Multiple epitopes in the murine cytomegalovirus early gene product M84 are efficiently presented in infected primary macrophages and contribute to strong CD8+-T-lymphocyte responses and protection following DNA immunization

Abstract

We previously demonstrated that after vaccination of BALB/c mice with DNA encoding murine cytomegalovirus (MCMV) IE1 or M84, a similar level of protection against MCMV infection was achieved. However, the percentage of antigen-specific CD8(+) T cells elicited by IE1 was higher than that by M84 as measured by intracellular cytokine staining when splenocytes were stimulated with an epitope peptide (M. Ye at al., J. Virol. 76:2100-2112, 2002). We show here that after DNA vaccination with M84, a higher percentage of M84-specific CD8(+) T cells was detected when splenocytes were stimulated with J774 cells expressing full-length M84. When the defined M84 epitope 297-305 was deleted, the mutant DNA vaccine was still protective against MCMV replication and induced strong M84-specific CD8(+)-T-cell responses. The M84 gene was subsequently subcloned into three fragments encoding overlapping protein fragments. When mice were immunized with each of the M84 subfragment DNAs, at least two additional protective CD8(+)-T-cell epitopes were detected. In contrast to strong responses after DNA vaccination, M84-specific CD8(+)-T-cell responses were poorly induced during MCMV infection. The weak M84-specific response after MCMV infection was not due to poor antigen presentation in antigen-presenting cells, since both J774 macrophages and primary peritoneal macrophages infected with MCMV in vitro were able to efficiently and constitutively present M84-specific epitopes starting at the early phase of infection. These results indicate that antigen presentation by macrophages is not sufficient for M84-specific CD8(+)-T-cell responses during MCMV infection.

FIG. 1.

Detection of M84-specific CD8⁺ T cells with a J774 cell-mediated ICCS assay. (A and B) Three BALB/c mice per group were immunized i.d. three times with 10 μg of pc3Δneo, pc3-M84, or pc3-M84+pc3-pp89 at 10-day intervals. Ten days after the last immunization, splenocytes from individual mice were stimulated with 1 μM M84 epitope 297-305 peptide or J774 macrophage cells infected with an M84-expressing vaccinia virus at an MOI of 10 for 10 h. The resulting cells were processed for the ICCS as described in Materials and Methods. Fluorescein isothiocyanate-anti-CD8 and phycoerythrin-anti-IFN-γ antibodies were used to stain the cells. CD8⁺ T cells that had accumulated intracellular IFN-γ were detected and enumerated by flow cytometry. Panel A shows representative dot plots displaying IFN-γ-positive CD8⁺ T cells detected from various mouse groups by using either M84 peptide or M84-expressing J774 cells (J774-M84) as stimulators in the ICCS assay. The numbers in quadrant C2 represent the percentages of IFN-γ positive CD8⁺ T cells after exclusion of the nonspecific cells in gate B (small rectangle in quadrant C2). The symbols around the columns in panel B represent the percentages of IFN-γ-positive CD8⁺ T cells in individual mice. (C) Two mice per group were immunized i.d. three times with 10 μg of pc3-M84 or pc3-pp89 plasmids at 10-day intervals. Ten days after the last immunization, mouse splenocytes were incubated with the IE1 epitope 168-176 peptide, the M84 epitope 297-305 peptide, or J774 cells infected for 10 h with recombinant vaccinia virus expressing IE1 or M84 and then analyzed by ICCS assay. Percentages of antigen-specific CD8⁺ T cells from a mouse immunized with pc3-pp89 (⧫), from the other pc3-pp89-immunized mouse (◊), from a mouse immunized with pc3-M84 (▪), and for the other M84-immunized mouse (□) are shown.

FIG. 2.

Detection of an additional protective CD8⁺-T-cell epitope in M84. Diagrams above panels A and B represent wild-type M84, IE1, and their mutant proteins, M84ΔKd and pp89ΔLd. The known CD8⁺-T-cell epitopes 297-305 (M84) or 168-176 (IE1) are indicated. They were deleted in M84ΔKd and pp89ΔLd. The numbers represent the amino acid position in the protein. BALB/c mice were immunized i.d. three times with DNA vaccines pc3Δneo (vector), pc3-M84, pc3-M84ΔKd, pc3-pp89, or pc3-pp89ΔLd. Ten days after the last immunization, three mice per group were examined for M84-specific CD8⁺ T cells by using a peptide-mediated or M84-expressing-J774 cell-mediated ICCS assay. The other four mice per group were challenged i.p. with 3.5 × 10⁵ PFU (0.5 LD₅₀) of salivary gland-derived MCMV (A and B). Five days later, the virus titer in the spleen was examined (C). (A) M84-specific CD8⁺-T-cell responses measured with ICCS assay by using M84 297-305 peptide (left three columns) or M84-expressing-J774 cells (right three columns) as a stimulator. (B) The IE1-specific CD8⁺-T-cell responses measured with ICCS with IE1 168-176 peptide (left three columns) or IE1-expressing-J774 cells as a stimulator. (C) Titer of MCMV per spleen after challenge of immunized mice. Solid symbols stand for measurement of CD8⁺-T-cell responses or virus titers in individual mice.

FIG. 3.

Expression of M84 subfragments in COS-7 cells. (A) Schematic diagram of M84 and its overlapping subfragments. The numbers mark the amino acid position in the protein. The known M84 297-305 epitope is indicated with dark shading. NFrg, amino-terminal fragment of M84; MFrg, middle fragment; CFrg, carboxy-terminal fragment; and ΔKdMFrg, middle fragment without the M84 297-305 epitope. (B) COS-7 cells were transfected with pc3Δneo (Vector), pc3-NFrg, pc3-MFrg, pc3-ΔKdMFrg, pc3-CFrg, or pc3-M84. Two days posttransfection, cells were lysed and cell lysates from an equal number of cells were subjected to SDS-PAGE. After transfer of the proteins to nitrocellulose, M84 protein or individual subfragments were examined by Western blotting with a rabbit polyclonal antibody against bacterially expressed GST-M84. Size markers (in kilodaltons) are on the left side of the figure.

FIG. 4.

M84-specific CD8⁺-T-cell responses and immune protection after vaccination of mice with M84 DNA or M84 DNA subfragments. BALB/c mice were immunized three times i.d. with pc3Δneo (Vector), pc3-M84, pc3-M84Δ Kd, pc3-NFrg, pc3-MFrg, pc3-ΔKdMFrg, or pc3-CFrg DNA vaccines. Ten days after the last immunization, three mice per group were examined for M84-specific CD8⁺-T-cell responses by using the ICCS assay with either M84 297-305 peptide or M84-expressing J774 cells as stimulators. The other four mice per group were challenged with 3.5 × 10⁵ PFU (0.5 LD₅₀) of salivary gland-derived MCMV. Five days later, the virus titer in the spleen was determined. (A) M84-specific CD8⁺-T-cell responses measured by ICCS assay with M84-expressing J774 cells (left) or M84 297-305 peptide (right) as a stimulator. (B) Titer of MCMV per spleen after viral challenge. The limit of virus detection was 200 PFU per spleen, and virus titers below the detection limit were arbitrarily set to 100 PFU for display purposes and mean calculation. Each individual mouse is represented by a solid symbol.

FIG. 5.

M84- and IE1-specific CD8⁺-T-cell responses after MCMV infection. Three BALB/c mice per group were infected with 1.2 × 10⁵ PFU of tissue culture-derived MCMV. Nine days or 1 month after infection, M84- or IE1-specific CD8⁺-T-cell responses were measured by ICCS assay after either peptide-based stimulation with the M84 297-305 or IE1 168-176 epitope peptides or cell-based stimulation with J774 cells infected with either the wild-type or M84-expressing vaccinia virus. Open or solid symbols represent the percentage of antigen-specific CD8⁺ T cells in each individual mouse except when indicated by an asterisk. In these cases, the splenocytes were pooled.

FIG. 6.

Presentation of M84 and IE1 antigens in MCMV-infected cells. Three BALB/c mice per group were immunized three times (A) or four times (B) i.d. with 10 μg of pc3-M84 or pc3-pp89 DNA vaccine at 7-day intervals. At 10 days (A) or 6 days (B) after the last immunization, mouse splenocytes were prepared and pooled as effector cells in the ICCS assay. BALB/c MEFs (A, top panel), J774 cells (A, lower panel), or freshly isolated primary BALB/c peritoneal macrophages (B) were infected with tissue culture-derived MCMV at an MOI of 5 for the indicated periods or conditions. The infected cells were subsequently used as stimulators in the ICCS assay to measure the level of antigen presentation as described in Materials and Methods. Uninfected cells were used as a negative control. The IE1 168-176 peptide or J774 cells infected with the M84-expressing recombinant vaccinia virus served as positive controls to measure the maximum level of M84- or IE1-specific CD8⁺-T-cell responses. Darkly shaded columns represent the measurement of M84-specific CD8⁺ T cells in splenocytes of M84-immunized mice. Lightly shaded columns stand for the measurement of IE1-specific CD8⁺ T cells in the splenocytes of IE1-immunized mice.