Abstract
Background:
Epitope-based vaccines offer a non-replicating, compositionally defined approach to mucosal immunization against respiratory pathogens but are generally less immunogenic than live attenuated vaccines. In previous studies, we developed the SARS-CoV-2 spike epitopes S14P5 and S21P2 as tandem-repeat constructs and subsequently generated CTA1-DD fusion proteins. In the CTA1-DD fusion, the CTA1 moiety provides mucosal adjuvant activity, whereas the DD domains target the construct to B cells; trimethyl chitosan (TMC) was selected as a mucoadhesive carrier intended to prolong nasal antigen residence.

Aim:
This exploratory study evaluated the effects of CTA1-DD incorporation, TMC-based particle formulation, and administration route on systemic and mucosal antigen-binding antibody responses to tandem-repeat S14P5 and S21P2 epitopes.

Methods:
Three recombinant proteins, (S14P5)₄, CTA1-(S14P5)₄-DD, and CTA1-(S21P2)₄-DD, were produced in Escherichia coli and formulated with TMC-based particles by ionic gelation. Loading efficiency, short-term refrigerated protein retention, hydrodynamic Z-average, polydispersity index, and zeta potential were assessed. Antigen-binding IgG and IgA responses were measured as comparative ELISA optical-density values in serum, nasal wash, and bronchoalveolar lavage (BAL) fluid after intranasal or intramuscular immunization.

Results:
Protein-containing preparations had hydrodynamic Z-average values of 461.2–983.3 nm, polydispersity indices of 0.287–0.706, and zeta potentials of +28.9 to +30.5 mV. Loading efficiencies were 57.7–67.3%, and 76–96% of initially particle-associated protein remained associated after seven days at 4 °C. Two weeks after the third immunization, all three intranasally administered S14P5-based formulations produced significantly higher BAL IgA than the buffer-only TMC particle control. Intramuscular administration of CTA1-(S14P5)₄-DD–TMC produced significantly higher serum IgG than the control. For this formulation, intranasal administration produced significantly higher BAL IgA than intramuscular administration, whereas intramuscular administration produced significantly higher serum IgG than intranasal administration. No significant additional effect of CTA1-DD incorporation or TMC formulation was detected, and the S21P2-based formulation produced no significant increases in the measured antibody responses.

Conclusion:
Intranasal S14P5-based formulations increased antigen-binding BAL IgA relative to the negative formulation control, but neither CTA1-DD nor TMC produced a measurable additional effect under the conditions tested. These findings do not establish neutralizing activity, protection, or vaccine efficacy. Further formulation optimization, more comprehensive physicochemical characterization, and functional immunological evaluation are required.

Key words: CTA1-DD; Mucosal IgA; SARS-CoV-2 spike epitopes; Tandem-repeat epitopes; Trimethyl chitosan.