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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 214-219, 2002
© 2002 American Thoracic Society

Inhibition of HIV-1 Replication in Alveolar Macrophages by Adenovirus Gene Transfer Vectors

Joshua Rice, Ruth Connor, Stefan Worgall, John P. Moore, Philip L. Leopold, Robert J. Kaner and Ronald G. Crystal

Division of Pulmonary and Critical Care Medicine, Department of Microbiology and Immunology, and Institute of Genetic Medicine, Weill Medical College of Cornell University, New York; and Aaron Diamond AIDS Research Center, The Rockefeller University, New York, New York

Address correspondence to: Ronald G. Crystal, M.D., Institute of Genetic Medicine, Weill Medical College of Cornell University, 515 East 71st Street, Suite 1000, New York, NY 10021. E-mail: geneticmedicine{at}med.cornell.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To assess the hypothesis that infection of alveolar macrophages (AM) with adenovirus (Ad) gene transfer vectors might prevent subsequent human immunodeficiency virus (HIV)-1 replication in AM, AM isolated from normal volunteers were infected with increasing doses of first generation (E1-) Ad vectors, followed 72 h later by infection with HIV-1JRFL, an R5/M-tropic strain that preferentially uses the CCR5 coreceptor. As a measure of HIV-1 replication, p24 Ag was quantified by enzyme-linked imunosorbent assay in supernatants on Days 4 to 14 after HIV-1infection. Pretreatment of the AM with an Ad vector resulted in a dose- and time-dependent suppression of subsequent HIV-1 replication. The Ad vector inhibition of HIV-1 replication was independent of the transgene in the Ad vector expression cassette and E4 genes in the Ad backbone. Moreover, it did not appear to be secondary to a soluble factor released by the AM, nor was it overridden by the concomitant transfer of the CCR5 or CXCR4 receptors to the AM before HIV-1 infection. These observations have implications regarding pulmonary host responses associated with HIV-1 infection, as well as possibly uncovering new therapeutic strategies against HIV-1 infection.

Abbreviations: adenovirus, Ad • alveolar macrophages, AM • enzyme-linked immunosorbent assay, ELISA • human immunodeficiency virus, HIV • phosphate-buffered saline, PBS • phycoerythrin, PE • tissue culture infectious doses, TCID


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Alveolar macrophages (AM) are important reservoirs of human immunodeficiency virus (HIV)-1 infection in the lung (1). Although antiretroviral therapy suppresses HIV-1 replication in blood cells, it is less successful in eliminating HIV-1 in reservoir cells such as AM (2). Not only do AM serve as a source of HIV-1 to infect other cells in the local milieu, but the AM are activated as a result of HIV-1 infection, contributing to HIV-1 induced pneumonitis (3, 4).

HIV-1 enters AM via interaction of the viral envelope glycoprotein, gp120, with CD4 and CC or CXC ß chemokine coreceptors (5–8). In addition to using the high-affinity CD4 receptor, "macrophage-tropic" strains of HIV-1 such as HIV-1JRFL use the coreceptor CCR5 (now designated R5), and to a much lesser extent, CXCR4, CCR3, and CCR2b (6–8). Interestingly, despite the fact that human AM exhibit low levels of CD4, CXCR4, and CCR5, and undetectable levels of the other coreceptors, HIV-1 readily infects AM in vitro and HIV-1 is easily recovered from AM from the lungs of HIV-1–positive individuals (1, 2, 5–10).

Because adenovirus (Ad) gene transfer vectors can be used to modify the genetic repertoire of AM (11–13), we initiated a study to assess whether HIV-1 infection of AM was influenced by prior use of Ad vectors that genetically modify AM to express higher levels of the HIV-1 coreceptors CCR5 or CXCR4. During the course of this study, we made the serendipitous observation that infection of AM with Ad gene transfer vectors per se appeared to suppress the ability of HIV-1 to replicate in AM. The focus of the present study was to further evaluate this observation. To do so, AM recovered from the lungs of normal individuals were infected with Ad gene transfer vectors, followed by infection of the AM with HIV-1JRFL. The data demonstrate that preinfection of AM with Ad vectors suppresses the ability of HIV-1 to replicate in AM in a dose- and time-dependent fashion. This effect is independent of the transgene in the Ad expression cassette and independent of the Ad E4 region. It is not associated with the induction of soluble inhibitors secreted by AM, and is not significantly influenced by the concomitant genetic transfer of the HIV-1 coreceptors CCR5 or CXCR4.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells
Human AM were obtained by bronchoalveolar lavage from healthy volunteers as previously described, following written informed consent under a protocol approved by the Committee for Human Rights in Research of the Weill Medical College of Cornell University (13, 14). The lavage fluid was filtered through gauze to remove debris. The cells were pelleted, washed with phosphate-buffered saline (PBS), pH 7.4, and resuspended in RPMI 1640 medium containing 10% fetal bovine serum, 2 mM glutamine, 100 U penicillin/ml, and 10 µg/ml streptomycin (GIBCO BRL, Gaithersburg, MD) at a final concentration of 2 x 105 AM/ml. AM were purified by adherence to plastic (2 h, 37°C) on a 48-well culture plate (COSTAR, Cambridge, MA). One milliliter of the cell suspension (2 x 105 cells) was added per well.

Ad Vectors
Except where noted, all Ad vectors were E1a-, partial E1b-, partial E3- based on the Ad5 backbone (15, 16). The transgenes used were the reporter genes ß-galactosidase (Adßgal) and green fluorescent protein (AdGFP), or the chemokine receptors CCR5 (AdCCR5) and CXCR4 (AdCXCR4) (17–25). Ad vectors expressing CCR5 and CXCR4 receptors were shown to be functional by infecting A549 cells and assessing the cells for CCR5 and CXCR4 expression after 24 h by flow cytometry using phycoerythrin (PE)-labeled mouse monoclonal antibodies recognizing CCR5 and CXCR4 (PharMingen, San Diego, CA; not shown). An identical vector containing no transgene (AdNull) was used as a control. All vectors, including AdNull, contained the cytomegalovirus immediate-early promoter/enhancer in the expression cassette in the E1 position. An E1, E3-, E4- vector expressing ßgal (Ad E4- ßgal) was used to assess possible contributions of the E4 region of Ad to HIV-1 replication (26, 27).

Ad Infection of AM
Twenty-four hours after plating the AM, the cells were washed twice with medium (RPMI 1640 medium containing 2% fetal bovine serum, 2 mM glutamine, 100 U penicillin/ml, and 10 µg/ml streptomycin [GIBCO BRL]) to remove red blood cells and other nonadherent cells. Infections were based on particle units (pu) of Ad (28). AM were infected with Ad at doses of 103, 104, or 2.5 x 104 pu/cell. Dilutions of thawed Ad stock were added to wells with a minimal volume of media (100 µl/well) to maximize contact of particles with AM. Infections were initiated by placing the plates on a rocker for 1 h, 37°C. The cells were then washed three times with culture medium to remove any residual Ad and replaced in the incubator with 1 ml culture medium per well.

HIV-1 Infection of AM
Seventy-two hours after Ad infection, the AM (2 x 105/well) were infected with the HIV-1JRFL at 103 tissue culture infectious doses (TCID)/well, a dose known to infect cells of monocytic origin (9, 11, 29). After 24 h incubation at 37°C, the cells were washed and 1 ml fresh culture media was added.

In experiments designed to assess whether a soluble inhibitor of HIV-1JRFL replication was induced in the AM by Ad infection, residual Ad particles from Ad-infected AM were neutralized or removed from the supernatant 48 h after Ad infection by two methods. First, supernatants from Ad-infected AM were collected, mixed, and centrifuged (1.5 x 105 x g, 90 min). The resulting supernatant was placed on naive AM, then 24 h later the cells were infected with HIV-1JRFL as described above. Second, anti-Ad serum was mixed with supernatant collected from Ad-infected AM in a 1:1,000 ratio. This supernatant was then placed on naive AM and after 24 h, the AM were infected with HIV-1JRFL as described above. Lack of Ad infectivity in the resulting supernatants was confirmed by viral plaque assay on 293 cells.

Quantification of HIV-1 Replication
Productive HIV-1JRFL infection was determined by measuring p24 antigen in the supernatant by enzyme-linked immunosorbent assay (ELISA; Beckman Coulter, Miami, FL) at 4–25 d after HIV-1JRFL infection. The effects of Ad infection of AM on subsequent HIV-1 infection and replication were compared on Day 14.

Morphologic Cell Count
Due to the avidity with which the macrophages adhered to culture dishes, AM number after infections was evaluated by performing cell counts in situ by analyzing the number of nuclei per microscopic field. Three conditions were evaluated: naive AM, AM infected with HIV-1 (103 TCID/well), and AM infected with AdNull (2.5 x 104 pu/well), 72 h before HIV-1 infection as described above. Fourteen days after infection with HIV-1, the AM were washed with PBS, fixed with 4% paraformaldehyde in PBS (20 min, 22°C), and stained with the DNA-intercalating stain, DAPI (Molecular Probes, Inc., Eugene, OR; 1 µM in PBS + 0.1% Triton X-100, 5 min, 22°C). Three independent cultures were evaluated for each condition. Twelve fields were selected in each culture at random using a Zeiss Axiovert 200M epifluorescence microscope equipped with a 20x PlanFluor objective and automated stage position and focus control systems (Universal Imaging, Inc., Downingtown, PA). The MetaMorph image analysis system (Universal Imaging, Inc.) was used to determine objectively the number of nuclei per field.

Analysis of AdCCR5 and AdCXCR4 Gene Transfer
To assess AM for expression of CCR5 and CXCR4 by flow cytometry, cells recovered by lavage were maintained in suspension in RPMI 1640 media containing 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, and 10 µg/ml streptomycin in Teflon chambers. The cells were incubated with PBS containing 2% goat serum and 2% human serum on ice followed by the PE-labeled anti-CCR5 antibody or PE-labeled anti-CXCR4 antibody (Pharmingen, La Jolla, CA) for 30 min on ice, washed in PBS, and analyzed by flow cytometry.

For Northern analysis, total cellular RNA (10 µg/lane) was transferred to nylon membranes after electrophoretic separation through a 1.0% agarose gel. Human CXCR4 cDNA was gel-purified and labeled with [32P]deoxycytidine triphosphate using a random-primer labeling kit (Stratagene, La Jolla, CA). Hybridizations were performed in QUICKHYB (Stratagene) for 2 h at 65°C using standard methods. Expression of CXCR4 mRNA was compared with 28S RNA to ensure equal loading of RNA on the gel.

Statistical Analysis
Data are expressed as mean ± SEM. Treatment group means were compared by analysis of variance (ANOVA). A P value < 0.05 was considered statistically significant. The Newman–Keuls post hoc test was performed when the ANOVA was significant. Statistics were compared with the Number Cruncher Statistical System software (NCSS, Kaysville, UT).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ad Vector Dose- and Time-Dependent Inhibition of HIV-1 Replication in AM
Preinfection of AM with increasing amounts of AdNull 72 h before HIV-1JRFL infection resulted in a marked dose-dependent inhibition of HIV-1 replication as measured by p24 antigen in the supernatant at 14 d (P < 0.001, ANOVA; Figure 1) . At a dose of 4 x 103 pu/cell, AdNull suppressed HIV-1 replication by 50% compared with the level in naive cells.



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  		Figure 1. Ad vector–mediated dose-dependent inhibition of HIV-1 replication in alveolar macrophages (AM). Human AM (2 x 105/well) were infected with an AdNull vector (0–2.5 x 104 pu/cell) and 72 h later, infected with the HIV-1JRFL (103 TCID/well). The culture supernatants were collected 14 d after HIV-1 infection. Ordinate, p24 level (pg x 103/ml), as a measure of HIV-1 replication; abscissa, increasing dose of AdNull (particle units [pu]). Each data point represents mean ± SE of five samples from different donors. *Significantly different from no Ad infection (ANOVA, P < 0.05; {alpha} = 0.05, Newman–Keuls).

 
Infection of naive AM with HIV-1 resulted in steadily increasing p24 antigen levels over time from a background p24 level of 1.7 x 102 ± 0.2 x 102 at Day 0 (Figure 2) . In contrast, AM preinfected with AdNull (2.5 x 104 pu/cell) displayed a plateau in HIV-1 replication at Day 15. By Day 26, AM preinfected with AdNull demonstrate greater than a 10-fold suppression in HIV-1 replication as compared with naive AM (P < 0.001, ANOVA), indicating a time-dependence of AdNull inhibition of HIV-1 replication in AM.



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  		Figure 2. Time dependence of AdNull inhibition of HIV-1 replication in alveolar macrophages. The upper curve represents AM that were not infected with an Ad vector before HIV-1 infection, whereas the lower curve represents AM that were infected with AdNull (2.5 x 104 pu/cell) before HIV-1 infection (103 TCID/well). Ordinate, p24 level (pg x 103/ml) in the supernatant; abscissa, time (d) after HIV-1 infection. Each data point represents mean ± SE of five samples. *Significantly different from no Ad infection (ANOVA, P < 0.05; {alpha} = 0.05, Newman–Keuls).

 
To determine whether AdNull was inhibiting HIV-1 replication in AM by reducing cell viability, cell number was quantified 14 d after HIV-1 infection. Preinfection with AdNull did not reduce cell number at Day 14 compared with HIV-1 infection alone (P > 0.05, ANOVA; Figure 3) .



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  		Figure 3. Cell number after HIV-1 infection is not affected by AdNull. AM were infected with AdNull (2.5 x 104 pu/cell) 72 h before HIV-1 infection (103 TCID/well). Cell number was quantified by computer-assisted imaging of DAPI-stained cells 14 d after HIV-1 infection. For each condition (HIV-1 alone, AdNull + HIV-1, no virus) 12 fields were evaluated in each well; the results are expressed as mean ± SEM of four separate wells.

 
Transgene Independence of Ad Vector Inhibition of HIV-1 Replication in AM
To assess the influence of the transgene on Ad vector–induced suppression of HIV-1 replication, AM were infected with AdNull, Adßgal, or AdGFP (2.5 x 104 pu/cell) 72 h before HIV-1 infection. HIV-1 infection was quantified by ELISA on Day 14 by measurement of p24 antigen in the supernatant. Whereas the p24 antigen produced in the naive AM supernatants ranged from 15–30 x 103 pg/ml at Day 14, those preinfected with AdNull, Adßgal, or AdGFP produced significantly less p24 antigen, indicating that Ad inhibition of subsequent HIV-1 replication in AM is independent of the transgene (Figure 4) . To assess the role of the E4 region of the Ad genome in Ad-mediated inhibition of HIV-1 replication, AM were infected with Adßgal E4- (2.5 x 104 pu/cell) 72 h before HIV-1 infection. HIV-1 replication was also suppressed as quantified by p24 in the culture supernatants (Figure 4). Therefore, it does not appear that the E4 region of the Ad contributes significantly to the Ad vector–mediated inhibition of HIV-1 replication.



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  		Figure 4. Transgene independence of adenovirus vector inhibition of HIV-1 replication in alveolar macrophages. AM were infected with Ad (2.5 x 104 pu/cell), the cells were infected 72 h later with HIV-1 (103 TCID/well), and supernatants were collected 14 d after HIV-1 infection. Ordinate, p24 level (pg x 103/ml); abscissa, various Ad vectors: no Ad; AdNull, Adßgal, AdGFP, and AdE4-ßgal. Each data point represents an individual sample.

 
Conditioned Media from AdNull-Infected AM Does Not Inhibit Subsequent HIV-1 Replication
To determine whether Ad infection of AM induces production of a soluble inhibitor of HIV-1 replication, naive AM were exposed to supernatants from Ad infected AM (2.5 x 104 pu/cell), or to the same supernatant from which residual Ad particles had been removed by ultracentrifugation or in which they had been neutralized by anti-Ad serum. The AM were then infected with HIV-1. As determined by the p24 level in the media at days 5, 8, and 12, after HIV-1JRFL infection, the AM supernatant did not suppress HIV-1 replication, and there was no significant difference in HIV-1 replication in AM between untreated AM and those which had been exposed to supernatant from AdNull-infected AM before HIV-1JRFL infection (overall ANOVA P < 0.001; {alpha} > 0.05 for naive versus supernatant-exposed groups; {alpha} < 0.05 for AdNull 250 versus supernatant-exposed groups; Figure 5) . These observations suggest no soluble inhibitor of HIV-1 replication was released from AM after Ad infection.



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  		Figure 5. Exposure of naive AM to supernatant from AdNull-infected AM before HIV-1 infection does not inhibit subsequent HIV-1 replication. Ordinate, p24 level (pg x 103/ml); abscissa, time (d) after HIV-1 infection. Each data point represents mean ± SE of four samples. "No Ad" (filled circles) = AM that were not infected with Ad before HIV-1 infection; "Naive with Ad Null supernatant post centrifugation" (filled triangles) = naive AM exposed to supernatant from AdNull (2.5 x 104 pu/cell) infected AM 24 h before HIV-1 infection in which residual Ad was removed by ultracentrifugation; "Naive with AdNull supernatant neutralized with anti-Ad serum" (filled squares) = naive AM exposed to supernatant from AdNull (2.5 x 104 pu/cell) infected AM 24 h before HIV-1 infection in which residual Ad was neutralized with human anti-Ad serum (1:1,000 serum:supernatant). "AdNull" (open triangles) = AM directly infected with AdNull (2.5 x 104 pu/cell) 72 h before HIV-1 infection.

 
Enhanced Expression of HIV-1 Coreceptors Does Not Rescue Ad-Mediated Inhibition of HIV-1 Replication
To determine whether the expression of HIV-1 coreceptors is related to the mechanism of Ad-mediated inhibition of HIV-1 replication in AM, Ad vectors containing the transgene for CCR5 or CXCR4 were used to transfer these coreceptors to AM before infection with HIV-1. Both vectors were known to be functional based on their ability to increase the expression of CCR5 and CXCR4 on A549 cells (not shown). Previous work from our laboratory demonstrated that Ad vector–mediated gene transfer to AM is feasible, although inefficient (12). AdCCR5 infection increased CCR5 expression by AM (Figure 6A) . Flow cytometry was not sufficiently sensitive to detect increased CXCR4 expression (Figure 6B). However, by Northern analysis AdCXCR4 infection of AM strongly increased CXCR4 mRNA in comparison to AdNull infection (Figure 6C). AdCXCR4 preinfection had no effect on subsequent HIV-1 replication at 14 d compared with no Ad or AdNull-infected AM (Figure 7) . AdCCR5 pretreatment moderately increased HIV-1 replication relative to AdNull. Despite this, there was greater than one log inhibition of HIV-1 replication as measured by p24 level in AM preinfected with AdCCR5 compared with naive cells. This suggests that reduction of HIV-1 coreceptor expression is not the major mechanism of Ad-mediated inhibition of HIV-1 replication.



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  		Figure 6. HIV-1 coreceptor reconstitution by gene transfer. AM were infected with either AdCCR5 (2.5 x 104 pu/cell; A, B) or AdCXCR4 (2.5 x 104 pu/cell; B, C). AM were stained with PE-labeled antibodies and flow cytometry (A, B) was performed 72 h after Ad infection (2.5 x 104 pu/cell). (A) Expression of AM CCR5 by flow cytometry; dotted line, AdNull; solid line, AdCCR5. (B) Expression of CXCR4 by flow cytometry; dotted line, AdNull; solid line, AdCXCR4. (C) Northern analysis of CXCR4 expression by AM.

 


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  		Figure 7. Ad vector inhibition of HIV-1 replication cannot be rescued by enhanced expression of HIV-1 coreceptors in AM. AM were infected with Ad at 2.5 x 104 pu/cell; supernatants were collected 14 d after HIV-1 infection (103 TCID/well). Ordinate, p24 level (pg x 103/ml); abscissa, various Ad vectors: no Ad; AdNull; AdCCR5; and AdCXCR4. Each data point represents an individual sample.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
AM are known to play an integral role in the pulmonary host response to HIV-1 infection. These pulmonary representatives of the mononuclear phagocyte system can serve as a reservoir for the virus and can contribute to HIV-1–related pneumonitis (1–4). In addition to using the CD4 receptor, R5/"macrophage (M)-tropic strains" of HIV-1 preferentially utilize the CCR5 chemokine coreceptor to enter AM, and to a much lesser extent the chemokine coreceptors CXCR4, CCR2, and CCR2b (5–8). Based on the development of technology to modify the genetic repertoire of AM using recombinant Ad vectors encoding the gene of interest (12, 13), we established a series of studies to evaluate the consequences of upregulation of HIV-1 coreceptors by pre-exposure of normal human AM to Ad vectors encoding CCR5 or CXCR4, followed by HIV-1 infection. To our surprise, we found that preinfection of AM with an E1--modified Ad alone before infection of AM with the M-tropic strain of HIV-1JRFL inhibited subsequent HIV-1 replication. Further investigation demonstrated that the suppression of HIV-1 replication by E1- Ad vectors was a function of time and dose, was independent of the transgene in the Ad vector, and was not associated with the E4 genes in the Ad backbone. Inhibition did not appear to be due to a soluble factor in the AM supernatant, and was not significantly overcome by addition of the CCR5 or CXCR4 coding sequences to the AM.

Influence of Infectious Agents and HIV-1 Replication in AM
In the context that HIV-1 infection of mononuclear phagocytes is an important component of HIV-1 disease, and that mononuclear phagocytes play a central role in host defenses against pathogens, there have been a variety of studies to assess the influences of infectious agents on the subsequent interaction of HIV-1 with mononuclear phagocytes.

Most in vitro studies show that copathogens, such as mycobacteria and cytomegalovirus, generally upregulate HIV-1 replication in AM, mononuclear cell lines, and peripheral blood mononuclear cells (30–32). Both Mycobacterium tuberculosis and M. avium complex infection of blood monocytes in vitro upregulates HIV-1 replication (31, 32). There are conflicting studies in the literature reporting either augmentation of HIV-1 replication or inhibition when monocyte-derived macrophages or AM are infected in vitro with M. tuberculosis (32, 33). The inhibitory effect is dependent on the state of differentiation of the monocytic cells (33). The inhibitory effect of M. tuberculosis was shown to be mediated via an interferon-ß–inducible inhibitory nuclear factor, C/EBP-ß, which suppressed transcription from the HIV-1 LTR (33, 34). In vivo, there is evidence that HIV-1 replication is enhanced in AM in lung segments with active M. tuberculosis infection (35). Macrophages in lymph nodes infected with atypical mycobacteria or pneumocystis also demonstrate upregulated HIV-1 replication (36). Influenza virus is known to inhibit subsequent HIV-1 replication via induction of release of soluble mediators (37).

Possible Mechanisms of Ad Vector-Mediated Suppression of HIV-1 Replication in AM
Attachment of the HIV-1 virion onto the cell is mediated by cell surface CD4 acting in concert with coreceptors CCR5, CXCR4, and, to a lesser extent, CCR3 and CCR2b (5, 17–22, 29). AM are readily infected by HIV-1 strains that predominantly use CCR5 as the coreceptor (6–8). In the present study, upregulation of expression of CCR5 or CXCR4 via gene transfer did not significantly abrogate Ad vector–mediated inhibition of HIV-1 replication, suggesting that the effect of Ad vectors is unlikely to involve depletion or blockade of cell surface coreceptors. Moreover, the plateau observed in HIV-1 replication as a function of time in AM preinfected with AdNull suggests that alteration of HIV-1 virion attachment or entry is not a predominant mechanism and that one or more subsequent steps in the viral life cycle may be actively suppressed.

After HIV-1 virion entry, the viral RNA genome is reverse transcribed into double-stranded DNA. The viral dsDNA is translocated to the nucleus, followed by the initiation of HIV-1 transcription leading to HIV-1 protein synthesis. Suppression of HIV-1 post-entry events might be due to either the direct effect of an Ad vector gene product or to the indirect effect of an inducible cellular factor(s) on nuclear import, integration, or transcription. Our data suggest that the adenovirus E1 and E4 genes are dispensable for inhibition of HIV-1 replication, and Ad vector transgenes are not involved. A secondary inducible cellular effect might be mediated by a soluble factor released extracellularly, a cell surface paracrine factor or direct activation or inhibition of an intracellular pathway (38). Examples of soluble factors known to inhibit HIV-1 replication by blocking entry include tumor necrosis factor-{alpha}–inducible CCR5 ligands such as RANTES, macrophage inflammatory protein-1{alpha}, and macrophage inflammatory protein-1ß (6, 39). An example of post-entry block is interferon-ß, which acts via repression of HIV-1 LTR transcription (34, 40). Other soluble cytokines, such as interleukin-4, interleukin-10, transforming growth factor-ß, CD8+ T-lymphocyte antiviral factor (CAF), and macrophage-derived chemokine inhibit HIV-1 replication at one or more post-entry levels (41–43). Our passive transfer experiment with conditioned media depleted of Ad vectors did not reveal a soluble inhibitor of HIV-1 replication. Further experiments will be needed to definitively rule out a cell membrane–associated paracrine factor and to determine whether Ad vectors upregulate an intracellular pathway inhibiting HIV-1 replication.

Implications of Ad Vector-Mediated Suppression of HIV-1 Replication in AM
Tissue macrophages remain a persistent source of latent HIV-1 infection, even in the face of successful highly active antiretroviral therapy (2, 44). Therefore, new methods will be needed to treat latent HIV-1 infection in organs such as the lung. In the context that genetic modification of AM with Ad vectors is feasible, it is important to know whether the gene delivery system itself induces HIV-1 replication (12, 13, 44). We have shown that Ad vectors suppress HIV-1 replication in AM in vitro. Further studies will be needed to address whether this will be a useful model for therapeutic suppression of HIV-1 replication in AM.


   Acknowledgments
 
The authors thank B.-G. Harvey, Division of Pulmonary and Critical Care Medicine, Weill Cornell, for the human anti-Ad neutralizing antiserum, and N. Mohamed for help in preparing this manuscript. The authors acknowledge the Tri-Institutional Chemical Biology Research Initiative for use of the automated microscopy system. These studies were supported, in part, by grants R01 HL59861, AI41420, and M01RR00047; by the Will Rogers Memorial Fund, Los Angeles, CA; and by GenVec, Inc., Gaithersburg, MD.

Received in original form August 7, 2001

Received in final form April 3, 2002


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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