Virus protein antigens of the JC virus

Details Virus protein antigens of the JC virus Virus protein antigens of the JC virus

1998-07-17

2001-05-29

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S069100, C435S235100, C435S320100, C435S455000, C435S456000, C435S366000, C435S348000, C435S006120, C435S007100, C435S236000, C530S350000

Reexamination Certificate

active

06238859

ABSTRACT:

DESCRIPTION
The present invention concerns new virus-like particles and their use in analytical, diagnostic and therapeutic methods.
The JC virus (JCV) belongs to the group of human polyoma viruses. JCV can cause a sub-acute demyelinizing disease of the brain by a lytic infection of myelin-forming oligodendrocytes and an abortive infection of astrocytes. This infection, which is referred to clinically as progressive multifocal leukoencephalopathy (PML), leads to the formation of demyelinizing foci in the cerebrum cerebellum and brain stem and usually ends lethally within a few months.
With PML there is usually no significant humoral or cellular immune response to JCV which makes it difficult to diagnose the disease. Although JCV appears to be present in about 80% of the adult population, PML generally only develops in connection with a weakening of the immune system. The increasing use of immuno-suppressive drugs and the increasing number of HIV-infected patients has led to a considerable increase in PML diseases in recent years. According to current estimations a PML develops in about 2-5% of AIDS patients.
The known methods of diagnosis for detecting a PML disease essentially comprise image forming methods such as CT (computer tomography) and MRI (magnetic resonance imaging) as well as immunocytochemical methods based on biopsies or autopsies. Recently PCR detection methods have increased in importance, virus DNA amplification from cerebrospinal fluid (CSF) yielding reliable and specific results (Weber et al., J. Infect. Dis. (1994), 1138-1141 and McGuire et al., Annals of Neurology 37 (1995), 395-399).
The disadvantages of the diagnostic methods known from the state of the art are that they can only be carried out with a large amount of work and are therefore unsuitable for a reliable routine diagnosis. This also applies to PCR methods in which there is a high contamination risk that can lead to a falsification of the results. It would therefore be desirable to have available a method and reagents which allow a reliable diagnosis of PML in a more simple manner. Furthermore there is a need for an agent that can be used for the therapeutic treatment of PML diseases.
Hence the object of the invention is to provide methods and reagents with which the above-mentioned goals can be achieved and which at least partially avoids the disadvantages of the state of the art.
It was surprisingly found that the virus protein 1 (VP1) of the JC virus is an excellent agent for the immunochemical detection of JCV. Furthermore it was found that VP1 can be produced in a large yield in the form of virus-like particles (VLP) by recombinant DNA techniques.
Hence one subject matter of the invention is a virus-like particle (VLP) which is composed of several molecules of the virus protein 1 (VP1), the main structural protein of the JC virus. This VLP is in particular characterized in that it has a structure that reacts immunologically with anti-JCV antisera and is free of nucleic acids associated with JCV.
It was surprisingly found that, after purification, recombinant VP1 of JCV can associate to form virus-like particles (VLP). Such VLP have an icosahedral structure with a diameter of about 50 nm. The advantage of VLP over an individual VP1 protein is above all that the properties or/and effects of the natural virus are more exactly simulated and that a broader field of application is opened up. Further advantages over the natural virus are, in particular, increased work safety and it was surprisingly also found that the VLP has a higher immunogenicity than the purified total virus.
The VP1 of JCV is the main structural protein of the capsid envelope of JCV e.g. from wild-type strains or mutagenized strains of JCV. In a special embodiment the VLP is composed of recombinantly produced VP1. In this case the term VP1 also encompasses proteins which differ from wild-type VP1 by mutations such as substitutions, insertions or/and deletions. In order to produce recombinant VP1 one preferably uses a nucleic acid which comprises the sequence shown in SEQ ID NO.1, a sequence corresponding to this within the scope of the degeneracy of the genetic code or a sequence hybridizing with it under stringent conditions wherein the nucleic acid sequence or a recombinant vector containing this sequence is introduced into a suitable host cell, the host cell is cultured under conditions in which an expression of the nucleic acid sequence takes place and the protein is isolated from the cell or cell supernatant. Stringent hybridization conditions are preferably defined according to Sambrook et al. (1989) Molecular Cloning A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, and comprise a wash step of 30 min in 0.1×SSC, 0.5% SDS at 68° C.
The VLP according to the invention can furthermore have one or several additional heterologous proteins in the capsid structure. This means that a heterologous protein is anchored in the capsid structure, at least part of this protein being preferably accessible from outside. In principle all proteins are suitable as this heterologous protein which can be incorporated into the capsid structure and do not impair the self-assembly of the VLP. For example it may be desirable to provide the VLP with an antigenic determinant by means of the heterologous protein that can be detected by a specific antibody. On the other hand the heterologous protein can be a binding partner for a cell surface receptor which enables an interaction of the VLP with a specific class or subclass of cells which carry the corresponding receptor. After binding to the receptor such an interaction is for example the triggering of a particular signal or the internalization of the VLP.
The origin of the proteins is not limited to a particular organism and, depending on the special type of application, it is possible to incorporate eukaryotic, in particular human proteins, and also prokaryotic or viral proteins into the VLP. Examples of suitable proteins are surface proteins or membrane proteins, explicit examples of which are CD4 for a human cell surface receptor and the envelope protein of an immuno-deficiency virus e.g. HIV-gp 120 for a viral surface protein. It goes without saying that the heterologous protein can be modified if necessary, e.g. shortened, using recombinant methods especially if the natural protein cannot be incorporated into the VLP or impairs the self-assembly.
In a further special embodiment the VLP can contain one or several active substances inside the capsid structure. In this description an active substance is understood as any molecule which is not usually present in the medium used for self-assembly. Such active substances include for example macromolecules such as nucleic acids i.e. RNA, DNA or artificial modified nucleic acids as well as proteins and other physiologically active substances that can be of a natural, synthetic or recombinant type. Examples of such physiologically active substances are e.g. lipids, phospholipids, peptides, drugs, toxins etc.
Yet a further subject matter of the present invention is a nucleic acid which codes for a VP1 protein and comprises
(a) the nucleotide sequence shown in SEQ ID NO. 1
(b) a nucleotide sequence corresponding to the sequence from (a) within the scope of the degeneracy of the genetic code or/and
(c) a nucleotide sequence hybridizing with one of the sequences from (a) or/and (b) under stringent conditions.
The nucleic acid according to the invention is preferably located on a recombinant vector, in particular under the control of an expression signal. Examples of suitable vectors are described in Sambrook et al., Supra, chapters 1, 2, 3, 4, 16 and 17. The invention also concerns a cell which is transformed with a vector according to the invention.
A further aspect of the invention also concerns a process for the production of a VLP in which VP1 is purified and converted into a form in which an assembly of several VP1 molecules to form a VLP takes place. Suitable conditions for assembly are for example present when VP1 is

Affiliated with

Also associated with

Rating

Virus protein antigens of the JC virus does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Virus protein antigens of the JC virus, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Virus protein antigens of the JC virus will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2514034