Bee venom phospholipase A2 induces a primary type 2 response that is dependent on the receptor ST2 and confers protective immunity.

Venoms consist of toxic components that are delivered to their victims via bites or stings. Venoms also represent a major class of allergens in humans. Phospholipase A2 (PLA2) is a conserved component of venoms from multiple species and is the major allergen in bee venom. Here we examined how bee venom PLA2 is sensed by the innate immune system and induces a type 2 immune response in mice. We found that bee venom PLA2 induced a T helper type 2 (Th2) cell-type response and group 2 innate lymphoid cell activation via the enzymatic cleavage of membrane phospholipids and release of interleukin-33. Furthermore, we showed that the IgE response to PLA2 could protect mice from future challenge with a near-lethal dose of PLA2. These data suggest that the innate immune system can detect the activity of a conserved component of venoms and induce a protective immune response against a venom toxin.

Role of the inflammasome in defense against venoms.

Venoms consist of a complex mixture of toxic components that are used by a variety of animal species for defense and predation. Envenomation of mammalian species leads to an acute inflammatory response and can lead to the development of IgE-dependent venom allergy. However, the mechanisms by which the innate immune system detects envenomation and initiates inflammatory and allergic responses to venoms remain largely unknown. Here we show that bee venom is detected by the NOD-like receptor family, pyrin domain-containing 3 inflammasome and can trigger activation of caspase-1 and the subsequent processing and unconventional secretion of the leaderless proinflammatory cytokine IL-1ß in macrophages. Whereas activation of the inflammasome by bee venom induces a caspase-1-dependent inflammatory response, characterized by recruitment of neutrophils to the site or envenomation, the inflammasome is dispensable for the allergic response to bee venom. Finally, we find that caspase-1-deficient mice are more susceptible to the noxious effects of bee and snake venoms, suggesting that a caspase-1-dependent immune response can protect against the damaging effects of envenomation.

In situ imaging of honeybee (Apis mellifera) venom components from aqueous and aluminum hydroxide-adsorbed venom immunotherapy preparations.

BACKGROUND: Treatment with aqueous and aluminum hydroxide (Al[OH](3))-adsorbed purified honeybee (Apis mellifera) venom (HBV) preparations can reduce the incidence of side effects associated with venom immunotherapy. OBJECTIVE: The aim of the present study was to assess these purified HBV immunotherapy preparations in situ. METHODS: Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used to visualize the distribution of HBV components. The preparations were administered on the back legs of naive Wistar rats. The rats were killed, and cryosectioned tissue sections were subjected to hematoxylin and eosin staining and MALDI-MSI analyses. RESULTS: Low-density maps of tissue distribution of HBV peptides, such as secapin, mast cell degranulating peptide, and melittin (Api m 4) were detected in the tissue after administration of HBV immunotherapy preparations. In addition, release of biogenic amines, cytokines, and leukotrienes was observed, and the distribution of HBV allergens, such as Api m 1 and Api m 2, was shown. At the 24-hour time point, the major HBV allergen Api m 1 was still detected at the site of Al(OH)(3)-adsorbed HVB injection, whereas in the case of aqueous HBV preparation, all the allergens, as well as most of the biogenic amines, were cleared at the 24-hour time point. CONCLUSION: The present study shows that the majority of low-molecular-weight HBV components are rapidly removed from the site of venom immunotherapy administration. Furthermore, Al(OH)(3)-adsorbed HBV preparation demonstrated a depot effect, prolonging the availability of bee venom allergens at the site of administration.

Episodic hemorrhage during honeybee venom anaphylaxis: potential mechanisms.

Episodic hemorrhage is not a typical symptom of anaphylactic reaction to insect stings. Cases of reactions to honeybee (HB) sting or venom immunotherapy in which the uterus is the main target organ are very rare. Hemorrhage can be induced by HB venom components, especially melittin, which interfere with complement cleavage and bradykinin release. Both mechanisms are directly or indirectly associated with coagulation, thrombolysis, hemolysis, and smooth muscle tone. Induction of episodic hemorrhage through pathway destabilization in a defective bradykinin system or vulnerable organ may not be compensated by appropriate regulatory mechanisms. The pathological role of effectors is generally offset by the interaction of various regulatory systems, and the probability of hemorrhage is minimized thanks to this compensatory capability. In endometrial bleeding, the uterus becomes more vulnerable as a result of postmenstrual vascular fragility and additional induction of anaphylaxis-related uterine contractions. Episodic hemorrhage, especially metrorrhagia, as a consequence of HB venom activity may be suspected by an allergologist, but not by a physician. Melittin-free or recombinant allergens of HB venom, as well as modulators of the biochemical systems involved, could help to reduce the likelihood of hemorrhage. However, further investigation is required before these strategies can be introduced in clinical practice.

Gangliosides inhibit bee venom melittin cytotoxicity but not phospholipase A(2)-induced degranulation in mast cells.

Sting accident by honeybee causes severe pain, inflammation and allergic reaction through IgE-mediated anaphylaxis. In addition to this hypersensitivity, an anaphylactoid reaction occurs by toxic effects even in a non-allergic person via cytolysis followed by similar clinical manifestations. Auto-injectable epinephrine might be effective for bee stings, but cannot inhibit mast cell lysis and degranulation by venom toxins. We used connective tissue type canine mast cell line (CM-MC) for finding an effective measure that might inhibit bee venom toxicity. We evaluated degranulation and cytotoxicity by measurement of ß-hexosaminidase release and MTT assay. Melittin and crude bee venom induced the degranulation and cytotoxicity, which were strongly inhibited by mono-sialoganglioside (G(M1)), di-sialoganglioside (G(D1a)) and tri-sialoganglioside (G(T1b)). In contrast, honeybee venom-derived phospholipase A(2) induced the net degranulation directly without cytotoxicity, which was not inhibited by G(M1), G(D1a) and G(T1b). For analysis of distribution of Gα(q) and Gα(i) protein by western blotting, lipid rafts were isolated by using discontinuous sucrose gradient centrifuge. Melittin disrupted the localization of Gα(q) and Gα(i) at lipid raft, but gangliosides stabilized the rafts. As a result from this cell-based study, bee venom-induced anaphylactoid reaction can be explained with melittin cytotoxicity and phospholipase A(2)-induced degranulation. Taken together, gangliosides inhibit the effect of melittin such as degranulation, cytotoxicity and lipid raft disruption but not phospholipase A(2)-induced degranulation in mast cells. Our study shows a potential of gangliosides as a therapeutic tool for anaphylactoid reaction by honeybee sting.

In vivo neutralization of bee venom lethality by IgY antibodies.

Bee venom is a complex mixture of molecules, among which melittin and phospholipase A2 (PLA2) are the toxic components involved in envenoming accidents with multiple honeybee stings. Traditionally, the treatment of envenomings has been based on the administration of specific antibodies to neutralize the deleterious effects of toxins. An alternative to mammalian polyclonal antibodies is the use of egg yolk immunoglobulins (IgY) due to their advantages regarding animal welfare and lower costs of production as compared to the conventional production methods. In this work, a novel composition containing specific IgY antibodies was developed. After four immunizations, IgY extracted from the egg yolks was able to recognize several components of the bee venom, including melittin and PLA2. The performance of IgY to neutralize the lethal activity was evaluated in a mouse model by using one median lethal dose (LD50) of the bee venom. The effective dose of the IgY extract was determined as 30.66 µg/mg. These results demonstrate the feasibility to produce IgY-based antivenoms to treat envenomings by multiple bee stings.

Recognition, neutralization, and clearance of target peptides in the bloodstream of living mice by molecularly imprinted polymer nanoparticles: a plastic antibody.

We report that simple, synthetic organic polymer nanoparticles (NPs) can capture and clear a target peptide toxin in the bloodstream of living mice. The protein-sized polymer nanoparticles, with a binding affinity and selectivity comparable to those of natural antibodies, were prepared by combining a functional monomer optimization strategy with molecular-imprinting nanoparticle synthesis. As a result of binding and removal of melittin by NPs in vivo, the mortality and peripheral toxic symptoms due to melittin were significantly diminished. In vivo imaging of the polymer nanoparticles (or "plastic antibodies") established that the NPs accelerate clearance of the peptide from blood and accumulate in the liver. Coupled with their biocompatibility and nontoxic characteristics, plastic antibodies offer the potential for neutralizing a wide range of biomacromolecules in vivo.

Melittin-lipid nanoparticles target to lymph nodes and elicit a systemic anti-tumor immune response.

Targeted delivery of a nanovaccine loaded with a tumor antigen and adjuvant to the lymph nodes (LNs) is an attractive approach for improving cancer immunotherapy outcomes. However, the application of this technique is restricted by the paucity of suitable tumor-associated antigens (TAAs) and the sophisticated technology required to identify tumor neoantigens. Here, we demonstrate that a self-assembling melittin-lipid nanoparticle (α-melittin-NP) that is not loaded with extra tumor antigens promotes whole tumor antigen release in situ and results in the activation of antigen-presenting cells (APCs) in LNs. Compared with free melittin, α-melittin-NPs markedly enhance LN accumulation and activation of APCs, leading to a 3.6-fold increase in antigen-specific CD8+ T cell responses. Furthermore, in a bilateral flank B16F10 tumor model, primary and distant tumor growth are significantly inhibited by α-melittin-NPs, with an inhibition rate of 95% and 92%, respectively. Thus, α-melittin-NPs induce a systemic anti-tumor response serving as an effective LN-targeted whole-cell nanovaccine.

Immune response to lytic peptides conjugated to a betaCG fragment in treated BALB/C mice.

Hecate-betaCG and Phor14-betaCG(ala) are relatively short, amphipathic alpha-helical cationic peptides with the ability to destroy selectively breast, prostate and ovarian cancer cells. Treatment with proteins and peptides frequently initiated antibody formation. Short peptides may minimize the risk of the immune system mobilization after treatment but it is necessary to investigate whether Hecate-betaCG and Phor14-betaCG(ala) induce the immune system to produce antibody and whether they affect the reproductive organs in normal wild-type mice. The results of our experiments showed that specific antibodies, tested by the enzyme-immunoassay, were not detected in the group treated with Hecate-betaCG and Phor14-betaCG(ala). The blood concentrations of both peptides begun to decrease from 60 minutes after injection and after 240 minutes its levels were undetectable. Histopatho-logical examination exhibited degenerative changes in the prostate glands and testes in males and in the ovaries and uteri of females treated with both peptides. In conclusion, our results indicate that both relatively small and rapidly metabolized peptides are not immunogenic and can be used for further investigation as a potential cancer treatment.

A biomimetic nanoparticle-enabled toxoid vaccine against melittin.

BACKGROUND: Melittin, the main active peptide ingredient of bee venom, can cause severe cell membrane lysis due to its robust interaction with negatively charged phospholipids. So far, no effective anti-melittin vaccine has been developed to protect people from undesired melittin intoxication. METHODS: Herein, we prepared a polydiacetylene (PDA) nanoparticle with cell membrane-mimic surface to complex melittin, forming an anti-melittin vaccine (PDA-melittin). RESULTS: PDA nanoparticles could effectively combine with melittin and neutralize its toxicity. PDA-melittin nanocomplex is demonstrated to enhance melittin uptake by DCs and stimulate strong melittin-specific immunity. Mice immunized with PDA-melittin nanocomplex showed higher survival rate after exposion to melittin than untreated mice. CONCLUSION: The PDA-melittin nanocomplex can efficiently and safely generate a specific immunity against melittin to protect body from melittin intoxication, providing a new method with potential clinical application for the treatment of melittin intoxication.

Acyl carrier protein interacts with melittin.

Acyl carrier protein (ACP) from Escherichia coli has been shown to form complexes with melittin, a cationic peptide from bee venom. ACP is a small (Mr 8847), acidic, Ca2(+)-binding protein, which possesses some characteristics resembling those of regulatory Ca2(+)-binding proteins including interaction with melittin. Complexing between melittin and ACP which occurred both in the presence and absence of Ca2+ was evident by chemical cross-linking the two peptides, fluorescence changes (including anisotropy measurements), and inhibition by melittin of the activity of a nonaggregated fatty acid synthetase from Euglena. Also, anti-Apis mellifera antibodies which contained antibodies against melittin specifically inhibited the same enzyme system activity relative to non-immune IgG.

Activation of bee venom phospholipase A2 through a peptide-enzyme complex.

Phospholipase A2 activation by membrane-bound peptides was investigated in order to understand the role of the membrane-induced conformation on activation, and to examine the occurrence of a peptide-enzyme complex at the lipid/water interface. For the peptides studies, bee venom phospholipase A2 was stimulated regardless of the membrane-bound conformation (alpha-helix, beta-sheet or random coil). Using antisera raised against melittin, we were able to demonstrate the occurrence of a calcium-dependent complex involving the enzyme, phospholipid substrate, and peptide.

Epitope analysis: biotinylated short peptides as inhibitors of anti-peptide antibody.

The interaction of anti-melittin antisera with melittin-coated ELISA plates could be inhibited by biotinylated peptides of the C terminal epitope which is one of three defined antigenic sites on the hexacosapeptide melittin. Non-biotinylated short peptides and peptide derivatives were inactive. It is suggested that biotinylation of epitopic peptides enhances their inhibitory properties in a methodologically useful way.

Lipopeptides as immunoadjuvants and immunostimulants in mucosal immunization.

In previous studies we have shown that lipopeptides constitute potent immunoadjuvants in mice, rabbits and other species: in parenteral immunization, lipopeptide adjuvants were comparable, or in some cases superior to Freund's adjuvant, and were devoid of the side effects of this additive. Here we demonstrate that lipopeptides also constitute adjuvants for mucosal immunizations. The serum antibody responses against the wheat storage protein gliadin, the bee venom constituent melittin, or the hen egg protein ovalbumin could in most cases be enhanced more than 100-fold by the lipopeptide P3CSK4, applied via the nasal route. An enhanced specific antibody level could also be detected in supernatants of cell cultures prepared from spleens, Peyer's patches, lungs and mesenteric lymph nodes of immunized mice. Moreover, the lipopeptide P3CSK4 enhanced chemiluminescence in mouse spleen cells and peritoneal macrophages in vitro, indicating a macrophage-activating effect. Finally, nasal application of lipopeptide increased protection against a lethal infection of influenza. Our findings are of importance for the improvement of immunizations and might lead to more effective vaccines.

Lipopeptide-polyoxyethylene conjugates as mitogens and adjuvants.

Two lipopeptide analogues of the Escherichia coli lipoprotein rendered water-soluble by polyoxyethylene were tested for mitogenicity in vitro in murine and human B lymphocytes and for adjuvant activity in vivo in mice. These highly amphiphilic lipopeptides retained the biological activity other lipopeptides usually exerted which supports the hypothesis of specific interactions of lipopeptides with membranes of reactive cells. The activation of human B lymphocytes by these lipopeptides was much less pronounced compared to that of murine cells. However, given in combination with anti-CD40 antibodies plus interleukin-4, human B lymphocytes could synergistically be stimulated to proliferate. As an adjuvant, the polyoxyethylene linked lipopeptides were almost as potent as Freund's adjuvants and other basic lipopeptides. Being water-soluble, these novel analogues are easy to apply and they are suitable for field studies as adjuvants when sonication can not usually be provided.

Quantity, analysis, and lethality of European and Africanized honey bee venoms.

Venom from Africanized honey bees (derived mainly from Apis mellifera scutellata) was compared with venom from domestic, European bees by study of lethality, immunological cross-reactivity, venom yield, isoelectric focusing (IEF) patterns, and melittin titers. The LD50s of European and Africanized bee venom by iv injection in mice were similar. In venom neutralization experiments, Africanized bee venom was mixed with antibodies from a beekeeper exposed only to European bees and used to challenge mice. Survival times of mice given these mixtures were significantly prolonged, indicating that human serum antibodies to European bee venom neutralized the lethal effects of Africanized bee venom. Reservoirs from Africanized bees contained less venom than European bees (94 and 147 micrograms venom/bee, respectively) and Africanized bee venom had a lower melittin content. The IEF patterns of venom from individual European bees varied considerably, as did IEF patterns of individual Africanized bees. Pools of venom from 1,000 bees of each population of A. mellifera showed noticeable but less obvious electrophoretic differences. The findings suggest that multiple stinging, and not increased venom potency or delivery, is the cause of serious reactions from Africanized bee attacks.

Neutralization of bee venom lethality by immune serum antibodies.

The lethal effects of Africanized honey bee venom depend on the absorption of venom delivered during simultaneous sting attacks by large numbers of bees. The hypothesis that antibodies to whole bee venom and bee venom components could neutralize the lethal effect of bee venom was tested. Antibodies from beekeepers and immunized rabbits were incubated with bee venom and neutralization was studied by survival of intravenously injected mice. Beekeeper serum antibodies were found effective in protecting mice challenged with whole venom, and serum from rabbits immunized with phospholipase A2 (PLA2) was effective in protection against lethal effects of PLA2. Serum antibodies from rabbits immunized with whole venom or melittin were ineffective in neutralizing whole venom in vivo and had low titers in a venom enzyme-linked immunosorbent assay. The results suggest the need for development of more effective methods for raising antitoxic antibodies to bee venom components in other animals as a means of developing an antiserum that would be effective for treatment of human victims of multiple bee stings.

Induction of an epitope-specific humoral immune response by lipopeptide-hapten conjugates: enhancement of the anti-melittin response by a synthetic T helper (Th)-cell epitope.

Lipopeptides of bacterial origin constitute potent immunoadjuvants when combined with antigens. After the immunization with lipopeptides covalently coupled to non-immunogenic low-molecular-mass antigens or haptens, a hapten-specific humoral immune response can often be obtained. The response against synthetically prepared melittin fragments was further enhanced by the additional introduction of a T helper (Th)-cell epitope into the lipopeptide-hapten conjugate. The Th-cell epitope applied, which is presented by the MHC class II molecule of the BALB/c (H-2d) haplotype, consisted of a synthetic 16-amino-acid oligopeptide derived from sperm whale myoglobin. The immune-enhancing effect was most pronounced for the melittin-derived peptide fragments [Mel(1-16)] and [Mel(17-26)-CONH2]. Antibodies obtained after 3 immunizations with the conjugates recognized the synthetic as well as the native melittin molecule. Our results show that it is possible to markedly enhance a weak hapten-specific immune response by coupling the haptens to a lipopeptide conjugated to a haplotype-specific T helper-cell epitope. The novel conjugates are well suited for the optimization of immunization procedures, and for the development of novel synthetic vaccines.