Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA.
Department of Chemistry, Wichita State University, Wichita, Kansas, USA.
mSphere. 2020 Jan 15;5(1):e00556-19. doi: 10.1128/mSphere.00556-19.
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. 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.
保护性抗原（PA）是炭疽毒素的一种成分，能够引发毒素中和抗体反应。PA 也是目前预防炭疽的疫苗中的主要抗原，但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。抗原物理稳定性与免疫原性之间的关系了解甚少，但有理论依据认为该参数可能会影响免疫反应。我们研究了炭疽 PA 在存在和不存在人形式的受体毛细血管形态发生蛋白 2 的可溶性 von Willebrand 因子 A 结构域（sCMG2）的情况下在 BALB/c 小鼠中的免疫原性，以引发针对 PA 的抗体。先前的研究表明，sCMG2 稳定 83kDa PA 结构对 pH、化学变性剂、温度和蛋白水解以及使远离结合界面的组氨酸残基的氢-氘交换率减慢。与不含佐剂的含有 PA 的疫苗相比，我们发现，用与 sCMG2 稳定复合物中的 PA 免疫的小鼠对 PA 的抗体反应明显降低，包括毒素中和抗体和针对结构域 4 的抗体，这与较少的毒素中和抗体相关。相比之下，用与非结合的 sCMG2 （D50A）突变体协同免疫的 PA 的小鼠表现出与单独使用 PA 时相似的抗 PA 抗体反应。我们的结果表明，将 sCMG2 添加到 PA 疫苗配方中可能会导致免疫反应明显减弱，但我们讨论了导致免疫原性降低的多种因素。炭疽毒素 PA 是当前炭疽疫苗（炭疽疫苗吸附剂）中的主要免疫原。为避免冷链，需要改进炭疽疫苗，这就需要提高 PA 的热力学稳定性。我们研究了使用 sCMG2 稳定 PA 如何影响 BALB/c 小鼠中的 PA 免疫原性。尽管 PA 与 sCMG2 结合后稳定性增加，但 PA 的免疫原性降低。这项研究强调，虽然配体的结合保留或改善了构象稳定性而不影响天然序列，但可能会影响表位识别或处理，从而破坏有效的免疫反应。
