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HIV Transmission and Pathogens in Human Tissues

Leonid Margolis, PhD
  • Leonid Margolis, PhD, Head, Section on Intercellular Interactions
  • Wendy Fitzgerald, BS, Technician
  • Jean-Charles Grivel, PhD, Staff Scientist
  • Andrea Lisco, MD, PhD, Visiting Fellow
  • Christophe Vanpuoille, PhD, Visiting Fellow
  • Anush Arakelyan, PhD, Visiting Fellow
  • Melanie Merbah, Postbaccalaureate Intramural Research Training Award Fellow

For the past several years, the main goal of the Section of Intercellular Interactions has been to understand the pathogenesis of human immunodeficiency virus (HIV) in human tissues. In particular, we suggested and now are testing our working hypothesis that non-HIV microbes (copathogens) associated with HIV infection largely determine the course of HIV disease by interacting with HIV-1 in the context of coinfected human tissues. Also, non-HIV microbes may play a significant role in HIV-1 transmission by changing the tissue microenvironment and selecting particular HIV-1 variants. Our objective is to decipher the mechanisms of these interactions and to develop ways to manipulate them in order to prevent and to contain HIV infection. We address these problems in a system of human tissues infected ex vivo with HIV and other microbes, recapitulating important aspects of HIV pathogenesis in vivo. A systematic and detailed study of the host-mediated interactions between non-HIV viruses and HIV is necessary both for the understanding of the complex basic mechanisms of HIV infection in vivo and for the development new anti-HIV therapies.

Toll-like receptors ligands modulate HIV-1 infection of human lymphoid tissue.

HIV-1 infection, as well as the progression of HIV-1 disease, usually occurs in the presence of other microbes. While the mechanisms by which microbes interact with HIV-1 are not fully understood, it is clear that their invasion into the human body triggers a cascade of events that can affect HIV-1 pathogenesis. One of the first events in the interaction of the human body with an invading microorganism is the engagement of the Toll-like receptors (TLRs) that modulate both the innate and adaptive immune responses. We tested whether engagement of TLRs by invading microorganisms affects HIV-1 replication in human lymphoid tissues. Because the effects of microbes on host cells are complicated, we used a reductionist approach: instead of whole microbes, we treated human lymphoid tissue ex vivo with TLR ligands. We administered various TLR ligands prior to and during HIV-1 infection and monitored HIV-1 replication, cytokine release, expression of selected surface molecules, and cell activation. For these experiments, we used blocks of ex vivo human tonsillar tissue in which cytoarchitecture and cellular repertoire, as well as some tissue functions, are preserved. We found that in human lymphoid tissue, some TLR ligands modulate HIV replication positively while others modulate it negatively and that a TLR-triggered cell activation and change of cytokine spectra are involved in these modulations. We focused on ligands to TLRs 5 and 9, and examined their effect on HIV-1 replication in lymphoid tissue ex vivo. We found that, while flagellin (TLR5 agonist) treatment enhanced replication of CC chemokine receptor 5 (CCR 5)–tropic and CXC chemokine receptor 4 (CXCR4)–tropic HIV-1, treatment with oligodeoxynucleotide (ODN) M362 (TLR9 agonist) suppressed both viral variants. The differential effects of these TLR ligands on HIV-1 replication correlated with changes in production of CC chemokines CCL3, CCL4, CCL5, and of CXC chemokines CXCL10, and CXCL12 in the ligand-treated HIV-1–infected tissues.

Our results suggest that binding of microbial ligands to TLRs is one of the mechanisms that mediate interactions between coinfected microbes and HIV-1 in human tissues. Thus, the engagement of appropriate TLRs by microbial molecules or their mimetic might become a new strategy for HIV therapy or prevention.

HIV-1 transmission to human cervico-vaginal tissue ex vivo

Heterosexual transmission of HIV-1 occurs predominantly through vaginal intercourse, meaning that the female lower genital tract mucosa an important gateway for virus entry. Despite the critical importance of cervico-vaginal tissue to HIV-1 infection, the key characteristics of cervico-vaginal T cells remain largely unknown. We addressed this problem by developing a technique to maintain cervico-vaginal tissue ex vivo and a protocol for flow-cytometry of tissue lymphocytes. We studied HIV-1 transmission to human cervico-vaginal tissue under controlled laboratory conditions. First, we investigated the status of CD4 T cells in cervico-vaginal tissue, given that, for other tissues, it has been shown that the status of these cells determines a tissue's ability to support productive HIV infection. We found that, unlike other tissues, virtually all T cells are of the effector memory phenotype, with an almost complete absence of naive T cells. Also, in cervico-vaginal tissue, the fraction of CD8 T cells was 3–4 times larger than in other tissues studied so far, including rectal, tonsillar, or lymph node tissues. The difference was accompanied by a higher expression of the activation markers CD38, HLA-DR, and CD57 in CD8 than in CD4 T cells. Such a composition of lymphocytes is associated with the tissue's ability to defend against incoming pathogens. However, these defense mechanisms are not sufficient to prevent HIV-1 infection. Cervico-vaginal tissue ex vivo supports HIV-1 replication that occurs preferentially in CD4 T cells that express the activation marker CD38. Surprisingly, the increase in activation of HIV-1–infected cells was associated with a concomitant increase in the expression of CD38 in HIV-1–uninfected (bystander) CD4 T cells, but not in CD8 T cells.  Such a vicious cycle of activation-infection-activation creates new targets for the virus and facilitates its dissemination. However not all HIV-1 variants are capable of efficiently replicating in cervico-vaginal tissue ex vivo. Similar to in vivo, human cervico-vaginal tissue ex vivo supports the productive infection preferentially of R5 HIV-1 rather than that of X4 HIV-1, despite broad expression of CXCR4. This "gatekeeping" mechanism is associated with the abundance of CD27−  CD28− effector memory CD4 T cells. It has been suggested that the "gatekeeping" mechanism is a superimposition of various incomplete restrictive barriers in the course of HIV-1 transmission, rather than a unique complete barrier. The system we developed permits us to decipher the mechanisms of the "gatekeeping" barriers. Our results provide new insights into the relationship between HIV-1 infection and the activation/differentiation status of the lymphocytic population in cervico-vaginal tissue, thus contributing to a better understanding of the early events of sexual HIV-1 transmission. Such an understanding and a planned future investigation of the role of non-HIV-1 microbes in HIV-1 transmission may lead to the development of new anti–HIV-1 strategies, in particular the expansion of the "gatekeeping" mechanisms to all HIV-1 variants and disruption of the vicious circle of HIV-1–triggered T-cell activation that facilitates HIV-1 dissemination.

Acyclovir as anti-HIV-1 drug

Compelling clinical evidence has demonstrated that treatment with acyclovir (ACV) or its prodrug valacyclovir (vACV) of HIV-1–infected patients results in reduction of HIV-1 load and delaying HIV-1 disease progression. Therefore, understanding the mechanisms of ACV-mediated reduction of HIV-1 load is crucial to evaluate its potential clinical use. Recently, we reported that phosphorylated ACV inhibits HIV-1 replication in human tissues ex vivo by inhibiting HIV-1 reverse transcriptase. We continue to study the breadth and efficiency of ACV anti–HIV-1 activity. In  particular, it is important to test the drug against primary isolates of different subtypes. Also, given that ACV acts as a nucleoside reverse transcriptase inhibitor (NRTI), it is important to investigate its activity against multidrug-resistant HIV-1. We addressed these problems using a system of human lymphoid tissues inoculated ex vivo with HIV-1 that recapitulates the critical events of HIV-1 pathogenesis in vivo. In this system we tested: 1) ACV suppression of infection by primary patient isolates of various clades rather than the HIV-1 laboratory strains we studied earlier, and 2) the sensitivity to ACV of HIV-1 variants that are resistant to approved anti–HIV-1 drugs, including non-NRTIs and protease and fusion inhibitors. ACV potently suppressed replication of all four tested HIV-1 primary isolates, irrespective of their clade origin (A, B, C) or of their coreceptor specificity (CCR5, CXCR4, CCR3, CCR2B, Bob, Bonzo), suggesting that the anti HIV-1 activity of ACV in human tissues is a general phenomenon. Also, we tested the activity of ACV against viruses that are resistant to two of the most common anti-HIV NRTIs—Zidovudine (AZT) and Lamivudine (3TC)—and found that ACV efficiently suppressed their replication. To further confirm the sensitivity of NRTI resistant viruses to ACV, we tested the sensitivity of a panel of prototypical infectious multidrug-resistant HIV-1 RT clones and found  that ACV suppresses infection of these HIV-1 variants as well. Lastly, although cross-resistance has been usually reported to occur predominantly between various drugs of the same class, we tested the sensitivity to ACV of HIV-1 variants that are resistant to non-NRTIs and found that they were also inhibited by ACV. Thus, in human lymphoid tissue, ACV suppresses replication of HIV-1 of different clades, of different coreceptor tropisms, and with mutations that confer resistance to some of the currently used NRTIs and non-NRTIs. Together with the reported absence of emergence of resistance of HIV to ACV in vivo and the well-documented beneficial effect of ACV on disease progression, one can consider the possibility of using ACV alone or in combination with anti-HIV drugs against this virus. Also, it may be possible to use this inexpensive drug, which is proven to be safe, to slow down disease progression and delay HAART in low-income countries, where access to HAART is limited.

Publications

  • Brichacek B, Vanpouille C, Kiselyeva Y, Biancotto A, Merbah M, Hirsch I, Lisco A, Grivel J-C, Margolis L. Contrasting roles for TLR ligands in HIV-1 pathogenesis. PloS One. 2010 5:1-12.
  • Saba E, Grivel JC, Vanpouille C, Brichacek B, Fitzgerald W, Margolis L, Lisco A. HIV-1 sexual transmission: early events of HIV-1 infection of human cervico-vaginal tissue in an optimized ex vivo model. Mucosal Immunol. 2010 3:280-290.
  • Grivel J-C, Shattock RJ, Margolis LB. Selective transmission of R5 HIV-1 variants: where is the gatekeeper? J Transl Med. 2010 in press.
  • Lisco A, Vanpouille C, Margolis L. Coinfecting viruses as determinants of HIV disease. Curr HIV/AIDS Rep. 2009 6:5-12.
  • Vanpouille C, Lisco A, Derudas M, Saba E, Grivel JC, Brichacek B, Scrimieri F, Schinazi R, Schols D, McGuigan C, Balzarini J, Margolis L. A new class of dual-targeted antivirals: monophosphorylated acyclovir prodrug derivatives suppress both human immunodeficiency virus type 1 and herpes simplex virus type 2. J Infect Dis. 2010 201:635-643.

Collaborators

  • Jan Balzarini, PhD, Rega Institute, Katholieke Universiteit Leuven, Leuven, Belgium
  • Matthias Götte, PhD, Université McGill, Montréal, Canada
  • Christopher McGuigan, PhD, Cardiff University, Cardiff, UK
  • Raymond Schinazi, PhD, DSc, Emory University, Atlanta, GA
  • Robin Shattock, PhD, University of London, London, UK

Contact

For more information, email margolis@helix.nih.gov.

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