Background: Antigen design beyond spike

The COVID-19 vaccine candidate CoV2-BMEPu induced antibody responses that neutralized four Omicron subvariants in mice, along with CD8⁺ T-cell responses that persisted through day 80 after the first dose. In a separate early-lineage virus challenge, all five vaccinated mice survived the 12-day observation period. This was a preclinical study published in Frontiers in Immunology; it did not establish preventive efficacy in humans or direct protection against Omicron infection.

Existing spike-based vaccines have reduced severe disease and death, but viral variation and waning immune responses remain challenges. Drawing on immunological data from convalescent patients and others, the researchers combined antigen regions from spike (S), membrane (M), and nucleocapsid (N) proteins. The approach aims to broaden the immune response by including regions recognized by T cells as well as antibodies.

Key findings: Cross-neutralization and CD8⁺ T-cell responses at day 80

The candidate antigen comprised 15 antigen patches in total: 10 from S, two from M, and three from N. These included a receptor-binding domain (RBD) region overlapping sites targeted by broadly neutralizing antibodies. A secretion signal sequence and a T4 fibritin foldon to promote trimerization were added, and mRNA encoding the construct was packaged in lipid nanoparticles (LNPs). In cell-culture supernatant, trimers as well as monomers and larger oligomers were observed.

The immunogenicity assessment used 10 female C57BL/6 mice per group. The vaccine was administered intramuscularly at 10 μg per dose on days 0 and 28, with LNPs containing luciferase mRNA used as the control. Sera collected on day 80 after the first dose—52 days after the booster—cross-neutralized Omicron XBB.1.5, XBB.1.16, KP.3, and JN.1. The neutralization titer (NT50), the serum dilution that inhibits viral infection by 50%, reached a maximum of approximately 1,000. This does not mean that the same titer was observed for every variant or every animal.

Cellular immunity was assessed on days 38 and 80 using five mice per group at each time point. At day 80, CD8⁺ T-cell responses displaying multiple functional markers were still present, particularly responses to S1 and N peptides. By contrast, general CD4⁺ T-cell responses assessed with S, M, and N peptide pools declined below the detection limit. RBD-specific CD4⁺ responses, assessed separately, were detected at both time points. T follicular helper (Tfh)-related responses were observed in the acute phase and should be distinguished from the CD8⁺ responses that persisted through day 80.

Protection was tested separately in K18-hACE2 mice expressing human ACE2. The same vaccination schedule was used with five mice per group, followed on day 62 by intranasal challenge with 100,000 plaque-forming units (PFU) of the early-lineage MAD6 virus. All vaccinated mice survived the subsequent 12 days and maintained their body weight. All control mice given phosphate-buffered saline (PBS) reached humane endpoints by day 7 after infection.

Meaning and outlook: Scope of protection and remaining validation

No infectious virus was detected in the lungs of vaccinated mice on day 12 after infection. However, control lungs were collected on day 7, and viral quantification was possible for only three animals. The lung virus and inflammation-related gene-expression results reflect observations at different time points across the groups. They therefore do not establish complete suppression of viral replication dynamics or the extent of inflammation reduction through a comparison at the same time point.

In macrophages differentiated from human THP-1 cells, expression of inflammation- and antiviral-related genes increased. This experiment did not include an empty-LNP control, making it difficult to separate the contributions of the antigen and the delivery vehicle. There is also insufficient evidence to conclude that the trimer design directly stimulated a particular receptor to produce protection.

These findings support a development strategy that combines antigen regions from multiple structural proteins in a single mRNA. Follow-up comparative studies and human research will need to determine whether it outperforms existing vaccines, prevents Omicron infection, and how long immune responses persist beyond day 80. The study design and results are available in the full paper.