Clapp Tanya, Munks Michael W, Trivedi Ruchit, Kompella Uday B, Braun LaToya Jones
Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Denver, 12850 E. Montview Boulevard, C238, Aurora, CO 80045, United States.
Integrated Department of Immunology, National Jewish Health, 1400 Jackson Street, Denver, CO 80206, United States.
Vaccine. 2014 Jun 24;32(30):3765-71. doi: 10.1016/j.vaccine.2014.05.037. Epub 2014 May 20.
Preventing losses in vaccine potency due to accidental freezing has recently become a topic of interest for improving vaccines. All vaccines with aluminum-containing adjuvants are susceptible to such potency losses. Recent studies have described excipients that protect the antigen from freeze-induced inactivation, prevent adjuvant agglomeration and retain potency. Although these strategies have demonstrated success, they do not provide a mechanistic understanding of freeze-thaw (FT) induced potency losses. In the current study, we investigated how adjuvant frozen in the absence of antigen affects vaccine immunogenicity and whether preventing damage to the freeze-sensitive recombinant hepatitis B surface antigen (rHBsAg) was sufficient for maintaining vaccine potency. The final vaccine formulation or Alhydrogel(®) alone was subjected to three FT-cycles. The vaccines were characterized for antigen adsorption, rHBsAg tertiary structure, particle size and charge, adjuvant elemental content and in-vivo potency. Particle agglomeration of either vaccine particles or adjuvant was observed following FT-stress. In vivo studies demonstrated no statistical differences in IgG responses between vaccines with FT-stressed adjuvant and no adjuvant. Adsorption of rHBsAg was achieved; regardless of adjuvant treatment, suggesting that the similar responses were not due to soluble antigen in the frozen adjuvant-containing formulations. All vaccines with adjuvant, including the non-frozen controls, yielded similar, blue-shifted fluorescence emission spectra. Immune response differences could not be traced to differences in the tertiary structure of the antigen in the formulations. Zeta potential measurements and elemental content analyses suggest that FT-stress resulted in a significant chemical alteration of the adjuvant surface. This data provides evidence that protecting a freeze-labile antigen from subzero exposure is insufficient to maintain vaccine potency. Future studies should focus on adjuvant protection. To our knowledge, this is the first study to systematically investigate how FT-stress to adjuvant alone affects immunogenicity. It provides definitive evidence that this damage is sufficient to reduce vaccine potency.
防止因意外冷冻导致疫苗效力损失最近已成为改善疫苗的一个关注话题。所有含铝佐剂的疫苗都易受此类效力损失的影响。最近的研究描述了一些辅料，它们可保护抗原免受冷冻诱导的失活，防止佐剂聚集并保持效力。尽管这些策略已取得成功，但它们并未提供对冻融（FT）诱导的效力损失的机制理解。在本研究中，我们调查了在无抗原情况下冷冻的佐剂如何影响疫苗免疫原性，以及防止对冻敏的重组乙型肝炎表面抗原（rHBsAg）造成损伤是否足以维持疫苗效力。最终疫苗制剂或单独的氢氧化铝佐剂（Alhydrogel®）经历了三个冻融循环。对疫苗进行了抗原吸附、rHBsAg三级结构、粒径和电荷、佐剂元素含量及体内效力的表征。在冻融应激后观察到疫苗颗粒或佐剂的颗粒聚集。体内研究表明，经冻融应激佐剂处理的疫苗与无佐剂疫苗之间的IgG反应无统计学差异。实现了rHBsAg的吸附；无论佐剂处理如何，这表明相似的反应并非由于含冷冻佐剂制剂中的可溶性抗原所致。所有含佐剂的疫苗，包括未冷冻的对照，都产生了相似的蓝移荧光发射光谱。免疫反应差异无法追溯到制剂中抗原三级结构的差异。zeta电位测量和元素含量分析表明，冻融应激导致佐剂表面发生显著化学变化。这些数据提供了证据，表明保护对冷冻敏感的抗原免于零下暴露不足以维持疫苗效力。未来的研究应聚焦于佐剂保护。据我们所知，这是第一项系统研究单独对佐剂进行冻融应激如何影响免疫原性的研究。它提供了确凿证据，证明这种损伤足以降低疫苗效力。
