ABSTRACT
While the role of CD8+ T cells in the control of herpes simplex virus 1 (HSV-1) infection and disease is gaining wider acceptance, a direct involvement of effector CD4+ T cells in this protection and the phenotype and function of HSV-specific human CD4+ T cell epitopes remain to be fully elucidated. In the present study, we report that several epitopes from the HSV-1 virion tegument protein (VP11/12) encoded by UL46 are targeted by CD4+ T cells from HSV-seropositive asymptomatic individuals (who, despite being infected, never develop any recurrent herpetic disease). Among these, we identified two immunodominant effector memory CD4+ TEM cell epitopes, amino acids (aa) 129 to 143 of VP11/12 (VP11/12129–143) and VP11/12483–497, using in silico, in vitro, and in vivo approaches based on the following: (i) a combination of the TEPITOPE algorithm and PepScan library scanning of the entire 718 aa of HSV-1 VP11/12 sequence; (ii) an in silico peptide-protein docking analysis and in vitro binding assay that identify epitopes with high affinity to soluble HLA-DRB1 molecules; and (iii) an ELISpot assay and intracellular detection of gamma interferon (IFN-γ), CD107a/b degranulation, and CD4+ T cell carboxyfluorescein succinimidyl ester (CFSE) proliferation assays. We demonstrated that native VP11/12129–143 and VP11/12483–497 epitopes presented by HSV-1-infected HLA-DR-positive target cells were recognized mainly by effector memory CD4+ TEM cells while being less targeted by FOXP3+ CD4+ CD25+ regulatory T cells. Furthermore, immunization of HLA-DR transgenic mice with a mixture of the two immunodominant human VP11/12 CD4+ TEM cell epitopes, but not with cryptic epitopes, induced HSV-specific polyfunctional IFN-γ-producing CD107ab+ CD4+ T cells associated with protective immunity against ocular herpes infection and disease.
IMPORTANCE We report that naturally protected HSV-1-seropositive asymptomatic individuals develop a higher frequency of antiviral effector memory CD4+ TEM cells specific to two immunodominant epitopes derived from the HSV-1 tegument protein VP11/12. Immunization of HLA-DR transgenic mice with a mixture of these two immunodominant CD4+ T cell epitopes induced a robust antiviral CD4+ T cell response in the cornea that was associated with protective immunity against ocular herpes. The emerging concept of developing an asymptomatic herpes vaccine that would boost effector memory CD4+ and CD8+ TEM cell responses is discussed.
INTRODUCTION
A staggering 3.72 billion individuals worldwide currently carry herpes simplex virus 1 (HSV-1), which causes a wide range of diseases throughout their lives (1). Ocular herpes, caused by HSV-1, is a potentially blinding disease impacting over 450,000 individuals in the United States (2–5). Current antiviral drug therapies (e.g., acyclovir and derivatives) reduce recurrent herpetic disease by ∼45% but does not eliminate the virus (6, 7). One such powerful and cost-effective strategy would be an effective vaccine able to eliminate the virus and the root of the disease as a means to prevent corneal herpetic disease and blindness (7–9).
There is substantial literature currently on the role of CD8+ T cells in the protection against herpes, whereas the role of CD4+ T cells in such protection is relatively less known (10–14). Activated CD4+ T cells are retained in latently infected trigeminal ganglia (TG) of mice and humans. In addition to their direct effect on virus replication, the CD4+ T helper cells are a source of large amounts of cytokines that promote the generation of primary and memory CD8+ T cell responses, thus indirectly contributing to protection (15, 16). Given the significant role of CD4+ T cells in herpes virus immunity, the identification and characterization of CD4+ T cell epitopes are crucial steps toward the design of an epitope-based herpes vaccine.
Several unsuccessful attempts to develop a herpes vaccine have concentrated around HSV glycoproteins (17–20). The HSV-1 and HSV-2 experimental vaccines designed to date were either purified virion products derived from infected cells (subunit vaccines), purified recombinant immunogenic HSV-coded proteins, focused mainly on the glycoprotein gD or attenuated live viruses lacking some of the virulence features, such as gH and gE (17, 21). The most prevalent CD4+ T cell responses in HSV-seropositive humans were detected against tegument proteins (22), suggesting their importance in preventing the infection. Tegument proteins are functional molecules located between the viral capsid enveloping the genome and the outer membrane (22–25). Previous reports have indicated that the genes encoding the tegument proteins are conserved between HSV-1 and HSV-2 and possess a high degree of similarity in viruses in the alpha subfamily. The tegument virion protein 11/12 (VP11/12) is encoded by the HSV open reading frame (ORF) UL46. Notably, CD4+ T cells from infected humans recognize a couple of epitopes in HSV-2 VP11/12 (26–28). However, the complete repertoire of HSV-1 VP11/12 CD4+ T cell epitopes remains to be fully elucidated.
In this study, we followed several in silico, in vitro, and in vivo epitope mapping approaches to identify the CD4+ T cell epitopes from the HSV-1 VP11/12. We discovered frequent, robust, and polyfunctional effector memory CD4+ TEM cell responses directed predominantly against two human epitopes, residues 129 to 143 of VP11/12 (VP11/12129–143) and VP11/12483–497, among HSV-seropositive healthy asymptomatic individuals. We further validated this finding in a “humanized” transgenic HLA-DR mouse model of ocular herpes by observing strong protective immunity associated with robust and polyfunctional VP11/12 epitope-specific CD4+ T cell responses upon immunization with a mixture of VP11/12129–143 and VP11/12483–497 CD4+ T cell epitopes against ocular herpes infection and disease. We demonstrated that induction of VP11/12129–143 and VP11/12483–497 epitope-specific CD4+ T cells in the cornea correlated with protection. Based on these findings, in this article, we discuss our emerging concept of peripheral epithelial immunity and central neuronal immunity in the development of a forthcoming future T cell epitope-based herpes vaccine.
RESULTS
In silico prediction models and in vitro assays identify two VP11/12 CD4+ T cell epitopes that bind with high affinity to the most common HLA-DR haplotypes.We first identified 10 potential CD4+ T cell epitopes within HSV-1 tegument protein VP11/12 (strain 17) using the TEPITOPE computer algorithm. The in silico TEPITOPE computational simulation-based prediction method predicts potential HLA-DRB1-restricted T cell epitopes. The amino acid sequence of HSV-1 VP11/12 tegument protein (strain 17) was screened for potential HLA-DRB1 binding regions, and the relative locations of 10 identified epitopes are illustrated in Fig. 1A.
Illustrations of the relative locations of VP11/12 CD4+ T cell epitopes and molecular docking for CD4+ T cell epitope peptide–HLA-DRB1 molecule. (A) The sequence of the HSV-1 (strain 17) tegument protein (VP11/12) was submitted to the screening of potential HLA-DR epitopes using the TEPITOPE computer algorithm. Ten potential CD4+ T cell epitopes were selected on the basis of high-affinity binding to multiple HLA-DR molecules. (B and C) Computational molecular docking of the 10 CD4+ T cell peptide epitopes into the groove of the HLA-DRB1 protein (PDB accession no. 4GBX) was performed using the GalaxyPepDock server. The peptides are shown in ball and stick structures, and the HLA-DRB1 protein is shown as a template. The prediction accuracy is estimated from a linear model as the relationship between the fraction of correctly predicted binding site residues and the template-target similarity measured by the protein structure similarity score (TM score) and interaction similarity score (SInter) obtained by linear regression. SInter shows the similarity of the amino acids of the CD4+ peptides aligned to the contacting residues in the amino acids of the HLA-DRB1 template structure. A higher SInter score represents a more significant binding affinity among the HLA-DRB1 molecule and CD4 peptides.
We then performed peptide-protein docking analysis that evaluated the interaction between each of the 10 CD4 peptide epitopes and HLA-DRB1 molecules at the atomic level. This structural characterization method identifies candidate peptide epitopes with high binding affinity to HLA-DRB1 molecules. Molecular docking of 10 individual CD4 peptide epitopes into the DRB1 region of the binding protein identified by PDB accession number 4GBX (crystal structure of HLA-DM-HLA-DR1) was performed using the GalaxyPepDock server. The prediction accuracy was estimated from a linear model as the relationship between the fraction of correctly predicted binding site residues and the template-target similarity measured by the protein structure similarity score (TM score) and by the interaction similarity score (SInter) obtained by linear regression. SInter shows the similarity of the amino acids of the CD4 peptides aligned to the contacting residues in the amino acids of the HLA-DRB1 template. A relatively higher SInter score of 157 was observed for VP11/12129–143, followed by an SInter score of 149 for VP11/12483–497 (Fig. 1B and C). These higher SInter scores indicate strong binding affinity of VP11/12129–143 and VP11/12483–497 peptide epitopes to the HLA-DRB1 molecule.
To verify the strong predicted affinity of VP11/12129–143 and VP11/12483–497 peptide epitopes to the HLA-DRB1 molecules, we then determined in vitro binding to soluble HLA-DR molecules. Peptides corresponding to the 10 potential peptide epitopes, predicted above in silico with high affinity to HLA-DRB1 molecules, were synthetized and further tested in vitro for binding to 10 soluble HLA-DR molecules (Table 1). This panel of available HLA-DR molecules consists of the most common HLA class II haplotypes, regardless of ethnicity. The relative binding capacity (50% inhibitory concentration [IC50], in nanomolars) for each peptide was calculated as the concentration of competitor peptide required to inhibit 50% of the binding of an allele-specific biotinylated peptide (indicator peptide), as described in Materials and Methods. Peptide epitopes with high-affinity binding to HLA-DR molecules have IC50 values below 100 nM. The peptides VP11/12129–143 and VP11/12483–497 bound to three or more different HLA-DR molecules, whereas the remaining peptides did not bind to even two or more HLA-DR molecules tested.
In vitro binding capacities of HSV-1 VP11/12-derived epitope peptides for soluble HLA-DR molecules
Altogether, the in vitro binding capacities of HSV-1 VP11/12-derived peptides for soluble HLA-DR molecules strengthen our in silico results of epitope prediction and molecular docking that predicted VP11/12129–143 and VP11/12483–497 as high-affinity binders of CD4+ T cell epitopes to most common HLA-DRB1 molecules.
High frequency of VP11/12129–143 and VP11/12483–497 peptide-specific T cell responses in HSV-1-seropositive asymptomatic individuals.To verify the in silico prediction models and the in vitro binding results above, we next assessed the ability of synthetic peptides, corresponding to the 10 predicted CD4+ T cell epitopes, to induce functional HSV-specific CD4+ T cell responses in HSV-1-seropositive asymptomatic individuals. Specifically, the ability of each VP11/12 peptide to recall proliferative gamma interferon (IFN-γ)-producing CD107a/b+ CD4+ T cells was examined in 10 HSV-seropositive asymptomatic individuals.
CD4+ T cells from HSV-seropositive asymptomatic individuals (n = 10) were stimulated with each of the 10 different VP11/12 peptides at a concentration of 10 μM in the presence of anti-CD28/49d, GolgiPlug, and GolgiStop for 6 h, and the frequency of IFN-γ-producing CD4+ T cells was quantified using a fluorescence-activated cell sorter (FACS) assay. The highest frequencies of IFN-γ-producing CD4+ T cell responses were detected against VP129–143 (10.7%) and VP11/12483–497 (8.6%) (Fig. 2A). The enzyme-linked immunosorbent spot assay (ELISpot) assay shown in Fig. 2B reveals the frequency distribution of IFN-γ-producing CD4+ T cells induced by the 10 VP11/2 peptides. Preliminary analysis of the IFN-γ-producing CD4+ T cells revealed that 20 or more spots/106 CD4+ T cells gave a 97% probability of defining a positive response. Based on the number of IFN-γ-producing CD4+ T cells, the response was ranked as follows: strong responses, >80 spot-forming cells (SFC) per half million peripheral blood mononuclear cells (PBMCs) (SFCs/0.5 × 106); medium responses, >40 and <80 SFCs; low responses, >20 and <40 SFCs. Similar to intracellular staining, the ELISpot assay confirmed that the highest IFN-γ-producing CD4+ T cell responses were directed to the VP129–143 (113 spots) and VP11/12483–497 (108 spots) epitopes (Fig. 2B). In contrast, a medium frequency of IFN-γ-producing CD4+ T cell responses was detected against VP11/12138-152 (52 spots), VP11/12264-278 (68 spots), and VP11/12519-533 (48 spots), while the lowest numbers of SFCs were detected against the remaining five VP11/12 peptides. As expected, all of the donors responded similarly to phytohemagglutinin (PHA; positive control) while no SFCs were recorded in the absence of stimulating peptides (negative controls).
Prevalence of CD4+ T cell responses specific to 10 VP11/12 epitopes in HSV-1-seropositive asymptomatic individuals. PBMCs from HSV-1-seropositive asymptomatic individuals (n = 10) were stimulated with 10 different VP11/12 peptides (10 μM) in the presence of anti-CD28/49d, anti-CD107a and -CD107b, GolgiPlug, and GolgiStop for 6 h. IFN-γ production and CD107a/b expression were then measured by FACS and ELISpot assay. (A) Representative FACS plot shows IFN-γ production by VP11/12 epitope-primed CD4+ T cells (upper panel). The numbers on the top of each dot plot indicate the percentage of IFN-γ+ CD4+ T cells. The average frequencies of VP11/12 epitope-specific IFN-γ+ CD4+ T cells from 10 HSV-1-seropositive asymptomatic individuals are shown in the lower panel. (B) IFN-γ ELISpot assay of CD4+ T cells from HSV-1-seropositive asymptomatic individuals following stimulation with 10 VP11/12 epitopes. Representative images of wells with IFN-γ-producing cells from PBMCs (0.5 × 106 cells/well) stimulated for 48 h with 10 μg of each VP11/12 peptide are depicted. The number on the top of each image represents the number of IFN-γ-producing spot-forming T cells per half a million total cells. PHA (1 μg/ml) and no peptide were used as positive and negative controls, respectively. (C) Representative FACS plots show the expression of CD107a/b from VP11/12 epitope peptide-primed CD4+ T cells. The numbers on the top of each dot plot indicate the percentage of CD107a/b+ CD4+ T cells (upper panels). Average percentages of VP11/12 epitope-specific CD107a/b+ CD4+ T cells from 10 HSV-seropositive asymptomatic individuals are shown in the lower panel. (D) Representative dot plots of the percentage of dividing CFSE+ CD4+ T cells from HSV-seropositive asymptomatic individuals following a six-day in vitro stimulation with each of the VP11/12 peptides. The results are representative of two experiments.
Furthermore, we tested whether VP11/12 CD4 peptides display any cytotoxic activity by performing a CD107a/b degranulation assay ex vivo on freshly isolated PBMCs. As shown in Fig. 2C, of the 10 VP11/12 peptide epitopes, VP11/12129–143 (11.4%) and VP11/12483–497 (9.2%) induced the most significant degranulation/mobilization of CD107a/b on the surface of CD4+ T cells from HSV-1-seropositive individuals. Since a lack of IFN-γ production and CD107a/b expression may not always reflect the lack of a T cell response, we also studied the proliferative response of CD4+ T cells to the 10 VP11/12 peptides from HSV-1-seropositive asymptomatic individuals. PBMCs were labeled with carboxyfluorescein succinimidyl ester (CFSE) and then stimulated in vitro for 6 days with each of the 10 VP11/12 CD4+ peptides. VP11/12129–143 and VP11/12483–497 induced the highest proliferation of CD4+ T cells (Fig. 2D).
Altogether, these functional results (i) confirm the in silico prediction and the in vitro binding results described above and (ii) suggest that VP11/12129–143 and VP11/12483–497 are immunodominant CD4+ T cell epitopes.
Human CD4+ T cells specific to VP11/12129–143 and VP11/12483–497 epitopes are of the TEM phenotype and recognize native epitopes naturally processed and presented by HSV-1-infected cells.Conventional circulating HSV-specific memory CD4+ T cells are categorized into two major phenotypically and functionally distinct subsets: the effector memory (TEM) and the central memory (TCM) cell subsets. We next investigated the phenotype and function of asymptomatic human CD4+ T cells specific to the VP11/12129–143 and VP11/12483–497 epitopes. CD4+ T cell lines specific to each of the two HSV-1 VP11/12 epitopes were generated. As shown in Fig. 3A, the vast majority of the CD4+ T cell lines specific to VP11/12129–143 (82.3%) and VP11/12483–497 (68.9%) are of a CD44high CC62Llow CD4+ TEM phenotype. These findings suggest that most of VP11/12129–143 and VP11/12483–497 epitope-specific CD4+ T cells from naturally protected HSV-1-seropositive asymptomatic individuals are of the TEM phenotype.
Phenotypic and functional characterization of CD4+ TEM cell lines specific to the immunodominant VP11/12129–143 and VP11/12483–497 epitopes. VP11/12129–143 and VP11/12483–497 peptide-specific CD4+ T cell lines were generated from HLA-DR-positive and HSV-1-seropositive asymptomatic individuals, and their TEM and TCM phenotype and function were analyzed. (A) FACS data show representative average frequencies (left panels) of CD44high CD62Llow CD4+ TEM cells and (right panels) CD44high CD62Lhigh CD4+ TCM cells specific to VP11/12129–143 and VP11/12483–497 epitopes. (B) ELISpot results show representative (left) and average (right) frequencies of IFN-γ-producing CD4+ TEM cells following stimulation with autologous DC pulsed with test peptides (VP11/12129–143 or VP11/12483–497) or control peptide (VP11/12107-121) or infected with HSV-1 (native epitopes). The number on the top of each image represents the number of IFN-γ-producing spot-forming T cells per half a million total cells. (C to F) Representative (left) and average (right) frequencies of CD107a/b+ CD4+ TEM cells (C), IFN-γ-producing CD4+ TEM cells (D), CD69+ CD4+ TEM cells (E), and CD44+ CD4+ TEM cells (F) following stimulation with autologous DC pulsed with test peptides (VP11/12129–143 or a VP11/12483–497) or control peptide (VP11/12107-121) or infected with HSV-1 (native epitopes). The results are representative of three experiments.
Because these CD4+ T cells specific to VP11/12129–143 and VP11/12483–497 epitopes were generated in vitro using artificial synthetic peptides, it was of interest to determine whether they recognized native viral epitopes that are naturally processed and presented by HSV-1-infected target cells. Therefore, we analyzed the ability of HLA-DR-positive HSV-1-infected target dendritic cells (DCs) (antigen-presenting dendritic cells [APCs]) to process and present native epitopes that would be recognized by the autologous VP11/12129–143 and VP11/12483–497 epitope-specific CD4+ T cell lines. CD4+ T cells specific to VP11/12129–143 and VP11/12483–497 peptides underwent significant IFN-γ production (Fig. 3B and D) and CD107a/b degranulation (Fig. 3C) when incubated with autologous HLA-DR-positive DC infected with HSV-1 (P < 0.05). Moreover, expression of both the CD69 and CD44 activation markers was induced on the VP11/12129–143 and VP11/12483–497 CD4+ T cell lines when they were incubated with HLA-DR-positive autologous DC infected with HSV-1 (P < 0.05) (Fig. 3E and F). As expected, significant functions were detected when target DC were pulsed with a test peptide epitope (VP11/12129–143 or VP11/12483–497). In contrast, no functional activities were detected in the VP11/12129–143 and VP11/12483–497 CD4+ T cell lines when incubated with autologous target DC pulsed with a control peptide (VP11/12107-121), indicating the epitope specificity of the responses. Altogether, these results demonstrate that the human CD4+ T cells specific to the VP11/12129–143 and VP11/12483–497 epitopes (i) were of effector memory phenotype (TEM) and (ii) recognized native epitopes naturally processed and presented by HSV-1-infected cells.
Three VP11/12 regions that contain immunodominant CD4+ T cell epitopes are identified using the PepScan library mapping strategy.To determine whether there were other potential VP11/12 CD4+ T epitopes, in addition to the VP11/12129–143 and VP11/12483–497 epitopes identified above, we used the PepScan library as an alternative mapping strategy; using this approach, 89 peptides were synthesized with 15-aa overlapping sequences, as illustrated in Fig. 4. This PepScan library of peptides was divided into nine distinct pools of peptides with 10 individual overlapping peptides forming each pool.
The PepScan library of peptides was designed from the HSV-1 tegument protein (VP11/12) sequence consisting of 716 aa (strain 17; NCBI accession number P10230). Shown are the sequences of nine pools of overlapping peptides (pool 1 to pool 9). Each pool contains 10 overlapping 15- to 21-mer VP11/12 peptides.
We next assessed the ability of the nine pools of peptides to induce functional HSV-specific CD4+ T cell responses in HSV-1-seropositive asymptomatic individuals. PBMCs from 10 HSV-seropositive asymptomatic individuals were stimulated with the nine different pools of VP11/12 peptides in the presence of anti-CD28/49d, GolgiPlug, and GolgiStop for 6 h. The highest frequency of IFN-γ-producing CD4+ T cells was detected from HSV-seropositive asymptomatic individuals against pool 2 (8.3%) and pool 5 (7.6%) (Fig. 5A). In contrast, a medium frequency of IFN-γ-producing CD4+ T cells was detected against pool 7 (4.9%) (Fig. 5A). Moreover, pool 2 (8.9%) and pool 5 (7.4%) induced a significantly higher frequency of CD107a/b+ CD4+ T cells (Fig. 5B). Similarly, significantly higher IFN-γ-producing CD4+ T cell responses were detected by the ELISpot assay for pool 2 (86 spots) and pool 5 (78 spots), while a medium frequency was detected in pool 7 (44 spots) (Fig. 5C). Lower frequencies of IFN-γ+ CD4+ T cells and CD107a/b+ CD4+ T cells were detected in pool 1, pool 3, pool 4, pool 6, pool 8, and pool 9. As expected, all the asymptomatic individuals responded similarly to PHA (positive control). Altogether, these results indicate that three VP11/12 regions, representing peptides from pool 2, pool 5, or pool 7, contain one or several immunodominant CD4+ T cell epitopes.
IFN-γ production and CD107a/b expression by HSV-specific CD4+ T cells following stimulation with nine pools of VP11/12 peptides. PBMCs from HSV-seropositive asymptomatic individuals were stimulated with 9 separate pools of VP11/12 peptides. Each pool contains 10 VP11/12 overlapping peptides. (A) Representative FACS plots (left) and average percentages (right) of IFN-γ-producing CD4+ T cells stimulated with 9 pools of VP11/12 peptides. The numbers on the top of each dot plot indicate the percentages of IFN-γ+ CD4+ T cells. (B) Representative FACS plots (left) and average percentages (right) of CD107a/b CD4+ T cells stimulated with 9 pools of VP11/12 peptides. The numbers on the top of each dot plot indicate the percentages of CD107a/b CD4+ T cells. (C) The 9 pools of VP11/12 peptides were analyzed through ELISpot assay for their capacity to elicit IFN-γ production (left). The numbers on the top of each image of representative ELISpot wells indicate the numbers of IFN-γ-producing spot-forming T cells per half a million total cells. Data are the average of IFN-γ-producing cells measured by ELISpot assay from five asymptomatic individuals (right). PHA (1 μg/ml) and no peptide used as positive and negative controls, respectively. The results are representative of two experiments.
The immunodominant VP11/12129–143 and VP11/12483–497 CD4+ T cell epitopes confirmed using the PepScan library mapping strategy.We next evaluated the ability of the 10 individual peptides forming each PepScan library mapping immunodominant pool 2, pool 5, and pool 7 to induce HSV-specific CD4+ T cell responses in 10 HSV-seropositive asymptomatic individuals. As shown in the representative FACS plots in Fig. 6A and average frequencies in Fig. 6C, the highest frequencies of IFN-γ+ CD4+ T cells were directed against four different peptide epitopes from pool 2: VP11/1289–106 (5.1%), VP11/12104–123 (4.2%), VP11/12126–145 (8.5%), and VP11/12135–150 (6.8%). Similarly, the highest frequency of IFN-γ+ CD4+ T cells was directed against two peptide epitopes from pool 5 and pool 7: VP11/12345–362 (5.6%) and VP11/12353–372 (8.9%) from pool 5 and VP11/12481–498 (8.1%) and VP11/12489–506 (4.5%) from pool 7. The sequences of the VP11/12126–145 and VP11/12481–498 epitopes identified using the PepScan library mapping strategy overlap the sequences of the two immunodominant VP11/12129–143 and VP11/12481–498 epitopes identified using the TEPITOPE mapping strategy described above.
IFN-γ production and CD107a/b expression by HSV-specific CD4+ T cells following stimulation with individual peptides from the dominant pools, as indicated. PBMCs from 10 HSV-seropositive asymptomatic individuals were stimulated in vitro with individual CD4+ T cell peptide epitopes derived from the indicated pools of HSV-1 VP11/12 protein (a total of 30 peptides at 10 μM each). Peptide simulation was performed in the presence of anti-CD28/49d, anti-CD107a and -CD107b, GolgiPlug, and GolgiStop for 6 h. IFN-γ production and CD107a/b expression were then measured by FACS. (A and B) Representative FACS plots showing the percentages of IFN-γ+ CD4+ T (A) and CD107a/b+ CD4+ T cells (B) detected from the immunodominant peptides from pools 2, 5, and 7. (C and D) Average frequencies of IFN-γ+ CD4+ T cells (C) and CD107a/b+ CD4+ T cells (D) detected in 10 HSV-seropositive asymptomatic individuals. The results are representative of two experiments.
Furthermore, as shown in the representative FACS plots in Fig. 6B and average frequencies in Fig. 6D, the highest frequencies of CD107a/b+ CD4+ T cells were directed against four different peptide epitopes from pool 2: VP11/1289–106 (4.9%), VP11/12104–123 (4.1%), VP11/12126–145 (7.7%), and VP11/12135–150 (5.4%). Similarly, the highest frequencies of IFN-γ+ CD4+ T cells were directed against two peptide epitopes from pool 5 and pool 7: VP11/12345–362 (4.1%) and VP11/12353–372 (8.1%) from pool 5 and VP11/12481–498 (7.2%) and VP11/12489–506 (3.9%) from pool 7.
Altogether, these results demonstrate that IFN-γ-producing CD4+ T cells expressing cytotoxic CD107a/b markers detected from HSV-1-seropositive asymptomatic individuals were directed against eight individual peptide epitopes derived from the whole VP11/12 protein: VP11/1289–106, VP11/12104–123, VP11/12126–145, VP11/12135–150, VP11/12345–362, VP11/12353–372, VP11/12481–498, and VP11/12489–506. In addition, the immunodominant VP11/12129–143 and VP11/12483–497 CD4+ T cell epitopes identified above were confirmed using the PepScan library mapping strategy. Moreover, the sequences of the VP11/12129–143 and VP11/12483–497 epitopes are highly conserved between HSV-1 (95 to 100%) strains (Table 2).
Overlapping sequences of HSV-1 VP11/12 CD4+ T cell epitopes between the HSV-1 and HSV-2 strains
The immunodominant VP11/12129–143 and VP11/12483–497 epitopes specific to effector CD4+ TEM cells are less targeted by FOXP3+ CD4+ CD25+ regulatory T cells.Furthermore, high frequencies of proliferative CD4+ T cells were induced in the majority of HSV-1-seropositive asymptomatic individuals by three peptide epitopes: VP11/12126–145 (up to 11.4%), VP11/12353–372 (up to 12.1%), and VP11/12481–498 (up to 9.1%) (Fig. 7A). The effector CD4+ T cells specific to these three peptides produced IFN-γ, as confirmed by the ELISpot assay (Fig. 7B). High frequencies of IFN-γ-producing CD4+ T cells were detected for VP11/12126–145 (133 spots), VP11/12135–150 (90 spots), VP11/12353–372 (123 spots), and VP11/12481–498 (100 spots), while medium frequencies of IFN-γ-producing CD4+ T cells were detected against VP11/1289–106 (69 spots), VP11/12104–123 (43 spots), VP11/12345–362 (61 spots), and VP11/12489–506 (42 spots). In contrast, the immunodominant VP11/12 epitopes were not targeted by FOXP3+ CD4+ CD25+ regulatory T (Treg) cells from HSV-seropositive asymptomatic individuals (Fig. 7C). Low frequencies of FOXP3+ CD4+ CD25+ Treg cells were detected against the immunodominant VP11/12 epitopes VP11/12126–145 (3.2%) and VP11/12353–372 (3.6%). However, medium frequencies of FOXP3+ CD4+ CD25+ Treg cells were detected against the subdominant epitopes VP11/12481–498 (5.3%) and VP11/12489–506 (6.2%) (Fig. 7C).
CFSE incorporation and frequency of regulatory cells in HSV-specific CD4+ T cells following stimulation with individual peptides from the dominant pools, as indicated. PBMCs were stained with carboxyfluorescein succinimidyl ester (CFSE) (2 mM) and then stimulated for 6 days with the immunodominant individual VP11/12 peptides selected from pools 2, 5, and 7. A total of eight peptides (four peptides from pool 2, two peptides from pool 5, and 2 peptides from pool 7 at 10 μM each). (A) Representative dot plots and histograms showing the percentages and intensity of dividing CFSE+ CD4+ T cells from HSV-seropositive asymptomatic individuals following in vitro stimulation with eight immunodominant VP11/12 peptides. (B) Immunodominant peptides from VP11/12 peptide pools were analyzed through ELISpot assay for their capacity to elicit IFN-γ production (right). The number on the top of each image of representative ELISpot wells indicates the number of IFN-γ-producing spot forming T cells per half a million total cells. (C) PBMCs were stimulated with eight individual VP11/12 peptides and then stained for CD4, CD25, and Foxp3 expression on gated CD25high and CD4+ T cells. Representative FACS plots (left) and average frequencies (right) of Foxp3+ CD4+ T cells specific to each of the immunodominant VP11/12 peptides detected in 10 HSV-1-seropositive asymptomatic individuals are shown. Data are representative of two independent experiments.
Altogether, these results(i) demonstrated that VP11/12126–145, VP11/12353–372, VP11/12481–498, and VP11/12489–506 peptide epitopes were major targets by multifunctional effector CD4+ T cells but were less targeted by FOXP3+ CD4+ CD25+ Treg cells and (ii) confirmed that VP11/12129–143 and VP11/12483–497 are immunodominant effector CD4+ T cell epitopes.
Protective immunity against ocular herpes virus infection and disease induced by the immunodominant VP11/12129–143 and VP11/12483–497 CD4+ T cell epitopes in HLA-DR mice.Finally, we set up a preclinical vaccine trial using our established HLA-DR transgenic mouse model of ocular herpes to evaluate whether immunization with the immunodominant VP11/12129–143 and VP11/12483–497 CD4+ T cell epitopes would confer protection against ocular herpes virus infection and disease. The protective efficacy of the two immunodominant epitopes was compared side by side with that induced by subdominant VP11/12107–121 and VP11/12603–617 epitopes. Three groups of HLA-DR transgenic mice (n = 10 mice/group) were immunized subcutaneously (s.c.) twice, at 21-day intervals, with either a mixture of immunodominant peptide epitopes (VP11/12129–143 and VP11/12483–497) or with a mixture of subdominant peptide epitopes (VP11/12107–121 and VP11/12603–617) emulsified in CpG1826 adjuvant (100 μg of each peptide plus 2 μg of CpG1826). The CpG1826 adjuvant alone was used as a control (mock vaccination).
Two weeks after the second and final immunization, animals from all groups received an ocular HSV-1 challenge (2 × 105 PFU of strain McKrae) without scarification. The mice were then assessed for up to 30 days postchallenge for ocular herpes pathology, ocular viral titers, and survival, as illustrated in Fig. 8A. On day 7 postinfection, the clinical scores of corneal diseases observed in the mice immunized with the two immunodominant peptide epitopes were significantly lower than clinical scores observed in mice immunized with the two subdominant peptide epitopes and than those of the mock-immunized group (P = 0.02) (Fig. 8B). Furthermore, a significantly lower virus titer was detected on day 7 postinfection (the peak of viral replication) in the eye swabs of the immunodominant group than in the mock-immunized group (P = 0.03) (Fig. 8C). Most animals in the immunodominant group survived infection (75%) whereas survival was 50% in the subdominant group and only 35% in the mock-immunized group (P = 0.03) (Fig. 8D). Representative images in Fig. 8E show lower ocular disease observed in HLA-DR transgenic mice immunized with immunodominant peptides than in those immunized with subdominant peptides and mock immunized. Moreover, significantly higher frequencies of IFN-γ-producing CD4+ T cells were detected in the cornea of in HLA-DR transgenic mice immunized with the mixture of the two immunodominant epitopes (10.5%) than in those immunized with subdominant epitopes (4.3%) or mock immunized (2.4%) (P = 0.03) (Fig. 8F). Similarly, the highest frequencies of CD107a/b-expressing CD4+ T cells were mainly detected in the immunodominant group (15.7%) than in the subdominant group (7.6%) or mock group (5.4%) (P = 0.02) (Fig. 8G).
Protective immunity against ocular herpes infection and disease induced by immunodominant VP11/12 CD4+ T epitopes in HLA-DR mice. (A) Timeline of immunization, HSV-1 infection, and immunological and virological analyses. Three groups of age-matched HLA-DR mice (n = 10 each) were immunized subcutaneously on days 0 and 21 with a mixture of two immunodominant human CD4+ T cell peptides (VP11/12129–143 and VP11/12483–497) or with a mixture of two subdominant human CD4+ T cell peptides (VP11/12107-121 and VP11/12603-617) emulsified in CpG1826 adjuvant (100 μg of each peptide plus 2 μg of CpG1826). The CpG1826 adjuvant alone was used as control (mock vaccination). Two weeks after the final immunization, all animals were challenged ocularly with 2 × 105 PFU of HSV-1 (strain McKrae). (B) Shown is the average eye disease in peptide-immunized and mock-immunized mice 30 days postchallenge scored after the infection. (C) Virus titrations were determined from eye swabs on day 7 postinfection (the peak of viral replication). (D) Survival was determined in a window of 30 days postchallenge. (E) Representative images of ocular disease in HLA-DR mice immunized with the immunodominant and the subdominant peptide epitopes and in mock-immunized control mice. The functions of HSV-specific CD4+ T cells were evaluated in the cornea from all three groups. (F) Representative FACS dot plots (left) and average (right) frequencies of IFN-γ+CD4+ T cells in the cornea of immunized and mock-immunized HLA-DR mice. (G) Representative FACS plots (left) and average (right) frequencies of CD107a/b+ CD4+ T cells in the cornea of immunized and mock-immunized HLA-DR mice. The P values (*, P < 0.05) indicate statistical significance between results for the mice immunized with immunodominant peptide epitopes and those of the mock control groups. The results are representative of two independent experiments.
Altogether, these in vivo results (i) confirm that the immunodominant VP11/12129–143 and VP11/12483–497 CD4+ T cell epitopes are targeted by protective T cells and (ii) demonstrate that immunization with the two immunodominant epitopes decreased ocular herpes infection and disease.
DISCUSSION
Evidence in both animal models and humans indicates that CD4+ T cell-mediated protective immunity is, directly or indirectly, related to the control of HSV infection (29–32). Mice with decreased CD4+ T cell counts are prone to developing severe herpetic diseases (31). CD4+ T helper cells play a major role in the development and maintenance of CD8+ T cells and antibody (Ab)-mediated protective responses (33). CD4+ T cells elicit various helper functions like priming and promotion of cytotoxic T lymphocytes and maintenance of effector memory CD8+ T cells (34). Moreover, the CD4+ T cells also help in the functioning and differentiation of B cells into plasmocytes that produce virus neutralizing antibodies (35, 36). However, in the past 2 decades, more attention has been given toward mapping and characterization of herpes CD8+ T cell epitopes, with only a few studies focusing on mapping and characterization of herpes CD4+ T cell epitopes. In the present study, we identified two previously unknown immunodominant VP11/12 CD4+ TEM cell epitopes, VP11/12129–143 and VP11/12483–497, using well-structured in silico, in vitro, and in vivo approaches. Remarkably, these effector VP11/12129–143 and VP11/12483–497 CD4+ T cell epitopes were found to be less targeted by FOXP3+ CD4+ CD25+ Treg cells. Moreover, immunization of HLA-DR transgenic mice with a mixture of the two immunodominant human VP11/12 CD4+ TEM cell epitopes, but not with cryptic epitopes which are major targets of Treg cells, gave protection from ocular herpes infection and disease. To the best of our knowledge, this is the first report that demonstrates the extent of balance between the effector memory CD4+ T cells and FOXP3+ CD4+ CD25+ Treg cells induced by a herpes vaccine, which determines protection from infection and disease.
Over the last 3 decades, clinical trials of herpes subunit vaccines have primarily focused on glycoproteins; results have been inconsistent, or the vaccines have failed to protect against herpes infection and disease (37). In addition to the 12 known herpes glycoproteins that are major targets of virus neutralizing antibodies, the tegument proteins appeared to be major targets of the HSV-specific CD4+ and CD8+ T cells. HSV tegument proteins are released into the cytoplasm during viral entry, making them one of the first viral proteins encountered by an infected cell and, therefore, an immediate target antigen (Ag) to induce host T cells. The HSV-1 virion protein 11/12 (VP11/12) is one of the most abundant tegument proteins and has emerged as an excellent antigen candidate for inducing protective immunity in animal models (38). In addition to their potential implication in a future herpes subunit immunotherapy and immunoprophylactic vaccine strategies, the VP11/12 CD4+ T cell epitopes identified and characterized in the present study would also contribute to a better understanding of the correlates of human CD4+ T cell responses with protection. Theoretically, numerous potential T cell epitopes could be generated from a protein antigen; however, in practice, T cells tend to focus upon just a few immunodominant regions. The VP11/12 CD4+ T cell epitopes identified in this study are confined to three major immunodominant regions of the VP11/12 proteins, confirming our previous reports (39–49).
Eckles et al. have reported that the specificities of T cell responses to class II HLA molecules are heterogeneous, which may be influenced by different peptides occupying the HLA combining site and by the diversity of antigen-specific receptors of CD4+ T cells recognizing the same HLA/peptide complex (50). Furthermore, a correlation between peptide binding to HLA-DR and CD4+ T cell responses to the peptide epitopes in subjects of similar HLA-DR haplotypes has been established (51). Peptide-protein interactions play an essential role in T cell responses, T cell regulation, and signal transduction. It has been found that nearly 40% of the protein-protein interactions are mediated by short peptides; hence, determining the structure of protein-peptide complexes involved in these interactions would be crucial for understanding the molecular mechanism and subsequently modulating the protein-protein interactions for therapeutic purposes in herpes infection. In this context, we performed the protein-peptide docking experiment, which strengthens our data showing in vitro binding capacities of HSV-1 VP11/12-derived epitope peptides for soluble HLA-DR molecules. The highest protein-peptide binding interaction similarities were observed for the VP11/12129–143 and VP11/12483–497 epitopes, an in silico observation which we later confirmed functionally both in vitro and in vivo. Based on these in silico analyses, we screened 10 potential peptide epitopes with high predicted affinity to HLA-DRB1 molecules for in vitro studies. There exist numerous immunological assays, such as the ELISpot assay, IFN-γ intracellular assay, CD107a/b degranulation cytotoxic assay, and CFSE proliferation assay, which have been used to validate the functional significance of computationally predicted CD4+ T cell epitopes (52–54). When used individually, each screen is not sufficient to conclusively identify functional CD4+ T cell epitopes (52). However, combinations of multiple immunological screens provide strong evidence of functional VP11/12129–143 and VP11/12483–497 epitopes, as shown in other studies (54).
While the identification of epitopes is important for the development of prophylactic and therapeutic herpes subunit T cell-based vaccines, the existing approaches may overlook the effects of natural processing on epitope selection. Identification of immunodominant CD4+ T cell epitopes using artificial peptides is challenging because they are often unable to recognize the native epitopes. This would be the result of multiple shortcomings, including the following: (i) the identified epitope may be cryptic; (ii) the artificial epitope is not processed or presented under natural conditions following HSV-1 infection of target cells; (iii) the T cell clone is inadvertently generated against contaminants within the peptide preparation; and/or (iv) the T cell clone is in fact directed to cross-reactive epitopes between viruses within or outside the alpha herpes family, a phenomenon called heterologous immunity (55–57). Thus, identification of an epitope by sole means of in vitro binding to soluble HLA molecules and the in vitro T cell responses does not provide a firm indication as to whether or not the identified epitope will be naturally processed and presented to T cells by target cells following natural HSV-1 infection. In this study, we used a combination of multiple in silico, in vitro, and in vivo immunological screens to confirm that CD4+ T cells specific to two identified epitopes, VP11/12129–143 and VP11/12483–497, specifically recognized native epitopes on HLA-DR-positive autologous dendritic cells when they were infected with HSV-1. We demonstrated in vitro binding capacities of VP11/12129–143 and VP11/12483–497 peptide epitopes to soluble HLA-DR molecules, a result that bolsters our in silico finding of epitope prediction and molecular docking that predicted the same epitopes as high-affinity binders to most common HLA-DRB1 molecules.
In the present study, the immunodominant VP11/12129–143 and VP11/12483–497 CD4+ T cell epitopes identified using the TEPITOPE strategy were confirmed using the PepScan library mapping strategy. Thus, using the traditional yet robust overlapping peptide screening approach to identify any missing epitopes from the entire sequence of a VP11/12 protein antigen, which was otherwise not predicted using computational tools, confirmed the same epitopes. Moreover, we generated CD4+ T cell lines specific to VP11/12129–143 and VP11/12483–497 epitopes and demonstrated that they undergo significant CD107a/b degranulation and produced higher levels of IFN-γ when incubated with HLA-DR-positive autologous target DC infected with HSV-1. Finally, we confirmed these in silico and in vitro results by demonstrating that immunization of HLA-DR transgenic mice with a mixture of the two immunodominant VP11/12129–143 and VP11/12483–497 CD4+ T cell epitopes induced protective multifunctional CD4+ T cells, associated with a decrease in ocular herpes infection and disease.
Over the last 25 years, several types of herpes vaccines have been attempted, eliciting mostly strong systemic antibody responses. However, in clinical trials, they failed to protect therapeutically or prophylactically. The TG and ocular epithelial tissues are the most obvious battlefield sites for the host’s B and T cells to control recurrent HSV-1. In addition to neutralizing antibodies and antiviral effector CD8+ T cells, CD4+ T cells appear to be critical in preventing or aborting virus reactivations from latently infected neurons of TG (designated central neuronal immunity) (49). Studies in the HSV-infected animal model and observations in HSV-1-infected asymptomatic individuals without recurrent herpetic diseases point to the presence of both CD4+ and CD8+ T cells at the healed sites of the epithelia. Antiviral effector CD4+ T cells play a direct role in preventing virus replication either through production of IFN-γ or through cytotoxic activity. Activated CD4+ T cells are also retained in the latently infected TG of mice and humans (49). Effector CD4+ T cells may also limit virus replication in the corneal epithelium (designated peripheral epithelial immunity). CD4+ T helper cells are a source of large amounts of cytokines that promote generation and maintenance of antiviral memory CD8+ T cells, thus indirectly contributing to protection (13).
The mechanisms by which HSV-specific CD4+ T cells gave protection from ocular herpes infection and disease remain to be fully determined. We demonstrated that induction of VP11/12129–143 and VP11/12483–497 epitope-specific CD4+ T cells in the cornea correlated with protection. Carbone’s group has demonstrated that, following skin infection, memory CD4+ T cells migrate between the blood and infected skin, while CD8+ memory cells remain localized in infected tissue (58–60). Therefore, increasing the number of circulating HSV-specific CD4+ T cells in the cornea and even in the TG, where they may contribute to preventing virus reactivation during latency, would be a prerequisite for an effective epitope-based herpes vaccine. We are currently investigating reopening the cornea (peripheral corneal immunity) and latently infected TG (central neuronal immunity) to infiltration by antiviral CD4+ and CD8+ T cells by increasing levels of T cell-attracting CXCL9, CXCL10, or CXCL11. We will accomplish this by inducing expression of these T cell-attracting chemokines locally, either in cornea or in TG, under tissue-specific epithelial and neurotropic promoters. Results from this approach, which we designate peripheral epithelial immunity and central neuronal immunity, respectively, will be the subject of future reports.
Safety concerns, such as autoimmunity, have been associated with systemic injections of monoclonal antibodies (MAbs) to block immune checkpoints. Thus, in addition to systemic injection of blockade MAbs, a direct delivery of single-chain variable-fragment Fc (scFv-Fc) Abs using engendered adeno-associated virus (AAV) vectors may avoid unwanted systemic immune-related adverse events (IRAE) associated with the use of systemically delivered checkpoint inhibitors (61). If local delivery of LAG-3 and PD-1 checkpoint inhibitors directly into cornea and TG causes uncontrolled local inflammation, we are currently considering using an inducible tissue-specific promoter that will be turned on/off (62) or selectively stimulated by reactivated HSV-1 locally within TG (63). Moreover, the timing of delivery and dosage of blockade MAbs that would result in clinical efficacy with little to no side effects are being considered, and the results will be the subject of future reports.
In conclusion, our findings report two, previously unknown epitopes derived from the HSV-1 VP11/12 protein that tend to recall polyfunctional effector memory CD4+ TEM cell responses in HSV-seropositive asymptomatic individuals. The results show quantitative and qualitative features of an effective HSV-specific CD4+ T cell response that should be considered in the next generation of herpes vaccine studies.
MATERIALS AND METHODS
Human study population.All clinical investigations in this study were conducted according to the Declaration of Helsinki. All subjects were enrolled at the University of California, Irvine, under an approved Institutional Review Board protocol (IRB 2009-6963). Written informed consent was received from all participants prior to inclusion in the study. During the last 15 years (i.e., January 2003 to September 2019), we screened 925 asymptomatic individuals for HSV-1 and HSV-2 seropositivity. A total of 587 were White, 338 were non-White (African, Asian, Hispanic, and other), 458 were female, and 467 were male. Among these participants, a cohort of 726 immuno-competent asymptomatic individuals, ranging from 21 to 67 years old (median age, 39 years), were seropositive for HSV-1 and seronegative for HSV-2. All patients were negative for HIV and hepatitis B virus (HBV), with no history of immunodeficiency. A total of 792 patients were HSV-1, HSV-2, or HSV-1/HSV-2 seropositive, among which 698 patients were healthy and defined as asymptomatic. Based on self-reporting and clinical examination, these patients never had any herpes disease (ocular, genital, or dermal). Even a single episode of any herpetic disease excluded the individual from this group. No attempt was made to assign specific T cell epitopes to the severity of recurrent lesions. Patients were also excluded if they (i) had an active ocular (or elsewhere) herpetic lesion or had had one within the past 30 days, (ii) were seropositive for HSV-2, (3) were pregnant or breastfeeding, or (4) were on acyclovir and other related antiviral drugs or any other immunosuppressive drugs at the time of blood draw. Among this large cohort of healthy seropositive asymptomatic individuals, 50 patients were enrolled in the current study (Table 3).
Cohorts of HLA-DR-positive, HSV-seropositive asymptomatic individuals enrolled in this study
HSV-specific serotyping.The serum samples collected from random donors were tested for anti-HSV antibodies. An enzyme-linked immunosorbent assay (ELISA) was performed on sterile 96-well flat-bottom microplates coated with the HSV-1 antigen in coating buffer overnight at 4°C. The next day, plates were washed with phosphate-buffered saline (PBS)–1% Tween 20 (PBST) five times, and nonspecific binding was blocked by incubation with a 5% solution of skimmed milk in PBS (200 μl/well) at 4°C for 1 h at room temperature (RT). The microplates were washed three times with PBS-Tween and incubated with various sera at 37°C for 2 h. Following five washes, biotinylated rabbit anti-human IgG, diluted 1:20,000 with PBST, was incubated at 37°C for 2 h. After five washes, streptavidin was added at a 1:5,000 dilution and incubated for 30 min at RT. After five additional washes, the color was developed by adding 100 μl of 3,3',5,5'-tetramethylbenzidine (TMB) substrate. The mixture was incubated for 5 to 15 min at RT in the absence of light. The reaction was terminated by adding 1 M H2SO4. Absorbance was measured at 450 nm.
Bioinformatics analyses.HSV-1 VP11/12 open reading frames used in this study were from strain 17 (NCBI accession no. P10230). The 718-aa sequence (Fig. 1) of the VP11/12 protein was screened for (HLA)-DR-restricted epitopes using different computational algorithms, as previously described (26, 27).
TEPITOPE algorithm.The VP11/12 sequence (HSV-1 strain 17) was loaded into the new prediction software (TEPITOPE) to predict CD4+ epitopes (2). The TEPITOPE algorithm is a Windows application that is based on 25 quantitative matrix-based motifs that cover a significant part of human HLA class II peptide binding specificity (2). The algorithm permits the prediction and parallel display of ligands for each of the 25 HLA-DR alleles. The TEPITOPE prediction threshold was set at 5%, and 10 regions, predicted to bind at least 50% of the major histocompatibility complex (MHC) class II molecules, were picked.
PepScan peptide library screening and synthesis.VP11/12 (strain 17; NCBI accession no. P10230) was used in this study. Eighty-nine synthetic peptides encompassing the entire sequence of HSV-1 VP11/12 (718 aa) were produced by using the Multipin DKP system (Mimotopes, San Diego, CA). Figure 4 show examples of the 10 overlapping peptides forming pool 1 to pool 9. Peptides were synthesized as 18- to 22-mers, overlapping by 10 amino acids with adjacent peptides. This synthesis was performed using classical Fmoc/tBu [(9-fluorenylmethoxy carbonyl)/tert-butyl] chemistry. After side-chain deprotection with a mixture of trifluoroacetic acid, ethanedithiol, and anisole (38:1:1), the peptides were cleaved in an ammonium bicarbonate buffer (0.1 M, pH 8.4) with 40% (vol/vol) acetonitrile via a cyclization mechanism, leaving a cyclo(lysylpropyl) moiety at the C terminus. Lastly, the peptides were freeze-dried and stored as a dry powder at −80°C. Working stock solutions of the peptides were made at 1 mg/ml in PBS–10% dimethyl sulfoxide (DMSO) and were stored at −20°C.
HLA-DR typing.The HLA-DR status was confirmed by staining PBMCs with 2 ml of anti-HLA-DR MAb (clone BB7.2; BD Pharmingen) at 4°C for 30 min. The cells were then washed and analyzed by flow cytometry using an LSRII instrument (Becton, Dickinson). The acquired data were analyzed with FlowJo software (BD Biosciences, San Jose, CA).
Molecular peptide docking.Computational peptide docking of CD4 peptides (VP11/12107–121, VP11/12129–143, VP11/12138–152, VP11/12144–158, VP11/12264–278, VP11/12465–479, VP11/12483–497, VP11/12493–507, VP11/12519–533, and VP11/12603–617) into the HLA-DRB1 complex (binding protein) was performed using GalaxyPepDock under GalaxyWEB (64). To retrieve the HLA-DR1 structure, we used the X-ray crystallographic structure of the HLA-DM-HLA-DR1 complex (PDB accession number 4GBX) (65). The 4GBX protein with a structural weight of 92,814.37 Da, possesses five unique protein chains, 811 residues, and 6,130 atoms. In this study, flexible target docking based on an energy optimization algorithm was carried out on the ligand-binding domain containing HLA-DRB1 within the 4GBX structure. Similarly, scores were calculated for protein-peptide interaction pairs for each residue. Subsequently, molecular docking models were built based on distance restraints for protein-peptide pairs using GalaxyPepDock (64). Docking models were ranked based on optimized energy scores.
Peptide binding assays specific for HLA-DR molecules.HLA-DR molecules were immunopurified from homologous Epstein-Barr virus (EBV) cell lines by affinity chromatography using the monomorphic MAbs L243 and B7/21, as previously reported (66–69). The binding to HLA-DR molecules was assessed by competitive ELISA using biotinylated VP11/12 peptides as previously reported (70). Unlabeled forms of nonherpes peptides were used as a reference to assess the validity of each experiment. The sequences of these reference peptides and IC50 values are as follows: hemagglutinin (HA) residues 306 to 318 (PKYVKQNTLKLAT), IC50s of 1 nM for DRB1*0101, 5 nM for DRB1*0401, and 11 nM for DRB1*1101; YKL (AAYAAAKAAALAA), IC50 of 10 nM for DRB1*0701; A3 residues 152 to 166 (EAEQLRAYLDGTGVE), IC50 of 35 nM for DRB1*1501; MT residues 2 to 16 (AKTIAYDEEARRGLE), IC50 of 129 nM for DRB1*0301; B1 residues 21 to 36 (TERVRLVTRHIYNREE), IC50 of 52 nM for DRB1*1301.
PBMCs: preparation and ELISpot assay.Peripheral blood mononuclear cells (PBMCs) were isolated, as we have previously described (25). Approximately 100 ml of the donor’s blood was drawn into yellow-top Vacutainer tubes (Becton, Dickinson, Franklin Lakes, NJ). The serum was isolated and centrifuged for 10 min at 800 × g. The PBMCs were isolated by gradient centrifugation using leukocyte separation medium (Corning, Tewksbury, MA). The cells were then washed in PBS and resuspended in complete culture medium. Aliquots of freshly isolated PBMCs were also cryopreserved in liquid nitrogen for future testing.
T cell stimulation was measured by IFN-γ production in peptide-stimulated PBMCs using a BD ELISpot IFN-γ kit (BD-Pharmingen, San Diego, CA), as we have previously described (25). Briefly, 3 × 105 PBMCs were stimulated for 2 days with 10 μg/ml of pooled peptides (10 peptides per pool), with 10 μg/ml of an individual VP11/12 peptide, with heat-inactivated HSV-1 (strain McKrae) (multiplicity of infection [MOI] of 5), or with 1 μg/ml PHA as a positive control, in ELISpot plates (Merck Millipore, Billerica, MA). These plates were precoated with anti-human IFN-γ capture Ab in a humidified incubator at 37°C with 5% CO2. The spot-forming cells were developed as described by the manufacturer (ELISpot IFN-γ kit; BD-Pharmingen) and counted using an automated ELISpot reader (ImmunoSpot).
T cell activation and proliferation.PBMCs were stained with CFSE (2 mM) and then incubated with individual VP11/12 peptides (10 μg/ml) for 5 days. The cells were then washed and stained for CD4 molecule expression. Cycling cells were then analyzed by flow cytometry, and their absolute numbers were calculated using the following formula: (number of events in double-positive cells) × (number of events in gated lymphocytes)/(number of total events acquired).
Generation of VP11/12 peptide-specific CD4+ T cell lines.PBMC-derived DCs were generated using the cytokines interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) and pulsed with different HSV-1 VP11/12 peptides (10 μg/ml). DC were then stimulated with autologous CD4+ T cells and irradiated autologous feeder cells. Recombinant human IL-2 (100 IU/ml; Peprotech) was added to the culture every fifth day starting on day 5. Fourteen days after initial contact, cells were restimulated using autologous immature DC (imDC) (1:20, DC/CD4+ T cell ratio) pulsed again with the same HSV-1 VP11/12 peptides and irradiated autologous feeder cells (5:1, feeder/CD4+ T cell ratio). Peptide specificities of the CD4+ T cell lines were tested 10 days later, and T cells were first put in coculture with autologous DC and pulsed with the two different HSV-1 peptides (VP11/12129–143 and (VP11/12483–497) or one control peptide (VP11/12107-121) under three separate conditions; the T-cell response was measured by IFN-γ ELISpot assay and intracellular FACS staining.
Generation of autologous target cells.To obtain autologous DC, mononuclear cells isolated from human blood samples were initially plated in serum-free medium (Life Technologies) for 2 h in a six-well plate at an initial density of 1 × 106 cells/ml in a final volume of 3 ml per well. Nonadherent cells were subsequently removed by three successive washes with 1× cold PBS, and the adherent monocytes were then cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS) supplemented with human GM-CSF (50 ng/ml) and IL-4 (5 ng/ml) (PeproTech Inc., Rocky Hill, NJ). After 5 to 6 days, over 90% of the nonadherent cells had acquired typical dendritic morphology. PBMC-derived autologous DC were then infected overnight with HSV-1, each at a multiplicity (MOI) of infection of 0.1 and 1. The next day, infected DC were washed three times and incubated with effector CD4+ T cell lines at an effector/target (E/T) ratio of 1:5 for 4 h. The cytotoxic activity was detected by a CD107a/b degranulation assay, and the T cell response was measured by IFN-γ ELISpot assay and intracellular FACS staining.
T cell assay.To detect cytolytic CD4+ T cells recognizing VP11/12 peptides in freshly isolated human PBMCs and mouse corneal cells, we used a CD107a/b cytotoxicity assay, as we have recently described (25). On the day of the assay, nonstimulated or VP11/12 peptide-stimulated PBMCs were incubated at 37°C for 5 to 6 h with BD GolgiStop, costimulatory Abs anti-CD28 and anti-CD49d (1 μg/ml), and 10 μl of CD107a-fluorescein isothiocyanate (FITC) and CD107b-FITC (clone LAMP; BioLegend, San Diego, CA). At the end of the incubation period, cells were harvested into separate tubes and washed once with FACS buffer and then stained with allophycocyanin (APC)-conjugated anti-human CD4 (clone OKT4) for 30 min. The cells were then rewashed and analyzed using an LSRII instrument (Becton, Dickinson, San Diego, CA). Cells from mouse cornea were also analyzed by flow cytometry after staining with phycoerythrin (PE)-Cy7 fluorochrome-conjugated mouse-specific CD4 MAb (clone GK1.5).
HLA-DR transgenic mice.All animal studies were conducted in facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care and according to an Institutional Animal Care and Use Committee-approved animal protocol (IACUC approval AUP19-111). HLA-DR transgenic mice, previously described (8, 71), were kindly provided by Francois Lemonier (Pasteur Institute, Paris, France) and were bred at the University of California, Irvine.
Virus production.HSV-1 (lab strain McKrae) was grown and titrated on rabbit skin (RS) cells. UV-inactivated HSV-1 was generated, and HSV inactivation was confirmed by the inability to produce plaques when virus was tested on RS cells.
Immunization of HLA-DR mice with VP11/12 peptide epitopes.Three groups of age-matched female HLA-DR mice (n = 10 each) were immunized subcutaneously with the immunodominant CD4+ T cell human epitopes (VP11/12129–143 and VP11/12483–497) emulsified in CpG1826 adjuvant, with the subdominant CD4+ T cell human epitopes (VP11/12107–121 and VP11/12603–617) emulsified in CpG1826 adjuvant or with the CpG1826 adjuvant alone (mock) on days 0 and 21. All immunizations were carried out with 100 μM each peptide. Two weeks after the second and final immunizations, animals from all groups received an ocular HSV-1 challenge (2 × 105 PFU, McKrae strain) without scarification. The mice were then assessed for up to 30 days postchallenge for ocular herpes pathology, ocular viral titers, and survival.
Monitoring of ocular herpes virus infection and disease.Animals were examined for signs of ocular disease by slit lamp. Clinical assessments were made immediately postinoculation and on days 1, 3, 5, 7, 10, 14, and 21 thereafter. The examination was performed by investigators blinded to the treatment regimen of the mice and scored according to a standard scale of 0 to 4: 0, no disease; 1, 25%; 2, 50%; 3, 75%; and 4, 100% staining. To quantify replication and clearance of HSV-1 from the eyes, mice were swabbed daily with moist, type 1 calcium alginate swabs. Swabs were placed in 1.0 ml of titration medium (Dulbecco’s modified Eagle medium [DMEM], 2% penicillin-streptomycin, 5% fetal bovine serum) and frozen at −80°C until titrated on RS cell monolayers. Mice were also examined for survival in a window of 30 days after the challenge.
Statistical analyses.Data for each assay were compared by analysis of variance (ANOVA) and Student's t test using GraphPad Prism, version 5 (La Jolla, CA). Differences between the groups were identified by ANOVA and multiple-comparison procedures, as we previously described (72). Data are expressed as the means ± standard deviations (SD). Results were considered statistically significant at a P value of <0.05.
ACKNOWLEDGMENTS
This work is supported by Public Health Service Research grants EY026103, EY019896, and EY024618 from the National Eye Institute, by grants AI143348, AI147499, AI143326, AI138764, AI124911, and AI110902 from the National Institutes of Allergy and Infectious Diseases (NIAID), and in part by a Discovery Center for Eye Research and Research to Prevent Blindness grant.
This work is dedicated to the memory of the late Steven L. Wechsler (1948–2016), whose numerous pioneering works on herpesvirus infection and immunity laid the foundation for this line of research.
We thank the NIH Tetramer Facility (Emory University, Atlanta, GA) for providing the tetramers used in this study and Angele Nalbandian (Gavin Herbert Eye Institute, University of California Irvine, Irvine, CA) for help editing the manuscript.
We declare that no conflict of interest exists.
FOOTNOTES
- Received 25 November 2019.
- Accepted 2 January 2020.
- Accepted manuscript posted online 8 January 2020.
- Copyright © 2020 American Society for Microbiology.
