A Plant-Produced in vivo deglycosylated full-length Pfs48/45 as a Transmission-Blocking Vaccine Candidate against malaria

Abstract

Pfs48/45 is a leading antigen candidate for a transmission blocking (TB) vaccine. However, efforts to produce affordable, safe and correctly folded full-length Pfs48/45 using different protein expression systems have not produced an antigen with satisfactory TB activity. Pfs48/45 has 16 cysteines involved in disulfide bond formation, and the correct formation is critical for proper folding and induction of TB antibodies. Moreover, Pfs48⁄45 is not a glycoprotein in the native hosts, but contains potential glycosylation sites, which are aberrantly glycosylated during expression in eukaryotic systems. Here, we demonstrate for the first time that full length, Endo H in vivo enzymatic deglycosylated Pfs48/45 antigen is produced at a high level in plants and is structurally stable at elevated temperatures. Sera from mice immunized with this antigen showed strong inhibition in SMFA. Thus, Endo H in vivo enzymatic deglycosylated Pfs48/45 is a promising candidate for the development of an affordable TB vaccine, which may have the potential to save millions.

Engineering, production and characterization of Spike and Nucleocapsid structural proteins of SARS–CoV-2 inNicotiana benthamianaas vaccine candidates against COVID-19

“…The stability analysis of plant produced glycosylated and Endo H in vivo deglycosylated forms of RBD and gS1 of SARC-CoV were examined after incubation at 37 °C for 48, 96, 72 and 96 hours. The procedure of stability analysis was similar to those described previously 21 .These analyses showed that dRBD degradation was very low at incubation at 37 ° C for 24 hours (less than 10 %), however, at the same condition, gRBD degraded significantly (more than 60%).…”

“…The stability analysis of plant produced gRBD, dRBD and gS1 of SARS-CoV-2 recombinant proteins were examined after incubation at 37 °C for 48, 96, 72 and 96 hours using similar procedure as described previously 21 . Protein samples were diluted to 0.5 mg/ mL with PBS and were transferred into polypropylene Eppendorf low-binding tubes.…”

“…This system has been successfully used for rapid and cost-effective production of a variety of recombinant proteins, vaccine candidates, therapeutic proteins, enzymes, antibodies etc. [15][16][17][18][19][20][21][22] . Recently, a number of flexible approaches have been developed, which enabled the successfully production of pharmaceutically important complex proteins including human proteins and enzymes in plants 15,[19][20][21] .…”

Bitki moleküler üretimli ilaçlar, veteriner aşılarının uygun maliyetli üretimi için umut verici bir sistemdir

“…However, it is challenging to produce functionally active antigens and associated with incorrect folding of some complex proteins (especially proteins with many disulfide bridges) produced in different host expression system. Therefore, for production of functionally active protein antigens more flexible approaches are required (Mamedov et al 2012;Mamedov et al 2017;Mamedov et al 2019a).…”

“…The problem has recently been addressed through the development of an economical and practical technology that provided production of proteins of interest in native-like, non-glycosylated forms. This was achieved by in vivo deglycosylation of target proteins of interest by co-expression with bacterial PNGase F (Mamedov et al 2012;Mamedov and Yusibov 2013;Mamedov et al 2016) or with Endo H enzymes (Mamedov et al 2017;Mamedov et al 2019b).Using this technology, a functional active Pfs48/45 antigen of Plasmodium falciparum has been produced in plants and mice immunized with this antigen showed strong inhibition in standard membrane-feeding assay (SMFA) analysis for the first time (Mamedov et al 2019a and b). This technology has been also applied for production of protective antigen of Bacillus anthracis (Mamedov et al 2012;Mamedov et al 2016;Mamedov et al 2017;Mamedov et al 2019a).…”