U.S. patent application number 11/669976 was filed with the patent office on 2007-06-14 for methods of assessing hiv integrase inhibitor therapy.
Invention is credited to Inge Dierynck, Lieve Emma Jan Michiels, Johan Hendrika Jozef Vingerhoets.
Application Number | 20070134766 11/669976 |
Document ID | / |
Family ID | 46150181 |
Filed Date | 2007-06-14 |
United States Patent
Application |
20070134766 |
Kind Code |
A1 |
Vingerhoets; Johan Hendrika Jozef ;
et al. |
June 14, 2007 |
METHODS OF ASSESSING HIV INTEGRASE INHIBITOR THERAPY
Abstract
The present invention relates to methods and products for the
evaluation of HIV treatment. The methods are based on evaluating
molecular events at the HIV integrase resulting in altered
therapeutic efficacy of the investigated compounds. The methods
rely on providing an integrase gene and evaluating either through
genotyping or phenotyping said integrase gene. The present
invention relates to the fields of diagnostics, drug screening,
pharmacogenetics and drug development.
Inventors: |
Vingerhoets; Johan Hendrika
Jozef; (Wijnegem, BE) ; Michiels; Lieve Emma Jan;
(Mol, BE) ; Dierynck; Inge; (Antwerp, BE) |
Correspondence
Address: |
PHILIP S. JOHNSON;JOHNSON & JOHNSON
ONE JOHNSON & JOHNSON PLAZA
NEW BRUNSWICK
NJ
08933-7003
US
|
Family ID: |
46150181 |
Appl. No.: |
11/669976 |
Filed: |
February 1, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11203768 |
Aug 15, 2005 |
7189505 |
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11669976 |
Feb 1, 2007 |
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10215158 |
Aug 8, 2002 |
6958211 |
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11203768 |
Aug 15, 2005 |
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60310480 |
Aug 8, 2001 |
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Current U.S.
Class: |
435/69.1 ;
435/5 |
Current CPC
Class: |
C12Q 1/703 20130101 |
Class at
Publication: |
435/069.1 ;
435/006 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12P 21/06 20060101 C12P021/06 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 8, 2001 |
EP |
01203012.8 |
Claims
1. to 8. (canceled)
9. A method of constructing a genotypic and phenotypic database of
IN sequences, comprising i) obtaining samples of HIV RNA comprising
the IN gene or a portion thereof; ii) reverse-transcribing and
amplifying said HIV RNA with primers specific for the IN region of
the HIV genome to obtain an amplicon comprising the IN gene or a
portion thereof; iii) determining the nucleotide sequence of the
amplicon or portions thereof; iv) generating a plasmid comprising
the wild-type HIV sequence with a deletion in the IN region of the
HIV genome characterized in that said deletion is generated using
nucleic acid amplification; v) preparing recombinant virus by
homologous recombination or ligation between the amplicon and a
plasmid comprising the wild-type HIV sequence with a deletion in
the IN region; vi) determining the relative replicative capacity of
the recombinant virus in the presence of anti-HIV drugs compared to
an HIV virus with a wild-type IN gene sequence.
10. A method of constructing a genotypic and phenotypic database of
IN sequences, comprising i) obtaining samples of HIV DNA comprising
the IN gene or a portion thereof; ii) amplifying said HIV DNA with
primers specific for the IN region of the HIV genome to obtain an
amplicon comprising the IN gene or a portion thereof; iii)
determining the nucleotide sequence of the amplicon or portions
thereof; iv) generating a plasmid comprising the wild-type HIV
sequence with a deletion in the IN region of the HIV genome
characterized in that said deletion is generated using nucleic acid
amplification v) preparing recombinant virus by homologous
recombination or ligation between the amplicon and a plasmid
comprising the wild-type HIV sequence with a deletion in the IN
region; vi) determining the relative replicative capacity of the
recombinant virus in the presence of anti-HIV drugs compared to an
HIV virus with a wild-type IN gene sequence.
11. A database comprising genotypic and phenotypic data of the HIV
integrase, wherein the database further provides a correlation
between genotypes and between genotypes and phenotypes, wherein the
correlation is indicative of efficacy of a given drug regimen.
12. to 17. (canceled)
Description
[0001] The present invention relates to methods and products for
evaluating treatment of human immunodeficiency virus (HIV). In
particular, molecular events at HIV integrase and their effect on
therapeutic efficacy of drugs are determined. Suitably, the events
are analysed by genotyping or phenotyping of HIV integrase. The
methods and products described herein find use in multiple fields
including diagnostics, drug screening, pharmacogenetics and drug
development.
[0002] Several different treatment regimens have been developed to
combat HIV infection. However, since the HIV virus is mutating
quickly, because reverse transcriptase (RT) duplicating the genetic
material has no proofreading capacity, it can counter the effects
of drugs or drug combinations used against it. Current HIV
chemotherapy involves inhibitors of the reverse transcriptase (RT)
and protease enzymes. Despite the development of novel classes of
inhibitors and complex drug regimens, drug resistance is
increasing. Thus, new types of anti-HIV drugs are continually
necessary. Development of compounds that inhibit other HIV gene
products in vivo such as the envelope, tat, and integrase (IN) is a
key area of investigation.
[0003] The integrase protein represents a target for HIV inhibitor
research. HIV integrase is required for integration of the viral
genome into the genome of the host cell, a step in the replicative
cycle of the virus. It is a protein of about 32 KDa encoded by the
pol gene, and is produced in vivo by protease cleavage of the
gag-pol precursor protein during the production of viral particles.
The integration process takes place following reverse transcription
of the viral RNA. First, the viral integrase binds to the viral DNA
and removes two nucleotides from the 3' end of the viral
long-terminal repeat (LTR) sequences on each strand. This step is
called 3' end processing and occurs in the cytoplasm within a
nucleoprotein complex termed the pre-integration complex (PIC).
Second, in a process called strand transfer, the two strands of the
cellular DNA into which the viral DNA will be inserted, i.e. the
target DNA, are cleaved in a staggered fashion. The 3' ends of the
viral DNA are ligated to the 5' ends of the cleaved target DNA.
Finally, remaining gaps are repaired, probably by host enzymes.
[0004] With the increasing number of available anti-HIV compounds,
the number of potential treatment protocols for HIV infected
patients will continue to increase. Many of the currently available
compounds are administered as part of a combination therapy. The
high complexity of treatment options coupled with the ability of
the virus to develop resistance to HIV inhibitors requires the
frequent assessment of treatment strategies. The ability to
accurately monitor the replicative capacity of viruses in patients
subjected to a drug regimen and to use that data to modify the
doses or combinations of inhibitors allows physicians to
effectively reduce the formation of drug resistant virus and
provide an optimal, tailored treatment for each patient.
[0005] Sophisticated patient monitoring techniques have been
developed for analysis of current therapies, e.g. such as
Antivirogram.RTM., (described in WO 97/27480 and U.S. Pat. No.
6,221,578 B1; incorporated herein by reference) and Phenosense.TM.
(WO 97/27319). These cellular based assays determine the resistance
of the patient borne virus towards a defined drug regimen by
providing information about the susceptibility of the patient's
virus strain to the treatment based on protease and reverse
transcriptase inhibitors treatment. Other monitoring strategies
include immunological means or sequencing techniques.
[0006] The Antivirogram.RTM. and Genseq.TM. assays determine the
phenotype and genotype respectively of a patient's reverse
transcriptase and protease genes. The relevant coding regions are
obtained from patient samples, reverse transcribed and amplified by
the polymerase chain reaction (PCR). Within lymphocyte cells the
relevant coding regions are combined with viral deletion constructs
to create chimeric viruses. The ability of these chimeric viruses
to invade and kill cells in culture is assessed in the presence of
HIV reverse transcriptase and protease inhibitors. A database
combining phenotypic and genotypic information can be developed, as
described in WO 00/73511 (incorporated herein by reference).
[0007] While phenotyping and genotyping assays such as
Antivirogram.RTM. and Genseq.TM. have been developed for reverse
transcriptase and protease genes, protocols for evaluation of drug
resistance at the integrase gene have not been successfully
developed.
DETAILED DESCRIPTION OF THE INVENTION
[0008] The instant invention provides techniques for evaluating
human immunodeficiency (HIV) drug effectiveness. Assays for wild
type or mutant HIV integrase are provided, using a set of primers
designed for the amplification and analysis of HIV genetic
material. The assessment of patient borne viral integrase leads to
a better prediction of the drugs suitable for treatment of the
strains present in the infected individual. The protocols and
products may be used for diverse diagnostic, clinical,
toxicological, research and forensic purposes including, drug
discovery, designing patient therapy, drug efficacy testing and
patient management. The assays described herein may be used in
combination with other assays. The results may be implemented in
computer models and databases. The products described herein may be
incorporated into kits.
[0009] The instant invention relates to a method for determining
the susceptibility of at least one HIV virus to at least one
treatment, comprising: i) obtaining at least one sample of HIV RNA,
wherein the sample comprises at least one IN gene or a portion
thereof, ii) reverse-transcribing and amplifying the HIV RNA with
primers specific for IN region of the HIV genome to obtain at least
one DNA construct comprising the at least one IN gene or a portion
thereof; iii) preparing at least one recombinant virus by
homologous recombination or ligation between the amplified at least
one DNA construct and a plasmid comprising the wild-type HIV
sequence with a deletion in the IN region of the HIV genome, and
iv) determining the phenotypic susceptibility of at least one HIV
virus to at least one treatment by monitoring the at least one
recombinant virus in the presence of the at least one
treatment.
[0010] In particular, the present invention relates to a method for
determining the susceptibility of at least one HIV virus to at
least one drug, comprising: i) obtaining at least one sample
comprising HIV RNA, wherein the sample comprises at least one IN
gene or a portion thereof; ii) reverse transcribing and amplifying
the HIV RNA with primers specific for IN region of the HIV genome
to obtain at least one amplicon comprising the at least one IN gene
or a portion thereof; iii) using nucleic acid amplification to
generate a plasmid comprising the wild-type HIV sequence with a
deletion in the IN region of the HIV genome; iv) preparing at least
one recombinant virus by homologous recombination or ligation
between the amplified at least one amplicon and a plasmid
comprising the wild-type HIV sequence with a deletion in the IN
region, and v) monitoring the at least one recombinant virus in the
presence of the at least one treatment to determine the phenotypic
susceptibility of at least one HIV virus to said at least one
drug.
[0011] Reverse transcription and amplification may be performed
with a single set of primers. Alternatively, more than one set of
primers may be used in a hemi-nested approach to reverse transcribe
and amplify the genetic material. Particularly, more than one set
of primer is used in a nested approach. Following the generation of
the recombinant construct, the chimeric virus may be grown and the
viral titer determined (expressed as multiplicity of infection,
MOI) before proceeding to the determination of the phenotypic
susceptibility. The indicator gene, encoding a signal indicative of
replication of the virus in the presence of a drug or indicative of
the susceptibility of the virus in the presence of a drug may be
present in the culturing cells such as MT-4 cells. In addition,
said indicator gene may be incorporated in the chimeric construct
introduced into the culturing cells or may be introduced
separately. Suitable indicator genes encode fluorescent proteins,
particularly green fluorescent protein or mutants thereof. In order
to allow homologous recombination, genetic material may be
introduced into the cells using a variety of techniques known in
the art including, calcium phosphate precipitation, liposomes,
viral infection, and electroporation. The monitoring may be
performed in high throughput.
[0012] A human immunodeficiency virus (HIV), as used herein refers
to any HIV including laboratory HIV strains, wild type HIV strains,
mutant HIV strains and any biological sample comprising at least
one HIV virus, such as, for example, an HIV clinical isolate. HIV
strains compatible with the present invention are any such strains
that are capable of infecting mammals, particularly humans.
Examples are HIV-1 and HIV-2. For reduction to practice of the
present invention, an HIV virus refers to any sample comprising at
least one HIV virus. As for instance a patient may have HIV viruses
in his body with different mutations in the integrase (IN) gene. It
is to be understood that a sample may contain a variety of
different HIV viruses containing different mutational profiles in
the IN gene. A sample may be obtained for example from an
individual, from cell cultures, or generated using recombinant
technology, or cloning. HIV strains compatible with the present
invention are any such strains that are capable of infecting
mammals, particularly humans. Viral strains used for obtaining a
plasmid are preferably HIV wild-type sequences, such as LAI or
HXB2D. LAI, also known as IIIB, is a wild type HIV strain. One is
particular clone thereof, this means one sequence, is HXB2D. This
sequence may be incorporated into a plasmid.
[0013] Instead of viral RNA, HIV DNA, e.g. proviral DNA, may be
used for the methods described herein. In case RNA is used, reverse
transcription into DNA by a suitable reverse transcriptase is
needed. The protocols describing the analysis of RNA are also
amenable for DNA analysis. However, if a protocol starts from DNA,
the person skilled in the art will know that no reverse
transcription is needed. The primers designed to amplify the RNA
strand, also anneal to, and amplify DNA. Reverse transcription and
amplification may be performed with a single set of primers.
Suitably a hemi-nested and more suitably a nested approach may be
used to reverse transcribe and amplify the genetic material.
[0014] Thus, the phenotyping method of the present invention may
also comprise: i) obtaining at least one sample comprising HIV DNA,
wherein the sample comprises at least one IN gene or a portion
thereof; ii) amplifying the HIV DNA with primers specific for IN
region of the HIV genome to obtain at least one amplicon comprising
the at least one IN gene or a portion thereof; iii) generating a
plasmid comprising the wild-type HIV sequence with a deletion in
the IN region of the HIV genome characterized in that said deletion
is generated using nucleic acid amplification; iv) preparing at
least one recombinant virus by homologous recombination or ligation
between the amplified at least one amplicon and a plasmid
comprising the wild-type HIV sequence with a deletion in the IN
region, and v) monitoring the at least one recombinant virus in the
presence of the at least one drug to determine the phenotypic
susceptibility of at least one HIV virus to at least one drug.
[0015] Nucleic acid may be amplified by techniques such as
polymerase chain reaction (PCR), nucleic acid sequence based
amplification (NASBA), self-sustained sequence replication (3SR),
transcription based amplification (TAS), ligation chain reaction
(LCR). Often PCR is used.
[0016] Any type of patient sample may be used to obtain the
integrase gene, such as, for example, serum and tissue. Viral RNA
may be isolated using known methods such as described in Boom, R.
et al. (J. Clin. Microbiol. 28(3): 495-503 (1990)). Alternatively,
a number of commercial methods such as the QIAAMP.RTM. viral RNA
kit (Qiagen, Inc.) may be used to obtain viral RNA from bodily
fluids such as plasma, serum, or cell-free fluids. DNA may be
obtained by procedures known in the art (e.g. Maniatis, 1989) and
commercial procedures (e.g. Qiagen).
[0017] The complete integrase (IN) or a portion of the IN gene may
be used. The complete IN gene comprises 864 nucleotides (nt),
coding for a 288 amino acid long integrase. A portion of the IN
gene is defined as a fragment of IN gene recovered from patient
borne virus, lab viruses including IIIB and NL4-3, or mutant
viruses. This fragment does not encompass the complete 864 nt. Said
fragment may be obtained directly from its source, including a
patient sample, or may be obtained using molecular biology tools
following the recovery of the complete IN sequence. Amplicon refers
to the amplified, and where necessary, reverse transcribed
integrase gene or portion thereof. It should be understood that
this IN may be of diverse origin including plasmids and patient
material. Suitably, the amplicon is obtained from patient material.
For the purpose of the present invention the amplicon is sometimes
referred to as "DNA construct". A viral sequence may contain one or
multiple mutations versus the consensus reference sequence given by
K03455. Said sequence, K03455, is present in Genbank and available
through the internet. A single mutation or a combination of IN
mutations may correlate to a change in drug efficacy. This
correlation may be indicative of an altered i.e. decreased or
increased susceptibility of the virus for a drug. Said mutations
may also influence the viral fitness.
[0018] "Chimeric" means a construct comprising nucleic acid
material from different origin such as for example a combination of
wild type HIV with a laboratory HIV virus, a combination of wild
type HIV sequence and patient derived HIV sequence.
[0019] A "drug" means any agent such as a chemotherapeutic,
peptide, antibody, antisense, ribozyme and any combination thereof.
Examples of drugs include protease inhibitors including ritonavir,
amprenavir, nelfinavir; reverse transcriptase inhibitors such as
nevirapine, delavirdine, AZT, zidovudine, didanosine; integrase
inhibitors; agents interfering with envelope (such as for example
T-20, T-1249). Treatment or treatment regimen refers to the
therapeutic management of an individual by the administration of
drugs. Different drug dosages, administration schemes,
administration routes and combinations may be used to treat an
individual.
[0020] An alteration in viral drug sensitivity is defined as a
change in susceptibility of a viral strain to said drug.
Susceptibilities are generally expressed as ratios of EC.sub.50 or
EC.sub.90 values (the EC.sub.50 or EC.sub.90 value being the drug
concentration at which 50% or 90% respectively of the viral
population is inhibited from replicating) of a viral strain under
investigation compared to the wild type strain. Hence, the
susceptibility of a viral strain towards a certain drug can be
expressed as a fold change in susceptibility, wherein the fold
change is derived from the ratio of for instance the EC.sub.50
values of a mutant viral strain compared to the wild type EC.sub.50
values. In particular, the susceptibility of a viral strain or
population may also be expressed as resistance of a viral strain,
wherein the result is indicated as a fold increase in EC.sub.50 as
compared to wild type EC.sub.50. The IC.sub.50 is the drug
concentration at which 50% of the enzyme activity is inhibited.
[0021] The susceptibility of at least one HIV virus to a drug may
be tested by determining the cytopathogenicity of the recombinant
virus to cells. In the context of this invention, the
cytopathogenic effect means the viability of the cells in culture
in the presence of chimeric viruses. The cells may be chosen from T
cells, monocytes, macrophages, dendritic cells, Langerhans cells,
hematopoetic stem cells or precursor cells, MT4 cells and PM-1
cells. Suitable host cells for homologous recombination of HIV
sequences include MT4 and PM-1. MT4 is a CD4.sup.+ T-cell line
containing the CXCR4 co-receptor. The PM-1 cell line expresses both
the CXCR4 and CCR5 co-receptors. All of the cells mentioned above
are capable of producing new infectious virus particles upon
recombination of the IN deletion vectors with IN sequences such as
those derived from patient samples. Thus, they can also be used for
testing the cytopathogenic effects of recombinant viruses. The
cytopathogenicity may, for example, be monitored by the presence of
any reporter molecule including reporter genes. A reporter gene is
defined as a gene whose product has reporting capabilities.
Suitable reporter molecules include tetrazolium salts, green
fluorescent proteins, beta-galactosidase, chloramfenicol
transferase, alkaline phophatase, and luciferase. Several methods
of cytopathogenic testing including phenotypic testing are
described in the literature comprising the recombinant virus assay
(Kellam and Larder, Antimicrob. Agents Chemotherap. 1994, 38,
23-30, Hertogs et al. Antimicrob. Agents Chemotherap. 1998, 42,
269-276; Pauwels et al. J. Virol Methods 1988, 20, 309-321)
[0022] The susceptibility of at least one HIV virus to at least one
drug may be determined by the replicative capacity of the
recombinant virus in the presence of at least one drug, relative to
the replicative capacity of an HIV virus with a wild-type IN gene
sequence. Replicative capacity means the ability of the virus or
chimeric construct to grow under culturing conditions. This is
sometimes referred to as viral fitness. The culturing conditions
may contain triggers that influence the growth of the virus,
examples of which are drugs. The methods for determining the
susceptibility may be useful for designing a treatment regimen for
an HIV-infected patient. For example, a method may comprise
determining the replicative capacity of a clinical isolate of a
patient and using said replicative capacity to determine an
appropriate drug regime for the patient. One approach is the
Antivirogram.RTM. assay.
[0023] The IN phenotyping assays of the present invention can be
performed at high throughput using, for example, a microtiter plate
containing a variety of anti-HIV drugs. The present assays may be
used to analyse the influence of changes at the HIV IN gene to any
type of drug useful to treat HIV. Examples of anti-HIV drugs that
can be tested in this assay include, nucleoside and non-nucleoside
reverse transcriptase inhibitors, nucleotide reverse transcriptase
inhibitors, protease inhibitors, membrane fusion inhibitors, and
integrase inhibitors, but those of skill in the art will appreciate
that other types of antiviral compounds may also be tested. The
results may be monitored by several approaches including but not
limited to morphology screening, microscopy, and optical methods,
such as, for example, absorbance and fluorescence. An IC.sub.50
value for each drug may be obtained in these assays and used to
determine viral replicative capacity in the presence of the drug.
Apart from IC.sub.50 also e.g. IC.sub.90 or EC.sub.50 (effective
concentrations) can be used. The replicative capacity of the
viruses may be compared to that of a wild-type HIV virus to
determine a relative replicative capacity value. Data from
phenotypic assays may further be used to predict the behaviour of a
particular HIV isolate to a given drug based on its genotype.
[0024] The assays of the present invention may be used for
therapeutic drug monitoring. Said approach includes a combination
of susceptibility testing, determination of drug level and
assessment of a threshold. Said threshold may be derived from
population based pharmacokinetic modelling (WO 02/23186). The
threshold is a drug concentration needed to obtain a beneficial
therapeutic effect in vivo. The in vivo drug level may be
determined using techniques such as high performance liquid
chromatography, liquid chromatography, mass spectroscopy or
combinations thereof. The susceptibility of the virus may be
derived from phenotyping or interpretation of genotyping results
i.e. virtual phenotyping (WO 01/79540).
[0025] The assays of the present invention may be useful to
discriminate an effective drug from an ineffective drug by
establishing cut-offs i.e. biological cut-offs (see e.g. WO
02/33402). A biological cutoff is drug specific. These cut-offs are
determined following phenotyping a large population of individuals
containing wild type viruses. The cut-off is derived from the
distribution of the fold increase in resistance of the virus for a
particular drug.
[0026] The instant invention also relates to a kit for phenotyping
HIV integrase. Such kit, useful for determining the susceptibility
of at least one HIV virus to at least one drug, may comprise: i) at
least one of the primers selected from SEQ ID N.degree.1-16, and
ii) a plasmid as described in the present invention. For the
purpose of performing the phenotyping assay, such kit may be
further completed with at least one inhibitor. Optionally, a
reference plasmid bearing a wild type HIV sequence may be added.
Optionally, cells susceptible of HIV transfection may be added to
the kit. In addition, at least one reagent for monitoring the
indicator genes, or reporter molecules such as enzyme substrates,
may be added.
[0027] The present invention also describes a method for
determining the susceptibility of at least one HIV virus to at
least one drug, comprising: i) obtaining at least one sample
comprising HIV RNA, wherein the sample comprises at least one IN
gene or a portion thereof; ii) reverse-transcribing and amplifying
said HIV RNA with primers specific for the IN region of the HIV
genome to obtain an amplicon comprising the IN gene or a portion
thereof; iii) determining the nucleotide sequence of the amplicon
or a portion thereof, and iv) comparing the nucleotide sequence of
the amplicon to the sequence of known sequences to determine the
susceptibility of at least one HIV virus to at least one drug. This
assay protocol is commonly referred to as genotyping.
[0028] The genotype of the patient-derived IN coding region may be
determined directly from the amplified DNA, i.e. the DNA construct,
by performing DNA sequencing during the amplification step.
Alternatively, the sequence may be obtained after sub-cloning into
a suitable vector. A variety of commercial sequencing enzymes and
equipment may be used in this process. The efficiency may be
increased by determining the sequence of the IN coding region in
several parallel reactions, each with a different set of primers.
Such a process could be performed at high throughput on a
multiple-well plate, for example. Commercially available detection
and analysis systems may be used to determine and store the
sequence information for later analysis. The nucleotide sequence
may be obtained using several approaches including sequencing
nucleic acids. This sequencing may be performed using techniques
including gel based approaches, mass spectroscopy and
hybridisation. However, as more resistance related mutations are
identified, the sequence at particular nucleic acids, codons or
short sequences may be obtained. If a particular resistance
associated mutation is known, the nucleotide sequence may be
determined using hybridisation assays (including Biochips,
LipA-assay), mass spectroscopy, allele specific PCR, or using
probes or primers discriminating between mutant and wild-type
sequence. For these purposes the probes or primers may be suitably
labelled for detection (e.g. Molecular beacons, TaqMan.RTM.,
SunRise primers). Suitably, fluorescent or quenched fluorescent
primers are used. The primer is present in a concentration ranging
from 0.01 pmol to 100 pmol, suitably between 0.10 and 10 pmol. The
cycling conditions include a denaturation step during 0.5 to 10
minutes, suitably, 1 to 5 minutes at a temperature ranging from 85
to 99.degree. C. Interestingly, the temperature is between 90 and
98.degree. C. Subsequently, the material is cycled during 14 to 45
cycles, suitably between 20 to 40 cycles, more suitably during 25
to 35 cycles. Nucleic acid is denatured at 90 to 98.degree. C.
during 5 seconds to 2 minutes. Suitably, denaturation periods range
from 15 seconds to 1 minute. Annealing is performed at 40 to
60.degree. C., specifically, between 45.degree. C. and 57.degree.
C. The annealing period is 5 seconds to 1 minute, especially
between 10 seconds and 35 seconds. Elongation is performed at
60.degree. C. to 75.degree. C. during 10 seconds to 10 minutes.
Preferably, the elongation period is 15 seconds to 5 minutes. A
selected set of sequencing primers includes SEQ ID 17-22. This
particular selection has the advantage that it enables the
sequencing of the complete HIV integrase gene. Consequently, using
this set of primers all possible mutations that may occur in the
HIV integrase gene may be resolved.
[0029] The patient IN genotype provides an additional means to
determine drug susceptibility of a virus strain. Phenotyping is a
lengthy process often requiring 2 or more weeks to accomplish.
Therefore, systems have been developed which enable the prediction
of the phenotype based on the genotypic results. The results of
genotyping may be interpreted in conjunction with phenotyping and
eventually be subjected to database interrogation. A suitable
system is virtual phenotyping (WO 01/79540). In the virtual
phenotyping process the complete IN genes may be used.
Alternatively, portions of the genes may be used. Also combinations
of mutations, preferentially mutations indicative of a change in
drug susceptibility, may be used. A combination of mutations is
sometimes referred to as a hot-spot (see e.g. WO 01/79540).
Briefly, in the process of virtual phenotyping, the genotype of a
patient derived IN sequence may be correlated to the phenotypic
response of said patient derived IN sequence. If no phenotyping is
performed, the sequence may be screened towards a collection of
sequences present in a database. Identical sequences are retrieved
and the database is further interrogated to identify if a
corresponding phenotype is known for any of the retrieved
sequences. In this latter case a virtual phenotype may be
determined. A report may be prepared including the EC.sub.50 of the
viral strain for one or more therapies, the sequence of the strain
under investigation, biological cut-offs.
[0030] The present invention also relates to a kit for genotyping
HIV integrase. Such kit useful for determining the susceptibility
of at least one HIV virus to at least one drug may comprise at
least one primer selected from SEQ ID N.degree. 1-12 and 17-22.
Optionally, additional reagents for performing the nucleic
amplification and subsequent sequence analysis may be added.
Reagents for cycle sequencing may be included. The primers may be
fluorescently labelled.
[0031] The instant invention provides a method of identifying a
drug effective against HIV integrase comprising: i) obtaining at
least one HIV integrase sequence, ii) determining the phenotypic
response of the integrase towards said drug, iii) using said
phenotypic response to determine the effectiveness of said drug.
The phenotypic response is determined according to the methods of
the instant invention.
[0032] The methods described in the instant invention may be used
in a method of identifying a drug effective against HIV integrase
comprising: i) obtaining at least one HIV integrase sequence,
determining the sequence of said HIV integrase, ii) comparing said
sequence with sequences present in a database of which the
susceptibility has been determined of the HIV integrase, iii) using
said sequence comparison to determine the effectiveness of said
drug. The susceptibility and the sequence of the HIV integrase gene
are determined according to the methods disclosed in the instant
invention.
[0033] The genotyping and phenotyping methods as described herein
can be used to create a genotypic and phenotypic database of IN
sequences, comprising: i) obtaining samples comprising HIV RNA
comprising the IN gene or a portion thereof; ii)
reverse-transcribing and amplifying said HIV RNA with primers
specific for the IN region of the HIV genome to obtain an amplicon
comprising the IN gene or a portion thereof; iii) determining the
nucleotide sequence of the amplicon or portions thereof; iv)
generating a plasmid comprising the wild-type HIV sequence with a
deletion in the IN region of the HIV genome characterized in that
said deletion is generated using nucleic acid amplification; v)
preparing recombinant virus by homologous recombination or ligation
between the amplicon and a plasmid comprising the wild-type HIV
sequence with a deletion in the IN region of the HIV genome,
characterised in that said deletion is introduced using PCR; vi)
determining the relative replicative capacity of the recombinant
virus in the presence of anti-HIV drugs compared to an HIV virus
with a wild-type IN gene sequence; vii) correlating the nucleotide
sequence and relative replicative capacity in a data table.
[0034] According to the methods described herein a database may be
constructed comprising genotypic and phenotypic data of the HIV
integrase, wherein the database further provides a correlation
between genotypes and between genotypes and phenotypes, wherein the
correlation is indicative of efficacy of a given drug regimen. A
database of IN sequences may be created and used as described in WO
01/79540. For example, such a database may be analysed in
combination with pol and pro sequence information and the results
used in the determination of appropriate treatment strategies. Said
database containing a collection of genotypes, phenotypes and
samples for which the combined genotype/phenotype are available may
be used to determine the virtual phenotype (see supra). In
addition, instead of interrogating the complete IN sequences,
particular codons correlating to a change in drug susceptibility of
the virus may be interrogated in such database.
[0035] A primer may be chosen from SEQ ID N.degree. 1-23. A
particular set of primers is SEQ ID 1-10, 13, 15, and 23. Primers
specific for the IN region of the HIV genome such as the primers
described herein and their homologs are claimed. The primer
sequences listed herein may be labelled. Suitably, this label may
be detected using fluorescence, luminescence or absorbance. The
primer for creating a deletion construct may contain a portion that
does not anneal to the HIV sequence. That portion may be used to
introduce a unique restriction site. Interestingly, primers may be
designed in which the unique restriction site is partially present
in the HIV sequence. The primers are chosen from those listed
herein or have at least 80% homology as determined by methods known
by the person skilled in the art such BLAST or FASTA. Specifically,
the homology is at least 90%, more specifically, at least 95%. In
addition, primers located in a region of 50 nucleotides (nt)
upstream or downstream from the sequences given herein constitute
part of the invention. Especially, said region is 20 nucleotides up
or downstream from the position in the HIV genome of the primer
sequences given herein. Alternatively, primers comprising at least
8 consecutive bases present in either of the primers described here
constitute one embodiment of the invention. Interestingly, the
primers comprise at least 12 consecutive bases present in either of
the primers described herein.
[0036] The present invention comprises the plasmids described in
the experimental part and the use of the plasmids in the methods
described herein. The HIV sequence incorporated in the plasmid may
be based on the K03455 sequence, The complete HIV sequence may be
incorporated or only part thereof. A suitable plasmid backbone may
be selected from the group including pUC, pSV or pGEM.
[0037] A plasmid comprising a deleted integrase, wherein the
deletion comprises at least 100 bp of the HIV integrase gene is
provided herein. Suitably, more that 500 bp of the integrase gene
are deleted, more suitably the complete IN gene is deleted. The
deletion may also comprise parts of flanking genes, or eventually
more than one gene, e.g. deletion of integrase and protease.
[0038] To prepare vectors containing recombinant IN coding
sequences, the patient derived IN RNA can be reverse transcribed
and amplified by the polymerase chain reaction (PCR), then inserted
into a vector containing the wild type HIV genome sequence but
lacking a complete IN coding region. Initially 36 different primer
combinations were used to obtain amplified DNA sequences from 16
patient samples. The 5' to 3' sequences and the primers identified
by SEQ ID's NO 1-10 of primers that can be successfully used to
reverse transcribe and PCR amplify IN coding regions are listed
below in Table 1.
[0039] A number of reverse transcription and PCR protocols known in
the art may be used in the context of the present invention. A
nested PCR approach to amplify the patient derived cDNA after
reverse transcription may be used as described in Kellam, P. and
Larder, B. A., (Antimicrobial Agents and Chemotherapy 38: 23-30
(1994)), which is incorporated herein by reference. The nested
approach of the instant invention utilizes two sets of primers, the
outer primers are 5'EGINT1 (SEQ ID NO 1) and 3'EGINT10 (SEQ ID NO
11), while the inner primers are 5'EGINT107 (SEQ ID NO 2) and
3'EGINT11 (SEQ ID NO 12). An additional inner 5' primer, 5'EGINT2
(SEQ ID NO 3), may also be used as a "rescue primer" to improve the
yield of amplified DNA. Amplification using these primers yields a
PCR product encompassing the complete IN coding sequence.
Alternatively, 5'EGINT3 (SEQ ID NO 4) and 3'EGINT10 (SEQ ID NO 11)
are used as outer PCR primers, while 5'EGINT4 (SEQ ID NO 5) or
5'EGINT5 (SEQ ID NO 6) and 3'EGINT6 (SEQ ID NO 7) are used as inner
primers, yielding a PCR product encompassing a portion of the IN
coding sequence. TABLE-US-00001 TABLE 1 Primers for IN reverse
transcription and PCR amplification. The underlined portions do not
anneal to the sequence to be amplified. Primer Name SEQ ID NO 5' to
3' sequence R-IN-vif and IN outer and inner primers 5'EGINT1 SEQ ID
NO:1 GGTACCAGTTAGAGAAAGAACCCA 5'EGINT107 SEQ ID NO:2
GGAGCAGAAACCTTCTATGTAGATG 5'EGINT2 SEQ ID NO:3
GGCAGCTAACAGGGAGACTAA 5'EGINT3 SEQ ID NO:4 GGAATCATTCAAGCACAACCAGA
5'EGINT4 SEQ ID NO:5 TCTGGCATGGGTACCAGCACA 5'EGINT5 SEQ ID NO:6
AGGAATTGGAGGAAATGAACAAGTA 3'EGINT6 SEQ ID NO:7
GTTCTAATCCTCATCCTGTCT 3'EGTNT7 SEQ ID NO:8 CCTCCATTCTATGGAGTGTCTATA
3'EGINT8 SEQ ID NO:9 GGGTCTACTTGTGTGCTATATCTC 3'EGINT9 SEQ ID NO:10
CAGATGAATTAGTTGGTCTGCTA 3'EGINT10 SEQ ID NO:11 CCT CCA TTC TAT GGA
GAC TCC CTG 3'EGINT11 SEQ ID NO:12 GCA TCC CCT AGT GGG ATG TG
R-IN-vif deletion-mutagenesis primers MUT-IN1 SEQ ID NO:13 GGG TGA
CAA CTT TTT GTC TTC CTC TAT MUT-IN2 SEQ ID NO:14 GGA TCC TGC AGC
CCG GGA AAG CTA GGG GAT GGT TTT ATA IN deletion-mutagenesis
primers: MUT-IN3 SEQ ID NO:15 GGG CCT TAT CTA TTC CAT CTA AAA ATA
GT MUT-IN4 SEQ ID NO:16 GGA TCC TGC AGC CCG GGA TTA TGG AAA ACA GAT
GGC A Sequencing primers IN_SEQ1F SEQ ID NO:17 AGT CAG TGC TGG AAT
CAG G IN_SEQ2F SEQ ID NO:18 TTC CAG CAG AAA CAG GGC AG IN_SEQ3F SEQ
ID NO:19 GTA GAC ATA ATA GCA ACA GAC IN_SEQ1R SEQ ID NO:20 CCC TGA
AAC ATA CAT ATG GTG IN_SEQ2R SEQ ID NO:21 CTG CCA TTT GTA CTG CTG
TC IN_SEQ1R SEQ ID NO:22 TGA ACT GCT ACC AGG ATA AC
[0040] To prepare recombinant vectors comprising the amplified
patient-derived IN sequences, these sequences can be inserted into
vectors comprising the wild-type HIV sequence and a deletion of all
or part of the IN coding region. The wild type HIV sequence can be
obtained from a plasmid such as pSV40HXB2D that is capable of
transfecting lymphocyte cells to produce viable virus particles. A
deletion of the entire IN coding region on the pSV40HXB2D vector
may effectively be created by PCR amplifying the plasmid using
primers annealing to sequences at or near the ends of the IN coding
region in the vector. The amplified product can be cleaved with a
restriction enzyme introduced into the primers, then re-ligated to
create a pSV40HXB2D-based IN deletion vector with a unique
restriction site at the location of the deletion. The IN deletion
vector can have a deletion of the complete IN coding sequence,
optionally with part of the preceding RNase and/or subsequent Vif
coding sequences also deleted. Alternatively, a partial deletion of
the IN coding sequence is created. This restriction site is unique
for the complete plasmid including the HIV gene. An example of such
restriction site is the SmaI restriction site. Interestingly, the
primers for creating a deletion construct are selected from SEQ ID
N.degree. 13-16.
[0041] Those of skill in the art will appreciate that several types
of HIV vectors and cloning procedures known in the art may be used
to create IN deletion plasmids for recombination or ligation with
patient derived sequences and creation of infectious viruses.
Generally, such vectors must be created to allow re-insertion of
the deleted sequences without disrupting the reading frame of the
gag-pol gene.
[0042] The amplified IN sequences may be inserted into the
appropriate vector by homologous recombination between overlapping
DNA segments in the vector and amplified sequence. Alternatively,
the amplified IN sequence can be incorporated into the vector at a
unique restriction site according to cloning procedures standard in
the art. This latter is a direct cloning strategy.
Experimental Part
EXAMPLE 1
Phenotyping HIV Integrase
1. PCR Amplification of Integrase Encoding Sequence
[0043] The integrase encoding sequence was amplified from either
wildtype HIV-1 (IIIB) or NL4.3 virus, or HXB2D site-directed mutant
viruses containing mutations in integrase (such as T66I, S153Y,
M154I, or combinations thereof) (Hazuda et al., Science 2000, 287,
646-650), or patient samples. Starting from RNA, extracted from
virus supernatant or plasma using the QIAamp.RTM. viral RNA
extraction kit (Qiagen), cDNA was synthesized by reverse
transcription Expand.TM. reverse transcriptase, 30 min at
42.degree. C.) with the primer 3'EGINT10 (SEQ ID NO 11), followed
by a nested PCR. The outer PCR was performed with the primers
5'EGINT1 (SEQ ID NO 1) and 3'EGINT10 (SEQ ID NO 11) (R-IN-vif
construct) or 5'EGINT3 (SEQ ID NO 4) and 3'EGINT10 (SEQ ID NO 11)
(IN construct) (Expand.TM. High Fidelity PCR system), and 5 .mu.l
of the outer product was used for an inner PCR with primers
5'EGINT2 (SEQ ID NO 3) and 3'EGINT11 (SEQ ID NO 12) (R-IN-vif
construct) or 5'EGINT4 (SEQ ID NO 5) and 3'EGINT6 (SEQ ID NO 7) (IN
construct). In a second protocol the outer primers were identical
as described above, the inner primers are 5'EGINT5 (SEQ ID NO 6)
and 3'EGINT6 (SEQ ID NO 7). The amplicons can be used for
genotyping and phenotyping. Cycling conditions for both PCRs are
denaturation for 3 min at 95.degree. C., followed by 30 cycles of 1
min 90.degree. C., 30 sec 55.degree. C., and 2 min 72.degree. C. A
final extension was performed at 72.degree. C. for 10 min. For
recombination, PCR products are purified using the QiaQuick.RTM. 96
PCR BioRobot kit (Qiagen), according to the manufacturer's
protocol. If the protocol starts from DNA containing the HIV
material such as proviral DNA, the reverse transcriptase step is
not needed. The nested approach is also not needed when starting
from proviral DNA. The obtained amplicons were sequenced using the
primers: In_seq1F (SEQ ID NO 17), In_seq2F (SEQ ID NO 18), In_seq3F
(SEQ ID NO 19), IN_seq1R (SEQ ID NO 20), In_seq2R (SEQ ID NO 21),
and IN_seq3R (SEQ ID NO 22). The sequence of the IIIB and patient
amplicon, and the NL4.3 amplicon were identical to the reference
IIIB and NL4.3 sequences respectively (data not shown).
2. Preparation of a IN Deletion Construct
[0044] A R-IN-vif or IN deletion construct was generated by
site-directed mutagenesis on the template pSV40HXB2D with the
primers MUT-IN1 (SEQ ID NO 13) and MUT-IN2 (R-IN-vif construct)
(SEQ ID NO 14) or MUT-IN3 (SEQ ID NO 15) and MUT-IN4 (SEQ ID NO 16)
(IN construct) (protocol Site-directed mutagenesis kit,
Stratagene). After DpnI digestion for removal of the methylated
template DNA, the construct was digested with SmaI and ligated to
circulize the plasmid. The plasmid was transformed into competent
cells such as Top10 cells, and colonies were screened for the
presence of the deletion construct. The IN-deletion construct was
checked by sequence analysis with primers 5'EGINT1 (SEQ ID NO 1) or
5'EGINT10 (SEQ ID NO 11) and 3'EGINT10 (SEQ ID NO 11) or 3'EGINT11
(SEQ ID NO 12). For use in recombination experiments, large-scale
plasmid DNA preparations were linearized by SmaI digestion and
recombined with PCR amplified integrase genes from wild type,
mutant, or patient viruses. The plasmid containing the integrase
deletion (IN) has been deposited pSV40HXB2D-IN. The sequence of
said plasmid is 14377 nucleotides long. The R-IN-vif deletion
construct is 13975 nucleotides long. The pSV40HXB2D-IN was
deposited at the Belgian Coordinated Collections of Micro-Organisms
located at the Universiteit Gent--Laboratorium voor Moleculaire
Biologie on Aug. 5, 2002 and the accession number is LMBP 4574.
3. Recombination of Integrase-Amplified Sequences with the
Corresponding Deletion Construct
[0045] Recombinant virus was produced by co-transfection by
electroporation of the SmaI-linearized IN-deletion construct and
the integrase amplicon into MT4 cells or MT4 cells equipped with an
LTR driven reporter gene construct. Production of recombinant virus
was evaluated by scoring the cytopathogenic effect (CPE) that is
induced by HIV-infection of MT4 cells or by the LTR-driven reporter
signal induced by HIV infection in MT4 reporter cells. Green
fluorescent protein was used as the reporter signal. Viruses are
harvested and titrated at maximum CPE. For recombination the
deletion construct pSV40HXB2D-IN was used. Recombination
experiments were performed with amplicon from wildtype HIV IIIB and
NL4.3, and patient sample 146514 generated by both primer sets. For
each recombination 2 .mu.g amplicon was co-transfected with 10
.mu.g SmaI-digested pSV40HXB2D-IN by electroporation into
MT4-LTR-EGFP cells. Virus stocks were titrated and tested in an
antiviral experiment on a reference panel including nucleoside
reverse transcriptase inhibitors (NRTI), non-nucleoside reverse
transcriptase inhibitors (NNRTI), protein inhibitor (PR), entry and
integrase (IN) inhibitors (Table 2).
[0046] Recombination was checked by nucleic acid sequence analysis
using protocols known to the person skilled in the art. Sequencing
primers which can be used are In_seq1F (SEQ ID NO 17), In_seq2F
(SEQ ID NO 18), In_seq3F (SEQ ID NO 19), IN_seq1R (SEQ ID NO 20),
IN_seq2R (SEQ ID NO 21), and IN_seq3R (SEQ ID NO 22). The
recombinant virus was evaluated in an anti-viral assay with a panel
of reference compounds including nucleoside RT inhibitors (NRTI)
Zidovudine (AZT) Lamivudine (3TC), Didanosine (DDI), non-nucleoside
RT inhibitors (NNRTI) Nevirapine (NVP),
4-[[6-amino-5-bromo-2-[(4-cyanophenyl)amino]-4-pyrimidinyl]oxy]-3,5-dimet-
hyl-benzonitrile also referred to as compound
1,4-[[6-amino-5-bromo-2-[(4-cyanophenyl)amino]-4-pyrimidinyl]oxy]-3,5-dim-
ethyl-Benzonitrile, also referred to as compound 2 protease
inhibitors (PR) Saquinavir (SQV), Amprenavir (APV), Indinavir
(IDV),
[(1S,2R)-3-[[(4-aminophenyl)sulfonyl](2-methylpropyl)amino]-2-hydroxy-1-(-
phenylmethyl)-propyl]-, (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl
ester carbamic acid also referred to as compound 3,
entry-inhibitors (Entry)(AMD3100, DS5000, ATA), and integrase
inhibitor (IN) 2-(1-methylethyloxy)-,
-dioxo-5-(phenylmethyl)-benzenebutanoic acid also referred to as
compound 4. The results are compiled in Table 2. AVE means
antiviral experiment. Type means the type of inhibitor
investigated. The fold change is the fold change in EC.sub.50. WT
III B means that a portion of the wild type IIIB strain has been
amplified and used in the antiviral experiment, including
transfection and generation of recombinant virus. NL 4-3 means the
integrase gene of this laboratory strain has been amplified and
subsequently used for the antiviral experiment. Patient 146514
means that the integrase gene of an HIV sample retrieved from said
patient has been amplified and used in the antiviral experiment.
pHXB2D has been used as a control. No recombination has been
effected using this HIV clone. pHXB2D has been used directly for
transfection and antiviral experiment. Primer set 3 consist of
outer primers 5'EGINT3 (SEQ ID NO 4) and 3'EGINT10 (SEQ ID NO 11),
and inner primers 5'EGINT4 (SEQ ID NO 5) and 3'EGINT6 (SEQ ID NO
7). Primer set 4 consist of outer primers 5'EGINT3 (SEQ ID NO 4)
and 3'EGINT10 (SEQ ID NO 11), and inner primers 5'EGINT5 (SEQ ID NO
5) and 3'EGINT6 (SEQ ID NO 7). Other suitable integrase inhibitors
include L-731,988, diketo-acids and S-1360.
[0047] The antiviral activity of these compounds against
recombinant virus from wildtype HIV-1 IIIB or NL4.3 was identical
to the activity against the HIV-1 IIIB and pHXB2D control strain,
where no recombination has been performed. Recombinant virus
generated from site-directed mutant virus gave a fold increase in
EC.sub.50 against compound 4 of respectively 2-fold (T66I
mutation), 5-fold (S153Y), 3-fold (M154I mutation), 10-fold
(T66I/S153Y mutations or T66I/M154I mutations). Recombinant virus
generated from patient samples without mutations in the integrase
coding sequence, displyed analogous results as the wildtype strains
in the antiviral assay. The panel of protease and reverse
transcriptase inhibitors were included in the list to prove that no
background resistance, expressed as a fold increase in EC.sub.50,
was detected. The reverse transcriptase and protease genes present
in the antiviral experiments were derived from wild type HIV
sequence, which does not confer resistance to the drugs included.
The instant results (Table 2) indicate that no change in
susceptibility for any of these compounds is found.
Table 2 Antiviral Experiment
EXAMPLE 2
Genotyping of Integrase
[0048] The methods and conditions used for sequence analysis of HIV
integrase gene are outlined below. The sequencing primers (cfr.
Table 1) cover the region of 864 nucleotides, from nucleotide 4230
until 5093 according to the sequence present in the HIV clone
HXB2D. The sequencing primers were diluted until 1 pmol/.mu.l and
used in the mix and conditions as described below. TABLE-US-00002
Reaction Mix Component Reaction Mix Big Dye Terminator Mix 4 .mu.l
2.5 Dilution Buffer 4 .mu.l Water 4.8 .mu.l Primer (1 pmol/.mu.l)
3.2 .mu.l Sample (200-500 ng/.mu.l) 4 .mu.l TOTAL 20 .mu.l
[0049] TABLE-US-00003 Thermal Cycle Conditions Initial Denaturation
3' on 96.degree. C. Denaturation 30'' on 96.degree. C. Annealing
15'' on 50.degree. C. {close oversize bracket} 30 cycles Elongation
4' on 60.degree. C. Hold on 4.degree. C.
After cycle sequencing the reaction products were purified and run
on the 3700 DNA analyzer.
EXAMPLE 3
Construction of a Recombinant IN Vector
A) Construction of pSV40HXB2D R-IN-vif
[0050] The pSV40HXB2D R-IN-vif vector has a deletion of the
complete IN coding sequence as well as part of the preceding RNase
and subsequent Vif coding sequences. It was constructed by PCR
amplification of pSV40HXB2D and religation of the amplified
fragment. The primers used for amplification were MUT IN1 (5' GGG
TGA CAA CTT TTT GTC TTC CTC TAT 3'; SEQ ID NO:13) and IN2 (5' GGA
TCC TGC AGC CCG GGA AAG CTA GGG GAT GGT TTT ATA GA 3'; SEQ ID
NO:23), which contain a SmaI site. Primer MUT IN1 (SEQ ID NO 13)
anneals to nucleotides 3954 to 3928, and primer IN2 (SEQ ID NO 23)
anneals to nucleotides 5137 to 5163. The first 14 nucleotides of
IN2 (SEQ ID NO 23) comprise the Sma I tail, which does not anneal
to the vector. The amplified product was cleaved with Sma I and
re-ligated to create pSV40HXB2D R-IN-vif; with a Sma I recognition
site at the location of the deletion,
B) Amplification of Patient Derived IN Sequences for Insertion into
pSV40HXB2D R-IN-vif
[0051] To amplify the complete IN coding region and the flanking
segments of the RNase and Vif coding regions for insertion into the
pSV40HXB2D R-IN-vif vector, a nested PCR method was used. The outer
primers were 5'EGINT1 (SEQ ID NO 1) and 3'EGINT10 (SEQ ID NO 11),
while the inner set was 5'EGINT107 (SEQ ID NO 2) and 3'EGINT11 (SEQ
ID NO 12). An additional inner 5' primer, 5'EGINT2(SEQ ID NO 3),
was used to improve the yield of amplified DNA. (The sequences of
these primers are given in Table 1, above.)
C) Construction of the pSV40HXB2D IN Vector
[0052] To create pSV40HXB2D IN, the pSV40HXB2D vector was PCR
amplified and re-ligated to effectively delete most of the IN
coding region, leaving the nucleotides coding for the N-terminal 8
amino acids and the C-terminal 20 amino acids in place. The
amplification was performed using the primers MUT IN3 (5' GGG CCT
TAT CTA TTC CAT CTA AAA ATA GT 3'; SEQ ID NO: 15) and MUT IN4 (5'
GGA TCC TGC AGC CCG GGA TTA TGG AAA ACA GAT GGC A 3'; SEQ ID
NO:16), containing a SmaI site. Primer MUT IN3 (SEQ ID NO 15)
anneals to nucleotides 4254 to 4226, and primer MUT IN4 (SEQ ID NO
16) anneals to nucleotides 5 create 036 to 5057. The resulting
amplified fragment can be cleaved with SmaI and re-ligated to
pSV40HXB2D IN.
D) Amplification of Patient Derived IN Sequences for Insertion into
pSV40HXB2D IN
[0053] Patient derived IN sequences was prepared for insertion into
the HIV deletion vector using a nested PCR approach as in part B
above. 5'EGINT3 (SEQ ID NO 4) and 3'EGINT10 (SEQ ID NO 11) were
used as outer PCR primers, while 5'EGINT4 (SEQ ID NO 5) or 5'3GINT5
(SEQ ID NO 6) and 3'EGINT6 (SEQ ID NO 7) were used as inner
primers. The sequences and SEQ ID NO 4-8 of these primers are given
in Table 1. The underlined portion of MUT IN4 (SEQ ID NO 16)
represents the SmaI tail that does not anneal to the vector.
E) Homologous Recombination and Ligation to Insert the PCR Products
into the Vectors.
[0054] The pSV40HXB2D IN or pSV40HXB2D.DELTA.R-IN-vif vectors was
linearized with SmaI. The vectors and the amplified IN DNA
fragments were transfected by electroporation into MT4 cells, MT4
cells equipped with a LTR reporter gene construct (MT4-rep) or PM-1
cells. By homologous recombination between overlapping portions of
the vector and IN amplicons, the HIV genome was reconstituted with
a patient derived IN coding region. The recombinant vectors were
capable of producing virus particles in infected cells. Virus
production was evaluated by scoring the cytopathogenic effect (CPE)
that was normally induced by HIV infection of MT4, MT4-rep, or PM-1
cells, or was evaluated by the induced LTR-driven reporter signal
in MT4-rep or PM-1 cells. Homologous recombination with wild type
IN sequences was used as a control.
[0055] The presence of recombinant IN DNA and RNA sequences in the
transfected cells was monitored by reverse transcription and PCR
analysis. The presence of PCR products corresponding to correctly
inserted IN sequences showed that recombination successfully
occurred and that viral RNA was produced in the cells.
[0056] Patient derived IN sequences and wild type controls were
alternatively inserted into SmaI-linearized pSV40HXB2D N or
pSV40HXB2D R-IN-vif vectors by a standard restriction digestion and
ligation procedure. The IN amplicons were modified to create SmaI
cleaved ends and were then inserted by ligation into the SmaI site
on the vectors.
EXAMPLE 4
Genotyping of Patient Derived IN Coding Sequences
A) Obtaining and Amplifying Patient Derived IN Sequences
[0057] RNA was isolated from 100 .mu.l of plasma according to the
method described by Boom et al. (1990), and reverse transcribed
with the GENEAMP.RTM. reverse transcriptase kit (Perkin Elmer) as
described by the manufacturer using an HIV-1 specific downstream
primer. Two subsequent nested PCRs were set up using specific outer
primers and inner primers, respectively, The outer primer reaction
were performed as described in WO97/27480 and U.S. Pat. No.
6,221,578 (which are incorporated herein by reference). The inner
amplification was performed in a 96 well plate as follows: 4 .mu.l
of the outer amplification product was diluted to a final volume of
50 .mu.l using a 10.times. amplification mix consisting of 5 .mu.l
10.times.PCR buffer containing 15 mM MgCl.sub.2, 1 .mu.l dNTP's (10
mM) 0.5 .mu.l each primer (0.25 .mu.g/ml), 0.4 .mu.l EXPAND.RTM.
High Fidelity polymerase (3.5 U/.mu.l; Roche) and deionized water.
Amplification was initiated after a short denaturation of the
amplification product made using the outer primers (2 min at
94.degree. C.). Ten amplification cycles were run, each consisting
of a 15 sec denaturation step at 94.degree. C., a 30 sec annealing
step at 60.degree. C. and a 2 min polymerization step at 72.degree.
C. This amplification was immediately followed by 25 cycles
consisting of a 15 sec denaturation step at 94.degree. C., a 30 sec
annealing step at 60.degree. C. and a variable time polymerization
step at 72.degree. C. The polymerization step was initially run for
2 min and 5 sec, then was increased by 5 seconds in each cycle.
Amplification was completed by an additional polymerization step of
7 min at 72.degree. C. The reactions were held at 4.degree. C.
until further analysis or stored at -20.degree. C. (for short
periods) or -70.degree. C. (for longer periods). The products can
be analysed on DNA agarose gels and visualised by UV-detection. The
products can be purified using the QIAQUICK.RTM. 96-well plate
system as described by the manufacturer (Qiagen).
B. Sequencing of IN Coding Region
[0058] The IN coding region present on the amplified fragments were
sequenced using techniques known in the art. The sequencing was
started by first distributing 4 .mu.l of the primer stocks (4.0
.mu.M) over a 96 well plate where each stock was pipetted down the
column. In a second step, master mixes were made consisting of 14
.mu.l deionized water, 17.5 .mu.l dilution buffer, 7 .mu.l sample
(PCR fragment) and 14 .mu.l Big Dye.TM. Terminator Mix (Perkin
Elmer). A fraction (7.5 .mu.l) of each master mix, containing a
specific PCR fragment, was transferred to a specific place into the
96 well plate so that each sample fraction was mixed with a
different PCR primer set. Samples were pipetted across the rows.
Samples were placed in a thermal cycler and sequencing cycles
started. The sequencing reaction consisted of 25 repetitive cycles
of 10 sec at 96.degree. C., 5 sec at 50.degree. C. and 4 min at
60.degree. C., respectively. Finally, sequence reactions were be
held at 4.degree. C. or frozen until further analysis. The
sequencing reactions were precipitated using a standard ethanol
precipitation procedure, resuspended in 2 .mu.l formamide and
heated for 2 minutes at 92.degree. C. in the thermal cycler.
Samples were cooled on ice until ready to load. 1 .mu.l of each
reaction was loaded on a 4.25% vertical acrylamide gel in a 377
sequencer system and gel was run until separation of the fragments
is complete.
C. Sequence Analysis of IN Coding Region
[0059] Sample sequences wer imported as a specific project into the
sequence manager of Sequencher.TM. (Genecodes) and compared to the
wild type reference sequence. Sequences were assembled
automatically and set at 85% minimum match. Secondary peaks were
searched and the minimum was set at 60%. Any sequence that extended
beyond the 5' end or the 3' end of the reference were deleted. When
a region of overlap between sequences from the same strand was
reached, the poorest quality of sequence was deleted leaving an
overlap of 5-10 bases. Ambiguous base calls were considered poor
matches to exact base calls. The sequence assembly was saved within
an editable contig.
[0060] Obtained sequences were edited to facilitate interpretation
of the base calls. Ambiguous sequences were retrieved and checked
for possible errors or points of heterogeneity. When the point of
ambiguity appeared correct (both strands of sequence agreed but
were different from the reference sequence) it was interpreted to
be a variant. The reference sequence was used as an aid for
building a contig and as a guide to overall size and for trimming.
The reference sequence was not used for deciding base calls. A
change was only made when both strands agreed. All gaps were
deleted or filled, unless they occurred in contiguous groups of
multiples of three (i.e., insertion or deletion of complete codons)
based on data form both sequence strands. Once the editing was
complete, the new contig sequence was saved as a consensus sequence
and used for further analysis.
[0061] Detailed sequence editing was performed following certain
rules: A) Applied Biosystems, Inc. primer blobs were trimmed at 5'
ends where 1 consecutive base remained off the scale, the sequence
was trimmed not more than 25% until the first 25 bases contained
less than 1 ambiguity, at least the first 10 bases from the 5' end
were removed, and B) 3' ends were trimmed starting 300 bases after
the 5' trim, the first 25 bases containing more than 2 ambiguities
were removed, the 3' end was trimmed until the last 25 bases
contained less than 1 ambiguity. The maximum length of the obtained
sequence fragment after trimming was 550 bases.
[0062] Sequences that failed to align were removed from the
assembly and replaced by data retrieved from new sequence analyses.
When further failures occur, PCR reactions were repeated.
Chromatograms were visualised using an IBM software system.
LEGENDS TO THE FIGURES
[0063] FIG. 1: Overview of the HIV genome indicating the primer
positions
Sequence CWU 1
1
23 1 24 DNA Human immunodeficiency virus 1 ggtaccagtt agagaaagaa
ccca 24 2 25 DNA Human immunodeficiency virus 2 ggagcagaaa
ccttctatgt agatg 25 3 21 DNA Human immunodeficiency virus 3
ggcagctaac agggagacta a 21 4 23 DNA Human immunodeficiency virus 4
ggaatcattc aagcacaacc aga 23 5 21 DNA Human immunodeficiency virus
5 tctggcatgg gtaccagcac a 21 6 25 DNA Human immunodeficiency virus
6 aggaattgga ggaaatgaac aagta 25 7 21 DNA Human immunodeficiency
virus 7 gttctaatcc tcatcctgtc t 21 8 24 DNA Human immunodeficiency
virus 8 cctccattct atggagtgtc tata 24 9 24 DNA Human
immunodeficiency virus 9 gggtctactt gtgtgctata tctc 24 10 23 DNA
Human immunodeficiency virus 10 cagatgaatt agttggtctg cta 23 11 24
DNA Human immunodeficiency virus 11 cctccattct atggagactc cctg 24
12 20 DNA Human immunodeficiency virus 12 gcatccccta gtgggatgtg 20
13 27 DNA Human immunodeficiency virus 13 gggtgacaac tttttgtctt
cctctat 27 14 39 DNA Human immunodeficiency virus 14 ggatcctgca
gcccgggaaa gctaggggat ggttttata 39 15 29 DNA Human immunodeficiency
virus 15 gggccttatc tattccatct aaaaatagt 29 16 37 DNA Human
immunodeficiency virus 16 ggatcctgca gcccgggatt atggaaaaca gatggca
37 17 19 DNA Human immunodeficiency virus 17 agtcagtgct ggaatcagg
19 18 20 DNA Human immunodeficiency virus 18 ttccagcaga aacagggcag
20 19 21 DNA Human immunodeficiency virus 19 gtagacataa tagcaacaga
c 21 20 21 DNA Human immunodeficiency virus 20 ccctgaaaca
tacatatggt g 21 21 20 DNA Human immunodeficiency virus 21
ctgccatttg tactgctgtc 20 22 20 DNA Human immunodeficiency virus 22
tgaactgcta ccaggataac 20 23 41 DNA Human immunodeficiency virus 23
ggatcctgca gcccgggaaa gctaggggat ggttttatag a 41
* * * * *