Randomised revaccination with pneumococcal polysaccharide or conjugate vaccine in asplenic children previously vaccinated with polysaccharide vaccine.

OBJECTIVE: Asplenic children are at high risk of invasive pneumococcal infection. In this group, the American Academy of Pediatrics recommends a single revaccination with the 23-valent polysaccharide vaccine (PSV23) 3-5 years after a previous PSV23 dose. Despite potential advantages, there are few data available regarding the safety and immunogenicity of the heptavalent pneumococcal conjugate vaccine (PCV7) in this population. The aim of the study was to prospectively determine and to compare, in asplenic children, the vaccine specific antibody titres against the seven serotypes included in the PCV7 after administration of one dose of PCV7 or of PSV23, 3 years or more after an initial vaccination with PSV23. PATIENTS AND METHODS: In this randomised, single-centre study, antibody titres were monitored at baseline, at 1 and 6 months after revaccination in 21 children with anatomic or functional asplenia. Response was considered as positive when there was a four-fold increase in antibody titres from baseline. RESULTS: The most frequently reported adverse events were local reactions in 7/11 of PCV7 subjects and in 5/8 of PSV23 subjects, and general reactions (loss of appetite, sleepiness) in 5/11 of PCV7 subjects and in 1/8 of PSV23 subjects; without any serious adverse events. One child in the PCV7 group had increased temperature (38.4 degrees C). At least half of the PCV7 children responded to four or five serotypes, while more than half of the PSV23 subjects responded to less than 3 serotypes (p=0.285). After 1 month, the immune response for serotype 23F was significantly greater after PCV7 vaccination than after PSV23 vaccination (p=0.036). CONCLUSIONS: PCV7 revaccination is safe and immunogenic in asplenic children previously vaccinated with PSV23, and could provide appropriate booster response in this high-risk population. The clinical repercussion on invasive pneumococcal diseases remains to be demonstrated.

Identification of a candidate vaccine peptide on the 37 kDa Schistosoma mansoni GAPDH.

A previous study performed in adolescents living in an area endemic for Schistosoma mansoni in Brazil has shown that a 37 kDa schistosome surface antigen is a selective target for antibodies in sera from those who were resistant to reinfection. This antigen was shown by molecular cloning to be the schistosome GAPDH. The aim of the present work was to assess whether peptides corresponding to GAPDH antigenic determinants could be used in a subunit vaccine. Five B cell and two T cell epitopic regions were identified on Sm37-GAPDH. One of the B cell determinants (Sm37-5, aa 268-289) is highly antigenic in human infections and antibody reactivity toward this determinant is associated with resistance to reinfection. Mice and rats immunized with Sm37-5 were partially protected against a challenge infection, indicating that this peptide can induce protective immunity. Analysis of Sm37-5 amino acid sequence indicated that this antigenic determinant is likely conserved among other pathogenic strains of schistosome (S. haematobium, S. intercalatum and S. japonicum), although it shows major amino acid differences with the corresponding human GAPDH sequence. All together these results indicate that Sm37-5 should be considered as a candidate component for an anti-schistosome subunit vaccine.

Production of Sm37-GAPDH, a major therapeutical target in human schistosomiasis.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the glycolytic metabolism and the production of energy. This probably explains why GAPDH was evidenced as a major therapeutical target in several parasitic diseases; either as a vaccine candidate or as a target for chemotherapeutic treatments. Schistosoma mansoni GAPDH (Sm37-GAPDH) is one of the main schistosome vaccine candidates. The production of recombinant Sm37-GAPDH is essential to evaluate the ability of this molecule to induce protective immunity in animals and possibly in humans. The cDNA encoding Sm37-GAPDH has been cloned and sequenced. In addition, five B cell (including the major B-cell epitope Sm35-5) and two T cell epitopes have been localized on the molecule. Different expression systems have been evaluated in respect with the production yield and the GAPDH enzymatic activity. Some of them have led to either a high production of insoluble material (E. coli) or to an inactive enzyme (Pischia pastoris). The present article describes the production setting of rSm37-GAPDH using the baculovirus-insect cell system. Large amounts of soluble rSm37-GAPDH with enzymatic activity were obtained. Most sera from individuals living in an area endemic for S. mansoni recognised the rSm37 molecule and inhibited its catalytic activity.

Induction of a protection against S. mansoni with a MAP containing epitopes of Sm37-GAPDH and Sm10-DLC. Effect of coadsorption with GM-CSF on alum.

Studies of anti-S. mansoni immunological responses in individuals living in endemic areas identified immunogens (Sm37-GAPDH and Sm10-DLC) with vaccine candidate properties. Analysis of the epitopes of these immunogens indicated that: (i) Sm37-5 is a major B-cell epitope of Sm37-GAPDH and the IgG antibody reactivity toward this determinant is associated with resistance to reinfection; (ii) Sm10-T is a T-cell epitope of the major T-cell immunogen Sm10-DLC. This led us to test a multiple antigen peptide (MAP) containing Sm37-5 and Sm10-T as an anti-schistosome vaccine. This MAP induced a significant protective immune response in mice when injected in Freund's adjuvant or coadsorbed with GM-CSF on aluminium hydroxide. In the latter case the physical link between the cytokine and the antigen via the coadsorption on alum was necessary to obtain a protective response. Results of the antibody response indicated that when the MAP and GM-CSF were coadsorbed on alum, the antibody response against the Sm10-T epitope located in the NH(2)-terminal position was significantly amplified up to 30% of the anti-Sm37-5 response.

Induction of a protective immunity against Schistosoma mansoni with ovalbumin-coupled Sm37-5 coadsorbed with granulocyte-macrophage colony stimulating factor (GM-CSF) or IL-12 on alum.

A previous study has shown that Sm37-5 is a major B cell epitope of Sm37-GAPDH. This epitope is highly antigenic in human infections and IgG antibody reactivity toward this determinant is associated with adolescent resistance to reinfection. This led us to test a synthetic peptide corresponding to Sm37-5, coupled to ovalbumin, as an anti-schistosome vaccine. Although mice injected with Sm37-5-OVA in Freund's adjuvant showed significant protection, immunization in aluminium hydroxide failed to induce protection. The adjuvant effect of cytokines (GM-CSF or IL-12) associated with the antigen on alum was investigated. With each of these two cytokines, significant reductions in the worm burden were obtained (32-38% with GM-CSF and 27% with IL-12, respectively). In addition, a reduction of the egg number trapped in the liver of immunized mice was also observed. Thus, protections were obtained with formulations that could potentially be used in humans.

Characterization of a schistosome T cell-stimulating antigen (Sm10) associated with protective immunity in humans.

Parasite antigens that are strong T cell immunogens represent potential candidates for vaccines against pathogens susceptible to T cell-mediated immunity. We have previously shown that chromatographic fractions of schistosomula extracts contain components that are major T cell immunogen(s) in natural schistosome infections in humans and might contribute to the induction of human protective immunity against this parasite. In the present study, we report on the molecular cloning and on the biochemical characterization of the active components of these fractions. The screening of a schistosomula cDNA expression library with antibodies raised against the fractions allowed the cloning of a cDNA that hybridized to a 0.56-kb mRNA of schistosomula and adult worms. This cDNA contains an open reading frame of 267 base pairs (bp) which encodes a 10-kDa polypeptide. The analysis of the cDNA sequence revealed 70% homology with the sequences of previously reported proteins of unknown function. The native molecules in the active fractions were analyzed by mass spectrometry after additional purification by reverse phase high-performance liquid chromatography (HPLC). This procedure revealed two components in the fractions of molecular mass 10383 +/- 2 Da and 10401 +/- 9 Da. Both polypeptides stimulated immune T cells and yielded tryptic peptides whose sequences matched the sequence of the cloned molecule. These two polypeptides probably correspond to different post-translationally modified forms of the polypeptide encoded by the cloned cDNA.

Identification of a major T cell immunogen in the anti-schistosome response of adult residents in an area endemic for Schistosoma mansoni.

Vaccine-induced immunity to Schistosoma mansoni infection depends on the specific priming of certain T cell subsets and on the recall of this response by natural infections months or years after vaccine administration. Thus, those schistosome proteins that activate T cells in individuals stimulated by natural infections are potential candidate vaccine antigens. In the present study, we identified and purified one such T cell-stimulating antigen and evaluated its immunological properties in subjects living in an area endemic for Schistosoma mansoni. Chromatography fractions (gel filtration, followed by ion exchange chromatography) of soluble extracts of schistosomula were screened for their ability to stimulate schistosome-specific T cell clones derived from a subject sensitized by natural infection. A fraction stimulating most clones was identified and characterized. A few nanograms of this fraction, containing a major 9-10-kDa component, stimulated the T helper cells of most adults living in an endemic area of Brazil, and was able to trigger a strong cutaneous immediate hypersensitivity reaction. In contrast, children reacted weakly to this antigen preparation both in blastogenesis and in skin tests, although they mounted a significant reaction to crude larval antigen preparations. In conclusion, this work identifies a schistosomula antigen that induces a strong T cell response in adults sensitized by natural infections. This T cell response develops gradually in children and adolescents, is apparently not restricted by the HLA haplotypes common in the study area, and allows the production of parasite-specific IgE antibodies. Thus, this T cell response has some features of the immune response that is believed to protect chronically exposed humans from reinfection.

Resistance to Schistosoma mansoni in humans: influence of the IgE/IgG4 balance and IgG2 in immunity to reinfection after chemotherapy.

The hypothesis of an association between human resistance to reinfection by the parasite Schistosoma mansoni and anti-larval immunoglobulin isotypes was tested by logistic regression in the presence of the explicative variables water contact, age, and sex. Of the seven isotypes tested (IgM, IgG1, IgG2, IgG3, IgG4, IgA, and IgE), only IgE, IgG4, and IgG2 showed an association (positive for IgE and negative for IgG2 and IgG4) with resistance to reinfection after chemotherapy. The opposite effects of IgE and IgG4 were undissociable in the analysis, indicating that these isotypes probably antagonize each other in protection. The negative association of IgG2 with resistance is consistent with the view that anti-carbohydrate antibodies may facilitate reinfection. Finally, epidemiologic and immunologic studies support the view that there is a progressive but slow development of acquired immunity in children and adolescents.

Environmental, genetic and immunological factors in human resistance to Schistosoma mansoni.

The design of programs for the control of endemies requires the knowledge of the principal factors that determine parasite transmission and infection levels in exposed populations. In the studies summarized in this article, the role of environmental and host specific factors in the infection by S. mansoni have been evaluated. It is shown that a limited number of factors actually influences infection intensity: water contacts, age, and sex were shown to account for 20 to 25% of infection variance, while 35 to 40% of it was accounted for by the effect of a major codominant gene. A remarkable fact is the important weighting (around 55% of the variance) of factors (the major gene and age) that influence human capacities of resistance. This observation strongly supports control measures aimed at increasing human resistance, such as vaccination. The effect of age on the development of resistance has now been observed in several studies on S. mansoni or S. haematobium. It is, therefore, a constant finding in schistosomiasis infections that resistance develops extremely slowly requiring a long period of exposure to the parasite and repeated infections. These studies provide strong incentives to increase efforts in the evaluation of the immune response of subjects living in endemic areas. Such evaluations are necessary to define vaccine and vaccination programs, and they are also urgently needed to evaluate the effects of chemotherapy on the development of immunity in children and adolescents, as well as on the persistence of protective immunity in adults. Immunological studies begin to provide a clearer picture of the role of acquired immunity in human protection against S. mansoni. It is increasingly clear that the slow development of resistance in children, as well as its alteration in certain age groups, are related to the maturation of parasite specific immunity and its alteration by specific immune factors. Thus, the development of resistance is associated with the maturation of IgE-dependent immunity, whereas blocking Ab may interfere in children and adolescents with the expression of full resistance. This finding raises the question as to whether a vaccine could include major allergens without triggering the well-known deleterious side effects associated with hypersensitivity reactions. The absence of such reactions in subjects with high parasite-specific IgE levels who are exposed to daily infections suggests that this may be feasible.

Strong serum inhibition of specific IgE correlated to competing IgG4, revealed by a new methodology in subjects from a S. mansoni endemic area.

A method allowing the immunopurification of human IgE from small volumes of sera with a yield close to 100% (mean = 97.8%; SEM = 0.7) has been developed. The immunopurification eluates were cleared of other class antibodies that could compete with IgE in specific assays. Immunopurification of IgE followed by specific IgE enzyme-linked immunosorbent assay (ELISA) (IMMEL) was then applied to sera of 160 individuals from an area endemic for Schistosoma mansoni. In comparison with radioimmunosorbent test (RAST) and ELISA performed on unfractionated sera, IMMEL provided the highest specific IgE signals. Furthermore, the best correlations between the specific IgE levels and either the specific basophil histamine release levels (r = 0.84; p less than 10(-4) or the anti-S. mansoni skin test values (r = 0.45; p = 10(-4)) were obtained with IMMEL. Measurement of anti-S. mansoni IgE levels in immunopurified fractions and in unfractionated sera of these 160 individuals revealed a strong serum inhibition (geometric means of 98.6% and 96.8% for the adult worms and the larvae, respectively) of the specific IgE reactivity in ELISA. This inhibition was correlated with the anti-adult worm and anti-larval IgG4 levels (r = 0.65; p less than 10(-4) and r = 0.58; p less than 10(-4), respectively). In contrast, this inhibition did not correlate with the specific IgG1, IgG2, IgG3 and IgM levels. Furthermore, the level of specific IgG4 was clearly lower than that of specific IgG1, suggesting that the major contribution of IgG4 in the competition effect is not due to higher levels but rather to a specificity spectrum close to that of the specific IgE. These results support the idea that a specific function of IgG4 in serum might be to control antigen recognition by IgE and consequently, to regulate anaphylactic reactions and IgE-mediated immunity.

Evidence for an association between human resistance to Schistosoma mansoni and high anti-larval IgE levels.

The anti-larval IgE antibody response of adolescents with high or low resistance to infection by Schistosoma mansoni was evaluated before parasitological cure with oxamniquine and over an extended post-treatment period during which the least resistant subjects regained high infections. IgE from most sera, taken at several bleeding times before and after treatment, reacted, on immunoblots, with a large number of antigens (Ag) in schistosomular tegument extract. A family of 120-165-kDa cross-reacting molecules and a 85-kDa Ag were the most prominent Ag. Some of these determinants were shown to be located on the outer tegumental membrane and to be accessible to IgE on living larvae. The comparison of IgE between the two study groups showed that IgE levels were on average six-to eightfold higher (p less than 0.01) in the sera of the most resistant adolescents whereas there was no difference in patterns of Ag recognition between study groups. In contrast to IgE, anti-larval IgG and IgM levels were either similar in both groups or higher in the least resistant subjects when these exhibited high reinfection levels. IgG that competed for the binding of IgE to larval Ag were detected in most sera and their levels were higher in the least resistant group after reinfection. Finally, the treatment had no observable long-lasting effects on the levels and on the specificity of the anti-larval IgE. Altogether, these observations can be taken as evidence supporting a role of IgE in human resistance to infection by S. mansoni.

Immunological analysis of porin polymorphism in Escherichia coli B and K-12.

Two sets of monoclonal antibodies (MoF type I and MoF type II) directed against the OmpF protein were used to analyze the immunological reactivity of the major outer membrane porins of E. coli B and K-12. All these antibodies present a specificity to the native OmpF protein. In addition, among the type II antibodies, MoF 18, 19 and 20 could recognize an epitope present on both monomeric and trimeric forms of the porin as demonstrated by immunoblotting analyses. The use of two different screening methods led to the isolation of two different sets of MoF, one specific for a native conformation accessible only on E. coli B strain and the second directed against epitopes present on OmpF of the two strains, B and K-12. These various responses are discussed in relation to the lipopolysaccharide binding to OmpF and with respect to the screening test used.

Immunopurification of human factor VIII/vWF complex from plasma.

In this study we describe a process for immunopurification of FVIII/vWF complex directly from plasma. A mAb against vWF has been selected that is able to bind, under physiologic conditions, the FVIII/vWF complex and to release it in slightly alkaline conditions while preserving its activity. After investigating the influence of solid supports and of coupling methods on the recovery of active FVIII we produced an immunoadsorbent by immobilisation of the selected mAb onto a Sephacryl S-1000 support using a benzoquinone coupling method. With this immunoadsorbent we developed a purification process directly from plasma with an excellent recovery (50%) of both FVIII and vWF activities. The product obtained is very enriched (the FVIII:C specific activity is 20 IU/mg of protein) and is stable after lyophilization.

Conformational differences of human IgE on hydrophobic and hydrophilic solid phases detected by monoclonal antibodies.

Very sensitive assays of IgE are required for determining prevalence of allergic reactions in children. In order to develop a sensitive two-site IRMA two kinds of mAb were produced. Antibodies specific for D epsilon 1 determinants were derived from immunization with a 40 kDa papain Fc fragment. They bound equally native and 56 degrees C heated IgE. D epsilon 2 specific mAb were obtained after immunization with IgE anti-D epsilon 1 complex and were selected on the basis of their inability to bind heated IgE. In a two-site assay on plastic plates, D epsilon 1 specific mAb led to the binding of IgE but always prevented further binding of anti-D epsilon 1 mAb, anti-human kappa chain mAb or allergen on bound IgE. This was not true when CNBr activated cellulose was used. The influence of the nature of the solid phase disappeared when D epsilon 2 specific mAb were coated on plastic tubes. In this case, the binding of a second mAb with identical or different fine specificity was observed. The best matched pair was E 164 (anti-D epsilon 2) on the solid phase and 6H10 (anti-D epsilon 1) as a tracer. As little as 0.2 UI/ml of IgE could be detected in a 2 hr test.

Electromicroelution and microfingerprinting of radiolabeled immunoglobulins from sodium dodecyl sulfate-polyacrylamide gels.

An apparatus for electromicroelution from gel slices is described. After 20 min 70-90% of radiolabeled molecules can be extracted from the gel and recovered in 100 mul of buffer. The extracted molecules could be analysed in SDS-PAGE after reduction or could be split to perform microfingerprints (4 cm X 4 cm). This method has been used in several studies on membrane molecules (Fc gamma receptor, surface IgD, T cell receptor).

Analysis of a fetal calf serum-induced T cell line. I. H-2 restricted growth and expression of membrane-bound and released molecules bearing 5936-idiotypic determinants.

It was previously established that a fetal calf serum-induced C57BL/6 T cell line that induces T and B cell differentiation could be kept proliferating in vitro only if cultured in the presence of irradiated syngeneic spleen cells and FCS. The present experiments were performed in order to investigate a) whether this cell line was a pure T cell line, b) whether the cells in this cell line (called line 12) were homogeneous with regard to Lyt phenotype, c) whether its growth was H-2 restricted, and d) whether line 12 cells reacted with our anti-idiotype (5936) and anti-T cell receptor allotype/isotype (6036) antisera. The results showed that line 12 consisted of T cells of Lyt 1+, 2.3- and phenotype. Its growth and proliferation was restricted to Kb and/or IAb alloantigen, and this phenomenon was observed with isolated Lyt 1+, 2.3- T cells. Line 12 cells reacted with both 5936 and 6036 antisera, and the positive cells were of Lyt 1+, 2.3- phenotype. Thus, our data indicate that Lyt 1+, 2.3- line 12 T cells interact with FCS and Kb/IAb alloantigen via receptors, which may bear 5936 and 6036 antisera-defined determinants. However, because these antisera only react with a subpopulation of Lyt 1+, 2.3- cells, proof that the same T cell has both MHC specificity and B cell idiotypic determinants will require further experimentation. 5936 and 6036 antisera-reactive molecules could be isolated from the supernatants of line 12 cells. Such molecules had characteristics similar to the 50,000 m.w. form of receptor molecules isolated from B6 anti-CBA T cell supernatants: a single chain polypeptide carrying both 5936 and 6036 antisera-defined determinants.

Rat IgD myeloma protein: cell-free translation of the delta mRNA and biochemical analysis of intracellular and membrane delta chain.

The analysis of IgD molecules produced by the tumor-derived IR 731 cell line showed that these molecules are not secreted and are only present at the cell surface as covalent dimers of 48 000 dalton delta chains. The original IR 731 tumor secreted complete IgD; thus, the disppearance of secretion in the cell line was correlated with the absence of light chain synthesis. The mRNA coding for the cell line delta chain was isolated and translated. The analysis of the translated delta chain (mol. wt.: 40 000) confirmed the absence of a region having the size of a domain as previously shown by partial amino acid sequence. The observation that the absence in delta chain of this region, which comprises most of the CH2 domain, could be a general feature of murine species is discussed.

Rabbit antisera with specificity for isolated mouse T-lymphocyte receptors.

Rabbit antibodies obtained after immunization of mouse immunoglobulin (MIg)-tolerant rabbits with B6 anti-CBA IgG and having specificity for B6 anti-CBA IgG and T-cell receptors (antiserum 5936) were used to isolate 5936-reactive molecules from B6 anti-CBA mixed lymphocyte culture supernatants. Such 5936-reactive molecules were produced by the B6 T cells, and they did not react with rabbit anti-MIg antisera. They had a mol. wt of 50,000-75,000, and were single-chain polypeptides that did not react with concanavalin A (Con A)--Sepharose. These molecules were in turn injected into rabbits, and the antisera thus obtained had the following characteristics: (1) they reacted against B6 anti-CBA T-cell receptor material but not against B6 anti-CBA IgG; (2) they reacted with about 35% of B6 (H-2b, Ig-1b) anti-CBA T cells, 25% of B6 Con A blasts and 0-10% of normal B6 T cells but not with B6 lipopolysaccharide (LPS) blasts, C3H.B10 (H-2b, Ig-1j) anti-CBA or CBA anti-B6 T cells, CBA Con A blasts or normal CBA T cells; and (3) they reacted with the same 50,000-75,000 mol. wt, T-cell-derived molecules as did antiserum 5936. The implications of these findings are discussed in relation to the nature of T-cell receptors.

Putative conformation of mouse Fc gamma-receptor.

Three cell surface molecules (m.w. = 115,000, 90,000, and 70,000) binding to the Fc portion of complexed IgG have been isolated from the murine mastocytoma line P815. Various results suggested that the 90,000 and 70,000 dalton components are generated from the 115,00 dalton molecule by spontaneous proteolytic clevages and release of 23,000 dalton fragments. It was demonstrated that these cleavages occur during cell culture and not when freshly harvested mouse spleen cells are used as an Fc gamma R cell source. The survey of the Fc gamma R molecular forms obtained from P815 and spleen cells together with their reduction products led us to conclude that the mouse Fc gamma-receptor for complexed IgG is a single chain molecule (115,000 daltons) folded into five globular subunits (m.w. eta 23,000) linked by loose connecting regions accessible to proteolytic enzymes. Three of these subunits that compose the 70,000-dalton fragment are linked by di-sulfide bonds. Furthermore, a 140,000-dalton Fc gamma-binding molecule, not identified after cell surface labeling, could be detected after internal labeling. This component could be a cytoplasmic precursor of the Fc gamma R molecule. The structural model we present here might in addition shed some light on the discrepancy that appears through the various biochemical studies performed so far on Fc gamma-receptors.