Design, production and characterization of Pfs48/45 R0.10C variants P. falciparum's in N. benthamiana

Although many efforts have been made toward developing malaria vaccines, however, no vaccine is currently available that provides a satisfactory level of protection against malaria. The native Pfs48/45 protein does not carry N-linked glycans but contains seven potential N-linked glycosylation sites which can be aberrantly glycosylated during expression in any eykaryotic system including plant expression systems. To solve the aberrant glycosylation problem, a strategy was developed by co-expression of the bacterial deglycosylation enzymes, PNGase F (Mamedov et al. 2012; Mamedov et al. 2013; Mamedov et al. 2016) or Endo H enzymes (Mamedov et al. 2017) with target proteins of interest in N. benthamiana plant by transient expression technology. In this study, we successfully produced different versions (glycosylated and deglycosylated) of the 10C domain of the Pfs48/45 protein in Nicotiana benthamiana plant by transient expression. Plant produced 10C-varants were purified and characterized. It should be noted that 10C domain of the Pfs48/45 protein is one of the most important candidates for the development of the transmission blocking vaccine (TB). Western blot analysis of Pfs48/45-10C variants using MRA-26 antibody (a conformational specific MRA-26 antibody, known as TB antibody), showed that MRA-26 antibody did not recognized or weakly recognized the aberrantly glycosylated Pfs48/45-10 in non-reduced samples, but strongly recognized plant produced Endo H and PNGase F in vivo deglycosylated Pfs48/45-10 proteins in non-reduced samples. These results suggest correct folding of plant produced, in vivo Endo H or PNGase F deglycosylated Pfs48/45-10C proteins, and also suggest that Pfs48/45-10C proteins contain epitopes present on native Pfs48/45. Taken together, our results collectevely support that plant produced Endo H in vivo deglycosylated Pfs48/45 and Pfs48/45-10C antigens have a potential for the development of a Pfs48/45-based TB malaria vaccine.