The mRNA vaccines against SARS-CoV-2 proved unexpectedly efficacious, but also require a series of booster immunizations to remain protective. Global deployment of these vaccines has been slow, particularly in low- and middle-income countries (LMIC), in part due to the logistical complications associated with multiple vaccinations. We therefore propose a new RNA vaccine design: a single-dose self-amplifying mRNA vaccine based on the SARS-CoV-2 coronavirus. Our long-term goal is to develop of a novel vaccine modality that consists of an RNA vaccine that feasibly delivers durable humoral and cellular immunity to SARS-CoV-2 in a single dose. Our novel design, named iDMV-1.0, utilizes RNA encoding the coronavirus replication machinery in order to produce Spike mRNA within double membrane vesicles (DMV). iDMV-1.0, a 20 kB RNA derived from the SARS-CoV-2 genome, consists of NSP1-16 of SARS CoV-2, mutations to stabilize the prefusion conformation of Spike protein (19), ORF6-10, M protein, and N protein, GFP and Luciferase for detection, and all non-coding 5’ and 3’ sequences of the SARS-CoV-2 genome. Because iDMV-1.0 permits self-amplification of Spike mRNA, this lipid nanoparticles-encapsulated RNA vaccine exhibits increased protein expression in tissue culture systems as compared to single round mRNA controls. The objective of this grant is to assess iDMV-1.0 in preclinical studies in mice. In Aim 1, the humoral immune response to the vaccine will be studied by assessing epitope choice of iDMV-1.0 as compared to mRNA-1273 (Moderna SARS-CoV-2 mRNA vaccine) using deep mutational scanning and traditional serology. We will assess durability of antigen expression and durability of humoral immune response for iDMV-1.0 as compared to conventional mRNA vaccination in mice. In Aim 2, the cellular immune response will be compared between both vaccine modalities to determine whether T cells specific to the SARS-CoV-2 proteome are generated by iDMV-1.0. Together, these studies will provide the foundation to understand durability and specificity of a potential single-shot SARS-CoV-2 mRNA vaccine, providing a paradigm that may facilitate greater vaccine uptake, particularly in LMIC settings.