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CSIC BIO037- New Procedure For The Identification Of Resistance To Antiviral Drugs (HIV).
Center of Molecular Biology “Severo Ochoa” of CSIC has developed a new method for the detection of minority genomes in viral infections, such as HIV, Hepatitis B and C, and the food-and-mouth disease virus. This method allows the detection, characterization and quantification of mutations, which might be implied in resistance to antiviral drugs and in the evasion to the host immune response. This group is looking for financing and for a license agreement with biotechnology companies.
Specification sheet:
DESCRIPTION

Virus with RNA as genetic material, and some virus with DNA genome which use RNA as a molecular intermediate during their infective cycle, replicate as complex distributions of related but nonidentical genomes, termed viral quasispecies. Some of the viral quasispecies infecting humans or animals have an outstanding clinical relevance, such as human immunodeficiency virus (HIV), hepatitis B and C virus (HBV and HCV) and foot-and-mouth disease virus (FMDV). It has been recently described that viral quasispecies posses a molecular memory in the form of minority components of their mutant spectra, that reflects the previous evolutionary history of the virus. The existence of such "historical record" of the genomes that were selected in the past allows viral quasispecies to modulate the nature and intensity of the response to new selective pressures.

Minority memory genomes (constituting from 0.1 to 10% of the total quasispecies) may be selected and become majority or dominant within the viral quasispecies if they show any replicative advantage. As an example, memory genomes could bear one mutation conferring resistance to a particular drug that is going to be administered to one patient (this clinical decision could have been taken after not detecting such resistance mutation in the majority genome, by means of a conventional genetic analysis). In this case, minority memory genomes would quickly become dominant in the presence of the drug, producing a lack of response to it (a situation that could not have been predicted through the analysis of the majority genome).

Therefore, in chronic viral infections, as those produced by HIV, HBV, HCV or FMDV, it is essential to characterize both majority and minority memory genomes, since both of them may bear mutations involved in the response to the treatment or in the response of the immune system of the host.

This invention incorporates a method for the detection of minority genomes in viral quasispecies which comprises three steps:

a) extraction of the nucleic acids (genomes) of a viral quasispecies, from a clinical sample or from an in vitro viral culture;

b) amplification of at least one genomic fragment from said quasispecies;

c) detection, characterization and quantification of minority genomes using techniques based on DNA microarrays, heroduplex tracking assay (HTA), or molecular cloning.

Six examples are given on the applicability of this invention. They describe the detection, characterization and quantification of minority FMDV genomes involved in the escape to a monoclonal antibody, and that of HIV, HBV and HCV minority memory genomes bearing drug-resistance mutations in pretreated patients.

INNOVATIVE ASPECTS

The main innovative aspect of this invention is the possibility for detecting, characterizing and quantifying minority genomes present in the viral population. Among these minority genomes are the memory genomes, which influence the nature, intensity and speed of the virus response to antiviral therapy, and the immune response of the host.

Therefore this method, unlike those currently used (which only recognize majority genomes in viral quasispecies) allows performing a deeper genetic diagnostics in the populations of HIV, HBV, HCV or FMDV present in an infection. The clinical interest of this invention may be summarized in two points:

a) the presence of drug-resistance mutations in minority memory genomes produces a loss of sensitivity to that drug, affecting the clinical outcome of the infected patient;

b) the presence of mutations involved in the escape to immune system in minority memory genomes may affect the immune response of the infected patient and the selection of vaccine-escape variants.

COMPETITIVE ADVANTAGES

The method included in this invention has a main advantage with respect to the genetic methodologies currently used for virus analysis: the possibility for detecting and characterizing both majority and minority genomes present in the viral quasispecies. Also, it allows to quantify those minority genomes within the quasispecies.

The preferred use of DNA microarrays technology provides a number of advantages with respect to the technologies based on sequencing or hybridisation to nitrocelulose strips. Such advantages include: i) the high sensitivity of the technique, together to some modifications developed in the method included in the invention, allows to detect minority genomes representing as little as 0.1% of the total population; ii) it allows a high speed of sample processing; iii) it allows to analyse clinical samples with very low viral load; iv) it allows to simultaneously analyse thousands of mutations in viral genomes, v) it allows to quantify the genomes.

KEYWORDS

006001 Medicine, Human Health; 006001005 Diagnostic, Diagnosis; 006001006 Diseases; 006001015 Virus, Virology / Antibiotics /Bacteriology.

PATENT

ES 200001068, applied for at 2000-04-27

CONTACT

Javier Etxabe
email: j.etxabe@orgc.csic.es
phone: +34 91 585 53 75

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