HAYNES: DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to methods of determining the degree of immune system destruction in HIV, of determining the prognosis and the course of the disease in AHI, and of determining the need for treatment in AHI. The present invention further relates to a method of monitoring the intensity of HIV infection and predicting the time to progression of AIDS. The methods are based on plasma tests of immune activation and/or apoptosis plus phenotypic and quantitative assays of plasma microparticles. In preferred embodiments, the methods comprise monitoring plasma levels of TRAIL, Fas Ligand and/or TNFR2. Other plasma markers of HIV-I destruction of the immune system can be used either alone or in combination with these tests (one such marker is nuclear protein HMGB-I that is released in plasma from apoptotic cells (Nowak et al, Cytokine 34:17-23 (2006), epub May 11, 2006)).
Microparticles can come from any immune or non-immune cells. Phenotypic characterization of microparticles has diagnostic use. HIV-I virion microparticles are CD45- and thus CD45 can be used to distinguish virions from immune cell microparticles (Esser et al, Virol. 75:6173-6182 (2001)). In general, elevated T cell microparticles indicate destruction of T cells in vivo.
It is important to note that there is heterogeneity in the degree of increase in TNFR2 and Fas Ligand. This indicates that there are patients with massive immune system apoptosis and those with little immune system apoptosis at the time of AHI. Thus, identification of patients who need treatment with anti-HIV drugs (i.e., those with elevated Fas Ligand, TNFR2, microparticles and TRAIL levels) versus those who do not (no or minimal Fas Ligand, TNFR2, TRAIL and CD45, CD3 T cell microparticle levels) is important for directing treatment in AHI.
The data provided in the Examples that follow demonstrate that, at the time of viral load ramp-up in AHI, when a protective antibody needs to be made, there are high levels of microparticles, high TRAIL, high Fas Ligand, and high TNFR2 levels in plasma in many patients. That there is heterogeneity in the levels of these proteins and microparticles indicates that measurement of these parameters individually or in combination provides a prognostic test suitable for use in the setting of AHI to predict the course of HIV infection and time to AIDS. It will be appreciated that one or more of these parameters can also be considered with other markers of apoptosis and cell death and/ or activation (e.g., soluble HMGB-I, CD25, CD69, or other soluble molecules of T cell activation/apoptosi s) .
While the details provided in the Examples below relate specifically to HIV, the invention includes within its scope methods of monitoring the progression and/or clinical course of other infectious diseases as well that are associated with apoptosis during acute infection (Bahl et al, J. Immunol. 176:4284-4295 (2006)) using these same parameters.
Certain aspects of the invention are described in greater detail in the non- limiting Examples that follows. This application is related to US Prov. Appln. No. 60/859,496, filed November 17, 2006, the entire content of which is incorporated herein by reference.
EXAMPLE 1
Several plasma markers of apoptosis have been studied in Blood Bank panels, including Fas Ligand, TNFR2, and TRAIL. TRAIL has been implicated in the activation induced cell death in HIV infection (Katsikis et al, J. Exp. Med. 186:1365-1372 (1997)). Fas Ligand has been found to be elevated in AHI
(Figs. 4 and 5). Elevations of Fas Ligand were observed in the plasma of 1 1/13 patients while it was not elevated in 2/13 — implying no or less apoptosis in some
patients and not others. Similarly, TNFR2 was elevated in 11/13 patients, and again, the same two patients with low Fas Ligand levels had no increases in plasma TNFR2 (Figs. 6 and 7). Finally, TRAIL levels were elevated in all thirteen patients tested, with the two patients that were low in the other markers of apoptosis, low in TRAIL (Figs. 8, 9).
Thus, the massive apoptosis that occurs with acute HIV infection with resulting release of TRAIL, mediation of apoptosis via FAS-FASL interactions, and release of PS containing viral and other particles, all conspire to initially immuno suppress the host, preventing rapid protective B cell responses. Next, flow cytometry phenotypic analysis of the microparticles in AHI sample 6246-15 was performed (Fig. 10). Fig. 11 shows the forward and side scatter of background with phosphate buffered saline, pH 7, purified microparticles from staurosporine treated Jurkat T cells, and the microparticles in patient 6246 plasma on day 11 at the time of peak viremia. Fig. 11 shows the microparticles in plasma with each panel counted for 2 minutes. Fig. 12 shows the phenotype of either purified Jurkat microparticles (pMP) or patient 6246 MP and shows that the microparticles can be phenotypically analyzed and that the AHI patient microparticles are 78.6 % CD3+, 53% CD45 +. CD45 is a surface molecule of T and monocyte cells that is not incorporated into HIV virions (Esser et al, J. Virol. 75:6173-6182 (2001)). Thus, about one half of the microparticles are likely virions in Fig. 12 and one half of the particles are apoptotic particles from HIV-uninfected cells or do not contain HIV genetic material. Of importance, most of the microparticles from patient 6246 are phosphatidylserine (PS) positive (Fig. 13). Similarly, HIV-infected cells are PS+, as shown in Fig. 14.
EXAMPLE 2
A Surface Plasmon Resonance (SPR) based proteomics assay has been developed for characterization of T cell derived microparticles. The assay allows characterization of micorparticles released from immune cells. Figs. 17 and 18 illustrate the application of the SPR assay for the characterization of T cell microparticles. The developed assay described here can be used to monitor the status of antigen-specific T cell responses in terms of TCR specificity, protein phosphorylation and functional markers of activation and apoptosis.
Characterization of antigen specific T cell microparticles using MHC class I and MHC class II tetramers.
Microparticles are released from immune cells like T or B cells or antigen presenting cells upon activation or apopotosis (Distler et al, Athritis & Rheumatism 52:3337-3348 (2005)). However, these microparticles differ quantitatively and qualitatively and vary depending upon the inducing stimulus (Jimenez et al, Thromb. Res. 109:175-180 (2003); Distler et al, Proc. Natl. Acad. Sci. 102:2892-2897 (2005); Kolowas et al, Scand. J. Immunol., 61 :226-233 (2005)). Since these particles carry membrane derived from the parental cell, characterization of surface antigens can be used to identify the source of the microparticles. Microparticles have been reported to contain surface proteins of the immunoglobulin family (TCR, BCR), glycosylphosphatidylinositol-(GPI-) anchored molecules and members of the tetraspan family (Denzer et al, J. Cell Sci. 113:3336-3374 (2000); Koopman et al, Blood 84:1415-1420 (1994); Heijnen et al, 94:3791-3799 (1999)).
The SPR assay described here is a highly sensitive assay for use in detecting antigen specific T cells using peptide-MHC tetramers. In the study described here, T cell micro vesicles were isolated from murine OTl T cells,
which express Vα2Vβ5 TCR specific for SIINFEKL-Kb (Ova-Kb) complex. These microvesicles are heterogenous in size and as assessed by dynamic light scattering and transmission electron microscopy their hydrodynamic radius vary form 400nm to 70nm (Fig. 17A). Microvesicles have been characterized that are spontaneously released in culture, upon cell lysis and following sucrose density centrifugation to harvest detergent resistant microdomains (DRM). The DRM have been reported to be enriched in TCR/CD3 complex and co-receptors (CD8/CD4) molecules (Montixi et al, The EMBO J. 17:5334-5348 (1998)). Reported here is the development of a SPR based assay in which the microvesicles were first anchored on a lipid linker immobilized on the surface of Ll sensor chip (BIAcore Inc.) (Fig. 17 1C). About 1600 Response Unit (RU) of OTl microvesicles were anchored on the Ll sensor chip and, following a brief period of stabilization of the surface, BSA (0.5 mg/mL) was injected for 5 min to block non-specific binding. Injection of Ova-Kb complex gave specific binding to the anchored OTl microvesicles when compared to the control, VSV-Kb complex (Fig. 18). This demonstrates that the SPR assay can be used to monitor the presence of microparticles released from T cells upon activation or apoptosis. Additionally, the capture of microvesicles on the Ll sensor surface in RU units can be quantitated using standard synthetic microvesicles of known size and total phospholipid content. Thus, it is possible to monitor the release of microvesicles from T cells in samples from HIV infected individual and determine the antigen specificity of the TCR on the T cells undergoing apopotosis. The T cell microvesicles will be selectively captured on anti-CD3 immobilized surface and then the antigen specificity of the captured microparticles will be determined using MHC class I or MHC class II tetramers.
The assay described above is not restricted to T cell microparticles but includes characterization of microparticles from B cells as well. B cell specific
tetramers have been developed by (see U.S. Prov. Appln. 60/840,423) and have been tested for specificity in SPR binding assays and by flow cytometry.
Characterization of protein phosphorylation status of T cell microparticles . Microparticles consist of shed plasma membrane fragments and include cytoplasmic elements (Distler et al, Athritis & Rheumatism 52:3337-3348 (2005)). For T cell microparticles, Src kinases, Lck and Fyn, and the adapter protein LAT are associated with lipid microdomains and are the key components involved in T cell signaling (Zhang et al, Immunity, 9:239-246 (1998); Resh et al, Nature, 387:617-620 (1997); Schade & Levine, Biochem. Biophys. Res.
Commun., 296:637-643 (2002)). Thus the methodologies described will serve to study the correlation of protein phosphorylation status identified in microparticles with immune T cell activation or HIV infection.
The strategies employed here include first the characterization of T cell microparticles in terms of antigen specificity using MHC tetramers as described above, and then to define the phoshorylation status of the identified antigen specific microparticles. The captured microparticles will be eluted from the BIAcore sensor surface using the BIAcore 300 recovery capability. The eluted particles will then be lysed and then the phosphorylation status determined by: a) immunoblotting with anti-signaling molecule antibodies (Src kinases, Lck, Fyn, LAT); and b) 2D-liquid chromatography followed by identification by mass spectrometry. Eluted material will be separated using 2D-LC and individual phosphorylated proteins will be analyzed using a MALDI-TOF/TOF. Proteins identified using this approach will be verified to be phosphorylated using conventional immunoblotting with anti-phospho antibodies. This application allows characterization of released microparticles in order to identify the activation state and signaling pathways in immune cells and their changes upon exposure to HIV infection.
Characterization of microparticles to determine the functional status of the parental cell.
As with HIV-I viruses that incorporate cell specific membrane proteins into its viral envelope in the process of budding from infected cells (Hioe et al, J. Virol. 75: 1077-1088 (2001); Laio et al, AIDS Res. Hum. Retro. 16:355-366 (2000)), budding microparticles carry on their cell surface protein markers derived from the parental cell. These surface markers are tell-tale sign of the functional status of the T cells from which they came from. In addition to flow cytometric based pheno typing of microparticles, the capture of microparticles from T cells using the SPR assay described above is a novel methodology for determining the activation status of the T cells. The functional status of the T cells is determined by monitoring the expression of activation markers (CD69, CD25), apopotosis markers (PDl, TRAIL receptors, FAS, Fas L).
EXAMPLE 3
A critical event in HIV-I and SIV infection is virus-induced massive CD4+, CCR5+ T cell loss that is severe in gut associated lymphoid tissues (GALT) (Guadalupe et al, J. Virol. 77:11708-11717 (2003), Brenchley et al, J. Exp. Med. 200:749-759 (2004), Mehandru et al, J. Exp. Med. 200:761-770 (2004)). Depletion of GALT CD4 T cells has been documented at peak viral load in acute SIVmac239 infection (Veazey et al, Science 280:427-431 (1998), Haase, Nat. Rev. Immunol. 5:783-792 (2005), Li et al, Nature 434:1148-1152 (2005), Mattapallil et al, Nature 434:1093-1097 (2005)), as well as within weeks of HIV- 1 transmission in man (Guadalupe et al, J. Virol. 77:11708-11717 (2003), Brenchley et al, J. Exp. Med. 200:749-759 (2004), Mehandru et al, J. Exp. Med. 200:761-770 (2004)). During acute SIV infection, 30-40% of memory CD4+ T cells are infected (Veazey et al, Science 280:427-431 (1998), Haase, Nat. Rev.
Immunol. 5:783-792 (2005), Li et al, Nature 434:1148-1152 (2005), Mattapallil et al, Nature 434:1093-1097 (2005)). The mechanisms of immune cell death during acute HIV-I infection are not known, but may involve induction of apoptotic pathways by HIV Tat, Vpr or gpl20 proteins (Badley et al, Blood 96:2951-1964 (2000), Chase et al, Trends Pharmacol. Sci. 27:4-7 (2006), Boya et al, Biochim. Biophys. Acta 1659:178-189 (2004)), HIV-I infection of CD4+ T cells (Guadalupe et al, J. Virol. 77:1 1708-11717 (2003), Mehandru et al, J. Exp. Med. 200:761-770 (2004), Veazey et al, Science 280:427-431 (1998), Li et al, Nature 434: 1148-1152 (2005)), and induction of uninfected cell death due to killing by molecules such as tumor necrosis factor related apoptotis inducing ligand (TRAIL) (Lum et al, J. Virol. 75:11128-11136 (2001), Herbeuval et al, Clin. Immunol. 123:121-128 (2007)).
The time from HIV-I transmission to establishment of the latently infected pool of CD4 T cells has been termed the window of opportunity within which a preventive HIV-I vaccine has to extinguish HIV-I (Johnston et al, N. Engl. J.
Med. 356:2073-2081 (2007, Wong et al, Biology of Early Infection and Impact on Vaccine Design, pgs. 17-22 (Caister Academic Press, Norfolk, UK (2007)). The latent pool is established at least by the time of symptomatic acute HIV-I infection at the time of seroconversion (-25 days after transmission), although the exact earliest time of establishment of the latent CD4 T cell pool is not known (Wong et al, Biology of Early Infection and Impact on Vaccine Design, pgs. 17- 22 (Caister Academic Press, Norfolk, UK (2007), Chun et al, Proc. Natl. Acad. Sci USA 95:8869-8873 (1998)). Adaptive CD4, CD8 and B cell antibody responses to HIV-I do not appear during the eclipse or viral load ramp-up phases of HIV-I infection, but rather appear coincident with the fall in viral load (VL) and appearance of acute infection symptoms at the end of the window of opportunity (Reynolds et al, J. Virol. 79:9228-9235 (2005), Abel et al, J. Virol. 79:12164-12172 (2005), Fiebig et al, AIDS 17:1871-1879 (2003)). Thus, study
of the events that transpire from transmission until the onset of plasma viremia (the eclipse phase) and during the viral load ramp-up phase of acute HIV-I infection are critical to understanding why immune responses do not occur earlier after HIV-I transmission, and to define what a successful vaccine must overcome to extinguish HIV-I.
In the study described below, the hypothesis is raised that, in addition to gut CD4 T cell loss, delay in HIV-I protective immune responses early on after HIV-I transmission may involve the production of elevated levels of immunosuppressive moieties such as TRAIL, TNFR2 and Fas ligand as well as plasma mi croparticles. If elevations in immunosuppressive molecules, coupled with early CD4+ T cell death, occur early on after HIV-I transmission, then this would define a protected time for HIV-I to replicate while anti-HIV-1 T or B cell responses were suppressed.
To study the eclipse and early viral load ramp-up phases of acute HIV-I infection, archived plasma of plasma donors with samples available before, during, and after HIV-I viral load ramp-up have been used (Fiebig et al (AIDS 17:1871-1879 (2003)). An initial burst of soluble TRAIL was found in plasma soon after the appearance of HIV-I in plasma, corresponding to ~17 days following transmission. Also observed were later elevated plasma TNFR2, Fas ligand and plasma microparticles (MP) levels around the peak of plasma VL. These data implicate TRAIL as an early mediator of cell death in acute HIV-I infection, and demonstrate a narrow window of opportunity in which a HIV-I vaccine must extinguish the transmitted virus.
Experimental Details Plasma Samples. Seroconversion panels (HIV- 1+/HCV-/HBV-, n=30,
HIV-1-/HCV-/HBV+, n=10, and HIV-I-, HCV+/HCV-, n=10) were obtained from ZeptoMetrix Corporation (Buffalo, NY). Each panel consisted of sequential
aliquots of plasma (range 4-30) collected approximately every 3 days during the time of acute infection with HIV-I (Huang et al, J. Immunol. 177:2304-1313 (2006)). HIV-I -/HC V-/HBV-human plasmas (n=25) were obtained from Innovative Research (Southfield, MI). All studies were approved by the Duke University human subjects institutional review board.
Viral Load Testing. Viral load testing of HIV-I plasma plasma donor panels was performed by Quest Diagnostics (Lyndhurst, NJ) (HIV-I RNA PCR Ultra). HCV and HBV viral loads were preformed by Zeptometrix; select HCV viral loads were provided by Philip Norris, Blood Systems Research Institute, San Francisco, CA.
ELISAs For Plasma Markers ofApoptosis. ELISAs for Fas, Fas Ligand, TRAIL, (Diaclone, Besancon Cedex, France), and TNFR2 (Hycult Biotechnology, Uden, The Netherlands) were performed according to the manufacturer's directions. Plasma was assayed undiluted (TRAIL), diluted 1:10 (TNFR2) or diluted 1 :2 (Fas Ligand). Increases in plasma analytes over time were defined as >20% increase of values after TO versus before TO.
Apoptotic Microparticle (MP) Quantification. The number of MP in each plasma sample was determined with flow cytometry. All flow cytometry analyses were performed on the LSRII Flow Cytometer (BD Biosciences, San Jose, CA), and data analyses were performed using FlowJo software (Ashland, OR). All buffers (PBS without calcium and magnesium (Cellgro, Herndon, VA) and formaldehyde (Sigma, St. Louis, MO)) were filtered with a 0.22 μm filter
(Millipore, Billerica, MA), before use in any MP experiment. The buffer used to dilute plasma samples (1% Formaldehyde in PBS without calcium and magnesium), was used to define the background MP count (-150 events counted
in 60 seconds on the flow cytometer). To define the MP gate, FluoSpheres Fluorescent Microspheres (Molecular Probes, Eugene, OR), ranging in size from 0.1 μm to 1 μm, were analyzed on the flow cytometer. The MP gate was drawn around the beads, encompassing the 0.1 μm, 0.2 μm, 0.5 μm, and 1.0 μm beads. Each plasma sample was diluted 1 : 100 and 1 : 1000 in 1 % formaldehyde/PBS, and data acquired for 60 seconds. Optimal sample dilutions were determined experimentally, with the acceptance criteria being the dilution of plasma with abort counts < 5%, and noise to signal ratios < 0.1 (noise to signal ratio ^background MP count in PBS/experimental plasma MP count).
Microparticle Phenotypic Analysis. MP were analyzed by flow cytometry for cell surface markers as described (Hosaka et al, J. Infect. Dis. 178:1030-1039 (1998), Stacey et al and the NIAID Centre for HIV/AIDS Vaccine Immunology. Elevations in plasma levels of innate cytokines prior to the peak in plasma viremia in acute HIV-I infection (2007), Clark et al, N. Engl. J. Med. 324:954- 960 (1991)). Plasma samples (l-2ml) were diluted in 5 ml of filtered saline, and then filtered through a 5 μm filter (Pall Corporation, East Hills, NY). The diluted samples were then centrifuged (lhr at 200,000xg at 40C) (Sorvall RC Ml 50 GX, Thermo Fisher Scientific, Waltham, MA). The top 2.5 ml of supernatant was removed, 2.5 ml of fresh saline added, and samples were centrifuged again (lhr at 200,000xg at 4°C). The pellet was washed X2 in ImI of filtered saline; after the last wash, 900 μl of the supernatant was removed, and the pellet resuspended in the remaining 200 μl of saline. Ten μl of MP suspension was incubated with an antibody and/or annexin V (total volume of 100 μl x 20 minutes, 20° C, in the dark). Saline with 1% BSA (Sigma) was used as staining buffer for incubation with antibodies, and 2.5 mM CaCl2 added to the buffer for annexin V staining. For annexin V controls, 50 mM EDTA was added to the buffer. After incubation,
the volume was adjusted to 500 μl with saline/formaldehyde, and analyzed by flow cytometry within 24 hours. Conjugated antibodies included mouse anti- human CD45-PE, CD3-PE, CD61-PerCp, CCR5-PE, and isotype controls (BD Biosciences, San Jose, CA) and annexin V conjugated to AlexaFluor 647 (Molecular Probes, Eugene, OR).
Electron Microscopy of Plasma Microparticles. Eight ml of plasma was diluted 1 :5 in filtered saline and MP were pelleted (200,000xg x 1 hr, 4°C). Pellets were washed x2 (100,000xg x 30 min.) in ImI of saline. The MP pellet was resuspended in 500 μl of saline and overlaid onto ImI of a 40% sucrose solution (in saline) and MP were centrifuged (100,000xg x 90 min.). The pellets were fixed (1% formaldehyde, 4°C overnight), pelleted (100,00xg x 60 min.), then fixed in 1 % osmium tetroxide. Ultrathin sections were cut and post stained with uranyl acetate and examined on a Philips CMl 2 electron microscope (FEI Co., Hillsboro, OR).
In Vitro Mucosal B cell Culture Model. Tonsils were obtained from pediatric and adult patients who underwent tonsillectomy at the Duke University Medical Center. Tonsils were placed in transport media (RPMI 1640 with L- Glutamine (Gibco, Carlsbad, CA), supplemented with 200U/ml penicillin G, 200 μg/ml streptomycin, 50 μg/ml gentamicin, and 1 μg/ml amphotericin B (Sigma) upon excision and transported to the laboratory for processing within 4 hours. The tonsils were washed extensively in RPMI 1640 with L-Glutamine supplemented with 100 U/ml penicillin G, 100 μg/ml streptomycin, 50 μg/ml gentamicin, and 2 μg/ml amphotericin B to prevent bacterial and fungal contamination. To isolate tonsillar lymphocytes, the specimens were mechanically minced and teased with sterile forceps, and the resulting single-cell
suspension was processed through a 70 μm nylon cell strainer (BD Biosciences). Lymphocytes were separated with lymphocyte separation media (Fisher Scientific, Pittsburg, PA) and washed twice. Cell number and viability were determined with a Guava EasyCyte Mini (Hayward, CA) per the manufacturer's instructions. Cells were cultured in RPMI 1640 supplemented withlOO U/ml penicillin G, 100 μg/ml streptomycin, 25 μg/ml gentamicin, 1 μg/ml amphotericin B, and 10% FBS (Gemini Bioproducts, West Sacramento, CA), at a density of 1x106 cells/ml in total volumes of ImI in polystyrene 5ml round-bottom tubes (BD Biosciences). To stimulate antibody production, cells were cultured with an optimized cocktail of oCpG (6 μg/ml) (Coley Pharmaceutical Group, Wellesley, MA), and pokeweed mitogen extract (PWM) 1/2000 (Crotty et al, J. Immunol. Methods 286:111-122 (2004)). After 5 days of culture, supernatants were harvested and total IgG and IgA present in culture supernatants was quantified by ELISA. Briefly, 96-well ELISA plates (Corning, Corning, NY) were coated with purified murine anti-human IgG Fc (HRL, Baltimore, MD) or purified murine anti-human IgA (BD Pharmingen) in 0.1 M NaHCO3 overnight. Wells were washed x3 with wash buffer (PBS with 0.1% Tween (Sigma)). Wells were blocked with dilution buffer (1% FBS, 0.5% BSA (Sigma) and 0.05% Tween) for 2 hours at 200C. Wells were washed x3, and supernatants were added diluted 1 :2 with dilution buffer. Plates were incubated overnight at 4°C, and washed x3.
Biotinylated murine anti-human IgG Fc (HRL) or biotinylated murine anti-human IgA (BD Pharmingen) was added, and plates were incubated for 2 hours at 200C. After washing x3, horseradish peroxidase streptavidin (Vector Laboratories, Burlingame, CA) was added and plates incubated for 45 minutes at 200C. Plates were washed x3, and the assay was developed using a 3,3',5,5' tetra- methylbenzidine (TMB), substrate and stop solution system (KPL, Gaithersburg,
MD). Standard curves were constructed using serial dilutions of purified human IgG and IgA (Sigma).
Microparticles were generated by treatment of normal donor PBMC or tonsil cells with staurosporine Bell et al, Am. J. Physiol. Cell Physiol. 291 :C1318- Cl 325 (2006)). Cell viability and cell counts were performed on the Guava EasyCyte Mini, and 5x107 cells were cultured in 5 ml RPMI 1640 with L- glutamine supplemented with 25 μg/ml of gentamicin, 10% FBS, and l μM staurosporine (Sigma). After culture overnight, the cells and supernatant were collected, and centrifuged X2 (5min at 400xg at 40C). The cell-free supernatant was then centrifuged in an ultracentrifuge to harvest the MP (30 min at 200,000xg at 4°C) and MP were washed Xl with RPMI 1640, resuspended in 1000 μl of fresh RPMI, and 100 μl of MP suspension was added to select tonsil cell cultures (or varying volumes to determine dose dependency).
Statistical Analyses. Statistical Analysis was performed as outlined in the figures and descriptions thereof using boxplot analyses, Student's t tests, and Wilcoxon Rank Sum analyses. To establish a reference point throughout all the plasma seroconversion panels, Time 0 (TO) was defined as the date when viral load reached 100 copies/ml for HIV-I, 600 copies/ml for HCV, and 700 copies/ml for HBV. To determine the percent increase in plasma markers of apoptosis during HIV-I, HBV, and HCV infections, the mean TRAIL, TNFR2, or Fas Ligand level before Day 0 was compared to the mean level after Day 0, and percent increase was calculated ([(mean after day 0 - mean before day 0)/mean after day 0] x 100).
Results
TRAIL, TNFR2 and Fas ligand plasma levels are elevated during acute HIV-I infection.
A timepoint (TO) was determined for each of 30 HIV-I, 10 HCV and 10 HBV patients, defined as the lower limits of detection for each viral load determination (Fig. 19). Soluble TRAIL, TNFR2, and Fas Ligand were next assayed in sequential plasma samples of each plasma donor (Fig. 20A). The percent change in plasma plasma TRAIL, TNFR2 and Fas ligand levels were determined by comparing the mean analyte level before T0 to the mean level after T0; 27/30 demonstrated increases in TRAIL, 26/30 had increased TNFR2, and 22/30 had increased Fas ligand levels by these criteria (Fig. 20B).
Hepatitis B and C infections have been reported to induce cell death of hepatocytes (Chase et al, Trends Pharmacol. Sci. 27:4-7 (2006), Chou et al, J. Immunol. 174:2160-2166 (2005)). As controls, HCV and HBV acutely infected subjects demonstrated a > 20% rise in TRAIL, TNFR2 or Fas ligand only in 0/10, 3/10, and 2/10 HBV acutely infected subjects, and in 1/10, 6/10 and 7/10 HCV acutely infected subjects, respectively (Figs. 2OC, 21B).
Second, cell death plasma analyte levels at the time of peak viral load were compared to samples drawn from subjects before viral load ramp-up, and as well, compared with uninfected plasmas. The mean TRAIL, TNFR2, and Fas ligand levels at the time of peak viral load were significantly different from the earliest plasma sample drawn from each acute HIV-I infected patient before To (p=0.0075 for TRAIL, p=1.18xlθ-5 for TNRF2 and p=3.88xlθ"6 for Fas ligand) (Fig. 21A). The peak TRAIL, TNFR2 and Fas ligand levels were also significantly different from the levels of TRAIL, TNFR2, and Fas ligand in uninfected plasma sample controls (p=2.16xlθ"8, p=6.16xlθ"9, and p=1.64xlθ"6 , respectively) (Fig. 21A).
Third, to investigate the timing of peak levels of TRAIL, TNFR2 and Fas ligand compared to peak plasma VL, a determination was made of the temporal relationship between an apoptotic analyte peak compared to the peak plasma VL. Also determined was the number of subjects that had peaks in plasma cell death analytes occurring before, coincident with or following the peak in HIV-I viral load (Fig. 21B). The majority of acute HIV-I infection subjects (30/30 for TRAIL, 27/30 for TNFR2, and 26/30 for Fas ligand) demonstrated peak analyte levels occurring within a 30-day time frame (i.e., 15 days before, at the time of, or within 15 days after the viral load peak). Of particular interest, the majority of subjects' TRAIL levels (21 /30) peaked before the peak viral load, while TNFR2 and Fas ligand levels more often peaked coincident with viral load (Fig. 21B).
Fourth, to analyze the timing of peak analyte levels relative to the rate of viral expansion during viral load ramp-up, paired Wilcoxon rank tests were performed (Fig. 21 C). The day of the peak viral expansion rate indicates the day following To on which the virus was replicating at the maximum rate. In 24 plasma donors in which the rate of VL expansion could be calculated, the peak viral expansion rate occurred on mean day 5.5 following T0 (Fig. 21C). Plasma TRAIL levels peaked 1.7 days after maximal viral expansion rate (day 7.2 after To) while TNFR2 levels peaked 7.5 days (day 13 after To), and Fas ligand levels peaked 9.8 days (day 15.3 after T0) after the time of maximal rate of viral expansion. Plasma donor HIV-I VL reached its peak an average of 13.9 days after TO (median 13 days, interquartile range 3 days), indicating that TRAIL levels peaked well before VL peaked, while TNFR2 and Fas ligand reached peak levels close to the time of highest VL levels. The mean of the peak plasma TRAIL levels was 201 lpg/ml with a range of 886-4138pg/ml. This level of TRAIL is well within the biologically relevant concentration range of an activity for induction apoptosis in immune cells21.
Quantitative flow cytometry analysis of plasma microparticles. Plasma MP are a normal by-product of a variety of types of activated or apoptotic cells, or are derived from multivesicular bodies (exosomes) (Distler et al, Autoimmunity 39:683-690 (2006), Piccin et al, Blood Rev. 21 :157-171 (2007) (Fig. 25). Eighteen of thirty (60%) plasma donors demonstrated peak MP levels near (within 15 days before or 15 days after T0) the peak in viral load, and 1 1 of these 18 peaks occurred immediately before the peak in viral load, while 4/18 peaked at the time of VL peak (Table 1 and Fig. 22A). Five often (50%) HCV donors had similar elevations in MP, but only two often (20%) HBV panels studied had elevations in MP levels near the peak VL (Fig. 22B).
Table 1
The morphology of MP at the time of peak viral load was studied from acute HIV-I infection patient 6244 on sucrose gradient purified MP from the time of both peak VL and peak MPs (day 10 in Fig. 22A) and found to be heterogeneous in size ranging from IOnm to lOOOnm (Fig. 23A).
Phenotypic characterization of plasma microparticles. Most studies of phenotypic analysis of MP used fresh plasma that was processed within hours (Jy
et al, J. Thromb. Haemost. 2:1842-1851 (2004)), whereas the plasma donor samples in this study have been frozen/thawed at least twice. It was found that two freeze/thaw cycles markedly decreased the percentage of CD3, CD45+, CD61+ and annexin+ MPs (Fig. 26). Thus, unable to accurately quantitate the 5 phenotype of plasma MP, MP was analyzed for annexin and CCR5 expression in a qualitative manner to determine if MP expressing annexin or CCR5 were present at the time of peak MP in five plasma donor peak MP samples vs. uninfected first samples. Both annexin+ and CCR5+ MP were present in the plasma donor plasmas; for annexin, the mean %+ at MP peak was 12%, range l o 2.3% - 38.0%, and for CCR5+ MP, %+ mean at the MP peak was 5.7,%, range 1.1% - 12.6% (Figs. 23B, 23C). For CCR5+ but not for annexin+ MP, there was a trend in higher MP numbers at the time of peak MP compared to the first panel sample (Fig. 23C).
While the average peak HIV-I VL level was 1,421,628 copies/ml, the
15 average peak of total MPs was 606,881,733/ml. Thus, there was an average of 427-fold more MP than virions present in plasma at their peaks during acute HIV- 1 infection.
Table 2
*Corrected MP levels - Quantitation of MP levels in fresh plasma vs. 2x frozen /thawed plasma showed a ~20% increase in MP levels in 2x frozen/thawed plasma. Thus, the mean MP level was corrected by -20%.
MP-Induced B Cell Suppression In Vitro. While plasma MPs have potent known suppressive effects on macrophages and DCs (Hoffmann et al, J. Immunol. 174:1393-1404 (2005), Huynh et al, J. Clin. Invest. 109:41-50 (2002)), only one study has suggested MP may inhibit B cell activation (Koppler et al, Eur. J. Immunol. 36:648-660 (2006)). There was particular interest in MP effects on human memory B cell activation, since what is desired is a rapid virus-induced memory B cell response after transmission. To determine if PBMC-derived or tonsil leukocyte-derived MP could be suppressive for memory B cell activation, a memory B cell Ig induction assay was used using pokeweed mitogen (PWM) + class B oCpG (Crotty et al, J. Immunol. Methods 286:11 1-122 (2004)). The addition of MP in PWM-stimulated tonsil cell cultures reduced total IgG and IgA production by 70.8 % +/- SEM for IgG (p=0.0064) and 94.2% +/- SEM for IgA (p=0.00004) (Fig. 24A); B cell suppression by MPs was dose-dependent (Fig. 24B). Similar results were observed when MP were generated from autologous tonsil leukocytes or from the Jurkat T cell line (data not shown).
In summary, a major finding in this study is the early appearance of a peak of TRAIL at 17 days of transmission in plasma donors, and implies the TRAIL/DR5 in a key pathway in HIV-I induced cell death immediately following transmission. An IFN-α, TRAIL, DR5 pathway of CD4+ T cell apoptosis has been proposed for chronic HIV-I infection based on in vitro studies and on studies in HIV-1+ progressor tonsillar tissues (Lum et al, J. Virol. 75:11128- 11136 (2001), Herbeuval et al, Clin. Immunol. 123:121-128 (2007), Herbeuvel et al, Blood 106:3524-3531 (2005)). CD4+ T cells in infected subjects are more sensitive to TRAIL-mediated apoptosis than are CD4+ T cells from uninfected subjects due to upregulated TRAIL receptor DR5 (Lum et al, J. Virol. 75:11128- 11136 (2001), Herbeuval et al, Clin. Immunol. 123:121-128 (2007), Herbeuvel et al, Blood 106:3524-3531 (2005), Jeremias et al, Eur. J. Immunol. 28:143-152
(1998)). In vitro, HIV-I gpl20 (Herveuval et al, Blood 105:2458-2464 (2005)) induces monocyte and plasmacytoid dendritic cell IFN-α, which in turn induces CD4+ T cell and monocyte/macrophage TRAIL (Lum et al, J. Virol. 75:11128- 11136 (2001), Herbeuval et al, Clin. Immunol. 123:121-128 (2007), Herbeuvel et al, Blood 106:3524-3531 (2005)). HIV-I Tat has also been reported to induce TRAIL as a mechanism of bystander killing of CD4+ T cells (Yang et al, J. Virol. 77:6700-6708 (2003)).
An important question is why do plasma TRAIL levels peak earlier after HIV-I transmission than do plasma Fas ligand, TNFR2 and MP? Plasma elevations of TRAIL, Fas ligand and TNFR2 occur in chronic HIV-I, and can be induced by immune cell activation, cell death, or both (Herveuval et al, Blood 105:2458-2464 (2005), Aukrust et al, J. Infect. Dis. 169:420-424 (1994), Hober et al, Infection 24:213-217 (1996), Hosaka et al, J. Infect. Dis. 178:1030-1039 (1998)). Stacy et al (Stacey et al and the NIAID Centre for HIV/ AIDS Vaccine Immunology. Elevations in plasma levels of innate cytokines prior to the peak in plasma viremia in acute HIV-I infection (2007)) have found a burst of IFN-α in the same plasma donors that coincides with the timing of the TRAIL peak seen in this study . Thus, the plasma TRAIL peak that precedes the VL plasma peak may be due either to early apoptosis, but may also result from immune activation and pDC production of IFN-αin response to rising VL. It is hypothesized that the later appearance of elevated plasma Fas ligand, TNFR2 and microparticles maybe the result of, or in response to, massive cell death, as this peak comes at an analogous time to the cell death peak documented in experimental SIV infection in rhesus macaques (Veazey et al, Science 280:427-431 (1998), Haase, Nat. Rev. Immunol. 5:783-792 (2005), Li et al, Nature 434:1148-1152 (2005), Mattapallil et al, Nature 434:1093-1097 (2005)).
Veazey (Mattapallil et al, Nature 434:1093-1097 (2005)) noted the onset of CD4+ gut T cell loss as early as 7 days after SIV infection . In humans,
Guadalupe et al (J. Virol. 77:11708-11717 (2003), Mehandru et al, J. Exp. Med. 200:761-770 (2004) and Mehandru and colleagues (Brenchley et al, J. Exp. Med. 200:749-759 (2004)) have studied 2, 1 and 9 patients, respectively, during the first month of HIV-I infection and found depletion of gut CD4+ T cells. The eclipse phase of HIV-I infection is the time from transmission until the appearance of plasma viremia, and is estimated to be 10 days with a range of 7-21 days (Clark et al, N. Engl. J. Med. 324:954-960 (1991), Gaines et al, BMJ 297:1363-1368 (1988), Littl et al, J. Exp. Med. 190:841-850 (1999), Schacker et al, Ann. Intern. Med. 125:257-264 (1996)). The time from appearance of HIV-I viremia until the first antibody response and symptomatic HIV-I infection (and therefore establishment of the latent pool) is approximately 14 days (Cooper et al, J. Infect. Dis. 155:1113-1118 (1987), Daar et al, N. Engl. J. med. 324:961-964 (1991), Gaines et al, Lancet 1 :1249-1253 (1987)). Thus, the maximal window of opportunity for preventive HIV-I vaccine efficacy without cell death-induced immune suppression is approximately 24 days. With mediators of apoptosis and immune suppression present as early as day 17 following transmission (10 days average eclipse phase + onset of TRAIL 7 days after T0), the window of opportunity is narrowed to ~14-17 days.
The presence of TRAIL, TNFR2 and elevated MP during this early period of acute HIV-I infection suggests at least four potential mechanisms of immunosuppression. First, direct HIV-I infection results in loss of a substantial proportion of CD4+ T cells, although the numbers of infected cells does not account for all CD4+ T cell depletion ((Guadalupe et al, J. Virol. 77:11708-11717 (2003), Brenchley et al, J. Exp. Med. 200:749-759 (2004), Mehandru et al, J. Exp. Med. 200:761-770 (2004), Fiebig et al, AIDS 17:1871-1879 (2003)). Second, in uninfected CD4+ T cells, TRAIL induces bystander killing ((Lum et al, J. Virol. 75:11128-1 1136 (2001), Herbeuval et al, Clin. Immunol. 123:121-128 (2007), Herveuval et al, Blood 105:2458-2464 (2005)). In this regard, Miura et al (J. Exp.
Med. 193:651-660 (2001)) have shown that administration of an anti-TRAIL mAb in HIV-I infected hu-PBL-NOD-SCID mice markedly reduces CD4+ T cell apoptosis.
Third, suppression of immune responses can be mediated by T cell MP (Huang et al, J. Immunol. 177:2304-1313 (2006), Distler et al, Arth. Rheum.
52:33337-3348 (2005), Krysko et al, Apoptosis 11:1709-1726 (2006)). CXCR4+ and CCR5+ MP can transfer co-receptors to co-receptor negative cells making them susceptible to HIV-I (Mack et al, Nat. Med. 6:769-775 (2000), Rozmyslowicz et al, AIDS 17:33-42 (2003)). Phagocytosis of MP by macrophages releases TGF- βprostaglandin E2 and IL-10 that can inhibit antigen-specific T and B cell responses (Huang et al, J. Immunol. 177:2304-1313 (2006), Hoffmann et al, J. Immunol. 174:1393-1404 (2005), Huynh et al, J. Clin. Invest. 109:41-50 (2002)). In this regard, Estes et al (J. Infect. Dis. 193:703-712 (2006)). have demonstrated dramatic increases in lymph node TGF-β and IL-10 on day 12 following SIV infection. Importantly, it has been directly shown that PBMC and tonsillar cell MP can directly inhibit memory B cell activation (Fig. 24).
Finally, both Fas ligand and TRAIL are incorporated into MP (Huynh et al, J. Clin. Invest. 109:41-50 (2002), Koppler et al, Eur. J. Immunol. 36:648-660 (2006), Crotty et al, J. Immunol. Methods 286: 111-122 (2004)). Fas ligand expressing MP can directly induce apoptosis in nearby cells (Huang et al, J. Immunol. 177:2304-1313 (2006), Jodo et al, J. Biol. Chem. 276:39938-39944 (2001), Monleon et al, J. Immunol. 167:6736-6744 (2001)) activated T cells can be the target of Fas ligand mediated proapoptotic microvesicles (Monleon et al, J. Immunol. 167:6736-6744 (2001)). Salvato et al (Clinical and Developmental Immunology (2008)) have recently suggested that treatment of SIV-infected macaques with a mAb against Fas ligand attenuates disease and may lead to elevated antibody responses to SIV.
Thus, the production of high levels of biologically active plasma mediators and byproducts of cell death during the first two to three weeks of HIV- 1 transmission raises the notion that the window of opportunity for a preventive vaccine to work may be shorter than previously thought, ie within the first 14-17 days of transmission, placing considerable constraints on the time available for development of robust anti-HIV-1 immunity following transmission. Preventive vaccine candidates may need to target HIV-I molecules that induce cell death and be designed to induce protective immune responses to HIV-I that will either be at maximum inhibitory levels at the time of transmission, or be boosted within hours to days as a secondary immune response to extinguish HIV-I before HIV-I- induced immunosuppression occurs.
Inhibition of cell death and immunosuppressive MP mediated effects by a vaccine for HIV or other infectious agents may be important as well. This could be accomplished, for example, by an HIV vaccine component inducing anti-lipid antibodies or antibodies against other components of microparticles to facilitate clearance of microparticles and/or to block microparticle toxic effects.
Another use of the data herein is as a rationale for the treatment of HIV-I. For example, antibodies against TNFR or TNF-α, antiphosphatidylserine antibodies or other inhibitors of cell death (Fas-Fc as an inhibitor of FAS-FAS ligand interactions and DR5-Fc as an inhibitor of TRAIL DR5 interactions) can be used to inhibit cell death in HIV as a therapy.
All documents and other information sources cited above are hereby incorporated in their entirety by reference.
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