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The coronavirus disease 2019 (COVID-19) pandemic requires the continued development of safe, long-lasting, and efficacious vaccines for preventive responses to major outbreaks around the world, and especially in isolated and developing countries. To combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we characterize a temperature-stable vaccine candidate (TOH-Vac1) that uses a replication-competent, attenuated vaccinia virus as a vector to express a membrane-tethered spike receptor binding domain (RBD) antigen. We evaluate the effects of dose escalation and administration routes on vaccine safety, efficacy, and immunogenicity in animal models. Our vaccine induces high levels of SARS-CoV-2 neutralizing antibodies and favorable T cell responses, while maintaining an optimal safety profile in mice and cynomolgus macaques. We demonstrate robust immune responses and protective immunity against SARS-CoV-2 variants after only a single dose. Together, these findings support further development of our novel and versatile vaccine platform as an alternative or complementary approach to current vaccines., Graphical abstract, To combat SARS-CoV-2, we characterize a novel vaccine candidate (TOH-Vac1) that uses a replication-competent, attenuated vaccinia virus as a vector to express a membrane-tethered spike receptor binding domain antigen. We evaluate the effects of dose escalation and administration routes on vaccine safety, efficacy, and immunogenicity in mice and cynomolgus macaques.

In eukaryotes, 45S ribosomal RNA (rRNA) genes are tandemly arrayed at nucleolus organizer regions (NORs) (see Fig. 1A). Their transcripts, generated by RNA polymerase I (Pol I) in the nucleolus, are processed into the 18S, 5.8S, and 25-28S (depending on species) catalytic RNAs of ribosomes (Moss et al. 2007; Kressler et al. 2010; Hannan et al. 2013). The number of active 45S rRNA genes changes with the physiological needs of the cell (McStay and Grummt 2008; Tucker et al. 2010). For instance, Arabidopsis thaliana has ∼1500 rRNA genes per diploid genome (Copenhaver et al. 1995; Copenhaver and Pikaard 1996), with subtypes distinguishable by insertions/deletions at their 3′ ends (Fig. 1A). All subtypes are expressed immediately following germination, but by ∼10 d of seedling growth, the variant 1 subtype (Fig. 1A), accounting for ∼50% of all rRNA genes, is silenced via epigenetic mechanisms that include changes in DNA methylation and histone modification (Earley et al. 2006, 2010; Pontvianne et al. 2010, 2012). Chromatin modifications mediate rRNA gene dosage control in yeast, mouse, and human cells as well (Sandmeier et al. 2002; McStay and Grummt 2008; Grummt and Langst 2013). Figure 1. Partitioning of active and silent rRNA genes between the nucleolus and nucleoplasm. (A) Relationships between the nucleolus, NORs, and 45S rRNA gene repeats. The drawing at the top depicts a metaphase chromosome with the remnants of a nucleolus associated ... To examine the subnuclear distribution of active and silenced rRNA genes, we adapted fluorescence-activated sorting technology to isolate whole A. thaliana nuclei or nucleoli liberated from sonicated nuclei. Exploiting sequence variation among differentially expressed rRNA gene subtypes and mutations that disrupt rRNA gene chromatin modifications or copy number, we show that rRNA genes occupy alternative subnuclear compartments depending on their activity state.

In genetic hybrids, the silencing of nucleolar rRNA genes inherited from one progenitor is the epigenetic phenomenon known as nucleolar dominance. An RNAi knockdown screen identified the Arabidopsis de novo cytosine methyltransferase, DRM2 and the methylcytosine binding domain proteins, MBD6 and MBD10 as activities required for nucleolar dominance. MBD10 localizes throughout the nucleus, but MBD6 preferentially associates with silenced rRNA genes, and does so in a DRM2-dependent manner. DRM2 methylation is thought to be guided by siRNAs whose biogenesis requires RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). Consistent with this hypothesis, knockdown of DCL3 or RDR2 disrupts nucleolar dominance. In genetic hybrids, the silencing of nucleolar rRNA genes inherited from one progenitor is the epigenetic phenomenon known as nucleolar dominance. An RNAi knockdown screen identified the Arabidopsis de novo cytosine methyltransferase, DRM2 and the methylcytosine binding domain proteins, MBD6 and MBD10 as activities required for nucleolar dominance. MBD10 localizes throughout the nucleus, but MBD6 preferentially associates with silenced rRNA genes, and does so in a DRM2-dependent manner. DRM2 methylation is thought to be guided by siRNAs whose biogenesis requires RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). Consistent with this hypothesis, knockdown of DCL3 or RDR2 disrupts nucleolar dominance. Collectively, these results indicate that in addition to directing the silencing of retrotransposons and noncoding repeats, siRNAs specify de novo cytosine methylation patterns that are recognized by MBD6 and MBD10 in the large-scale silencing of rRNA gene loci.

Eukaryotes have three multisubunit DNA-dependent RNA polymerases that are essential for viability, abbreviated as Pol I, Pol II, and Pol III. Remarkably, Arabidopsis thaliana and other higher plants contain two additional nuclear multisubunit RNA polymerases, Pol IV and Pol V. These plant-specific polymerases are not essential for viability but have nonredundant roles in RNA-mediated gene-silencing pathways. Proteomic analyses have revealed that Arabidopsis Pol IV and Pol V have a 12-subunit composition like Pol II. In fact, half of the subunits of Pols II, IV, and V are encoded by the same genes. The remaining Pol IV- or Pol V-specific subunit genes arose through duplication and subfunctionalization of ancestral Pol II subunit genes. These include the genes encoding the largest subunits unique to Pol IV or Pol V, the genes encoding the second- and the fourth-largest subunits that are used by both Pol IV and Pol V, the gene encoding the fifth-largest subunit unique to Pol V and the genes encoding the seventh-largest subunits that are unique to Pol IV and Pol V. On the basis of phylogenetic reconstructions, the gene duplication events giving rise to the first-, second-, fourth-, fifth-, and seventh-largest subunits of Pol IV and/or Pol V occurred independently. Interestingly, a cDNA-mediated duplication of the Pol II seventh-largest subunit gene via retro-tranposition was an early event in Pol IV evolution, preceded only by the duplications of the largest and second-largest subunit genes. Secondary duplication of this cDNA-like gene to generate Pol IV- and Pol V-specific seventh-largest subunits has occurred in Arabidopsis but not all dicotyledonous plants or monocots, indicative of the dynamic evolution of RNA Pol IV and Pol V in plants.

The Arabidopsis histone deacetylase HDA6 is required to silence transgenes, transposons, and ribosomal RNA (rRNA) genes subjected to nucleolar dominance in genetic hybrids. In nonhybrid Arabidopsis thaliana, we show that a class of 45S rRNA gene variants that is normally inactivated during development fails to be silenced in hda6 mutants. In these mutants, symmetric cytosine methylation at CG and CHG motifs is reduced, and spurious RNA polymerase II (Pol II) transcription occurs throughout the intergenic spacers. The resulting sense and antisense spacer transcripts facilitate a massive overproduction of siRNAs that, in turn, direct de novo cytosine methylation of corresponding gene sequences. However, the resulting de novo DNA methylation fails to suppress Pol I or Pol II transcription in the absence of HDA6 activity; instead, euchromatic histone modifications typical of active genes accumulate. Collectively, the data reveal a futile cycle of unregulated transcription, siRNA production, and siRNA-directed DNA methylation in the absence of HDA6-mediated histone deacetylation. We propose that spurious Pol II transcription throughout the intergenic spacers in hda6 mutants, combined with losses of histone deacetylase activity and/or maintenance DNA methylation, eliminates repressive chromatin modifications needed for developmental rRNA gene dosage control.

Nucleolar dominance is an epigenetic phenomenon that occurs in genetic hybrids and describes the expression of 45S rRNA genes inherited from one progenitor due to the silencing of the other progenitor's rRNA genes. Nucleolar dominance is a manifestation of rRNA gene dosage control, which also occurs in non-hybrids, regulating the number of active rRNA genes according to the cellular demand for ribosomes and protein synthesis. Ribosomal RNA gene silencing involves changes in DNA methylation and histone modifications, but the molecular basis for choosing which genes to silence remains unclear. Recent studies indicate a role for short interfering RNAs (siRNAs) or structured regulatory RNAs in rRNA gene silencing in plants or mammals, respectively, suggesting that RNA may impart specificity to the choice mechanism.