Project Summary
Vaccination represents the most effective means of preventing influenza A virus (IAV) infections. Current
approaches to IAV vaccines primarily target the HA protein, but HA undergoes significant antigenic drift and is
thus a poor target for lasting humoral immunity. An alternative approach to developing a universal IAV vaccine
is to target internal viral proteins (such as the nucleoprotein), which are also highly conserved between most IAV
strains. A major roadblock to this approach, however, is a lack of adjuvants that trigger the right kind of immune
response - a ‘type I’ (or Th1/CD8+ T-cell driven) response - to these internal antigens. In this proposal, we outline
a new strategy that seeks to harness immunogenic cell death as a means of activating type I immunity to IAV
vaccines. We have identified a host signaling pathway which accounts for almost all IAV-activated immunogenic
death in infected cells, including primary airway epithelial cells, macrophages and DCs. This cell death pathway
is initiated when the host sensor protein ZBP1 detects IAV genomic RNA and activates parallel pathways of
programmed necrosis (necroptosis) and apoptosis to kill the infected cell and instigate CD8+ T cell-mediated
adaptive immune responses. Adding to the attractiveness of ZBP1-initiated cell death as a vaccine approach are
our discoveries, first, that IAV produces a new form of double-stranded RNA - termed Z-RNA - to activate ZBP1.
Z-RNA is thus a new PAMP, and can be readily harnessed to trigger immunogenic cell death and adjuvant IAV
vaccines. Second, ZBP1 initiates necroptosis from the nucleus, resulting in nuclear envelope rupture and release
of highly-immunogenic nuclear DAMPs and IAV antigens. Nuclear necroptosis is even more immunogenic than
conventional (cytoplasm-initiated) necroptosis. As ZBP1-driven immunogenic cell death (both apoptosis and
nuclear necroptosis) is important for effective anti-IAV cytotoxic T cell responses, our discoveries allow us to
propose that Z-RNAs are potent new adjuvants for activating type I immune responses to IAV, and that
recombinant IAVs engineered to produce Z-RNAs represent new type I immunity-activating vaccines. In this
proposal, three labs with strong expertise in IAV cell death signaling (Balachandran), recombinant IAV
technology (Langlois), and pathogenesis/vaccine strategies (Lopez) will unite to identify IAV-generated Z-RNAs
that activate ZBP1 (Aim 1); determine which stromal and immune cell types die by ZBP1-mediated mechanisms
to initiate and regulate effective adaptive immune responses (Aim 2); and test if triggering ZBP1-mediated cell
death with synthetic and virus-generated Z-RNAs can be leveraged to inform and supply next-generation
vaccines capable of providing effective cross-protective immunity to IAV (Aim 3).