Use of new T-cell-based cell lines expressing two luciferase reporters for accurately evaluating susceptibility to anti-human immunodeficiency virus type 1 drugs

National Institute of Infectious Diseases, Tokyo, Edo, Tōkyō, Japan
Journal of Clinical Microbiology (Impact Factor: 4.23). 03/2007; 45(2):477-87. DOI: 10.1128/JCM.01708-06
Source: PubMed

ABSTRACT Two new T-cell-based reporter cell lines were established to measure human immunodeficiency virus type 1 (HIV-1) infectivity. One cell line naturally expresses CD4 and CXCR4, making it susceptible to X4-tropic viruses, and the other cell line, in which a CCR5 expression vector was introduced, is susceptible to both X4- and R5-tropic viruses. Reporter cells were constructed by transfecting the human T-cell line HPB-Ma, which demonstrates high susceptibility to HIV-1, with genomes expressing two different luciferase reporters, HIV-1 long terminal repeat-driven firefly luciferase and cytomegalovirus promoter-driven renilla luciferase. Upon HIV infection, the cells expressed firefly luciferase at levels that were highly correlated (r2=0.91 to 0.98) with the production of the capsid antigen p24. The cells also constitutively expressed renilla luciferase, which was used to monitor cell numbers and viability. The reliability of the cell lines for two in vitro applications, drug resistance phenotyping and drug screening, was confirmed. As HIV-1 efficiently replicated in these cells, they could be used for multiple-round replication assays as an alternative method to a single-cycle replication protocol. Coefficients of variation for drug susceptibility evaluated with the cell lines ranged from 17 to 41%. The new cell lines were beneficial for evaluating antiretroviral drug resistance. Firefly luciferase gave a wider dynamic range for evaluating virus infectivity, and the introduction of renilla luciferase improved assay reproducibility. The cell lines were also beneficial for screening new antiretroviral agents, as false inhibition caused by the cytotoxicity of test compounds was easily detected by monitoring renilla luciferase activity.

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    ABSTRACT: Limited HIV-1 drug-resistance surveillance has been carried out in Ghana since the implementation of antiretroviral therapy (ART). This study sought to provide data on the profile of HIV-1 drug resistance in ART-experienced and newly diagnosed individuals in Ghana. Samples were collected from 101 HIV-1-infected patients (32 ART-experienced cases with virological failure and 69 newly diagnosed ART-naïve cases, including 11 children), in Koforidua, Eastern region of Ghana, from February 2009 to January 2010. The pol gene sequences were analyzed by in-house HIV-1 drug-resistance testing. The most prevalent HIV-1 subtype was CRF02_AG (66.3%, 67/101) followed by unique recombinant forms (25.7%, 26/101). Among 31 ART-experienced adults, 22 (71.0%) possessed at least one drug-resistance mutation, and 14 (45.2%) had two-class-resistance to nucleoside and non-nucleoside reverse-transcriptase inhibitors used in their first ART regimen. Importantly, the number of accumulated mutations clearly correlated with the duration of ART. The most prevalent mutation was lamivudine-resistance M184V (n = 12, 38.7%) followed by efavirenz/nevirapine-resistance K103N (n = 9, 29.0%), and zidovudine/stavudine-resistance T215Y/F (n = 6, 19.4%). Within the viral protease, the major nelfinavir-resistance mutation L90M was found in one case. No transmitted HIV-1 drug-resistance mutation was found in 59 ART-naïve adults, but K103N and G190S mutations were observed in one ART-naïve child. Despite expanding accessibility to ART in Eastern Ghana, the prevalence of transmitted HIV-1 drug resistance presently appears to be low. As ART provision with limited options is scaled up nationwide in Ghana, careful monitoring of transmitted HIV-1 drug resistance is necessary.
    PLoS ONE 08/2013; 8(8):e71972. DOI:10.1371/journal.pone.0071972 · 3.53 Impact Factor
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    PLoS Pathogens 10/2014; 10(10):e1004453. DOI:10.1371/journal.ppat.1004453 · 8.14 Impact Factor
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    ABSTRACT: Introduction: Till today, HIV remains one of the most serious infectious diseases with over 34 million people infected around the world. Semen is the most important vehicle of HIV, both in homosexual and heterosexual transmission. Semen contains several substances like amines, nutrients and enzymes that protect HIV and could even increase viral infectivity. Despite decades of research, HIV transmission mechanisms are not completely understood yet because there is no consensus about the source for transmission, i.e. cell free virus (CFV) or cell-associated virus (CAV). Therefore, we want to isolate infectious seminal HIV, determine its source in semen and characterize the virus from this compartment phenotypically. Material and methods: Several cell lines (CEM-SS, Jurkat, SupT1, R5MaRBLE and Ghost) were tested for sensitivity to pick up and propagate both CFV and CAV HIV. The cell lines were investigated for their susceptibility to R5-tropic and X4-tropic viruses. Semen samples, received from untreated HIV patients consulting the AIDS Reference Center of the Institute of Tropical Medicine in Antwerp, were fractionized in seminal plasma (SP), which contains only CFV, and non-sperm cells (NSC), containing CAV, by centrifugation over a Nycodenz gradient. SP HIV was further isolated using the μMACSTM VitalVirus HIV Isolation kit. Both separated fractions were cocultured with Ghost and R5MaRBLE cells. Infection after 7 days incubation is assessed by p24 ELISA and Luciferase activity, respectively. Blood from the same patients was also collected to isolate HIV for comparison with the seminal viruses. Characterization was performed by sequencing both seminal and blood-derived HIV. A comparison of replicative fitness between two seminal, blood-derived and transmitted/founder (T/F) viruses was made using a competition/dual infection assay. Results: R5MaRBLE and Ghost showed to be the most sensitive cell lines for HIV isolation, both for the SP and the NSC fraction. R5MaRBLE is also capable of picking up X4-strains in very low concentrations. Unfortunately, no CFV or CAV virus could be isolated from the obtained semen samples. No significant differences in replicative fitness were observed between two previously isolated seminal samples, the blood-derived viruses from the same patients and two T/F viruses. Discussion: We have optimized a previously used method to isolate infectious HIV from semen. The low success rate is related to the low viral load (VL) in semen of the patients tested. Estimation of seminal viral loads in advance is difficult due to low correlation between VL in blood and semen. The fact that we did not observe any significant difference in replicative fitness between the seminal, blood-derived and T/F viruses might be due to the model used. Also the used statistical test had too few power because only three replicates were used. Next, we will test the replicative fitness in a model that is more relevant for sexual transmission and also include more replicates so that a parametric test with more power can be used.
    06/2014, Degree: Magna Cum Laude, Supervisor: Dr. Kevin Ariën

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