Active and Passive Immunity Against Emergent Infectious

针对突发传染病的主动和被动免疫

• 批准号: 6839884
• 负责人: Basil Golding
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6839884
• 关键词: AIDS therapy; Brucella abortus; HIV infections; T lymphocyte; anthrax; antibody; immunity; immunoglobulin G; interleukin 12; toll like receptor; tumor necrosis factor alpha

We have previously shown that Brucella abortus can provide immune carrier function for HIV peptides and induce potent anti-HIV neutralizing antibody and killer T cell responses. We are now inserting cloned HIV genes into plasmids which are then used to transform B. abortus. This will allow us to deliver multiple HIV epitopes to the immune system of HIV at risk or infected individuals. We have succeeded in expressing HIV pol genes in B. abortus thus far. In a separate project we are exploring the interaction of B. abortus with the innate immune system, in particular in terms of activating dendritic cells via their Toll-like receptors (TLR) We have shown that B. abortus uses two distinct pathways i.e. TLR2 to induce TNF and TLR9 to induce IL-12. These findings will enable us to use B. abortus in a more optimal way in developing an immunotherapeutic approach to HIV infection. Immunoglobulin subclasses: roles in activity of IGIV and in adverse reactions. Previously we showed that IgG subclasses differ in their ability to neutralize HIV-1. All three subclasses showed binding to all major HIV-1 proteins (IgG1 >IgG2 > IgG3). In contrast, IgG3 was more active than other subclasses in its ability to neutralize HIV-1 as assayed by syncytia inhibition and cell-free virus neutralization (IgG3 > IgG1 > IgG2). IgG3 differs from IgG1 and IgG2 by virtue of a longer and more flexible hinge region. IgG subclasses will also be tested for ability to neutralize other infectious agents. Firstly, they will be tested for anthrax. Human subclasses, and sheep and human anti-anthrax antibodies will be purified as intact, Fab, and Fab2 fragments and studied in terms of ability to bind and neutralize or enhance anthrax toxins using the Toxin Neutralization Assay. The findings should indicate which subclasses or antibody fragments are preferable as a product for treatment of anthrax infection. Secondly, a similar approach will be applied to SARS using convalescent plasma from SARS patients. In the case of SARS there is evidence that feline coronavirus infection can be enhanced by antibodies. The ability of human anti-SARS antibody to protect or enhance will be tested using an invitro neutralization assay in collaboration with Drs. D. Taylor and S. Feinstone in OVRR. Selecting an Immunogen/Vaccine for Production of High-Titer Anti-Anthrax Immune Globulin in Sheep. The objectives were to determine which of four anthrax immunogens, AVA Vaccine, CAMR Vaccine, Sterne Vaccine, and recombinant PA protein (rPA), elicit the most potent antibodies, as measured by binding affinity/avidity and toxin neutralization in vitro, and protection in vivo. The results show rPA and Sterne Vaccine have similar ability to induce high titer antibodies that neutralize anthrax toxins in vitro and in vivo. Combined antibiotic and antibody administration potentiated survival in mice challenged with the Sterne strain compared to antibiotic or antibody alone. We have also entered into a CRADA with Hematech to collaborate with them in studying the ability of cows that have been engineered to express human immune globulin genes to make antibodies against anthrax toxins. The human antibodies from cows will be purified and tested for ability to bind rPA and neutralize lethal toxin (rPA + LF)in the Toxin Neutralization Assay. In addition, the antibodies will be tested for avidity to rPA by plasmon resonance and in a mouse challenge model. Studies of Immunity and Tolerance Relating to Factor VIII. The objective are (i) to determine whether early exposure to F. VIII in the fetus or neonate abrogates antibody development in mouse hemophiliacs; and (ii) whether induction of cytotoxic T cells (CTL) can abrogate inhibitor antibody production following Factor VIII treatment. B-domain-deleted F. VIII has been expressed in our laboratory in several vectors: adenovirus, adeno-associated virus and as DNA plasmids. All three have been shown to express functional F. VIII in vitro. Preliminary data show that the constructs can correct clotting times in hemophiliac mice. Future experiments will determine whether these constructs induce antibody responses in adult mice as expected. If antibodies are induced the constructs will be introduced earlier in the life of these mice to see whether they can be tolerized rather than immunized. First neonatal treatment will be tried and if this fails fetal treatment will be attempted to induce tolerance. It has been possible to generate CTL in mice using minigenes expressing CTL epitopes in vaccinia virus. In a model system we showed that an ovalbumin (OVA) epitope, SIINFEKL, expressed in vaccinia, could induce CTL which targeted antige-presenting-cells (APC) pulsed with OVA. We now have identified Factor VIII epitopes which will be tested for ability to induce CTL against APC pulsed with Factor VIII. In preliminary experiments CD8+ cells that secrete interferon-gamma were induced by this approach. This project incorporates FY2002 projects 1Z01BQ004019-07, 1Z01BQ004026-01, and 1Z01BQ004027-01.

我们之前已经证明，流产布鲁氏菌可以为 HIV 肽提供免疫载体功能，并诱导有效的抗 HIV 中和抗体和杀伤性 T 细胞反应。我们现在将克隆的 HIV 基因插入质粒中，然后用该质粒转化流产布鲁氏菌。 这将使我们能够向艾滋病毒高危人群或感染者的免疫系统提供多个艾滋病毒表位。 迄今为止，我们已成功在流产芽孢杆菌中表达 HIV pol 基因。 在一个单独的项目中，我们正在探索流产布鲁氏菌与先天免疫系统的相互作用，特别是在通过其 Toll 样受体 (TLR) 激活树突状细胞方面。我们已经证明流产布鲁氏菌使用两种不同的途径，即 TLR2 诱导 TNF 和 TLR9 诱导 IL-12。 这些发现将使我们能够以更优化的方式使用流产布鲁氏菌来开发针对 HIV 感染的免疫治疗方法。 免疫球蛋白亚类：在 IGIV 活性和不良反应中的作用。之前我们表明 IgG 亚类中和 HIV-1 的能力不同。所有三个亚类均显示与所有主要 HIV-1 蛋白结合（IgG1 > IgG2 > IgG3）。 相比之下，通过合胞体抑制和无细胞病毒中和测定，IgG3 中和 HIV-1 的能力比其他亚类更活跃 (IgG3 > IgG1 > IgG2)。 IgG3 与 IgG1 和 IgG2 的不同之处在于具有更长且更灵活的铰链区。 还将测试 IgG 亚类中和其他感染因子的能力。 首先，他们将对炭疽病进行检测。 人类亚类、绵羊和人类抗炭疽抗体将被纯化为完整片段、Fab 和 Fab2 片段，并使用毒素中和测定研究结合、中和或增强炭疽毒素的能力。研究结果应表明哪些亚类或抗体片段更适合作为治疗炭疽感染的产品。 其次，类似的方法将适用于 SARS，使用 SARS 患者的恢复期血浆。 就 SARS 而言，有证据表明抗体可以增强猫冠状病毒的感染。人类抗 SARS 抗体的保护或增强能力将与 Drs 合作使用体外中和测定进行测试。 OVRR 中的 D. Taylor 和 S. Feinstone。 选择用于在绵羊中生产高滴度抗炭疽免疫球蛋白的免疫原/疫苗。目的是确定四种炭疽免疫原（AVA 疫苗、CAMR 疫苗、Sterne 疫苗和重组 PA 蛋白 (rPA)）中哪一种能够引发最有效的抗体（通过体外结合亲和力/亲和力和毒素中和以及体内保护来测量）。 结果表明，rPA 和 Sterne 疫苗具有相似的能力，可在体外和体内诱导中和炭疽毒素的高效价抗体。 与单独使用抗生素或抗体相比，联合施用抗生素和抗体可增强接受 Sterne 菌株攻击的小鼠的存活率。 我们还与 Hematech 签订了 CRADA 协议，与他们合作研究经过改造的奶牛表达人类免疫球蛋白基因以产生抗炭疽毒素抗体的能力。 来自奶牛的人类抗体将被纯化并在毒素中和测定中测试结合 rPA 和中和致命毒素 (rPA + LF) 的能力。 此外，还将通过等离振子共振和小鼠攻击模型来测试这些抗体对 rPA 的亲和力。 与因子 VIII 相关的免疫和耐受性研究。目的是 (i) 确定胎儿或新生儿早期接触 F.VIII 是否会消除小鼠血友病患者的抗体发育； (ii) 细胞毒性 T 细胞 (CTL) 的诱导是否可以消除因子 VIII 治疗后抑制剂抗体的产生。 B 结构域缺失的 F.VIII 已在我们的实验室中以多种载体表达：腺病毒、腺相关病毒和 DNA 质粒。 这三者均已被证明能够在体外表达功能性 F.VIII。 初步数据表明，该构建体可以纠正血友病小鼠的凝血时间。 未来的实验将确定这些构建体是否如预期的那样在成年小鼠中诱导抗体反应。 如果诱导产生抗体，则将在这些小鼠生命早期引入构建体，以观察它们是否可以耐受而不是免疫。 将尝试首次新生儿治疗，如果失败，将尝试胎儿治疗以诱导耐受。 使用在牛痘病毒中表达 CTL 表位的小基因，可以在小鼠中产生 CTL。 在模型系统中，我们表明，在牛痘中表达的卵清蛋白 (OVA) 表位 SIINFEKL 可以诱导针对用 OVA 脉冲的抗原呈递细胞 (APC) 的 CTL。 我们现在已经鉴定了因子 VIII 表位，将测试其诱导针对因子 VIII 脉冲的 APC 的 CTL 的能力。 在初步实验中，通过这种方法诱导分泌干扰素-γ的CD8+细胞。 该项目包括 2002 财年项目 1Z01BQ004019-07、1Z01BQ004026-01 和 1Z01BQ004027-01。