Novel plant virus-based vaccine induces protective cytotoxic T-lymphocyte-mediated antiviral immunity through dendritic cell maturation

Abstract

Currently used vaccines protect mainly through the production of neutralizing antibodies. However, antibodies confer little or no protection for a majority of chronic viral infections that require active involvement of cytotoxic T lymphocytes (CTLs). Virus-like particles (VLPs) have been shown to be efficient inducers of cell-mediated immune responses, but administration of an adjuvant is generally required. We recently reported the generation of a novel VLP system exploiting the self-assembly property of the papaya mosaic virus (PapMV) coat protein. We show here that uptake of PapMV-like particles by murine splenic dendritic cells (DCs) in vivo leads to their maturation, suggesting that they possess intrinsic adjuvant-like properties. DCs pulsed with PapMV-like particles displaying the lymphocytic choriomeningitis virus (LCMV) p33 immunodominant CTL epitope (PapMV-p33) efficiently process and cross-present the viral epitope to p33-specific transgenic T cells. Importantly, the CTL epitope is also properly processed and presented in vivo, since immunization of p33-specific T-cell receptor transgenic mice with PapMV-p33 induces the activation of large numbers of specific CTLs. C57BL/6 mice immunized with PapMV-p33 VLPs in the absence of adjuvant develop p33-specific effector CTLs that rapidly expand following LCMV challenge and protect vaccinated mice against LCMV infection in a dose-dependent manner. These results demonstrate the efficiency of this novel plant virus-based vaccination platform in inducing DC maturation leading to protective CTL responses.

FIG. 1.

Expression and purification of PapMV-like particles. (A) The LCMV immunodominant p33 peptide (underlined) was fused between PapMV CP and a six-His tag located at the C terminus of the protein. (B) Purification of recombinant proteins. The size and purity of the recombinant proteins were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lanes: 1, molecular weight markers; 2, total bacterial lysates without plasmid; 3, total bacterial lysates expressing the recombinant protein; 4, proteins purified by nickel-affinity chromatography; 5, purified VLPs concentrated by ultracentrifugation. (C) Electron microscopy of PapMV native virions, PapMV VLPs, and PapMV-p33 VLPs. Bars, 100 nm.

FIG. 2.

Splenic DCs take up PapMV-like particles in vivo and acquire a mature phenotype. (A) C57BL/6 mice were injected i.v. with 100 μg of Alexa Fluor 647-labeled VLPs in PBS or with 100 μl of sterile PBS as a negative control. Spleens were collected at 2 h postimmunization, and total CD11c⁺ splenic DCs were isolated by magnetic bead separation and stained with FITC-labeled CD11c-specific MAbs. The negative fraction obtain after magnetic purification of CD11c⁺ cells was stained with FITC-coupled B220 or F4/80 specific MAb. Cells were analyzed by flow cytometry. Values shown in the upper right quadrants represent the percentages of given cell populations associated with labeled VLPs within the total population. (B) In vivo capture of VLPs was visualized by confocal microscopy analysis. Mice were injected with 100 μg of Alexa Fluor 647-labeled VLPs (red) in PBS or with 100 μl of sterile PBS as a negative control, and CD11c⁺ spleen cells were purified 2 h later. Purified CD11c⁺ spleen cells were allowed to adhere for 12 h to glass slides coated with poly-l-lysine and were fixed with 4% paraformaldehyde. Cells were labeled with wheat germ agglutinin conjugated to Alexa Fluor 488 (green) to visualize the plasma membrane. (C) C57BL/6 mice were injected i.v. with 100 μg VLPs in PBS. Spleens were collected at 6 h postimmunization, and CD11c⁺ cells were isolated by magnetic separation. Mice injected with 25 μg LPS served as positive controls. Purified CD11c⁺ DCs were stained with FITC-labeled CD11c-specific antibodies in combination with PE-labeled anti-CD40, -CD80, or -CD86 or isotypic control antibodies. Data are gated on the CD11c⁺ population. Data are shown for isotypic controls (dotted lines), PBS-injected mice (filled histograms), and VLP-injected mice (bold lines). These results are representative of three independent experiments.

FIG. 3.

In vitro and in vivo processing of the LCMV p33 CTL epitope displayed on PapMV-like particles by splenic DCs and cross-priming of naïve T cells. (A) Splenic DCs were purified by CD11c magnetic bead separation and pulsed for 8 h with the indicated concentrations of VLPs in 96-well culture plates. DCs pulsed with the p33 synthetic peptide served as a positive control. Splenic p33-specific CD8⁺ T lymphocytes purified from TCR transgenic P14 mice were then added to the pulsed DC cultures. Proliferation of p33-specific CD8⁺ T lymphocytes was assayed 48 h later by [³H]thymidine incorporation into cellular DNA. (B) Activation of p33-specific CD8⁺ T lymphocytes was analyzed by flow cytometry following 24 h of coculture between specific T cells and DCs pulsed for 8 h with 1 μg of VLPs. Cells were stained with FITC-labeled anti-CD8 antibodies, PerCP-labeled anti-CD69 antibodies, and PE-labeled p33-specific tetramers. (C) P14 mice were injected i.v. with 100 μg of VLPs in PBS, and 24 h later, total spleen cells were stained with FITC-labeled anti-CD8 antibodies, PerCP-labeled anti-CD69 antibodies, and PE-labeled p33-specific tetramers. Data are gated on the CD8⁺ T-cell population. Values shown in the upper right quadrants represent the percentages of activated p33-specific CD8⁺ T lymphocytes within the total spleen p33-specific CD8⁺ T-lymphocyte population. These results are representative of three independent experiments.

FIG. 4.

Immunization with PapMV-p33 VLPs generates p33-specific CD8⁺ T lymphocytes. C57BL/6 mice were injected i.v. with 100 μg of VLPs in PBS, and identical recall injections were given 10, 20, and 30 days later. Total spleen cells were then double stained with FITC-labeled anti-CD8 antibodies and PE-labeled p33-specific tetramers. 7-AAD was used for dead cell exclusion. Data are gated on the total lymphocyte population. Values shown in the upper right quadrants represent the percentages of p33-specific CD8⁺ T lymphocytes within the total CD8⁺ T-lymphocyte population. These results are representative of four independent experiments.

FIG. 5.

Expansion of effector p33-specific CTLs following LCMV infection. C57BL/6 mice were injected i.v. with 100 μg of VLPs in PBS, followed by three recall injections at 10-day intervals. Seven days after the last recall injection, immunized mice were infected i.v. with 200 PFU of LCMV. (A) Expansion of p33-specific CD8⁺ T lymphocytes was assayed at 5 days postinfection by flow cytometry. Total spleen cells were double stained with FITC-labeled anti-CD8 antibodies and PE-labeled p33-specific tetramers. 7-AAD was used for dead cell exclusion. Data are gated on the total lymphocyte population. Values shown in the upper right quadrants represent percentages of p33-specific CD8⁺ T lymphocytes within the total CD8⁺ T-lymphocyte population. (B) Production of IFN-γ by CD8⁺ T lymphocytes was also assayed at 5 days postinfection by flow cytometry. Total spleen cells were restimulated with the p33 peptide for 5 h in vitro. Spleen cells were then surface stained with a PE-labeled anti-CD8 antibody. After fixation and permeabilization, cells were stained with an anti-IFN-γ-FITC MAb. 7-AAD was used to exclude dead cells. Data are gated on the total lymphocyte population. Values shown in the upper right quadrants represent percentages of CD8⁺ IFN-γ⁺ T lymphocytes within the total CD8⁺ T-lymphocyte population. (C) Spleen cells were tested for cytotoxic activity in a standard 5-h ⁵¹Cr release assay of unpulsed (open circles) and p33-pulsed (closed circles) EL-4 target cells. Results are representative of four independent experiments.

FIG. 6.

Immunization with PapMV-p33 VLPs induces protective immunity against LCMV infection. C57BL/6 mice were injected i.v. with 100 μg of VLPs in PBS, followed by identical recall injections 10, 20, and 30 days later. Seven days after the last recall injection, immunized mice were infected i.v. with 200 PFU of LCMV. Spleens from infected mice were removed 5 days after infection, and LCMV titers were determined by a standard focus-forming assay. Results are representative of three identical and independent experiments.