Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1999-12-23
2003-04-22
Wortman, Donna (Department: 1648)
Chemistry: molecular biology and microbiology
Vector, per se
C435S069300, C435S071200, C536S023400
Reexamination Certificate
active
06551820
ABSTRACT:
TECHNICAL FIELD
The present invention relates to oral vaccines, particularly those provided by edible plants. The invention employs genetic engineering techniques to produce transgenic plants capable of expressing immunogenic polypeptides, including hepatitis B antigen (HBsAg) in quantities sufficient to elicit an immune response in a human or animal that consumes all or a part of the plant.
BACKGROUND OF THE INVENTION
A vaccine for hepatitis B was the first “new generation” recombinant vaccine licensed by the FDA for human use. The immunogenic subunit in this formulation is produced by expressing the gene encoding HBsAg in recombinant yeast; the protein is purified from the genetically engineered yeast and is used for parenteral delivery. In the developed world, the recombinant vaccine has displaced the use of an earlier vaccine derived from the plasma of infected individuals. Both plasma-derived and rHBsAg vaccines are shown to be reasonably safe and effective in high-risk adult populations and newborn infants. However, the cost of the rHBsAg vaccine prevents its extensive availability in developing countries.
The envelope of the hepatitis B virus (HBV) contains three size classes of proteins that share carboxy-terminal sequences. These proteins, called hepatitis B surface antigens (HBsAgs), include large (L, containing a pre-S
2
domain), medium (M
1
containing a pre-S
1
domain), and small (S, containing only the S domain) size classes. All three proteins are found in infectious virions (often referred to as Dane particles) recovered as 42 nm spheres from the serum of infected patients. Serum samples also contain empty spherical particles averaging 22 nm, which contain primarily S class proteins. Mammalian cell lines transfected exclusively with DNA encoding the S protein release 20 nm empty spheres similar to those from infected cells. Moreover, yeast cells transformed with the same gene form analogous spheres, which are found to be equally immunogenic as the 22 nm spheres from infected cells. The yeast-derived material forms the active constituents of the currently available commercial vaccines ENGERIX (SKB) and RECOMBIVAX (Merck).
In mammalian cells, newly synthesized L, M, or S proteins insert into the membrane of the endoplasmic reticulum (ER). The S protein consists of 226 amino acids; its N and C-termini are thought to be on the ER lumen side and it has four transmembrane helices. All three proteins have a glycosylation site at position 146 of the S domain. The three proteins differ in the available sites for and the extent of glycosylation. Improper glycosylation can prevent virion formation, presumably due to misfolding of the proteins. Also, multiple disulfide bonds among cysteines in the proteins are known to be important to the structure of the assembled virion or particles and to the structure of antigenic loops in the protein. Incorrectly assembled particles may interact with cellular chaperones because of the incorrect folding to prevent secretion.
Higher levels of preS1 and preS2 are present in HBV than in 17-25 nm HBsAg particles therefore, immunization with HBsAg particles may not generate high titer antibodies to the preS sequences expressed on HBV. During the course of HBV infection in humans the levels of preS proteins increase during active replication, and anti-preS antibodies and T cells are generated prior to S protein-specific responses. Once anti-HBsAg antibodies rise, anti-preS antibodies decline.
The roles of preS1 and preS2 in virus attachment and neutralization have led to the development of vaccines containing these sequences as well as the entire S region. Vaccines incorporating preS sequences include HEPAGEN (Merck) and BIO-HEP-B (BTG); both are produced in mammalian cell lines. In formulating whole particles that contain S and preS proteins it is important to note that the relative amounts of S, M, and L proteins affect HBsAg assembly, e.g., high levels of L protein reduce the amount of HBsAg particle formation and secretion.
The assembly of the S, M and L surface proteins into particles occurs during budding of the complex into the ER, followed by transport of the particles through the Golgi apparatus to the exterior of the cells. Nanometer scale biological structures, such as viral capsids, assemble through polymerization of similarly folded protein subunits using a small number of well-defined bonding contacts. The driving force for polymerization is the formation of favorable bonding interactions as free subunits are incorporated into the growing polymer. For envelope proteins such as HBsAg, essential steps in the polymerization process are appropriate integration of the polypeptide into the ER membrane followed by establishment of contact among the protein subunits. Normal cellular transport and sorting of proteins in the endomembrane system may contribute to this process.
U.S. Pat. No. 4,710,463 to Murray proposes a method of producing a polypeptide having the antigenicity of a hepatitis B core or surface antigen, which employs a unicellular host. U.S. Pat. No. 5,738,855 to Szu et al. proposes a modified oligosaccharide immunogen similar to the Vi antigen of
Salmonella typhi
, which can be conjugated to a carrier, such as hepatitis B surface antigen. U.S. Pat. No. 4,847,080, EU 0154902 B1, and subsequent papers of Neurath et al. identify peptide epitopes in the preS1 and preS2 regions for both hepatocyte binding and neutralization, as well as peptides that can be included in a vaccine.
Mammals infected by a pathogen mount an immune response when overcoming the invading microorganism by initiating at least one of three branches of the immune system: mucosal, humoral, or cellular immunity. Mucosal immunity largely results from the production of secretory IgA antibodies in secretions that bathe mucosal surfaces in the respiratory tract, the gastrointestinal tract, the genitourinary tract, and the secretory glands. These mucosal antibodies act to limit colonization of the pathogen on mucosal surfaces, thus establishing a first line of defense against invasion. The production of mucosal antibodies can be initiated by local immunization of the secretory gland or tissue or by presentation of the antigen to either the gut-associated lymphoid tissues (GALT; Peyer's Patches) or the bronchial-associated lymphoid tissue (BALT).
Mucosal immunization can be achieved by oral presentation of antigens. Specialized epithelial cells (M cells) overlying organized mucosal lymphoid tissues along the intestinal tract sample the antigens by taking up (by endocytosis) infectious bacteria, viruses, and macromolecules. These are passed to the underlying follicles where immune responses are initiated and cells are dispersed to both mucosal and systemic immune compartments. Epithelial cells are also an integral component of the regulatory cytokine network, including those that are important in the differentiation of B cells.
Oral immunization also induces strong humoral immune responses. Humoral immunity results from the production of circulating antibodies in the serum (especially IgG and IgM), precipitating phagocytosis of invading pathogens, neutralization of viruses, or complement-mediated cytotoxicity against the pathogen. A well-documented relationship exists between HBV protection and the amplitude of the systemic antibody and T cell response to HBsAg proteins, and this protection is likely to be achieved by oral immunization.
In contrast to the large variety of currently available injectable vaccines that provide systemic immunity, vaccines administered non-systemically to stimulate mucosal immunity are rare. Recently, however, there has been a surge of interest in developing novel strategies for vaccine development with oral delivery as the preferred route of delivery. In the design of a successful oral vaccine, two aspects deserve special attention—the use of an appropriate adjuvant and the development of an appropriate antigen delivery system.
Most protein antigens studied for use as adjuvants when administered orally in large doses fail to p
Arntzen Charles Joel
Mason Hugh S.
Richter Elizabeth
Thanavala Yasmin
Boyce Thompson Institute for Plant Research
Palmer & Dodge LLP
Spar Elizabeth N.
Williams Kathleen M.
Wortman Donna
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