Multivalent presentation of antihantavirus peptides on nanoparticles enhances infection blockade

Abstract

Viral entry into susceptible host cells typically results from multivalent interactions between viral surface proteins and host entry receptors. In the case of Sin Nombre virus (SNV), a New World hantavirus that causes hantavirus cardiopulmonary syndrome, infection involves the interaction between viral membrane surface glycoproteins and the human integrin alpha(v)beta(3). Currently, there are no therapeutic agents available which specifically target SNV. To address this problem, we used phage display selection of cyclic nonapeptides to identify peptides that bound SNV and specifically prevented SNV infection in vitro. We synthesized cyclic nonapeptides based on peptide sequences of phage demonstrating the strongest inhibition of infection, and in all cases, the isolated peptides were less effective at blocking infection (9.0% to 27.6% inhibition) than were the same peptides presented by phage (74.0% to 82.6% inhibition). Since peptides presented by the phage were pentavalent, we determined whether the identified peptides would show greater inhibition if presented in a multivalent format. We used carboxyl linkages to conjugate selected cyclic peptides to multivalent nanoparticles and tested infection inhibition. Two of the peptides, CLVRNLAWC and CQATTARNC, showed inhibition that was improved over that of the free format when presented on nanoparticles at a 4:1 nanoparticle-to-virus ratio (9.0% to 32.5% and 27.6% to 37.6%, respectively), with CQATTARNC inhibition surpassing 50% when nanoparticles were used at a 20:1 ratio versus virus. These data illustrate that multivalent inhibitors may disrupt polyvalent protein-protein interactions, such as those utilized for viral infection of host cells, and may represent a useful therapeutic approach.

FIG. 1.

Inhibitory peptides identified by phage display show homology to the βA domain of integrin β₃. (A) Pairwise alignment of peptide sequences from phage showing greater than 60% inhibition of SNV infection versus the cellular entry receptor integrin β₃. Residues comprising the signal peptide, transmembrane region, and cytoplasmic tail of β₃ were not included in the pairwise alignment (shown in italics). The PSI domain of integrin β₃ is shown in orange, the hybrid domain is shown in gray, and the βA domain is underlined. β₃ residues outlining the ReoPro binding site are shown in green. (B) Side and frontal view of integrin α_vβ₃ (PDB 1U8C [49]). β₃ is shown in surface representation (salmon), and α_v is shown as a light blue ribbon diagram. The βA domain of β₃ is circled. Regions corresponding to the aligned peptides and the ReoPro binding site are colored as in panel A. (C) Enlargement of βA domain oriented as depicted on the right side of panel B. Graphics for panels B and C were prepared with Pymol (DeLano Scientific LLC, San Carlos, CA).

FIG. 2.

Dose-response curves showing the activites of peptide-bearing phage in blocking SNV infection. Along with HCP (diluted 1:400), controls included ReoPro at 40 μg/ml and a phage bearing the control peptide (CFSSARLSC) used at 10⁹ phage/μl. Buffer controls include medium and the phosphate buffer used to dilute ReoPro. IC₅₀ values for phage-bearing peptides CLVRNLAWC, CQTTNWNTC, and CSASTESLC were 1.0 × 10⁶, 3.5 × 10⁵, and 7 × 10⁸, respectively, as determined by nonlinear regression analysis. Data points represent the mean of experiments performed in duplicate, and error bars show ± the standard errors of the mean. The dotted line indicates 50% inhibition.

FIG. 3.

Focus reduction assay demonstrating specificities of hantavirus inhibition by peptide-bearing phage. The five peptide-bearing phage that were the most potent inhibitors of SNV were tested in a parallel focus assay for their abilities to inhibit infection of Vero E6 cells by SNV, HTNV, and PHV. Virus (2,000 PFU) and 50 μl of 1 × 10⁹ phage were added to each well. Controls include HCP (1:400) and ReoPro (40 μg/ml). The mean percent inhibition of infection is indicated, and the standard errors (error bars) of the means are shown. There were two to four samples tested for each condition.

FIG. 4.

Focus reduction assay comparing the abilities of monovalent peptides versus those of multivalent peptide-coated nanoparticles to block SNV infection of Vero E6 cells. Prior to being added to confluent monolayers of Vero E6 cells, SNV was treated with medium, 1 mM peptide, or peptide-coated nanoparticles (NP) at a nanoparticle:virus ratio of 4:1. ReoPro was added directly to Vero cells at a final concentration of 80 μg/ml, and HCP (1:400) and Gn-BSA antibody (1:40) were added directly to virus. The mean degrees of inhibition are indicated, and the standard errors (error bars) of the means are shown. The asterisk indicates statistical significance (P = 0.001). There were four samples for each condition.

FIG. 5.

Activity and specificity of peptide-coated nanoparticles in blocking SNV infection. (A) Dose-response curves showing the peptide-coated nanoparticles (CLVRNLAWC, CSASTESLC, CQTTNWNTC, CQATTARNC, and random) used at various concentrations and incubated with SNV for 1 h prior to being added to confluent monolayers of Vero E6 cells. Controls included ReoPro, medium only, and uncoated nanoparticles. ReoPro control was added directly to Vero cells at a final concentration of 80 μg/ml. After 24 to 36 h, monolayers were stained with polyclonal rabbit anti-SNV N (nucleocapsid) antibody, followed by FITC-conjugated anti-rabbit IgG antibody, and foci were counted. Each condition represents an N of 4 to 6. (B) Peptides and peptide-coated nanoparticle inhibitors of SNV were tested in a parallel focus assay for their abilities to inhibit infection of Vero E6 cells by SNV, HTNV, and PHV. Virus (2,000 PFU) and peptide (1 mM) or peptide-coated nanoparticles (at a nanoparticle-to-virus ratio of 4:1 or 20:1) were added to each well. Controls include medium, uncoated nanoparticles, and ReoPro (40 μg/ml). The mean degrees of inhibition are indicated, and the standard errors (error bars) of the means are shown. There were two samples tested for each condition.

FIG. 6.

Focus reduction assay comparing the abilities of anti-SNV monovalent peptides and multivalent peptide-coated nanoparticles versus those of anti-integrin β₃ peptides and coated nanoparticles to block SNV infection either alone or in combination treatments. In the case of the anti-SNV peptides, SNV was treated with medium, 1 mM peptide, or peptide-coated nanoparticles (NP), at a nanoparticle-to-virus ratio of 4:1 or 20:1, prior to being added to confluent monolayers of Vero E6 cells. In the case of the anti-integrin peptides, medium, 1 mM peptide, or peptide-coated nanoparticles, at a nanoparticle-to-virus ratio of 4:1 or 20:1, were added to the cells prior to addition of virus. ReoPro was added directly to Vero cells at a final concentration of 80 μg/ml. Mean degrees of inhibition are indicated, and the standard errors (error bars) of the means are shown. Each condition represents an N of 2. For the combination treatments shown in the last six groupings of the graph, the indicated anti-integrin treatment (shown below the bars) was paired with an anti-SNV treatment (shown above the bars).