New Approaches To Passive Immunoprophylaxis

抗体的被动免疫预防是一种重要的公共卫生手段。例如，正常的免疫球蛋白在预防甲型肝炎中一直是重要的。然而，单克隆制剂可能更有效，针对特定的中和表位定制，并且效力高度一致。我们已经从黑猩猩的骨髓中制备了组合文库，这些黑猩猩已经被实验性地依次感染了五种人类肝炎病毒中的每一种。黑猩猩球蛋白几乎与人免疫球蛋白相同，使其成为免疫预防剂和免疫抑制剂的有吸引力的选择。迄今为止，我们已经分离出与HAV、HBV、HDV和HEV反应的单克隆免疫球蛋白。在其他研究中，我们已经回收了与HCV反应的人单克隆抗体。上述许多单克隆抗体都是中和性的，它们的生产正在扩大，用于黑猩猩和最终人类的被动免疫预防试验。这些研究和以下研究已延长至2008年。从骨髓中构建的组合文库也已在实验感染登革病毒1至4型的黑猩猩中进行：这些黑猩猩产生了针对登革病毒1、2和4型的重要中和单克隆抗体。更重要的是，我们重新建立了抗体介导的登革病毒感染增强的动物模型，该模型可导致更严重形式的登革病毒感染：登革出血热和登革休克综合征。此外，我们已经确定了抗体分子中的缺失，消除了增强，可能使抗体治疗首次实用。我们已经将我们的抗体研究扩展到其他感兴趣的病毒和细菌，这些病毒和细菌可以实验性地给予黑猩猩。例如，针对生物恐怖主义的新问题，我们制备了牛痘病毒的中和单克隆抗体，用作免疫预防/免疫抑制剂，用于需要牛痘免疫但易受这种免疫副作用影响的人。更重要的是，在与CDC的合作中，我们已经证明了中和牛痘的单克隆抗体也可以中和天花病毒。因此，这些中和性单克隆抗体应该不仅可用于预防和治疗疫苗接种的副作用，而且可用于预防和治疗天花，如果天花被释放到人群中的话。同样，我们用炭疽毒素免疫黑猩猩，试图制造能够在体内立即中和炭疽的单克隆抗体，并分离出了能够中和所有三种炭疽毒素（PA、LF和EF）的高效单克隆抗体，以及结合到炭疽B的荚膜上并调理细菌的单克隆抗体。这些抗体和中和牛痘和天花的单克隆抗体是与MacroGenics合作的CRADA的主题。我们还在制备针对脊髓灰质炎病毒、狂犬病病毒、西尼罗河病毒和蜱传脑炎病毒复合体的三种血清型的黑猩猩单克隆抗体。我们还增加了肉毒梭菌的七种毒素。其中一些将在对抗生物恐怖主义的努力中具有潜在的效用，所有这些都将在对抗新出现和重新出现的病原体的斗争中具有免疫预防和免疫增强潜力。 理解对丙型肝炎病毒（HCV）的免疫应答的一个障碍是无法测量中和抗体，因为大多数HCV毒株在细胞培养中不复制。我们以前证明中和抗体在体内中和试验利用黑猩猩。这一直是唯一被接受的中和试验，直到最近，当在体外试验的基础上的中和重组逆转录病毒携带的包膜糖蛋白的HCV被开发出来。我们证明了该测定通常与体内测定相关，并且中和抗体的反应性比以前认为的更广泛。 我们已经将假型病毒中和试验应用于实验性接种了两种基于抗体的候选HCV疫苗的黑猩猩的血清，并且已经表明，一种疫苗在用强毒HCV激发后在大多数动物中有效预防感染、肝炎和慢性化，从而刺激高水平的广泛中和抗体，而另一种疫苗在预防感染方面完全无效，通过假型病毒检测，肝炎或慢性完全无法刺激中和抗体。已通过与Innogenetics（根特，比利时）的CRADA鉴定了HCV的广泛中和单克隆抗体。这些单克隆抗体，针对E1包膜糖蛋白的HCV，回收的患者谁已成功治疗慢性丙型肝炎与干扰素。单克隆抗体在假型病毒试验中具有高度中和性和广泛反应性。 用它们（以及用其他多克隆血清）获得的结果表明，由六种基因型组成的丙型肝炎病毒可能包含两种或三种血清型。这些单克隆中和抗体有望在HCV感染的预防和治疗中找到临床应用。

Antibodies. Passive immunoprophylaxis has been an important public health tool. For example, normal immunoglobulin has been important in the prevention of hepatitis A. However, monoclonal preparations could be more potent, tailored to specific neutralization epitopes and highly consistent in potency. We have prepared combinatorial libraries from the bone marrow of chimpanzees that had been experimentally infected in sequence with each of the five human hepatitis viruses. Chimpanzee globulins are virtually identical to human immunoglobulins, making them attractive choices for immunoprophylactic and immunotherapeutic agents. To date, we have isolated monoclonal immunoglobulins that react with HAV, HBV, HDV and HEV. In other studies, we have recovered human monoclonal antibodies that react with HCV. Many of the monoclonal antibodies described above are neutralizing and their production is being scaled up for tests of passive immunoprophylaxis in chimpanzees and, eventually, humans. These and the following studies have been extended through 2008. Construction of combinatorial libraries from bone marrow has also been carried out for chimpanzees that have been experimentally infected with dengue viruses 1 through 4: these have yielded important neutralizing monoclonal antibodies to dengue virus types 1, 2 and 4. More importantly, we have reestablished an animal model for antibody-mediated enhancement of dengue virus infections, which can lead to the more severe forms of dengue virus infection: dengue hemorrhagic fever and dengue shock syndrome. Furthermore, we have identified a deletion in the antibody molecule that abrogates enhancement, possibly making antibody therapy practical for the first time. We have extended our antibody studies to other viruses and bacteria of interest that can be experimentally administered to chimpanzees. For example, in response to new concerns about bioterrorism, we have prepared neutralizing monoclonal antibodies to vaccinia virus for use as immunoprophylactic/immunotherapeutic agents in those who require immunization with vaccinia but who are susceptible to the side-effects of such immunization. More importantly, in collaboration with the CDC, we have demonstrated that monoclonal antibodies that neutralize vaccinia can also neutralize variola (the smallpox virus). Thus, these neutralizing monoclonal antibodies should be useful not only for prophylaxis and therapy of the side effects of vaccination but also for the prevention and therapy of smallpox, should it ever be released into populations. Similarly, we have immunized chimpanzees with anthrax toxin in an attempt to make monoclonal antibodies that could immediately neutralize anthrax in vivo and have isolated highly potent monoclonal antibodies that can neutralize all three anthrax toxins (PA, LF and EF), as well as bind to the capsule of B.anthracis and opsonize the bacterium. These and monoclonal antibodies that neutralize vaccinia and smallpox are the subjects of a CRADA with MacroGenics. We are also preparing chimpanzee monoclonal antibodies to the three serotypes of poliovirus, to rabies virus, to West Nile virus and to the tick-borne encephalitis virus complex. We have also added the seven toxins of Clostridium botulinum. Some of these will have potential utility in efforts to counteract bioterrorism and all will have immunoprophylactic and immunotherapeutic potential in the battle against emerging and re-emerging pathogens. An impediment to understanding the immune response to hepatitis C virus (HCV) has been the inability to measure neutralizing antibodies because most HCV strains do not replicate in cell culture. We previously demonstrated neutralizing antibodies in an in vivo neutralization assay utilizing chimpanzees. This has been the only accepted neutralization assay until recently, when an in vitro assay based on the neutralization of recombinant retroviruses bearing the envelope glycoproteins of HCV was developed. We demonstrated that this assay generally correlated with the in vivo assay and that the neutralizing antibodies were more broadly reactive than previously thought. We have applied the pseudo-typed virus neutralization assay to sera from chimpanzees experimentally vaccinated with two candidate antibody-based HCV vaccines and have shown that one vaccine, which was effective in preventing infection, hepatitis and chronicity in most animals following challenge with virulent HCV stimulated high levels of broadly neutralizing antibody, whereas the other vaccine, which was completely ineffective in preventing infection, hepatitis or chronicity, completely failed to stimulate neutralizing antibodies as measured by the pseudo-typed virus assay. Broadly neutralizing monoclonal antibodies have been identified for HCV through a CRADA with Innogenetics, Ghent, Belgium. These monoclonal antibodies, directed against the E1 envelope glycoprotein of HCV, were recovered from a patient who had been successfully treated for chronic hepatitis C with interferon. The monoclonal antibodies were highly neutralizing and broadly reactive in the pseudo-typed virus assay. The results obtained with them (and with other polyclonal sera) suggest that hepatitis C viruses, which consist of six genotypes, may comprise two or three serotypes. It is hoped that these monoclonal neutralizing antibodies will find clinical utility in the prevention and therapy of HCV infections.