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Key Message: Rice-produced SD1 retains its physicochemical properties and provides efficient pre-exposure HIV-1 prophylaxis against infection in vitro. Scytovirin (SVN) is an HIV-neutralizing lectin that features two structural domains (SD1 and SD2) that bind to HIV-1 envelope glycoproteins. We expressed SD1 in rice seeds as a potential large-scale production platform and confirmed that rice-derived SD1 binds the HIV-1 envelope glycoprotein gp120 in vitro. We analyzed the thermodynamic properties of SD1 compared to full-size SVN (produced in E. coli) by isothermal titration and differential scanning calorimetry to characterize the specific interactions between SVN/SD1 and gp120 as well as to high-mannose oligosaccharides. SVN bound with moderate affinity (K d  = 1.5 µM) to recombinant gp120, with 2.5-fold weaker affinity to nonamannoside (K d of 3.9 µM), and with tenfold weaker affinity to tetramannoside (13.8 µM). The melting temperature (T m ) of full-size SVN was 59.1 °C and the enthalpy of unfolding (ΔH unf ) was 16.4 kcal/mol, but the T m fell when SVN bound to nonamannoside (56.5 °C) and twice as much energy was required for unfolding (ΔH unf  = 33.5 kcal/mol). Interestingly, binding to tetramannoside destabilized the structure of SD1 (ΔT m  ~ 11.5 °C) and doubled the enthalpy of unfolding, suggesting a dimerization event. The similar melting phenomenon shared by SVN and SD1 in the presence of oligomannose confirmed their conserved oligosaccharide-binding mechanisms. SD1 expressed in transgenic rice was able to neutralize HIV-1 in vitro. SD1 expressed in rice, therefore, is suitable as a microbicide component., (© 2022. The Author(s).)