Scientists learn why even treated genital herpes
sores boost the risk of HIV infection
New research helps explain why infection with herpes simplex virus-2 (HSV-2), which causes genital herpes, increases the risk for HIV infection even after successful treatment heals the genital skin sores and breaks that often result from HSV-2.
Scientists have uncovered details of an immune-cell environment conducive to HIV infection that persists at the location of HSV-2 genital skin lesions long after they have been treated with oral doses of the drug acyclovir and have healed and the skin appears normal. These findings are published in the advance online edition of Nature Medicine on Aug. 2.
Led by Lawrence Corey, M.D., and Jia Zhu, Ph.D., of the Fred Hutchinson Cancer Research Center and Anna Wald, M.D., M.P.H., of the University of Washington, both in Seattle, the study was funded mainly by the National Institute of Allergy and Infectious Diseases (NIAID) with support from the Eunice Kennedy Shriver National Institute of Child Health and Human Development, both part of the National Institutes of Health.
"The findings of this study mark an important step toward understanding why HSV-2 infection increases the risk of acquiring HIV and why acyclovir treatment does not reduce that risk," says NIAID Director Anthony S. Fauci, M.D. "Understanding that even treated HSV-2 infections provide a cellular environment conducive to HIV infection suggests new directions for HIV prevention research, including more powerful anti-HSV therapies and ideally an HSV-2 vaccine."
One of the most common sexually transmitted infections worldwide, HSV-2 is associated with a two- to three-fold increased risk for HIV infection. Some HSV-2-infected people have recurring sores and breaks in genital skin, and it has been hypothesized that these lesions account for the higher risk of HIV acquisition. However, recent clinical trials, including an NIAID-funded study completed last year, demonstrated that successful treatment of such genital herpes lesions with the drug acyclovir does not reduce the risk of HIV infection posed by HSV-2 (http://www3.niaid.nih.gov/news/newsreleases/2008/hptn039.htm). The current study sought to understand why this is so and to test an alternative theory.
"We hypothesized that sores and breaks in the skin from HSV-2 are associated with a long-lasting immune response at those locations, and that the response consists of an influx of cells that are a perfect storm for HIV infection," says Dr. Corey, co-director of the Vaccine and Infectious Diseases Institute at The Hutchinson Center and head of the Virology Division in the Department of Laboratory Medicine at the University of Washington. "We believe HIV gains access to these cells mainly through microscopic breaks in the skin that occur during sex."
The research team took biopsies of genital skin tissue from eight HIV-negative men and women who were infected with HSV-2. These biopsies were taken at multiple time points: when the patients had genital herpes sores and breaks in the skin, when these lesions had healed, and at two, four and eight weeks after healing. The researchers also took biopsies from four of the patients when herpes lesions reappeared and the patients underwent treatment with oral acyclovir. The scientists continued to take biopsies at regular intervals for 20 weeks after the lesions had healed. For comparison, the investigators also took biopsies from genital tissue that did not have herpes lesions from the same patients.
Previous research has demonstrated that immune cells involved in the body's response to infection remain at the site of genital herpes lesions even after they have healed. The scientists conducting the current study made several important findings about the nature of these immune cells. First, they found that CD4+ T cells-the cells that HIV primarily infects-populate tissue at the sites of healed genital HSV-2 lesions at concentrations 2 to 37 times greater than in unaffected genital skin. Treatment with acyclovir did not reduce this long-lasting, high concentration of HSV-2-specific CD4+ T cells at the sites of healed herpes lesions.
Second, the scientists discovered that a significant proportion of these CD4+ T cells carried CCR5 or CXCR4, the cell-surface proteins that HIV uses (in addition to CD4) to enter cells. The percentage of CD4+ T cells expressing CCR5 during acute HSV-2 infection and after healing of genital sores was twice as high in biopsies from the sites of these sores as from unaffected control skin. Moreover, the level of CCR5 expression in CD4+ T cells at the sites of healed genital herpes lesions was similar for patients who had been treated with acyclovir as for those who had not.
Third, the scientists found a significantly higher concentration of immune cells called dendritic cells with the surface protein called DC-SIGN at the sites of healed genital herpes lesions than in control tissue, whether or not the patient was treated with acyclovir. Dendritic cells with DC-SIGN ferry HIV particles to CD4+ T cells, which the virus infects. The DC-SIGN cells often were near CD4+ T cells at the sites of healed lesions-an ideal scenario for the rapid spread of HIV infection.
Finally, using biopsies from two study participants, the scientists found laboratory evidence that HIV replicates three to five times as quickly in cultured tissue from the sites of healed HSV-2 lesions than in cultured tissue from control sites.
All four of these findings help explain why people infected with HSV-2 are at greater risk of acquiring HIV than people who are not infected with HSV-2, even after successful acyclovir treatment of genital lesions.
"HSV-2 infection provides a wide surface area and long duration of time for allowing HIV access to more target cells, providing a greater chance for the initial 'spark' of infection," the authors write. This spark likely ignites once HIV penetrates tiny breaks in genital skin that commonly occur during sex. "Additionally," the authors continue, "the close proximity to DC-SIGN-expressing DCs [dendritic cells] is likely to fuel these embers and provide a mechanism for more efficient localized spread of initial infection." The investigators conclude that reducing the HSV-2-associated risk of HIV infection will require diminishing or eliminating the long-lived immune-cell environment created by HSV-2 infection in the genital tract, ideally through an HSV vaccine. Further, they hypothesize that other sexually transmitted infections (STIs) may create similar cellular environments conducive to HIV infection, explaining why STIs in general are a risk factor for acquiring HIV.
Published online: 2 August 2009 | doi:10.1038/nm.2006
Persistence of HIV-1 receptor-positive cells after HSV-2 reactivation is a potential mechanism for increased HIV-1 acquisition
Jia Zhu1,2, Florian Hladik1,3,4,6, Amanda Woodward1,3,6, Alexis Klock1,2, Tao Peng1,2, Christine Johnston1,3, Michael Remington2, Amalia Magaret1,2, David M Koelle1,2,3, Anna Wald1,2,3,5 & Lawrence Corey1,2,3,5
To explore the mechanism by which herpes simplex virus (HSV)-2 infection is related to HIV-1 acquisition, we conducted in situ analysis of the cellular infiltrate from sequential biopsies of HSV-2 lesions from patients on and off antiviral therapy. CD4+ and CD8+ T cells and a mixed population of plasmacytoid and myeloid dendritic cells (DCs), including cells expressing the C-type lectin receptor DC-SIGN, persisted at sites of HSV-2 reactivation for months after healing, even with daily antiviral therapy. The CD4+ T cells that persisted reacted to HSV-2 antigen, were enriched for expression of the chemokine receptor CCR5, and were contiguous to DCs expressing the interleukin-3 receptor CD123 or DC-SIGN. Ex vivo infection with a CCR5-tropic strain of HIV-1 revealed greater concentrations of integrated HIV-1 DNA in cells derived from healed genital lesion biopsies than in cells from control skin biopsies. The persistence and enrichment of HIV receptor-positive inflammatory cells in the genitalia help explain the inability of anti-HSV-2 therapy to reduce HIV acquisition.
Both incident and prevalent HSV-2 infections are associated with an increased risk of HIV acquisition1, 2, presumably owing to frequent infectious HSV-2 ulcerations and the associated influx of activated CD4+ T cells3 that provide HIV easier access to large numbers of potential target cells. Antiviral drugs, such as acyclovir, reduce the frequency of both clinical and subclinical herpetic ulcerations by 75-85% (refs. 4,5,6), findings that provided the impetus for conducting two large-scale international trials to determine whether daily suppressive anti-HSV-2 therapy reduces HIV acquisition. Despite significant reductions in HSV-2 genital ulcer disease, a reduction in HIV acquisition was not observed in either study7, 8. The biological explanation for these findings is unknown.
In previous studies, we showed that HSV-2-specific CD8+ T cells persist in a very localized area at the dermal-epidermal junction after lesion healing, suggesting that localized inflammatory cells persist in genital skin despite healing of the epithelial surface and the appearance of a clinically normal epithelium9. We undertook a detailed in situ study of sequential biopsies of genital skin to characterize whether HIV receptor-positive cells persist in genital skin after HSV reactivation and whether this would be affected by antiviral therapy for HSV-2.
Localized persistence of CD4+ T cells in genital skin
We studied eight healthy, HIV-negative subjects (seven women, one man) with culture-proven recurrent symptomatic genital HSV-2 infection. Subjects underwent 3-mm punch biopsies at the time of a clinically symptomatic ulcerative lesion; at the time of lesion resolution (7-10 d later); and at 2, 4 and 8 weeks after healing. Four of the eight subjects then participated in the second phase of the study, in which 400 mg of acyclovir twice daily was initiated at the start of an acute episode and continued for 20 consecutive weeks. This was the dose used in both of the clinical trials that evaluated whether HSV-2 therapy could reduce HIV-1 acquisition7, 8. During the acyclovir phase, we took biopsies from the lesion site during the acute lesion; at lesion healing; and at 2, 4, 8, 12, 16 and 20 weeks after healing (Table 1)10. In both phases of the study, we collected biopsies from the unaffected contralateral genital area at the same time points. We also obtained peripheral blood mononuclear cells (PBMCs) at enrollment and at 4- to 8-week intervals.
During an acute HSV-2 lesion when HSV-2 antigen was present, CD4+ and CD8+ T cells infiltrated the epidermis and dermis at the ulcer site (Fig. 1a,b). The acute lesion was associated with a massive localized infiltration of cells. The mean numbers of CD4+ and CD8+ cells per mm2 in HSV-infected skin were 655 and 618, respectively, versus 68 and 55 cells per mm2 in uninvolved genital skin (P less than or equal to 0.003 for comparison between HSV-2 lesion biopsies and control biopsies; Fig. 1c,d). Upon healing (day 7), a new epidermal layer replaced the ulcer, and both CD4+ and CD8+ cells were relatively restricted to the upper dermis (Fig. 1a). Follow-up biopsies showed clearance of HSV antigen and gradual reduction of inflammation. However, localized foci of CD4+ and CD8+ T cells persisted for months (Fig. 1c,d) despite complete healing of lesions, normal skin thickness and appearance, and the absence of any clinical evidence of genital herpes. The number of CD4+ and CD8+ cells in lesion-site biopsies obtained 2-8 weeks after healing was significantly higher than in skin biopsies from uninvolved genital skin (Fig. 1c; P less than or equal to 0.001). This enrichment in CD4+ T cells ranged from 2- to 37-fold higher than in contralateral uninvolved genital skin (median, 8-fold). These higher concentrations of CD4 cells in the skin biopsies from HSV-affected versus unaffected areas were statistically significant at all time points after healing (P = 0.01 at week 2 after healing; P = 0.03 at weeks 4-8 after healing).
These persisting CD4+ and CD8+ cells showed distinct spatial distributions (Fig. 1d). CD4+ cells were present in the upper dermis and concentrated perivascularly, whereas CD8+ cells were localized at the dermal-epidermal junction in close contact with basal keratinocytes (Fig. 1d). Simultaneous staining for CD3 and CD4, or CD3 and CD8, revealed that 58 plusminus 6% of the CD4+ cells and 74 plusminus 6% of the CD8+ cells were CD3+ T cells (Fig. 1d).
Acyclovir therapy does not affect CD4+ T cell persistence
We evaluated the persistence of CD4+ and CD8+ T cells in genital skin in serial biopsies from subjects treated with daily acyclovir. Acyclovir therapy reduced the median healing time of the herpetic ulcers (Table 1) and prevented subsequent clinical and subclinical mucosal alternations; none of the lesion-site biopsies taken after healing while participants were on acyclovir had detectable HSV DNA or HSV antigen (n = 24). Similarly, all of the control biopsies were negative for HSV DNA11, 12.
Acyclovir treatment did not significantly alter the spatial distribution or the magnitude of the CD4+ and CD8+ T cell infiltration during either the acute biopsies or those taken after healing. The time course of CD4+ or CD8+ T cell infiltration in skin tissue, from the onset of genital disease to 20 weeks after the lesion completely healed in a representative subject, showed persistence of CD4+ or CD8+ T cells after HSV-2 reactivation, despite the 20 consecutive weeks of acyclovir treatment (Fig. 2a-c). We observed persisting CD4+ or CD8+ T cells in all subjects (Fig. 2d,e). The intensity of the persisting CD4+ T cell infiltration in the dermis at 4-20 weeks after healing on suppressive acyclovir was similar to that seen with no antiviral therapy (Fig. 2e). Even after 20 continuous weeks of acyclovir therapy, the number of CD4+ T cells in the dermis was significantly higher in lesion-site biopsies than in uninvolved genital skin (P = 0.01 at 4-20 weeks after healing; Fig. 2e).
HSV-2 specificity of persisting CD4+ T cells
To evaluate the specificity of the resident CD4+ T cells in genital skin, we isolated the lymphocytes from the biopsies, expanded the lymphocytes in vitro and tested their reactivity to HSV antigen10. Representative experiments from two separate biopsies taken at weeks 2 and 12 after healing, from patients on acyclovir, showed that persisting CD4+ T cells reacted to HSV-2 antigen (Fig. 2c,f). We did not detect HSV DNA in these biopsies, and their histological appearance was normal (data not shown). CD4+ T cells derived from lesion-site biopsies from patients on daily suppressive acyclovir secreted interferon (IFN)-gamma after in vitro stimulation with UV-inactivated HSV-2. We did not find HSV-2-specific CD4+ T cells in control genital skin biopsies from the same subjects, indicating that persisting CD4+ T cells in areas of prior HSV-2 reactivation were enriched for HSV-2 antigen specificity (Fig. 2f). We obtained similar findings from biopsies taken after healing in untreated patients (data not shown).
The mean IFN-gamma response to inactivated HSV-2 antigen in CD4+ T cells obtained from PBMCs from HSV-2-infected individuals was 0.42 plusminus 0.3% (n = 29), 8- to 10-fold lower than was shown in these lesion biopsies, indicating that HSV-2-specific CD4+ T cells were enriched in genital skin compared to circulating PBMCs13.
Enrichment of CCR5+CD4+ T cells persisting in genital skin
The CD4+ T cells that persisted in the genital skin expressed the HIV co-receptors CCR5 and CXCR4 (Fig. 3a). On average, 55% (range, 42-81%) of the CD4+ T cells detected in lesion-site biopsies expressed CCR5 (ref. 14), sixfold higher than levels found among circulating CD3+CD4+ lymphocytes in peripheral blood obtained at the same time (P = 0.04) and twofold higher than levels from uninvolved genital skin biopsies obtained at the same time (P = 0.04; Fig. 3b). The percentage of CD4+ T cells expressing CCR5 in dermal skin during the acute period and after healing was higher in biopsies taken from HSV-infected skin compared to control skin in 14 of the 15 biopsies evaluated (Fig. 3b). The degree of CCR5 expression in dermal CD4+ T cells that persisted after healing of HSV-2 was similar between patients on daily acyclovir (n = 12) and those on no therapy (n = 8; Fig. 3c).
Thus, prolonged acyclovir treatment did not alter the persistence, quantity, spatial distribution or degree of CCR5 enrichment, or the HSV-2 specificity of dermal CD4+ T cells detected at the site of genital herpes infection after lesion healing.
Persistence of DCs and interaction with CD4+ T cells
As DCs have been detected in HSV lesions of mice and humans15, 16, we used a variety of cell surface markers to determine the anatomic distribution, quantity and type of DCs present in human HSV lesions. Genital herpes lesions were associated with a marked infiltration of cells expressing CD123 or DC-SIGN, the HIV-1 receptor capable of transferring HIV-1 to CD4+ T cells. We detected both CD123- and DC-SIGN-expressing cells in all skin biopsies from healed genital lesions (Fig. 4a,b). Notably, both CD123+ and DC-SIGN+ cells persisted at significantly higher concentrations in the dermis of HSV-2-involved skin at all measured time points after healing, compared to control skin (average 3.3- and 5.6-fold, respectively; P = 0.01 and P = 0.035, respectively; Fig. 4a). We observed persistent enrichment in CD123+ or DC-SIGN+ DCs in skin biopsies from HSV-2-infected regions compared to normal control skin even during acyclovir therapy (average 2.0- and 2.9-fold, respectively; P = 0.013 and P = 0.019, respectively; Fig. 4a,b). Furthermore, acyclovir had no statistically significant effect on the persistence of CD123+ or DC-SIGN+ cells over time (P = 0.96 and P = 0.64, respectively).
The persisting CD123+ cells were both CD11c+ and CD11c- and did not express CD14 (Fig. 4c). The CD11c+ cells largely costained with the blood dendritic cell antigen BDCA1 (Fig. 4c). A portion of the CD123+ cells also costained with BDCA2 (Fig. 4c). These data indicate that both plasmacytoid and myeloid DCs persist in the skin after HSV-2 reactivation17, 18, 19, 20. DC-SIGN+ cells tended not to express CD123 or CD11c (Fig. 4c)20, 21. The above data indicate that a large and diverse population of DCs, including plasmacytoid and myeloid DCs and DC-SIGN-expressing cells, persist in the localized areas of previously HSV-2-infected genital skin for weeks after lesion healing, and that this enrichment is not mitigated by treatment with acyclovir.
Because the anatomic distribution of dermal DCs seemed similar to that seen for CD4+ T cells (Figs. 2c,3a and 4b), we more closely investigated the association between CD123+ or DC-SIGN+ DCs with dermal CD4+ T cells (Fig. 5a-c). Dual staining for CD4 or CD3 with CD123 showed direct contact between CD123+ DCs and T cells (Fig. 5a and c, top two rows). We frequently observed contiguous appearance of DCs (CD3-CD4low) and CD4+ T cells (CD3+CD4high) in the upper dermis of skin after healing (Fig. 5c, third row). We consistently found evidence of interaction between DC-SIGN+ cells and CD4+ T cells in the upper dermis of lesion biopsies taken after healing (Fig. 5b and c, fourth row). These interactions were present whether the biopsy was taken on or off acyclovir.
Increased HIV-1 infectivity in healed genital herpes lesion
To determine whether the persistence of the increased target cell environment for CCR5-tropic HIV-1, which we observed in healed herpes lesions, results in enhanced susceptibility to HIV-1 infection, we conducted ex vivo HIV infection of skin biopsies. We placed biopsies from healed genital herpes lesions into organ culture and challenged them ex vivo with the CCR5-tropic HIV-1 JRCSF isolate. Similarly, we obtained biopsies from unaffected control sites on the same day and challenged them in the same experiment. After 2 d in culture, we lysed the cells, extracted the DNA, and measured the amount of integrated HIV-1 DNA by quantitative PCR (normalized to ACTB (beta-actin) DNA)22, 23. This measurement allowed us to evaluate the replicative portion of the HIV-1 life cycle, which could reflect increased local replication from a larger number of targets or increased infectious synapse activity in cells contained in the lesion biopsies.
In two subjects studied, the skin biopsies from healed genital herpes lesions (2 weeks after healing) supported a substantially greater amount of HIV replication than did skin biopsies from the unaffected control area. The amount of integrated HIV-1 DNA per cell was 2.7- and 4.8-fold as high, respectively, in the ex vivo-infected cells obtained from each of the two genital herpes skin biopsies relative to cells from uninvolved skin tissue (1.53 and 2.87 HIV-1 DNA copies per cell versus 0.56 and 0.60 copies per cell, respectively). These data provide experimental evidence that tissue contained within healed genital herpes lesions is more susceptible to HIV-1 infection than is unaffected genital skin.
Our data provide several insights into the immunobiology of HSV-2 reactivation and offer a possible explanation for the observation that, although HSV-2 increases the likelihood of HIV acquisition1, suppression of HSV-2 reactivation with systemic antiviral drugs does not reduce HIV infection7, 8. Notably, HSV-2 infection results in a profound, persistent localized inflammatory response in the dermis below the healed lesion. This inflammatory focus, which is detected in clinically normal-appearing skin, consists of HSV-2-specific CD4+ T cells that express CCR5 or CXCR4, as well as a mixed population of DCs, including inflammatory cells that express DC-SIGN. These cells persist in spatial closeness at a density that ranges from 2- to 37-fold higher than in genital skin not previously infected by HSV-2. Unfortunately, even prolonged antiviral therapy for HSV-2 does not significantly influence this dense, localized inflammatory infiltrate. Ex vivo infection of skin biopsies containing this dense inflammatory infiltrate supports three- to fivefold higher levels of localized HIV-1 replication than do skin biopsies from control areas that do not contain these inflammatory infiltrates.
The mechanism behind this persistent inflammatory infiltrate requires further study. It has been known for decades that acyclovir is not completely effective in abolishing neuronal release of virus into the periphery4, 5, 6, but to our knowledge, our recent studies are the first to show that lymphocytes taken from the site of prior HSV-2 reactivation that is devoid of detectable HSV-2 antigen or DNA contain a high percentage of resident HSV-specific CD4+ and CD8+ T cells9. Compared to the lack of such cells in non-HSV-involved genital skin, our data suggest that the localized dermal inflammation that persists in genital skin after healing of the ulcer is related either to long-term immunologic memory or to continuously renewed immune responses owing to the intermittent expression of HSV antigen into these localized areas of skin13, 24. T cell-DC interactions in the peripheral tissue are important for orchestrating a rapid adaptive immune response and in maintaining persistent immunologic memory to HSV-2 antigen in the genital skin and mucosa15. However, these CD4+ T cell-DC interactions also provide an increased opportunity for HIV to infect additional CD4+ T cells25.
The wide anatomical distribution of HSV-2 in the male and female genital tracts underscores the importance that these localized reservoirs of inflammatory cells are likely to have in HIV acquisition26, 27. The persistence and spatial closeness of CD4+ T cells with DCs that could either support or enhance HIV-1 infection are likely to increase the potential for HIV-1 to initiate a focus of infection and increase the speed and likelihood of dissemination when deposited onto genital skin25, 26, 27, 28. Although antiviral therapy for HSV-2 is effective in reducing breaches in the epithelial barrier caused by HSV-2 (ref. 5), noninfectious ulcerations in the epithelium can initiate lentivirus infection29. Coitus is associated with a high number of breaks in the epithelial barrier, which would provide HIV access to this persistent nidus of CCR5-expressing CD4+ T cells and DC-SIGN-expressing DCs in the dermis and genital mucosa17, 20. Thus, HSV-2 infection provides a wide surface area and long duration of time, owing to its lifelong infection and reactivation frequency, for allowing HIV access to more target cells26, 30, providing a greater chance for the initial 'spark' of infection. Additionally, the close proximity to DC-SIGN-expressing DCs is likely to fuel these embers and provide a mechanism for more efficient localized spread of initial infection. This potential sequence of events is supported by our ex vivo experiments, in which we show greater integration of HIV-1 into organ cultures taken from genital lesions containing these inflammatory cells compared to control skin. Keeping the anatomic and spatial characteristics of the inflammatory cells that we found in vivo in an ex vivo environment is difficult and makes further mechanistic studies difficult. Similarly, the complexities of organ culture limit the ability to determine the role of increased CCR5 expression in the CCR5 tropism of HIV-1 strains associated with acute infection. Unfortunately, there is no well-developed nonhuman primate model of HSV-2 reactivation with which to study the detailed interaction between reactivating HSV-2 and a replicating lentivirus in vivo.
We recognize that we studied only a limited number of individuals. However, the demographic and clinical characteristics of the participants were similar to those with recurrent genital herpes studied in our clinic for the last two decades4, 5, 6, 26, 30, 31. The detailed clinical evaluations, biopsies and laboratory studies required preclude this protocol's widespread applicability to large patient cohorts. However, given the consistency of our findings between individuals and the typical appearance of histological and clinical findings from acute lesions, we feel that our central finding-that HSV reactivation leaves a residual inflammatory response not appreciated clinically-is typical of HSV-2 genital lesions. There is a large body of epidemiological data to indicate that ulcerations in the skin are an important point of entry for HIV-1, especially in heterosexual males6, 32, 33. Recent data indicating that circumcision reduces HIV acquisition provide direct experimental support of this concept34, 35, 36. It remains to be studied whether the persistent T cell infiltration we described in genital skin also occurs on mucosal surfaces such as the cervix. Sequential sampling of the cervix is possible, but clinically visible ulcerations and subclinical viral shedding at the cervix resulting from HSV-2 reactivation are infrequent in established HSV-2 infection26. In addition, the ability to anatomically define an area of the cervix that is unaffected by HSV-2 as the 'control' region is problematic.
Interventions designed to interrupt the association between HSV-2 and HIV must be directed at diminishing the chronic inflammatory milieu caused by HSV-2 infection in the genital tract through a reduction of HSV-2 reactivation from sacral ganglia or, perhaps more important, at addressing the need to prevent the acquisition of HSV-2 infection through the development of an effective HSV-2 vaccine.