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Rationale: Albumin-binding carriers have been shown to target cytotoxic T lymphocyte (CTL) vaccines to lymph nodes (LNs) and improve the efficacy of the vaccines. However, it was not clear whether the improved efficacy is solely due to the LN targeting, which prompted this study. Methods: First, we generated a fusion protein consisting of an albumin-binding domain (ABD) and an immune-tolerant elastin-like polypeptide (iTEP). Then, we examined the binding between this fusion protein, termed ABD-iTEP, and mouse serum albumin (MSA). Next, we evaluated the accumulation of ABD-iTEP in LNs and dendritic cells (DCs) in the LNs. We also analyzed antigen presentation and in vitro T cell activation of vaccines that were delivered by ABD-iTEP and investigated possible underlying mechanisms of the presentation and activation results. Last, we measured CTL responses induced by ABD-iTEP-delivered vaccines in vivo. Results: ABD-iTEP bound with MSA strongly with an affinity of 1.41 nM. This albumin-binding carrier, ABD-iTEP, accumulated in LNs 3-fold more than iTEP, a control carrier that did not bind with albumin. ABD-iTEP also resulted in 4-fold more accumulation in DCs in the LNs than iTEP. Most importantly, ABD-iTEP drastically enhanced the antigen presentation of its vaccine payloads and the T cell activation induced by its payloads. The enhancement was dependent on the formation of the complex between MSA and ABD-iTEP. Meanwhile, the MSA/ABD-iTEP complex was found to have increased stability in acidic subcellular compartments and increased cytosolic accumulation in DCs, which might explain the enhanced vaccine presentation resulting from the complex. Finally, when ABD-iTEP was used to deliver CTL vaccines derived from both self- and non-self-antigens, it boosted the vaccine-induced responses by 2-fold in either case. Conclusion: ABD-iTEP not only targets vaccines to LNs but also promotes the presentation of the vaccines by DCs. Albumin-binding carriers have more than one mechanism to boost the efficacy of CTL vaccines.

Vaccine carriers have been shown to enhance cytotoxic T lymphocyte (CTL) epitope peptide vaccines by addressing intrinsic limitations of the vaccines. We have previously developed an immune-tolerant elastin-like polypeptide (iTEP)-based nanoparticle (NP) as an effective and unique CTL vaccine carrier. The NP is unique for its humoral immune tolerance, flexible structure, and ability to deliver CTL vaccines as polypeptide fusions. Here, we aimed to improve the NP by increasing its stability since we found it was not stable. We thus generated a more stable iTEP NP (ST-NP) and used it to deliver a CTL peptide vaccine, SIINFEKL. However, we surprisingly found that the ST-NP had a lower efficiency than the previously developed, marginally stable iTEP NP (MS-NP) in terms of promoting vaccine presentation and vaccine-induced CTL responses. On the other hand, dendritic cells (DCs) showed preferential uptake of the ST-NP but not the MS-NP. To develop an iTEP vaccine carrier that outperforms both the MS-NP and the ST-NP, we devised an iTEP NP that has a changeable stability responsive to a cytosolic, reductive environment, termed reductive environment-dependent NP or RED-NP. The RED-NP showed an intermediate ability to promote vaccine presentation and T cell responses in vitro between the MS-NP and the ST-NP. However, the RED-NP induced the strongest CTL responses in vivo among all three NPs. In conclusion, iTEP NPs that have a dynamically changeable stability are most effective to deliver and enhance CTL peptide vaccines. The work also demonstrated the versatile nature of iTEP vaccine carriers.