Oral delivery of human biopharmaceuticals, autoantigens and vaccine antigens bioencapsulated in plant cells

Abstract

Among 12billion injections administered annually, unsafe delivery leads to >20million infections and >100million reactions. In an emerging new concept, freeze-dried plant cells (lettuce) expressing vaccine antigens/biopharmaceuticals are protected in the stomach from acids/enzymes but are released to the immune or blood circulatory system when plant cell walls are digested by microbes that colonize the gut. Vaccine antigens bioencapsulated in plant cells upon oral delivery after priming, conferred both mucosal and systemic immunity and protection against bacterial, viral or protozoan pathogens or toxin challenge. Oral delivery of autoantigens was effective against complications of type 1 diabetes and hemophilia, by developing tolerance. Oral delivery of proinsulin or exendin-4 expressed in plant cells regulated blood glucose levels similar to injections. Therefore, this new platform offers a low cost alternative to deliver different therapeutic proteins to combat infectious or inherited diseases by eliminating inactivated pathogens, expensive purification, cold storage/transportation and sterile injections.

Fig. 1

An outline of the process of oral delivery of plant-derived vaccine antigens and biopharmaceuticals. Foreign genes are first expressed in lettuce chloroplasts by bombardment of leaves with chloroplast vectors using the gene gun. After confirmation of stable integration of foreign genes into all of the chloroplast genomes in each plant cell and expression of the correct size protein and functionality, genetically modified lines are transferred to the greenhouse for increasing biomass. Harvested leaves are lyophilized, powdered and stored in moisture free environment. Machines are commercially available for processing lyophilized leaf materials into desired particle size and packaging into capsules. Evaluation process includes microbial count in lyophilized materials, integrity of therapeutic proteins after prolonged storage (folding with disulfide bonds, pentameric or multimeric structures) and functionality by conferring immunity with vaccine antigens (protective immunoglobulins IgG1, IgA, cytokines, pathogen/toxin challenge) or developing tolerance with autoantigens (suppression of allergy, formation of IgE, inhibitory antibodies, destruction of pancreatic islets, etc) or conferring desired functions (regulating blood glucose with insulin, exendin-4, etc).

Fig. 2

Functional evaluation of CTB-proinsulin and CTB-exendin-4 delivered by injection or oral gavage in mice. (a) Blood glucose level after intraperitoneal (IP) injection of purified CTBP-Fx3, commercial insulin (INS Comm.) and oral delivery of control (WT), plant cells expressing CTB-PFx3 (Tg tobacco, Tg lettuce). © Plant Biotechnology Journal, Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal (2010) 9: 585–598 [20]. (b) Blood glucose level after intraperitoneal (IP) injection of purified commercial EX4 and oral administration of untransformed tobacco (WT), tobacco expressing CTB-EX4 (EX4). PBS was also injected as negative control. Arrows indicate the time point of glucose spike. *P < 0.05, **P < 0.01, ***P<0.001. © Plant Biotechnology Journal, Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal (2012): In Press [25].

Fig. 3

Pancreatic lymphocyte infiltration and insulin production in non-obese diabetic mice. (a) Haematoxylin and eosin staining of a cryosections of the pancreas (showing an islet, isl) of a NOD mouse receiving cholera toxin B subunit–human proinsulin (CTB-Pins) for 7 weeks with lymphocytes seen outside the islet (arrow) compared to a large islet with severe lymphocytic infiltration in a mouse receiving untransformed plant leaf material (UN-Tr). Fifty sections per animal were analyzed and CTB-Pins showed significantly less cellular infiltration than untreated group. Insulitis score was ~1 in CTB-pins treated group and ~5 in untreated control group (*P < 0.05). Scale bar, 50 µm. (b) Merged image of insulin and caspase-3 double immunostaining in Langerhans islets of a NOD mouse receiving CTB-Pins and untransformed plant leaf material. © Plant Biotechnology Journal, Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal (2007) 5: 495–510 [19].

Fig. 4

Suppression of fatal anaphylaxis and IgE immunoglobulin response in Hemophilia B mice through induction of oral tolerance. (a) hF.IX antigen delivery to the GALT. Peyer's patch and villi of ileum of CTB-FFIX-fed mouse stained for hF.IX (red), M cells (UEA-1, green), and CD11c (blue) are shown. (b) Mice survival in orally fed wild-type (WT, n= 10 mice at the onset of protein therapy), CTB-FIX (n=17), or CTB-FFIX (n=15) plant material after 0–12 intravenous injections of hF.IX protein. (c) IgE titers in WT-, CTB-, CTB-FFIX-fed mice, unfed mice that received antihistamine/anti-PAF before a sixth injection of hF.IX, and mice that received four hF.IX administrations. Arrows next to data points from unfed mice with four administrations of hF.IX protein indicate animals that died after a subsequent fifth injection. i.e., after 8 weekly i.v. injections of hF.IX. Copyright © by National Academy of Sciences of the United States of America, Proc. Natl. Acad. Sci. U.S.A. (2010) 107: 7101–7106 [112].

Fig. 5

Protection against Yersinia pestis challege after immunization. Mice receiving oral boosts of chloroplast-derived F1-V survived longer than mice receiving subcutaneous boosts Animals were challenged with 15 LD₅₀ of Y. pestis CO92 (whole-body LD₅₀, 6.8 × 10⁴ CFU) While control mice had up to 10¹⁰ Y. pestis counts in their spleen, survivors had no detectable Y. pestis, confirming 10-log reduction in the mean bacterial burden among survivors. Copyright © American Society for Microbiology, Infection and Immunity (2008) 76: 3640–3650 doi:10.1128/IAI.00050-08 [18].

Fig. 6

Evaluation of cholera toxin (CT) challenge and cross-reactivity of antisera generated in mice immunized with cholera and malarial vaccine antigens. (a) CT challenge in control and vaccinated mice. (b) Determination of effectiveness of numbers of boosters to generate CTB-specific serum IgA in orally gavaged mice with plant cells expressing CTB. Ten-week-old mice were boosted subcutaneously (until 189 days) or orally (until 219 days). Sera were collected until 197 days post-immunization.(c & d) CTB-antigen-specific serum and intestinal IgA in different groups of mice. Control (Ctrl), adjuvant (ADJ), subcutaneous vaccinated (SQV) and orally vaccinated (ORV) mice (e) Immunoblot analysis: Sera collected from immunized mice recognized the native 83-kDa apical membraneantigen-1 (AMA1) protein (lanes 1–3) and the native 190 kDa MSP-1protein (lanes 4–6) respectively. (f) Immunofluorescence analysis: The AMA1 antibodies recognized the apical end of the parasite in the ring developmental stage of intraerythrocytic growth (1, 2 and 3). The MSP-1 sera from immunized mice (bottom row) detected the developing merozoites at the schizont stage of the parasitic growth (4, 5 and 6). Bar size = 10 um. © Plant Biotechnology Journal, Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal (2010) 8: 223–242 [16].

Fig. 7

Evaluation of stability of antigens in lyophilized lettuce after prolonged storage. (a) Western blot analysis to evaluate stability of lyophilized anthrax protective antigen (PA) expressed in lettuce leaves after storage at room temperature for 2 (1), 4 (2), and 6 (3) months. PA, standard (100 ng), WT, untransformed lettuce. (b) PA stability after 3 months of storage, and lyophilization for different durations: (1) 24, (2) 48 and (3) 72 hrs. (c) Western blot analysis of fresh (F) and lyophilized (L) leaves expressing PA. Total soluble protein (10 µl) was loaded on an equal quantity basis (50 mg from each sample were extracted in 300 ul of extraction buffer) to examine the increase of PA content after lyophilization. (d) Fold increase of specific antigen after lyophilization. PA, lettuce transplastomic plant expressing PA; CTB-Pins, lettuce transplastomic plant expressing CTB-Proinsulin; white bar, fresh material; grey bar, lyophilized material. (e) Evaluation of long-term stability of PA in lyophilized lettuce leaves after storage at room temperature for 15 (1), 13 (2), 3 months (3), and 1 week (4). F, fresh leaf; total soluble protein (20 ng) was loaded in each lane for immunoblot assay. (f) Microbial burden of leaves expressing PA. 1, fresh leaf; 2, lyophilized leaf; 3, commercially available freeze-dried alfalfa capsules. © Plant Biotechnology Journal, Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal (2012): In Press [25].