HIV Pathogens in Human Lymphoid Tissue

Leonid Margolis, PhD, Head, Section on Intercellular Interactions
Jean-Charles Grivel, PhD, Staff Scientist
Beda Brichacek, PhD, Senior Research Fellow
Angelique Biancotto, PhD, Visiting Fellow
Andrea Lisco, MD, PhD, Visiting Fellow
Christophe Vanpuoille, PhD, Visiting Fellow
Wendy Fitzgerald, BS, Technician

Host and viral factors largely determine the course of HIV infection. Among host factors, tissue immuno-activation plays a critical role. Among viral factors, viral phenotype is important, as illustrated by accelerated HIV disease progression associated with the evolution of CXCR4-using HI V-1 at the late stages of disease and by the exclusive transmission of CC R5-using HI V-1 clade B. Non–HIV pathogens are important host factors that affect HIV infection. To investigate the role of immuno-activation in HIV infection, to decipher the mechanisms of HIV interactions with other microbes, and to use the interactions to contain HI V infection in complex tissue microenvironments, we studied viral pathogenesis in various human tissues infected ex vivo with HIV-1 alone or in combination with other viruses. The first study focused on the role of immuno-activation in HIV-1 pathogenesis; the second study investigated the mechanisms of interactions of non–HIV microbes with HIV-1 in co-infected tissues; and the third study exploited the ability of herpesviruses to transform an antiherpetic drug into an HIV suppressor. The results of these studies provide an understanding of basic mechanisms of HIV transmission, evolution, and interactions with other viruses in human tissues and lay the foundation for designing new strategies to prevent and contain HIV infection.

Critical role of tissue activation in HIV infection

Biancotto, Grivel, Lisco, Vanpouille, Margolis; in collaboration with Lederman

Paradoxically, infection with HIV, the virus that eventually leads to immunodeficiency, depends on tissue immuno-activation, and HIV itself immuno-activates tissues. We propose that immune activation in secondary lymphoid tissues is central to the pathogenesis of immune deficiency in chronic HIV-1 infection. In vivo, the level of activation may predict the pace of CD4 depletion and the onset of clinical immune deficiency. Earlier, we found significant perturbation of cytokine secretion in HIV-infected lymph nodes. We also observed a dramatic alteration in the normal pattern of constitutive cytokine expression, with some cytokines such as interleukin-1β, interleukin-2, interferon-γ, and interleukin-15 characteristically found in increased concentrations and some others such as MIP-1α and SDF-1β substantially diminished.

In addition, we found a profound upregulation of the activation marker CD38 in naive, central-memory, and effector CD4⁺ and CD8⁺ T cells. The distorted activation patterns of lymphocytes in lymph nodes and tonsils from HI V-1–infected individuals emphasize the important role of tissue activation in HIV-1 disease progression. To study this phenomenon in a laboratory setting, we used a system of blocks of human lymphoid tissue developed earlier and a newly developed system of cervico-vaginal tissue ex vivo. (In vivo, both tissues are critical in HIV transmission and pathogenesis.) Both types of tissue revealed a similar response to HIV infection: HIV-1 infection was associated with the activation of both HIV-1-infected and uninfected (bystander) T cells in infected tissues. Given that productive HIV infection is limited to activated cells, HIV-1 infection facilitates its own production by creating new cellular targets for viral infection. Moreover, with a selective increase in apoptosis in productively infected T cells expressing the activation markers CD 25/HLA -DR, the vicious circle of HI V infection-activation-infection contributes to T-cell depletion, the hallmark of HIV infection. Such a mechanism, especially in cervico-vaginal tissue, the main site for HIV transmission, may play a critical role in susceptibility of the vagina to HIV transmission. The targeting of individual elements of this cycle may constitute a new basis for preventive and therapeutic strategies against HIV infection.

Biancotto A, Grivel J-C, Iglehart SJ, Vanpouille C, Lisco A, Sieg SF, Debernardo R, Garate K, Rodriguez B, Margolis LB, Lederman MM. Abnormal activation and cytokine spectra in lymph nodes of people chronically infected with HIV-1. Blood 2007;109:4272-4279.
Biancotto A, Iglehart SJ, Vanpouille C, Condack CE, Lisco A, Ruecker E, Hirsch I, Margolis LB, Grivel J-C. HIV-1-induced activation of CD4+ T cells creates new targets for HIV-1 infection in human lymphoid tissue ex vivo. Blood 2008;111:699-704.
Grivel J-C, Margolis L. Use of human tissue explants to study human infectious agents. Nat Protocols 2008, in press.
Lederman M, Margolis L. The lymph node in HIV pathogenesis. Semin Immunol 2008;20:187-195.

Effect of non–HIV pathogens on HIV infection in human lymphoid tissue

Biancotto, Sassi,¹ Grivel, Vanpouille, Brichacek, Margolis; in collaboration with Sher, Golding

We continue to study the effect of non–HIV pathogens on HIV infection in human lymphoid tissue. Earlier, on the basis of our studies on HIV interactions with human herpesviruses (HHV) 6 and HHV7 and vaccinia and measles viruses, we found that these microbes affect HIV-1 by inducing the release of various cytokines (chemokines) in tissue as well as by altering the expression of cellsurface molecules relevant to HIV infection. In this study, we investigated HIV interactions with two HIV-1 co-pathogens, human cytomegalovirus (HCMV) or HHV-5 and the cellular parasite Toxoplasma gondii. In vivo, the interactions occur in the context of co-infected tissue. To investigate these phenomena under controlled laboratory conditions, we co-infected human lymphoid tissue ex vivo with HIV-1 of different co-receptor usages and with one of the non–HIV pathogens.

HCMV is typically latent in healthy individuals; however, reactivation frequently occurs in immunocompromised individuals and can lead to severe organ dysfunction. Therefore, HCMV can become an important HIV co-pathogen. HCMV induces disease only in humans; thus, there is currently no adequate model to study the replication of HCMV in tissues. To support HCMV replication, we optimized the system of human tonsillar tissue ex vivo that was developed earlier to study HIV-1 pathogenesis in human tissue. To monitor HCM V infection, we used a recombinant strain of HCM V that expresses the green fluorescent protein CM VPT 30-gfp. In addition, we used detection of HCM V DNA to characterize chronic infection in controls and analyzed infection of individual cells by flow cytometry. HCMV readily replicated in tissue blocks, as revealed by the release of HCMV viral DNA and a rise in the number of viral-positive cells. Immuno-phenotyping of HCM V-infected cells showed the preferential infection of activated lymphocytes (CD3⁺, HLA-DR⁺, and CD3⁺ CD25⁺) and of plasmocytoid and myeloid dendritic cells (CD3^- CD123⁺ CD11c⁺). The number of these cells significantly increased in HI V-1 co-infected tissues. Accordingly, HCM V replication was enhanced two- to three-fold; such upregulation occurred in tissues infected with either CXCR4- or CC R5-using HIV-1. Thus, HIV-1 creates new targets for HCMV, perhaps explaining the strong association of HCMV with HIV-1 infection in vivo. Human lymphoid tissue infected ex vivo constitutes a model for the study of mechanisms of HCMV tissue pathogenesis and its interactions with HIV-1 and may provide new targets for anti–HIV-1 therapy.

Human lymphoid tissue ex vivo also provides a model to study HIV interaction with non-viral HIV pathogens. The apicomplexan parasite Toxoplasma gondii is a common HI V co-pathogen associated with AIDS development. We examined the interaction of T. gondii and HIV in co-infected human lymphoid tissue ex vivo. Both pathogens readily replicate in ex vivo–infected blocks of human tonsillar tissue. Surprisingly, we found that live T. gondii preferentially inhibits replication of CCR5- using HIV-1 in co-infected tissues. This effect is reproduced in treatment of the tissue blocks with recombinant C-18, a T. gondii–encoded cyclophilin that binds to CC R5. These ex vivo findings raise the possibility that, in addition to functioning as a co-factor in HIV disease, T. gondii may influence the outcome of viral infection by preferentially suppressing CCR5-using HIV-1 variants.

Our studies of HIV interactions with non–HIV pathogens in human tissues, together with earlier published results, revealed diverse mechanisms of local HIV interactions with non–HIV viruses in lymphoid tissues. The mechanisms involve activation of T lymphocytes, thereby providing for new targets for HIV, competition for T cells between HIV and other microbes, downregulation of HIV receptors and co-receptors as well as upregulation of HIV co-receptor ligands (both tissue- and pathogen-encoded), and depletion of target cells. HIV co-pathogens may differentially generate positive and negative signals for HIV replication. We attempted to mimic one of the negative signals for HIV-1 of different phenotypes, thus affecting selection of particular HIV variants and eventually changing the course of HIV disease.

Biancotto A, Iglehart SJ, Lisco A, Vanpouille C, Grivel J-C, Lurain NS, Reichelderfer PS, Margolis LB. Upregulation of human cytomegalovirus by HIV type 1 in human lymphoid tissue ex vivo. AIDS Res Hum Retroviruses 2008;24:453-462.
Sassi A, Brichacek B, Hieny S, Yarovinsky F, Golding H, Grivel J-C, Sher A, Margolis L. Toxoplasma gondii inhibits R5 HIV-1 replication in human lymphoid tissues ex vivo. AIDS Res Hum Retroviruses 2008, in press.
Vanpouille C, Biancotto A, Lisco A, Brichacek B. Interactions between HIV-1 and vaccinia virus in human lymphoid tissue ex vivo. J Virol 2007;81:12458-12464.

Up- and downregulation of HIV-1 replication in cervico-vaginal tissue

Lisco, Vanpouille, Grivel, Biancotto, Margolis; in collaboration with Anton, Balzarini, McGuigan, Schinazi, Shattock, Münch, Kirchhoff

Sexual intercourse is the major route of HIV transmission. Therefore, it is important to identify the endogenous factors that affect the efficiency of sexual viral transmission and to develop strategies to prevent and contain infection at the critical sites of HIV transmission and pathogenesis. We participated in the identification of a semen factor that facilitates HIV replication and showed that the factor upregulates HIV replication in infected T cells, macrophages, ex vivo human tonsillar tissues, and cervico-vaginal tissues. The factor consists of naturally occurring fragments of the abundant semen marker prostatic acidic phosphatase, which form amyloid fibrils. Thus, the fibrils may play an important role in sexual transmission of HIV and could represent new targets for its prevention.

Earlier, we found that, in HI V-infected tissues, it is also possible to generate factors that downregulate HIV-1 infection. These factors may be generated by other pathogens (see above). Here, we suggest a new strategy for generating such factors: we exploit the unique ability of herpesvirus kinases to phosphorylate acyclovir (ACV), the first step in transforming this otherwise inert compound into an inhibitor of herpesvirus DNA polymerases. Surprisingly, we found that in a cell-free system, phosphorylated ACV directly suppresses HIV reverse transcriptase (RT). We showed that HIV-1 RT incorporates ACV-TP into the nascent HIV-1 DNA chain with a level of efficiency similar to that of its natural equivalent dGTP. Incorporation of ACV-TP results in the formation of a dead-end complex and traps RT at the site of termination, leading to the complete termination of reverse transcription. Accordingly, we found that, in tissues co-infected with HHVs that are capable of phosphorylating ACV, HIV-1 was inhibited. We demonstrated such inhibition in cervico-vaginal tissue as well as in tonsils, lymph nodes, and colorectal tissue, where the critical events of HIV-1 pathogenesis and transmission occur in vivo. Various HHVs, including the ubiquitous HHV-6 (detected in all but one tissue ex vivo), may mediate HIV-1 suppression by ACV. We could detect no anti–HIV ACV activity in either the HHV-free tissue or HHV-free cell lines. However, when we added HH V-6–infected cells to HI V-1–infected cultures, AC V became a potent suppressor of HI V-1 replication. In summary, the following mechanism seems to be responsible for ACV suppression of HIV-1 in human tissues ex vivo, the majority of which carry one or several HHVs, including HHV-6: ACV is monophosphorylated by herpesviral enzymes in HHV-infected cells, and then cellular kinases convert it into ACV-TP, which suppresses HIV in co-infected tissues by inhibiting HIV-1 RT. Our results suggest that ACV may be therapeutically beneficial for various HIV-1–infected patients, as the majority of humans are already infected with various HHVs that transform ACV, at least during reactivation. However, clinical trials are needed to test this concept in vivo. In general, the combination of ACV with an endogenous HHV infection to suppress HIV may constitute a new principle of anti–HIV therapy: a “binary weapon” in which one inert component is converted by another, endogenous component into an active therapeutic compound.

Lisco A, Vanpouille C. HSV-2 suppression and the incidence on HIV. N Engl J Med 2008;359:535.
Lisco A, Vanpouille C, Tchesnokov EP , Grivel J-C, Biancotto A, Brichacek B, Elliott J, Fromentin E, Shattock R, Anton P, Gorelick R, Balzarini J, McGuigan C, Derudas M, Götte M, Schinazi RF, Margolis L. Acyclovir is activated into a HIV-1 reverse transcriptase inhibitor in herpesvirus-infected human tissues. Cell Host Microbe 2008;4:260-270.
Münch J, Rücker1 E, Ständker L, Adermann K, Goffinet C, Schindler M, Wildum S, Chinnadurai R, Rajan D, Specht A, Giménez-Gallego G, Cuevas Sánchez P, Fowler DM, Koulov A, Kelly JW, Mothes W, Grivel J-C, Margolis L, Keppler OT, Forssmann W-G, Kirchhoff F. Semen-derived amyloid fibrils drastically enhance HIV infection. Cell 2007;131:1059-1071.

¹ Atfa Sassi, PhD, former Visiting Fellow

Collaborators

Peter Anton, MD, David Geffen School of Medicine at UCLA, Los Angeles, CA
Jan Balzarini, PhD, Rega Institute, Katholieke Universiteit Leuven, Leuven, Belgium
Hana Golding, PhD, Center for Biologics Evaluation and Research, FDA, Rockville, MD
Matthias Götte, PhD, Université McGill, Montréal, Canada
Frank Kirchhoff, PhD, Universität Ulm, Ulm, Germany
Michael Lederman, MD, Case Western Reserve University/University Hospitals of Cleveland, Cleveland, OH
Christopher McGuigan, PhD, Cardiff University, Cardiff, UK
Jan Münch, PhD, Universität Ulm, Ulm, Germany
Raymond Schinazi, PhD, DSc, Emory University, Atlanta, GA
Robin Shattock, MD, St. George’s Hospital Medical School, University of London, London, UK
Alan Sher, PhD, Laboratory of Parasitic Diseases, NIAID, Bethesda, MD

For further information, contact margolis@helix.nih.gov.