Two overlapping subdominant epitopes identified by DNA immunization induce protective CD8(+) T-cell populations with differing cytolytic activities

Abstract

Subdominant CD8(+) T-cell responses contribute to control of several viral infections and to vaccine-induced immunity. Here, using the lymphocytic choriomeningitis virus model, we demonstrate that subdominant epitopes can be more reliably identified by DNA immunization than by other methods, permitting the identification, in the virus nucleoprotein, of two overlapping subdominant epitopes: one presented by L(d) and the other presented by K(d). This subdominant sequence confers immunity as effective as that induced by the dominant epitope, against which >90% of the antiviral CD8(+) T cells are normally directed. We compare the kinetics of the dominant and subdominant responses after vaccination with those following subsequent viral infection. The dominant CD8(+) response expands more rapidly than the subdominant responses, but after virus infection is cleared, mice which had been immunized with the "dominant" vaccine have a pool of memory T cells focused almost entirely upon the dominant epitope. In contrast, after virus infection, mice which had been immunized with the "subdominant" vaccine retain both dominant and subdominant memory cells. During the acute phase of the immune response, the acquisition of cytokine responsiveness by subdominant CD8(+) T cells precedes their development of lytic activity. Furthermore, in both dominant and subdominant populations, lytic activity declines more rapidly than cytokine responsiveness. Thus, the lysis(low)-cytokine(competent) phenotype associated with most memory CD8(+) T cells appears to develop soon after antigen clearance. Finally, lytic activity differs among CD8(+) T-cell populations with different epitope specificities, suggesting that vaccines can be designed to selectively induce CD8(+) T cells with distinct functional attributes.

FIG. 1

Deletion of the dominant CTL epitope has minimal effect on vaccine efficacy. BALB/c mice (eight per group) were immunized as indicated with LCMV (positive control), or with DNA vaccines pCMV (negative control), pCMV-NP, or pCMV-NPΔ (NPd). Six weeks later mice were challenged in one of two ways. Four mice per vaccine group were given a nonlethal dose of LCMV (2 × 10⁵ PFU i.p.), and 4 days later these mice were sacrificed and titers of LCMV in the spleens were determined. Results are shown for individual mice (open circles) as PFU per gram of spleen (left axis). The remaining four mice in each vaccine group were challenged i.c. with a normally lethal dose of LCMV (20 LD₅₀). All deaths occurred between days 6 and 8 after the infection, and the percentage of surviving mice is shown (right axis) as vertical grey bars.

FIG. 2

Fragments used to identify protective sequences in LCMV NP. The gene encoding the 558-residue NP is shown, with the dominant epitope (NP_118–126) as a solid black box; this epitope is not present in NPΔ, from which the five fragments were generated, and thus is absent from fragment I. The location of a sequence which binds to K^d (NP_314–322) (see text) is shown as a grey box in NP and in fragment III.

FIG. 3

Sequence X protects against two modes of virus challenge. (A) BALB/c mice (12 mice per group) were immunized with plasmids encoding the ubiquitinated NP fratments I to V, with pCMV-U (negative control) or with LCMV (L) (positive control). Six weeks later, four mice per group were challenged with a nonlethal dose of LCMV (2 × 10⁵ PFU i.p.) and were sacrificed 4 days thereafter. LCMV titers in the spleens were determined, and the results for individual mice (closed circles) and the mean of each group (open triangles) are shown in the lefthand panel. Virus was undetectable in LCMV-immunized mice. The remaining eight mice in each group were challenged with a normally lethal dose of LCMV (20 LD₅₀ i.c.). All deaths occurred between days 6 and 8 after the infection, and the percentages of mice which survived are shown in the right-hand panel. (B) A similar experiment was carried out, this time using BALB/c mice (eight mice per group) immunized with the minigene plasmids pCMV-UMGX or pCMV-UMG34 or (as a negative control) with pCMV-U. Six weeks later, four mice per group were challenged i.p., and the virus titers 4 days later are shown in the left-hand panel. The remaining mice (four per group) were challenged with a lethal dose of LCMV (20 LS₅₀ i.c.), and the percentages of surviving mice are shown (right-hand panel).

FIG. 4

Following vaccination and challenge, subdominant CD8⁺ T-cell populations expand more slowly than dominant ones. BALB/c mice were immunized with pCMV, pCMV-NPΔ, or pCMV-NP. Six weeks later mice were challenged with LCMV i.p. and at 4, 5, and 7 days p.i. were sacrificed (4 mice per vaccine and time point). LCMV- specific CD8⁺ T-cell responses were evaluated by ICCS, using as stimulators BALB cl7 cells transfected with plasmids pCMV-UMGX, pCMV-UMG4, or pCMV. The percentages of CD8⁺ T cells producing IFN-γ are shown with standard errors (error bars).

FIG. 5

Peptide X is more efficiently presented by transfected cells than by peptide-coated cells. Two different groups of effector cells were used, as shown on the y axis: (i) splenocytes harvested 7 days after infection of naïve mice and (ii) splenocytes harvested 5 days after infection of mice which had been immunized 6 weeks previously with pCMV-UMGX. Stimulator cells were BALB cl7 cells transfected with pCMV-UMGX or coated with peptide X (PYIACRTSI). The percentages of CD8⁺ cells producing IFN-γ are shown, with standard errors (error bars).

FIG. 6

Region X contains two overlapping epitopes: one presented by K^d, and the other presented by L^d. Four mice were immunized with pCMV-UMGX and 6 weeks later were infected with LCMV. Seven days thereafter splenocytes were harvested and stimulated for 5 h on BALB c17 (K^d, D^d, and L^d) T2-Kd (K^d) or T2-Ld (L^d) cells precoated with peptide X (NP_314–322) (PYIACRTSI), peptide WX (NP_313–322) (WPYIACRTSI) or the dominant peptide D (NP_118–126) (RPQASGVYM). Epitope-specific responses were evaluated by ICCS (see Materials and Methods).

FIG. 7

Induction of X- and WX-specific responses by LCMV infection and DNA immunization. Responses to peptides D, WX, and X were evaluated by ICCS 7 days after virus infection of naïve mice (top row) and 15 days after pCMV-U-MGX immunization (bottom row). The location of CD8⁺ IFN-γ-positive cells is indicated by an ellipse, and for levels above background, the percentages of CD8⁺ T cells which produce IFN-γ are shown.

FIG. 8

Protective immunity induced by immunization with peptide-coated cells. Groups of mice (eight mice per group) were immunized with spleen cells, either uncoated (none), or coated with peptide D, WX, or X (see Materials and Methods). Three weeks later, mice were challenged i.c. with 20 LD₅₀ of LCMV and were observed daily for 21 days. All deaths occurred between days 7 and 90. The percent surviving mice is shown.

FIG. 9

Delayed development of lytic activity by CD8⁺ T cells specific for the subdominant epitopes WX and X. BALB/c mice were immunized with pCMV-UMGX or pCMV-UMG4 and 6 weeks later were infected with LCMV. Four, five, or seven days later, mice were sacrificed; their spleens were harvested; and splenocytes were used in an ICCS using the indicated peptides as stimulators and also in a classical in vitro cytotoxicity assay using as targets BALB c17 cells coated with the indicated peptides. The number of lytic units per million epitope-specific cells was calculated as described in Materials and Methods. (A) ICCS and cytotoxicity data from selected p-CMV-UMGX vaccinees. ICCS dot plots are shown (x axis, CD8; y axis, IFN-γ) for CD8⁺ T cells responsive to peptide X or D, at the indicated times after in vivo secondary stimulation with LCMV; arrows point to their related cytoxocity assays. The day 4 cells (bottom dot plot) could produce IFN-γ but were very weakly lytic (filled circles); the low level of lysis was not due to a low number of X-specific cells, since fewer cells specific for peptide D (center dot plot) showed higher lytic activity (open squares). X-specific cells eventually acquired lytic activity (top dot plot and filled triangles). (B) Summary of ICCS and cytotoxicity data from all vacinees. For CD8⁺ T cells specific for peptides X, WX, and D, the lytic activity (in lytic units) is compared to the percentage of CD8⁺ T cells producing IFN-γ.