Effective induction of simian immunodeficiency virus-specific cytotoxic T lymphocytes in macaques by using a multiepitope gene and DNA prime-modified vaccinia virus Ankara boost vaccination regimen

Abstract

DNA and modified vaccinia virus Ankara (MVA) are vaccine vehicles suitable and safe for use in humans. Here, by using a multicytotoxic T-lymphocyte (CTL) epitope gene and a DNA prime-MVA boost vaccination regimen, high levels of CTLs specific for a single simian immunodeficiency virus (SIV) gag-derived epitope were elicited in rhesus macaques. These vaccine-induced CTLs were capable of killing SIV-infected cells in vitro. Fluorescence-activated cell sorter analysis using soluble tetrameric major histocompatibility complex-peptide complexes showed that the vaccinated animals had 1 to 5% circulating CD8(+) lymphocytes specific for the vaccine epitope, frequencies comparable to those in SIV-infected monkeys. Upon intrarectal challenge with pathogenic SIVmac251, no evidence for protection was observed in at least two of the three vaccinated animals. This study does not attempt to define correlates of protective immunity nor design a protective vaccine against immunodeficiency viruses, but it demonstrates clearly that the DNA prime-MVA boost regimen is an effective protocol for induction of CTLs in macaques. It also shows that powerful tools for studying the role of CTLs in the control of SIV and human immunodeficiency virus infections are now available: epitope-based vaccines, a protocol for an effective induction of CTLs in primates, and a simple and sensitive method for quantitation of epitope-specific T cells. The advantages of the DNA prime-MVA boost regimen as well as the correlations of tetramer staining of peripheral blood lymphocytes with CTL killing in vitro and postchallenge control of viremia are discussed.

Figures

FIG. 1

Cytolytic T-cell responses determined by a standard 51Cr-release assay. PBLs were isolated, and the bulk CTL cultures were treated in vitro with the following reagents: peptide CTPYDINQM and IL-7 on day 0, Lymphocult-T on day 3, peptide-pulsed irradiated autologous B-LCL on day 8, and Lymphocult-T on day 11. The CTL assays were carried out on day 14. (A) The restimulated effectors were incubated for 6 h with 51Cr-labelled peptide or unpulsed (the first two bars for each animal) or pulsed (the last two bars for each animal) autologous B-LCL cells at the effector/target ratios indicated below the graphs. (B) Restimulated PBLs drawn from Doris on week 35 were tested on 51Cr-labelled autologous B-LCL cells (left) or 6-day PHA-stimulated PBLs (right), which had been treated as indicated, at effector/target ratios of 54:1, 27:1, and 13:1.

FIG. 2

Specificity of the soluble MamuA*01–p11C, C-M tetrameric complexes. The peptide-grown CTL line (A) or frozen blood samples containing Mamu-A*01-negative rhesus macaque PBLs (B), and PBLs isolated from Di at weeks 0 (C) and 23 (D) were double stained for the CD8 marker and Mamu-A*01–p11C, C-M-specific T-cell receptors and examined by FACS.

FIG. 3

Longitudinal analysis of vaccine-induced SIV-specific T cells. Groups of rhesus macaques were immunized with 8 μg of pTH.HW DNA with a gene gun or 5 × 108 PFU of MVA.HW i.d. at the times indicated below each graph. Frozen isolated PBL samples were thawed, double stained with PE-conjugated tetrameric MHC-peptide complexes, and human CD8-specific Tri-color-conjugated MAb for the CD8 marker, and examined by FACS. An n indicates a sample measured, but negative.

FIG. 4

Longitudinal analysis of peripheral blood after SIV challenge. The left panels show DNA prime-MVA boost-vaccinated macaques Cyd (⧫), Di (▴), and Doris (■), and the right panels show naive macaques X23 (◊), X32 (□), and V48 (×). (A) Plasma virus loads were determined with a quantitative competitive RT-PCR. A negative result meant there were less than 40 genome equivalents (Eq) per ml of plasma. (B) Cell-associated virus loads were determined by coculturing multiple dilutions of isolated PBLs with the C8166 cell line and assessing the cytopathic effect resulting from SIV infection. A negative result meant there were less than 1.6 infected cells per 106 PBLs. (C and D) CD8+ (C) and CD4+ (D) lymphocytes were enumerated by a whole-blood lysis method whereby PBLs were first incubated with a CD8- or CD4-specific MAb followed by Immunolyse, and their counts were established from the difference between untreated and treated PBLs.

FIG. 5

Postchallenge analysis of tetramer-specific lymphocytes. Cyd, Di, and Doris were immunized by the DNA-MVA regimen, and the X23 and X32 monkeys are two Mamu-A*01+ naive controls. Frozen isolated PBLs were double stained with fluorochrome-conjugated Mamu-A*01–p11C, C-M tetramer and a CD8-specific MAb and analyzed with a FACScan. To facilitate the correlation, the plasma virus loads from Fig. 4A were overlaid. An n indicates a sample measured, but negative. Eq, genome equivalents.

FIG. 6

p11C, C-M-specific CTL responses 4 weeks after the SIVmac challenge (week 49). PBLs were isolated, restimulated in vitro, and tested on peptide-unpulsed (the first bar for each animal) or pulsed (the last three bars for each animal) PBLs for cytolytic activity as described in the legend to Fig. 1. The effector/target ratios were 25:1, 12:1, and 6:1.