Enhanced Generation of Cytotoxic T Lymphocytes by Heat Shock Protein 70 Fusion Proteins Harboring Both CD8⁺ T Cell and CD4⁺ T Cell Epitopes

Abstract

Heat shock protein 70 (Hsp70) can be a potent carrier of antigens because it is effectively delivered to antigen presenting cells (APCs) and activates innate immunity. To induce a potent cytotoxic T lymphocyte (CTL) response, a Hsp70 fusion protein harboring both CD8(+) and CD4(+) T cell epitopes was developed based on the recent understanding of the importance of the role of CD4(+) T cells in inducing the CTL response following vaccination. OVA(257-264) (pepI) and OVA(323-339) (pepII) were selected as the CD8(+) and CD4(+) T cell epitope of a model antigen, ovalbumin (OVA), respectively. Hsp70 and its fusion proteins, Hsp70-pepI, pepII-Hsp70, Hsp70-pepII and pepII-Hsp70-pepI, were developed. pepII-Hsp70 and pepII-Hsp70-pepI were effectively presented on MHC class II of macrophages compared with Hsp70-pepII, suggesting that pepII conjugation to the N-terminus of Hsp70 is better than the C-terminus for more effective MHC class II antigen presentation. Immunization with pepII-Hsp70-pepI resulted in a higher CTL activity than immunization with the mixture of Hsp70-pepI and pepII-Hsp70. Furthermore, pepII-Hsp70-pepI exhibited a greater antitumor effect in the mice bearing EG7 tumor cells than the physical mixture of Hsp70-pepI and pepII-Hsp70. In addition, immunization with the DCs pulsed with the fusion proteins also suggested that APCs which present both pepI and pepII can induce the highest CTL generation. These results demonstrated that Hsp70 fusion protein harboring both pepI and pepII is a useful option for Hsp70-based antigen delivery systems.

Heat shock proteins: A dual carrier-adjuvant for an anti-drug vaccine against heroin

“…The endotoxin level was measured with the Limulus amebocyte lysate assay (Thermo Scientific) and determined to be >6EU/μg of protein before column treatments and <1.1 EU/μg of protein (Table A3). 25 The purified protein was stored in PBS with 50% glycerol (v/v) at −80 °C.…”

Dendritic Cells in Anticancer Vaccination: Rationale for Ex Vivo Loading or In Vivo Targeting

“…Therefore, a number of strategies utilizing HSPs to target the SRs of DCs have been developed [158]. Different studies reported different variants of HSP-based vaccines designed to target SRs, which included the harvesting of autologous HSP-TAA complexes [159], in vitro constructed HSP-TSA complexes [160,161] and fusions of HSPs to cancer peptides [162]. Eventually, all of these methods aim to develop a DC-targeting vaccine that will activate DCs in vivo after its administration to the patient.…”

Hsp70 in cancer: A double agent in the battle between survival and death

“…Furthermore, the eHsp70 in complex with tumor antigenic peptides can interact with surface receptors on APCs such as DC, macrophages and monocytes as well as endothelial/epithelial cells including CD36, CD91, CD14, CD40, TLR2/4 and scavenger receptors such as LOX‐1, SR‐A, SREC‐1 and FEEL‐1, which recently have been considered as putative eHsp70 receptors (Thériault et al, 2005). This interaction facilitates the internalization of Hsp70 by receptor‐mediated endocytosis leading to the antigen cross‐presentation onto MHC class I molecules (Noessner et al, 2002) which, in turn, elicit an antitumor CTL response (Takemoto et al, 2010), Figure 7. It is noteworthy that, the SBD of Hsp70 plays an important role in transporting antigens for binding to MHC class I molecules (Rohrer et al, 2014).…”