# Patent

## Title
Quantitative HIV phenotype or tropism assay

## Abstract

The present invention concerns a method for predicting quantitative phenotype, e.g. gag-phenotype, integrase phenotype or tropism in a patient infected by Human Immunodeficiency Virus (HIV).

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a national stage of International Application No. PCT/EP2007/051035 filed Feb. 2, 2007, which claims priority to European Patent Application No. 06101294 filed Feb. 3, 2006 and European Patent Application No. 06115363.1 filed Jun. 13, 2006. The complete disclosures of the aforementioned related patent application are hereby incorporated herein by reference for all purposes.
BRIEF SUMMARY
The present invention relates to a method for prediction of a quantitative phenotype, e.g. gag phenotype, integrase phenotype or tropism in a patient infected by Human Immunodeficiency Virus (HIV).
The human immunodeficiency virus, commonly called HIV, is a retrovirus that primarily infects vital components of the human immune system such as CD4+ T cells, macrophages and dendritic cells. HIV even, directly or indirectly, destroys CD4+ T cells. When enough CD4+ cells have been destroyed by HIV, the immune system barely works, which leads to AIDS (Acquired ImmunoDeficiency Syndrome). Further, HIV directly attacks organs, such as the kidneys, the heart and the brain, leading to acute renal failure, cardiomyopathy, dementia and encephalopathy. Many of the problems faced by people infected with HIV, result from the failure of the immune system to protect them from opportunistic infections and cancers.
AIDS is thought to have originated in sub-Saharan Africa during the twentieth century and it is now a global epidemic. At the end of 2004, UNAIDS estimated that nearly 40 million people were living with HIV. The World Health Organization estimated that the AIDS epidemic had claimed more than 3 million people and that 5 million people had acquired HIV in the same year. Currently it is estimated that 28 million people have died and that it is set to infect 90 million Africans alone, resulting in a minimum estimate of 18 million orphans in the African continent alone.
To infect a cell, a virus must first be able to enter it. HIV is an enveloped virus and accomplishes cell entry by fusing the viral membrane with the cellular plasma membrane. This process is carried out by the viral envelope proteins gp120 and gp41, which are synthesized as a single 160 kD protein before cleavage. The products of this cleavage remain associated until the process of viral entry into the cell begins. gp120 binds to CD4 on CD4+ T lymphocytes and cells of the monocyte/macrophage lineage. This binding event and further interaction between gp120 and cellular co-receptors lead to gp120 dissociation from gp41. The dissociation of gp120 occurs as part of a conformational change in gp41 that leaves it in a “fusion-active” form. This form of gp41 can then mediate fusion between the cellular and viral membranes.
The primary cellular receptor for HIV entry is CD4. However, expression of CD4 on a target cell is necessary but not sufficient for HIV entry and infection. Several chemokine receptors act as co-factors that allow HIV entry when co-expressed with CD4 on a cell surface.
CCR5 and CXCR4 are the major chemokine co-receptors used by HIV to enter into human cells. Based on this co-receptor usage, a new HIV classification was established in 1998, i.e., CCR5-tropic (R5), CXCR4-tropic (X4), or dual tropic (R5/X4) HIV strains. Several years earlier a relationship between viral phenotype (i.e., non-syncytium-inducing, NSI or syncytium-inducing, SI) and the virulence of HIV strains had been identified.
Current knowledge show that, in vitro, R5 viruses usually correspond to NSI on T-cell lines and are able to replicate in monocyte-macrophages (M-tropic), all features previously linked to less virulent strains. In contrast, X4 strains are SI on T-cell lines and replicate preferably on T lymphocytes (T-tropic), all characteristics of more pathogenic virus strains. Based on this knowledge it is believed that HIV co-receptor usage is associated with disease progression.
The first of these co-factors to be identified was CXCR4, or fusin, which is expressed on T cells (Feng et al., HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science 1996 May 10; 272 (5263):872-877.) Co-expression of CXCR4 and CD4 on a cell allow T-tropic HIV isolates to fuse with and infect the cell. CXCR4 is expressed on many T cells, but usually not on macrophages and hence does not allow fusion with macrophage-tropic (M-tropic) HIV isolates (Feng et al., 1996).
Shortly after the identification of CXCR4, another co-receptor was identified. CCR5, which is expressed on macrophages and on some populations of T cells, can also function in concert with CD4 to allow HIV membrane fusion (Deng et al., Identification of a major co-receptor for primary isolates of HIV-1. Nature 1996 Jun. 20; 381(6584):661-6.) HIV gp120 binding to CCR5 is CD4-dependent, as antibody inhibition of CD4 can reduce binding to CCR5 by 87% (Trkola et al., CD4-dependent, antibody-sensitive interactions between HIV-1 and its co-receptor CCR-5. Nature 1996 Nov. 14; 384(6605):184-7). M-tropic HIV isolates appear to use CCR5 as their co-receptor for infection both of macrophages and of some T cells.
The existence of these two larger receptors of HIV, known as CCR5 and CXCR4 as mentioned above, means that the different viral variants are classified into three categories: R5, X4 and R5X4 in line with their capacity to enter the cell by one of the two receptors exclusively or by both receptors.
CCR5 and CXCR4 belong to the seven-transmembrane G protein-coupled receptor family. They present an α-helix structure composed of four transmembrane domains, three extracellular loops and one N-terminal domain. The CD4-gp120 complex binds to co-receptors through the V3 variable domain of gp120, although other gp120 regions such as V1/V2 and C4 are also involved in this interaction. However, the amino acid sequence of V3 seems to be the major determinant of CCR5 or CXCR4 usage.
The term viral tropism refers to the cell type that the virus infects and replicates in. Nowadays, determination of viral tropism is not performed as a diagnostic test but it does represent a highly useful parameter in certain areas of HIV research. Furthermore, the introduction of specific drugs targeting HIV entry, and more specifically targeting the co-receptors CCR5 or CXCR4, implies that characterization of the viral tropism of an HIV-infected patient will become very important before starting treatment.
Co-receptor antagonists constitute a promising new class of anti-HIV-1 drugs, with several lead compounds being currently in full clinical development.
Several assays have been developed to determine HIV tropism. At the moment it remains unclear which is the most convenient and reliable method.
The MT-2 assay was widely used during the late 1980s to test the cytopathic effect of HIV isolates and served to establish the classification of HIV strains into SI and NSI viruses. The MT-2 cell assay is based on the unique expression of CXCR4 but not CCR5 on the surface of those cells. The main disadvantage though is the need for viral stocks from stimulated patient PBMC (Peripheral Blood Mononuclear Cell). The MT-2 assay may not be the most appropriate for use in patients being treated with co-receptor antagonists.
Another tool for viral tropism determination is the use of recombinant virus tropism assays such as Phenoscript (VIRalliance, Paris) and PhenoSense (Monogram Biosciences, San Francisco). Both assays amplify the HIV-1 envelope glycoprotein gene sequence from patient's plasma samples to produce either replication-competent or replication-defective recombinant viruses, respectively. These viruses are then used to infect cell lines that express CD4 in combination with either CCR5 or CXCR4 co-receptors, which permits determination of viral tropism. The severe limitation of these assays is the threshold for detection of X4 viruses in mixed population (R5+X4) i.e., the threshold for detection of minority quasispecies in the presence of mixed viral populations.
This limitation might have important implications in patients undergoing treatment with CCR5 antagonists, in whom emergence of X4 viruses present as a minor population at baseline could be favoured.
Testing for co-receptor utilization (or tropism) prior to initiating therapy with a CCR5 antagonist will be critical to avoid the use of these compounds in patients that are infected with CXCR4 or dual tropic strains.
The molecular basis of HIV tropism is still under investigation, although some investigators showed that probably the V3 loop of the gp120 envelope protein could be involved. There were efforts made to identify which residues within the V3 domain could be involved in determining viral co-receptor usage. No single changes seem to be responsible for tropism, although several clusters of genotypes could determine viral tropism. Several algorithms have been produced to predict HIV co-receptor usage based on the V3 genetic sequence.
However, there is an urgent need for a viral envelope tropism determination assay, which can accurately and with high sensitivity determine the co-receptor usage of a virus strain. Furthermore, because of the development of successful entry inhibitors, assays aimed at evaluating the impact of viral envelope variation on resistance to entry inhibitors and fusion inhibitors will undoubtedly become very important for guiding HIV therapy.
Since patients infected with HIV harbors a diversity of viral subspecies, each with their own co-receptor usage, it is important to analyze the distribution of tropism phenotypes in the entire patient viral population. Furthermore, since methods to predict tropism phenotypes are based on nucleotide and/or amino acid sequences of highly variable regions, it is necessary to determine sequences at clonal level.
So, there definitely exists an unmet high need to have reliable methods in place permitting the characterization of viral tropism in HIV infection in a patient to contribute substantially to our knowledge of the variability and distribution of CCR5 and CXCR4-tropic quasi-species within clinical isolates by means of techniques that are simple and accessible to any analysis laboratory, systems which are so far unavailable.
The present disclosure describes a method and accordingly a tropism test to identify HIV co-receptor usage as a marker for disease progression.
At least two tropism prediction algorithms PSSM (University of Washington, Webpssm) and Geno2-Pheno (G2P), indicated for the tool which is a support vector machine approach (SVM) (Max Plank Institut) are publicly available, both based on the analysis of specific amino acid characteristics of the V3-loop of HIV-1 env. The predictive value however of these algorithms is still limited.
Using clonal V3 env sequences, a comparison was made between the predictions yielded by the PSSM and those obtained by the SVM model. A high concordance was found for R5-tropic isolates between both program models, while X4 predictions were significantly less concordant.
More specifically the instant disclosure describes a method for predicting quantitative phenotype, e.g. gag phenotype, integrase phenotype or tropism, in a patient infected by HIV comprising

    
    
        a) using a sample comprising viral genetic material from the patient;
        b) extraction of viral genetic material from said sample followed by single genome sequencing comprising the following steps:
        
            1. amplification of the viral genetic material of a specific HIV region
            2. analysis of amplicon integrity and pooling of samples
            3. purification of the pooled amplicons
            4. ligation of the pool of amplicons into a vector and transformation of the ligated product into competent cells
            5. analysis of individual transformants obtained
            6. sequencing the resulting single clones to obtain a single clone genotype sequence;
        
        
        c) prediction of a specific phenotype, e.g. gag phenotype, integrase phenotype or tropism, using said genotype sequence with a predictive algorithm comprising the following steps:
        
            1. identifying the genetic pattern in said genotype sequence wherein at least one natural variability, acquired variability, drug selected mutation or mutation pattern is associated with the quantitative phenotypic outcome, e.g. gag phenotype, integrase phenotype or tropism,
            2. searching a genotype/phenotype correlative database for at least one genotype entry with a similar genetic pattern to at least one of the natural variability, acquired variability, drug selected mutation or mutation pattern identified in the genetic sequence in step c1,
            3. obtaining the said at least one genotype entry with a similar genetic pattern with a matched phenotype in the correlative genotype/phenotype database, and,
            4. predicting the HIV phenotype from the database of the at least one genotype entry with a similar genetic pattern;
        
        
        d) prediction of the quantitative phenotype, e.g. gag phenotype, integrase phenotype or tropism, based on the information obtained in steps c1 to c4 for every single sequence clone present in a sample of a HIV infected patient.
    
    


The method according to the invention may further comprise the following two additional steps after step (c) and before step (d) wherein

    
    
        1. clonal sequences without predictable phenotype are analyzed in a single clone biological phenotyping assay and
        2. the information obtained after said analysis is loaded in the correlative genotype-phenotype database used in step (c).
    
    


The single clone biological phenotyping assay above-mentioned comprises the following steps:

    
    
        1. generation of clonal partial or full-length HIV genome
        2. transfection of mammalian cells with said genome either together with a suitable backbone to obtain recombinant HIV or directly as a full length HIV-1 genome
        3. infection of cell lines by said recombinant HIV to determine their biological phenotype wherein the infection process is occurring
        4. whereafter the information obtained is loaded in the correlative genotype-phenotype database used in step (c).
    
    


Two of the above-mentioned mentioned steps viz. step 2 (transfection) and step 3 (infection) may be performed in a single step.
The amplification of the viral genetic material of a specific HIV region in step (b1) is either performed by RT-PCR or by PCR. The competent cells used in step (b4) are E. Coli, Bacillus or yeast.
The infection process above mentioned can be monitored either by a marker gene introduced in the full-length HIV genome or by a marker gene introduced in an indicator cell line, or microscopically by cytopathic effect scoring, or by syncitia formation.
In another embodiment of the current invention, HIV sequences obtained and sequenced from samples of patients infected by HIV are loaded into the correlative genotype-phenotype database following the algorithm for prediction of quantitative phenotype, e.g. gag phenotype, integrase phenotype or tropism where after said phenotype or tropism is reported.
With gag phenotype is meant e.g. protease or gag inhibitor resistance, while with integrase phenotype is meant e.g. entry inhibitor resistance.
Samples from a patient used for performing the method are obtained from a biological sample chosen from a blood sample, a biopsy sample, a plasma sample, a saliva sample, a tissue sample, and a bodily fluid or mucous sample.
In addition to the current method and according to the invention, viral load is determined in the sample of a patient infected by HIV.
Part of the invention is the prediction of the quantitative tropism as the quantitative shift in HIV-1 co-receptor usage e.g. from either CCR5 to CXCR4, from CXCR4 to CCR5, or from dual tropic viruses to either CCR5 or CXCR4.
In another embodiment of the instant invention, the prediction of the quantitative gag-phenotype is brought into relation of HIV-1 protease enzymatic activity as a consequence of natural variability or drug-induced/selected variability in the gag open reading frame and/or at the gag-cleavage site.
Alternatively part of the invention is the prediction of the quantitative integrase-phenotype which is brought into relation of HIV-1 integrase enzymatic activity as a consequence of natural variability or drug-induced/selected variability in the integrase open reading frame and/or integrase donor/acceptor sites.
As a final result of the method according to the invention a report is generated wherein said report comprises the predicted phenotype or tropism providing the treating physician with guidance for HIV therapy or treatment.
Part of the invention is also a computer readable medium comprising the predicted phenotype or tropism using any of the methods performed according to the current invention.
To the current invention also belongs the vector pHXB2D-ΔNH2-V4-eGFP having SEQ ID NO: 6 and the use of said vector pHXB2D-ΔNH2-V4-eGFP having SEQ ID NO: 6 in any of the above mentioned methods.



DESCRIPTION OF THE DRAWINGS
In FIG. 1 a flow-chart shows the method outlined as used in the current invention.
FIG. 2 shows the schematic presentation of recombination clinical env NH2-V4 amplicon into pHXB2D-ΔNH2-V4-eGFP (SEQ ID NO: 6) backbone, cloning into bacteria, nucleofection of full length HIV genome recombinant plasmid in 293 T cells and infection of recombinant virus into U87-CD4 (-CXCR4 or -CCR5) cells.
FIG. 3. PSSM scores according to clone number. Clones were subdivided into groups according to their prediction by PSSM and SVM. Clones selected for phenotyping are marked by their corresponding numbers.
FIG. 4. Sequence logo representing the variability of the V3 loop present in the 60 clones. The overall height of each stack in the logo indicates the sequence conservation at that position, whereas the height of each letter within the stack is proportional to its relative frequency at that position.
FIG. 5. Phylogenetic tree for the HIV-infected subject under investigation. Branches were colored according to the classification based on the prediction by PSSM and SVM: dark green: predicted R5 by both programs; light green: predicted R5 by PSSM and no prediction by SVM; blue: R5 prediction by PSSM and X4 prediction by SVM; red: X4 prediction by both programs. Bootstrap values of the X4 sequences are shown at the base of its branches. Scale indicates genetic distances based on the nucleotide alignment.
FIG. 6. Nucleotide alignment of the NH2-V4 region of 12 selected clones and HXB2D Conservative base pairs are shown in blue, while identical base pairs are presented in yellow. Env V1, V2, V3 and V4 loops are indicated by red lines.
FIG. 7. Fluorescent microscopic images of U87-CD4-CXCR4 and U87-CD4-CCR5 infected with 12 selected clones and positive (HXB2D-eGFP and HXB2D-JRCSF-eGFP) and negative controls (HXB2D-ΔNH2-V4-eGFP) (SEQ ID NO: 6).



DETAILED DESCRIPTION
Examples
Example 1
RNA Extraction
Three clinical plasma samples were randomly selected and were referred to as patient 1, 2 & 3. From a total of 300 μl plasma, total RNA was extracted using the EASYMAGTM RNA extraction platform (Biomérieux, Boxtel, The Netherlands). After elution in 25 μl elution buffer, 5 μl of the eluate was used for viral load measurement using the NucliSens EasyQ® HIV-1 v1.1 system (Biomérieux, Boxtel, The Netherlands). The remainder of the RNA samples was used for amplicon generation.
Amplicon Generation
The remaining 20 μl RNA was mixed with 2× reaction buffer, 0.2 μM primer Env—6210F (CAGAAGACAGTGGCAATGAGAGTGA) (SEQ ID NO: 1), 0.2 μM primer HMA_R3 (ATGGGAGGGGCATACATTGCT) (SEQ ID NO: 2) and 2 units Platinum® Taq High Fidelity from the SuperScript® III One-Step RT-PCR System (Invitrogen, Merelbeke, Belgium) in a total volume of 120 μl. This mixture was divided over eight reactions of 15 μl each and reverse transcription took place at 53° C. for 30 min. Initial denaturation was 94° C. for 2 min and thermal cycling consisted out of 50 cycles of denaturation at 92° C. for 15 s, annealing at 55° C. for 30 s and elongation at 68° C. for 1 min 20 s. Final extension took place at 68° C. for 7 min. The resulting amplicons were pooled, analyzed using the LC90 platform (Caliper, Mountainview, Calif.) and subsequently purified using the QiaQuick® gel purification kit (Qiagen, Hilden, Germany). Final volume of the purified amplicon pools was 30 μl.
TOPO-TA Cloning®
A total of 2 μl of the purified amplicon pools was used for ligation into the pCR®4-TOPO® vector (commercially available) from the TOPO TA Cloning® Kit for Sequencing (Invitrogen, Merelbeke, Belgium) and one aliquot One Shot® TOP10 chemically competent cells (Invitrogen, Merelbeke, Belgium) was transformed with 2 μl of the cloning reaction mixture according to manufacturers instructions.
Colony PCR
Using a sterile tip, a total of 95 colonies (plus one blanc control reaction) was picked (manually or using a robot) per clinical sample to inoculate 50 μl PCR reaction mixture. The latter consisted out of 10×PCR buffer, 25 mM dNTPs, 0.33 μM primer T3 (ATTAACCCTCACTAAAGGGA) (SEQ ID NO: 3), 0.33 μM primer T7 (TAATACGACTCACTATA GGG) (SEQ ID NO: 4) and 0.03 units Expand High Fidelity Enzyme Mix (Roche, Penzberg, Germany). Thermal cycling started with 10 min denaturation at 94° C., 10 cycles of denaturation at 94° C. for 15 s, annealing at 50° C. for 30 s and elongation at 72° C. for 2 min. This was followed by 20 cycles of denaturation at 94° C. for 15 s, annealing at 50° C. for 30 s and elongation at 72° C. for 2 min with an increment of 5 s per cycle. Final extension took place at 72° C. for 7 min. Colony PCR products were purified using the Qiagen 9600 PCR purification platform, eluting in 50 μl (Qiagen, Hilden, Germany).
Cycle Sequencing
From each purified colony PCR product, 1 μl was mixed with 2.5× dilution buffer, 1 μl BigDye® Terminator Mix and 0.2 μM sequencing primer in a total volume of 11.5 μl. Each product was sequenced using primer T3 and T7 in a separate reaction. Thermal cycling consisted out of 25 cycles of denaturation at 96° C. for 10 s, annealing at 50° C. for 5 s and elongation at 60° C. for 4 min. Excess BigDye® was removed using ethanol/sodium acetate precipitation and products were denatured for 2 min at 95° C. and analyzed on the ABI3730 capillary sequencer.
Raw Sequencing Analysis
Electropherograms were retrieved from the ABI3730 capillary sequencer and imported into Seqscape v2 (Applied Biosystems, Foster City, Calif., USA). Sequence ends were trimmed based on quality values and the length of the JR-CSF reference sequence; the latter spanned the region between the amplification primers. Certain clones were removed from the analysis when the generated sequence:

    
    
        did not span the entire region of interest between the amplification primer sequences
        contained a STOP codon

Tropism Prediction

1. V3-Loop Amino Acid Sequence Extraction

    
    


Because the PSSM prediction algorithm requires amino acid sequences, correct translation of the V3-region out of the nucleotide sequences spanning the entire range from the amino terminal part of Env up to the V4-loop was performed. By performing a BLAST search of the translated nucleotide sequences (in all 6 frames) vs. a small database containing the HXB2 V3-loop amino acid sequence, the region with the highest match with V3 could be demarcated. Subsequently these regions were extracted and translated.
2. PSSM Tropism Prediction
The position specific scoring matrix (PSSM) prediction was generated by uploading the V3-loop amino acid sequences (PSSM algorithm, University of Washington, Webpssm) according to Jensen, M. A., F. S. Li, A. B. van 't Wout, D. C. Nickle, D. Shriner, H. X. He, S. McLaughlin, R. Shankarappa, J. B. Margolick, and J. I. Mullins. 2003. Improved coreceptor usage prediction and genotypic monitoring of R5-to-X4 transition by motif analysis of human immunodeficiency virus type 1 env V3 loop sequences. J Virol 77:13376-88.
3. Support Vector Machine (SVM) Algoritm as Available in the Geno2-Pheno Tropism Prediction Tool
Since the geno2-pheno co receptor prediction tool (indicated as SVM) does not allow batch submitting of nucleotide sequences, a Perl script was written that automates submission of all the sequences and an HTML output (SVM) was then parsed with another perl script to yield geno2-pheno tropism predictions per patient.
4. Comparison of SVM and PSSM Tropism Predictions
A SAS script puts all predictions into 1 dataset and makes contingency tables for each patient.
Results.
Three clinical isolates were randomly selected. From each isolate, viral RNA was reverse transcribed, amplified several times, and the obtained amplicons pooled, purified and cloned in bacterial cells. More than 50 randomly selected clones were sequenced and submitted to the two prediction programs. The result of this analysis is shown in table 1.







TABLE 1







Tropism prediction on individual V3 clones obtained from clinical isolates









G2P














PSSM
CCR5
CXCR4
DUAL
NONE
TOTAL
















PATIENT 1
R5



55
55



X4

1





PATIENT 2
R5
7
7

36
50



X4

10


10


PATIENT 3
R5
2
19
1
27
49



X4

5


5





G2P: prediction tool using a SVM approach (Max Plank Institut);


PSSM: prediction tool (University of Washington, Webpssm).


DUAL: V3 sequences predicted to infect both CCR5 and CXCR4 expressing cells.


NONE: no prediction available in SVM at the standard settings.






For every patient tested in this study, there is a significant amount of clonal sequences that resulted in no prediction in the SVM algorithm, while a prediction was obtained in the PSSM method. Further improvements of the prediction tools that are based on larger relational databases are needed to fine-tune these predictions. A single clone phenotyping assay is instrumental to build such database.
Example 2
Single Clone Phenotyping Assay
Patient-derived clonal sequences constituting complete gp160 or part of gp160 were introduced via the BD In Fusion system into hXB2D-eGFP backbone in which complete gp160 or part of it, respectively, was deleted (SEQ ID NO: 5). HXB2D-eGFP is a vector containing GFP instead of nef (Chen et al (1997), J Virol 71: 5495-5504). Instead of eGFP as marker other well known markers such as luciferase or other commercially available fluorescent proteins, can be used in the current assay. For every patient-derived full-length recombinant HIV-eGFP clone generated in this way, DNA was prepared and checked by restriction analysis. One μg of positive clones was transfected to 293T cells using the Amaxa nucleofection technique. Supernatant virus cultures were harvested 24-48 h after transfection and used to infect U87 cells (U87 parental, U87-CD4, U87-CD4-CXCR4 and U87-CD4-CCR5 cells). Co receptor usage was determined 24-96 h after infection by fluorescence microscopy. Alternatively, supernatant virus cultures were used to infect U87 containing CXCR4-CCR5 chimeric receptors (Karlsson et al (2003) AIDS 17: 2561-2569). In this way, predictions concerning the potency of a CCR5-using virus to shift to a CXCR4-tropic virus are performed.
Example 3
Clonal Phenotypic Confirmation of Genotypic V3-Loop Tropism Prediction on a Treatment-Naïve HIV-1 Infected Subject Sample
RNA Extraction and VIRCOTYPETM
From a total of 300 μl plasma obtained from a randomly selected HIV-1-infected subject, RNA was extracted using the EASYMAGTM RNA extraction platform (Biomérieux, Boxtel, The Netherlands). Viral load was determined by NucliSens EasyQ® HIV-1 v1.1 system (Biomérieux; output in IU/ml). A VIRCOTYPETM resistance analysis was generated.
Amplification
RNA was reverse transcribed and amplified using SuperScriptTM III One-Step RT-PCR System with Platinum® Taq High Fidelity (Invitrogen, Merelbeke, Belgium) in 7-fold. The forward primer was situated before the start codon of Env, the reverse primer in the Env C4 region.
The PCR fragment was called NH2-V4 amplicon.
Clonal Sequencing
After pooling, the amplicon was cloned into pCR4-TOPO® vector (Invitrogen). After transformation into competent TOP10 E. coli cells, individual clones were picked and inserts amplified by colony PCR using forward and reverse plasmid primers. After purification, colony PCR products were sequenced using the BigDye® Terminator cycle sequencing kit (Applied Biosystems, Foster City, Calif., USA), and run on an ABI 3730 XL automated sequencer. Sequence editing and contig assembly were performed using SeqScape v2.5 (Applied Biosystems).
Data Analysis
Alignments were constructed using ClustalW (European Bioinformatics Institute), and used as input for creating a sequence logo in the WebLogo sequence logo generator program (University of California, Berkeley). Viral tropism was predicted based on the V3 loop sequence by the PSSM algorithm (University of Washington, Webpssm) and SVM (Max Plank Institut) using the standard settings as provided with the programs on the websites. Phylogenetic analysis was based on a nucleotide alignment of the full NH2-V4 sequence ( ̃1260 bp). Distances were calculated (DNADIST), trees were constructed (NEIGHBOR), and finally, a consensus tree was built (CONSENSE). N-linked glycosylation of the V3-loop was assessed by the EMBOSS program patmatdb (Institut Pasteur, France). NH2-V4 sequence-based clading was performed by alignment of all clonal sequences with the same region of 66 HIV-1 clade reference strains downloaded from the Los Alamos HIV electronic database (Los Alamos National Library). Similarity tables were retrieved and the reference strains showing the highest percentage of identity with all clones were recorded.
Clonal Phenotyping
Clonal NH2-V4 amplicons were recombined into pHXB2D-ΔNH2-V4-eGFP an hXB2D-based eGFP-containing NH2-V4-deleted backbone (SEQ ID NO: 6), using In-Fusion® CF Dry-Down Cloning Kit (BD Biosciences, Erembodegem, Belgium). Instead of eGFP as marker other well known markers such as luciferase or other commercially available fluorescent proteins, can be used in the current assay. After transformation into MAX Efficiency Stbl cells (Invitrogen), DNA was prepared using QiaPrep® Spin Miniprep Kit (Qiagen, Hilden, Germany). After transfection of the recombinant plasmids into 293T cells using the Amaxa nucleofection technique, produced viruses were used to infect U87-CD4, U87-CD4-CXCR4 and U87-CD4-CCR5 cells. (FIG. 2). After 120 h incubation at 37° C., infection was visualized by fluorescence microscopy. Recombinant plasmids and virus stocks were sequenced using the BigDye® Terminator cycle sequencing kit (Applied Biosystems), and run on an ABI 3730 XL automated sequencer. Sequence editing and contig assembly were performed using SeqScape v2.5 (Applied Biosystems).
Results
Clonal Genotypic V3-Loop Tropism Prediction
One HIV-1-infected subject was randomly selected for clonal genotypic and phenotypic tropism analysis. VIRCOTYPETM analysis showed that the selected HIV-1 strain was susceptible to all FDA-approved proteases and RT inhibitors. Furthermore, the plasma sample contained a viral load of 5.48 log IU/ml indicating that the subject was treatment-naïve. Both GPRT-based (VIRCOTYPETM) and Env NH2-V4-sequence-based clading showed that the selected strain was clade B.
After RNA extraction, the NH2-V4 region was amplified in 7-fold in a single round RT-PCR reaction. After pooling and cloning the NH2-V4 amplicon into the pCR4-TOPO vector, a total of 95 colonies were picked for sequencing. Two clones contained no NH2-V4 insert and 4 clones contained a premature stop codon.
Out of the 89 remaining clones, 60 were selected for tropism prediction by algorithms PSSM (Position Specific Scoring Matrix) and SVM (Support Vector Machine). Four different categories were characterized: RR, XX, RX, and RU with R=R5-tropic, X=X4-tropic, U=unpredictable whereby the first letter in each duplet represents the prediction by PSSM and the second by SVM. Between the 2 algorithms, 11.7% concordant R5 and 16.7% concordant X4 predictions were observed. Almost 12% of the clones showed a discordant prediction (R5 by PSSM and X4 by SVM) and 60% of the clones yielded no prediction by SVM. The PSSM plot (FIG. 3) and sequence logo (FIG. 4) demonstrated a great variability in the selected HIV-1 strain. Further, it was observed that the PSSM scores gradually increased from 1) clones that were predicted R5 by both programs (RR group) to 2) clones with R5 by PSSM versus no prediction by SVM (RU group) to 3) clones with discordant results (R5 by PSSM and X4 by SVM, RX group) to 4) concordant X4 clones (XX group). Finally, it could be observed that only one clone (from the RX group) was situated in the intermediate zone between the cut-off for R5 prediction (−7.3) and the cut-off for X4 prediction (−3.2).
To demonstrate the relatedness among the sequences, phylogenetic analysis was performed for the complete NH2-V4 nucleotide region (FIG. 5). Besides the great variability, it was clear that X4 clones cluster together and the genetic distance between X4 clusters and R5 clusters is relatively short. X4 clustering was significant as assessed by bootstrapping.
All clones were screened for the presence of the N-linked glycosylation motif N {P} S/T {P}, which might be involved in the interaction of R5 gp120 with CCR5, while it might preclude CXCR4 usage. In total, 11 clones lacked the glycosylation motif: 10 clones from the XX group and 1 clone from the RX group, which was located in the intermediate zone of PSSM scoring (clone 30).
Clonal Phenotypic Tropism Determination
Twelve clones were selected for phenotypic tropism determination: clone 1 and 74 (RR group), clone 14 and 83 (RU group), clone 27 and 30 (RX group) and clone 23, 54, 59, 72, 80 and 87 (XX group). An NH2-V4 nucleotide alignment including some characteristics of the selected clones was performed and shown in FIG. 6.
Each clonal NH2-V4 region was recombined into pHXB2D-ΔNH2-V4-eGFP (SEQ ID NO: 6) backbone vector to obtain HIV full genome plasmids, carrying eGFP in nef. After transfection into 293T cells, replication-competent recombinant virus stocks were obtained. Sequencing the NH2-V4 region, including the recombination sites, of both recombinant plasmids and recombinant virus stocks revealed no mismatches when compared to the original clonal sequences obtained in the clonal genotyping experiments.
Recombinant virus stocks were tested phenotypically by infection of U87-CD4, U87-CD4-CXCR4 and U87-CD4-CCR5 cells (FIG. 7). Clones selected from the RR group and the RU group were R5-tropic only, while clones selected from the XX group showed CXCR4 usage only. One clone selected from the RX group showed CCR5 usage (clone 27), while another clone from this group was phenotyped as being dual-tropic (clone 30). Interestingly, the latter clone showed an intermediate PSSM score of −7.11.
Clonal genotypic and phenotypic tropism analysis on a treatment-naïve HIV-1-infected subject revealed the presence of both R5-, dual-, and X4-tropic virus strains. Tropism algorithms were accurate for isolates with clear affinity for their co-receptor (RR and XX group, possibly also the RU group), and need refinement for isolates showing discordant predictions (RX and possibly the RU group).
The above demonstrates that this platform allows quantitative (NH2-V4 clonal sequencing and NH2-V4 clonal phenotyping) tropism testing with accurate reproduction of the viral quasi-species present in the original patient's sample. In addition NH2-V4 population phenotyping was performed on 40 different HIV-1 samples and a good correlation was observed between V3 population sequencing and said NH2-V4 population phenotyping.

## Claims

1. A method for predicting quantitative tropism, quantitative tropism being the quantitative shift in HIV-1 co-receptor usage from CCR5 to CXCR4, from CXCR4 to CCR5, or from dual trophic virus to either CCR5 or CXCR4, in a patient infected by HIV comprising:
a) using a sample comprising viral genetic material from the patient;
b) extraction of viral genetic material from said sample followed by single genome sequencing comprising the following steps:
1. amplification of the viral genetic material of a specific HIV region;
2. analysis of amplicon integrity and pooling of samples;
3. purification of the pooled amplicons;
4. ligation of the pool of amplicons into a vector and transformation of the ligated product into competent cells;
5. analysis of individual transformants obtained; and
6. sequencing the resulting single clones to obtain a single clone genotype sequence;

c) prediction of the specific tropism using said genotype sequence with a predictive algorithm comprising the following steps:
1. identifying the genetic pattern in said genotype sequence wherein at least one natural variability, acquired variability, drug selected mutation or mutation pattern is associated with the quantitative tropism,
2. searching a genotype/phenotype correlative database for at least one genotype entry with a similar genetic pattern to at least one of the natural variability, acquired variability, drug selected mutation or mutation pattern identified in the genetic sequence in step c)1,
3. obtaining the said at least one genotype entry with a similar genetic pattern with a matched phenotype in the correlative genotype/phenotype database, and
4. predicting the HIV tropism from the database of the at least one genotype entry with a similar genetic pattern;

d) analysis of clonal sequences without predictable phenotype in a single clone biological phenotyping assay, wherein the information obtained after said analysis is loaded into the correlative genotype-phenotype database used in step c), wherein the single clone biological phenotyping assay comprises the following steps:
1. generation of clonal partial or full-length HIV genome by using vector pHXB2D-≢NH2-V4-eGFP having SEQ ID NO: 6;
2. transfection of mammalian cells with said genome either together with a suitable backbone to obtain recombinant HIV or directly as a full length HIV-1 genome; and
3. infection of cell lines by said recombinant HIV to determine their biological phenotype wherein the infection process is occurring;

e) prediction of the quantitative tropism based on the information obtained in steps c)1 to c)4 for every single sequence clone present in a sample of a HIV infected patient.

2. The method according to claim 1 wherein step d)2 (transfection) and step d)3 (infection) of the single clone biological phenotyping assay are performed in a single step.

3. The method according to claim 1 wherein the amplification of the viral genetic material of a specific HIV region in step b)1 is either performed by reverse transcription polymerase chain reaction (RT-PCR) or by polymerase chain reaction (PCR).

4. The method according to claim 1 wherein the competent cells used in step b)4 are E. Coli, yeast or Bacillus.

5. The method according to claim 1, step d) wherein the infection process is monitored either by a marker gene introduced in the full-length HIV genome or by a marker gene introduced in an indicator cell line, or microscopically by cytopathic effect scoring or by syncitia formation.

6. The method according to claim 1 whereby HIV sequences obtained and sequenced from samples of patients infected by HIV are loaded into the correlative genotype-phenotype database following the algorithm for prediction of quantitative tropism where after said tropism is reported.

7. The method according to claim 1 wherein said sample from the patient is obtained from a biological sample chosen from a blood sample, a biopsy sample, a plasma sample, a saliva sample, a tissue sample, and a bodily fluid or mucous sample.

8. The method according to claim 1 where in addition viral load is determined in the sample of a patient infected by HIV.

9. The method according to claim 1 wherein the prediction of the quantitative tropism is the quantitative shift in HIV-1 co-receptor usage from either CCR5 to CXCR4 or from CXCR4 to CCR5.

10. A method of generating a report wherein said report comprises the predicted tropism using the method of claim 1.

11. A pHXB2D-ΔNH2-V4-eGFP vector having SEQ ID NO: 6.