Efficacy of silk fibroin biomaterial vehicle for in vivo mucosal delivery of Griffithsin and protection against HIV and SHIV infection ex vivo

Abstract

Introduction: The majority of new HIV infections occur through mucosal transmission. The availability of readily applicable and accessible platforms for anti-retroviral (ARV) delivery is critical for the prevention of HIV acquisition through sexual transmission in both women and men. There is a compelling need for developing new topical delivery systems that have advantages over the pills, gels and rings, which currently fail to guarantee protection against mucosal viral transmission in vulnerable populations due to lack of user compliance. The silk fibroin (SF) platform offers another option that may be better suited to individual circumstances and preferences to increase efficacy through user compliance. The objective of this study was to test safety and efficacy of SF for anti-HIV drug delivery to mucosal sites and for viral prevention.

Methods: We formulated a potent HIV inhibitor Griffithsin (Grft) in a mucoadhesive silk fibroin (SF) drug delivery platform and tested the application in a non-human primate model in vivo and a pre-clinical human cervical and colorectal tissue explant model. Both vaginal and rectal compartments were assessed in rhesus macaques (Mucaca mulatta) that received SF (n = 4), no SF (n = 7) and SF-Grft (n = 11). In this study, we evaluated the composition of local microbiota, inflammatory cytokine production, histopathological changes in the vaginal and rectal compartments and mucosal protection after ex vivo SHIV challenge.

Results: Effective Grft release and retention in mucosal tissues from the SF-Grft platform resulted in protection against HIV in human cervical and colorectal tissue as well as against SHIV challenge in both rhesus macaque vaginal and rectal tissues. Mucoadhesion of SF-Grft inserts did not cause any inflammatory responses or changes in local microbiota.

Conclusions: We demonstrated that in vivo delivery of SF-Grft in rhesus macaques fully protects against SHIV challenge ex vivo after two hours of application and is safe to use in both the vaginal and rectal compartments. Our study provides support for the development of silk fibroin as a highly promising, user-friendly HIV prevention modality to address the global disparity in HIV infection.

Keywords: Mucaca mulatta; Griffithsin; HIV infections; biocompatible materials; silk fibroin; vulnerable populations.

Figure 1

HIV inhibition following ex vivo dosing of Griffithsin in human cervical and colorectal tissue in the presence of biological fluids. Human explants were incubated in the presence of cervical mucous (CM) or seminal fluid (SM) and treated with serial dilutions of Grft. The inhibitory capacity in (A) cervical and (B) colorectal tissue was evaluated in dose–response curves. Data are means (± standard deviations) from three independent experiments performed in triplicate.

Figure 2

Griffithsin release and dispersal in vivo. (A) For PK evaluation, SF‐Grft discs were inserted vaginally and rectally in macaques. The Grft concentration was determined in fluids collected from each compartment after 2 hours. Each data point represents the Grft concentration measured for each animal in the vaginal (left) and rectal (right) compartments, and the mean ± SEM (n = 5) is indicated by a bold horizontal line with error bars for each compartment. The limit of detection (LOD) for the Grft assay (blue line) and the 100‐fold EC₉₀ level for Griffithsin (724.4 ng/mL, red dashed line) are indicated. (B‐D) Vaginal and rectal tissues were sampled after exposure to SF containing Grft conjugated to AF‐610. Tiled 10X images on confocal microscopy revealed (B) accumulation of Grft on the epithelial surface and small amounts of Grft located in the lamina propria (yellow arrow). Confocal images of (C) vaginal tissue and (D) rectal tissue showed coverage of the epithelial surface by Grft.

Figure 3

Safety profile of SF‐Grft delivery in vaginal and rectal compartments. (A) H&E stains were obtained from vaginal and rectal tissue biopsies in the following groups: (1) control, (2) following exposure to SF (Silk) at and away from the placement site, and (3) following exposure to SF‐Grft (Silk + Grft) at and away from the placement site. Inflammatory signatures were detected by a multi‐plex Luminex assay in culture supernatant of vaginal explants following (B) in vivo exposure to SF or (C) SF‐Grft and rectal explants following (D) in vivo exposure to SF or (E) SF‐Grft. All were compared to the culture supernatant of tissue explants from control animals.

Figure 4

Ex vivo protection after SHIV challenge. Vaginal (A, B) and rectal (C, D) explants were obtained from rhesus macaques after 2 hours of exposure to SF‐only or SF‐Grft discs. Unchallenged explants from all animals were used as viral negative controls (Ctrl), while ex vivo SHIV‐challenged explants from macaques that received SF‐only were used as virus‐positive controls (SHIV). Explants obtained from animals that received SF‐only (Silk + SHIV) or to SF‐Grft (Silk + Grft + SHIV) were challenged ex vivo with SHIV. Inhibitory activity of Grft was assessed in tissue explant culture supernatants by ELISA at (A, C) peak of p27 concentration and (B, D) across 15 days of culture as area under the curve of p27 concentration between days 3 and 15 (AUCp27 d3‐d15). Data shown are means (± standard deviations) of at least triplicate explants for each condition for each animal from each group (n = 5 control animals, n = 3 SF‐disc exposed animals, n = 5 SF‐Grft dosed animals).

Figure 5

Microbial changes following SF‐Grft insertion. Vaginal microbiota comparing pre‐ and post‐silk placement was analysed through (A) alpha diversity and (B) taxonomic classification at the phylum level. (C) Beta diversity of vaginal microbiota was analysed using PCoA plots with ellipses representing 95% confidence. Similarly, rectal microbiota pre‐ and post‐silk placement compared using (D) alpha diversity, (E) taxonomic classification and (F) PCoA of beta diversity plots.