Hapivirins and diprovirins: novel θ-defensin analogs with potent activity against influenza A virus

Abstract

θ-Defensins are cyclic octadecapeptides found in nonhuman primates whose broad antiviral spectrum includes HIV-1, HSV-1, severe acute respiratory syndrome coronavirus, and influenza A virus (IAV). We previously reported that synthetic θ-defensins called retrocyclins can neutralize and aggregate various strains of IAV and increase IAV uptake by neutrophils. This study describes two families of peptides, hapivirins and diprovirins, whose design was inspired by retrocyclins. The goal was to develop smaller partially cyclic peptides that retain the antiviral activity of retrocyclins, while being easier to synthesize. The novel peptides also allowed for systemic substitution of key residues to evaluate the role of charge or hydrophobicity on antiviral activity. Seventy-two hapivirin or diprovirin peptides are described in this work, including several whose anti-IAV activity equals or exceeds that of normal α- or θ-defensins. Some of these also had strong antibacterial and antifungal activity. These new peptides were active against H3N2 and H1N1 strains of IAV. Structural features imparting strong antiviral activity were identified through iterative cycles of synthesis and testing. Our findings show the importance of hydrophobic residues for antiviral activity and show that pegylation, which often increases a peptide's serum t(1/2) in vivo, can increase the antiviral activity of DpVs. The new peptides acted at an early phase of viral infection, and, when combined with pulmonary surfactant protein D, their antiviral effects were additive. The peptides strongly increased neutrophil and macrophage uptake of IAV, while inhibiting monocyte cytokine generation. Development of modified θ-defensin analogs provides an approach for creating novel antiviral agents for IAV infections.

Figure 1. Schematic representation of synthesized HpV and DpV peptides

All peptides were synthesized as a C-terminal amides as described in Methods.

Figure 2

Comparison of some amino acids used as substituents in position 7 of HpVs.

Figure 3. IAV neutralizing activity of Hapivirins

Aliquots of the Phil82 strain of IAV were pre-incubated with 1 or 2μg/ml of the indicated Hapivirins. Results are mean±SEM of 3 experiments using the fluorescent focus assay on MDCK cells. All of the tested HpVs significantly reduced infectious foci as compared to virus alone (p<0.05).

Figure 4. Dose response for viral neutralization by HpV11, HpV17, HpV18, and HpV19

Neutralizing activity of the most potent Hapivirins was compared using A549 and MDCK cells. Panels A–C used the Phil82 viral strains and panel D used the PR-8 strain. Ther results shown are mean±SEM of 4 experiments. All concentrations of the peptides tested significantly inhibited IAV replication as compared to control (p<0.05).

Figure 5. Dose response curves for neutralization of IAV by selected Diprovirins

The neutralizing activity of different doses of DpVs that had high (panels A and B) or intermediate (panels C and D) activity on screening assays were tested. Mean±SEM of 4 experiments. All concentrations of the peptides tested in panel A and of peptide 1623 in panel B significantly inhibited IAV replication as compared to control (p<0.05). For the other peptides shown in panel B and all shown in panels C and D, significant inhibition was achieved at the highest concentration of peptide tested i.e. 40μg/ml.

Figure 6. Effect of delaying addition of defensins on neutralizing activity

All the peptides were used at concentrations of 1 μg/ml. The defensins were either pre-incubated with IAV (Phil82 strain) as in figure 5 (Group 1), or added to the MDCK or A549 cells 15 (Group 1) or 45 (Group 3) minutes after viral infection of the cells. In Group 1 all defensins significantly inhibited viral nucleoprotein expression 7 hours after infection (with the single exception of DpV21 in A549 cells). In contrast, no defensin inhibited IAV in groups 2 or 3. All of the peptides tested in panel A significantly inhibited IAV replication as compared to control (p<0.05), with the exception of DpV21.

Figure 7. Viral aggregating activity of selected HpVs, DpVs and RCs

Panels A–C show results of viral aggregation assays using light scattering, in which control buffer (IAV alone) or HpVs, DpVs or RCs were added at time 0. The results are expressed as % of light absorption at time zero and are mean±SEM of 3 or more experiments in each case. In panels A and B 8μg/ml of peptides were used, and in panel C 10μg/ml was used. Aggregation induced by HpV11 and 17 was highly significant compared to control (p<0.001) and was significantly greater than aggregation induced by RC2 by ANOVA. Aggregation induced by HpV15 and RC2 was also significant compared to IAV alone (p<0.05). Aggregation induced by DpV13 was significantly greater than that induced by RC1 as assessed by ANOVA (panel C). The lower panels show results of electron micrographs of untreated virus, or virus treated with the indicated HpVs or DpVs demonstrating virus aggregation. The electron microscopy results were representative of at least three similar experiments using 40μg/ml of the peptides.

Figure 8. Aggregation of S. aureus and Zymosan particles by HpV11 and DpV1607

Fluorescently labeled S. aureus and Zymosan particles were obtained from Molecular Probes and incubated with various HpVs and DpVs, followed by examination using a fluorescent microscope. Representative pictures (from 3 or more experiments) showing aggregation of S. aureus by HpV11 (24μg/ml) and DpV1607 (60 μg/ml) are shown.

Figure 9. Comparison the ability of HpVs or DpVs to increase viral uptake by neutrophils or RAW cells

FITC labeled Phil 82 virus was used to detect viral uptake by flow cytometry. Panel A shows uptake of virus that was treated with various concentrations of HpV 17 and 18. Panel B shows similar experiments using RAW cells. Results are mean±SEM of 7 experiments for panels A and B using separate neutrophil donors or RAW cells harvested on different days. Panel C shows neutrophil uptake of virus after pre-incubation with the indicated DpVs. Results are mean±SEM of 3 experiments using separate neutrophil donors. All of the tested DpVs caused significant increases in viral uptake by neutrophils at the 30μg/ml concentration (p<0.03), but only DpV1632 caused a significant increase in uptake at 15 μg/ml. All of the tested peptides caused significant increases in neutrophil or RAW cell uptake of IAV (p<0.05).

Figure 10. Ability of HpVs or DpVs to inhibit IAV – induced TNF generation by human monocytes

Human peripheral blood monocytes were infected with IAV (Phil82 strain) followed by culture in vitro for 18 hrs. TNF was measured by ELISA on culture supernatants as described. Pre-treatment of IAV with the indicated peptides reduced cytokine production as compared to virus alone (* indicates significant reduction at p<0.05). Results are mean±SEM of 4–5 experiments using separate monocyte donors.