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Anti-HIV-1 activity of indolicidin, an antimicrobial peptide from neutrophils W. Edward Robinson, Jr.,*† Brenda McDougall,* Dat Tran,*† and Michael E. Selsted*† Departments of *Pathology and †Microbiology and Molecular Genetics, University of California, Irvine

Abstract: Indolicidin is a tridecapeptide amide isolated from the cytoplasmic granules of bovine neutrophils. It has potent, broad spectrum microbicidal activities in vitro that are thought to be related to the membrane-disruptive properties of the peptide. Based on the putative membrane-targeted mode of action, we postulated that indolicidin would be active against HIV-1, an enveloped virus. Indolicidin was reproducibly virucidal against HIV-1 at a concentration of 333 mg/mL (174 mM) with a 50% inhibitory dose between 67 and 100 mg/mL. At 37°C, killing was rapid with G50% killing of HIV occurring within 5 min, and nearly 100% viral inactivation achieved by 60 min. The anti-HIV activity of indolicidin was temperature-sensitive, a finding consistent with a membrane-mediated antiviral mechanism. Parallel experiments revealed that indolicidin lysed cultured lymphoblastoid cells at concentrations similar to those required for antiviral activity. However, a des-R13-amide indolicidin analog (R12-OH), previously shown to have less antibacterial activity than indolicidin, was significantly less active against HIV and was non-toxic to lymphoid target cells at concentrations up to 333 mg/mL, the highest level tested. J. Leukoc. Biol. 63: 94–100; 1998. Key Words: AIDS · defensin · host defense · innate immunity

INTRODUCTION Infection with the human immunodeficiency virus (HIV) is a major health problem both in the United States and, in particular, in developing countries; in 1996 the Centers for Disease Control (CDC) predicted that over 20 million persons worldwide were infected by HIV. The primary routes of infection are sexual, vertical (from mother to offspring), and intravenous, either through intravenous drug use or transfusion of contaminated blood or blood products [1–3]. Sexually transmitted diseases are frequently associated with a significant local infiltration of neutrophils and other acute inflammatory cells. Although it is likely that neutrophils can kill HIV, the mechanism of such killing has not been fully elucidated. Neutrophils have been implicated in antibody-dependent cellular cytotoxicity (ADCC) against HIV-infected cells [4], although the specific mechanism of ADCC remains unclear. Moreover, direct interactions between neutrophils and free HIV virions have not been characterized. 94

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In addition to their production of antimicrobial oxygen-derived intermediates (e.g., superoxide, hydrogen peroxide, hypochlorous acid), neutrophils utilize microbicidal polypeptides that are stored in and released from cytoplasmic granules [5–9]. Among the numerous antimicrobial peptides characterized to date are defensins [10–13], b-defensins [14], and cathelicidins [15–18]. Previous studies have demonstrated that defensins are microbicidal for a wide range of organisms, including bacteria, fungi, and some enveloped viruses [reviewed in refs. 19 and 20]. Certain defensins have been shown to kill herpes simplex virus types 1 and 2, cytomegalovirus, vesicular stomatitis virus, and influenza virus but not two nonenveloped viruses, echovirus and reovirus [21, 22]. In a recent report, defensins were shown to inhibit infection by another enveloped virus, HIV [12]. Indolicidin is an antimicrobial tridecapeptide amide (H2N-IleLeu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-CONH2) isolated from the large granules of bovine neutrophils [10, 23, 24]. It is a member of the cathelicidin family of antimicrobial peptides [18]. Previous work has demonstrated that natural or synthetic indolicidin was bactericidal and fungicidal at low micromolar concentrations in vitro [14, 23, 25]. Like the defensins, the effects of indolicidin on microorganisms are thought to be mediated through binding and permeabilization of the cell membrane [26]. Ahmad et al. recently demonstrated that a liposomal formulation of indolicidin had an anti-infective therapeutic effect on mice challenged with Aspergillus fumigatus spores [27]. This study suggested that indolicidin might be adapted for systemic administration in the treatment of opportunistic fungal infections. To determine whether the antimicrobial spectrum of indolicidin might include enveloped viruses, we characterized the virucidal activity of this peptide against HIV-1. We also evaluated the virucidal potency of a des-Arg-amide indolicidin analog (R12-OH; H2N-Ile-Leu-Pro-Trp-Lys-Trp-ProTrp-Trp-Pro-Trp-Arg-COOH), a peptide that possesses much less antibacterial and antifungal activity than indolicidin. Our studies demonstrate that indolicidin directly kills HIV-1 at concentrations that parallel those required to lyse target cells. The temperature dependence of killing was consistent with a membrane-disruptive mechanism. The R12-OH analog was an order of magnitude less potent than indolicidin against HIV but was

Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; CDC, Centers for Disease Control; HIV, human immunodeficiency virus; RT, reverse transcriptase. Correspondence: W. Edward Robinson, Jr., M.D., Ph.D., Dept. of Pathology, D440 Med Sci I, University of California, Irvine, CA 92697-4800. E-mail: [email protected]. Received May 1, 1997; revised August 7, 1997; accepted August 11, 1997.

also found to be completely non-toxic for lymphoid cells at all concentrations tested.

MATERIALS AND METHODS Solid-phase peptide synthesis of indolicidin and indolicidin-des-R13-amide Indolicidin and indolicidin-des-R13-amide (R12-OH) were synthesized on a 9050 Milligen peptide synthesizer essentially as described previously [25]. Briefly, chain assembly was carried out at a 0.2 mmol scale with the use of BOP/HOBt in situ activation. Cleavage and deprotection reactions were carried out using Reagent K as described [25]. Each of the crude peptide preparations was extracted with 30% acetic acid and dichloromethane. The peptides were purified by C18 reversed-phase high-performance liquid chromatography (RPHPLC) on a 22.5 3 250-mm Vydac C18 column [25]. Peptides were determined to be homogeneous by analytical RP-HPLC, acid-urea polyacrylamide gel electrophoresis, electrospray mass spectroscopy, and amino acid analysis. Peptides were stored lyophilized at 270°C. Before use, peptides were dissolved in 0.9 mL of deionized water and 0.1 mL of 9% (w/v) NaCl was added to bring the final NaCl concentration to 0.9% (w/v). Working solutions of peptides were stored at 220°C for up to 3 years without any loss of anti-HIV activity.

indirect immunofluorescence microscopy after staining of fixed cells with pooled human anti-HIV serum followed by fluorescein-conjugated goat anti-human IgG as described previously [29].

Cytotoxicity assays Peptide-mediated cytotoxicity was determined by two methods. Peptides were serially diluted in microtiter plates and an equal volume of MT-2 cells in growth medium (5 3 105 cells/mL) was added to each well. The cells were incubated for 2 days at 37°C and the number of viable cells was determined with the use of Finter’s neutral red dye as described [28]. The fifty percent growth inhibitory concentration (IC50) was calculated relative to eight cell control replicates (100% viable) and eight blank wells (0% viable). In other experiments, H9 cells were labeled with Na251CrO4, washed, and plated with diluted peptide in triplicate wells of a 96-well microdilution plate. Cells were pelleted by centrifugation at 1200 g after various times and the 51Cr release was determined with a Beckman b-scintillation counter using the 3H window. Specific release was calculated relative to eight spontaneous-release wells and eight total-release wells (treatment with 3% Triton X-100) according to the following formula: % specific release 5 (sample release 2 spontaneous release)/(total release 2 spontaneous release).

RESULTS

Cells and virus The CD41 lymphoblastoid cell lines MT-2 and H9 were passaged in growth medium (RPMI-1640 containing 25 mM N-2-hydroxyethylpiperazine-N8-2ethanesulfonic acid supplemented with L-glutamine and 11.5% heat-inactivated fetal bovine serum). The HIVLAI isolate of HIV-1 was propagated in H9 lymphoblastoid cells in growth medium. Infectivity assays were performed essentially as described [28]. HIVLAI culture supernatants were clarified by low-speed centrifugation followed by filtration through 0.45-µm cellulose acetate syringe filters immediately before inoculation of target cell cultures.

Anti-HIV assays For anti-HIV assays, 30 µL of HIVLAI-containing culture supernatant was incubated with an equal volume of 23 peptide for intervals of 0–60 min at 4, 12, 24, or 37°C. At t0 the viral titer in the incubation mixture was 1 to 2 3 106 infection particles per milliliter. Aliquots of the incubation mixture were diluted 1:500 in growth medium and 1 mL of the peptide/virus mixture was added to triplicate wells of a 24-well microtiter plate. Finally, 1 mL of MT-2 cell suspension (,5 3 105 cells/mL) was added to each well. Virus control wells included HIV incubated with an equal volume of RPMI-1640 before dilution and final addition to MT-2 cells. The final concentration of peptide to which the cells were exposed was 2,000-fold less than the concentration used to treat virus. Cells were incubated for 24 h at 37°C with virus and peptide. Culture fluids were then removed and cells recultured in 2 mL of fresh growth medium. At intervals ranging from 3 to 13 days after HIV infection, 0.75 mL of culture supernatant was removed for reverse transcriptase (RT) assays; cells were then resuspended in the remaining medium and 0.5 mL of cell suspension was removed for immunofluorescence assay. Next, 1.3 mL of fresh growth medium was added to each well and the cells incubated at 37°C. Reculturing of cells was carried out for 10–13 days, by which time cells incubated with all peptide concentrations, except 333 µg/mL indolicidin, were completely lysed.

RT assay and immunofluorescence analysis Each culture supernatant was precipitated with 0.42 mL of 30% polyethylene glycol as described previously [29]. Precipitated virus was lysed and incorporation of [3H]thymidine into poly rA-oligo-dT templates was measured according to a modification [29] of the method first described by Poiesz et al. [30]. Trichloroacetic acid-precipitable raw cpms were determined on a Beckman b-scintillation counter. The mean cpm for the triplicate infections was determined and mean background cpm from three cell control cultures, run in parallel with each assay, was subtracted. The resultant corrected cpm was multiplied by 8 to convert to cpm/mL of culture supernatant fluid. For immunofluorescence analysis, cells from triplicate wells were combined, washed, and spotted onto glass slides. The percentage of cells expressing HIV antigens was estimated by

Indolicidin and other antimicrobial peptides are known to have microbicidal activity against a variety of prokaryotic and eukaryotic microorganisms. The mechanism of action appears to rely on processes that include binding and permeabilization of target cell membrane(s). This mechanistic model would predict that indolicidin might be virucidal for HIV, an enveloped virus. Similarly, indolicidin might also be toxic for mammalian cells under the conditions of the assays. Therefore, to eliminate potential peptide-mediated cytocidal or cytostatic effects on the producer cells, the primary incubation mixtures, containing indolicidin and HIV, were diluted 1000 times in growth medium before addition to target cells. As indicated in Figure 1, incubation of HIV with varied concentrations of indolicidin resulted in direct viral inactivation within 1 h at 37°C. The IC50 of indolicidin under these conditions was between 67 and 100 µg/mL. At the highest peptide concentration tested, 333 µg/mL, one or two of the triplicate wells was rendered sterile, resulting in the very low levels of surviving virus observed even at day 13 (Fig. 1). These data indicate that the average number of infectious virions present in the incubation at the time of dilution was ,1000/mL, representing a greater-than-3-log reduction in infectious particles. Because the RT assay provides only one measure of HIV replication, we also performed indirect immunofluorescence analysis of cell-associated HIV antigens. In all experiments, the percentage of HIV-antigen-positive cells correlated with RT release as previously observed [29]. To more accurately determine the interval required for the anti-HIV effect, incubation of HIV with 333 µg/mL of indolicidin was carried out for intervals ranging from 0 to 60 min. At the highest concentration tested (333 µg/mL), complete inactivation of HIV occurred within 60 min of incubation at 37°C (Fig. 2A). However, incubation times as short as 20 min were adequate to produce a significant anti-HIV effect. For example, the level of progeny virus at post-infection day 8 was reduced approximately 30-fold compared to the virus control. The effect was not instantaneous, however, because virus levels resulting from incubation mixtures that were immediately diluted and mixed Robinson et al.

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Fig. 1. Anti-HIV activity of indolicidin. HIV was incubated with 0 (circles), 10 (squares), 33 (triangles), 67 (inverted triangles), 100 (diamonds), or 333 µg/mL (hexagons) indolicidin for 1 h at 37°C. The peptide and virus mixture was then diluted, added to triplicate wells of a 24-well plate, and MT-2 target cells were added to each well. After 24 h the entire culture supernatant was removed and cells were resuspended in 2 mL of growth medium. Cells were harvested for immunofluorescence assays and supernatant fluids harvested for RT assay on the indicated days. Each point represents the mean RT release for triplicate infections; error bars are one standard deviation. Percent values represent the fraction of cells positive for HIV antigens as measured by indirect immunofluorescence.

with MT-2 indicator cells were not substantially different than those produced in incubations lacking indolicidin (Fig. 2A). To further our understanding of the relationship between the membrane-mediated effects of indolicidin on bacterial or fungal targets and its antiviral effects, we conducted similar anti-HIV assays using R12-OH indolicidin, an analog shown previously to be much less active than indolicidin against Staphylococcus aureus, Escherichia coli, and Cryptococcus neoformans [31]. As illustrated in Figure 3, R12-OH was much less effective in killing HIV when compared to indolicidin. Whereas 333 µg/mL of indolicidin virtually sterilized the virus inoculum (Figs. 1 and 3), 333 µg/mL of R12-OH indolicidin was only slightly more active than a log lower concentration of indolicidin (33 µg/mL; Fig. 3). Similarly, the reduced activity of R12-OH was also observed in a time course analysis of antiviral activity (Fig. 2B) performed as described above for indolicidin (Fig. 2A). To further characterize the interaction of indolicidin and R12-OH with HIV, we assayed virucidal activity in incubations carried out at temperatures ranging from 4 to 37°C. As illustrated in Figure 4, indolicidin completely sterilized HIV-containing culture fluid after a 1-h incubation at a concentration of 333 µg/mL at 37°C. However, when the incubation was performed at 12 or 24°C, the anti-HIV activity of the indolicidin was substantially reduced. Moreover, when HIV was incubated with indolicidin at 4°C, the anti-HIV activity was virtually abolished. To assess the susceptibility of the lymphoid target cells to indolicidin we determined the cytotoxicity of the peptide against MT-2 target cells. As illustrated in Figure 5, the concentration of indolicidin that killed 50% of the cells (LD50) for indolicidin 96



Fig. 2. Time course of anti-HIV activity of indolicidin and the R12-OH indolicidin analogue. HIV was incubated with (A) indolicidin or (B) R12-OH indolicidin at 333 µg/mL for 60 (circles), 40 (squares), 20 (triangles), or 0 min (inverted triangles), or in the absence (diamonds) of indolicidin at 37°C. The peptide and HIV mixture was then diluted and added to triplicate wells of a 24-well plate as described in the legend to Figure 1. Culture supernatant fluids were harvested for RT assay and cells were obtained for immunofluorescence analysis at the indicated day after initial virus infection as described in the legend to Figure 1. Each point is the mean RT activity from triplicate wells; error bars represent one standard deviation. Percent values correspond to the fraction of cells positive for HIV antigens as measured by immunofluorescence.

Fig. 3. Comparative anti-HIV activities of indolicidin and its R12-OH analog. HIV-1 was pretreated with indolicidin at 333 (circles), 100 (squares), and 33 µg/mL (triangles) or with R12-OH indolicidin at 333 (inverted triangles), 100 (diamonds), and 33 µg/mL (hexagons) for 1 h at 37°C as described in the legend to Figure 1. Culture supernatants were harvested for RT activity and cells for immunofluorescence assay as described in legend to Figure 1. Each point is the mean of RT assays from triplicate infections; error bars are one standard deviation. Virus controls were not significantly different from the 33 µg/mL R12-OH analog (data not shown). Percent values represent the fraction of cells positive for HIV antigens as measured by immunofluorescence.

tions ranged from 30 to 40 µg/mL [12]. Tamamura et al. reported that synthetic protegrin peptides were approximately 3 to 10 times as active as the defensins in a similar in vitro assay system [32]. In this study we investigated the virucidal activity of indolicidin against HIV as a model for investigating the interaction of membrane-active antimicrobial peptides with an enveloped virus. Although indolicidin was cytotoxic for producer cells, we were able to demonstrate a direct, dose-dependent virucidal effect of the peptide by incorporating a large dilution step into the assay, thus avoiding the cytotoxic activity against lymphoid target cells. As illustrated in Figures 1–4, indolicidin was capable of virtually sterilizing an HIV-containing preparation. Furthermore, this activity was both time- and temperature-dependent (Figs. 2 and 4, respectively). Indolicidin, but not R12-OH indolicidin, rapidly caused chromium release from labeled HIV producer cells, H9 (Fig. 6, A and B). Peptide-mediated cell death was rapid (within 7 min) and demonstrated a sharp, nearly asymptotic dilution profile (Fig. 5). These data differ markedly from the cytotoxicity data reported for the human defensins, HNP 1-3, where chromium release was detectable only after 3 h and increased to a maximum level 6 h later [41]. Although R12-OH was virucidal for HIV at concentrations above 100 µg/mL, it was significantly less potent against the virus than indolicidin. Indeed, a log lower concentration of indolicidin was required to achieve a similar anti-HIV effect as R12-OH (Fig. 3). However, R12-OH had no effect on chromium

was approximately 33 µg/mL. This concentration is similar to the observed IC50 of indolicidin for HIV (approximately 67–100 µg/mL) and is within the variability of the assay. These results suggest that, under the conditions of the assay, there is little selectivity of indolicidin for viral particles versus animal cells as targets. To assess whether indolicidin-induced cytotoxicity involved a cytolytic process, H9 cells (the HIV producer cell line) were labeled with Na251CrO4 and peptide-induced chromium release was measured. As illustrated in Figure 6A, indolicidin induced rapid, dose-dependent chromium release. Indeed, nearly 50% of the chromium was released within 20 min at a peptide concentration of 167 µg/mL, and concentrations of indolicidin as low as 83 µg/mL caused significant release of 51Cr from the cells. R12-OH, on the other hand, had no cytolytic activity against H9 cells at all concentrations tested (Fig. 6B). These data suggest that a membrane-disruptive mechanism is operative in the cytotoxic and anti-HIV activities mediated by indolicidin.

DISCUSSION Despite previous reports that phagocyte-derived antimicrobial peptides kill some enveloped viruses, there have been few reported studies on the anti-HIV activity of these molecules. In this regard, the only two studies we are aware of have reported on the anti-HIV effects of non-human defensins [12] and protegrinrelated peptides [32]. In the former study, Nakashima and co-workers synthesized three defensins produced in rabbit, rat, or guinea pig neutrophils, and demonstrated a dose-dependent antiviral effect in which the 50% inhibitory peptide concentra-

Fig. 4. Temperature-dependent anti-HIV activity of indolicidin. HIV was incubated in the presence or absence of indolicidin at 4, 12, 24, or 37°C. The infection kinetics for HIV alone at 4 (circles) and 37°C (squares) are shown; curves for the 12 and 24°C treated virus were not significantly different from the 37°C treated virus. HIV was incubated with indolicidin at a final concentration of 333 µg/mL for 60 min at temperatures of 37 (triangles), 24 (inverted triangles), 12 (diamonds), and 4°C (hexagons). After the 1-h incubation, virus was diluted 1:500 in growth medium, incubated at 37°C for 15 min, and added to MT-2 cells as described in Materials and Methods. Cells were harvested to determine the fraction positive for HIV antigens by immunofluorescence microscopy percent values and supernatants were harvested for RT assay at the indicated times after infection. Each point represents the mean of triplicate infections; error bars are one standard deviation.

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proline (3/13 residues), and possesses no detectable secondary structure [26]. Although the indole side chains endow the peptide with hydrophobic character, three basic amino acids confer substantial side-chain polarity. Substitution of tryptophan with phenylalanine residues was reported to abolish the hemolytic activity of indolicidin, although there was no effect of these substitutions on the antimicrobial activity of the resultant analogs [40]. The combined side chain properties probably create an amphipathic topology in indolicidin that is well suited to binding of biological membranes. Recent studies demonstrate that indoli-

Fig. 5. Cell toxicity of indolicidin against MT-2 cells. Fifty microliters of a 3000 µg/mL stock solution of indolicidin in normal saline was added to triplicate wells of a 96-well microtiter plate. Peptide was twofold serially diluted in an equal volume of growth medium. To each well, 100 µL of an MT-2 cell suspension containing approximately 5 3 106 cells/mL were added and the cells allowed to incubate at 37°C for 48 h, then harvested for cytopathic effect as described previously [28]. A540 of the cells was measured and the percent viable cells calculated based on eight-cell controls (no peptide) and eight blank wells (no cells and thus no viable cells). Each point represents the mean percent viable cells for triplicate wells; error bars are one standard deviation.

release from H9 cells, even at the highest levels tested (Fig. 6B). Although the molecular details underlying the difference in activities of indolicidin and R12-OH are not understood, these data indicate that a moderate degree of antiviral selectivity (approximately threefold compared to indolicidin) was produced by alteration of the indolicidin carboxyl terminus. There is growing evidence that several antimicrobial peptides kill microorganisms by a mechanism that includes disruption of the membrane and subsequent lysis of the target [34–40]. The molecular features that endow indolicidin with lytic activity probably differ from those of disulfide-containing defensins and protegrins, peptides in which the structures are predominated by b-sheet and turns [37]. Unlike those peptides, indolicidin lacks disulfides, is unusually rich in tryptophan (5/13 residues) and

Fig. 6. Cytolytic potencies of indolicidin and its R12-OH analog. Indolicidin (A) and R12-OH (B) were diluted in triplicate wells of a 96-well round-bottom plate. Na251CrO4-labeled H9 cells, the HIV producer cell line, were added at a final concentration of 5 3 103 cells per well. Final concentrations of indolicidin and R12-OH indolicidin per well were: 333 (circles), 167 (squares), 83 (triangles), 42 (inverted triangles), 21 (diamonds), and 10 µg/mL (hexagons). Cells were incubated for the indicated times before removal of 100 µL of the supernatant fluid. Percent specific 51Cr release was calculated. Each point represents the mean of triplicate samples; error bars are one standard deviation. Data from two separate experiments (time 7–30 min and 30–120 min) are illustrated by the two separate curves at each concentration in panel A. Total 51Cr release (eight replicates) for the short time-course incubation reaction was 47,880 6 1587 cpm and 33,058 6 1127 cpm for the long time-course incubation experiment. Spontaneous release (eight replicates) was different for each individual time point but ranged from a low of 272 6 26 cpm (0.6% of total) for the 7-min incubation to 962 6 54 cpm (3.13% of total) for the 120-min incubation.

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cidin binds strongly, but reversibly, to phospholipid vesicles, and that the vesicles are permeabilized in a dose-dependent manner [26]. These observations are consistent with the hypothesis that indolicidin kills target microbes by disrupting membranes constituting the microbial envelope. Although the in vitro studies reported here do not replicate the complex environment in which antimicrobial peptides interact with microbial pathogens and host cells in vivo, it is quite possible that indolicidin is relatively non-selective in its action against enveloped viruses and lymphoid cells in inflammatory foci. This lack of specificity has many precedents in acute inflammation, since reactive oxygen species, proteases, and nitric oxide are known to be toxic to both microbial and host cells. In this regard, animals have evolved tissue repair mechanisms that compensate for inflammatory injury during responses to infection, an acceptable tradeoff for the ability of the host to eliminate potentially lethal pathogens. The current study suggests that antimicrobial peptides may play a role in the inactivation of HIV through mechanisms that act directly against enveloped virions, by lysis of virus-infected cells, or a combination of both effects.

ACKNOWLEDGMENTS This work was supported in part by NIH AI22931 and Biosource Technologies. The authors would like to thank Peter King for thoughtful comments on the manuscript.

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