As a barrier and immune organ, the gastrointestinal tract, lung and skin play a key role in protecting the body from a hostile environment 1. The low incidence of infection at normal epithelial surfaces reflects the presence of innate, broad-spectrum antimicrobial defense mechanisms 2. Host defense peptides (HDPs) of the innate immune response play an important role in the protective barrier function of the epithelia 3. Host defense peptides have been isolated from diverse organisms, including plants, insects, bacteria and vertebrates 4. Several classes of mammalian peptide antibiotics have been ascribed pivotal roles in innate immunity 5. Among these are various cysteine-rich peptides such as defensins 67 and the more structurally diverse cathelicidins 8. Produced as precursors, they require proteolytic processing to liberate the mature functional antimicrobial peptide. Cathelicidins contain a conserved N-terminal cathelin domain, and a structurally diverse C-terminal domain that possesses the peptide's antimicrobial activity. Rabbit CAP18 was the first cathelicidin precursor described, and its mature peptide has broad-spectrum antimicrobial activity 9. Cathelicidins have since been identified in many other species including hCAP18/LL37 in humans 10, protegrins in swine 111213, CRAMP in mice 1415 and SMAP29 in sheep 16. Many of these peptides demonstrate extremely broad-spectrum antimicrobial activity, including Gram positive and Gram negative bacteria and fungi 415. In addition, they achieve bacterial killing much more rapidly than any commercially available antibiotic 17. Recently, a new family of synthetic, α-helical HDPs called "ovispirins" was described 181920. Although some of these modified peptides had similar antimicrobial activity of naturally occurring peptides, they manifested appreciable cytotoxicity. We have demonstrated recently that variants of Ovispirin, the so called Novispirin peptides, displayed more favorable toxic/ therapeutic ratios in vitro and broad spectrum activity in infected rat burn model 2122.

Some of these peptides are induced at epithelial surfaces in response to invading organisms 232425. Many HDPs kill microorganisms by causing membrane permeabilization, although not necessarily as their sole mode of action 26. Some HDPs also direct chemotaxis, promote wound healing, angiogenesis and contribute to adaptive immunity by mobilizing memory T cells and immature dendritic cells 2527. Recent studies have also demonstrated antitumor activity after treatment with HDPs 28.

In addition, several antiviral activities were reported. Recently it has been demonstrated that rabbit neutrophil peptide alpha-defensin NP1 protects cells from infection with HSV-1 and 2 29. Other studies revealed that human neutrophil peptide HNP1 to 3 and Theta-defensins also inhibit HSV infection although by different mechanisms 303132. The ancestral human theta-defensins retrocyclin blocked HSV attachment 33. The inhibition of adenovirus replication by the antimicrobial peptide awaits identification of a mechanism of action 30. Anti-HIV activity of defensins were first reported 1993 by Nakashima and coworkers 34. The alpha-defensins exhibited anti-HIV activity on at least two levels: directly inactivating virus particles; and affecting the ability of target CD4 cells to replicate the virus 353637. Binding to gp120 of HIV-1 and inhibition of HIV entry has also been identified as the mechanism of inhibition of HIV infection by theta defensins 38. Due to their inhibitory effect on HIV-1 replication and due to an association of a single-nucleotide polymorphism in a beta defensin gene human beta-defensins might also play an important role in host defense against HIV-1 39.

The antibacterial activity of HDPs is largely mediated by pore formation leading to permeablization of the bacterial membrane. Although some selectivity for bacterial membranes has been described, the lipid membrane of enveloped viruses might also be a target of antimicrobial peptides 3240. This might allow development of antiviral effector molecules for topical application against a broad spectrum of enveloped viruses. Targeting host cell-derived membrane components might be a particularly interesting approach to inhibit viruses that rapidly develop resistance to compounds directed against viral proteins such as HIV. Therefore, we screened a panel of different natural occurring and designer HDPs for Env-independent inhibition of HIV infection at an early step in the viral replication cycle.

Given the potential cytotoxicity of HDPs, it was important to discriminate between modulation of cell metabolism and direct antiviral effects. We were concerned that HDPs could modulate host cell metabolism without affecting cell viability as assayed for example by standard MTT assays. We therefore decided to use immunodeficiency virus-based vectors transferring the luciferase gene to determine both, the HDP antiviral activity and modulation of cell metabolism. The luciferase activity of target cells, stably transduced with the lentiviral vector prior to HDP treatment should reveal any effect of HDPs on cellular transcriptional and translational efficacy. Treating cells with HDPs during infection with the lentiviral vector and comparison with results obtained with the stably transduced cells should then allow identifying HDPs that inhibit early steps in the viral replication cycle. To validate the luciferase-based assay for modulation of cell metabolism, 293 cells were stably transduced with a lentiviral vector transferring the luciferase gene. Incubation of transduced cells with increasing concentrations of Protegrin-1 and LL37 led to a dose-dependent inhibition of luciferase activity (Fig. 1). Comparison to the MTT test revealed a similar dose response curve, although the MTT test might be less sensitive at lower concentrations of the HDPs. Testing cell proliferation by a BrdU incorporation assay revealed a threshold level above which proliferation is strongly reduced. To allow side by side evaluation of cytotoxic and antiviral effects of HDPs with the same read-out the luciferase-based assay was used in most subsequent experiments.

A panel of HDPs containing members of the major classes of antimicrobial peptides were analyzed for inhibitory effects against lentiviral vectors. Given the variability of the viral envelope protein, we focused on identifying Env-independent inhibitory activities by using VSV-G pseudotyped lentiviral vectors. All antimicrobial peptides used in this study, human cathelicidin LL37, recombinant human β-Defensin-2, porcine Protegrin-1 (PG-1), fungal Plectasin and Novispirin G10, inhibited gram positive and gram negative bacteria revealing antimicrobial activity in the expected range (Table 1) and confirmed bioactivity of the peptides used.

The inhibitory effect against the lentiviral vector was determined by preincubation of the vector with human cathelicidin LL37, recombinant human β-Defensin-2, porcine PG-1, fungal Plectasin and Novispirin G10 for 30 minutes in increasing concentrations of peptide prior to the addition of vectors with antimicrobial peptide to the target cells. Stably transduced target cells were incubated in parallel with the HDPs to detect effects on cell metabolism. Luciferase activities were determined 2 days after infection. All HDPs led to a reduction in luciferase activity of cells transduced with the lentiviral vector (Fig. 2A–E), but most of them also reduced the luciferase activity of stably transduced target cells. Specific inhibition of early steps of infection was only seen for the cathelicidin LL37 and PG-1. As an additional control for the specificity of inhibition, a non-enveloped adenoviral vector transferring the luciferase gene was also incubated with the same panel of HDPs. None of the HDPs led to a dose-dependent inhibition of early steps of the adenoviral replication cycle (Fig. 2F–J). Two-fold serial dilutions were used to determine 50% inhibitory concentrations (IC₅₀) of LL37 and PG-1, resulting in IC₅₀s of approximately 30 μg/ml and 16.8 μg/ml, respectively (Fig. 3).

In initial attempts to characterize the mechanism of inhibition of lentiviral vector infectivity, LL37 and PG-1 were added at different time points during infection: both HDPs were either preincubated with the vector preparation for 30 minutes prior to addition to the cells or the HDPs and the vector were added simultaneously to the cells (Fig. 4A+B). In addition, cells were first infected with the lentiviral vector for two hours prior to addition of the HDPs. LL37 and PG-1 exerted the strongest inhibition after preincubation of HDPs and the lentiviral vectors, indicating a direct effect on infectivity of the vector particles. However, adding LL37 and PG-1 two hours after incubation of cells with the lentiviral vector also led to a stronger reduction of luciferase activity than observed after incubation of cells stably transduced with the luciferase gene suggesting a second target in the infection cycle that is affected by LL37 and PG-1. To further discriminate between direct inhibitory effects on vector particles and effects mediated by potential HDP cell interactions lentiviral vector particles were first incubated with LL37 and PG-1 for 30 minutes and then added either undiluted or at a 1:10 dilution to the target cells. Due to a 10-fold lower HDP concentration in the latter cultures, vector infectivity should be reduced to a lesser extent, if inhibitory effects are mediated by cellular targets. Comparable dose-dependent inhibition curves (Fig 4C,D) of diluted and undiluted vectors demonstrate that the inhibitory effects of LL37 and PG-1 depend on the HDP concentration during preincubation of the vector particles and not on the HDP concentration during subsequent cell culture.

The lentiviral vector used in this study had been pseudotyped with the G protein of vesicular stomatitis virus (VSV-G). To evaluate whether LL37 and PG-1 directly target VSV-G or a lentiviral protein, the inhibitory effect of these HDP against the lentiviral vector was compared side by side to their effect on a retroviral vector containing the amphotropic MLV Env for entry into target cells. The dose dependent reduction in the luciferase activity in the target cells was very similar in cells transduced with the lentiviral or the MLV vector (Fig. 5).

The inhibition of HIV vectors containing the HIV-1 envelope by LL37 and PG-1 were studied on P4CCR5 cells expressing CD4 and coreceptors. IC₅₀s of 25 μg/ml and 14 μg/ml were observed for LL37 and PG-1, respectively (Fig 6A,B), while only minimal inhibitory effects on cell proliferation were detected at these concentrations. Inhibition of early steps of wild type HIV-1 infection by LL37 and PG-1 was also evaluated on P4CCR5 indicator cells, which produce beta-Galactosidase upon expression of the viral tat gene after infection. The BrdU incorporation assay was used to evaluate modulation of cell function. A dose dependent reduction of the titer of HIV-1 on P4CCR5 cells was observed for both HDPs. However, the IC₅₀ of LL37 was approximately 3-fold higher than the IC₅₀ previously determined for the lentiviral vectors resulting in a narrow gap between antiviral and antiproliferative effects of LL37. In contrast, the IC₅₀ of PG-1 was below 10 μg/ml, while inhibitory effects on cell proliferation were not observed up to concentrations of 50 μg/ml.

From the panel of five HDP studied, the cathelicidin LL37 and PG-1 were found to specifically inhibit lentiviral and retroviral vector, but not adenoviral vector infectivity. The strongest inhibition was seen if the lentiviral vectors were preincubated with LL37 and PG-1. This suggests that these HDPs directly interacted with the vector particles, which is consistent with our observation that inhibition was dependent on the HDP concentration during preincubation of the vectors with HDPs, but not on the HDP concentration during infection of the cells. Since lentiviral vectors and retroviral vectors were inhibited to a similar degree although they do not share any viral protein, the target for the HDP on the particles is probably cell-derived. This could either be the lipid membrane derived from the cell, which surrounds the vector particles or cellular membrane proteins that are frequently incorporated in lentiviral and retroviral particles during budding 41. A permeabilizing effect of LL37 and PG-1 on the viral particles would be consistent with our data, but other mechanisms of inhibition cannot be excluded. While the inhibitory effect of PG-1 was also detected with wild type HIV-1 on P4CCR5 cells, LL37 inhibited HIV-1 to lesser degree then the lentiviral vectors. Due an IC₅₀ of 88 μg/ml against wild type HIV-1, it is questionable whether LL37 concentrations are sufficiently high at mucosal membranes to play a role in host defense against HIV-1.

Modulation of cell metabolism was generally seen at concentrations of HDPs exceeding 50 μg/ml, while the MEC of the antibacterial activity ranged from 1 to 10 μg/ml. This might leave a sufficient window for therapeutic intervention of bacterial infection. However, for the treatment of HIV-1, the therapeutic window of LL37 and PG-1 is rather narrow. It should also be noted that the HDP-induced modulation of cell metabolism and cytotoxicity can be cell type dependent. Therefore, increasing the selectivity of HDPs for early steps in the viral replication cycle seems to be necessary for further development of the human cathelicidin LL37 and the porcine Protegrin-1 as antiviral agents for systemic or topical applications.

The author(s) declare that they have no competing interests.

LS, BT and JM performed most of the experiments. LS, OW, ML, EL, HH, HS and KU participated in the experimental design, data interpretation and writing of the manuscript.