ES2434191A1 - Method of determining tropism of HIV-1 and associated kit - Google Patents

Abstract

HIV-1 tropism determination method and associated kit. The present invention relates to a method for determining the tropism of the human immunodeficiency virus 1 (HIV-1) comprising PCR amplification of the env gene. Furthermore, the present invention relates to a kit for carrying out said method and to the use of said kit for the determination of HIV-1 tropism.

Description

METHOD OF DETERMINATION OF HIV-1 TROPISM AND KIT ASSOCIATED

The present invention relates to a method of determining the tropism of human immunodeficiency virus 1 (HIV-1) comprising PCR amplification of the env gene. Furthermore, the present invention relates to a kit for carrying out said method and the use of said kit for the determination of the tropism of HIV-1.

STATE OF THE PREVIOUS TECHNIQUE

Human immunodeficiency virus 1 (HIV-1) is a lentivirus of the Retroviridae family that infects cells of the immune system. The virus interacts with the target cell through the envelope gene product (env), which corresponds to the gp120 / gp41 envelope protein. This process requires binding to the CD4 receptor followed by a conformational change that allows the interaction of a second co-receptor that can be CCR5, or CXCR4. Depending on the correceptor involved in the interaction, the virus will present a viral or other tropism.

Initially, viral tropism was classified into syncytium-inducing strains (SI) and non-syncytium-inducing strains (NSI), depending on whether the virus caused said cytopathic effect. In the case of HIV, a syncytium is a multinucleated cell due to the fusion of individual cells. It has been shown that the differentiation between SI and NSI strains is due to differential use of co-receptors: SI strains have CXCR4 as co-receptor and NSI strains have CCR5. It is considered that there are three types of viral tropism: the R5 strains, whose viruses use the CCR5 correceptor, the X4 strains that use CXCR4 as a correceptor, and the X4R5 strains capable of using both co-receptors.

According to viral tropism, the most appropriate medical treatment for the patient will vary. Currently, the only drug marketed as a CCR5 receptor antagonist capable of blocking viral entry is Maraviroc (Pfeizer), but there are other drugs from the same family in different stages of clinical development such as Vicriviroc (Schering-Plow), SPRO 140 ( Progenics Pharmaceuticals) and PF-232798 (ViiV Healthcare). The use of these drugs is limited to patients infected with viral strains dependent on CCR5. The next appearance of new treatments predicts a significant increase in the number of determinations that will have to be made to select the patients who can benefit from the treatment.

According to the annual report of the Commission of Security and Change of the United States, on the companies Monogram and Quest, the technologies to identify genetic resistance of HIV-1 to the new treatments with inhibitors of the viral entrance are essential for a better management of the treatment of the infection and provide critical information that allows personalized prescription. This diagnostic system also helps the development and clinical evaluation of new drugs under development by pharmaceutical companies, especially in the selection of patients who can benefit most from the therapy.

The determination of HIV-1 viral tropism can be performed by phenotypic and genotypic methods (Rose et al., 2009 Curr Opin HIV HIV 4: 136-142). There are several phenotypic methods for determining viral tropism. The oldest is known as MT2 and consists of the detection of the presence or absence of syncytia in cells that express the CD4 receptor. Among the most current methods, Trofile stands out (Monogram Biosciences). The Trofile method is based on the generation of recombinant pseudoviruses to infect cell lines that express on its surface CD4, one of the co-receptors and also contain a marker gene. The presence of the marker gene allows measurement of the level of infection of target cells, as well as the inhibition of infection by antagonists, in case the recombinant pseudoviruses are capable of infecting the cells. For the generation of recombinant pseudoviruses, the patient's envelope gene is amplified by PCR and cloned into an expression vector that is co-transfected into cells containing the defective virus with a deletion in the envelope gene itself. In this way, a phenomenon of trans complementation is achieved by the cloned envelope protein and recombinant pseudoviruses are generated that have only one infective cycle (Rose et al., 2009 Curr Opin HIV AIDS 4: 136-142).

An approach to the generation of recombinant pseudoviruses without cloning is the construction of a recombinant DNA that contains a CMV promoter and the envelope gene that is transfected to a cell line to generate the recombinant pseudoviruses (Kirchherr et al., 2007 J Virol Methods 143 (1): 104-111; Lin et al. Journal of Virological Methods 169 (2010) 39–46).

Genotypic methods are the main alternative thanks to the application of in silico methodologies. The use of disruptive technologies such as the direct sequencing of the viral genome and the prediction of the strain type using specific algorithms currently present significant sensitivity and specificity problems (Rose et al., 2009 Curr Opin HIV AIDS 4: 136-142).

Other methods are those described in González et al., 2010 J Antimicrob Chemother. 65 (12): 2493-501; Raymond et al., 2010 J Clin Virol. 47 (2): 126-30; o Saliou et al., 2011 Antimicrob Agents Chemother. 55 (6): 2831-6.

Currently, the diagnosis of the type of viral strain is mainly carried out at Monogram Biosciences (San Francisco, USA) with a high cost per sample. In Europe the offer is limited, highlighting Lab21 (United Kingdom). Quest Diagnostics has developed a diagnostic system based on viral DNA amplification and sequencing (US7566532). Hospitals and research centers make numerous diagnoses a year, and it is highly likely that the volume will continue to grow in the coming years due to the incorporation of new drugs from the same family, which makes it necessary a simpler method of less cost that can be performed in the hospitals and research centers themselves.

DESCRIPTION OF THE INVENTION

The authors of the present invention have developed a method of expression of PCR products directly in the cytoplasm of transfected mammalian cells, without the need to clone said product into a eukaryotic expression plasmid. The method is based on the following technical premises:

one.

The 5 'end of any PCR primer can accept a DNA fragment of sequence not complementary to the fragment to be amplified without significant loss of amplification performance. This may include a promoter, restriction enzyme targets or polyadenine tails, etc ...

2.

Two PCR products with a small overlapping region can be mixed and amplified again with two external primers that bind to the 5'end of the first product and 3'end of the second. Generating a fusion product of both DNA fragments.

3.

The so-called Internal Ribosome Entries (IRES) of several viruses with cytoplasmic replication allow translation of mRNA in the absence of a standard capping at the 5 'end of the mRNA.

Four.

The bacteriophage T7 RNA polymerase can be efficiently functionally expressed in the mammalian cell cytoplasm.

This technique allows the determination of the viral tropism of HIV-1 in which the use of cloning vectors is not necessary and, therefore, simplifies said method. The method of the present invention allows the isolation and amplification of the env gene from an isolated sample of a patient infected with HIV-1, and it does so more directly and simply and in sufficient quantities as for the determination of tropism viral in cell-cell fusion studies (syncytium formation).

The method of the present invention is simple and based on 7 primers that are used in successive PCR reactions. The sequences of said primers allow the generation of an amplicon comprising a strong cytoplasmic promoter, an IRES sequence, the HIV-1 env gene and a polyA.

The diagnostic method of the present invention requires a shorter processing time than the methods known in the prior art and its cost is less. In addition, the genotypic methods used to date, such as sequencing, are not very specific.

The method of determining the viral tropism of HIV-1 of the present invention, briefly, is to obtain a DNA copy of the genome of the virus that infects a patient, from the isolated sample of said patient.

From this DNA copy and by PCR, an amplicon is generated comprising a promoter, an IRES sequence, the HIV-1 env gene that infects the patient in question and a polyA tail. Said amplicon is transfected into a cell line containing the bacteriophage T7 polymerase, so that the expression of the env gene in these cells is possible, and the TAT viral activity transactivator protein.

With the transfected cells a cell fusion assay is performed. For this, it is necessary to co-cultivate the previous cells with another cell line that contains the β-galactosidase gene flanked by LTR sequences, the CD4 receptor and, either present the CCR5 correceptor, or present the CXCR4 correceptor.

The result of co-cultivation may be the presence or absence of fusion. The presence of syncytia is a consequence of the fusion mediated by the interaction of the env gene with the CD4 receptor and the appropriate co-receptor. In addition, due to the fusion of the two cells, the transactivator protein of the viral activity can interact with the LTRs that flank the β-galactosidase and allow its transcription and translation, which allows the detection of the fusion by colored or luminescent staining of β-galactosidase activity.

If there is no fusion, this implies that the co-receptor presented by the cells does not allow the correct interaction between the env1 protein of the patient's HIV1 gene, the CD4 and the co-receptor.

To determine viral tropism, a β-galactosidase staining and / or a measure of the luminescence of β-galactosidase can be performed. With the staining, it can be determined visually by using an inverted microscope because the cells that form syncytia will have a characteristic blue color of the processing of the β-galactosidase substrate (see Figures 2 and 3).

Therefore, a first aspect of the present invention relates to a method for the determination of HIV-1 viral tropism comprising the following steps:

(i)

 PCR amplifying a promoter and an IRES sequence from a template nucleic acid comprising said promoter and said IRES sequence, by means of a direct primer A, wherein said primer A hybridizes with the 5 'end of the promoter, and a reverse primer B, wherein said primer B hybridizes at its 3 'end with the 3' end of the IRES sequence and at its 5 'end is complementary to the 5' end of the env gene of the human immunodeficiency virus 1;

(ii)

 PCR amplifying a sequence comprising the HIV-1 env gene from HIV-1 copy DNA by a direct primer C and a reverse primer D, wherein said primers C and D hybridize with the sequences flanking said env gene;

(iii) PCR amplify the sequence of the env gene from the product obtained in step (ii), by means of a direct primer E, where the 5 'end of said primer E is complementary to the IRES sequence of step (i) and wherein the 3 'end of said primer E hybridizes with the 5' end of the env gene, and a reverse primer F, which hybridizes with the 3 'end of the env gene;

(iv)

 PCR amplify the products of steps (i) and (iii) by the direct primer A and the reverse primer G whose 5 'end has a polyimine tail and the 3' zone inhibited with the 3 'end of the env gene.

(v)

 transfect the amplicon obtained in step (iv) in cells expressing the transactivator protein of viral activity as well as the bacteriophage T7 RNA polymerase.

(saw)

co-culturing the cells of step (v) with cells containing a marker gene flanked by LTR sequences and expressing the CD4 receptor and either the CXCR4 correceptor or the CCR5 correceptor.

(vii) detect and / or quantify expression of the marker gene, and

(viii) associate the levels of expression of the marker gene detected and / or quantified in step (vii) to the viral tropism CCR5 or double tropism.

The term "viral tropism," as used herein, refers to the ability of a virus to selectively infect certain cell populations, which may be influenced by viral and host factors. The viral tropism to be determined by the method of the present invention will be CCR5, or double tropism depending on the correceptor that the virus requires to infect the cells. When the virus can infect both CXCR4 cells and CCR5 cells, its tropism is said to be twofold.

The term "human immunodeficiency virus 1 (HIV-1)", as used herein, refers to the lentivirus (of the Retroviridae family), which causes acquired immunodeficiency syndrome (AIDS). Its genome is a chain of single stranded RNA that must be provisionally copied to DNA in order to multiply and integrate into the genome of the cell it infects. The genome of this virus has the gag, pol and env genes, and five additional genes (tat, rev, vpu, vif and nef). The viral envelope is based on a lipid bilayer, and its basic structural components come from the plasma membrane of the parasitized cell. But the envelope also carries regularly spaced 72 spicules, which are protein complexes integrated in the membrane formed by viral proteins encoded by the env gene.

The term "promoter," as used herein, refers to a transcriptional regulatory nucleotide sequence of a gene, which directs its expression. A promoter can be strong or weak if it induces a strong or weak expression, respectively, which means that the gene it regulates expresses much or little. In addition, a promoter can be constitutive, so that transcription of the gene it regulates is always activated, or it can be dependent on conditions of the cellular environment, such as tissue type, temperature, salinity, etc. Moreover, a promoter can induce gene expression in the cell nucleus or in the cell cytoplasm. In the present invention, the promoter is preferably a strong promoter. Preferably it is a cytoplasmic promoter. Preferably it is the T7 bacteriophage RNA polymerase promoter. The advantage of using a strong promoter that is also cytoplasmic is that the expression of the env gene is greater and thus the sensitivity of the method for the determination of viral tropism is increased.

The term "IRES sequence", as used herein, refers to a nucleotide sequence that allows messenger RNA (mRNA) to enter the ribosome and thus favors translation of the protein encoded by said mRNA. In the present invention, the IRES sequence is preferably the 5’UTR (untranslated region) sequence of the encephalomyocarditis virus (ECMV). The combination between the strong cytoplasmic promoter and the IRES sequence further enhances the transcription of the env gene, and also the sensitivity of the method for the determination of viral tropism. In addition to the advantages already mentioned of the strong and cytoplasmic promoter and the IRES sequence, the presence of the polyA tail, thanks to primer F, increases the stability of the transcript, which allows, together with the greater expression thereof, a considerable increased translation of the env protein, which in general translates into an unexpected increase in the effectiveness of the method of the invention as well as its cytoplasmic expression.

The template nucleic acid of the present invention comprising a promoter and an IRES sequence can be, for example, but not limited to, a plasmid.

The term "plasmid", as used herein, refers to a double-stranded, circular or linear extrachromosomal DNA molecule, with replication independent of that of the cellular genome. The plasmid of the present invention comprises at least one origin of replication, a promoter and an IRES sequence and may optionally comprise an antibiotic resistance gene or other type of marker genes, and other sequences widely known in the state of the technique. In addition, in biotechnology, a plasmid can be used as an expression vector of a gene of interest. In the present invention, a gene of interest may be the TAT viral activity transactivator protein gene, that of the T7 bacteriophage RNA polymerase, that of the rev protein, the envelope protein gp160 and / or that of The green fluorescent protein.

The term "primer", as used herein, refers to an oligonucleotide, that is, a nucleotide sequence of between about 10 and 100 nucleotides. In the present invention, preferably primers from A to G are between 18 and 100 nucleotides in length. More preferably, they have between 20 and 70 nucleotides.

In a preferred embodiment of the first aspect of the present invention, the sequence of primer A is SEQ ID NO: 1, the sequence of primer B is SEQ ID NO: 2, the sequence of primer C is SEQ ID NO: 3, the sequence of primer D is SEQ ID NO: 4, the sequence of primer E is SEQ ID NO: 5, the sequence of primer F is SEQ ID NO: 6, and sequence of primer G is SEQ ID NO: 7.

SEQ ID NO: 1 hybridizes with the 5 ′ end of the T7 bacteriophage RNA polymerase promoter.

SEQ ID NO: 2 hybridizes at its 3 ’end with the 3’ end of the 5 ’UTR sequence of the encephalomyocarditis virus and at its 5’ end is complementary to the 5 ’end of the env gene of the human immunodeficiency virus 1.

SEQ ID NO: 3 hybridizes in the sequence adjacent to the env gene at its 5 ’end.

SEQ ID NO: 4 hybridizes in the sequence adjacent to the env gene at its 3 ’end.

SEQ ID NO: 5 at its 5 ’end is complementary to the 5’ UTR sequence of the encephalomyocarditis enterovirus and its 3 ’end hybridizes to the 5’ end of the env gene of the human immunodeficiency virus 1.

SEQ ID NO: 6 hybridizes in the sequence adjacent to the env gene at its 3 ’end.

SEQ ID NO: 7 comprises a poly-T at its 5 ′ end and at its 3 ′ end hybridizes with the 3 ′ end of the env gene of the human immunodeficiency virus 1.

The term "env gene", as used herein, refers to the gene encoding the gp160 viral protein that is subsequently proteolyzed and gives rise to the gp41 and gp120 proteins, which form the spicules of the viral envelope. These proteins are essential for the coupling of the virus with the outside of the cell that is going to infect.

The term "flanking", as used herein, refers to the nucleotide sequences that are located on both sides (at the 5 'and 3' end) of the nucleotide sequence in question, in this case the env gene

The term "transfect", as used herein, refers to introducing an exogenous nucleic acid into a cell. Transfection can be carried out by any of the methods known in the state of the art, among which are electroporation, lipofection, and calcium phosphate.

The term "amplicon," as used herein, refers to the nucleotide sequence that is obtained as a product of an amplification reaction, such as PCR. The nucleotide sequence of the amplicon will be defined by the nucleotide sequence used as a template and the nucleotide sequences of the primers used in the amplification reaction.

The term "transactivator protein of viral activity", as used herein, refers to the gene or Tat viral protein, which is essential for the transactivation of the viral promoter. The protein binds to a region of 59 nucleotides located at the 5 'end of the viral RNA called TAR (from the English "Transactivator Active Region") and acts as a transactivator, promoting elongation of the viral genome, during its transcription.

In a preferred embodiment of the first aspect of the present invention, the cells expressing transactivator protein of viral activity are the U-251 MG cell line. Preferably, said cells comprise the bacteriophage T7 RNA polymerase. More preferably, said cells are obtained by transfecting an expression plasmid of bacteriophage T7 RNA polymerase.

The term "U-251 MG", as used herein, refers to a cell line formerly known as U-373 MG derived from a human glioblastoma astrocytoma, whose reference number in the ECCAC (European Crop Collection cellular) is 09063001. In the present invention, U-251 MG cells are transfected with a bacteriophage T7 polymerase expression plasmid.

In a preferred embodiment of the first aspect of the invention, the cells that contain a marker gene flanked by LTR sequences, express CD4 and CCR5 are the P4-R5 MAGI cell line. P4-R5 MAGI cells are HeLa cells that express a copy of the human CD4 gene integrated into their genome and the HIV-1 LTRs fused with the β-galactosidase marker gene, and also express the CCR5 coreceptor. These cells are commercially available.

In a preferred embodiment of the first aspect of the invention, cells containing a marker gene flanked by LTR sequences, express the CD4 receptor and the CXCR4 correceptor are the HeLa-CD4 cell line. HeLa cells are a human cell line derived from cervical cancer commonly used in research and available in cell culture collections and that has been genetically engineered to express the CD4 receptor.

The term "marker gene", as used herein, refers to a gene that codes for a marker peptide or protein. Said peptide or marker protein allows easy detection and / or quantification, either because it is easily detectable by immunological methods, or because it has an enzymatic activity, or because it is fluorescent or luminescent. The expression of the marker gene makes it possible to detect the presence and / or quantify the marker protein, which is associated with the presence of that gene in the cell presenting the marker peptide or protein. In the event that the marker gene is an enzyme, its activity will allow the transformation of a particular substrate into a product that can be either colored or luminescent. The detection and / or quantification of said colored product

or luminescent will allow to determine if a cell comprises the marker gene. In a preferred embodiment, the marker gene is β-galactosidase, luciferase, amylase or green fluorescent protein. In an even more preferred embodiment, the marker gene is that of β-galactosidase.

The term "β-galactosidase," as used herein, refers to a hydrolytic enzyme that catalyzes the hydrolysis of β-galactosides in monosaccharides. This enzyme is encoded by the E. coli lacZ gene, forming part of the lac operon, which is activated in the presence of lactose and low glucose levels. Β-Galactosidase can use as lactose substrate, lactosylceramides, GM1 ganglioside, 5-bromo-4-chloro-3-indolyl-β-Dgalactopyranoside (X-Gal) and various glycoproteins, among other substrates. The β-galactosidase gene is widely used as a marker gene in conjunction with the X-Gal substrate in various applications, since its combination results in a blue product that allows easy detection.

The term "luciferase", as used herein, refers to an oxidative enzyme capable of producing light by a reaction that uses luciferin as a substrate and sometimes requires the presence of cofactors such as calcium ions or ATP. The best known example of luciferase is that of the firefly Photinus spyralis, although there are other recombinant luciferases.

The term "amylase", as used herein, refers to an enzyme that catalyzes the transformation of starch into monosaccharides acting on α-1-4-glycosidic bonds. As a marker gene, amylase is used since the presence of this enzyme can be detected, which prevents staining of cell starch with iodine.

The term "green fluorescent protein", as used herein, refers to a protein commonly known as GFP ("green fluorescent protein"), which emits green fluorescence when exposed to blue ultraviolet light. The gene encoding this protein is isolated and is commonly used in molecular biology as a marker.

In a preferred embodiment, the detection and / or quantification of the expression of the marker gene is performed by fluorescence, colorimetric labeling, immunological methods, spectroscopic methods and / or PCR. In another even more preferred embodiment, the detection and / or quantification of the expression of the marker gene is performed by staining of β-galactosidase and / or luminescent β-galactosidase activity.

The term "fluorescence," as used herein, refers to the use of a particular type of luminescence, which characterizes substances that are capable of absorbing energy in the form of electromagnetic radiation and then emitting some of that energy in form of electromagnetic radiation of different wavelength, for the detection and / or quantification of a marker gene. The total energy emitted in the form of light is always less than the total energy absorbed and the difference between the two is dissipated in the form of heat. In general, fluorescent substances absorb energy in the form of short-wave electromagnetic radiation, such as gamma radiation, X-rays or ultraviolet. After this they emit it again at a longer wavelength, that is to say it has less energy. The characteristics of fluorescent substances can be used in a laboratory to perform different techniques. Examples of the use of fluorescence include, among others, fluorescence microscopy, sequencing, detection and cytometry.

The term "colorimetric labeling", as used herein, refers to the detection of the presence or absence, or to the quantification of a marker peptide or protein when it can be associated with the presence of a colored compound, such as for example, but not limited to, the case of the detection of β-galactosidase by the presence of the colored product after hydrolysis of the X-Gal substrate.

The term "immunological methods", as used herein, refers to methods based on the use of antibodies to detect and / or quantify the presence of an antigen in a sample. Examples of immunological methods, among others, are immunohistochemistry, immunocytochemistry, immunodiffusion, immunoelectrophoresis, radioimmunoassay and ELISA.

The term "spectroscopic methods", as used herein, refers to a method based on the interaction of electromagnetic radiation, or other particles, with an analyte to identify it or determine its concentration. The analyte undergoes absorption, emission or luminescence processes. Examples of spectroscopic methods, among others, are: absorption spectroscopy, X-ray spectroscopy, infrared spectroscopy, nuclear magnetic resonance spectroscopy, mass spectrometry, X-ray diffraction and spectrophotometry. The β-galactosidase activity can be detected by spectroscopic methods, such as a luminometer, when an enzyme substrate that generates a luminescent product is used.

In a preferred embodiment of the first aspect of the invention, cells containing a marker gene flanked by LTR sequences express the CD4 receptor and the CXCR4 correceptor are the HeLa line.

The term "LTR sequences", as used herein, refers to the characteristic nucleotide sequences found at each end of the retroviral genome. These sequences are called LTR (long terminal repetitions) because they have hundreds or thousands of repetitions and are involved in the process of integration into the genome of the virus-infected cell.

The term "CD4 receptor," as used herein, refers to the glycoprotein of the immunoglobulin superfamily that participates in the adhesion of T cells to the target cell. , being involved in thymic maturation and in signal transmission during interaction with the antigen. It is expressed on the surface of T helper lymphocytes, monocytes, macrophages and dendritic cells. The CD4 receptor is the gene described in the “Gene” database of the NCBI (National Center for Biotechnology Information) with reference (Gene ID) 920.

The term "CXCR4 correceptor" (as "CXC chemokine receptor type 4") as used herein, refers to an α-chemokine receptor involved in chemotaxis that some HIV-1 use as a receptor to infect the cells of the immune system. The CXCR4 co-receptor is the gene described in the NCBI "Gene" database with reference (Gene ID) 7852.

The term "CCR5 correceptor", (in English, "CC chemokine receptor 5") as used herein, refers to a β-chemokine receptor whose natural ligands are RANTES (English, regulated upon Activation, normal T- cell expressed, and secreted) and MIP (macrophage inflammatory protein). It is expressed mainly in T lymphocytes, macrophages, dendritic cells and microglia. The CCR5 co-receptor is one of the co-receptors that HIV-1 uses to infect immune system cells.

The CCR5 co-receptor is the gene described in the NCBI "Gene" database with reference (Gene ID) 1234.

A second aspect of the present invention relates to primers SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6. SEQ ID NO: 7

A third aspect of the present invention relates to a kit comprising:

(i)

 a direct primer A that hybridizes with the 5 'end of the T7 bacteriophage RNA polymerase promoter;

(ii)

a reverse primer B that hybridizes at its 3 'end with the 3' end of an IRES sequence and at its 5 'end is complementary to the 5' end of the env gene of the human immunodeficiency virus 1;

(iii) a direct primer C that hybridizes with the 5 ’flanking sequence of the env gene;

(iv)

 a reverse primer D that hybridizes with the 3 ’flanking sequence of the env gene;

(v)

 a direct primer E which at its 5 ’end is complementary to the IRES sequence and at its 3’ end hybridizes to the 5 ’end of the env gene; Y

(saw)

 an inverse primer F that hybridizes at its 3 ’end with the 3’ end of the env gene

(vii) a reverse primer G that hybridizes at its 3 ’end with the 3’ end of the env gene and whose 5 ’end comprises a poly-T.

In a preferred embodiment, primer A is SEQ ID NO: 1, primer B is SEQ ID NO: 2, primer C is SEQ ID NO: 3, primer D is SEQ ID NO: 4, primer E is SEQ ID NO: 5 primer F is SEQ ID NO: 6 and primer G is SEQ ID NO: 7.

In a preferred embodiment, the kit further comprises a TAT viral activity transactivator protein expression plasmid, a T7 bacteriophage RNA polymerase expression plasmid, as controls include a protein rev expression plasmid (regulator of the virion), a gp160 envelope protein expression plasmid and / or a fluorescent green protein expression plasmid.

The term "virion regulator" as used herein refers to the gene or rev protein, which regulates the transport of the mRNA from the viral envelope of the nucleus to the cytoplasm.

In general, the kit of the invention comprises all those reagents necessary to carry out the method of the invention as described above. The kit can also include, without any limitation, buffers, enzymes, such as, but not limited to, polymerases such as Taq polymerase, cofactors to obtain an optimal activity of polymerases, dNTPs, a magnesium salt, probes, fluorochromes, agents to prevent contamination, etc. The kit may also contain other molecules, genes, proteins or probes of interest, which serve as positive and / or negative controls; as well as another pair (s) of primers for the amplification of one or several control nucleotide sequences. On the other hand, the kit can include all the supports and containers necessary for its start-up. Preferably, the kit further comprises instructions for carrying out the method of the invention.

A fourth aspect of the present invention relates to the use of primers SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or the kit of the third aspect of the invention, to determine the viral tropism of the human immunodeficiency virus 1.

With the primers and the kit of the present invention it is also possible to obtain pseudovirus. These pseudoviruses can be produced by transfection of the amplicon comprising the T7 promoter, IRES, env and polyA in a packaging cell line carrying a retrovirus such as MMLV whose env gene has been replaced by a reporter gene such as luciferase. Once the pseudoviruses have been generated, they can also be used for the typing of HIV-1 by means of an assay in which they can enter cells with CD4 and either the CXCR4 correceptor or the CCR5 correceptor. In this case it is not necessary to use TAT. And REV is only included for the expression of a viral control envelope from a plasmid.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Figure 1.-Scheme of the method of the first aspect of the present invention. In the upper part you can see how the final amplicon will be and the origin of each of its elements. In primers B and E, and in PCR products 1 and 2, black boxes indicate overlapping regions.

Figure 2.- Shows a microscope photograph of the cell co-culture and syncytia at 40X magnification. The marking with X-Gal shows that there has been

cell fusion with the line that expresses the CCR5 co-receptor and therefore the viral tropism is CCR5.

Figure 3.-Shows a microscope photograph of the cell co-culture and syncytia at 40X magnification. The X-Gal marking demonstrates that there has been cell fusion with the line that expresses the CCR5 correceptor and therefore the viral tropism is CCR5.

EXAMPLES

The invention will now be illustrated by tests carried out by the inventors demonstrating the effectiveness of the method of the invention, the primers and the kit. The terms primer, oligo and oligonucleotide are used interchangeably.

Example Typing of HIV-1 by the method of the present invention

Material and methods

Simplified process of determining the viral tropism of HIV-1.

The simplified process consists of the following stages, which are shown in Figure 1:

(i)

Amplify the T7 bacteriophage RNA polymerase promoter along with the 5’UTR of the encephalomyocarditis enterovirus from a plasmid where this structure is cloned. The zone 3 primer has an overlapping sequence with the region 5 of the HIV-1 envelope gene. At the end of this PCR, the PCR product 1 is obtained.

(ii)

PCR amplified the nested HIV-1 envelope gene. For this, two PCRs are performed. Primer 5 ’of the second PCR performed overlaps with the product of PCR 1 and primer 3’ contains a 40-base polyimine region in addition to the specific sequence of the viral envelope region. At the end of this stage the PCR product 2 is obtained.

(iii) Mix the PCR products 1 and 2 and reamplify with the end primers. A DNA fragment with the following characteristics (from 5 'to 3') is obtained: T7 bacteriophage RNA polymerase promoter, 5’UTR region of the encephalomyocarditis enterovirus, HIV-1 envelope and polyadenin tail.

(iv)

 Transfect the amplicon in human cells that express the bacteriophage T7 RNA polymerase, resulting in massive expression of the envelope protein of HIV-1. This level of expression allows rapid diagnosis of tropism through a fusion test.

(v)

 Perform a fusion assay by co-culturing the cells transfected with cells expressing CD4 / CCR5 / lacZ gene surrounded by LTR or CD4 / CXCR4 / lacZ gene surrounded by LTR or any other marker gene. In these examples in particular, fusion has been evaluated through the use of TAT, which acts in trans on the target cell, specifically controls the viral LTR and responds strongly to the presence of TAT.

Materials for the generation of T7-IRES-env-PoliA PCR products.

one.

Primers for amplification of PCR product 1 (100 pmol / μl): Primer A (SEQ ID NO: 1):

2.

5´-TAATACGACTCACTATAGGCTAGCCTCGAGGA-3´

3.

Primer B (SEQ ID NO2): 5´-TTCCTGCCATAGGAGATGCCTAAGTCGACTCTAGAGGATCCCGGGT TG-3´

Four.

Primers for amplification of PCR product 2 (100 pmol / μl) C

5 (SEQ ID NO: 3): 5'-TACAGTGCAGGGGAAAGAATAATAGACATAATA3 ', D (SEQ ID NO: 4): 5'-AGACCCAGTACAGGCRARAAGC3´, E (SEQ ID NO: 5): 5'-CAACCCGGGATCCTCTAGAGTCGACTTGGATCAGAGGGATCAGGAGGAGGAGGAGGAGGGG SEQ ID NO: 6): 5 '

10 CCAGTCCCCCCTTTTCTTTTAAAAAG -3 ’

5.

G (SEQ ID NO: 7): 5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCCAGTCCCCCC TTTTCTTTTAAAAAG -3 ’

6.

.Kit “Expand long template PCR system” (Roche)

15 7. Conventional PCR material (dNTPs (mixture of deoxyribonucleotide triphosphate: ATP, GTP, CTP and TTP), pipette tips with filter, tube racks, etc.)

Protocol for the generation of PCR products T7-IRES-env-Poli-A.

T7 + IRES amplification (PCR product 1): Prepare the following reaction mixtures A and B, stir and then mix:

Mix A:

Material

Volume (μl) per sample

10 mM dNTPs

2

Primer A (SEQ ID NO: 1)

one

Primer B (SEQ ID NO: 2)

one

Plasmid DNA (100 ng / μl)

one

Water

twenty

The promoter template DNA and IRES is the pIRES plasmid DNA (Clontech 10 Catalog No. 631605).

Mix B:

Material

Volume (μl) per sample

Buffer 1 (Expand long template PCR system, Roche)

5

Taq polymerase

0.5

Water

19.5

15 Once the reaction mixture is prepared, perform the following PCR cycles:

Number of times

Temperature (ºC) -time

one

94-2 minutes

30

94-10 seconds 55-30 seconds

68-30 seconds

one

68-10 minutes

one

4-indefinitely

The product of PCR 1 is a DNA fragment of about 600 bp (base pairs) to be identified by 2% agarose electrophoresis and purified by affinity chromatography.

5 Amplification of the env gene (PCR product 2): In this step two consecutive PCRs are performed. For the first prepare the following reaction mixtures, stir and then mix: First PCR of the shell

10 Mix A:

Material

Volume (μl) per sample

10 mM dNTPs

2

Primer C (SEQ ID NO: 3)

one

Primer D (SEQ ID NO: 4)

one

DNA copy (100 ng / ul)

5

Water

16

The cDNA (DNA copy) is obtained from the plasma samples of patients infected with HIV-1, from the viral RNA, using the 15 Superscript III kit by Invitrogen, following the manufacturer's instructions (Superscript III, Catalog No. 18080- 044).

B mix

Material

Volume (μl) per sample

Buffer 1 (Expand long template PCR system Roche)

5

Taq polymerase

0.5

Water

19.5

Once the reaction mixture is prepared, perform the following PCR cycles:

Number of times

Temperature (ºC) -time

one

94-2 minutes

10

94-10 seconds 55-30 seconds 68-2 minutes 30 seconds

twenty

94-10 seconds 55-30 seconds 68-2 minutes 30 seconds + 20 extra seconds per cycle

one

68-10 minutes

one

4-indefinitely

Second PCR of the envelope

Material

Volume (μl) per sample

10 mM dNTPs

2

Primer E (SEQ ID NO: 5)

one

Primer F (SEQ ID NO: 6)

one

DNA product first PCR envelope

0.5

Water

20.5

B mix

Material

Volume (μl) per sample

Buffer 1 (Expand long template PCR system Roche)

5

Taq polymerase

0.5

Water

19.5

Once the reaction mixture is prepared, perform the following PCR cycles:

Number of times

Temperature (ºC) -time

one

94-2 minutes

10

94-10 seconds 55-30 seconds 68-2 minutes 30 seconds

twenty

94-10 seconds 55-30 seconds 68-2 minutes 30 seconds + 20 extra seconds per cycle

one

68-10 minutes

one

4-indefinitely

The final product is a DNA fragment of about 3 Kb to be characterized by 0.8% agarose electrophoresis and purified by affinity chromatography, according to the manufacturer's instructions Macherey-Nagel (NucleoSpin® Gel and PCR Clean-up, Catalog No. 740609.10) before proceeding to the next step.

5 Assembly PCR: The result of both amplifications (T7-IRES and HIV-1 env gene) are used as a template for the last PCR. Dilute both PCR products 1/100, mix as described below and amplify with primers A (SEQ ID NO: 1) and G

10 (SEQ ID NO: 7).

Prepare the following reaction mixtures, stir and then mix:

Mix A: 15

Material

Volume (μl) per sample

10 mM dNTPs

2

Primer A (SEQ ID NO: 1)

one

Primer G (SEQ ID NO: 7)

one

PCR product DNA 1 (1/100 dilution)

one

PCR product DNA 2 (1/100 dilution)

one

Water

19

Mix B:

Material

Volume (μl) per sample

Buffer 1 (Expand long template PCR system, Roche)

5

Taq polymerase

0.5

Water

19.5

20 Once the reaction mixture is prepared, the following PCR cycles are performed:

Number of times

Temperature (ºC) -time

one

94-2 minutes

10

94-10 seconds 55-30 seconds 68-2 minutes 30 seconds

twenty

94-10 seconds 55-30 seconds 68-2 minutes 30 seconds + 20 extra seconds per cycle

one

68-10 minutes

one

4-indefinitely

This product has a size of about 3.6 Kb to be identified by 0.8% agarose electrophoresis and purified by affinity chromatography in ion exchange resins.

Materials for the evaluation of cell fusion activity mediated by the product of the env gene.

Cell cultures:

10 1. P4-R5 MAGI cell line (from NIH AIDS Research and Reference Reagent Program, described in Chackerian B et al., 1997 J Virol 71: 3932–3939). They are constitutive expression cells of CXCR4 and induced expression of CCR5 and CD4. They also have integrated a construction with the HIV LTR that controls the expression of β

15 galactosidase. In the presence of TAT (from the NIH AIDS Research and Reference Reagent Program, described in Frankel AD and Pabo CO, 1988 Cell 55: 1189-1193) a mass expression of β-gal can be measured by staining in situ or by the kit de Roche! -gal reporter gene chemiluminescent assay (Catalog No. 11758241001).

20 2. HELA-CD4 cell line constitutively expressing CXCR4 and CD4 induced expression. They also have integrated a construction with the HIV LTR that controls the expression of β-galactosidase. In the presence of TAT, a mass expression of measurable β-gal is produced by staining in situ or by chemiluminescent systems

25 (Roche). They do not allow the entry of CCR5 dependent viruses.

3.

U-251 MG cell line. They are used to express the Env viral envelope protein plus the TAT viral transactivator by transient transfection by, for example, electroporation.

Four.

Culture medium for P4-R5 MAGI: RPMI 1640 with HEPES and Lglutamine (Gibco), supplemented with 10% fetal cow serum (Linus), Penicillin (100 U / ml) (Roche), Streptomycin (100 ∀g / ml) (Roche), G418 (0.2 mg / ml) (Roche), Hygromycin B (0.2 mg / ml) (Roche) and Puromycin (1 ∀g / ml) (Sigma).

5.

Culture medium for HELA-CD4: RPMI 1640 with HEPES and L-glutamine (Gibco), supplemented with 10% fetal cow serum (Linus), Penicillin (100 U / ml) (Roche), Streptomycin (100 ∀g / ml) (Roche), G418 (0.2 mg / ml) (Roche), and Puromycin (1 /g / ml) (Sigma).

6.

Culture medium for U-251 MG: RPMI 1640 (Gibco) with HEPES and Lglutamine, supplemented with 10% fetal cow serum (Linus), Penicillin (100 U / ml) (Roche), Streptomycin (100 ∀g / ml) (Roche).

7.

Biorad electroporator.

8.

REV expression plasmid: pREV (NIH AIDS Research and Reference Reagent Program: https://www.aidsreagent.org/reagentdetail.cfm?t=expression_vectors&id= 63). It is used in transfections of viral envelope control plasmids, since it is necessary in the presence of this protein so that the envelope mRNA can be exported outside the cell nucleus. This plasmid does not need to be included in the transfections of envelope PCR products fused with the T7 plus IRES promoter, since protein expression takes place in the cytoplasm.

9.

HIV-1 GP160 expression plasmid: pCAGGS SF 162 GP160 (NIH AIDS Research and Reference Reagent Program:

https://www.aidsreagent.org/reagentdetail.cfm?t=expression_vectors&id= 302).

10. TAT expression plasmid: pCMV-TAT (NIH AIDS Research and Reference Reagent Program:

https://www.aidsreagent.org/reagentdetail.cfm?t=expression_vectors&id= 86).

eleven.

T7 polymerase eukaryotic expression plasmid (T7 bacteriophage RNA polymerase cloned into the invitrogen eukaryotic expression vector pCDNA3.1).

12.

T7-IRES-ENV fusion PCR product of strains NL43 (Adachi A et al., 1986 J Virol 59: 284-291) and JRCSF (Koyanagi Y et al., 1987 Science 236: 819-822, and Cann AJ et al. 1990 J Virol 64: 4735-4742), produced according to the previous protocol and purified with the PCR product purification kit from the manufacturer Macheray-Nagel.

13.

Electroporation cuvettes.

14.

Diethylaminoethyl-Dextran (DEAE-Dextran) stock solution at 20 mg / ml (1000X).

fifteen.

TAK 779: stock solution at 1 mM (1000X) antiviral agent inhibiting shell fusion with CCR5.

16.

1X PBS buffer (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4).

17.

Dimethyl sulfoxide (DMSO).

18.

1% trypsin solution (GIBCO).

19.

Conventional cell culture material (culture bottles, inverted microscope, 96-well plates).

twenty.

Bradford reagent to measure protein concentration.

Staining! -Galactosidase

one.

Fixing solution: Formaldehyde (1%) and glutaraldehyde (0.2%) in PBS.

2.

Staining solution (per ml): 949 µl of PBS, 20 µl of potassium ferrocyanine, 20 µl of potassium ferricyanine, 1 µl of 2M MgCl2 and 10 µl of X-GAL (40 mg / ml).

Measurement of the luminescent! -Galactosidase activity According to the Roche kit protocol! -Gal reporter gene assay chemiluminescent (Catalog number 11758241001).

Protocol for evaluation of cell fusion activity mediated by the product of the env gene.

Preparation of P4-R5 MAGI and HELA-CD4 cells: Cultivate the P4-R5 MAGI or HELA-CD4 cells with their specific medium in culture bottles until they reach 50% confluence. Calculate the total number of cells needed based on the number of patients to be evaluated knowing that 50,000 cells are used per fusion assay.

Wash the cells with PBS on the day of the experiment. Take off from the bottom of the Trypsin bottle, count and adjust the concentration to 50,000 cells in 50 µl. Sow the 50 μl in a well of a 96-well plate in RPMI medium with penicillin and streptomycin but without the rest of antibiotics. Add DEAE- Dextran (20 ∀g / ml).

Preparation of U-251 MG cells: Cultivate the U-251 MG cells in their medium until confluence. Make a medium change the day before the trial.

Wash with PBS and detach the trypsin cells on the day of the experiments. Count and adjust the concentration to 75,000 cells per assay. Resuspend the total cells per assay in 350 µl of RPMI medium with 50% fetal bovine serum in a 4 mm diameter electroporation cuvette and add the plasmid DNA corresponding to each sample: pREV (0.5 ∀g / million cells, positive fusion control with envelope plasmids), pCAGGS SF162 GP160 (2 ∀g / million cells, positive fusion control with envelope plasmids), PCR product T7-IRES-ENV strain NL43 and JRCSF strain (2 µg / million cells, T7-IRES promoter positive fusion controls (0.5 µg / million cells) and pTAT (0.5 µg / million cells).

Table 1. Shows the transfected elements in the different tests performed.

Transfected element

Plasmid Env Fusion Control Fusion with PCR product-T7-IRES-ENV_NL43 Fusion with PCR-T7-IRESENV_patients product Negative fusion control with IRES PCR-T7

Pcaggs-SF162 GP160

YES NO NO NO

pREV

YES NO NO NO

pTAT

YES YES YES YES

pT7-IRES-ENV NL43 or JRCSF

NO YES NO NO

pT7-IRESENV_patient

NO NO YES NO

pT7-IRES WITHOUT WRAPPING

NO NO NO YES

The electroporation conditions are: 260 Volts, 960 aFaradios and infinite resistance. Resuspend the cells in RPMI medium: 50 X number of points to be evaluated (in ∀l) and 50 ∀l is added in each of the wells of the

5 96-well plate that we have previously prepared with the P4-R5 MAGI cells and in parallel with HeLa-CD4.

Co-cultivate the cells for 48 hours and after this time proceed to detect the chemiluminescent! -Galactosidase activity and / or in situ.

10 Staining! -Galactosidase At 48 hours after starting cell fusion, remove the culture medium and wash the wells twice with 100 µl of PBS. Add 100 µl of fixation solution and incubate at room temperature for 5 minutes. Remove fixer

15 and wash twice with PBS. Add 100 µl of staining solution and incubate 50 minutes at 37 ° C. Finally wash twice with PBS.

Count the number of syncytiums per well under the microscope. The presence of syncytia indicates that there has been cell fusion, and therefore that the env gene has used the co-receptor that expressed the cell. When syncytia are formed with P4-R5 MAGI cells, this implies that the virus of the patient from which it came

The initial sample is using CCR5 as a correceptor, that is, the typing of said virus is CCR5, as long as no syncytia are formed in Hela-CD4 cells. When syncytia are formed in both co-cultures, the typing is of envelopes that use both co-receptors. The absence of syncytia reveals inhibition of cell fusion mediated by the HIV-1 envelope and the result is undetermined. The typing algorithm is summarized in the following table:

Symptoms or activity! Galactosidase in MAGI P4C5 cells

Symptoms or activity! Galactosidase in HELA-CD4 cells Diagnosis of viral strain type

Positive

Negative CCR5

Positive

Positive

CCR5 / CXCR4

Negative

Negative

Indeterminate

They do not exist in isolated samples of patients, pure viral populations for

The use of CXCR4 is composed of viral quasi species that include mixed CCR5 and CXCR4 strains. Therefore, this system determines whether there are CXCR4 dependent strains in the viral population compared to patients with homogeneous strains exclusively dependent on CCR5.

15 In parallel, cell viability and morphology can be evaluated.

Measurement of luminescent! -Galactosidase activity According to the protocol corresponding to the Roche kit, it is carried out in triplicate and normalized by concentration of protein measured with Bradford (Biorad).

RESULTS

The envelope amplification protocol was performed based on genomic DNA from 4 HIV-1 infected patients. The viral genome is integrated into the genome of infected cells.

Fusion tests have yielded the results represented in Table 2. The third column corresponds to the average of the relative luminescence unit, calculated with the data obtained from the luminescence measurement and the amount of total protein in duplicate for each sample. The fourth column corresponds to the values in the previous column to which the value of CONTROL -1 has been subtracted. Finally, the last column represents the% of fusion taking as reference the transfection of the positive control expression plasmid pCAGGS SF162 GP160.

For control +1 the HIV envelope expression plasmid is used as production control by a standard protein expression system. It is a CCR5 dependent envelope. It is the positive control and is used to calibrate the rest of the experiments. The control with pT7-IRES-ENV NL43 carries a CXCR4 dependent envelope and expressed using the T7-IRES system. The control with pT7-IRES-ENV JRFL carries a CCR5 dependent envelope and expressed using the T7-IRES system. The control with pT7-IRES is a control

of spontaneous fusion independent of the expression of the envelope or by aberrant induction of the LTR of the reporter cells (the value of this group must be zero). A control similar to the previous one is that of untransfected cells. Another control to measure the effectiveness of transfection is that which transfects pCMV-GFP, which should be like the two previous cases.

Most patients generate a fusion-capable envelope at levels similar to those achieved with the envelope expression plasmid. Since P4-R5 MAGI cells produce βgalactosidase activity under the control of the viral LTR promoter, which is sensitive to various stimuli or possibly soluble TAT protein, we proceeded to determine if the formation of multinucleated syncytia formed from a cell was detected which expresses the envelope functionally and merges with the P4-R5 MAGI or HeLa-CD4 cells, which express the viral receptors.

Table 2. It shows the levels of β-galactosidase activity in relative luminescence units (URL) for the indicated assay and the% fusion with P4-R5 MAGI cells.

TRANSFECTED ELEMENT

TEST URL URL CONTROL -1 % level of fusion

ENV1 pCAGGS SF162 GP160

CONTROL + 1 96,685 92,576 100

pT7-IRES-ENV NL43

CONTROL + 2 88,811 84,702 91

pT7-IRES-ENV JRFL

CONTROL + 3 65,716 61,607 67

pT7-IRES-

CONTROL -1 4,109 0 0

WITHOUT TRANSFECTING

CONTROL -2 3,595 -514 -one

pCMV-GFP

CONTROL -3 4,587 478 one

pT7-IRESENV_patient 1

SAMPLE 1 86,343 82,234 89

pT7-IRESENV_patient 2

SAMPLE 2 104,053 99,944 108

pT7-IRESENV_patient 3

SAMPLE 3 104,651 100,542 109

pT7-IRESENV_patient 4

SAMPLE 4 111,504 107,395 116

In the co-cultures with P4-R5 MAGI cells, in all cases the production of multinucleated syncytia with β-galactosidase activity that generates a blue stain was detected. Syncytia were evaluated by conventional microscopy and image capture with specific software

5 (Olympus) (Figures 2 and 3).

Fusion with the HELA-CD4 cell line did not produce β-galactosidase activity above baseline (mean of 7,250 URLs) in any of the clinical samples and only fusion was detected in transfection with the pT7 PCR product

10 IRES-ENV NL43 (mean 135,000 URL), indicating that all clinical samples evaluated have viruses with CCR5 tropism.

Claims (21)

1.-Method for the determination of the viral tropism of the human immunodeficiency virus 1 comprising the following stages:

(i)

 PCR amplifying a promoter and an IRES sequence from a template nucleic acid comprising said promoter and said IRES sequence, by means of a direct primer A, wherein said primer A hybridizes with the 5 'end of the promoter, and a reverse primer B, wherein said primer B hybridizes at its 3 'end with the 3' end of the IRES sequence and at its 5 'end is complementary to the 5' end of the env gene of the human immunodeficiency virus 1;

(ii)

 PCR amplifying a sequence comprising a fragment of the human immunodeficiency virus 1 that includes the env gene, from copy DNA or genomic DNA by a direct primer C and a reverse primer D, where said primers C and D hybridize with the sequences flanking said env gene;

(iii) PCR amplify the sequence of the env gene from the product obtained in step (ii), by means of a direct primer E, where the 5 'end of said primer E is complementary to the IRES sequence of step (i) and wherein the 3 'end of said primer E hybridizes with the 5' end of the env gene, and a reverse primer F, which hybridizes with the 3 'end of the env gene;

(iv)

assemble the products of steps (i) and (iii) by PCR using a direct primer A that hybridizes with the 5'-end of the product of step (i) and a reverse primer G whose 3'-end hybridizes with the 3'-end of the product of step (iii) and whose end 5 'comprises a poly-T;

(v)

 transfect the amplicon obtained in step (iv) in cells expressing the transactivator protein of viral activity;

(saw)

 co-cultivate the cells of step (v) with cells that contain a marker gene flanked by LTR sequences and express:

to.

The CD4 receiver and the CXCR4 correceptor,

b.

The CD4 receiver and the CCR5 correceptor

C.

The CD4 receiver and jointly the CXCR4 and CCR5 co-receptors.

(vii) detect and / or quantify expression of the marker gene, and (viii) associate the expression levels of the marker gene detected and / or quantified in step (vii) to the viral tropism CXCR4, CCR5 or double tropism. 2. Method according to claim 1, wherein the promoter is the T7 bacteriophage RNA polymerase promoter. 3. Method according to any one of claims 1 or 2, wherein the IRES sequence is the 5 ’UTR region of the encephalomyocarditis enterovirus. 4. Method according to any of claims 1 to 3, wherein the sequence of primer A is SEQ ID NO: 1, the sequence of primer B is SEQ ID NO: 2, the sequence of primer C is SEQ ID NO: 3, the sequence of primer D is SEQ ID NO: 4, the sequence of primer E is SEQ ID NO: 5, the sequence of primer F is SEQ ID NO: 6 and the sequence of primer G is SEQ ID NO: 7. 5. Method according to any of claims 1 to 4, wherein the cells expressing viral activity transactivator protein are the U251 MG cell line. 6. Method according to any of claims 1 to 5, wherein the cells expressing viral activity transactivator protein comprise the bacteriophage T7 RNA polymerase. 7. Method according to claim 6, wherein the cells are obtained by transfecting an expression plasmid of the bacteriophage T7 RNA polymerase. 8. Method according to any of claims 1 to 7, wherein the cells containing a marker gene flanked by LTR sequences express the CD4 receptor and the CXCR4 correceptor are the HeLa cell line. 9. Method according to any one of claims 1 to 8, wherein the cells containing a marker gene flanked by LTR sequences express the CD4 receptor and the CCR5 receptor are the P4-R5 MAGI cell line. 10. Method according to any of claims 1 to 9, wherein the cells contain a marker gene that is that of β-galactosidase, luciferase, amylase or fluorescent green protein. 11. Method according to claim 10, wherein the marker gene is that of βgalactosidase. 12. Method according to any of claims 10 or 11, wherein the detection and / or quantification of the expression of the marker gene is performed by fluorescence, colorimetric labeling, immunological methods, spectroscopic methods and / or PCR. 13. Method according to any of claims 10 to 12, wherein the detection and / or quantification of the expression of the marker gene is performed by staining of β-galactosidase and / or luminescent β-galactosidase activity. 14.-Primers SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 7. 15.-Kit comprising:

(i)

 an artificial DNA fragment produced by PCR that includes at the 5 ’end the T7 bacteriophage RNA polymerase promoter and at the 3 ′ end an IRES sequence and a fragment of the env gene of the human immunodeficiency virus 1;

(ii)

 a direct primer A, SEQ ID NO: 1, which hybridizes with the 5 ’end flanking sequence, the T7 bacteriophage RNA polymerase promoter

(iii) a reverse primer B, SEQ ID NO: 2, which hybridizes at the 3'-end an IRES sequence and at the 5'-end with a fragment of the env gene of the human immunodeficiency virus 1;

(iv)

 a direct primer C, SEQ ID NO: 3, which hybridizes with the 5 ’flanking sequence of the env gene;

(v)

 a reverse primer D, SEQ ID NO: 4, which hybridizes with the 3 ’flanking sequence of the env gene;

(saw)

 a direct primer E, SEQ ID NO: 5, which at its 5 'end is complementary to the IRES sequence and at its 3' end hybridizes to the 5 'end of the env gene; Y

(vii) a reverse primer F, SEQ ID NO: 6, which hybridizes through its 3 ’zone with the 3’ end of the env gene. (viii) a reverse primer G, SEQ ID NO: 7, which hybridizes through its 3 ’zone with the 3’ end of the env gene and whose 5 ′ end comprises a poly-T. 16. Kit according to claim 15, further comprising an expression plasmid of the TAT viral activity transactivator protein, an expression plasmid of the bacteriophage T7 RNA polymerase, a plasmid of 5 expression of the rev protein, a plasmid of gp160 envelope protein expression and / or a fluorescent green protein expression plasmid. 17.-Use of primers SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 or the kit according to any of claims 15 to 10 16 to determine the viral tropism of the human immunodeficiency virus one. FIG. one FIG. 2 FIG. 3  SEQUENCE LIST <110> Jaen University <120> METHOD OF DETERMINATION OF TROPISM OF HIV-1 AND ASSOCIATED KIT <130> ES1881.10 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 32 <212> DNA <213> Artificial sequence <220> <223> T7-IRES-FORWARD <400> 1 taatacgact cactataggc tagcctcgag ga <210> 2 <211> 48 <212> DNA <213> Artificial sequence <220> <223> T7-IRES-REVERSE <400> 2 ttcctgccat aggagatgcc taagtcgact ctagaggatc ccgggttg <210> 3 <211> 33 <212> DNA <213> Env-1 oligonucleotide <400> 3 tacagtgcag gggaaagaat aatagacata ata <210> 4 <211> 22 <212> DNA <213> Env-2 oligonucleotide <400> 4 agacccagta caggcraraa gc <210> 5 <211> 52 <212> DNA <213> Artificial sequence <220> <223> Oligonule6tido Env-3 <400> 5 caacccggga tcctctagag tcgacttagg catctcctat ggcaggaaga ag <210> 6 <211> 26 <212> DNA 1 <213> Artificial sequence <220> <223> Env-4 oligonucleotide <400> 6 ccagtccccc cttttctttt aaaaag 26 <210> 7 <211> 66 <212> DNA <213> Artificial sequence <220> <223> Env-4-POLYT oligonucleotide <400> 7 tttttttttt tttttttttt tttttttttt tttttttttt ccagtccccc cttttctttt 60 aaaaag 66 SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 201331182 SPAIN Date of submission of the application: 07/31/2013 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: C12Q1 / 68 (2006.01) C12Q1 / 04 (2006.01) RELEVANT DOCUMENTS

Category

56 Documents cited Claims Affected

Y

LIN, N. H. et al. The design and validation of a novel phenotypic assay to determine HIV-1 coreceptor usage of clinical isolates. Journal of Virological Methods. October 2010, Vol. 169, No. 1, pages: 39-46. ISSN 0166-0934. <Doi: 10.1016 / j.jviromet.2010.06.012> 1-17

Y

JENKINSON, S. et al. Development of a novel high-throughput surrogate assay to measure HIV envelope / CCR5 / CD4-mediated viral / cell fusion using BacMam baculovirus technology. Journal of Biomolecular Screening. August 2003, Vol. 8, No. 4, pages 463-470. ISSN 1087-0571 (printed). ISSN 1552-454X (electronic). <Doi: 10.1177 / 1087057103255747> 1-17

TO

KIRCHHERR, J. L. et al. High throughput functional analysis of HIV-1 env genes without cloning. 1-4,6,7,10,12,

Journal of Virological Methods. July 2007, Vol. 143, No. 1, pages: 104-111. ISSN 0166-0934. <Doi: 10.1016 / j.jviromet. 2007.02.015>

14-17

TO

TROUPLIN, V. et al. Determination of coreceptor usage of human immunodeficiency virus type 1 from patient plasma samples by using as recombinant phenotypic assay.Journal of Virology. January 2001, Vol. 75, No. 1, pages: 251-259. ISSN 0022-538X. <Doi: 10.1128 / JVI.75.1.251-259.2001> 1,4,10,12,14-17

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 29.11.2013

Examiner E. Relaño Reyes Page 1/6

REPORT OF THE STATE OF THE TECHNIQUE Application number: 201331182 Minimum documentation searched (classification system followed by classification symbols) C12Q Electronic databases consulted during the search (name of the database and, if possible, terms of search used) INVENES, EPODOC, WPI, EMBASE, MEDLINE, BIOSIS, INSPEC, COMPDX, XPESP, XPESP2, REGISTRY, HCAPLUS, EMBL, IMGT, NR PATENTS DNA State of the Art Report Page 2/6  WRITTEN OPINION Application number: 201331182 Date of Written Opinion: 29.11.2013 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims 1-17 Claims IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims Claims 1-17 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/6  WRITTEN OPINION Application number: 201331182 1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

LIN, N. H. et al. Journal of Virological Methods. October 2010, Vol. 169, No. 1, pages: 39-46. 01.10.2010

D02

JENKINSON, S. et al. Journal of Biomolecular Screening. August 2003, Vol. 8, No. 4, pages 463-470. 08.2003

D04

KIRCHHERR, J. L. et al. Journal of Virological Methods. July 2007, Vol. 143, No. 1, pages: 104-111. 01.07.2007

D03

TROUPLIN, V. et al. Journal of Virology. January 2001, Vol. 75, No. 1, pages: 251-259. 01.01.2001

D01 optimizes a method of determining HIV-1 viral tropism. To do this, it amplifies the HIV-1 env gene of infected patients by RT-PCR, performs a second CMV promoter PCR and assembles both products using a third PCR. Next, co-transfects 293T cells, said amplicons with a plasmid containing the HIV-1 genome with a deletion in env and the luciferase reporter gene in the nef region. The virions obtained are used to infect two cell lines separately, the U87-CD4-CCR5 and the U87-CD4-CXCR4, determining the entry of the viruses and therefore their tropism, by detecting luciferase. In D02 a cell fusion assay is developed in which HEK-263 cells are transduced with BacMan viral vectors expressing gp160, REV and TAT. These cells are co-cultured with HOS cells that stably express CCR5 and CD4, and the luciferase reporter gene under the control of HIV-1 LTR. This method is used in the selection of drugs that inhibit viral fusion. D03 presents a procedure to determine the viral tropism of HIV-1. To this end, the HIV-1 env gene of infected patients is amplified by RT-PCR, a second CMV promoter PCR is performed and both products are assembled by a third PCR. Subsequently, amplicons with a plasmid containing the HIV genome with a deletion in env are co-transfected into 293T cells. The virions obtained are used to infect TZM-bl cells and viral infection is determined by the detection of luciferase. In D04 a method of determining tropism of HIV-1 is described, which comprises amplification of the env gene of the patient by RT-PCR. 293T cells are co-transfected with this amplicon and with a vector that presents the HIV genome with a deletion in the env gene, obtaining recombinant viruses by complementing both sequences. These viruses were used to infect indicator cells, which express CD4 together with CXCR4 (U373MG-CD4-CXCCR4) or CCR5 (U373MG-CD4-CCR5) and that have the lacZ operon under the control of HIV-1 LTR. 2. Statement motivated according to articles 29.6 and 29.7 of the Regulation of execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The present application aims at a method for the determination of HIV-1 viral tropism comprising: (i) PCR amplifying the T7 bacteriophage RNA polymerase promoter and the 5 'UTR of the encephalomyocarditis enterovirus, from of a nucleic acid comprising both, by a SEQ primer. ID. NO. 1 that hybridizes with the 5 ’end of the promoter, and a SEQ primer. ID. NO. 2 whose 3 ’end hybridizes with the 3’ end of the UTR and its 5 ’end is complementary to the 5’ end of the HIV-1 env gene; (ii) PCR amplify the env gene with the SEQ primers. ID. NO. 3 and 4; (iii) perform a second PCR of the amplified env product with a SEQ primer. ID. NO. 5, whose 5 ’is complementary to the 5’ UTR and its 3 ’hybridizes with the 5’ of env, and a SEQ primer. ID. NO. 6 complementary to 3 ’of env; (iv) assemble the products of steps (i) and (iii) with the SEQ primers. ID. NO. 1 and SEQ. ID. NO. 7, whose 3 'hybridizes with the 3' of the product of stage (iii) and its 5 'has a poly-T tail; (v) transfect the amplicon obtained in (iv) in U-251 MG cells that express the viral activity transactivator protein (TAT) and that comprise the bacteriophage T7 RNA polymerase; (vi) co-culturing the cells of step (v) with cells having a marker gene, preferably that of 1-galactosidase, flanked by LTR sequences and expressing the CD4 receptor together with the CXCR4 (HeLa), CCR5 co-receptor. (P4-R5 MAGI) or both correceptors; (vii) detect the expression of the marker gene and (viii) associate the expression levels of the marker gene with the viral tropism CXCR4, CCR5 or double tropism (claims 1 to 13). In addition, SEQ sequence primers are subject of the invention. ID. NO. 1, 2, 5 and 7 (claim 14). Also included is a kit comprising: an artificial DNA fragment comprising the T7 bacteriophage RNA polymerase promoter, an IRES sequence and an HIV-1 env gene fragment, together with the SEQ sequence primers. ID. NO 1, 2, 3, 4, 5, 6 and 7, which may additionally include a TAT expression plasmid, a T7 RNA polymerase expression plasmid, a REV expression plasmid, an expression plasmid of the gp160 protein and / or an expression plasmid of the green fluorescent protein (claims 15 and 16). Finally, the use of the primers or the kit in the determination of the tropism of HIV-1 is also an object of the invention. State of the Art Report Page 4/6  WRITTEN OPINION Application number: 201331182 1.NOVEDAD (Art. 6.1 LP 11/1986) Claims 1 to 17 meet the requirement of novelty (Art. 6.1 LP 11/1986). 2. INVENTIVE ACTIVITY (Art. 8.1 LP 11/1986) 2.1. CLAIMS FROM 1 TO 13 The object of claim 1 is a method for determining viral tropism of HIV-1 comprising: (i) PCR amplifying a promoter and an IRES sequence from a nucleic acid comprising both, by a primer A which hybridizes with the 5 'end of the promoter, and a primer B whose 3' end hybridizes with the 3 'end of the IRES and its 5' end is complementary to the 5 'end of the HIV-1 env gene; (ii) amplify by PCR the env gene with primers C and D; (iii) perform a second PCR of the amplified env product with an E primer whose 5 ’is complementary to the IRES sequence and the 3’ hybridizes with the 5 ’of env, and an F primer complementary to the 3’ of env; (iv) assemble the products of the stages (i) and (iii) with primers A and G, whose 3 ’of this last hybrid with 3’ of the product of stage (iii) and its 5 ’has a poly-T tail; (v) transfect the amplicon obtained in (iv) in cells expressing TAT; (vi) co-culturing the cells of step (v) with cells that have a marker gene flanked by LTR sequences and that express the CD4 receptor together with the CXCR4, CCR5 or both co-receptors; (vii) detect the expression of the marker gene and (viii) associate the expression levels of the marker gene with the viral tropism CXCR4, CCR5 or double tropism. In D01, a method of determining HIV-1 viral tropism is optimized. To this end, the HIV-1 env gene of infected patients is amplified by RT-PCR, a CMV promoter PCR is performed and both products are assembled by a third PCR. Next, 293T cells are co-transfected with the amplicon obtained and a plasmid containing the HIV genome with a deletion in the env gene, and the luciferase reporter gene in the nef region. The virions produced are used to infect two cell lines separately, the U87-CD4-CCR5 and the U87-CD4-CXCR4. Virus entry is measured by luciferase detection. The difference between D01 and the request is that, although a nucleotide sequence expressing the HIV-1 gp160 protein of the patient under the control of a promoter is obtained by PCR, in D01 the determination of viral tropism is performed by co-transfection the amplicons with a plasmid containing the HIV genome, thereby obtaining defective virions that will be used in viral infection studies, while in the method of the invention, the patient's env protein is expressed in the cell. transfected, which is co-cultured directly with the cell line expressing CD4 and one of the co-receptors. The technical effect is the development of a faster viral tropism detection method in which the intermediate stage of obtaining defective viruses and the subsequent infection of the corresponding cell lines is avoided. In D02 a cell fusion assay is developed in which HEK-263 cells are transduced with BacMan viral vectors expressing gp160, REV and TAT. These cells are co-cultured with HOS cells that stably express CCR5 and CD4, and that have the luciferase gene under the control of LTR expression. It is considered that one skilled in the art would attempt to develop a method of detection of viral tropism that comprises: obtaining by PCR a nucleotide sequence that expresses the gp160 protein of HIV-1 of the patient under the control of a promoter (D01 ); its co-transfection in a cell that expresses TAT and; co-culture of these cells with another cell line that stably expresses CD4 and one of the two co-receptors and a marker gene under the control of the HIV LTR (D02), with a reasonable expectation of success. Consequently, in the light of D01 together with D02, it is believed that claim 1 has no inventive activity (Art. 8.1 LP 11/1986). The choice of the T7 bacteriophage RNA polymerase promoter (claim 2), the 5 'UTR region of the encephalomyocarditis enterovirus (claim 3), the cell lines (claims 5, 8 and 9), or the marker gene (claims 10 and 11) as well as the method of detecting it (claims 12 and 13), are one of the numerous alternatives known to the person skilled in the art when developing this type of procedures, not deducing from the request that they provide any effect Surprising technician regarding other available options. In addition, it is obvious for the development of the procedure, that TAT-expressing cells express the bacteriophage T7 RNA polymerase (claims 6 and 7). Therefore, given documents D01 and D02, claims 3, and 5 through 13 have no inventive activity (Art. 8.1 LP 11/1986). The sequence of the primers (claim 4) is a direct consequence of the process of the invention, having not solved a technical problem in addition to obtaining the desired amplifications. Thus, it is understood that claim 4 does not meet the requirement of inventive activity in light of D01 together with D02 (Art. 8.1 LP 11/1986). State of the Art Report Page 5/6 WRITTEN OPINION Application number: 201331182 2.2. CLAIMS FROM 14 TO 17 Claim 14, is directed to sequence primers SEQ. ID. NO. 1, 2, 5 and 7, and claims 15 and 16 a kit comprising: an artificial DNA fragment, comprising the bacteriophage T7 RNA polymerase promoter, an IRES sequence and a fragment of the HIV-1 env gene , along with the SEQ sequence primers. ID. NO 1, 2, 3, 4, 5, 6 and 7, which may additionally include a TAT expression plasmid, a T7 RNA polymerase expression plasmid, a REV expression plasmid, an expression plasmid of gp160 and / or an expression plasmid of the green fluorescent protein. The sequences of the primers, as well as the reagents included in the kit, are considered a direct consequence of the development of the HIV tropism detection method, not solving any additional problem to the implementation of the procedure per se. Therefore, it is understood that claims 14 to 16 have no inventive activity in light of the combination of D01 with D02 (Art. 8.1 LP 11/1986). The object of the application, according to claim 17, is the use of the primers or kit of the invention in the determination of viral tropism. Since the design of the sequences of the primers and the kit reagents are a direct consequence of the optimization of the method of the invention, their use in said procedure does not meet the requirement of inventive activity in view of the combination of documents D01 and D02 . Consequently, claim 17 has no inventive activity (Art. 8.1 LP 11/1986). State of the Art Report Page 6/6