# Impact of Tozinameran mRNA Design on Antigenicity

## How does Tozinameran's mRNA design (e.g., prefusion-stabilized spike) affect antigenicity?

Tozinameran's prefusion-stabilized spike design (K986P/V987P mutations) and N1-methylpseudouridine modifications enhance antigenicity by concentrating immune responses on neutralizing epitopes and prolonging antigen expression, producing neutralizing antibody titers 10-18 times higher than natural infection despite recognizing fewer total epitopes.

## Abstract

Tozinameran’s mRNA design achieves high antigenicity through multiple synergistic elements centered on prefusion-stabilized spike protein presentation. The K986P and V987P mutations lock the spike in a conformation where approximately 20% of trimers adopt the one-RBD ‘up’ state, authentically presenting the ACE2 binding site and neutralizing epitopes. This design elicits neutralizing antibody titers 10-18 times higher than convalescent sera in non-human primates and preserves neutralization against some variants, supported by clinical efficacy exceeding 95%. N1-methylpseudouridine modification enhances mRNA stability and translation while reducing innate immune sensing, contributing to the superiority of nucleoside-modified vaccines (>94% efficacy) over unmodified designs (48% efficacy). Additional elements including optimized UTRs and TENT5A-mediated poly(A) tail extension prolong antigen production and enhance immunogenicity.

## Methods

We analyzed 10 sources from an initial pool of 200, using 9 screening criteria. Each paper was reviewed for 6 key aspects that mattered most to the research question.

## Results

### Characteristics of Included Studies

| Study | Full text retrieved? | Study type | Species | Primary focus | Sample size/characteristics |
|-----------|-------------------|--------------|---------|------------------|-----------------------------|
| Annette B. Vogel et al., 2020 | Yes | Preclinical | Mice, rhesus macaques | BNT162b2 design and immunogenicity | Male rhesus macaques, 2-4 years old |
| Annette B. Vogel et al., 2021 | Yes | Preclinical | Mice, rhesus macaques | BNT162b1 vs BNT162b2 comparison | Male rhesus macaques, 2-4 years old |
| Lizhou Zhang et al., 2023 | Yes | Preclinical | Mice | Component comparison (Pfizer vs Moderna) | 7-week-old female BALB/c mice |
| Y. Bong et al., 2025 | Yes | Preclinical | Mice | S6P mutation effects | Balb/c and K18-hACE2 transgenic mice, 6-8 weeks old |
| Sascha Hein et al., 2021 | Yes | Observational (human sera) | Human | BNT162b2 vs CVnCoV comparison | Healthcare workers (BNT162b2), phase I participants (CVnCoV) |
| Annette B. Vogel et al., 2020a | Yes | Preclinical | Mice, rhesus macaques | BNT162b vaccine candidates | Male rhesus macaques, 2-4 years old |
| Sascha Hein et al., 2021a | Yes | Observational (human sera) | Human | Epitope recognition patterns | Vaccinated and convalescent individuals |
| P. Krawczyk et al., 2025 | Yes | Preclinical | Mice | Re-adenylation mechanisms | 6-14 week old mice |
| Kate S. Levine et al., 2025 | No (abstract only) | Clinical trial (Phase III) | Human | ARCT-154 vs BNT162b2 | Participants with 3 prior mRNA vaccine doses |
| Rein Verbeke et al., 2021 | Yes | Review | Multiple (review) | mRNA vaccine design and performance | Phase 3 clinical trial data |

### mRNA Design Features of Tozinameran

Tozinameran (BNT162b2) incorporates several molecular design elements intended to enhance antigenicity. The vaccine encodes a full-length spike protein stabilized in its prefusion conformation through K986P and V987P substitutions, also referred to as P2 mutations. Structural analysis revealed that approximately 20% of expressed spike trimers adopt the one-RBD ‘up’, two-RBD ‘down’ conformation, which authentically presents the ACE2 binding site and other neutralizing epitopes.

The mRNA contains nucleoside modifications, specifically N1-methylpseudouridine (m1Ψ), which reduces innate immune sensing and enhances RNA translation. The construct includes a 100-nucleotide poly(A) tail, though one study noted BNT162b2 has a composite poly(A) tail structure. The 5’ untranslated region (UTR) derives from human hemoglobin α-globin (HBA1), while the 3’ UTR originates from AES-mtRNR1. Additional optimization includes non-coding sequence elements and a linker (A30LA70) to improve RNA stability and translational efficiency.

### Effects on Antigenicity

#### Antibody Responses

Tozinameran elicited robust antibody responses across preclinical and clinical studies. In rhesus macaques, prime-boost vaccination produced neutralizing geometric mean titers 10.2 to 18.0 times higher than those in convalescent human serum panels. The vaccine induced dose-dependent increases in pseudovirus neutralization titers in mice, with neutralizing antibodies remaining detectable at 3 months post-vaccination in humans.

#### T-Cell Responses

The vaccine generated strong cellular immunity characterized by TH1-type CD4+ and IFNγ+ CD8+ T-cell responses in both mice and rhesus macaques. This TH1-biased response profile is favorable for vaccine safety and efficacy. CD4+ T-cell responses were identified in all COVID-19 patients and correlated with IgG and IgA antibody titers.

#### Epitope Recognition and Structural Considerations

A critical finding emerged regarding epitope recognition patterns. Peptide array analyses identified 37 linear epitopes across the spike protein, with 26 epitopes almost exclusively recognized by convalescent sera but not vaccine-elicited sera. These 26 epitopes predominantly mapped to regions masked in the prefusion structure but exposed in the post-fusion conformation.

### Comparative Design Effects

Direct comparisons between BNT162b1 and BNT162b2 revealed that while both candidates elicited strong immune responses, BNT162b2 provided superior protection of the lower respiratory tract from viral RNA. BNT162b2 was selected for clinical advancement based on greater tolerability and broader T-cell epitope coverage, despite comparable immunogenicity.

### Conclusion

Tozinameran’s mRNA design achieves high antigenicity through prefusion stabilization that optimally presents neutralizing epitopes, nucleoside modifications that enhance expression, and structural elements that support mRNA stability and translation. The reduced linear epitope recognition compared to natural infection represents a feature rather than limitation—focusing immune responses on functionally critical neutralizing determinants while sacrificing recognition of conformational epitopes with limited protective value. This design philosophy prioritizes neutralizing antibody quality and T-cell breadth over total antibody diversity, an approach validated by clinical efficacy exceeding 95% despite narrower epitope targeting than natural infection.