HK1038191A - Oral immunology using plant product containing a non-enteric pathogen antigen - Google Patents

Description

Oral immunization using plant products containing antigens of parenteral pathogens

It is well known that pathogenic microorganisms do not elicit a protective intestinal immune response (parenteral pathogens) in mammals. Until now, it was thought that protection against infection by this parenteral pathogen could not be obtained by oral immunization, especially when antigens from this pathogen were used against fully live or attenuated pathogens.

These pathogenic microorganisms are often infected by parenteral routes, in particular through small punctures in the skin, abrasions, incisions, or other breaches, for example, by blood transfusions, and the like.

The diseases caused by parenteral pathogens are the following: hepatitis B, hepatitis C, hepatitis D, yellow fever, Lassa fever, dengue fever, rabies, tetanus, Staphylococcus aureus infection, Yask disease, relapsing fever, rat bite fever, bubonic plague, typhoid fever and macula fever.

As one of the above examples, Hepatitis B Virus (HBV) is an important pathogenic virus causing a high morbidity and mortality, although there are many effective parenteral vaccines. It is estimated that in 1996, approximately 115 million people become infected with hepatitis B virus. The resulting data of deaths is estimated to be 100 million cases per year. In developed countries such as the united states, the immune rate against hepatitis b virus remains below established targets and over 30 million new cases occur each year, with 5000 deaths caused by hepatitis b virus infection each year. Furthermore, as can be seen in a review of the prevalence of hepatitis B virus infection in the United states from 1976 to 1994, the rate of hepatitis B virus infection did not significantly decrease during this period despite the use of hepatitis B vaccine. Thus, there remains a need for improvements and improvements in existing parenteral vaccine alternatives and approaches in developed countries. This is particularly important as more and more vaccines become part of childhood immunizations, as we must consider how to safely and effectively use the numerous antigens in pediatric immunization programs.

Another notable concern is that a significant percentage of hepatitis b morbidity and mortality worldwide is present in the developing world where hepatitis b viruses are spread. For example, in the country of maraviroc, 72% of the hospital parturients have significant HBV infection and 13% are chronic carriers. In these places, the supply of existing parenteral vaccines is very limited because of the need for cryopreservation and the high price of the vaccine. Although some significant activities have been initiated to provide hepatitis b vaccines to developing countries, there is still a need for alternative approaches. Despite the ever-increasing immune rates in developed countries, the entry of hepatitis b from developing countries into developed countries will continue in the absence of an effective global hepatitis b immunization program.

An alternative route to parenteral immunization against certain diseases is vaccines that can be administered orally. As discussed previously, oral vaccines are generally ineffective against parenteral pathogens.

One particular approach to oral immunization has been proposed, namely expression of antigens in transgenic plant tissues, followed by ingestion. This technique makes it possible to provide both a simple production method and an antigen in a matrix suitable for oral immunization. Furthermore, plant tissues such as potato tubers have significant advantages, since vegetables, even in the raw state, have a long safety period in the market. Finally, transgenic plant tissues expressing oral antigens may have the additional advantage of stimulating both humoral and mucosal immunity, with the result that they protect mucosal surfaces more effectively than parenteral immunization. Until now, it has not been found that transgenic plant tissues expressing antigenic material of parenteral pathogens are more effective as oral vaccines than the oral ingestion of such purified antigens directly.

Plants expressing hepatitis b surface antigen (HBsAg) have in fact been developed, but at the same time disappointing that only small or unacceptably low immune responses are produced by mammalian ingestion of the plant, whereas HBsAg isolated from plants has been found to stimulate an immune response by parenteral administration. Throughout this specification HBsAg is exemplary and represents other parenteral pathogen antigens known to produce an immune response when administered parenterally.

Transgenic plants such as potato have been developed that express the hepatitis b surface antigen, an antigen known to produce an immune response to hepatitis b by parenteral administration. Unfortunately, such an immune response is not stimulated to an acceptable level when plants such as potatoes are simply used to feed animals.

However, it has now been unexpectedly found that when an animal is immunoreceptive to HBsAg, an immune response to a non-enteric pathogen antigen, such as hepatitis B surface antigen (HBsAg), can be obtained when the animal is fed with the antigen in a plant material. It has now been found that animals can be made immunoreceptive to a non-enteric pathogen antigen such as HBsAg by co-administering an antigen-containing plant material and a suitable adjuvant. The animal may also be immune-sensitive as a result of a prior immunization, e.g., a primary immunization, in which case the immune response to the parenteral antigen, e.g., HBsAg, may be boosted in the animal by feeding the animal plant material containing the antigen. In this case no immunological adjuvant was found to be required. But immunological adjuvants may be used for the purpose of obtaining a higher immune response. For example, an animal such as a human may be raised to an HBsAg immune response by feeding plant material containing the antigen if it has previously responded positively to a primary anti-hepatitis B immunity. These plant materials are substances which contain physiologically acceptable plant materials from plants (e.g. juices, pulp, leaves, stems, roots, fruit seeds, plant parts or whole parts), in particular potatoes, wherein they contain hepatitis B surface antigen (HBsAg). The HBsAg in these plants is derived from HBsAg expressed by the genetically altered plant.

"parenteral pathogen antigen" (NEPA) refers to an antigen that stimulates the body's immune response to a parenteral pathogen via the parenteral route.

HBsAg as used herein refers to hepatitis B surface antigen and is used herein as an example of an antigen of a parenteral pathogen.

The plant from which the plant material of interest is derived may be any plant, provided that it contains a parenteral patient antigen such as HBsAg. Plants can be genetically engineered to express HBsAg and other parenteral pathogen antigens. Almost any plant that can be ingested can be engineered to express HBsAg and other parenteral pathogen antigens, but the most preferred plants are food plants, such as fruit, grain and vegetable producing plants, such as banana, potato and tomato. Particularly preferred plant materials are plant materials which do not contain large amounts of acid, such as tuber plants like potatoes, since acid in certain plant materials, such as the fruits of tomatoes or citrus, can cause degradation of HBsAg. In addition, plant materials such as papaya that contain large amounts of proteases may be undesirable because these enzymes may also degrade HBsAg. As used herein, "substantial amount" refers to an amount that results in degradation of the antigen to a degree that significantly reduces the immune response.

Genetic engineering methods are known to those skilled in the art to allow expression of HBsAg and other antigens in tobacco plants, such as Mason et al, "expression of hepatitis B surface antigen in transgenic plants", Proc. Natl. Acad. Sci USA, Vol.89, pp.11749, month 12 1992. This article is cited as background. Unfortunately, tobacco is not suitable for ingestion and is therefore not physiologically acceptable. Similar methods can be used to genetically engineer other plants to express HBsAg and other parenteral pathogen antigens in accordance with the present invention. Suitable plants are in particular plants of the solanaceae family, in particular potatoes. The process of genetic alteration with respect to potato will be described in detail below.

The plants used in the present invention should contain at least 5. mu.g, preferably 7. mu.g to 15. mu.g HBsAg per gram of the plant product to be digested. Animals such as humans should ingest sufficient amounts of plant material to provide from about 10 to about 100 micrograms of hepatitis b surface antigen per kilogram of body weight. Animals such as humans should generally ingest sufficient amounts of plant material to provide about 2-5 grams of plant material per kilogram of body weight.

The immune response may be enhanced if the plant material is ingested in a series of intervals, for example 2 or 3 series of intakes each of which should be separated by at least 5 days, preferably at least 7 to 14 days.

The plant material of the present invention does not produce a significant immune response, i.e. protection from an immune response, when administered orally in the absence of the process steps claimed in the present invention. According to the invention, plant material containing HBsAg or other antigens of parenteral pathogens must be administered orally either to individuals who have previously been initially immunized, for example by parenteral injection, or in combination with an immunological adjuvant which is effective in causing a protective immune response to HBsAg or antigens of parenteral pathogens. Prior to the present invention, it was not possible to predict that an immune response against NEPA such as HBsAg could be generated by ingestion of plant material containing NEPA by individuals who had previously been primed or in combination with an immunological adjuvant.

Possible effective adjuvants include bacterial plasmid DNA, anti-HB antibodies, oligodeoxynucleotide molecules containing the immunostimulatory agent CpG, modified Cholera Toxin (CT), modified E.coli heat stable lymphotoxin, lipophilic derivatives of muramyl dipeptide (MDP-Lys (L18)), aluminum phosphate or sulfate, cytokines, or hepatitis C core protein. A large number of subjects who have received immunity against hepatitis b exhibit an immune-enhancing response when treated by the method of the present invention, e.g., 60% or more of the subjects. However, it should be recognized that there are some subjects who for unknown reasons do not develop a measurable immune enhancing response. Possible reasons for this are that these subjects, despite having previously received a primary immunotherapy, do not actually develop a strong primary immune response or have too few memory cells in the subject because the time after the primary immunization is too long. Similar results occur in existing vaccines, regardless of the mode of use, in other words, some subjects do not respond at all.

The invention can be demonstrated by the following examples.

The animals are fed with potatoes expressing and containing HBsAg and the resulting immune response against hepatitis B can be measured by enzyme-linked immunosorbent assay.

The use of potatoes as preferred examples of plants useful in the present invention is based on the following considerations. Firstly because potatoes are relatively neutral compared to other plant materials, especially certain fruits; in addition, there has been a great deal of research into the genetic characteristics of potatoes or possible transgenic modifications; more importantly, potatoes are the major food for humans, and it is estimated that 1 to 100 kilograms of potatoes are consumed annually by everyone worldwide, and that 36 kilograms are consumed annually by the U.S. average population. Further, in the united states, potatoes are listed as one of the 20 most commonly used vegetable sources under federal regulation [21 CFR 101.44(b) ] as a vegetable source. The particular cultivar of potato used to produce the currently common HBV-EPV transgenic plants, according to the particular embodiment of the invention, also transgenic plants expressing other antigens can be produced. Unprocessed or peeled potatoes from these plants are as safe as untransformed potatoes from the same potato parent and are well tolerated in other phase i clinical trials expressing intestinal antigens.

Methods for transforming plants to express HBsAg and other antigens are known to those skilled in the art, for example, U.S. Pat. nos. 5,484,719; 5,914,123 and 5,612,487, which are incorporated herein by reference.

HBsAg has previously been expressed in transgenic tobacco plants (one of the members of the solanaceae family (potato)). In this system, the expression rate of HBsAg was 0.01% of total soluble leaf protein. HBsAg particles corresponding to those derived from recombinant yeast are found in extracts of leaf tissue. When this material was administered intraperitoneally (i.p.) to mice with Complete Freund's Adjuvant (CFA), it produced anti-HBS with no significant side effects.

The HBsAg-expressing potatoes selected for use in these examples were transformed lines from S.tuberosum L.c.v.Frito-Lay 1607 HB-7. These transformation lines were designated FL-1607 HB-7 and HB 114-16. To obtain these transformation lines, the HBsAg gene of the pMT-SA clone from one HBV Chinese adr isolate was inserted into transformation plasmid vectors (pHB-7 and pHB114), which were carried into Agrobacterium tumefaciens (LBA4404), and then Solanum tuberosum L cv. "Frito-Lay 1607" was transformed therewith. The plasmid vectors used to construct the potato lines pHB-7 and pHB114-16 used in these examples both contained a gene expressing neomycin phosphotransferase (NPTII, also known as APH (3') II). The gene can likewise be integrated into the potato genome and expressed in potato cells. Coli-derived NPTII has been shown to have the same biochemical properties as plant-expressed NPTII. The E.coli-derived NPTII degrades rapidly under conditions that mimic the digestive system of mammals and does not have any deleterious effects when fed to mice with purified protein up to 5g/kg body weight. The transformed FL-1607 was treated with Agrobacterium tumefaciens and asexually propagated, and FL-1607 HB-7 and HB114-16 lines were selected based on their high-level HBsAg expression ability. Extracts of FL-1607 transformed lines were tested for HBsAg concentration by ELISA technique, with HB-7 averaging 1100ng HBsAg per gram of tuber, and HB114-16 averaging 8500ng + -2100 ng HBsAg per gram of tuber.

In addition, the extracted HBsAg spontaneously forms virus-like particles (VLPs), which settle at the same density as HBsAg VLPs from yeast. Both electrophoretic migration and Western blot analysis showed that tuber-expressed antigens were indistinguishable from those from yeast.

The number of the plants was multiplied by clonal propagation of the cell lines and potted in soil to obtain tubers to be used in the examples. The transformants were maintained by in vitro clonal propagation.

The untransformed parental potato line FL-1607 was maintained by vegetative propagation and tubers for use in the control group were produced in a pot culture. The tissues from these tubers do not express any protein that reacts with the reagents for detecting HBsAg.

Example 1

BALB/c mice were fed either peeled HB-7 potato slices or control untransformed potatoes. Each group of mice was fed with 5g of potatoes on days 0,7 and 14, respectively. The B subunit of Cholera Toxin (CT) (Sigma) was used as an oral adjuvant. 10 μ g of adjuvant was placed on potato sections (in test and control groups) and consumed with antigen for the animals. Animals thus fed HB-7 received an average of 5.5 μ g HBsAg per serving, or received an average total of 16.5 μ g HBsAg over the three servings provided.

Mice fed HB-7 developed HBsAg-specific serum IgM and IgG responses, while controls fed untransformed potatoes did not produce any antibodies. An immune response peak around 70mIU/ml was observed with the third feeding. A secondary response reaching a peak at 1700mIU/ml was observed after a single intraperitoneal inoculation of 0.5. mu.g yeast derived recombinant HBsAg (rHBsAg) in alum (normal sub-immunizing dose). The primary immunoglobulin for this response is IgG. However, no primary and secondary responses were observed in the control group fed with untransformed potatoes and CT. Without oral adjuvant no significant response to HBsAg was produced.

Example 2

The next experiment used the Frito-Lay 1607HB114-16 cell line, in which expression was driven by the 35S promoter, and the tuber expression used in these experiments averaged 8.37. mu.g HBsAg/g tuber wet weight.

BALB/c mice (5/group) were fed either HB114-16 or control non-transgenic potatoes. 10 μ g CT was added to potatoes in both groups. Feeding was repeated after one and two weeks. The average dose of HBsAg given to each mouse was 125.5. mu.g in 3 weeks. Then, 70 days after the first feeding, or 3 to 6 weeks after the primary immune response, the mice were immunized with a sub-immune dose (0.5 μ g) of rHBsAg (Merck) by subcutaneous injection administered with aluminum as an adjuvant, and returned to baseline.

At these dosage levels, an initial immune response often occurs immediately after a second meal. These immune responses continued to rise and reached a peak of 100mIU/ml around 6 weeks. The currently licensed injectable hepatitis B vaccine is considered successfully immunized in humans after three administrations to a titer of only 10 mIU/ml. These immune responses returned to baseline after 13 weeks, and the animals were given an enhanced dose of rHBsAg at 16 weeks. This will result in a rapid rise in the immune titer to greater than 3000mIU/ml, which will remain at levels in excess of 1000mIU/ml for the remainder of the experiment (40 weeks). This demonstrates that antigen-specific immune memory cells generated during primary immunization can be activated rapidly during secondary immunization (boosting).

The control animals of this experiment given non-transformed potato + CT did not produce an immune response against HBsAg and no secondary response during a secondary challenge at sub-immune doses (as described above), demonstrating the specificity of the results. Control animals given only transgenic potatoes without CT produced only low levels of primary immune response, e.g., titers of 10mIU/ml, and returned to baseline after one week. A secondary immune response of only 50mIU/ml was generated during the secondary challenge with the sub-immune dose described above and returned to the level of 10mIU/ml after only two weeks.

Example 3

Transgenic potatoes have also been used to boost pre-existing sub-immune doses of rHBsAg in mice. In this experiment BALB/c mice (5/group) were immunized subcutaneously with sub-immunizing doses of rhbsag (merck) with alum as adjuvant. After 5 weeks, each mouse was fed either HB114-16 or control non-transgenic potatoes. 10 μ g CT was added to the potatoes in both groups. The feeding was repeated one and two weeks later, and the total average dose per mouse was 125.55 μ g over a three-week period.

A secondary immune response began to appear at the third feeding and increased to a level of about 1000mIU/ml at 11 weeks after the primary prime and not yet declined. No immune response was seen when non-transgenic potatoes were fed to the control group.

Example 4

42 volunteers were tested without HIV and previously received a primary immunization with a commercial hepatitis B vaccine and had an anti-HBsAg titer of less than 115mIU/ml over time. In a randomized, double-blind study, these volunteers were fed potatoes with HBsAg and in the control group were fed potatoes without HBsAg. Group 1 was identified as a control group after unlocking the code and received three doses of 100 grams of non-transgenic potato FL-1607. Group 2 received two doses of 100 grams of transgenic potato FL-1607HB114-16 and one dose of non-transgenic potato FL-1607. Group 3 received three doses of 100 grams of transgenic potato FL-1607HB 114-16.

The current preclinical data show that (1) mice freely consume up to 25% of their body weight of potatoes by weight without any toxic effects; and (2) HBsAg in about 15 grams of potato at a dose of about 16 μ g together with an oral adjuvant is immunogenic in a prime immunization experiment. Current clinical data from other potato vaccines indicate that (1) consumption of 100 grams of raw potatoes is generally well tolerated; and (2) a person who weighs 70 kg consumes 100 g of the corresponding 0.14% of his body weight by weight. This amount is about 178 times less (by weight) than the amount consumed by the mice in the preclinical trial.

Thus, for this human test, 100 to 110 grams of potatoes were consumed per volunteer. The clinical material used in the study was designed to contain 8.5. + -. 2.1. mu.g HBsAg per gram potato. Throughout 28 days, subjects receiving two 100 gram doses of transgenic potatoes received a total dose of 1,280 to 2,120 μ g of HBsAg; subjects receiving three doses received a total of 1,920 to 3,180ug of HBsAg.

On each day of dosing (days 0,14 and 28), the dispenser took a number of potatoes per group (test and control) using a clean technique and processed to dispense a dose of 100 to 110 grams per person. Briefly, selected potatoes were washed clean, peeled, sliced and placed in a water bath of ice-water mixture. The peeling of potatoes is intended to remove potato peels containing the alkaloid solanine, which may cause abdominal discomfort or nausea and a certain bitterness. After peeling and slicing, 100 to 110 g of potatoes are weighed into each individual study according to the divided groups and the identification number (SID) of the individual to be treated. The potato peels and unused portions were collected and disposed of. The potatoes for each subject were stored in water to prevent oxidative discoloration from the time they were sliced to the time they were consumed by the subject. A portion of the treated potato samples for each set of each feeding was kept frozen for further processing to verify antigen content.

anti-HBsAg titers were measured in the subjects on the days shown in tables 1,2, and 3. The results clearly show that consumption of genetically transformed potatoes results in an increased immune response to the given HBsAg NEPA antigen. More than 60% of the subjects receiving three doses of potatoes containing the HBSAg NEPA antigen showed a significant enhancement in the immune response. It is clearly shown in the table that in many cases ingestion of plant material containing genetically expressed HBsAg NEPA can effectively boost primary hepatitis b immunity. None of the subjects in the control group who received three doses of non-transgenic potatoes had any change in antibody titer throughout the test.

TABLE 1 first group (receiving three doses of non-transgenic potato tubers) Titers (1m/ml)

Volunteers	Day 0	Day 7	Day 14	Day 21	Day 28	Day 35	Day 42	Day 56	Day 70
										1	63	51	56	67	69	74	88	89	93
2	66	78	52	62	54	74	67	69	80
										3	12	9	12	18	18	16	17	19	16
4	34	28	24	32	33	29	34	33	30
										5	104	99	83	110	120	100	99	92	92
6	72	64	73	74	78	78	63	57	62
										7	17	14	12	12	2	5	10	9	6
8	O	0	0	1	0	0	0	7	11
										9	9	11	12	11	8	7	9	9	8

TABLE 2 second group (receiving two doses of transgenic potato tubers and one dose of non-transgenic potato tubers)

Titer (1m/ml)

Volunteers	Day 0	Day 7	Day 14	Day 21	Day 28	Day 35	Day 42	Day 56	Day 70
										1	29	29	29	29	29	29	47	93	105
2	8	15	27	49	41	40	73	79	66
										3	170	161	158	144	130	144	144	132	178
4	32	32	31	34	33	23	23	42	60
										5	43	37	46	77	69	85	85	78	81
6	67	37	47	57	80	89	77	73	75
										7	11	7	114	114	136	176	191	200	136
8	104	126	262	269	318	313	357	390	445
										9	33	26	22	21	21	25	25	29	31
10	107	92	96	89	93	83	95	90	100
										11	21	22	55	112	120	219	395	458	462
12	65	68	66	63	89	103	137	258	304
										13	20	24	18	15	12	12	15	20	17
14	0	0	0	0	0	0	0	0	0
										15	97	93	112	109	128	294	454	432	347
16	26	34	197	330	353	360	707	863	790
										17	13	15	15	14	11	11	17	17	18

TABLE 3 third group (receiving three doses of transgenic potato tubers) Titers (1m/ml)

Volunteers	Day 0	Day 7	Day 14	Day 21	Day 28	Day 35	Day 42	Day 56	Day 70
										1	17	20	70	140	269	428	401	463	496
2	94	87	100	99	88	79	87	88	99
										3	33	34	32	33	27	34	31	32	34
4	9	9	53	74	74	85	64	61	60
										5	20	41	57	84	452	475	897	652	745
6	85	76	496	1212	3058	3572	4152	4526	4788
										7	13	19	19	15	28	14	20	21	24
8	120	236	282	390	605	667	1583	1717	1712
										9	72	77	137	270	349	523	1098	1226	1225
10	85	76	84	74	111	215	175	163	108
										11	40	35	39	71	119	122	330	430	342
12	56	51	59	85	252	407	520	745	834
										13	115	213	511	1054	1964	3069	2966	3449	3266
14	0	0	0	0	0	0	0	0	0
										15	9	11	14	13	13	18	11	15	18
16	0	0	0	0	0	0	0	0	0

Claims (12)

1. A method of generating an immune response to a non-enteric pathogen antigen (NEPA) in an animal that is immunoreceptive to the NEPA, characterized in that the animal is fed with a substance comprising a physiologically acceptable plant material containing the NEPA.

2. The method of claim 1, wherein the NEPA is hepatitis b surface antigen (HBsAg).

3. The method of claim 1, wherein the animal is made immunoreceptive to the NEPA by feeding the animal with the agent and an adjuvant that immunoreceptions the animal to the NEPA.

4. The method of claim 3, wherein the NEPA is HBsAg.

5. The method of claim 1, wherein the animal is a human.

6. A method according to claim 5, wherein the plant material is derived from a plant genetically engineered to express said antigen.

7. The method of claim 6, wherein the human ingests a sufficient amount of plant material to obtain about 10 to 100 micrograms of NEPA per kilogram of body weight of the human.

8. A method according to claim 7, wherein the human ingests a sufficient amount of plant material to obtain about 2 to 5 grams of plant material per kilogram of body weight of the human.

9. The method of claim 8, wherein said plant material is ingested by the human in a plurality of times, each of said times being separated from each other by at least 5 days.

10. The method of claim 9, wherein said plurality of times is 3 times.

11. A method according to claim 10, wherein the plant material is material from a plant of the solanaceae family.

12. The method of claim 11, wherein the plant is potato.