PROJECT SUMMARY Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in late 2019 causing Coronavirus Disease 2019 (COVID-19) and within months became a worldwide pandemic - as of the writing of this proposal, at least 82.5M people have been infected and more than 1.8M have died in addition to having a significant impact on the worldwide economy. Multiple vaccines are in development for COVID-19 and two of these have been awarded emergency use approval from the US FDA based on RNA platforms. With our partners at HDT Bio Corp. we are soon to enter phase I clinical trials with an RNA-based vaccine, HDT-301. While the development and deployment of these vaccines is exciting and has moved with unprecedented speed, it is unclear how durable the immune responses will be and whether or not they will induce broad protection against emerging strains. Therefore, the ability to boost these vaccines should be investigated to determine if a prime / boost regimen can be deployed in the face of waning immunity or newly emerging viral variants like the recently identified UK strain. As next generation candidates we are now producing 4 different adjuvants containing a non-GLA based TLR4 active ingredient derived from MPL referred to as 3D(6acyl)-PHAD (“3D-PHAD”). These adjuvants are called AlT4™, EmT4™, LiT4™, and MiT4. In this proposal, we will test each of these adjuvants in combination with recombinant Covid-19 spike protein in mice as adjuvanted protein vaccines and as RNA vaccine boosters. Following immunization with protein / adjuvant, potential protection against multiple Covid-19 spike protein variants will be confirmed using both ELISA and viral neutralization assays. Lead combinations will then be tested using a prime-boost strategy using a proprietary mRNA prime vaccination with HDT-301 followed by a adjuvanted protein boost. Specifically, we propose to (1) Down-select a lead adjuvant in mice and (2) Determine protection from Covid-19 using an RNA prime protein boost immunization strategy. When this research is completed, we will have protocols and data supporting the use of these vaccines in further studies in higher animal models leading to human trials as the data warrant.