1a.Objectives (from AD-416):
The objective is to evaluate the immunopathology induced by sequential prime and challenge with influenza A viruses containing antigenically divergent HA or NP proteins in a swine model to recapitulate the 1918 pandemic.
1b.Approach (from AD-416):
Our hypothesis is that an immune response elicited by a virus with antigenic divergence in the HA protein compared to the subsequent challenge virus will induce aggravated pneumonia due to cross-reacting adaptive immune mediators, such as non-neutralizing HA antibody and/ or CTL recognition of conserved NP epitopes. We propose that targeted substitution of the immunodominant CTL epitope in the NP can abrogate the immune-mediated enhancement by altering CTL recognition. Using our established swine model, our approach will be to generate isogenic viruses that differ only in the HA and NP by cloning the HA of A/swine/Minnesota/02011/2008 H1N2 (MN/08) and A/California/04/2009 pH1N1 (CA/09) onto a well-characterized swine IAV backbone (OH/04) via reverse genetics. The swine lineage NP will be substituted with a divergent NP and mutations will be engineered at positions 4, 5 and 6 of the swine NP418-426 CTL epitope shown to abolish CTL recognition. The NP variants will be paired with the MN/08-HA and CA/09-HA viruses. Groups of pigs (n=15) will be vaccinated twice with adjuvanted inactivated vaccine prepared with MN/08-HA with wild type NP and MN/08-HA with mutated NPs. Pigs will then be challenged via an intratracheal route with virus containing CA/09-HA with wild type NP. Pigs will be humanely euthanized for necropsy on days 0 and 5 post infection to evaluate mucosal immune responses and lung lesions. Immune parameters against the vaccine and challenge strains will be evaluated for humoral and cell mediated immunity induced by the vaccine in peripheral blood and from the respiratory tract.
A well-characterized swine IAV backbone A/turkey/OH/313053/04 (H3N2) (OH/04) was used to engineer viruses with mismatched HA and NP. Reverse engineered viruses containing the HA of A/swine/Minnesota/02011/2008 H1N2 (rMN/08) or A/California/04/2009 H1N1pdm09 (rCA/09) using the bidirectional reverse genetics system pDP-2002 were generated. Recombinant viruses with these HA on the OH/04 backbone were successfully rescued and verified by sequencing. A divergent NP from an avian H9 virus was selected based on overall divergence from the OH/04 NP gene, cloned, and a recombinant virus rescued with the MN/08 HA and isogenic backbone virus genes. Viruses were transferred from University of Maryland to USDA-ARS-NADC for use in the in vivo pig study and were prepared for use in vaccines. The in vivo swine study using the engineered rMN/08 strains in vaccines followed with rCA/09 challenge has been completed. The laboratory assays with experimental clinical samples are in progress.