Scaleable manufacture of HIV-1 entry inhibitor griffithsin and validation of its safety and efficacy as a topical microbicide component

Abstract

To prevent sexually transmitted HIV, the most desirable active ingredients of microbicides are antiretrovirals (ARVs) that directly target viral entry and avert infection at mucosal surfaces. However, most promising ARV entry inhibitors are biologicals, which are costly to manufacture and deliver to resource-poor areas where effective microbicides are urgently needed. Here, we report a manufacturing breakthrough for griffithsin (GRFT), one of the most potent HIV entry inhibitors. This red algal protein was produced in multigram quantities after extraction from Nicotiana benthamiana plants transduced with a tobacco mosaic virus vector expressing GRFT. Plant-produced GRFT (GRFT-P) was shown as active against HIV at picomolar concentrations, directly virucidal via binding to HIV envelope glycoproteins, and capable of blocking cell-to-cell HIV transmission. GRFT-P has broad-spectrum activity against HIV clades A, B, and C, with utility as a microbicide component for HIV prevention in established epidemics in sub-Saharan Africa, South Asia, China, and the industrialized West. Cognizant of the imperative that microbicides not induce epithelial damage or inflammatory responses, we also show that GRFT-P is nonirritating and noninflammatory in human cervical explants and in vivo in the rabbit vaginal irritation model. Moreover, GRFT-P is potently active in preventing infection of cervical explants by HIV-1 and has no mitogenic activity on cultured human lymphocytes.

Fig. 1.

A summary of the purification of GRFT-P from N. benthamiana plants infected with an rTMV vector expressing GRFT cDNA. The SDS-PAGE gel shows molecular weight marker in lane M. The initial GJ extract is shown in lane 1; the ceramic membrane permeate is shown in lane 2; lane 3 shows the UF concentrate; lane 4 is the SP-Sepharose column eluate; lane 5 contains the reversed-phase column chromatography eluate; and lane 6 shows 10 μg of the final GRFT-P product.

Fig. 2.

Comparison of HIV-1_IIIB gp120 binding GRFT-E and GRFT-P. HIV-1_IIIB gp120 was bound to the wells of a 96-well plate and subsequently incubated with various dilutions of either GRFT-E (filled circles) or GRFT-P (open circles). Binding was visualized by HRP-labeled anti-GRFT rabbit polyclonal Abs and measured by absorbance at 650 nm. All test samples were measured in triplicate.

Fig. 3.

Production of proinflammatory cytokines and chemokines in human cervical explants exposed to GRFT-P. Cervical explants were treated for 3 h with media alone (control, gray bar) and 2 μM GRFT-P (white bar). Expression of a panel of cytokines and chemokines was measured by Luminex-based immunoassays. GCSF, granulocyte colony-stimulating factor; IP10, Interferon inducible protein-10; MIG, monocyte induced by gamma interferon; SDF, stromal cell-derived factor.

Fig. 4.

Proliferative effects of GRFT-P on PBMCs. Proliferation was assayed in PBMCs after 3 days of culture. Cells were exposed to phytohemagglutinin A (PhA) or GRFT-P at different concentration for 2 h or 3 days. The stimulation index was calculated by dividing the mean cpm value of stimulated samples by the mean cpm of unstimulated ones.

Fig. 5.

Inhibition of HIV-1 BaL infection of cervical explants and dissemination by migratory cells. Infection of cervical explants was determined at postchallenge days 3, 7, and 11 by detection of p24 antigen in the tissue culture supernatants (A). GRFT-P was administered on day 0 and removed by washing after 2 h. Migratory cells were cocultured with PM1 cells, and infection was monitored by p24 ELISA at the same time points (B). GRFT-P was tested in cervical tissue deriving from 3 donors. Each condition was tested in triplicate. Data represent the mean ± SEM.

Fig. 6.

RVI trial of GRFT-P. Bars represent the MIS derived from the sum of the contributions of the individual classes of pathology.