by Dr. Mark Edwards
Algae Science Articles — Stephen P. Mayfield and the UCSD Laboratory
series of articles by Stephen Mayfield and the UCSD Laboratory deserve recognition for their articles on algae-based medicines for malaria and cancer. Mayfield and his team have worked for seven years to create complex protein-based drugs from green algae Chlamydomonas reinhardtii. Their work shows algae can produce medicines in greater quantity, faster and at a fraction of the cost of similar drugs derived from mammalian or terrestrial plant cells.
An algae production platform can dramatically cut the costs of making complex proteins that are the primary components in drugs such as:
- An affordable oral malarial vaccine (under development by the UCSD team).
- The use of antibody drug conjugates to target cancer cells combined with the application of protein toxins that inhibit cancer-cell proliferation by killing cancer cells.
- All mammalian mothers make colostrum as the first milk for their newborns. Colostrum protects newborns from bacterial and viral infections, and colostrum proteins produced in algae could be made available at large scale and very low cost.
Complex drugs are currently produced from the cells of mammals, terrestrial plants or bacterial cells. Stephen Mayfield’s team is exploring methods for faster, cheaper and better medical algae-based compounds.
The parasitic, mosquito-borne, infectious disease malaria threatens nearly half of the global population. Many of the over 3 billion people most vulnerable to malaria are children and families that are extremely poor. Eradication of malaria requires low-cost, easily administered vaccines that work in concert with current control methods. Current medicines require refrigeration, which is difficult and expensive in hot rural areas. A short, low cost supply chain and ease of administration will be essential components of a malaria vaccine, because malaria endemic regions are poor and often lack an adequate healthcare infrastructure.
Mayfield’s team collaborated with a medical team led by Joseph Vinetz, from UC San Diego’s School of Medicine, to create the precursor to a low cost algae-based malaria vaccine that does not need refrigeration.
Recent work has focused on identifying specific parasite antigens that elicit the desired cellular and humoral immunity. These subunit vaccines are generally made in recombinant systems, purified, and delivered via injection. Production and purification of subunit vaccines are often complex and expensive. Bacteria, yeast, insect, and mammalian cells are most commonly used for producing recombinant proteins but they are slow to produce and expensive to extract.
Authors James Gregory and Stephen Mayfield in “Developing inexpensive malaria vaccines from plants and algae,” (Applied Microbiology & Biotechnology, March 2014) note the expression platform must be capable of producing a recombinant antigen that faithfully mimics native protein structure. Doing so ensures that the immune response confers protection to the corresponding pathogen. This is difficult to achieve because predicting whether a heterologous platform can replicate the three-dimensional structure of a foreign protein is nearly impossible, particularly for unique or structurally complex antigens.
The authors describe mosquito stage vaccines, called transmission-blocking vaccines, (TBVs) which focus on antigens from sexual stage parasites. Antibodies to several of these proteins block parasite sexual development when taken up with Plasmodium gametocytes during a mosquito bloodmeal, thus preventing mosquito infection and subsequent transmission to the next human host. Antibodies raised in mice to TBV candidate antigens have successfully blocked transmission in both animal models and standard membrane feeding assays but have not advanced beyond safety tests in human clinical trials.
Malaria parasite life cycle and potential points of vaccine intervention include the following.
- Mosquito bloodmeal introduced sporozoites into the bloodstream.
- Sporozoites enter the liver.
- Asexual division into merozoites within liver cells.
- Merozoite filled vesicles release parasites into the circulatory system via the lungs.
- Cycles of red blood cell invasion by merozoites causing repeated bouts of symptoms.
- A subset of merozoites develop into sexual stage parasites.
- Mosquitoes take up sexual stage parasites during a bloodmeal.
- Motile ookinetes burrow through the midgut and develop into oocysts.
- Thousands of sporozoites travel to the mosquito salivary glands when the oocysts burst.
- The parasite life cycle repeats after being transferred to a new human host via the mosquito (Image credit: PATH Malaria Vaccine Initiative)
The UCSD team recently produced Pfs25 and Pfs45/48 in the chloroplast of the green alga Chlamydomonas reinhardtii. In collaboration with Joseph Vinetz’s laboratory, they demonstrated that alga-produced Pfs25 (CrPfs25) elicits TB antibodies. C. reinhardtii is an extensively researched single-celled eukaryotic alga that has only recently been exploited as a platform for producing recombinant proteins. Algae have been used to produce industrial enzymes, vaccine antigens, and complex immunotoxins on an academic scale. Depending on the desired posttranslational modifications, transgenes can be expressed from the nuclear or chloroplast genome.
Terrestrial plants such as tobacco, tomatoes and others require a year to propagate and substantial cost to extract the target compounds. Stable transgenic algal strains of Chlamydomonas reinhardtii can be constructed in a few weeks. Oral administration of the whole cell eliminates the costly extraction activities. Algae proteins are easily scalable in closed algae production systems or in outdoor ponds.
The ideal malaria vaccine must be extremely inexpensive, heat-stable and easily administered. Using a whole cell, oral vaccine for malaria could avoid substantial costs. Currently, oral vaccines are available for polio, rotavirus, cholera, and typhoid, but these vaccines are based on attenuated or heat-killed pathogens. Novel strategies are necessary to overcome the obstacles that block orally available subunit vaccines, especially for pathogens like malaria than cannot easily be cultured and primarily affect poor regions of the world.
The UCSD team investigated a simple strategy utilizing whole C. reinhardtii cells. CTB-Pfs25 was produced as a fusion protein in C. reinhardtii chloroplasts and orally delivered to balb/c mice in freeze-dried whole cells. While this strategy was not sufficiently effective, the team learned several insights that hold promise for future oral malaria medicine. This work has attracted funding from the Bill & Melinda Gates Foundation.
Algae offer an ideal production platform for protein-based drugs that fight cancer. The therapeutic strategy Mayfield’s team use is described in “Production of unique immunotoxin cancer therapeutics in algal chloroplasts, Proceedings of The National Academy of Sciences, January 2, 2013.
The team uses a hunt and kill strategy that required genetically engineering designer algae for the production of special compounds for both searching for cancer cells and killing them. The first step introduces an antibody that hunts down a cancer cell. Then a designer toxin is sent to kill the cancer cell while leaving healthy cells uninjured. Mayfield calls these “dual-domain drugs,” which offer better cancer therapy. These drugs are currently produced through a complex process so costly that a course of treatments for lymphoma can cost over $100,000. The new algae-based production method could cut cost by 90%.
The UCSD team demonstrated that the green algae Chlamydomonas reinhardtii are capable of expressing, folding, and accumulating a range of human therapeutic proteins in the chloroplast. They also showed that recombinant proteins could be secreted from algae. Cost is a critical factor in the production of protein-based therapies. Algae reduce production costs because growing algae requires only trace minerals, fertilizer, and sunlight. Algae have the potential to produce a wide variety of recombinant proteins for various therapeutic applications. An algae platform can produce desirable classes of therapeutically relevant proteins quickly. Algae also offer the potential to produce a number of novel proteins because of the unique biochemical environment of the chloroplast.
Algae’s ability to fold, assemble and accumulate multiple domain proteins as soluble molecules offers significant advantages. The attributes that truly distinguish algae from other recombinant expression platforms are the presence of chloroplasts and the ability to produce and accumulate immunotoxin proteins in these compartments. Chloroplasts of higher plants such as tobacco show a high degree of conservation of algal chloroplasts and could be a viable option for expressing immunotoxins as well. However, algae offer better quality control because cultures can be grown in closed systems, avoiding problems that might arise through cross-contamination with native species.
No other recombinant protein production system is capable of accumulating these complex eukaryotic toxin molecules as soluble and enzymatically active proteins. These traits set algae apart from other expression platforms. The potential of immunotoxins as potent and specific anticancer therapeutics is enormous. The use of antibody drug conjugates, using small-molecule drugs, to target and kill cancer cells and to minimize the exposure of healthy cells is already a reality. Some (expensive) therapies are in late-stage clinical trials or already are approved by the US Food and Drug Administration.
Protein toxins are effective in inhibiting cancer-cell proliferation, but their production is limited to bacterial expression platforms that require the protein to be denatured and subsequently refolded. The process increases production time and cost substantially. Algae provide a faster; less costly production platform that adds the ability to create more complex molecules than presently can be produced in bacterial systems. Although additional work needs to be done to determine whether larger or smaller immunotoxins are more effective for specific cancers, the αCD22PE40 and αCD22HCH23PE40 produced by the UCSD team demonstrated that C. reinhardtii chloroplasts are capable of producing complex immunotoxins and provide a effective means to produce next-generation therapeutics.
The UCSD team proved this year that algae-based drugs are effective in shrinking tumors in mice. They plan next to show that these two-domain proteins can target and kill human breast cancer cells, again using animal models, a move that could lead to clinical trials on breast cancer patients as early as 2015.