New Approaches To Passive Immunoprophylaxis

抗体。被动免疫预防已成为一种重要的公共卫生工具。例如，正常的免疫球蛋白在预防甲型肝炎方面一直很重要。然而，单抗制剂可能更有效，为特定的中和表位量身定做，效力高度一致。我们从实验中先后感染了五种人类肝炎病毒的黑猩猩的骨髓中制备了组合文库。黑猩猩的球蛋白与人类的免疫球蛋白几乎完全相同，这使它们成为免疫预防和免疫治疗药物的诱人选择。到目前为止，我们已经分离出与甲型肝炎病毒、乙肝病毒、丁型肝炎病毒和戊型肝炎病毒发生反应的单抗免疫球蛋白。在其他研究中，我们已经恢复了与丙型肝炎病毒反应的人类单抗。上面描述的许多单抗都是中和的，它们的生产正在扩大规模，用于在黑猩猩以及最终人类身上进行被动免疫预防测试。这些研究和以下研究已延长至2008年。我们还为实验感染登革病毒1至4的黑猩猩进行了骨髓组合库的构建：这些黑猩猩产生了针对登革病毒1、2和4型的重要中和单抗。更重要的是，我们重建了一个通过抗体介导的增强登革病毒感染的动物模型，这可能导致更严重的登革病毒感染形式：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一个缺失，这可能使抗体治疗第一次变得实用。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地应用于黑猩猩。例如，为了应对对生物恐怖主义的新关切，我们制备了针对牛痘病毒的中和单抗，作为免疫预防/免疫治疗剂，用于需要牛痘免疫但容易受到这种免疫副作用影响的人。更重要的是，我们与疾控中心合作，证明了中和牛痘的单抗也可以中和天花(天花病毒)。因此，这些中和单抗不仅可用于预防和治疗疫苗接种的副作用，而且还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抗，并分离出高度有效的单抗，可以中和所有三种炭疽毒素(PA、LF和EF)，以及结合到炭疽杆菌的胶囊上并使细菌调理。这些和中和牛痘和天花的单抗是与MacroGenics合作的CRADA的主题。我们还在制备针对三种血清型脊髓灰质炎病毒、狂犬病病毒、西尼罗河病毒和森林脑炎病毒复合体的黑猩猩单抗。我们还添加了肉毒梭菌的七种毒素。其中一些将在努力对抗生物恐怖主义方面具有潜在的效用，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治疗的潜力。 丙型肝炎病毒免疫应答的一个障碍是无法测量中和抗体，因为大多数丙型肝炎病毒株不能在细胞培养中复制。我们之前在利用黑猩猩进行的体内中和试验中展示了中和抗体。直到最近，基于携带丙型肝炎病毒包膜糖蛋白的重组逆转录病毒的体外中和试验，这一直是唯一被接受的中和试验。我们证明了这种试验与体内试验大体相关，并且中和抗体比之前认为的更具广泛的反应性。我们将伪型病毒中和试验应用于实验接种了两种候选抗体的丙型肝炎病毒疫苗的黑猩猩血清中，结果表明，其中一种疫苗在与强毒丙型肝炎病毒攻击后有效地预防大多数动物的感染、肝炎和慢性化，而另一种疫苗在预防感染、肝炎或慢性化方面完全无效，根据伪型病毒检测，完全未能激发中和抗体。通过比利时根特的创新CRADA，已经确定了丙型肝炎病毒的广泛中和单抗。这些针对丙型肝炎病毒包膜糖蛋白的单抗是从一名成功接受干扰素治疗的慢性丙型肝炎患者身上回收的。在伪型病毒检测中，这些单抗具有高度的中和性和广泛的反应性。用它们(和其他多克隆血清)获得的结果表明，丙型肝炎病毒由六个基因型组成，可能包括两个或三个血清型。这些单克隆中和抗体有望在丙型肝炎病毒感染的预防和治疗中获得临床应用。

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.