Chemokine receptor-mediated delivery of mycobacterial MPT51 protein efficiently induces antigen-specific T-cell responses
Introduction
Mycobacterium tuberculosis, primary agent of tuberculosis (TB), is responsible for the three million deaths annually worldwide [1]. The only TB vaccine currently available is the attenuated Mycobacterium bovis strain bacillus Calmette-Guerin (BCG) which has been reported to have a variable protective efficiency [2]. The emergence of multi-drug-resistant strains of M. tuberculosis has given urgency to the need for novel agents and development of more effective vaccines.
Chemokines play an essential role in induction of inflammatory responses by trafficking of immune cells [3]. Chemokines bind to specific cell-surface receptors which are internalized after binding with ligands [4], [5]. Chemokine receptors are differentially expressed on a variety of immune cells. Sentinel antigen-presenting cells (APCs), such as immature dendritic cells (DCs), express chemokine receptors such as CC chemokine receptor 5 (CCR5). CCR5 has been identified as the receptor for macrophage inflammatory protein-1α (MIP-1α), regulated on activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), -2, -3, -4, and geotaxis [6]. CCR5 has been transported to early endosomes and subsequently recycled back to the cell surface or targeted for degradation [4]. Therefore, it should be possible to harness the receptor binding and internalization of chemokine to increase the immunogenecity of vaccines. MIP-1α binds to two kinds of receptors, CCR1 and CCR5, In contrast, RANTES or MCPs bind to more than three receptors. In this study, we focused on the CCR5 and its ligand, MIP-1α as a simple molecular and cellular targeting model. The efficacy of MIP-1α-antigen fusion was examined by using DNA vaccine against M. tuberculosis. Antigen-specific T-cell responses appeared to be significantly enhanced by genetic fusion of MIP-1α to MPT51, one of major protective antigens of M. tuberculosis[7].
Section snippets
Fusion gene cloning and plasmid constructions
The eukaryotic expression vector, pCI (Promega, Madison, WI, USA) containing a cytomegalovirus (CMV) immediate-early promoter, chimeric intron, and SV40 late polyadenilation signal, was used for construction for DNA vaccines. Murine MIP-1α gene was cloned by reverse transcription (RT)-PCR from total RNA of DCs. MIP-1α gene was fused with MPT51 gene via 14-amino acids (GTNDAQAPKSLEGT) spacer sequence and cloned into the EcoRI/XbaI sites of pCI vector (pCI-MIP-1α-MPT51). A plasmid expressing
Receptor binding and internalization of MIP-1α fusion protein
To investigate receptor binding and internalization of chemokine fusion protein, we constructed a MIP-1α-GFP expression plasmid (Fig. 1A). HEK293T cells were transiently transfected with pCI-MIP-1α-GFP plasmid and the cell lysates were used for receptor binding assay by using confocal microscopy. Most of the MIP-1α-GFP fusion proteins localized on the surface of murine macrophage-like RAW264.7 cells (Fig. 1B, left). Co-staining of the cells with PE-labeled ant-CCR5 antibody showed
Discussion
The potency of vaccine presumably relies on the ability to recruit APCs and deliver antigens to them, leading to efficient antigen presentation to specific T cells. DCs are crucial in the activation of naïve T cells and induction of T cell-dependent immune responses. For this reason, experimental modification of vaccines, in particular genetic antigen delivery, has attracted much interest. Immature DCs, which are known as sentinel APCs, preferentially express CCR1, CCR2, CCR5, and CCR6 [11],
Acknowledgements
We are grateful to Kiyoshi Shibata (Hamamatsu Univ. Sch. Med.) for excellent technical assistance. This work was supported by a grant-in-aid for scientific research and a grant-in-aid for centers of excellence (CoE) research program from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
References (21)
Chemokines
Blood
(1997)- et al.
Pathways for internalization and recycling of the chemokine receptor CCR5
Blood
(2002) - et al.
Regulation of the human chemokine receptor CCR1: cross-regulation by CXCR1 and CXCR2
J Biol Chem
(2000) - et al.
CCR5 binds multiple CC-chemokines: MCP-3 acts as a natural antagonist
Blood
(1999) - et al.
Expression mapping using a retroviral vector for CD8+ T cell epitopes: definition of a Mycobacterium tuberculosis peptide presented by H2-Dd
J Immunol Meth
(2005) - et al.
Chemokine receptor targeting efficiently directs antigens to MHC class I pathways and elicits antigen-specific CD8+ T-cell responses
Blood
(2006) - et al.
Advantage of gene gun-mediated over intramuscular inoculation of plasmid DNA vaccine in reproducible induction of specific immune responses
Vaccine
(2000) - et al.
Expression kinetics of the interleukin-2/immunoglobulin (IL-2/Ig) plasmid cytokine adjuvant
Vaccine
(2004) - et al.
Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country
JAMA
(1999) - et al.
Efficacy of BCG vaccine in the prevention of tuberculosis
JAMA
(1994)
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