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Transmission and Pathogenesis of HIV-1 and its Copathogens in Human Tissues

Leonid Margolis, PhD, Head, Section on Intercellular Interactions
Jean-Charles Grivel, PhD, Staff Scientist
Christophe Vanpuoille, PhD, Staff Scientist
Anush Arakelyan, PhD, Visiting Fellow
Andrea Introini, MS, PhD Student
Wendy Fitzgerald, BS, Technician

The general goal of the Section of Intercellular Interactions is to understand the mechanisms of pathogenesis and sexual transmission of human pathogens, including the human immunodeficiency virus (HIV). To achieve this goal, we require a comprehensive understanding of the mechanisms of pathogenesis in human tissues, where the critical events of this process occur.

During the past year, we pursued three aims: (i) to investigate the effects of HIV-1 on the network of cytokines in semen of HIV-1–infected men that may play an important role in HIV-1 sexual transmission; (ii) to study the development of multi-targeted drugs to contain HIV-I infection and transmission; and (iii) to design new technologies to study immunological mechanisms associated with the pathogen's transmission and pathogenesis.

Our research of the past year provided new insights into HIV-1 transmission and pathogenesis, leading to new concepts in anti-HIV-1 strategies. In particular, we found that, in addition to modulating the concentrations of individual cytokines, HIV infection in men alters the entire pattern of the seminal cytokine network. The HIV-1–triggered rearrangement of the seminal cytokine network may contribute to the association of common opportunistic infections as well as to the efficiency of male-to-female HIV-1 transmission. Also, in continuation of our translational research, we found that the anti-herpetic drug acyclovir (ACV) inhibits replication of various clinical HIV-1 variants, including ones resistant to commonly approved antivirals. Moreover, based on our knowledge of the molecular mechanisms of anti-HIV activity of ACV, we developed a way to enhance this activity with ribavirin, another clinically approved drug. Finally, an understanding of the basic mechanisms of infection by HIV and other pathogens requires the development of new experimental and diagnostic technologies. We report on one of them, which is based on nanotechnology. Specifically, we developed a new method to identify cells that secrete particular cytokines.

Human immunodeficiency virus imposes rigidity on the cytokine network in semen of chronically HIV-1 infected men.

As HIV-1 infection progresses to acquired immunodeficiency syndrome (AIDS), associated changes in the levels of soluble immunoregulators—cytokines—take place. The disruption of the cytokine network is believed to contribute to immunodeficiency. In previous studies, we found that, upon HIV infection, levels of seminal (and blood) cytokines are significantly altered, reflecting a profound dysregulation of the functional state of immune cells resident in the male genital tract. Although it is well established that the levels of individual cytokines are drastically altered by HIV-1 infection, the interrelations of cytokines that organize cytokines into a network have been studied only in a fragmentary fashion. Indeed, although important, the changes in the levels of individual cytokines give no information as to the global pattern of their concerted interactions. To evaluate the latter, we determined whether cytokines change in a coordinated way by measuring the correlations between the production of each cytokine and those of each of the other cytokines. We investigated the organization of cytokine networks in semen and blood and their alterations upon HIV-1 infection. Specifically, semen was collected at the All-India Institute of Medical Sciences (India) from forty seven therapy-naive HIV-1–infected individuals and HIV-1–uninfected control patients. We measured the levels of 21 cytokines using a multiplex bead array assay and assessed the linear association of continuous variables using the non-parametric Spearman`s rank correlation coefficient. We found that in HIV-1–infected individuals the cytokine networks were more interlocked than in uninfected controls. Indeed, in blood and semen of HIV-1–infected individuals there were, respectively, 68 and 72 statistically significant correlations between cytokines, while in uninfected individuals there were 18 and 21 such correlations. HIV infection resulted in the establishment of new correlations and the strengthening of pre-existing correlations between various cytokines.

In summary, we found that HIV-1 infection had a counter-intuitive effect on cytokines: rather than creating disarray in the cytokine network, HIV-1 imposes rigidity on the network, thus decreasing its degree of freedom. The high rigidity imposed by HIV-1 infection on the cytokine network may contribute to the inability of the immune system to adapt to new microbial challenges, leading to the eventual failure of a fixed and ordered immune system. Therefore, the rigidity triggered by HIV-1 in the network may be one of several important factors leading to immunodeficiency.

Potentiation of anti-HIV-1 activity of an antiherpetic drug acyclovir: the suppression of primary and drug-resistant HIV isolates

Several clinical studies have demonstrated that ACV treatment of HIV-1–infected patients co-infected with herpes simplex virus 2 (HSV-2) delays progression to AIDS and suppresses not only HSV-2, but also HIV-1. We found that ACV, which is metabolized into ACV-triphosphate (ACV-TP) in herpesvirus-infected cells, is incorporated in the form of ACV-monophosphate into the nascent DNA chain instead of dGMP, resulting in the termination of viral DNA elongation, and thus directly inhibits the retrotranscription of HIV-1 laboratory strains. We investigated the anti–HIV-1 activity of ACV not only against laboratory HIV-1 strains, but also against primary HIV-1 clinical isolates of different subtypes and against multidrug-resistant variants. Also, by using ribavirin, which is known to deplete the pool of dGTP, the intracellular counterpart of ACV-triphosphate (ACV-TP), we successfully potentiated anti-HIV activity of ACV.

We first tested the susceptibility of four clinical HIV-1 isolates to concentrations of ACV similar to those achieved in patients treated with oral ACV. In these ex vivo experiments, ACV suppressed the replication of all tested HIV-1 primary isolates, including two of clade C, one of clade A, and one of clade B. Also, the inhibition occurred irrespective of HIV-1 co-receptor usage (CCR5, CXCR4, CCR3, CCR2B, Bob, Bonzo). In general, there was no difference in the suppression of viral replication in ex vivo tonsillar tissues between clinical HIV-1 isolates and laboratory strains, although one of the two clade C isolates (HIV-1_96USNG31) was not as susceptible to as low an ACV concentration as the laboratory isolate HIV-1_LAI.04.

For the future use of ACV or its derivatives in anti-HIV-1 therapy, it is important that ACV remains active against common drug-resistant HIV-1 variants that evolve in the course of regular anti-HIV therapy. We found that ACV efficiently suppressed replication of viruses resistant to the common drugs zidovudine (AZT) and lamivudine (3TC). We also tested a panel of six prototypical infectious multidrug-resistant HIV-1 RT molecular clones. Each clone carries several mutations that occur most frequently in HIV–infected individuals treated with the nucleoside analogue reverse transcriptase inhibitor (NRTI) antiretrovirals. ACV suppressed equally well the replication of the six multidrug-resistant clones and the laboratory strain HIV-1_LAI.04, but an HIV-1 variant that carries K65R RT mutation showed a lower susceptibility to ACV inhibition. Finally, understanding the molecular mechanism of ACV suppression of HIV-1 allowed us to hypothesize that, by increasing the intracellular ACV-TP/dGTP ratio, we would increase the probability of incorporation of ACV-TP rather than dGTP by RT, thus enhancing the anti-HIV activity of ACV. We tested this hypothesis by using ribavirin, which inhibits a key enzyme in the de novo biosynthesis of GTP and dGTP, and found that ribavirin significantly potentiated the anti–HIV-1 activity of ACV in human tissues ex vivo and in single cell cultures.

In summary, we found that in human lymphoid tissue, ACV suppresses the replication of HIV-1 of different clades, of different coreceptor tropisms, and of HIV-1 variants with mutations conferring resistance to the anti-virals currently used in HIV-1 treatment. The combination of ACV with ribavirin may be envisioned as an addition to commonly used anti-viral cocktails.

Development of a universal nanoparticle cell secretion capture assay

Cytokines play an important role in the coordination of the immune response to various pathogens. The identification and full characterization of cells that secrete the cytokines is paramount to the understanding of basic mechanisms of immunity. Currently, immunohistochemistry and flow cytometry are the two main techniques allowing the identification of individual cells secreting a particular protein in general and cytokines in particular. However, because both techniques identify secretory proteins inside the cell, they do not distinguish between cells that secrete the proteins and cells that store them. Moreover, identification by flow cytometry of cells harboring potentially secreted proteins requires additional manipulations that include artificial blocking of the secretory pathway to accumulate the secreted protein inside the cell and the permeabilization of the cell membrane, which compromises cell viability.

We developed a novel, easy, inexpensive, and versatile method that allows the identification of living cells secreting any protein of interest. Our method is based on targeting to the cell surface of nanoparticles that capture the secreted protein on the surface of the secreting cell. The method allows further characterization of a secreting cell by multi-color flow cytometry and does not compromise cell viability. Specifically, we prepared complexes of goat anti-mouse antibodies bound to magnetic nanoparticles with anti-CD45 as a cell-anchoring moiety. Mouse anti-human antibodies against particular cytokines bound to these complexes to capture cytokines on the cell surface. Nanoparticles were separated from unbound antibodies in a magnetic field. The complexes were bound to human PBMCs, which were activated with a polyclonal activator to stimulate secretion. Using our assay, we were able to identify cells secreting IL-2, IFN-γ, as well as MIP-1α, MIP-1β, and RANTES. Thus, our assay allows the identification of cells that secrete both de novo synthesized or stored proteins. The versatility of the technique allows the identification of virtually any cell based on its secreted protein, and the technique's simplicity and ease of application mean that it can be used outside a traditional well-equipped laboratory focused on molecules of immunological interest.

Additional Funding

Intramural-to-India (I-to-I) Program
Intramural-to-Russia (I-to-R) Program

Publications

Graciela A, Lisco A, Vanpouille C, Introini A, Balestra E, van den Oord J, Cihlar T, Perno C, Snoeck R, Margolis LB, Balzarini J. Topical tenofovir as dual-targeted anti-human immunodeficiency virus and anti-herpesvirus microbicide. Cell Host Microbes 2011;10(4):379-389.
Lisco A, Munawwar A, Introini A, Vanpouille C, Saba E, Feng X, Grivel J, Singh S, Margolis LB. Semen of HIV-1-infected individuals: local shedding of herpesviruses and reprogrammed cytokine network. J Infect Dis 2012;205(1):97-105.
Vanpouille C, Arakelyan A, Margolis L. Microbicides: still a long road to success. Trends Microbiol 2012;8:369-375.
Lisco A, Introini A, Munawwar A, Vanpouile C, Grivel J-C, Blank P, Singh S, Margolis LB. HIV-1 imposes rigidity on blood and semen cytokine network. Am J Reprod Immunol 2012;DOI:10.11:1-7.
Fitzgerald W, Grivel J-C. A universal nanoparticle cell secretion capture assay (see Editorial Comment "Beyond surface markers with a universal cell secretion assay"). Cytometry 2012;E-pub ahead of print.

Collaborators

Jan Balzarini, PhD, Rega Institute, Katholieke Universiteit Leuven, Leuven, Belgium
Christopher McGuigan, PhD, Cardiff University, Cardiff, UK
Robin Shattock, PhD, Imperial College, University of London, London, UK
Alexander Shpektor, MD, Moscow State University of Medicine and Dentistry, Moscow, Russia
Sarman Singh, MD, All India Institute of Medical Sciences, New Delhi, India
Elena Vasilieva, MD, Moscow State University of Medicine and Dentistry, Moscow, Russia

Contact

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

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