PROJECT SUMMARY The deployment of efficacious SARS-CoV-2 vaccines has prevented the death of millions of people. New SARS- CoV-2 variants that are more transmissible exhibit increased resistance to neutralizing antibodies elicited by either the current vaccine or by prior infection with the original strain. To address this, we have developed a 2nd generation COVID19 vaccine that employs a self-amplifying replicon RNA (repRNA) and is delivered using a novel nanolipid carrier formulation (LION) that can be rapidly scaled up and is more stable at warmer temperatures to support effective worldwide distribution. Our repRNA vaccine expresses a structurally intact receptor binding domain (RBD) immunogen (called SHARP) that matches variants exhibiting resistance to neutralization by current vaccines. Second generation vaccines like this one will be administered to people that are already pre-immune to earlier variants of SARS-CoV-2 due to prior immunization or infection but, to date, the impact of pre-immunity to the original variants on these new vaccines is not known. Pre-immunity may enable the new vaccines to induce even broader antibody responses against different variants. On the other hand, original antigenic sin (OAS), wherein the immune response to an infection preferentially recalls memory cells primed by the first antigenic exposure, could dampen efficacy of 2nd generation vaccines if upon exposure to the new variant, B cell responses primed by the original variant are preferentially recalled. To gain a better understanding of the impact of pre-existing immunity on 2nd generation COVID-19 vaccines, we propose a pilot study using a repRNA vaccine expressing SHARP to immunize macaques with pre-existing immunity to the original D614G variant due to prior immunization. Our goal is to determine if pre-immunity improves or, alternatively, dampens the immunogenicity and/or protective efficacy of 2nd generation vaccines designed to protect against new variants. These questions are best addressed in NHPs that closely model the repertoire of innate and adaptive immune responses in humans and can be challenged with SARS-CoV-2 to investigate the impact on protective efficacy and recall responses. Our Aims are: 1) Characterize the magnitude, specificity and type of immune responses induced in macaques pre-immune to the original D614G variant and boosted with a a repRNA vaccine expressing the B.1351 variant immunogen. 2) Investigate pathogenesis of B.1351 infection in pigtail macaques, protective efficacy of the repRNA B.1351 SHARP vaccine in pre-immune macaques, and immune correlates of protection. 3) Determine the impact of prior immunization with the original D614G variant on innate immune responses and their role in the immunogenicity and efficacy of the 2nd generation repRNA B.1351 SHARP vaccine. Knowledge gained on the impact of pre-immunity from this study will have broader implications for the development of this and other 2nd generation vaccines. I...