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. 2005 Jul;79(14):9197–9205. doi: 10.1128/JVI.79.14.9197-9205.2005

In Vitro Suppression of Human Immunodeficiency Virus Type 1 Replication by Measles Virus

Mayra García 1,2, Xiao-Fang Yu 1, Diane E Griffin 1, William J Moss 1,3,*
PMCID: PMC1168732  PMID: 15994814

Abstract

During the acute phase of measles, human immunodeficiency virus type 1 (HIV-1)-infected children have a transient, but dramatic, decrease in plasma HIV-1 RNA levels (W. J. Moss, J. J. Ryon, M. Monze, F. Cutts, T. C. Quinn, and D. E. Griffin, J. Infect. Dis. 185:1035-1042, 2002). To determine the mechanism(s) by which coinfection with measles virus (MV) decreases HIV-1 replication, we established an in vitro culture system that reproduces this effect. The addition of MV to CCR5- or CXCR4-tropic HIV-1-infected human peripheral blood mononuclear cells (PBMCs) decreased HIV-1 p24 antigen production in a dose-dependent manner. This decrease occurred with the addition of MV before or after HIV-1. The inhibition of HIV-1 p24 antigen production was decreased when UV-inactivated MV or virus-free supernatant fluid from MV-infected PBMCs was used. Inhibition was not due to increased production of chemokines known to block coreceptor usage by HIV-1, a decrease in the percentage of CD4+ T cells, or a decrease in chemokine receptor expression by CD4+ T cells. Viability of PBMCs was decreased only 10 to 20% by MV coinfection; however, lymphocyte proliferation was decreased by 60 to 90% and correlated with decreased production of p24 antigen. These studies showed that an in vitro system of coinfected PBMCs could be used to dissect the mechanism(s) by which MV suppresses HIV-1 replication in coinfected children and suggest that inhibition of lymphocyte proliferation by MV may play a role in the suppression of HIV-1 p24 antigen production.

Measles virus (MV) and human immunodeficiency virus type 1 (HIV-1) infection are important causes of morbidity and mortality in many parts of the world. Both viruses induce immunosuppression, and disease is associated with an increased susceptibility to secondary infections. In individuals infected with HIV-1, coinfections and vaccinations are often associated with transient increases in plasma HIV-1 RNA levels (26), presumably due to an increase in the number of activated CD4+ T-lymphocyte target cells and the induction of proinflammatory cytokines that favor HIV-1 replication.

Surprisingly, MV infection, which results in both immune activation and induction of proinflammatory cytokines (12), is associated with a transient but profound reduction in plasma levels of HIV-1 in coinfected children (19). Decreased plasma HIV-1 RNA levels also have been reported following coinfection with Orientia tsutsugamushi (29), the etiologic agent of scrub typhus, and dengue virus (30) and with chronic infection with GB virus C (GBV-C) (32). The mechanisms for in vivo reduction in HIV-1 replication by these various pathogens have not been identified.

Several viruses inhibit HIV-1 replication in vitro. Supernatant fluids from peripheral blood mononuclear cells (PBMCs) stimulated with live or inactivated influenza A virus inhibit replication of CCR5- and CXCR4-tropic HIV-1 isolates, an effect partially mediated by alpha interferon (22). Human herpesvirus 6 inhibits replication of CCR5-tropic but not CXCR-4-tropic HIV-1 in human lymphoid tissue, an effect mediated by RANTES (13). Similarly, GBV-C was shown to inhibit HIV-1 replication and induce chemokine production by PBMCs in vitro (35). Studies of MV-coinfected children suggest that the decrease in plasma HIV RNA levels is not due to a reduction in the number of CD4+ T lymphocytes but that an increase in soluble suppressive factors, such as β-chemokines, may contribute to the inhibition of HIV-1 replication (19). To better understand the reduction in plasma HIV-1 levels in children with measles, we developed an in vitro system to investigate the mechanisms by which MV suppresses HIV-1 replication.

MATERIALS AND METHODS

Cells.

Peripheral blood samples were obtained from healthy, anonymous donors through the blood bank of the Johns Hopkins Hospital. PBMCs were separated by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density gradient centrifugation and cultured in RPMI 1640 supplemented with 20% fetal bovine serum (FBS) and 10 U/ml interleukin 2 (IL-2) (ICN, Aurora, Ohio) (RPMI-20% FBS-IL-2). Vero cells were obtained from the American Type Culture Collection (Manassas, VA).

Viruses and controls.

The Chicago-1 and Edmonston strains of MV were obtained from lysates of infected Vero cells and assayed by plaque formation on Vero cells. MV stocks and Vero cell lysates were prepared by freezing MV-infected or uninfected Vero cells at −80°C for at least 2 hours and thawing at 37°C. The lysates were pelleted at 12,000 rpm for 5 min, passed through a 0.22-μm filter, and frozen in aliquots at −80°C. The Chicago-1 strain was isolated in 1989 during a measles outbreak in Chicago, Illinois, and has been classified as genotype group D3 (33). The wild-type Edmonston strain (American Type Culture Collection) is a minimally passaged derivative of the original genotype A Edmonston strain of MV isolated by Enders and Peebles (11). MV stocks and Vero cell lysate controls were tested for lipopolysaccharide (LPS) by the Limulus amebocyte lysate assay QCL-1000 Chromogenic LAL (BioWhittaker, Walkersville, MD). The concentration of LPS was 0.01 to 0.04 ng/ml for Chicago-1 MV, 0.005 to 0.0005 ng/ml for Edmonston MV, and 0.008 ng/ml for Vero cell lysates. For some experiments, MV was inactivated with 3,000 W/cm2 UV radiation for 2 min. Free radicals were allowed to decay for at least 1 week prior to use. Inactivation was confirmed by culture on Vero cells.

Stocks of HIV-1BaL, a CCR5-tropic laboratory strain, were grown on PBMCs obtained from healthy donors that were cultured for 10 days in RPMI-20% FBS-IL-2. Stocks of HIV-1IIIB, a CXCR4-tropic laboratory strain, were obtained from chronically infected MOLT-3 cells. HIV-1-infected cells were pelleted at 3,000 rpm for 20 min, and supernatant fluid was passed through a 0.2-μm filter and frozen in aliquots at −80°C. The titers of stocks were determined using TZM cells (9).

HIV-1 and MV infection of PBMCs.

HIV-1Ba-Lwas incubated with 107 PBMCs/ml in RPMI-20% FBS-IL-2 at a multiplicity of infection (MOI) of 0.00005 for 48 h. For HIV-1IIIB, PBMCs were stimulated with 2 μg/ml of phytohemagglutinin (PHA-P; Sigma, St. Louis, MO) for 24 h in RPMI-20% FBS-IL-2 with 1× glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin before the addition of HIV-1IIIB at a MOI of 0.0004 for 24 h in RPMI-20% FBS-IL-2.

Serial 10-fold dilutions of MV were added to 2 × 106 PBMCs infected with HIV-1BaL or HIV-1IIIB, starting with 3 × 106 PFU/ml of Chicago-1 MV (MOI of 1.5) or 3 × 107 PFU/ml of Edmonston MV (MOI of 15) (Fig. 1). Lysates of uninfected Vero cells and media alone served as controls. For later experiments, only a single MOI (0.15) of MV was used. Vero cell lysates were diluted to correspond with the dilution of the MV stock. To assess the impact of the relative timing of MV and HIV-1 infection, MV was added 3 h before, concurrently, or 15 h after HIV-1 infection. For these experiments, HIV-1BaL was used at a MOI of 0.00007.

FIG. 1.

FIG. 1.

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Scheme for the in vitro evaluation of interactions between measles virus and HIV-1. MV, Vero cell (VC) lysate, or media were added to HIV-1-infected cells and incubated for 96 h. Alternatively, PBMCs were incubated with MV or VC lysate for 24, 48, or 72 h, and supernatant fluids were collected and incubated with HIV-1-infected PBMCs for 96 h. The levels of p24 antigen were determined by enzyme immunoassay. For HIV-1 infection with CCR5-tropic HIV-1BaL, PBMCs were infected for 48 h in the presence of 10 U IL-2/ml. For CXCR4-tropic HIV-1IIIB, PBMCs were stimulated with 10 U IL-2/ml and 2 μg phytohemagglutinin/ml for 24 h before the addition of HIV-1. The medium was removed, and the cells were infected for 24 h in the presence of 10 U/ml of IL-2.

Supernatant fluid was harvested at 4, 7, and 12 days after the addition of MV (corresponding to 6, 9, and 14 total days in culture) and assayed for HIV-1 p24 antigen by enzyme immunoassay (EIA) (NEN Life Science Products, Boston, MA) (Fig. 1). Control samples consisted of supernatant fluids from uninfected PBMCs (negative control) and from PBMCs infected with HIV-1 alone (positive control). Percent inhibition was calculated as follows: 100 − [(concentration of p24 antigen in the test sample × 100)/(concentration of p24 antigen in the positive-control sample)].

Supernatant fluid from PBMCs infected with MV.

PBMCs (2 × 106) were incubated with 3 × 105 PFU of Chicago-1 MV or Vero cell lysate (1:10) in RPMI-10% FBS (Fig. 1). Supernatant fluid was collected at 24, 48, and 72 h after infection and added to PBMCs infected with HIV-1BaL or HIVIIIB. Supernatant fluid obtained at 24 and 72 h was passed through a 0.1-μm filter, and the absence of MV was confirmed on Vero cells. After 96 h, supernatant fluid from the coculture was collected and assayed for HIV-1 p24 antigen by EIA.

Cell viability.

Cell viability was determined by propidium iodide (PI) exclusion or staining with 7-amino actinomycin D (7AAD) (21) 4 days after the addition of MV to HIV-infected PBMCs. Cells were incubated on ice with a 1:5,000 dilution of PI for 10 min and analyzed by flow cytometry. For experiments involving costaining for CD4, CD8, and CXCR4, 7AAD (1:100) was used. Cell death attributed specifically to MV was calculated as the percentage of coinfected, PI-positive cells − the percentage of HIV-1-infected, PI-positive cells.

Cell surface expression of CD4, CD8, CCR5, and CXCR4.

PBMCs harvested 4 days after the addition of MV to HIV-infected cells were studied. Cells were incubated with a 1:5 dilution of human serum, washed with 1× phosphate-buffered saline supplemented with 2% FBS, stained with fluorescein isothiocyanate-conjugated mouse monoclonal antibodies against human CD4 and CD8, phycoerythrin-conjugated mouse monoclonal antibodies against human CXCR4 and CCR5, or isotype control antibodies (Pharmingen, BD Biosciences, San Diego, CA), washed, and fixed with BD Cytofix/Cytoperm (Pharmingen, BD Biosciences, San Diego, CA). Cells were analyzed on a FACS Calibur using Cell Quest software (Becton Dickinson).

Cytokines and chemokines.

The levels of RANTES, stromal-cell-derived factor 1 (SDF-1) (R&D, Minneapolis, MN), and macrophage inflammatory protein 1α (MIP-1α) and MIP-1β (Biosource International, Camarillo, CA) in supernatant fluids from HIV-1-infected PBMCs cocultured with MV or Vero cell lysates were measured by EIA according to the manufacturer's instructions. To assess the effects of β-chemokines in the culture on HIV-1 p24 antigen production, neutralizing antibodies to RANTES, MIP-1α, and MIP-1β (R&D, Minneapolis, MN) were added at 0.5, 1, or 5 μg/ml to coinfected PBMCs. Isotype control antibodies or media alone were used as controls. After 96 h, p24 antigen was measured by EIA.

Cell depletion experiments.

PBMCs were stimulated with Chicago-1 and Edmonston MV strains after removal of CD14+ cells. PBMCs were incubated with microbead-labeled antibody to CD14 (MACS; Miltenyi Biotec, Auburn, CA), resuspended, and passed over a magnetic column. CD14+ cells remained bound to the column, and the CD14− cells were collected. Cell purity was confirmed by flow cytometry. The depleted cell population was infected with MV and HIV-1, and p24 antigen was measured after 96 h.

Lymphoproliferation assays.

HIV-1-infected PBMCs were plated in triplicate in 96-well round-bottom plates at a concentration of 105 cells/well in 100 μl of RPMI-20% FBS-IL-2. Four test samples were added to the wells: (i) Chicago-1 MV at a MOI of 1.5, 0.15, and 0.015; (ii) Edmonston MV at a MOI of 15, 1.5, and 0.15; (iii) Vero cell lysate appropriately diluted; and (iv) media. The cells were incubated at 37°C for 72 h, and 1 μCi/well (10 μl) of [3H]thymidine was added overnight. DNA was harvested onto filter paper and read in a scintillation counter. The reduction in lymphoproliferation attributed to MV was calculated as follows: 100 − (cpm of the test samples ×100)/(cpm of HIV-infected cells), where cpm is counts per minute.

Statistical analysis.

Paired comparisons were made with the Wilcoxon signed-rank test. The relationship between numerical variables was measured by estimating the correlation coefficient, with confidence intervals calculated on the basis of Fisher's transformation. Statistical analyses were conducted using Stata statistical software release 7.0 (Stata Corporation, College Station, TX).

RESULTS

Reduction in HIV-1 p24 antigen production following the addition of MV.

The addition of MV to CCR5- and CXCR4-tropic HIV-1-infected PBMCs resulted in a dose-dependent decrease in p24 antigen production after 96 h of coculture (Fig. 2A and B). A 10-fold-higher concentration of the Edmonston strain of MV was required to achieve a decrease in p24 antigen production comparable to that of the Chicago-1 strain. When MV was added at a MOI lower than 0.015, the decrease in p24 antigen production was not significant and was similar to that observed with lysates of uninfected Vero cells. The reduction in p24 antigen production persisted for at least 12 days of coculture (Fig. 2C and D). Inhibition of HIV-1 replication was independent of the relative timing of the addition of MV. Reduction in p24 antigen production by PBMCs infected with HIV-1BaL (Fig. 3A) or HIV-1IIIB (Fig. 3B) was observed when MV was added 3 h before HIV-1, concurrently with HIV-1, or 15 h after HIV-1. Similar reductions in p24 antigen production were observed using cell populations depleted of CD14+ cells. The reductions in p24 antigen production by PMBCs infected with HIV-1BaL were 83% for Chicago-1 and 59% for Edmonston MV strains. For HIV-1IIIB, the reductions in p24 antigen production by Chicago-1 and Edmonston MV strains were 78% and 44%, respectively.

FIG. 2.

FIG. 2.

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Effect of MV on HIV-1 p24 antigen production. Effects of different concentrations of MV on p24 antigen (Ag) production by CCR5-tropic HIV-1BaL-infected PBMCs (A) or CXCR4-tropic HIV-1IIIB-infected PBMCs (B) using Chicago-1 or Edmonston MV at a MOI of 15 or 1.5 to 0.00015 and Vero cell lysates at dilutions comparable to those used for MV. Data are presented as means ± standard errors of the means (SEM) (error bars) of duplicate wells with PBMCs from 4 to 13 donors. Values that are significantly different (P < 0.05) between MV and Vero cell lysates are indicated by an asterisk. ND, not done. (C and D) Time course of p24 antigen production by HIV-1-infected PBMCs after the addition of MV. PBMCs were infected as described above with CCR5-tropic HIV-1BaL (C) or CXCR4-tropic HIV-1IIIB (D) and Chicago-1 and Edmonston MV at a MOI of 0.15. Vero cell lysates (1:10) served as control. Data are presented as means ± SEM (error bars) of duplicate wells with PBMCs from 7 to 11 donors. Values that are significantly different (P < 0.05) between MV and media are indicated by an asterisk. Ag conc., antigen concentration.

FIG. 3.

FIG. 3.

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Inhibition of p24 antigen production in HIV-1-infected PBMCs after the addition of measles virus (MV) at different time points. Chicago-1 MV or Edmonston MV (MOI of 0.15) was added to PBMCs infected with CCR5-tropic HIV-1BaL (A) or CXCR4-tropic HIV-1IIIB (B) 3 h before HIV-1 infection, concurrently with HIV-infection (0 h), or 15 h after HIV-1 infection. The levels of p24 antigen (Ag) were measured after 96 h. Experiments were performed in duplicate with PBMCs from three to five donors, and data are presented as means ± standard errors of the means (error bars). The value that is significantly different (P < 0.05) between MV and the corresponding dilution of Vero cell lysate is indicated by an asterisk.

MV replication and inactivation.

To determine whether MV replicated in the presence of HIV-1, MV in supernatant fluids was assayed (Fig. 4). MV replicated in HIV-infected PBMCs, and virus production 4 days after infection was highest at an inoculating MOI of 0.015 or 0.0015 (Fig. 4A and B). Replication of Edmonston MV was maintained over 12 days in cell culture, but the amounts of Chicago-1 MV declined 10- to 100-fold (Fig. 4C and D). To assess whether replicating MV was necessary for the reduction in p24 antigen production, the experiments were repeated using UV-inactivated MV. Inactivated Chicago-1 and Edmonston MV reduced p24 antigen production by PBMCs infected with HIV-1BaL and HIV-1IIIB but to a lesser extent than live virus did (Fig. 4E and F).

FIG. 4.

FIG. 4.

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Replication of MV in HIV-1-infected PBMCs and requirement for live MV. Chicago-1 or Edmonston MV was added to CCR5-tropic HIV-1BaL-infected PBMCs (A) or CXCR4-tropic HIV-1IIIB-infected PBMCs (B) at a MOI of 15 or 1.5 to 0.00015. Production of MV was measured after 96 h of coculture by plaque formation on Vero cells. Data are presented as means ± standard errors of the means (SEM) (error bars) of duplicate wells with PBMCs from two to six donors. Values that are significantly different (P < 0.05) between Chicago-1 and Edmonston strains of MV are indicated by an asterisk. ND, not done. (C and D) Time course of MV production by HIV-1-infected PBMCs. PBMCs were infected with HIV-1 CCR5-tropic HIV-1BaL (C) or CXCR4-tropic HIV-1IIIB (D), and Chicago-1 or Edmonston MV was added on day 2 of cells in culture at a MOI of 0.15. Data are presented as means ± SEM (error bars) of duplicate wells with PBMCs from three to seven donors. Values that are significantly different (P < 0.05) between Chicago-1 and Edmonston strains of MV are indicated by an asterisk. MV and Vero cell lysates were UV irradiated for 2 min and added to HIV-1BaL-infected PBMCs (E) or HIV-1IIIB-infected PBMCs (F) and tested at 96 h. p24 antigen (Ag) production was determined by EIA. Values that are significantly different (P < 0.05) between Chicago-1 and Edmonston MV strains are indicated by an asterisk and bracket. Data are presented as means ± SEM (error bars) of duplicate wells with PBMCs from 10 donors. Values that are significantly different (P < 0.05) between live or inactivated MV and Vero cell lysate are indicated by an asterisk.

Effect of MV on cell viability.

To assess whether the reduction in p24 antigen production following the addition of MV was due to a decrease in the number of cells capable of supporting HIV-1 replication, the viability of coinfected PBMCs was measured (Fig. 5). Death due to HIV infection (range, 3 to 9%) was subtracted to determine excess cell death due to MV. The percentage of cells that failed to exclude PI due to MV increased with the MV MOI and was higher for the Chicago-1 strain than for the Edmonston strain (Fig. 5A and B), although these differences were not statistically significant. The percentage of PI-staining cells ranged from 0 to 17% for the MV MOI used in most experiments (0.15).

FIG. 5.

FIG. 5.

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Effect of MV on cell death. Proportion of propidium iodide-staining HIV-1BaL-infected PBMCs (A) or HIV-1IIIB-infected PBMCs (B) 4 days after the addition of Chicago-1 MV (MOI of 1.5 to 0.015) or Edmonston MV (MOI of 15 to 0.15). Controls were equivalent 10-fold dilutions of Vero cell lysate. Background cell death due to HIV-1 alone (3 to 9%) was subtracted. Data are presented as means ± standard errors of the means (error bars) of triplicate wells of PBMCs from two or three donors. ND, not done.

Effects of MV on cell surface expression of CD4, CCR5, and CXCR4.

To determine whether MV coinfection decreased the number of target cells for HIV-1 replication by reducing availability of the HIV-1 cellular receptors, surface expression of CD4, CCR5, and CXCR4 was assessed by flow cytometry (Table 1). The proportion of CD4+ and CD8+ cells was not altered by coinfection with MV, and cell surface expression of the HIV coreceptors CCR5 and CXC4 did not decrease. In fact, surface expression of CXCR4 increased after the addition of Chicago-1 MV to PBMCs infected with HIV-1IIIB (Table 1). The number of nonviable cells expressing the surface markers CD4, CD8, and CXCR4 was assessed by costaining for 7AAD and did not demonstrate preferential death of a specific lymphocyte subset (data not shown).

TABLE 1.

Surface expression of CD4, CD8, CCR5, and CXCR4 4 days after the addition of MV (MOI of 0.15) to HIV-1-infected PBMCsa

HIV-1 strain MV or control % CD4+ % CD8+ % CCR5+ % CXCR4+
HIV-1BaL None 51.5 ± 2.9 29.8 ± 3.3 4.1 ± 2.6 ND
Chicago-1 MV 54.0 ± 3.3 28.9 ± 3.8 4.1 ± 2.9 ND
Edmonston MV 53.7 ± 2.7 28.2 ± 3.6 6.1 ± 3.3 ND
Vero cell lysate 50.7 ± 3.3 28.8 ± 3.3 3.4 ± 2.2 ND
HIV-1IIIB None 49.3 ± 3.3 34.2 ± 5.8 NDb 57.9 ± 3.8
Chicago-1 MV 48.0 ± 3.3 35.8 ± 3.6 ND 77.9 ± 3.2c
Edmonston MV 50.5 ± 2.6 37.3 ± 5.0 ND 64.1 ± 6.3
Vero cell lysate 49.6 ± 3.7 33.3 ± 5.7 ND 63.1 ± 2.6
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a

Vero cell lysate (1:10) was used as control. Data represent the means ± standard errors of the means of the percentage of positive cells from five to nine experiments.

b

ND, not determined.

c

P < 0.05 compared to Vero cell lysate.

Effect of MV on lymphoproliferation.

Although the addition of MV did not result in the loss of potential target cells, the ability of cells to proliferate could be impaired in the presence of MV (14, 28). MV inhibited the proliferation of PBMCs infected with HIV-1 as measured by incorporation of [3H]thymidine (Fig. 6). Inhibition increased with the MV MOI and was greater with the Chicago-1 strain than with the Edmonston strain. The reduction in p24 antigen production was strongly correlated with the extent to which lymphoproliferation was inhibited when the mean values for the two MV strains (at four different MOIs) and the two HIV-1 strains were compared (r = 0.72; 95% confidence interval [CI], 0.35 to 0.90). The correlation between p24 antigen production and lymphoproliferation was strongest for Chicago-1 MV (r = 0.91; 95% CI, 0.55 to 0.98) and weakest for CXCR4-tropic HIV-1 (r = 0.57; 95% CI, 0.23 to 0.91).

FIG. 6.

FIG. 6.

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Effect of MV on proliferation of HIV-infected PBMCs. Incorporation of [3H]thymidine by HIV-1BaL-infected PBMCs (A) or HIV-1IIIB-infected PBMCs (B) after the addition of Chicago-1 (MOI of 1.5 to 0.015) or Edmonston (MOI of 15 to 0.15) MV or Vero cell lysate at an equivalent dilution. Data are presented as means ± standard errors of the means (error bars) of triplicate wells with PBMCs from 6 to 10 donors. Values that are significantly different (P < 0.05) between MV and Vero cell lysate are indicated by an asterisk. ND, not done.

Effects of supernatant fluids from MV-infected PBMCs on HIV-1 p24 antigen production.

To assess whether soluble factors produced by MV-infected PMBCs inhibit HIV-1 replication, supernatant fluids were harvested from PBMCs infected with Chicago-1 MV (MOI of 0.15) at 1, 2, and 3 days following infection and added to PBMCs infected with HIV-1BaL or HIVIIIB (Fig. 7). Supernatant fluids from MV-infected PBMCs reduced p24 antigen production by PBMCs infected with HIV-1BaL (Fig. 7A) and HIVIIIB (Fig. 7B). Similar reductions in p24 antigen production were observed when the supernatant fluids were filtered to remove possible contaminating viral particles and when PBMCs were infected with the wild-type Bilthoven strain of MV (data not shown). The reduction in p24 antigen production following the addition of supernatant fluid from MV-infected PBMCs was not as marked (maximum, 70 to 80%) as following the direct addition of MV to PBMCs (maximum, 85 to 95% [Fig. 2A and B]).

FIG. 7.

FIG. 7.

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Effect of supernatant fluid on p24 antigen production. Supernatant fluid from PBMCs incubated with Chicago-1 MV (MOI of 0.15) or Vero cell lysate at 10-fold dilutions were added to HIV-1BaL-infected PBMCs (A) or HIV-1IIIB-infected PBMCs (B). Supernatant fluids were harvested on day 1, day 2, and day 3 of stimulation, and p24 antigen (Ag) was assayed after 96 h. Data are presented as means ± standard errors of the means (error bars) of duplicate wells with PBMCs from seven or eight donors. Values that are significantly different (P < 0.05) between MV and the corresponding dilution of Vero cell lysate are indicated by an asterisk.

Measurement of chemokines in supernatant fluids.

To determine whether MV coinfection increased production of HIV-inhibitory chemokines, the levels of chemokines were measured in the supernatant fluids from PBMCs coinfected with MV and HIV-1 (Table 2). The levels of the β-chemokines RANTES, MIP-1α, and MIP-1β were measured for cultures infected with CCR5-tropic HIVBaL and of the α-chemokine SDF-1 for CXCR4-tropic HIVIIIB. The levels of MIP-1α, MIP-1β, and SDF-1 were increased, but these increases were similar for MV-infected PBMCs and Vero cell lysate controls and did not correlate with decreases in p24 antigen production. Furthermore, the addition of high concentrations of neutralizing antibodies to the β-chemokines RANTES, MIP-1α, and MIP-1β had no effect on the inhibition of p24 antigen production by MV (data not shown).

TABLE 2.

Chemokine levels in supernatant fluid from PBMCs infected with HIV-1 and MV (MOI of 0.15) for 4 days

HIV-1 strain MV or controla Chemokine levelb (pg/ml)
MIP-1α MIP-1β RANTES SDF-1α
HIV-1BaL None 6,576 ± 4,729 7,741 ± 4,997 5,218 ± 3,510 ND
Chicago-1 MV 12,845 ± 9,306 10,227 ± 7,389 3,064 ± 1,608 ND
Vero cell lysate (1:10) 14,142 ± 10,131 11,015 ± 9,019 2,567 ± 1,326 ND
Edmonston MV 17,216 ± 13,277 12,834 ± 9,172 3,679 ± 1,549 ND
Vero cell lysate (1:100) 18,388 ± 18,050 13,314 ± 11,699 4,080 ± 2,111 ND
HIV-1IIIB None ND ND ND 0 ± 0
Chicago-1 MV ND ND ND 83 ± 83
Vero cell lysate (1:10) ND ND ND 552 ± 249
Edmonston MV ND ND ND 260 ± 232
Vero cell lysate (1:100) ND ND ND 290 ± 230
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a

Vero cell lysate served as control.

b

Data represent the means ± standard errors of the means of duplicate wells from three to eight experiments. ND, not determined.

DISCUSSION

During measles there is a transient, but profound, reduction in plasma HIV-1 RNA levels in coinfected children (19). In these studies, we have shown that two different strains of MV inhibited replication of both CCR5- and CXCR4-tropic strains of HIV-1 in PBMCs cultured in vitro. This inhibitory effect persisted for at least 12 days, was dose dependent, and was independent of the relative timing of the addition of MV. Inactivated MV and supernatant fluid from PBMCs stimulated with MV also inhibited HIV-1 replication, although not to the same degree as for replication-competent MV. This suppression was not associated with MV-induced death of HIV-susceptible cells or decreased expression of HIV-1 coreceptors and could not be correlated with production of HIV-inhibitory chemokines. Rather, MV-induced suppression of lymphocyte proliferation correlated with decreased p24 antigen production and may contribute to inhibition of HIV-1 replication.

We explored several potential mechanisms by which MV could suppress HIV-1 replication. Measles is accompanied by a transient lymphopenia, with a reduction in the number of circulating CD4+ and CD8+ T lymphocytes. Increased surface expression of Fas (CD95) and annexin V staining suggests that apoptosis of uninfected lymphocytes may contribute to the lymphopenia (23). However, the number of viable cells was not markedly reduced in our in vitro system. Only 10 to 20% of cells were nonviable after 4 days of culture with MV. This degree of cell death is insufficient to explain the marked reduction in p24 antigen production. In addition, cell surface expression of CD4, CD8, CCR5, or CXCR4 was not down-regulated following MV infection, further evidence that loss of target cells did not account for the reduction in p24 antigen production.

The inhibitory effect was reduced but not abrogated by inactivation of MV, suggesting that MV replication is necessary for maximal suppression of HIV-1 replication but that suppression may be mediated in part by MV proteins or factors induced by MV. The MV immune status of our anonymous donors of PBMCs was not known, but it is assumed that most, if not all, healthy adults in the United States are MV immune.

MV antigens can induce synthesis of IL-4 and gamma interferon in the absence of replicating virus (10, 15). Supernatant fluid obtained from PBMCs infected with MV also reduced p24 antigen production, suggesting the presence of soluble factors induced by MV capable of suppressing HIV-1 replication. Several classes of factors with this ability have been identified, including the β-chemokines (MIP-1α, MIP-1β, RANTES, and monocyte chemoattractant protein-2) (8, 36), CD8+ T-cell antiviral factor (17, 27), type 1/2 interferons (24), and the cytokines IL-10, IL-13, IL-16, and IL-18 (1, 2, 5-7). Furthermore, suppression of HIV-1 replication by human herpesvirus 6 has been linked to production of RANTES (13) and by GBV-C to production of both α- and β-chemokines (35). However, we were not able to identify elevated levels of chemokines (Table 2) or cytokines (data not shown) in the supernatant fluids of PBMCs stimulated with MV that could account for the suppression, and the addition of neutralizing antibodies to β-chemokines did not block the inhibitory effect of MV on p24 antigen production. These results, however, do not exclude the possibility that MV induces as yet unidentified soluble factor(s) that suppresses HIV-1 replication.

Productive HIV-1 replication requires target cell activation and proliferation (25, 37). Specifically, progression to the G1b phase of the cell cycle is necessary for nuclear importation, integration of the viral genome, and production of viral progeny (16). MV inhibits lymphoproliferation (14, 28), an effect that can be mediated by the hemagglutinin (H) and fusion (F) surface glycoproteins (31) and the nucleocapsid protein (18). Coexpression of MV H and F glycoproteins on nonlymphoid cells inhibits proliferation of peripheral blood lymphocytes through a mechanism that requires cleavage of the F protein (31) and impaired protein kinase B (Akt kinase) activation (4), disrupting the downstream pathway critical to IL-2-dependent T-lymphocyte survival and proliferation. In our system, MV inhibited the proliferation of HIV-1-infected PBMCs as measured by incorporation of tritiated thymidine. This inhibitory effect was correlated with the reduction in p24 antigen production and was more marked with the less attenuated MV strain and with a higher inoculating dose of MV. Thus, the inhibitory effect of MV on the proliferation of CD4+ lymphocytes may contribute to the reduction in p24 antigen production.

We observed some differences between the two MV strains in their ability to replicate in this system and in their effects on p24 antigen production, cell viability, and lymphoproliferation. Differences in both structural and nonstructural proteins have been described in attenuated vaccine strains of MV compared to wild-type MV strains (20). Attenuating mutations have not been definitively identified, and differences of unknown importance exist between the D and A clades of MV (34). The fact that the more virulent Chicago-1 strain was more effective in suppressing HIV-1 replication could be related to the induction of a more robust immune response or altered target cell interactions. The Chicago-1 strain replicates better in the human thymus than the Edmonston strain (3), but the Edmonston strain replicated better in cultured PBMCs. We cannot exclude the fact that differences in the concentrations of LPS of the two MV strains play a role in some of these differences. However, when LPS was added to HIV-1-infected PMBCs in concentrations similar to those detected in the two MV strains, the effect on p24 antigen production was markedly less, demonstrating that LPS was not responsible for the inhibition of p24 antigen production (data not shown).

Multiple mechanisms are likely responsible for the inhibition of HIV-1 replication by MV. Although this in vitro system provides insights into the potential mechanisms, such a system clearly does not capture the immunologic and virologic complexities of a coinfected child. The immune responses to both viruses are dynamic, changing substantially over the course of infection. In addition, cell types necessary for the induction of chemokines or cytokines may not be present in this system. Nevertheless, our in vitro system allows for the investigation of several of these potential mechanisms that is not feasible in coinfected children and suggests that suppression of HIV-1 replication may be mediated by a block in lymphoproliferation induced by MV.

Acknowledgments

This work was funded by the Elizabeth Glaser Pediatric AIDS Foundation (PG-51331), the Gustave J. Martin Innovative Research Fund of the Johns Hopkins Bloomberg School of Public Health, and research grant AI23047 from the National Institutes of Health.

We are grateful to Robert Siliciano for his many helpful suggestions and provision of reagents.

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