Unexpected synergistic HIV neutralization by a triple microbicide produced in rice endosperm

Significance Our paper provides an approach for the durable deployment of anti-HIV agents in the developing world. We developed a transgenic rice line expressing three microbicidal proteins (the HIV-neutralizing antibody 2G12 and the lectins griffithsin and cyanovirin-N). Simultaneous expression in the same plant allows the crude seed extract to be used directly as a topical microbicide cocktail, avoiding the costs of multiple downstream processes. This groundbreaking strategy is realistically the only way that microbicidal cocktails can be manufactured at a cost low enough for the developing world, where HIV prophylaxis is most in demand. The transmission of HIV can be prevented by the application of neutralizing monoclonal antibodies and lectins. Traditional recombinant protein manufacturing platforms lack sufficient capacity and are too expensive for developing countries, which suffer the greatest disease burden. Plants offer an inexpensive and scalable alternative manufacturing platform that can produce multiple components in a single plant, which is important because multiple components are required to avoid the rapid emergence of HIV-1 strains resistant to single microbicides. Furthermore, crude extracts can be used directly for prophylaxis to avoid the massive costs of downstream processing and purification. We investigated whether rice could simultaneously produce three functional HIV-neutralizing proteins (the monoclonal antibody 2G12, and the lectins griffithsin and cyanovirin-N). Preliminary in vitro tests showed that the cocktail of three proteins bound to gp120 and achieved HIV-1 neutralization. Remarkably, when we mixed the components with crude extracts of wild-type rice endosperm, we observed enhanced binding to gp120 in vitro and synergistic neutralization when all three components were present. Extracts of transgenic plants expressing all three proteins also showed enhanced in vitro binding to gp120 and synergistic HIV-1 neutralization. Fractionation of the rice extracts suggested that the enhanced gp120 binding was dependent on rice proteins, primarily the globulin fraction. Therefore, the production of HIV-1 microbicides in rice may not only reduce costs compared to traditional platforms but may also provide functional benefits in terms of microbicidal potency.