Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Conjugate or complex
Reexamination Certificate
1998-09-09
2001-09-11
Stucker, Jeffrey (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Conjugate or complex
C424S188100, C424S208100, C424S804000, C424S812000, C530S350000, C530S387500, C530S388350, C435S329000, C435S339100
Reexamination Certificate
active
06287568
ABSTRACT:
Throughout this application, various references are referred to with arabic numbers. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this specification, preceding the claims.
BACKGROUND OF THE INVENTION
Recognition of polysaccharides as antigens began with the study in 1917 by Dochez and Avery(1) who found that when pneumococci are grown in fluid media, there is a substance in the culture fluid which precipitated specifically with antisera to the same pneumococcus. Heidelberger and Avery(2) showed that this substance was polysaccharide and not protein as thought previously.
Microbial polysaccharides as antigens gained renewed interest when it was found that strains of microorganisms resistant to antibiotics and/or chemotherapeutic agents appeared increasingly and became a worldwide problem. Vaccines with microbial polysaccharides were developed to meet the requirements.
Given the feature of host immune responses induced by polysaccharides, they were classified as the “T-independent”(TI) antigen. The concept of T-independency arose from the observations that neonatally thymectomized (3,4) and nude mice(5) gave unimpaired antibody responses to large polymeric molecules, although they were not able to mount a humoral response to T-dependent (TD) antigens, such as proteins. They are further divided into type I(TI-I) and type II(TI-II), based on the ability to elicit antibodies in the CBA/N mouse strain, with an X-chromosome linked immunodeficiency (xid) (6). Lipopolysaccharides (LPS) of gram-negative bacteria which induce antibodies in such strains are TI-I antigens; capsular polysaccharides of gram positive bacteria and exopolysaccharides, induced no response in xid strains, are TI-II antigens.
T-dependent and T-independent antigens may induce different pathways of B cell activation and differentiation, germinal center reactions and antibody-secreting cell responses. T-dependent antigens can induce germinal center formation as well as the antibody-secreting cell responses. In germinal centers, B cells may undergo somatic hypermutation, IgH class-switching and memory cell induction(7-9). These molecular events are believed to be T cell dependent. In contrast to the T-dependent patterns of B cell activation which are associated with germinal center development, many T-independent antigens, such as the TI-I antigen LPS and the TI-II antigens, polyvinylpyrrolidone and DNP-Ficoll, are reported to induce only minimal or no germinal center development (72).
The onset of full response to polysaccharide TI-II antigens in both mice and humans is strikingly delayed. In mice(10) antibody responses to TI-I antigens(LPS and other) and protein antigens reach adult levels within 1-2 weeks; antibodies to TI-II antigens can be detected only at 2-3 weeks. For pneumococcal polysaccharides SSS-III and dextran B1355S, full development of the antibody response is not reached until 4 weeks; with levan and &agr;(1,6)dextran, 7 and 13 weeks respectively, are needed. In humans, children younger than 18 months of age fail to respond to microbial polysaccharides or produce antibodies at levels too low to be protective. Such poor responsiveness generally lasts until 5 years of age. Thus, there is a period when maternal-derived protective antibodies have declined, yet the age-related development of immunity to bacterial infection remains immature. Pathogens causing severe problems during this high-risk period have long posed the need for developing efficient vaccines.
Thus, the “T-independent” property of polysaccharide antigens has been considered as a limitation to their application for vaccination. In 1990, Robbins and Schneerson introduced a conjugate strategy for vaccine development(11). By coupling purified capsular polysaccharides of
Haemophilus influenzae
B with tetanus toxoid, the first polysaccharide-protein conjugate against this bacterium was made(12). Instead of large capsular polysaccharides, its oligosaccharide was conjugated with different protein carriers for vaccinations (13,14), so that the conjugate-vaccines preserve the antigenic specificities of the original polysaccharides but gain the T-dependent property in addition. A significant shift in the age at which the anti-carbohydrate response can be induced and Ig class switches to the protective IgG isotypes were observed with these vaccines, resulting in better protection of high-risk populations from
Haemophilus influenzae
B. The principles underlying the approaches to
Haemophilus influenzae
B vaccines, have been extended to microbial polysaccharides of other bacteria, viruses and parasites, etc.(15).
The magnitude of the worldwide AIDS epidemic presents current challenges for developing effective vaccines and therapeutic strategies These efforts are hampered, however, by the targeted elimination of T-cells by the retrovirus(16, 17), by difficulties in inducing effective neutralizing antibodies to HIV1(7-19), and by the lack of an effective strategy for the induction of mucosal immune response, which may eliminate the invasion on the mucosal surface before a systemic infection occurs. The center of the difficulties in developing HIV-vaccines is a paradox caused by the retrovirus: effective vaccinations require the functions of T-cell which are however destroyed directly by the virus.
An effective vaccination against HIV must fulfill the following requirements: 1) It must elicit an effective immune response to HIV in the presence and absence of functional T-cells; 2) It can induce anti-HIV antibody of the IgA isotype to enhance the mucosal protection; and 3) It is safe, non-toxic and clinically acceptable. In addition, a vaccination strategy applying such T-independent Vaccines must be able to inherit all the advantages of the current T-dependent “conjugate-vaccines”.
Our invention described here provides the concept, materials and the methodology for the development of such vaccination strategies against HIV, and other infectious agents.
SUMMARY OF THE INVENTION
This invention includes the conception of the T-independent conjugate-vaccines, their application in the induction of antigen specific IgA and in reshaping the repertoire of responding B-cells. We demonstrated for the first time that a specific polysaccharide antigen &agr;(1,6)dextran can elicit a markedly enhanced IgA response in T-cell free mice; co-injection of the molecule with other antigens can enhance the IgA response to the co-antigen; and its conjugates with the Gag protein of HIV-1 can elicit Gag-specific IgA in the presence and absence of functional T-cells.
The invention identified and illustrated specifically: 1. &agr;(1,6)dextran can serve as a carrier molecule for producing the T-independent conjugate-vaccines; 2. The polysaccharide can serve as an adjuvant to enhance IgA production for other specificities and to reshape the repertoire of responding B-cells; 3. The technical details for producing the first generation of the TI-conjugate-vaccines and their in vitro identification are described; and 4. A combinatory vaccination strategy, incorporating the advantage of both “TD-conjugate” and our “TI-conjugates”, is proposed and experimentally illustrated.
The T-independent property of these conjugates makes it especially useful in vaccinations against HIV, AIDS-associated opportunistic infections, and other infectious- and non-infectious diseases with T-cell deficiency (including tumors). Its application is, however, not restricted in the T-cell deficient situation.
This invention provides a method of identifying an &agr;(1,6) dextran molecule as a potent IgA-B cell activator comprising the steps of:
(a) conjugating a first and a second &agr;(1,6) dextran molecule, each molecule having a different molecular weight of ≧90 kd, to an HIV-1 gp120 glycoprotein, an HIV-1 protein or epitopes thereof to produce a T-independent &agr;(1,6) dextran conju
Erlanger Bernard F.
Kabat Elvin A.
Wang Denong
Cooper & Dunham LLP
Stucker Jeffrey
The Trustees of Columbia University in the City of New York
White John P.
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