Jack Jumper Allergy Program, Royal Hobart Hospital, GPO Box 1061L, Hobart, Tasmania, 7001, Australia; Division of Pharmacy, School of Medicine, University of Tasmania, Private Bag 26, Hobart, Tasmania, 7001, Australia; School of Medicine, University of Tasmania, Private Bag 68, Hobart, Tasmania, 7001, Australia; Department of Pharmacy, Royal Hobart Hospital, GPO Box 1061L, Hobart, Tasmania, 7001, Australia.
Flinders University, Bedford Park, South Australia, 5042, Australia; Vaxine Pty Ltd, Bedford Park, Adelaide, 5042, Australia.
J Pharm Biomed Anal. 2019 Aug 5;172:1-8. doi: 10.1016/j.jpba.2019.04.017. Epub 2019 Apr 9.
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.
在更广泛的临床应用中，跳蚁毒液免疫疗法（JJA VIT）面临的一个主要挑战是抗毒液的稀缺，这种毒液需要从野外蚂蚁中手动采集。佐剂常用于其他疫苗中的抗原节省，因此如果它们能够类似地增强 JJA VIT 免疫原性，那么它们有可能为扩大 JJA 供应带来重大益处。本研究的目的是评估低剂量 JJA VIT 与新型多糖佐剂 delta 菊粉或 Advax™联合使用的物理化学和微生物稳定性以及小鼠免疫原性。通过 UPLC-UV、SDS-PAGE、SDS-PAGE 免疫印迹和 ELISA 抑制评估跳蚁毒液（JJAV）蛋白稳定性。使用衍射光散射评估 Advax 的粒径分布；通过 UPLC-UV 评估 JJAV 稀释剂的物理化学稳定性，使用内毒素含量和防腐剂效力测试评估佐剂 VIT 制剂的微生物特性。为了评估佐剂对 JJA 毒液免疫原性的影响，用 JJAV 单独或与 Advax 佐剂联合免疫小鼠四次。Advax 佐剂配制的 JJA VIT 在 4°C 和 25°C 下储存至少 2 天，其物理化学和微生物稳定性良好，储存超过 2 天，观察到变应原效力增加的趋势。与单独使用 5 倍剂量的 JJAV 相比，Advax 佐剂配制的低剂量 JJAV 诱导的 JJAV 特异性 IgG 显著更高，这与强大的抗原节省效应一致。药物数据为 Advax 佐剂配制的 JJA VIT 的配方、储存和使用提供了重要指导，而小鼠免疫原性研究为计划进行的临床试验提供了有力的依据，以测试 Advax 佐剂在 JJA 过敏人类患者中实现 4 倍 JJAV 剂量节省的能力。
