Vaccines and sera through plant biotechnology.

During the last 50 years, more vaccines and sera have become available for prophylaxis or treatment of disease than during the preceding century. Yet at the same time, the number of obstacles preventing the use of those biomedical discoveries for the benefit of mankind has risen almost proportionately. Stricter regulations for safety and efficacy have led to increased cost of production of biomedicals, but secondly the price to the consumer has risen to a degree that now precludes prophylactic vaccination against some diseases on a global scale. In addition to the extraordinarily high price of new vaccines and sera, the method of administration of these products through syringe and needle presents another burden in their worldwide use. In 1958, I organized a mass vaccination trial with my oral polio vaccine in the then Belgian Congo. Two hundred and fifty thousand individuals were vaccinated in 6 weeks by oral spraying. This became a model for worldwide polio vaccination—a project requiring only one oral administration of a product—to eradicate polio in the world. The global use of vaccine requires that the vaccines be inexpensive and easily maintained and distributed. After considering various alternatives of fulfilling the criteria established for a global approach to immunization, it has become clear that our only choice is the production of vaccines or other materials of biomedical importance in plants.

The advantages of plants are quite clear. Plants are very inexpensive to grow in mass; some can grow in almost any soil or climate in the world. Various parts of plants (leaves, seeds, fruits, and chloroplasts) can also be used as vehicles for the biomedical products. Production of biomedicals in plants and their administration to humans and animals also provides an added margin of safety as compared to biologicals produced in animal tissues. Early concerns that plants might not be capable of producing biomedicals of animal origin, and that plant-specific glycosylation patterns might functionally alter plant-produced antibodies have proven unwarranted.

Basic methods to transfer foreign genese into plants are well established. In the first approach, the plant is transfected with a foreign gene in combination with the plant parasite, Agrobacterium tumefaciens. In this constitutive approach, future generations of the same transgenic plant species heritably retain the capacity to express the original product.

The product is isolated from the extract of leaves, seeds, etc. (Table 1 ). In the second approach, the gene is inserted into a plant virus (for example, alfalfa or tobacco mosaic virus), which is used in combination with a foreign antigen to infect plants. Here, the final product is harvested from the plant in tandem with the plant virus. In both cases, purification of the vaccine or antibody is a rather simple procedure.

Table 1

Plant production: virus vectors vs. transgenic plants

	Virus	Transgenic
Construct design to testing	Weeks	Months
Stable plant genotype	No	Yes
Nuclear transcript processing	No	Yes
Translational optimisation	Yes	Yes
Post-translational modification	Yes	Yes
Yield, mg per 1 kg of plant tissue	Up to 800	Up to 10

The achievements in production of plant-derived vaccines and sera, can be classified in two categories:

vaccines and antibodies for infectious diseases;

vaccines and antibodies for cancer.

Table 2 shows the list of infectious disease agents against which plant-derived products were made in the Biotechnology Foundation Laboratories, in chronological order, respiratory syncytial virus (RSV), hepatitis B, HIV, rabies, anthrax, diphtheria, SARS, and smallpox virus.

Table 2

Plant-derived biomedicals for treatment of infectious diseases

Diseases	Vaccines	Plant	Antibody	Plant
Respiratory syncytial		Tobacco
Hepatitis B		Lettuce
HIV		Spinach
Rabies		Spinach		Tobacco
		Tobacco
Anthrax		Tobacco
		Tomato
Diphtheria		Tobacco
SARS		Tobacco
		Tomato
Smallpox		Tobacco
		Tomato

One of the first vaccines produced in this system was against RSV, which causes disease of newborn children. We produced the vaccine antigen as a fusion protein with the capsid protein of alfalfa mosaic virus in tobacco. Immunogenicity was tested in mice, which were either injected with or fed the plant-produced vaccine (Table 3 ). RSV infection causes distinct lesions in mice. Evidence of protection by plant RSV vaccines against challenge with wild-type virus was evidenced by low infectivity of lungs of vaccinated mice as compared to controls; high-titer antibodies against RSV were also induced.

Table 3

Serum titers of RSV G-protein-specific IgG induced by plant virus containing RSV G peptide and protection from challenge (Belanger et al., 2000)

Groups	ELISA titer	Challenge virus replication (log TCID50/g of lung tissue)	Protectiona
NF1-RSV	1/720	< 2.0 ± 0.37	3/5
NF2-RSV	1/61440	< 1.7 ± 0.0	4/4
A1MV	1/120	03.05 ± 0.46	0/5
RSV-Long	1/29440	< 1.7 ± 0.0	5/5
PBS	1/100	< 3.1 ± 0.41	0/5

Undetectable virus titers in the lungs.

Plant-derived vaccines for HIV and anthrax will be presented by Dr. Alexander Karasev. Scientists under the leadership of Professor Andrzej Legocki in Poznan, Poland, have produced a vaccine against hepatitis B in transgenic lettuce. These scientists have shown that vaccines expressed in lettuce leaves can be ground, frozen, dried and powdered without loss of immunogenicity. Observation of immune response in mice fed the plant-derived vaccine indicates that two feedings at 2-month intervals produce optimal immunogenic responses.

Rabies is a worldwide disease of humans and warm-blooded animals. Except for the time-honored Pasteur type vaccine, all modern vaccines are too expensive for use on a worldwide scale. Rabies antibody is essential in treating severe bites but is unavailable because it is not sufficiently lucrative for production by the pharmaceutical industry. Rabies in dogs is a major, but not unique, source of infection in Southeast Asia and in many African countries. An inexpensive bait vaccine for dogs could lead to the eradication of rabies in those parts of the world.

To express rabies vaccine in plants, we have used a recombinant alfalfa mosaic virus in spinach leaves. This virus expressed a chimeric peptide containing antigenic determinants of rabies virus glycoprotein, which elicits immune responses; and of nucleoprotein, which increases glycoprotein immunogenicity. The chimeric gene antigen was PCR-amplified and cloned for fusion with the gene encoding the coat protein of alfalfa mosaic virus. Tobacco and spinach were the plants used to express the antigen. The presence of rabies virus protein in spinach leaves was checked by Western blot and immunogenicity was tested in mice. Then volunteers were fed 20 g of spinach extract recombinant virus twice at 2-week intervals. Half of the subjects responded to the oral administration of spinach as indicated by a vigorous booster response after a single injection of commercial vaccine.

There is a global crisis in post-exposure treatment of rabies because of worldwide unavailability of the antirabies serum required for severe bites. Thus, it was decided on an emergency basis to produce rabies antibody in plants. Research conducted by Dr. Kisung Ko, led to the production of a transgenic tobacco plant containing the heavy and light chains of human rabies antibody. The two chains recombined in the plants to produce a complete antirabies antibody, which was as effective as the original antibody in animals, before and after exposure to rabies (Table 4 ). Two very interesting results emerged from the research with this antibody. First, leaf extract of the transgenic tobacco was found to be virtually free of nicotine and other alkaloids (Fig. 1 ). Second, the glycosylation pattern of the plant-derived antibody was considerably different from that of the animal tissue-derived antibody (Fig. 2 ). Mannose is the only glycan prevalent in the plantibody, yet the antibody was at least as efficacious as the native antibody produced in animals.

Table 4

In vivo efficacy of mAbP for post-exposure prophylaxis of hamster injected with a lethal dose of coyote rabies street viruses (Dr. C. Rupprecht, CDC)

Post-exposure treatment
Antibodya (IU/animal)	Vaccine (HDCV)b
	−	+
mAbP (3)	5/9c	9/9
mAbP (0.7)	1/9	8/9
mAbP (0.4)	2/9	8/9
HRIG (2)	4/9	8/9
Untreated control	0/9	0/3

Ko et al. [1], PNAS.

IU: international unit.

HDCV: (Imovax, lot MO475) human diploid cell culture rabies virus vaccine, “−” and “+” indicate treatments without or with HDCV, respectively.

Number of surviving hamsters/number of hamsters tested.

Fig. 1

GC/MS analysis of nicotine contents in purified anti-rabies mAbP (Dr. P. Crooks, University of Kentucky).

Fig. 2

N-glycan structures of mAbP and mAbM (Dr. P. Rudd, Oxford, UK).

Strict guidelines regarding the amount and potency of antirabies antibody for use in post-exposure treatment has enabled calculation-based on the yield of antibody per tobacco plant-of plant acreage needed to produce a given number of antibody international units. If, as expected, 1 kg of tobacco produces 5–6 mg of antibody, 1 ha or 2.4 acres is needed to produce enough antibody for vaccination of 10,000 people. I leave it to accountants to calculate the production cost of such an antibody as compared to antibody produced in animal tissue.

One of the many advantages of plants for production of biological products is their ability to express multiple genes in the same plant. Vaccines against diphtheria, tetanus, and oral pertussis are the triad injected into infants during their first 2–3 months of life. A plan to substitute oral products for injectable vaccines was initiated through production of transgenic tobacco (as a model) and transgenic carrots expressing all three vaccines (Table 5 ). Plants expressing current diphtheria toxoid have already been produced (Fig. 3 ).

Table 5

Expression of anti-diphtheria, tetanus and pertussis (DTP) vaccine components in plants

Bacteria	Antigen
Corynebacterium diphtheriae	Diphtheria toxin (DT 535 aa)
Clostridium tetanus	Tetanus toxin C fragment (TetC 452 aa)
Bordattela pertussis	S1 subunit of pertussis toxin (200aa) + hemaglutinine (HMA) + pertactin

Fig. 3

Detection of diphtheria toxin (DT) in tobacco (T0 generation).

The spike protein of SARS coronavirus is presumed to be immunogenic. Transgenic tobacco and tomato expressing the spike protein were generated (Fig. 4 ). Interestingly, only 3 months elapsed between the introduction of the spike construct into the plant and the first observation of SARS coronavirus mRNA expression by the plant (Fig. 5 ). Theoretically, the vaccine could be obtained by growing large numbers of transgenic plants. It is doubtful whether other methods currently available would produce vaccine material against a newly discovered disease in such a short time.

Fig. 4

Development of edible vaccine against SARS coronavirus tobacco transformation.

Fig. 5

Development of edible vaccine against SARS coronavirus tomato transformation.

After decades of neglect, we are now witnessing a revived interest in immunological approaches to cancer treatment. Thus, we sought expression of colorectal cancer antigen GA 733 and of the corresponding 17-1A antibody in plants. Attempts were also made to express antibodies recognizing epidermal growth factor (EGF) which is present at the surface of epithelial tumors in plants. The tobacco mosaic virus-based gene vector (p30B) was used for expressing the 733-2 GA antigen. Nicotiana betthamiana was infected with the construct and checked expression by Western blot analysis of the leaf extract. A group of mice were then immunized with the plant-produced 733-2 antigen and, for comparison, another group with the same antigen expressed in baculovirus systems. Sera obtained from both groups of mice recognized human colorectal cell lines expressing this antigen (Fig. 6 ).

Fig. 6

Antigens expressed in sera obtained from mice recognizing human colorectal cell lines.

A cross reactivity test capable of discriminating the two antigens, that produced in plants and that produced in baculovirus vector, ruled out any “contaminant” basis for the immune response. Complement-dependent cytotoxicity assays of sera obtained from mice immunized with the plant-derived 733-2 antigen showed lysis of breast cancer cells, but not of antigenically unrelated melanoma cells (Fig. 7 ). The plant-derived 733-2 antigen also induced antigen-specific proliferation of T cells. Using the same technique as for antirabies antibodies, we expressed 733-2-reactive antibody 17-1A in tobacco plants. Purified leaf extract of these plants specifically recognized receptors present in human colorectal cancer cells.

Fig. 7

Complement-dependent cytotoxicity assays of sera obtained from mice immunized with the plant-derived 733-2 antigen.

Perhaps the most interesting results obtained from the study of the two plantibodies against rabies and cancer relate to the glycosylation pattern. The plant-derived antirabies heavy chain was fused to KDEL (lysine–asperagine–glycosamine–leucine) endoplasmic reticulum retention signal. As a result, the anti-rabies plantibody contained abundant mannose, whereas the plant-derived anticancer antibody contained less mannose and more N-acetylglucosamine (Fig. 8 ).

Fig. 8

Effect of KDEL on glycan structures of plant derived mAb.

Despite the different glycosylation patterns of the two plantibodies and the difference of both patterns from those of the native antibody, their biological activity was equivalent to each other and to that of the native antibody. Antibodies reactive with the epidermal growth factor were recently licensed to treat certain types of epithelial cancers. Such antibodies were first developed at the Wistar Institute many years ago. They have recently been adapted to production in tobacco plants and within the near future might provide an inexpensive therapeutic tool in human cancer.

However, plant production of vaccines and sera is not a simple procedure with assured success in each undertaking. One of the most important remaining problems is the low yield of the bioproduct in plants. To date, no magical solution to this problem has been found. Codon optimization, careful approaches to harvesting and purifying plant products, use of plant parts such as chloroplasts to increase uptake of the material are but a few potential avenues to help increase the yield of the final product. At present, it is still difficult to produce sizeable amounts of plant-derived products.

Great strides have been made in our knowledge and expertise in the use of plants as hosts for production of biomedicals. As shown in the last table (Table 6 ), a segment of the population remains to be convinced that the future of biologicals is linked with production in plants. Some people will consider the facts and ultimately opt for acceptance. Others will remain “true believers” in harmful effects caused by transgenic plants. Their voices may become subdued or even silenced by increasing evidence that the future of global health improvement rests strictly in the progress made on the worldwide use of plants to combat diseases.

Table 6

There are four stages of adopting new ideas

The first is “It's impossible”

The second is “Maybe it's possible, but it's weak and uninteresting”

The third is, “It's true and I told you so”

And the fourth is, “I thought of it first”