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.

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Subvisible aggregates of proteins are suspected to cause adverse immune response, and a recent FDA guideline has recommended the monitoring of micrometer-sized aggregates (2–10 μm) though recognizing that the underlying mechanism behind aggregation and immunogenicity remains unclear. Here, we report a correlation between the immunogenicity and the size of nanometer-scaled aggregates of a small 6.5 kDa model protein, bovine pancreatic trypsin inhibitor (BPTI) variant. BPTI-19A, a monomeric and nonimmunogenic protein, was oligomerized into subvisible aggregates with hydrodynamic radii (R h) of 3–4 nm by attaching hydrophobic solubility controlling peptide (SCP) tags to its C-terminus. The results showed that the association of nonimmunogenic BPTI into nanometer-sized subvisible aggregates made it highly immunogenic, as assessed by the IgG antibody titers of the mice’s sera. Overall, the study emphasizes that subvisible aggregates, as small as a few nanometers, which are presently ignored, are worth monitoring for deciphering the origin of undesired immunogenicity of therapeutic proteins.

Abbreviations AUC, analytical ultra-centrifugation; BPTI-19A (untagged BPTI-19A), a simplified variant of bovine pancreatic trypsin inhibitor containing 19 alanines; BPTI-C5P, a BPTI-19A with C5P tag at its C terminus; other tags are named correspondingly; DLS, dynamic light scattering; SCP-tags, solubility controlling peptide tags; SLS, static light scattering.

Nomenclature: SCP-tags: solubility controlling peptide tags; We use "aggregates" and "oligomer" interchangeably. Sub-visible aggregates/oligomers: Aggregates/oligomers ~<100 µm that are not visible to the naked eye and remain in the soluble fraction upon 2 centrifugation; : A simplified variant of bovine pancreatic trypsin inhibitor containing 19 alanines; BPTI-C5X: 5 amino acid residues as SCP-tags added at the C-terminus of BPTI, where X stands for amino acid identity.3 Abstract (150 words)Sub-visible aggregates of proteins are suspected to cause adverse immune response, and a recent FDA guideline has recommended the monitoring of micrometer-size aggregates (2-10 µm) though recognizing that the underlying mechanism behind aggregation and immunogenicity remains unclear. Here, we report a correlation between the immunogenicity and the size of nanometer-scale aggregates of a small 6.5 kDa model protein, Bovine Pancreatic Trypsin Inhibitor (BPTI) variant. BPTI-19A, a monomeric and non-immunogenic protein, was oligomerized into sub-visible aggregates with hydrodynamic radii (Rh) of 3~4 nm by attaching hydrophobic solubility controlling peptide (SCP) tags to its C-terminus. The results showed that the association of non-immunogenic BPTI into nanometer-size aggregates made it highly immunogenic, as assessed by the IgG antibody titers of the mice's sera. Overall, the study emphasizes that sub-visible aggregates, as small as a few nanometers, which are presently ignored, are worth monitoring for deciphering the origin of undesired immunogenicity of therapeutic proteins.