Highly efficient antiviral CD8+ T-cell induction by peptides coupled to the surfaces of liposomes

Abstract

In previous studies, we have demonstrated that liposomes with differential lipid components display differential adjuvant effects when antigens (Ags) are chemically coupled to their surfaces. When ovalbumin was coupled to liposomes made by using unsaturated fatty acids, it was found to be presented not only to CD4(+) T cells but also to CD8(+) T cells and induced cytotoxic T lymphocytes (CTLs) which effectively eradicated the tumor from mice. In this study, we coupled liposomes to immunodominant CTL epitope peptides derived from lymphocytic choriomeningitis virus (LCMV) and evaluated its potency as an antiviral vaccine. The intramuscular immunization of mice with the peptide-liposome conjugates along with CpG resulted in the efficient induction of antiviral CD8(+) T-cell responses which conferred complete protection against not only LCMV Armstrong but also a highly virulent mutant strain, clone 13, that establishes persistent infections in immunocompetent mice. The intranasal vaccination induced mucosal immunity effective enough to protect mice from the virus challenge via the same route. Complete protection was achieved in mice even when the Ag dose was reduced to as low as 280 ng of liposomal peptide. This form of vaccination with a single CTL epitope induced Ag-specific memory CD8(+) T cells in the absence of CD4(+) T-cell help, which could be shown by the complete protection of CD4-knockout mice in 10 weeks as well as by the analysis of recall responses. Thus, surface-linked liposomal peptide might have a potential advantage for the induction of antiviral immunity.

FIG. 1.

Comparison of three different routes of immunization with Lip-GP33. Each C57BL/6 mouse received 20 μl of Lip-GP33 diluted in 100 μl of PBS containing 5 μg of CpG. Spleen cells were prepared 7 days after immunization for the ⁵¹Cr-release assays (A) and ELISPOT assays (B). (A) ⁵¹Cr-release assays were performed to detect GP33-specific CTL activity at various E/T ratios by using MC57G cells pulsed with peptide GP33 as targets. (B) IFN-γ-producing cells responding to peptide GP33 were detected by ELISPOT assays, as described in Materials and Methods. Spleen cells from nonimmunized (naive) mice were used as a negative control. The data are representative of those from two independent and reproducible experiments. The results are shown as the means of four mice per group ± standard errors of the means. *, P < 0.05; **, P < 0.01; ns, not statistically significant.

FIG. 2.

Confirmation of GP33-specific CD8⁺ T-cell responses of immunized mice. C57BL/6 mice were injected i.m. with 20 μl of Lip-GP33 or Lip-GP283 diluted in 100 μl of PBS containing 5 μg of CpG, and spleen cells were prepared 7 days later for analysis. (A) The numbers of IFN-γ-producing CD8⁺ T cells specific to GP33 were determined by intracellular IFN-γ staining. Spleen cells from mice immunized with Lip-GP33 or Lip-GP283 were stimulated with either GP33-pulsed or unpulsed syngeneic spleen cells for 5 h and were then stained to detect their surface expression of CD8 (x axis) with FITC-conjugated MAb and their intracellular expression of IFN-γ (y axis) with PE-conjugated MAb. All lymphocytes were gated and analyzed on a FACScan flow cytometer by the use of CellQuest software (BD Biosciences). The values shown in the upper right quadrants indicate the percentage of CD8⁺ cells that are positive for intracellular IFN-γ after stimulation with GP33-pulsed spleen cells. The results of stimulation with unpulsed spleen cells were almost zero (data not shown). Each experiment used four mice per group, and the spleen cells of the mice in each group were pooled. The data shown are representative of those from three independent and reproducible experiments. (B) ⁵¹Cr-release assays with MC57G cells infected with VVGP33 as targets were performed to detect CTL activity against the endogenously expressed GP33 epitope (gray bars). As control targets, MC57G cells were infected with wild-type vaccinia virus. The same effector cells were used as GP33 peptide-pulsed and unpulsed targets as well for comparison (black bar). Lysis was also tested in the presence of an anti-CD4 or an anti-CD8 MAb (5 μg/ml) in the CTL assay. Specific lysis is demonstrated at an E/T ratio of 50. Each experiment used three mice, and the spleen cells of the three mice were pooled. The data are representative of those from two independent and reproducible experiments.

FIG. 3.

Challenge experiments with vaccinated and control mice. C57BL/6 mice were immunized by injecting Lip-GP33 or Lip-GP283, as described in the legends to Fig. 1 and 2, or by i.p. inoculation of 2 × 10⁵ PFU of LCMV Arm. After 2 weeks, the mice received 2 × 10⁵ PFU of LCMV Arm i.p. to initiate acute infection (A) or 2 × 10⁶ PFU of Cl.13 intravenously to initiate chronic infection (B). The virus titers in the spleens were quantitated by plaque assay on Vero cells at day 4 (A) or day 8 (B) postchallenge. Virus titers are indicated for each animal in the study. A dotted line represents the lower limit of detection (2 × 10¹ PFU/g [spleen]). The data are representative of those from three independent and reproducible experiments.

FIG. 4.

Comparison of GP33 and NP396 peptides as vaccine components. C57BL/6 mice were immunized i.m. with either Lip-GP33, Lip-NP396, Lip-GP283, or Lip-GP33/NP396 (20 μl each) or with Lip-GP33-Lip-NP396 (10 μl each) in 100 μl of PBS containing 5 μg of CpG. Seven days later, ⁵¹Cr-release assays (A), in vivo CTL assays (B), ELISPOT assays (C), and challenge experiments with LCMV Arm (D) were performed. The ⁵¹Cr-release assays (A) and ELISPOT assays (C) were done as described in the legend to Fig. 1. The in vivo CTL assays (B) were performed only for the mice immunized with either Lip-GP33, Lip-NP396, or Lip-GP283. One week after immunization, the mice received an equal number of a relevant peptide (GP33 or NP396)-pulsed CFSE^high targets and unpulsed CFSE^low targets. The numbers show the percentages of specific lysis. The experiment was repeated twice. The challenge experiments with LCMV Arm (D) were performed by the methods described in the legend to Fig. 3A. *, P < 0.05; **, P < 0.01. No statistically significant difference was observed between the four groups by the ELISPOT assays (C).

FIG. 5.

i.n. immunizations with Lip-GP33. C57BL/6 mice were immunized by the i.n. or i.m. route with 20 μl of Lip-GP33 or Lip-GP283 and 5 μg of CpG. One week later, lymphocytes from cervical lymph nodes and spleens were prepared and subjected to ELISPOT (IFN-γ) assays (A). The immunized mice were also challenged with 2 × 10⁵ PFU of LCMV Arm via the i.n. route, and the virus titers in the spleens were quantitated (B). (A) ELISPOT assays of IFN-γ-producing CD8⁺ T cells from cervical lymph nodes (left) and spleens (right) of the mice immunized via the i.n. or i.m. route with Lip-GP33 or Lip-GP283. The data are representative of those from two independent and reproducible experiments. The results for one pool of cells from the lymph nodes and the means for spleen cells from four mice per group ± standard errors of the means are shown. *, P < 0.05 compared with the results obtained with Lip-GP283 immunization; ns, not statistically significant. (B) One week after immunization, the mice received 2 × 10⁵ PFU of LCMV Arm i.n., and the virus titers in the spleens were quantitated by plaque assay on Vero cells at day 4 postchallenge. The virus titers are indicated for each animal in the study. A dotted line represents the lower limit of detection (2 × 10¹ PFU/g [spleen]). The data are representative of those from three independent and reproducible experiments.

FIG. 6.

Dose-response experiments. Serial fivefold dilutions of Lip-GP33 starting at 50 μl were prepared in 100 μl of PBS containing 5 μg of CpG and injected i.m. to each C57BL/6 mouse. Seven days later, ELISPOT (IFN-γ) assays (A) and challenge experiments with LCMV Arm (B) were performed. (A) ELISPOT assays were performed with spleen cells from immunized mice. One group of mice received peptide GP33 (35 μg) without (w/o) conjugation, and one group received GP33 at the same dose conjugated with 5 μg of CpG as one of the Lip-GP33 immunizations. The data are representative of those from three independent and reproducible experiments. The results are shown as the means for four mice per group ± standard errors of the means. (B) The immunized mice received 2 × 10⁵ PFU of LCMV Arm i.p., and the virus titers in the spleens were quantitated by plaque assay on Vero cells at day 4 postchallenge. Virus titers are indicated for each animal in the study. The dotted line represents the lower limit of detection (2 × 10¹ PFU/g [spleen]). The data are representative of those from two independent and reproducible experiments.

FIG. 7.

Analyses of effector and memory CD8⁺ T-cell induction in the absence of CD4 help. Each of C57BL/6 and CD4-KO mice received 20 μl of Lip-GP33 or Lip-GP283 diluted in 100 μl of PBS containing 5 μg of CpG; and ELISPOT assays (A), tetramer and CD62L staining (B), and challenge experiments with LCMV Arm (C) were performed at different times. (A) At 1 and 12 weeks postimmunization, IFN-γ-producing cells responding to peptide GP33 were detected by ELISPOT assays of spleen cells from C57BL/6 (gray bars) and CD4-KO (black bars) mice. At week (w) 12, the mice were boosted in the same manner in which they received the primary immunizations, and ELISPOT assays were performed 1 week later. (B) GP33 H-2D^b tetramer staining was performed 1 and 18 weeks postimmunization for C57BL/6 mice (gray bars). For comparison, mice immunized by inoculating 2 × 10⁵ PFU of LCMV Arm i.p. were included in these experiments (shaded bars). At week 18, both groups of mice were boosted with Lip-GP33 and were analyzed for their recall responses 1 week later. The expression of CD62L on gated GP33 H-2D^b tetramer-positive CD8⁺ T cells was also determined by flow cytometry. (C) Challenge experiments were performed by the i.p. injection of 2 × 10⁵ PFU of LCMV Arm at the indicated times. The virus titers are shown for each of the C57BL/6 (open circles) and the CD4-KO (closed circles) mice. The data are shown as the means for at least three and five mice per group ± standard errors of the means for the ELISPOT and tetramer assays, respectively. *, P < 0.05; **, P < 0.01.