Alga-Produced Cholera Toxin-Pfs25 Fusion Proteins as Oral Vaccines

Abstract

Infectious diseases disproportionately affect indigent regions and are the greatest cause of childhood mortality in developing countries. Practical, low-cost vaccines for use in these countries are paramount to reducing disease burdens and concomitant poverty. Algae are a promising low-cost system for producing vaccines that can be orally delivered, thereby avoiding expensive purification and injectable delivery. We engineered the chloroplast of the eukaryotic alga Chlamydomonas reinhardtii to produce a chimeric protein consisting of the 25-kDa Plasmodium falciparum surface protein (Pfs25) fused to the ␤ subunit of the cholera toxin (CtxB) to investigate an alga-based whole-cell oral vaccine. Pfs25 is a promising malaria transmission-blocking vaccine candidate that has been difficult to produce in traditional recombinant systems due to its structurally complex tandem repeats of epidermal growth factor-like domains. The noncatalytic CtxB domain of the cholera holotoxin assembles into a pentameric structure and acts as a mucosal adjuvant by binding GM1 ganglioside receptors on gut epithelial cells. We demonstrate that CtxB-Pfs25 accumulates as a soluble, properly folded and functional protein within algal chloroplasts, and it is stable in freeze-dried alga cells at ambient temperatures. In mice, oral vaccination using freeze-dried algae that produce CtxB-Pfs25 elicited CtxB-specific serum IgG antibodies and both CtxB-and Pfs25-specific secretory IgA antibodies. These data suggest that algae are a promising system for production and oral delivery of vaccine antigens, but as an orally delivered adjuvant, CtxB is best suited for eliciting secretory IgA antibodies for vaccine antigens against pathogens that invade mucosal surfaces using this strategy.

Alga-Produced Malaria Transmission-Blocking Vaccine Candidate Pfs25 Formulated with a Human Use-Compatible Potent Adjuvant Induces High-Affinity Antibodies That Block Plasmodium falciparum Infection of Mosquitoes

“…reinhardtii-produced recombinant Pfs25 has certain biotechnological advantages as the basis for vaccine development. Production is as simple as growing the alga in minimal growth medium plus light (17,18). Photosynthetic growth, combined with high recombinant protein yields and the ease of scaling up production, results in a relatively inexpensive production platform (49).…”

“…The microalga C. reinhardtii has previously been reported to produce nonglycosylated, immunogenic recombinant Pfs25 that induces transmission-blocking antibodies in combination with complete Freund's adjuvant (17) and mucosal IgA antibodies when fused with cholera toxin beta subunit (CtxB) and administered orally (18). Production in algal chloroplasts is advantageous from an economic and biological perspective.…”

“…Parasite-produced Pfs25 contains 4 epidermal growth factor (EGF)-like domains and is not glycosylated, which makes it biotechnologically challenging to produce the properly folded, conformationally correct recombinant protein(s) required for the induction of effective transmission-blocking antibodies (7-9). Heterologous expression systems used to produce Pfs25 as a recombinant subunit immunogen include Escherichia coli (10), Saccharomyces cerevisiae (11), Pichia pastoris (7, 12), baculovirus (13), Nicotiana benthamiana (14), Triticum vulgare (wheat germ) extract (6,15,16), and most recently, the chloroplast of the microalga Chlamydomonas reinhardtii (17,18).…”

Rare codons effect on expression of recombinant gene cassette in Escherichia coli BL21(DE3)

“…For facilitated entry of these proteins into the intestine, GM1 and globotriaosylceramide receptors on the intestinal epithelial cells have been used by several pathogens such as Vibrio cholerae and Shigella dysenteriae [14,15]. protein such as CTB is needed [2].…”

Fertilization Mechanisms of the Rodent Malarial Parasite Plasmodium berghei

“…This failure to completely inhibit reproduction may arise from PbGCS1 possessing many cysteine residues and displaying a complex conformation that is not recapitulated by the recombinant protein, resulting in less effective antibody production. Such a requirement for correct folding of recombinant TBV proteins for effective antibody production has been observed in cases in which the TBV candidate is produced in yeast and algal systems (Kaslow et al 1994 ;Gregory et al 2013 ). In addition to this problem, the immunization protocol needs to be improved to generate higher antibody titers (Kubler-Kielb et al 2007 ;Outchkourov et al 2008 ).…”