PROJECT SUMMARY – RP3 RNA vaccines are one of the most promising vaccine platforms available to rapidly deploy in response to emerging viral infections with pandemic potential. However, mechanistic investigations of how various mRNA vaccine approaches induce protective immune responses are warranted. The order Bunyavirales comprises multiple families of viruses causing severe human disease. No FDA-licensed vaccines currently exist for any Bunyavirales virus, and medical countermeasures for infection are limited. For example, Andes virus (ANDV) caused a human outbreak with aggressive human-to-human transmission and multiple fatalities in 2019, highlighting the pandemic potential of hantaviruses. This proposal includes development and preclinical testing of innovative RNA vaccines against multiple bunyavirales and investigation of mechanisms of protection of three forms of RNA vaccines against representative New and Old World hantaviruses: ANDV, Sin Nombre virus (SNV) and Hantaan virus (HTNV). The following Aims will be pursued. Specific Aim 1. To convert existing DNA/JET vaccines to Hantaviridae, Arenaviridae, and Nairoviridae viruses to lipid nanoparticle (LNP)-mRNA vaccines (industrial partner, Moderna) and evaluate immunogenicity and protective efficacy of homologous and heterologous prime-boost vaccination strategies in established in- house small animal models. This aim will be based on extensive preliminary data, including clinical trials, on bunyavirales DNA vaccines and unsurpassed LNP-mRNA industrial experience of Moderna. Specific Aim 2. To generate LNP and polymer nanoparticle (PNP)-formulated vaccines against SNV and HTNV based on non-modified mRNA, modified mRNA or circular RNA. In addition to the non-modified and modified mRNA and circular RNA vaccines against ANDV we already developed, we will use these three platforms to generate vaccines against SNV and HTNV. We will use the platforms to generate the constructs expressing the N protein of ANDV, SNV and HTNV. The most promising constructs will be selected for encapsulation in PNP. As PNP formulations are expected to be less stringent for low-temperature requirements, we will test stability at various temperatures. Specific Aim 3. To perform an in-depth comparative investigation of the protective efficacy and immunogenicity of the new RNA vaccine constructs and formulations. We will perform in-depth studies of the immune response to the three vaccine platforms and two nanoparticle formulations in animal models. By the end of the funding period, we expect to have developed effective, innovative RNA vaccines against multiple Bunyavirales viruses and justified their promise to advance to clinical trials. The proposed studies will result in unparalleled insights into the antibody responses to mRNA vaccines and differences in antibody responses to vaccines based on modified versus non-modified RNA and linear versus circular RNA platforms. This knowledge will be critical for developing...