Oral edible vaccines

ORAL EDIBLE VACCINES (i.e. vaccines containing an oral adjuvant, e.g. B subunit of cholera exotoxin

)

advantages :

safer than those produced in animal tissues because the chances of unknown human pathogens hitching a ride would be extremely small
economical advantages (obstacle to interests of vaccine industry !)

cheap to produce => costs per dose would be low
scaling up would just mean planting a larger crop
no refrigeration required by vaccine-containing seeds or dried leaves, a significant advantage in developing countries
no purification required
no skilled medical personnel required to deliver jabs

no injection required => no risk of accidental parenteral infections from contaminated needles
elicit mucosal immunity in addition to systemic immunity : anyway nasal immunization is more efficient than oral immunization at stimulating effector immunity in the reproductive tract.

Peripheral and mucosal immunity : critical issues for oral vaccine design by Arthur Anderson

obstacles and risks

resistance to degradation in gastroenteric apparatus : the tough plant cell wall apparently serves as temporary armor for the antigens, keeping them relatively safe from gastric secretions. When the wall finally begins to break up in the intestines, the cells gradually release their antigenic cargo.
induction of oral tolerance due to regular intake (uncontrolled dose) : a very severe risk ! (but also a feature that can be used for immunotherapy of autoimmune diseases ). Researchers say they have not yet seen signs of oral tolerance, but experiments to convince regulators have yet to be designed : immunologists will have to figure out how gut can do this, and do it right 99.9999% of the time. Producing veterinary vaccines first, followed by human booster vaccines, could be the sensible way forward. They could be administered as juices or tablets to circumvent dose variability
requirements of "vector" plants

able to produce functional copies of the specified protein(s)
able to produce a substantial amount per cell (up to 0.05% of total soluble proteins) of the specified protein(s) : add regulatory elements to switch on gene-expression. In general, the technology is limited by low expression levels for nuclear-integrated transgenes, but recent progress in plant organelle transformation shows promise for enhanced expression^ref
able to grow in volume (plants sometimes grow poorly when they start producing large amounts of a foreign protein) : add regulatory elements to switch off gene expression at earlier stages of plant growth.
able to produce a defined amount of the specified protein(s) to ensure that any given amount of vaccine food provides a predictable dose of antigen. Regulatory agencies will not approve vaccines with variable dosing
cheap
able to be grown locally
able to be regenerated readily without the growers having to purchase more seeds or plants year after year

other components of transgenic plants may have inhibiting or promoting effects on immunogenicity
allergy to antigens that until now only have been applied parenterally
possible recombination events of viral sequences of plant and animal pathogenic viruses
underfunding due to targeting primarily to markets outside the lucrative West
falling under the rubric of genetically modified (GM) plants : convincing the general public that it is safe to grow vaccines in fields poses a bigger challenge. Citing fears over supermarket shelves stocked with vaccine-contaminated foods, consumer groups have called for a ban on using food crops to produce pharmaceuticals. Some companies are avoiding the issue by developing injectable plant-based vaccines,by using nonfood crops or by not using genetically modified crops. For instance, California-based Large Scale Biology Corp. at Stanford University uses genetically engineered mosaic viruses to infect tobacco plants. A few weeks later antibodies can be purified from the harvested leaves. The company has produced 16 patient-specific antibodies against lymphoma cells just months after biopsies were taken.

vectors (with respective advantages and disadvantages)

transgenic bacterial spores^ref

Bacillus subtilis (transgenic proteins coat spores) : easy to manipulate, resist for millions of years !

anti-Clostridium tetani vaccine

transgenic plants : stable integration of a gene into the plant nuclear or chloroplast genome can transform higher plants (e.g. tobacco, potato, tomato, banana) into bioreactors for the production of subunit vaccines for oral (but also for parental !) administration. This can also be achieved by using recombinant plant viruses as transient expression vectors in infected plant. A minimalized version of the bean yellow dwarf geminivirus (BeYDV)-based replicon was designed consisting of the cis-acting elements required for BeYDV replication as a means to express foreign genes in a plant cell line : replication can be switched on at high levels upon expression of the BeYDV Rep protein, and gene expression enhanced enormously^ref.

tobacco (Nicotiana spp.) : easy to manipulate, but high levels of toxic alkaloids in the leaves don't allow studies on animal feeding. For experimental purposes antigens have to be substantially purified and then tested for their immunogenicity.
potato (Solanum tuberosum)^ref : easily propagated from "eyes", generated within a few months, stored for long periods without refrigeration, but they have to be cooked to be palatable (apart from toleration by volunteers ! Anyway mice accept raw potato tubers and further some kinds of potatoes are actually eaten raw in South America populations), and heating can sometimes (but not always) denature antigens.
banana (Musa acuminata) : need no cooking, but banana trees take at least 2 years to mature, the fruit has low protein content and spoils fairly rapidly after ripening.
tomato (Lycopersicon esculentum) : grow more quickly, but their fruit too has low protein content and may rot readly.
lettuce (Lactuca spp.)
carrot (Daucus carota)
peanut (Arachis hypogaea)
rice (Oryza sativa)
wheat (Triticum aestivum)
corn / maize (Zea mays subsp. mays) (if for animal vaccines : alfalfa, grains and beans). Transgenic corn is particularly attractive for this purpose since the recombinant antigen is stable and homogeneous, and corn can be formulated in several edible forms without destroying the cloned antigen
lupine (Lupinus spp.)
thale cress (Arabidopsis thaliana)
seeds (high protein content and preservable for long periods !)

soybean seeds (Glycine max)
corn / maize seeds (Zea mays subsp. mays)

... human pathogens :

viruses :

anti-Norwalk virus vaccine : capsid protein in GM tobacco and potatoes^ref
anti-rotaviruses vaccine :

VP7 DNA vaccine can induce high levels of IgG in mice but cannot protect mice against challenge. Mice immunized with transgenic potato successfully elicited serum IgG and mucosal IgA specific for VP7. The mucosal IgA titer was as high as 1000, while serum IgG titer was only 600. Neutralizing assays indicate that IgA could neutralize rotavirus^ref
cholera toxin (CT) B and A2 subunit complementary DNAs (cDNAs) fused to a rotavirus enterotoxin and enterotoxigenic Escherichia coli fimbrial antigen genes and transferred into potato. Fusion antigens are synthesized in transformed tuber tissues and assembled into cholera holotoxin-like structures that retained enterocyte-binding affinity. Orally immunized mice generated detectable levels of serum and intestinal antibodies against the pathogen antigens^ref.

anti-HAV vaccine : the construction of the plant expression vector pBI121-A was reported, which contained a fusion gene encoding hepatitis A capsid proteins. The gene was located between the left and right Ti border sequences under the control of CaMV35S promoter.The vector was identified via PCR and restriction enzyme analysis and was introduced into Agrobacterium tumerifacience LBA4404.The transgenic Citrus plants were produced by Agrobacterium-mediated transformation of epicotyl segments.13 putatively transformed plants through the kanamycin selection were micrografted onto the seedlings.The presence and integration of the transgene had been verified by PCR analysis.The result showed that five transformants were integrated and the transformation efficiency was 4.1%^ref.
anti-HBV vaccine : HBsAg using Agrobacterium mediated transformation in transgenic ... :

potatoes accumulate intracellularly as tubular structures, with a complex size distribution, differing substantially from the virus-like particle (VLP) preparations of the current commercial vaccines : natural bioencapsulation of the antigen may provide protection from degradation in the digestive tract until plant cell degradation occurs near an immune effector site in the gut^ref. Extensive disulfide-bond cross-linking, which is important for immunogenicity, was evident and 21-37% of total HBsAg protein displayed epitopes which correlate with vaccine potency^ref. The most striking improvements result from (1) alternative polyadenylation signals, and (2) fusion proteins containing targeting signals designed to enhance integration or retention of HBsAg in the endoplasmic reticulum (ER) of plant cells^ref. Although this approach is unlikely to supersede initial vaccinations. Participants in a study had already received the primary injections against HBV between 1 and 15 years ago : 19 of the 33 people (60%) in their study produced more antibodies against HBV after eating the potatoes (1 subject's protective antibodies increased 56-fold). Even the commercial vaccine, which contains an adjuvant, does not work in 10% of subjects. Unlike travellers' diarrhoea and the Norwalk virus, the hepatitis B virus did not evolve to survive in the gut, which makes the success of this edible vaccine all the more surprising. For the hepatitis B vaccine to work, it must survive digestion before acting on the immune system. But raw potatoes do not make an appetising dish and they contain relatively inconsistent vaccine doses.
tomatoes : Arntzen and his colleagues are focusing on creaing them and converting them into pills
banana cv. Rasthali (AAB) : 4 different expression cassettes (pHBS, pHER, pEFEHBS and pEFEHER) were utilized to optimize the expression of HBsAg in banana. The transgenic nature of the plants and expression of the antigen was confirmed by PCR, Southern hybridization and RT-PCR. The expression levels of the antigen in the plants grown under in vitro conditions as well as the green house hardened plants were estimated by ELISA for all the four constructs. Maximum expression level of 38 ng/g F.W. of leaves was noted in plants transformed with pEFEHBS grown under in vitro conditions, whereas pHER transformed plants grown in the green house showed the maximum expression level of 19.92 ng/g F.W. of leaves. Higher mAb binding of 67.87% of the antigen was observed when it was expressed with a C-terminal ER retention signal. The buoyant density in CsCl of HBsAg derived from transgenic banana leaves was determined and found to be 1.146 g/ml. HBsAg obtained from transgenic banana plants is similar to human serum derived one in buoyant density properties. The transgenic plants were grown up to maturity in the green house and the expression of HBsAg in the fruits was confirmed by RT-PCR. These transgenic plants were multiplied under in vitro using floral apex cultures. Attempts were also made to enhance the expression of HBsAg in the leaves of transgenic banana plants by wounding and/or treatment with plant growth regulators. This is the first report on the expression of HBsAg in transgenic banana fruits^ref.
tobacco plants were genetically transformed with HBsAg gene linked to a nominally constitutive promoter. Enzyme-linked immunoassays using a monoclonal antibody directed against human serum-derived HBsAg revealed the presence of HBsAg in extracts of transformed leaves at levels that correlated with mRNA abundance. This suggests that there were no major inherent limitations of transcription or translation of this foreign gene in plants. Recombinant HBsAg was purified from transgenic plants by immunoaffinity chromatography and examined by electron microscopy. Spherical particles with an average diameter of 22 nm were observed in negatively stained preparations. Sedimentation of transgenic plant extracts in sucrose and cesium chloride density gradients showed that the recombinant HBsAg and human serum-derived HBsAg had similar physical properties. Because the HBsAg produced in transgenic plants is antigenically and physically similar to the HBsAg particles derived from human serum and recombinant yeast, which are used as vaccines, we conclude that transgenic plants hold promise as low-cost vaccine production systems^ref. HBsAg gene was cloned into plant transformation vectors pHER100 and pHBs100 with and without endoplasmic reticulum retention signal, respectively. Transformed tobacco cell lines were analyzed for the integration of the transgene by PCR and Southern blot hybridization. Expression levels as determined by ELISA showed maximum expression levels of 2 mg HBsAg/g fresh weight and 10 ng/mL of spent medium in pHER100 transformed cells. Western blot analysis confirmed the presence of 24 kDa band specific to HBsAg in the transformed cells. HBsAg was expressed both as intracellular and secreted forms in pHER100 transformed cells. The buoyant density in CsCl of HBsAg derived from pHBs100 transformed tobacco cells was determined and found to be 1.095 g/mL. HBsAg obtained from transformed tobacco cells is similar to the human serum derived one in buoyant density properties. This is the first report on the secretion of HBsAg particles by plant cells into the cell culture medium^ref.
Imani J, Berting A, Nitsche S, Schaefer S, Gerlich WH, Neumann KH (2002) The integration of a major hepatitis B virus gene into cell-cycle synchronized carrot cell suspension cultures and its expression in regenerated carrot plants. Plant Cell Tissue Org Cult 71:157–164

anti-HHV-2 / HSV-2 vaccine
anti-HPVs vaccine :

transgenic tobacco and potato plants carrying the HPV type 16 major structural gene L1 under the control of the cauliflower mosaic virus 35S promoter. The plant-derived L1 protein displayed conformation-specific epitopes and assembled into virus-like particles. Furthermore, we did not find any indications of protein modification of the L1 protein produced in plants. Plant-derived L1 was as immunogenic as L1 expressed in baculovirus-infected insect cells. Feeding of tubers from transgenic potatoes to mice induced an anti-L1 antibody response in 3 out of 24 mice, although this response was only transient in two of the mice. However an anti-L1 response was primed in about half of the 24 animals^ref1,
ref2
expression of the human papillomavirus 16 E7 protein in Nicotiana benthamiana plant using a potato virus X-derived vector. C57BL/6 mice immunized with E7-containing crude foliar extracts developed both humoral and cell-mediated immune responses and were protected from tumor development after challenge with the E7-expressing C3 tumoral cell line. There is possibility of producing a cost-effective anticancer vaccine in plant with intrinsic adjuvant-like properties^ref

anti-rabies virus vaccine
anti-measles virus vaccine
anti-HIV-1 vaccine : chimeric virus particles (CVPs) in which gp41 is expressed as an N-terminal translational fusion with the potato virus X (PVX) coat protein. Transgenic tomatoes expressing the Tat protein. 2 independent plants testing positives to transgene detection analysis were selected and grown to maturity. Monoclonal antibodies against tat recognized a protein of the expected size. Interestingly, expression of Tat seemed to be toxic to the plant as in all cases the fruit presented underdeveloped reproductive structures and no seeds. 9 groups of ten pathogen-free Balb/c male mice were primed either, orally, intraperitoneally (i.p.) or intramuscularly (i.m.) with 10 mg of tomato fruit extract derived from transgenic or wild type plants and with 10 mg of Tat86 recombinant protein. Mice were immunized at days 0, 14, 28 and boosted after 15 weeks; sera were drawn 7 days after each boost and the antibody titer determined by ELISA. All 3 immunization approaches induced the development of a strong anti-Tat immunological response, which increased over time. Isotype subclasses determination showed the presence of mucosal (IgA) immunity soon after the beginning of the oral immunization protocol and the data were confirmed by the presence of anti-Tat antibodies in fecal pellets and in vaginal washes. Sera from immunized mice inhibited with high efficiency recombinant tat-dependent transactivation of the HIV-1 LTR promoter. This neutralization activity might be of relevance for the suppression of extracellular Tat activities, which play an important role in HIV disease development^ref
anti-HIV-1 + HBV vaccine : the synthetic chimeric gene TBI-HBS encoding the synthesis of immunogenic ENV and GAC epitopes of HIV-1 (immunogenes of T- and B-lymphocytes) and of the surface protein (HBsAg) of the hepatitis B virus was introduced into tomato plants var. Ventura by agrobacterial vector pBIN35TBI-HBS; transgenic tomato plants with the integrated gene TBI-HBS were generated. The integration of the TBI-HBS target gene was confirmed by PCR. The synthesis of antigenic proteins of TBI and HBsAg in fruits of transgenic tomato plants was displayed by immunoassay. The fruits of transgenic tomato plants were fed to experimental mice with a 1-week interval. On days 14 and 28, there was discovered a sufficiently high content of antibodies to the antigenic proteins of HBV and HIV-1 in serum of experimental animals. Antibodies were found in feces of experimental mice; no antibodies were found in the control group of mice. Hence, it was established that the TBI (HIV-1) and HBsAg (HBV) antigens were synthesized in transgenic tomato fruits due to the integrated construction of pBINNp35TBI-HBS in an amount that was enough to induce the immunogenic response in mice to the oral delivery of edible vaccine^ref.

Bacteria :

anti-Vibrio cholerae vaccine : short cholera toxin B subunit (CTB) in rice^ref
anti-ETECs vaccine :

Escherichia coli heat-labile enterotoxin (LT)

B subunit (LT-B) in

potatoes^ref
thale cress (fused with Mycobacterium tuberculosis ESAT-6 retained both its native antigenicity and the ability to form pentamers)^ref
transgenic corn expressing 1 mg of LT-B of Escherichia coli without buffer was fed to adult volunteers in 3 doses, each consisting of 2.1 g of plant material. 7 (78%) of 9 volunteers developed rises in both serum IgG anti-LT and numbers of specific antibody secreting cells after vaccination. 4 (44%) of 9 volunteers also developed stool IgA^ref

chloroplast expression of LTK63 (change Ser-->Lys at position 63 in the A subunit), the mutant of Escherichia coli heat-labile toxin. LTK63 is devoid of any toxic activity, but still retains its mucosal adjuvanticity. The LTK63 was cloned into chloroplast targeting vector and transformed to tobacco chloroplasts by particle bombardment. PCR and Southern blot analyses confirmed stable homologous recombination of the LTK63 gene into the chloroplast genome. The amount of LTK63 protein detected in tobacco chloroplasts was approximately 3.7% of the total soluble protein. The GM₁-ganglioside binding assay confirmed that chloroplast-synthesized LTB of LTK63 binds to the intestinal membrane GM₁-ganglioside receptor^ref.

as a potential alternative to traditional needle vaccination of cattle, the major K99 fimbrial subunit, FanC, was expressed in soybean (Glycine max) for use as an edible subunit vaccine. As a first step in this developmental process, a synthetic version of fanC was optimized for expression in the cytosol and transferred to soybean via Agrobacterium-mediated transformation. Western analysis of T(0) events revealed the presence of a peptide with the expected mobility for FanC in transgenic protein extracts, and immunofluorescense confirmed localization to the cytosol. 2 T(0) lines, which accumulated FanC to levels near 0.5% of total soluble protein, were chosen for further molecular characterization in the T(1) and T(2) generations. Mice immunized intraperitoneally with protein extract derived from transgenic leaves expressing synthetic FanC developed significant antibody titers against bacterially derived FanC and produced antigen-specific CD4⁺ T lymphocytes, demonstrating the ability of transgenic FanC to function as an immunogen. These experiments are the first to demonstrate the expression and immunogenicity of a model subunit antigen in the soybean system, and mark the first steps toward the development of a K99 edible vaccine to protect against ETEC^ref

anti-Helicobacter pylori vaccine

... non-human pathogens :

anti-foot-and-mouth disease virus (FMDV) vaccine : transgenic potatoes plants containing the VP1 gene cloned under the regulatory activity of either a single (pRok2) copy of the S35 cauliflower mosaic virus (CaMV 35S) promoter : use of double CaMV 35S promoter does not cause a significant increase in the level of the VP1 expressed^ref
anti-infectious bronchitis virus (IBV) vaccine : S1 glycoprotein in potatoes^ref
anti-rabbit hemorrhagic disease virus (RHDV) vaccine : major structural protein VP60 in transgenic tubers of potatoes plants^ref
anti-SIV vaccine : cholera toxin B subunit (CTB) was linked 5' to the simian immunodeficiency virus (SIVmac) Gag p27 capsid gene (CTB-Gag). The fusion gene was transferred into potato cells by Agrobacterium tumefaciens-mediated transformation methods and transformed plants regenerated. The CTB-Gag gene fusion was detected in transformed potato leaf genomic DNA by polymerase chain reaction-mediated DNA amplification. The results of immunoblot analysis with anti-CTB and anti-Gag antibodies verified the synthesis of biologically active CTB-Gag fusion protein in transformed leaf and tuber tissues. Synthesis and assembly of the CTB-Gag fusion protein into oligomeric structures of pentamer size was confirmed by GM1-ganglioside-enzyme-linked immunosorbent assay (GM1-ELISA) of transformed potato tuber tissue extracts. The binding of CTB-Gag fusion protein oligomers to intestinal epithelial cell membrane receptors quantified by GM1-ELISA showed that CTB-Gag fusion protein made up approx 0.016-0.022% of the total soluble tuber protein. The synthesis of CTB-Gag monomers and their assembly into biologically active CTB-Gag fusion protein oligomers in potato tuber tissues provides the opportunity for employment of the carrier and adjuvant properties of CTB for the development of edible plant-based subunit mucosal vaccines for enhanced mucosal immunity against SIV in macaques^ref

Since 1992, when biologist Charles Arntzen proposed genetically modifying bananas to serve as cheap vaccines against infectious diseases, research on plant-based pharmaceuticals has grown rapidly. In July 2004, the European Union promised € 12 million to European and South African scientists developing vaccines or antibodies against HIV-1

, rabies virus and tuberculosis. Work is further ahead in the US, where several acres of crops, most of them still experimental, are planted each year. Researchers have thus far produced > 45 different antigens in a wide range of plants. Arntzen is collaborating with companies in Egypt, South Africa and South Korea, but outside developing nations, where there is an urgent need for such vaccines, finding manufacturers willing to finance larger trials to demonstrate efficacy has been a formidable challenge. Vaccine manufacturers have little reason to replace existing production lines, as most vaccines are economically unattractive. This area is evolving from a food strategy to a strategy for easier local manifacturing, still convenient to industry interests. The medical community is also focused on high-tech approaches, making farm-grown vaccines a tough sell. But smaller companies, led by young people willing to take risks, could challenge the current thinking. Part of the hesitation stems from the fact that plant-based oral vaccines constitute a new technology from both a regulatory and scientific perspective.
Web resources : Edible vaccines by Scientific American