Abstract
Malaria is a parasitic, mosquito-borne, infectious disease that threatens nearly half of the global population. The last decade has seen a dramatic drop in the number of malaria-related deaths because of vector control methods and anti-malarial drugs. Unfortunately, this strategy is not sustainable because of the emergence of insecticide-resistant mosquitoes and drug-resistant Plasmodium parasites. Eradication of malaria will ultimately require low-cost easily administered vaccines that work in concert with current control methods. Low cost and ease of administration will be essential components of any vaccine, because malaria endemic regions are poor and often lack an adequate healthcare infrastructure. Recently, several groups have begun addressing these issues using inexpensive photosynthetic organisms for producing vaccine antigens and exploring oral delivery strategies. Immune responses from plant-based injectable malaria vaccines are promising, but attempts to adapt these for oral delivery suggest we are far from a feasible strategy. Here, we review examples of these technologies and discuss the progress and potential of this research, as well as the obstacles ahead.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
Human malaria is caused by any of four different species of protozoan parasites from the genus Plasmodium: falciparum, vivax, malariae, and ovale. Plasmodium falciparum is responsible for the majority of the over half a million malaria deaths per year, which are predominantly children under the age of five that live in indigent African nations (WHO 2012). These nations lack sufficient resources to effectively combat malaria, which contributes to continued poverty (Ingstad et al. 2012). Bed nets, insecticides against the Anopheles mosquito vector, and anti-malarial drugs have been instrumental in reducing disease burdens over the last decade, but these measures are not sustainable. The incidence of insecticide-resistant mosquitoes (malERA 2011b), drug-resistant malaria (Eastman and Fidock 2009), and counterfeit drugs is increasing (Nayyar et al. 2012). The best way forward is to develop efficacious vaccines, but progress has been slow, primarily because the malaria parasite life cycle is complex (Riley and Stewart 2013), involving several distinct cell types, and evades the immune system through antigenic variation (Kirkman and Deitsch 2012). Complicating matters further, malaria vaccines must be extremely inexpensive to produce and easy to administer. It is not feasible to implement a large-scale vaccination campaign if the cost is similar to recent subunit vaccines like the HPV vaccine ($130/dose (CDC 2013)). Realistically, a malaria vaccine needs to be a tiny fraction of that cost. Reaching these goals requires new strategies for both producing and delivering vaccines.
Individuals that live in regions with high malaria transmission gradually acquire immunity only after repeated infections over an extended period of time (Mueller et al. 2013). These infections induce a diverse set of malaria parasite specific antibodies as well as CD4 and CD8 T cells that together confer immunity. Paramount to developing malaria vaccines that can elicit a similar immune response is a better understanding of the parasite life cycle (Fig. 1). There are three distinct stages—liver, blood, and mosquito. All three stages are potential points of vaccine intervention (Riley and Stewart 2013). Pre-erythrocytic vaccines target sporozoites, the infectious cells of malaria that are injected into the bloodstream during a mosquito bloodmeal. Early attempts at pre-erythrocytic vaccines demonstrated that radiation-attenuated sporozoites can confer sterile immunity to malaria when injected via mosquitoes (Duffy et al. 2012). A recent study demonstrated that intravenously injected, attenuated, and cryopreserved P. falciparum sporozoites can provide complete protection (Seder et al. 2013). While extremely promising, production and administration of sufficient in vitro-cultured sporozoites using this strategy are technically challenging and not scalable using current technology.
Recent work has focused on identifying specific parasite antigens that elicit the desired cellular and humoral immunity (Patarroyo et al. 2012). These subunit vaccines are generally made in recombinant systems, purified, and delivered via injection. Antigens can also be delivered using viral vectors (Hill et al. 2010; Schuldt and Amalfitano 2012). Subunit vaccines are safer than attenuated pathogens because they cannot cause disease and do not require large-scale cultivation of pathogens. The most studied pre-erythrocytic subunit vaccine, RTS/S, uses a 189 amino acid peptide from the circumsporozoite protein (CSP) (Duffy et al. 2012). The aim of this vaccine is to elicit circulating antibodies and a T cell response that confers protection to the vaccinated individual. Thus far, data from phase 3 RTS/S clinical trials suggests modest, but significant protection (RTS/S Clinical Trials Partnership 2012).
Unlike pre-erythrocytic vaccines, blood and mosquito stage vaccines would not prevent initial infection. Instead they would reduce malaria symptoms and transmission, respectively. Sporozoites asexually multiply into merozoites after reaching liver over a 7–10-day period. These cells are released within vesicles that travel through the circulatory system to the lungs where they dissolve and release the merozoites into the bloodstream and attack red blood cells (Baer et al. 2007). Blood stage vaccines focus on merozoite antigens and aim to reduce red blood cell invasion and eliminate intraerythrocytic parasites. Mosquito stage vaccines, more commonly called transmission blocking vaccines (TBVs), focus on antigens from sexual stage parasites (malERA 2011a). 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 (SMFA, discussed below), but unfortunately have not advanced beyond safety tests in human clinical trials.
Malaria vaccines from photosynthetic organisms
Production and purification of subunit vaccines are often complex and expensive. Once a promising vaccine antigen has been identified, it is made using one of several possible heterologous systems. Bacteria, yeast, insect, and mammalian cells are most commonly used for producing recombinant proteins, and each has advantages and disadvantages with respect to cost and ease of use. Importantly, the expression platform that is used 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, and several malaria vaccine antigens are prime examples of this dilemma. Plasmodium proteins are rarely glycosylated and often contain highly repetitive sequences or complex tertiary structures. For example, the leading TBV candidate, Pfs25, contains tandem repeats of epidermal growth factor (EGF)-like motifs, each containing multiple disulfide bonds, and Pfs25 is not glycosylated. Pfs25 made in Escherichia coli did not elicit transmission-blocking (TB) antibodies (Kaslow et al. 1992), most likely because E. coli cannot efficiently form disulfide bonds in Pfs25 even when targeted to the periplasm. There are two distinct isoforms of yeast-produced Pfs25, only one of which has the correct conformation, and the primary sequence must be mutated in order to prevent glycosylation (Zou et al. 2003). Merozoite surface protein 1 (MSP1), a blood stage vaccine candidate, also contains EGF-like domains that are required for eliciting protective antibodies (Egan et al. 1996). Correct disulfide bond formation in another blood stage vaccine antigen, apical membrane antigen 1 (AMA1), is required for eliciting protective antibodies (Anders et al. 1998). Unfortunately, alternative mammalian and insect cell platforms are too costly to be considered for producing malaria vaccines.
Photosynthetic organisms, including terrestrial plants and algae, offer several advantages over conventional heterologous protein production systems and are a potential solution (Daniell et al. 2009; Melnik and Stoger 2013). From a production standpoint, they are safe, inexpensive, and easily scalable. Biomass can be generated for pennies per gram or even fractions of a penny in the case of algae (Georgianna and Mayfield 2012), and plant or algae biomass can be freeze dried for long-term storage at ambient temperatures. The absence of human pathogens, prions, or other harmful toxins in many photosynthetic organisms greatly reduces the risk of residual contamination following purification. The earliest examples of plant-produced recombinant proteins were made in tobacco in the late 1980s; plant-produced Hepatitis B virus vaccine followed shortly thereafter (Mason et al. 1992). In 2006, Dow AgroSciences licensed the first plant-produced vaccine. Several more from other groups are presently in clinical trials (Rosales-Mendoza et al. 2012; Yusibov et al. 2011).
After over two decades of research, the close relative of tobacco, Nicotiana benthamiana, has established methods for both nuclear and plastid transformation, defined promoters and regulatory elements, and has the highest yields of any plant system to date. Arguably, the most popular transformation method is Agrobacterium infiltration of leaf tissue, which uses transient nuclear expression from viral vectors (Giritch et al. 2006). Rather than taking more than a year to generate a stable transgenic plant, recombinant protein can be recovered in less than 1 month. Using viral vectors can cause genetic drift of the transgene over generations, which makes scaling up a significant challenge (Kearney et al. 1993), but academic and commercial entities have made significant progress in recent years (Davies 2010).
Several malaria vaccines have been produced using transient expression or stably transformed plants, including P. falciparum blood stage and TBV candidates (Clemente and Corigliano 2012). In studies conducted by Fraunhofer USA, N. benthamiana-produced Pfs2522-193 (NbPfs25) (Farrance et al. 2011a) and Pfs230444-730 (NbPfs230) (Farrance et al. 2011b) elicited TB antibodies in mice and rabbits, respectively, as measured by SMFA. More recently, they demonstrated that fusing Pfs25 to virus-like particles can reduce the amount of antigen needed to elicit TB antibodies (Jones et al. 2013). Because P. falciparum does not infect mice or other animal models, SMFAs are used to emulate the process of a mosquito bloodmeal. P. falciparum gametocytes and sera from vaccinated or unvaccinated animals are added to human blood, which is then fed to female Anopeheles stephensi mosquitoes. Mosquito midguts are later dissected and analyzed for the presence of ookinetes; a reduction of oocysts in mosquitoes that received sera from vaccinated animals indicates the presence of TB antibodies.
We recently produced Pfs2522-193 and Pfs48/45178-448 in the chloroplast of the green alga Chlamydomonas reinhardtii and, in collaboration with Joseph Vinetz's laboratory at the University of California, San Diego, demonstrated that alga-produced Pfs25 (CrPfs25) elicits TB antibodies by SMFA (Gregory et al. 2012). C. reinhardtii is a single-celled eukaryotic alga that has long been used as a model system to study photosynthesis and flagella function, but has only recently been exploited as a platform for producing recombinant proteins. Although still in its infancy compared to N. benthamiana, algae have been used to produce industrial enzymes (Rasala et al. 2012), vaccine antigens (Surzycki et al. 2009), and complex immunotoxins (Tran et al. 2013) on an academic scale. Depending on the desired posttranslational modifications, transgenes can be expressed from the nuclear or chloroplast genome. Unlike tobacco and other terrestrial plants, stable transgenic algal strains can be constructed in a few weeks time. They are also easily scaled in fully contained photobioreactors or in outdoor ponds, although large-scale production is currently limited to biofuel and nutraceutical production (e.g., Solazyme, Sapphire Energy, and Nutrex) rather than recombinant proteins.
Expression in C. reinhardtii chloroplasts is markedly different than transient nuclear expression in N. benthamiana. Chloroplasts have prokaryotic-like ribosomes and importantly, lack the machinery for post-translational glycosylation, thus the peptide sequence of CrPfs25 is identical to native Pfs25. In contrast, NbPfs25 elicits transmission-blocking antibodies only when produced as a fusion with lichenase or when potential N-linked glycosylation sites are mutated (Farrance et al. 2011a). This drawback could potentially be overcome by co-expressing bacterial PNGase F (Peptide: N-glycosidase F) to remove posttranslational glycosylation (Mamedov et al. 2012), but the impact of PNGase on the host cell viability, Pfs25 folding, and the ability of Pfs25 to elicit TB antibodies is unknown. Algal chloroplasts contain eukaryotic chaperones (Liu et al. 2007), peptidylprolyl isomerases (Ingelsson et al. 2009), and disulfide isomerases (Kim and Mayfield 1997), which presumably facilitate production of the complex EGF-like domains in CrPfs25. A high priority for TBV development should be a head-to-head comparison of TB activity in sera from animals vaccinated with CrPfs25 or NbPfs25 of equivalent purity.
Oral vaccines
Biomass from plants and algae is a promising low-cost feedstock for injectable vaccines, but a large portion of the cost is due to purification, cold storage, and administration by injection. For example, RTS/S is produced in yeast, which is a relatively inexpensive platform, but purification and the complex adjuvant significantly increases production costs (Geels et al. 2011). The ideal malaria vaccine must be extremely inexpensive, heat-stable, and easily administered without a skilled medical worker. Using a whole cell, oral vaccine for malaria could avoid all of these costs. To that end, several groups have begun investigating strategies for plant-based orally available vaccines that require little or no pre-processing, but there are currently no approved oral subunit vaccines, and their development faces immense challenges (Renukuntla et al. 2013). 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.
Antigen uptake by the gut associate lymphoid tissue (GALT) is inefficient partly because of the proteolytic and acidic stomach environment. To ensure bioavailability of orally delivered proteins, antigens must be sufficiently protected by encapsulation, which could be accomplished with either synthetic nanoparticles or biological cells with protective cell walls. Several groups are working to overcome the challenges associated with oral delivery using nanoparticles (Ensign et al. 2012). As this technology develops, it could be a viable option for oral delivery, but antigen–nanoparticle formulation will always be more complex and costly than using whole cells because the payload protein must first be purified from the expression platform. Ideally, proteins for vaccines will be both produced and delivered using the same cells, completely eliminating the cost of purification and formulation.
Many plants and algae could be used as a delivery vehicle for oral vaccines because they are edible, and therefore generally recognized as safe (GRAS) by the US Food and Drug Administration (FDA). GRAS-approved organisms lack microbial toxins and they are not susceptible to human pathogens. The first plant-based oral vaccine, which used tobacco and potato, found that oral administration of plant tissue containing recombinant heat-labile enterotoxin subunit β (LTB) from E. coli induces low levels of both serum IgG and secretory IgA (sIgA) antibodies (Haq et al. 1995). Unfortunately, the complexity of the mucosal immune system, which must discriminate between pathogenic and harmless organisms, has slowed oral vaccine development (Mowat 2003). Repeated, large oral doses of antigen prior to systemic immunization can mute T cell-mediated responses to the same antigen. Indeed, the default immune response to most soluble antigens in the gut is one of non-responsiveness or tolerance (Mason et al. 2008). As a result, orally delivered protein subunit antigens elicit little or no adaptive immune response.
Initial strategies to overcome these obstacles combined high doses of plant tissue with co-administered or conjugated mucosal adjuvants, or prior parenteral vaccination. Several of these studies took advantage of the β subunit of the cholera toxin (CTB), a well-characterized mucosal adjuvant, for malaria vaccines (Clemente and Corigliano 2012; Kumar et al. 2012; Kwon et al. 2013). In one example, AMA1 and MSP1 fusions to CTB produced in tobacco chloroplasts elicited antibodies in mice that inhibited parasite invasion of human red blood cells in vitro and provided protection against cholera toxin challenge (Davoodi-Semiromi et al. 2010). This result was a major step forward that provides a springboard for future development. One area that needs improvement is the immunization regime. First, all of the mice were initially primed by subcutaneous injection using soluble protein fractions that were enriched for CTB fusions. Second, ground up leaf material was prepared under liquid nitrogen and stored at −80 °C. Whether or not cold storage was necessary is unclear. Freeze-dried leaf material can often be stored at ambient temperatures while maintaining antigen integrity. Even if cold storage of leaf material is not necessary, purification and injection negatively impact vaccine cost and ease of administration. Importantly, this study used stable transgenic chloroplasts as opposed to transient expression through Agrobacterium-mediated transformation (Davoodi-Semiromi et al. 2010). Despite being the best choice for high protein yields, transient expression from Agrobacterium transformation is not ideal for oral vaccines because of potentially harmful contaminants that must be removed.
There are only two examples of alga-based oral vaccines for malaria. The first looked at AMA1 and MSP1 from the rodent malaria, Plasmodium berghei (Dauvillee et al. 2010). Rodent malaria provides a safe, convenient model system for studying the entire parasite lifecycle and vaccine efficacy because of its similarity to human malaria. MSP1 or AMA1 peptides were fused to the granule bound starch synthase (GBSS), expressed from the C. reinhardtii nuclear genome, and successfully targeted to starch granules. Mice that were orally vaccinated with LTB and purified starch granules containing GBSS-MSP1and GBSS-AMA1 showed a delay in mortality and reduced parasitemia after a lethal challenge of P. berghei that was comparable to intraperitoneal (IP) injection. Unfortunately, the dose of antigen in starch granules is unclear, and the function of antibodies from oral vaccination was not tested further. It would be interesting to compare the inhibitory activity of antibodies from oral and IP vaccination in red blood cell invasion assays, which is commonly done to assess the efficacy of blood stage vaccine candidates. Nevertheless, this strategy warrants further investigation because specialized microfold cells (M cells) found in Peyer's patches (PP), or organized bundles of lymphoid tissue found in the ileum that are primary sites for adaptive immune induction, preferentially take up particulate matter. Starch granules, if they remain intact with antigen in the gut, may facilitate an adaptive immune response via PPs.
We investigated a simpler 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 (Gregory et al. 2013). This strategy elicited CTB specific IgG antibodies and secretory IgA antibodies to both CTB and Pfs25. Pfs25-specific serum IgG antibodies that can be taken up during a mosquito blood meal are necessary for transmission blocking activity. Thus, orally delivered algae containing CTB-Pfs25 is not a suitable TBV. An identical strategy examining the Staphylococcus aureus D2 fibronectin binding domain (D2) detected serum IgG and IgA antibodies to both CTB and D2 (Dreesen et al. 2010). There are two potential explanations for the discrepancy between the two studies. First, expression of CTB-D2 was significantly higher than CTB-Pfs25 (0.7 vs 0.09 %, respectively), so it could simply be a dosing issue. Second, D2 is inherently more immunogenic than Pfs25 (Brennan et al. 1999; Qian et al. 2007). Together, these studies suggest that the immune response to oral vaccines is, at least in part, dependent on the antigen itself. It is conceivable that alternative mucosal adjuvants might elicit different immune responses. In the case of Pfs25, an adjuvant that facilitates a Pfs25 specific IgG response would be more appropriate than the IgA only response elicited by CTB-Pfs25. Identifying and characterizing mucosal adjuvants is therefore critical to the success of oral vaccines.
Exploring mucosal adjuvants
Cholera toxin (CT) and the E. coli heat-labile toxin (LT) are arguably the best-characterized mucosal adjuvants to date(Sanchez and Holmgren 2011). They consist of a catalytically active α subunit (CTA and LTA) and a targeting β subunit pentamer (CTB and LTB), which binds GM1 ganglioside receptors on gut epithelial cells. Ultimately, CT causes massive fluid loss in the intestine through ADP-ribosylation of G proteins by CTA (Sanchez and Holmgren 2011). Removing CTA, and its associated toxicity, and replacing it with antigen provide a convenient targeting mechanism. Conjugation to CTB facilitates transport across the mucosal barrier and increases antigen uptake by antigen presenting cells (APCs). Despite extensive investigation of CTB, the immunomodulatory activity of CTB is still debated. Some studies suggest that it promotes immunity (Holmgren et al. 2005; Miyata et al. 2012), while others find that it promotes tolerance (Stanford et al. 2004; Sun et al. 2013). This contrast exemplifies our lack of understanding of how mucosal adjuvants facilitate an immune response to fused antigens. It is likely that the combined immunomodulatory effect of both the mucosal adjuvant and the fused antigen determines the overall outcome, which might be different depending on the immunization regime, the vehicle used for oral delivery, and the immune cells that are responsible for mediating the immune response. Our understanding of the interplay between these factors is currently limited for most mucosal adjuvants.
Mechanistic studies of potential mucosal adjuvants may eventually allow oral vaccines to be tailored to specific pathogens. For example, pre-erythrocytic malaria vaccines require both circulating IgG antibodies and cytotoxic T cells while transmission blocking vaccines require only circulating IgG antibodies. Secretory IgA antibodies at mucosal surfaces, while less important for malaria, can help to mediate protection from gastrointestinal (polio, typhoid, E. coli), genital (HIV, HPV), and respiratory (Mycobacterium tuberculosis, influenza virus) pathogens. Importantly, systemic immunization is particularly poor at activating mucosal immunity compared to oral and intranasal routes of immunization (Cerutti 2008).
There are several proposed mucosal adjuvants other than CT and LT that may amplify an adaptive immune response (partial list in Table 1). These proteins act at different stages of the cascade of events that usually occur during a natural gastrointestinal infection. The first step is antigen introduction to the GALT. M cells are a primary port of entry to the GALT for many pathogens and are a proposed site of immune induction (Yamamoto et al. 2012). The hypothesis is that targeting to M cells more efficiently delivers antigen to APCs, which can then migrate to the mesenteric lymph node and induce an adaptive immune response through B and T cells. However, M cell ligands have been shown to induce tolerance and immunity depending on the ligand, antigen, and method of delivery, suggesting that targeting alone is not sufficient. Pathogen associated molecular pattern (PAMP) proteins induce cytokine production through TLRs, which impacts antigen presentation by DCs. For example, Flagellin is a potent activator of TLR5 and induces a mixed Th1 and Th2 immune response. TLR5 is particularly relevant to oral vaccines because it is highly expressed on gut DCs and resident macrophages (Uematsu and Akira 2009). Orally delivered plant tissue containing ovalbumin (OVA), the main protein found in egg white, fused to Flagellin, increased cellular and humoral immunity to OVA compared to OVA alone (Girard et al. 2011). Using specific cytokines rather than relying on induction by PAMPs has also been proposed (Tovey and Lallemand 2010). Deconvolving the relative adjuvanticity will require side-by-side comparisons using the same antigen, dosing, and delivery method. A particularly attractive approach would be to combine M cell targeting with TLR agonists. This would ensure that antigen and adjuvant were delivered to APCs.
Concluding remarks
Malaria has the greatest impact on many of the poorest regions of the globe. Vaccines are the most cost effective means for disease prevention and will be the key to eradicating malaria, but expensive injectable vaccines that require cold storage are not a practical option. Production of vaccines in plants and algae can drastically reduce costs, and making these vaccines orally available will further reduce costs and make administration easier if the many obstacles to oral delivery can be overcome. Future development of oral vaccines will depend on a better understanding of the immune response to orally delivered proteins and adjuvants.
References
Anders RF, Crewther PE, Edwards S, Margetts M, Matthew ML, Pollock B, Pye D (1998) Immunisation with recombinant AMA-1 protects mice against infection with Plasmodium chabaudi. Vaccine 16(2–3):240–247
Autenrieth SE, Autenrieth IB (2008) Yersinia enterocolitica: subversion of adaptive immunity and implications for vaccine development. Int J Med Microbiol 298(1-2):69–77. doi:10.1016/j.ijmm.2007.07.010
Baer K, Klotz C, Kappe SH, Schnieder T, Frevert U (2007) Release of hepatic Plasmodium yoelii merozoites into the pulmonary microvasculature. PLoS Pathog 3(11):e171. doi:10.1371/journal.ppat.0030171
Brennan FR, Jones TD, Longstaff M, Chapman S, Bellaby T, Smith H, Xu F, Hamilton WD, Flock JI (1999) Immunogenicity of peptides derived from a fibronectin-binding protein of S. aureus expressed on two different plant viruses. Vaccine 17(15–16):1846–1857
Cerutti A (2008) The regulation of IgA class switching. Nat Rev Immunol 8(6):421–434. doi:10.1038/nri2322
Clemente M, Corigliano MG (2012) Overview of plant-made vaccine antigens against malaria. J Biomed Biotechnol 2012:206918. doi:10.1155/2012/206918
Center for Disease Control (2013) Pediatric/VFC Vaccine Price List.
Daniell H, Singh ND, Mason H, Streatfield SJ (2009) Plant-made vaccine antigens and biopharmaceuticals. Trends Plant Sci 14(12):669–679. doi:10.1016/j.tplants.2009.09.009
Dauvillee D, Delhaye S, Gruyer S, Slomianny C, Moretz SE, d’Hulst C, Long CA, Ball SG, S T (2010) Engineering the chloroplast targeted malarial vaccine antigens in Chlamydomonas starch granules. PLoS One 5(12):e15424. doi:10.1371/journal.pone.0015424
Davies HM (2010) Review article: commercialization of whole-plant systems for biomanufacturing of protein products: evolution and prospects. Plant Biotechnol J 8(8):845–861. doi:10.1111/j.1467-7652.2010.00550.x
Davoodi-Semiromi A, Schreiber M, Nalapalli S, Verma D, Singh ND, Banks RK, Chakrabarti D, Daniell H (2010) Chloroplast-derived vaccine antigens confer dual immunity against cholera and malaria by oral or injectable delivery. Plant Biotechnol J 8(2):223–242. doi:10.1111/j.1467-7652.2009.00479.x
Dreesen IA, Charpin-El Hamri G, Fussenegger M (2010) Heat-stable oral alga-based vaccine protects mice from Staphylococcus aureus infection. J Biotechnol 145(3):273–280. doi:10.1016/j.jbiotec.2009.12.006
Duffy PE, Sahu T, Akue A, Milman N, Anderson C (2012) Pre-erythrocytic malaria vaccines: identifying the targets. Expert Rev Vaccines 11(10):1261–1280. doi:10.1586/erv.12.92
Eastman RT, Fidock DA (2009) Artemisinin-based combination therapies: a vital tool in efforts to eliminate malaria. Nat Rev Microbiol 7(12):864–874. doi:10.1038/nrmicro2239
Egan AF, Morris J, Barnish G, Allen S, Greenwood BM, Kaslow DC, Holder AA, Riley EM (1996) Clinical immunity to Plasmodium falciparum malaria is associated with serum antibodies to the 19-kDa C-terminal fragment of the merozoite surface antigen, PfMSP-1. J Infect Dis 173(3):765–769
Ensign LM, Cone R, Hanes J (2012) Oral drug delivery with polymeric nanoparticles: the gastrointestinal mucus barriers. Adv Drug Deliv Rev 64(6):557–570. doi:10.1016/j.addr.2011.12.009
Farrance CE, Chichester JA, Musiychuk K, Shamloul M, Rhee A, Manceva SD, Jones RM, Mamedov T, Sharma S, Mett V, Streatfield SJ, Roeffen W, van de Vegte-Bolmer M, Sauerwein RW, Wu Y, Muratova O, Miller L, Duffy P, Sinden R, Yusibov V (2011a) Antibodies to plant-produced Plasmodium falciparum sexual stage protein Pfs25 exhibit transmission blocking activity. Hum Vaccin 7(Suppl):191–198
Farrance CE, Rhee A, Jones RM, Musiychuk K, Shamloul M, Sharma S, Mett V, Chichester JA, Streatfield SJ, Roeffen W, van de Vegte-Bolmer M, Sauerwein RW, Tsuboi T, Muratova OV, Wu Y, Yusibov V (2011b) A plant-produced Pfs230 vaccine candidate blocks transmission of Plasmodium falciparum. Clin Vaccine Immunol 18(8):1351–1357. doi:10.1128/CVI.05105-11
Geels MJ, Imoukhuede EB, Imbault N, van Schooten H, McWade T, Troye-Blomberg M, Dobbelaer R, Craig AG, Leroy O (2011) European Vaccine Initiative: lessons from developing malaria vaccines. Expert Rev Vaccines 10(12):1697–1708. doi:10.1586/erv.11.158
Georgianna DR, Mayfield SP (2012) Exploiting diversity and synthetic biology for the production of algal biofuels. Nature 488(7411):329–335. doi:10.1038/nature11479
Girard A, Saron W, Bergeron-Sandoval LP, Sarhan F, Archambault D (2011) Flagellin produced in plants is a potent adjuvant for oral immunization. Vaccine 29(38):6695–6703. doi:10.1016/j.vaccine.2011.06.092
Giritch A, Marillonnet S, Engler C, van Eldik G, Botterman J, Klimyuk V, Gleba Y (2006) Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors. Proc Natl Acad Sci U S A 103(40):14701–14706. doi:10.1073/pnas.0606631103
Gregory JA, Li F, Tomosada LM, Cox CJ, Topol AB, Vinetz JM, Mayfield S (2012) Algae-produced Pfs25 elicits antibodies that inhibit malaria transmission. PLoS One 7(5):e37179. doi:10.1371/journal.pone.0037179
Gregory JA, Topol AB, Doerner DZ, Mayfield S (2013) Alga-produced cholera toxin-Pfs25 fusion proteins as oral vaccines. Appl Environ Microbiol 79(13):3917–3925. doi:10.1128/AEM.00714-13
Haq TA, Mason HS, Clements JD, Arntzen CJ (1995) Oral immunization with a recombinant bacterial antigen produced in transgenic plants. Science 268(5211):714–716
Hill AV, Reyes-Sandoval A, O'Hara G, Ewer K, Lawrie A, Goodman A, Nicosia A, Folgori A, Colloca S, Cortese R, Gilbert SC, Draper SJ (2010) Prime-boost vectored malaria vaccines: progress and prospects. Hum Vaccin 6(1):78–83
Holmgren J, Adamsson J, Anjuere F, Clemens J, Czerkinsky C, Eriksson K, Flach CF, George-Chandy A, Harandi AM, Lebens M, Lehner T, Lindblad M, Nygren E, Raghavan S, Sanchez J, Stanford M, Sun JB, Svennerholm AM, Tengvall S (2005) Mucosal adjuvants and anti-infection and anti-immunopathology vaccines based on cholera toxin, cholera toxin B subunit and CpG DNA. Immunol Lett 97(2):181–188. doi:10.1016/j.imlet.2004.11.009
Ingelsson B, Shapiguzov A, Kieselbach T, Vener AV (2009) Peptidyl-prolyl isomerase activity in chloroplast thylakoid lumen is a dispensable function of immunophilins in Arabidopsis thaliana. Plant Cell Physiol 50(10):1801–1814. doi:10.1093/pcp/pcp122
Ingstad B, Munthali AC, Braathen SH, Grut L (2012) The evil circle of poverty: a qualitative study of malaria and disability. Malar J 11:15. doi:10.1186/1475-2875-11-15
Jones RM, Chichester JA, Mett V, Jaje J, Tottey S, Manceva S, Casta LJ, Gibbs SK, Musiychuk K, Shamloul M, Norikane J, Mett V, Streatfield SJ, van de Vegte-Bolmer M, Roeffen W, Sauerwein RW, Yusibov V (2013) A Plant-Produced Pfs25 VLP Malaria vaccine candidate induces persistent transmission blocking antibodies against Plasmodium falciparum in immunized mice. PLoS One 8(11):e79538. doi:10.1371/journal.pone.0079538
Kaminski RW, Turbyfill KR, Oaks EV (2006) Mucosal adjuvant properties of the Shigella invasin complex. Infect Immun 74(5):2856–2866. doi:10.1128/IAI.74.5.2856-2866.2006
Kaslow DC, Bathurst IC, Isaacs SN, Keister DB, Moss B, Barr PJ (1992) Induction of Plasmodium falciparum transmission-blocking antibodies by recombinant Pfs25. Mem Inst Oswaldo Cruz 87(Suppl 3):175–177
Kearney CM, Donson J, Jones GE, Dawson WO (1993) Low level of genetic drift in foreign sequences replicating in an RNA virus in plants. Virology 192(1):11–17. doi:10.1006/viro.1993.1002
Kim J, Mayfield SP (1997) Protein disulfide isomerase as a regulator of chloroplast translational activation. Science 278(5345):1954–1957
Kim SH, Seo KW, Kim J, Lee KY, Jang YS (2010) The M cell-targeting ligand promotes antigen delivery and induces antigen-specific immune responses in mucosal vaccination. J Immunol 185(10):5787–5795. doi:10.4049/jimmunol.0903184
Kirkman LA, Deitsch KW (2012) Antigenic variation and the generation of diversity in malaria parasites. Curr Opin Microbiol 15(4):456–462. doi:10.1016/j.mib.2012.03.003
Kumar CS, Deepesh G, Mahavir Y, Archana T (2012) Edible vaccine: a new platform for the development of malaria vaccine. Crit Rev Eukaryot Gene Expr 22(3):243–248
Kwon KC, Verma D, Singh ND, Herzog R, Daniell H (2013) Oral delivery of human biopharmaceuticals, autoantigens and vaccine antigens bioencapsulated in plant cells. Adv Drug Deliv Rev 65(6):782–799. doi:10.1016/j.addr.2012.10.005
Liu C, Willmund F, Golecki JR, Cacace S, Hess B, Markert C, Schroda M (2007) The chloroplast HSP70B-CDJ2-CGE1 chaperones catalyse assembly and disassembly of VIPP1 oligomers in Chlamydomonas. Plant J 50(2):265–277. doi:10.1111/j.1365-313X.2007.03047.x
Malaria Eradication Research Agenda (2011a) A research agenda for malaria eradication: vaccines. PLoS medicine 8(1):e1000398. doi:10.1371/journal.pmed.1000398
Malaria Eradication Research Agenda (2011b) A research agenda for malaria eradication: vector control. PLoS medicine 8(1):e1000401. doi:10.1371/journal.pmed.1000401
Mamedov T, Ghosh A, Jones RM, Mett V, Farrance CE, Musiychuk K, Horsey A, Yusibov V (2012) Production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expressing bacterial PNGase F. Plant Biotechnol J 10(7):773–782. doi:10.1111/j.1467-7652.2012.00694.x
Mason HS, Lam DM, Arntzen CJ (1992) Expression of hepatitis B surface antigen in transgenic plants. Proc Natl Acad Sci U S A 89(24):11745–11749
Mason KL, Huffnagle GB, Noverr MC, Kao JY (2008) Overview of gut immunology. Adv Exp Med Biol 635:1–14. doi:10.1007/978-0-387-09550-9_1
Melnik S, Stoger E (2013) Green factories for biopharmaceuticals. Curr Med Chem 20(8):1038–1046
Miyata T, Harakuni T, Taira T, Matsuzaki G, Arakawa T (2012) Merozoite surface protein-1 of Plasmodium yoelii fused via an oligosaccharide moiety of cholera toxin B subunit glycoprotein expressed in yeast induced protective immunity against lethal malaria infection in mice. Vaccine 30(5):948–958. doi:10.1016/j.vaccine.2011.11.059
Mizel SB, Bates JT (2010) Flagellin as an adjuvant: cellular mechanisms and potential. J Immunol 185(10):5677–5682. doi:10.4049/jimmunol.1002156
Mowat AM (2003) Anatomical basis of tolerance and immunity to intestinal antigens. Nat Rev Immunol 3(4):331–341. doi:10.1038/nri1057
Mueller I, Galinski MR, Tsuboi T, Arevalo-Herrera M, Collins WE, King CL (2013) Natural acquisition of immunity to Plasmodium vivax: epidemiological observations and potential targets. Adv Parasitol 81:77–131. doi:10.1016/B978-0-12-407826-0.00003-5
Nayyar GM, Breman JG, Newton PN, Herrington J (2012) Poor-quality antimalarial drugs in southeast Asia and sub-Saharan Africa. Lancet Infect Dis 12(6):488–496. doi:10.1016/S1473-3099(12)70064-6
Patarroyo ME, Bermudez A, Moreno-Vranich A (2012) Towards the development of a fully protective Plasmodium falciparum antimalarial vaccine. Expert Rev Vaccines 11(9):1057–1070. doi:10.1586/erv.12.57
Qian F, Wu Y, Muratova O, Zhou H, Dobrescu G, Duggan P, Lynn L, Song G, Zhang Y, Reiter K, MacDonald N, Narum DL, Long CA, Miller LH, Saul A, Mullen GE (2007) Conjugating recombinant proteins to Pseudomonas aeruginosa ExoProtein A: a strategy for enhancing immunogenicity of malaria vaccine candidates. Vaccine 25(20):3923–3933. doi:10.1016/j.vaccine.2007.02.073
Rasala BA, Lee PA, Shen Z, Briggs SP, Mendez M, Mayfield SP (2012) Robust expression and secretion of Xylanase1 in Chlamydomonas reinhardtii by fusion to a selection gene and processing with the FMDV 2A peptide. PLoS One 7(8):e43349. doi:10.1371/journal.pone.0043349
Renukuntla J, Vadlapudi AD, Patel A, Boddu SH, Mitra AK (2013) Approaches for enhancing oral bioavailability of peptides and proteins. Int J Pharm 447(1–2):75–93. doi:10.1016/j.ijpharm.2013.02.030
Riley EM, Stewart VA (2013) Immune mechanisms in malaria: new insights in vaccine development. Nat Med 19(2):168–178. doi:10.1038/nm.3083
Rosales-Mendoza S, Rubio-Infante N, Govea-Alonso DO, Moreno-Fierros L (2012) Current status and perspectives of plant-based candidate vaccines against the human immunodeficiency virus (HIV). Plant Cell Rep 31(3):495–511. doi:10.1007/s00299-011-1194-8
RTS, S Clinical Trials Partnership (2012) A phase 3 trial of RTS, S/AS01 malaria vaccine in African infants. The New England journal of medicine 367(24):2284–2295. doi:10.1056/NEJMoa1208394
Rynda A, Maddaloni M, Mierzejewska D, Ochoa-Reparaz J, Maslanka T, Crist K, Riccardi C, Barszczewska B, Fujihashi K, McGhee JR, Pascual DW (2008) Low-dose tolerance is mediated by the microfold cell ligand, reovirus protein sigma1. J Immunol 180(8):5187–5200
Sanchez J, Holmgren J (2011) Cholera toxin - a foe & a friend. Indian J Med Res 133:153–163
Schuldt NJ, Amalfitano A (2012) Malaria vaccines: focus on adenovirus based vectors. Vaccine 30(35):5191–5198. doi:10.1016/j.vaccine.2012.05.048
Seder RA, Chang LJ, Enama ME, Zephir KL, Sarwar UN, Gordon IJ, Holman LA, James ER, Billingsley PF, Gunasekera A, Richman A, Chakravarty S, Manoj A, Velmurugan S, Li M, Ruben AJ, Li T, Eappen AG, Stafford RE, Plummer SH, Hendel CS, Novik L, Costner PJ, Mendoza FH, Saunders JG, Nason MC, Richardson JH, Murphy J, Davidson SA, Richie TL, Sedegah M, Sutamihardja A, Fahle GA, Lyke KE, Laurens MB, Roederer M, Tewari K, Epstein JE, Sim BK, Ledgerwood JE, Graham BS, Hoffman SL, the VRCST (2013) Protection against malaria by intravenous immunization with a nonreplicating sporozoite vaccine. Science. doi:10.1126/science.1241800
Stanford M, Whittall T, Bergmeier LA, Lindblad M, Lundin S, Shinnick T, Mizushima Y, Holmgren J, Lehner T (2004) Oral tolerization with peptide 336–351 linked to cholera toxin B subunit in preventing relapses of uveitis in Behcet's disease. Clin Exp Immunol 137(1):201–208. doi:10.1111/j.1365-2249.2004.02520.x
Sun JB, Xiang Z, Smith KG, Holmgren J (2013) Important role for FcgammaRIIB on B lymphocytes for mucosal antigen-induced tolerance and Foxp3+ Regulatory T Cells. J Immunol 191(8):4412–4422. doi:10.4049/jimmunol.1301324
Surzycki R, Greenham K, Kitayama K, Dibal F, Wagner R, Rochaix JD, Ajam T, Surzycki S (2009) Factors effecting expression of vaccines in microalgae. Biologicals 37(3):133–138. doi:10.1016/j.biologicals.2009.02.005
Tovey MG, Lallemand C (2010) Adjuvant activity of cytokines. Methods Mol Biol 626:287–309. doi:10.1007/978-1-60761-585-9_19
Tran M, Van C, Barrera DJ, Pettersson PL, Peinado CD, Bui J, Mayfield SP (2013) Production of unique immunotoxin cancer therapeutics in algal chloroplasts. Proc Natl Acad Sci U S A 110(1):E15–E22. doi:10.1073/pnas.1214638110
Uematsu S, Akira S (2009) Immune responses of TLR5(+) lamina propria dendritic cells in enterobacterial infection. J Gastroenterol 44(8):803–811. doi:10.1007/s00535-009-0094-y
Vignali DA, Kuchroo VK (2012) IL-12 family cytokines: immunological playmakers. Nat Immunol 13(8):722–728. doi:10.1038/ni.2366
World Health Organization (2012) World Malaria Report. World Health Organization, Geneva, Switzerland
Yamamoto M, Pascual DW, Kiyono H (2012) M cell-targeted mucosal vaccine strategies. Curr Top Microbiol Immunol 354:39–52. doi:10.1007/82_2011_134
Yarovinsky F, Zhang D, Andersen JF, Bannenberg GL, Serhan CN, Hayden MS, Hieny S, Sutterwala FS, Flavell RA, Ghosh S, Sher A (2005) TLR11 activation of dendritic cells by a protozoan profilin-like protein. Science 308(5728):1626–1629. doi:10.1126/science.1109893
Yusibov V, Streatfield SJ, Kushnir N (2011) Clinical development of plant-produced recombinant pharmaceuticals: vaccines, antibodies and beyond. Hum Vaccin 7(3):313–321
Zhan Y, Xu Y, Lew AM (2012) The regulation of the development and function of dendritic cell subsets by GM-CSF: more than a hematopoietic growth factor. Mol Immunol 52(1):30–37. doi:10.1016/j.molimm.2012.04.009
Zou L, Miles AP, Wang J, Stowers AW (2003) Expression of malaria transmission-blocking vaccine antigen Pfs25 in Pichia pastoris for use in human clinical trials. Vaccine 21(15):1650–1657
Acknowledgements
Support was provided by the San Diego Foundation, the California Energy Commission (500-10-039) and the National Science Foundation (CBET-1160184).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Gregory, J.A., Mayfield, S.P. Developing inexpensive malaria vaccines from plants and algae. Appl Microbiol Biotechnol 98, 1983–1990 (2014). https://doi.org/10.1007/s00253-013-5477-6
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-013-5477-6