Margaret Tisdale

Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement.

Abstract Background: The antiviral effect of anti-influenza drugs such as zanamivir may be demonstrated in patients as an increased rate of decline in viral load over a time course of treatment as compared with placebo. Historically this was measured using plaque assays, or Culture Enhanced Enzyme Linked Immunosorbent Assay (CE-ELISA). Objectives: to develop and characterise real time quantitative PCR (qPCR) assays to measure influenza A and B viral load in clinical samples, that offer improvements over existing methods, in particular virus infectivity assays. Study design: The dynamic range and robustness were established for the real time qPCR assays along with stability of the assay components. Cross validation of the real time PCR assays with CE-ELISA was performed by parallel testing of both serial dilutions of three different subtypes of cultured virus and a panel of influenza positive throat swab specimens. Results: the assays were specific for influenza A and B and the dynamic ranges were at least seven logs. The assay variability was within acceptable limits but increased towards the lower limit of quantification, which was 3.33 log10 viral cDNA copies/ml of virus transport medium (ten viral RNA copies/PCR). The components of the assay were robust enough to withstand extended storage and several freeze–thaw cycles. For the real time PCR assays the limit of quantification was equivalent to the virus infectivity cut off, which equates to a 93-fold increase in sensitivity. Conclusion: Well characterised real time PCR assays offer significant improvements over the existing methods for measuring the viral load of strains of influenza A and B in clinical specimens.

Changes in human immunodeficiency virus type 1 Gag at positions L449 and P453 are linked to I50V protease mutants in vivo and cause reduction of sensitivity to amprenavir and improved viral fitness in vitro.

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Gag protease cleavage sites (CS) undergo sequence changes during the development of resistance to several protease inhibitors (PIs). We have analyzed the association of sequence variation at the p7/p1 and p1/p6 CS in conjunction with amprenavir (APV)-specific protease mutations following PI combination therapy with APV. Querying a central resistance data repository resulted in the detection of significant associations (P < 0.001) between the presence of APV protease signature mutations and Gag L449F (p1/p6 LP1′F) and P453L (p1/p6 PP5′L) CS changes. In population-based sequence analyses the I50V mutant was invariably linked to either L449F or P453L. Clonal analysis revealed that both CS mutations were never present in the same genome. Sequential plasma samples from one patient revealed a transition from I50V M46L P453L viruses at early time points to I50V M46I L449F viruses in later samples. Various combinations of the protease and Gag mutations were introduced into the HXB2 laboratory strain of HIV-1. In both single- and multiple-cycle assay systems and in the context of I50V, the L449F and P453L changes consistently increased the 50% inhibitory concentration of APV, while the CS changes alone had no measurable effect on inhibitor sensitivity. The decreased in vitro fitness of the I50V mutant was only partially improved by addition of either CS change (I50V M46I L449F mutant replicative capacity ≈ 16% of that of wild-type virus). Western blot analysis of Pr55 Gag precursor cleavage products from infected-cell cultures indicated accumulation of uncleaved Gag p1-p6 in all I50V viruses without coexisting CS changes. Purified I50V protease catalyzed cleavage of decapeptides incorporating the L449F or P453L change 10-fold and 22-fold more efficiently than cleavage of the wild-type substrate, respectively. HIV-1 protease CS changes are selected during PI therapy and can have effects on both viral fitness and phenotypic resistance to PIs.

Resistance Profile of the Human Immunodeficiency Virus Type 1 Reverse Transcriptase Inhibitor Abacavir (1592U89) after Monotherapy and Combination Therapy

Abacavir (1592U89) is a nucleoside inhibitor of human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT). Resistance to abacavir was studied with abacavir alone and with abacavir in combination with other nucleoside analogues in cell culture, in virus isolates from zidovudine/lamivudine clinical trials, and in the first dose-escalating 12-week clinical trial (CNA2001) to evaluate abacavir clinical potency. Abacavir alone in vitro selected for mutations at HIV RT codons K65R, L74V, Y115F, and M184V. However, abacavir combined with zidovudine selected against virus with the M184V mutation. Abacavir therapy in vivo resulted in large decreases in HIV load (>1 log), even in 1 subject who had the M184V mutation at baseline. A total of 51% of subjects showed new mutations at any of codons K65R, L74V, and M184V after abacavir monotherapy, compared with 11% who received zidovudine/abacavir. Small changes (2- to 4-fold) in abacavir susceptibility were detected. On stopping therapy, reselection of the pretherapy sequence occurred within 4 weeks.

HIV-1 reverse transcriptase (RT) genotype and susceptibility to RT inhibitors during abacavir monotherapy and combination therapy.

ObjectiveTo examine changes in HIV-1 susceptibility (genotype and phenotype) during an initial abacavir monotherapy phase followed by the addition of zidovudine and lamivudine. DesignSixty HIV-1 infected, antiretroviral therapy-na]ve subjects were randomized to receive 100, 300 or 600 mg abacavir twice daily. Subjects completing 24 weeks of randomized therapy or meeting a protocol defined switch criterion could switch to open label abacavir/zidovudine/lamivudine. MethodsPlasma HIV-1 reverse transcriptase was genotyped at baseline, week 12, and at the last time point on ABC monotherapy. Drug susceptibility was analysed at baseline and on subsequent samples with sufficient HIV-1 RNA levels using the recombinant virus assay. Virological responses (week 24) were correlated to week 24 genotypes. ResultsMutant viruses were not detected before week 12 with the exception of one subject. At the latest time point on abacavir monotherapy (range, weeks 6–48), 21 out of 43 subjects harboured virus with resistance conferring mutations including single, double and triple combinations of K65R, L74V, Y115F and M184V. The most common mutational pattern was L74V + M184V (11/21 cases). Twenty of the 21 subjects with isolates containing abacavir-associated mutations reached week 48, and upon addition of lamivudine/zidovudiine, 16 out of 20 (80%) had week 48 plasma HIV-1-RNA below 400 copies/ml. At week 48, 16 out of 46 genotypes were obtained; one of these was wild-type; 15 contained M184V either alone, in combination with K65R and/or L74V and/or Y115F or with thymidine analogue-associated mutations. Week 48 viral load levels for these 15 subjects was low (median 3.43 log10 copies/ml or −1.99 log10 copies reduction from baseline). Genotype correlated well with phenotypic resistance to ABC; four samples with three abacavir-associated mutations had high level abacavir resistance (> 8-fold) and six samples with two or three mutations showed intermediate (4–8-fold) resistance. All samples with single mutations retained full ABC susceptibility. ConclusionsResistance conferring mutations to abacavir were relatively slow to develop during the monotherapy phase, and did not preclude durable efficacy of abacavir/lamivudine/zidovudine up to 48 weeks.

Zanamivir Susceptibility Monitoring and Characterization of Influenza Virus Clinical Isolates Obtained during Phase II Clinical Efficacy Studies

ABSTRACT Zanamivir is a highly selective neuraminidase (NA) inhibitor with demonstrated clinical efficacy against influenza A and B virus infections. In phase II clinical efficacy trials (NAIB2005 and NAIB2008), virological substudies showed mean reductions in virus shedding after 24 h of treatment of 1.5 to 2.0 log1050% tissue culture infective doses compared to a placebo, with no reemergence of virus after the completion of therapy. Paired isolates (n = 41) obtained before and during therapy with zanamivir demonstrated no shifts in susceptibility to zanamivir when measured by NA assays, although for a few isolates NA activity was too low to evaluate. In plaque reduction assays in MDCK cells, the susceptibility of isolates to zanamivir was extremely variable even at baseline and did not correlate with the speed of resolution of virus shedding. Isolates with apparent limited susceptibility to zanamivir by plaque reduction proved highly susceptible in vivo in the ferret model. Further sequence analysis of paired isolates revealed no changes in the hemagglutinin and NA genes in the majority of isolates. The few changes observed were all natural variants. No amino acid changes that had previously been identified in vitro as being involved with reduced susceptibility to zanamivir were observed. These studies highlighted problems associated with monitoring susceptibility to NA inhibitors in the clinic, in that no reliable cell-based assay is available. At present the NA assay is the best available predictor of susceptibility to NA inhibitors in vivo, as measured in the validated ferret model of infection.

Emergence of Resistance to Protease Inhibitor Amprenavir in Human Immunodeficiency Virus Type 1-Infected Patients: Selection of Four Alternative Viral Protease Genotypes and Influence of Viral Susceptibility to Coadministered Reverse Transcriptase Nucleoside Inhibitors

ABSTRACT Previous data have indicated that the development of resistance to amprenavir, an inhibitor of the human immunodeficiency virus type 1 protease, is associated with the substitution of valine for isoleucine at residue 50 (I50V) in the viral protease. We present further findings from retrospective genotypic and phenotypic analyses of plasma samples from protease inhibitor-naïve and nucleoside reverse transcriptase inhibitor (NRTI)-experienced patients who experienced virological failure while participating in a clinical trial where they had been randomized to receive either amprenavir or indinavir in combination with NRTIs. Paired baseline and on-therapy isolates from 31 of 48 (65%) amprenavir-treated patients analyzed demonstrated the selection of protease mutations. These mutations fell into four distinct categories, characterized by the presence of either I50V, I54L/I54M, I84V, or V32I+I47V and often included accessory mutations, commonly M46I/L. The I50V and I84V genotypes displayed the greatest reductions in susceptibility to amprenavir, although each of the amprenavir-selected genotypes conferred little or no cross-resistance to other protease inhibitors. There was a significant association, for both amprenavir and indinavir, between preexisting baseline resistance to NRTIs subsequently received during the study and development of protease mutations (P = 0.014 and P = 0.031, respectively). Our data provide a comprehensive analysis of the mechanisms by which amprenavir resistance develops during clinical use and present evidence that resistance to concomitant agents in the treatment regimen predisposes to the development of mutations associated with protease inhibitor resistance and treatment failure.

Absence of zidovudine resistance in antiretroviral-naive patients following zidovudine/lamivudine/protease inhibitor combination therapy: virological evaluation of the AVANTI 2 and AVANTI 3 studies.

ObjectivesTo assess the role of resistance mutations in subjects experiencing virological failure on zidovudine (ZDV) and lamivudine (3TC) combined with a protease inhibitor (PI) to those failing on ZDV/3TC alone. Design and methodsSamples were obtained from previously antiretroviral therapy-naive subjects enrolled into two studies, AVANTI 2 and AVANTI 3. Subjects were randomized to receive either: ZDV/3TC or ZDV/3TC plus indinavir (IDV) for 52 weeks (AVANTI 2), and ZDV/3TC or ZDV/3TC and nelfinavir (NFV) for 28 weeks (AVANTI 3). Emergence of viral resistance mutations was monitored by population sequencing and phenotypic resistance was determined by the recombinant virus assay. ResultsGenotypic data were obtained for subjects with plasma HIV-1 RNA > 400 copies/ml. In AVANTI 2, ZDV mutations were detected in 27% of ZDV/3TC-treated patients at week 52, but were absent in subjects treated with ZDV/3TC/IDV. No subjects from either arm of AVANTI 3 developed ZDV resistance mutations at week 28. The M184V mutation developed in most ZDV/3TC-treated subjects from both studies. The presence of M184V was, however, associated with significantly lower plasma viral RNA levels when compared with values obtained before initiation of treatment. There was a high frequency (4 of 11) of the protease L10F substitution in ZDV/3TC/IDV-treated patients that was associated with virological failure but did not result in phenotypic resistance to any of the PIs tested. ConclusionsZDV mutations were not detected in ZDV/3TC/PI-treated patients and they developed slowly in those treated with ZDV/3TC. Few protease mutations known to confer phenotypic PI resistance developed in the ZDV/3TC/PI arms of either study. The low prevalence of ZDV and PI mutations is encouraging regarding the future treatment options of these patients.

HIV type 1 protease cleavage site mutations and viral fitness : Implications for drug susceptibility phenotyping assays

The recombinant virus assay (RVA) is a method for assessing the susceptibility of human immunodeficiency virus type 1 (HIV-1) plasma isolates to antiretroviral drugs. The RVA involves the production of viable virus in vitro by homologous recombination of RT-PCR products from plasma virus with a noninfectious reverse transcriptase (RT) or protease (PR)-deleted cloned HIV-1 provirus. In this study, we have constructed RVA plasmids with contiguous deletions in RT, PR, and the p7/p1 and p1/6 gag protease cleavage sites (CS). The deletions in these plasmids allow generation of recombinant viruses with all loci currently identified as important for resistance to anti-HIV-1 drugs being derived from the clinical isolate, including CS mutations that compensate for the reduced fitness of viruses resistant to protease inhibitors (Doyon et al., J Virol 1996:70:3763-3769). We have also used these new constructs to generate viruses with or without compensatory CS mutations, and examined the effects on fitness. In the case of an indinavir-selected virus, fitness was restored close to that of a wild type virus when a vector deleted in the CS and PR was used. With an amprenavir-selected isolate, virus fitness was incompletely restored by including the CS, and this defect appeared to be partially due to reduced infectivity of the virions. We conclude that the CS mutations were required for optimum detection of resistance in the RVA, but that virus fitness can remain compromised even in the presence of compensatory CS mutations.

Efficacy of zanamivir against avian influenza A viruses that possess genes encoding H5N1 internal proteins and are pathogenic in mammals.

ABSTRACT In 1997, an avian H5N1 influenza virus, A/Hong Kong/156/97 (A/HK/156/97), caused six deaths in Hong Kong, and in 1999, an avian H9N2 influenza virus infected two children in Hong Kong. These viruses and a third avian virus [A/Teal/HK/W312/97 (H6N1)] have six highly related genes encoding internal proteins. Additionally, A/Chicken/HK/G9/97 (H9N2) virus has PB1 and PB2 genes that are highly related to those of A/HK/156/97 (H5N1), A/Teal/HK/W312/97 (H6N1), and A/Quail/HK/G1/97 (H9N2) viruses. Because of their similarities with the H5N1 virus, these H6N1 and H9N2 viruses may have the potential for interspecies transmission. We demonstrate that these H6N1 and H9N2 viruses are pathogenic in mice but that their pathogenicities are less than that of A/HK/156/97 (H5N1). Unadapted virus replicated in lungs, but only A/HK/156/97 (H5N1) was found in the brain. After three passages (P3) in mouse lungs, the pathogenicity of the viruses increased, with both A/Teal/HK/W312/97 (H6N1) (P3) and A/Quail/HK/G1/97 (H9N2) (P3) viruses being found in the brain. The neuraminidase inhibitor zanamivir inhibited viral replication in Madin-Darby canine kidney cells in virus yield assays (50% effective concentration, 8.5 to 14.0 μM) and inhibited viral neuraminidase activity (50% inhibitory concentration, 5 to 10 nM). Twice daily intranasal administration of zanamivir (50 and 100 mg/kg of body weight) completely protected infected mice from death. At a dose of 10 mg/kg, zanamivir completely protected mice from infection with H9N2 viruses and increased the mean survival day and the number of survivors infected with H6N1 and H5N1 viruses. Zanamivir, at all doses tested, significantly reduced the virus titers in the lungs and completely blocked the spread of virus to the brain. Thus, zanamivir is efficacious in treating avian influenza viruses that can be transmitted to mammals.

M184V is associated with a low incidence of thymidine analogue mutations and low phenotypic resistance to zidovudine and stavudine

The resistance of HIV clinical isolates with or without M184V was analysed in relation to plasma HIV-1-RNA level and time on therapy. The number of thymidine analogue mutations (TAMs) was lower in isolates with M184V, this was independent of plasma HIV-1-RNA level and time on therapy for T215F/Y, D67N and L210W. This suggests a direct effect of M184V on the reduced selection of TAMs. Lamivudine use was significantly associated with lower median fold resistance to zidovudine and stavudine.