An algal system for manufacturing recombinant proteins has provided scientists with the means to generate a malaria-blocking vaccine candidate based on two native unglycosylated Plasmodium falciparum surface proteins, Pfs25 and Pfs28. The University of California, San Diego researchers produced the two P. falciparum proteins in the chloroplasts of the widely used model alga Chlamydomonas reinhardtii, and then injected the purified proteins into mice. The treated animals generated antibodies that bound to the surface of in vitro-cultured P. falciparum sexual stage parasites that blocked the ability of the parasite to be transmitted from mosquitoes to humans.
Reporting their work in PLoS One, Stephen Mayfield, Ph.D., and colleagues say the algal system is ideally suited to the cost-effective mass production of complex, unglycosylated malarial proteins that can’t be generated cheaply using existing technologies. The researchers published their results in a paper titled “Algae-Produced Pfs25 Elicits Antibodies That Inhibit Malaria Transmission.”
This paper tells us two things, Dr. Mayfield states. “The proteins that we made here are viable vaccine candidates, and we at least have the opportunity to produce enough of this vaccine that we can think about inoculating two billion people. In no other system could you even begin to think about that.”
Every system used to produce recombinant proteins has its own advantages and disadvantages with respect to cost, protein folding, yield, ease of manipulation, and scalability, the authors explain. These features must be carefully weighed against the specific application and structure of the antigen. In the case of malarial proteins that could form the basis of subunit vaccines, production is hampered by the fact that the proteins are complex and unglycosylated. Eukaryotic production systems glycosylate recombinant proteins, and bacterial systems aren’t capable of generating proteins with the complexity of Pfs25 and Pfs28, which contain tandem repeats of structurally complex epidermal growth factor-like domains. “Attempts to produce conformationally correct Pfs25 in Escherichia coli failed, and yeast-produced Pfs25 has multiple conformations and caused an allergic reaction during human clinical trials,” the authors note.
One strategy for reducing the cost of manufacturing subunit vaccines is to use plants as the expression system. Production in algal chloroplasts avoids issues with other bacterial and eukaryotic systems because chloroplasts can fold complex eukaryotic proteins and do not glycosylate proteins. Indeed, the chloroplast of the eukaryotic green microalgae C. reinhardtii has already been used to generate human monoclonal antibodies, human protein therapeutics, and subunit vaccines.
Working with colleagues at the UCSD’s Division of Infectious Diseases, Dr. Mayfield’s team at the San Diego Center for Algal Biotechnology turned to the C. reinhardtii system to produce two P. falciparum proteins that represent promising candidates as a potential malaria vaccine. Previous work had already demonstrated that antibodies against Pfs25 and other conserved proteins critical to the parasite’s sexual reproduction process prevent parasite maturation that leads to sporozoites, and so there is no transmission of the parasite from the mosquito to the human host.
Analyses of Pfs25 and Pfs28 produced in C. reinhardtii confirmed that the proteins were structurally very close to those of the native proteins, down to the disulphide bridges that hold the protein in its 3-D shape. Mice receiving injections of either alga-produced Pfs25 or Pfs28 raised high levels of antibodies to the respective proteins. The resulting Pfs25 or Pfs28 antibodies and antisera bound to the native proteins in sexual stage parasite lysates. Importantly, when the antisera from immunized mice were fed to malaria-infected Anopheles stephensi mosquitoes, animals receiving the Pfs25 antisera didn’t generate malarial oocysts, and so wouldn’t be able to transmit the parasite to a future human host.