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Abstract

A Chlamydomonas reinhardtii chloroplast expression vector, pACTBVP1, containing the fusion of the foot and mouth disease virus (FMDV) VP1 gene and the cholera toxin B subunit (CTB) gene was constructed and transfered to the chloroplast genome of C. reinhardtii by the biolistic method. The transformants were identified by PCR, Southern blot, Western blot and ELISA assays after selection on resistant medium and incubation in the dark. The CTBVP1 fusion protein was expressed in C. reinhardtii chloroplast and accounted for up to 3% of the total soluble protein. The fusion protein also retained both GM1-ganglioside binding affinity and antigenicity of the FMDV VP1 and CTB proteins. These experimental results support the possibility of using transgenic chloroplasts of green alga as a mucosal vaccine source.

Micro-algae come of age as a platform for recombinant protein production

“…A fusion protein containing the foot-and-mouth disease virus VP1 gene and the cholera toxin B subunit (CTBVP1) was produced in the chloroplast as well (Sun et al 2003). The protein was reported to accumulate to 3% total protein by ELISA quantification, and retained GM1-ganglioside binding activity and antigenicity.…”

“…Furthermore, fusions of antigens with the cholera toxin B subunit show promise for eliciting immune response from mucosal delivery alone, as the cholera toxin B subunit allows a fused protein to penetrate the intestinal lining (Sun et al 2003; Harakuni et al 2005). …”

Algae-based oral recombinant vaccines

“…The first reported algal-synthesized vaccine antigen was a chimeric molecule comprising the foot-and-mouth disease virus structural protein VP1 and the beta subunit of cholera toxin (CTB), a known mucosal adjuvant (Sun et al, 2003). This antigen had been previously expressed in plants and had demonstrated oral immunity in mice (Wigdorovitz et al, 1999), but advancement of trials was hindered by low expression levels.…”

“…This antigen had been previously expressed in plants and had demonstrated oral immunity in mice (Wigdorovitz et al, 1999), but advancement of trials was hindered by low expression levels. In C. reinhardtii , 3–4% TSP was reported, but higher yields may be possible because the strains examined were not completely homoplasmic (Sun et al, 2003). …”

“…It is also clear that identifying alternative mucosal adjuvants to complement these antigens is critical, whether for co-administration with algal-produced antigens or for incorporation into chimeric fusion proteins. It has been suggested that antigenic fusions with CTB, one of the preferred adjuvants, may interfere with the CTB subunit’s ability to form the pentameric structure essential for strong GM1 ganglioside binding (Sun et al, 2003). Many alternatives to CTB are under investigation for oral vaccination in other production platforms, including CpG-containing oligodeoxynucleotides, saponins, and subunits from heat-labile enterotoxin and ricin toxin (Pelosi et al, 2012).…”