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.

The protective effects of melittin on Propionibacterium acnes-induced inflammatory responses in vitro and in vivo.

Melittin is the main component in the venom of the honey bee (Apis mellifera). It has multiple effects including antibacterial, antiviral, and anti-inflammatory activities in various cell types. However, the anti-inflammatory mechanisms of melittin have not been elucidated in Propionibactierium acnes (P. acnes)-induced keratinocyte or inflammatory skin disease animal models. In this study, we examined the effects of melittin on the production of inflammatory cytokines in heat-killed P. acnes-induced HaCaT cells. Heat-killed P. acnes-treated keratinocytes increased the expression of pro-inflammatory cytokines and Toll-like receptor 2. However, melittin treatment significantly suppressed the expression of these cytokines through regulation of the NF-κB and MAPK signaling pathways. Subsequently, the living P. acnes (1 × 10(7) CFU) were intradermally injected into the ear of mice. Living P. acnes-injected ears showed cutaneous erythema, swelling, and granulomatous response at 24 hours after injection. However, melittin-treated ears showed markedly reduced swelling and granulomatous responses compared with ears injected with only living P. acnes. These results demonstrate the feasibility of applying melittin for the prevention of inflammatory skin diseases induced by P. acnes.

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.

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 protein whose binding to Na,K-ATPase is regulated by ouabain.

Immunoprecipitation of Na,K-ATPase from kidney homogenate by antibodies against alpha1-subunit results in the precipitation of several proteins together with the Na,K-ATPase. A protein with molecular mass of about 67 kD interacting with antibodies against melittin (melittin-like protein, MLP) was found in the precipitate when immunoprecipitation was done in the presence of ouabain. If immunoprecipitation was done using antibodies against melittin, MLP and Na,K-ATPase alpha1-subunit were detected in the precipitate, and the amount of alpha1-subunit in the precipitate was increased after the addition of ouabain to the immunoprecipitation medium. MLP was purified from mouse kidney homogenate using immunoaffinity chromatography with antibodies against melittin. The addition of MLP to purified FITC-labeled Na,K-ATPase decreases fluorescence in medium with K+ and increases it in medium with Na+. The enhancement of fluorescence depends upon the MLP concentration. The N-terminal sequence of MLP determined by the Edman method is the following: HPPKRVRSRLNG. No proteins with such N-terminal sequence were found in the protein sequence databases. However, we revealed five amino acid sequences that contain this peptide in the middle part of the chain at distance 553 amino acids from the C-terminus (that corresponds to protein with molecular mass of about 67 kD). Analysis of amino acid sequence located between C-terminus and HPPKRVRSRLNG in all found sequences has shown that they were highly conservative and include WD40 repeats. It is suggested that the 67-kD MLP either belongs to the found protein family or was a product of proteolysis of one of them.

Increased sensitivity of early apoptotic cells to complement-mediated lysis.

Opsonization of apoptotic cells with complement proteins contributes to their clearance by phagocytes. Little is known about the lytic effects of complement on apoptotic cells. Sensitivity of cells treated with anti-Fas antibody (Jurkat cells), staurosporine or etoposide (Raji cells) to lysis by complement was examined. As shown here, early apoptotic cells are more sensitive to lysis by antibody and complement than control cells. More complement C3 and C9 bound to apoptotic than to control cells, even though antibody binding was similar. Enhanced killing and C3/C9 deposition were blocked by benzyloxy-Val-Ala-Asp-fluoromethylketone, a pan-caspase inhibitor. Complement-mediated lysis of early apoptotic cells was also prevented by inhibitors of caspases 6, 8, 9 or 10. In contrast, caspase inhibitors had no effect on the lysis of non-apoptotic Jurkat and Raji cells. Early apoptotic Jurkat cells were also more sensitive to lysis by the pore formers streptolysin O and melittin. Sensitivity of Jurkat Bcl-2 transfectants to lysis by complement was analyzed. Enhanced Bcl-2 expression was associated with reduced C3 deposition and lower sensitivity to complement-mediated lysis. These results demonstrate that at an early stage in apoptosis, following caspase activation, cells become sensitive to necrotic-type death by complement and other pore formers. Furthermore, they suggest that Bcl-2 is actively protecting Jurkat cells from complement-mediated lysis.

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.

Antibody responses to bee melittin (Api m 4) and hornet antigen 5 (Dol m 5) in mice treated with the dominant T-cell epitope peptides.

BACKGROUND: Mice treated with the dominant T-cell epitope peptides of allergens were reported to have reduced peptide or allergen-specific T-cell responses on subsequent immunization, but the extent of reduction of allergen-specific antibodies is not clear. OBJECTIVE: This study was done to compare the extent of reduction of T-cell and antibody responses in peptide-treated mice. Two allergens were tested. Bee melittin (Api m 4), an allergen of 26 amino acid residues, has a single dominant T- or B-cell epitope. Hornet antigen 5 (Dol m 5), an allergen of 204 amino acid residues, has multiple dominant T- or B-cell epitopes. METHODS: Mice were treated with T-cell peptides of Api m 4 or Dol m 5 and then immunized biweekly with their respective allergen with alum adjuvant. T-cell peptides tested were residues 7-19 of Api m 4 and residues 41-60, 141-160, and 176-195 of Dol m 5. T-cell responses at week 9 or 11 were assayed by proliferation of spleen cell cultures. Antibody responses of different isotypes were measured biweekly by ELISA. RESULTS: Partial reduction of 30% to 50% of T-cell responses to peptide or allergen was observed in bee and hornet peptide-treated mice. About 65% reduction of Api m 4-specific antibody response was observed early in the immune response but gradually subsided to about 40% late in the response. Partial reduction of about 40% of Dol m 5-specific antibody response was only observed early in the immune response. CONCLUSION: Peptide treatment is partially effective in the reduction of T-cell responses of univalent or multivalent allergens. It is also partially effective in the reduction of antibody response of a univalent allergen, but it is poorly effective for a multivalent allergen.

Immunogenicity studies with haptenated melittin peptides: implication for membrane involvement during the recognition step.

Melittin peptides carrying 2,4-dinitro-6-carboxyphenyl (Dncp) haptenic groups regularly evoked anti-hapten IgG responses in mice or guinea pigs when the hapten was C-terminally attached. Single haptens on the N-terminal helix in several positions gave poor or no responses in the early stages but adequate titres after prolonged immunization. Peptides with Dncp at the C-terminus as an invariant feature and a second Dncp in various positions along the peptide chain did not fail to produce adequate responses. The hampering effect is not due to a defect at the T-cell level but involves the recognition step on the B-cell. It is implied that the haptenic interaction with the paratope of the recognizing immunoglobulin on the B-cell involves the cell membrane in an important way. It is also suggested that late antibody responses should not be overlooked during the development of proteinaceous immunogens for vaccination.

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.

Immunogenicity of dinitrocarboxyphenylated melittin: the influence of C-terminal chain shortening, N-terminal substitution and prolin insertion at positions 5 and 10.

Peptides derived from the bee-venom melittin were fitted with the haptenic group dinitrocarboxyphenyl (Dncp) and tested in out-bred guinea pigs for immunogenicity by measuring the IgG anti-Dncp antibody response by ELISA. Dncp-conjugates comprising virtually the entire melittin proved to be strong immunogens producing antibody responses comparable to those of proteins. Weak responses were obtained with considerably shortened sequences. Conjugates with N-terminal Dncp gave markedly reduced antibody responses compared to peptides with C-terminal Dncp. An N-terminal biotinyl substituent abolished the immune response whereas N-terminal lauryl and caprylyl had little effect. Insertion of L-proline into a hexadecapeptide conjugate abolishing the possibility of helix formation gave an immunogen to which individual animals clearly responded on a low level. Oligomerisation, but not the cytolytic activity of melittin peptides, may contribute to the immunogenicities observed.

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.

Stimulus-associated protein in gastric parietal cell detected using antimelittin antibody.

The bee venom polypeptide melittin binds to and inhibits the gastric hydrogen-potassium-adenosinetriphosphatase (H(+)-K(+)-ATPase). A search for parietal cell proteins with a melittin-like structure was carried out. A 67-kDa (doublet) protein, which reacted with a polyclonal antimelittin antibody, was found in purified rabbit parietal cells. The protein exhibited reversible stimulus-dependent redistribution from cytosol to (total) membranes. It was also found to be associated with H(+)-K(+)-ATPase-containing membranes when isolated from the gastric mucosae of rabbits treated with histamine, but not with cimetidine. The presence of the protein correlated with the ability of the membrane preparations to exhibit ionophore-independent HCl accumulation, a characteristic of gastric membranes from histamine-stimulated animals. The 67-kDa melittin-like protein may play a role in the functional changes in the gastric parietal cell that are involved in stimulation of HCl secretion.

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.

Immunological studies on bee-keepers: specific IgG and subclass typing IgG against bee venom and bee venom components.

Specific IgE antibodies against bee venom and its components were studied in 23 bee-keepers. The highest IgG serum levels were observed for whole bee venom followed by phospholipase A. The serum levels of specific IgG antibodies against melittin and MCD-peptide were lower, the lowest serum levels being observed for apamin. After a 5 month absence from bee-keeping a fall in the serum levels of IgG antibodies was observed in all the bee-keepers studied. The investigation of the IgG subclass antibodies 1-4 against bee venom and phospholipase A demonstrated the highest serum levels for IgG 4 and IgG 2, the lowest levels were observed for IgG 1. The lowest IgG serum levels were associated with the least effective protection to bee stings. These findings support the concept that specific IgG antibodies prevent the development of allergic symptoms after bee sting.