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. 1998 Feb;72(2):1403-10.
doi: 10.1128/JVI.72.2.1403-1410.1998.

Selection of virus variants and emergence of virus escape mutants after immunization with an epitope vaccine

L Mortara  1 , F Letourneur, H Gras-Masse, A Venet, J G Guillet, I Bourgault-Villada
Affiliations

Selection of virus variants and emergence of virus escape mutants after immunization with an epitope vaccine

L Mortara et al. J Virol. 1998 Feb.

Abstract

In this report, we assessed the evolution of the cytotoxic T-lymphocyte (CTL) response induced by an epitope vaccine. In two macaques immunized with a mixture of lipopeptides derived from simian immunodeficiency virus (SIV) Nef and Gag proteins, CTL responses were directed against the same, single epitope of the Nef protein (amino acids 128 to 137) presenting an alanine at position 136 (Nef 128-137/136A). However, after 5 months of SIV infection, peripheral blood mononuclear cells from both macaques lost their ability to be stimulated by autologous SIV-infected cells while still retaining their capacity to generate cytotoxic responses after specific Nef 128-137/136A peptide stimulation. The sequences of the pathogenic viral isolate used for the challenge showed a mixture of several variants. Within the Nef epitopic sequence from amino acids 128 to 137, 82% of viral variants expressed the epitopic peptide Nef 128-137/136A; the remaining variants presented a threonine at position 136 (Nef 128-137/136T). In contrast, sequence analysis of cloned proviral DNA obtained from both macaques 5 months after SIV challenge showed a different pattern of quasi-species variants; 100% of clones presented a threonine at position 136 (Nef 128-137/136T), suggesting the disappearance of viral variants with an alanine at this position under antiviral pressure exerted by Nef 128-137/136A-specific CTLs. In addition, 12 months after SIV challenge, six of eight clones from one macaque presented a glutamic acid at position 131 (Nef 128-137/131E+136T), which was not found in the infecting isolate. Furthermore, CTLs generated very early after SIV challenge were able to lyse cells sensitized with the Nef 128-137/136A epitope. In contrast, lysis was significantly less effective or even did not occur when either the selected peptide Nef 128-137/136T or the escape variant peptide Nef 128-137/131E+136T was used in a target cell sensitization assay. Dose analysis of peptides used to sensitize target cells as well as a major histocompatibility complex (MHC)-peptide stability assay suggested that the selected peptide Nef 128-137/136T has an altered capacity to bind to the MHC. These data suggest that CTL pressure leads to the selection of viral variants and to the emergence of escape mutants and supports the fact that immunization should elicit broad CTL responses.

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Figures

FIG. 1
FIG. 1
Cytotoxic activities were tested with bulk cultured cells of macaques C16 (a and c) and C18 (b and d). These effector cells were CTL specific for immunodominant epitopic peptide Nef 128-137/136A (GLEGIYYSAR) as described in Materials and Methods. Target cells were autologous B-LCLs alone (○) or incubated with peptide Nef 128-137/136A (•) (a and b) and infected with wild-type vaccinia virus (▵) or with Vac-Nef recombinant vaccinia virus (▴) (c and d). Mean values for 51Cr release from target cells are expressed as percent specific lysis.
FIG. 2
FIG. 2
Cytotoxic activities were assayed with CTL obtained by stimulating PBMC of both macaques (C16 [a and c] and C18 [b and d]) with autologous SIV-infected cells after infection, as described in Materials and Methods. Target cells were autologous B-LCLs infected with wild-type vaccinia virus (○) or with Vac-Nef recombinant vaccinia virus (•) (a and b) and incubated with various peptides or without peptide (+) (c and d).
FIG. 3
FIG. 3
Cytotoxic activities were assayed with CTL stimulated by autologous SIV-infected cells after 5 months of experimental infection. Target cells were autologous B-LCLs infected with wild-type vaccinia virus (○) or with Vac-Nef recombinant vaccinia virus (•). (a) Macaque C16. (b) Macaque C18.
FIG. 4
FIG. 4
Comparison of different sequences of viral clones isolated from PBMC of infected macaques at 5 and 12 months after SIV challenge with pathogenic viral isolate SIVmac251. Analysis was performed within the epitopic sequence Nef 128-137.
FIG. 5
FIG. 5
(a) Anti-Nef 128-137/136A CTL cell lines from macaque C16 were tested against autologous target cells pulsed with the original peptide Nef 128-137/136A (○), with the selected peptide Nef 128-137/136T (▵), or without peptide (+). (b) Identical data for macaque C18 with autologous target cells pulsed with the original peptide Nef 128-137/136A (○), with the selected peptide Nef 128-137/136T (▵), with the mutant peptide Nef 128-137/131E+136T (□), or without peptide (+).
FIG. 6
FIG. 6
Specific lysis observed on target cells sensitized with different peptides: the original peptide Nef 128-137/136A (○), the selected peptide Nef 128-137/136T (▵), and the mutant peptide Nef 128-137/131E+136T (□). Target cells were autologous B-LCLs preincubated for 1 h with 10-fold serial dilutions of the different peptides. The CTL cell lines were obtained by stimulation of PBMC with the original Nef 128-137/136A peptide from macaque C16 (a) and macaque C18 (b). The E/T cell ratio was 10:1.
FIG. 7
FIG. 7
Time dependence of the peptide sensitization of targets. The CTL cell line was obtained by stimulating PBMC from macaque C16 with the original peptide Nef 128-137/136A. Autologous target cells were sensitized with the original peptide Nef 128-137/136A (•) or with the selected peptide Nef 128-137/136T (○) for 1 h, washed, and incubated for a further 1 h (a) or for 14 h (b) prior to the addition of effector cells. +, no peptide. (c) Data obtained at an E/T cell ratio of 100:1 with different incubation times for peptides with target cells (range, 1 to 14 h).
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