CD8(+) lymphocytes from simian immunodeficiency virus-infected rhesus macaques recognize 14 different epitopes bound by the major histocompatibility complex class I molecule mamu-A*01: implications for vaccine design and testing

Abstract

It is becoming increasingly clear that any human immunodeficiency virus (HIV) vaccine should induce a strong CD8(+) response. Additional desirable elements are multispecificity and a focus on conserved epitopes. The use of multiple conserved epitopes arranged in an artificial gene (or EpiGene) is a potential means to achieve these goals. To test this concept in a relevant disease model we sought to identify multiple simian immunodeficiency virus (SIV)-derived CD8(+) epitopes bound by a single nonhuman primate major histocompatibility complex (MHC) class I molecule. We had previously identified the peptide binding motif of Mamu-A*01(2), a common rhesus macaque MHC class I molecule that presents the immunodominant SIV gag-derived cytotoxic T lymphocyte (CTL) epitope Gag_CM9 (CTPYDINQM). Herein, we scanned SIV proteins for the presence of Mamu-A*01 motifs. The binding capacity of 221 motif-positive peptides was determined using purified Mamu-A*01 molecules. Thirty-seven peptides bound with apparent K(d) values of 500 nM or lower, with 21 peptides binding better than the Gag_CM9 peptide. Peripheral blood mononuclear cells from SIV-infected Mamu-A*01(+) macaques recognized 14 of these peptides in ELISPOT, CTL, or tetramer analyses. This study reveals an unprecedented complexity and diversity of anti-SIV CTL responses. Furthermore, it represents an important step toward the design of a multiepitope vaccine for SIV and HIV.

FIG. 1

Detection of IFN-γ production by PBMC using the ELISPOT assay. PBMC from various Mamu-A*01⁺ SIV-infected animals were tested with the Mamu-A*01 peptides in 16-h ELISPOT assays. (A) Animal 96031 (Gag_CM9 vaccinated) and animal 95024. (B) Animal 95114. (C) Animal 95115. PBMC were plated in 96-well plates at 2 × 10⁵ cells/well and stimulated with various peptides (1 to 10 μM concentration). Mean values and standard deviations from triplicate wells were averaged for each assay, and SFCs were enumerated as described in Materials and Methods. Asterisks indicate statistically significant responses (see the text). Responses to concanavalin A were always greater than 200 SFCs per 2 × 10⁵ cells. The ELGDYKLV peptide represents an irrelevant SIV Env peptide (negative control).

FIG. 1

Detection of IFN-γ production by PBMC using the ELISPOT assay. PBMC from various Mamu-A*01⁺ SIV-infected animals were tested with the Mamu-A*01 peptides in 16-h ELISPOT assays. (A) Animal 96031 (Gag_CM9 vaccinated) and animal 95024. (B) Animal 95114. (C) Animal 95115. PBMC were plated in 96-well plates at 2 × 10⁵ cells/well and stimulated with various peptides (1 to 10 μM concentration). Mean values and standard deviations from triplicate wells were averaged for each assay, and SFCs were enumerated as described in Materials and Methods. Asterisks indicate statistically significant responses (see the text). Responses to concanavalin A were always greater than 200 SFCs per 2 × 10⁵ cells. The ELGDYKLV peptide represents an irrelevant SIV Env peptide (negative control).

FIG. 2

Mamu-A*01 tetramers refolded with individual peptides stain unique populations of lymphocytes. The specificity of the tetramers was assessed through double staining of fresh PBMC (animal 95114) with the Gag_CM9-PE-labeled tetramer and one of three APC-labeled Mamu-A*01 tetramers. Each of the tetramers stained a unique population of CD3⁺ CD8⁺ T lymphocytes, with no cells staining with more than one tetramer. Background levels of tetramer staining in fresh PBMC from a naive Mamu-A*01⁺ animal (96078) were below 0.06%.

FIG. 3

Locations of Mamu-A*01-bound peptides recognized in SIV-infected rhesus macaques. Boxes within each of the proteins correspond to the position of the Mamu-A*01-restricted CTL epitopes which were identified by ELISPOT or CTL assays.