Identification of a highly conserved surface on Tat variants

Abstract

Extracellular Tat is suspected to protect HIV-1-infected cells from cellular immunity. Seropositive patients are unable to produce neutralizing antibodies against Tat, and Tat is still secreted under antiviral treatment. In mice, the Tat OYI vaccine candidate generates neutralizing antibodies such as the mAb 7G12. A peptide called MIMOOX was designed from fragments of Tat OYI identified as the possible binding site for mAb 7G12. MIMOOX was chemically synthesized, and its structure was stabilized with a disulfide bridge. Circular dichroism spectra showed that MIMOOX had mainly β turns but no α helix as Tat OYI. MIMOOX was recognized by mAb 7G12 in ELISA only in reduced conditions. Moreover, a competitive recognition assay with mAb 7G12 between MIMOOX and Tat variants showed that MIMOOX mimics a highly conserved surface in Tat variants. Rat immunizations with MIMOOX induce antibodies recognizing Tat variants from the main HIV-1 subtypes and confirm the Tat OYI vaccine approach.

Keywords: Cellular Immune Response; Epitope Mapping; HIV-1; Protein Design; Tat; Vaccine; Vaccine Development.

FIGURE 1.

Tat OYI and MIMOOX molecular modeling. Tat OYI (A, C) is divided into six regions: region I (residues 1–21) is depicted in red, region II (cysteine-rich region, residues 22–37) in orange, region III (residues 38–48) in yellow, region IV (basic region, residues 49–59) in green, region V (residues 60–72) in light blue, region VI (residues 73–101) in blue. MIMOOX (B and C) comprises the N-terminal region (red), followed by the inverted sequence of the C terminus (blue) and then region III (yellow), and a part of the arginine-rich region (green). Glycines serve as a linker between the three Tat regions are in purple. Two of the three cysteines create a disulfide bridge that is essential to have a three-dimensional epitope in the MIMO equivalent to the three-dimensional epitope in Tat OYI.

FIGURE 2.

Control of MIMOOX folding. A, oxidation of MIMOOX was verified in HPLC method between T = 0 h (dashed line) and T = 6 h (continuous line). B, mass spectrometry after alkylation with iodoacetamide of MIMO (black), MIMOOX in oxidation conditions at T = 0 h (dashed lines) and at T = 6 h (gray). In C, CD spectra of MIMOOX and Tat OYI were measured from 260 to 178 nm with a 50-mm path length in 20 mm phosphate buffer (pH 4.5). The same percentage of secondary structures of MIMOOX and Tat OYI was determined from the CD data analysis with no α helix, 23% extended structure, 30% β turns, and 47% of other structures. The absence of the α helix in MIMOOX is in accordance with a similar structure of the conserved surface of Tat variants that contain only β turn secondary structure. Furthermore, MIMOOX having 56 residues, 30% β turns correspond to four β turns. This is compatible with the presence of at least β turns in the MIMOOX structure. mAU, milliabsorbance units.

FIGURE 3.

MIMOOX was recognized by the conformational mAb 7G12 able to cross-neutralize Tat variants. In A, the affinity curves (mean ± S.D., n ≥ 3) of mAb 7G12 with Tat OYI (black) and MIMOOX (gray) were measured in ELISA. In B, the affinity curves (mean ± S.D., n ≥ 3) of mAbs 7G12 and 6E7 with native (dark gray) or denatured (light gray) MIMOOX were measured in ELISA. Different dilutions of mAbs were used. In bar graphs, statistical significant differences (p < 0.05) between the affinity of mAbs 7G12 with native and denatured MIMOOX were indicated by an asterisk. In C, the recognition of native or denatured MIMOOX by mAbs 7G12 and 6E7 in dot blot assay was represented (n = 3). Lane 1, denatured MIMOOX; lane 2, denatured MIMOOX; lane 3, native MIMOOX.

FIGURE 4.

Competitive ELISA (mean ± S. D.; n ≥ 3) with mAb 7G12 between MIMOOX in solution and coated Tat OYI (A) or Tat in solution and coated MIMOOX (B) are shown. Native (continuous line) and denatured (dashed line) form of proteins in solution were shown. In C, competitive ELISA (mean ± S.D., n ≥ 3) with mAb 7G12 between different coated Tat variants and native (dark gray) or denatured (light gray) MIMOOX or coated MIMOOX and native (dark gray) or denatured (light gray) Tat from different clades (D). Signal without competitor represented the 100% (black) for each Tat tested. These experiments were done in triplicate. In bar graphs, statistical significant differences (p < 0.05) between the condition without competitor and with native competitor were indicated by an asterisk.

FIGURE 5.

MIMOOX and Tat variants recognition by anti-MIMOOX serum dilution. ELISA (n = 2) was performed with four dilutions of the anti-MIMOOX rat serum with coated MIMOOX (black squares), Tat OYI (white squares), Tat UG11RP (white triangles), Tat 96BW (white circles), Tat Eli (black triangles), and Tat CM240 (black circles). ELISA was performed also with a negative control (white star) made of a pool of three peptides corresponding respectively to regions 1, 3, and 6 of Tat OYI. The anti-MIMOOX serum recognizes the five Tat variants and MIMOOX. Tat OYI was recognized as well as MIMOOX. Serum dilution shows that Tat Eli is better recognized compared with the other Tat variants. A similar result was observed with a previously published Tat OYI immunization (14).