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HIV Pathogens in Human Lymphoid Tissue

Leonid Margolis, PhD, Head, Section on Intercellular Interactions
Angelique Biancotto, PhD, Visiting Fellow
Brida Brichacek, PhD, Senior Research Fellow
Wendy Fitzgerald, BS, Technician
Jean-Charles Grivel, PhD, Staff Scientist
Andrea Lisco, MD, PhD, Visiting Fellow
Elisa Saba, PhD, Visiting Fellow
Christophe Vanpuoille, PhD, Visiting 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. Our objective is to decipher the mechanisms of these interactions and to develop ways to manipulate them in order to contain HIV replication. 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.

The human host exists in a dynamic equilibrium with multiple viruses, in particular herpesviruses (HHV), that remain under continuous immunosurveillance. Upon HIV infection, the system is no longer in equilibrium, leading to infections by new viruses and reactivation of the “latent viruses.” A systematic and detailed study of the host-mediated interactions between reactivated 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.

In one project, we investigated the role of a common HIV-associated pathogen, HHV-6, in HIV-1 infection using an SIV/macaque model; in a second project, we continued to develop strategies aimed at exploiting herpesviruses to contain HIV-1 infections in human tissues by using derivatives of the antiherpetic drug acyclovir, which suppresses HIV reverse transcriptase; our third project aims to develop new models and assays to study HIV infection of human tissues under controlled laboratory conditions ex vivo.

HIV interactions with coinfecting viruses

Over the past few years, we and others have identified several microbes that, upon coinfection, suppress HIV-1 replication. In particular, we found that HHV-6 suppresses HIV in coinfected human tissues by upregulating CCL-5 (RANTES). However, HHV-6 is also known as a cofactor in HIV disease progression. This apparent paradox has now been resolved in our experiments with coinfected macaques. SIV isolates were obtained from pig-tailed macaques (M. nemestrina) after approximately 1 year of single infection with SIVsmE660 or dual infection SIV_smE660−HHV-6_AGS. All HHV-6 animals progressed to AIDS within 2 years of infection and were found to harbor SIV variants with a reduced sensitivity to both HHV-6A and RANTES, despite maintaining an exclusive CCR5 specificity. Viruses derived from two of these animals replicated even more vigorously in the presence of exogenous HHV-6A or CCL5. The SIV variants that emerged in HHV-6–coinfected macaques showed overall a reduced ex vivo replication capacity that was partially reversed upon addition of exogenous CCL5. SIV isolates from singly SIV-infected macaques maintained HHV-6A/CCL5 sensitivity. These results provide the first demonstration of SIV evolution towards RANTES resistance under the influence of a coinfecting microbe, illustrating a potential mechanism for the accelerated progression to full-blown AIDS seen in HHV-6–coinfected macaques. CCL5 resistance may represent a common virulence factor allowing primate immunodeficiency retroviruses to evade a critical mechanism of host antiviral defense. In conclusion, our study illustrates a novel mechanism whereby coinfection with a putative AIDS-progression cofactor, the T-lymphotropic HHV-6A, may affect the in vivo evolution of SIV, leading to accelerated development of AIDS. Understanding the complex interactions between HHV-6A and primate immunodeficiency viruses may provide important information not only on mechanisms of HIV pathogenesis, but also for the development of novel preventive and therapeutic strategies against HIV-1.

Acyclovir derivatives as anti-HIV-1 drugs

Recently, we reported that phosphorylated acyclovir (ACV) inhibits HIV-1 reverse transcriptase in a cell-free system. Also, ACV suppresses HIV-1 replication in human lymphoid tissues coinfected ex vivo with herpesviruses that are capable of phosphorylating this compound. In view of these data, we suggested new studies and new targeted clinical trials to understand these newly discovered features of the herpes-suppressive drugs in HIV-1/HSV–coinfected patients; we thus launched new trials. Also, we started to develop ACV-based drugs that work against HIV-1 without being phosphorylated by HHV in spite of the lack of the HIV-encoded enzyme that recognizes ACV as a substrate for phosphorylation. Unfortunately, the monophosphate itself cannot be used as an efficient anti–HIV agent to bypass the first limiting phosphorylation step because of its instability in biological media and its poor penetration through plasma membranes. To deliver phosphorylated ACV into HIV-infected cells, we applied a ProTide technology that masks the negative charges of the monophosphate with lipophilic groups. In this approach, the nucleoside monophosphate is masked with an aryl moiety and an amino acid ester. Once inside the cell, the phosphoramidate prodrug is activated and converted to the monophosphorylated ACV. We analyzed the effects of the aryl and ester moiety variations as well as that of variation of the amino acid of the ACV ProTides on their anti-HIV activity in isolated cells. L-alanine derivative–containing ProTides showed anti-HIV activity at concentrations far below any that caused cytotoxicity. ACV ProTides with other amino acids, with the exception of L-phenylalanine, were inactive against HIV in cell culture. Enzymatic and molecular modeling studies have been performed in order to better understand the antiviral behavior of these compounds. The next step in this project is to test ACV ProTides in human tissues.

Meanwhile, we studied HIV-1 evolution under the pressure of ACV ProTides. We found that V75I was the dominant mutation of the emerged ACV-resistant HIV-1. We studied the biochemical mechanism underlying this resistance and found that the incorporated ACV monophosphate is vulnerable to excision in the presence of the pyrophosphate donor ATP. V75I compromises binding of the next nucleotide, which can otherwise provide a protection from excision. Further studies of the development and mechanisms of HIV-1 resistance to ACV are necessary for assessing its potential clinical utility in combination with established NRTIs (nucleoside reverse transcriptase inhibitors).

Development of experimental models for studying HIV pathogenesis in human tissues ex vivo

The study of human cell-cell and cell-pathogen interactions that occur in the context of tissue cytoarchitecture is critical for deciphering the mechanisms of many normal and pathogenic processes. Notwithstanding the complexity of HIV pathogenesis in vivo, HIV infection is usually studied in vitro in a relatively primitive system of isolated cells. Earlier, we developed a system of human tonsillar tissue ex vivo and studied the pathogenesis of HIV as well as that of other infectious agents in this system. Now we have extended this system to cervico-vaginal and recto-sigmoid tissues, as these tissues are critical in HIV transmission and pathogenesis. Currently we are adapting ex vivo cervico-vaginal tissue in order to study HIV transmission through the cervical mucus. Also, we found that the developed system supports productive infection by HHV-6, HHV-7, HCMV (HHV-5), HSV-2 (HHV-2), vaccinia virus, measles virus, and West Nile virus as well by the parasite Toxoplasma gondii. In principle, this technique can be adapted to the study of other viruses as well as to that of various normal physiological processes.

To compare various aspects of HIV-1 pathogenesis ex vivo as well as to combine data from different laboratories and apply this system to evaluate microbicides, it is necessary to standardize the procedure of tissue culture and the assays for monitoring HIV. In collaboration with other laboratories, we developed a novel “soft endpoint” method to provide an objective measure of virus replication. The applicability of the soft endpoint has been demonstrated across several different ex vivo tissue types, cultured in different laboratories, and for a candidate microbicide. Statistical analysis showed that different laboratories can provide consistent measurements of anti-HIV-1 microbicide efficacy using the new assay.

Finally, we developed a new methodology to measure HIV p24 in biological samples. This method relies on beads coupled to a high-affinity monoclonal antibody against HIV-1 p24 and to a second complementary monoclonal antibody against another epitope of the same antigen. The developed bead-based assay is simple, sensitive, and inexpensive, offering a wide dynamic measurement range that allows the detection of p24 concentrations over five orders of magnitude. It is in the research community that we currently see the most promising application of this assay because of its low cost and its wide response range. However, this assay may be adapted for diagnostic purposes.

Publications

Grivel JC, Margolis L. Use of human tissue explants to study human infectious agents. Nat Protoc 2009 4:256-269.
Lisco A, Vanpouille C, Margolis LB. A missed point in deciphering the viral synergy between herpes simplex virus and HIV. Lancet Infect Dis 2009 9:522-523.
Biancotto A, Grivel JC, Lisco A, Vanpouille C, Markham PD, Gallo RC, Margolis LB, Lusso P. Evolution of SIV toward RANTES resistance in macaques rapidly progressing to AIDS upon coinfection with HHV-6A. Retrovirology 2009 6:61.
Tchesnokov EP, Obikhod A, Massud I, Lisco A, Vanpouille C, Brichacek B, Balzarini J, McGuigan C, Derudas M, Margolis L, Schinazi RF, Gotte M. Mechanisms associated with HIV-1 resistance to acyclovir by the V75I mutation in reverse transcriptase. J Biol Chem 2009 284:21496-21504.
Lisco A, Vanpouille C, Margolis L. Coinfecting viruses as determinants of HIV disease. Curr HIV/AIDS Rep 2009 6:5-12.

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
Michael Lederman, MD, Case Western Reserve University/University Hospitals of Cleveland, Cleveland, OH
Christopher McGuigan, PhD, Cardiff University, Cardiff, UK
Raymond Schinazi, PhD, DSc, Emory University, Atlanta, GA
Alan Sher, PhD, Laboratory of Parasitic Diseases, NIAID, Bethesda, MD

Contact

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

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