Cyanovirin-N produced in rice endosperm offers effective pre-exposure prophylaxis against HIV-1BaL infection in vitro
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Cyanovirin-N produced in rice endosperm provides efficient pre-exposure prophylaxis against HIV-1 BaL infection in vitro.
Cyanovirin-N (CV-N) is a lectin with potent antiviral activity that has been proposed as a component of microbicides for the prevention of infection with Human immunodeficiency virus (HIV). The production of protein-based microbicide components requires a platform that is sufficiently economical and scalable to meet the demands of the large at-risk population, particularly in resource poor developing countries. We, therefore, expressed CV-N in rice endosperm, because the dried seed is ideal for storage and transport and crude extracts could be prepared locally and used as a microbicide component without further purification. We found that crude extracts from rice seeds expressing up to 10 µg CV-N per gram dry seed weight showed dose-dependent gp120 binding activity, confirming that the protein was soluble, correctly folded and active. The recombinant lectin (OSCV-N) reduced the infectivity of HIV-1BaL (an R5 virus strain representing the majority of transmitted infections) by ~90 % but showed only weak neutralization activity against HIV-1RF (representative of X4 virus, rarely associated with transmission), suggesting it would be highly effective for pre-exposure prophylaxis against the vast majority of transmitted strains. Crude extracts expressing OSCV-N showed no toxicity towards human cells at working dilutions indicating that microbicide components produced in rice endosperm are safe for direct application as topical microbicides in humans.
KeywordsCyanovirin-N Rice crude extract Endosperm Anti-HIV Microbicide
The authors are grateful to Dr. Maite Sabalza Gallués for cloning the CV-N gene in the pRP5 plasmid and Ms. Jennifer Wilson for technical assistance with live-virus anti-HIV assays. In addition, the authors would like to acknowledge funding from the Ministerio de Ciencia e Innovación, Spain (BIO2012-35359), the Center CONSOLIDER on Agrigenomics funded by MICINN, Spain, and COST Action FA0804 (Molecular farming: plants as a production platform for high value proteins). This project was funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract HHSN26120080001E, and supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest.
- Boyd MR, Gustafson KR, McMahon JB, Shoemaker RH, O’Keefe BR, Mori T, Gulakowski RJ, Wu L, Rivera MI, Laurencot CM, Currens MJ, Cardellina JH, Buckheit RW, Nara PL, Pannell LK, Sowder RC, Henderson LE (1997) Discovery of cyanovirin-N, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development. Antimicrob Agents Chemother 41:1521–1530PubMedPubMedCentralGoogle Scholar
- Carlson JM, Schaefer M, Monaco DC, Batorsky R, Claiborne DT, Prince J, Deymier MJ, Ende ZS, Klatt NR, DeZiel CE, Lin TH, Peng J, Seese AM, Shapiro R, Frater J, Ndung’u T, Tang J, Goepfert P, Gilmour J, Price MA, Kilembe W, Heckerman D, Goulder PJ, Allen TM, Allen S, Hunter E (2014) HIV transmission. Selection bias at the heterosexual HIV-1 transmission bottleneck. Science 345:1254031CrossRefPubMedPubMedCentralGoogle Scholar
- Colleluori DM, Tien D, Kang F, Pagliei T, Kuss R, McCormick T, Watson K, McFadden K, Chaiken I, Buckheit RW, Romano JW (2005) Expression, purification, and characterization of recombinant cyanovirin-N for vaginal anti-HIV microbicide development. Protein Expr Purif 39:229–236CrossRefPubMedGoogle Scholar
- Dey B, Lerner DL, Lusso P, Boyd MR, Elder JH, Berger EA (2000) Multiple antiviral activities of cyanovirin-N: blocking of human immunodeficiency virus type 1 gp120 interaction with CD4 and coreceptor and inhibition of diverse enveloped viruses. J Virol 74:4562–4569CrossRefPubMedPubMedCentralGoogle Scholar
- Este JA, Schols D, De Vreese K, Van Laethem K, Vandamme AM, Desmyter J, De Clercq E (1997) Development of resistance of human immunodeficiency virus type 1 to dextran sulfate associated with the emergence of specific mutations in the envelope gp120 glycoprotein. Mol Pharmacol 52:98–104PubMedGoogle Scholar
- Goldstein IJ, Poretz RD (1986) Isolation, physicochemical characterization, and carbohydrate binding specificity of lectins. In: Liener IE, Sharon N, Goldstein IJ (eds) The lectins: properties, functions, and applications in biology and medicine. Academic Press, New York, pp 33–248CrossRefGoogle Scholar
- Gustafson KR, Sowder RC, Henderson LE, Cardellina JH, McMahon JB, Rajamani U, Pannell LK, Boyd MR (1997) Isolation, primary sequence determination, and disulfide bond structure of cyanovirin-N, an anti-HIV (human immunodeficiency virus) protein from the cyanobacterium Nostoc ellipsosporum. Biochem Biophys Res Commun 238:223–228CrossRefPubMedGoogle Scholar
- Liu X, Lagenaur LA, Simpson DA, Essenmacher KP, Frazier-Parker CL, Liu Y, Tsai D, Rao SS, Hamer DH, Parks TP, Lee PP, Xu Q (2006) Engineered vaginal lactobacillus strain for mucosal delivery of the human immunodeficiency virus inhibitor cyanovirin-N. Antimicrob Agents Chemother 50:3250–3259CrossRefPubMedPubMedCentralGoogle Scholar
- Madan RP, Mesquita PM, Cheshenko N, Jing B, Shende V, Guzman E, Heald T, Keller MJ, Regen SL, Shattock RJ, Herold BC (2007) Molecular umbrellas: a novel class of candidate topical microbicides to prevent human immunodeficiency virus and herpes simplex virus infections. J Virol 81:7636–7646CrossRefPubMedPubMedCentralGoogle Scholar
- Mesquita PM, Wilson SS, Manlow P, Fischetti L, Keller MJ, Herold BC, Shattock RJ (2008) Candidate microbicide PPCM blocks human immunodeficiency virus type 1 infection in cell and tissue cultures and prevents genital herpes in a murine model. J Virol 82:6576–6584CrossRefPubMedPubMedCentralGoogle Scholar
- Mori T, Shoemaker RH, Gulakowski RJ, Krepps BL, McMahon JB, Gustafson KR, Pannell LK, Boyd MR (1997) Analysis of sequence requirements for biological activity of cyanovirin-N, a potent HIV (human immunodeficiency virus)-inactivating protein. Biochem Biophys Res Commun 238:218–222CrossRefPubMedGoogle Scholar
- Naqvi S, Zhu C, Farre G, Ramessar K, Bassie L, Breitenbach J, Perez Conesa D, Ros G, Sandmann G, Capell T, Christou P (2009) Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways. Proc Natl Acad Sci USA 106:7762–7767CrossRefPubMedPubMedCentralGoogle Scholar
- O’Keefe BR, Murad AM, Vianna GR, Ramessar K, Saucedo CJ, Wilson, J, Buckheit KW, da Cunha NB, Araújo AC, Lacorte CC, Madeira L, McMahon JB, Rech EL (2015) Engineering soya bean seeds as a scalable platform to produce cyanovirin-N, a non-ARV microbicide against HIV. Plant Biotechnol J 1–9 [pub ahead of print]Google Scholar
- Parrish NF, Gao F, Li H, Giorgi EE, Barbian HJ, Parrish EH, Zajic L, Iyer SS, Decker JM, Kumar A, Hora B, Berg A, Cai F, Hopper J, Denny TN, Ding H, Ochsenbauer C, Kappes JC, Galimidi RP, West AP Jr, Bjorkman PJ, Wilen CB, Doms RW, O’Brien M, Bhardwaj N, Borrow P, Haynes BF, Muldoon M, Theiler JP, Korber B, Shaw GM, Hahn BH (2013) Phenotypic properties of transmitted founder HIV-1. Proc Natl Acad Sci USA 110:6626–6633CrossRefPubMedPubMedCentralGoogle Scholar
- Schuitemaker H, Koot M, Kootstra NA, Dercksen MW, de Goede RE, van Steenwijk RP, Lange JM, Schattenkerk JK, Miedema F, Tersmette M (1992) Biological phenotype of human immunodeficiency virus type 1 clones at different stages of infection: progression of disease is associated with a shift from monocytotropic to T-cell-tropic virus population. J Virol 66:1354–1360PubMedPubMedCentralGoogle Scholar
- Valdez M, Cabrera-Ponce JL, Sudhakar D, Herrera-Estrella L, Christou P (1998) Transgenic central American, west African and Asian elite rice varieties resulting from particle bombardment of foreign DNA into mature seed-derived explants utilizing three different bombardment devices. Ann Bot 82:795–801CrossRefGoogle Scholar
- Wei X, Hunt G, Abdool Karim SS, Naranbhai V, Sibeko S, Abdool Karim Q, Li JF, Kashuba AD, Werner L, Passmore JA, Morris L, Heneine W, Johnson JA (2014) Sensitive tenofovir resistance screening of HIV-1 from the genital and blood compartments of women with breakthrough infections in the CAPRISA 004 tenofovir gel trial. J Infect Dis 209:1916–1920CrossRefPubMedPubMedCentralGoogle Scholar
- Yamagata H, Tanaka K (1986) The site of synthesis and accumulation of rice storage proteins. Plant Cell Physiol 27:135–145Google Scholar