i213 Api m 4

Summary

Api m 4, or melittin, is a marker allergen for genuine sensitization to Apis mellifera (honeybee) venom (HBV). Api m 4 sensitization is observed in 17 - 54% of HBV-allergic patients. Accounting for 50% of HBV dry weight, Api m 4 is also a powerful cytolytic, pain-inducing, and proinflammatory peptide acting in synergy with Api m 1, the HBV phospholipase A2 (PLA2).

Epidemiology

Worldwide distribution

The prevalence of sensitization to Api m 4 varies from 17% to 54% in HBV-allergic patients [1]. Api m 4 is usually considered a minor allergen in HBV-allergic patients but has been reported as a major allergen in studies from Spain [2,3,4].

Environmental characteristics                       

Source and tissue

Api m 4 is secreted into the venom sac of Apis mellifera, where it contributes 50% of HBV dry weight [1]. Its production exhibits seasonal variations, with a maximum during the winter months, as well as variations related to the category and age of bees  [1].

Risk factors

The risk of becoming sensitized to Api m 4 is related to Api m 4 injection during a bee sting. Beekeepers are at higher risk of being stung and hence of becoming sensitized to HBV allergens, including Api m 4. The induction of sensitization to Api m 4 is apparently unrelated to its direct cytolytic and proinflammatory effects as a toxin [5].

The risk of systemic manifestations in both sensitized and non-sensitized subjects increases with the amount of injected Api m 4, as seen upon simultaneous stinging by multiple bees. Larger concentrations of Api m 4 increase its cytolytic, pain-inducing, and proinflammatory effects, further enhanced by synergistic toxicity with Api m 1, the HBV PLA2 [1, 5, 6].

Clinical relevance

Specific molecules

Api m 4 is a marker allergen for genuine HBV sensitization [6]. Apparent monosensitization to Api m 4 is infrequent, reported at 1-2% in HBV-allergic patients [7].

Cross-reactive molecules

Api m 4 homologues with high sequence identity are present in other Hymenoptera venoms from the Apis (bee) and Bombus (bumblebee) genera, called bombolittins for the latter [8], as well as from Vespids, however, an allergenic activity has not been reported for these homologues [6, 9, 10].

Disease severity

An Api m 4-related phenotype with increased severity and decreased tolerance to venom immunotherapy (VIT) has been described in studies from Spain [3,4]. Further studies are warranted since these associations were not observed in subsequent studies within other locations.

Systemic reactions and Anaphylaxis

Among HBV-allergic patients with Api m 4-specific IgE at 0.98 kU_A/L or greater, 83% developed systemic reactions grade 3 or 4 in Müller’s classification following field stings, compared with 42% in the control group comprising HBV-allergic patients with Api m 4-specific IgE lower than 0.98 kU_A/L [4].

Prevention and therapy

Experimental trials

Api m 4 immunogenicity is low, due to its small size and high water solubility; moreover, the safe therapeutic interval is narrow, due to its cytotoxicity. Current therapeutic applications of Api m 4 aim to harnessing its anti-tumor effects, both direct via cytotoxicity and indirect via immune response elicitation   [8, 11].

Molecular aspects                  

Biochemistry

Api m 4 is a small, 26-aminoacid cationic amphiphilic peptide with a molecular weight of 3 kDa [6, 8]. Api m 4 is found as a tetramer during storage in the venom sac or at high concentrations, or as a monomer in solution or following its injection into the host’s tissues [8]. Api m 4 inserts into lipid bilayers either as a monomer or as a tetramer, disrupting membranes through enhanced ion transport or by forming pores  [8, 12].

Cleavage of the Api m 4 pro-protein is performed by dipeptidyl-peptidase IV (DPPIV, Api m 2) within the venom sac [6, 8]. Api m 4 activates Api m 1, the HBV PLA2, resulting in coordinated effects of Api m 1 targeting cellular, bacterial, and surfactant phospholipids and Api m 4 disrupting lipid bilayers. The small size and high water-solubility of Api m 4 explain its systemic diffusion and the risk of systemic reaction in case of massive envenomation [8,13].

Api m 4 targets lipid bilayers of viral, bacterial, fungal, protozoan, and animal cells, including cancer cells; its ability to insert into membranes, its cytolytic effects and the resulting activation of the nociceptive, inflammatory and adaptive immune responses are currently under research for new pathways of drug delivery [8]. Api m 4 also exerts anti-inflammatory and immune modulation effects [5,8,11].

Isoforms, epitopes, antibodies

As of December 12, 2023, a unique isoallergen, Api m 4.0101, has been included in the World Health Organization (WHO) and International Union of Immunological Societies (IUIS) Allergen Nomenclature [9].

 Cross-reactivity due to structural similarity

 Api m 4 shares aminoacid sequence identity of 90% or higher with venom peptides from other Hymenoptera species, including Vespids, without clinically relevant allergen cross-reactivity [6,10].

Diagnostic relevance              

Diagnosis of genuine sensitization to Apis mellifera venom

Api m 4 is a marker allergen for HBV sensitization. It is especially useful as an additional marker allergen, in combination with Api m 3 and Api m 10, in populations with lower prevalence of IgE to Api m 1, since demonstrated genuine sensitization to HBV supports the initiation of HBV VIT in eligible patients [2,6,7]. Since Vespid venoms lack clinically relevant Api m 4 homologues, Api m 4 is also useful for discrimination between HBV and Vespid sensitization [2].

Disease severity

In Hymenoptera venom IgE testing, the quantitative result of specific IgE to a molecular allergen or whole venom extract is currently considered as neither predictive of, nor correlated to the severity of the reaction [6].  This view may change in the future, since reports of identifiable phenotypes associated with certain molecular sensitization patterns and levels of allergen-specific IgE, such as the presence and high levels of Api m 4-specific IgE, are available for some populations [3,4]. An increased severity at diagnosis and during VIT has been reported in Spanish HBV-allergic patients [3,4].

Sensitivity of in vitro assays

The prevalence of sensitization to individual HBV allergens, including Api m 4, in HBV-allergic patients varies depending on multiple factors such as geography, patient inclusion criteria, single or double positivity to HBV and Vespid venoms, use of a purified, recombinant or synthetic allergen, and assay format [6]. Api m 4-specific antibodies have been measured using purified, recombinant, and synthetic peptides, with the latter reportedly displaying greater detection sensitivity, e.g. 43% with the purified peptide versus 54% with the synthetic peptide [2].

Functional studies with basophil activation assays are scarce. An early study of HBV molecular allergens reported the lack of Api m 4-induced basophil upregulation of the activation marker CD203c, however, actual Api m 4 sensitization of the recruited HBV-allergic patients had not been assessed [14].

Api m 4 sensitization can be detected with commercially available singleplex and multiplex methods, with variable availability among manufacturers and over time.

Diagnostic specificity

The diagnostic specificity of Api m 4-specific IgE for HBV sensitization has been consistently reported at very high levels, estimated at 100% since no healthy controls and no Vespid-only allergic patient displayed Api m 4-specific IgE [4,7].

AIT Prescription

Api m 4 is a marker of genuine sensitization to HBV, thus supporting the choice of HBV AIT in eligible patients [6]. An association between Api m 4 sensitization greater than 0.98 kU_A/L, increased risk of adverse systemic reactions to VIT and a success rate of controlled sting challenge of 82% (compared to 100% in the control group) after one or two years of VIT were reported in Spanish HBV-allergic patients [3,4]. Thus, elevated levels of Api m 4-specific IgE before VIT initiation have been proposed as predictive for poor tolerance of HBV VIT [3].

A robust increase in Api m 4-specific IgG4 levels was observed during VIT, a finding also used to confirm the presence of Api m 4 in VIT preparations [4,7].

Explained results

Allergen Information

Api m 4 or melittin, a small cationic and amphiphilic defense peptide, is the most abundant HBV component, accounting for half of HBV dry weight. Api m 4 exerts IgE-dependent (allergic) and IgE-independent (Api m 1 activation, lipid membrane disruption, cytolysis, pain, immune modulation) effects.

Clinical information

Api m 4-specific IgE identifies genuine HBV sensitization and discriminates it from Vespid sensitization in patients who are double positive to HBV and Vespid venom extracts. In HBV-allergic patients, the prevalence of Api m 4 sensitization ranges from 17 to 54% and its diagnostic specificity is 100%.

Cross-reactivity

Api m 4 does not cross-react with Vespid venom homologues.

Author: Dr. Joana Vitte

Reviewed by: Dr. Michael Spangfort