The development of Jack Jumper ant venom immunotherapy: our 25 years' experience.

Jack Jumper ant venom allergy is a uniquely Australian medical issue. The stinging ant is a leading cause of insect venom allergy in south-eastern Australia. An effective venom immunotherapy-based treatment was successfully developed by the Tasmanian Jack Jumper Allergy Research group. This paper provides a synopsis of our 25 years' research journey in developing this evidence-based treatment modality.

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.

Immediate-type hypersensitivity drug reactions.

Hypersensitivity reactions including anaphylaxis have been reported for nearly all classes of therapeutic reagents and these reactions can occur within minutes to hours of exposure. These reactions are unpredictable, not directly related to dose or the pharmacological action of the drug and have a relatively high mortality risk. This review will focus on the clinical presentation, immune mechanisms, diagnosis and prevention of the most serious form of immediate onset drug hypersensitivity reaction, anaphylaxis. The incidence of drug-induced anaphylaxis deaths appears to be increasing and our understanding of the multiple and complex reasons for the unpredictable nature of anaphylaxis to drugs is also expanding. This review highlights the importance of enhancing our understanding of the biology of the patient (i.e. immune response, genetics) as well as the pharmacology and chemistry of the drug when investigating, diagnosing and treating drug hypersensitivity. Misdiagnosis of drug hypersensitivity leads to substantial patient risk and cost. Although oral provocation is often considered the gold standard of diagnosis, it can pose a potential risk to the patient. There is an urgent need to improve and standardize diagnostic testing and desensitization protocols as other diagnostic tests currently available for assessment of immediate drug allergy are not highly predictive.

Ultrarush versus semirush initiation of insect venom immunotherapy: a randomized controlled trial.

BACKGROUND: Venom immunotherapy can be initiated by different schedules, but randomized comparisons have not been performed. OBJECTIVE: We aimed to compare the safety of 2 initiation schedules. METHODS: Patients of any age with prior immediate generalized reactions to jack jumper ant (Myrmecia pilosula) stings were randomized to venom immunotherapy initiation by a semirush schedule over 10 visits (9 weeks) or an ultrarush schedule over 3 visits (2 weeks). In a concurrent treatment efficacy study, the target maintenance dose was randomized to either 50 µg or 100 µg. The primary outcome was the occurrence of 1 or more objective systemic reactions during venom immunotherapy initiation. Analyses were by intention to treat. We also assessed outcomes in patients who declined randomization. RESULTS: Of 213 eligible patients, 93 were randomized to semirush (44 patients) or ultrarush (49 patients) initiation. Objective systemic reactions were more likely during ultrarush initiation (65% vs 29%; P < .001), as were severe reactions (12% vs 0%; P= .029). Times to maximal increases in venom-specific IgG(4) were no different between treatments, whereas the maximal increase in venom-specific IgE occurred earlier with ultrarush treatment. Similar differences between methods were observed in patients who declined randomization. One hundred seventy-eight patients were randomized to maintenance doses of either 50 µg (90 patients) or 100 µg (88 patients). The target maintenance dose had no effect on the primary outcome, but multiple-failure-per-subject analysis found that the 50 µg dose reduced the likelihood of reactions. CONCLUSION: Ultrarush initiation increases the risk of systemic reactions. A lower maintenance dose reduces the risk of repeated reactions, but the effect on treatment efficacy is unknown.

Causes of ant sting anaphylaxis in Australia: the Australian Ant Venom Allergy Study.

OBJECTIVE: To determine the Australian native ant species associated with ant sting anaphylaxis, geographical distribution of allergic reactions, and feasibility of diagnostic venom-specific IgE (sIgE) testing. DESIGN, SETTING AND PARTICIPANTS: Descriptive clinical, entomological and immunological study of Australians with a history of ant sting anaphylaxis, recruited in 2006-2007 through media exposure and referrals from allergy practices and emergency physicians nationwide. We interviewed participants, collected entomological specimens, prepared reference venom extracts, and conducted serum sIgE testing against ant venom panels relevant to the species found in each geographical region. MAIN OUTCOME MEASURES: Reaction causation attributed using a combination of ant identification and sIgE testing. RESULTS: 376 participants reported 735 systemic reactions. Of 299 participants for whom a cause was determined, 265 (89%; 95% CI, 84%-92%) had reacted clinically to Myrmecia species and 34 (11%; 95% CI, 8%-16%) to green-head ant (Rhytidoponera metallica). Of those with reactions to Myrmecia species, 176 reacted to jack jumper ant (Myrmecia pilosula species complex), 18 to other jumper ants (15 to Myrmecia nigrocincta, three to Myrmecia ludlowi) and 56 to a variety of bulldog ants, with some participants reacting to more than one type of bulldog ant. Variable serological cross-reactivity between bulldog ant species was observed, and sera from patients with bulldog ant allergy were all positive to one or more venoms extracted from Myrmecia forficata, Myrmecia pyriformis and Myrmecia nigriceps. CONCLUSION: Four main groups of Australian ants cause anaphylaxis. Serum sIgE testing enhances the accuracy of diagnosis and is a prerequisite for administering species-specific venom immunotherapy.

Stability of Myrmecia pilosula (Jack Jumper) Ant venom for use in immunotherapy.

Allergy to Myrmecia pilosula (Jack Jumper Ant) venom is common in Australia, affecting ∼2.7% of some communities. Venom immunotherapy is a highly effective treatment, but for the venom to be widely distributed for clinical use, the stability and shelf-life of formulated Jack Jumper Ant venom must be demonstrated. HPLC-UV, ELISA Inhibition, SDS-PAGE and SDS-PAGE Immunoblot were used to assess venom stability under conditions of varying temperature, pH and in the presence of various stabilising agents. Optimal stability occurred between pH 8 and 10, however the presence of benzyl alcohol within this pH range resulted in a cloudy appearance within 3 days, so a pH of 6 was used. Increasing polysorbate 80 concentrations accelerated the degradation of allergenic peptides in 100 µg/mL venom, but improved stability at concentrations of 1 µg/mL or less. Sucrose reduced degradation of allergens Myr p 1 and Myr p 3, whilst glycerol was destabilizing. In the presence of 22% sucrose, 1.1mg/mL Jack Jumper Ant venom was stable at -18 °C and 4 °C for 12 months; following dilution to 100 µg/mL with 0.9% sodium chloride, 10mM phosphate (pH 6), 0.05% polysorbate 80 and 0.9% benzyl alcohol (giving 2% sucrose), venom was stable for 7 days when stored at 4 °C. Concentrated Jack Jumper Ant venom can be stored in 22% sucrose for 12 months, and after dilution to 100 µg/mL for clinical use, it should be discarded after 7 days.

Myrmecia pilosula (Jack Jumper) ant venom: validation of a procedure to standardise an allergy vaccine.

Ant sting allergy is relatively common within south-eastern Australia and is predominantly due to Myrmecia pilosula (Jack Jumper Ant, JJA). Venom immunotherapy has been shown to be effective in preventing anaphylaxis to the sting of the JJA, but analytical techniques to standardise the venom have not been validated. The purpose of this study was to develop assays to analyse JJA venom and apply these to the standardisation of venom prior to new batches being used for the diagnosis and treatment of JJA sting allergy. Venom was analysed by protein content, HPLC-UV, enzyme-linked immunosorbent assay (ELISA) inhibition, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and SDS-PAGE immunoblot. The protein content in JJA venom was adjusted so that all batches were equivalent. A HPLC-UV assay was used to quantify the relative amount of the major allergen Myr p 2 and two minor allergens Myr p 1 and Myr p 3 and allergenic potency was determined by ELISA inhibition. SDS-PAGE and SDS-PAGE immunoblot were used as qualitative tools to determine the protein profile and presence or absence of additional high molecular weight allergens not quantifiable by HPLC-UV. A standardisation procedure has been developed that complies with the requirements described in the European Pharmacopoeia. Techniques used to determine the content of some of the other minor allergens could be developed, which would further improve the standardisation methodology.

Proteomic analysis of Myrmecia pilosula (jack jumper) ant venom.

Ant sting allergy in Australia is predominantly due to the Myrmecia pilosula species complex. Gel separation of M. pilosula venom is necessary so that the allergenic importance of each component can be defined by western blotting. However, previous PAGE methods produced suboptimal resolution and the components of each band were not precisely defined. Venom was resolved in both non-reduced and reduced form by one-dimensional acid urea PAGE, SDS-PAGE and two-dimensional acid urea-SDS PAGE. Resolved peptides were extracted and analysed by HPLC-MS. Acid urea PAGE and acid urea-SDS PAGE proved more effective than SDS-PAGE for resolution of peptides smaller than 10 kDa. All of the major peptides previously observed in M. pilosula venom were observed in gel resolved venom. Venom was found to primarily consist of peptides with molecular weight <10 kDa, most of which contain disulfide bridges. SDS-PAGE of non-reduced venom clearly defined six higher molecular weight proteins between 26 and 90 kDa. An 8546 Da dimer named pilosulin 5 was observed, but pilosulin 4, a peptide recently proposed to be present in venom was not. A variant of pilosulin 4 here named pilosulin 4.1a, existing as an 8198 Da dimer, was observed and has been characterised.

Non-conventional approaches to allergy testing: reconciling patient autonomy with medical practitioners' concerns.

It may be difficult for patients to distinguish current concepts of immune function from other, non-conventional explanations of illness.

Optimising the management of allergic rhinitis: an Australian perspective.

Allergic rhinitis (AR) is one of the most prevalent medical conditions. It has significant effects on quality of life and can have considerable socioeconomic effects. The traditional classification of perennial and seasonal rhinitis does not distinguish between provoking factors, nor does it indicate the most appropriate treatment. A more useful classification is based on symptoms, which may be intermittent or persistent, and vary widely in severity. The goal of management is to achieve optimal symptom control. Therapeutic options include allergen avoidance, pharmacotherapy and immunotherapy. Antihistamines and intranasal corticosteroids (INCS) have become the cornerstones of therapy. A variety of effective treatments are available for consumers to self-select, without the advice of a doctor or pharmacist. INCS are widely recognised as the most effective pharmacotherapy for AR, in both adults and children. The efficacy of various preparations is similar, but those with low systemic bioavailability are preferred for children and for patients who are also receiving inhaled, topical or systemic corticosteroids.

Can serum mast cell tryptase help diagnose anaphylaxis?

OBJECTIVE: We aimed to determine the utility of serum mast cell tryptase to diagnose anaphylaxis. METHODS: As part of a venom immunotherapy trial, we performed 64 sting challenges. Blood samples were taken before the sting (baseline), and 15 min and 60 min after the sting. Tryptase was measured in baseline, 15 minute and 60 minute serum samples. Histamine was measured in baseline and 15 minute plasma samples. Eleven people had undisputed severe anaphylactic reactions; tryptase and histamine levels were assessed against this clinical gold standard diagnosis. RESULTS: Excluding mild reactions from the analysis, peak tryptase readings had sensitivity of 0.36 and specificity of 0.93 using the recommended cut-off range (< 12.0 microg/L). Receiver-operator curve analysis found a cut-off of 9.0 microg/L would improve diagnostic performance (sensitivity 0.55, specificity 0.93). Serial tryptase measurement was significantly more discriminatory; an increase in tryptase of 2.0 microg/L or greater had a sensitivity of 0.73 and specificity of 0.98. The addition of histamine measurements, defining a positive result by either a rise in tryptase or a rise in histamine, appeared to further increase sensitivity (0.90). CONCLUSIONS: Clinicians should use caution when using serum tryptase to refute or support a diagnosis of anaphylaxis. Serial tryptase measurement increases sensitivity and specificity. Further studies using serial tryptase determinations in general emergency department practice, perhaps supplemented by histamine determinations, are warranted.

Prevention of anaphylaxis with ant venom immunotherapy.

PURPOSE OF REVIEW: Worldwide, eight genera of ants have been associated with sting allergy. Until recently only whole ant body extracts have been used for immunotherapy. The purpose of this review is to examine recent advances in the understanding of ant venom allergy and treatment using venom immunotherapy. RECENT FINDINGS: Public health problems due to severe ant sting anaphylaxis are not confined to the imported fire ant of North America. Pachycondyla sennaarensis (samsum ant), Pachycondyla chinensis, and Myrmecia pilosula (jack jumper ant) also appear to pose notable threats. The risk to humans from a particular species probably depends on complex interactions between likelihood of human contact, insect aggression, efficiency of the venom delivery apparatus, and venom allergenicity. The highest population prevalence of clinical ant sting allergy so far (3.0%) was reported from south-eastern Australia, due mainly to M. pilosula. Prospective follow-up of untreated people suggests that those older than 30 years with a history of severe reactions (respiratory compromise or hypotension) will benefit most from venom immunotherapy. Whereas the efficacy of ant whole body extract immunotherapy remains to be proven, ant venom immunotherapy has been demonstrated to reduce the risk of systemic reactions to M. pilosula from 72% to 3%. Although a simple method of venom extraction has been developed, small market size means that the treatment may never become widely available. SUMMARY: Ant venom immunotherapy is feasible and highly efficacious. However, the limited geographical distribution of each species presents a major challenge to making venom extracts available for clinical use.

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.

Prevalence, severity, and natural history of jack jumper ant venom allergy in Tasmania.

BACKGROUND: The jack jumper ant (Myrmecia pilosula) is responsible for greater than 90% of Australian ant venom allergy. However, deaths have only been recorded in the island of Tasmania. OBJECTIVES: We sought to determine the prevalence, clinical features, natural history, and predictors of severity of M pilosula sting allergy in Tasmania. METHODS: We performed a random telephone survey supported by serum venom-specific IgE analysis, review of emergency department presentations, and follow-up of allergic volunteers. RESULTS: M pilosula, honeybee (Apis mellifera), and yellow jacket wasp (Vespula germanica) sting allergy prevalences were 2.7%, 1.4%, and 0.6% compared with annual sting exposure rates of 12%, 7%, and 2%, respectively. Similarly, emergency department presentations with anaphylaxis to M pilosula were double those for honeybee. M pilosula allergy prevalence increased with age of 35 years or greater (odds ratio [OR], 2.4) and bee sting allergy (OR, 16.9). Patients 35 years of age or older had a greater risk of hypotensive reactions (OR, 2.9). Mueller reaction grades correlated well with adrenaline use. During follow-up, 79 (70%) of 113 jack jumper stings caused anaphylaxis. Prior worst reaction severity predicted the likelihood and severity of follow-up reactions; only 3 subjects had more severe reactions. Venom-specific IgE levels and other clinical features, including comorbidities, were not predictive of severity. CONCLUSIONS: Sting allergy prevalence is determined by age and exposure rate. M pilosula sting exposure in Tasmania is excessive compared with that found in mainland Australia, and there is a high systemic reaction risk in allergic people on re-sting. Prior worst reaction severity (Mueller grade) and age predict reaction severity and might be used to guide management.