Asymptomatic HLA-A*02:01-restricted epitopes from herpes simplex virus glycoprotein B preferentially recall polyfunctional CD8+ T cells from seropositive asymptomatic individuals and protect HLA transgenic mice against ocular herpes

Abstract

Evidence from C57BL/6 mice suggests that CD8(+) T cells, specific to the immunodominant HSV-1 glycoprotein B (gB) H-2(b)-restricted epitope (gB498-505), protect against ocular herpes infection and disease. However, the possible role of CD8(+) T cells, specific to HLA-restricted gB epitopes, in protective immunity seen in HSV-1-seropositive asymptomatic (ASYMP) healthy individuals (who have never had clinical herpes) remains to be determined. In this study, we used multiple prediction algorithms to identify 10 potential HLA-A*02:01-restricted CD8(+) T cell epitopes from the HSV-1 gB amino acid sequence. Six of these epitopes exhibited high-affinity binding to HLA-A*02:01 molecules. In 10 sequentially studied HLA-A*02:01-positive, HSV-1-seropositive ASYMP individuals, the most frequent, robust, and polyfunctional CD8(+) T cell responses, as assessed by a combination of tetramer, IFN-γ-ELISPOT, CFSE proliferation, CD107a/b cytotoxic degranulation, and multiplex cytokine assays, were directed mainly against epitopes gB342-350 and gB561-569. In contrast, in 10 HLA-A*02:01-positive, HSV-1-seropositive symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent clinical herpes disease) frequent, but less robust, CD8(+) T cell responses were directed mainly against nonoverlapping epitopes (gB183-191 and gB441-449). ASYMP individuals had a significantly higher proportion of HSV-gB-specific CD8(+) T cells expressing CD107a/b degranulation marker and producing effector cytokines IL-2, IFN-γ, and TNF-α than did SYMP individuals. Moreover, immunization of a novel herpes-susceptible HLA-A*02:01 transgenic mouse model with ASYMP epitopes, but not with SYMP epitopes, induced strong CD8(+) T cell-dependent protective immunity against ocular herpes infection and disease. These findings should guide the development of a safe and effective T cell-based herpes vaccine.

FIGURE 1

Schematic representation showing the relative location within HSV-1 gB of the potential CD8⁺ T cell epitopes studied. HSV-1 (strain 17) gB regions carrying potential HLA A*0201 (HLA-A*0201)–restricted T cell epitopes were predicted using computer-assisted algorithms based on known HLA/peptide/TCR interactions, as described in Materials and Methods. The amino acid sequence, in a single letter code, and the peptide positions based on the 904-aa sequence of gB are shown. The high-affinity immunodominant epitopes identified in this study are shown in bold. ID, Intracellular domain; SS, signal sequence; TM, transmembrane domain.

FIGURE 2

Binding capacities of HSV-1 gB-derived epitope peptides to HLA-A*0201 molecules. (A) In vitro binding capacities of HSV-1 gB-derived epitope peptides to soluble HLA-A*0201 molecules. HSV-1 gB-derived peptides were tested by an ELISA binding assay specific for HLA-A*0201 molecules as described in Materials and Methods. A reference nonherpes peptide was used to validate each assay. Data are expressed as relative activity (ratio of the IC₅₀ of the test peptide to the IC₅₀ of the reference peptide) and are the means of two experiments. Six peptide epitopes with high-affinity binding to HLA-A*0201 molecules (IC₅₀ < 100) are indicated by an asterisk. The columns show four peptide epitopes that failed to bind HLA-A*0201 molecules. (B) Stabilization of HLA-A*02:01 molecules on the surface of T₂ cells by gB peptides. T₂ cells (3 × 10⁵) were incubated with individual gB peptides at various concentrations (20, 10, 5, and 2.5 µM), as described in Materials and Methods. T₂ cells were then stained with FITC-conjugated anti–HLA-A2 mAb (BB7.2). The graph represents the percentage of MFI of HLA-A*02:01 molecules on the surface of T₂ cells following incubation with peptides. MFI was calculated as follows: percentage of MFI increase = [(MFI with the given peptide − MFI without peptide)/(MFI without peptide)] × 100. Error bars show SD for three independent experiments. Solid lines represent high levels of HLA-A*02:01 expression on the surface of T₂ cells incubated with gB peptides. Broken lines represent low levels of stabilized HLA-A*02:01 expression.

FIGURE 3

High frequency of IFN-γ–producing CD8⁺ T cells specific to gB_17–25, gB_183–191, gB_342–350, gB_441–449, and gB_561–569 epitopes detected in HLA-A*02:01–positive, HSV-seropositive individuals. PBMC-derived CD8⁺ T cells were analyzed ex vivo, after in vitro expansion, for the frequency of CD8⁺ T cells specific to 10 gB peptide/tetramer complexes representing each of the 10 potential gB epitopes shown in Fig. 1. (A) Representative FACS data of the frequencies of CD8⁺ T cells in an HSV-1–seropositive individual following a 5-d expansion with the indicated peptide. (B) Average frequency of PBMC-derived CD8⁺ T cells that recognize each of the indicated gB epitopes from 10 HLA-A*02:01–positive, HSV-1–seropositive individuals (HSV⁺) compared with 10 HLA-A*02:01–positive, HSV-seronegative individuals (HSV⁻). (C) gB epitope-specific IFN-γ–producing CD8⁺ T cells detected in healthy HLA-A*02:01–positive, HSV-seropositive individuals. PBMC-derived CD8⁺ T cells, either from HSV⁺ or from HSV⁻ individuals, were stimulated with individual gB peptides. The number of gB epitope-specific, IFN-γ–producing T cells was determined by ELISPOT assay. Tests were performed in duplicates for each experiment. Spots were developed as described in Materials and Methods and calculated as spot-forming cells = mean number of spots in the presence of Ag − mean number of spots in the absence of stimulation. Open circles represent HSV⁻ individuals whereas filled circles represent HSV⁺ individuals. (D) Profile of T cell proliferation with individual gB peptides. The graphs represent the proliferation of CD8⁺ T cells in the presence of 10 µM of each individual gB peptide detected from HSV⁺ individuals using a CFSE assay. (E) Absolute numbers of dividing CD8⁺ T cells per 300,000 total cells after 5 d stimulation. The results are representative of two independent experiments. *p < 0.005, comparing HSV⁺ to HSV⁻ individuals using one-way ANOVA test.

FIGURE 4

Frequent and strong IFN-γ–producing CD8⁺ T cells preferentially induced by gB_342–350 and gB_561–569 epitopes in asymptomatic individuals. (A) PBMC-derived CD8⁺ T cells, either from 10 ASYMP or from 10 SYMP individuals, were stimulated with individual gB peptides. The number of gB epitope-specific, IFN-γ–producing T cells was determined by ELISPOT assay, as in Fig. 4. (B) Representative data from one SYMP and one ASYMP individual showing the number of gB epitope-specific CD8⁺ T cells per 100,000 cells detected ex vivo using several concentrations of tetramer. (C) The average number of gB epitope-specific, PBMC-derived CD8⁺ T cells per 100,000 cells from five HLA-A*02:01–positive, HSV-1–seropositive SYMP individuals versus five ASYMP individuals. The results are representative of two independent experiments. *p < 0.005, comparing ASYMP to SYMP individuals using one-way ANOVA test.

FIGURE 5

Asymptomatic epitopes preferentially induced polyfunctional CD8⁺ T cells. (A) ASYMP gB epitope-primed CD8⁺ T cells displayed lytic activity. CD8⁺ T cell lines specific to gB_342–350 and gB_561–569, gB_183–191 and gB_441–449 epitopes were derived from HLA-A*02:01–positive ASYMP (n = 5) and SYMP (n = 5) individuals. Each CD8⁺ T cell line was incubated with HSV-1– or VVgB-infected autologous target monocyte-derived dendritic cells (moDCs) in the presence of anti-CD28/49d, FITC-conjugated anti-CD107a and CD107b, and GolgiStop for 6 h. Uninfected target cells were used as control. FACS was used to analyze CD107 expression, as described in Materials and Methods. The graph represents the means ± SD of the percentage of CD107a/b and CD8⁺ T cells in the presence of uninfected (mock)-, HSV-1–, VVgB-, or control VVgD-infected target cells (moDCs). Samples were acquired on a BD LSR II, and data analysis was performed using FlowJo. *p < 0.005, comparing ASYMP to SYMP individuals using one-way ANOVA test. (B) Summary pie charts showing the average amount of each cytokine produced by CD8⁺ T cells from ASYMP patients (n = 10, top row) and SYMP patients (n = 8, bottom row), as detected by Luminex assay. The average frequency of different cytokines producing CD8⁺ T cells is shown under each pie chart. (C) Each pie chart represents the overall mean of CD8⁺ T cell functions from five HLA-A*02:01–positive ASYMP and five SYMP individuals in responses to stimulation with either SYMP or ASYMP gB peptides. Each sector of the pie chart represents the number of CD8⁺ T cell functions produced. (D) Representative data showing the expression of HLA-A0201 molecule by dendritic cells from an HLA-A*02:01–positive, HSV-1–seropositive SYMP individual versus an ASYMP individual and by dendritic cells from an HLA-A*02:01–negative, HSV-1–seronegative healthy control (HC).

FIGURE 6

CD8⁺ T cell–dependent protective immunity against ocular herpes induced by ASYMP epitopes in humanized HLA transgenic mice. Three groups of age-matched female HLA-A*02:01 transgenic mice (n = 10 each) were immunized s.c. with the ASYMP CD8⁺ T cell human epitopes (gB_342–350 and gB_561–569) delivered with the CD4⁺ T cell PADRE epitope emulsified in CpG₁₈₂₆ adjuvant (ASYMP), with the SYMP CD8⁺ T cell human epitopes (gB_183–191 and gB_441–449) delivered with the CD4⁺ T cell PADRE epitope emulsified in CpG₁₈₂₆ adjuvant (SYMP), or with the CpG₁₈₂₆ adjuvant alone (mock) on days 0 and 21. Two weeks after the final immunization, all animals were challenged ocularly with 2 × 10⁵ PFU of HSV-1 (strain McKrae). (A) The eye disease was detected and scored 2 wk after immunization as described in Materials and Methods. (B) Viral loads were detected on day 7 postinfection in eye swabs, as described in Materials and Methods. (C) Immunized and infected mice were examined for survival in a window of 30 d postchallenge, as described in Materials and Methods. (D) Scattergram and linear regression analysis of mouse survival (%) and HSV-specific CD8⁺ T cell responses after challenge with HSV-1. Correlation was performed using the Pearson test with two-tailed p value analysis (r² = 0.7996; p < 0.0001). (E) The protective immunity against ocular herpes induced by the ASYMP CD8⁺ T cell human epitope immunization is abrogated following depletion of CD8⁺ T cells, but not of CD4⁺ T cells. Following the second immunization, and before challenge with HSV-1, mice were injected i.p. with six doses (i.e., 1 every other day) of 100 µl saline containing anti-CD4, anti-CD8, or isotype control. Flow cytometry analysis confirmed a decrease in spleen CD4⁺ and CD8⁺ T cells in treated mice to consistently <2% after mAb treatment. The p values compare protection achieved in mAb treated versus untreated mice using the ANOVA test. Immunized, mAb-treated, and infected mice were examined for survival in a window of 30 d after challenge. Results are representative of two independent experiments.

FIGURE 7

A proposed model of phenotypic and functional characteristics of HSV-specific CD8⁺ T cells in HSV-seropositive SYMP versus ASYMP individuals. After stimulation with HSV-1 ASYMP epitopes, CD8⁺ T cells (purple) from HSV-seropositive ASYMP individuals develop more protective CD8⁺ T cells that prevent herpes disease. In contrast, after stimulation with HSV-1 SYMP epitopes, CD8⁺ T cells (orange) from SYMP individuals develop more pathogenic CD8⁺ T cells that lead to inflammation. CD8⁺ T cells from SYMP individuals secrete substantial amounts of proinflammatory cytokines IL-6, IL-8, and IL-17 (small red circles) and have low proliferation and low lytic granules. In contrast, CD8⁺ T cells from ASYMP individuals have efficient proliferation and clonal expansion, and they produce more lytic granules and more IL-2, IFN-γ, and TNF-α (small blue circles), resulting in decreased or subclinical disease. In SYMP individuals, lower amounts of lytic granules likely account for inefficient killing of HSV-infected target cells.