Human Plasma Enhances the Infectivity of ... - Journal of Virology

JOURNAL OF VIROLOGY, Oct. 1995, p. 6054–6062 0022-538X/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 69, No. 10

Human Plasma Enhances the Infectivity of Primary Human Immunodeficiency Virus Type 1 Isolates in Peripheral Blood Mononuclear Cells and Monocyte-Derived Macrophages SUH-CHIN WU,1,2* JOHN L. SPOUGE,1 SHAWN R. CONLEY,3 WEN-PO TSAI,4 MICHAEL J. MERGES,2 AND PETER L. NARA2 National Center for Biotechnology Information, National Library of Medicine, Bethesda, Maryland 20894,1 and Virus Biology Section, Laboratory of Tumor Cell Biology,2 and Laboratory of Biochemical Physiology,4 National Cancer Institute-Frederick Cancer Research and Development Center, and Biological Carcinogenesis and Development Program, PRI/DynCorp,3 Frederick, Maryland 21702 Received 15 March 1995/Accepted 22 June 1995

Physiological microenvironments such as blood, seminal plasma, mucosal secretions, or lymphatic fluids may influence the biology of the virus-host cell and immune interactions for human immunodeficiency virus type 1 (HIV-1). Relative to media, physiological levels of human plasma were found to enhance the infectivity of HIV-1 primary isolates in both phytohemagglutinin-stimulated peripheral blood mononuclear cells and monocyte-derived macrophages. Enhancement was observed only when plasma was present during the viruscell incubation and resulted in a 3- to 30-fold increase in virus titers in all of the four primary isolates tested. Both infectivity and virion binding experiments demonstrated a slow, time-dependent process generally requiring between 1 and 10 h. Human plasma collected in anticoagulants CPDA-1 and heparin, but not EDTA, exhibited this effect at concentrations from 90 to 40%. Furthermore, heat-inactivated plasma resulted in a loss of enhancement in peripheral blood mononuclear cells but not in monocyte-derived macrophages. Physiological concentrations of human plasma appear to recruit additional infectivity, thus increasing the infectious potential of the virus inoculum. to recruit more infectious viral particles in both peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDMs).

The major targets for human immunodeficiency virus type 1 (HIV-1) infection are CD41 T lymphocytes and cells of monocyte/macrophage lineage. Viral infectivity and tropism is a complex and poorly understood process. HIV-1 entry via the CD4 ligand (8, 23) and some other possible additional surface molecule(s) (3, 6, 16, 19, 29) involves viral adsorption and cooperative recruitment of host cell receptors (25), followed by either direct viral fusion with cell membrane (28, 53) or receptor-mediated endocytosis (41) before uncoating of the virus into host cell cytosol and the synthesis of viral DNA. Much insight into virus-cell interactions has been gained from these studies; however, the in vitro systems used for these analyses studied the phenomena in the absence of various other physiological host factors as might occur in vivo. Physiological microenvironments from which infectious HIV-1 has been isolated such as whole blood, blood plasma, seminal plasma, cerebrospinal fluid, mucosal secretions, milk, or lymphatic fluids may influence the biology of the virus-host cell and immune interactions for HIV-1 (reviewed in reference 27). Of these, only a few examples such as seminal plasma (57) and vaginal secretions (32) have been reported and found to enhance virus infectivity. Recently human plasma was observed to increase the infectivity of HIV-1 (36), whereas numerous fresh serum samples from other species failed to enhance infectivity (20). We have extended this earlier work to further investigate the role of human plasma on infections of HIV-1 primary isolates by using the primary cells. The results showed that human plasma increased the maximal infectious titers for primary isolates. Therefore, human plasma appears

MATERIALS AND METHODS Cells. T-cell lines H9 and CEM-SS were grown in RPMI 1640 (Gibco, Grand Island, N.Y.) containing 10% fetal bovine serum (FBS; Gibco) and 1% PSN antibiotic mixture (Gibco) and used only during log-phase growth as previously described (26). PBMCs from normal human blood donors (Clinical Center, National Institutes of Health [NIH], Bethesda, Md.) were collected through Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) density gradients, washed in phosphate-buffered saline (PBS) and ACK lysing buffer (Biofluids, Rockville, Md.), and then subjected to 2 days of stimulation in 2 mg of phytohemagglutinin (Boehringer Mannheim, Indianapolis, Ind.) per ml and 1 day of culture in medium (RPMI 1640, 20% FBS, 1% PSN) containing 32 U of interleukin 2 per ml. MDMs were obtained from the adherent cells of Ficoll-collected fresh PBMCs in RPMI 1640 containing 2% FBS for 1 to 2 h and incubated in the same medium containing 5% FBS for an additional 16 to 20 h. These cells were then transferred to polypropylene boxes for 5 days of activation of adherence to macrophages in RPMI 1640 containing 10% human type AB serum (HAB; Sigma, St. Louis, Mo.) as previously described (55). Viruses. Laboratory-adapted (lab) strains HIV-1HXB3, HIV-1MN, and HIV1RF were grown in H9 cells and collected by an acute harvest protocol (26). Primary isolates HIV-1108 and HIV-1MR452 were provided by A. J. Conley and E. A. Emini, Merck Research Laboratories, West Point, Pa. (5), and harvested from PBMCs. Primary isolate HIV-1ADA was isolated from a male with AIDS-related Kaposi’s sarcoma (15) (provided by H. E. Gendelman, University of Nebraska Medical Center, Omaha). Primary isolate HIV-1D117III was isolated from an infant transmitted by the mother (47) (provided by Horst Ruppach and H. Rubsamen-Waigmann, Georg-Speyer-Haus Chemotherapy Research Institute, Frankfurt, Germany) and prepared from MDMs. All primary isolates from both primary cell types were maintained at low passage numbers and harvested 3 days following a complete replacement of medium. The titer of each virus stock was determined by endpoint titration on the cell type used for production of the virus stock. A series of fourfold dilutions was made from virus stock in medium, mixed with the cells for 1 h, subsequently washed three times to remove the residual viruses, and replaced with fresh medium. Virus titers were calculated as 50% tissue culture infective doses per milliliter of inoculum, using a computer ID50 (50% infectious dilution) package based on Fisher’s statistical model (12, 13) (available through John Spouge, National Center for Biotechnology Information, National Library of Medicine, NIH) as shown in Table 1.

* Corresponding author. Mailing address: Virus Biology Section, Laboratory of Tumor Cell Biology, NCI-FCRDC, Building 560, R 12-92, Frederick, MD 21702. Phone: (301) 846-1336 Fax: (301) 8466194. Electronic mail address: [email protected]. 6054

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TABLE 1. Infections of PBMC, MDM, and T-cell lines by primary isolates and lab strains of HIV-1 Virus

Cell type for virus stock production

Cell susceptibilityb

Titera

Classification PBMC

MDM

H9 or CEM-SS

HIV-1HXB3 HIV-1MN HIV-1RF

H9 H9 H9

8.0 3 10 2.2 3 104 1.0 3 106

Lab strain Lab strain Lab strain

111 111 111

1 2 1

111 111 111

HIV-1108 HIV-1MR452 HIV-1ADA HIV-1D117III

PBMC PBMC MDM MDM

5.9 3 104 7.5 3 104 3.1 3 103 4.2 3 103

Primary Primary Primary Primary

111 111 111 111

2 2 111 111

2 2 2 2

4

isolate isolate isolate isolate

a

Determined as 50% tissue culture infective dose per milliliter on the cell type used for virus stock production. Determined by measuring HIV-1 p24 antigen in culture supernatants. Data represent the peak titers of p24 during 4 weeks postinfection. 111, p24 . 50 ng/ml; 1, 5 ng/ml . p24 . 0.5 ng/ml; 2, undetectable p24 in our assay system. b

Human and chimpanzee plasma. Human plasma from normal HIV-1-negative blood donors serving the blood bank at the NIH Clinical Center were collected by a standard clinical protocol, using the anticoagulant CPDA-1 (containing citrate, phosphate, dextrose, and adenine; Baxter Healthcare Corp., Deerfield, Ill.) (provided by Connie Lowe, Blood Services Section, Department of Transfusion Medicine, NIH Clinical Center). Human plasma samples from more than six donors were collected, pooled, aliquoted, and immediately frozen at 2868C (stored for less than 3 months). Each donor plasma sample was screened for hepatitis C virus and HIV-1 and found to be negative. Two additional anticoagulants (heparin and EDTA) were used to collect human plasma from four healthy donors through the Occupation Health Service Blood Bank Program at the National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Md. Chimpanzee plasma samples from three animals were collected and pooled, again using the anticoagulant CPDA-1 (provided by Max Shapiro, BIOQUAL, Rockville, Md.). Primary cell-based endpoint infectivity assay. To quantify infectivity on a per-cell basis, 3 3 105 phytohemagglutin-stimulated PBMCs or MDMs from a normal donor were centrifuged (8,000 3 g for 1 min) in Eppendorf tubes, resuspended in 540 ml of undiluted human plasma or culture medium (RPMI 1640–20% FBS–1% PSN–interleukin 2 for PBMCs or RPMI 1640–10% HAB–1% PSN for MDMs). A fixed volume (60 ml) of each undiluted virus stock was added directly to the resuspended cells, resulting in a final virus concentration of 10% (vol/vol) and a final plasma or medium concentration of 90% (vol/vol). The cell-virus suspension containing either plasma or medium was subsequently mixed on a roller apparatus at 1 vessel rpm at 378C. At designated time intervals (1 to 30 h), virus-cell mixtures were removed from the roller apparatus, centrifuged (8,000 3 g for 1 min), washed with medium, and resuspended in 938 ml of medium. After the various virus-cell-plasma and/or medium incubations, cell viability was measured by trypan blue exclusion with a starting final concentration of approximately 32,000 viable cells per well. These cells were then endpoint titrated in triplicate, starting from approximately 32,000 cells per well and proceeding to 32 cells per well by fourfold dilutions of 100 ml. Approximately 2.5 3 105 cells of uninfected PBMCs and 1.4 3 105 cells of uninfected MDMs were added to each well to amplify the endpoint infection. Cultures of PBMCs and MDMs were then incubated at 378C for 2 and 4 weeks, respectively, without further addition of uninfected cells. PBMC cultures were maintained during 2 weeks of incubation without replacement with fresh medium. MDM cultures were refed 50% (vol/vol) once a week for the entire length of the experiment. Endpoints of virus infectivity were determined by screening for p24 core antigen. Endpoint infectivity was calculated as the log reduction of ID50 (log ID50) through a computer ID50 package based on Fisher’s statistical model (12, 13) (see above). The numerical value of ID50 was calculated from the dilution which was the 50% point of the cell titration. From this, the number of target cells at the 50% point was calculated, and from that the volume of virus inoculum (in milliliters) required to infect a single target cell 50% of the time was calculated. The negative log value of this inoculum volume is represented in the data as log ID50 (in milliliters). All infectivity assays were repeated two to six times. An alternative method for determining enhancement of infectivity in MDMs was a quantitative cytopathic assay in which virus-induced changes in monolayers were measured at 1, 3, 7, and 10 h by the enumeration of syncytia per well at the target cell numbers described above. p24 antigen capture assay. Microtiter plates coated with an anti-p24 monoclonal antibody were obtained from Larry Arthur (National Cancer Institute, Frederick Cancer Research and Development Center) and stored at 48C. Before the assay, plates were washed three times with PBS–0.5% Tween 20 (Sigma) (PBST) and blotted dry. Samples containing a final concentration of 0.5% Triton X-100 were added to the plates (100 ml per well) and incubated at 378C for 2 h. The wells were then washed three times with PBST, incubated with 100 ml of 1:13,000 rabbit anti-p24 polyclonal antibody (provided by Larry Arthur) in PBST–1% bovine serum albumin (Sigma), and incubated at 378C for 2 h. Again the wells were washed three times with PBST and incubated with 100 ml of a

1:40,000 dilution of horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) diluted in PBST– 10% normal goat serum (Gibco) for 1 h at 378C. The plates were finally washed six times, and 100 ml of o-phenylenediamine dihydrochloride solution (Sigma) was added to each well. The plate was placed in the dark for 30 min before being read on a microplate reader at 450 nm. For cell-associated p24 experiments, p24 standards were run in identical concentrations of plasma and media, with similar results being obtained. Virus binding/internalization assay. To more directly measure the effect of human plasma on the binding of HIV-1 to primary cells, the amounts of cellbound viral particles were measured by a quantitative p24 enzyme-linked immunosorbent assay (ELISA) as previously described (40, 52). In each tube, 6 3 105 cells were incubated with 80% (vol/vol) human plasma or medium, with the addition of 20% (vol/vol) virus inoculum at a total volume of 600 ml. At each time point, infected cells were centrifuged, washed two times with PBS, resuspended in 200 ml of 1% Triton X-100, and incubated for 1 h at 378C. The virus core antigen p24 concentrations were determined from the linear ranges of standard p24 solutions at the concentrations from 0.1 to 4 ng/ml. Kinetic analysis. Viral infectivity data were fit to a curve corresponding to a standard first-order kinetic model (10, 43). The model can be derived from the following assumptions: (i) cells are in excess over infectious virions (low multiplicities of infection); (ii) the rates of the process(es) limiting infection are similar for most infectious virions; and (iii) each rate is constant over time. Data in conflict with these assumptions will not fit the following equation (50): I 5 Imax(1 2 e2lt)

(1)

where I is the infectivity, Imax is the maximal value of infectivity, t is the virus-cell contact time, and l does not depend on time. Since half-maximal infection is achieved when t 5 t1/2 5 ln2/(l), equation 1 is equivalent to I 5 Imax (1 2 22t/t1/2)

(2)

Experimental data were fit to equation 2 by using nonlinear regression (45) to determine Imax, t1/2, and their errors. Goodness of fit to the model was assessed by the chi-square test.

RESULTS Characteristics of HIV-1 isolates. To evaluate the effects of plasma on a wide spectrum of HIV-1 isolates, viral isolates chosen were to be as representative as possible of lab strains and primary isolates tropic for both T-cell lines and the primary cell types, PBMCs and MDMs. Cell permissivity to HIV-1 infection was determined by detecting p24 concentrations in cultured cell supernatants in PBMCs, MDMs, and two T-cell lines (summarized in Table 1). All lab strains and primary isolates were able to infect PBMCs. As expected, only lab strains productively infected H9 and CEM-SS. All primary isolates replicated in PBMCs to p24 levels of greater than 50 ng/ml by 1 to 2 weeks postinfection. Two (HIV-1ADA and HIV-1D117III) of the four primary isolates replicated to similarly high levels of p24 in both PBMCs and MDMs. The two remaining primary isolates (HIV-1108 and HIV-1MR452) failed to replicate in MDMs (even with initial plasma incubation) as determined by p24 after 4 weeks in culture (data not shown).

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FIG. 1. Effects of human plasma on HIV-1 lab strains in PBMCs. Endpoints of virus infectivity (log ID50s) were plotted at different virus-cell contact times in the presence (F) or absence (E) of human plasma for HIV-1HXB3 (A), HIV-1MN (B), and HIV-1RF (C).

Effects of human plasma on HIV-1 lab strains entering PBMCs. The log reduction assay assesses the infectivity of viral inoculum on a per-cell basis. An increase of infectivity of 0.3 log unit in this assay is equivalent to a twofold reduction of the inoculum volume required to infect a fixed number of cells (i.e., log 2 5 0.3). Calculated statistical errors in our hands are consistent with variations actually encountered in experimental repetition. Relative to medium controls, no effect was observed for HIV-1MN (Fig. 1B). However, human plasma increased HIV-1HXB3 and HIV-1RF infectivity by 0.7 log unit (Fig. 1A and C) within the first 1 to 10 h. Further incubation for up to

J. VIROL.

30 h in plasma did not result in a significant increase of infectivity (Fig. 1). Effects of human plasma on HIV-1 primary isolates entering PBMCs. As biological differences are known to exist between lab strains and primary isolates, the effects of plasma on infectivity were further studied with the isolates exhibiting both PBMC-restricted (HIV-1108 and HIV-1MR452) and broader (HIV-1ADA and HIV-1D117III) tropism. Compared with the previous results for three lab strains, notable differences were observed for primary isolates (Fig. 2). No significant difference in infectivity was observed at the initial (1-h) time point between plasma and media among the four primary isolates. In three (HIV-1108, HIV-1MR452, and HIV-1ADA) of the four samples in media, the log ID50 values changed little during the 30 h (Fig. 2). However, in two cases (HIV-1108 and HIV1MR452), although not statistically significant, an overall decline of the mean log ID50 was observed in media at the initial time points. In the fourth case, a time-dependent increase in the log ID50 was observed for HIV-1D117III in medium during the first 1 to 6 h (Fig. 2D). However, after the initial (1-h) time point, in the presence of human plasma the four primary isolates tested significantly enhanced infectivity in a time-dependent manner (Fig. 2). HIV-1ADA demonstrated the most extreme example of plasma enhancement; in this case, the fourth time point (approximately 10 h) showed about a 1.5-log-unit enhancement of infectivity over that of the medium control (Fig. 3C). Overall, human plasma increased the infectivity between 0.5 to 1.5 log units for all four primary isolates (Fig. 2). In three (HIV-1108, HIV-1452, and HIV-1ADA) of four isolates, human plasma resulted in statistical enhancement of infectivity over that of the medium control by as early as 3 h, whereas in the fourth isolate (HIV1D117III), enhancement was not statistically different until the 8-h time point. Furthermore, when the levels of plasma-mediated enhancement observed among the HIV-1 isolates were compared, no correlation related to the initial virus stock infectivity titers was found (Table 1). The shapes of the infectivity curves in the presence of plasma were fit and demonstrated a first-order kinetic behavior (discussed below). The virus isolate (HIV-1D117III) that demonstrated a more obvious delay of plasma-mediated enhancement also exhibited a first-order kinetic behavior in medium (Fig. 2D). Effects of human plasma on HIV-1 primary isolates entering MDMs. Striking enhancement of virus infectivity was observed in both of the macrophage-tropic primary isolates in plasma. More specifically, HIV-1ADA (Fig. 3A) demonstrated a pronounced time-dependent enhancement of infectivity. Maximum infectivity of 1.0 log unit was observed by 10 h as compared with the medium control (Fig. 3A). A significant difference of 0.5 log ID50 as compared with the medium control was observed by 3 h. As seen previously in PBMCs, although not statistically significant, a decline of mean values of log ID50 was again repeatedly observed in media. In contrast, the other macrophage-tropic isolate, HIV-1D117III, demonstrated no apparent difference in the log ID50s as compared with the medium control until after approximately 10 h (Fig. 3B). Beyond 10 h of incubation in the presence of plasma, enhancement of 0.5 to 1.0 log unit was observed. Furthermore, the 0.25- to 0.5-log-unit increase of HIV-1ADA over that of HIV-1D117III in MDMs could not be ascribed to the initial virus stock infectivity titer (Table 1). Coincident with the observed enhancement of ID50 was a marked increase in the numbers of syncytia formed in MDM monolayers. When HIV-1ADA was incubated in plasma, a time-dependent increase of quantifiable syncytia occurred, from 3-fold at a 1-h, incubation (Fig. 4A), through 3- and 7-h

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FIG. 2. Effects of human plasma on HIV-1 primary isolates in PBMCs. Endpoints of virus infectivity (log ID50s) were plotted at different virus-cell contact times in the presence (F) or absence (E) of human plasma for HIV-1108 (A), HIV-1MR452 (B), HIV-1ADA (C), and HIV-1D117III (D).

incubations (Fig. 4B and C), to a maximal value of 18-fold at 10 h of incubation (Fig. 4D), at the target cell concentrations tested. The kinetics of this enhanced cytopathic effect in MDMs was similar to that of the ID50 infectivity measurement shown in Fig. 3A. A time-dependent increase of viral cytopathology by plasma incubation was also seen in HIV-1D117 (data not shown). Titration effects of plasma on enhancement. To determine whether the enhancing properties were concentration dependent, 5-fold serial dilutions of plasma starting from 90 to 50% were used, after which 10-fold dilutions were made to 30%. These titration studies used HIV-1ADA in MDMs for a fixed 10-h virus-cell incubation period. The enhancing effects were examined and presented as the ratio of each concentration of plasma to the concentration of medium. Plasma-mediated enhancement was observed at human plasma concentrations of 40% (vol/vol) or more (Fig. 5). Concentrations of human plasma lower than 50% resulted in a linear decline of viral enhancement which approached the infectivity values of medium controls at or below 30% (Fig. 5). No enhancement was observed when either the virus or the cells were preincubated alone with 90% plasma for up to 6 and 3 h, respectively (data not shown). Human plasma increases bound virus. To establish a relationship between plasma-mediated enhancement and bound virus, experiments measured cell-associated p24 in PBMCs over time. In the presence of plasma, the concentration of

cell-associated HIV-1ADA p24 was approximately the same as in the medium at 1 h (Fig. 6), suggesting that plasma fluids did not interfere with the detection of cell-bound viral particles. Beyond 1 h, however a time-dependent increase of HIV-1ADA cell-associated p24 in the presence of plasma was observed (Fig. 6). The peak level of cell-associated p24 was observed after 10 h of incubation (approximately 1-log-unit increase) compared with the medium control, which coincided with the maximum infectivity described previously (Fig. 2C). Maximal infectivity and half-maximal time analyses. To compare the effects of plasma on the entry kinetics of different HIV-1 isolates in different primary cell types, the Imax and t1/2 were determined from equation 2. Imax and t1/2 values for individual HIV-1 primary isolates are given in Table 2. Imax corresponds to the y intercept of the plateau of the log ID50 value; t1/2 is the time taken to reach 50% of this value. In media, five of the six data sets plateaued by 1 h, before the first experimental measurement, and so their t1/2 was taken to be 1 h or less. All primary isolates in all primary cells had significant increases in the Imax value in plasma as compared with the medium control, ranging from 3-fold (HIV-1D117III in PBMCs) to 30-fold (HIV-1ADA in PBMCs). As well, simultaneous incubation of HIV-1 primary isolates with cells in the presence of human plasma resulted in a significant increase of the t1/2 values of all primary cell types. The increases of t1/2 values in plasma compared with medium control ranged from 0.5 h

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origin (chimpanzee) or commercial source human serum. Heparin-collected human plasma exhibited approximately the same levels of enhancements as CPDA-1-collected plasma in both PBMCs and MDMs. EDTA-collected human plasma, however, resulted in a significant loss of the enhancement property in both cell types. In addition to the control for FBS used in the PBMC culture medium, FBS concentrations of as high as 90% failed to result in enhancement (data not shown). DISCUSSION

FIG. 3. Effects of human plasma on HIV-1 primary isolates in MDMs. Virus infectivity (log ID50) was plotted at each virus-cell contact time in the presence (F) or absence (E) of human plasma for HIV-1ADA (A), and HIV-1D117III (B).

(HIV-1108 in PBMCs) to 13 h (HIV-1ADA in PBMCs), indicating a slow and accumulative process of infection (Table 2). Specificity controls and physical properties. To further characterize the specificity of CPDA-1-collected plasma enhancing factor(s) with HIV-1 primary isolates, HIV-1ADA was used to infect both PBMCs and MDMs at a fixed virus-cell incubation time of 10 h. The virus-cell mixtures contained the same numbers of virus and cells and 90% (vol/vol) of the following samples: (i) CPDA-1-collected human plasma (same as used in all previous experiments), (ii) CPDA-1 medium (iii) standard heat-inactivated (568C, 0.5 h) CPDA-1-collected human plasma, (iv) CPDA-1-collected chimpanzee plasma, (v) commercial source human serum (HAB; Sigma), (vi) heparincollected human plasma, and (vii) EDTA-collected human plasma. Compared with the culture medium with respect to each sample or treatment, the enhancement ratio (in log scale) was obtained (Fig. 7). The enhancement ratio of CPDA-1 human plasma was about 0.8 log unit in PBMCs and 1.2 log units in MDMs. The CPDA-1 medium control did not cause any enhancement of infectivity in either primary cell type. When human plasma was heat inactivated, the mean enhancement ratio in PBMCs showed a 0.6-log-unit reduction (from 0.8 to 0.2 log unit) (statistically significant at P , 0.05 by t test) (Fig. 7A, bar c). Surprisingly, heat-inactivated human plasma retained its viral enhancing activity in MDMs (Fig. 7B, bar c). In both PBMCs and MDMs, the enhancements were less pronounced in CPDA-1-collected samples of nonhuman species

Since HIV-1 is known to exist at relative high infectious titers in plasma during the acute phase of infection, we have investigated the role of physiological levels of normal human plasma on the in vitro infectivity of HIV-1. Our results show that human plasma at concentrations found in blood can increase the in vitro infectious titers for HIV-1 primary isolates entering PBMCs and MDMs. HIV-1 infectivity and entry kinetics have been studied only in tissue culture media containing low concentrations (10 to 20%) FBS or calf serum, using either established T-cell lines or PBMCs (9, 11, 17, 34, 41, 51). The infectivity and tropism of viruses are known to be altered by various physiological factors. Examples include human cytomegalovirus and murine retrovirus, which in the presence of b2-microglobulin and serum lipoproteins, respectively, can enhance infectivity (18) and alter cellular tropism (35). Previous experiments showed that incubating HIV-1 in plasma for 1 h with either T-cell lines or PBMCs usually increased syncytium-forming units or end point infectivity about two- to fivefold (36). Primary isolates and lab strains are known to exhibit differences in soluble CD4 (7), antibody-mediated neutralization (30, 56), cellular CD4 dependency (21), and per-virion envelope density (39) and were therefore compared in the presence of more physiological levels of human plasma for their effects on infectivity and entry kinetics. For a 1-h incubation at the cell concentrations tested, the presence of CPDA-1-collected plasma with virus and cell had little influence on altering primary isolate infectivity titers. Indeed, the effects of plasma on infectivity would not have been apparent without an extended incubation of the primary isolates with the target cells. Extended incubation in media, however, made little difference, as 70 to 100% of the maximal infectivity was found to occur by 1 h, with subtle decreases in infectivity thereafter being observed. By contrast, infectivity in CPDA-1-collected plasma increased over the next 1 to 10 h and varied depending on the isolate, with resultant enhancements of up to 0.5 to 1.5 log units in the infectivity in both PBMCs and MDMs. In other words, the infection of a fixed number of cells in a fixed volume of plasma required approximately 3- to 30-fold less volume of viral inoculum than of medium to infect the same numbers of cells. Further evidence for enhancement of infectivity was suggested by the approximately 18-fold-greater numbers of MDM syncytia occurring (after 2 weeks postinfection) compared with the infected MDM monolayer in medium alone. The similarity of the kinetics and magnitude of the increases in both ID50s and numbers of syncytia observed 2 weeks after the initial removal of the plasma or virus incubation can be explained only as a direct result of the infection of more MDMs during the initial incubation. Furthermore, the enhancement of infectivity among the various isolates tested was not related to either the initial virus stock infectivity titers or the different multiplicities of infection (data not shown). To further characterize the plasma-mediated enhancement, we conducted time course virus binding experiments using a

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FIG. 4. Effects of human plasma on viral cytopathology of HIV-1ADA in MDMs. Virus-induced MDM-derived syncytium-forming units were counted at 2 weeks postinfection and plotted at each target cell number-per-well point with virus-cell incubation for 1 h (A), 3 h (B), 7 h (C), and 10 h (D) in the presence (F) or absence (E) of human plasma.

well-characterized HIV-1ADA stock in PBMCs in a cell-associated p24 assay. Cell-associated p24 values and ID50 infectivity values in the presence of plasma displayed concomitant time-dependent increases and plateaued together (approximately 10 h), suggesting that the cell-associated p24 was measured before viral replication produced secondary p24. In addition, viral proteins synthesized following infection are generally produced only after 24 h postinfection (22, 42). Ad-

FIG. 5. Effects of human plasma concentration on HIV-1ADA in MDMs. The plasma enhancement ratio was calculated as the infectivity obtained from each plasma concentration with respect to the medium control at 10 h of virus-cell incubation.

ditionally, since the virus attachment can be significantly affected by the cell density (26, 50), preliminary studies using the primary isolate HIV-1108 at a lower PBMC density and shorter incubation times (1 h) also demonstrated enhanced rates of infectivity (33). These results taken together suggest that the effect of plasma-mediated enhancement is most likely on virus entry; however, we cannot completely rule out contributions from other postentry steps and/or viral stability. The apparent relationship between the cell-associated p24

FIG. 6. Effects of human plasma on the binding of HIV-1ADA to PBMCs. Cell-associated viral core antigen p24 concentrations were plotted at different virus-cell contact times in the presence (F) or absence (E) of human plasma.

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J. VIROL. TABLE 2. Determination of virus entry kinetics for HIV-1 primary isolatesa Cell type

Human plasma incubation

Imax (log ID50 [ml]) (mean 6 1 SE)

HIV-1108 HIV-1108

PBMC PBMC

Yes No

4.216 6 0.126 3.385 6 0.229

1.5 (0.7–3.1) ,1.0

HIV-1MR452 HIV-1MR452

PBMC PBMC

Yes No

4.852 6 0.153 4.071 6 0.233

2.4 (1.2–5.1) ,1.0

HIV-1ADA HIV-1ADA HIV-1ADA HIV-1ADA

PBMC PBMC MDM MDM

Yes No Yes No

4.560 6 0.299 3.086 6 0.179 5.557 6 0.268 4.411 6 0.181

14.4 (5.9–35.0) ,1.0 6.6 (2.8–15.5) ,1.0

HIV-1D117III HIV-1D117III HIV-1D117III HIV-1D117III

PBMC PBMC MDM MDM

Yes No Yes No

5.221 6 0.241 4.817 6 0.177 3.689 6 0.185 3.098 6 0.194

7.0 (3.3–15.0) 5.2 (2.6–10.4) 12.5 (7.0–22.4) 4.9 (2.2–10.9)

Virus

a b

t1/2b (h)

Fittings of experimental data to equation 2 pass the chi-square test for goodness of fit with P . 0.05. Presented as mean (range of values below or above 1 standard error of the mean).

FIG. 7. Plasma specificity and physical properties. HIV-1ADA was used to infect PBMCs (A) and MDMs (B) at a fixed virus-cell incubation time period of 10 h. The enhancement ratios were plotted in a log scale as the infectivity in the presence of 90% (vol/vol) CPDA-1-collected human plasma (bar a), CPDA-1 medium (bar b), heat-inactivated CPDA-1-collected human plasma (bar c), CPDA-1-collected chimpanzee plasma (bar d), commercial source human serum (HAB; Sigma) (bar e), heparin-collected human plasma (bar f), and EDTAcollected human plasma (bar g) compared with medium controls. The data are presented as the mean values 6 standard errors. Comparisons were based on the enhancement ratio of CPDA-1-collected human plasma, and statistically significant differences (P , 0.05; unbiased t test) are marked with asterisks.

and infectivity observed previously can be further estimated through additional quantitative virological methods. Having previously determined the concentration of infectious and noninfectious virus particles by electron microscopy quantitation of p24, and determination of the ID50 of the HIV-1ADA stock, we quantitated the relative proportions of bound virus particles and infectivity on a per-cell basis. Since the HIV-1ADA stock contained about 3.83 3 109 physical particles per ml of virus stock (54) and 1 ID50 is equal to 0.693 infectious units, the ID50 shift of 3.1 log to 4.6 log units in Fig. 2C increases the number of infectious units from 0.693 3 103.1 to 0.693 3 104.6/ml of virus inoculum. Thus, the viral infectious-to-noninfectious unit ratio increased from 1:6 3 106 to 1:2 3 105. Also, assuming a p24 content of about 5 3 1028 ng per virion (24, 54), Fig. 2C and 5 give the fraction of bound virions at 10 h as 2.6 and 0.3% in plasma and media, respectively. The cell-associated p24 enhancement is 2.6/0.3, a nearly 10-fold ratio, compared with the 30-fold infectivity enhancement (Table 2; 4.6 to 3.1 5 1.5 log) for HIV-1ADA in PBMCs, suggesting that plasma may be selectively acting on a unique fraction of the total virus, resulting in a threefold increase of infectivity. The threefold ratio was reproducible and consistent with the amount of infectivity observed, although it is at the limit of the assay’s sensitivity (0.05 to 0.1 ng/ml of p24 concentration). Previous studies describing changes in both infectivity and cellular tropism by physiological factors have demonstrated that these factors interact primarily with the virus (18, 35, 36). Enveloped viruses such as HIV-1 are well known to incorporate various cellular membrane proteins into their envelopes (2, 14, 40, 48). The incorporation of these molecules into the virus envelope may influence subsequent interactions with other physiological factors, i.e., plasma, leading to new viruscell interactions. Our results suggested that plasma-mediated enhancement required the presence of plasma during virus-cell incubation. Evidence for this was demonstrated by studies in which either virus or cells when preincubated separately with plasma for up to 6 h or 3 h, respectively, failed relative to their medium controls to mediate a similar enhancement. In addition, postinfection incubation with plasma also resulted in no relative enhancement (data not shown). Therefore, in the one case studied, enhancement was probably not due to a stabilization effect on the virion or its envelope, as well as the induction of various cellular factors. Plasma-mediated enhancement also did not depend either on the primary cell type from which

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the virus was produced or the primary cell type used for infection. This finding was evidenced by the fact that plasma-mediated enhancement occurred in primary viruses derived in both PBMCs and MDMs. Two macrophage-tropic isolates (HIV1ADA and HIV-1D117III) harvested from identical MDMs in the presence of plasma resulted in 14- and 4-fold increases in maximal infectivity in MDMs, respectively. When PBMCs were used as target cells with the same two primary isolates, similar plasma-mediated enhancements were observed (30and 3-fold increases in maximal infectivity, respectively). Therefore, the different amounts of maximal infectivity observed among the isolates suggest that some other viral isolatespecific factor(s) may contribute to these complex interactions and therefore may be gp120/41 specific. Preliminary evidence from both native virion and solid-phase CD4 capture ELISAs suggested that a direct interaction occurred between the envelope of HIV-1 and some plasma factor(s) (36). The mechanism(s) or receptor(s) reported to date for HIV-1 infection of MDMs involves primarily CD4; however, other receptors, such as phagocytosis, Fc receptor-mediated endocytosis (reviewed in reference 31), and CD44 (46), may also be involved. MDMs have markedly (approximately 10-fold) lower levels of CD4 on their surfaces than do PBMCs (4). Despite the difference in cellular CD4 levels, similar levels of enhancements of HIV1ADA of between 1.0 and 1.5 log units in both PBMCs and MDMs were observed. Therefore, plasma-mediated enhancement may not primarily involve the CD4 molecule. These findings suggest that plasma-mediated HIV-1 infection and fusion in MDMs may be less dependent on the CD4 receptor. The use of human plasma in these experiments was an in vitro attempt to mimic the in vivo physiological condition in which HIV-1 exists in blood. Human plasma under normal conditions when removed from the blood vascular system initiates a clotting reaction. Thus, the plasma must be collected in the presence of various anticoagulants. These anticoagulants may target either divalent cations or proteins responsible for the clotting cascades. Our results showed that plasma collected in the anticoagulants CPDA-1 and heparin enhanced viral infectivity, while plasma collected in EDTA had little effect. Crude fractionation experiments of CPDA-1-collected plasma demonstrated that the inactivation of clotting cascade proteins by heat and removal by calcium chloride precipitation (33) failed to reduce the infectivity-enhancing properties in MDMs. Heat inactivation, however, was found to reduce the infectivity-enhancing effect in PBMCs, suggesting that the enhancement in PBMCs and not MDMs may be related to a heat-labile factor(s), e.g., complement (reviewed in reference 49). Interestingly, fresh serum derived from calcium chloride-precipitated CPDA-1-collected plasma retained this property in MDMs (33), whereas a commercially obtained serum demonstrated a significantly lower level of enhancement. Collectively, these results suggest the factors or mechanisms of plasmamediated enhancements may differ between PBMCs and MDMs. To further define the physiologic capacity of plasma-mediated enhancement, dose titration of plasma between 90 and 30% demonstrated enhancement to occur from 90 to 40% in MDMs, suggesting that the enhancement phenomenon would occur in blood. The specificity of CPDA-1-collected human plasma was further supported by the reduced levels of enhancement in both fresh CPDA-1-collected chimpanzee plasma and commercial human serum. Human plasma at physiological concentrations as examined in this study obviously may have a profound impact on a wide spectrum of virus-cell interactions. Some examples of plasma factors known to interact with HIV-1 and cells include complement with or without

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fibronectin and complement-reactive protein, mannose-binding protein, and naturally occurring plasma polyanions, e.g., heparan, chondroitin, and keratan sulfate (reviewed in reference 49). In addition, another plasma protein, complement factor H, was recently found to directly bind to the C1 domain of HIV-1 gp120 and to enhance, in a CD4-dependent manner, heterologous syncytium formation (44). Furthermore, as a negative regulator of complement activation, complement factor H with an as yet undefined plasma factor(s) may protect HIV-1 virions and infected cells from lysis. These reports as well as our findings suggest that various factors in normal human plasma in addition to complement may naturally promote HIV-1 infection rather than augmenting natural antiviral defenses to the primary viremia. Primary isolates characterized to date exhibit an array of cellular and biological phenotypes, i.e., PBMC tropism, macrophage tropism, syncytium induction, and non-syncytium induction, which may be correlated with immunopathogenesis, such as cytopathology, soluble CD4, and/or antibody neutralization resistance. Early studies have suggested that a slow rate of viral entry may be correlated with pathogenesis (11, 37) which may be secondary to selection for neutralization-resistant variants having a slower or low viral phenotype (1, 38). Interestingly, our study found that the plasma-mediated enhancement was associated with a slow time-dependent increase of infectious particles. A similar plasma-mediated enhancement has been observed for 10 other international primary B-clade isolates (33). Current studies are aimed at determining the molecular mechanism(s) for the specific interaction of the human plasma component(s) with additional isolates and clades of HIV-1 from acute to symptomatic stages in primary lymphoreticular cells. ACKNOWLEDGMENT We thank N. M. Dunlop for testing the infections in H9 and CEM-SS cell lines. REFERENCES 1. Albert, J., B. Abrahamsson, K. Nagy, E. Aurelius, H. Graines, G. Nystrom, and E. M. Fenyo. 1990. Rapid development of isolate-specific neutralizing antibodies after primary HIV-1 infection and consequent emergence of virus variants which resist neutralization by autologous sera. AIDS 4:107–112. 2. Arthur, L. O., J. W. Bess, R. C. Sowder II, R. E. Benveniste, D. L. Mann, J.-C. Chermann, and L. E. Henderson. 1992. Cellular proteins bound to immunodeficiency viruses: implications for pathogenesis and vaccines. Science 258:1935–1938. 3. Callebaut, C., B. Kurst, E. Jacotot, and A. G. Hovanessian. 1993. T cell activation antigen CD26 as a cofactor for entry of HIV in CD41 cells. Science 262:2045–2050. 4. Collman, R. 1992. Human immunodeficiency virus type 1 tropism from human macrophages. Pathobiology 60:213–218. 5. Conley, A. J., J. A. Kessler II, L. J. Boots, J.-S. Tung, B. A. Arnold, P. M. Keller, A. R. Shaw, and E. A. Emini. 1994. Neutralization of divergent human immunodeficiency virus type 1 variants an primary isolates by IAM41-2F5, an anti-gp41 human monoclonal antibody. Proc. Natl. Acad. Sci. USA 91:3348–3352. 6. Corbeau, P., D. Olive, and C. Deveaux. 1991. Anti-HLA class-I heavy chain monoclonal antibodies inhibit human immunodeficiency virus production by peripheral blood mononuclear cells. Eur. J. Immunol. 21:865–871. 7. Daar, E. S., X. L. Li, T. Moudgil, and D. D. Ho. 1990. High concentrations of recombinant soluble CD4 are required to neutralize primary HIV-1 isolates. Proc. Natl. Acad. Sci. USA 87:6574–6578. 8. Dalgleish, A. G., P. C. L. Beverley, P. R. Clapham, D. H. Crawford, M. F. Greaves, and R. A. Weiss. 1984. The CD4 (T4) antigen is an essential component of the receptor of the AIDS retrovirus. Nature (London) 312: 763–767. 9. Dimitrov, D. S., R. L. Willey, M. A. Martin, and R. Blumenthal. 1992. Kinetics of HIV-1 interactions with sCD4 and CD41 cells: implications for inhibition of virus infection and initial steps of virus entry into cells. Virology 187:398–406. 10. Ellis, E. L., and M. Delbruck. 1939. The growth of bacteriophage. J. Gen. Physiol. 22:365–384.

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