A super TLR agonist to improve efficacy of dendritic cell vaccine in induction of anti-HCV immunity

Abstract

Persistent infections caused by pathogens such as hepatitis C virus are major human diseases with limited or suboptimal prophylactic and therapeutic options. Given the critical role of dendritic cell (DC) in inducing immune responses, DC vaccination is an attractive means to prevent and control the occurrence and persistence of the infections. However, DCs are built-in with inherent negative regulation mechanisms which attenuate their immune stimulatory activity and lead to their ineffectiveness in clinical application. In this study, we developed a super DC stimulant that consists of a modified, secretory Toll-like Receptor (TLR)-5 ligand and an inhibitor of the negative regulator, suppressor of cytokine sinaling-1 (SOCS1). We found that expressing the super stimulant in DCs is drastically more potent and persistent than using the commonly used DC stimuli to enhance the level and duration of inflammatory cytokine production by both murine and human DCs. Moreover, the DCs expressing the super stimulant are more potent to provoke both cellular and humoral immune responses against hepatitis C virus (HCV) antigen in vivo. Thus, the strategy capable of triggering and sustaining proinflammatory status of DCs may be used to boost efficiency of DC vaccine in preventing and combating the persistent infection of HCV or other chronic viruses.

Figure 1. The expression of shS1/FliC rather than shS1 or FliC enhances the levels and duration of proinflammatory cytokine production by DCs.

Murine BMDCs were transduced with the recombinant Ad vectors at an MOI of 250. A, 24 h later, culture media were collected for evaluation of the representative cytokines by ELISA. B, cells were analyzed for expression of costimulatory and MHC class-II molecules by flow cytometry. C, 24 h later, cultures were washed and replaced with fresh medium that does not contain stimuli. The concentrations of representative cytokines in culture medium 3 days after washing were examined by ELISA. Data are representative of three repeated experiments. *p<0.01; **p<0.05.

Figure 2. The expression of shS1/FliC is more potent than using TLR agonist as a maturation agent to enhance the levels and duration of proinflammatory cytokine production by DCs.

Murine BMDCs were transduced with Ad-shS1/FliC or stimulated with commonly used TLR agonist LPS (100 ng/ml), CpG (1 µM), or Poly I:C (1 µg/ml), respectively. A, 24 h later, culture media were collected for evaluation of the representative cytokines by ELISA. B, 24 h later, cultures were washed and replaced with fresh medium that does not contain the corresponding stimuli. The concentrations of representative cytokines were examined by ELISA in culture medium 72 hr after the wash. C&D, 24 h later, cultures were washed and replaced with fresh medium that contains the corresponding stimuli or not (for Ad-shS1/FliC-transduced DCs). The concentrations of representative cytokines were examined by ELISA 24 hr (C) or 72 hr (D) after the wash. Data are representative of three repeated experiments. *p<0.01.

Figure 3. The expression of shS1/FliC enhances the acetylation of histone H3 at Lys9/14 and binding of transcription factor sp1 to the promoter of TNF-α in DCs.

Murine BMDCs were transduced with the recombinant Ad vectors or stimulated with LPS (100 ng/ml). 24 h later, cultures were washed and replaced with fresh medium that contains LPS or not (for Ad-transduced DCs) for 72 hr. The cells were fixed with formaldehyde and a ChIP assay was performed using anti-acetyl histone H3 (Lys9/14) antibody (A) or anti-sp1 antibody (B). The precipitated DNA was amplified by real-time PCR using a pair of TNF-α promoter primers. The results were normalized to the input DNA and expressed as a ratio of Ad-shS1/FliC DC or LPS-stimulated DC to Ad-shGFP DC. *p<0.01.

Figure 4. shS1/FliC-expressing DC immunization induces more potent HCV E2-specific T cell immune responses in mice.

Murine BMDCs were transduced with the recombinant Ad vectors followed by pulse with recombinant HCV-E2 protein (20 µg/ml) for overnight, or pulsed with HCV-E2 for 6 hr prior to addition with LPS (100 ng/ml) and then cultured for overnight. Groups of C57BL/6 mice (6 mice/group) were immunized via footpads with the transduced or LPS-stimulated DCs (1×10⁶ cells per mouse) twice. 2 weeks after the 2^nd immunization, splenocytes were isolated for activation marker CD69 staining (A), and intracellular IFN-γ staining (B). Data are representative of three repeated experiments.

Figure 5. shS1/FliC-expressing DC immunization induces more potent HCV E2-specific humoral immune responses in mice.

The above immunized mice were bled and sera were isolated for evaluation of HCV E2-specific IgG level by ELISA (A), and HCV E2-specific neutralizing antibody activity by HCVpp neutralization assay (B), splenocytes were isolated for evaluation of HCV E2-specific B cell response by ASC assay (C). Data are representative of three repeated experiments. *p<0.01 (Chi-square test).

Figure 6. The expression of the human version shhS1/FliC potently activates human monocyte-derived DCs.

Human monocyte-derived DCs were transduced with Ad vectors at an MOI of 250 or stimulated with different TLR agonists. A & B. 24 h later, culture media were collected from Ad-transduced DCs (A) or TLR-transduced DCs (B) for evaluation of the representative cytokines by ELISA. C & D. 24 h later, cultures were washed and replaced with fresh medium. The concentrations of representative cytokines in culture medium 3 days after the wash were examined by ELISA. Data are representative of three repeated experiments. *p<0.01, **p<0.05.