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| **Ab      | Influenza pandemics are caused by viruses with novel     |
| stract:** | hemagglutinin (HA) and neuraminidase (NA) genes. Because |
|           | of the lack of existing immunity in the human            |
|           | population, such viruses can spread globally. A CDC      |
|           | mathematical model estimated 89,000 - 207,000 deaths and |
|           | 314,000 - 734,000 hospitalizations in the U.S. in the    |
|           | first year of a moderate pandemic in the absence of      |
|           | effective interventions.                                 |
|           |                                                          |
|           | H7 subtype influenza viruses are widely distributed in   |
|           | avian populations. Outbreaks of H7 viruses have caused   |
|           | serious economic impact in poultry industries in the     |
|           | U.S. as well as in Europe and H7 human infections have   |
|           | been documented. A recent outbreak of highly pathogenic  |
|           | H7N7 virus in poultry in the Netherlands also caused 83  |
|           | human infections including one fatality. During an       |
|           | outbreak of low pathogenic H7N2 in the U.S. in 2002, at  |
|           | least one human infection was detected by serology.      |
|           |                                                          |
|           | In response to the increasing threat of H7 influenza     |
|           | viruses in poultry and human populations, we propose to  |
|           | generate reassortant vaccine viruses against the H7N7    |
|           | and H7N2 subtypes using the conventional reassortment    |
|           | method with A/Puerto Rico/8/34 (PR8) as a donor of       |
|           | internal genes. The protocol to generate and evaluate    |
|           | such reassortant vaccine virus is well established in    |
|           | our laboratory and we have already produced vaccine      |
|           | reference viruses against the H5 and H9 subtypes.        |
|           | Pathogenicity of H7 vaccines in chickens and mice will   |
|           | be tested to assure the safety of reassortant virus.     |
|           | Small lots of formalin inactivated vaccines will be      |
|           | prepared and tested for its protective efficacy in the   |
|           | mouse model.                                             |
|           |                                                          |
|           | Production of an effective vaccine against H7 subtype    |
|           | viruses is a high priority for national and              |
|           | international pandemic preparedness.                     |
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| *         | 1)  To generate a reassortant virus with Eurasian H7N7   |
| *Specific |     surface protein genes with the internal genes of     |
| aims:**   |     A/Puerto Rico/8/34.                                  |
|           |                                                          |
|           | 2)  To generate reassortant virus with North American    |
|           |     H7N2 surface protein genes with the internal genes   |
|           |     of A/Puerto Rico/8/34.                               |
|           |                                                          |
|           | 3)  To evaluate the two candidate vaccines for           |
|           |     immunogenicity, pathogenicity and protective         |
|           |     efficacy using a standard protocol established in    |
|           |     the Influenza Branch.                                |
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| **Back    | Influenza is a highly contagious respiratory disease     |
| ground:** | that is responsible for considerable morbidity and       |
|           | mortality each year. In addition to causing annual       |
|           | epidemics, type A influenza viruses have periodically    |
|           | caused serious pandemics that spread rapidly in all age  |
|           | groups on a global scale. These pandemics are caused by  |
|           | introduction of novel surface protein genes,             |
|           | hemagglutinin (HA) and neuraminidase (NA), from          |
|           | non-human viruses by reassortment. An increased number   |
|           | of human infections with various subtypes of avian       |
|           | influenza viruses including H5N1, H9N2 and H7N7, have    |
|           | been reported in recent years, providing clear evidence  |
|           | that certain avian influenza viruses can directly        |
|           | transmit to humans.                                      |
+-----------+----------------------------------------------------------+
| **Back    | Most recently, an outbreak of highly pathogenic H7N7     |
| ground:** | virus among chicken farms in Netherlands resulted in 83  |
|           | human infections including a death of an attending       |
| **(con    | veterinarian. In the U.S., serological data suggested    |
| tinued)** | that a human infection occurred during an outbreak of    |
|           | H7N2 virus in poultry farms in Virginia in 2002.         |
|           |                                                          |
|           | Because there are no licensed vaccines to prevent avian  |
|           | influenza virus infections in humans, the Influenza      |
|           | Branch has initiated a program to develop candidate      |
|           | vaccines against various subtypes of avian influenza     |
|           | viruses. Currently licensed influenza vaccines are       |
|           | formalin-inactivated vaccines prepared from viruses      |
|           | grown in embryonated eggs using high-growth reassortant  |
|           | viruses that derive their HA and NA genes from           |
|           | circulating influenza A viruses and internal genes from  |
|           | a vaccine donor strain, influenza A/Puerto Rico/8/34     |
|           | (PR8). We have established a protocol to produce and     |
|           | evaluate avian-PR8 reassortant virus vaccine for future  |
|           | pandemic influenza. During the past three years, we      |
|           | prepared reassortant vaccine seed viruses against H9N2   |
|           | subtype virus, the North American lineage of H5N2        |
|           | subtype and the Eurasian lineage of H5N1 subtype         |
|           | viruses. The resulting viruses were fully analyzed for   |
|           | their safety and efficacy in animal models and were      |
|           | demonstrated to be satisfactory for the use as vaccine   |
|           | reference viruses. Plans to use our H9N2 vaccine         |
|           | candidate for clinical trials in humans are underway.    |
|           | Thus we have established a protocol to produce and       |
|           | evaluate avian-PR8 reassortant virus vaccines for future |
|           | pandemic influenza.                                      |
|           |                                                          |
|           | Avian influenza A viruses of the H7 subtype are widely   |
|           | distributed among avian populations. Low pathogenic H7   |
|           | viruses may change to a highly pathogenic form by        |
|           | acquiring certain HA mutations. An outbreak caused by a  |
|           | highly pathogenic virus virus can cause a devastating    |
|           | impact on poultry industries and it can pose a severe    |
|           | threat to the human population as experienced during the |
|           | H7N7 outbreak in the Netherlands in 2003. An effective   |
|           | human vaccine against the H7 subtype virus is urgently   |
|           | needed. Using our expertise in the production of         |
|           | PR8-based avian-human reassortant vaccines, we proposed  |
|           | to generate H7 vaccine reference viruses using the       |
|           | conventional reassortment method. We will safety test    |
|           | these two candidate vaccine strains using guidelines     |
|           | recently developed by WHO. We have chosen classical      |
|           | reassortment methods rather than a reverse genetics      |
|           | approach for the generation of H7 candidate vaccines     |
|           | because significant intellectual property issues remain  |
|           | unresolved for vaccine candidates produced using reverse |
|           | genetics. Development of candidate pandemic vaccine that |
|           | can be distributed without restriction is a high         |
|           | priority for the H7 subtypes that poses an immediate     |
|           | threat to human health.                                  |
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| **Study   | **Specific Aim 1:** A reassortant virus containing       |
| desig     | Eurasian H7 HA and N7 NA surface glycoprotein genes and  |
| n/Plan:** | 6 internal genes from PR8 (H7N7/PR8) will be generated   |
|           | by conventional reassortment. The recent influenza       |
|           | outbreak in poultry in the Netherlands was caused by     |
|           | highly pathogenic H7N7 strain. In order to prepare a     |
|           | safe reassortant vaccine seed virus, we have selected    |
|           | two low pathogenic avian virus isolates,                 |
|           | A/mallard/Netherlands/12/00 (H7N3) and                   |
|           | A/mallard/Netherlands/02/00 (H10N7), as donor strains    |
|           | for HA and NA genes, respectively.                       |
+-----------+----------------------------------------------------------+
| **Study   | The HA and NA genes of these isolates are genetically    |
| desig     | closely related to H7N7 viruses isolated from humans and |
| n/Plan:** | chickens. H7N3 and H10N7 viruses will be co-inoculated   |
|           | into 10-day-old embryonated eggs. The allantoic fluid    |
| **(con    | will be harvested after 24 hours and treated with ferret |
| tinued)** | antisera raised against H10N8 and H9N3 subtype viruses.  |
|           | After two rounds of antibody selection, it is expected   |
|           | that the majority of the virus population will contain   |
|           | H7N7 surface protein genes and such virus will be        |
|           | screened by reverse transcription-polymerase chain       |
|           | reaction (RT-PCR) using subtype specific primers. The    |
|           | resulting H7N7 virus will be co-inoculated with PR8      |
|           | virus into embryonated eggs. After an antibody selection |
|           | process using antiserum specific for PR8 surface         |
|           | proteins, a reassortant virus containing H7 HA, N7 NA    |
|           | and the 6 internal genes from PR8 will be selected by    |
|           | RT-PCR screening and will be biologically cloned by      |
|           | limiting dilution.                                       |
|           |                                                          |
|           | **Specific Aim 2:** A PR8 reassortant virus containing a |
|           | North American linage H7 HA and N2 NA will be generated. |
|           | One of the most recent H7N2 low pathogenic viruses,      |
|           | A/turkey/Virginia/158512/02 (H7N2) will be used as a     |
|           | donor strain of HA and NA genes for reassortment. The    |
|           | H7N2 viruses recently circulating in the U.S. are        |
|           | genetically closely related. On phylogenetic analysis,   |
|           | there is only 2-3% divergence between the HAs of 1999    |
|           | and 2002 isolates at the amino acid level. Thus, the     |
|           | resulting H7N2/PR8 reassortant vaccine is expected to be |
|           | effective against North American linage H7 viruses. This |
|           | reassortant virus will be generated by methods similar   |
|           | to those described in Specific Aim 1.                    |
|           |                                                          |
|           | **Specific Aim 3:** Pathogenicity of reassortant viruses |
|           | and immunogenicity and protective efficacy of formalin   |
|           | inactivated vaccines will be evaluated. Pathogenicity of |
|           | reassortant viruses in mice will be tested by            |
|           | determining 50% mouse lethal doses (MLD~50~) and         |
|           | comparing them with those of parent viruses. In          |
|           | collaboration with Dr. David Swayne at Southeast Poultry |
|           | Research Laboratory, USDA in Athens, GA, the             |
|           | pathogenicity of the reassortant viruses will be         |
|           | evaluated in chickens using the standard pathotyping     |
|           | methods. To evaluate immunogenicity and protective       |
|           | efficacy of vaccines, formalin inactivated vaccines will |
|           | be prepared from these reassortant viruses. Groups of    |
|           | mice will be immunized with two doses of vaccines with   |
|           | or without alum. Four weeks after the second dose, mice  |
|           | will be challenged with wild-type parent viruses as well |
|           | as a highly pathogenic H7N7 isolate. Mice will be bled   |
|           | periodically to test for serum antibody by HI test.      |
|           | Three days after challenge infection, mice from each     |
|           | group will be sacrificed, lung tissue will be collected  |
|           | for virus isolation. The remaining mice will be observed |
|           | for additional 2 weeks to determine the ability of the   |
|           | vaccine to prevent deaths from lethal challenge.         |
|           | Protective efficacy will be evaluated by survival rate   |
|           | and virus titer in lung. Candidate vaccine viruses that  |
|           | are determined to be safe and effective in animal models |
|           | will be provided to industrial partners for vaccine      |
|           | preparation. These viruses will be sent to Food and Drug |
|           | Administration for production of purified HA antigen and |
|           | sheep serum antibody for potency test.                   |
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