Low-dose self-amplifying mRNA COVID-19 vaccine drives strong protective immunity in non-human primates against SARS-CoV-2 infection.

The coronavirus disease 2019 (COVID-19) pandemic continues to spread globally, highlighting the urgent need for safe and effective vaccines that could be rapidly mobilized to immunize large populations. We report the preclinical development of a self-amplifying mRNA (SAM) vaccine encoding a prefusion stabilized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein and demonstrate strong cellular and humoral immune responses at low doses in mice and rhesus macaques. The homologous prime-boost vaccination regimen of SAM at 3, 10 and 30 µg induced potent neutralizing antibody (nAb) titers in rhesus macaques following two SAM vaccinations at all dose levels, with the 10 µg dose generating geometric mean titers (GMT) 48-fold greater than the GMT of a panel of SARS-CoV-2 convalescent human sera. Spike-specific T cell responses were observed with all tested vaccine regimens. SAM vaccination provided protective efficacy against SARS-CoV-2 challenge as both a homologous prime-boost and as a single boost following ChAd prime, demonstrating reduction of viral replication in both the upper and lower airways. The SAM vaccine is currently being evaluated in clinical trials as both a homologous prime-boost regimen at low doses and as a boost following heterologous prime.

A GTP-synthesizing ribozyme selected by metabolic coupling to an RNA polymerase ribozyme.

Synthesis of RNA in early life forms required chemically activated nucleotides, perhaps in the same form of nucleoside 5'-triphosphates (NTPs) as in the contemporary biosphere. We show the development of a catalytic RNA (ribozyme) that generates the nucleoside triphosphate guanosine 5'-triphosphate (GTP) from the nucleoside guanosine and the prebiotically plausible cyclic trimetaphosphate. Ribozymes were selected from 1.6 × 1014 different randomized sequences by metabolically coupling 6-thio GTP synthesis to primer extension by an RNA polymerase ribozyme within 1016 emulsion droplets. Several functional RNAs were identified, one of which was characterized in more detail. Under optimized reaction conditions, this ribozyme produced GTP at a rate 18,000-fold higher than the uncatalyzed rate, with a turnover of 1.7-fold, and supported the incorporation of GTP into RNA oligomers in tandem with an RNA polymerase ribozyme. These results are discussed in the context of early life forms.

Water/Oil Emulsions with Controlled Droplet Sizes for In Vitro Selection Experiments.

In the early history of life, RNA might have had many catalytic functions as ribozymes that do not exist today. To explore this possibility, catalytically active RNAs can be identified by in vitro selection experiments. Some of these experiments are best performed in nanodroplets to prevent diffusion between individual RNA sequences. In order to explore the suitability for the large-scale in emulsio selection of water-in-oil emulsions made by passing a mixture of mineral oil, the emulsifier ABIL-EM90, and a few percent of an aqueous phase through a microfluidizer, we used dynamic light scattering to characterize the size of aqueous droplets dispersed throughout the oil. We found that seven or more passes through the microfluidizer at 8000 psi with close to half molar inorganic salts and 10% polyethylene glycol produced droplets with sizes below 100 nm that were ideal for our purposes. We also identified conditions that would produce larger or smaller droplets, and we demonstrate that the emulsions are stable over weeks and months, which is desirable for different types of in vitro selection experiments.

The NTP binding site of the polymerase ribozyme.

A previously developed RNA polymerase ribozyme uses nucleoside triphosphates (NTPs) to extend a primer 3'-terminus, templated by an RNA template with good fidelity, forming 3'-5'-phosphordiester bonds. Indirect evidence has suggested that the ribozyme's accessory domain binds the NTP with a highly conserved purine-rich loop. To determine the NTP binding site more precisely we evolved the ribozyme for efficient use of 6-thio guanosine triphosphate (6sGTP). 6sGTP never appeared in the evolutionary history of the ribozyme, therefore it was expected that mutations would appear at the NTP binding site, adapting to more efficient binding of 6sGTP. Indeed, the evolution identified three mutations that mediate 200-fold improved incorporation kinetics for 6sGTP. A >50-fold effect resulted from mutation A156U in the purine-rich loop, identifying the NTP binding site. This mutation acted weakly cooperative with two other beneficial mutations, C113U in the P2 stem near the catalytic site, and C79U on the surface of the catalytic domain. The preference pattern of the ribozyme for different NTPs changed when position 156 was mutated, confirming a direct contact between position 156 and the NTP. The results suggest that A156 stabilizes the NTP in the active site by a hydrogen bond to the Hoogsteen face of the NTP.

Cotranscriptional 3'-End Processing of T7 RNA Polymerase Transcripts by a Smaller HDV Ribozyme.

In vitro run-off transcription by T7 RNA polymerase generates heterogeneous 3'-ends because the enzyme tends to add untemplated adenylates. To generate homogeneous 3'-termini, HDV ribozymes have been used widely. Their sequences are added to the 3'-terminus such that co-transcriptional self-cleavage generates homogeneous 3'-ends. A shorter HDV sequence that cleaves itself efficiently would be advantageous. Here we show that a recently discovered, small HDV ribozyme is a good alternative to the previously used HDV ribozyme. The new HDV ribozyme is more efficient in some sequence contexts, and less efficient in other sequence contexts than the previously used HDV ribozyme. The smaller size makes the new HDV ribozyme a good alternative for transcript 3'-end processing.

Lower temperature optimum of a smaller, fragmented triphosphorylation ribozyme.

The RNA world hypothesis describes a stage in the early evolution of life in which catalytic RNAs mediated the replication of RNA world organisms. One challenge to this hypothesis is that most existing ribozymes are much longer than what may be expected to originate from prebiotically plausible methods, or from the polymerization by currently existing polymerase ribozymes. We previously developed a 96-nucleotide long ribozyme, which generates a chemically activated 5'-phosphate (a 5'-triphosphate) from a prebiotically plausible molecule, trimetaphosphate, and an RNA 5'-hydroxyl group. Analogous ribozymes may have been important in the RNA world to access an energy source for the earliest life forms. Here we reduce the length of this ribozyme by fragmenting the ribozyme into multiple RNA strands, and by successively removing its longest double strand. The resulting ribozyme is composed of RNA fragments with none longer than 34 nucleotides. The temperature optimum was ∼20 °C, compared to ∼40 °C for the parent ribozyme. This shift in temperature dependence may be a more general phenomenon for fragmented ribozymes, and may have helped RNA world organisms to emerge at low temperature.

A Faster Triphosphorylation Ribozyme.

In support of the RNA world hypothesis, previous studies identified trimetaphosphate (Tmp) as a plausible energy source for RNA world organisms. In one of these studies, catalytic RNAs (ribozymes) that catalyze the triphosphorylation of RNA 5'-hydroxyl groups using Tmp were obtained by in vitro selection. One ribozyme (TPR1) was analyzed in more detail. TPR1 catalyzes the triphosphorylation reaction to a rate of 0.013 min-1 under selection conditions (50 mM Tmp, 100 mM MgCl2, 22°C). To identify a triphosphorylation ribozyme that catalyzes faster triphosphorylation, and possibly learn about its secondary structure TPR1 was subjected to a doped selection. The resulting ribozyme, TPR1e, contains seven mutations relative to TPR1, displays a previously unidentified duplex that constrains the ribozyme's structure, and reacts at a 24-fold faster rate than the parent ribozyme. Under optimal conditions (150 mM Tmp, 650 mM MgCl2, 40°C), the triphosphorylation rate of TRP1e reaches 6.8 min-1.