Multiepitopic B- and T-cell responses induced in humans by a human immunodeficiency virus type 1 lipopeptide vaccine

Abstract

We have attempted to develop an anti-human immunodeficiency virus (HIV) lipopeptide vaccine with several HIV-specific long peptides modified by C-terminal addition of a single palmitoyl chain. A mixture of six lipopeptides derived from regulatory or structural HIV-1 proteins (Nef, Gag, and Env) was prepared. A phase I study was conducted to evaluate immunogenicity and tolerance in lipopeptide vaccination of HIV-1-seronegative volunteers given three injections of either 100, 250, or 500 microg of each lipopeptide, with or without immunoadjuvant (QS21). This report analyzes in detail B- and T-cell responses induced by vaccination. The lipopeptide vaccine elicited strong and multiepitopic B- and T-cell responses. Vaccinated subjects produced specific immunoglobulin G antibodies that recognized the Nef and Gag proteins. After the third injection, helper CD4(+)-T-cell responses as well as specific cytotoxic CD8(+) T cells were also obtained. These CD8(+) T cells were able to recognize naturally processed viral proteins. Finally, specific gamma interferon-secreting CD8(+) T cells were also detected ex vivo.

FIG. 1

Immunoblot pattern of serum specific for Nef protein. Suspensions of recombinant NEF protein (10 μl) harvested from E. coli production were separated by SDS-PAGE. Sera (diluted 1/100) collected from volunteers before (W0) and after (W20) immunization were tested and revealed with a sheep horseradish peroxidase-labeled anti-human immunoglobulin conjugate (1/2,000).

FIG. 2

Patterns of IgG specific for Gag protein were detected by using a commercial kit (New Lav Blot I; see Materials and Methods). Sera (diluted 1/100) collected from volunteers at different time points (W0 and W20) were tested for the presence of anti-HIV-1 Gag protein IgG antibodies. We could detect the mature Gag protein (P25) and the two precursors (P40 and P55).

FIG. 3

The positive anti-N1, anti-G2, and anti-E CTL obtained after four stimulations with PBMC (W20) from volunteer V4.1 (results shown in Table 5) were stimulated one more time with the respective long peptides to obtain more cells. To determine the fine nature of effector cells in response to these peptides, T-cell depletion was performed with anti-CD4 or anti-CD8 antibodies and complement 1 week after the last stimulation. Target cells (EBV infected) were sensitized with the N1 (A), G2 (B), or E (C) long peptides. The HIV-1-specific CTL from volunteer V4.1 were analyzed at an E/T ratio of 1.5:1 to 40:1.

FIG. 4

Anti-NEF CTL obtained with PBMC from volunteers V4.5 were tested for their capacities to recognize autologous EBV-infected targets infected with different recombinant Nef vaccinia viruses. For the same E/T ratio (16:1), the Nef peptide-specific CTL were tested against recombinant Nef proteins derived from HIV-1 LAI and MN (clade B), HIV-1 Bangui (clade A), and HIV-2 ROD.