The invention incorporates a method for the detection of minority memory genomes in viral quasispecies. Such genomes contain, at least, one mutation with respect to the majority genomes and include those involved in resistance to antiviral drugs and viral

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 non identical 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, using FMDV as model system, that viral quasispecies posses a molecular memory in the form of minority components of their mutant spectra, which 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 module 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 characterise 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 also allows a deeper knowledge of the genetic complexity of the viral quasispecies.

This invention incorporates a method for the detection of minority genomes in viral quasispecies:

1. The method 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, characterisation and quantification of minority genomes using techniques based on DNA microarrays, heroduplex tracking assay (HTA), or molecular cloning.

In case the detection of minority genomes uses DNA microarrays, the method comprises:

a) extraction of the 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) labelling of said amplified fragment/s with a labelling marker (fluorescent, radioactive or other);

d) construction of a microarray containing the following DNA (or 'peptide-nucleic acid', PNA) probes: i) at least one oligonucleotide (ON) useful as a positive control; ii) at least one ON as a negative control; iii) at least one ON useful for the specific detection of a mutation present in a minority genome; iv) the necessary ON for the construction of a calibration curve;

e) interaction of said amplified and labelled DNA fragment/s with the probes attached to the microarray, in the appropriate conditions so that hybridisation only takes place if all nucleotides of an ON in the microarray match to their complementary sequence in the genomic fragment;

f) identification of all ON showing positive hybridisations, discarding negative results and substracting background level (inespecific hybridisations);

g) estimation of the relative amount of hybridised genomes with respect to the total number of genomes in the viral quasispecies, by means of the calibration curve.

h) characterisation and quantification of the minority genomes, defined as those represented in less than 50% of the population;

Said minority genomes are minority memory genomes, which reflect the past evolutionary story of the virus and may constitute from 0.1 to 10% of the viral quasispecies.

Six examples are given on the applicability of this invention. They describe the detection, characterisation 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. It is given a list of 181 HIV drug-resistance mutations in reverse transcriptase and protease genes, which can be detected and quantified by means of the method included in the invention.

This method, unlike those currently used (which only recognise 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 summarised 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.

 

 

 

European patent granted (No. 2001929654; published as EP1284296), and patent applications filed in USA (No. 10/258,765), in Australia (No. 56362/01), in Brazil (BR 0110671) and in Canada (CA 2407753).

Technical cooperation; commercial agreement with technical assistance; license agreement...