Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector
Reexamination Certificate
2001-05-11
2003-12-02
Saunders, David (Department: 1644)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
C424S489000, C424S490000, C424S493000, C424S499000
Reexamination Certificate
active
06656470
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates generally to a composition and method for vaccinating vertebrate species, and to a method of preparing the composition. More specifically, the invention relates to a vaccine composition including an antigen in modified alginate microparticles, a method of making the vaccine composition, and a method of vaccinating using the composition to induce immunity.
2. Brief Description of Related Technology
Historically, immunization has relied upon the induction of humoral immunity by parenteral administration of vaccines. Antibodies induced by parenteral administration, however, do not necessarily reach mucosal surfaces, the sites of entry of most infectious agents. Mucosal immunity, which develops at mucosal surfaces as a result of contact of antigen with mucosal lymphoid tissues, is an important first line of defense against infectious agents.
Induction of immunity at mucosal surfaces requires direct contact of antigen with a mucosal surface. This is not always possible or practical, however, because of handling and delivery problems and because of the toxicity of some antigens to mucosal surfaces. An alternative way to induce protective immunity at mucosal surfaces is by stimulation of the common mucosal immune system (CMIS), a network immunologically linking all mucosal sites to each other. The CMIS consists of lymphoid tissue at mucosal sites that sample antigens to induce an immune response. Microfold cells (M cells) are the specialized cells that sample antigens so that they can be processed by the lymphocytes and macrophages. Major concentrations of mucosal associated lymphoid tissue are found in the upper respiratory tract, the lower respiratory tract, and the gastrointestinal tract.
The gastrointestinal site contains the greatest concentration of lymphocytes in the body, primarily in areas referred to as Peyer's patches. Stimulation of the lymphoid tissue in the gut (gut associated lymphoid tissue, or GALT) by oral vaccines can result in antigen specific lymphocytes that enter the lymph and general circulation. These lymphocytes home to (migrate back to) the lamina propria of the site of their origin where they then produce antibody that coats the mucosal surface. As part of the CMIS, a significant population of these lymphocytes migrate to other mucosal sites. In this manner, oral administration of antigens can be used to induce mucosal response to provide systemic protection from infection and to prevent infection at a variety of mucosal sites in the body.
The oral route of delivery is the most attractive way to deliver antigens, but at the same time very challenging. For proper immunogenicity by oral antigen delivery using microparticles, the microparticles must be taken up by M cells, transferred to lymphoid follicles, and processed by lymphocytes and macrophages underlying these follicles. Uptake of microparticles by the M cells depends heavily on the nature of the microparticles such as size, hydrophobicity, and possibly surface charge.
Oral vaccine carriers are being studied for delivery of vaccines useful in treatment of a variety of human diseases. Often, oral vaccines are developed using animal models. Information gained from oral vaccines developed for one species can be used for more efficient development of vaccines for other species.
Oral administration of vaccines is easy and economical, with little labor required. Oral administration reduces the chance of adverse reactions and eliminates injection site reactions that can damages the carcass or hide of an animal. However, orally administered antigens must be protected from the low pH and enzymes of the stomach until they reach the GALT in the small intestine. Otherwise, antigens can be damaged or altered, resulting in reduced stimulation and a less effective immune response.
Generally, it is known that alginate gel microparticles (or microspheres) can be used as a matrix for oral delivery of vaccine-relevant antigens. See Bowersock et al., U.S. Pat. No. 5,674,495, issued on Oct. 7, 1997. The advantages of this matrix include compatibility with a wide variety of antigens, including fragile antigens, compatibility with live organisms, compatibility with nucleic acids, and provision of an adjuvant effect by the alginate system itself. Although the basic technique is known, there are many variables that can affect the formation of alginate microparticles, their resulting size, their efficiency in loading antigen, their hydrophobicity, their uptake by antigen sampling cells, their antigen-release characteristics, and the overall immunological response generated when using the microparticles.
While it is known that antigen encapsulated in smaller (e.g., diameter less than 10 &mgr;m) alginate microparticles have a greater chance of uptake (phagocytosis) by GALT, formation of microparticles with diameters less than 10 &mgr;m using traditional methods can compromise antigen loading. Thus, reducing the size of alginate microparticles while maintaining, or increasing, antigen loading can allow for administration of smaller volumes of vaccine, leading to material conservation.
While it is known that phagocytosis of microparticles increases as the hydrophobicity of the microparticles increases and as the charge on the molecule is more positive, traditional vaccines made with alginate-encapsulated antigen are somewhat hydrophilic, which hinders uptake (phagocytosis) by GALT, and alginate naturally carries a negative charge. Traditional antigen-containing alginate microparticles require coating with polymers, including poly-cations such as poly-l-lysine, to give the microparticles a positive charge and to increase their hydrophobicity, to produce any immunological response. Thus, increasing the hydrophobicity and positive surface charge of antigen-containing alginate microparticles would eliminate or reduce the necessity of polymer coatings, increase uptake by GALT, and improve immunological response.
Finally, traditional vaccines using alginate microparticles require multiple administrations over time to produce sustained antigen delivery to the desired target because the timing of immune system stimulation following administration of vaccines made by traditional methods is unpredictable and is not subject to control. For example, alginate microparticles made by Cho et al.,
J. Control. Rel.,
53, p. 215-224 (1998), released 80% of their antigen within 24 hours. Thus, antigen-containing alginate microparticles which have predictable and controllable antigen-release profiles can eliminate or reduce the necessity for multiple vaccine administrations and can provide sustained antigen delivery to the desired target.
Accordingly, it would be desirable to provide methods and compositions for the treatment of diseases in vertebrate species which have improved antigen loading, reduced microparticle size, increased hydrophobicity, improved uptake by antigen sampling cells, controlled antigen release characteristics, and improved immunogenicity.
SUMMARY OF THE INVENTION
Accordingly, the invention provides a vaccine composition and a method of preparation including the steps of: forming a water-in-oil emulsion including an alginate in water, an oil, an antigen, and at least one of (a) a cellulose ether and at least one nonionic surfactant; and (b) a poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer surfactant and at least one nonionic surfactant; followed by crosslinking the alginate in the emulsion with at least two cations selected from the group consisting of aluminum, barium, calcium, lithium, manganese, strontium, and zinc, to form antigen-containing, crosslinked alginate microparticles; and harvesting the microparticles.
Another aspect of the invention is a method of vaccinating a vertebrate species including the step of administering to the species a vaccine composition prepared according to the method of the invention.
Another aspect of the invention is a method of provoking an immune response in a vertebrate spec
Bowersock Terry L.
Guimond Paul
Ju Tzu-Chi R.
Kidane Argaw
Pharmacia & Upjohn Company
Pharmacia & Upjohn Company
Rehberg Edward F.
Saunders David
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