Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1999-04-27
2002-10-08
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S003000, C435S004000, C435S007100, C435S007200, C435S007230, C435S007240, C435S007920, C436S512000, C436S513000, C436S518000, C436S525000, C436S526000
Reexamination Certificate
active
06461809
ABSTRACT:
BACKGROUND OF THE INVENTION
There is historical precedent for the use of flow cytometry to identify and quantitate HIV-infected cells. Investigators in the late eighties and early nineties demonstrated flow cytometry's ability to identify and quantitate HIV-1 infected mononuclear cells at low frequency in lymphoid cell lines and whole blood.
The means of identification used at first, however, was to treat HIV- infected cells, derived from human T lymphoid cell lines H9 and A3.01, with an HIV- inactivating, permeabilizing fixative. This was followed by binding with a monoclonal antibody specific for the major core protein p24, and then by binding with FITC-conjugated F(ab′)
2
fragments of goat anti-mouse immunoglobulin antibody (Cory, J. M., et al. 1987). The method allowed determination of the percentage of the cell population that was infected and the relative amount of p24 antigen per cell. The first of such work utilized the T-cell adapted strain HIV in H-9 cells, and was able to detect as few as 1 HIV-1 infected cell in 10,000.
Shortly thereafter, a number of workers studied HIV-1 infected mononuclear cells using staining procedures for intracellular p24, and flow cytometric analysis of peripheral blood from seropositive patients throughout the spectrum of HIV disease progression. Using Centers for Disease Control (CDC) criteria, they were able to demonstrate that HIV-1 infected peripheral blood mononuclear cells (PBMCs) could be separated from whole blood. This was done by utilizing fixation and permeabilization and/or live cell immunofluorescence to gain entry to the core protein p24 with monoclonal anti-p24 antibodies. FITC labeling was accomplished with a goat/human anti-globulin conjugate. Flow cytometric analysis revealed clear and statistically significant differences in quantities of HIV-1 infected PBMCs between seropositive patients in CDC Classes I, II, and III, but not between patients in Classes III and IV.
The percent of HIV-1 infected PBMCs in seropositive individuals, when detectable, ranged form 4% to as high as 25%, while seronegative controls never exceeded 0.1% (autofluorescence). The consensus from several authors was that there was a correlation between the fall in CD4 counts and the sharp increase in PBMC virus loading. Thus flow cytometry, used to quantitate PBMCs productively or latently infected by HIV-1, has been recognized as a valuable tool. This tool can be used most effectively to further the understanding of various pathogenic aspects of the disease, better define the stages of the disease, and enhance the capacity to tailor therapeutic strategies.
Comparisons of both the technical and rather large percentage differences reported by different authors in various studies highlight the limitations of PCR as a means to label HIV-infected cells. A current limitation of PCR used on extracted DNA is the difficulty in correlating the presence of the viral genome with a single cell, and thus determine the exact percentage of HIV-1-producing cells.
On the other hand, in situ hybridization procedures suffer from low sensitivity. Although they are capable of identifying the single infected cell, these procedures are further compromised by requiring the microscopic screening of a large number of cells to determine the exact percentage of HIV-1 infected mononuclear cells in peripheral blood utilized the product of and in situ PCR assay to demonstrate the presence of proviral DNA.
Determination of the efficacy of vaccine candidates for the treatment and/or prevention of HIV disease relies on the ability of the candidate agent to substantially reduce HIV reproduction. This is true also for passive immunotherapeutic agents (engineered neutralizing monoclonal and polyclonal antibodies). Within the past few years, it has become clear that primary HIV-1 isolates, while relatively resistant to neutralization by antibodies as well as by CD4-based reagents, can be strongly neutralized by certain monoclonal antibodies (mAbs) and by some sera from HIV-1 infected people.
Primate retroviruses, such as HIV-1 and simian immunodeficiency virus (SIV), share a primary cellular receptor, the CD4 molecule. Isolates of HIV-1 have been generally characterized on the basis of their replication patterns in peripheral blood mononuclear cells (PBMCs), primary macrophages, and immortalized T-cell lines. All HIV-1 isolates are able to replicate to some degree in PBMC cultures; M-tropic viruses also replicate in macrophages, but T-tropic viruses replicate in T cell lines, and dual tropic viruses replicate in all three types of cells.
Additional (secondary) determinants for HIV-1 fusion and entry have recently been identified on the basis of their interactions with chemokines called macrophage inflammatory protein 1&agr; (MIP-1&agr;), MIP-1&bgr;, and RANTES, which act as powerful modulators of HIV-1 infection. T- and M-tropic isolates have been shown to require the chemokine receptors CXCR4 (fusin) and CCR5 as the major HIV-1 coreceptors respectively. The above-named chemokines have been shown to block M-tropic HIV1 strains because they are competitive ligands for the CCR5 receptor.
The key to performing a highly sensitive, accurate, and consistent neutralization assay depends upon the assay conditions used. These may include: the type and strain of virus used, the multiplicity of infection, the ratio of antibody to virus, the length of exposure of the antibody-treated virus to target cells, the type of target cell used for infection, the kinetics of virus growth, the length of the assay, the type of read-out used, the method of data analysis, and the criteria used to define neutralization. The neutralization and the infectivity reduction assays remain in a paradoxically underdeveloped state, in spite of increased efforts to develop new neutralizing agents. It is not surprising, therefore, that studies of HIV-1 neutralization have shown significant differences in results because of dependence on many of the variables listed above, for which there are no uniformly accepted standards.
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Chacherian et al.; Human Immunodeficiency Virus Type 1 Coreceptors Participate in Postentry Stages in the Virus Replication Cycle and Function in the Simian Immunodificency Virus Infection; Journal of Virology; vol. 71, No. 5; pp. 3932-3939, May 1997.*
Deng et al.; Identification of a Major Co-receptor for Primary Isolates of HIV-1; Nature; vol. 381; pp. 661-666, Jun. 1996.*
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Hallowitz Robert A.
King Chester
Young Susan
Bio-Tech Imaging, INC
Brown Stacy S.
Millen White Zelano & Branigan P.C.
Park Hankyel T.
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