Antigenicity of putative phospholipid membrane-binding residues in factor VIII.

Most inhibitory antibodies to human factor VIII (fVIII) bind to epitopes in the A2, ap-A3, or C2 domains. The anticoagulant action of antibodies to the C2 domain is due to inhibition of binding of fVIII to phospholipid. The x-ray structure of the human fVIII C2 domain shows a putative hydrophobic, 3-prong, phospholipid membrane-binding site consisting of Met2199/Phe2200, Val2223, and Leu2251/Leu2252. Additionally, Lys2227, near Val2223, is part of a ring of positively charged residues that may contribute to electrostatic interaction of fVIII with negatively charged phosphatidylserine. In this study, 8 active mutants of human fVIII (Met2199Ile, Leu2252Phe, Phe2200Leu, Val2223Ala, Lys2227Glu, Met2199Ile/Phe2200Leu, Val2223Ala/Lys2227Glu, and Met2199Ile/Phe2200Leu/Val2223Ala/Lys2227Glu), which were constructed on the basis of differences between human, porcine, murine, and canine fVIII at proposed phospholipid binding sites, were expressed. The antigenicity of the mutants toward 5 C2-specific polyclonal human antibodies was measured by using the Bethesda assay. A human monoclonal anti-C2 antibody, BO2C11, and a murine C2-specific monoclonal antibody, NMC VIII-5, were also included in the analysis. In comparison with wild-type, B-domainless fVIII, the Met2199Ile, Phe2200Leu, and Leu2252 single mutants had lower antigenicity toward most of the inhibitors. In contrast, the Val2223Ala and Lys2227Glu mutants usually showed increased antigenicity. These results suggest that C2 inhibitors frequently target the Met2199/Phe2200 and Leu2251/Leu2252 beta-hairpins and are consistent with the hypothesis that these residues participate in binding to phospholipid membranes. In contrast, Val2223 and Lys2227 may oppose antibody binding sterically or through stabilization of a low-affinity membrane-binding conformation of the C2 domain.

Major T cell epitope-containing peptides can elicit strong antibody responses.

Peptides containing major T cell epitopes have the capacity to induce T cell anergy and have therefore been proposed for the treatment of allergic and autoimmune diseases. Such peptides should not be immunogenic, i. e. should not contain a B cell recognition site. We have evaluated in BALB/c mice the therapeutic potential of a 15-mer peptide (p21 - 35) derived from Der p2, a major allergen of the house dust mite Dermatophagoides pteronyssinus, which contains a dominant T cell epitope but is not recognized by antibodies to Der p2. Unexpectedly, p21 - 35 elicited strong immune responses, suggesting the presence of a cryptic B cell epitope. Similar results were obtained with mice of three additional MHC haplotypes. A core sequence of four amino acids (Ile-Ile-His-Arg) corresponding to residues 28 - 31 was shared by the B and T cell epitopes. Critical residues for B cell recognition were Arg31 and Lys33, while Ile28 was essential for T cell recognition. A Lys33Ala mutant of p21 - 35 still activated T cells but had much reduced immunogenic properties, making it a suitable alternative peptide for T cell anergy induction. Careful investigation of the immunogenic potential of peptides used to induce T cell anergy should be carried out prior to their clinical application.

Anti-idiotypic antibodies: from regulation to therapy of factor VIII inhibitors.

Evidence has recently accumulated showing that anti-idiotypic antibodies specific to anti-FVIII antibodies are present in the plasma of healthy individuals and of haemophilia A patients with or without inhibitors, where they can neutralise the FVIII inhibitory activity. Additionally, patients successfully desensitised towards FVIII have an increased production of anti-idiotypic antibodies with no significant reduction in anti-FVIII antibodies. We review here possible strategies for modulating the anti-FVIII immune response by idiotypic interactions.

Some factor VIII (FVIII) inhibitors recognise a FVIII epitope(s) that is present only on FVIII-vWF complexes.

A mild haemophilia A patient (LE) with an Arg2150His mutation in the C1 domain of the factor VIII (FVIII) light chain was shown to have anti-FVIII antibodies inhibiting wild type but not self FVIII. Polyclonal anti-FVIII antibodies of this patient were purified by affinity adsorption using recombinant FVIII (rFVIII) and/or plasma-derived FVIII-von Willebrand factor (vWF) complexes. A distinct population of antibodies was obtained that bound to FVIII-vWF complexes but not to rFVIII, indicating that an epitope was created by the association of FVIII to vWF. Such antibodies belonged to the IgG2 isotype, but the FVIII epitopes to which they bind could not be mapped with precision due to vWF dependency. Depletion experiments showed that anti-FVIII antibodies recognising FVIII-vWF complex also distinguished wildtype from mutated self FVIII, indicating that the Arg2150His mutation alters the B cell epitope formed by the association of FVIII to vWF. To determine whether the Arg2150His substitution also alters the formation of the FVIII-vWF complex, the interaction between mutated or normal FVIII with vWF was evaluated in plasma. The dissociation rate of mutated FVIII from vWF was found to be significantly increased. The presence of an Arg2150His mutation therefore results in the disappearance of a FVIII B cell epitope generated by the association of FVIII with vWF. Patients carrying such an Arg2150His mutation and receiving infusion of wild-type FVIII may therefore be at risk of developing inhibitors to allogeneic FVIII only.

Antifactor VIII antibody inhibiting allogeneic but not autologous factor VIII in patients with mild hemophilia A.

Two unrelated patients with the same Arg2150His mutation in the factor VIII (FVIII) C1 domain, a residual FVIII activity of 0.09 IU/mL, and inhibitor titres of 300 and 6 Bethesda Units, respectively, were studied. Further analysis of patient LE, with the highest inhibitor titer, showed that (1) plasma or polyclonal IgG antibodies prepared from LE plasma inhibited the activity of allogeneic (wild-type) but not of self FVIII; (2) the presence of von Willebrand factor (vWF) increased by over 10-fold the inhibitory activity on wild-type FVIII; (3) the kinetics of FVIII inhibition followed a type II pattern, but in contrast to previously described type II inhibitors, LE IgG was potentiated by the presence of vWF instead of being in competition with it; (4) polyclonal LE IgG recognized the FVIII light chain in enzyme-linked immunosorbent assay and the recombinant A3-C1 domains in an immunoprecipitation assay, indicating that at least part of LE antibodies reacted with the FVIII domain encompassing the mutation site; and (5) LE IgG inhibited FVIII activity by decreasing the rate of FVIIIa release from vWF, but LE IgG recognized an epitope distinct from ESH8, a murine monoclonal antibody exhibiting the same property. We conclude that the present inhibitors are unique in that they clearly distinguish wild-type from self, mutated FVIII. The inhibition of wild-type FVIII by LE antibody is enhanced by vWF and is associated with an antibody-dependent reduced rate of FVIIIa release from vWF.

Mechanism and kinetics of factor VIII inactivation: study with an IgG4 monoclonal antibody derived from a hemophilia A patient with inhibitor.

The development of an immune response towards factor VIII (fVIII) remains a major complication for hemophilia A patients receiving fVIII infusions. The design of a specific therapy to restore unresponsiveness to fVIII has been hampered by the diversity of the anti-fVIII antibody. Molecular analysis of the specific immune response is therefore required. To this end, we have characterized an fVIII-specific human IgG4kappa monoclonal antibody (BO2C11) produced by a cell line derived from the memory B-cell repertoire of a hemophilia A patient with inhibitor. BO2C11 recognizes the C2 domain of fVIII and inhibits its binding to both von Willebrand factor (vWF) and phospholipids. It completely inhibits the procoagulant activity of native and activated fVIII, with a specific activity of approximately 7,000 Bethesda units/mg. vWF reduces the rate of fVIII inactivation by BO2C11. The antibody-fVIII association rate constant (kass approximately 7.4 x 10(5) M-1 s-1) is eightfold lower than that for vWF-fVIII association, whereas its dissociation rate constant (kdiss < or = 1 x 10(-5) s-1) is 100-fold lower than that for the vWF-fVIII complex, which suggests that BO2C11 almost irreversibly neutralizes fVIII after its dissociation from vWF. BO2C11 is the first human monoclonal anti-fVIII IgG antibody that has been isolated and allows the study of fVIII inactivation at the molecular level.

Factor VIII immunogenicity.

The immunogenicity of factor VIII depends on the interaction of multiple parametres including host susceptibility and characteristics of the factor VIII preparations. We briefly review here the basic mechanisms by which tolerance to self is established, maintained, and possibly broken in the context of haemophilia A, with special emphasis on the severity of the disease. Reference is also made to the situation observed in healthy individuals and patients with autoantibodies to factor VIII.

Epitope-specific down-regulation of anti-allergen antibodies following injection of allergen-antibody complexes in hypersensitive patients.

Administration to allergic patients of complexes made of allergen and anti-allergen antibodies results in a reduction in the levels of specific IgE and IgG antibodies that is limited to antibodies present in the complexes. The epitope-specific nature of this reduction is demonstrated by taking advantage of the cross-reactivity between Der p I and Der f I. In addition, an increased production of anti-idiotypic antibodies is demonstrated. As such treatment significantly improves patients with allergic asthma or atopic dermatitis, it may represent a valuable alternative to conventional immunotherapy.

Allergen-sensitive atopic dermatitis is improved by injections of allergen combined with F(ab')2 fragments of specific antibodies.

Symptoms of atopic dermatitis (AD) can be provoked by exposure to airborne allergens. We have previously shown that patients hypersensitive to D. pteronyssinus (Dpt) allergens were improved by administration of complexes composed of specific antibodies and allergen, which reduce the allergen-specific immune response. We now report that similar results can be achieved by using F(ab')2 fragments of specific antibodies instead of whole antibody molecules. Eight adult patients with severe AD were included in a single-blind study. During the first 11 months patients were maintained on injections of carrier buffer alone, in an effort to evaluate the extent of spontaneous improvement. They were then treated with intradermal injections of allergen-F(ab')2 complexes made from autologous specific antibodies and Dpt allergens. The majority of the patients improved spontaneously during the summer months, with an average 30% reduction of symptoms. However, a much more pronounced improvement was observed after 3 months on active therapy, corresponding to a cumulative amount of 60 micrograms F(ab')2 and 15 micrograms allergens. The patients continued to improve over the next 5 months, showing an average 83% reduction of severity scores. The use of F(ab')2 antibody fragments reduces the risk of inducing an anti-allotypic immune response, and raises the possibility of adding adjuvants to allergen-antibody complexes and/or using specific antibodies isolated from pooled gammaglobulins.

Efficient EBV superinfection of group I Burkitt's lymphoma cells distinguishes requirements for expression of the Cp viral promoter and can activate the EBV productive cycle.

Group I Burkitt's lymphoma cell lines conditionally expressing the CD21 receptor for EBV infection were superinfected with EBV. The incoming EBV entered its normal program of gene expression, producing EBNA-2 and LMP-1 and activating the Cp EBNA promoter, but the endogenous virus in the BL lines was not induced to express EBNA-2 or transcribe RNA from Cp. LMP-1 was, however, expressed from the endogenous genome in response to superinfection. In a proportion of the superinfected Akata cells, the productive cycle antigen BZLF1 was induced and the ability of infecting virus to cause this was sensitive to inactivation by uv light. The results show that the restricted latent pattern of EBV gene expression observed in Group I BL cells is not a consequence of lack of appropriate transcription factor activity but results from inactivation of part of the viral genome, probably by methylation. Induction of BZLF1 in some of the cells also indicates a novel pathway for activation of the virus productive cycle.

Specific down-regulation of anti-allergen IgE and IgG antibodies in humans associated with injections of allergen-specific antibody complexes.

Several approaches have recently been put forward describing attempts to suppress the IgE immune response towards allergens, which is thought to be the key event in allergic diseases. In a series of clinical trials we have shown that injections of complexes made up from allergen and specific antibodies are an effective treatment for allergic bronchial asthma and atopic dermatitis. In the work presented here we have examined the humoral immunity changes associated with the use of such complexes in a group of 19 adult patients suffering from atopic dermatitis and hypersensitive to Dermatophagoides pteronyssinus (Dp), and in whom a significant clinical improvement was observed. By comparing serum samples taken prior to and after 4 months of therapy, we show that the administration of immune complexes is associated with: (i) a significant and selective reduction of IgG and IgE antibodies specific for Dp allergens; (ii) a down-regulation that affects only the antibodies present in the complexes; (iii) the induction of corresponding anti-idiotypic antibodies. To our knowledge, this is the first demonstration in humans that an anti-allergen antibody response can be down-regulated in a highly selective manner and that this is accompanied by significant clinical improvement. Moreover, the selective reduction of IgG antibodies could be of value in the treatment of some forms of auto-immune diseases.

Reduced signal transduction through glucocorticoid receptor in Burkitt's lymphoma cell lines.

Signal transduction through the glucocorticoid receptor (GR) was shown to be significantly reduced in Epstein-Barr virus (EBV)-positive group I Burkitt's lymphoma (BL) cell lines compared to human B-lymphocytes immortalized by EBV (LCLs). On the basis of hormone binding assays, nuclear DNA binding activity, and transactivation assays the response was reduced 5- to 10-fold. Direct sequence analysis of the expressed glucocorticoid receptor mRNA in two BL cell lines indicated that the phenotype did not result from mutation of the GR gene. By preparing a high-titer polyclonal antiserum against the t-1 region of the human GR, we further showed that the deficient GR response in BLs is largely reflected in reduced GR steady-state protein levels in BL cells compared to LCLs. However, the level of GR mRNA varies less between the BL cell lines and the LCLs. The Cp promoter of EBV which normally drives expression of the EBNA gene family in EBV-immortalized LCLs contains a functional glucocorticoid response element. Transfection of GR expression constructs to group I BL cells converted the GR response to approximately LCL levels both with respect to hormone binding and glucocorticoid-dependent transcription of a glucocorticoid-dependent promoter. A modest activation of EBNA-2 expression was seen in some such cell lines, suggesting that the lower GR response contributes to the down-regulation of EBNA expression observed in BL.

Significant reduction of nonspecific bronchial reactivity in patients with Dermatophagoides pteronyssinus-sensitive allergic asthma under therapy with allergen-antibody complexes.

Thirty-nine asthmatic patients hypersensitive to Dermatophagoides pteronyssinus were treated for a total of 4 yr with injections of complexes made of allergen and autologous specific antibodies. The results obtained throughout the first 2 yr of a double-blind placebo-controlled trial have been published (1) and we now report the results of such therapy during an additional 2 yr. Three groups of patients had been defined: Groups A and B were comprised of patients treated with either "higher" doses of complexes (Group A) or "lower" doses (Group B), whereas Group C received the placebo preparation. Four injections of complexes were performed during the third yr and none during the fourth yr. The clinical benefit resulting from such injections was maintained until the end of the study, whereas medication intake, especially systemic or high doses of inhaled corticosteroids, was much reduced. Skin reactivity to allergen was significantly decreased in both treated groups. Bronchial provocation tests were carried out at 1-yr intervals with either allergen or acetylcholine (ACh). Reactivity to allergen inhalation was significantly decreased at each time point. Reactivity to ACh was significantly decreased at the end of Years 3 and 4. Fifty percent of treated patients who underwent bronchial challenges lost their bronchial reactivity to the highest concentrations of both allergen and ACh. A significant improvement in the basal lung function was observed in both treated groups. The long-term effects of immunotherapy with allergen-antibody complexes in allergic asthma patients thus include reduction in nonspecific bronchial reactivity.

A novel therapy for atopic dermatitis with allergen-antibody complexes: a double-blind, placebo-controlled study.

BACKGROUND: Exposure to airborne allergens exacerbates symptoms of atopic dermatitis (AD) in hypersensitive patients. OBJECTIVE: Our purpose was to determine whether the administration of allergen-antibody complexes would improve the symptoms of AD. METHODS: Twenty-four adults with AD and hypersensitivity to Dermatophagoides pteronyssinus (Dpt) were treated in a double-blind, placebo-controlled trial by intradermal injections of complexes containing autologous specific antibodies and Dpt allergens. After 4 months, placebo-treated patients started receiving active treatment. All patients were treated for a full year. Clinical status and Dpt-specific IgG and IgE antibody levels were monitored. RESULTS: Symptoms of AD subsided within a few weeks after starting therapy, with significant reduction after 4 months in treated patients only. After 1 year, 82% of the patients exhibited a mean improvement of 83%, associated with reduction of Dpt-specific IgG antibodies. CONCLUSION: The treatment of Dpt-sensitive patients with AD by injections of allergen-antibody complexes is safe and effective in a majority of patients.

Allergen-antibody complexes in the treatment of atopic dermatitis: preliminary results of a double-blind placebo-controlled study.

Twenty-three adult patients suffering from chronic atopic dermatitis (AD) have been treated by regular injections of complexes made of D. pteronyssinus allergens and specific autologous antibodies. A double-blind placebo-controlled protocol was followed for 4 months, then the patients were treated openly to complete a full year on active therapy. Preliminary results are presented for the first 8 months. Seventy-three percent of patients improved when treated with complexes, showing a mean improvement of more than 70% after 4 months. This study suggests that injections of allergen-antibody complexes is an effective treatment of at least some forms of AD and confirms that airborne allergens are significant exacerbating factors of AD.

Allergen-antibody complexes can efficiently prevent seasonal rhinitis and asthma in grass pollen hypersensitive patients. Allergen-antibody complex immunotherapy.

We have prepared antigen-antibody complexes from grass pollen allergens and autologous specific antibodies isolated by immunoadsorption from the serum of allergic patients. These complexes were inoculated into patients in a double-blind trial to evaluate their effect on grass pollen-related rhinitis and bronchial asthma. Thirty-eight grass pollen-hypersensitive patients were allocated to three groups; patients in the first two groups were treated with antigen-antibody complexes at different ratios and dosages and were compared with the third group who received the placebo carrier buffer alone. In addition, we treated a fourth group who had already received antigen-antibody complex inoculation during the previous pollen season. Injections were given every 2 weeks during the pollen season, starting 5 weeks prior to it. Tolerance was excellent with no signs of local or systemic side effects. The treatment prevented nasal symptoms while enabling the patients to reduce antihistamine intake. Bronchial asthma was virtually absent in the treated groups even though no bronchodilators or corticosteroids had to be taken. Specific IgE antibodies did not increase during the pollen season nor did IgG "blocking" antibodies. Inoculation of allergen-antibody complexes could provide a valuable alternative for the treatment of immediate hypersensitivity to airborne allergens as it appears to be safe and rapidly efficacious. This treatment offers several advantages compared to conventional hyposensitization and is characterized by the absence of an increase in specific IgG antibodies.

Injection of allergen-antibody complexes is an effective treatment of atopic dermatitis.

Atopic dermatitis (AD) can be exacerbated by contact with airborne allergens, amongst which Dermatophagoides pteronyssinus (Dpt) appears to be potentially important. Specific IgE antibodies towards Dpt are often found in AD, and it can therefore be speculated that suppression of the production of anti-Dpt IgE might result in a significant clinical improvement. Complexes of antigen and specific antibodies have been shown to suppress the production of antibody in other systems; we report here the evaluation in an open trial of the capacity of such complexes to improve symptoms of AD. Ten adult patients were enrolled in this study. In addition to satisfying the criteria of AD, they all suffered from a severe disease (more than 20% of the body surface involved) that had been stable for at least the last 2 years. The patients had high titers of total IgE antibodies and specific anti-Dpt antibodies. Allergen-antibody complexes were prepared from Dpt allergens and an excess of autologous specific anti-Dpt antibodies obtained by immunoadsorption. The patients received regular injections of these complexes throughout 1 year, during which clinical parameters of disease intensity, percentage of body surface affected and intensity of pruritus were regularly monitored. A significant clinical improvement was obtained after 3-4 months of therapy and was maintained through the 9th month. After 1 year of treatment, 2 patients were completely free of disease, 4 had residual lesions which continued to improve and 4 patients had a partial recurrence of dermatitis.(ABSTRACT TRUNCATED AT 250 WORDS)