Toward the discovery of vaccine adjuvants: coupling in silico screening and in vitro analysis of antagonist binding to human and mouse CCR4 receptors

Abstract

Background: Adjuvants enhance or modify an immune response that is made to an antigen. An antagonist of the chemokine CCR4 receptor can display adjuvant-like properties by diminishing the ability of CD4+CD25+ regulatory T cells (Tregs) to down-regulate immune responses.

Methodology: Here, we have used protein modelling to create a plausible chemokine receptor model with the aim of using virtual screening to identify potential small molecule chemokine antagonists. A combination of homology modelling and molecular docking was used to create a model of the CCR4 receptor in order to investigate potential lead compounds that display antagonistic properties. Three-dimensional structure-based virtual screening of the CCR4 receptor identified 116 small molecules that were calculated to have a high affinity for the receptor; these were tested experimentally for CCR4 antagonism. Fifteen of these small molecules were shown to inhibit specifically CCR4-mediated cell migration, including that of CCR4(+) Tregs.

Significance: Our CCR4 antagonists act as adjuvants augmenting human T cell proliferation in an in vitro immune response model and compound SP50 increases T cell and antibody responses in vivo when combined with vaccine antigens of Mycobacterium tuberculosis and Plasmodium yoelii in mice.

Figure 1. Structure of the thiazoline (a) and lactam (b) derivatives.

Figure 2. Structure of the quinazoline, quinoline and isoquinoline derivatives where either X or Y or both equal N.

Figure 3. The crystal structure of bovine rhodopsin.

Figure 4. The CCR4 homology model inserted into a LMP and fully solvated.

The water atoms (red) form a box around the protein and DPP atoms (blue).

Figure 5. The conventional 2D representation of the six selected ligands showing inhibitory properties for CCR4-mediated migration.

Figure 6. Assessment of CCRF-CEM cell viability by propidium iodide (PI) and annexin V labeling following treatment with CCR4 antagonists.

Propidium iodide (PI) labels cells lacking intact plasma membranes and hence at an advanced stage of cell death. Annexin V labels cells at early and late stages of apoptosis. Plots show representative data for cells cultured in medium alone or in medium to which solvent (DMSO) or antagonist has been added. Numbers indicate percentage of cells in each quadrant.

Figure 7. CCR4 antagonists enhance cellular and humoral responses in mice.

(a) Effect of SP50 on T cell priming to Mycobacterium tuberculosis protein antigens Rv1818c or Rv3812. Splenocytes (0.5×10⁶) from mice immunised with the indicated proteins+SP50 or DMSO were cultured in vitro in the presence of 5 µg/ml of either Rv1818c or Rv3812 recombinant proteins for 5 days. Cells from three mice per group were tested individually in quadruplicate wells. Antigen-specific T cell proliferation was measured by thymidine incorporation assay and data are presented as stimulation index (SI, mean cpm of the antigen stimulated wells/mean cpm of control wells). Significant differences are indicated by asterisks * p<0.05. The results are from one experiment. (b) IgG serum antibody responses against MSP-1₁₉ measured by ELISA 14 days post vaccination with Ad-MSP-1₄₂ plus the indicated compounds or DMSO control. Three mice per group were tested individually. Columns represent the mean±SD. Significant differences are indicated by asterisks * p<0.05. Similar results were obtained in two independent experiments.

Figure 8. CB20 ligand docked into the CCR4 homology model.