Novel approaches to immunotherapy: epitopes, determinants, activators, or modulators?

The immunological mechanism through which immunotherapy (IT) acts is not certainly known. The participation of the so-called "blocking antibodies" has not been proved and how it intervenes in the regulation of the production of IgE is still to be cleared, as it affects the action of lymphocytes Th1 and Th2. Nowadays, IT is based in the concept that the allergic reaction is somehow an antigen (allergen)-antibody (reagin) reaction. The possible modifications of IT are also based on the possibility to interfere in the antigen-antibody interaction. It has been proved in vitro that aqueous extracts of some allergens produce the consumption of complement, by its usual via, in which the C1 component is involved, without the mediation of antibodies, generating anaphylotoxin C3a, which is a powerful releaser of histamine, as well as C3b, which participates in the regulation of the cellular immunitary system. The term atopen should therefore be used when referring to the unspecific activation or adjuvant activity of antigenically different allergens, due to their common structural or functional characteristics. The term allergen should then be used when describing those structural traits of the carrier molecule, which preferently produces the induction, and possible recognition of IgE antibodies. The action atopen and allergen as separate characteristics of a same molecule could theoretically become important in the future of therapeutics. The receptor of membrane CD21 for C3b, in B cells is a link for lectine CD23 of lymphocytes B, which has been identified with the low affinity receptor IgE (Fce RII-CD23). In summary, the basic defect in atopy may reside in a defect of the receptor portion of reagin for atopen, but not in IgE. This means that in the nearby future, this fact should be taken into account in the search of drugs which participate in this kind of activation of allergic reaction, trying to modify or modulate the low affinity receptor IgE or even the high affinity receptor IgE.

The role of IgG in type-I allergy: an unsolved problem.

The role of antiallergen IgG antibodies in allergy remains unclear. In this review we present evidence for and against the hypothesis that IgG (IgG4) could act as a sensitizing antibody. After considering the available data, we conclude that the possible sensitizing ability of IgG4 may depend on the nature and origin of the antibodies and cells used in the various experiments and/or is related to the differential induction of two subtypes of IgG4 (anaphylactic vs. blocking). It still remains to be settled whether the observed increase in specific IgG during immunotherapy is causally related to the relief of symptoms, or whether it merely represents an epiphenomenon due to high antigen exposure. Furthermore, the presence of IgG anti-IgE antibodies in the serum of allergic patients and their possible pathophysiological relevance is discussed in the light of recent evidence suggesting that the raised levels of these antibodies may likewise represent an epiphenomenon of the immune response. There exists a tight relationship between antiallergen IgE and IgG antibodies. This correlation has led to the suggestion that IgG determinations might be useful for diagnostic purposes. There also exists a good correlation between these antiallergen isotypes in inhibition assays. It has therefore been proposed to employ inhibition of IgG binding for the standardization of allergenic extracts. More recent studies explore the relevance of the binding affinities or avidities of allergen-specific IgG antibodies. Antibody affinity may have important repercussions with respect to the biological effects. It has been pointed out that the affinity of specific IgG subclasses in allergic patients depends, among other things, on the nature of the sensitizing allergen. Further studies on IgG binding to the so-called "major" and "minor" allergens may help clarify the role of IgG in allergic disorders.

Comparative study of the complement-activating and specific IgE-binding properties of ragweed pollen allergen.

Previous reports have defined the capacity of ragweed pollen extract (RWA) to activate human complement (C) in fluid phase through the classical pathway and have ascertained a strong correlation between the extent of complement activation and the severity of symptoms of allergic rhinoconjunctivitis during the ragweed blooming season. In the present study the complement-activating and specific IgE-binding capacities of various ragweed allergen preparations were compared. Elimination of physically adsorbed (flavonoid) pigments from the allergenic proteins had no significant effect on their complement-consuming capacity, although the process strongly diminished specific IgE binding. Removal of an IgE-binding trypsin inhibitor from RWA significantly enhanced RWA-induced complement activation, whereas it did not change IgE binding. These findings indicate that neither the physically adsorbed pigments nor the trypsin inhibitor are involved in complement activation by ragweed pollen allergens, and suggest that complement activation and specific IgE binding are distinct molecular properties of ragweed pollen allergen.

Haemolytic complement consumption by Parietaria pollen extracts in relation to peptide-bound flavonoids.

Inhaled allergens from house dust, mites or animal danders activate human complement in vitro by engaging the C1-component through a non-antibody-dependent mechanism. These earlier findings are extended by showing that the allergenic components in extracts of Parietaria pollen are almost equally potent complement activators as those from house dust or mites. Spectroscopic evidence indicates that haemolytic complement consumption by the Parietaria allergens and their enzymic fragments is most likely related to post-translational side-chains comprising flavonoid derivatives. These adsorbed and/or peptide-bound tannin-like structures may also explain the exceptional stability of the high- and low-molecular mass allergenic components in Parietaria pollen extracts.

Complement inactivation by allergenic plant pollen extracts.

Dialyzed aqueous extracts of plant pollen are widely used in the clinical practice of allergy for diagnostic and therapeutic purposes. The present investigation shows that such allergenic extracts are capable of consuming complement in every human serum, independent of the clinical condition. Complement is engaged by way of the first component C1, but without the participation of allergen-specific antibodies. The capacity of distinct pollen extracts to inactivate haemolytic complement was found to depend on the plant species, the most potent extracts being from the pollen of the weeds and trees. Analysis by UV-spectroscopy of the flavonoids remaining firmly bound to the proteins gives rise to the proposal that complement inactivation by allergenic and non-allergenic pollen extracts is due to polyphenolic (flavonoid) structures complexed with, or chemically conjugated to, the pollen proteins.

Complement activating agents in allergenic extracts.

OBJECTIVE: Extracts of environmental allergens, moulds and plant pollens are known to consume haemolytic complement (huC) in human serum in vitro through the antibody-independent engagement of the first component C1 of the classical pathway. The present work was undertaken to establish the nature and characteristics of the complement activating agents in allergenic extracts and to probe their relationship with the IgE-binding allergens. MATERIALS AND METHODS: A large series of different > 10 kDa allergenic products was investigated for their capacity to consume haemolytic complement in the sera of allergic patients with specific anti-allergen IgE antibodies, as well as in normal control sera. UV-spectroscopy was used for categorizing the non-protein components in the extracts. RESULTS: The experiments confirmed that huC is consumed in an antibody-independent fashion and in a qualitatively similar, but quantitatively distinct manner. The ratio of the complement activating potencies among the different allergenic preparations thereby remained constant and independent of the serum source, while an overt relationship with specific IgE-antibodies could not be established. UV-spectroscopy of the allergenic preparations revealed the presence of chemical decomposition products in nearly every extract and roughly in proportion to the complement activating potencies. CONCLUSION: The data support the idea that huC-activation by traditional allergenic extracts is mainly due to by-stander degradation products of the melanoidin (Maillard) or tannin type, which may or may not occur in physical association with the IgE-binding protein allergens.

Extraordinary stability of IgE-binding Parietaria pollen allergens in relation to chemically bound flavonoids.

It is known that the skin-active and IgE-binding components in Parietaria pollen extracts are not restricted to the predominant protein allergens of M(r) 12000-15000, but are present as well among the naturally occurring constituents of M(r) < 10000. Indeed, the IgE-binding Parietaria pollen components are quite heterogeneous, ranging from high- to low-molecular mass, whereby the IgE-binding epitopes display an unusual chemical stability. Furthermore, the pollen of Parietaria species demonstrably contain a high proportion of flavonoid pigments. Since these pollen grains cannot be collected entirely free from non-pollen plant parts, the usual allergenic extracts of Parietaria encompass both the polyphenolic substrate molecules and the enzyme polyphenoloxidase as ingredients for the oxidative generation of flavonol-protein conjugates during the extraction process. In the present work this is illustrated by spectroscopic analyses of the free and bound flavonoids in Parietaria pollen extracts, as well as of the peptide fragments produced from the allergenic proteins by enzymatic or chemical hydrolysis. None of these relatively harsh treatments had a significant effect on the IgE-binding properties of the allergenic (sub-)components, even though detectable proteins in isoelectric focusing and immunoblotting were lost. It is proposed that the extraordinary stability of IgE-binding Parietaria components over a wide molecular range may be attributed to chromophoric flavonoid side-chains as (parts of) the corresponding B-cell epitopes.

Binding affinities of allergens from pollen, mites, and house dust for specific IgG subclass antibodies.

The distribution and affinity of IgG subclasses against various aeroallergens were assessed by inhibition of specific antibody binding. Two parameters from the dose-response curves were taken as indicative of antibody affinity: the point of 50% inhibition and the value of the slopes on double-log plots. It was found that IgG4 antibody specific for aeroallergens (i.e., from pollens of several species of Gramineae, Olea europaea, and Parietaria judaica and from house dust) usually exhibits high affinity, except for Dermatophagoides pteronyssinus. High binding affinity was also displayed by IgG1 subclass antibodies against the allergens of O. europaea and P. judaica. Distinct IgG subclass affinity profiles were observed for the allergens of grass pollen (i.e., Holcus lanatus) and dust mites (i.e., D. pteronyssinus). These results demonstrate that IgG subclass distribution, as well as antibody affinity, depends on the nature of the sensitizing allergen.

Monoclonal antibodies against Dermatophagoides group I allergens as pseudo-cystatins blocking the catalytic site of cysteine proteinases.

The enzyme allergens Der p I and Der f I produced by the house dust mites Dermatophagoides pteronyssinus and D. farinae display partial sequence homology with other members of the cysteine proteinase superfamily. We report that certain widely used mouse mAbs against these Group I allergens indeed crossreact with the plant enzymes papain, bromelain and ficin. The recognition sites of these anti Group I mAbs comprise conformational and thermolabile epitopes involved in molding the catalytic center of the proteinases. Thus, the mAbs inhibit the enzymatic hydrolysis of specific chromogenic substrates by the Group I allergens, while specific cysteine proteinase inhibitors abolish the recognition of the enzymes by the mAbs. Similarly, activation of the thiol-proteases with L-cysteine abrogates their binding in the two-site mAb system, indicating that the mAbs recognize a proenzyme conformational peptide epitope. It follows that mAb-based assays for mite Group I components can neither detect the allergens after inactivation, nor in their fully activated forms.

IgE-binding trypsin inhibitors in plant pollen extracts.

In an attempt to access the possible role of protease-antiprotease mechanisms of non-immune defence in pollinosis, only low levels of trypsin-, kallikrein- or plasmin-like proteinases could be detected in aqueous pollen extracts. In contrast, several pollen species displayed appreciable trypsin inhibitory activity, e.g. Parietaria, Olea, Ambrosia, Rumex, Chenopodium, Holcus and Poa spp. These proteins of the serpin family of anti-proteinases were found to bind specific IgE-antibodies from the serum of hay fever patients. As examples, the IgE-binding trypsin inhibitors from the pollen of Parietaria judaica and Ambrosia elatior were purified and characterized as acidic proteins with pI 4.2 and a molecular weight of 20-24 kDa.

An IgE-binding TAME esterase in the urine of the mouse, Mus musculus domesticus.

Among the highly heterogeneous components of the mouse urinary protein complex (MUP) a hydrolase was detected which was capable of spliting the proteinase ester substrate TAME as well as a synthetic chromogenic tripeptide specific for tissue and urinary kallikreins. The binding of mouse-specific IgE antibodies from the serum of a highly mouse-allergic patient occurred preferentially to this kallikrein-like enzyme. This finding underscores the possible association of significant biological activities with predominant IgE-binding allergens, especially in view of the strongly sensitizing potential and the known messenger functions of the MUP proteins.

Comparative studies of allergenic extracts prepared from freshly collected, dried, or dried and defatted pollen.

Fresh pollen samples of Parietaria judaica, Betula pubescens, Dactylis glomerata and Olea europaea were collected and aliquot portions of these pollens were stored after drying in hot air, while other portions were dried and then defatted with diethylether. The different pollen materials were extracted with aqueous media to obtain the non-dialyzable allergenic protein constituents. Review of the production data, together with comparative analyses of the UV-absorption spectra and of IgE binding potencies did not reveal appreciable differences in allergen composition among extracts of fresh, dried, or dried and defatted pollen materials. However, microscopic examination showed the fresh pollen to be no longer viable.

Do IgE-IgG complexes occur in the circulation?

IgG anti-IgE autoantibodies in human allergic sera have been investigated using an enzyme immunoassay on microplates coated with anti-human IgE MoAb. After incubation with serum, the plates were developed with peroxidase-labelled anti-human IgE MoAb for the determination of total IgE levels, or with anti-IgG MoAb for evaluating IgG anti-IgE autoantibodies. Using this methodology, no correlation was found between total IgE and IgG anti-IgE levels in groups of sera of allergic individuals. Although the results obtained with enzyme-labelled anti-IgG are often interpreted as indicative of IgE-IgG complexes captured from the serum, molecular sieving on gel columns as well as direct ultrafiltration experiments through 300-kD membranes demonstrate that such complexes do not occur preformed in the circulation, but arise de novo on the anti-IgE-coated solid phase during in vitro incubation with human serum. It is suggested that IgG anti-IgE autoantibodies react with IgE only after the latter has undergone a conformational change, either by colloidal manipulation or after reaction with allergen.

Investigation of acid phosphatase as a possible IgE-binding marker for pollen allergens and their polymerized derivatives.

The enzyme acid phosphatase represents an important component among the allergenic proteins in most pollen extracts. However, determination of the level of this enzyme in extracts of various pollen species, as well as protein separation by molecular sieving shows that acid phosphatase cannot be used as a marker for IgE-binding protein allergens. A conspicuous discrepancy was observed between the heat- and acid-sensitivities of the phosphatase enzyme relative to the overall IgE-binding properties of the pollen extracts. Remarkably, the enzyme remained unaffected by cross-linking the pollen proteins with glutardialdehyde, whereas this process of polymerization caused the IgE-binding potency to diminish considerably. It is concluded that the enzyme acid phosphatase is not a suitable marker for the potency assessment of pollen allergens, but may be useful for monitoring the production process of polymerized allergoids.

Evaluation of the potency of allergenic extracts by inhibition of IgG-antibody binding.

A method is described for the in vitro potency evaluation of allergenic extracts by using their capacity of binding to specific human IgG antibodies. The results obtained with this inhibition-type enzyme immunoassay are compared with the analyses by the customary method of IgE antibody binding. A large series of allergenic protein fractions from the pollen of the Gramineae, Olea europea, Parietaria judaica, and from the dust mite Dermatophagoides pteronyssinus as well as from cat dander and peanuts were examined for inhibition of specific antibodies of both IgG and IgE isotypes. Potency evaluation by inhibition assays for IgE- and IgG-binding showed a significant correlation for the points of 50% inhibition (r = 0.92, p = 0.0001), but not for the slopes of the inhibition curves, i.e. the respective antibody avidities. Evidence is provided that the strict relationship between IgE- and IgG-inhibition by allergens could not be explained by possible cross-contaminations of the anti IgE- or anti IgG-reagents employed in the immunoassays. It is concluded that the inhibition of IgG-antibody binding presents a fast, reliable and low-cost alternative for the potency control of allergens used in clinical practice.