Chloroplast-derived anthrax and other vaccine antigens: their immunogenic and immunoprotective properties

Review

10.1586/14760584.5.6.839 © 2006 Future Drugs Ltd ISSN 1476-0584 839www.future-drugs.com

Chloroplast-derived anthrax and other vaccine antigens: their immunogenic and immunoprotective propertiesSushama Kamarajugadda and Henry Daniell†

†Author for correspondenceDepartment of Molecular Biology and Microbiology, University of Central Florida, Bimolecular Science Building 20, room 336, Orlando, FL 32816–2364, USATel.: +1 407 823 0952Fax: +1 407 823 [email protected]

KEYWORDS: anthrax vaccine, chloroplast genetic engineering, genetically modified crops, immunization, mucosal immunity, oral delivery, protective antigens, systemic immunity, vaccines

Transgenic plants offer many advantages, including low cost of production (by elimination of fermenters), storage and transportation, heat stability, absence of human pathogens, protection of antigens in the stomach through bioencapsulation (when delivered orally), elimination of the need for expensive purification and sterile injections and generation of both systemic and mucosal immunity. Recent studies have demonstrated that chloroplast-derived anthrax-protective antigen elicits effective immune responses, develops neutralizing antibodies, confers complete protection against anthrax lethal toxin challenge and produces 360 million doses of vaccine in one acre of transgenic plants. Chloroplast-derived vaccine antigens are efficacious against bacterial, fungal, viral and protozoan pathogens.

Expert Rev. Vaccines 5(6), 839–849 (2006)

The concept of vaccination discovered byEdward Jenner in 1796 has helped mankind infighting against many infectious diseases. Wehave come a long way in global eradication ofdeadly diseases, such as small pox, polio andmeasles. The daunting challenge of fightingagainst emerging infectious diseases can bemet by further advancements in vaccinedevelopment. Although it has been 200 yearssince its discovery, vaccine developmentneeds continuous improvement. Many ele-ments must be considered for an effectivevaccine development. A vaccine must:

• Elicit protective immunity against aninfection;

• Be potent enough even at lower doses so thatit can be practical and affordable;

• Be safe and not cause any side effects;

• Be stable and retain its functional efficacystarting from production, transport, storageto the time of delivery into the host;

• Be able to elicit both humoral and cell medi-ated immunity depending on the typeof organism;

• Be able to elicit long term immune responsewith few booster doses;

• Be cost effective [1].

None of the current vaccines meet all ofthese criteria. The conventional method ofproducing vaccines using whole or partialcomponents of the organism, whether dead oralive, has raised concerns regarding their safetyand efficacy.

The need for larger populations at lowercosts demands alternative approaches for vac-cine production. For example, hundreds ofmillions of people living in developing coun-tries are infected with Hepatitis, but the dailyincome of a third of the world population isless than US$2 per day [2]. With the onset ofrecombinant gene technologies, the vaccineindustry has revolutionized the production ofvaccines in more effective expression systemsthat are safer, cheaper and provide protectionto the host organisms against bacterial, viral orother pathogens [1]. Among the various expres-sion systems, plants are increasingly recognizedas a safe and inexpensive system for the

CONTENTS

Chloroplast-derived bacterial vaccine antigens

Chloroplast-derived viral vaccine antigens

Chloroplast-derived protozoan vaccine antigens

Expert commentary & five-year view

Key issues

References

Affiliations

For reprint orders, please contact:[email protected]

Kamarajugadda & Daniell

840 Expert Rev. Vaccines 5(6), (2006)

production of recombinant proteins and antigens. The produc-tion of recombinant vaccine antigens in plants has manyunique advantages, such as:

• The culture and the processing technology of plants isalready available, which is straightforward and cost-effectivewhen compared with fermentation processes;

• Plant system is much safer to human health since it mini-mizes the risks arising from the contamination with humanpathogens or toxins;

• Purification processes can be avoided if the plant tissue withthe recombinant protein is used for oral delivery;

• Plant-based vaccines can be targeted into the intracellularcompartments or expressed directly in certain compartments,such as chloroplasts, where they are more stable;

• Plant-based vaccines used for oral delivery can be used toinduce mucosal immune response against pathogens thatcolonize mucosal surfaces [3].

In general, three approaches have been used for the expres-sion of vaccine antigens in plants: stable expression via nuclearor chloroplast genomes or transient expression via recombinantplant viral sequences. Expression levels are often low and varygreatly in stable nuclear transgenic plants, and transmission offoreign genes via pollen are major concerns. In the case of viralsystems, larger proteins are not highly expressed; therefore, afew epitopes of a vaccine antigen are generally expressed,instead of the full-length antigen [4,5]. Nuclear transformation ismost commonly achieved by using a modified plant pathogen,Agrobacterium tumefaciens, that can target the foreign DNA tothe nucleus, causing chromosomal integration at random sitesin the plant cells [4].

Stable genetic transformation of the plants with higherexpression levels of the foreign antigens is often achieved bychloroplast transformation. Stable integration of a foreign geneinto the chloroplast genome results in accumulation of excep-tionally high levels of recombinant proteins up to 46% of thetotal leaf protein [6] or 500–4000-fold more vaccine antigensthan expression of the same vaccine antigens via the nucleargenome [7]. Apart from higher expression levels of foreign pro-teins, chloroplast transformation has several other uniqueadvantages [8]. Nuclear integration of foreign genes is randomand occurs by illegitimate recombination, resulting in variablelevels of transgene expression (position effect) and foreign tran-scripts are often recognized and silenced [9]. The integration ofa transgene into a chloroplast genome is site specific by homol-ogous recombination, thus eliminating the ‘position effects’. Todate, gene silencing has not been observed in transgenic chloro-plasts, in spite of expression of foreign proteins up to 47% ofthe total leaf protein [6] or accumulation transcripts 150-foldhigher than nuclear transcripts [10,11]. Chloroplast transforma-tion eliminates the risk of transgene escape via pollen owingto the maternal inheritance of the plastids [12] and, thus, offersthe advantage of gene containment [13]. In addition, cytoplas-mic male sterility engineered via the chloroplast genomeoffers yet another tool to eliminate transmission of foreign

genes via pollen [14]. Multigene engineering is achieved bychloroplast transformation owing to the ability of chloroplaststo process the polycistrons [15,16], in contrast to translation ofmonocistrons via the nuclear genome. This should facilitate theproduction of multicomponent vaccine antigens. Also, thechloroplast genome has been used in molecular farming toexpress many therapeutic proteins [17], such as human serumalbumin [18], interferons [19] and human somatotropin [20] withcorrect folding and formation of disulfide bonds [21,22]. Expres-sion of vaccine antigens within the chloroplast are not toxic,even at high expression levels, whereas even very low accumula-tion levels are toxic in the cytosol [23]; oral delivery of vaccinesinduces high mucosal and systemic titer values and helps incombating the disease response at their portals of entry.

The main objective of this review is to provide an overviewon the recently developed chloroplast-derived anthrax vaccine,the exceptional production capabilities (360–400 million dosesper acre), the high levels of immnunity observed in mice andthe survival of immunized mice after high levels of lethalanthrax toxin challenge. In addition, an overview of recentadvancements in other chloroplast-derived vaccine antigens isprovided (TABLE 1).

Chloroplast-derived bacterial vaccine antigensAnthrax vaccineBacillus anthracis, a gram-positive spore-forming organism, islisted as a category A biological agent due to severity of infection,leading to death, acuteness of disease and its adverse impact onhuman health. The terrorist attacks on the USA on September11th, 2001, the mailing of letters containing anthrax spores andits aftermath had created a terror among US citizens, leading to agreater responsibility on the scientific community for the devel-opment of an effective vaccine. Bacillus anthracis carries pagA, lefand cya genes coding for the protective antigen (PA), lethal factor(LF) and edema factor (EF), respectively. Together, these proteinsform toxic components, whereas PA alone is considered to behighly immunogenic and a key component in vaccine produc-tion [24,25]. Apart from using different expression systems, such asbacteria and yeast, alternative approaches, such as gene ther-apy/DNA vaccine technology [26], have been used for the success-ful development of anthrax vaccines. Although the effortsresulted in reasonable outcome, the methodologies used werevery tedious, expensive, demanded high technical know-how,purification and fermentation facilities. In order to overcomethese disadvantages, plants were used as safe and ideal expressionsystems. Transgenic PA lines were produced in tobacco [27] andtomato [28] by nuclear transformation, confirming that plant-expressing PA antigen is similar to rPA of Bacillus anthracis.However, the low expression level of recombinant PA (rPA) was amajor concern in this approach.

The enhanced expression levels of the recombinant protein inchloroplasts has enabled the production of the PA in tobaccoplastids. The pag gene, along with light-regulated psbA regula-tory sequences, was integrated into the chloroplast genomeusing trnI/trnA-flanking sequences. The yield from transgenic

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lines was approximately 172 mg of PA from each plant with92.7% contribution from mature leaves [29]. The PA proteinlevels were approxmately 2.7% of total soluble protein (TSP)from the mature leaves and, under continuous light for 3–5days, a maximum of 18.1% of TSP was observed in transgenicplants. It has been reported that 2.5 mg of PA per gram offresh leaf and 172 mg of PA per plant should yield 400 milliondoses of vaccine per acre of land [29]. A full length 83 kDa PAwas observed, indicating stability and intactness of the PAwithin transgenic chloroplasts. The biological function of PA,along with lethal toxin, was shown by cytolytic activity ofmacrophage cell lines.

In a follow-up study, PA with a His tag was expressed intransgenic chloroplasts and further characterization of the PAwas performed [30]. PA expression was confirmed by immunoblotassays, which demonstrated that the antigen was highly stable.The yield from transgenic lines was approximately 150 mg of PAfrom a single plant and the overall PA protein levels were approx-imately 4.5% of TSP under normal light conditions, whereasunder 5-day continuous illumination, it reached a maximumlevel of 14.2% of TSP. At this expression level, 360 million dosesof anthrax vaccine could be obtained from 1 acre of land, whichis sufficient for the entire US population [30]. The biologicalfunctions of chloroplast-derived PA in crude extracts and in par-tially purified forms were tested in vitro using the macrophagelysis assay, which confirmed that PA in transgenic chloroplastswas fully active (FIGURE 1). The immunization studies of micewith partially purified and the crude extracts of transgenic PAplants were performed in several groups of mice, injecting subcu-taneously with 5 µg of antigen four times on days 0, 14, 28 and140, along with an alhydrogel adjuvant. The chloroplast-derivedpartially purified PA and B. anthracis-derived PA demonstrated

similar titer values of 1:300,000, whereas, without adjuvant, thetiter values were 1:10,000–1:40,000 and 1:80,000–1:160,000,respectively (FIGURE 2). The sera from the immunized mice weretested for their ability to neutralize PA and protect the macro-phage lysis from labile toxin (LT) in toxin neutralization assays(TNA). The TNA for chloroplast-derived PA and B. anthracis-derived PA with adjuvant were similar for the sera collected afterthe third dose, whereas some differences were observed after thefourth dose. When mice were subjected to a toxin challenge with1.5-times the 100% lethal toxin, all the animals immunized witheither source of recombinant PA plus adjuvant survived the chal-lenge (100%), confirming the immunoprotective and immuno-genic properties of the plant-derived anthrax vaccine (FIGURE 3).Thus, high expression levels of PA without using fermenters, freefrom edema and lethal toxins holds promise for the developmentof a cleaner and safer anthrax vaccine.

Cholera toxin B subunit vaccineCholera is a disease that causes acute watery diarrhea by coloniza-tion of Vibrio cholerae in the small intestine and produces theenterotoxin, cholera toxin (CT). Heat labile toxin (LT) is anotherform of enterotoxin produced by E. coli and is very much similarto CT, both immunologically and physichochemically. Both LTand CT are pentameric proteins consisting of a 27 kDa A subu-nit, which is toxic and consists of ADP ribosyl transferase activ-ity. The nontoxic 11.6 kDa B subunit binds to the A subunit andfacilitates its entry into intestinal epithelial cells. CT-B is consid-ered to be a potential vaccine candidate, since, when adminis-tered orally, it elicits strong mucosal immunity by binding toeukaryotic cell surfaces via GM1 receptors (lipid-base membranereceptor) present on the intestinal epithelial surfaces [23] and alsoenhances the immune response when administered together with

Table 1. Chloroplast-derived vaccine antigens.

Vaccine name Protein/peptide used

% of TSP Animal studies Ref.

Anthrax PA(Protective antigen)

4.5– 18.1% Systemic immune response – anti-PA IgG titer values up to 1:300,000; 100% survival after lethal anthrax toxin challenge

[29,30]

Cholera toxin CTB CTB-GFP

4.1% (CTB)19.09–21.3 %(CTB-GFP)

Receptor-mediated oral delivery of GFP using CTB as transmucosal carrier; GM1 binding

[23,38]

Canine parvovirus

2L21 peptide 31.1 %( CTB-2L21)22.6 %( GFP-2L21)

Systemic and mucosal immune response [37,49]

Plague F1/V fusion 14.8% Not yet tested [21]

Rotavirus CTB-NSP4VP6

2.5%0.06%

Not yet testedNot yet tested

[21,58]

Amebiasis Gal/GalNAc LecA 6.3% Systemic immune response – anti-LecA IgG titers up to 1:10,000

[61]

Tetanus toxin Tet C (native & synthetic)

10–25% Systemic immune response [68]

TSP: Total soluble protein.

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other antigens [31,32]. Therefore, CT-B was expressed at differentlevels in transgenic plants via nuclear transformation. To ensureproper folding, a C-terminal SEKDEL sequence was introducedto the CT-B gene, targeting the protein to the endoplasmic reticu-lum, which increased the CT-B expression to 0.3% of TSP inauxin-induced potato tubers [33]. However, even at such lowexpression levels, the plant-based CT-B vaccine antigen proved tobe an efficient vaccine candidate in eliciting the immune response.LT-B (synthetic gene) expressed in potatoes was protective againstE. coli heat-labile enterotoxin in mice upon oral delivery [34] andwas immunogenic in humans clinical trials [35]. These studies havebeen extended to corn. In these trials, volunteers were fed withtransgenic corn containing 1 mg of LT-B in three doses, each con-sisting of 2.1 g of plant material. In total, 78% of patients devel-oped immunoglobulin (Ig)G and 44% of volunteers developedIgA in stools [36]. These studies demonstrate that even at lowexpression levels, CT-B/LT-B are highly immunogenic. In order toincrease expression levels, CT-B and several fusion proteins wereintegrated into the chloroplast genome. Expression levels varieddepending upon the regulatory sequences used or the fusion pro-tein, such as 4.1% of TSP with CT-B [23], 31.1% of TSP withCT-B–canine parpovirus (CPV) [37], 19.09–21.3% of TSP withCT-B–green fluorescent protein (GFP) [38]. The GM1 bindingassays confirmed the functionality of chloroplast-derived CT-B,although the protective capacity of all these antigens has not yet

been tested, except for the CT-B-CPV. LT-B has also been expressed via the tobaccochloroplast genome, with a recordedexpression level of 2.5% TSP [39]. Unlikenuclear-expressed LT-B plants, no signifi-cant pleiotropic changes were observed inthe chloroplast-derived transgenic plants.

Oral delivery using CT-B as a transmucosal carrierSsuccessful expression of CT-B in chloro-plasts facilitated oral delivery studies, as didits use as a transmucosal carrier in deliveringforeign proteins orally to enhance mucosalimmunity or induce oral tolerance [33]. Inorder to study the receptor-mediated trans-mucosal carrier function of CT-B in deliver-ing foreign antigens, the CT-B gene wasfused with GFP with a furin cleavage sitebetween the two proteins. The construct wasexpressed via the chloroplast genome to pro-duce transgenic plants [38]. The transgenicCT-B–GFP plants showed expression levelsranging from 19.09 to 21.3% of TSP. Themice were orally fed with leaf materials ofCT-B–GFP, interferon (IFN)α5–GFP andwild-type plants. Both fusion proteins con-tain a furin cleavage site between the twoproteins. Experimental investigations con-ducted by performing immunohistochemis-

try on intestine, liver and spleen tissues to locate GFP and/orCT-B, and fluorescence microscopic tests for GFP in varioustissues, suggested that:

• CT-B–GFP was bound to the intestinal mucosal epitheliumand also the lymphoid tissue via GM1 receptors;

• As it passed across the intestinal mucosa, GFP was cleavedfrom CT-B in the intestine by the action of a furin protease;

• GFP alone was found in the liver and spleen, with no tracesof CT-B in either of these tissues, confirming the cleavage ofGFP from CT-B by furin in the intestine;

• No significant amount of GFP was observed in either liver orspleen of chloroplast-derived IFNα5–GFP-fed mice, confirmingthat a transmucosal carrier (e.g., CT-B) was required for efficientdelivery of foreign protein across intestinal lumen (FIGURE 4).

This study demonstrates the internalization of CT-B–GFPby the mouse intestinal mucosal cells as well as the antigen-presenting cells in the intestinal mucosa and submucosa. Wealso showed the presence of GFP but not CT-B in the liver ofmice following oral delivery of CT-B–GFP leaf material.Detection of both CT-B and GFP in mouse intestinal cells fol-lowing oral administration of CT-B-GFP expressing leaf mate-rial demonstrates that the recombinant protein has been pro-tected from peptidases and/or acids by bioencapsulationwithin the plant cells. The ability to express high levels of

Figure 1. Functional analysis of PA with macrophage cytotoxicity assay. The cytotoxicities of various PA preparations for mouse macrophage RAW264.7 cells were assayed in the presence of LF. Samples that were diluted serially were as follows: crude extract of plant leaves expressing PA with His tag, wild-type (WT) plant leaf crude extract, 20-µg/ml stock of purified chloroplast-derived PA, 20-µg/ml stock of purified PA derived from B.acillus anthracis, and plant protein extraction buffer. The cytotoxicities of various PA preparations for mouse macrophage RAW264.7 cells were assayed in the presence of LF. Samples that were diluted serially were as follows: crude extract of plant leaves expressing PA with His tag, wild-type (WT) plant leaf crude extract, 20-µg/ml stock of purified chloroplast-derived PA, 20-µg/ml stock of purified PA derived from B.acillus anthracis, and plant protein extraction buffer.Figure from Koya et al., 2005 (permission granted by ASM) [30].

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foreign proteins in plastids present within edible plant parts[40,41,42] and the rapid turnover of intestinal epithelial cells [43]

for recycling GM1 receptors, make this approach a reality. Thisstudy opens the door for further studies in delivery of humantherapeutic proteins.

Plague vaccinePlague is a bacterial disease caused byYersinia pestis, a gram-negative bacteria.It has three different forms: bubonic,septicemic and pneumoni. Of these, thebubonic form is the most common andis caused by infected fleas. The bacte-rium enters into the lymph nodes andcauses swelling of the nodes, formingbubos [44].

Several groups have been working on theproduction of a plague vaccine and the cur-rently available vaccine for plague is a killedwhole vaccine, which is moderately effectiveagainst the bubonic form and ineffectiveagainst other forms. CaF1 and LcrV areconsidered to be the most effective antigensfor Y. pestis. F1 is a capsular protein presenton the surface of bacterium with antiphago-cytic properties and V is a part of the Y. pes-tis type III secretion system and may formpart of the injectosome. Owing to the highimmunogenic nature of the F1/V antigen, ithas been expressed in tomato by nucleartransformation and its immunogenicity wasconfirmed by priming subcutaneously withbacterially expressed F1-V antigen and

boosted orally with transgenic tomatoes. These experiments dem-onstrated an IgG response in all the immunized mice, whereas anIgA response was observed in two out of six mice [45]. Anothergroup has expressed F1-V antigen in tobacco leaves transientlyusing tobacco mosaic virus and reported a systemic immune

response in guinea pigs [45]. The F1/V antigen has also been

expressed via the chloroplast genome andthe expression levels of F1/V fusion pro-tein was approximately 14.8% of TSP [21].Such high expression levels of F1/V trans-genic plants should help in inducinghigher titer values when immunizationstudies are carried out.

Chloroplast-derived viral vaccine antigensCPV vaccineCPV is a viral disease in dogs, causing acutegastroenteritis and myocarditis. CPV is asingle-stranded DNA virus with the capsidprotein formed by various other compo-nents. The 2L21 peptide is a 21 amino acidslong, B cell epitope and is the amino termi-nus region of the VP2 protein (capsid pro-tein of CPV). The 2L21 peptide coupling asa hapten is important to induce humoralresponse and its coupling with KLH has

Figure 2. IgG antibody titers and toxin neutralization assay titers in serum samples obtained from mice after third and fourth doses. Comparison of immune responses in serum samples of mice administered subcutaneously with chloroplast-derived PA (CpPA) with adjuvant (column 1), chloroplast-derived PA (CpPA) alone (column 2), Std-PA derived from Bacillus anthracis with adjuvant (column 3), Std-PA alone (column 4), PA plant leaf crude extract with adjuvant (column 5), wild-type plant leaf crude extract with adjuvant (column 6) and unimmunized mice (column 7). Figure from Koya et al., 2005 (permission granted by ASM) [30].PA: Protective antigen

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Figure 3. Toxin challenge of the mice with systemic anthrax lethal toxin. Shown is survival over time for different groups of mice after challenge with a 150-µg dose of lethal toxin. ADJ: Adjuvant; CHLPST: Chloroplast; IP: Intraperitoneal; LT: Labile enterotoxin; PA: Protective antigen; PBS: Phosphate buffered saline; WT: Wild type.Fig from Koya et al., 2005 (permission granted by ASM) [30]..

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provided protection to dogs against this virus [46]. The 2L21 pep-tide was expressed in various plant viruses eliciting CPV-neutraliz-ing antibodies in mice [47]. It has also been expressed in Arabidop-sis via nuclear transformation as an N-terminal fusion withβ-glucuronidase (GUS) [48].

In order to increase the expression levels, the 2L21 peptidewas expressed via the chloroplast genome as a fusion proteinwith CT-B and GFP, respectively. CT-B was used as an adju-vant to enhance the immune response and GFP was used as areporter gene for an easy visible selection of transgenic plants[37]. Transgenic plants obtained from the two constructs of CT-B–CPV and GFP–CPV showed expression levels of approxi-mately 31.1 and 22.6% of TSP, respectively. Mice immunizedwith transgenic lines by the intraperitoneal route elicited anti-2L21 antibodies with the titer values ranging from 200 to25000 for CT-B–CPV and very low titers were observed withGFP–CPV. These results confirmed the ability of CT-B toenhance the immune response against fused peptides.

A further investigation was carried out with the same trans-genic lines to study the immune responses in mice by per-forming parenteral, oral and combined immunizations [49].Mice immunized with leaf crude extracts of CT-B–2l21 bythe parenteral route induced higher titers of antibodiesagainst 2L21 and VP2, whereas mice immunized withGFP–2L21 crude extracts could not induce significant titers.The antibodies induced in rabbit by chloroplast-derivedCT-B–2L21 were able to efficiently neutralize the CPV infec-tion in Crandell feline kidney (CRFK) cell lines. The pulver-ized CT-B–2L21 leaf tissues were able to induce anti-2L21IgG and IgA, when administered through oral routes,whereas, in the combined parenteral and oral dosages, the oralboosters helped to maintain the anti-2L21 IgG response aftersingle injection and the parenteral administration of CT-B-2L21 primed the subsequent oral boosters to induce andmaintain anti-2L21 IgA antibodies in mice. Although theinduced humoral response could demonstrate neutralizationactivity, the orally delivered mice could not induce the neu-tralization activity, which suggests that more effort is neces-sary for a proper protection against orally delivered antigens.For a proper mucosal immunity, immunoregulation should beexploited to enhance activity [50].

Rotavirus vaccineRotaviruses are the important causes of viral-based diarrhealillness in infants and children aged under 3 years. Theyaccount for 6% of the deaths among young children in devel-oping countries. The nucleocapsids of rotaviruses are com-posed of three concentric layers of proteins, enclosing 11 seg-ments of double-stranded RNA. VP7 and VP4 are theproteins present on the outermost capsid and are consideredas potential targets for neutralizing antibodies. The VP6 pro-tein is present on the intermediate layer of the capsid, andantibodies against it have a critical role in inducing protectionagainst rotavirus infection [51]. Therefore, the proteinsexpressed on the three layers of capsids are potential candi-dates for vaccine production and have been expressed via thenuclear transformation in plants such as tomato [52], potato[53–56] and alfalfa [51]. The immunization studies performed bythese groups independently in mice demonstrated measurabletiters of serum IgG and mucosal IgA. In spite of a reasonable

Figure 4. Immunohistochemistry of ileum, liver and spleen tissues of mice fed with CTB-GFP expressing leaves or IFN-GFP expressing leaves or wild-type leaves. (A) Shows a section of the intestine of a CTB-GFP treated mouse. The arrows indicate CTB in the submucosa of the intestinal villi. (B) Shows a section of mouse ileum fed with wild-type plant, immunostained for CTB. (C-F) Double staining for macrophage (red) and CTB (green) in mouse intestine and liver. (C) Arrows show macrophages in the submucosa of the intestine containing CTB, in a mouse fed with CTB-GFP expressing plant leaf material. The merged color is yellow. (D) Arrows indicate F4/80-positive cells (macrophages, in red) in a merged picture in the intestine of a mouse fed with WT leaf material. (E) A merged picture showing double staining for macrophage (Kupffer cells) and CTB in mouse liver. Arrows show macrophages (red) in the liver. No sign of CTB (green) was found in the liver of CTB-GFP fed mouse. (F) Liver section of an IFN-GFP fed mouse used as a negative control for CTB. Macrophages are seen in red. (G) F4/80 Ab was used as a marker of macrophages in the intestine. Arrows indicate macrophages, which have entrapped GFP (yellow after merging the red and the green). Many of the macrophages are not associated with GFP. (H) Many macrophages are seen in the intestine of mouse fed with IFN-GFP expressing plant leaf material, which do not show GFP immunoreactivity.Figure from Limaye et al., 2006 (permission granted by FASEB Journal) [38].CTB: Cholera-toxin B; GFP: Green fluorescent protein; IFN: Interferon: WT: Wild-type.

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immune response in mice, the low expression level of the anti-gen in transgenic plants is the major constraint in extendingthe study to nonprimates and humans.

The nonstructural protein (NSP4) encoded by 11 seg-ments of double-stranded RNA of rotavirus, is also consid-ered as a potential vaccine candidate in inducing humoraland cellular responses. The CT-B–NSP4 fusion protein hasbeen expressed via nuclear transformation in potato and itsassembly into biologically active oligomers in transgenicpotato demonstrated the feasibility of developing edibleplants for the synthesis of fully functional rotavirus entero-toxin antigens [57].

In order to increase the expression levels, the rotavirus VP6gene is inserted with prrn or psbA promoters, between therbcL and accD genes of the tobacco plastid genome [58]. Theoverall expression level of the VP6 protein in transgenic plantswas low (0.6% TSP) and was undetectable using trc promoter.The CT-B–NSP4 fusion protein has also been expressed viachloroplast transformation in tobacco, although the gene ofinterest is inserted with prrn and psbA promoters, between thetrnI and trnA genes of the chloroplast genome and thereported expression levels of CT-B–NSP4 in transgenictobacco plants is 2.5% of TSP [21]. Therefore, such highexpression levels of VP6/CT-B–NSP4 in transgenic plantscompared with other reports and synthesis of biologicallyactive rotavirus antigens may provide promising immuneresponses, when immunization studies are conducted.

Chloroplast-derived protozoan vaccine antigensAmebiasis vaccineAmebiasis is caused by Entamoeba histolytica, a protozoan para-site that causes diarrhea in many developing countries. It rankssecond to malarial diseases in causing death. E. histolytica causesdamage to the human tissues and infection is initiated by thecystic form of the pathogen, which is highly resistant to gastricacidity and chlorination and survives well in moist environ-ments. Motile trophozoites are released from the bowel lumen,which adhere to colonic mucins and colonize in the largeintestine with the help of galactose and N-acetyl-D-galactos-amine (Gal/GalNAc)-specific lectin. The proteolytic enzymessecreted by trophozoites disrupt the intestinal epithelial layerand facilitate deeper penetration of the pathogen into the tis-sues and, ultimately, lead to ulcers in the respective tissues.Finally, the parasite survives host immune response and leadsto a prolonged intestinal infection called amoebic liverabscesses [59]. The carbohydrate recognition domain (CRD)was identified in the heavy subunit of Gal/GalNAc lectin,which is a potential candidate for blocking colonization of thispathogen. It has been shown that the recombinant fragmentsof the cysteine-rich region of lectin (termed as LecA) contain-ing CRD of the Gal/GalNAc lectin provides protectionagainst amebiasis [60].

In order to produce an efficient, low-cost vaccine againstamoebiasis, the LecA gene was integrated into the tobacco chlo-roplast genome with an expression level up to 6.3% of TSP [61].

Table 2. List of important disease outbreaks in the last 5 years and their vaccine availability.

Disease Year of outbreak Vaccine availability Affected countries

Avian influenza virus/bird flu 2002,2003,2004,2005,2006 Effective vaccine candidate is required. Asia

Cholera 2002,2003,2004,2005 and 2006 Vaccines are being developed with most of them under clinical trials

West Africa, South Africa, Africa, Afghanistan

Dengue fever 2002,2003,2004 Vaccine are being developed Indonesia,India,Brazil

Ebola haemorrhagic fever 2002,2003,2004,2005,2006 Vaccine is required African countries

Hepatitis E 2004 Vaccines are under development Sudan and Chad

HIV Reports almost every year Vaccines are under under development Developing and under-developed countries

Malaria Reports almost every year Vaccines are under develoment Asia,Africa and Latin America

Meningitis 2002,2003,2004,2005,2006 Vaccines are under development Africa and Asia

Plague 2003,2005 Vaccines are under development Africa and Asia

Poliomylitis 2003,2005,2006 Vaccines under development Mostly African countries and some parts of China

Severe acute respiratory syndrome (SARS)

2003,2004 Vaccines under development Asian countries like China, Taiwan, Singapore, Toronto

Typhoid 2003,2004,2005 Vaccines under development Republican countries of Cango, Haiti

Yellow fever 2003,2004,2005 Vaccine is required African countries

Data from [101].

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The crude extracts of LecA with and without aldhydrogel adju-vant were injected subcutaneously into different groups ofmice and the immunization results suggested that the miceimmunized with transgenic LecA crude extracts with adjuvantshowed titers up to 1:10000. The recent report on the clinicaltrials of amebic vaccine antigen with CT-B observed a partialprotection against recurrent intestinal infection, but could notprovide protection after amebic liver abcess [62]. Since a chlo-roplast-derived LecA vaccine antigen could induce high anti-body titers in mice, this antigen may be targeted as a potentialvaccine candidate for human clinical trials.

Expert commentary & five-year viewOne of the most challenging problems of human health man-agement is the high cost of prescription drugs in developedcountries and their lack of availability in developing countries.Such high cost of therapeutic proteins can be attributed to theirproduction in fermentation-based systems, expensive purifica-tion and processing methods, low temperature storage, trans-portation and sterile delivery using syringes by health profes-sionals. Most of these expenses could be avoided by expressingtherapeutic proteins in plant cells, thus allowing oral delivery.

The ability to express several vaccine antigens in chloroplastshas been well established. Many of these antigens (e.g., CT-B)require post-translational modifications, including formationof disulfide bonds. Fully functional human blood proteins with2–17 disulfide bonds, including human serum albumin [18],interferon [19], somatotropin [20] and vaccine antigens [37,38,49]

have already been expressed in transgenic chloroplasts, withappropriate post-translational modifications, including lipidmodifications (OspA lipoprotein) [63]. However, chloroplastsare glycosylation-free zones and, to date, no glycoproteins havebeen expressed in chloroplast, and this is one of the limitationsof this system. The efficacy of several chloroplast-derived vac-cine antigens against bacterial, viral and protozoan pathogenshas also been demonstrated through preclinical studies.

Another limitation of the chloroplast-expression system isthat the trangenes should be integrated into all 10,000 copiesof the chloroplast genomes in each plant cell to achievehomoplasmy, eliminating all native chloroplast genomes. Thisrequires several rounds of selection and at least 25–30 celldivisions under heavy selection. Such an efficient system hasbeen very well worked for tobacco, a nonfood and nonfeedcrop. While this is ideal for production of therapeutic pro-teins that require purification, chloroplast transformation sys-tems should be established for edible crops to facilitate theoral delivery of therapeutic proteins. Therefore, the next chal-lenge is to extend the chloroplast technology to edible cropspecies to facilitate oral delivery. Successful transformation ofchloroplast genomes in carrot [40] and lettuce [41,42] are quitepromising and vaccine antigens should be expressed in thesesystems for oral delivery. The recent reports on completechloroplast genomic analysis of different edible crop species,such as grape [64], potato and tomato [65], soybean [66] and car-rot [67], should provide the basic understanding of the chloro-plast genome in different species, which may help in design-ing appropriate vectors with endogenous regulatory sequencesand optimize the regeneration protocols for these crops.

Several vaccine antigens have been expressed using chloro-plasts as expression systems. However, these studies should beextended to nonhuman primates and, ultimately, to humanclinical trials. The recent reports of the WHO on outbreaks ofavian influenza virus, yellow fever and poliomyelitis, amongothers, in the past 5 years show them to be devastating anddetrimental [101]. Although the diseases are being monitored,no effective vaccine is available to prevent them. Avian influ-enza virus is not only infecting birds but is also spreading dis-ease among humans, causing great concern. Therefore, thereis an urgent need for the development of vaccines for suchemerging diseases, which cause major losses to mankind andanimals (TABLES 2 & 3). The concept of chloroplast technologyin producing vaccine antigens should be exploited further to

Table 3. List of diseases causing death and their vaccine availability as per DALY.

Serial No Disease % of people effected worldwide according to DALY

Vaccine availability

1 HIV/AIDS 1.36 Vaccine in development

2 Amebiasis 0.99 Vaccine in development

3 Cholera 0.99 Vaccine in development

4 Rotavirus 0.99 Vaccine in development

5 Malaria 0.74 Vaccine in development

6 Tuberculosis 0.55 BCG vaccine available

7 Neonatal tetanus 0.1 Vaccine in development

8 Filaria 0.09 Vaccine in development

9 Kala Azar-Leishmaniasis 0.03 Vaccine in development

DALY: Disability adjusted life years = sum of the years lost due to premature death and years lost due to disability.

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develop less expensive vaccines for all of these diseases. Thiswill provide a new hope for people in developing nations in

the fight against emerging infectious diseases or bioterrorismagents in developed and developing nations.

Key issues

• Chloroplast-derived anthrax vaccine showed exceptionally high capacity, producing 360 million doses of antigen per acre of tobacco, which is sufficient for the entire US population. Transgenic plants offer a low cost of production by the elimination of expensive fermenters.

• Anthrax protective antigen expressed in chloroplasts is functionally similar to PA of Bacillus anthracis, in both in vitro and in vivo studies, but it is cleaner than current vaccines (free of toxins). Absence of human pathogens in plants is a major advantage.

• Several chloroplast-derived vaccine antigens have been shown to be efficacious against bacterial, viral and protozoan pathogens.

• The receptor-mediated oral delivery of the chloroplast-derived cholera toxin (CT)-B–green fluorescent protein (GFP) to target tissues in mice via the circulatory system shows the feasibility of oral delivery of various therapeutic proteins in humans. Antigens or therapeutic proteins are protected in the stomach from acids and digestive enzymes through bioencapsulation in plant cells. Most importantly, the need for expensive purification, cold storage and transportation is eliminated. Both systemic and mucosal immunity can be achieved through oral delivery.

• This concept and the technology should be extended to edible crop species, such as tomato, carrot and lettuce, to facilitate the oral delivery of therapeutic proteins.

• Chloroplast-derived vaccine antigens should be tested in nonhuman primates and in human clinical trials.

ReferencesPapers of special note have been highlighted as:• of interest•• of considerable interest

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3 Daniell H, Streatfield SJ, Wycoff K. Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci. 6, 219–226 (2001).

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•• Reports the highest expression level of any foreign protein ever expressed in tranasgenic plants and the first

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•• Reports expression of a fully functional human blood protein in transgenic chloroplasts with proper folding and disulfide bonds.

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•• First report of a vaccine antigen expressed in transgenic chloroplasts. Formation of disulfide bonds and assembly of pentamers were demonstrated by polyacrylamide gel electrophoresis (PAGE) and GM1 binding assays.

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•• First report of a functional vaccine antigen against a pathogen used in bioterrorism. Reports high expression levels of anthrax protective antigen, confirms its biological functionality in in vitro assays and also shows the intactness and stability of full length PA protein within chloroplasts.

30 Koya V, Moayeri M, Leppla SH, Daniell H. Plant-based vaccine: mice immunized with chloroplast-derived anthrax protective antigen survive anthrax lethal toxin challenge. Infect. Immun. 73(12), 8266–8274 (2005).

•• Reports exceptionally high expression levels of chloroplast-derived anthrax protective antigen and immunogenic and immunoprotective properties in immunized mice. This article has been highlighted by numerous articles in the public press and broadcasting stations, including the Discovery channel and scientific journals.

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•• First report of oral tolerance from a plant-derived autoantigen.

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35 Tacket CO, Mason HS, Losonsky G, Clements JD, Levine MM, Arntzen CJ. Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato. Nat. Med. 4, 607–609.

•• First report of human clinical trials conducted using a plant-derived vaccine antigen.

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37 Molina A, Hervás-Stubbs S, Daniell H, Mingo-Castel AM, Veramendi A. High-yield expression of a viral peptide animal vaccine in transgenic tobacco chloroplasts. Plant Biotechnol. J. 2(2), 141–153 (2004).

•• First report of animal vaccine via the chloroplast genome, confirming expression and immunogenic properties in mice.

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•• First demonstration of the transmucosal activity of plant-derived CTB in delivering a foreign protein to target tissues via the circulatory system in orally fed mice and confirmation of protection of recombinant proteins from proteases/pepdidases by bioencapsulation within plant cells.

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•• First chloroplast transformation via somatic embryogenesis using nongreen tissues for the expression of recombinant proteins.

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Website

101 World Health Organization www.who.int/en/

Affiliations

• Sushama Kamarajugadda, MS,

Graduate student, Department of Molecular Biology and Microbiology, University of Central Florida, Bimolecular science Building 20, room 336, Orlando, FL 32816–2364, USATel.: +1 407 823 0952Fax: +1 407 823 0956

• Henry Daniell, PhD,

Pegasus Professor and Trustee Chair, Department of Molecular Biology and Microbiology, University of Central Florida, Bimolecular Science Building 20, room 336, Orlando, FL 32816–2364, USATel.: +1 407 823 0952Fax: +1 407 823 [email protected]