PL217229B1 - Expression cassettes, T-DNA particles, plant expression vectors, transgenic plant cells and their application to produce the vaccine - Google Patents

Description

Description of the invention

The invention concerns transgenic plant cells and the preparation of a vaccine containing the medium HBV surface antigen - M-HBsAg, directed against hepatitis B (hepatitis B), administered orally in the form of a fixed plant material and administered parenterally or intranasally in the form of a purified preparation extracted from plant material .

Obtaining a recombinant vaccine against hepatitis B, i.e. viral hepatitis B (hepatitis B) in the early 1980s was a significant success of medicine and vaccinology, as it enabled mass vaccination against hepatitis B. Despite the high effectiveness of the vaccine, reaching 90 - 95% , the global number of people chronically ill as a result of HBV (Hepatitis B Virus) infection is growing every year and now amounts to over 400 million. It is estimated that nearly 100 million of these patients may die of diseases and other complications, such as cirrhosis or primary liver cancer. At the same time, the chronic hepatitis B carrier population is a great reservoir for new infections. Epidemiological data show that nearly 1/3 of the world's population has been infected with hepatitis B. While in highly developed countries the incidence of hepatitis B is rather low - less than 0.5%, in developing countries and China the incidence rate is from 5 to 50% (Hollinger 1996, Young 2001). The incidence of hepatitis B has been and remains a serious epidemiological problem also in developed countries, where up to 20% of the population shows markers of HBV infection. In Poland, more than 50% of viral liver diseases are caused by HBV (Walewska-Zielecka 1996).

The number of chronically ill people and carriers of HBV is increasing despite the introduction of recombinant vaccines (eg Engerix-B®. Huvax B®) based on the small S subunit of surface antigen (HBsAg, Hepatitis B surface Antigen), in short the so-called small viral surface antigen, ie S-HBsAg, produced in recombinant yeast cells (rHBsAg). These vaccines, while allowing mass vaccination, do not provide sufficient protection. It has been found that a number of vaccinated people become infected and replicated the virus as a result of the emergence of mutant strains of HBV. These strains are characterized by an altered amino acid sequence within the epitope "a" of the S-HBs antigen, neutralizing all virus subtypes described so far. It is presumed that as a result of the selective pressure of vaccination antibodies, mutant variants of the "a" epitope appear, for which the vaccinated organism does not produce neutralizing antibodies. In the absence of sufficient immune protection, chronic disease develops, despite a prior response of> 10 mlU / ml of anti-HBs antibodies, considered sufficient in most countries to provide immune protection against virus infection (Cooreman 2001, Huang 2004).

Moreover, some patients after parenteral administration of the vaccine containing the small surface antigen S-HBsAg do not develop an immune response or it is insufficient. Especially the elderly, smokers, obese, men, people infected with viruses lowering immunocompetence (eg HIV) or suffering from immunological deficiencies of other causes, and patients with renal insufficiency react much worse to the classic vaccine. In extreme cases, the effectiveness of vaccination in these groups is only a few percent despite repeated vaccination with increased doses of the preparation (Singh 2003). As a consequence, these people remain susceptible to developing the disease. Therefore, for some time now, research has been conducted on the development of a new vaccine, the so-called third generation, against hepatitis B, containing S-HBsAg and one or two other HBV surface antigens, i.e. medium M-HBsAg and large L - HBsAg (Madaliński 2002, Yamada 2001, Young 2001 ). As these antigens are highly immunogenic, third generation vaccines (eg Hepacare®, Bio-Hep-B ™) induce a much larger spectrum of anti-viral antibodies compared to S-HBsAg only vaccines. Clinical trials conducted with the participation of adults, children and newborns confirm the strong immunostimulatory properties of preparations containing the full set of HBV surface antigens. The titer of anti-HBV antibodies after two immunization with such a preparation was up to three times higher compared to the results obtained after three administration of the classic vaccine containing only the S-HBs antigen. More importantly, this preparation induced an immune response in a much greater proportion of patients who responded worse to vaccines against hepatitis B: elderly, men,

And overweight people (Young 2001). In other experiments, it was shown that people who did not respond initially to the small surface antigen vaccine developed appropriate antibodies after administration of the preparation in which the medium M-HBs and large L-HBs antigens were also present. After a single dose, almost 70% (Zuckerman 1997), and after two applications of the drug, nearly 80% of patients seroconverted (Yap 1996). The greater effectiveness of the vaccine containing all envelope antigens is also confirmed by studies in which the use of a dose of such a preparation several times lower (2.5 μg) than the standard dose of the vaccine based on the S-HBs antigen caused the development of appropriate antibodies in 100% of vaccinated children (Madaliński 2002).

In addition to work on obtaining an improved prophylactic vaccine against hepatitis B, efforts are being made to develop drugs for the treatment of the chronic form of the disease, which affects up to 10% of adults, 40% of children and as many as 90% of newborns. The currently used interferon therapy brings moderately positive effects in about 30-50% of patients (Juszczyk 2000, Martin-Vivaldi 2000), and a significant percentage of people experience severe side effects (Deres 2003). In view of these problems, an approach based on attempts to modulate the immune response with preparations containing all of the HBV envelope surface antigens and / or the core antigen, ie HBcAg (Hepatitis B core Antigen), is of interest. So far, attempts to immunotherapy of chronic hepatitis B, carried out with the preparation of S-, Μ and L-HBsAg surface antigens, have shown promising results, as immunogenicity was 100 times higher than that of the standard vaccine (Yuen 2001) or the induction of a humoral immune response to HBs antigen and a certain cytotoxic response were found. in 50% of chronic HBV carriers (Couillin 1999, Pol 1998). In addition to the envelope proteins, an important role in the future therapeutic vaccine is assigned to the HBcAg core antigen, which is the capsid of the hepatitis B virus. HBc is a strong immunogen, both T-lymphocyte-dependent and T-independent, and therefore an excellent candidate for vaccine design. therapeutic agents by inducing both a strong cytotoxic response as well as a humoral anti-HBc antibody response (Heathcote 1999, Bocher 2001).

The growing number of HBV carriers, the need for mass vaccinations, as well as the insufficient effectiveness of existing preparations mean that the development of cheap prophylactic vaccines with increased effectiveness and therapeutic vaccines against hepatitis B is widely expected.

Based on the data to date, it can be assumed that the immunogenicity of a preparation containing all envelope antigens, i.e. S-, M- and L-HBsAg and / or the HBcAg core antigen, will be very similar to that caused by complete HBV virions, the so-called Dane particles. Such a preparation, being a "vaccine analogue" of the whole virus, would induce a strong immune response by eliciting antibodies against all antigens of the virus. Vaccination with this type of preparation would create a situation similar to immunization with native virus, but safe, because this preparation, being devoid of genetic material and therefore non-infectious, would not cause disease symptoms. The creation of a panel of transgenic plants producing a complete set of HBV antigens from various strains of the virus will allow for the development of an alternative vaccine of plant origin, both prophylactic and therapeutic against many strains (subtypes, serotypes) of HBV.

The main assumption of obtaining a prophylactic vaccine with increased effectiveness and therapeutic against hepatitis B administered orally, parenterally or intranasally is the expression and efficient production of highly immunogenic subunits of the S-HBs, M-HBs and L-HBs surface antigen and the HBc core antigen of various HBV strains in a transgenic plant edible for an oral vaccine or fast growing, high biomass and easy to extract for a parenteral or intranasal vaccine. Work on this issue was carried out over a number of years in several research groups (see cited literature) around the world. The so far obtained transgenic plants or tissue / cell cultures were in each case characterized by resistance to antibiotics, mainly kanamycin. At the same time, the expression level of HBsAg proteins was not high and for S-HBsAg it was in the range of 1 - 3 μg / gram of fresh weight (g FW), and occasionally it was a maximum of 16 μg / g FW, for M-HBsAg 0.01-1 μg / g FW, and for L-HBsAg in 0.01 μg / g FW. In addition, only fresh, unprocessed, ie raw, plant material was used in studies on the oral immunization of animals, possibly volunteers.

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The aim of the invention is to obtain an alternative oral, parenteral or intranasal prophylactic and therapeutic 3rd generation vaccine against hepatitis B, developed with the use of transgenic plants.

In particular, a first object of the invention is the efficient expression of the medium M-HBsAg surface antigen, belonging to the different HBV subtypes, in edible transgenic plants, e.g. lettuce, enabling the development of an orally administered vaccine, e.g. in the form of a suspension, syrup, granule, tablet or capsules. An oral vaccine in this form would be significantly easier to apply on a large scale, both as a primary vaccine and as a booster vaccine for pre-vaccinated people whose anti-HBs antibody titer has decreased, or anti-HBs antibodies are no longer detectable.

In particular, a second object of the invention is the efficient expression of the medium M-HBsAg surface antigen, belonging to different HBV subtypes, in tobacco as a species characterized by rapid growth and high biomass, allowing for efficient extraction and purification of antigens as basic components of parenterally injected vaccine. or nasally in the form of an aerosol or drops. The use of transgenic plants for the production of vaccine components would be assumed to be significantly cheaper than the currently used industrial methods of microbiological production.

Unexpectedly, the objects described above have been achieved with the present invention. Further objects of the invention are defined in the appended claims.

The subject of the invention is a transgenic plant cell capable of expressing the average M-HBsAg surface antigen of HBV strain ayw4 or adwA at a level> 4 μg / g of St. m., where it contains a T-DNA molecule consisting of T-DNA border sequences and two expression cassettes placed between them:

- a first expression cassette consisting of the sequence encoding the M-HBsAg protein of the HBV strain aywA or adw4 and controlling its expression of the CaMV 35S RNA promoter sequences with a single enhancer and the synthinopaline transcription terminator (NOSt), preferably containing at least one of the sequences: SEQ ID No . 4 and SEQ ID No. 5, and

- a second expression cassette consisting of a sequence encoding a phosphinothricin herbicide resistance gene, preferably the bar gene, and regulatory sequences controlling its expression, preferably a nopaline synthase promoter (PNOS) sequence and a g7t transcription terminator, preferably containing the sequence SEQ ID No. 16.

The cell according to the invention is preferably a lettuce or tobacco cell.

Another object of the invention is the use of a plant cell according to the invention for the production of an oral vaccine against hepatitis B.

The use according to the invention is characterized in that the plant cells used are preferably in the form of plant biomass, in particular freeze-dried plant material, preferably lettuce.

Preferably, the use according to the invention is characterized in that the produced vaccine has a form selected from: suspension, syrup, granules, tablets, capsules.

Another object of the invention is the use of a plant cell according to the invention for the preparation of a parenterally or intranasally administered vaccine against hepatitis B.

In one embodiment, the plant cells of the invention, preferably tobacco, used, produce HBV mean M-HBsAg surface antigen proteins that can be extracted and purified.

In a further embodiment of the invention, the produced vaccine is preferably in the form selected from parenteral injection or intranasal aerosols or drops.

Detailed Description of the Invention

Disclosed was a transgenic plant cell and regenerated and descendant plants of subsequent generations of lettuce and tobacco, as well as their vegetative clones, having phosphinothricin resistance and herbicide derivatives and capable of expressing the M-HBsAg protein of HBV strain ayw4 or adw4 at a level> 4 μg / g st. m. (FW), wherein the cells contain a T-DNA molecule from the expression vector composed of border sequences and two expression cassettes interposed between them:

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- The first, consisting of the sequence coding the mean surface antigen subunit (M-HBsAg) of HBV strain ayw4 or adw4 and regulatory sequences controlling their expression, preferably the Cauliflower mosaic virus 35S RNA promoter (CaMV) and the nopaline synthase terminator (NOSt). They preferably have the sequences shown as SEQ ID No. 4. SEQ ID No. 5.

- The second, consisting of a sequence encoding a phosphinothricin resistance gene, preferably the bar gene, and regulatory sequences controlling its expression, preferably a nopaline synthase (PNOS) promoter sequence and a g7t transcription terminator shown as SEQ ID No. 16.

The plant material derived from lettuce can be used as an oral vaccine against HBV and consequently against hepatitis B. The plant material according to the invention, after dehydration by freeze-drying under deep freeze conditions, maintains at room temperature the native structure of the M-HBsAg antigen protein and immunogenicity for a period of at least 12 months and can be used to obtain an oral hepatitis B vaccine in the form of a suspension, syrup, granules, tablets or capsules. Tobacco-derived plant material can be used as a raw material for the extraction and purification of the M-HBsAg antigen as a component of the vaccine against HBV and consequently against hepatitis B, either parenterally in the form of intramuscular or intraperitoneal or subcutaneous injection, or in the form of nasal aerosol or droplets.

Furthermore, the use of a lettuce plant cell according to the invention for the production of an oral hepatitis B vaccine was disclosed. The lyophilized plant material containing the S-HBsAg antigen administered orally in the form of a suspension to experimental animals elicited an immune response of the mucous membranes characterized by the production of IgA anti-HBs antibodies. and a systemic response characterized by the production of anti-SHBs IgA and IgG antibodies. In particular, the lyophilized plant material according to the invention, administered orally to experimental animals once previously immunized orally, enhances the immune response of the mucous membranes, characterized by the production of anti-HBs IgA antibodies, and enhances the systemic response characterized by the production of anti-HBs IgA and IgG antibodies. It can be assumed with high probability that a similar immunizing effect will take place for plant material containing M-HBsAg ayw4 / adw4.

Preferably, the produced oral vaccine is in a form selected from: suspension, syrup, granules, tablets and capsules. Preferably, the granules, tablets and capsules formed from the transgenic lettuce freeze-dried powder according to the invention retain at room temperature the native structure of the M-HBsAg antigenic protein and immunogenicity for a period of at least 12 months.

The use of a tobacco plant cell of the invention for the preparation of a parenteral or intranasal vaccine against hepatitis B is also disclosed.

On the basis of the current state of knowledge, it can be assumed with high probability that the M-HBsAg antigen subjected to extraction and purification, preferably from plant material according to the invention, administered by parenteral injection (intramuscular, intraperitoneal, subcutaneous) will induce a systemic immune response characterized by the production of antibodies anti-HBs, mainly from the IgG class and other classes, as well as this antigen administered intranasally in the form of an aerosol or droplets through the mucosa of the nasal cavity will induce an immune response of the mucous membranes characterized by the production of IgA anti-HBs antibodies and a systemic response characterized by production of anti-HBs antibodies, mainly IgG and other classes.

With respect to the results of the prior art, the present invention represents a significant novelty and substantially innovative approach to the preparation of a 3rd generation oral, parenteral or nasal hepatitis B vaccine, comprising the following elements:

- transgenic lettuce plants that produce M-HBsAg at levels above 4 μg MHBsAg / g FW and up to 35 μg / g FW and do not contain marker genes for antibiotic resistance but resistant to phosphinothricin herbicides, e.g. Basta, and therefore do not pose a risk of potential extension antibiotic resistance of the human microflora

- a method of vegetative propagation of transgenic lettuce plants producing the vaccine M-HBsAg protein in a way that ensures reproducible and multiple generation of plant clones with at least 80% of the variation in the vaccine protein content does not exceed 20%, and in addition for at least 80% of the clones the protein content HBV does not change from the starting plant. The obtained vegetative clones are therefore statistically equal6

The specific content of vaccine proteins, which allows the industrial production of plants with a high and constant content of M-HBsAg protein as a material for the production of an oral vaccine

- obtaining for the first time a prototype of an oral vaccine in the condensed form of a lyophilisate from the transgenic lettuce producing M-HBsAg, the initial intermediate for the oral form of the vaccine, such as: lyophilisate suspension, syrup, granules, tablets or capsules

- transgenic tobacco plants producing M-HBsAg at levels exceeding 4 pg M-HBsAg / g FW and up to 35 μg / g FW, which is the basis for efficient production of the M-HBsAg antigen by extraction and purification for parenteral vaccine on an industrial scale or intranasal, and at the same time resistant to phosphinothricin herbicides, e.g. Basta, which allows them to be cultivated under field conditions.

One of the preferred embodiments of the invention are the pKHBMBAR and pKHBMadwBAR vectors [Fig. 1] for obtaining plants resistant to phosphonotricin herbicides, e.g. Basta, and simultaneously expressing the heterologous M-HBsAg protein constitutively, mainly in leaves.

An important feature of the vectors is the T-DNA containing two expression cassettes, determining the expression of the immunogenic protein of the M subunit of the HBs surface antigen (subtype ayw4 / adw4) of the hepatitis B virus (HBV) and phosphinothricin acetyltransferase, which determines resistance to phosphinothricin, which is a component of some herbicides o non-selective action, eg Basta. The first cassette contains the sequence encoding the antigenic M-HBsAg protein under the control of the regulatory sequences: the CaMV 35S RNA promoter with a single enhancer and the nopaline synthase transcription terminator (NOSt). The second cassette contains the coding sequence for the bar gene under the control of the nopaline synthase (PNOS) promoter and the g7t transcription terminator. Another important feature of the constructed T-DNA is the presence of said coding or regulatory sequences only in one copy and in a specific orientation with respect to each other [Fig. 1]. The lack of repetitions of sequence motifs eliminates recombination within the introduced T-DNA and the balanced transcription of individual transgenes. The structural features of T-DNA thus contribute to reducing the phenomenon of gene silencing, and thus to greater stability of T-DNA within the genome of transgenic plants, which is particularly important in relation to lettuce, the genome of which is relatively variable and rearranged (McCabe 1999) . Consequently, the structural features of T-DNA contribute to the stable expression of M-HBs and bar transgenes in plant cells.

The invention discloses transformed lettuce and tobacco cells and plants regenerated therefrom, and plants of subsequent generative and / or vegetative generations, characterized by simultaneous expression of the HBV ayw4 / adw4 M-HBsAg surface antigen subunit protein and resistance to phosphinothricin herbicides. The resistance of plants to herbicides enables the use of such a product as a starting material for an oral vaccine or the cultivation of plants in field conditions in accordance with the applicable requirements and recommendations. Transformation of lettuce and tobacco with the vectors described in the invention [Fig. 1] provides for obtaining lines of transgenic plants [Fig. 2], which are characterized by resistance to phosphinothricin herbicides, e.g. Basta, and the expression of the M-HBsAg protein at the level of 1 4 μg / g FW to 35 μg / g FW [Fig. 3-5]. These features persist both in the primary regenerants (T0 generation) and in the first (T1) and in subsequent generations, as well as in clones of lettuce plants [Fig. 5, Tab. 1] vegetatively propagated according to the developed method [Fig. 5]. Lines of transgenic plants were analyzed by PCR to initially confirm the presence of the transgene sequence in genomic DNA as well as to determine / confirm the Mendelian 3: 1 mechanism of inheritance of the transgene in transgenic plants [Fig. 2A]. Then, the genomic DNA of plants from individual lines was hybridized with a probe complementary to the sequence of the M-HBs transgene according to the Southern blot method [Fig. 2B] to determine the number of T-DNA integration sites containing transgenes. Plants of the T0 and T1 generations and their vegetative clones were also tested for the expression of the M subunit protein of the HBs surface antigen by means of qualitative and quantitative sandwich enzyme immunoassay or sandwich ELISA using specific antibodies from Biodesign and a specialized kit from Abbott [Fig. 3], as well as using the Western-blot method [Fig. 4]. The M-HBsAg antigen protein produced in plants is characterized by a native structure, the ability to glycosylate and retains the ability to assemble into highly immunogenic subviral particles, or virus-like (VLPs), or smaller aggregates, as evidenced by the ELISA results. , including by an Abbott kit targeting the detection of the epitope "a" of HBsAg proteins structured into VLPs / aggregates. In turn, among those observed in the Western-blot

There are bands corresponding to the different forms of the M-HBsAg protein, and glycosylated forms and dimers are also found, which are the first step in the assembly of VLPs. Such particles were also observed using the Jeol JEM1200EXII transmission electron microscope directly in the mesophyll cells of tobacco leaves [Fig. 6], prepared using standard serial section techniques and contrasting with slight modifications (Kocjan 1996).

An aspect of the invention is also a method of vegetative propagation (micro-propagation, propagation) in an in vitro culture of transgenic lettuce plants ensuring reproducible and multiple production of plants with high yield, i.e. 8-13 progeny obtained from one starting plant [Fig. 5]. In the obtained clones, the expression level of the M-HBsAg protein is balanced, i.e. with non-statistically significant deviations or the differences in the content of the vaccine protein do not exceed 20% [Tab. 1], and in addition, for at least 80% of the clones, the level of HBV proteins is comparable with the starting plant and with plants obtained from seeds [Fig. 5, Tab. 1]. The method of micro-propagation according to the invention enables the industrial multiplication of plants characterized by the desired high level of expression, ensuring at least several dozen-fold increase in the amount of material with a high and statistically constant amount of antigenic proteins, as a starting material for the production of an oral vaccine against hepatitis B.

In addition, the invention discloses the use of a transgenic lettuce resistant to phosphinothricin and producing the M-HBsAg antigen protein as a component to obtain a 3rd generation prophylactic and therapeutic oral hepatitis B vaccine, i.e. containing the full complement of HBV structural proteins. Lettuce (Lactuca sativa L.) is a species which, unlike the plant species used so far, is characterized by properties essential for the preparation of an orally administered vaccine. Lettuce leaves, unlike the vast majority of cultivated plants, are suitable for direct consumption without the need for treatment, especially heat treatment. They also do not contain any anti-nutritional or allergenic substances, the presence of which could be a factor limiting consumption. A significant feature of the transgenic lettuce producing M-HBsAg and at the same time resistant to herbicide is the suitability and convenience of its use as a starting material for the production of a vaccine against hepatitis B administered orally.

The invention also discloses lyophilized plant material, its use to obtain a third generation oral vaccine in the form of derivatives: suspension, syrup, granules, tablets and capsules containing, inter alia, M-HBsAg vaccine protein, as well as the mentioned forms of lyophilisate derivatives and the method of their preparation from plant material.

An effective third generation oral vaccine against hepatitis B, in contrast to the solutions used so far [see the cited literature], has a condensed form of a powdered lyophilisate applied in the form of a suspension, syrup, granules, tablets or capsules. The starting material for the preparation of the condensate are leaves of individual lettuce lines, containing antigenic proteins, including M-HBsAg, which are lyophilized. The material from individual lines is ground into a powder, and then can be formulated into a preparation containing specific amounts of antigenic proteins, including M-HBsAg, of various HBV subtypes. suitable for eliciting the desired immune response. This preparation using a physiological saline solution or a similar buffer, e.g. PBS, or after the addition of excipients, can be converted into a suspension or syrup, granules, tablets or capsules [Fig. 6A]. Powdered, freeze-dried plant material and a suspension / syrup or granular / tablet / capsule form of the 3rd generation oral vaccine against hepatitis B [Fig. 6A], in contrast to raw plant material, are characterized by significant features that enable:

- fixation of plant material while maintaining a high content of M-HBsAg vaccine protein of various subtypes,

- concentrating the dose of the vaccine agent, i.e. the M-HBsAg protein of various subtypes, to a level sufficient for oral immunization,

- administration of a constant standardized dose of M-HBsAg thanks to a) control of the level of antigenic protein at individual stages of preparation of the intermediate - lyophilisate containing antigen, b) control of the level of antigenic protein at the stage of suspension or syrup preparation, granules and tablets or capsules, and c) selection of appropriate starting materials and filling components,

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- determining the appropriate proportions of M-HBsAg of various subtypes to obtain an appropriate level of protective immune response, or to obtain an appropriate therapeutic effect of the chronic form of hepatitis B, caused by the most common strains of HBV,

- establishing an effective vaccination protocol by controlling the dose and timing of immunization,

- easy storage of powdered lyophilisate as well as granules and tablets or capsules at room temperature while maintaining the stability of M-HBsAg for a period of at least 12 months,

- a simple way to vaccinate by mouth by drinking or swallowing.

The invention further discloses the use of a transgenic tobacco resistant to phosphinothricin and producing the antigenic M-HBsAg protein for the preparation of a prophylactic and therapeutic 3rd generation parenteral or intranasal vaccine against hepatitis B. Tobacco (Nicotiana tabacum L.) is a plant species characterized by the properties of essential for protein production. The production of heterologous proteins in herbicide-resistant tobacco is characterized by high profitability, which results from low cultivation costs in both greenhouse and field conditions, rapid growth, high biomass, and the stability and efficiency of the expression level of heterologous proteins. HBV antigen proteins, including M-HBsAg, forming VLPs or aggregates in tobacco leaves [Fig. 6B] can be extracted and purified from the plant material using standard physical methods, e.g. centrifugation and physicochemical methods, e.g. affinity chromatography. The purified M-HBsAg antigen of various subtypes can then be used to prepare a preparation containing specific proportions of individual antigens to obtain an appropriate level of immune response to protect against hepatitis B, or to obtain an appropriate therapeutic effect of chronic hepatitis B. Preparation of vaccine proteins after the addition of adjuvants, stabilizers and other substances can be used for vaccination according to the procedures used by parenteral injection (intramuscular, intraperitoneal, subcutaneous) or through the mucous membranes in the form of an aerosol or intranasal drops [Fig. 6B]. A significant feature of the transgenic tobacco producing M-HBsAg and at the same time resistant to herbicides is the suitability and convenience of its use as a starting material for the production of a third generation prophylactic and therapeutic hepatitis B vaccine containing purified antigens and administered parenterally or intranasally.

The 3rd generation vaccines obtained according to the invention can be used for oral vaccination against hepatitis B with suspension, syrup, granules, tablets or capsules made of powdered lyophilisate derived from lettuce containing M-HBsAg antigen, or parenterally by injection or intranasally with an aerosol or drops containing M-HBsAg antigen extracted and purified from tobacco tissues. The obtained preparations can also be used in combined orally-parenteral or oral-intranasal or intranasal-parenteral immunization.

The M-HBsAg surface antigen and other HBV antigens are potent immunogens that elicit an immune response in a variety of mammalian species, including mice, chimpanzees, and humans. The immune response may be of the cellular type involving the generation of specific cytotoxic lymphocytes, or the humoral type involving the generation of specific antibodies. It is assumed that in humans and chimpanzees, an appropriately induced humoral response is sufficient to protect against the disease after exposure to the virus. Depending on the method of administering the immunogen, the immune reaction to HBV antigens includes the formation of antibodies mainly of the IgG class and other classes during parenteral immunization (intramuscular, intraperitoneal or subcutaneous injection) and IgG and IgA during immunization through the mucous membranes of the gastrointestinal tract, i.e. by the oral method (see by suspension, syrup, granules, tablets or capsules) and through the epithelium of the respiratory tract, i.e. by the nasal method (by means of an aerosol or drops), possibly also by the vaginal, rectal or skin routes.

An essential element of the method of vaccination with the M-HBsAg antigen produced in plants according to the invention is immunization through the intestinal mucosa in the case of an oral vaccine made from lettuce or the nasal mucosa in the case of a nasal vaccine prepared on the basis of an antigen extracted and purified from tobacco.

According to the available knowledge (Mowat and Viney 1997, Langridge 2000, Mowat 2003, Poonam 2007), antigenic proteins in contact with mucous membranes induce locally associated cells of the immune system, including GALT (Gut Associated Lyphoid Tissue) in the intestine, NALT (Nasal Associated Lymphoid Tissue) in the nasal mucosa and BALT (Bronchus Associated Lymhoid Tissue)

Distal airways and others. In the structure of GALT, NALT or BALT there are specialized cells (e.g. M cells in Peyer's patches in the intestinal mucosa) which take up degraded and complete antigen proteins and viruses from the external environment, i.e. the lumen of the intestine or the nasal cavity and distal respiratory tract, bacteria or protozoa. The antigens or microbes are then transported to cells of the immune system, including Antigen Presenting Cells (APCs), i.e. dendritic cells and macrophages, and T and B lymphocytes, both locally within the mucosa and to the peripheral lymphoid organs in the result of systemic circulation. As a result of the presentation of antigens to Th lymphocytes present in the mucosa of the intestine or airways, a local humoral immune response occurs through the activation of Th lymphocytes, the secretion of cytokines and the subsequent activation of B lymphocytes manifested by the secretion of antigen-specific IgA antibodies by the mucosa. In subsequent stages, Th lymphocytes pass into the blood and, after reaching the peripheral organs of the immune system, by a similar activation path they induce a systemic humoral response in the IgG class and other classes (IgM, IgA), and possibly a cellular response manifested by the formation of a subpopulation of antigen-activated Tc cytotoxic lymphocytes, as well as immune memory.

As previously shown, the S-HBsAg antigen is immunogenic in mice after the lettuce plant material containing S-HBsAg has been fixed by lyophilization and made into suspension immediately prior to administration. The immune response to the S-HBsAg antigen arises from the primary oral administration of the antigen (priming) followed by repeated one or more immunizations that result in a boosting response. It can be assumed with high probability that a similar immune response after oral administration will be induced by the third generation vaccine preparation, i.e. containing the S-HBsAg antigen and other HBV antigens, including M-HBsAg. The premise of this assumption is the structural relationship of HBsAg surface antigens, manifested by the ability to create higher order structures, i.e. VLPs or aggregates, including those formed by M-HBsAg. The similarity of the mucosal immune response mechanisms within GALT and NALT / BALT (Brandtzaeg 2007, Poonam 2007) suggests that the third generation nasally administered vaccine preparation containing purified M-HBsAg and other HBV antigens will induce a local and systemic immune response.

The M-HBsAg antigen produced in plants according to the invention and purified can also be used as a component of conventional vaccines, i.e. by parenteral injection according to the procedures used. An alternative method of producing vaccine antigens in plants is essential for this method of vaccination. The production of the M-HBsAg antigen of different subtypes in the distinct lines of transgenic plants according to the invention allows the optimal determination and standardization of the proportions of the individual vaccine immunogens for a highly effective 3rd generation prophylactic and therapeutic hepatitis B vaccine, whether oral, intranasal or parenteral. The possibility of using M-HBsAg and other antigens, the two most common (ayw4, adw4) or other common HBV strains will allow to determine the optimal parameters of effective immunization, i.e. the number and amount of antigen doses, periods between individual immunizations, the use of adjuvants, etc. vaccines will take place by appropriate selection of the proportions of the M-HBsAg antigen and the remaining, purified, in the case of an intranasal or parenteral vaccine, or in the case of an oral vaccine, present in fixed plant materials, i.e. powdered lyophilisates containing specific amounts of antigens.

The type of vaccine according to the invention, i.e. oral in the form of a suspension, syrup, granules, tablets, capsules, or intranasally in the form of an aerosol or drops, or parenteral in the form of an injection, allows a wide range of immunization methods depending on the age of the immunized animals and humans, general condition and immunocompetence, sex, body weight, and other parameters influencing the immune response. The diversity of the types of vaccine according to the invention also allows the use of one method or combination of different immunization methods, i.e. orally-intranasal, oro-parenteral, intranasal-parenteral, etc., in order to obtain the maximum level or range of the humoral immune response, i.e. elicitation of IgG or IgG antibodies. and IgA, or the cellular response. As vertical transmission of HBV is assumed to involve the mucosa as gateway to infection, a combination of immunization methods linking the entero-mucosal, respiratory-mucosal and parenteral pathways with different forms of prophylactic vaccine

The invention will potentially provide a more tight immune defense in families of people infected with HBV or in chronic HBV carriers where vertical transmission of the infection may occur.

To better illustrate the essence of the invention, the present description has been enriched with the sequence listing and figures.

Sequence No. 4 (SEQ ID No. 4) shows the nucleotide sequence of the P35S-MHBsayw4-NOSt expression cassette contained in the binary vector pKHBMBAR for plant transformation described in the examples.

Sequence No. 5 (SEQ ID No. 5) shows the nucleotide sequence of the P35S-MHBsadw4-NOSt expression cassette contained in the binary vector pKHBMadwBAR for plant transformation described in the examples.

Sequence No. 16 (SEQ ID No. 16) shows the nucleotide sequence of the PNOS-bar-g7t expression cassette found in the binary vectors pKHBMBAR and pKHBMadwBAR described in the examples.

₂

Table 1 presents the results of the analysis (test χ, test F) of the significance of changes in the M-HBsAg protein level in plants-vegetative clones obtained during multiplication (micro-propagation, micropropagation) and the results of the analysis of the variability of the M-HBsAg level in clones and control plants with a natural development cycle . The results were analyzed statistically (after log transformation, separately for each parental line) by analysis of variance according to the mixed model taking into account the constant effects of propagated progeny (T1 generation) and the random effects of vegetative clones and allowing heterogeneity of variance between clones.

Figure 1 shows the construction scheme of the pKHBMBAR and pKHBMadwBAR vector used to transform lettuce and tobacco containing the M-HBs coding sequence of the ayw4 or adw4 strain ayw4 or adw4 hepatitis B medium surface antigen (HBV) under the control of the constitutive CaMV 35S RNA promoter (P35S) and the terminator nopaline synthase (NOSt) and the bar gene sequence encoding the phosphinothricin acetyltransferase under the control of the nopaline synthase (PNOS) promoter and the g7t terminator, determining the resistance of transgenic plants to phosphinothricin herbicides.

Designations: M-HBs, M-HBsAg - sequence coding the HBV ayw4 or adw4 medium surface antigen, P35S - 35S RNA promoter of cauliflower mosaic virus (CaMV), NOSt - nopaline synthase gene terminator, BAR - coding sequence for the bar gene - phosphine acetyltransferase, PNOSotricin nopaline synthase gene promoter, g7t - g7 terminator, RB, LB - right and left T-DNA border sequence, NPT III - neomycin phosphotransferase gene, GUS - β-glucuronidase coding sequence, GUS-INT - β-glucuronidase coding sequence with intron; (ayw / adw) - identical plasmid construction with the sequence coding the HBV antigen of the ayw4 and adw4 strains, the name of the binary vector without the index - the vector contains the sequence coding the HBV antigen of the ayw4 strain, the name of the binary vector with the index "adw" - the vector contains the sequence coding the HBV antigen of the strain adw4.

Figure 2 shows the results of the analysis of the presence of transgenes containing the M-HBsAg antigen coding sequences in the genomic DNA of the progeny lettuce transformants (T1 generation) and tobacco by PCR using primers specific to the coding sequence and by the Southern-blot method using specific probes. to individual coding sequences.

A - electrophoregram of the products of the M-HBs transgene amplification reaction in lettuce plants of the lineage

LT9A-16B.

Marks of electrophoregram paths: M - DNA fragment size marker (200 bp DNA Ladder, MBI Fermentas), 1 - 27 - analyzed plants, N - negative control - DNA of non-transgenic plant, P - positive control - pKHBMBAR plasmid.

B- Southern-blot analysis of the Tt8 tobacco line for the M-HBs transgene.

Blot lane markings: 8-4 / 1 - 8-9 / 15 - plants analyzed, N - negative control - DNA of non-transgenic plant, P - positive control - pKHBMBAR plasmid of grass. EcoR I and EcoR I and Kpn I.

Figure 3 is a graph of the analysis of M-HBsAg antigen protein content by enzyme immunoassay ELISA in progeny of lettuce transformants (T1 generation) of LT9A-16B (A) and tobacco lines Tt8 (B) containing the M-HBsAg coding sequence under the control of the constitutive promoter 35S. Protein content was expressed in μg / g fresh weight as the arithmetic mean with standard deviation from 3 determinations.

PL 217 229 B1

Explanations: T0 - antigen content for a T0 generation plant (parent), T1 - arithmetic mean of HBV antigen content for T1 generation plants (progeny).

Figure 4 shows the result of the analysis of the M-HBsAg antigen protein forms in the leaves of the progeny of lettuce transformants (A) and tobacco (B) by Western-blot using specific rabbit polyclonal antibodies.

A - Western-blot analysis of the M-HBsAg protein in leaf extracts of lettuce plants (1E / 4 - 18A / 8) using the murine monoclonal antibody Mab C86600 (Biodesign), specific for the S domain of HBsAg proteins.

B - Western-blot analysis of the M-HBsAg protein in leaf extracts of tobacco plants (8-4 / 1 - 8-9 / 19) using the rabbit polyclonal Pab B65811R antibody (Biodesign), specific for the S domain of HBsAg proteins.

Blot path markings: M - protein mass standard (MBI Fermentas), N - non-transgenic plant - negative control, P - M / L-HBs antigen obtained from prof. R. Schirmbeck from the University of Ulm, Germany - positive control

Forms of M-HBsAg protein:

p31 - monomer of non-glycosylated M-HBs protein, 31 kDa gp33 - monomer of glycosylated M-HBs protein, 33 kDa

Figure 5 shows the process and yield of vegetative propagation in an in vitro culture, and the production level of M-HBsAg protein in transgenic lettuce for the industrial propagation of plants as starting vaccine materials.

A - vegetative reproduction of transgenic lettuce in in vitro culture: left side - culture on LRM2 medium, in the center - single polyclinic, right side - rooted vegetative clones.

B - efficiency of vegetative reproduction in an in vitro culture of a transgenic lettuce line producing M-HBsAg.

Marking for B: mean - arithmetic mean for a given culture passage, MG - geometric mean for all culture passages, control - geometric mean for non-transgenic lettuce.

C - content of the M-HBsAg antigen protein, expressed in μg / g fresh weight as the arithmetic mean together with the standard deviation from 3 determinations, performed by means of an enzyme-linked immunosorbent assay, in leaves of vegetatively propagated progeny lettuce transformants of the LT9A-15E line.

Figure 6 illustrates the use of transgenic plants producing the M-HBsAg antigen protein for the preparation of prophylactic and therapeutic derivatives of third generation hepatitis B vaccines.

A - the use of transgenic lettuce to obtain a powdered lyophilisate as a semi-finished product for the production of an oral vaccine in the form of a suspension or syrup and in the form of granules, tablets or capsules

B - the use of subviral particles (VLPs) produced in the mesophyll cells of tobacco leaves, which, after extraction and purification and the addition of excipients, can be used in the form of a preparation administered by parenteral injection or in the form of a nasal preparation applied in the form of an aerosol or droplets. In plant cells, subviral particles accumulate in vesicles, then closed in cisterns of the endoplasmic reticulum - ER. The micrographs were made using the Jeol JEM 1200 EXII transmission electron microscope.

The following embodiments are provided merely for the purpose of better elucidating particular aspects of the invention and should not be equated with the entire scope thereof, as defined in the appended claims.

Example 1. Construction of pKHBMBAR and pKHBMadwBAR vectors for transformation of lettuce and tobacco

The preparation of vectors containing the sequences encoding the M-HBsAg antigen protein of the HBV ayw4 and adw4 subtypes under the control of the 35S promoter included the following steps:

The nopaline synthase (NOSt) terminator (NOSt) was amplified on a template of total DNA of Agrobacterium tumefaciens from a nopaline strain C58 by PCR (Croy 1993). The sequences recognized by the restriction enzymes Pst I, Xho I from the 5 'end and Hind III from the 3' end were simultaneously introduced into the NOSt sequence. The terminator was then cloned into the pUC18 plasmid (MBI Fermentas, Yanisch-Perron 1985, Genebank L09136) to obtain p18PNOSt and then sequenced.

PL 217 229 B1

The previously cloned CaMV 35S promoter (P35S) from the p35SGUS-INT vector (Vannaceyt 1990) was adapted in the pBluescript KS plasmid (Stratagene, Alting-Mees and Short 1989, Genebank X52327) by removing the Pst I restriction site at the 5 'end of the promoter by digestion with the restriction enzyme Pst I, degradation of 3 'sticky overhangs with T4 DNA polymerase followed by religion - plasmid pKSP35SGI was finally obtained.

The 35S promoter from plasmid pKSP35SGI was cloned into p18PNOSt - resulting in the vector pMG2A.

Using the PCR method, the M-HBs coding sequence (pos. 3173-837) was amplified on the template of the previously obtained plasmid pHBV312, containing the complete genome of the Polish HBV ayw4 isolate (Płucienniczak 1994a). Similarly, the M-HBs coding sequence of the adw4 strain (Płucienniczak 1994b) - M-HBsadw (positions 3212-837) was amplified. At the same time, with the help of appropriate oligonucleotides, substitutions of certain nucleotides were made, thus removing the motifs located within the sequence, recognized by the restriction enzymes EcoR I, BamH I and Pst I, while maintaining the original codon system for amino acids. BamH I restriction sites from the 5 'end and Pst I from the 3' end were simultaneously introduced into the antigen coding sequences of the individual subtypes. The modified sequences were cloned into pGEM-T (Promega, Marcus 1996) to give rise to pGTHBM and pGTHB-Madw and then sequenced.

The individual subtype antigen coding sequences from pGTHBM and pGTHBMadw were cloned into the pMG2A vector, yielding pMG2AHBM and pMG2AHBMadw, respectively.

Prepared expression cassettes: P35S-MHBsayw-NOSt and P35S-MHBsadw-NOSt, were transferred to the pGPTV-BAR vector (Becker 1992) while removing the GUS-NOSt fragment, obtaining the pKHBMBAR and pKHBMadwBAR vectors for plant transformation.

Plant transformation vectors were prepared using restriction enzymes, Taq DNA polymerase and other reagents from MBI Fermentas. The schematic diagram of the construction of binary vectors according to the invention is shown in detail in the diagram [Fig. 1].

The prepared binary vectors were introduced into Agrobacterium tumefaciens cells of LBA4404 and EHA105 strains. The presence of the plasmid in the Agrobacterium clones was checked by PCR using primers specific to the sequences encoding the individual M-HBsAg protein subtypes.

Example 2. Determination of M-HBsAg antigen in transgenic lettuce and tobacco plants by ELISA method

The content of the M-HBsAg antigen in plant tissues was determined by the ELISA method with the use of: primary anti-preS2 C8A031M monoclonal antibody (Mab) (Biodesign) and anti-HBs B65811R primary polyclonal antibody (Pab) (Biodesign). The developed test enables the determination of the total level of the M-HBsAg protein in the tested material, both formed into VLP particles or aggregates of several dozen molecules, as well as individual subunits.

For the determination of M-HBsAg in transgenic plants, extracts were prepared by grinding leaf samples in a gradual added buffer in a volume equal to fifty times the weight of the sample (1 mg = 50 μΐ buffer). The extraction used a buffer with the following composition: 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, 10.3 mM Na2SO3, 2% PVP40000, 0.2% BSA, 1% Tween® 20, pH = 7.4. The grinded samples were centrifuged for 5 min at 10,000 rpm at room temperature. 20 μl of the extract for determinations was taken from the supernatant.

ELISAs were performed on Nunc-Immuno Plate F96 Maxisorb from NUNC ™ using a Model 1575 washer from Bio-Rad. The plate was coated overnight at 4 ° C with the monoclonal antibody C8A031M in carbonate buffer at a concentration of 0.5 µg / ml. The plate was washed 3 times in PBST buffer and then blocked for 60 - 90 minutes at 25 ° C with a 5% solution of skim milk in PBS buffer. The plate was washed as above and samples of the plant extracts were applied which were then diluted in PBS in a ratio of 1/1 to obtain a 50-, 100-, 200- and 400-fold dilution, respectively. The plate was incubated for 45 min at RT. on a shaker at 400 rpm. After removing the reagents and rinsing three times as above. Anti-HBs Pab B65811R diluted 5000x in PBS was applied and incubated for 45 min at RT. 400 rpm After removing the reagents and washing three times as above, biotinylated monoclonal anti-rabbit Fc antibody (Sigma) diluted 20,000x in PBS was applied to the plate and incubated for 45 min at RT. 400 rpm After removing the reagents and washing three times as above, the plate was loaded with alkaline phosphatase-conjugated extravidin (Sigma) diluted 4000x in PBS buffer and incubated for 45 min.

PL 217 229 B1 at room temperature 400 rpm After removing the reagents and washing it three times, the substrate for alkaline phosphatase, ie p-nitrophenyl phosphate (pNPP, Sigma), was applied. Most of the reagents were applied in a volume of 100 µl / well, except for the blocking and rinsing step where a volume of 400 µl was applied. After 1 h and the reaction was stopped, the absorbance was read at 405 nm with a Model 680 microplate reader from Bio-Rad. The absorbance was converted into the content of a given antigenic protein according to the pattern of a calibration curve performed for each determination with the use of an appropriate standard, i.e. the M / L-HBsAg preparation obtained from prof. R. Schirmbeck from the University of Ulm in Germany.

The content of M-HBsAg antigen proteins was determined at least three times during plant development in ex vitro conditions. The average level of a given protein was calculated as the arithmetic mean together with the standard deviation from partial determinations [Fig. 3, 5].

The reagents for the analyzes were, in addition to the antibodies mentioned, from POCh and Sigma.

Example 3. Detection of M-HBsAg antigen in transgenic lettuce and tobacco by Western-blot method

The M-HBsAg antigen protein was detected in the transgenic plants by Western-blot method using specific antibodies. M-HBsAg protein was detected with monoclonal antibody (Mab) C86600M and polyclonal antibody (Pab) B65811R (Biodesign).

For Western-blot analysis, plant extracts were prepared by grinding lettuce leaf samples five times in volume (1 mg = 5 µl) in PBS buffers with 0.5% Tween® 20 detergent. Denaturing sample buffer was added to the ground tissues (Laemmli 1970) with 50 mM addition of DTT and incubated for 15 min at 65 ° C. Samples of extracts along with the M / LHBsAg protein standard (obtained from Prof. R. Schirmbeck, University of Ulm, Germany) and a protein size marker (MBI Fermentas) were applied to a 12.5% denaturing polyacrylamide gel and ejectrophoresis was carried out in Laemmli buffer. Proteins were transferred from the gel to a nitrocellulose membrane by the "wet" electrotransfer method in a Bio-Rad apparatus. After washing three times in TBST buffer (TBS buffer with 0.05% Tween® 20), the protein transferred membrane was blocked with 3% BSA in buffer

TBS. After washing three times in TBST, the membrane was incubated in an antibody solution in TBS: 1 µg / ml Mab or 0.5 µg / ml Pab. After washing three times in TBST, the membrane was incubated in a biotin-labeled secondary antibody solution in TBS buffer: 10000x diluted mouse anti-rabbit Fc monoclonal antibody (Sigma) or 10000x diluted goat poly-ionic whole molecule anti-rabbit (Sigma) andub 10,000x diluted goat anti-mouse whole molecule polyclonal antibody (Sigma). After washing three times in TBST, the membrane was incubated in a 4000x diluted solution of extravidin labeled with horseradish peroxidase or alkaline phosphatase (Sigma). After washing five times in TBST, the membrane was incubated with diaminobenzidine (DAB, Sigma), i.e. a peroxidase substrate or 5-bromo-4-chioro-3-indolylphosphate / nitro tetrazolium blue (BCIP / NBT, Sigma), i.e. a dia aikai substrate. phosphatase. All antibody incubations and membrane washes were performed with shaking at approximately 100 rpm.

Observed in Western blot [Fig. 4] the bands correspond to the different forms of the M-HBsAg protein, ie, glycosylated and unmodified monomers.

Buffer salts, BSA, etc. were purchased from POCh and Sigma.

Example 4. Propagation of transgenic lettuce producing the antigenic protein M-HBsAg

In order to obtain significant amounts of starting vaccine plant material, selected lettuce lines efficiently producing the M-HBsAg surface antigen were vegetatively propagated in the chicken coop in vitro, and the resulting cions were cultivated ex vitro. After the M-HBsAg protein content was analyzed, material was obtained for further work.

Lettuce lines characterized by the presence of a transgene in genomic DNA, confirmed by PCR and Southern blot methods, were selected for vegetative propagation (micro-propagation, propagation) [Fig. 2] and efficiently expressing the M-HBsAg antigen protein [Fig. 3-5]. In order to germinate butter lettuce seeds of the Syrena variety from selected transgenic lines producing the M-HBsAg protein, sterilized 12 min in 20% Clorox® bleach with 0.01% addition of Tween® 20 detergent, and then rinsed 5 - 6 times in sterile deionized water in to remove residual sterilizing agent. Seeds were germinated in partial shade under 16/8 h light / dark photoperiod conditions. The starting material for micro-propagation were 2-3-day-old seedlings, which were placed on the LRM1 selection medium with the composition: macro- and microelements according to the SH medium

PL 217 229 B1 (Schenk and Hildebrandt 1972), vitamins according to the B5 medium (Gamborg 1968), sucrose 3%, kinetin -1 mg ^- , pH 5.75, agar - 0.8%, with the addition of phosphinothricin (Riedel de Haen) , of the active ingredient of Basta herbi-1 cide at a concentration of 2.5 mgl ^-1 . The seedlings and the multiplants developing from them were kept at a temperature of approx. 24 ° C with a 16/8 day / night photoperiod and lighting of approx. 3,000 - 4,000 lux during the micro-multiplication.

During approx. 4-6 weeks of culture, the activation of lateral meristems was observed on the LRMl medium, followed by the development of lettuce plants - vegetative clones. The obtained multi-plants were then transferred to the LRM2 selection medium with the following composition: macro- and microelements according to SH medium-1, vitamin B5, sucrose - 3%, kinetin - 0.5 mgl ^-1 , pH 5.75, agar - 0.8% ), with phosphinothricin

- 2.5 mgl ^-1 . Some of the developing plants were cut off and transferred back to LRM2 medium for further propagation, and some were transferred for rooting to 1 / 2SH selection medium containing half the amount of macro elements according to SH and full dose of micro elements according to SH, vitamin B5, sucrose - 3%, pH 5.75, agar - 0.8% with phosphinothricin as above. Transgenic plants rooted on 1 / 2SH selection medium were transferred to ex vitro conditions in a greenhouse and adapted to soil conditions, i.e. horticultural soil under phytotron conditions (day / night photoperiod 16/8 h and temperature 22 ° C, lighting at plant level approx. 15 - 20 klx). The procedure of micro-propagation of lettuce, i.e. vegetative propagation in in vitro culture, enables obtaining at least several dozen vegetative clones for one plant from a given transgenic lettuce line, which results from the high multiplication efficiency, i.e. 8 - 13 clones from one passage with at least 8 passages [Fig. 5]. During greenhouse cultivation, the M-HBsAg protein content of the plants was analyzed by ELISA (see Example 2). The variability of the M-HBsAg level in vegetative clones of individual plants, between clones of successive plants of a given line, and the significance of differences in the level of

M-HBsAg between clone and control plants, i.e. not vegetatively propagated. The above ₂ parameters were statistically analyzed (χ test, F test) (after log transformation, separately for each parental line) by analysis of variance according to the mixed model taking into account the constant effects of propagated progeny plants (T1 generation) and the random effects of vegetative clones and allowing heterogeneity of variance between clones. The calculations were performed with the GenStat 10® program (VSN International Ltd.). The obtained plant clones were characterized by a high level of M-HBsAg protein, for at least 80% of the clones did not differ from the control plants obtained from seeds and did not undergo in vitro culture and the micro-propagation process. Moreover, the content of the M-HBsAg protein in individual clones of a given plant was similar, and the observed deviations in most cases were statistically insignificant, including for 80% or more of the clones the differences in the content of the vaccine M-HBsAg protein did not exceed 20% [Fig. 5].

The procedure of micro-propagation of lettuce, i.e. vegetative propagation in in vitro culture, enables the multiplication of plants on a semi-industrial and industrial scale, ensuring the production of significant amounts of starting vaccine material with the desired properties, i.e. high and even content of the M-HBsAg antigen protein. Salts, growth and development regulators, and other plant media reagents were obtained from POCh and Sigma.

Claims (62)

Example 5. Preparation of lyophilisates containing M-HBsAg vaccine antigen based on transgenic lettuce leaves Plants of selected transgenic lettuce lines, characterized by a relatively high content of M-HBsAg, i.e. 15 μg / g of St. the masses were grown in a greenhouse under natural photoperiod conditions and at a temperature of 20 - 22 ° C during the day and 14 - 16 ° C at night. The leaves from the well-developed heads were harvested, frozen in liquid nitrogen, partially disintegrating the tissue, and then stored at -80 ° C. The frozen material was placed in a CHRIST® BETA 1-16 freeze dryer and freeze-dried for 24 - 36 h under a vacuum of 0.2 mbar at a freezing temperature - 80 ° C and 55 ° C shelf temperature, where the material was placed on metal trays. The lyophilized material was ground into a powder and stored in sealed containers in the presence of a desiccator at room temperature as a starting material for the preparation of individual oral vaccine forms [Fig. 6A]. 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