LID scientists are collaborating with scientists from Medimmune, Inc., under a CRADA to generate candidate vaccines against H5N1 viruses that have caused human infections in Asia in 1997, 2003 and 2004. The vaccines were generated using plasmid based reverse genetics and each contains the hemagglutinin and neuraminidase genes from an avian H5N1 influenza virus and six internal gene segments from the AA ca virus. H5N1 ca vaccine candidates are attenuated in ferrets, chickens and mice. The vaccines are immunogenic and efficacious in mice and ferrets. The toxicity of the vaccine candidates at doses comparable to seasonal LAIV and at doses up to 100-fold higher was characterized in the ferret model. The results demonstrate that H5N1 ca vaccines are restricted in replication in the lower respiratory tract of ferrets, even when administered at high doses. However, intranasal administration of 0.5 mL can result in deposition of H5N1 vaccine in the ferret lung, where it induces a pulmonary inflammatory response in the absence of significant local replication of the vaccine virus. Based on promising preclinical data in mice and ferrets, clinical lots of the 2004 and 2003 H5N1 ca vaccines were generated and Phase I clinical trials of the safety and immunogenicity of the vaccines for healthy adults were undertaken under an IND. ? Avian influenza A H7 subtype viruses also pose a significant threat to human health because of their ability to transmit directly from domestic poultry to humans and to cause disease and sometimes death. Although it is important to develop vaccines against viruses of this subtype, very limited information is available on the immune response and pathogenesis of H7 viruses in animal models such as mice and ferrets. A group of 10 H7 viruses were selected for possible vaccine development based on their phylogenetic relationships and geographical location. The virulence of the 10 viruses for mice was determined, and the immunogenicity of the viruses in mice and ferrets were evaluated to study the extent of antigenic relatedness and the level of cross-reactivity of the antibodies that were induced by infection. Most of the viruses showed similar patterns of cross-reactivity with mice and ferret antisera. The Eurasian viruses elicited broadly cross-reactive antibodies that neutralized viruses from both Eurasian and North American lineages but the converse was not true. A subset of the viruses was also evaluated for their ability to replicate and cause disease in BALB/c mice following intranasal administration. H7 subtype viruses were able to infect mice without adaptation and manifested different levels of lethality and kinetics of replication indicating that the mouse model can be used for preclinical evaluation of vaccines against H7 subtype viruses. We have selected A/Netherlands/219/03 (H7N7) and A/chicken/BC/CN-7/04 (H7N3) viruses for vaccine development based on phylogenetic data, the induction of broadly cross-neutralizing antibodies in mouse and ferret antisera, and the ability to replicate in mice. ? We applied plasmid-based reverse genetics techniques to generate an H7N3 ca vaccine virus that derived its HA and NA genes from the low pathogenicity influenza A/chicken/BC/CN-6/04 (H7N3) virus that was the precursor of the highly pathogenic influenza A A/chicken/BC/CN-7/04 (H7N3) virus that caused the outbreak of disease in poultry and 2 human infections in Canada in 2004. The candidate H7N3 ca vaccine virus possessed the ts and att phenotypes specified by the internal protein genes of the AA ca virus, and the vaccine virus was immunogenic in mice. Four weeks after receiving a single dose of vaccine, mice developed neutralizing antibodies in the serum and they were fully protected from lethality following challenge with homologous and antigenically distinct heterologous wild-type highly pathogenic H7 viruses isolated from diverse locations. Four weeks after receiving two doses of the vaccine, mice and ferrets were fully protected from pulmonary replication of the highly pathogenic homologous wild-type H7N3 virus. Based on promising preclinical data in mice and ferrets, a clinical lot of the 2004 H7N3 ca vaccine was generated and an IND was submitted. ? In addition to the development and evaluation of vaccines against avian influenza viruses, we have investigated the prophylactic and therapeutic efficacy of human monoclonal antibodies against H5N1 influenza. In collaboration with investigators from Vietnam and Switzerland, we generated neutralizing anti-H5N1 human monoclonal antibodies (mAbs) and tested their efficacy for prophylaxis and therapy in a murine model of infection. Memory B cells from Vietnamese adults who had recovered from infections with HPAI H5N1 viruses were immortalised using Epstein Barr virus (EBV) and supernatants from B cell lines were screened in a virus neutralization assay. B cell lines secreting neutralizing antibodies were cloned and the mAbs purified. The cross-reactivity of these antibodies for different strains of H5N1 was tested in vitro by neutralization assays, and their prophylactic and therapeutic efficacy in vivo was tested in mice. In vivo, the mAbs conferred protection from lethality in mice challenged with A/Viet Nam/1203/04 (H5N1) in a dose-dependent manner. mAb prophylaxis provided a statistically significant reduction in pulmonary virus titer, reduced associated inflammation in the lungs, and restricted extrapulmonary dissemination of the virus. Therapeutic doses of 3 mAbs provided robust protection from lethality at least up to 72 h postinfection with A/Viet Nam/1203/04 (H5N1) and were also therapeutically active in vivo against the A/Indonesia/5/2005 (H5N1), a virus that belongs to a different genetic clade. These studies provide proof of concept that fully human mAbs with neutralizing activity can be rapidly generated from the peripheral blood of convalescent patients and that these mAbs are effective for the prevention and treatment of H5N1 infection in a mouse model. A panel of neutralizing, cross-reactive mAbs might be useful for prophylaxis or adjunctive treatment of human cases of H5N1 influenza.
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