Methods, plasmid vectors and primers for assessing HIV viral fitness
View Patent ↗The present invention relates to methods and means for the evaluation of HIV replicative capacity in a given environment. In particular, the invention provides a growth competition assay that can determine relative viral fitness using a recombinant tagged HIV-1 virus system. The methods rely on plasmid vectors, amplicons, primers and probes, and the generation of replication-competent viruses therefrom. Said methods and materials may find use in multiple fields including diagnostics, drug screening, pharmacogenetics and drug development.
1. An ex vivo or in vitro method for determining the replicative capacity of two HIVs in an environment, said method comprising the steps of:
a) generating a recombinant infectious virus comprising a tagged HIV genome having a polynucleotide comprising:
i) a polynucleotide sequence derived from a first HIV comprising all, or part of, one or more of the gag, pol or env gene(s); and
ii) polynucleotide sequences encoding the tat, rev, nef, vif, vpr, and vpu genes;
wherein said polynucleotide further comprises polynucleotide sequences encoding any part of the gag, pol or env gene(s) that is not encoded by the polynucleotide sequence derived from said first HIV, wherein the tagged HIV genome includes one or more silent mutations in one of the HIV genes not comprised by the polynucleotide sequence derived from the first HIV,
wherein the polynucleotide in subparts i) and ii) are from different HIVs;
b) generating a non-tagged recombinant infectious virus comprising a non-tagged HIV genome having a polynucleotide comprising:
i) a polynucleotide sequence derived from a second HIV comprising all, or part of, one or more of the gag, pol or env gene(s); and
ii) polynucleotide sequences encoding the tat, rev, nef, vif, vpr, and vpu genes;
wherein said polynucleotide further comprises polynucleotide sequences encoding any part of the gag, pol or env gene(s) that is not encoded by the polynucleotide sequence derived from said second HIV,
wherein the polynucleotide in subparts i) and ii) are from different HIVs;
c) mixing the recombinant infectious viruses obtained in steps a) and b) in a cell culture in a given environment; and
d) performing quantitative amplification to determine the proportion of the tagged recombinant infectious virus and of the non-tagged recombinant infectious virus within the overall viral population, by detecting the one or more silent mutations in the tagged recombinant infectious virus, and by detecting the lack of the one or more silent mutations in the non-tagged recombinant infectious virus.
2. The method according to claim 1 wherein the amplification is performed by polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription-mediated amplification (TMA), nucleic acid sequence based amplification (NASBA), self-sustained sequence replication (3SR), transcription-based amplification (TAS), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP) or ligase chain reaction (LCR).
3. The method according to claim 1 where the quantitative amplification is monitored using fluorogenic primers, probes cleavable by deoxyribonucleic acid enzymes (DNAzymes) or multi-component nucleic acid enzymes (MNAzymes), fluorogenic probes, molecular beacons, scorpions or any other fluorescent resonance energy transfer (FRET) probes.
4. The method according to claim 1 wherein the quantitative amplification is performed by polymerase chain reaction, employing primers in the vicinity of MNAzymes, and probes that are cleaved by the MNAzymes.
5. The method according to claim 1 wherein each of the tagged HIV genome and the non-tagged HIV genome comprises the backbone of a wild-type or a mutant strain.
6. The method according to claim 5 wherein each of the tagged HIV genome and the non-tagged HIV genome is obtained from a proviral HIV genome or a HIV genome incorporated into a plasmid.
7. The method according to claim 6 wherein the plasmid comprises the genome of the HXB2D wild-type strain.
8. The method according to claim 1 wherein the polynucleotide sequence derived from the first HIV comprises one of the sequences selected from gag-PR-RT-int, gag-PR-RT, gag-PR, gag, PR-RT-int, RT-int, int, PR-RT, PR, RT, or env, and portions thereof.
9. The method according to claim 1 wherein the environment comprises one or more drugs, one or more binding proteins, or a mixture thereof.
10. The method according to claim 1 wherein the tagged HIV genome is generated by introducing one or more silent mutations in the integrase encoding region of the pol gene and wherein said introduction of one or more silent mutations is performed with primers SEQ ID NOs: 1-2.