Human Asymptomatic Epitopes Identified from the Herpes Simplex Virus Tegument Protein VP13/14 (UL47) Preferentially Recall Polyfunctional Effector Memory CD44high CD62Llow CD8+ TEM Cells and Protect Humanized HLA-A*02:01 Transgenic Mice against Ocular Herpesvirus Infection

Abstract

Herpes simplex virus 1 (HSV-1) infection is widespread among humans. The HSV-1 virion protein 13/14 (VP13/14), also known as UL47, is a tegument antigen targeted by CD8⁺ T cells from HSV-seropositive individuals. However, whether VP13/14-specific CD8⁺ T cells play a role in the natural protection seen in asymptomatic (ASYMP) individuals (individuals who have never had a clinical herpetic disease) has not been elucidated. Using predictive computer-assisted algorithms, we identified 10 potential HLA-A*02:01-restricted CD8⁺ T-cell epitopes from the 693-amino-acid sequence of the VP13/14 protein. Three out of 10 epitopes exhibited a high to moderate affinity of binding to soluble HLA-A*02:01 molecules. The phenotype and function of CD8⁺ T cells specific for each epitope were compared in HLA-A*02:01-positive ASYMP individuals and symptomatic (SYMP) individuals (individuals who have frequent clinical herpetic diseases) using determination of a combination of tetramer frequency and the levels of granzyme B, granzyme K, perforin, gamma interferon, tumor necrosis factor alpha, and interleukin-2 production and CD107^a/b cytotoxic degranulation. High frequencies of multifunctional CD8⁺ T cells directed against three epitopes, VP13/14 from amino acids 286 to 294 (VP13/14_286-294), VP13/14 from amino acids 504 to 512 (VP13/14_504-512), and VP13/14 from amino acids 544 to 552 (VP13/14_544-552), were detected in ASYMP individuals, while only low frequencies were detected in SYMP individuals. The three epitopes also predominantly recalled more CD45RA^low CD44^high CCR7^low CD62L^low CD8⁺ effector memory T cells (T_EM cells) in ASYMP individuals than SYMP individuals. Moreover, immunization of HLA-A*02:01 transgenic mice with the three CD8⁺ T_EM-cell epitopes from ASYMP individuals induced robust and polyfunctional HSV-specific CD8⁺ T_EM cells associated with strong protective immunity against ocular herpesvirus infection and disease. Our findings outline the phenotypic and functional features of protective HSV-specific CD8⁺ T cells that should guide the development of a safe and effective T-cell-based herpes simplex vaccine.

Importance: Although most herpes simplex virus 1 (HSV-1)-infected individuals shed the virus in their body fluids following reactivation from latently infected sensory ganglia, the majority never develop a recurrent herpetic disease and remain asymptomatic (ASYMP). In contrast, small proportions of individuals are symptomatic (SYMP) and develop frequent bouts of recurrent disease. The present study demonstrates that naturally protected ASYMP individuals have a higher frequency of effector memory CD8⁺ T cells (CD8⁺ T_EM cells) specific to three epitopes derived from the HSV-1 tegument protein VP13/14 (VP13/14_286-294,VP13/14_504-512, and VP13/14_544-552) than SYMP patients. Moreover, immunization of humanized HLA-A*02:01 transgenic mice with the three CD8⁺ T_EM-cell epitopes from ASYMP individuals induced robust and polyfunctional HSV-specific CD8⁺ T cells associated with strong protective immunity against ocular herpesvirus infection and disease. The findings support the emerging concept of the development of a safe and effective asymptomatic herpes simplex vaccine that is selectively based on CD8⁺ T-cell epitopes from ASYMP individuals.

Keywords: HSV; granzyme B; granzyme K; herpes simplex virus; human leukocyte antigen; humans; immunization; memory CD8+ T cells; perforin; transgenic; virion phosphoprotein.

FIG 1

Stabilization of HLA-A*02:01 molecules on the surface of T2 cells by VP13/14 peptide epitopes. T2 cells (3 × 10⁵) were incubated with serial dilutions of the indicated VP13/14 peptide, as described in Materials and Methods. Cells were then stained with an FITC-conjugated anti-HLA-A2 MAb (BB7.2). The graph represents the increase in the expression of HLA-A2 molecules on the surface of T2 cells triggered by various concentrations of VP13/14 peptides, and the data represent the percent MFI increase, which was calculated as follows: [(MFI with the given peptide − MFI without peptide)/(MFI without peptide)] × 100. Solid lines, peptides that bind to HLA-A02:01 molecules with a moderate to high affinity; broken lines, peptides that bind to HLA-A02:01 molecules with a low affinity. Error bars show SDs obtained from 3 independent experiments.

FIG 2

CD8⁺ T cells specific to VP13/14_286–294, VP13/14_504–512, and VP13/14_544–552 epitopes are detected at higher frequencies in ASYMP than in SYMP individuals. PBMCs (∼10 × 10⁶) derived from 10 HLA-A*02:01-positive, HSV-1-seropositive ASYMP individuals and from 10 HLA-A*02:01-positive, HSV-1-seropositive SYMP individuals were analyzed ex vivo by FACS for the frequency of CD8⁺ T cells specific to three VP13/14 epitopes using HLA-A*02:01 VP13/14 peptide-tetramer complexes representing each of the three peptides with medium to high levels of binding (VP13/14_286–294, VP13/14_504–512, and VP13/14_544–552 epitopes), as determined in the assay whose results are presented in Fig. 1. (A) Representative FACS data on the frequencies of CD8⁺ T cells specific to the VP13/14_286–294, VP13/14_504–512, and VP13/14_544–552 epitopes detected in PBMCs from one ASYMP individual (top) and one SYMP individual (bottom). +Ve, positive; –Ve, negative. Values within the panels represent percentages of VP13/14 epitope-specific CD8⁺ T cells. (B) Average frequencies of PBMC-derived CD8⁺ T cells specific to VP13/14_286–294, VP13/14_504–512, and VP13/14_544–552 epitopes detected from 10 ASYMP individuals and 10 SYMP individuals. The results are representative of those from 2 independent experiments. The indicated P values, calculated using an unpaired t test, show statistically significant differences between SYMP and ASYMP individuals.

FIG 3

More VP13/14 epitope-specific CD44^high CD62L^low CD8⁺ T_EM cells are detected in ASYMP individuals than in SYMP individuals. The T_EM- and T_CM-cell phenotypes of CD8⁺ T cells specific to VP13/14_286–294, VP13/14_504–512, and VP13/14_544–552 epitopes among PBMCs of 10 HLA-A*02:01-positive, HSV-1-seropositive SYMP individuals and 10 HLA-A*02:01-positive, HSV-1-seropositive ASYMP individuals were analyzed by FACS. (A, C, and E) Representative FACS data on the frequencies of CD44^high CD62L^low CD8⁺ T_EM cells and CD44^high CD62L^high CD8⁺ T_CM cells in PBMCs from one SYMP individual and one ASYMP individual. (B, D, and F) Average frequencies of VP13/14-specific T_EM or T_CM CD8⁺ T cells from 10 SYMP and 10 ASYMP individuals. (G, I, and K) Representative FACS data on the frequencies of CD8⁺ CD45RA⁻ CCR7⁻ T_EM cells and CD8⁺ CD45RA⁻ CCR7⁺ T_CM cells in one ASYMP individual and one SYMP individual. (H, J, and L) Average frequencies of VP13/14-specific T_EM and T_CM CD8⁺ cells from 10 ASYMP and 10 SYMP individuals. The results are representative of those from 2 independent experiments. The indicated P values, calculated using an unpaired t test, show statistically significant differences between ASYMP and SYMP individuals.

FIG 4

Higher proportions of polyfunctional HSV-1 VP13/14 epitope-specific CD8⁺ T cells are detected in asymptomatic individuals. (A) Representative FACS data on the levels expression of GzmB, GzmK, and PFN on tetramer-gated CD8⁺ T cells specific for the VP13/14_286–294, VP13/14_504–512, and VP13/14_544–552 epitopes, determined by FACS. Samples were acquired on a BD LSRII flow cytometer, and data analysis was performed using FlowJo software. The numbers on the top of each histogram represent MFIs, depicting the level of expression of each cytotoxic molecule. Numbers in bold represent the MFIs for ASYMP individuals, and numbers in nonbold represent the MFIs for SYMP individuals. (B) Average MFI for the levels of VP13/14_504–512 epitope-specific GzmB, GzmK, and PFN expression for 10 ASYMP and 10 SYMP individuals. (C) Representative FACS data on VP13/14_286–294, VP13/14_504–512, and VP13/14_544–552 epitope-specific CD107^a/bhigh CD8⁺ T cells from one ASYMP individuals and one SYMP individual. (D and E) Average percentage (D) and average absolute number (N^br) (E) of VP13/14_504–512 epitope-specific CD107a/b^high CD8⁺ T cells from 10 ASYMP and 10 SYMP individuals. (F and G) Average amounts of IL-2 (F) and TNF-α (G) effector cytokines produced by CD8⁺ T cells from ASYMP and SYMP individuals, as detected by the Luminex assay. (H) Representative FACS data for VP13/14_286–294, VP13/14_504–512, and VP13/14_544–552 epitope-specific IFN-γ^high CD8⁺ T cells from one ASYMP individual and one SYMP individual. (I and J) Average percentage (I) and average absolute number (J) of VP13/14_504–512 epitope-specific IFN-γ^high CD8⁺ T cells from 10 ASYMP and 10 SYMP individuals. (K) Pie charts representing the overall mean number of CD8⁺ T-cell functions detected from 10 ASYMP and 10 SYMP individuals in response to stimulation with VP13/14 peptides. The results are representative of those from 2 independent experiments. The indicated P values, calculated using an unpaired t test, show statistically significant differences between ASYMP and SYMP individuals. NS, no significant difference.

FIG 4

Higher proportions of polyfunctional HSV-1 VP13/14 epitope-specific CD8⁺ T cells are detected in asymptomatic individuals. (A) Representative FACS data on the levels expression of GzmB, GzmK, and PFN on tetramer-gated CD8⁺ T cells specific for the VP13/14_286–294, VP13/14_504–512, and VP13/14_544–552 epitopes, determined by FACS. Samples were acquired on a BD LSRII flow cytometer, and data analysis was performed using FlowJo software. The numbers on the top of each histogram represent MFIs, depicting the level of expression of each cytotoxic molecule. Numbers in bold represent the MFIs for ASYMP individuals, and numbers in nonbold represent the MFIs for SYMP individuals. (B) Average MFI for the levels of VP13/14_504–512 epitope-specific GzmB, GzmK, and PFN expression for 10 ASYMP and 10 SYMP individuals. (C) Representative FACS data on VP13/14_286–294, VP13/14_504–512, and VP13/14_544–552 epitope-specific CD107^a/bhigh CD8⁺ T cells from one ASYMP individuals and one SYMP individual. (D and E) Average percentage (D) and average absolute number (N^br) (E) of VP13/14_504–512 epitope-specific CD107a/b^high CD8⁺ T cells from 10 ASYMP and 10 SYMP individuals. (F and G) Average amounts of IL-2 (F) and TNF-α (G) effector cytokines produced by CD8⁺ T cells from ASYMP and SYMP individuals, as detected by the Luminex assay. (H) Representative FACS data for VP13/14_286–294, VP13/14_504–512, and VP13/14_544–552 epitope-specific IFN-γ^high CD8⁺ T cells from one ASYMP individual and one SYMP individual. (I and J) Average percentage (I) and average absolute number (J) of VP13/14_504–512 epitope-specific IFN-γ^high CD8⁺ T cells from 10 ASYMP and 10 SYMP individuals. (K) Pie charts representing the overall mean number of CD8⁺ T-cell functions detected from 10 ASYMP and 10 SYMP individuals in response to stimulation with VP13/14 peptides. The results are representative of those from 2 independent experiments. The indicated P values, calculated using an unpaired t test, show statistically significant differences between ASYMP and SYMP individuals. NS, no significant difference.

FIG 5

Asymptomatic individuals had a significantly higher proportion of differentiated and functional VP13/14 epitope-specific CD8⁺ T cells. (A and B) Representative FACS data (A) and average frequencies (B) of VP_504–512-specific Ki-67-positive [Ki-67⁽⁺⁾] CD8⁺ T cells from 10 ASYMP and 10 SYMP individuals. (C) Representative histograms showing the numbers of VP_504–512-specific PD-1^high CD8⁺ T cells from one ASYMP individual (closed histogram and nonbold data) and one SYMP individual (open histogram and bold data). (D) Average frequencies of VP_504–512-specific PD-1^high CD8⁺ T cells from 10 ASYMP and 10 SYMP individuals. (E and H) The expression patterns of the T-bet (H) and Eomes (E) transcription factors from CD8⁺ T cells derived from SYMP and ASYMP individuals were analyzed at the RNA level, using RT-PCR. (F and I) Representative FACS data on the percentage of Eomes-positive (F) and T-bet-positive (I) HSV-1 VP13/14_504–512-specific CD8⁺ T cells derived from one SYMP individual and one ASYMP individual. SSC, side scatter. (G and J) Average frequencies of the expression patterns of the Eomes (G) and T-bet (J) transcription factors at the protein level from gated VP13/14_504–512-specific CD8⁺ T_EM cells derived from 10 SYMP and 10 ASYMP individuals analyzed by FACS. The results are representative of those from 3 independent experiments. The indicated P values, calculated using an unpaired t test, show statistically significant differences between SYMP and ASYMP individuals.

FIG 6

Protective immunity against ocular herpes induced by ASYMP VP13/14 CD8⁺ T_EM-cell epitopes in humanized HLA transgenic mice. Three groups of age- and sex-matched HLA Tg mice (n = 10 each) were immunized subcutaneously on days 0 and 21 with a mixture of ASYMP CD8⁺ T-cell peptide epitopes (VP13/14_286–294, VP13/14_504–512, and VP13/14_544–552) and a mixture of SYMP CD8⁺ T-cell human epitopes (VP13/14_410–418,VP13/14_497–505 and VP13/14_657–665). These were delivered together with a promiscuous CD4⁺ T-cell epitope (PADRE) emulsified in CpG₁₈₂₆ adjuvant. CpG₁₈₂₆ adjuvant alone was used for mock vaccination. Two weeks after the final immunization, all animals were challenged ocularly with 2 × 10⁵ PFU of HSV-1 (strain McKrae). (A and B) The severity of herpetic eye disease was followed for 2 weeks after immunization and scored on a scale of from 1 to 5. (C) Virus titrations were determined from eye swab specimens collected on day 7 postinfection. (D) Survival was determined within a window of 30 days postchallenge. Corneas were harvested from each group, and the frequencies of CD44^high CD62L^low CD8⁺ T_EM cells and CD44^high CD62L^high CD8⁺ T_CM cells were analyzed by FACS using specific MAbs. (E) Representative data on the frequencies of CD8⁺ T_EM and CD8⁺ T_CM cells detected from the corneas of one protected ASYMP HLA Tg mouse, one nonprotected SYMP HLA Tg mouse, and one mock-immunized HLA Tg mouse. (F) Average frequencies of T_CM and T_EM CD8⁺ cells detected in the corneas of four ASYMP, four SYMP, and four mock-immunized HLA Tg mice. The results are representative of those from 2 independent experiments. The indicated P values, calculated using an unpaired t test, show statistically significant differences between ASYMP, SYMP, and mock-immunized mice.

FIG 7

The corneas of protected ASYMP HLA transgenic mice have more VP13/14-specific polyfunctional CD8⁺ T_EM cells than the corneas of nonprotected SYMP mice. Mice were infected with HSV-1 and then segregated into two groups, (i) ASYMP mice and (ii) SYMP mice, on the basis of the clinical score for their ocular herpetic disease. Corneas were harvested from each group on day 9 postinfection, and the frequencies of CD44^high CD62L^low CD8⁺ T_EM cells and CD44^high CD62L^high CD8⁺ T_CM cells specific to the VP13/14_286–294, VP13/14_504–512, and VP13/14_544–552 epitopes were analyzed by FACS using tetramers and specific MAbs, as described in Materials and Methods. (A, C, and E) Representative data on the frequencies of VP13/14_286–294-specific (A), VP13/14_504–512-specific (C), and VP13/14_544–552-specific (E) T_EM cells and CD8⁺ T_CM cells detected from the corneas of one ASYMP HLA Tg mouse and one SYMP HLA Tg mouse. (B, D, and F) Average frequencies of VP13/14_286–294-specific (B), VP13/14_504–512- specific (D), and VP13/14_544–552-specific (F) T_CM and T_EM CD8⁺ cells detected in the corneas of five ASYMP HLA Tg mice and five SYMP HLA Tg mice. (G) Representative data on the percentage of IFN-γ-positive CD8⁺ T cells specific to VP13/14_286–294, VP13/14_504–512, and VP13/14_544–552 epitopes detected from ASYMP and SYMP mice. (H) Average frequencies of IFN-γ-positive CD8⁺ T cells specific to the VP13/14_504–512 peptide. The results are representative of those from 2 independent experiments. The indicated P values, calculated using an unpaired t test, show a statistically significant difference between SYMP and ASYMP mice.