Plant-Based Vaccine: Mice Immunized with Chloroplast-Derived Anthrax Protective Antigen Survive Anthrax Lethal Toxin Challenge
http://www.100md.com
感染与免疫杂志 2005年第12期
Department of Molecular Biology and Microbiology, University of Central Florida, Biomolecular Science Building 20, Room 336, Orlando, Florida 32816-2364
Microbial Pathogenesis Section, National Institute of Allergy and Infectious Diseases, NIH, Building 30, Room 303, 30 Convent Dr., Bethesda, Maryland 20892-4349
ABSTRACT
The currently available human vaccine for anthrax, derived from the culture supernatant of Bacillus anthracis, contains the protective antigen (PA) and traces of the lethal and edema factors, which may contribute to adverse side effects associated with this vaccine. Therefore, an effective expression system that can provide a clean, safe, and efficacious vaccine is required. In an effort to produce anthrax vaccine in large quantities and free of extraneous bacterial contaminants, PA was expressed in transgenic tobacco chloroplasts by inserting the pagA gene into the chloroplast genome. Chloroplast integration of the pagA gene was confirmed by PCR and Southern analysis. Mature leaves grown under continuous illumination contained PA as up to 14.2% of the total soluble protein. Cytotoxicity measurements in macrophage lysis assays showed that chloroplast-derived PA was equal in potency to PA produced in B. anthracis. Subcutaneous immunization of mice with partially purified chloroplast-derived or B. anthracis-derived PA with adjuvant yielded immunoglobulin G titers up to 1:320,000, and both groups of mice survived (100%) challenge with lethal doses of toxin. An average yield of about 150 mg of PA per plant should produce 360 million doses of a purified vaccine free of bacterial toxins edema factor and lethal factor from 1 acre of land. Such high expression levels without using fermenters and the immunoprotection offered by the chloroplast-derived PA should facilitate development of a cleaner and safer anthrax vaccine at a lower production cost. These results demonstrate the immunogenic and immunoprotective properties of plant-derived anthrax vaccine antigen.
INTRODUCTION
Anthrax, a fatal bacterial infection, is caused by Bacillus anthracis, a gram-positive spore-forming organism. It is a zoonotic disease transmitted from animals to humans. CDC lists Bacillus anthracis as a category A biological agent due to its severity of impact on human health, high mortality rate, acuteness of disease, and potential for delivery as a biological weapon. The disease is acquired when spores enter the body through the skin or by inhalation or ingestion. Virulent strains of B. anthracis contain plasmids pX01, which carries genes encoding the toxins, and pX02, which encodes the poly-D-glutamic acid capsule. Plasmid pX01 carries the genes pagA, lef, and cya that encode the protective antigen (PA), lethal factor (LF), and edema factor (EF), respectively. The term "protective antigen" is derived because of this protein's ability to elicit a protective immune response against anthrax. None of these proteins is toxic when administered individually to cells or animals. However, PA in combination with EF, known as edema toxin, causes edema. Similarly, PA in combination with LF forms lethal toxin (LT) (1, 5).
PA is the primary immunogen and key component of human vaccines produced and licensed in the United Kingdom and United States. The current U.S. vaccine (BioThrax; BioPort Corp.) consists of an alum-absorbed, formalin-treated culture supernatant of a toxigenic, nonencapsulated strain of B. anthracis. The British anthrax vaccine is produced from supernatant of a static culture of the Sterne strain, a nonencapsulated toxigenic variant of B. anthracis, adsorbed to aluminum salts. These vaccines contain predominantly PA, but also small quantities of LF and trace amounts of EF (31). These traces of LF and EF may contribute to the vaccine side effects, such as local pain and edema (19), and relatively high rates of local and systemic reactions, including inflammation, flu-like symptoms, malaise, rash, arthralgia, and headache (14, 29). Therefore, an effective expression system that can provide a clean, safe, and efficacious vaccine is required.
Recombinant PA has been expressed in Escherichia coli (15), Lactobacillus casei (32), and Salmonella enterica serovar Typhimurium (6). Expression of PA in plants through chloroplast transformation has several advantages over bacterial and mammalian expression systems. Foreign proteins have been expressed at extraordinarily high levels in transgenic chloroplasts due to the presence of 10,000 copies of the chloroplast genomes per cell. These include AT-rich proteins such as Cry2a (67% AT) at 47% of the total soluble protein (TSP) (11), cholera toxin B chain fusion protein (59% AT) at 33% TSP (23), and human serum albumin (66% AT) up to 11.1% TSP (12). Therefore, we first tested the feasibility of expressing PA in transgenic chloroplasts (30), but no further studies were possible because no tag was used in that study to facilitate purification. In addition to high levels of transgene expression, there are several other advantages to chloroplast genetic engineering. Several genes can be introduced in a single transformation event to facilitate development of multivalent vaccines (11, 28). Gene silencing is a common concern in nuclear transformation, but this has not been observed in transgenic chloroplasts in spite of hyperexpression of transgenes (11). There is minimal risk of animal or human pathogens contaminating the vaccine as seen with mammalian expression systems. Additionally, chloroplast expression systems minimize cross-pollination of the transgene due to the maternal inheritance of the chloroplast genome (8).
In this study, we expressed PA with a histidine tag in transgenic chloroplasts, characterized the resultant transgenic plants, and performed immunization studies. We compared the efficacy of the plant-derived PA with that of PA derived from B. anthracis in both in vitro and in vivo studies.
MATERIALS AND METHODS
Construction of pLD-VK1 vector for chloroplast transformation. The six-histidine tag and the factor Xa cleavage site with NdeI and XhoI restriction sites were introduced N terminal to pagA using PCR (Fig. 1a). The PCR-amplified region was sequenced and shown to match corresponding pagA database sequences (accession no. AY700758). The PCR product was then cloned into pCR2.1 vector containing the psbA 5' untranslated region (UTR). Finally, the fragment containing the 5' UTR, His tag, and pagA was cloned into tobacco universal vector pLD-ctv to produce pLD-VK1 (Fig. 1a).
Leaf bombardment and selection protocol. Microprojectiles coated with plasmid DNA (pLD-VK1) were bombarded into Nicotiana tabacum var. petit Havana leaves using the biolistic device PDS1000/He (Bio-Rad) as described elsewhere (9). Following incubation at 24°C in the dark for 2 days, the leaves were cut into small (5 mm by 5 mm) pieces and placed abaxial side up (five pieces/plate) on selection medium (RMOP [regeneration medium of plants] containing 500 mg/liter spectinomycin dihydrochloride [9]). Spectinomycin-resistant shoots obtained after about 6 weeks were cut into small pieces (2 mm by 2 mm) and placed on plates containing the same selection medium.
Confirmation of transgene integration into the chloroplast genome. To confirm the transgene cassette integration into the chloroplast genome, PCR was performed using the primer pairs 3P (5'-AAAACCCGTCCTCEGTTCGGATTGC-3') and 3 M (5'-CCGCGTTGTTTCATCAAG-CCTTACG-3') (10), and to confirm the integration of gene of interest, PCR was performed using primer pairs 5P (5'-CTGTAGAAGTC-ACCATTGTTGTGC-3') and 2 M (5'-TGACTGCCCACCTGA-GAGCGGACA-3') (10).
Southern blot analysis. Two micrograms of plant DNA per sample (isolated using DNeasy kit) digested with BglII was separated on a 0.7% (wt/vol) agarose gel and transferred to a nylon membrane. The chloroplast vector DNA digested with BglII and BamHI generated a 0.8-kb probe homologous to the flanking sequences. Hybridization was performed using the Ready-To-Go protocol (Pharmacia).
Immunoblot analysis. Transformed and untransformed leaves (100 mg) were ground in liquid nitrogen and resuspended in 500 μl of extraction buffer (200 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10 mM EDTA, 2 mM phenylmethylsulfonyl fluoride). Leaf crude extracts, boiled (4 min) or unboiled, in sample buffer (Bio-Rad) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Thirty percent acrylamide Bis solution (Bio-Rad) was used to make the 10% gels. The gel was run in 1x electrode buffer (10x electrode buffer is 30.3 g Tris base, 144.0 g glycine, and 10.0 g SDS added to 1,000 ml distilled water). The separated proteins were then transferred to nitrocellulose, and Western blot analyses was performed using anti-PA primary antibody (Immunochemical labs) diluted in phosphate-buffered saline (PBS)-0.1% Tween-3% milk powder (PTM) (1:20,000) and secondary horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G (IgG) (Sigma) diluted in PTM (1:5,000) followed by washing with PBS and finally incubated with Lumiphos WB (Pierce) as a substrate for HRP at room temperature for 5 min for chemiluminescence.
ELISA for PA. Leaf samples (100 mg of young, mature, or old leaves) were collected from plants exposed to regular (16 h of light and 8 h of dark) or continuous illumination. The extraction buffer (15 mM Na2CO3, 35 mM NaHCO3, 3 mM NaN3, pH 9.6, 0.1% Tween, 5 mM phenylmethylsulfonyl fluoride) was used to isolate plant protein. All dilutions were made in the coating buffer (15 mM Na2CO3, 35 mM NaHCO3, 3 mM NaN3, pH 9.6). Antibodies were used at dilutions similar to those in the Western blotting protocol. Wells were then loaded with 100 μl of 3,3,5,5-tetramethylbenzidine (TMB; American Qualex) substrate and incubated for 10 to 15 min at room temperature. The reaction was terminated by adding 50 μl of 2 N H2SO4 per well, and the plate was read on a plate reader (Dynex Technologies) at 450 nm.
Purification of His-tagged PA by affinity chromatography. His affinity chromatography using nickel-chelate-charged columns (Amersham Biosciences) was used to purify His-tagged PA as per the manufacturer's protocol. The buffers used for purification include the following: binding buffer, 20 mM Na2HPO4, 0.5 M NaCl, 10 mM imidazole, pH 7.4; elution buffer, 20 mM Na2HPO4, 0.5 M NaCl, 0.5 M imidazole, pH 7.4; and Ni-loading eluent, 100 mM NiSO4 solution (Sigma). Protein samples were analyzed for PA using enzyme-linked immunosorbent assay (ELISA). Eluate fractions containing purified PA were pooled together and dialyzed against PBS, pH 7.4, using dialysis cassettes (molecular weight, 10,000; Pierce) and concentrated using Centricon 10,000-molecular-weight-cutoff ultrafiltration units (Millipore) following the manufacturer's protocols.
Macrophage lysis assay. Macrophage lysis assays were performed on the crude leaf extracts, partially purified chloroplast-derived PA, and B. anthracis-derived PA. RAW264.7 macrophage cells were plated in 96-well plates in 120 μl Dulbecco's modified Eagle's medium and grown to 50% confluence. The plant samples or solutions containing 20 μg/ml of the purified PA proteins were diluted serially 3.14-fold in a separate 96-well plate and then transferred onto the RAW264.7 cells in such a way that the top row had plant extract at a 1:50 dilution and PA at 0.4 μg/ml. Cells were incubated with LT for 2.5 h, and the cell viability was assessed by addition of MTT [3-(4, 5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma, St. Louis, MO) at a final concentration of 0.5 mg/ml. Cells were then further incubated with MTT for 40 min, and the blue pigment produced by viable cells was dissolved by aspirating the medium and adding 50 μl/well of a mixture containing 0.5% (wt/vol) SDS and 25 mM HCl in 90% (vol/vol) isopropanol and shaking the plates for 5 min prior to reading at 570 nm using a microplate reader. Control plates received medium with no LF to test toxicity of plant material and buffers.
Immunization studies in mice. The immunization studies were conducted in accordance with federal and institutional guidelines. Seven groups of five female 6- to 7-week-old BALB/c mice (Charles River) were immunized subcutaneously (s.c.; 5 μg PA) at two sites (100 μl per site) on day 0. The groups include mice immunized with (i) chloroplast-derived PA (CpPA) with adjuvant, (ii) chloroplast-derived PA (CpPA) alone, (iii) Std.PA derived from B. anthracis with adjuvant, (iv) Std-PA alone (26), (v) PA plant leaf crude extract with adjuvant, or (vi) wild-type plant leaf crude extract with adjuvant and (vii) unimmunized mice. The measurement of PA adsorbed to alhydrogel was done as described previously (20). Booster doses were administered on day 14, day 28, and day 140. Blood was drawn from the retro-orbital plexus 15 days after the third and fourth doses (i.e., on days 43 and 155 of post-initial immunization). The blood samples were allowed to stay undisturbed for 2 h at room temperature, stored at 4°C overnight, and centrifuged at 3,000 rpm for 10 min to extract the serum.
ELISA to detect the anti-PA IgG antibodies in the serum samples. Ninety-six-well microtiter ELISA plates were coated with 100 μl/well of PA standard at a concentration of 2.0 μg/ml in PBS, pH 7.4. The plates were stored overnight at 4°C. The serum samples from the mouse were serially diluted (1:100 to 1:640,000). Plates were incubated with 100 μl of diluted serum samples for 1 h at 37°C followed by washing with PBS-Tween. The plates were then incubated for 1 h at 37°C with 100 μl of HRP-conjugated goat anti-mouse IgG (1:5,000 dilution of 1-mg/ml stock). TMB was used as the substrate, and the reaction was stopped by adding 50 μl of 2 M sulfuric acid. The plates were read on a plate reader (Dynex Technologies) at 450 nm. Titer values were calculated using a cutoff value equal to an absorbance difference of 0.5 between immunized and unimmunized mice (25).
Toxin neutralization assays. Sera from immunized mice were tested for neutralization in the macrophage cytotoxicity assay described above. LT (PA plus LF) was added at 50 ng/ml in Dulbecco's modified Eagle's medium to 96-well plates (100 μl/well, except 150 μl in first well). Serum from each mouse was diluted directly into the LT plates starting at 1:150 and proceeding in 3.14-fold dilutions. Each serum was tested in triplicate. Following a 30-min incubation of sera with toxin, 90 μl of the mixture was moved to a 96-well plate containing RAW264.7 cells grown to 90% confluence and incubated for 5 h at 37°C. MTT was then added (final concentration, 0.5 mg/ml), and cell death was assessed as described above. Neutralization curves were plotted, and 50% effective concentrations (EC50s) were calculated for the averaged data from each mouse serum using GraphPad Prism 4.0 software.
Toxin challenge in mice. Groups of five mice with various immunization treatments described above were injected intraperitoneally with 150 μg LT (150 μg LF plus 150 μg PA) in sterile PBS (1 ml). Mice were monitored every 8 h for signs of malaise and mortality.
RESULTS AND DISCUSSION
Chloroplast vector design. The pLD-VK1 vector (Fig. 1a) contains homologous sequences that facilitate recombination of the pag gene cassette between the trnI and trnA genes of the native chloroplast genome (9). The constitutive 16S rRNA promoter regulates expression of the aadA (aminoglycoside 3' adenyltransferase) gene. The pagA gene is regulated by the psbA promoter and 5' and 3' UTRs. The psbA 5' UTR has several sequences for ribosomal binding that act as a scaffold for the light-regulated proteins involved in ribosomal binding to enhance translation (12), and the psbA 3' UTR serves to stabilize the transcript.
Demonstration of transgene integration. Several shoots appeared 5 to 6 weeks after the bombardment of tobacco leaves with gold particles coated with the pLD-VK1 plasmid DNA (Fig. 1a). There are three genetic events that can lead to survival of shoots on the selective medium: chloroplast integration, nuclear integration, or spontaneous mutation of the 16S rRNA gene to confer resistance to spectinomycin in the ribosome. True chloroplast transformants were distinguished from nuclear transformants and spontaneous spectinomycin resistance mutants by PCR. Previously described primers, 3P and 3M, were used to test for chloroplast integration of transgenes (9). The 3P primer anneals to the native chloroplast genome within the 16S rRNA gene. The 3M primer anneals to the aadA gene (Fig. 1a). Nuclear transformants could be distinguished because 3P will not anneal and mutants were identified because 3M will not anneal. Thus, the 3P and 3M primers will only yield a product (1.65 kb) from true chloroplast integrants (Fig. 1c).
The integration of the transgenes was further tested by using the 5P and 2M primer pairs for PCR analysis. The 5P and 2M primers anneal to the internal region of the aadA gene and the internal region of the trnA gene, respectively, as shown in Fig. 1a (9). The product size of a positive clone is 3.9 kb for PA, while the mutants and the control do not show any product. Figure 1d shows the result of the 5P/2M PCR analysis. After PCR analysis using both primer pairs, the transgenic plants were subsequently transferred through different rounds of selection to obtain mature plants and reach homoplasmy.
Southern blot analysis of transgenic plants. The plants that tested positive by PCR analysis were moved through three rounds of selection and were then evaluated by Southern analysis. The flanking sequence probe (0.81 kb, Fig. 1b) allowed detection of the site-specific integration of the gene cassette into the chloroplast genome (9). Figure 1a shows the BglII sites used for the restriction digestion of the chloroplast DNA for pLD-VK1. The transformed chloroplast genome digested with BglII produced fragments of 5.2 kb and 3.0 kb for pLD-VK1 (Fig. 1e), while the untransformed chloroplast genome that had been digested with BglII formed a 4.4-kb fragment. The flanking sequence probe can also show if homoplasmy of the chloroplast genome had been achieved through the three rounds of selection. The plants expressing PA showed slight degree of heteroplasmy in one or two transgenic lines, as few of the wild-type genomes were not transformed. This is not uncommon and could be eliminated by germinating seeds on stringent selection medium containing 500 μg/ml spectinomycin. The gene-specific probe with a size of approximately 0.52 kb was used to show the specific gene integration producing a 3-kb fragment containing the pagA gene as shown in Fig. 1f.
Immunoblot detection of PA expression. To determine whether the transgenic plants were producing PA, immunoblot analysis was performed on leaf extracts. Probing blots with anti-PA monoclonal antibody revealed full-length 83-kDa protein (Fig. 2a). PA has protease-sensitive sequences at residues 164 and 314 that are easily cleaved by trypsin and chymotrypsin, respectively, resulting in polypeptides of 63 kDa and 20 kDa (for trypsin) or 47 kDa and 37 kDa (for chymotrypsin). The absence of these or other such bands demonstrates that PA is intact within the chloroplast (Fig. 2a). The supernatant samples from wild-type plants did not show any band, indicating that anti-PA antibodies did not cross-react with any plant proteins in the crude extract.
Quantification of PA using ELISA. The PA protein expression levels of pLD-VK1 plants of T0 generation reached up to 4.5% of TSP in mature leaves under normal illumination conditions (16 h of light and 8 h of dark, Fig. 2b). The psbA regulatory sequences, including the promoters and UTRs, have been shown to enhance translation and accumulation of foreign proteins under continuous light (12). Therefore, the pLD-VK1 transgenic lines were exposed to continuous light and expression patterns were determined on days 1, 3, 5, and 7 (Fig. 2b). PA expression levels reached a maximum of 14.2% of the TSP in mature leaves at the end of day 5 and the expression levels declined to 11.7% TSP on day 7. The larger amount of PA in mature leaves is probably due to the high number of chloroplasts in mature leaves and the high copy number of chloroplast genomes (up to 10,000 copies per cell). The decrease in PA expression in bleached old leaves could be due to degradation of the proteins during senescence. These results show that approximately 1.8 mg PA can be obtained per gram fresh weight of mature leaf upon exposure to 5-day continuous illumination. Thus, approximately 150 mg of PA can be obtained from a single plant and with 8,000 tobacco plants on an acre of land, 1.2 kg of PA can be obtained per single cutting of tobacco plant (petit Havana variety, Table 1). Upon three cuttings in a year, a total of 3.6 kg of PA can be obtained. Assuming a loss of 50% during purification and 5 μg PA per dose (current vaccine dose is in a range of 1.75 to 7 μg PA) (17), a total of 360 million doses of vaccine can be obtained per acre of land. The commercial cultivar yields 40 metric tons biomass of fresh leaves as opposed to 2.2 tons in experimental cultivar petit Havana (7). Therefore, the commercial cultivar is expected to give 18-fold-higher yields than the experimental cultivar. Thus an acre of land grown with transgenic tobacco plants would yield vaccine sufficient for a very large population.
Functional analysis of PA with macrophage cytotoxicity assay. Figure 3a shows the Coomassie-stained gel of crude leaf extracts and various purification fractions and the absence of PA in the flowthrough. The expression level of PA is so high that it can be observed in a Coomassie-stained gel even in crude plant extracts. Figure 3b is a Coomassie-stained gel showing fractions of purified and concentrated chloroplast-derived PA used for immunization studies. Supernatant samples from crude extracts of plant leaves expressing PA and partially purified chloroplast-derived PA were tested for functionality in vitro using the well-defined macrophage lysis assay (16). The transgenic plants were shown to produce fully functional PA (Fig. 4). Crude extracts of wild-type tobacco plant and plant extraction buffer were used as negative controls. The crude extract of plant leaves expressing PA had activity equal to that of a 20-μg/ml solution of purified B. anthracis-derived PA. These results show that the PA expressed in plants has high functional activity.
Immunization of BALB/c mice. Having confirmed that the chloroplast-derived PA has in vitro biological activity comparable to that of the B. anthracis-derived PA, we proceeded further to investigate the functionality in vivo. For this, seven groups each consisting of five mice were injected s.c. with 5 μg of the antigen on days 0, 14, 28, and 140. The group 1 and group 2 mice, immunized with chloroplast-derived partially purified PA and with B. anthracis-derived fully purified PA, respectively, both adsorbed to alhydrogel adjuvant, showed comparable IgG immune titers of about 1:300,000 (Fig. 5a). These observations are comparable to those of earlier studies where anti-PA titers up to 1:250,000 were observed in guinea pigs immunized with PA along with adjuvant (4). The observation that the chloroplast-derived PA and PA derived from B. anthracis show comparable immune responses suggests that the plant-derived PA has been properly folded and was fully functional. The group that received partially purified chloroplast-derived PA without adjuvant showed titers ranging from 1:10,000 to 1:40,000, while the mice that received PA derived from B. anthracis without adjuvant showed titers of 1:80,000 to 1:160,000. Previous studies showed that mice immunized s.c. with recombinant PA (rPA) derived from B. anthracis along with the adjuvant had significant antibody titers, while no significant immune response was observed in the group immunized with PA alone (13). Similarly, guinea pigs immunized s.c. with rPA derived from B. subtilis did not elicit a significant IgG immune response, while rPA with alhydrogel adjuvant showed significant levels of IgG titers above 1:15,000 (20). Taken together, these studies show that PA alone may not be a potent immunogen to elicit a significant immune response and therefore all currently used anthrax vaccines contain an adjuvant.
The difference between the immune responses between the two groups immunized with chloroplast-derived PA and B. anthracis-derived PA could be due to differences in the purities of the proteins. The level of purity was extremely high in PA derived from B. anthracis because of the use of anion-exchange and gel filtration chromatography and fast protein liquid chromatography (FPLC) to eliminate the breakdown products of the PA (21), whereas chloroplast-derived PA was purified by affinity chromatography without using protease inhibitors. In the presence of adjuvant, PA binds to the alhydrogel via electrostatic forces (21), making it more stable against proteolytic degradation. Differences in the titer values of the groups that received PA with and without adjuvant were probably due to depot effect (2) and due to the alhydrogel's nonspecific priming of the immune system. The group that received transgenic plant crude extracts expressing PA with adjuvant showed IgG titers ranging from 1:40,000 to 1:80,000. In spite of significant levels of impurities in the crude extract, this group showed good immune titers, confirming high expression levels of PA in transgenic leaves.
Toxin neutralization assay of serum samples. In order to evaluate the functionality of the IgG antibodies produced in response to the immunization, sera from the mice were tested for their ability to neutralize PA and thereby protect macrophages against LT killing. Toxin neutralization assays were performed on two different sets of sera. The first set was drawn 15 days after the third immunization dose (day 43 of post-initial immunization), and the second set was drawn 15 days after the fourth immunization dose (day 155 of post-initial immunization). Sera obtained after the third dose (Fig. 5b) showed similar neutralization titers for the mice immunized with chloroplast-derived PA or B. anthracis-derived PA when both proteins were administered with adjuvant (1:10,000 to 1:100,000). These observations are in agreement with the results obtained in earlier studies where neutralization titers of 20,000 to 70,000 were obtained when guinea pigs were immunized with PA derived from B. anthracis along with adjuvant (4). However, titers were slightly higher for B. anthracis-derived PA used in conjunction with adjuvant in bleeds after the fourth immunization (Fig. 5c). The mice immunized with chloroplast-derived PA alone showed significantly smaller neutralization titers (between 1:100 and 1:1,000) than the mice immunized with B. anthracis-derived PA alone (1:10,000 to 1:200,000 after the third immunization and 1:10,000 to 1:50,000 after the fourth immunization, Fig. 5b and c). Mice immunized with the crude extracts of PA-expressing leaves showed strong neutralization titers, ranging from 1:500 to 1;7500, with the exception of a single mouse after the fourth immunization (Fig. 5c). Control mice immunized with wild-type plant leaf crude extract or PBS did not show any immune response or neutralization ability. Generally, the average neutralization titers compared among different groups showed similar distribution patterns to that of the average anti-PA immune titers determined by the ELISA. These results show that there is good correlation between the anti-PA antibody levels and neutralization titers.
Toxin challenge of BALB/c mice. We proceeded to test the immunized mice for their ability to survive challenge with 1.5 x 100% lethal dose (LD100) of LT (22). Mice immunized with the chloroplast or B. anthracis-derived PA with adjuvant survived the toxin challenge. Mice immunized with crude extracts of plants expressing PA showed a significant survival rate of 80%, confirming high PA expression levels. In this group, 4 out of 5 mice showed neutralization titers above 1:1,000. These studies demonstrate the immunoprotective properties of chloroplast-derived PA against anthrax LT challenge. The single mouse in this group that showed a neutralization titer below 1:150 may have been the one to succumb. None of the mice immunized with chloroplast-derived PA without adjuvant survived (Fig. 6), as expected from their low neutralization titers (Fig. 5b and c). The comparison of neutralization titers to mouse challenge survival for all the groups seems to indicate neutralization titers at and above 1:1,000 result in protection against challenge with greater than LD100 doses of LT.
These results prove the immunogenic and immunoprotective properties of plant-derived B. anthracis PA. Prior studies did not investigate functionality of plant-derived PA in animal studies (3, 30). The production of anti-PA IgG antibodies combined with in vitro neutralization and toxin challenge studies shows that immunization with transgenic chloroplast-derived PA is highly effective. Plant-derived recombinant PA is free of EF and LF and easy to produce, without the need for expensive fermenters. Even with 50% loss during purification, 1 acre of transgenic plants can produce 360 million doses of functional anthrax vaccine. Our studies open the door for possible oral immunization through feeding of edible plant parts like carrot roots, which should effectively stimulate the mucosal immune system as well as a systemic immune response, thereby offering better protection against pathogens that attack through mucosa. Delivering vaccines in edible plants can potentially eliminate existing vaccine purification and processing steps, cold storage and transportation requirements, and the need for health professionals for vaccine delivery. Although foreign genes have been expressed in chromoplasts of edible plant parts (18), there is no report of expressing vaccine antigens in non-green plastids present within edible tissues so far. In addition to maternal inheritance of transgenes engineered via the chloroplast genomes (8), cytoplasmic male sterility has been developed as another fail-safe mechanism for biological containment of transgenes (27). Furthermore, successful engineering of several foreign operons via the chloroplast genome (24) has opened the door for development of multivalent vaccines.
ACKNOWLEDGMENTS
The authors gratefully acknowledge R. Bhatnagar for providing the pMS1 plasmid containing the pagA gene, S. B. Lee for technical help during early stages of this investigation, Bob Banks for help with the handling and shipment of animals, and Jason Wiggins for assistance in toxin challenge of mice.
This investigation was supported in part by the USDA 3611-21000-017-00D and NIH R01GM 63879 award to H.D and in part by the intramural research program of the National Institute of Allergy and Infectious Diseases, NIH.
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Microbial Pathogenesis Section, National Institute of Allergy and Infectious Diseases, NIH, Building 30, Room 303, 30 Convent Dr., Bethesda, Maryland 20892-4349
ABSTRACT
The currently available human vaccine for anthrax, derived from the culture supernatant of Bacillus anthracis, contains the protective antigen (PA) and traces of the lethal and edema factors, which may contribute to adverse side effects associated with this vaccine. Therefore, an effective expression system that can provide a clean, safe, and efficacious vaccine is required. In an effort to produce anthrax vaccine in large quantities and free of extraneous bacterial contaminants, PA was expressed in transgenic tobacco chloroplasts by inserting the pagA gene into the chloroplast genome. Chloroplast integration of the pagA gene was confirmed by PCR and Southern analysis. Mature leaves grown under continuous illumination contained PA as up to 14.2% of the total soluble protein. Cytotoxicity measurements in macrophage lysis assays showed that chloroplast-derived PA was equal in potency to PA produced in B. anthracis. Subcutaneous immunization of mice with partially purified chloroplast-derived or B. anthracis-derived PA with adjuvant yielded immunoglobulin G titers up to 1:320,000, and both groups of mice survived (100%) challenge with lethal doses of toxin. An average yield of about 150 mg of PA per plant should produce 360 million doses of a purified vaccine free of bacterial toxins edema factor and lethal factor from 1 acre of land. Such high expression levels without using fermenters and the immunoprotection offered by the chloroplast-derived PA should facilitate development of a cleaner and safer anthrax vaccine at a lower production cost. These results demonstrate the immunogenic and immunoprotective properties of plant-derived anthrax vaccine antigen.
INTRODUCTION
Anthrax, a fatal bacterial infection, is caused by Bacillus anthracis, a gram-positive spore-forming organism. It is a zoonotic disease transmitted from animals to humans. CDC lists Bacillus anthracis as a category A biological agent due to its severity of impact on human health, high mortality rate, acuteness of disease, and potential for delivery as a biological weapon. The disease is acquired when spores enter the body through the skin or by inhalation or ingestion. Virulent strains of B. anthracis contain plasmids pX01, which carries genes encoding the toxins, and pX02, which encodes the poly-D-glutamic acid capsule. Plasmid pX01 carries the genes pagA, lef, and cya that encode the protective antigen (PA), lethal factor (LF), and edema factor (EF), respectively. The term "protective antigen" is derived because of this protein's ability to elicit a protective immune response against anthrax. None of these proteins is toxic when administered individually to cells or animals. However, PA in combination with EF, known as edema toxin, causes edema. Similarly, PA in combination with LF forms lethal toxin (LT) (1, 5).
PA is the primary immunogen and key component of human vaccines produced and licensed in the United Kingdom and United States. The current U.S. vaccine (BioThrax; BioPort Corp.) consists of an alum-absorbed, formalin-treated culture supernatant of a toxigenic, nonencapsulated strain of B. anthracis. The British anthrax vaccine is produced from supernatant of a static culture of the Sterne strain, a nonencapsulated toxigenic variant of B. anthracis, adsorbed to aluminum salts. These vaccines contain predominantly PA, but also small quantities of LF and trace amounts of EF (31). These traces of LF and EF may contribute to the vaccine side effects, such as local pain and edema (19), and relatively high rates of local and systemic reactions, including inflammation, flu-like symptoms, malaise, rash, arthralgia, and headache (14, 29). Therefore, an effective expression system that can provide a clean, safe, and efficacious vaccine is required.
Recombinant PA has been expressed in Escherichia coli (15), Lactobacillus casei (32), and Salmonella enterica serovar Typhimurium (6). Expression of PA in plants through chloroplast transformation has several advantages over bacterial and mammalian expression systems. Foreign proteins have been expressed at extraordinarily high levels in transgenic chloroplasts due to the presence of 10,000 copies of the chloroplast genomes per cell. These include AT-rich proteins such as Cry2a (67% AT) at 47% of the total soluble protein (TSP) (11), cholera toxin B chain fusion protein (59% AT) at 33% TSP (23), and human serum albumin (66% AT) up to 11.1% TSP (12). Therefore, we first tested the feasibility of expressing PA in transgenic chloroplasts (30), but no further studies were possible because no tag was used in that study to facilitate purification. In addition to high levels of transgene expression, there are several other advantages to chloroplast genetic engineering. Several genes can be introduced in a single transformation event to facilitate development of multivalent vaccines (11, 28). Gene silencing is a common concern in nuclear transformation, but this has not been observed in transgenic chloroplasts in spite of hyperexpression of transgenes (11). There is minimal risk of animal or human pathogens contaminating the vaccine as seen with mammalian expression systems. Additionally, chloroplast expression systems minimize cross-pollination of the transgene due to the maternal inheritance of the chloroplast genome (8).
In this study, we expressed PA with a histidine tag in transgenic chloroplasts, characterized the resultant transgenic plants, and performed immunization studies. We compared the efficacy of the plant-derived PA with that of PA derived from B. anthracis in both in vitro and in vivo studies.
MATERIALS AND METHODS
Construction of pLD-VK1 vector for chloroplast transformation. The six-histidine tag and the factor Xa cleavage site with NdeI and XhoI restriction sites were introduced N terminal to pagA using PCR (Fig. 1a). The PCR-amplified region was sequenced and shown to match corresponding pagA database sequences (accession no. AY700758). The PCR product was then cloned into pCR2.1 vector containing the psbA 5' untranslated region (UTR). Finally, the fragment containing the 5' UTR, His tag, and pagA was cloned into tobacco universal vector pLD-ctv to produce pLD-VK1 (Fig. 1a).
Leaf bombardment and selection protocol. Microprojectiles coated with plasmid DNA (pLD-VK1) were bombarded into Nicotiana tabacum var. petit Havana leaves using the biolistic device PDS1000/He (Bio-Rad) as described elsewhere (9). Following incubation at 24°C in the dark for 2 days, the leaves were cut into small (5 mm by 5 mm) pieces and placed abaxial side up (five pieces/plate) on selection medium (RMOP [regeneration medium of plants] containing 500 mg/liter spectinomycin dihydrochloride [9]). Spectinomycin-resistant shoots obtained after about 6 weeks were cut into small pieces (2 mm by 2 mm) and placed on plates containing the same selection medium.
Confirmation of transgene integration into the chloroplast genome. To confirm the transgene cassette integration into the chloroplast genome, PCR was performed using the primer pairs 3P (5'-AAAACCCGTCCTCEGTTCGGATTGC-3') and 3 M (5'-CCGCGTTGTTTCATCAAG-CCTTACG-3') (10), and to confirm the integration of gene of interest, PCR was performed using primer pairs 5P (5'-CTGTAGAAGTC-ACCATTGTTGTGC-3') and 2 M (5'-TGACTGCCCACCTGA-GAGCGGACA-3') (10).
Southern blot analysis. Two micrograms of plant DNA per sample (isolated using DNeasy kit) digested with BglII was separated on a 0.7% (wt/vol) agarose gel and transferred to a nylon membrane. The chloroplast vector DNA digested with BglII and BamHI generated a 0.8-kb probe homologous to the flanking sequences. Hybridization was performed using the Ready-To-Go protocol (Pharmacia).
Immunoblot analysis. Transformed and untransformed leaves (100 mg) were ground in liquid nitrogen and resuspended in 500 μl of extraction buffer (200 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10 mM EDTA, 2 mM phenylmethylsulfonyl fluoride). Leaf crude extracts, boiled (4 min) or unboiled, in sample buffer (Bio-Rad) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Thirty percent acrylamide Bis solution (Bio-Rad) was used to make the 10% gels. The gel was run in 1x electrode buffer (10x electrode buffer is 30.3 g Tris base, 144.0 g glycine, and 10.0 g SDS added to 1,000 ml distilled water). The separated proteins were then transferred to nitrocellulose, and Western blot analyses was performed using anti-PA primary antibody (Immunochemical labs) diluted in phosphate-buffered saline (PBS)-0.1% Tween-3% milk powder (PTM) (1:20,000) and secondary horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G (IgG) (Sigma) diluted in PTM (1:5,000) followed by washing with PBS and finally incubated with Lumiphos WB (Pierce) as a substrate for HRP at room temperature for 5 min for chemiluminescence.
ELISA for PA. Leaf samples (100 mg of young, mature, or old leaves) were collected from plants exposed to regular (16 h of light and 8 h of dark) or continuous illumination. The extraction buffer (15 mM Na2CO3, 35 mM NaHCO3, 3 mM NaN3, pH 9.6, 0.1% Tween, 5 mM phenylmethylsulfonyl fluoride) was used to isolate plant protein. All dilutions were made in the coating buffer (15 mM Na2CO3, 35 mM NaHCO3, 3 mM NaN3, pH 9.6). Antibodies were used at dilutions similar to those in the Western blotting protocol. Wells were then loaded with 100 μl of 3,3,5,5-tetramethylbenzidine (TMB; American Qualex) substrate and incubated for 10 to 15 min at room temperature. The reaction was terminated by adding 50 μl of 2 N H2SO4 per well, and the plate was read on a plate reader (Dynex Technologies) at 450 nm.
Purification of His-tagged PA by affinity chromatography. His affinity chromatography using nickel-chelate-charged columns (Amersham Biosciences) was used to purify His-tagged PA as per the manufacturer's protocol. The buffers used for purification include the following: binding buffer, 20 mM Na2HPO4, 0.5 M NaCl, 10 mM imidazole, pH 7.4; elution buffer, 20 mM Na2HPO4, 0.5 M NaCl, 0.5 M imidazole, pH 7.4; and Ni-loading eluent, 100 mM NiSO4 solution (Sigma). Protein samples were analyzed for PA using enzyme-linked immunosorbent assay (ELISA). Eluate fractions containing purified PA were pooled together and dialyzed against PBS, pH 7.4, using dialysis cassettes (molecular weight, 10,000; Pierce) and concentrated using Centricon 10,000-molecular-weight-cutoff ultrafiltration units (Millipore) following the manufacturer's protocols.
Macrophage lysis assay. Macrophage lysis assays were performed on the crude leaf extracts, partially purified chloroplast-derived PA, and B. anthracis-derived PA. RAW264.7 macrophage cells were plated in 96-well plates in 120 μl Dulbecco's modified Eagle's medium and grown to 50% confluence. The plant samples or solutions containing 20 μg/ml of the purified PA proteins were diluted serially 3.14-fold in a separate 96-well plate and then transferred onto the RAW264.7 cells in such a way that the top row had plant extract at a 1:50 dilution and PA at 0.4 μg/ml. Cells were incubated with LT for 2.5 h, and the cell viability was assessed by addition of MTT [3-(4, 5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma, St. Louis, MO) at a final concentration of 0.5 mg/ml. Cells were then further incubated with MTT for 40 min, and the blue pigment produced by viable cells was dissolved by aspirating the medium and adding 50 μl/well of a mixture containing 0.5% (wt/vol) SDS and 25 mM HCl in 90% (vol/vol) isopropanol and shaking the plates for 5 min prior to reading at 570 nm using a microplate reader. Control plates received medium with no LF to test toxicity of plant material and buffers.
Immunization studies in mice. The immunization studies were conducted in accordance with federal and institutional guidelines. Seven groups of five female 6- to 7-week-old BALB/c mice (Charles River) were immunized subcutaneously (s.c.; 5 μg PA) at two sites (100 μl per site) on day 0. The groups include mice immunized with (i) chloroplast-derived PA (CpPA) with adjuvant, (ii) chloroplast-derived PA (CpPA) alone, (iii) Std.PA derived from B. anthracis with adjuvant, (iv) Std-PA alone (26), (v) PA plant leaf crude extract with adjuvant, or (vi) wild-type plant leaf crude extract with adjuvant and (vii) unimmunized mice. The measurement of PA adsorbed to alhydrogel was done as described previously (20). Booster doses were administered on day 14, day 28, and day 140. Blood was drawn from the retro-orbital plexus 15 days after the third and fourth doses (i.e., on days 43 and 155 of post-initial immunization). The blood samples were allowed to stay undisturbed for 2 h at room temperature, stored at 4°C overnight, and centrifuged at 3,000 rpm for 10 min to extract the serum.
ELISA to detect the anti-PA IgG antibodies in the serum samples. Ninety-six-well microtiter ELISA plates were coated with 100 μl/well of PA standard at a concentration of 2.0 μg/ml in PBS, pH 7.4. The plates were stored overnight at 4°C. The serum samples from the mouse were serially diluted (1:100 to 1:640,000). Plates were incubated with 100 μl of diluted serum samples for 1 h at 37°C followed by washing with PBS-Tween. The plates were then incubated for 1 h at 37°C with 100 μl of HRP-conjugated goat anti-mouse IgG (1:5,000 dilution of 1-mg/ml stock). TMB was used as the substrate, and the reaction was stopped by adding 50 μl of 2 M sulfuric acid. The plates were read on a plate reader (Dynex Technologies) at 450 nm. Titer values were calculated using a cutoff value equal to an absorbance difference of 0.5 between immunized and unimmunized mice (25).
Toxin neutralization assays. Sera from immunized mice were tested for neutralization in the macrophage cytotoxicity assay described above. LT (PA plus LF) was added at 50 ng/ml in Dulbecco's modified Eagle's medium to 96-well plates (100 μl/well, except 150 μl in first well). Serum from each mouse was diluted directly into the LT plates starting at 1:150 and proceeding in 3.14-fold dilutions. Each serum was tested in triplicate. Following a 30-min incubation of sera with toxin, 90 μl of the mixture was moved to a 96-well plate containing RAW264.7 cells grown to 90% confluence and incubated for 5 h at 37°C. MTT was then added (final concentration, 0.5 mg/ml), and cell death was assessed as described above. Neutralization curves were plotted, and 50% effective concentrations (EC50s) were calculated for the averaged data from each mouse serum using GraphPad Prism 4.0 software.
Toxin challenge in mice. Groups of five mice with various immunization treatments described above were injected intraperitoneally with 150 μg LT (150 μg LF plus 150 μg PA) in sterile PBS (1 ml). Mice were monitored every 8 h for signs of malaise and mortality.
RESULTS AND DISCUSSION
Chloroplast vector design. The pLD-VK1 vector (Fig. 1a) contains homologous sequences that facilitate recombination of the pag gene cassette between the trnI and trnA genes of the native chloroplast genome (9). The constitutive 16S rRNA promoter regulates expression of the aadA (aminoglycoside 3' adenyltransferase) gene. The pagA gene is regulated by the psbA promoter and 5' and 3' UTRs. The psbA 5' UTR has several sequences for ribosomal binding that act as a scaffold for the light-regulated proteins involved in ribosomal binding to enhance translation (12), and the psbA 3' UTR serves to stabilize the transcript.
Demonstration of transgene integration. Several shoots appeared 5 to 6 weeks after the bombardment of tobacco leaves with gold particles coated with the pLD-VK1 plasmid DNA (Fig. 1a). There are three genetic events that can lead to survival of shoots on the selective medium: chloroplast integration, nuclear integration, or spontaneous mutation of the 16S rRNA gene to confer resistance to spectinomycin in the ribosome. True chloroplast transformants were distinguished from nuclear transformants and spontaneous spectinomycin resistance mutants by PCR. Previously described primers, 3P and 3M, were used to test for chloroplast integration of transgenes (9). The 3P primer anneals to the native chloroplast genome within the 16S rRNA gene. The 3M primer anneals to the aadA gene (Fig. 1a). Nuclear transformants could be distinguished because 3P will not anneal and mutants were identified because 3M will not anneal. Thus, the 3P and 3M primers will only yield a product (1.65 kb) from true chloroplast integrants (Fig. 1c).
The integration of the transgenes was further tested by using the 5P and 2M primer pairs for PCR analysis. The 5P and 2M primers anneal to the internal region of the aadA gene and the internal region of the trnA gene, respectively, as shown in Fig. 1a (9). The product size of a positive clone is 3.9 kb for PA, while the mutants and the control do not show any product. Figure 1d shows the result of the 5P/2M PCR analysis. After PCR analysis using both primer pairs, the transgenic plants were subsequently transferred through different rounds of selection to obtain mature plants and reach homoplasmy.
Southern blot analysis of transgenic plants. The plants that tested positive by PCR analysis were moved through three rounds of selection and were then evaluated by Southern analysis. The flanking sequence probe (0.81 kb, Fig. 1b) allowed detection of the site-specific integration of the gene cassette into the chloroplast genome (9). Figure 1a shows the BglII sites used for the restriction digestion of the chloroplast DNA for pLD-VK1. The transformed chloroplast genome digested with BglII produced fragments of 5.2 kb and 3.0 kb for pLD-VK1 (Fig. 1e), while the untransformed chloroplast genome that had been digested with BglII formed a 4.4-kb fragment. The flanking sequence probe can also show if homoplasmy of the chloroplast genome had been achieved through the three rounds of selection. The plants expressing PA showed slight degree of heteroplasmy in one or two transgenic lines, as few of the wild-type genomes were not transformed. This is not uncommon and could be eliminated by germinating seeds on stringent selection medium containing 500 μg/ml spectinomycin. The gene-specific probe with a size of approximately 0.52 kb was used to show the specific gene integration producing a 3-kb fragment containing the pagA gene as shown in Fig. 1f.
Immunoblot detection of PA expression. To determine whether the transgenic plants were producing PA, immunoblot analysis was performed on leaf extracts. Probing blots with anti-PA monoclonal antibody revealed full-length 83-kDa protein (Fig. 2a). PA has protease-sensitive sequences at residues 164 and 314 that are easily cleaved by trypsin and chymotrypsin, respectively, resulting in polypeptides of 63 kDa and 20 kDa (for trypsin) or 47 kDa and 37 kDa (for chymotrypsin). The absence of these or other such bands demonstrates that PA is intact within the chloroplast (Fig. 2a). The supernatant samples from wild-type plants did not show any band, indicating that anti-PA antibodies did not cross-react with any plant proteins in the crude extract.
Quantification of PA using ELISA. The PA protein expression levels of pLD-VK1 plants of T0 generation reached up to 4.5% of TSP in mature leaves under normal illumination conditions (16 h of light and 8 h of dark, Fig. 2b). The psbA regulatory sequences, including the promoters and UTRs, have been shown to enhance translation and accumulation of foreign proteins under continuous light (12). Therefore, the pLD-VK1 transgenic lines were exposed to continuous light and expression patterns were determined on days 1, 3, 5, and 7 (Fig. 2b). PA expression levels reached a maximum of 14.2% of the TSP in mature leaves at the end of day 5 and the expression levels declined to 11.7% TSP on day 7. The larger amount of PA in mature leaves is probably due to the high number of chloroplasts in mature leaves and the high copy number of chloroplast genomes (up to 10,000 copies per cell). The decrease in PA expression in bleached old leaves could be due to degradation of the proteins during senescence. These results show that approximately 1.8 mg PA can be obtained per gram fresh weight of mature leaf upon exposure to 5-day continuous illumination. Thus, approximately 150 mg of PA can be obtained from a single plant and with 8,000 tobacco plants on an acre of land, 1.2 kg of PA can be obtained per single cutting of tobacco plant (petit Havana variety, Table 1). Upon three cuttings in a year, a total of 3.6 kg of PA can be obtained. Assuming a loss of 50% during purification and 5 μg PA per dose (current vaccine dose is in a range of 1.75 to 7 μg PA) (17), a total of 360 million doses of vaccine can be obtained per acre of land. The commercial cultivar yields 40 metric tons biomass of fresh leaves as opposed to 2.2 tons in experimental cultivar petit Havana (7). Therefore, the commercial cultivar is expected to give 18-fold-higher yields than the experimental cultivar. Thus an acre of land grown with transgenic tobacco plants would yield vaccine sufficient for a very large population.
Functional analysis of PA with macrophage cytotoxicity assay. Figure 3a shows the Coomassie-stained gel of crude leaf extracts and various purification fractions and the absence of PA in the flowthrough. The expression level of PA is so high that it can be observed in a Coomassie-stained gel even in crude plant extracts. Figure 3b is a Coomassie-stained gel showing fractions of purified and concentrated chloroplast-derived PA used for immunization studies. Supernatant samples from crude extracts of plant leaves expressing PA and partially purified chloroplast-derived PA were tested for functionality in vitro using the well-defined macrophage lysis assay (16). The transgenic plants were shown to produce fully functional PA (Fig. 4). Crude extracts of wild-type tobacco plant and plant extraction buffer were used as negative controls. The crude extract of plant leaves expressing PA had activity equal to that of a 20-μg/ml solution of purified B. anthracis-derived PA. These results show that the PA expressed in plants has high functional activity.
Immunization of BALB/c mice. Having confirmed that the chloroplast-derived PA has in vitro biological activity comparable to that of the B. anthracis-derived PA, we proceeded further to investigate the functionality in vivo. For this, seven groups each consisting of five mice were injected s.c. with 5 μg of the antigen on days 0, 14, 28, and 140. The group 1 and group 2 mice, immunized with chloroplast-derived partially purified PA and with B. anthracis-derived fully purified PA, respectively, both adsorbed to alhydrogel adjuvant, showed comparable IgG immune titers of about 1:300,000 (Fig. 5a). These observations are comparable to those of earlier studies where anti-PA titers up to 1:250,000 were observed in guinea pigs immunized with PA along with adjuvant (4). The observation that the chloroplast-derived PA and PA derived from B. anthracis show comparable immune responses suggests that the plant-derived PA has been properly folded and was fully functional. The group that received partially purified chloroplast-derived PA without adjuvant showed titers ranging from 1:10,000 to 1:40,000, while the mice that received PA derived from B. anthracis without adjuvant showed titers of 1:80,000 to 1:160,000. Previous studies showed that mice immunized s.c. with recombinant PA (rPA) derived from B. anthracis along with the adjuvant had significant antibody titers, while no significant immune response was observed in the group immunized with PA alone (13). Similarly, guinea pigs immunized s.c. with rPA derived from B. subtilis did not elicit a significant IgG immune response, while rPA with alhydrogel adjuvant showed significant levels of IgG titers above 1:15,000 (20). Taken together, these studies show that PA alone may not be a potent immunogen to elicit a significant immune response and therefore all currently used anthrax vaccines contain an adjuvant.
The difference between the immune responses between the two groups immunized with chloroplast-derived PA and B. anthracis-derived PA could be due to differences in the purities of the proteins. The level of purity was extremely high in PA derived from B. anthracis because of the use of anion-exchange and gel filtration chromatography and fast protein liquid chromatography (FPLC) to eliminate the breakdown products of the PA (21), whereas chloroplast-derived PA was purified by affinity chromatography without using protease inhibitors. In the presence of adjuvant, PA binds to the alhydrogel via electrostatic forces (21), making it more stable against proteolytic degradation. Differences in the titer values of the groups that received PA with and without adjuvant were probably due to depot effect (2) and due to the alhydrogel's nonspecific priming of the immune system. The group that received transgenic plant crude extracts expressing PA with adjuvant showed IgG titers ranging from 1:40,000 to 1:80,000. In spite of significant levels of impurities in the crude extract, this group showed good immune titers, confirming high expression levels of PA in transgenic leaves.
Toxin neutralization assay of serum samples. In order to evaluate the functionality of the IgG antibodies produced in response to the immunization, sera from the mice were tested for their ability to neutralize PA and thereby protect macrophages against LT killing. Toxin neutralization assays were performed on two different sets of sera. The first set was drawn 15 days after the third immunization dose (day 43 of post-initial immunization), and the second set was drawn 15 days after the fourth immunization dose (day 155 of post-initial immunization). Sera obtained after the third dose (Fig. 5b) showed similar neutralization titers for the mice immunized with chloroplast-derived PA or B. anthracis-derived PA when both proteins were administered with adjuvant (1:10,000 to 1:100,000). These observations are in agreement with the results obtained in earlier studies where neutralization titers of 20,000 to 70,000 were obtained when guinea pigs were immunized with PA derived from B. anthracis along with adjuvant (4). However, titers were slightly higher for B. anthracis-derived PA used in conjunction with adjuvant in bleeds after the fourth immunization (Fig. 5c). The mice immunized with chloroplast-derived PA alone showed significantly smaller neutralization titers (between 1:100 and 1:1,000) than the mice immunized with B. anthracis-derived PA alone (1:10,000 to 1:200,000 after the third immunization and 1:10,000 to 1:50,000 after the fourth immunization, Fig. 5b and c). Mice immunized with the crude extracts of PA-expressing leaves showed strong neutralization titers, ranging from 1:500 to 1;7500, with the exception of a single mouse after the fourth immunization (Fig. 5c). Control mice immunized with wild-type plant leaf crude extract or PBS did not show any immune response or neutralization ability. Generally, the average neutralization titers compared among different groups showed similar distribution patterns to that of the average anti-PA immune titers determined by the ELISA. These results show that there is good correlation between the anti-PA antibody levels and neutralization titers.
Toxin challenge of BALB/c mice. We proceeded to test the immunized mice for their ability to survive challenge with 1.5 x 100% lethal dose (LD100) of LT (22). Mice immunized with the chloroplast or B. anthracis-derived PA with adjuvant survived the toxin challenge. Mice immunized with crude extracts of plants expressing PA showed a significant survival rate of 80%, confirming high PA expression levels. In this group, 4 out of 5 mice showed neutralization titers above 1:1,000. These studies demonstrate the immunoprotective properties of chloroplast-derived PA against anthrax LT challenge. The single mouse in this group that showed a neutralization titer below 1:150 may have been the one to succumb. None of the mice immunized with chloroplast-derived PA without adjuvant survived (Fig. 6), as expected from their low neutralization titers (Fig. 5b and c). The comparison of neutralization titers to mouse challenge survival for all the groups seems to indicate neutralization titers at and above 1:1,000 result in protection against challenge with greater than LD100 doses of LT.
These results prove the immunogenic and immunoprotective properties of plant-derived B. anthracis PA. Prior studies did not investigate functionality of plant-derived PA in animal studies (3, 30). The production of anti-PA IgG antibodies combined with in vitro neutralization and toxin challenge studies shows that immunization with transgenic chloroplast-derived PA is highly effective. Plant-derived recombinant PA is free of EF and LF and easy to produce, without the need for expensive fermenters. Even with 50% loss during purification, 1 acre of transgenic plants can produce 360 million doses of functional anthrax vaccine. Our studies open the door for possible oral immunization through feeding of edible plant parts like carrot roots, which should effectively stimulate the mucosal immune system as well as a systemic immune response, thereby offering better protection against pathogens that attack through mucosa. Delivering vaccines in edible plants can potentially eliminate existing vaccine purification and processing steps, cold storage and transportation requirements, and the need for health professionals for vaccine delivery. Although foreign genes have been expressed in chromoplasts of edible plant parts (18), there is no report of expressing vaccine antigens in non-green plastids present within edible tissues so far. In addition to maternal inheritance of transgenes engineered via the chloroplast genomes (8), cytoplasmic male sterility has been developed as another fail-safe mechanism for biological containment of transgenes (27). Furthermore, successful engineering of several foreign operons via the chloroplast genome (24) has opened the door for development of multivalent vaccines.
ACKNOWLEDGMENTS
The authors gratefully acknowledge R. Bhatnagar for providing the pMS1 plasmid containing the pagA gene, S. B. Lee for technical help during early stages of this investigation, Bob Banks for help with the handling and shipment of animals, and Jason Wiggins for assistance in toxin challenge of mice.
This investigation was supported in part by the USDA 3611-21000-017-00D and NIH R01GM 63879 award to H.D and in part by the intramural research program of the National Institute of Allergy and Infectious Diseases, NIH.
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