Immunogenicity of a hexavalent combination vaccine in rhesus monkeys.

Preclinical immunogenicity studies were conducted in rhesus monkeys to determine whether there is immune interference in the response to one or more components of a hexavalent vaccine (Hexavac) that contains antigens from Haemophilus influenzae (Hib), hepatitis B (HB), diphtheria (D), tetanus (T), acellular pertussis (aP) and inactivated polio virus (IPV). Antibody responses were measured following co-administration of the components at three separate anatomical sites or administration as a hexavalent combination in a single site. After three injections of the hexavalent vaccine, the peak antibody responses to each component of the vaccine were >100-fold above pre-immune titers and persisted at levels >10-fold above pre-immune titers at approximately 1 year. Immune interference was observed in the peak response to HB, D and pertussis toxin, but was not seen at later time points. The results indicate that the rhesus monkey model may be useful for pre-clinical evaluation of combination vaccines.

Immunogenicity and safety of a liquid combination of DTP-PRP-T [corrected] vs lyophilized PRP-T reconstituted with DTP.

The immunogenicity and safety of a combined diphtheria, tetanus, pertussis and Haemophilus influenzae type b-tetanus conjugate vaccine (DTP-PRP-T) was compared to the same combination obtained by the reconstitution of H. influenzae type b-tetanus conjugate vaccine lyophilized (PRP-T) with liquid diphtheria-tetanus-pertussis vaccine (DTP). Two hundred and sixty-two healthy infants were randomized to receive a intramuscular injection of 0.5 ml of one of the above combination vaccines at 2, 4 and 6 months of age, and a subgroup of 134 infants received a booster dose at 12 months. Serum antibody levels to each vaccine component were measured at ages 2, 6, 7, 12 and 13 months. Systemic and local reactions were assessed during the first 3 days after each injection by diary cards distributed to the parents. After the third dose and booster administered at 12 months of age, significant equivalence between the groups was observed, and the geometric mean titer of anti H. influenzae type b capsular polysaccharide (Hib-CP) antibodies were 5.9 and 32.6 micrograms ml-1 for the liquid combination group and 5.8 and 19.4 for the lyophilized group, respectively. After the third dose, anti-Hib-PC antibody levels of > or = 1.0 microgram ml-1 and 0.15 microgram ml-1 were seen in 94% and 100%, respectively, of the liquid combination group and 90 and 99%, respectively of the lyophilized group. After the booster dose, levels of > or = 1.0 microgram ml-1 were observed in 100% and 93.5% of the liquid combination group and the lyophilized combination group, respectively. Systemic and local reactions to the vaccination were generally mild and did not differ significantly between the groups. We conclude that the liquid combination of DTP-PRP-T is safe and at least as immunogenic as the lyophilized preparation. This liquid preparation, like other combined vaccines may be helpful for planning vaccination programs with a reduced number of injections.

Biochemical and immunochemical studies of proteolytic fragments of exotoxin A from Pseudomonas aeruginosa.

Limited proteolysis of Pseudomonas aeruginosa exotoxin A by four proteases (chymotrypsin, Staphylococcal serine proteinase, pepsin A and subtilisin) resulted in the formation of polypeptides having a molecular mass of approximately 25 kDa. They possessed both enzymatic activity and residual antigenicity. Their N-terminal sequence analysis showed that the different proteases cleaved exotoxin A in a very restricted area within domain Ib (amino acids 365-404). As a result, the polypeptides contained a large portion (13-34 amino acids) of domain Ib linked to the adjacent C-terminal domain III (amino acids 405-613). The major fragment derived from subtilisin cleavage, at a final yield of 35% (S-fragment; residues 392-613; 24201 Da; pI 4.7) possessed the same level of ADP-ribosyltransferase activity as uncleaved exotoxin A (by mass), and a 37-fold higher NAD-glycohydrolase activity. Polyclonal antibodies from rabbits against exotoxin A completely inhibited the ADP-ribosyltransferase activity of both exotoxin A and the S-fragment, but not the NAD-glycohydrolase activity of the S-fragment. Antibodies against the S-fragment neutralized the ADP-ribosyltransferase activity of exotoxin A. These data determine the primary proteolytic cleavage site of exotoxin A, suggest that some residues in the amino acid sequence 392-404 of exotoxin A seem to have a role in binding or positioning elongation factor 2 (EF-2) and show that antibodies recognize the EF-2-binding site but not the NAD(+)-binding site.

Molecular size characterization of bacterial capsular polysaccharide vaccines by high performance liquid chromatography.

Bacterial capsular polysaccharides are major virulence factors and some are used as vaccinal antigens. Their molecular size is an important physicochemical criterion which correlates with immunogenicity. This article describes a new application of high performance liquid chromatography (HPLC), based on molecular sieving, for such an evaluation. This HPLC method is rapid, accurate, reproducible, requires only very low amounts of product and presents good correlation with conventional gel permeation chromatography.

Serum antibody response to filamentous hemagglutinin in patients with clinical pertussis measured by an enzyme-linked immunosorbent assay.

Titers of antibodies to filamentous hemagglutinin (FHA) were determined by enzyme-linked immunosorbent assay in acute and convalescent phase serum samples from 158 patients with clinical symptoms typical of whooping-cough. In 96 of the patients the diagnosis was verified by culture. Significant changes in serum levels of IgG, IgM and/or IgA antibodies against FHA were demonstrated in 126 patients (80%). Thus, demonstration of significant changes in FHA antibody titers in serum can be used for serological diagnosis of pertussis. The results also show that high levels of IgG, IgM and/or IgA antibodies in a single serum sample suggest current pertussis infection, but if the diagnosis is based on determinations of FHA antibody titers in a single serum sample the sensitivity is low. The levels of antibody to FHA were compared with previously determined levels of antibodies to pertussis toxin. A significant antibody response against both FHA and pertussis toxin was seen in 111 patients (70%) while 147 patients (93%) developed a significant increase in antibodies against one or both antigens.

Acellular pertussis vaccines: evaluation of reversion in a nude mouse model.

An animal model has been developed to assess the safety of acellular pertussis vaccines in terms of reversion to toxicity. Adsorbed pertussis toxoid preparations, alone or combined in a DTP formulation, were administered to nude mice intraperitoneally. In parallel, groups of positive and negative control mice received pertussis toxin and buffer, respectively. The circulating white blood cells of the animals were monitored for 28 days. Mice immunized with glutaraldehyde toxoid preparations did not develop a lymphocytosis during the observation period, whereas mice immunized with an experimental formalin pertussis toxoid vaccine exhibited a high lymphocytosis six days after vaccine administration, demonstrating, in this model, a reversion of the toxoid. The nude mouse model thus appears to reveal the in-vivo reversion of pertussis toxoids and could be included in the quality control panel for the assessment of the safety of acellular pertussis vaccine.

Tetravalent (A, C, Y, W 135) meningococcal vaccine in children: immunogenicity and safety.

Tetravalent meningococcal polysaccharide vaccine (groups A, C, Y and W 135) was administered (s.c.) to 26 healthy children, aged three to 13 years old, in order to assess safety and immunogenicity of the vaccine. Side effects were very mild in all children, generally consisting of local soreness for a few hours following injection. A fourfold increase in bactericidal antibody titres was obtained for the four components in 100% of initially seronegative children. This vaccine thus appears as safe and immunogenic in children as it is in adults.

A new 23 valent pneumococcal vaccine: immunogenicity and reactogenicity in adults.

A 23 valent pneumococcal vaccine containing 25 micrograms of each polysaccharide was administered to 30 healthy adults. Adverse reactions were always mild, consisting only of local pain and erythema in most subjects. Pre- and four weeks post-vaccination sera were analysed by radioimmunoassay. Geometric mean titre (GMT) ratios were between 2.3 and 14.3 (overall mean X 5.7) according to type. A level of 300 ng of antibody N/ml, the supposedly protective level, was achieved in at least 86.6% of the subjects for all the serotypes.

Specificity profiles of the membrane-bound gamma-D-glutamyl-(L)meso-diaminopimelateendopeptidase and LD-carboxypeptidase from Bacillus sphaericus 9602.

Immunological studies on the carboxypeptidase Y mutant prel-1 of Saccharomyces cerevisiae revealed the origin of mutation in the structural gene of carboxypeptidase Y. The absence of carboxypeptidase Y has no effect on growth, even after drastic changes of growth conditions...

[Characterization and separation of exocellular gamma-D-glutamyl-(L)meso-diaminopimelate endopeptidase and LD-carboxypeptidase from Bacillus sphaericus 9602]. / Mise en évidence et séparation d'une gamma-D-glutamyl-(L)meso-diaminopimélate endopeptidase et d'une LD-carboxypeptidase exocellulaires à partir de Bacillus sphaericus 9602

Two exocellular enzymes have been characterized in the culture media of sporulating Bacillus sphaericus 9602 : a gamma-D-glutamyl-(L) meso-diaminopimelate endopeptidase and a L-lysyl-D-alanine carboxypeptidase. These two enzymes and the corresponding membrane-bound peptidases found in Bacillus sphaericus and Bacillus subtilis strains have similar activities. Their separation is described. Both enzymes were precipitated between 25 and 65 per cent (NH4)2SO4 saturation and a first chromatography was carried out on a column of DEAE-cellulose. The separation was performed by chromatography on hydroxyapatite, each enzyme was finally filtered through Ultrogel AcA 34. After separation, the endopeptidase activity and the carboxypeptidase activity increased respectively 93 and 11 fold. Both enzymes have a molecular weight near 200 000. By gel electrophoresis at pH 8.5, they were shown to have different mobilities : the carboxypeptidase is more anionic than the endopeptidase.

[Enzymatic preparation of peptides of the general type: L-Ala-D-Glu-L-mesodiaminopimelyl (L)-D (140) Ala by transfer reactions between non radioactive corresponding peptides and D-alanine (140)]. / Préparation enzymatique de peptides du type L-Ala-D-Glu (L)meso-diaminopimélyl(L)-D-Ala(14C) par réactions d'échange entre les peptides correspondants non radioactifs et la D-alanine (14C)

Membrane bound LD-transpeptidase of Streptococcus faecalis ATCC 9790 was used to catalyse transpeptidation reactions between non radioactive peptide donors of the general type: L-Ala-D-Glu (L)meso-A2pm(L)-D-Ala and D-(14C) alanine acceptor. The presence of one or two amide residues on the carboxyl groups of glutamic acid and meso-diaminopimelic acid increases the transfer reactions and subsequently the yield in radioactive peptides L-Ala-D-Glu (L)meso-A2pm(L)-D-(14C) Ala.