Protective antigen (PA) is a component of anthrax toxin that can elicit toxin-neutralizing antibody responses. PA is also the major antigen in the current vaccine to prevent anthrax, but stability problems with recombinant proteins have complicated the development of new vaccines containing recombinant PA. The relationship between antigen physical stability and immunogenicity is poorly understood, but there are theoretical reasons to think that this parameter can affect immune responses. We investigated the immunogenicity of anthrax PA, in the presence and absence of the soluble von Willebrand factor A domain of the human form of receptor capillary morphogenesis protein 2 (sCMG2), to elicit antibodies to PA in BALB/c mice. Prior studies showed that sCMG2 stabilizes the 83-kDa PA structure to pH, chemical denaturants, temperature, and proteolysis and slows the hydrogen-deuterium exchange rate of histidine residues far from the binding interface. In contrast to a vaccine containing PA without adjuvant, we found that mice immunized with PA in stable complex with sCMG2 showed markedly reduced antibody responses to PA, including toxin-neutralizing antibodies and antibodies to domain 4, which correlated with fewer toxin-neutralizing antibodies. In contrast, mice immunized with PA in concert with a nonbinding mutant of sCMG2 (D50A) showed anti-PA antibody responses similar to those observed with PA alone. Our results suggest that addition of sCMG2 to a PA vaccine formulation is likely to result in a significantly diminished immune response, but we discuss the multitude of factors that could contribute to reduced immunogenicity.IMPORTANCE The anthrax toxin PA is the major immunogen in the current anthrax vaccine (anthrax vaccine adsorbed). Improving the anthrax vaccine for avoidance of a cold chain necessitates improvements in the thermodynamic stability of PA. We address how stabilizing PA using sCMG2 affects PA immunogenicity in BALB/c mice. Although the stability of PA is increased by binding to sCMG2, PA immunogenicity is decreased. This study emphasizes that, while binding of a ligand retains or improves conformational stability without affecting the native sequence, epitope recognition or processing may be affected, abrogating an effective immune response.

中文翻译：

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毛细血管生成蛋白2的血管性假血友病因子A域与炭疽保护性抗原疫苗的结合降低了小鼠的免疫原性。

保护性抗原（PA）是炭疽毒素的一种成分，可引起毒素中和抗体反应。PA也是目前预防炭疽病的疫苗中的主要抗原，但是重组蛋白的稳定性问题使含有重组PA的新疫苗的开发变得复杂。抗原物理稳定性和免疫原性之间的关系了解甚少，但是有理论上的理由认为该参数会影响免疫反应。我们调查了炭疽PA的免疫原性，在人类毛细血管形态发生蛋白2（sCMG2）的人类形式的可溶性von Willebrand因子A域的存在和不存在下，在BALB / c小鼠中引发针对PA的抗体。先前的研究表明，sCMG2可将83 kDa的PA结构稳定在pH，化学变性剂，温度，和蛋白水解作用，减慢了远离结合界面的组氨酸残基的氢-氘交换速率。与不含佐剂的PA疫苗相比，我们发现用sCMG2稳定复合物的PA免疫的小鼠对PA的抗体反应显着降低，包括毒素中和抗体和结构域4的抗体，这与较少的毒素中和抗体相关。相反，与sCMG2的非结合突变体（D50A）一起用PA免疫的小鼠显示出与仅用PA观察到的相似的抗PA抗体应答。我们的结果表明，向PA疫苗制剂中添加sCMG2可能会导致免疫应答显着降低，但是我们讨论了许多可能导致免疫原性降低的因素。重要事项炭疽毒素PA是当前炭疽疫苗（吸附的炭疽疫苗）中的主要免疫原。改进炭疽疫苗以避免冷链需要改进PA的热力学稳定性。我们探讨了如何使用sCMG2稳定PA对BALB / c小鼠的PA免疫原性。尽管通过与sCMG2结合而提高了PA的稳定性，但PA的免疫原性却降低了。这项研究强调，尽管配体的结合可以保留或改善构象稳定性，而不影响天然序列，但可能会影响表位的识别或加工，从而废除了有效的免疫反应。我们探讨了如何使用sCMG2稳定PA对BALB / c小鼠的PA免疫原性。尽管通过与sCMG2结合而提高了PA的稳定性，但PA的免疫原性却降低了。这项研究强调，尽管配体的结合可以保留或改善构象稳定性而不影响天然序列，但可能会影响表位的识别或加工，从而废除有效的免疫反应。我们探讨了如何使用sCMG2稳定PA对BALB / c小鼠的PA免疫原性。尽管通过与sCMG2结合而提高了PA的稳定性，但PA的免疫原性却降低了。这项研究强调，尽管配体的结合可以保留或改善构象稳定性，而不影响天然序列，但可能会影响表位的识别或加工，从而废除了有效的免疫反应。