Rice endosperm is cost-effective for the production of recombinant griffithsin with potent activity against HIV

Abstract

Protein microbicides containing neutralizing antibodies and antiviral lectins may help to reduce the rate of infection with human immunodeficiency virus (HIV) if it is possible to manufacture the components in large quantities at a cost affordable in HIV-endemic regions such as sub-Saharan Africa. We expressed the antiviral lectin griffithsin (GRFT), which shows potent neutralizing activity against HIV, in the endosperm of transgenic rice plants (Oryza sativa), to determine whether rice can be used to produce inexpensive GRFT as a microbicide ingredient. The yield of (OS) GRFT in the best-performing plants was 223 μg/g dry seed weight. We also established a one-step purification protocol, achieving a recovery of 74% and a purity of 80%, which potentially could be developed into a larger-scale process to facilitate inexpensive downstream processing. (OS) GRFT bound to HIV glycans with similar efficiency to GRFT produced in Escherichia coli. Whole-cell assays using purified (OS) GRFT and infectivity assays using crude extracts of transgenic rice endosperm confirmed that both crude and pure (OS) GRFT showed potent activity against HIV and the crude extracts were not toxic towards human cell lines, suggesting they could be administered as a microbicide with only minimal processing. A freedom-to-operate analysis confirmed that GRFT produced in rice is suitable for commercial development, and an economic evaluation suggested that 1.8 kg/ha of pure GRFT could be produced from rice seeds. Our data therefore indicate that rice could be developed as an inexpensive production platform for GRFT as a microbicide component.

Keywords: HIV; endosperm; griffithsin; lectin; microbicides; rice.

Figure 1

(a) GRFT accumulation in rice endosperm. An immunoglobulin‐specific sandwich ELISA was used to screen the three best‐performing independent events. The ELISA plate was coated with gp120, and GRFT protein assembly was confirmed using a primary rabbit anti‐GRFT polyclonal antiserum and a secondary HRP‐conjugated anti‐rabbit IgG antiserum. Four serial dilutions per sample are shown (neat, 1/2, 1/4 and 1/8). WT, wild‐type extracts; C⁻, negative control (PBS); C⁺, GRFT purified from E. coli (500 ng/mL) as a positive control. OD, optical density at 450 nm. (b) Transformation construct pgZ63‐GRFT for the stable expression of GRFT in rice endosperm. The expression cassette comprised the endosperm‐specific maize zein promoter, the rice α‐amylase 3A signal peptide sequence (SP), a His₆ tag, the GRFT coding region (grft) and the nos terminator (t‐nos).

Figure 2

(a) Separation of the purified GRFT fraction by SDS‐PAGE under reducing conditions showing the 14.6 kDa band representing GRFT (arrow). M = Precision Plus Protein^™ All Blue Standards (Bio‐Rad), lane A = 3.5 μg GRFT and lane B = 3.8 μg of GRFT protein. (b) Analysis of ^OSGRFT by SDS‐PAGE under reducing conditions and immunoblotting using a primary rabbit anti‐GRFT polyclonal antiserum and a secondary HRP‐conjugated anti‐rabbit IgG. C⁺ = positive control (50 ng ^ECGRFT), M = protein size marker Precision Plus Protein^™ All Blue Standards (Bio‐Rad). The arrow shows the anticipated size of the GRFT‐His₆ protein.

Figure 3

Antigen‐binding activity of crude rice endosperm extracts containing ^OSGRFT and the purified ^ECGRFT determined by ELISA. Ordinate: Optical density at 450 nm. Abscissa: Serial dilutions. Values are the average of two experiments ± SE. Plates coated with HIV‐1 gp120 were tested with different concentrations of crude extracts or fixed concentrations of purified GRFT from E. coli, and binding activity was measured using a primary rabbit anti‐GRFT polyclonal antiserum and a secondary HRP‐conjugated anti‐rabbit IgG. WT, wild‐type plants; C⁻, negative control (PBS); C⁺, ^ECGRFT (concentration 500 ng/mL) as a positive control; 2, 6, 10, independent events expressing ^OSGRFT; OD, optical density at 450 nm.

Figure 4

Localization of ^OSGRFT in immature rice seeds, 15–20 days after pollination. (a) Interference contrast microscopy. (b) Fluorescence microscopy. The outlined area in (a) is shown as an inset panel in (b) with merged channels. (c) Immuno‐electron microscopy. Outlined area is shown enlarged twofold along each axis in the inset panel. Arrowheads in (a) and (b) represent storage organelles. Abbreviations: SG, starch granule; AL, aleurone layer; PRO, protein body; PSV, protein storage vacuole. Bars = 5 μm (a, b, including inset panel), 0.5 μm (c).

Figure 5

Concentration‐dependent effects of GRFT on cellular viability. The in vitro HIV‐neutralizing activity of (a) ^OSGRFT, (b) ^ESGRFT and (c) wild‐type rice extracts against CEM‐SS cells infected with HIV‐1_RF is presented as cell viability relative to uninfected and untreated controls. Cell viability was assessed using the XTT assay. All points are averages (±SE) of triplicate measurements.

Figure 6

(a) HIV‐1 infectivity assay. TZM‐bl cells were incubated with serial dilutions of the extracts (in triplicate) and cultured for 72 h with HIV‐1BaL using a concentration optimized for infectivity. The extent of viral replication was determined by measuring the luciferase activity of cell lysates. Ordinate: Percentage of relative infection. Abscissa: Serial dilutions. Results are shown as the percentage of infection relative to the virus‐only control ± SE. (b) Effect of different concentrations of crude extracts on cell viability. Cytotoxicity was determined using an MTT assay and was expressed as the percentage of dead cells (mean ± SD, n = 3). Ordinate: Percentage relative viability. Abscissa: Serial dilutions. WT, untransformed rice seed extract; C⁻, negative (medium‐only) control; C⁺, cytotoxic positive control (nonoxynol‐9).