Institute of Immunology, Faculty of Veterinary Medicine, Leipzig University, Leipzig, Germany.
Institute of Bioanalytical Chemistry, Leipzig University, Leipzig, Germany.
Front Immunol. 2021 Jul 23;12:709695. doi: 10.3389/fimmu.2021.709695. eCollection 2021.
, an opportunistic fungal pathogen ubiquitously present in the environment, causes cryptococcal meningitis (CM) mainly in immunocompromised patients, such as AIDS patients. We aimed to identify disease-associated cryptococcal protein antigens targeted by the human humoral immune response. Therefore, we used sera from Colombian CM patients, with or without HIV infection, and from healthy individuals living in the same region. Serological analysis revealed increased titers of anti-cryptococcal IgG in HIV-negative CM patients, but not HIV-positive CM patients, compared to healthy controls. In contrast, titers of anti-cryptococcal IgM were not affected by CM. Furthermore, we detected pre-existing IgG and IgM antibodies even in sera from healthy individuals. The observed induction of anti-cryptococcal IgG but not IgM during CM was supported by analysis of sera from -infected mice. Stronger increase in IgG was found in wild type mice with high lung fungal burden compared to IL-4Rα-deficient mice showing low lung fungal burden. To identify the proteins targeted by human anti-cryptococcal IgG antibodies, we applied a quantitative 2D immunoproteome approach identifying cryptococcal protein spots preferentially recognized by sera from CM patients or healthy individuals followed by mass spectrometry analysis. Twenty-three cryptococcal proteins were recombinantly expressed and confirmed to be immunoreactive with human sera. Fourteen of them were newly described as immunoreactive proteins. Twelve proteins were classified as disease-associated antigens, based on significantly stronger immunoreactivity with sera from CM patients compared to healthy individuals. The proteins identified in our screen significantly expand the pool of cryptococcal proteins with potential for (i) development of novel anti-cryptococcal agents based on implications in cryptococcal virulence or survival, or (ii) development of an anti-cryptococcal vaccine, as several candidates lack homology to human proteins and are localized extracellularly. Furthermore, this study defines pre-existing anti-cryptococcal immunoreactivity in healthy individuals at a molecular level, identifying target antigens recognized by sera from healthy control persons.
新型隐球菌是一种普遍存在于环境中的机会性真菌病原体，主要引起免疫功能低下患者（如艾滋病患者）的隐球菌性脑膜炎（CM）。我们旨在鉴定人类体液免疫反应靶向的疾病相关隐球菌蛋白抗原。因此，我们使用了来自哥伦比亚有或没有 HIV 感染的 CM 患者以及来自同一地区的健康个体的血清。血清学分析显示，与健康对照组相比，HIV 阴性 CM 患者的抗隐球菌 IgG 滴度升高，但 HIV 阳性 CM 患者的抗隐球菌 IgG 滴度没有升高。相比之下，CM 并不影响抗隐球菌 IgM 的滴度。此外，我们甚至在健康个体的血清中检测到了预先存在的 IgG 和 IgM 抗体。从感染小鼠的分析中证实了在 CM 期间观察到的抗隐球菌 IgG 但不是 IgM 的诱导。与 IL-4Rα 缺陷型小鼠（肺部真菌负担低）相比，肺部真菌负担高的野生型小鼠中 IgG 的增加更为明显。为了鉴定人抗隐球菌 IgG 抗体靶向的蛋白，我们应用了一种定量的 2D 免疫蛋白质组学方法，该方法可鉴定 CM 患者或健康个体的血清优先识别的隐球菌蛋白斑点，然后进行质谱分析。重组表达了 23 种隐球菌蛋白，并证实它们与 CM 患者或健康个体的血清具有免疫反应性。其中 14 种被新描述为具有免疫反应性的蛋白。基于与健康个体相比，CM 患者血清的免疫反应性显著增强，将 12 种蛋白归类为疾病相关抗原。在我们的筛选中鉴定的蛋白显著扩展了具有潜在（i）基于隐球菌毒力或存活的新型抗隐球菌药物开发或（ii）抗隐球菌疫苗开发的隐球菌蛋白库，因为几种候选蛋白缺乏与人蛋白的同源性，并且位于细胞外。此外，这项研究在分子水平上定义了健康个体中预先存在的抗隐球菌反应性，确定了由健康对照个体的血清识别的靶抗原。
