Updated and revised versions of COVID-19 vaccines are vital due to genetic variations of the SARS-CoV-2 spike antigen. Furthermore, vaccines that are safe, cost-effective, and logistic-friendly are critically needed for global equity, especially for middle- to low-income countries. Recombinant protein-based subunit vaccines against SARS-CoV-2 have been reported using the receptor-binding domain (RBD) and the prefusion spike trimers (S-2P). Recently, a new version of prefusion spike trimers, named HexaPro, has been shown to possess two RBD in the “up” conformation, due to its physical property, as opposed to just one exposed RBD found in S-2P. Importantly, this HexaPro spike antigen is more stable than S-2P, raising its feasibility for global logistics and supply chain. Here, we report that the spike protein HexaPro offers a promising candidate for the SARS-CoV-2 vaccine. Mice immunized by the recombinant HexaPro adjuvanted with aluminum hydroxide using a prime-boost regimen produced high-titer neutralizing antibodies for up to 56 days after initial immunization against live SARS-CoV-2 infection. Also, the level of neutralization activity is comparable to that of convalescence sera. Our results indicate that the HexaPro subunit vaccine confers neutralization activity in sera collected from mice receiving the prime-boost regimen.

Next-generation COVID-19 vaccines are critical due to the ongoing evolution of SARS-CoV-2 virus and waning duration of the neutralizing antibody response against current vaccines. The mRNA vaccines mRNA-1273 and BNT162b2 were developed using linear transcripts encoding the prefusion-stabilized trimers (S-2P) of the wildtype spike, which have shown a reduced neutralizing activity against the variants of concern B.1.617.2 and B.1.1.529. Recently, a new version of spike trimer, termed VFLIP has been suggested to possess native-like glycosylation, and greater pre-fusion trimeric stability as opposed to S-2P. Here, we report that the spike protein VFLIP-X, containing six rationally substituted amino acids to reflect emerging variants (K417N, L452R, T478K, E484K, N501Y and D614G), offers a promising candidate for a next-generation SARS-CoV-2 vaccine. Mice immunized by a circular mRNA (circRNA) vaccine prototype producing VFLIP-X elicited neutralizing antibodies for up to 7 weeks post-boost against SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs). In addition, a balance in T _H 1 and T _H 2 responses was achieved by immunization with VFLIP-X. Our results indicate that the VFLIP-X delivered by circRNA confers humoral and cellular immune responses, as well as neutralizing activity against broad SARS-CoV-2 variants.

The COVID-19 mRNA vaccines demonstrated the power of mRNA medicines. Despite advancements in sequence design, evidence regarding the preferential use of synonymous codons on cellular stress and innate immune responses is lacking. To this end, we developed a proprietary codon optimality matrix to re-engineer the coding sequences of three luciferase reporters. We demonstrate that optimal mRNAs elicited dramatic increases in luciferase activities compared to non-optimal sequences. Notably, transfecting an optimal RNA affects the translation of other RNAs in the cell including control transcripts in dual luciferase assays. This held true in multiple cell lines and for an unrelated reporter. Further, non-optimal mRNAs preferentially activated innate immune pathways and the phosphorylation of the translation initiation factor eIF2α, a central event of the integrated stress response. Using nucleoside-modified or circular RNAs partially or fully abrogated these responses. Finally, we show that circularizing RNAs enhances both RNA lifespan and durability of protein expression. Our results show that RNA sequence, composition, and structure all govern RNA translatability. However, we also show that RNA sequences with poor codon optimality are immunogenic and induce cellular stress. Hence, RNA sequence engineering, chemical, and topological modifications must all be combined to elicit favorable therapeutic outcomes.

Key findings: Circular RNAs (circRNAs) have emerged as promising platforms for stable RNA expression and therapeutic applications, but methods for their generation remain limited. Here, we engineered the Lactococcus lactis LtrB group II intron into a permuted intron-exon (PIE) configuration to enable efficient circRNA formation (circLtrB). Among the tested split designs, LtrB domain IV cleavage yielded the highest circularization efficiency. Although LtrB encodes LtrA maturase required for efficient in vivo splicing, in vitro application of LtrA to the PIE constructs resulted in lower circularization efficiency than self-splicing conditions. We further examined circularization under distinct salt conditions and showed that NH ₄ Cl- and KCl-based splicing buffers independently support high circularization efficiencies (>80%), while producing distinct splicing intermediates, indicating ion-specific mechanistic effects on circularization. Extending outer homology arms did not enhance circularization efficiency, demonstrating that efficient circRNA formation in the circLtrB platform is independent of extended exon pairing interactions. Using this system, we generated circRNAs across a range of coding sequence lengths, highlighting its payload flexibility. To optimize translation, we evaluated multiple internal ribosome entry sites (IRES), including Aichivirus, Crohivirus, CVB3, improved EMCV, HRV, and Salivirus, and found that the CVB3 IRES drove the highest translation efficiency. Linear NLuc showed faster decay kinetics, while circLtrB-NLuc exhibited more sustained expression over time. Transcriptome-wide RNA sequencing revealed comparable innate immune responses between circular and linear RNAs. Together, these results establish circLtrB as a versatile and efficient platform for circRNA production, providing mechanistic insight into group II intron-mediated circularization and expanding the toolkit for functional circRNA engineering.