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The peptide TAT-I24, composed of the 9-mer peptide I24 and the TAT (48-60) peptide, exerts broad-spectrum antiviral activity against several DNA viruses. The current model of the mode of action suggests a reduction of viral entry and also a possible interaction with the viral DNA upon virus entry. To further support this model, the present study investigates the DNA binding properties of TAT-I24. DNA binding was analysed by gel retardation of a peptide-complexed DNA, fluorescence reduction of DNA labelled with intercalating dyes and determination of binding kinetics by surface plasmon resonance. Molecular dynamics simulations of DNA-peptide complexes predict high-affinity binding and destabilization of the DNA by TAT-I24. The effect on viral DNA levels of infected cells were studied by real-time PCR and staining of viral DNA by bromodeoxyuridine. TAT-I24 binds double-stranded DNA with high affinity, leading to inhibition of polymerase binding and thereby blocking of de novo nucleic acid synthesis. Analysis of early steps of virus entry using a bromodeoxyuridine-labelled virus as well as quantification of viral genomes in the cells indicate direct binding of the peptide to the viral DNA. Saturation of the peptide with exogenous DNA can fully neutralize the inhibitory effect. The antiviral activity of TAT-I24 is linked to its ability to bind DNA with high affinity. This mechanism could be the basis for the development of novel antiviral agents.

According to the network theory antibodies may act as antigens thus generating anti-idiotypic antibodies that can function as regulators of immune responses. Designed ankyrin repeat proteins (DARPins) are a new class of binding proteins and may serve as an alternative to antibodies. Selections from large DARPin libraries against the variable regions of a murine monoclonal anti-human IgE antibody, termed BSW17, yielded two highly specific anti-idiotypic DARPins both with high affinity. Their binding characteristics were comparable with these of a previously selected anti-idiotypic antibody. In vitro cell assays showed that the anti-idiotypic DARPins were able to inhibit the binding of BSW17 to cell-bound IgE and prevented BSW17 functional activity. These experiments demonstrate the possibility to isolate anti-idiotypic DARPins recognizing idiotypic determinants analogous to antibodies. In the future these DARPins may be further analyzed for their potential as putative vaccine candidates.

Background: Intradermal injection of autologous serum elicits a wheal-and-flare response in about 60% of patients with chronic idiopathic urticaria (CIU). This reactivity has been attributed to the presence of IgG autoantibodies directed against IgE or the α-chain of the high-affinity IgE receptor (FcϵRIα) expressed on basophils and mast cells, leading to the hypothesis that at least some forms of CIU could be sustained by an autoimmune process. Objectives: The aim of this study was to investigate the relationship between the presence of anti-IgE or anti-FcϵRI antibodies and the ability to induce wheal-and-flare responses in CIU sera selected for the capacity to give a positive skin test response. Methods: Fifteen patients with CIU and a positive skin test response to autologous serum were injected intradermally with native serum and with serum heated at 56°C for 30 minutes and then adsorbed on Sepharose-protein G to obtain IgG depletion. Serum levels of anti-IgE and anti-FcϵRIα antibodies were measured by ELISA by using purified IgE and recombinant RIα-soluble double-fusion protein RIα-human serum albumin-RIα, respectively. The histamine-releasing activity of sera was tested by using ELISA with whole human blood from a healthy donor. Results: All patients had positive cutaneous responses to native serum injection. Anti-FcϵRIα antibodies were present in 14 of 15 native sera, only two of which were able to induce in vitro basophil degranulation. On the contrary, detectable amounts of anti-IgE antibodies were not found in any serum. IgG depletion by protein G resulted in complete (10/14 samples) or considerable (4/14 samples) removal of anti-FcϵRIα antibodies. The two sera endowed with functional activity lost their capacity to trigger histamine release from basophils after heating and protein G adsorption. Nonetheless, heat-decomplemented/IgG-depleted sera elicited wheal-and-flare reactions comparable with those observed with untreated sera. Conclusions: These results strongly suggest that skin reactivity to autologous serum could be due to as yet unidentified non–Ig reactants present in the sera of patients with CIU. (J Allergy Clin Immunol 2000;106:567-72.)

IgE Abs mediate allergic responses by binding to specific high affinity receptors (FcεRI) on mast cells and basophils. Therefore, the IgE/FcεRI interaction is a target for clinical intervention in allergic disease. An anti-IgE mAb, termed BSW17, is nonanaphylactogenic, although recognizing IgE bound to FcεRI, and interferes with binding of IgE to FcεRI. Thus, BSW17 represents a candidate Ab for treatment of IgE-mediated disorders. By panning BSW17 against random peptide libraries displayed on phages, we defined mimotopes that mimic the conformational epitope recognized on human IgE. Two types of mimotopes, one within the Cε3 and one within the Cε4 domain, were identified, indicating that this mAb may recognize either a large conformational epitope or eventually two distinct epitopes on IgE. On the basis of alignments of the two mimotopes with the human IgE sequence, we postulate that binding of BSW17 to the Cε3 region predominantly blocks binding of IgE to FcεRI, leading to neutralization of IgE. Moreover, binding of BSW17 to the Cε4 region may explain how BSW17 recognizes FcεRI-bound IgE, and binding to this region may also interfere with degranulation of IgE sensitized cells (basophils and mast cells). As a practical application of these findings, mimotope peptides coupled to a carrier protein may be used for the development of a peptide-based anti–allergy vaccine by induction of anti-IgE Abs similar to the current approach of using humanized nonanaphylactogenic anti-IgE Abs as a passive vaccine.

According to Jerne’s network hypothesis, the binding site of an anti-idiotypic antibody also represents the internal image of an epitope present on a foreign, or even a self antigen. In recent years, antigen mimicry has been defined at the molecular level for some xeno-antigens. However, until now there has been no demonstration of structural mimicry between a human anti-idiotypic antibody and a self structure. To address this question, we used human IgE as the self structure and a well-defined anti-human IgE mAb (BSW17). We describe the isolation of two anti- idiotypic antibodies specific for the anti-IgE antibody BSW17 from a non-immune human Fab phage display library. Interestingly, these two anti-idiotypic antibodies mimic the same molecular surface region as a previously described IgE peptide mimotope isolated by panning on BSW17, but they cover a much larger epitope on the IgE molecule. Accordingly, immunisation of rabbits with the two anti-idiotypic antibodies induced high-affinity antibodies with the same characteristics as BSW17. Thus, our data demonstrate that it is possible to isolate anti-idiotypic antibodies derived from the human genome without the need for hyperimmunisation, and confirm Jerne’s hypothesis that both foreign antigens and self structures can be mimicked by our own immunoglobulins.

Background: Binding of human IgE via the heavy–chain constant region domain 3 (Cε3) to the α–chain of its high affinity receptor (FcεRIα) is a key event in mediating allergic reactions. We wanted to identify epitopes within Cε3 that are stable to denaturation and to evaluate whether such structures are involved in receptor binding. The existence of stable epitopes would facilitate the generation of compounds that inhibit the IgE–FcεRIα interaction. Methods: Monoclonal anti–human IgE–antibodies against recombinant bacterially synthesized Cε3, which is known to be partly misfolded, were raised in mice. These antibodies were probed for binding to native, immobilized and receptor–bound IgE, respectively, providing tools for the identification of the indicated stable epitopes. Results: Two of the generated antibodies (8E7, 3G9) discriminate between IgE in solution and IgE attached to FcεRIα, pointing towards a steric rearrangement within Cε3 induced upon receptor binding. The described antibodies represent tools for studying the mechanism of the Fcε–FcεRIα interaction and may be of diagnostic value since serum IgE from various human donors was differently recognized by 8E7, which is indicative for naturally occurring IgE molecules with different steric conformation. Conclusion: The presented data support the hypothesis of a conformational change within IgE Cε3 upon receptor binding by showing that monoclonal antibodies raised against recombinant Cε3 differently recognize soluble and receptor–bound IgE. The presence of an IgE portion in sera of human donors that is recognized by 8E7 indicates the existence of IgE molecules in different steric conformations in human blood, which may be related to pathologic parameters.

Injection of mice with purified goat anti-mouse IgD (GAMD) leads to an interleukin (IL)-4-dependent increase of serum IgE levels. Challenge of GAMD-primed mice with goat IgG (GIG) initiates a secondary immune response with elevated serum IgE. In this report, kinetic cytokine transcript profiles of murine lymphoid tissues in response to primary i.v. GAMD treatment, as well as GIG challenge are presented. For the first time, gene transcription patterns of the recently described cytokines IL-12 and IL-13 are shown and compared with the corresponding patterns for other cytokine genes involved in IgE regulation, i.e. IL-4, and interferon (IFN)-gamma. After GAMD injection, two groups of induction profiles were observed in spleen, mesenteric lymph nodes and Peyer's patches: while IL-4 and IL-12p35 gene transcription was strongly enhanced, IFN-gamma, IL-12p40 and IL-13 mRNA were only moderately induced. Generally, maximal mRNA induction was measured on days 3 to 4 after GAMD treatment. The data demonstrate a clear-cut difference between the IL-4 and IL-13 response on the transcriptional level although both gene products show similar biological activities. The cytokine mRNA profiles support the assumption of IL-4 playing the central role in generating an IgE response. However, they do not reflect a strict Th1 versus Th2 cytokine gene transcription pattern but rather point towards a concerted action of various, partially antagonizing cytokines with respect to the regulation of IgE synthesis. IL-12 may, possibly via stimulation of IFN-gamma synthesis, represent a counterbalancing factor in the IL-4-mediated IgE response.

Modulation of IgE isotype expression on B cells is one of the numerous effects of muramyl peptides on the regulation of the immune system. A non toxic diacyl glycerol derivative of muramyl dipeptide (MDP), in which the l -alanine is replaced by l -threonine (MDP-Threo-GDP; SDZ 280.636), is currently under investigation as lead compound for the development of an anti-allergic drug. In this report, the modulatory effect of orally administered SDZ 280.636 in a murine model on polyclonally induced IgE levels is described. In this model, mice are injected i.v. with goat anti mouse IgD (GAMD) and challenged three to four weeks later with goat IgG (GIG). Both the primary and secondary immune responses lead to an increase of serum IgE levels. We demonstrate the efficacy of this muramyl dipeptide derivative in selectively inhibiting a polyclonal IgE response in GAMD-primed, GIG challenged mice without affecting the levels of other immunoglobulin classes. It is further shown that the induction of interleukin 4 (IL-4) gene transcript levels in lymphoid organs, which is observed as a consequence of boosting GAMD pretreated mice with GIG, is selectively suppressed in gut associated lymphoid tissues (GALT) and mesenteric lymph nodes but not in spleen. In contrast, interleukin 13 (IL-13) mRNA levels are not affected by SDZ 280.636. The findings that SDZ 280.636 inhibits polyclonal IgE responses and suppresses IL-4, but not IL-13 mRNA expression point towards differences in the regulatory pathways of IL-4 and IL-13 gene transcription in lymphoid organs. Thus the mechanism of action appears to involve a specific suppression of IL-4 gene transcription in cells occurring in Peyer's patches and mesenteric lymph nodes which are among the first constituents of the immune system encountered by an orally administered drug.

The genetic and evolutionary origin of the resistance determinants of R-factors is unknown. The genes which code for the synthesis of resistance functions are expressed in different bacterial hosts at high levels. This could mean that the transcriptional and translational machinery of bacterial species is a priori capable of efficiently expressing genes which may have evolved in other organisms. Alternatively, the rate at which the foreign genes are expressed in bacteria may depend on the complementation of the chromosomally coded protein synthesis system of the host with components coded for by the R-factor. One type of molecule that may have to be supplied by the R-factor in order to promote optimal translation of its genes might be tRNA because the tRNA composition is in a given species probably related to the overall spectrum of DNA code words.