Characterization of human immunodeficiency virus Gag-specific gamma interferon-expressing cells following protective mucosal immunization with alphavirus replicon particles

Abstract

A safe, replication-defective viral vector that can induce mucosal and systemic immune responses and confer protection against many infectious pathogens, such as human immunodeficiency virus type 1 (HIV-1), may be an ideal vaccine platform. Accordingly, we have generated and tested alphavirus replicon particles encoding HIV-1 Gag from Sindbis virus (SIN-Gag) and Venezuelan equine encephalitis virus (VEE-Gag), as well as chimeras between the two (VEE/SIN-Gag). Following intramuscular (i.m.), intranasal (i.n.), or intravaginal (IVAG) immunization with VEE/SIN-Gag and an IVAG challenge with vaccinia virus encoding HIV Gag (VV-Gag), a larger number of Gag-specific CD8+ intracellular gamma interferon-expressing cells (iIFNEC) were detected in iliac lymph nodes (ILN), which drain the vaginal/uterine mucosa (VUM), than were observed after immunizations with SIN-Gag. Moreover, a single i.n. or IVAG immunization with VEE/SIN-Gag induced a larger number of cells expressing HIV Gag in ILN, and immunizations with VEE/SIN-Gag through any route induced better protective responses than immunizations with SIN-Gag. In VUM, a larger percentage of iIFNEC expressed alpha4beta7 or alpha(Ebeta)7 integrin than expressed CD62L integrin. However, in spleens (SP), a larger percentage of iIFNEC expressed alpha4beta7 or CD62L than expressed alpha(Ebeta)7. Moreover, a larger percentage of iIFNEC expressed the chemokine receptor CCR5 in VUM and ILN than in SP. These results demonstrate a better induction of cellular and protective responses following immunizations with VEE/SIN-Gag than that following immunizations with SIN-Gag and also indicate a differential expression of homing and chemokine receptors on iIFNEC in mucosal effector and inductive sites versus systemic lymphoid tissues.

FIG. 1.

CD8⁺ IFN-γ-secreting cells in SP. Groups of five female BALB/c mice were immunized with VEE/SIN or PBS three times through the i.m. route and then were challenged IVAG with VV-Gag. Single-cell suspensions from the various SP were pooled from five immunized/challenged mice restimulated with medium alone (A) or from naïve mice challenged IVAG with VV-Gag and restimulated with p7g (B) or the HIV Gag peptide p7g, representing a single MHC I-restricted epitope (C). The cells were analyzed by intracellular staining for IFN-γ and surface staining for CD8. The data are presented as mean total percentages of CD8⁺ IFN-γ⁺ cells from one pool of five mice. The data are representative of four similar results from two independent experiments and from various tissues.

FIG. 2.

CD8⁺ intracellular IFN-γ-expressing cells (iIFNEC) in VUM, ILN, and SP. Groups of five female BALB/c mice were immunized with VEE, VEE/SIN, or SIN three times through the i.m. (A), i.n. (B), or IVAG (C) route and then challenged IVAG with VV-Gag. Single-cell suspensions from the various tissues were pooled from five mice per group, restimulated with an HIV Gag peptide representing a single MHC I-restricted epitope, and analyzed by intracellular staining for IFN-γ and surface staining for CD8. The data are presented as mean total percentages of CD8⁺ IFN-γ⁺ cells ± SD of four pools of five mice each from two independent experiments.

FIG. 3.

Expression of mucosal and peripheral homing receptors of IFN-γ⁺ cells in VUM, ILN, and SP following immunizations with VEE/SIN-Gag. Groups of five female BALB/c mice were immunized with VEE/SIN-Gag three times through the i.m. (A), i.n. (B), or IVAG (C) route and then challenged IVAG with VV-Gag. Single-cell suspensions from the various tissues were pooled from five mice per group, restimulated with an HIV Gag peptide representing an MHC I-restricted single epitope, and analyzed by intracellular staining for IFN-γ and surface staining for α4β7, α_Eβ7, or CD62L. The data are presented as mean total percentages of iIFNEC cells coexpressing α4β7, α_Eβ7, or CD62L ± SD of two pools of five mice each from one experiment. Data from one representative experiment of two with similar results are shown.

FIG. 4.

Surface expression of α_Eβ7 by iIFNEC in VUM, ILN, and SP following immunizations with VEE-Gag, VEE/SIN-Gag, or SIN-Gag. Groups of five female BALB/c mice were immunized with VEE-Gag, VEE/SIN-Gag, or SIN-Gag three times through the i.m. (A), i.n. (B), or IVAG (C) route and then challenged IVAG with VV-Gag. Single-cell suspensions from the various tissues were pooled from five mice per group, restimulated with an HIV Gag peptide representing a single MHC I-restricted epitope, and analyzed by intracellular staining for IFN-γ and surface staining for α_Eβ7. The data are presented as mean total percentages of iIFNEC cells per tissue coexpressing α_Eβ7 ± SD of two pools of five mice each from one experiment. Data from one representative experiment of two with similar results are shown.

FIG. 5.

Flow cytometric plots demonstrating expression of mucosal and peripheral homing receptors of IFN-γ⁺ cells in VUM, ILN, and SP following immunizations with VEE/SIN-Gag. Groups of five female BALB/c mice were immunized with VEE/SIN-Gag three times through the i.n. route and then challenged IVAG with VV-Gag. Single-cell suspensions from the VUM (A, D, and G), ILN (B, E, and H), and SP (C, F, and I) were pooled from five mice per group, restimulated with an HIV Gag peptide representing an MHC I-restricted single epitope, and analyzed by intracellular staining for IFN-γ and surface staining for CD62L (A, B, and C), α_Eβ7 (D, E, and F), and α4β7 (G, H, and I). The data are presented as mean total percentages of iIFNEC cells per tissue coexpressing α4β7, α_Eβ7, or CD62L ± SD of two pools of five mice each from one experiment. Data from one representative experiment of two with similar results are shown.

FIG. 6.

Surface expression of CCR5 by iIFNEC in VUM, ILN, and SP following immunizations with VEE-Gag, VEE/SIN-Gag, or SIN-Gag. Groups of five female BALB/c mice were immunized with VEE-Gag, VEE/SIN-Gag, or SIN-Gag three times through the i.m. (A), i.n. (B), or IVAG (C) route and then challenged IVAG with VV-Gag. Single-cell suspensions from the various tissues were pooled from five mice per group, restimulated with an HIV Gag peptide representing a single MHC I-restricted epitope, and analyzed by intracellular staining for IFN-γ and surface staining for CCR5. The data are presented as mean total percentages of iIFNEC cells coexpressing CCR5 ± SD of two pools of five mice each from one experiment. Data from one representative experiment of two with similar results are shown.

FIG. 7.

Gag-expressing cells in ILN following immunization with VEE/SIN-Gag or SIN-Gag. Groups of three female BALB/c mice were immunized i.m., i.n., or IVAG with a single dose of VEE/SIN-Gag or SIN-Gag. After 16 h, ILN were removed and snap-frozen. Immunohistological staining was then performed on 7-μm thin sections with a FITC-conjugated anti-HIV Gag antigen. The data are presented as mean numbers of Gag-expressing cells per high-power field ± SD for three mice per group.

FIG. 8.

Representative photomicrographs of Gag-expressing cells in ILN following immunization with VEE/SIN-Gag or SIN-Gag. Groups of three female BALB/c mice were immunized i.n. with a single dose of VEE/SIN-Gag (A) or SIN-Gag (B). After 16 h, ILN were removed and snap-frozen. Immunohistological staining was then performed on 7-μm thin sections with a FITC-conjugated anti-HIV Gag antigen or with a control antibody from the same species but with a different specificity (C).

FIG. 9.

Relative percentages of CD11b⁺ and CD11c⁺ monocyte-lineage cells in VUM and ILN. Single-cell suspensions from the various tissues were pooled from five female BALB/c mice per group and then analyzed for surface expression of CD11c or CD11b by three-color flow cytometry. The data are presented as mean relative percentages of CD11b⁺ or CD11c⁺ cells ± SD for two experiments.

FIG. 10.

Protective efficacy of alphavirus-based replicon particles against IVAG challenge with VV-Gag. Two groups of five female BALB/c mice were immunized with VEE, VEE/SIN, or SIN three times through the i.m., i.n., or IVAG route and then challenged IVAG with VV-Gag. Five days after the challenge, the ovaries were removed and a PFU assay was performed. The data are presented as PFU from pairs of ovaries from individual mice, with horizontal bars representing mean PFU per pair of ovaries from 5 (naïve group) or 10 (all other groups) mice per group. The numbers on the x axis show the numbers of mice with no PFU, i.e., protected mice. The statistical significance between groups is shown by the P values on dotted lines between the groups.