Active and Passive Immunity Against Emergent Infectious

我们先前已经证明，流产布鲁氏菌可以为HIV多肽提供免疫载体功能，并诱导强大的抗HIV中和抗体和杀伤T细胞反应。我们现在正在将克隆的HIV基因插入到质粒中，然后用来转化流产杆菌。这将使我们能够向艾滋病毒高危或感染者的免疫系统传递多个艾滋病毒表位。到目前为止，我们已经成功地在流产杆菌中表达了HIV Poll基因。 在另一个单独的项目中，我们正在探索流产杆菌与先天免疫系统的相互作用，特别是通过其Toll样受体(TLR)激活树突状细胞。我们已经证明，流产杆菌使用两条不同的途径，即TLR2诱导肿瘤坏死因子和TLR9诱导IL-12。这些发现将使我们能够以更理想的方式使用流产杆菌来开发针对艾滋病毒感染的免疫治疗方法。 免疫球蛋白亚类：在免疫球蛋白活性和不良反应中的作用。在此之前，我们发现不同的免疫球蛋白亚类在中和HIV-1的能力上有所不同。所有三个亚类都显示与所有主要的HIV-1蛋白(IgG1和GT；IgG2和GT；IgG3)结合。相反，通过合胞体抑制和无细胞病毒中和检测，IgG3在中和HIV-1的能力方面比其他亚类更有活性(IgG3和GT；IgG1和GT；IgG2)。IgG3不同于IgG1和IgG2，因为它的铰链区更长、更灵活。还将测试免疫球蛋白亚类中和其他感染性病原体的能力。首先，他们将接受炭疽病检测。人、羊和人的抗炭疽抗体将被纯化为完整的、Fab和Fab2片段，并利用毒素中和试验研究结合和中和或增强炭疽毒素的能力。这些发现应该指出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，类似的方法将应用于SARS患者的恢复期血浆。在SARS的案例中，有证据表明猫冠状病毒感染可以通过抗体增强。人类抗SARS抗体保护或增强的能力将通过与D.Taylor博士和S.Feinstone博士在OVRR中合作的体外中和试验来测试。 绵羊高效价抗炭疽免疫球蛋白免疫原/疫苗的筛选目的是确定AVA疫苗、CAMR疫苗、Sterne疫苗和重组PA蛋白(RPA)这四种炭疽免疫原中哪一种在体外通过结合亲和力/亲和力和毒素中和来检测最有效的抗体，并在体内进行保护。 结果表明，RPA疫苗和斯特恩疫苗在体外和体内诱导高滴度抗体中和炭疽毒素的能力相似。与单独使用抗生素或抗体相比，联合使用抗生素和抗体可以提高接受Sterne菌株攻击的小鼠的存活率。 我们还与血液科技公司达成了一项CRADA协议，与他们合作研究经过改造表达人类免疫球蛋白基因的奶牛的能力，以制造针对炭疽毒素的抗体。来自奶牛的人类抗体将被提纯，并在毒素中和试验中测试结合RPA和中和致命毒素(RPA+LF)的能力。此外，这些抗体将通过等离子共振和小鼠挑战模型来测试对RPA的亲和力。 与第VIII因子有关的免疫和耐受性研究。目的是(I)确定胎儿或新生儿早期暴露于第VIII因子是否能消除血友病小鼠的抗体形成；以及(Ii)诱导细胞毒性T细胞(CTL)是否可以在第VIII因子治疗后消除抑制抗体的产生。 我们实验室已经在几种载体中表达了B结构域缺失的F.VIII：腺病毒、腺相关病毒和DNA质粒。这三种细胞在体外都能表达功能性F-VIII。初步数据显示，这些构建物可以纠正血友病小鼠的凝血时间。未来的实验将确定这些结构是否会像预期的那样在成年小鼠中诱导抗体反应。如果抗体被诱导，这些构建物将在这些小鼠的生命早期被引入，看看它们是否可以被耐受而不是免疫。首先将尝试新生儿治疗，如果失败，将尝试胎儿治疗以诱导耐受性。 利用表达痘苗病毒CTL表位的迷你基因在小鼠体内产生CTL是可能的。在一个模型系统中，我们证明了在牛痘中表达的卵清蛋白(OVA)表位SIINFEKL可以诱导以OVA冲击的抗提呈细胞(APC)为靶点的CTL。我们现在已经确定了因子VIII表位，将测试其诱导CTL的能力，以对抗因子VIII冲击的APC。在初步实验中，通过这种方法诱导了分泌干扰素-γ的CD8+细胞。 本项目包含2002财年项目1Z01BQ004019-07、1Z01BQ004026-01和1Z01BQ004027-01。

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.