Anti-Dengue ED3 Long-Term Immune Response With T-Cell Memory Generated Using Solubility Controlling Peptide Tags

Abstract

Recombinant proteins are an attractive choice as a safe alternative to traditional live attenuated vaccines. However, most small-size proteins are poorly immunogenic, and adjuvants, whose mode of action remain to be fully clarified, are needed for increasing their immunogenicity. Here, we report the effects of short solubility controlling peptide tags (SCP-tags) on the immunogenicity of DENV3 envelope protein domain 3 (3ED3; 103 residues, 11.46 kDa) in ICR and Swiss albino model mice. The attachment of a 4-Ile SCP-tag (C4I-tag) increased the hydrodynamic radius of 3ED3 from 2.2 ± 0.09 to 111 ± 146 nm as assessed by dynamic light scattering in phosphate buffered saline at 37 • C, indicating that the C4I-tag oligomerized 3ED3. Immunization at 30 µg/dose showed that the untagged 3ED3 was not or poorly immunogenic, whereas the C4I-tag increased its immunogenicity by up to 39-fold as assessed by the IgG level measured using ELISA. Moreover, the increased antibody level was sustained for over 6 months after immunization and a high number of effector and central memory T cells were generated. These observations provide solid and quantitative evidence for the hypothesis that subvisible aggregates with hydrodynamic radii of 100 nm can increase immunogenicity and that SCP-tag can establish a long-term, target-specific immune response in a way adequate for the development of a peptide/protein-based DENV vaccine.

A systematic mutational analysis identifies a 5‐residue proline tag that enhances the in vivo immunogenicity of a non‐immunogenic model protein

“…In previous reports, we showed that short solubility controlling peptide tags (SCP‐tags) [11–13] attached at the C terminus of a host protein could be used to induce the formation of subvisible amorphous aggregates and thereby increase the immunogenicity of non‐immunogenic proteins [14,15]. In particular, a hydrophobic isoleucine tag attached to the C terminus of two model proteins, BPTI‐19A (58 residues; MW: 5.98 kDa) and DEN3ED3 (103 residues; MW 11.46 kDa), increased their immunogenicity by oligomerizing the proteins into small nanometer‐scale aggregates, and the immune response was long‐lasting with a T‐cell‐dependent activation of the B cells [16,17].…”

“…Thus, in addition to the previously reported C5I tag, which increased immunogenicity through protein oligomerization, C5P and C5R tags are expected to provide a tool for increasing a protein's immunogenicity without any oligomerization. [14].…”

A systematic mutational analysis identifies a 5-residue proline tag that enhances thein vivoimmunogenicity of a non-immunogenic model protein 240 folds

“…ELISA was performed as previously reported [14]. In short, anti-BPTI IgG levels were evaluated using untagged BPTI-19A (2.5 µg/mL in PBS) as coating antigen on the 96-well microtiter plates (TPP microtiter plates, Japan).…”

“…In previous reports, we showed that short Solubility Controlling Peptide tags (SCP-tags) [11,12,13] attached at the C-terminus of a host protein could be used to induce the formation of sub-visible amorphous aggregates and thereby increase the immunogenicity of nonimmunogenic proteins [14,15]. In particular, a hydrophobic isoleucine tag attached to the Cterminus of two model proteins, BPTI-19A (58 residues; MW: 5.98 kDa) and DEN3ED3 (103 residues; MW 11.46 kDa) increased their immunogenicity by oligomerizing the proteins into small nanometer-scale aggregates, and the immune response was long-lasting with a T-celldependent activation of the B-cells [16,17].…”

“…We then measured their immunogenicity in mice and found that C5P and C5R tags significantly enhanced the anti-BPTI-19A IgG titer, which lasted for several weeks. Thus, in addition to the previously reported C5I tag, which increased immunogenicity through protein oligomerization, C5P and C5R tags are expected to provide a tool for the effective production of biopharmaceutical antibodies and possibly for developing protein-based vaccines [14].…”