Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or... – Recovery or purification
Reexamination Certificate
1998-07-28
2001-03-06
Allen, Marianne P. (Department: 1631)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
Recovery or purification
C435S005000, C435S007100, C435S007800, C435S029000, C435S803000, C435S948000, C424S185100, C424S193100, C424S196110, C424S197110, C424S279100, C514S023000, C514S054000, C514S055000, C514S056000, C514S057000, C514S058000, C514S059000, C514S060000, C514S061000, C514S062000
Reexamination Certificate
active
06197568
ABSTRACT:
BACKGROUND OF THE INVENTION
Methods and compositions for the isolation, diagnosis and treatment of microorganisms such as flaviviruses and other hemorrhagic fever viruses are based on the sulfated polyanion-dependent interaction of the microorganisms with target cells. The cellular receptors used by flaviviruses and hemorrhagic fever viruses, in particular dengue virus, have been identified as sulfated polyanionic glycoproteins, that include highly sulfated heparan sulfate glycosaminoglycans for some target cell types, and as a sulfated mucin on vascular endothelium. Compounds such as heparin, highly sulfated heparan sulfate, and synthetic polyanions such as Suramin, inhibit the interaction between the virus and target cells, thereby disrupting the infective process.
The family of flaviviridae include pathogens causing disease in both humans and animals. Deleterious effects of the pathogens are of global health, agricultural, and economic significance. The viral family of flaviviruses includes members such as the arthropod-borne dengue virus that causes a serious febrile illness in humans. The failure to maintain programs to control the mosquito vector for dengue virus transmission has been associated with an alarming escalation of infection rates that is of increasing medical, public health, and military concern. It has been estimated that two and a half billion people in over 100 countries are at risk of infection, and there are a minimum of 20 million infections per year, mostly in children. There is no vaccine for dengue virus, few vaccines for other flaviviruses are available, and there is no specific or successful treatment for any flavivirus infection.
Dengue virus is a representative member of the family flaviviridae genus flavivirus, is also an arbovirus, and is also classified as one of a group of hemorrhagic fever viruses (HFV's) that cause prominent vascular pathology. Hemorrhagic fever viruses are an otherwise diverse grouping of viruses that include Yellow Fever virus (another flavivirus); the arenaviridae Lassa, Junin, and Machupo; bunyaviridae Rift Valley, Congo-Crimean, and Hantaan; and filoviridae Marburg and Ebola. Because these viruses share characteristics of short incubation, acute onset, debilitation and high mortality, and untreatability, they are considered likely choices for development of biological warfare agents. It is important that the molecular basis of the pathogenesis of these viruses be understood, and in particular that any common pathogenic features be exploited, in order to develop generic approaches to detection and treatment. An important unanswered question about the pathogenesis of dengue virus, other flaviviruses and other HFV's is the mechanism whereby they bind target cells. It has been difficult to find an answer to this question due to limitations inherent in investigations of these microorganisms. For example, because there is no valid animal model for dengue virus infection, studies of dengue pathophysiology usually involve infection of cultured cells, such as Vero cells.
The initial binding of a virus to a target cell is a critical determinant of cell and tissue tropism, and thus infectivity and pathogenesis. Binding occurs as a result of an adhesion receptor-like interaction between a viral ectodomain molecule and a corresponding co-receptor expressed on the surface of target cells. Little is understood about the pathogenesis of flavivirus or hemorrhagic fever virus infection, and there is no information about the molecular basis of the binding of any of these viruses to target cells. It has been suggested that infection of some cells may involve anti-viral antibody mediated immune-adherence; the Fc domain of antibody that is bound to virus may mediate binding to cells such as monocytes that express Fc receptors. However, this mechanism does not explain primary infection in patients without antibody, or infection of cells lacking Fc receptors.
Currently, viral diagnosis depends on indirect serological analysis (i.e. development of antibodies), or direct assessment of patient-derived specimens for the presence of virus (culture, molecular detection). Serology is probably the most sensitive diagnostic test, but the time required for development of significant titers of antibodies leads to delays in diagnosis. Direct assessment for the presence of virus is definitive, but often insensitive, probably due to inadequately low concentrations of virus in specimens. Elucidation of the binding mechanism of microorganisms such as flaviviruses, to target cells would facilitate diagnostic and therapeutic strategies.
SUMMARY OF THE INVENTION
Methods and compositions based on the interaction of microorganism ectodomain proteins with sulfated polyanions, in particular some glycosaminoglycans (GAGs) such as heparin and highly sulfated heparan sulfate, allow isolation, diagnosis and treatment of the microorganism. Suitable microorganisms include bacteria, flaviviruses and hemorrhagic fever viruses.
Flaviviruses have a relatively simple structure with only a single major external protein, the envelope protein. An aspect of the invention is that the cellular receptor utilized by flavivirus envelope proteins to bind to some target cells, as exemplified by dengue virus, is a highly sulfated type of heparan sulfate (HS). The cellular receptor on vascular endothelial cells is a related type of sulfated polyanionic glycoprotein, a mucin. The interaction between virus and host cell is a critical determinant of infectivity. Highly sulfated HS and heparin and synthetic polyanions, but not other glycosaminoglycans (GAGs), were effective competitive antagonists of dengue virus envelope protein binding to target cells. Pre-treatment of cells with GAG-lyases, and with the sulfation inhibitor sodium chlorate, prevented envelope protein binding. Mutant cell lines with defective GAG expression, including a mutant cell line specifically deficient only in HS expression, also failed to bind envelope protein. GAGs such as heparin and HS failed to competitively inhibit dengue virus envelope protein binding to endothelial cells, and treating endothelial cells with GAG-lyases also failed to inhibit subsequent binding of envelope protein, indicating that the endothelial receptor was not a GAG. However, treatment of endothelial cells with the mucin-specific lyase, O-sialoglycopeptidase did prevent subsequent envelope protein binding, indicating that the endothelial receptor was a mucin. Recombinant envelope protein bound to immobilized heparin, and eluted with 0.5 M NaCl, indicating a high affinity interaction.
Examination of the dengue virus envelope protein sequence revealed two GAG-binding motifs at the carboxy-terminus; the first could be structurally modeled and formed an unusual extended binding surface of basic amino acids, sufficiently large and accessible to interact with a GAG. Similar motifs were also identified in the envelope proteins of other flaviviridae and other hemorrhagic fever viruses. Many microorganisms including bacteria, protozoa and other viruses are known to utilize GAGs as binding targets, therefore, similar motifs are expected to be a general phenomenon. Heparin, highly sulfated HS, and the polysulfonate pharmaceutical Suramin effectively prevented dengue virus infection of target cells. Therefore, blockade of virus-target cell interactions is an effective strategy for treating flavivirus infections. This conclusion extends to all microorganisms.
In view of their common feature of vascular pathology, it is expected that other flaviviruses and hemorrhagic fever viruses all use the GAG-binding domains expressed in their envelope proteins, to bind to a similar vascular endothelial cell receptor. Supporting evidence consists of identifying putative GAG-binding domains in the peptide sequences of the envelope proteins of all flaviviruses examined and in four hemorrhagic fever viruses other than dengue virus: Yellow Fever, Ebola, Marburg and Lassa fever viruses.
An aspect of the invention is a method for isolating a microorganism such as
Chen Yaping
Linhardt Robert J.
Maguire Terence
Marks Rory M.
Allen Marianne P.
Brinks Hofer Gilson & Lione
Feman Mary K
The Regents of the University of Michigan
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