Herpes simplex virus (HSV)-specific T-cell immunity is affected by HIV-1 infection leading to more frequent HSV reactivations and shedding.1 In turn, HIV-1/HSV-coinfected individuals have a lower absolute CD4+ T-cell count and an increased HIV-1 RNA load than HIV-1-infected individuals without HSV infection.2,3 The exact immune mechanisms involved in this HIV-1/HSV synergy are unknown.
Some clinical trials showed that HSV suppressive drug therapies indirectly contribute in reducing HIV-1 RNA in plasma and genital tract.4-7 This finding supports the concept that HSV infection enhances HIV-1 replication and hence HIV-1 transmission or HIV-1 disease progression.8 Additionally to current HSV suppressive drug therapies observations, we hypothesize that immune responses to HSV antigens, such as glycoprotein D (gD), might indirectly affect HIV-1 replication and HIV-1 disease progression. Therefore, efficient immunotherapeutic strategies that boost HSV-specific immunity and limit HSV reactivations might also reduce HIV-1 transmission and disease progression.9-12 Although vaccines would represent an ideal tool to control HSV infections and reactivations by inducing a strong cell-mediated immune response, with potentially benefits for HIV-1 control, effectiveness of prophylactic or therapeutic vaccine against HSV has not been substantially approved in humans for the moment. However, the development of efficient immunotherapeutic strategies against these 2 sexually transmitted pathogens is crucial and relies on a better knowledge of the adaptive immune correlates of HIV-1 and HSV infections.5,6,13
The aim of this study is to investigate the impact of HIV-1 infection on the HSV-specific T-cell immunity by comparing the T-cell responses to a panel of HSV-1 gD peptide epitopes between HIV-1/HSV-coinfected and HIV-1-uninfected/HSV-infected individuals. We particularly focused on the Th1, Th2, and Th17 cytokine production and the CCR5 ligand expression because of the potential involvement of these immune effectors in the HIV-1/HSV immunosynergy.8,10,11
A total of 20 HIV-1/HSV-coinfected and 12 HIV-1-uninfected/HSV-infected individuals were enrolled in this study after written informed consent was obtained in compliance with our institutional ethical committee. HIV-1-infected individuals were treated with antiretroviral therapy (ART) for at least 12 months, had a median CD4+ T-cell count of 425 cells per cubic millimeter [interquartile range (IQR): 274-569], and a median plasma viral load of 20 copies per milliliter (IQR: <20-179). Sera were collected from all individuals and tested for HSV-1 and HSV-2 status using the HerpeSelect IgG1 and IgG2 ELISA kits (Eurobio, Les Ullis, France). Individuals were considered positive when index values were greater than 1.10 and negative when lesser than 0.90 according to the manufacturer's instructions. No samples were considered as equivocal with an index value between 0.90 and 1.10. The clinical characteristics of the participants are detailed in Table 1. All of the individuals included in this study have a controlled chronic HSV infection and were negative for HSV IgM antibodies (data not shown).
Isolation of Peripheral Blood Mononuclear Cells
Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of individuals, as previously described.13,14 Briefly, approximately 30 mL of blood was drawn into EDTA Vacutainer tubes (BD Biosciences, Le Pont de Claix, France). The PBMC were isolated by gradient centrifugation at 1200g for 20 minutes using leukocyte separation medium (Eurobio). The cells were washed in phosphate-buffered saline plus 5% of fetal calf serum and resuspended in 1 mL complete culture medium consisting of RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin, (all reagents from Eurobio).
Enumeration of HSV-Specific Interferon-γ-Producing Memory T Cells
T cells stimulation by HSV peptide epitopes was measured by interferon gamma (IFN-γ) production as previously described14 and using an IFN-γ ELISpot kit (Diaclone, Besancon, France). Briefly, 105 PBMC were stimulated with individual gD peptide epitopes (20 μM each) or with 1 μg/mL phytohemagglutinin (PHA) for 6 days at 37°C with 5% CO2. Cells in culture medium without HSV gD peptide epitopes were used as negative controls. The HSV gD peptide epitopes consisted of well-characterized CD4+ (n = 12) and CD8+ (n = 9) T-cell peptide epitopes highly conserved between HSV-1 and HSV-2.14-16 These peptide epitopes have been previously selected after screening of the amino acid sequences of HSV-1 gD and using several computational algorithms and immunological assays for the potential use as vaccine candidates eliciting T cell-mediated immunity in humans.14-16 At day 6, PBMC were harvested and restimulated with the respective peptide epitopes or PHA for 24 hours at 37°C with 5% CO2 on ELISpot plates (Millipore, Molsheim, France) that were precoated with antihuman IFN-γ capture antibodies. The number of IFN-γ-secreting cells (ISC) was normalized per 106 PBMC. The response to each peptide epitope was considered as positive if the number of ISC was (1) greater than twice the response without antigen stimulation, after deduction of the background level with unstimulated cells, and (2) equal or superior to the mean plus 2 SD of the responses observed in healthy HIV-1/HSV-uninfected controls. The intra-assay coefficient of variation for each peptide was defined as: (standard deviation of duplicate wells/mean value of individual assays) × 100. The median (IQR) coefficient of variation was 57.8% (44.2-87.5).
Evaluation of the Cytokine Profile in PBMC Culture
After the 7-day expansion of PBMC with HSV peptide epitopes stimulation (20 μM each, 100,000 PBMC/100 μL), supernatants were collected from the ELISpot experiments and stored at −20°C. Then, the secreted cytokines and chemokines were quantified using a multiplex microbeads assay (FIDIS human cytokine twenty-five-plex kit, MLX-Booster program, Marne-la-Vallée, France) and a Luminex 100 apparatus (Luminex, Oosterhout, The Netherlands) according to the manufacturer's instructions. The cytokine secretion analysis was performed on 18 HIV-1/HSV-coinfected and 11 HIV-1-uninfected/HSV-infected individuals who were also previously tested for the IFN-γ ELISpot assay. Data were analyzed using the MLX-Booster program, and standard curves were established to determine concentrations. Mean concentrations (pg/mL) of cytokines and chemokines were all superior to the detection limits, defined as the mean background value plus 2 SD.
Mean values ± standard errors of the mean (SEM) were compared using the nonparametric Mann-Whitney U test (P < 0.05 was considered significant). The nonparametric Spearman rank correlation coefficient was used to evaluate linear associations.
The IFN-γ Production is Impaired in HIV-1/HSV-Coinfected Individuals
Each CD4+ and CD8+ HSV peptide epitopes was recognized by T cells from 10.0% to 40.0% of HIV-1/HSV-coinfected individuals and from 16.7% to 58.3% of HIV-1-uninfected/HSV-infected individuals (see Table, Supplemental Digital Content 1, http://links.lww.com/QAI/A184). The most frequently recognized T-cell peptide epitopes among HSV-infected individuals were CD4+ gD121-152, CD8+ gD53-61, CD8+ gD95-103, and CD8+ gD278-286 (43,8% of individuals overall). A significantly lower rate of HIV-1/HSV-coinfected individuals than HIV-1-uninfected/HSV-infected individuals had positive IFN-γ responses after stimulation with both the CD4+ (P = 0.014) and CD8+ peptide epitopes (P = 0.029). Breadth of the response was defined as the number of HSV peptide epitopes recognized by each individual. Overall, there is no statistically significant differences in breadth of the response between HIV-1/HSV-coinfected individuals (mean = 4, n = 20) and HIV-1-uninfected/HSV-infected individuals (mean = 8, n = 12 and P = 0.18) (see Table, Supplemental Digital Content 1, http://links.lww.com/QAI/A184).
Mean value (± SEM) of the different ISC means observed in Fig.1 was significantly lower in HIV-1/HSV-coinfected individuals than in HIV-1-uninfected/HSV-infected individuals, after stimulation with either CD4+ peptide epitopes (70 ISC ± 10 versus 280 ISC ± 25/106 PBMC, P < 0.001) or CD8+ peptide epitopes (60 ISC ± 8 versus 234 ISC ± 23/106 PBMC, P < 0.001), whereas it remains unchanged in HIV-1/HSV-coinfected individuals (1465 ± 462 ISC/106 PBMC) and HIV-1-uninfected/HSV-infected individuals (1478 ± 244 ISC/106 PBMC, P > 0.73) following stimulation with PHA (Fig. 1). ISC results presented in Fig. 1 were derived from those defined as positive in the supplemental table (see Supplemental Digital Content 1, http://links.lww.com/QAI/A184).
Assessing the effect of immunosuppression on the capacity of T cells to produce IFN-γ after stimulation with synthetic HSV peptide epitopes, we did not find any statistical correlation between the frequency of ISC among responders after stimulation with the CD4+ or the CD8+ HSV peptide epitopes, and the CD4+ T-cell count in HIV-1/HSV-coinfected individuals (ρ = 0.15, P = 0.55 and ρ = 0.13, P = 0.63, respectively) (Fig. 2).
HIV-1 Modulates Cytokine and Chemokine Production by T Lymphocytes in Response to HSV Peptide Epitope Stimulation
We quantified the secretion of cytokines (Fig. 3A) and chemokines (Fig. 3B) after PBMC stimulation with immunodominant CD4+ gD228-258 and CD8+ gD53-61 T-cell peptide epitopes. These 2 peptides epitopes were selected for their ability to induce distinctive IFN-γ response in HIV-1/HSV-coinfected individuals and HIV-1-uninfected/HSV-infected individuals (Fig. 1). As suggested above, we observed that the amount of IFN-γ was 2.2-fold lower (P = 0.02) after stimulation with the CD4+ HSV gD228-258 peptide epitope and 13.9-fold lower after stimulation with the CD8+ HSV gD53-61 peptide epitope (P = 0.001) in T-cell cultures from HIV-1/HSV-coinfected individuals than in T-cell cultures from HIV-1-uninfected/HSV-infected individuals (Fig. 3A). In addition, the secretion of the IFN-γ-inducible protein 10 kDa (IP-10) was also lower in T-cell cultures from HIV-1/HSV-coinfected individuals than in T-cell cultures from HIV-1-uninfected/HSV-infected individuals (123-fold reduction for the CD4+ peptide epitope, P < 0.0001 and 29.1-fold reduction for the CD8+ peptide epitope, P < 0.0001) (Fig. 3B). In contrast, cells from HIV-1/HSV-coinfected individuals produced higher amount of interleukin (IL)-6 (1.6-fold, P = 0.003) compared with T cells from HIV-1-uninfected individuals after stimulation with the CD4+ gD228-258 peptide epitope and IL-4 (1.2-fold, P < 0.001), IL-5 (1.2-fold, P < 0.001), IL-6 (3.3-fold, P = 0.002), IL-12p70 (2.0-fold, P = 0.002), IL-17 (1.8-fold, P < 0.001), TNF-α (3.1-fold, P < 0.001), GM-CSF (1.4-fold, P = 0.001), and IFN-α (1.8-fold, P = 0.001) after stimulation with the CD8+ gD53-61 peptide epitope (Fig. 3A). Despite levels of the Th2 cytokines, IL-4 (1.2 fold, P < 0.001) and IL-5 (1.2 fold, P < 0.001) were also significantly increased in the coinfected group after stimulation with the CD8+ gD53-61 peptide epitope, they tend to be decreased after stimulation with the CD4+ peptide epitope (6.8-fold, P = 0.67 and 3.1-fold, P = 0.60, respectively) so as IL-13 (31.0-fold, P = 0.91), another Th2 cytokine. Furthermore, IL-4 and IL-5 secretion remained in a very low range of concentration (<10 pg/mL) (Fig. 3A) suggesting that results concerning Th2 cytokines are unclear and need further investigations.
In HIV-1/HSV-coinfected individuals, a significant reduction in the secretion of soluble CCR5 ligands [i.e. macrophage inflammatory protein (MIP)-1α (12.5-fold and 8.1-fold, P = 0.004 and P = 0.006), MIP-1β (3.7-fold and 6.3-fold, P = 0.005 and P = 0.003), and RANTES (9.9-fold and 2.9-fold, P < 0.001 and P = 0.01)], was observed in response to HSV peptide epitopes (gD228-258 and gD53-61, respectively) (Fig. 3B). Monokine induced by IFN-γ (MIG) was also significantly reduced after stimulation with the CD4+ gD228-258 (71.5-fold, P = 0.005) and the CD8+ gD53-61 peptide epitope (6.9 fold, P = 0.03), whereas monocyte chemotactic protein 1 (MCP-1) was reduced after stimulation with the CD8+ gD53-61 peptide epitope (4.6-fold, P = 0.002) (Fig. 3B). Therefore, after stimulation with the CD8+ gD53-61 epitope, we observed an increased production of Th17 cytokines (defined as IL-6 and IL-17—in green) with a concomitant decrease in the secretion of Th1 cytokines (defined as IL-2, IFN-γ, IP-10 and MIG—in red) and CCR5 ligands (defined as MIP-1α, MIP-1β, RANTES, and Eotaxin—in yellow) in the HIV-1/HSV-coinfected group suggesting a favorable environment for viral replication (Fig. 3C). We also found that IL-8 was the predominant cytokine produced after restimulation of anti-HSV memory T cells with the CD8+ gD53-61 epitope in the HIV-1/HSV-coinfected group, whereas it was MCP-1 in the HIV-1-uninfected/HIV-1-infected group (Fig. 3C). A drastic decrease of MCP-1 (mean value = 669 ± 124 pg/mL) was observed in HIV-1/HSV-coinfected individuals compared with HIV-1-uninfected individuals (mean value = 9948 ± 3612 pg/mL, P = 0.002) after stimulation with the HSV CD8+ gD53-61 epitope.
In addition, a correlation was observed between the secretion of MIP-1α and MIP-1β (both P < 0.001), MIP-1α and RANTES (P < 0.001 and P = 0.004, respectively), and MIP-1β and RANTES (P < 0.001 and P = 0.005, respectively) in HIV-1/HSV-coinfected individuals in response to either gD228-258 or gD53-61 epitope stimulation (Fig. 4A). There was also a correlation between the mean concentration of Th1-related molecules (IL-2, IFN-γ, MIG, and IP-10) and CCR5 ligands (RANTES, MIP-1α, MIP-1β and Eotaxin) in response to either CD4+ (ρ = 0.56, P = 0.02) or CD8+ peptide epitopes (ρ = 0.52, P = 0.03) (Fig. 4B). In contrast, no statistical correlation was observed between the mean concentration of Th2-related cytokines (IL-4, IL-5, IL-10, and IL-13) and CCR5 ligands after stimulation with CD4+ gD228-258 epitope (ρ = 0.30, P = 0.22) or CD8+ gD53-61 epitope (ρ = 0.14, P = 0.36) (Fig. 4C).
To investigate the immunosynergy between HIV-1 and HSV, 2 sexually transmitted pathogens, the present study addresses how HIV-1 infection affects the HSV-specific CD4+ and CD8+ T-cell response after in vitro restimulation with HSV peptide epitopes. Generally, the majority of HSV gD peptide epitopes elicits a specific T-cell response, but here we observed an impairment of T-cell immunity directed to HSV in HIV-1-infected individuals, both qualitatively and quantitatively. The lower number of cells secreting IFN-γ in response to HSV peptide epitopes in HIV-1/HSV-coinfected individuals were observed by ELISpot assay at a single cell level and confirmed by quantification in cell supernatant. This suggests that HIV-1 limits anti-HSV immunity and therefore, facilitates HSV replication and/or reactivations (see Figure, Supplemental Digital Content 2, http://links.lww.com/QAI/A185). CD4+ T lymphocytes secreting IFN-γ play a key role in the control of herpes virus reactivations as previously observed for HSV, cytomegalovirus, and Epstein-Barr virus.17 In addition, ART administered to HIV-1/HSV-coinfected individuals enables the restoration of the HSV-specific IFN-γ responses and then leads to improved control of HSV disease.18 This suggests that the CD4+ T-cell count in HIV-1-infected individuals is likely associated to the enhanced HSV-specific immune response. Our results do not fit with those data, as we have observed no statistical correlation between the CD4+ T lymphocytes count and the HSV-specific IFN-γ response. The main reason of this discrepancy likely originates in the differences between the populations studied. For example, Ramaswamy et al18 showed that the improve of HSV response was related to the CD4+ T-cell recovery in 9 individuals with a median CD4+ T-cell count of only 208 cells per cubic millimeter at baseline. By contrast, we have explored patients on ART for at least 12 months and with a higher median CD4+ T-cell count (ie, 425 cells/mm3).
The Th1 response is pivotal in the protective immunity against HSV reactivation.19,20 Overall, the levels of Th1 cytokine secretion in HIV-1-infected individuals is reduced after in vitro stimulation with gD peptide epitopes by comparison with HIV-1-uninfected individuals. The dysfunction in the HSV-specific immune response due to HIV-1 infection facilitates HSV reactivations that may induce the CD4+ T-cell activation.21 CD4+ T-cell activation is required to trigger the HIV-1 replication cycle, then the facilitation of HSV reactivation may increase, in turn, HIV-1 replication (see Figure, Supplemental Digital Content 3, http://links.lww.com/QAI/A186). In addition, our results may potentially support the statement that, beside targeting CD4+ T cells, HIV-1 infection might also switch the Th1/Th2 cytokine balance toward a less protective Th2 profile.22 The Th2-type cytokine environment in response to HSV reactivation would restrain the circulating CD8+ cytotoxic T lymphocytes responses against HIV-123 and limit the capacity of proliferative responses to p24 essential for the HSV immune response to p24.24,25 Nevertheless, the Th2 cytokine results obtained in our study are not convincing as levels of secretion in this range of concentration can lack reproducibility and may only reflect assay variability. These arguments suggest that further substantiation would lend stronger support to the interpretation of the data.
We also observed a higher secretion of IL-17 in response to the CD8+ HSV gD53-61 epitope in the HIV-1-infected group. These findings should be further confirmed but are consistent with previous reports of an association between the IL-17 response and HIV-126 or HSV infection.27 The low IL-13 production observed in HIV-1-infected individuals, unlike other Th2 cytokines, and the increase of IL-6 cytokine are other results suggesting a switch of the HSV-specific T-cell response toward a Th17 type: IL-13 has a direct antagonistic effect on Th17 cells, mainly by inhibiting the production of IL-23 and IL-6, both involved in Th17 cell polarization.28 The decreased IFN-γ secretion, and the overall reduction of Th1 cytokines in HIV-1/HSV-coinfected individuals, may also favor Th17 cell development as IFN-γ inhibits Th17 cell differentiation from naive precursors.29 During HIV-1 infection, the switch of the HSV-specific Th17 profile suggests an immunoevasive strategy that would facilitate the replication of both HSV and HIV-1. These data emphasize the need of further investigations on Th17 regulation in the case of HIV-1/HSV coinfection and reinforce the hypothesis that the modulation of the Th17 response during vaccination may be beneficial for triggering the recruitment of Th1 cells and restricting pathogen replication.30 As a result of this HIV-1/HSV vicious circle, the subversion of the immune system by HIV-1 seems advantageous for the replication of both viruses.
In addition to the reduction of Th1 cytokine production after HSV epitope stimulation, HIV-1-infected individuals secreted reduced levels of RANTES, MIP-1α, and MIP-1β, the main CCR5 ligands, but also MCP-1, that can compete for binding on CCR5 receptor, so as Eotaxin.31,32 The binding of CCR5 ligands to their specific receptor results in a fast internalization of the receptor-ligand complex, thus depriving the cell surface from a coreceptor that is essential for HIV-1 entry.33 The reduced secretion of CCR5 ligands and its imbalance with CCR5 receptors may therefore increase the availability of CCR5 receptors at the cell surface, thereby promoting HIV-1 replication and accelerating HIV-1 disease progression.34,35 The ineffectiveness of acyclovir therapy in preventing HIV-1 transmission despite reduced frequency of genital ulcers might be due to a persistent genital tissue infiltration by HIV-1 target cells expressing a high CCR5 level.4,11 Likewise, the greater ability of HIV-1 to replicate and spread in a Th2 environment36 may be also related to the limited secretion of CCR5 ligands by Th2 cells.37 In support to these speculation, we observed a statistically significant correlation in the secretion of CCR5 ligands MIP-1α, MIP-1β, and RANTES suggesting their possible synergistic action during HIV-1/HSV coinfection. We also found a correlation between the secretion of CCR5 ligands and the Th1-related cytokines, but not with the Th2-related molecules, which clearly indicates the role of Th1 cytokines in maintaining an elevated level of β-chemokines such as MIP-1α, MIP-1β, and RANTES for reducing HIV-1 accessibility to CCR5 at the surface of CD4+ T cells. Our findings are consistent with a previous report of a CCR5 ligand secretion that correlates with the Th1-like phenotype indicating that CCR5 ligands may be considered as a component of the Th1 response against pathogens. CCR5 ligands can mediate the recruitment of specific effector cells to inflammatory areas thanks to their chemoattractant properties.38 Therefore, the reduction of cellular protective immune response observed in HIV-1/HSV-coinfected individuals contributes to facilitate HIV-1 cell infection through the reduction of CCR5 ligand production. The decreased secretion of Th1 cytokines and CCR5 ligands is possibly due to the suppression of CD4+ T lymphocytes characterizing HIV-1-infected individuals. We showed here a tendency to a reduced proportion of T cells capable to produce IFN-γ after restimulation of HSV-specific memory T cells by CD4+ and CD8+ gD peptide epitopes in HIV-1/HSV-coinfected individuals. Our results are consistent with previous investigations showing that CD4+ T cells are the source of the Th1-related molecule secretion involved in the protective cellular immune response against HIV-1 such as IFN-γ and CCR5 ligands.24,25 The lack of significant correlation observed between the proportion of ISC and the CD4+ T-cell count in coinfected individuals after stimulation with gD peptide epitopes may be mainly due to the current ART that have been taken for several months enhancing rapidly the number of CD4+ T cells in HIV-1-infected individuals. Thus, despite a median CD4+ T-cell recovery of 425 cells per cubic millimeter and a HIV-1 plasma viral load <20 copies per milliliter, the Th1 immune response remains lower in HIV-1/HSV-coinfected individuals by contrast to the HIV-1-uninfected group highlighting an immune scar due to HIV-1 infection that contributes to the suboptimal HSV-specific immune response in coinfected participants. The reduced levels of IFN-γ, IP-10, and CCR5 ligands observed in the coinfected group by comparison to the HIV-uninfected group may be view as other evidence of the lower number of HSV-specific Th1 response observed by ELISpot. By contrast, the Th2 cytokines secretion seems similar in both groups or moderately elevated in HIV-1-infected individuals, suggesting that the Th1 response against HSV is preferentially impaired in the latter population. Also, we cannot strictly rule out that cells producing cytokines after HSV peptide stimulation may not be HSV-specific T cells. Nevertheless, the differences of cytokine and chemokine concentration observed after cell stimulation with HSV peptides by contrast to unstimulated control wells strongly suggest that most of these soluble factors are specifically secreted in response to antigens stimulation and unlikely resulting of bystander activation.
In conclusion, our results indicate that HSV-specific T-cell response is impaired in HIV-1/HSV-coinfected individuals, which probably facilitates HSV reactivations and consequently CD4+ T cells activation (see Figure, Supplemental Digital Content 3, http://links.lww.com/QAI/A186). This viral interaction may involve a decline of HSV-specific ISC, a downregulation of other Th1 cytokines and CCR5 ligands secretion that, in turn, promotes HIV-1 replication. Successful HSV therapeutic strategies, such as therapeutic vaccines, should not only reduce HSV reactivations but also target the HIV-1/HSV immunosynergy to significantly reduce HIV-1 transmission and disease progression.
We would like to thank all the patients who provided blood specimens, Cara-Chan Manville for her careful review of this article, and Sophie Bendriss who provided technical assistance. We are also grateful to the French Agency for AIDS Research (ANRS, director: Jean-François Delfraissy) and especially to Professor Yves Lévy (who is in charge of the ANRS vaccine research program) for his support and constant encouragements.
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