New Approaches To Passive Immunoprophylaxis

点击翻译按钮获取中文摘要

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 a variety of animals and, eventually, in humans. These and the following studies have been extended through 2009. 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. Also in FY 2009, we have initiated a program whereby chimpanzees are being immunized with candidate vaccines against Ebola virus, Marburg virus and Lassa fever virus. Briefly, we are utilizing vesicular stomatitis virus (VSV) that has been modified by replacing the VSV envelope with glycoproteins of the BSL4 viruses. These chimeric viruses, developed by Heinz Feldmann, have been shown to be highly protective as vaccines in animal studies. Our goal is to recover potent neutralizing monoclonal antibodies that can be tested for immunoprophylaxis and immunotherapy. Previous attempts to generate such antibodies have yielded mixed results for reasons that are not completely clear but may be related to enhancing activity of certain antibodies. We plan to evaluate both the neutralizing and the enhancing activity of recovered antibodies and, if necessary, modify their FC portions to abolish enhancement but not neutralization, similar to what we have done with anti-dengue monoclonal antibodies.