Pharmaceutical and preclinical evaluation of Advax adjuvant as a dose-sparing strategy for ant venom immunotherapy.

A major challenge in broader clinical application of Jack Jumper ant venom immunotherapy (JJA VIT) is the scarcity of ant venom which needs to be manually harvested from wild ants. Adjuvants are commonly used for antigen sparing in other vaccines, and thereby could potentially have major benefits to extend JJA supplies if they were to similarly enhance JJA VIT immunogenicity. The purpose of this study was to evaluate the physicochemical and microbiological stability and murine immunogenicity of low-dose JJA VIT formulated with a novel polysaccharide adjuvant referred to as delta inulin or Advax™. Jack Jumper ant venom (JJAV) protein stability was assessed by UPLC-UV, SDS-PAGE, SDS-PAGE immunoblot, and ELISA inhibition. Diffraction light scattering was used to assess particle size distribution of Advax; pH and benzyl alcohol quantification by UPLC-UV were used to assess the physicochemical stability of JJAV diluent, and endotoxin content and preservative efficacy test was used to investigate the microbiological properties of the adjuvanted VIT formulation. To assess the effect of adjuvant on JJA venom immunogenicity, mice were immunised four times with JJAV alone or formulated with Advax adjuvant. JJA VIT formulated with Advax was found to be physicochemically and microbiologically stable for at least 2 days when stored at 4 and 25 °C with a trend for an increase in allergenic potency observed beyond 2 days of storage. Low-dose JJAV formulated with Advax adjuvant induced significantly higher JJAV-specific IgG than a 5-fold higher dose of JJAV alone, consistent with a powerful allergen-sparing effect. The pharmaceutical data provides important guidance on the formulation, storage and use of JJA VIT formulated with Advax adjuvant, with the murine immunogenicity studies providing a strong rationale for a planned clinical trial to test the ability of Advax adjuvant to achieve 4-fold JJAV dose sparing in JJA-allergic human patients.

Factors influencing the quality of Myrmecia pilosula (Jack Jumper) ant venom for use in in vitro and in vivo diagnoses of allergen sensitization and in allergen immunotherapy.

BACKGROUND: Allergen immunotherapy uses pharmaceutical preparations derived from naturally occurring source materials, which contain water-soluble allergenic components responsible for allergic reactions. The success of in vivo and in vitro diagnoses in allergen sensitization and allergen immunotherapy largely depends on the quality, composition and uniformity of allergenic materials used to produce the active ingredients, and the formulation employed to prepare finished products. OBJECTIVES: We aimed to examine the factors influencing batch-to-batch consistency of Jack Jumper (Myrmecia pilosula) ant venom (JJAV) in the form of active pharmaceutical ingredient (AI) and informed whether factors such as temperature, artificial light and container materials influence the quality of JJAV AIs. We also aimed to establish handling and storage requirements of JJAV AIs to ensure preservation of allergenic activities during usage in the diagnosis of allergen sensitization and in allergen immunotherapy. METHODS: The quality and consistency of JJAV AIs were analysed using a combination of bicinchoninic acid assay for total protein quantification, HPLC-UV for JJAV allergen peptides quantification, ELISA inhibition for total allergenic potency, SDS-PAGE, AU-PAGE and immunoblot for qualitative assessment of JJAV components, and Limulus Amebocyte Lysate assay for the quantification of endotoxin concentration. API-ZYM and Zymogram assays were used to probe the presence of enzymatic activities in JJAV. RESULTS: Pharmaceutical-grade JJAV for allergen immunotherapy has good batch-to-batch consistency. Temporary storage at 4°C and light exposure do not affect the quality of JJAV. Exposure to temperature above 40°C degrades high MW allergens in JJAV. Vials containing JJAV must be stored frozen and in upright position during long-term storage. CONCLUSIONS AND CLINICAL RELEVANCE: We have identified factors, which can influence the quality and consistency of JJAV AIs, and provided a framework for appropriate handling, transporting and storage of JJAV to be used for the diagnosis of allergen sensitization and in AIT.

Antiparasitic effect of Dinoponera quadriceps giant ant venom.

Neglected tropical diseases (NTD) are treated with toxic therapy of limited efficacy. Previously, we studied the antimicrobial effect of Dinoponera quadriceps venom (DqV) against bacteria. To continue the study, we report in this short communication the antimicrobial effect of DqV against Leishmania amazonensis and Trypanosoma cruzi. DqV inhibits the promastigote forms of L. amazonensis and all T. cruzi developmental forms, with low toxicity in host cells. DqV causes cell death in T. cruzi through necrotic and apoptotic mechanisms observed by staining the cells with annexin V-FITC (AX) and propidium iodide (PI), loss of mitochondrial membrane potential by flow cytometry analyses and confocal microscopy and morphological alterations, such as loss of membrane integrity and cell shrinkage by scanning electron microscopy (SEM). In conclusion, we suggest there is an antimicrobial effect also on parasites.

Influence of dose and frequency of antigen injection on IgE development in young children: a comparison of fire ant stings and tetanus immunizations.

BACKGROUND: Previous studies suggest that small antigen doses given frequently are more likely to induce IgE production than are large antigen doses given infrequently. OBJECTIVE: To compare the prevalence of antitetanus IgE resulting from the relatively large dose of tetanus toxoid delivered by standard immunizations at 2, 4, 6, and 18 months of age with the previously reported prevalence of anti-fire ant venom IgE resulting from the relatively small dose of venom delivered sporadically by accidental fire ant stings in children younger than 5 years. METHODS: This study uses previously published data on the prevalence of IgE antibodies to imported fire ant venom among children living in an imported fire ant endemic area of Georgia and antitetanus IgE measurements of children recruited between August 1, 2003, and December 30, 2007, as part of the Wayne County Health, Environment, Allergy, and Asthma Longitudinal Study in Michigan, where there are no imported fire ants. The prevalence of anti-fire ant venom IgE antibodies was compared with the prevalence of antitetanus IgE antibodies in these 2 cohorts of children. RESULTS: The reported prevalence of IgE to fire ant venom among 42 children 2 to 5 years old was 57.1% using a cutoff of 0.1 IU/mL and 35.7% using a cutoff of 0.35 IU/mL. The prevalence of antitetanus IgE in 395 children 2 years old was 52.9% using a cutoff of 0.1 IU/mL and 42.7% using a cutoff of 0.35 IU/mL. The proportion of children with detectable anti-fire ant venom IgE was not statistically significantly different from the proportion of those with antitetanus IgE at either cutoff level (P = .74 and .50 at 0.1 and 0.35 IU/mL, respectively). CONCLUSIONS: The relatively large dose of tetanus toxoid delivered 4 times during the first 24 months of life produces detectable tetanus specific IgE antibodies as frequently as the smaller doses of venom delivered sporadically by fire ant stings in young children.

Imported fire ant immunotherapy prescribing patterns in a large health care system during a 17-year period.

BACKGROUND: No large evaluation has been performed of the maintenance vial concentration commonly used by physicians when prescribing imported fire ant (IFA) immunotherapy since the publication of the first Stinging Insect Hypersensitivity Practice Parameter 10 years ago. OBJECTIVE: To describe the prescribing patterns for IFA immunotherapy among practicing allergists in a large health care setting and the impact of published Practice Parameter recommendations. METHODS: Data from the US Army Centralized Allergen Extract Laboratory were analyzed to determine IFA immunotherapy prescribing patterns from 1990 to May 2007. This extract laboratory provides prescriptions for more than 320 US Department of Defense, US Department of Veterans Affairs, and US Public Health Service clinics. RESULTS: A total of 1,091 patients were given 1,437 new or revised prescriptions for IFA immunotherapy. Monotherapy for Solenopsis invicta and Solenopsis richteri was prescribed in 169 (11.8%) and 3 (0.1%) instances, respectively, with the remainder of patients given both IFA antigens. The most commonly prescribed maintenance vial dose was 0.5 mL of a 1:200 (wt/vol) dilution, accounting for 36.3% of prescriptions. A total of 17.3% of prescriptions had a maintenance vial dose of 0.5 mL of a 1:100 (wt/vol) dilution, 4.6% had a dilution of 1:10 (wt/vol), and 50.6% had a dilution between 1:10 and 1:100 (wt/vol). The mean starting dose was 4.4 10-fold dilutions below the maintenance dose (5.4 vials per treatment set). CONCLUSIONS: The most commonly prescribed maintenance dose was 0.5 mL of a 1:200 (wt/vol) dilution, although most prescriptions used a maintenance dose consistent with recommended dosing in the Stinging Insect Practice Parameters. Both IFA antigens were used by most physicians. Further study evaluating the effective dose range for IFA immunotherapy is needed.

Ant venom immunotherapy: a double-blind, placebo-controlled, crossover trial.

BACKGROUND: The jack jumper ant Myrmecia pilosula is responsible for about 90% of ant venom anaphylaxis in southeastern Australia. We aimed to establish whether M pilosula venom immunotherapy (VIT) prevents lifethreatening sting anaphylaxis in otherwise healthy adults. METHODS: We did a double-blind, placebo-controlled crossover trial of M pilosula VIT. Participants were randomly allocated either immunotherapy, in accordance with the semirush hyposensitisation regimen, or placebo. The primary endpoint was systemic reaction after a deliberate sting challenge. Analysis was per protocol. FINDINGS: We randomly allocated 68 healthy volunteers (aged 20-63 years) who were allergic to M pilosula venom to placebo (33) and VIT (35). Four on placebo were stopped early and 12 on VIT had their treatment allocations revealed before the sting challenge, thus 29 on placebo and 23 on VIT were included in the primary analysis. Objectively defined systemic reactions to sting challenges arose in 21 of 29 participants (72%) on placebo (8 reactions were associated with hypotension) and none of 23 on VIT (p<0.0001). Of the remaining 12 on VIT who underwent sting challenges after treatment allocations were revealed, only one reacted to sting challenge with transient urticaria that did not require treatment. After crossover of the placebo group to VIT, one of 26 had a reaction to sting challenge (transient urticaria). In all patients who had VIT, we recorded objective systemic reactions in 22 of 64 (34%) during VIT; two of which were hypotensive. INTERPRETATION: In well motivated, highly allergic, but otherwise healthy adults, VIT is highly effective in prevention of M pilosula sting anaphylaxis. The risk of systemic reactions during VIT means that treatment should be given where there is immediate access to resuscitation facilities.

Comparison of in vivo and in vitro tests in the diagnosis of imported fire ant sting allergy.

The specificities and sensitivities of skin test reactivity to imported fire ant (IFA) whole body extract (WBE) and IFA venom were compared with IFA WBE RAST and IFA venom RAST in the diagnosis of IFA allergy. Study groups consisted of 18 IFA allergic patients and 21 control subjects with no history of allergy to insect stings. All IFA allergic patients had positive skin tests to both IFA WBE and IFA venom. Six of 21 (29%) control subjects also had positive skin tests to both IFA WBE and IFA venom. A commercial IFA WBE RAST was positive in 10 of 18 (56%) IFA-allergic patients and 2 of 21 (10%) control subjects. Imported fire ant aqueous venom RAST was positive in 11 of 11 (100%) IFA-allergic patients and three of ten (30%) control subjects. Vespa IFA venom RAST was positive in 16 of 18 (89%) IFA-allergic patients and 5 of 21 (24%) controls. The sensitivities and specificities of IFA WBE skin testing, IFA venom skin testing, and IFA venom RAST did not differ significantly. Imported fire ant WBE RAST was less sensitive than the other diagnostic methods.