A genital tract peptide epitope vaccine targeting TLR-2 efficiently induces local and systemic CD8+ T cells and protects against herpes simplex virus type 2 challenge

Abstract

The next generation of needle-free mucosal vaccines is being rationally designed according to rules that govern the way in which the epitopes are recognized by and stimulate the genital mucosal immune system. We hypothesized that synthetic peptide epitopes extended with an agonist of Toll-like receptor 2 (TLR-2), that are abundantly expressed by dendritic and epithelial cells of the vaginal mucosa, would lead to induction of protective immunity against genital herpes. To test this hypothesis, we intravaginally (IVAG) immunized wild-type B6, TLR-2 (TLR2(-/-)) or myeloid differentiation factor 88 deficient (MyD88(-/-)) mice with a herpes simplex virus type 2 (HSV-2) CD8+ T-cell peptide epitope extended by a palmitic acid moiety (a TLR-2 agonist). IVAG delivery of the lipopeptide generated HSV-2-specific memory CD8+ cytotoxic T cells both locally in the genital tract draining lymph nodes and systemically in the spleen. Moreover, lipopeptide-immunized TLR2(-/-) and MyD88(-/-) mice developed significantly less HSV-specific CD8+ T-cell response, earlier death, faster disease progression, and higher vaginal HSV-2 titers compared to lipopeptide-immunized wild-type B6 mice. IVAG immunization with self-adjuvanting lipid-tailed peptides appears to be a novel mucosal vaccine approach, which has attractive practical and immunological features.

Figure 1

Glycoprotein B (gB)_498–505- and herpes simplex virus (HSV)-2-specific CD8⁺ T cells induced in the draining lymph nodes following intravaginal lipopeptide immunization. B6 mice (n=10) were immunized twice with helper-cytotoxic-T-lymphocyte chimeric epitopes (Th-CTL lipopeptide; LIPO), Th-CTL peptide (PEP), CTL peptide alone or Th peptide alone with an interval of 2 weeks between immunizations. Ten days after the second immunization, inguinal lymph nodes were harvested and gB_498–505- and HSV-specific interferon (IFN)-γ-producing CD8⁺ T cells and their CTL activity were measured. (a) Shows the IFN-γ producing CD8⁺ T cells as measured by enzyme-linked immunosorbent spot (ELISpot) assay and; (b) shows the CTL activity specific to either gB_498–505 peptide (top) or the vaccinia virus expressing gB (VVgB) (bottom) as measured by a ⁵¹Cr release assay using EL-4 as target cells at effector–target ratios (E:T) of 3, 10, 30, and 90 respectively. Bars represent mean values for each group (± s.e.m.). Asterisk (*) indicates P<0.05. PowerPoint slide

Figure 2

Induction of herpes simplex virus (HSV)-specific CD8⁺ TC₁ cells in the spleen following intravaginal immunization with helper-cytotoxic-T-lymphocyte chimeric epitopes (Th-CTL lipopeptide). B6 mice (n=5) were immunized as described in Figure 1. Ten days after the second immunization spleens were harvested and in (a) vaccinia virus expressing glycoprotein B (VVgB)-specific CD8⁺ CTL was measured in spleen by ⁵¹Cr release assay at an effector–target (E:T) ratio of 30 in (b) frequency of HSV-gB_498–505-specific CD8⁺ T cells were measured by tetramer. CD8⁺ T cells were stimulated in vitro with heat-inactivated virus pulsed Ag-presenting cells (APCs) and stained with a PE-labeled anti-CD8 mAb followed by an FITC-labeled HSV-gB_498–505/H2-K^b tetramer. Cells were analyzed using a FACSCalibur with a total of 4 × 10⁵ events collected for each point. Histograms of the percentage of CD8⁺/Tetramer⁺ cells representative of two independent experiments is shown. Bars represent mean values for each group (± s.e.m.). Asterisk (*) indicates significant difference between groups (P≤0.05). (c) Represents the cytokines profile produced by CD8⁺ T cells. Spleen derived CD8⁺ T cells were stimulated in vitro with the target gB_498–505 peptide-loaded H2^b-irradiated splenocytes for 5 days at 37°C in 5% CO₂. The amounts of interleukin (IL)-2, IL-4, IL-12, and interferon (IFN)-γ secreted into the culture medium were measured in a specific sandwich ELISA according to the manufacturer's instructions. The data are the representative of two independent experiments. The P values compare the amounts of each cytokine between Th-CTL lipopeptide PEP, CTL, and Th peptide-immunized mice. PowerPoint slide

Figure 3

Functional characterization of herpes simplex virus (HSV)-specific CD8⁺ T cells induced following intravaginally (IVAG) immunization of self-adjuvanting lipopeptide. (a) Syngeneic spleen cells were infected with HSV-2 and labeled with CFSE (2.5 μm). To control for antigen specificity, noninfected syngeneic spleen cells were labeled with a lower concentration of CFSE (0.25 μm). A 1:1 mixture of each target cell population was injected i.v. into helper-cytotoxic-T-lymphocyte chimeric epitopes (Th-CTL lipopeptide) immunized and control groups of B6 mice 30 d post-IVAG immunization. One hour after injection, spleen cells were harvested from individual mice and acquired on a FACS. The percentage of specific lysis was determined as mentioned in Methods. The number shown in each plot is the mean of percent antigen specific lysis, determined from gate M1, and that was observed in four mice per group. (b and c) Single cell suspensions of spleen were analyzed for expression of early and late marker of activation; CD44 and CD69 (dashed lines), or isotype control (dark line). Following FACS analysis, the forward angle and side scatter gates were set on the CD3⁺ population. Gating on the CD8⁺ population determined the proportion of CD8⁺ T cells that expressed the activation markers. These data are representative of three independent experiments. PowerPoint slide

Figure 4

Intravaginal (IVAG) immunization of helper-cytotoxic-T-lymphocyte chimeric epitopes (Th-CTL lipopeptide) induced interferon (IFN)-γ-producing T cells via toll-like receptor-2 signaling pathway. (a) Groups of wild-type B6 and TLR-2 knockout (TLR2^−/−) H2^b mice (n=5) were immunized IVAG with equimolar amounts of Th-CTL lipopeptide in saline. Twelve weeks after the second immunization, spleen (SPL)-derived CD8⁺ T cells in each group were stimulated in vitro for 5 days with glycoprotein B (gB)_498–505-pulsed or heat-inactivated herpes simplex virus (HSV)-2-infected autologous H2^b cells. The number of IFN-γ producing cells in each group of mice was determined by enzyme-linked immunosorbent spot (ELISpot) assay. Bars represent the mean values from quadruplicate wells in two independent experiments with ±s.e.m. Asterisk (*) indicates P<0.05. (b) SPL derived CD8⁺ T cells were stimulated in vitro with heat-inactivated virus pulsed Ag-presenting cells (APCs) and stained with a PE-labeled anti-CD8 mAb followed by an FITC-labeled HSV-gB_498–505/H2-K^b tetramer. Cells were analyzed using a FACSCalibur with a total of 4 × 10⁵ events collected for each point. Density plot shows the percentage of CD8⁺/Tetramer⁺ cells representative of two independent experiments. (c) Compares the in vitro proliferation of CFSE labeled gB_498–505-specific CD8⁺ T cell in B6 and TLR-2 knockout (TLR2^−/−) mice following IVAG immunization with Th-CTL lipopeptide (left) or saline alone (right). PowerPoint slide

Figure 5

Protection against herpes simplex virus (HSV)-2 by intravaginal (IVAG) immunization of mice with helper-cytotoxic-T-lymphocyte chimeric epitopes (Th-CTL lipopeptide). Four groups of sex- and age-matched B6 mice (n=20) received an IVAG immunization with the Th-CTL lipopeptide, Th-CTL parental nonlipidated peptide, Th or CTL peptides alone. Two weeks after the second immunization (acute phase) mice were intravaginally challenged with HSV-2 (333, 2 × 10⁴ pfu). (a) Survival following genital HSV-2 challenge. (b) Mean genital pathology scores following intravaginal HSV-2 challenge. Signs of disease were monitored daily and animals with pathology scores ≥4 were killed. (c) Average viral titer following HSV-2 challenge. Vaginal washes were collected daily following infection. Viral titers were measured by plaque assay on rabbit skin (RS) cell monolayers. Data show average virus titers in each group, detected on day 5, 7, 9, and 11 postinfection. The mean±s.e.m. of four mice per group are shown. PowerPoint slide

Figure 6

Intravaginal (IVAG) immunization with helper-cytotoxic-T-lymphocyte chimeric epitopes (Th-CTL) lipopeptide elicits protective memory CD8⁺ T-cell response. Three d groups of sex- and age-matched B6 mice (n=20) received an IVAG immunization with the Th-CTL lipopeptide, herpes simplex virus (HSV)-2 TK⁻−, or administered with phosphate-buffered saline (PBS) alone (Mock). Twelve weeks (memory phase) after the final immunization, local CD8⁺ T cells were harvested from draining lymph nodes (DLNs) of five B6 mice and stimulated for 5 days with HSV-infected autologous spleen cells. The cells were tested for (a) CTL activity following incubation with glycoprotein B (gB)_498–505 peptide pulsed target cells by ⁵¹Cr release assay; (b) interferon (IFN)-γ-ELISpot assay. B6 mice (n=15) were first treated with Depo-Provera, to synchronize the estrous cycle, and then intravaginally challenged with HSV-2 (333, 2 × 10⁴ pfu). (c) Survival. (d) Mean genital pathology scores and (e) Maximal viral titers in each group detected on day 5 postinfection were followed as in Figure 5. PowerPoint slide

Figure 7

Myeloid differentiation factor 88 (MyD88) signaling is critical in helper-cytotoxic-T-lymphocyte chimeric epitopes (Th-CTL lipopeptide) immunogenicity and protective efficacy. (a) Groups of wild-type B6 and MyD88^−/−-deficient mice (n=10) were immunized intravaginally (IVAG) with equimolar amounts of Th-CTL lipopeptide in saline. Two weeks after the second immunization, draining lymph nodes (DLNs) derived CD8⁺ T cells from each group were stimulated in vitro for 5 days with glycoprotein B (gB)_498–505-pulsed or heat-inactivated herpes simplex virus (HSV)-2-infected autologous H2^b cells. The numbers of interferon (IFN)-γ-producing cells were measured by enzyme-linked immunosorbent spot (ELISpot) assay. Asterisk (*) indicates P<0.003. (b) Survival. (c) Mean genital pathology scores and (d) Viral titers in each group detected on day 5 postinfection were followed as in Figures 5 and 6. PowerPoint slide

Figure 8

Illustration of the helper-cytotoxic-T-lymphocyte chimeric epitopes (Th-CTL lipopeptide) used in this study. (a) Structure of the lipidated Th-CTL lipopeptide (LIPO). The C-terminal region of the Pan DR peptide (PADRE; center) was fused to the N-terminal region of the herpes simplex virus (HSV) glycoprotein B (gB)_498–505 target peptide (right). The resulting Th-CTL chimeric peptide backbone, was modified by N-terminal addition of three palmitoyl lysines (TLR-2 agonist) shown in the box. The amino acid sequences of CD4⁺ T helper epitope (Th or PADRE) and CD8⁺ T-cell epitope (CTL or gB_498–505) are indicated in a single-letter code. dA, L-alanine; Cha, L-cyclohexyl alanine; Ahx, aminocaproic acid. (b) Structure of nonlipidated Th-CTL peptide analog (PEP). PowerPoint slide