Self Assembled Peptide Nanoparticles as Multi-Antigen Universal Influenza Vaccines - The goal of this proposal is to use self-assembled protein nanocages (SAPNs) as a universal flu vaccine platform and evaluate and enhance vaccine effectiveness. Despite vaccines, seasonal influenza viruses have continuously caused annual widespread disease and deadly flu pandemics occasionally arise. The limited success in preventative flu vaccines is due in large part to frequent reassortment and mutation of genes to generate different influenza strains. To address this, highly conserved regions of viral proteins are desirable targets for subunit vaccines. However, conserved antigens alone are usually poorly immunogenic. Vaccine nanoparticles can enhance immunogenicity via multivalency, tissue retention, and uptake by antigen presenting cells. Protein nanoparticles, in particular, enable controlled design of (multi)antigen presentation and provide other benefits over lipid and polymer nanoparticles. However, commonly used virus like particles (VLPs) and other protein scaffolds contain significant off-target immunogenic proteins that can misdirect the immune system and limit re-administration. As an alternative, SAPNs were created using coiled coil motifs that present multiple copies of three highly conserved antigens with good structural stability in proper orientation and oligomeric state: trimeric head-removed hemagglutinin stalk (HrHA) from H1N1 and four tandem consensus sequences (human, avian, swine, fowl) of matrix protein 2 ectodomain (4M2e) on the outer surface and nucleoprotein (NP) peptide T cell antigens inside the cage. Mice vaccinated intramuscularly with SAPNs without adjuvants showed potent humoral and cellular immune responses, including IgG with cross-recognition for HA from other strains. The coiled coils did not induce immune responses, suggesting that SAPNs can prevent irrelevant immune responses, maximize subunit specific immunity, and be a platform for other vaccines. The hypothesis is that broadly protective immunity against challenge with influenza viruses will be improved by presenting conserved HrHA and 4M2e domains accessible to B cells in highly repetitive arrays and controlled orientation on the SAPN outer surface with NP CD8 and CD4 T cell antigen epitopes inside administered by intramuscular and intranasal routes. Aim 1. Assembly and characterization of SAPNs with HrHA and 4M2e functionalized external surface and NP functionalized inner surface. Complete physicochemical and stability characterization will be performed. Aim 2. Assessment of SAPN vaccine effectiveness via different routes of vaccine delivery. Mice will be vaccinated by intramuscular, intranasal and mixed administration with assessment of IgG, IgG isotypes, and IgA responses, activation of T cell subsets, and breadth of protection following challenge with different flu strains. Aim 3. Assessment of the effectiveness of SAPN cross-protective vaccine candidates for aged mice and long- term protection. Also, SAPN stability and efficacy outside the cold chain will be assessed.
