TRANSGENE IL-21-ENGINEERED ANTIGEN-SPECIFIC EXOSOME TARGETED T CELL-BASED VACCINE POTENTLY CONVERTS CTL EXHAUSTION IN CHRONIC INFECTION

Abstract

CD8+ cytotoxic T lymphocytes (CTLs), the potent effector T cells, capable of directly destroying virus-infected cells, correlate with acute viral control and long-term non-progression in virus-mediated infectious diseases, play an important role in controlling viral infections. However, CD8+ CTLs due to persistent viral stimulation showed functional exhaustion in virally induced chronic infections, which expressed inhibitory molecules such as inhibitory programmed death (PD)-1, programmed death ligand (PDL)-1, T-cell Ig and mucin protein-3 (TIM3) and lymphocyte-activation gene 3 (LAG-3), and were functionally exhausted such as defect in effector cytokine IFN- production, lack of cytolytic effect and reduction of recall responses upon the pathogen reencounter. Therefore, CTL exhaustion has become one of the major obstacles for the ineffectiveness of viral control in chronic infectious diseases such as human immunodeficiency virus (HIV)-1. We previously generated novel ovalbumin (OVA)-specific 41BBL-expressing OVA-TEXO and HIV-1 Gag-specific Gag-TEXO vaccines inducing therapeutic immunity in B6 mice and converting CTL exhaustion via its CD40L signaling activation of the PI3K-Akt-mTORC1 pathway in recombinant OVA-specific adenovirus AdVOVA-infected B6 (AdVOVA-B6) mice with chronic infection. In AdVOVA-B6 mice, OVA-specific CTLs expressing IL-7R, IL-21R and inhibitory PD-1, PDL-1 and LAG-3 were inflated and functionally exhausted. Cytokine IL-21, a member of the common -chain cytokine family, produced by CD4+ helper T cells, plays an important role in controlling chronic infections. IL-21 promotes CTL activation and survival by activation of the phosphatidylinositol-3 kinase (PI3K) and the mTORC1-regulated T-bet pathway. In this study, we constructed recombinant transgene IL-21-expressing AdVIL-21 by recombinant DNA technology, generated IL-21-expressing OVA-TEXO/IL-21 and Gag-TEXO/IL21 vaccines or the control OVA-TEXO/Null and Gag-TEXO/Null vaccines by infection of OVA-TEXO and Gag-TEXO cells with AdVIL-21 or the control AdVNull without transgene, and assessed their stimulatory immunogenicity in wild-type B6 or AdVOVA-B6 mice, respectively. We demonstrate that both OVA-TEXO/IL-21 and the control OVA-TEXO/Null vaccines are capable of converting CTL exhaustion in chronic infection. However, IL-21-expressing OVA-TEXO/IL-21 vaccine more efficiently rescues exhausted CTLs through increasing CTL proliferation and effector cytokine IFN-ɤ expression by 6-fold than the 3-fold in OVA-TEXO/Null-vaccinated AdVOVA-B6 mice, though these two vaccines stimulated comparable OVA-specific responses and immunity against OVA-expressing BL6-10OVA melanoma in B6 mice. In vivo OVA-TEXO/IL-21-stimulated CTLs more efficiently up-regulate phosphorylation of mTORC1-regulated EIF4E and expression of mTORC1-controlled T-bet molecules as well as Ki67 (a protein associated with cell-cycle progression) than the control OVA-TEXO/Null-stimulated CTLs, indicating that enhancement of converting CTL exhaustion in chronic infection by OVA-TEXO/IL-21 vaccination is mostly through the stronger activation of the PI3K-Akt-mTORC1 pathway derived from both its endogenous CD40L and transgenic IL-21 signaling. Importantly, Gag-TEXO/IL21 vaccine also induces stronger Gag-specific therapeutic immunity against established Gag-expressing BL6-10Gag melanoma lung metastases than Gag-TEXO/Null vaccine in chronic infection. Therefore, this study should have a strong impact on developing new therapeutic vaccines for chronic infectious diseases such as HIV-1 infection.