Aboutorabian Sepideh, Hakimi Jalil, Boudet Florence, Montano Sandrine, Dookie Annie, Roque Cristopher, Ausar Salvador F, Rahman Nausheen, Brookes Roger H
a Sanofi Pasteur; Bioprocess Research and Development ; Toronto , Canada.
Hum Vaccin Immunother. 2015;11(6):1449-55. doi: 10.1080/21645515.2015.1023970.
A tuberculosis (TB) vaccine consisting of a recombinant fusion protein (H4) and a novel TLR9 adjuvant (IC31) is in clinical development. To better understand the H4-IC31 ratio, we measured the binding capacity of IC31 for H4 protein and immunized mice with formulations that contained limiting to excess ratios of IC31 to H4. An immunomodulated H4-specific IFNγ response was only observed when IC31 was present in excess of H4. Since TLR expression is species-specific and the vaccine is intended to boost BCG-primed immunity, we questioned whether data in mice would translate to humans. To address this question, we used the fresh human Whole Blood (hWB) recovered from BCG-vaccinated subjects to screen H4-IC31 formulations. We found IC31 modulation in hWB to be quite distinct from the TLR4-Adjuvant. Unlike TLR4-Adjuvant, IC31 formulations did not induce the pro-inflammatory cytokine TNFα, but modulated a robust H4-specific IFNγ response after 12 d of culture. We then re-stimulated the fresh hWB of 5 BCG-primed subjects with formulations that had excess or limiting IC31 binding for H4 protein and again found that an immunomodulated H4-specific IFNγ response needed an excess of IC31. Finally, we monitored the zeta (ζ) potential of H4-IC31 formulations and found that the overall charge of H4-IC31 particles changes from negative to positive once IC31 is in greater than 9-fold excess. Using two diverse yet mutually supportive approaches, we confirm the need for an excess of IC31 adjuvant in H4 TB vaccine formulations and suggest surface potential may be an important factor.
一种由重组融合蛋白（H4）和新型TLR9佐剂（IC31）组成的结核病（TB）疫苗正处于临床开发阶段。为了更好地了解H4与IC31的比例，我们测量了IC31与H4蛋白的结合能力，并用含有从IC31与H4的限量到过量比例的制剂免疫小鼠。仅当IC31过量于H4时才观察到免疫调节的H4特异性IFNγ反应。由于TLR表达具有物种特异性，且该疫苗旨在增强卡介苗引发的免疫力，我们质疑小鼠的数据是否能转化到人类身上。为了解决这个问题，我们使用从接种卡介苗的受试者中回收的新鲜人全血（hWB）来筛选H4-IC31制剂。我们发现hWB中的IC31调节与TLR4佐剂截然不同。与TLR4佐剂不同，IC31制剂不会诱导促炎细胞因子TNFα，但在培养12天后调节了强烈的H4特异性IFNγ反应。然后，我们用对H4蛋白具有过量或限量IC31结合的制剂再次刺激5名接种卡介苗受试者的新鲜hWB，再次发现免疫调节的H4特异性IFNγ反应需要过量的IC31。最后，我们监测了H4-IC31制剂的zeta（ζ）电位，发现一旦IC31过量超过9倍，H4-IC31颗粒的总体电荷就会从负变为正。通过两种不同但相互支持的方法，我们证实了H4结核病疫苗制剂中需要过量的IC31佐剂，并表明表面电位可能是一个重要因素。
