National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2015 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
  • Jean-Charles Grivel, PhD, Staff Scientist
  • Christophe Vanpouille, PhD, Staff Scientist
  • Anush Arakelyan, PhD, Visiting Fellow
  • Victor Silva, PhD, Visiting Fellow
  • Sonia Zicary, PhD, Visiting Fellow
  • Wendy Fitzgerald, BS, Technician

The general goal of the Section of Intercellular Interactions is to understand the mechanisms of pathogenesis of various pathogens in human tissues, including the human immunodeficiency virus (HIV). Given that the critical events of this pathogenesis occur in tissues, and thus difficult to study in vivo, for our study we used a system of human tissues ex vivo, developed in our Section and now adopted by many investigators, to study viral infections and to test antivirals.

During the last year, we focused on three aims, to: (1) investigate pathogens' interactions mediated by cytokine network; (2) study HIV-1 infectivity in human tissue by analyzing individual virions' individual spikes, that mediate virus-cell fusion; (3) extend our flow virometry technology developed for HIV-1 analysis to other viruses, in particular to Dengue virus; and (4) investigate whether the immunoactivation that we demonstrated to be the consequence of HIV-1 infection plays a role in other human diseases. The research conducted during the last year provided new insights into general mechanisms of the interaction of human pathogens with the infected host, leading to new concepts in therapeutic strategies.

Mosaics of spikes on HIV-1: analysis of individual Envs on individual HIV-1 virions

Owing to its high mutation rate, HIV remains one of the most variable viruses, in particular in the envelope encoded by env. As a result, such quick changes allow the virus to evade host immune responses that target predominantly viral surface proteins. The HIV envelope plays a critical role in HIV infection. Functional HIV envelope protein (Env) or spike is a non-covalently linked trimer of heterodimers, consisting of three surface homodimers of gp120 and trans-membrane gp41 subunits (stumps). The correct conformation of Env is critical for the virus to bind to cell receptors (CD4) and coreceptors (CCR5/CXCR4) and to undergo the complex conformational changes required for plasma membrane fusion and viral entry. Malfunctioning of this machinery renders virions incapable of binding/fusing with cells. The malfunctioning may be mediated by incorporation of dysfunctional forms of Env into the virion membrane or by the instability of incorporated functional Env. Indeed, a significant proportion of virions within any viral population are thought to be non-infectious. Several ways in which the HIV Env can be dysfunctional include uncleaved Env, dimeric or monomeric Env, or gp41 stumps. Each HIV-1 virion carries 7–14 spikes and, in principle, it is possible that on a given virion all spikes are either defective or are all functional, rendering the former virion defective and the latter virion infectious. Alternatively, virions may carry both functional and non-functional Envs in different conformations, as is currently thought. Understanding the extent of this mosaicism is important both for understanding of the basic mechanisms of HIV infection and for the development of new therapeutic and prevention strategies, in particular vaccines. To answer this question it is necessary to analyze the conformation of individual Env proteins on individual virions. To date, however, the majority of the biochemical and immunochemical analysis of HIV surface proteins is performed in bulk so that the diversity of individual virions is lost. Recently, we developed a nanoparticle-based technique called flow virometry, which permits the analysis of antigenicity on individual viral particles. We applied flow virometry to investigate the functional and non-functional conformations of Envs on individual virions, using a panel of antibodies that discriminate between various gp120 conformations. Individual virions were immuno-captured with 15–nm magnetic nanoparticles and stained with antibodies recognizing different conformations of Envs. The resulting complexes were separated from non-bound fluorescent antibodies and their aggregates in a magnetic field and analyzed on a regular commercial flow cytometer.

We found that viruses exhibit little mosaicism: on the majority of virions either all the spikes are functional or all are defective. Only a small subfraction of the virions were mosaic with virions carrying both functional and non-functional Envs. Contribution of this minor fraction in HIV infection of human tissue ex vivo appears to be small. The results of our study suggest that spikes may not be formed independently, and our approach can be used to describe mosaicism of HIV-1 in plasma of individual patients, thus determining individual treatment.

This all-or-nothing viral strategy is likely to aid immune evasion by subverting the focus of humoral responses to generate multiple non-neutralizing antibodies at no cost to infectious virions. Therefore, only the induction of antibodies that target functional Envs, and thus target predominantly infectious viruses, appears to be critical for the development of effective prophylactic strategies.

Maturity of individual dengue virions

The dengue virus (DENV) is a positive single-stranded RNA–positive virus that belongs to the family of Flaviviridae, genus Flavivirus. The diameter of the virus varies between 50–60nm, depending on the maturation state. The virus is enveloped with a lipid membrane, and carries on its surface 180 copies of the envelope (E) glycoprotein, which is responsible for cell attachment and fusion to the plasma membrane, and 180 copies of the structural membrane (M) protein. Viral maturation of DENV is determined by the cleavage of the prM precursor into M protein and pr peptide. Although based on bulk analysis, it is possible to evaluate the presence of prM on DENV virions and thus to determine the average degree of maturation of a viral preparation. Only the analysis of individual viral particles would allow us to distinguish immature from mosaic virions, and thus carry prM, or determine whether there is a fraction of fully mature viruses that do not carry prM. We performed a flow virometry analysis. We collected DENV virions from supernatant of infected BHK-21 and LoVo cells and labeled them with the fluorescent lipidic dye DiI. The labeled viral preparations were purified from floating dye in a discontinuous Optiprep density gradient then filtered through 0.22μm filters. To identify DENV virions among other membrane particles produced by BHK-21 and LoVo cells, we captured them with fluorescently labeled Zenon Alexa Fluor 488 3H5-1-MNPs, specific for the E protein of DENV that is present on all DENV viral envelopes. The MNPs are magnetic nanoparticles with a biopolymer coating consisting of a monolayer of oleic acid and a monolayer of amphiphilic polymer that contains carboxyl groups and can be coupled to amine-containing proteins via a two-step carbodiimide process. We coupled them with 3H5-1 antibodies and then labeled them with Zenon Alexa Fluor 488. We defined a DENV virus as a particle that is labeled with DiI and positive for 3H5-1. After the Optiprep purification gradient, all the membrane particles isolated in our viral preparations carried E protein, thus representing DENV particles. To be able to distinguish between mature and immature/partially mature virions, we used another antibody, 2H2, that is specifically against the prM protein. To validate our technology, we compared maturation of DENV produced in BHK-21 and LoVo cells. The latter are furin-deficient and should thus produce more immature virions, given that furin is required for prM cleavage and therefore for the maturation process. For our flow virometry study, DiI–labeled virus produced from both cell types was stained with 2H2 Alexa Fluor 647 antibodies, then captured by Zenon Alexa Fluor 488 3H5-1-MNPs. In a direct flow analysis of this preparation it would be difficult to distinguish virions from antibodies (or their aggregates) by size or by fluorescence making it impossible to attribute any detected fluorescent signal to labeled viruses or to free-floating antibodies occupying the cytometer interrogation chamber. Therefore, it was critical to separate them physically before the flow analysis. Towards this goal, we ran the preparation in magnetic column, which allowed us to exploit the magnetic properties of our MNPs so that our viral particles captured by 3H5-1-MNPs were retained in the column while free-floating antibodies were washed away during the washing step. Such a separation removes free antibody almost entirely.

We found that, in DENV produced in BHK-21 cells, prM is present on about 45% of DENV particles, while in DENV produced in LoVo cells about 85% of DENV virions carry uncleaved prM on their surface, indicating their non-fully mature state; the difference was statistically highly significant. The existence of mosaic flaviviruses had been documented in earlier electron microscopy studies that revealed co-existing smooth and spiky elements on viral surface. The specificity of DENV capture was evident from the comparison of the number of complexes captured with MNPs coupled to specific 3H5-1 antibodies with those captured with MNPs coupled to non-specific isotype control MNPs.

Flow virometry, earlier applied to the analysis of single HIV virions and used more recently for the analysis of single DENV virions, appears to be a universal method for the analysis of heterogeneity of different viruses, which allows quantification of virions carrying particular surface proteins. Their physical separation will allow us to correlate the antigenic composition of virions with their biological functions.

Cytokines as mediators of immunoactivation by human pathogens.

The change in the cytokine network in response to a pathogen invasion is common for various infections. Previous studies on cytokine concentrations and cytokine networks in spontaneous labor at term and preterm labor have been based on data derived from bioassays for these molecules and from specific individual immunoassays. Given that biological functions are the expression of integrated and interdependent networks of cells and molecules, the study of biological networks, rather than individual cells/molecules, is considered necessary to improve the understanding of the pathophysiology of disease. The objective of this study was to characterize the behavior of the cytokine network in the amniotic fluid of women in preterm labor, according to the presence/absence of intra-amniotic inflammation and microorganisms in the amniotic cavity. Earlier, we had performed such an analysis for HIV infection. We extended and further developed such an analysis to the intra-amniotic infection/inflammation that may lead to spontaneous preterm labor/delivery. We found that: (1) patients with preterm labor and intact membranes who had microbial-associated intra-amniotic inflammation had a higher amniotic fluid cytokine concentration correlation than those without intra-amniotic inflammation. IL-1β, IL-6, MIP-1α, and IL-1α were highly connected nodes (highest degree) in this differential correlation network; (2) patients with sterile intra-amniotic inflammation had correlation patterns of inflammatory-related proteins that were both increased and decreased when compared with those without intra-amniotic inflammation. IL-1α, MIP-1α, and IL-1β were the most connected nodes in this differential correlation network; and (3) there were more coordinated inflammatory-related protein concentrations in the amniotic fluid of women with microbial-associated intra-amniotic inflammation than in those with sterile intra-amniotic inflammation. IL-4 and IL-33 had the largest number of perturbed correlations with other inflammatory-related proteins in the differential correlation network. The observations provide evidence that the cytokine network behaves differently in women with preterm labor according to the presence or absence of intra-amniotic inflammation and/or microorganisms.

In general, our observations show that the cytokine network connectivity with microbial-associated intra-amniotic inflammation is denser and more coordinated than in women with sterile inflammation or without intra-amniotic inflammation. Our network analysis provides deeper insight into understanding the pathophysiological mechanisms of intra-amniotic infection/inflammation in preterm labor, as well as identifying potentially relevant modules of cytokines that correspond to distinct disease pathways in preterm labor. Also, such an approach may help to minimize the number of individual cytokines measured in order to characterize pathologic states, given that some elements of the network may have key roles in the regulation of the entire network/modules. A similar network analysis can now be applied to other pathogens. In particular, we studied the cytokine perturbation in cohorts of HIV–infected individuals receiving antiretroviral therapy. We found that a particular cytokine pattern associated with coinfection with human cytomegalovirus may predispose these individuals to syphilis acquisition.

Immunoactivation as a common pattern for various human diseases

We undertook a broad analysis of the biomedical literature and reviewed our own published results to determine the possible role of immunoactivation in human disease. It appears that many human diseases, including general aging, have one common driving mechanism, an inappropriate and dysregulated chronic immunoactivation. The analysis led us to conclude that immunoactivation can become dysregulated and promote the pathogenesis of diverse diseases with both known and unknown etiologies. Immunoactivation appears to be a common denominator or general mechanism of pathogenesis and may explain the association and similarities in pathology among otherwise unrelated human diseases. For example, immunoactivation may link rheumatoid arthritis, type 2 diabetes, or end-stage renal disease to atherosclerosis. It is possible that immunoactivation caused by irritation of the airway by cigarette smoke may explain why smoking is a common risk factor not only in cancer of the lung, where the products of smoking are deposited, but also in many apparently unrelated pathologies: cardiovascular diseases, preeclampsia, type 2 diabetes, and others. Also, the well known link of stress to cardiovascular disease seems to involve immunoactivation, as suggested earlier, and was evidenced by a recently described stress-induced activation of haemopoetic stem cells and release of high numbers of neutrophils, monocytes, and lymphocytes into blood. Not only the links between apparently unrelated diseases may be explained in the framework of immunoactivation but also similarities in the pathogenic details. For example, as mentioned above, induction of angiogenesis is typical for solid tumors. Similarly, abnormal angiogenesis, albeit of leaky vessels, is the main feature of the wet form of age-related macular degeneration (AMD). In an apparent recognition of this similarity, the same angiogenesis inhibitors are currently used in treatment of AMD and of some solid tumors.

The idea that immunoactivation contributes to pathogenesis and disease progression (the "immunoactivation hypothesis") is not new. At different times, it was introduced in various fields of medicine. However, in most cases the concept of damaging immunoactivation remained confined to the field of research where it was formulated. Now, it is becoming clear that the immunoactivation hypothesis is applicable generally. Thus, immunoactivation, which evolved as a system of host defense against pathogens, can become dysregulated and promote the pathogenesis of diverse diseases with both known and unknown etiologies and even be an important factor in general aging. Identification of general mechanisms of immunoactivation may lead to the development of new therapeutic strategies applicable to many diseases, even before detailed knowledge of their specific etiology and pathogenesis may be available.

Additional Funding

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

Publications

  1. Arakelyan A, Ivanova O, Vasilieva E, Grivel J-C, Margolis L. Antigenic composition of single nano-sized extra-cellular vesicles. Nanomedicine 2015; 11:489-498.
  2. Vanpouille C, Lisco A, Grivel JC, Bassit LC, Kauffman RC, Sanchez J, Schinazi RF, Lederman MM, Rodriguez B, Margolis L. Valacyclovir decreases plasma HIV-1 RNA in HSV-2 seronegative individuals: a randomized placebo-controlled crossover trial. Clin Infect Dis 2015; 60:1708-1714.
  3. Gianella S, Smith DM, Daar ES, Dube MP, Lisco A, Vanpouille C, Margolis L, Haubrich RH, Morris SR. Cytomegalovirus replication predicts syphilis acquisition among HIV-1 infected men who have sex with men. PLoS One 2015; 10:e0130410.
  4. Margolis L. Immunoactivation at the crossroads of human disease. Am J Med 2015; 128:652-656.
  5. Romero R, Grivel J-C, Tarca AL, Chaemsaithong P, Xu Z, Fitzgerald W, Hassan S, Chaiworapongsa T, Margolis L. Evidence of perturbations of the cytokine network in preterm labor. Am J Obstet Gynecol 2015; 213(6):836.e1-836.e18.

Collaborators

  • Jan Balzarini, PhD, Rega Institute, Katholieke Universiteit Leuven, Leuven, Belgium
  • Sergey Kochetkov, PhD, Engelhard Institute of Molecular Biology, Moscow, Russia
  • Michael Lederman, MD, Case Western University, Cleveland, OH
  • Roberto Romero-Galue, MD, DMedSci, Program in Perinatal Research and Obstetrics, NICHD, Detroit, MI
  • Alexandr Shpektor, MD, Moscow Medical University, Moscow, Russia
  • Sarman Singh, MD, All India Institute of Medical Sciences, New Delhi, India
  • 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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