method. Thus two simple, reproducible methods for serological typing of group B meningococci have been developed. These methods developed for the serological typing of group B meningococci will aid in epidemiological studies of meningococcal disease. They may also be of value for selecting suitable strains for vaccine production.

Neisseria meningitidisgroup B was subdivided into a number of different serotypes. A sensitive microbactericidal assay procedure was developed, and, with this method, over 10 different serotypes were provisionally established. The method by which distinct serotype designations were assigned is described. Almost half of the strains examined thus far contained a common serotype antigen which was distinct from the grouping antigen. Differentiation of group B meningococci into distinct types will aid in epidemiological studies and in the selection of appropriate strains for vaccines against group B meningococci.