National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2017 Annual Report of the Division of Intramural Research

Pathogenesis of HIV-1 and Its Copathogens in Human Tissues

Leonid Chernomordik
  • Leonid Margolis, PhD, Head, Section on Intercellular Interactions
  • Christophe Vanpouille, PhD, Staff Scientist
  • Anush Arakelyan, PhD, Staff Scientist
  • Rogers Nahui Palomino, BA, Visiting Fellow
  • Sonia Zicary, PhD, Visiting Fellow
  • Wendy Fitzgerald, BS, Technician

The general goal of the Section on Intercellular Interactions is to understand the mechanisms of pathogenesis and of the transmission of human pathogens, in particular of the human immunodeficiency virus (HIV). This year, we continued our studies on viral pathogenesis using our nanotechnology “flow virometry,” developed in our laboratory for the analysis of individual virions and extracellular vesicles (EVs). We studied the distribution of the envelope glycoprotein (Env) in functional and non-functional conformations on the surface of individual HIV-1 virions. Such distribution determines the infectivity of the viral preparation. Also, we analyzed viral proteins on individual EVs released by HIV–infected cells and studied their roles in HIV pathogenesis. We also continued to study defense mechanisms against HIV infection.

During the past year, we (1) analyzed the functionality of Env on individual HIV virions;( 2) identified EVs carrying HIV Env and investigated their role in HIV infection of human tissues ex vivo; (3) determined the mechanisms of inhibitory activity of an anti-cytomegalovirus (CMV) drug against HIV; and (4) investigated the mechanisms of lactobacillus-mediated prevention of HIV vaginal transmission.

Env conformation on individual HIV virions

HIV Env plays a major role in HIV infection, given that the correct trimeric conformation of Env is critical for the virus to bind to cell receptors and co-receptors and fuse with the plasma membrane. Dysfunctional forms of Env render virions incapable of binding to/fusion with cells. Each HIV-1 virion carries 10–14 Env spikes, and in principle it is possible that, on a given virion, all spikes are either defective or functional, rendering the former virion defective and the latter virion infectious. Alternatively, virions may carry both functional and non-functional Envs in various conformations. Determining which of these possibilities exists is important both for understanding the basic mechanisms of HIV infection and for the development of new therapeutic and prevention strategies. In this project, we analyzed the distribution of different forms of Env on single HIV-1 virions, using flow virometry. Specifically, individual HIV virions were captured with 15-nm magnetic nanoparticles decorated with ('capture') antibodies that recognize different conformations of Env.

The results obtained from staining of individual virions with mixtures of antibodies that recognize trimeric (“functional”) and “defective” conformations of Env indicate that only a minor fraction of virions carry both trimeric and defective Envs, while most virions carry either exclusively trimeric or exclusively defective Envs. Accordingly, depletion of virions that carry defective Envs only mildly reduced the infection of human lymphoid tissues. In conclusion, using flow virometry, we demonstrated that most HIV virions do not appear to be mosaic but rather to carry either only functional or only defective Envs.

The observed lack of Env mosaicism for the majority of infectious virions suggests that this all-or-nothing viral strategy likely aids immune evasion by subverting the focus of humoral responses to generate multiple non-neutralizing antibodies at no cost to infectious virions. In contrast, induction of antibodies that target functional Env and thus target predominantly infectious viruses appears to be critical for the development of effective prophylactic strategies.

Extracellular vesicles containing HIV env facilitate HIV infection.

Various cells in vivo and in vitro release extracellular vesicles (EVs), many of which are generated along pathways similar to the ones used by retroviruses, in particular HIV. Consequently, EVs are of the same size and physical properties as this virus and it is therefore almost impossible to separate such EVs from HIV virions. We overcame some of these problems by segregating EVs through the glycoprotein CD45 and/or acetylcholinesterase (AChE), two proteins that are not incorporated into HIV membranes and thus can be used to distinguish EVs from HIV virions. To capture and further identify these EVs, we applied our flow virometry nanotechnology (see #1).

In the present work, we addressed two questions regarding EVs released by HIV-infected cells: first, whether these EVs carry viral Envs, and second, whether EVs affect HIV infection. Using the tetraspanin CD81, which is shared by EVs and HIV, we captured both HIV virions and EVs with magnetic nanoparticles coupled with anti-CD81 antibodies and then identified EVs by the presence of either CD45 or AChE. When we stained our preparation with fluorescent anti-Env antibodies, approximately 50% of the events were positive both for EV markers and for Env. The results were similar whether we used CD45 or AChE for identification of EVs or whether we used prototypical CXCR4 or CCR5 HIV viral preparations. Thus, EVs released by HIV–infected cells carry HIV Env.

The question rises as to whether these EVs, which appear to be a part of HIV preparations, affect viral infection. We addressed this question by inoculating human lymphoid tissue ex vivo with a viral suspension depleted of specific EVs. Depletion of viral preparations of EVs, in particular of those that carry Env, reduced viral infection of human lymphoid tissue ex vivo. The reduction occurred because of EV depletion rather than concomitant depletion of viruses; the amount of p24 or HIV genomic RNA depleted by CD45 magnetic nanoparticles (MNPs) was negligible and did not differ from the depletion of p24 or HIV RNA by isotype control MNPs, that also served as a control for tissue infection.

Thus, our study indicates that HIV–infected cells release not only virions but also EVs and that some EVs carry viral Env, making them indistinguishable not only physically but also semantically from virions, in particular from those that are defective and are not capable of replication. EVs that carry Env identified in our work appear to facilitate HIV infection and may therefore constitute a new therapeutic target for antiviral strategy.

Mechanisms of HIV suppression by anti-CMV drug

Cytomegalovirus (CMV) is a common HIV-1 copathogen. Given that CMV infection is an important contributor to immune activation, the driving force of HIV disease, CMV–suppressive strategies have been investigated. Recent studies showed that valganciclovir, a common anti-CMV drug, is beneficial to CMV/HIV–coinfected individuals by reducing HIV viral load. The anti-HIV effect of this anti-herpetic drug was considered to be indirect and was ascribed to decreasing immunoactivation caused by CMV infection. However, we also investigated whether there was a direct effect of the drug on HIV infection. Towards this goal, we used ganciclovir (GCV), the active form of valganciclovir, and tested the effect of GCV on HIV replication. We treated tonsillar and cervico-vaginal tissues ex vivo with GCV and then inoculated tissues with HIV. On average, we found that GCV suppressed replication of HIV-1 in these tissues by 85–90%.

We deciphered the mechanism of this suppression. GCV is a synthetic purine nucleoside analog of guanine, which must undergo triphosphorylation to become active, with the initial monophosphorylation catalyzed by herpesvirus (HHV)–encoded kinase rather than by cellular kinases. HHVs appear to be necessary to inhibit HIV-1, as GCV did not inhibit HIV-1 in MT-4 cell cultures, which are free of endogenous HHVs. Moreover, we showed that the EC50 of GCV for HIV-1 was approximately 5 μM, whether human tissues were exogenously co-infected with CMV or not. Thus, it seems that kinases expressed by endogenous HHVs present in human tissues activate GCV by adding the first phosphate. The anti-HIV activity of the GCV occurs at clinically relevant concentrations. Indeed, although GCV penetration efficiency and drug clearance were unknown for ex-vivo tissues, the calculated EC50s were in the range of what has been reported in vivo. Using an exogenous template reverse transcriptase (RT) assay, we showed that GCV-monophosphate inhibits HIV-1 RT by acting as a delayed chain terminator.

In conclusion, our results suggest that an anti-CMV strategy using valganciclovir in HIV-1–infected individuals may reduce HIV-1 viral load directly by inhibiting HIV-1 RT. Future trials should evaluate the relative contributions both of indirect mechanisms of HIV-1 suppression mediated by CMV reduction and of the direct suppression of HIV-1 RT by phosphorylated GCV.

Lactobacillus-mediated prevention of HIV-1 vaginal transmission

The vaginal microbiota of healthy reproductive-age women is generally dominated by Lactobacillus species that are involved in maintaining vaginal homeostasis and have been reported to protect against vaginal transmission of HIV. However, the exact mechanism of HIV inhibition by vaginal lactobacilli remains to be fully elucidated. We studied the protective mechanisms of lactobacilli against HIV-1 infection in the context of human cervico-vaginal and lymphoid tissues ex vivo. To address these effects in the context of human tissues, we first colonized them ex vivo with different strains of Lactobacillus that were isolated from vaginal swabs of healthy premenopausal women. Lactobacilli colonized and grew in human tissues ex vivo to densities comparable with those observed in vaginal specimens. To investigate whether lactobacilli release suppressive factors that inhibit HIV-1 replication, we applied bacteria-conditioned culture medium to human tissues ex vivo and infected tissues with HIV. We found that HIV-1 replication was significantly suppressed when human tissues were cultured in Lactobacillus-conditioned medium in both human cervico-vaginal and tonsillar tissues. Although such a medium may contain multiple inhibitory factors, we first focused on two, pH and lactic acid, whose roles in suppressing HIV infection were hypothesized in earlier studies. The pH of conditioned medium of all tested lactobacilli ranged from 6.3 to 6.9. Although acidification may be directly responsible for HIV-1 inhibition, no HIV-1 suppression was observed when the pH was adjusted to 6.9 with HCl in control experiments, which suggested that other factors beyond lowered pH may also be important for HIV-1 inhibition by lactobacilli. One of these factors may be the major Lactobacillus metabolite, namely lactic acid. We found a correlation between the concentration of lactic acid in the Lactobacillus-conditioned medium and its ability to suppress HIV-1 infection in human tissues ex vivo. Addition of lactic acid isomers D and L to tissue culture medium at the concentration that corresponded to their amount released by lactobacilli resulted in HIV-1 inhibition. We found that the L-isomer rather than the D-isomer was predominantly responsible for HIV-1 inhibition. The results thus indicate that lactic acid, in particular its L-isomer, inhibits HIV-1 independently of lowering of the pH.

Moreover, we investigated whether Lactobacillus could have a direct virucidal effect on HIV-1. We incubated an HIV-1 preparation in Lactobacillus-conditioned medium and then tested for HIV-1 infectivity in human tissue culture. We found that incubation of HIV-1 in Lactobacillus–conditioned medium significantly suppressed viral infectivity in cervico-vaginal tissues. We also investigated whether a direct interaction of lactobacilli with HIV may affect the virus and found that virions adhere to lactobacilli. Thus, lactobacilli can directly inactivate HIV-1 virions and also serve as a sink decreasing the amount of free virions.

In summary, in ex vivo systems we identified several mechanisms by which lactobacilli inhibit HIV infection. Extrapolated to in vivo conditions, the mechanisms may explain the protective effect of vaginal Lactobacillus on HIV infection. Further studies are needed to evaluate the potential of altering the spectra of the vaginal microbiota as effective strategies to enhance vaginal health. Human tissues ex vivo may serve as a test system for these strategies.

Additional Funding

  • Office of AIDS Research (OAR), NIH, Intramural Award

Publications

  1. Arakelyan A, Fitzgerald W, King DF, Rogers P, Cheeseman HM, Grivel JC, Shattock RJ, Margolis L. Flow virometry analysis of envelope glycoprotein conformations on individual HIV virions. Sci Rep 2017 7:948.
  2. Arakelyan A, Fitzgerald W, Zicari S, Vanpouille C, Margolis L. Extracellular vesicles carry HIV Env and facilitate Hiv infection of human lymphoid tissue. Sci Rep 2017 7:1695.
  3. Vanpouille C, Bernatchez JA, Lisco A, Arakelyan A, Saba E, Götte M, Margolis L. A common anti-cytomegalovirus drug, ganciclovir, inhibits HIV-1 replication in human tissues ex vivo. AIDS 2017 31:1519.
  4. Ñahui Palomino RA, Zicari S, Vanpouille C, Vitali B, Margolis L. Vaginal lactobacillus inhibits HIV-1 replication in human tissues ex vivo. Front Microbiol 2017 8:906.
  5. Nolte-‘t Hoen E, Cremer T, Gallo R, Margolis L. Extracellular vesicles and viruses: are they close relatives? Proc Natl Acad Sci USA 2016 113:9155-9161.

Collaborators

  • Leonid Chernomordik, PhD, Section on Membrane Biology, NICHD, Bethesda, MD
  • Robert Gallo, MD, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD
  • Matthias Götte, PhD, McGill University, Montreal, Canada
  • Sergey Kochetkov, PhD, Engelhard Institute of Molecular Biology, Moscow, Russia
  • Michael Lederman, MD, Case Western University, Cleveland, OH
  • Roberto Romero-Galue, MD, DMedSci, Perinatology Research Branch, NICHD, Detroit, MI
  • Robin Shattock, PhD, St. George's Hospital Medical School, University of London, United Kingdom
  • Alexandr Shpektor, MD, Moscow Medical University, Moscow, Russia
  • Elena Vasilieva, MD, Moscow Medical University, Moscow, Russia

Contact

For more information, email margolis@helix.nih.gov or visit http://irp.nih.gov/pi/leonid-margolis.

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