Immune-tolerant elastin-like polypeptides (iTEPs) and their application as CTL vaccine carriers

Abstract

Background: Cytotoxic T lymphocyte (CTL) vaccine carriers are known to enhance the efficacy of vaccines, but a search for more effective carriers is warranted. Elastin-like polypeptides (ELPs) have been examined for many medical applications but not as CTL vaccine carriers.

Purpose: We aimed to create immune tolerant ELPs using a new polypeptide engineering practice and create CTL vaccine carriers using the ELPs.

Results: Four sets of novel ELPs, termed immune-tolerant elastin-like polypeptide (iTEP) were generated according to the principles dictating humoral immunogenicity of polypeptides and phase transition property of ELPs. The iTEPs were non-immunogenic in mice. Their phase transition feature was confirmed through a turbidity assay. An iTEP nanoparticle (NP) was assembled from an amphiphilic iTEP copolymer plus a CTL peptide vaccine, SIINFEKL. The NP facilitated the presentation of the vaccine by dendritic cells (DCs) and enhanced vaccine-induced CTL responses.

Discussion: A new ELP design and development practice was established. The non-canonical motif and the immune tolerant nature of the iTEPs broaden our insights about ELPs. ELPs, for the first time, were successfully used as carriers for CTL vaccines.

Conclusion: It is feasible to concurrently engineer both immune-tolerant and functional peptide materials. ELPs are a promising type of CTL vaccine carriers.

Keywords: Cytotoxic T lymphocyte (CTL) vaccine; immune-tolerant elastin-like polypeptide; inverse phase transition; non-canonical elastin-like polypeptide motifs; reversible; thermally-induced; vaccine carrier.

Figure 1

An outline of iTEP monomers obtained from a homology analysis between mouse tropoelastin and human elastin. Blue and red letters indicate amino acids from mouse and human elastins, respectively. Green letters indicate the same amino acids across both species. The numbers indicate the positions of these building blocks in their parent elastin proteins.

Figure 2

(A) Schematics showing the approach to double the length of the coding genes of iTEPs. (B) iTEP coding genes on agarose gel after they were cleaved from pET25b(+) vector by XbaI and BamHI. Sizes of these iTEP genes confirmed they have the right lengths, suggesting these genes would code iTEPs of expected lengths. (C) SDS-PAGE gel showing MWs and purity of individual iTEPs.

Figure 3

(A–D) Turbidity profiles (OD350) of iTEP_A, iTEP_B, iTEP_C, and iTEP_D as they were heated and then cooled between 20 °C and 80 °C. (E) The turbidity profiles of iTEP_B in 2.5M NaCl as a function of temperature. Each curve represented an average of three measurements.

Figure 4

(A) The immunization schedule and the time point of the assessment of humoral responses. (B) The summary of IgG titers of OVA, MSA, and iTEP-immunized mice. Each dot represents one mouse’s result. The medians and interquartile ranges of the titers are shown. (C–F) Absorbance (OD450) of sera that were collected from iTEP_A (C), iTEP_B (D), iTEP_C (E), and iTEP_D(F) immunized mice after they were diluted and assayed by ELISA. Each data point corresponds to the mean value of three absorbance measurements per serum dilution. The data of each mouse were linked together with a line. The cut-off ranges for positive absorbance values were shown as a blue shade.

Figure 5

(A) Schematics showing that the iTEP_B-iTEP_A-pOVA fusion self-assembles into a NP. (B) SDS-PAGE gel showing MWs and purity of two fusions, iTEP_B-pOVA and iTEP_B-iTEP_A-pOVA.(C) Turbidity profile of the iTEP_B-iTEP_A-pOVA fusion as a function of temperature. The solution has a sharp increase of the turbidity at 70 °C indicating the formation of aggregates at that temperature. Before that, there is a slow, mild increase of turbidity suggesting the formation of micelles⁽⁸⁾ (D) Size distributions of the iTEP_B-pOVA and iTEP_B-iTEP_A-pOVA fusions (25 µM) obtained from DLS measurement. (E) A representative micrograph of a negatively stained iTEP_B-iTEP_A-pOVA fusion confirming the NP size of the fusion.

Figure 6

(A) Presentation of SIINFEKL by DC 2.4 cells after the SIINFEKL was delivered by OVA, soluble iTEP_B-pOVA fusion, or iTEP-pOVA NP. Data are presented as means of normalized MFI ± SD of the entire DC population used in the experiments (n ≥ 4 independent experiments). (B) Activation of B3Z cells after they were incubated with DCs that presented SIINFEKL. The DCs were pre-incubated with different forms of antigens as noted in the picture. Data are presented as mean ± SD 3 independent experiments). (C) Ex vivo analysis of active, SIINFEKL-restricted splenocytes cells from mice (n=3–5) immunized with OVA, free SIINFEKL peptide, or iTEP-pOVA NP. The activation of the cells was characterized by using an INF-γ-based ELISPOT assay. Data were presented as Spot Forming Units (SFU)/million cells ± SD. For all panels, ★ indicates p< 0.05, t-test between the paired treatments.