A self-amplifying RNA vaccine against COVID-19 with long-term room-temperature stability.

mRNA vaccines were the first to be authorized for use against SARS-CoV-2 and have since demonstrated high efficacy against serious illness and death. However, limitations in these vaccines have been recognized due to their requirement for cold storage, short durability of protection, and lack of access in low-resource regions. We have developed an easily-manufactured, potent self-amplifying RNA (saRNA) vaccine against SARS-CoV-2 that is stable at room temperature. This saRNA vaccine is formulated with a nanostructured lipid carrier (NLC), providing stability, ease of manufacturing, and protection against degradation. In preclinical studies, this saRNA/NLC vaccine induced strong humoral immunity, as demonstrated by high pseudovirus neutralization titers to the Alpha, Beta, and Delta variants of concern and induction of bone marrow-resident antibody-secreting cells. Robust Th1-biased T-cell responses were also observed after prime or homologous prime-boost in mice. Notably, the saRNA/NLC platform demonstrated thermostability when stored lyophilized at room temperature for at least 6 months and at refrigerated temperatures for at least 10 months. Taken together, this saRNA delivered by NLC represents a potential improvement in RNA technology that could allow wider access to RNA vaccines for the current COVID-19 and future pandemics.

Heterologous saRNA Prime, DNA Dual-Antigen Boost SARS-CoV-2 Vaccination Elicits Robust Cellular Immunogenicity and Cross-Variant Neutralizing Antibodies.

We assessed if immune responses are enhanced in CD-1 mice by heterologous vaccination with two different nucleic acid-based COVID-19 vaccines: a next-generation human adenovirus serotype 5 (hAd5)-vectored dual-antigen spike (S) and nucleocapsid (N) vaccine (AdS+N) and a self-amplifying and -adjuvanted S RNA vaccine (AAHI-SC2) delivered by a nanostructured lipid carrier. The AdS+N vaccine encodes S modified with a fusion motif to increase cell-surface expression and an N antigen modified with an Enhanced T-cell Stimulation Domain (N-ETSD) to direct N to the endosomal/lysosomal compartment and increase MHC class I and II stimulation potential. The S sequence in the AAHI-SC2 vaccine comprises the D614G mutation, two prolines to stabilize S in the prefusion conformation, and 3 glutamines in the furin cleavage region to confer protease resistance. CD-1 mice received vaccination by homologous and heterologous prime > boost combinations. Humoral responses to S were the highest with any regimen that included the AAHI-SC2 vaccine, and IgG bound to wild type and Delta (B.1.617.2) variant S1 at similar levels. An AAHI-SC2 prime followed by an AdS+N boost particularly enhanced CD4+ and CD8+ T-cell responses to both wild type and Delta S peptides relative to all other vaccine regimens. Sera from mice receiving AAHI-SC2 homologous or heterologous vaccination were found to be highly neutralizing for all pseudovirus strains tested: Wuhan, Beta, Delta, and Omicron strains. The findings here, taken in consideration with the availability of both vaccines in thermostable formulations, support the testing of heterologous vaccination by an AAHI-SC2 > AdS+N regimen in animal models of SARS-CoV-2 infection to assess its potential to provide increased protection against emerging SARS-CoV-2 variants particularly in regions of the world where the need for cold-chain storage has limited the distribution of other vaccines.

A flexible, thermostable nanostructured lipid carrier platform for RNA vaccine delivery.

Current RNA vaccines against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are limited by instability of both the RNA and the lipid nanoparticle delivery system, requiring storage at -20°C or -70°C and compromising universally accessible vaccine distribution. This study demonstrates the thermostability and adaptability of a nanostructured lipid carrier (NLC) delivery system for RNA vaccines that has the potential to address these concerns. Liquid NLC alone is stable at refrigerated temperatures for ≥1 year, enabling stockpiling and rapid deployment by point-of-care mixing with any vaccine RNA. Alternatively, NLC complexed with RNA may be readily lyophilized and stored at room temperature for ≥8 months or refrigerated temperature for ≥21 months while still retaining the ability to express protein in vivo. The thermostability of this NLC/RNA vaccine delivery platform could significantly improve distribution of current and future pandemic response vaccines, particularly in low-resource settings.