Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-12-07
2004-11-09
Smith, Lynette R. F. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S007100, C435S007200, C435S007320, C435S007330, C435S007340, C435S007350, C435S007370, C435S007920, C435S029000, C435S034000, C435S069300, C435S332000, C435S340000, C436S172000, C436S501000, C436S507000, C436S546000, C436S800000
Reexamination Certificate
active
06815172
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of immunologic detection of functional antibodies.
BACKGROUND OF THE INVENTION
Within the last decade, the emergence of multi-drug resistant pathogens has led to the increased use of vaccines as a method of treating infectious disease. Vaccine development and immunization has moved to the forefront of both international and national concerns as evidenced by the recent establishment of organizations such as the International Vaccine Institute established in 1996, and the Vaccine and Immunization Division of the Pan American Health Organization established in 1999. To date, the United States alone has more than twenty different federal government agencies that play a role in the development of vaccines.
With this increase in importance, new vaccines are being developed at a rapid pace, and assays to determine the in vivo efficacy of these vaccines are also being developed. Several assays have been developed that determine the efficacy of vaccines by detecting vaccine-specific antibodies generated in vivo after vaccine administration. These assays include radioantigen binding assays and enzyme-linked immunosorbant assays (ELISAs) (Schiffman et al., 1980; Nahm et al., 1996; Quataert et al., 1995). One notable problem with these assays, is that they measure the total amount of antibody generated in response to administration of the vaccine without regard to whether the antibodies actually provide a protective response in the immunized individual. Therefore, traditional assays do not provide an accurate picture as to the in vivo efficacy of a vaccine.
Whether a vaccine provides protective immunity or simply generates an antibody response depends upon the type of infection the vaccine seeks to prevent as well as the type of antibodies generated in response to administration of the vaccine. For example, a protective immune response to pathogens such as
Streptococcus pneumoniae
and
Neisseria meningitidis
involves opsonophagocytosis of the infectious agents. Opsonophagocytosis is the binding (or opsonization) of antibodies and complement or complement components to the infectious agent and the subsequent uptake of the infectious agent by effector cells via the binding of the effector cells to the antibody/antigen complex. Therefore, a protective immune response against such pathogens requires more than the mere generation of antibodies that bind the pathogen. A protective immune response requires the generation of “functional” antibodies that bind to the infectious agents and also provide a means for uptake and clearance by effector cells. Another aspect of functionality, is the ability to interact with complement reactions that may also be necessary for opsonphagocytosis.
Since functional antibodies play a major role in the generation of a protective response against infection by certain pathogens, vaccines for these pathogens must also generate functional antibodies in order to protect against infection. Assays that determine the effectiveness of vaccines of this nature should test for the production of functional antibodies. Notably, the Federal Drug Administration has taken this stance with regard to all future pneumococcal vaccines and will require measurement of antibody levels using functional assays which more closely approximate biologic function of the host.
One functional assay that has been developed to measure the efficacy of vaccines is the opsonophagocytic assay. Opsonophagocytic assays are more attractive than other measures of in vitro protective immunity because they more closely resemble animal models, and appear to provide a closer correlation with serotype-specific vaccine efficacy than other prior art assays such as ELISAs (Wenger et al., 1996). Opsonophagocytic assays have been performed with peripheral blood leukocytes (PBLs) as effector cells and have used a variety of techniques such as radioisotope labeling, flow cytometry, microscopic evaluation and viability assays (Esposito et al., 1990; Guckian et al., 1980; Kaniuk et al., 1992; Lortan et al., 1993; Obaro et al., 1996; Sveum et al., 1986; Vioarsson et al., 1994; Winkelstein et al., 1975). Differentiated HL-60 cells have also been used as an alternative to PBL effector cells, eliminating the need for human donors and decreasing the inter-assay variability that occurs with random PBL donors (Romero-Steiner et al., 1997).
Although functional assays have been developed to measure vaccine efficacy, none of the prior art methods allow the simultaneous detection of functional antibodies generated by multiple serotypes of a pathogen. Detection of functional antibodies to multiple serotypes is essential to the determination of the efficacy of vaccines that include antigens from multiple serotypes. For example, there are multiple serotypes of
Streptococcus pneumoniae. S. pneumoniae
is the leading cause of meningitis, septicemia, pneumonia and acute otitis media in young children and an important cause of illness and death in the elderly and persons with certain underlying conditions. Host protection against invasive pneumococcal disease is primarily mediated by anti-capsular antibodies and complement-mediated phagocytosis. Similarly,
Neisseria meningitidis
has several serotypes and protection from disease caused by
Neisseria meningitidis
serogroup A and C is critically dependent upon capsular polysaccharide antibodies. Immunization with meningococcal polysaccharide vaccines elicits complement-dependent serum bactericidal and opsonophagocytic antibodies. Both mechanisms operate simultaneously and function to clear meningococci. However there are individuals incapable of mounting a complement-dependent serum bactericidal response, and for those individuals, opsonophagocytic antibodies serve as the only protection against meningococcal disease. Since functional opsonophagocytic activity appears to correlate with protection against the pneumococcal and meningococcal diseases described above, functional assays will best determine the efficacy of vaccines for these diseases.
Measurement of the effectiveness of a vaccine designed to provide protection against infection by multiple serotypes of
S. pneumoniae
or
N. meningitidis
would require measurement of multiple functional antibodies generated toward each different serotype. Although the functional assays described above could be run multiple times for each different serotype present in the vaccine, such a practice would be arduous and expensive. Therefore, what is needed in the art is a rapid, automated, fictional assay that can simultaneously measure antibodies directed against multiple serotypes of pathogens, particularly those such as
S. pneumoniae
and
N. meningitidis.
SUMMARY OF THE INVENTION
The present invention relates to methods and compositions directed to measurement of a functional antibody response. These methods and compositions address the continuing need for determination of vaccine protection against pathogens and the ability to test for multiple serotypes.
The present invention comprises methods and compositions for opsonophagocytic assays to measure the presence of functional antibodies. The methods of the present invention comprise the addition of a biological sample to compositions comprising fluorescent beads coated with antigens. Other embodiments of the present invention comprise methods of measuring the presence of functional antibodies comprising addition of a biological sample to compositions of fluorescently labeled bacteria. The compositions of the antibodies of the sample and the fluorescent beads or the labeled bacteria are then added to phagocytic cells for opsonization. A preferred embodiment of the present invention further comprises the addition of complement to complete or enhance the opsonization of the compositions by the phagocytic cells.
Accordingly, it is an object of the present invention to provide methods and compositions for the measurement of the presence of antibodies to a pathogen.
It is another object of the present invention to provide met
Carlone George M.
Hickey Michael H.
Martinez Joseph E.
Steiner Sandra
Hines Ja'Na
Klarquist & Sparkman, LLP
Smith Lynette R. F.
The United States of America as represented by the Department of
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