Simultaneous modification of <i>Thermotoga maritima</i> encapsulin subunits to produce multivalent nanoparticle vaccines for non‐typhoidal <i>Salmonella enterica</i>

Protein nanoparticles are increasingly used in medical applications such as drug delivery and antigen display. To fully harness their potential, it is essential to assess their capacity for surface modification and optimize their stability and production. In this study, we investigated the immunogenicity of Thermotoga maritima encapsulin (TmENC) displaying a 28 amino acid peptide from FepA, a Salmonella outer membrane protein, which we had previously characterized. In BALB/c mice, immunization induced a specific antibody response against the surface-displayed epitope. We also evaluated the modularity of TmENC by introducing individual modifications at four distinct sites within the protein subunit, as well as simultaneous modification of up to four sites per subunit. All TmENC variants were transiently expressed in Nicotiana benthamiana, where they successfully assembled into nanoparticles; however, their accumulation and stability varied depending on the number and type of modifications introduced. These results demonstrate the versatility of TmENC as an antigen display platform and underscore its potential for developing multivalent vaccines through genetic modification of multiple sites within a single protein subunit.