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MARK C. EVANS,
AGNES C. PAQUET,
WEI HUANG,
LAURA NAPOLITANO,
ARNE FRANTZELL,
JONATHAN TOMA,
ERIC W. STAWISKI,
MATTHEW BIDWELL GOETZ, CHRISTOS J. PETROPOULOS,
JEANNETTE WHITCOMB,
EOIN COAKLEY,
MOJGAN HADDAD
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ABSTRACT: Accurate co-receptor tropism (CRT) determination is critical for making treatment decisions in HIV management. We created a genotypic tropism prediction tool by utilizing the case-based reasoning (CBR) technique that attempts to solve new problems through applying the solution from similar past problems. V3 loop sequences from 732 clinical samples with diverse characteristics were used to build a case library. Additional sequence and molecular properties of the V3 loop were examined and used for similarity assessment. A similarity metric was defined based on each attribute's frequency in the CXCR4-using viruses. We implemented three other genotype-based tropism predictors, support vector machines (SVM), position specific scoring matrices (PSSM), and the 11/25 rule, and evaluated their performance as the ability to predict CRT compared to Monogram's enhanced sensitivity Trofile® assay (ESTA). Overall concordance of the CBR based tropism prediction algorithm was 81%, as compared to ESTA. Sensitivity to detect CXCR4 usage was 90% and specificity was at 73%. In comparison, sensitivity of the SVM, PSSM, and the 11/25 rule were 85%, 81%, and 36% respectively while achieving a specificity of 90% by SVM, 75% by PSSM, and 97% by the 11/25 rule. When we evaluated these predictors in an unseen dataset, higher sensitivity was achieved by the CBR algorithm (87%), compared to SVM (82%), PSSM (76%), and the 11/25 rule (33%), while maintaining similar level of specificity. Overall this study suggests that CBR can be utilized as a genotypic tropism prediction tool, and can achieve improved performance in independent datasets compared to model or rule based methods.
Keywords: Tropism prediction; genotypic algorithm; HIV-1 co-receptor; V3
Read More: http://www.worldscientific.com/doi/abs/10.1142/S0219720013500066
Journal of Bioinformatics and Computational Biology 04/2013;
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ABSTRACT: Mutations at amino acids 143, 148, and 155 in HIV-1 integrase (IN) define primary resistance pathways in subjects failing raltegravir (RAL)-containing treatments. Although each pathway appears to be genetically distinct, shifts in the predominant resistant virus population have been reported under continued drug pressure. To better understand this dynamic, we characterized the RAL susceptibility of 200 resistant viruses, and we performed sequential clonal analysis for selected cases. Patient viruses containing Y143R, Q148R, or Q148H mutations consistently exhibited larger reductions in RAL susceptibility than patient viruses containing N155H mutations. Sequential analyses of virus populations from three subjects revealed temporal shifts in subpopulations representing N155H, Y143R, or Q148H escape pathways. Evaluation of molecular clones isolated from different time points demonstrated that Y143R and Q148H variants exhibited larger reductions in RAL susceptibility and higher IN-mediated replication capacity (RC) than N155H variants within the same subject. Furthermore, shifts from the N155H pathway to either the Q148R or H pathway or the Y143R pathway were dependent on the amino acid substitution at position 148 and the secondary mutations in Y143R- or Q148R- or H-containing variants and correlated with reductions in RAL susceptibility and restorations in RC. Our observations in patient viruses were confirmed by analyzing site-directed mutations. In summary, viruses that acquire mutations defining the 143 or 148 escape pathways are less susceptible to RAL and exhibit greater RC than viruses containing 155 pathway mutations. These selective pressures result in the displacement of N155H variants by 143 or 148 variants under continued drug exposure.
Journal of Virology 05/2012; 86(13):7249-55. · 5.40 Impact Factor
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ABSTRACT: Human immunodeficiency virus (HIV-1) is, like most pathogens, under selective pressure to escape the immune system of its host. In particular, HIV-1 can avoid recognition by cytotoxic T lymphocytes (CTLs) by altering the binding affinity of viral peptides to human leukocyte antigen (HLA) molecules, the role of which is to present those peptides to the immune system. It is generally assumed that HLA escape mutations carry a replicative fitness cost, but these costs have not been quantified. In this study, we assess the replicative cost of mutations which are likely to escape presentation by HLA molecules in the region of HIV-1 protease and reverse transcriptase. Specifically, we combine computational approaches for prediction of in vitro replicative fitness and peptide binding affinity to HLA molecules. We find that mutations which impair binding to HLA-A molecules tend to have lower in vitro replicative fitness than mutations which do not impair binding to HLA-A molecules, suggesting that HLA-A escape mutations carry higher fitness costs than non-escape mutations. We argue that the association between fitness and HLA-A binding impairment is probably due to an intrinsic cost of escape from HLA-A molecules, and these costs are particularly strong for HLA-A alleles associated with efficient virus control. Counter-intuitively, we do not observe a significant effect in the case of HLA-B, but, as discussed, this does not argue against the relevance of HLA-B in virus control. Overall, this article points to the intriguing possibility that HLA-A molecules preferentially target more conserved regions of HIV-1, emphasizing the importance of HLA-A genes in the evolution of HIV-1 and RNA viruses in general.
PLoS Computational Biology 05/2012; 8(5):e1002525. · 5.22 Impact Factor
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Roger D Kouyos,
Viktor von Wyl,
Trevor Hinkley, Christos J Petropoulos,
Mojgan Haddad,
Jeannette M Whitcomb,
Jürg Böni,
Sabine Yerly,
Cristina Cellerai,
Thomas Klimkait,
Huldrych F Günthard,
Sebastian Bonhoeffer
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ABSTRACT: HIV-1 replicative capacity (RC) provides a measure of within-host fitness and is determined in the context of phenotypic drug resistance testing. However it is unclear how these in-vitro measurements relate to in-vivo processes. Here we assess RCs in a clinical setting by combining a previously published machine-learning tool, which predicts RC values from partial pol sequences with genotypic and clinical data from the Swiss HIV Cohort Study. The machine-learning tool is based on a training set consisting of 65000 RC measurements paired with their corresponding partial pol sequences. We find that predicted RC values (pRCs) correlate significantly with the virus load measured in 2073 infected but drug naïve individuals. Furthermore, we find that, for 53 pairs of sequences, each pair sampled in the same infected individual, the pRC was significantly higher for the sequence sampled later in the infection and that the increase in pRC was also significantly correlated with the increase in plasma viral load and with the length of the time-interval between the sampling points. These findings indicate that selection within a patient favors the evolution of higher replicative capacities and that these in-vitro fitness measures are indicative of in-vivo HIV virus load.
PLoS Pathogens 11/2011; 7(11):e1002321. · 9.13 Impact Factor
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Evelien M Bunnik,
Luke C Swenson,
Diana Edo-Matas,
Wei Huang,
Winnie Dong,
Arne Frantzell, Christos J Petropoulos,
Eoin Coakley,
Hanneke Schuitemaker,
P Richard Harrigan,
Angélique B van 't Wout
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ABSTRACT: The emergence of CXCR4-using human immunodeficiency virus type 1 (HIV-1) variants is associated with accelerated disease progression. CXCR4-using variants are believed to evolve from CCR5-using variants, but due to the extremely low frequency at which transitional intermediate variants are often present, the kinetics and mutational pathways involved in this process have been difficult to study and are therefore poorly understood. Here, we used ultra-deep sequencing of the V3 loop of the viral envelope in combination with the V3-based coreceptor prediction tools PSSM(NSI/SI) and geno2pheno([coreceptor]) to detect HIV-1 variants during the transition from CCR5- to CXCR4-usage. We analyzed PBMC and serum samples obtained from eight HIV-1-infected individuals at three-month intervals up to one year prior to the first phenotypic detection of CXCR4-using variants in the MT-2 assay. Between 3,482 and 10,521 reads were generated from each sample. In all individuals, V3 sequences of predicted CXCR4-using HIV-1 were detected at least three months prior to phenotypic detection of CXCR4-using variants in the MT-2 assay. Subsequent analysis of the genetic relationships of these V3 sequences using minimum spanning trees revealed that the transition in coreceptor usage followed a stepwise mutational pathway involving sequential intermediate variants, which were generally present at relatively low frequencies compared to the major predicted CCR5- and CXCR4-using variants. In addition, we observed differences between individuals with respect to the number of predicted CXCR4-using variants, the diversity among major predicted CCR5-using variants, and the presence or absence of intermediate variants with discordant phenotype predictions. These results provide the first detailed description of the mutational pathways in V3 during the transition from CCR5- to CXCR4-usage in natural HIV-1 infection.
PLoS Pathogens 06/2011; 7(6):e1002106. · 9.13 Impact Factor
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Soumi Gupta,
Johan Vingerhoets,
Signe Fransen,
Lotke Tambuyzer,
Hilde Azijn,
Arne Frantzell,
Roger Paredes,
Eoin Coakley,
Steven Nijs,
Bonaventura Clotet, Christos J Petropoulos,
Jonathan Schapiro,
Wei Huang,
Gaston Picchio
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ABSTRACT: Connection domain mutations (CDMs) in HIV-1 reverse transcriptase (RT) alter susceptibility to some nucleoside/nonnucleoside RT inhibitors (NRTIs/NNRTIs). Their effects on susceptibility and virologic responses to etravirine were analyzed. Seventeen CDMs were evaluated: L283I, E312Q, G333D, G333E, G335C, G335D, N348I, A360I, A360T, A360V, V365I, T369I, A371V, A376S, I393L, E399D, and E399G. CDM prevalence and effects on virologic responses were analyzed retrospectively using clinical data. The effects on etravirine susceptibility were assessed in clinical samples and confirmed using site-directed mutants. The most prevalent CDMs (>10%) were A371V, E399D, A376S, N348I, A360T, G333E, and L283I. CDM presence was positively correlated with thymidine analogue-associated mutations, but not with NNRTI resistance-associated mutations (RAMs). The presence or number of CDMs did not significantly reduce etravirine susceptibility, although small reductions were seen in samples with G333D, N348I, A360V, T369I, and A376S. N348I, E399G, and N348I/T369I were associated with reduced etravirine susceptibility when present with K103N, L100I, or Y181C. N348I or T369I was associated with reduced etravirine susceptibility when present with K101P or K103R/V179D. Virologic responses to an etravirine-containing regimen were slightly diminished when G333D, G335D, or A376S was present, but this was not confirmed in subgroups with higher baseline resistance or without etravirine RAMs. CDMs alone do not confer substantial reductions in etravirine susceptibility but can further reduce etravirine susceptibility in combination with certain NNRTI mutations. Since virologic responses to etravirine were not affected by CDMs, the clinical impacts of these mutations on etravirine susceptibility appear to be minimal.
Antimicrobial Agents and Chemotherapy 04/2011; 55(6):2872-9. · 4.84 Impact Factor
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Nature Genetics 03/2011; 43(5):487-489. · 35.53 Impact Factor
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ABSTRACT: The development of a quantitative understanding of viral evolution and the fitness landscape in HIV-1 drug resistance is a formidable challenge given the large number of available drugs and drug resistance mutations. We analyzed a dataset measuring the in vitro fitness of 70,081 virus samples isolated from HIV-1 subtype B infected individuals undergoing routine drug resistance testing. We assayed virus samples for in vitro replicative capacity in the absence of drugs as well as in the presence of 15 individual drugs. We employed a generalized kernel ridge regression to estimate main fitness effects and epistatic interactions of 1,859 single amino acid variants found within the HIV-1 protease and reverse transcriptase sequences. Models including epistatic interactions predict an average of 54.8% of the variance in replicative capacity across the 16 different environments and substantially outperform models based on main fitness effects only. We find that the fitness landscape of HIV-1 protease and reverse transcriptase is characterized by strong epistasis.
Nature Genetics 03/2011; 43(5):487-9. · 35.53 Impact Factor
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ABSTRACT: Ibalizumab (formerly TNX-355) is a first-in-class, monoclonal antibody inhibitor of CD4-mediated human immunodeficiency type 1 (HIV-1) entry. Multiple clinical trials with HIV-infected patients have demonstrated the antiviral activity, safety, and tolerability of ibalizumab treatment. A 9-week phase Ib study adding ibalizumab monotherapy to failing drug regimens led to transient reductions in HIV viral loads and the evolution of HIV-1 variants with reduced susceptibility to ibalizumab. This report characterizes these variants by comparing the phenotypic susceptibilities and envelope (env) sequences of (i) paired baseline and on-treatment virus populations, (ii) individual env clones from selected paired samples, and (iii) env clones containing site-directed mutations. Viruses with reduced susceptibility to ibalizumab were found to exhibit reduced susceptibility to the anti-CD4 antibody RPA-T4. Conversely, susceptibility to soluble CD4, which targets the HIV-1 gp120 envelope protein, was enhanced. No changes in susceptibility to the fusion inhibitor enfuvirtide or the CCR5 antagonist maraviroc were observed. Functionally, viruses with reduced ibalizumab susceptibility also displayed high levels of infectivity relative to those of paired baseline viruses. Individual env clones exhibiting reduced ibalizumab susceptibility contained multiple amino acid changes in different regions relative to the paired baseline clones. In particular, clones with reduced susceptibility to ibalizumab contained fewer potential asparagine-linked glycosylation sites (PNGSs) in variable region 5 (V5) than did paired ibalizumab-susceptible clones. The reduction in ibalizumab susceptibility due to the loss of V5 PNGSs was confirmed by site-directed mutagenesis. Taken together, these findings provide important insights into resistance to this new class of antiretroviral drug.
Journal of Virology 02/2011; 85(8):3872-80. · 5.40 Impact Factor
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Sean X Du,
Li Xu,
Wenge Zhang,
Susan Tang,
Rebecca I Boenig,
Helen Chen,
Ellaine B Mariano,
Michael B Zwick,
Paul W H I Parren,
Dennis R Burton,
Terri Wrin, Christos J Petropoulos,
John A Ballantyne,
Michael Chambers,
Robert G Whalen
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ABSTRACT: A prophylactic vaccine is needed to slow the spread of HIV-1 infection. Optimization of the wild-type envelope glycoproteins to create immunogens that can elicit effective neutralizing antibodies is a high priority. Starting with ten genes encoding subtype B HIV-1 gp120 envelope glycoproteins and using in vitro homologous DNA recombination, we created chimeric gp120 variants that were screened for their ability to bind neutralizing monoclonal antibodies. Hundreds of variants were identified with novel antigenic phenotypes that exhibit considerable sequence diversity. Immunization of rabbits with these gp120 variants demonstrated that the majority can induce neutralizing antibodies to HIV-1. One novel variant, called ST-008, induced significantly improved neutralizing antibody responses when assayed against a large panel of primary HIV-1 isolates. Further study of various deletion constructs of ST-008 showed that the enhanced immunogenicity results from a combination of effective DNA priming, an enhanced V3-based response, and an improved response to the constant backbone sequences.
PLoS ONE 01/2011; 6(6):e20927. · 4.09 Impact Factor
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ABSTRACT: To examine mutational pathways that lead to CXCR4 use of HIV-1, we analyzed the genotypic and phenotypic characteristics of envelope sequences from a large panel of patient virus populations and individual clones containing different V3 mutations. Basic amino acid substitutions at position 11 were strong determinants of CXCR4-mediated entry but required multiple compensatory mutations to overcome associated reductions in infectivity. In contrast, basic amino acid substitutions at position 25, or substitutions at positions 6-8 resulting in the loss of a potential N-linked glycosylation site, contributed to CXCR4-mediated entry but required additional substitutions acting cooperatively to confer efficient CXCR4 use. Our assumptions, based upon examination of patient viruses, were largely confirmed by characterizing the coreceptor utilization of five distinct panels of isogenic envelope sequences containing V3 amino acid substitutions introduced by site-directed mutagenesis. These results further define the mutational pathways leading to CXCR4 use and their associated genetic barriers.
Virology 11/2010; 409(2):308-18. · 3.35 Impact Factor
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ABSTRACT: Dual HIV-1 utilizes cellular CCR5 and CXCR4 coreceptors to enter host cells. Recent studies indicate that the ability of these viruses to use both coreceptors varies significantly in cell lines expressing CXCR4 or CCR5; however, it is not clear whether differences in coreceptor mediated infection in vitro reflect infection of primary cells in vivo.
We evaluated coreceptor usage of dual envelope clones from patient viruses using a single-cycle pseudovirus assay conducted in cell lines and a replication-competent assay performed using peripheral blood mononuclear cells. Dual envelope clones were selected and classified into three groups, R5>X4, R5 approximately X4, and X4>R5, based on their ability to mediate entry by using CXCR4 and CCR5 in a pseudovirus assay.
We observed a high degree of concordance between measurements of coreceptor-mediated entry in pseudovirus and peripheral blood mononuclear cell assays. R5>X4 viruses were efficiently inhibited by a CCR5 antagonist, but not a CXCR4 antagonist, whereas X4>R5 viruses were efficiently inhibited by a CXCR4 antagonist, but not a CCR5 antagonist. R5 approximately X4 viruses were not inhibited, or only partially inhibited, by either a CCR5 or a CXCR4 antagonist alone.
These observations indicate that measurements of coreceptor use determined using pseudoviruses and coreceptor-expressing cell lines are generally concordant with the results obtained using replication-competent assays and peripheral blood mononuclear cell. This suggests that a considerable fraction of dual viruses preferentially infect either CCR5 or CXCR4 target cells in vivo. The clinical implications of preferential coreceptor utilization by dual viruses, that is, HIV-1 pathogenesis and response to coreceptor antagonists, require additional studies.
AIDS (London, England) 09/2010; 24(14):2181-6. · 4.91 Impact Factor
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ABSTRACT: To detect general patterns and temporal trends of HIV-1 resistance, we apply principal component analysis (PCA) to in vitro fitness data. Twenty-eight thousand virus samples, obtained between 2002 and 2008, were assayed for fitness in 16 to 21 selective environments. Fitness measurements are based on replication capacity (RC), which quantifies in vitro viral replication in a single cycle of infection. RC is determined both in the absence of drugs and in the presence of 6-7 nucleoside analog reverse transcriptase inhibitors (NRTIs), 3-4 non-nucleoside reverse transcriptase inhibitors (NNRTIs), and 6-9 protease inhibitors (PIs). PCA shows remarkable structure in RC across the different environments, which reveals differences in the patterns of resistance and cross-resistance between drugs or between drug classes. To probe the causes of the observed patterns, we develop a model to generate simulated data and subject these simulated data to an equivalent analysis. By comparing the outcomes of PCA on the original and the simulated data, we quantify which part of the total variance of the original data is due to non-specific effects, class-specific effects, and drug-specific effects of resistance mutations. We find that relative fitness is mainly drug-independent and that drug-specific effects are substantially bigger than class-specific effects for NRTIs, but not for NNRTIs or PIs. The observed patterns remain remarkably stable over the period of observation. Comparison with known potent combination therapies suggests that PCA helps to identify combinations that act synergistically in preventing the emergence of resistance.
Epidemics. 06/2010; 2(2):85-91.
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ABSTRACT: Recent reports have described the effect of mutations in the connection and RNase H domains of reverse transcriptase (RT) on nucleoside and nonnucleoside reverse transcriptase inhibitor (NRTI and NNRTI, respectively) resistance in the presence of thymidine analog resistance mutations (TAMs) and NNRTI mutations (J. H. Brehm, D. Koontz, J. D. Meteer, V. Pathak, N. Sluis-Cremer, and J. W. Mellors, J. Virol. 81:7852-7859, 2007; K. A. Delviks-Frankenberry, G. N. Nikolenko, R. Barr, and V. K. Pathak, J. Virol. 81:6837-6845, 2007; G. N. Nikolenko, K. A. Delviks-Frankenberry, S. Palmer, F. Maldarelli, M. J. Fivash, Jr., J. M. Coffin, and V. K. Pathak, Proc. Natl. Acad. Sci. U. S. A. 104:317-322, 2007; G. N. Nikolenko, S. Palmer, F. Maldarelli, J. W. Mellors, J. M. Coffin, and V. K. Pathak, Proc. Natl. Acad. Sci. U. S. A. 102:2093-2098, 2005; and S. H. Yap, C. W. Sheen, J. Fahey, M. Zanin, D. Tyssen, V. D. Lima, B. Wynhoven, M. Kuiper, N. Sluis-Cremer, P. R. Harrigan, and G. Tachedjian, PLoS Med. 4:e335, 2007). In the present study, novel mutations in the connection domain of RT (T369I/V), first identified in patient-derived viruses, were characterized, and their effects on NNRTI and NNRTI susceptibility were determined. Furthermore, the effect of N348I on NRTI and NNRTI resistance was confirmed. HIV-1 with either N348I or T369I/V demonstrated reduced susceptibility to nevirapine (NVP), efavirenz (EFV), delaviridine (DLV), and zidovudine (ZDV) compared to wild-type HIV-1. However, HIV-1 with T369I and N348I demonstrated 10- to 60-fold resistance to these same drugs. In clinical samples, these two connection domain RT mutations were predominantly observed in viruses containing TAMs and NNRTI mutations and did not alter the susceptible-resistant classifications of these samples. Introduction of T369I, N348I, or T369I/N348I also reduced replication capacity (RC). These observations suggest that it may be of scientific interest to test these mutations against new NNRTI candidates.
Antimicrobial Agents and Chemotherapy 03/2010; 54(5):1973-80. · 4.84 Impact Factor
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Jeffrey S Larson,
Laurie J Goodman,
Yuping Tan,
Lisa Defazio-Eli,
Agnes C Paquet,
Jennifer W Cook,
Amber Rivera,
Kristi Frankson,
Jolly Bose,
Lili Chen, [......],
Linda D B Kiss,
Weidong Huang,
Shannon Utter,
Thomas Sherwood,
Michael Bates,
Jodi Weidler,
Gordon Parry,
John Winslow, Christos J Petropoulos,
Jeannette M Whitcomb
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ABSTRACT: We report here the results of the analytical validation of assays that measure HER2 total protein (H2T) and HER2 homodimer (H2D) expression in Formalin Fixed Paraffin Embedded (FFPE) breast cancer tumors as well as cell line controls. The assays are based on the VeraTag technology platform and are commercially available through a central CAP-accredited clinical reference laboratory. The accuracy of H2T measurements spans a broad dynamic range (2-3 logs) as evaluated by comparison with cross-validating technologies. The measurement of H2T expression demonstrates a sensitivity that is approximately 7-10 times greater than conventional immunohistochemistry (IHC) (HercepTest). The HERmark assay is a quantitative assay that sensitively and reproducibly measures continuous H2T and H2D protein expression levels and therefore may have the potential to stratify patients more accurately with respect to response to HER2-targeted therapies than current methods which rely on semiquantitative protein measurements (IHC) or on indirect assessments of gene amplification (FISH).
Pathology research international. 01/2010; 2010:814176.
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Sean X Du,
Rebecca J Idiart,
Ellaine B Mariano,
Helen Chen,
Peifeng Jiang,
Li Xu,
Kristin M Ostrow,
Terri Wrin,
Pham Phung,
James M Binley, Christos J Petropoulos,
John A Ballantyne,
Robert G Whalen
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ABSTRACT: The external domains of the HIV-1 envelope glycoprotein (gp120 and the gp41 ectodomain, collectively known as gp140) contain all known viral neutralization epitopes. Various strategies have been used to create soluble trimers of the envelope to mimic the structure of the native viral protein, including mutation of the gp120-gp41 cleavage site, introduction of disulfide bonds, and fusion to heterologous trimerization motifs. We compared the effects on quaternary structure, antigenicity, and immunogenicity of three such motifs: T4 fibritin, a GCN4 variant, and the Escherichia coli aspartate transcarbamoylase catalytic subunit. Fusion of each motif to the C-terminus of a noncleavable JRCSF gp140(-) envelope protein led to enhanced trimerization but had limited effects on the antigenic profile and CD4-binding ability of the trimers. Immunization of rabbits provided no evidence that the trimerized gp140(-) constructs induced significantly improved neutralizing antibodies to several HIV-1 pseudoviruses, compared to gp140 lacking a trimerization motif. However, modest differences in both binding specificity and neutralizing antibody responses were observed among the various immunogens.
Virology 10/2009; 395(1):33-44. · 3.35 Impact Factor
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ABSTRACT: We characterized the raltegravir (RAL) susceptibility and the integrase (IN)-mediated replication capacity (RC) of RAL-resistant human immunodeficiency virus type 1 from two patients experiencing virologic failure during continuous RAL salvage therapy. The following two distinct outcomes were observed: (i) the selective outgrowth of virus with high-level RAL resistance and high IN-mediated RC leading to significant viral load rebound and (ii) the selection of virus with a slight reduction in RAL susceptibility and low IN-mediated RC resulting in sustained low-level viremia.
Antimicrobial Agents and Chemotherapy 09/2009; 53(10):4522-4. · 4.84 Impact Factor
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Wei Huang,
Jonathan Toma,
Eric Stawiski,
Signe Fransen,
Terri Wrin,
Neil Parkin,
Jeannette M Whitcomb,
Eoin Coakley,
Frederick M Hecht,
Steven G Deeks,
Rajesh T Gandhi,
Susan H Eshleman, Christos J Petropoulos
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ABSTRACT: We screened 150 individuals from two recent seroconverter cohorts and found that six (4%) had CXCR4-using viruses. Clonal analysis of these six individuals, along with a seventh individual identified during clinical care as a recent seroconverter, revealed the presence of both X4- and dual-tropic variants in these recently infected adults. The ability of individual CXCR4-using variants to infect cells expressing CD4/CXCR4 or CD4/CCR5 varied dramatically. These data demonstrate that virus populations in some newly infected individuals can consist of either heterogeneous populations containing both CXCR4-using and CCR5-tropic viruses, or homogeneous populations containing only CXCR4-using viruses. The presence of CXCR4-using viruses at early stages of infection suggests that testing for viral tropism before using CCR5 antagonists may be important even in persons with known recent infection. The presence of CXCR4-using viruses in a subset of newly infected individuals could impact the efficacies of vaccine and microbicide strategies that target CCR5-tropic viruses.
AIDS research and human retroviruses 09/2009; 25(8):795-802. · 2.18 Impact Factor
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ABSTRACT: The human immunodeficiency virus type 1 (HIV-1) integrase mutations N155H and Q148R(H)(K) that reduce susceptibility to the integrase inhibitor raltegravir have been identified in patients failing treatment regimens containing raltegravir. Whether these resistance mutations occur individually or in combination within a single virus genome has not been defined, nor do we fully understand the impact of these primary mutations and other secondary mutations on raltegravir susceptibility and viral replication capacity. To address these important questions, we investigated the raltegravir susceptibility and replication capacity of viruses containing mutations at positions 155 and 148 separately or in combination with secondary mutations selected in subjects failing treatment regimens containing raltegravir. Clonal analysis demonstrated that N155H and Q148R(H)(K) occur independently, not in combination. Viruses containing a Q148R(H)(K) mutation generally displayed larger reductions in raltegravir susceptibility than viruses with an N155H mutation. Analysis of site-directed mutants indicated that E92Q in combination with N155H resulted in a higher level of resistance to raltegravir than N155H alone. Viruses containing a Q148R(H) mutation together with a G140S mutation were more resistant to raltegravir than viruses containing a Q148R(H) mutation alone; however, viruses containing G140S and Q148K were more susceptible to raltegravir than viruses containing a Q148K mutation alone. Both N155H and Q148R(H)(K) mutations reduced the replication capacity, while the addition of secondary mutations either improved or reduced the replication capacity depending on the primary mutation. This study demonstrates distinct genetic pathways to resistance in subjects failing raltegravir regimens and defines the effects of primary and secondary resistance mutations on raltegravir susceptibility and replication capacity.
Journal of Virology 09/2009; 83(22):11440-6. · 5.40 Impact Factor
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Eoin Coakley,
Jacqueline D Reeves,
Wei Huang,
Marga Mangas-Ruiz,
Irma Maurer,
Agnes M Harskamp,
Soumi Gupta,
Yolanda Lie, Christos J Petropoulos,
Hanneke Schuitemaker,
Angélique B van 't Wout
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ABSTRACT: The recent availability of CCR5 antagonists as anti-human immunodeficiency virus (anti-HIV) therapeutics has highlighted the need to accurately identify CXCR4-using variants in patient samples when use of this new drug class is considered. The Trofile assay (Monogram Biosciences) has become the method that is the most widely used to define tropism in the clinic prior to the use of a CCR5 antagonist. By comparison, the MT-2 assay has been used since early in the HIV epidemic to define tropism in clinical specimens. Given that there are few data from direct comparisons of these two assays, we evaluated the performance of the plasma-based Trofile assay and the peripheral blood mononuclear cell (PBMC)-based MT-2 assay for the detection of CXCR4 use in defining the tropism of HIV isolates derived from clinical samples. The various samples used for this comparison were derived from participants of the Amsterdam Cohort Studies on HIV infection and AIDS who underwent consecutive MT-2 assay testing of their PBMCs at approximately 3-month intervals. This unique sample set was specifically selected because consecutive MT-2 assays had demonstrated a shift from negative to positive in PBMCs, reflecting the first emergence of CXCR4-using virus in PBMCs above the level of detection of the assay in these individuals. Trofile testing was performed with clonal HIV type 1 (HIV-1) variants (n = 21), MT-2 cell culture-derived cells (n = 20) and supernatants (n = 42), and plasma samples (n = 76). Among the clonal HIV-1 variants and MT-2 cell culture-derived samples, the results of the Trofile and MT-2 assays demonstrated a high degree of concordance (95% to 98%). Among consecutive plasma samples, detection of CXCR4-using virus was at or before the time of first detection by the MT-2 assay in 5/10 patients by the original Trofile assay and in 9/10 patients by the enhanced-sensitivity Trofile assay. Differences in the time to the first detection of CXCR4 use between the MT-2 assay (PBMCs) and the original Trofile assay (plasma) were greatly reduced by the enhanced-sensitivity Trofile assay, suggesting that sensitivity for the detection of minor CXCR4-using variants may be a more important determinant of discordant findings than compartmentalization. The similarities in performance of the enhanced-sensitivity Trofile and MT-2 assays suggest that either may be an appropriate methodology to define tropism in patient specimens.
Antimicrobial Agents and Chemotherapy 09/2009; 53(11):4686-93. · 4.84 Impact Factor
