Hepatitis A and E vaccines. The HVS in collaboration with GlaxoSmithKline (GSK), Rixensart, Belgium, has developed several candidate live attenuated HAV vaccines. One such candidate was modified to become the currently licensed GSK inactivated HAV vaccine. In addition, the HVS has developed a candidate recombinant hepatitis E vaccine that is highly promising and that has recently completed clinical trials. In studies to further characterize this candidate hepatitis E vaccine, we have performed extensive pre-clinical trials to determine the potency of the vaccine, the duration of protection, the optimum regimen for administration, its protective efficacy against homologous versus heterologous virus strains, its ability to prevent infection as well as hepatitis and the minimum antibody titer that was effective in preventing infection and hepatitis, respectively. In the clinical trial, the vaccine was 96% efficacious in preventing hepatitis E. ? Hepatitis B and C vaccines. The HVS is studying the technology of DNA vaccines with a model system based upon hepatitis B virus (HBV) vaccine, a vaccine with which the HVS has had extensive experience. We have tested the efficacy of an immunostimulant (CpG) as an adjuvant for DNA vaccines, as well as for protein vaccines. Protein vaccines were found to be superior to DNA vaccines when compared in chimpanzees, a surrogate of man. CpG provided a greater but short-lived antibody response when compared to alum adjuvant. In addition, the utility of DNA vaccines for the control of hepatitis C virus (HCV) has been explored. A DNA vaccine based on the E2 envelope protein of HCV proved to be highly immunogenic in mice and rhesus monkeys and moderately immunogenic in chimpanzees, but the chimpanzees were not fully protected when they were challenged with live HCV. A similar approach has been utilized in the study of a DNA vaccine based on the E1 envelope protein of HCV: various constructs of the E1 gene were prepared as DNA vaccines (expression vector plasmids) and as vectored vaccines (recombinant vaccinia) and tested in mice. The mice had excellent immune responses to the DNA vaccine as well as to the vaccinia boost. In other studies, recombinant HCV E1 envelope glycoprotein is being tested in chimpanzees as an immunoprophylactic and immunotherapeutic vaccine. ? Antibodies. Passive immunoprophylaxis has also 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. Similar construction of combinatorial libraries from bone marrow has 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. We have now extended these 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 are preparing neutralizing monoclonal antibodies to vaccinia virus for use as an immunoprophylactic/immunotherapeutic agent in those who require immunization with vaccinia but who are susceptible to the side-effects of such immunization. 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 a highly potent monoclonal antibody that can neutralize the toxin. We are also preparing chimpanzee monoclonal antibodies to the three serotypes of poliovirus, to rabies virus, Japanese encephalitis virus, to West Nile virus and to the tick-borne encephalitis virus complex. Most recently we have 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. ? Neutralization Assays. 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 applied this knowledge to the testing of lots of commercial immune globulin that were manufactured before or after the screening of donor plasma for evidence of HCV infection and found that those lots manufactured before HCV screening contained high levels of neutralizing antibody and were not associated with hepatitis C virus infections in recipients. In contrast, immune globulins manufactured after initiation of screening for HCV lacked neutralizing antibodies and were associated with many cases of hepatitis C in recipients. This study provides, for the first time, a rational basis for passive immunoprophylaxis against hepatitis C and provides a means whereby protective neutralizing antibody can be detected and quantified. This information is being used to prepare hepatitis C immune globulin for the prevention of hepatitis C. Furthermore, for the first time, the mapping of neutralization epitopes of HCV can be facilitated by an in vitro assay. Broadly neutralizing monoclonal antibodies have been identified for HCV through a CRADA with Innogenetics, Ghent, Belgium. Identification of neutralization epitopes will be a key factor in developing an effective hepatitis C vaccine.
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