The potential association of HSV2 infection status in HIV-negative FSWs with changes in genital CD4/CCR5+ T-cell or CD1a/DC-SIGN+ iDC populations was assessed (Fig. 2a; representative FACS plot of cervical cell populations). There were no differences in the absolute number of cervical CD4+ T cells, but there was a 0.3 log10 increase (three-fold higher absolute cell numbers) of CD4/CCR5+ T cells in the cervix of HSV2-infected FSWs (P < 0.05; Fig. 2b). Cervical CD4+ T cells were also more activated, with a greater absolute number and proportion (46% in HSV2+ versus 16% in HSV2) expressing CD69 (P < 0.001 for both; Fig. 2b). Strikingly, there was an entire log10 increase (ten-fold higher absolute cell numbers) in DC-SIGN+ iDCs (P < 0.001; Fig. 2c), and an increased proportion of cervical iDCs expressed DC-SIGN (11.9% in HSV2+ versus 4.6% in HSV2–, P < 0.05; Fig. 2c). Further characterization demonstrated that over 90% of cervical CD1a+ iDCs co-expressed CD11c, suggesting that these cells were of myeloid origin (data not shown).
Overall, HSV2 infection was associated with increases in genital HIV target cell populations, even in the absence of genital ulceration or HSV2 reactivation. We did not find any significant association between HSV2 infection and cervical cytokine or chemokine levels in HIV-uninfected FSWs. Since HSV2 shedding was not detected in HIV-negative FSWs, changes in the genital immune milieu related to HSV2 reactivation could not be examined.
In comparison with HIV-negative women, there was a profound depletion of iDC populations in the genital mucosa of HIV–HSV2 co-infected FSWs. Cervical CD1a+ iDCs were reduced (P < 0.05; Fig. 3a), particularly iDC subsets expressing either DC-SIGN (P < 0.05) or Toll-like receptor 9 (TLR9; P = 0.05), an innate immune receptor that binds CpG motifs on HSV2 DNA to trigger signaling cascades that culminate in type 1 interferon and inflammatory cytokine release, and a potentially important mediator of HSV2 immune control [36,37]. This iDC depletion was independent of systemic immune status (blood CD4+ T-cell counts or CD4/CD8 ratio; data not shown). HIV-infected FSWs had increases in cervical CD3+ T cells (P < 0.05) and CD8+ T cells (P = 0.001; Fig. 3a), without differences in activation levels. Cervical CD4+ T-cell numbers did not vary with HIV status (P = 0.7), but the CD4/CD8+ T-cell ratio was decreased in both the blood (P < 0.001) and cervix (P < 0.001) of HIV-infected FSWs, and to a similar degree at both sites (ratio 0.5 in blood versus 0.6 in the cervix; P = 0.5). There were no significant differences in CCR5+CD4+ cervical cell populations (absolute numbers, log10 transformed or % CCR5 expression by CD4 cells) between HIV-uninfected and infected women, although there was a trend (P = 0.07) for a decrease in the proportion of CCR5 expressing CD4+ T cells in HIV-infected FSWs. Although there were no significant differences in cervical cytokines/chemokines levels between HIV-infected and uninfected women, there were some trends to increased cytokines/chemokines in HIV-infected FSWs (MCP, IP-10, IL-10, IL-5, IL-2; P < 0.1 for all). HIV infection status was not associated with overall differences in the expression of TLRs 1–10 or FoxP3.
Cervical HIV shedding was detected in 10 of 36 FSWs (28%), and was strongly associated with immune activation in the genital tract. Levels of chemokines (IP-10, MCP, RANTES; all P < 0.001 and MIG; P < 0.05) and inflammatory cytokines (IL-1β, IL-8, IL-6 and IFN-γ; all P < 0.05; Fig. 3b) were elevated in the cervico-vaginal secretions of the 10 HIV shedders. In this subgroup, absolute HIV levels were inversely correlated with TLR9+ iDC numbers (r 2 = −0.7; P < 0.05) and with mRNA expression of TLR8 (r 2 = −0.9; P = 0.001) and TLR9 (r 2 = −0.7; P < 0.05). Cervical HIV load correlated with both the number and proportion of activated cervical CD4+ T cells expressing CD69 (r 2 = 0.7; P < 0.05; and r 2 = 0.7; P < 0.05, respectively). Neither blood CD4 cell counts, blood HIV viral load, nor cervical immune cell populations varied significantly with HIV RNA shedding status.
Cervical HSV2 shedding was only detected in HIV-infected sex workers, and the level of cervical HIV RNA was strongly correlated with HSV2 DNA levels (r 2 = 0.9; P < 0.001: Fig. 3c). Activated cervical CD4+ CD69+ T cells were increased in the genital mucosa of FSWs shedding HSV2 (202 versus 68 cells/cytobrush; P < 0.05, Fig. 4a), as were cervical levels of chemokines (P < 0.05 for all; Fig. 4b), but not pro-inflammatory cytokines. HSV2 levels correlated with the number and proportion of CD4 T cells expressing CCR5 (r 2 = 0.7; P < 0.05; and r 2 = 0.7; P = 0.02; respectively) (Fig. 4c), and with levels of the chemokines IP-10, MCP, MIG and RANTES (all r 2 > 0.6; all P ≤ 0.05). Cervical iDC numbers were unchanged in FSWs shedding HSV2 (13 588 versus 10 696 cells/cytobrush; P = 0.2), although fewer iDCs expressed TLR9 (3.1 versus 11.6%; P = 0.001). HSV2 DNA levels correlated inversely with the number of DC-SIGN+ iDCs (r 2 = −0.8; P < 0.05) and TLR9+ iDCs (r 2 = −0.8; P < 0.05) (Fig. 3d, e). This suggests that TLR9+ and DC-SIGN+ iDCs may mediate local immune control of HSV2 reactivation at a genital level.
This study confirms the substantial epidemiological synergy reported between HIV and HSV2, and is the first to demonstrate that this may be underpinned by a profound negative ‘mucosal synergy’ between these viruses in the female genital tract. In HIV-negative at-risk women, HSV2 infection was associated with increases in genital mucosal target cell populations that would be expected to increase susceptibility to HIV infection. These mucosal immune changes were evident even in the absence of genital ulceration or HSV2 reactivation, implying that HSV2 induces a persistent state of increased mucosal HIV susceptibility. The ten-fold increase in cervical iDCs expressing the DC-SIGN lectin, and the three-fold increase in cervical CD4+ T cells expressing the HIV co-receptor CCR5, provides a putative biological explanation for the observation that HSV2 seropositivity is a strong independent risk factor for HIV acquisition [6–9] even in the absence of symptomatic ulceration.
Both DC-SIGN and CD1a are important in mucosal immune responses. Upon antigen encounter, immature dendritic cells mount innate immune responses and undergo a coordinated series of dynamic cellular events that lead to maturation, antigen peptide loading onto MHC class II molecules, migration to lymph nodes and antigen presentation to T cells to induce adaptive immune responses. DC-SIGN binding of various viral and microbial pathogens triggers DC activation and/or maturation, as well as signaling via Toll-like receptors [25,39]. In addition, CD1a molecules can bind and efficiently present antigen to T cells in a maturation-independent manner . Intravaginal inoculation of mice with HSV2 led to rapid recruitment of submucosal DCs to the infected epithelium, followed by local stimulation of IFNγ production from HSV-specific CD4+ T cells  and submucosal DCs were the primary cells responsible for priming and mounting protective T-cell helper 1 responses during HSV2 infection . HSV2 infection of rhesus macaques impaired iDC maturation and promoted the release of chemokines RANTES and MIP1α, offering another possible explanation for the association of increased mucosal iDCs with HSV2 infection, and for increased genital chemokine levels during HSV2 reactivation. Together with these studies, our in vivo demonstration that HSV2 infection was associated with increased cervical DC-SIGN+ CD1a+ iDCs suggests that these cells may be important in mediating local immune control of HSV2.
However, DC-SIGN has also been demonstrated to permit the propagation of productive HIV infection in CD4+ T cells at low viral titers , such as are present in the genital mucosa soon after sexual acquisition of HIV . Therefore, HIV may exploit the local increases in DC-SIGN+ iDCs mediating HSV2 immune control in the genital mucosa, to enhance host susceptibility to HIV infection after sexual exposure.
HIV infection was associated with an overall depletion of cervical iDCs, as well as depletion of iDC subpopulations expressing DC-SIGN+ and/or TLR9+, regardless of HIV disease stage. HIV nef has been shown to reduce surface expression of CD1a on iDCs through redistribution into late endosomal/lysosomal compartments . Therefore, we cannot determine whether HIV infection was associated with a true depletion of iDCs, or a downregulation of CD1a surface expression by iDCs. Regardless, the reduced numbers of cervical iDCs expressing CD1a was strongly associated with local HSV2 reactivation in HIV-infected FSWs, again implying that these iDCs mediate local HSV2 immune control, and that their depletion in the context of HIV infection may further enhance the sexual transmission of both HSV2 and HIV.
The association of HSV2 and HIV shedding levels in FSWs co-infected by both HIV and HSV2 suggests that HSV2 infection may increase HIV transmission from this core group to their male clients. During reactivation, HSV2-encoded proteins may directly increase transcription from HIV-LTR [17,19–21]. HSV2 shedding was also associated with increased chemokine levels and the amount of HSV2 viral DNA was positively correlated with CCR5+ CD4+ cells. CCR5 receptor engagement by HIV envelope glycoproteins activates signalling that culminates in the release of various chemokines that generate a chemokine gradient that recruits T cells to sites of inflammation [45–47]. The increase in CCR5+ cells and chemokines during episodes of HSV2 shedding may recruit activated CD4+ T cells to the genital tract, and therefore enhance local replication of HIV  and consequently increase HIV transmission during sexual intercourse.
Overall, we observed strong immune synergy between HSV2 and HIV specifically in the mucosal genital compartment that may have direct implications for HIV prevention strategies. Our ex vivo approach allowed direct characterization of the female genital immune milieu, and elucidation of the immediate HSV2-associated mucosal changes that may underlie increased sexual HIV transmission. The resulting static ‘snap shot’ of the FGT immune milieu does not permit study of the extremely dynamic processes of immune cell migration to and from the genital mucosa, but does allow precise definition of the cell populations present in the mucosa at the time of sexual exposure to HIV, which is likely to be a critical determinant of transmission.
In sub-Saharan Africa, over half of new HIV infections occur in women, with young women from 15–24 years of age being at particularly high risk . Given the expected lack of a protective HIV vaccine in the near future, these statistics highlight the urgent need for an accessible alternative to prevent HIV infection . Our finding of increased DC-SIGN+ iDCs and CCR5+CD4+ T cells in the cervix of HSV2-infected women implies that microbicides targeting these molecules may hold promise, and such compounds are in early clinical testing [50,51]. Since DC-SIGN+ dendritic cells in the rectal mucosa also bind HIV avidly, an effective microbicide targeting this molecule might be important for both men and women practicing anal intercourse . Our results strongly suggest that HSV2 suppression, both in HIV-infected and uninfected individuals, holds promise as an HIV prevention strategy, and supports the rationale for large-scale clinical trials that are currently testing this hypothesis .
We thank Dr. Mario Ostrowski for helpful discussions and critical reading of the manuscript, Jane Kamene and the Pumwani clinic nurses for study recruitment and providing treatment, Ann Miangi, Nyakio Chinga and the laboratory staff at the University of Nairobi Microbiology Annex for specimen processing and performing diagnostic assays. Above all, we thank the women of the Pumwani ML cohort for their continued participation and support of our studies.
1. Mbopi-Keou FX, Gresenguet G, Mayaud P, Weiss HA, Gopal R, Matta M, et al
. Interactions between herpes simplex virus type 2 and human immunodeficiency virus type 1 infection in African women: opportunities for intervention. J Infect Dis 2000; 182:1090–1096.
2. Weiss HA, Buve A, Robinson NJ, Van Dyck E, Kahindo M, Anagonou S, et al
. The epidemiology of HSV-2 infection and its association with HIV infection in four urban African populations. AIDS 2001; 15(Suppl 4):S97–S108.
3. MasCasullo V, Fam E, Keller MJ, Herold BC. Role of mucosal immunity in preventing genital herpes infection. Viral Immunol 2005; 18:595–606.
4. Barton S, Celum C, Shacker TW. The role of anti-HSV therapeutics in the HIV-infected host and in controlling the HIV epidemic. Herpes 2005; 12:15–22.
5. Wald A, Link K. Risk of human immunodeficiency virus Infection in herpes simplex virus type 2–seropositive persons: a meta-analysis. J Infect Dis 2002; 185:45–52.
6. Kaul R, Kimani J, Nagelkerke NJ, Fonck K, Ngugi EN, Keli F, et al
. Monthly antibiotic chemoprophylaxis and incidence of sexually transmitted infections and HIV-1 infection in Kenyan sex workers: a randomized controlled trial. JAMA 2004; 291:2555–2562.
7. Corey L, Wald A, Celum CL, Quinn TC. The effects of herpes simplex virus-2 on HIV-1 acquisition and transmission: a review of two overlapping epidemics. J Acquir Immune Defic Syndr 2004; 35:435–445.
8. Freeman EE, Weiss HA, Glynn JR, Cross PL, Whitworth JA, Hayes RJ. Herpes simplex virus 2 infection increases HIV acquisition in men and women: systematic review and meta-analysis of longitudinal studies. AIDS 2006; 20:73–83.
9. Freeman EE, Glynn JR. Factors affecting HIV concordancy in married couples in four African cities. AIDS 2004; 18:1715–1721.
10. Kebede Y, Dorigo-Zetsma W, Mengistu Y, Mekonnen Y, Schaap A, Wolday D, et al
. Transmission of herpes simplex virus type 2 among factory workers in Ethiopia. J Infect Dis 2004; 190:365–372.
11. Schacker T, Zeh J, Hu H, Shaughnessy M, Corey L. Changes in plasma human immunodeficiency virus type 1 RNA associated with herpes simplex virus reactivation and suppression. J Infect Dis 2002; 186:1718–1725.
12. Wald A. Synergistic interactions between herpes simplex virus type-2 and human immunodeficiency virus epidemics. Herpes 2004; 11:70–76.
13. Celum CL. The interaction between herpes simplex virus and human immunodeficiency virus. Herpes 2004; 11(Suppl 1):36A–45A.
14. Plummer FA, Nagelkerke NJ, Moses S, Ndinya-Achola JO, Bwayo J, Ngugi E. The importance of core groups in the epidemiology and control of HIV-1 infection. AIDS 1991; 5(Suppl 1):S169–S176.
15. Cote AM, Sobela F, Dzokoto A, Nzambi K, Asamoah-Adu C, Labbe AC, et al
. Transactional sex is the driving force in the dynamics of HIV in Accra, Ghana. AIDS 2004; 18:917–925.
16. Mbopi-Keou FX, Legoff J, Gresenguet G, Si-Mohamed A, Matta M, Mayaud P, et al
. Genital shedding of herpes simplex virus-2 DNA and HIV-1 RNA and proviral DNA in HIV-1- and herpes simplex virus-2-coinfected African women. J Acquir Immune Defic Syndr 2003; 33:121–124.
17. Mosca JD, Bednarik DP, Raj NB, Rosen CA, Sodroski JG, Haseltine WA, Pitha PM. Herpes simplex virus type-1 can reactivate transcription of latent human immunodeficiency virus. Nature 1987; 325:67–70.
18. Moriuchi M, Moriuchi H. In vitro reactivation of HIV-1 by stimulation with herpes simplex virus infection. Sex Transm Dis 2002; 29:308–309.
19. Golden MP, Kim S, Hammer SM, Ladd EA, Schaffer PA, DeLuca N, Albrecht MA. Activation of human immunodeficiency virus by herpes simplex virus. J Infect Dis 1992; 166:494–499.
20. Albrecht MA, DeLuca NA, Byrn RA, Schaffer PA, Hammer SM. The herpes simplex virus immediate-early protein, ICP4, is required to potentiate replication of human immunodeficiency virus in CD4+ lymphocytes. J Virol 1989; 63:1861–1868.
21. Kucera LS, Leake E, Iyer N, Raben D, Myrvik QN. Human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus type 2 (HSV-2) can coinfect and simultaneously replicate in the same human CD4+ cell: effect of coinfection on infectious HSV-2 and HIV-1 replication. AIDS Res Hum Retroviruses 1990; 6:641–647.
22. Samson M, Libert F, Doranz BJ, Rucker J, Liesnard C, Farber CM, et al
. Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature 1996; 382:722–725.
23. Deng H, Liu R, Ellmeier W, Choe S, Unutmaz D, Burkhart M, et al
. Identification of a major co-receptor for primary isolates of HIV-1. Nature 1996; 381:661–666.
24. Geijtenbeek TB, Kwon DS, Torensma R, van Vliet SJ, van Duijnhoven GC, Middel J, et al
. DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells. Cell 2000; 100:587–597.
25. Cambi A, Koopman M, Figdor CG. How C-type lectins detect pathogens. Cell Microbiol 2005; 7:481–488.
26. Moris A, Nobile C, Buseyne F, Porrot F, Abastado JP, Schwartz O. DC-SIGN promotes exogenous MHC-I-restricted HIV-1 antigen presentation. Blood 2004; 103:2648–2654.
27. van Kooyk Y, Geijtenbeek TB. DC-SIGN: Escape mechanism for pathogens. Nat Rev Immunol 2003; 3:697–709.
28. Nugent RP, Krohn MA, Hillier SL. Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation. J Clin Microbiol 1991; 29:297–301.
29. Mostad SB, Kreiss JK, Ryncarz AJ, Overbaugh J, Mandaliya K, Chohan B, et al
. Cervical shedding of cytomegalovirus in human immunodeficiency virus type 1-infected women. J Med Virol 1999; 59:469–473.
30. Lurain NS, Robert ES, Xu J, Camarca M, Landay A, Kovacs AA, Reichelderfer PS. HIV type 1 and cytomegalovirus coinfection in the female genital tract. J Infect Dis 2004; 190:619–623.
31. Sheth PM, Danesh A, Sheung A, Rebbapragada A, Shahabi K, Kovacs C, et al
. Disproportionately high semen shedding of HIV is associated with compartmentalized cytomegalovirus reactivation. J Infect Dis 2006; 193:45–48.
32. Sheth PM, Danesh A, Shahabi K, Rebbapragada A, Kovacs C, Dimayuga R, et al
. HIV-specific CD8+ lymphocytes in semen are not associated with reduced HIV shedding. J Immunol 2005; 175:4789–4796.
33. Prakash M, Patterson S, Kapembwa MS. Evaluation of the cervical cytobrush sampling technique for the preparation of CD45+ mononuclear cells from the human cervix. J Immunol Methods 2001; 258:37–46.
34. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162:156–159.
35. Bird TG, Kaul R, Rostron T, Kimani J, Embree J, Dunn PP, et al
. HLA typing in a Kenyan cohort identifies novel class I alleles that restrict cytotoxic T-cell responses to local HIV-1 clades. AIDS 2002; 16:1899–1904.
36. Lund J, Sato A, Akira S, Medzhitov R, Iwasaki A. Toll-like receptor 9-mediated recognition of Herpes simplex virus-2 by plasmacytoid dendritic cells. J Exp Med 2003; 198:513–520.
37. Liew FY, Xu D, Brint EK, O'Neill LA. Negative regulation of toll-like receptor-mediated immune responses. Nat Rev Immunol 2005; 5:446–458.
38. Lawn SD. AIDS in Africa: the impact of coinfections on the pathogenesis of HIV-1 infection. J Infect 2004; 48:1–12.
39. Garcia-Pineres AJ, Hildesheim A, Trivett M, Williams M, Wu L, Kewalramani VN, Pinto LA. Role of DC-SIGN in the activation of dendritic cells by HPV-16 L1 virus-like particle vaccine. Eur J Immunol 2006; 36:437–445.
40. Cao X, Sugita M, Van Der Wel N, Lai J, Rogers RA, Peters PJ, Brenner MB. CD1 molecules efficiently present antigen in immature dendritic cells and traffic independently of MHC class II during dendritic cell maturation. J Immunol 2002; 169:4770–4777.
41. Linehan MM, Richman S, Krummenacher C, Eisenberg RJ, Cohen GH, Iwasaki A. In vivo role of nectin-1 in entry of herpes simplex virus type 1 (HSV-1) and HSV-2 through the vaginal mucosa. J Virol 2004; 78:2530–2536.
42. King NJ, Parr EL, Parr MB. Migration of lymphoid cells from vaginal epithelium to iliac lymph nodes in relation to vaginal infection by herpes simplex virus type 2. J Immunol 1998; 160:1173–1180.
43. Haase AT. Perils at mucosal front lines for HIV and SIV and their hosts. Nat Rev Immunol 2005; 5:783–792.
44. Shinya E, Owaki A, Shimizu M, Takeuchi J, Kawashima T, Hidaka C, et al
. Endogenously expressed HIV-1 nef down-regulates antigen-presenting molecules, not only class I MHC but also CD1a, in immature dendritic cells. Virology 2004; 326:79–89.
45. Weissman D, Rabin RL, Arthos J, Rubbert A, Dybul M, Swofford R, et al
. Macrophage-tropic HIV and SIV envelope proteins induce a signal through the CCR5 chemokine receptor. Nature 1997; 389:981–985.
46. Davis CB, Dikic I, Unutmaz D, Hill CM, Arthos J, Siani MA, et al
. Signal transduction due to HIV-1 envelope interactions with chemokine receptors CXCR4 or CCR5. J Exp Med 1997; 186:1793–1798.
47. Lee C, Liu QH, Tomkowicz B, Yi Y, Freedman BD, Collman RG. Macrophage activation through CCR5- and CXCR4-mediated gp120-elicited signaling pathways. J Leukoc Biol 2003; 74:676–682.
48. Quinn TC, Overbaugh J. HIV/AIDS in women: an expanding epidemic. Science 2005; 308:1582–1583.
49. Lederman MM, Offord RE, Hartley O. Microbicides and other topical strategies to prevent vaginal transmission of HIV. Nat Rev Immunol 2006; 6:371–382.
50. Lederman MM, Veazey RS, Offord R, Mosier DE, Dufour J, Mefford M, et al
. Prevention of vaginal SHIV transmission in rhesus macaques through inhibition of CCR5. Science 2004; 306:485–487.
51. Veazey RS, Klasse PJ, Schader SM, Hu Q, Ketas TJ, Lu M, et al
. Protection of macaques from vaginal SHIV challenge by vaginally delivered inhibitors of virus-cell fusion. Nature 2005; 438:99–102.
52. Gurney KB, Elliott J, Nassanian H, Song C, Soilleux E, McGowan I, et al
. Binding and transfer of human immunodeficiency virus by DC-SIGN+ cells in human rectal mucosa. J Virol 2005; 79:5762–5773.