clotet.qxp 28/1/09 4:43 pm Page 56
HIV and AIDS
Table 5: Principles of the Commercial Phenotypic Assays
Assay Characteristics
Antivirogram
®
(Virco BVBA) Recombinant viruses containing the patient insert are transfected into MT4 cells. Cell cultures are monitored for the appearance of
cytopathic effect (CPE). Infectivity is determined by the viral CPE assay by using a 50% end-point method (50% cell culture infectious
dose). HIV-1 drug susceptibility is determined by -3(4,5-dimethylthiazol-2-yl)-2,5-dyphenyltetrazolium bromide (MTT) (MT4-MTT)-
based CPE protection assay. MT4 cells are infected with 50% cell culture infective doses of recombinant viruses in the presence of
five-fold dilutions of the different antiretroviral drugs. In general, the wildtype HXB2 viruses are tested in parallel with clinical
samples for each assay. Fold-resistance values are calculated by dividing the mean IC
50
of the patient-derived recombinant virus by
the mean IC
50
of the wildtype control.
PhenoSense™ (Monogram Biosciences) The genes of interest are amplified from HIV sequence pools and incorporated into an indicator gene viral vector (IGVV) by conventional
cloning methods using ApaI and PinAI restriction sites to construct a resistance test vector (RTV). Host cells are co-transfected with RTV
DNA and a plasmid that expresses the envelope protein of amphotropic murine leukaemia virus (MLV). Following transfection, virus
particles are harvested and used to infect fresh target cells. The completion of a single cycle of viral replication results in the production
of luciferase. Serial dilutions of PIs are added at the transfection step and RT inhibitors at the infection step. For the measurement of
susceptibility to entry inhibitors (EIs), indicator cells expressing CCR-5 or CXCR-4 co-receptors are treated with serial dilutions of drugs
and infected with recombinant viruses harvested from the producer cell. Drug susceptibility is measured by comparing the luciferase
activity in the presence and absence of drugs. Susceptible viruses result in decreased levels of luciferase activity in the presence of
drugs, whereas viruses with reduced susceptibility produce comparable levels to the wild-type control.
PhenoScript™ (Viralliance) The Phenoscript is based on a single cycle of in vitro replication and measures viral capacity of replication in the presence of drugs.
Plasma is obtained from the patient’s blood sample. Viral RNA is extracted and three regions – gag-protease (GP), reverse-
transcriptase (RT) and envelope (ENV) – are separately amplified to test PIs, RTIs and EIs, respectively. Each PCR product is then
separately co-transfected into producer cells along with the corresponding PHENOSCRIPT™ plasmid. For the PI and RTI assays, the
single cycle of infection is ensured by the deletion of the envelope-encoding region of the HIV plasmid. The envelope of the
recombinant virus is provided by the G protein of the vesicular stomitis virus (VSV-G protein), for which the genetic information is
carried on a separate plasmid. Serial dilutions of PIs are added at the transfection step and RT inhibitors at the infection step. For the
measurement of susceptibility to EIs, indicator cells expressing CCR-5 or CXCR-4 co-receptors are treated with serial dilutions of
drugs and infected with recombinant viruses harvested from the producer cell. The reporter cells used contain a LacZ gene under
control of the HIV LTR. Once cells are infected, b-galacosidase is produced, the amount of which is detected using a CPRG-based
colorimetric assay and measured by optical density.
Figure 1: Phenotypic Inhibition Curves or Resistant Viruses
(Virco
®
TYPE HIV-1, Virco BVBA; Geno2Pheno, www.geno2pheno.org)
correlates genotypic data obtained from the plasma HIV-1 RNA of a
candidate gene with a large database of paired phenotypes and
genotypes.
5–8
Such linkage assigns calculated fold-changes in IC
50
to
query genotypes. Virtual and actual phenotypes show good
correlation for most drugs. However, the superiority of the virtual
phenotype over genotype alone could not be demonstrated in
predicting clinical response to salvage regimens.
7,9,10
The main
r
c
ent inhibition
r
c
ent inhibition
Pe Pe
limitation of virtual phenotype is that its predictive power depends on
the number of matched data sets available. Thus, variation is
frequently higher in smaller data sets, as well as for newer drugs or
Drug concentration (log scale) Drug concentration (log scale)
complex resistant patterns. Moreover, matches are based on pre-
The left panel shows a typical inhibition curve of a susceptible virus (solid line) with a typical
selected codons, not on the entire nucleotide sequence, and most
competitive inhibitor (e.g. a protease inhibitor). The IC50 value of the resistant virus (dotted
genotype–phenotype pairs in the database were obtained on subtype
line) is shifted to the right (arrow). The right panel shows an example of a non-competitive
inhibitor (e.g. a chemokine receptor 5 antagonist). The susceptible virus (solid line) shows a
B viruses. This warrants caution when inferring phenotypes from non-
typical inhibition curve, but in this case the resistant virus (dotted line) reaches a plateau. The
B-subtype genotypic data using virtual phenotype.
maximum achievable per cent inhibition is shifted downwards (arrow), but the curve does
not shift to the right; hence, the IC50 value remains unchanged. Source: Hirsch et al., 2008.
1
Viral Tropism Assays
a virus to replicate modifies therapy outcomes in a clinically The incorporation of CCR-5 antagonists into clinical practice has renewed
meaningful manner. Moreover, the overall RC of a virus in vivo is interest in viral tropism assays. Co-receptor tropism determination is
determined not only by the genomic regions studied, but also by mandatory before initiating CCR-5 antagonist therapy. Most subjects failing
epistatic effects of other viral genes and complex interactions with the CCR-5 antagonist therapy show rebounds of X4-using HIV-1 at the time of
other viral variants in the quasispecies and between the quasispecies virological failure. Switches in co-receptor use from CCR-5 to CXCR-4 have
as a whole and the particular environment in which viruses replicate. been associated with accelerated CD4+ count decay and an increased risk
Current guidelines for resistance testing in HIV-infected subjects do of AIDS-defining diseases and death. Several studies have found that most
not recommend using RC measurements for guiding ART choices. X4-using viruses emerging at the time of CCR-5 antagonist failure were
already present before therapy as minority species and went undetected by
Virtual Phenotype standard tropism assays. Therefore, the presence of low levels of X4 virus is
The virtual phenotype is an alternative approach to interpreting a challenge to all assay methods, resulting in reduced sensitivity in clinical
genotypic drug resistance information. The virtual phenotype patient-derived samples compared with clonally derived samples.
56 EUROPEAN INFECTIOUS DISEASE
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