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Methods for the detection of jc polyoma virus

a technology of jc polyomavirus and detection method, which is applied in the field of detection of human jc polyomavirus, can solve the problems of pml, death, and serious conditions in some subjects, and achieve the effect of increasing the risk profile of pml and achieving the effect of higher risk profil

Inactive Publication Date: 2012-10-11
BIOGEN MA INC
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention provides methods and compositions for determining whether a person is susceptible to PML (a brain infection caused by JCV) by analyzing the person's immune system. The invention identifies specific mutations in the viral surface protein (VP1) that are associated with an increased risk of PML. The invention also provides a panel of variants that are associated with PML risk. The invention can help healthcare professionals identify individuals at risk for PML and provide targeted treatment options. The invention also includes methods for detecting and analyzing JCV variants associated with PML. Overall, the invention helps to better understand and prevent PML infections in immunocompromised individuals.

Problems solved by technology

While infection by JCV is asymptomatic in most subjects, infection may result in serious conditions (like PML) and even death in some subjects.
However, since publicly available PML sequences were obtained from CSF or brain tissues of PML patients whereas non-PML sequences were obtained from the urine of healthy subjects, just based on the analysis of Genebank samples it could not unequivocally be demonstrated whether these amino acid variants were produced via a viral mutation in the patients or represented one or more rare viral variants that occur in all JCV clades and are enriched (e.g., positively selected) in PML cases as being more likely to be causing PML.

Method used

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  • Methods for the detection of jc polyoma virus
  • Methods for the detection of jc polyoma virus
  • Methods for the detection of jc polyoma virus

Examples

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example 1

Detection of JCV Variants by PCR

[0282]Nucleic acids are isolated from a biological sample using established protocols (e.g., cell lysis). Because the viral DNA may have integrated in the genomic DNA or may still be present as a smaller entity, both genomic DNA and shorter DNA sequences are isolated and subjected to PCR analysis. Upon isolation the nucleic acids are resuspended in a buffer that will facilitate PCR analysis. Buffers that facilitate PCR analysis are known to the skilled artisan (e.g., Maniatis) and are also commercially available from manufacturers of PCR enzymes (e.g., New England Biolabs, Beverly, Mass.). Nucleotide primers are designed to result in the amplification of the JCV-VP1 gene. PCR amplification is an established laboratory technique and comprises the addition of nucleotide primers, a polymerase and single nucleotides, and polymerase buffer and subjection this mixture to cycles of annealing, amplification and dissociation resulting in the amplification of a...

example 2

Detection of JCV Variants Using ELISA

[0283]Proteins and peptides are isolated from a biological sample using standard laboratory techniques (e.g., Maniatis). Both the cellular proteins and proteins of non-cellular components are subjected to the analysis. In one assay the sample is interrogated for the presence of JCV-VP1 polypeptides comprising one or more variants of the invention. The polypeptides are detected using sandwich ELISA comprising antibodies specific for JCV-VP1 polypeptides of the invention. The antibodies are generated by inoculating animals (e.g., rabbits) with the JCV-VP1 polypeptides of the invention resulting in polyclonal antibodies. If so desired, cells can be harvested from the inoculated animal to generate monoclonal antibodies. Methods for the generation of both polyclonal and monoclonal antibodies are routine in the art. The antibodies against JCV-VP1 polypeptide variants are immobilized on a solid surface (e.g., a 96-well plate), with one antibody type per...

example 3

Determining Solvent Accessible Surface Area Composition of VP1 Protein

[0285]Accessible surface area calculations require the knowledge of the 3D coordinates of biomolecule. A homology model of JCV VP1 virus-like particle was constructed using structure of CoAl of SV40 virus-like particle as a template (PDB ID: 1SVA). MODELER (A. Sali & T. L. Blundell. Comparative protein modelling by satisfaction of spatial restraints. J Mol. Biol. 234, 779-815, 1993) algorithm was used for model building and the SCWRL3 (A. A. Canutescu, A. A. Shelenkov, and R. L. Dunbrack, Jr. A graph theory algorithm for protein side-chain prediction. Protein Science 12, 2001-2014 (2003)) approach was used for side-chain position refinement. The polar and non-polar solvent accessible surface areas of amino acid sidechains were calculated using Lee and Richards's method (B. Lee B & F. M. Richards. The Interpretation of Protein Structures: Estimation of Static Accessibility. J. Mol. Biol 55, 379-400 (1971)). Subsequ...

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Abstract

Methods and compositions for determining whether a subject is at risk for PML, including subjects being treated with immunosuppressants, by determining whether the subject harbors a JCV variant with reduced binding for sialic acid relative to a normal JCV, are presented. Furthermore, combinations of JCV-VP1 sequence variations that are associated with PML and that can be used as a basis of an assay for identifying subjects susceptible to PML, subjects with PML (e.g., early stage PML), or subjects at risk of developing PML in response to an immunosuppressive treatment are provided.

Description

RELATED APPLICATIONS[0001]This application claims the benefit under 35 U.S.C. §119(e) from U.S. provisional application Ser. No. 61 / 150,310 entitled “Methods for the Detection of JC Polyoma Virus” filed Feb. 5, 2009, the disclosure of which is incorporated herein by reference.FIELD OF THE INVENTION[0002]The invention relates to methods for the detection of the human JC polyomavirus virus, diagnosis of progressive multifocal leukoencephalopathy (PML), and the development of therapeutics for PML.BACKGROUND OF THE INVENTION[0003]JC polyomavirus (JCV) infection in humans can cause a demyelinating disease of the central nervous system, progressive multifocal leukoencephalopathy (PML). However, JCV infection usually does not result in PML in healthy subjects. JCV infection is prevalent in many human populations without causing widespread PML. PML typically only develops in JCV-infected subjects that also have a weakened immune system. Subjects that are immuno-compromised due to a disease ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/70G01N33/569
CPCG01N33/56983G01N2333/025C07K2317/33G01N2800/50C07K16/081G01N2800/28G01N2469/20G01N2800/52
Inventor GORELIK, LEONIDLUGOVSKOY, ALEXEY
Owner BIOGEN MA INC
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