Last data update: Apr 29, 2024. (Total: 46658 publications since 2009)
Records 1-17 (of 17 Records) |
Query Trace: Youngpairoj AS [original query] |
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A monoclonal antibody for the detection of the antiretroviral drug emtricitabine
Youngpairoj AS , Vanderford TH , Reed MS , Granade TC , Pau CP , Pohl J , Switzer WM , Heneine W . AIDS 2022 36 (13) 1890-1893 Antibody-based testing for emtricitabine (FTC), a critical component of pre-exposure prophylaxis and antiretroviral therapy would provide low-cost detection for clinical monitoring to improve adherence. We developed a monoclonal antibody (5D2) to FTC and demonstrated its high specificity and physiologically relevant linear range of detection in a competitive enzyme immunoassay (EIA). Thus, this monoclonal antibody is a key reagent that will enable simple and low-cost lateral flow assays and EIAs for adherence monitoring. |
Development and optimization of thermal contrast amplification lateral flow immunoassays for ultrasensitive HIV p24 protein detection
Zhan L , Granade T , Liu Y , Wei X , Youngpairoj A , Sullivan V , Johnson J , Bischof J . Microsyst Nanoeng 2020 6 (1) 54 Detection of human immunodeficiency virus (HIV) p24 protein at a single pg/ml concentration in point-of-care (POC) settings is important because it can facilitate acute HIV infection diagnosis with a detection sensitivity approaching that of laboratory-based assays. However, the limit of detection (LOD) of lateral flow immunoassays (LFAs), the most prominent POC diagnostic platform, falls short of that of laboratory protein detection methods such as enzyme-linked immunosorbent assay (ELISA). Here, we report the development and optimization of a thermal contrast amplification (TCA) LFA that will allow ultrasensitive detection of 8 pg/ml p24 protein spiked into human serum at POC, approaching the LOD of a laboratory test. To achieve this aim, we pursued several innovations as follows: (a) defining a new quantitative figure of merit for LFA design based on the specific to nonspecific binding ratio (BR); (b) using different sizes and shapes of gold nanoparticles (GNPs) in the systematic optimization of TCA LFA designs; and (c) exploring new laser wavelengths and power regimes for TCA LFA designs. First, we optimized the blocking buffer for the membrane and running buffer by quantitatively measuring the BR using a TCA reader. The TCA reader interprets the thermal signal (i.e., temperature) of GNPs within the membrane when irradiated by a laser at the plasmon resonance wavelength of the particle. This process results in higher detection and quantitation of GNPs than in traditional visual detection (i.e., color intensity). Further, we investigated the effect of laser power (30, 100, 200 mW), GNP size and shape (30 and 100 nm gold spheres, 150 nm gold-silica shells), and laser wavelength (532, 800 nm). Applying these innovations to a new TCA LFA design, we demonstrated that 100 nm spheres with a 100 mW 532 nm laser provided the best performance (i.e., LOD = 8 pg/ml). This LOD is significantly better than that of the current colorimetric LFA and is in the range of the laboratory-based p24 ELISA. In summary, this TCA LFA for p24 protein shows promise for detecting acute HIV infection in POC settings. |
Characterization of real-time microarrays for simultaneous detection of HIV-1, HIV-2, and hepatitis viruses.
Granade TC , Kodani M , Wells SK , Youngpairoj AS , Masciotra S , Curtis KM , Kamili S , Owen SM . J Virol Methods 2018 259 60-65 Real-time PCR assays for nucleic acid testing (NAT) of hepatitis viruses A-E and for HIV-1 and HIV-2 have been developed; however, a multiplex assay that can simultaneously detect all of these agents is not yet available. Standardized TaqMan assays for detection of hepatitis viruses A-E have been described and applied to TaqMan Array Cards (TAC) which are capable of multiple pathogen detection using a single set of optimized PCR conditions. Assays for three gene regions of HIV-1 (long-terminal repeat (LTR), gag, and polymerase) and HIV-2 (overlap of LTR and gag, protease and integrase) were designed using the hepatitis assay conditions. Nucleic acid extracts of HIV-1-infected samples (44 plasma, 41 whole blood, 20 HIV-1 viral stocks) were tested on the TAC cards; 98 were reactive (92%) with 70 in multiple gene regions. Twenty-four of the 27 (89%) HIV-2 specimens (10 plasma, 1 PBMC lysate, 6 whole blood and 10 plasmids containing HIV-2 polymerase) were detected on TAC. No HIV or hepatitis virus sequences were detected in 30 HIV-negative samples (specificity 100%). Three HBV and 18 HCV co-infections were identified in the HIV-1-infected specimens. Multi-pathogen detection using TAC could provide a rapid, sensitive and more efficient method of surveying for a variety of infectious disease nucleic acids. |
Reference panel of cloned HIV-2 plasmid DNA for nucleic acid assay development, evaluation, and quality monitoring.
Youngpairoj AS , Curtis KA , Wells SK , Pau CP , Granade TC , Owen SM . J Clin Virol 2014 61 (2) 293-7 BACKGROUND: Currently, no FDA-approved HIV-2 nucleic acid assay is commercially available in the United States, although several laboratories have developed in-house assays to confirm HIV-2 infections. A major limitation in the development of novel HIV-2 diagnostic assays is the lack of reference materials that can be used to evaluate, optimize, and monitor assay performance. STUDY DESIGN: Eleven viral stocks of HIV-2 isolates from various West African countries, including the Ivory Coast, Senegal, and Guinea-Bissau, were used to clone the entire LTR and pol regions from each virus. RESULTS: We successfully cloned, sequenced, and group classified 22 HIV-2 DNA plasmids including 11 full length LTR ( approximately 849bp) and 11 pol ( approximately 2995bp) sequences. There were eight HIV-2 group A and three group B in both the LTR and pol regions. CONCLUSIONS: This reference panel provides a robust, quantifiable, renewable, and non-infectious set of reagents that can be used for the development and evaluation of new HIV-2 molecular diagnostic assays and quality assurance and quality control reagents for use in the clinical laboratories. |
Real-Time Detection of HIV-2 by Reverse Transcription-Loop-Mediated Isothermal Amplification.
Curtis KA , Niedzwiedz P , Youngpairoj AS , Rudolph DL , Owen SM . J Clin Microbiol 2014 52 (7) 2674-6 Currently, there are no FDA-approved, nucleic acid amplification tests (NAATs) for the detection or confirmation of HIV-2 infection. We describe the development of a real-time assay for detection of HIV-2 DNA and RNA, using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) and the ESEQuant Tube Scanner, a portable isothermal amplification/detection device. |
Evaluation of the CDC proposed laboratory HIV testing algorithm among men who have sex with men (MSM) from five US metropolitan statistical areas using specimens collected in 2011
Masciotra S , Smith AJ , Youngpairoj AS , Sprinkle P , Miles I , Sionean C , Paz-Bailey G , Johnson JA , Owen SM . J Clin Virol 2013 58 Suppl 1 e8-e12 BACKGROUND: Until recently most testing algorithms in the United States (US) utilized Western blot (WB) as the supplemental test. CDC has proposed an algorithm for HIV diagnosis which includes an initial screen with a Combo Antigen/Antibody 4th generation-immunoassay (IA), followed by an HIV-1/2 discriminatory IA of initially reactive-IA specimens. Discordant results in the proposed algorithm are resolved by nucleic acid-amplification testing (NAAT). OBJECTIVES: Evaluate the results obtained with the CDC proposed laboratory-based algorithm using specimens from men who have sex with men (MSM) obtained in five metropolitan statistical areas (MSAs). STUDY DESIGN: Specimens from 992 MSM from five MSAs participating in the CDC's National HIV Behavioral Surveillance System in 2011 were tested at local facilities and CDC. The five MSAs utilized algorithms of various screening assays and specimen types, and WB as the supplemental test. At the CDC, serum/plasma specimens were screened with 4th generation-IA and the Multispot HIV-1/HIV-2 discriminatory assay was used as the supplemental test. NAAT was used to resolve discordant results and to further identify acute HIV infections from all screened-non-reactive missed by the proposed algorithm. Performance of the proposed algorithm was compared to site-specific WB-based algorithms. RESULTS: The proposed algorithm detected 254 infections. The WB-based algorithms detected 19 fewer infections; 4 by oral fluid (OF) rapid testing and 15 by WB supplemental testing (12 OF and 3 blood). One acute infection was identified by NAAT from all screened-non-reactive specimens. CONCLUSIONS: The proposed algorithm identified more infections than the WB-based algorithms in a high-risk MSM population. OF testing was associated with most of the discordant results between algorithms. HIV testing with the proposed algorithm can increase diagnosis of infected individuals, including early infections. |
Performance of the Alere Determine HIV-1/2 Ag/Ab Combo Rapid Test with specimens from HIV-1 seroconverters from the US and HIV-2 infected individuals from Ivory Coast
Masciotra S , Luo W , Youngpairoj AS , Kennedy MS , Wells S , Ambrose K , Sprinkle P , Owen SM . J Clin Virol 2013 58 Suppl 1 e54-8 BACKGROUND: FDA-approved HIV Antigen/Antibody combo (4th generation) immunoassays (IAs) can identify HIV-1 infections before the Western blot (WB) becomes positive. In the US, increased detection of acute HIV infections has been facilitated by using 4th generation IAs, but there is no FDA-approved 4th generation rapid test (RT). The Alere Determine HIV-1/2 Ag/Ab Combo (Determine Combo) RT detects and distinguishes HIV p24 Antigen (Ag) from Antibody (Ab) to HIV-1+HIV-2 and thus has the potential to improve diagnosis of acute HIV infection. OBJECTIVE: To evaluate the ability of Determine Combo RT to detect acute/early HIV-1 infections and HIV-2 antibody in well-characterized plasma specimens. STUDY DESIGN: In HIV-1 seroconverters from the US, Determine Combo reactivity was evaluated by performing the 50% cumulative frequency analysis and by comparing with 3rd and 4th generation IAs' reactivity. HIV-2 plasma specimens from Ivory Coast were tested with Determine Combo. RESULTS: The 50% cumulative frequency analysis in 17 seroconverters placed Determine Combo (Ag+/Ab-, Ag+Ab+, Ag-/Ab+) and Ab-component reactivity at 15.5 and 7 days before WB positivity, respectively. In 26 seroconverters, Determine Combo was reactive in 99.0% and 92.5% of 3rd and 4th generation IAs-reactive specimens, respectively. All HIV-2 plasma specimens were Ab-reactive/Ag-non-reactive by Determine Combo. CONCLUSIONS: Based on previous results with the same seroconversion panels, combined Ag/Ab reactivity of the Determine Combo appears between FDA-approved 4th and 3rd generation laboratory IAs. These data indicate that this RT could detect HIV-1 infection earlier than other RTs and it performs well in HIV-2 specimens. |
Reduced inflammation and CD4 loss in acute SHIV infection during oral PrEP
Kersh EN , Luo W , Zheng Q , Adams DR , Hanson D , Youngpairoj AS , Cong ME , Butler K , Hendry RM , McNicholl JM , Heneine W , Garcia-Lerma JG . J Infect Dis 2012 206 (5) 770-9 BACKGROUND: The impact of Pre-exposure Prophylaxis (PrEP) with anti-retrovirals on breakthrough HIV or SHIV infection is not fully documented. We addressed the hypothesis that SHIV(SF162P3) infection despite active PrEP results in altered early immune parameters compared to untreated infection. METHODS: Eleven rhesus macaques were infected during repeated, rectal, low-dose SHIV(SF162P3) exposures while receiving concurrent, oral PrEP (Truvada, n=2, or GS7340, n=4), or as untreated controls (n=5). We measured SHIV RNA, inflammatory cytokines, CD4 cells, and SHIV-specific and memory T cells until 20 weeks post peak viremia. RESULTS: SHIV infection during PrEP resulted in 100-fold lower peak viremia and lower IL-15, IL-18, and IL-1Ra levels compared to controls (p<0.05; Wilcoxon rank-sum test). Unlike controls, PrEP-treated macaques showed no significant CD4 reduction during acute infection, and developed more SHIV-specific central memory T cells relative to controls. Following in-vivo CD8(+) cell depletion, viremia rose to similar levels, indicating that CD8(+) cells were critical for viral control in both groups. CONCLUSIONS: PrEP with anti-retrovirals has beneficial effects on early SHIV infection even when infection is not prevented. While long-term immune control could not be examined in this SHIV infection model, our results suggest that PrEP results in improved early disease parameters in breakthrough infections. |
Prevalence of drug resistance-related polymorphisms in treatment-naive individuals infected with nonsubtype B HIV type 1 in Cameroon.
Fonjungo PN , Youngpairoj AS , Alemnji GA , Eno LT , Lyonga EJ , Eloundou MA , Shanmugam V , Mpoudi-Ngole E , Kalish ML , Folks TM , Pieniazek D . AIDS Res Hum Retroviruses 2011 28 (7) 675-84 Mutations associated with the use of protease (PR) and reverse transcriptase (RT) inhibitors have been mostly mapped for HIV-1 subtype B. The prevalence of these mutations in drug-naive HIV-1 subtype B infected individuals is low but occurs at high frequencies in treated individuals. To determine the prevalence of treatment-associated mutations in non-B viruses, we analyzed a 1613bp pol region of specimens collected from 57 HIV-1 infected treatment-naive individuals from Cameroon. Of the 57 HIV-1 sequences, 43 belonged to CRF02-AG, two to CRF11-cpx, six to subtype A, one to subtype D and five were unclassifiable. Of the 57 PR sequences, 100% contained at least one codon change giving substitutions at positions 10, 11, 16, 20, 33, 36, 60, 62, 64, 69, 77, and 89. These substitutions gave the following prevalence pattern, 36I/L (100%, 57/57) > 89M/I (98%, 56/57) > 69K/R (93%, 53/57) > 20I/R (89%, 51/57) > 16E (16%, 9/57) > 64M (12%, 7/57) > 10I (11%, 6/57) > 11V (5%, 3/57) = 62V (5%, 3/57) = 77I (5%, 3/57) > 233F/V (4%, 2/57) = 60E (4%), which differed significantly from subtype B at positions 20, 36, 69 and 89. All but one (98%) of the 57 RT sequences (438 amino acid residues) carried substitutions located at codons 39A (7%), 43E (7%), 122E (7%), 312Q (2%), 333E (2%), 335C/D (89%), 356K (89%), 358K (14%), 365I (2%), 371V (81%), 376S (11%) or 399D (4%); the frequency of these substitutions ranged from <0.5% to 4% in RT of subtype B. The high prevalence of minor mutations associated with protease inhibitors (PI) and reverse transcriptase inhibitors (RTI) represent natural polymorphisms. HIV-1 PR and RT sequences from ARV-naive HIV-infected persons in Cameroon are important for monitoring the development of resistance to PIs and RTIs as such mutations could lead to treatment failures in individuals undergoing ARV therapy. |
Natural substrate concentrations can modulate the prophylactic efficacy of nucleotide HIV reverse transcriptase inhibitors
Garcia-Lerma JG , Aung W , Cong ME , Zheng Q , Youngpairoj AS , Mitchell J , Holder A , Martin A , Kuklenyik S , Luo W , Lin CY , Hanson DL , Kersh E , Pau CP , Ray AS , Rooney JF , Lee WA , Heneine W . J Virol 2011 85 (13) 6610-7 Preexposure prophylaxis (PrEP) with antiretroviral drugs is a novel human immunodeficiency virus (HIV) prevention strategy. It is generally thought that high systemic and mucosal drug levels are sufficient for protection. We investigated whether GS7340, a next-generation tenofovir (TFV) prodrug that effectively delivers tenofovir diphosphate (TFV-DP) to lymphoid cells and tissues, could protect macaques against repeated weekly rectal simian-human immunodeficiency virus (SHIV) exposures. Macaques received prophylactic GS7340 treatment 3 days prior to each virus exposure. At 3 days postdosing, TFV-DP concentrations in peripheral blood mononuclear cells (PBMCs) were about 50-fold higher than those seen with TFV disoproxil fumarate (TDF), and they remained above 1,000 fmol/10(6) cells for as long as 7 days. TFV-DP accumulated in lymphoid and rectal tissues, with concentrations at 3 days exceeding 500 fmol/10(6) mononuclear cells. Despite high mucosal and systemic TFV levels, GS7340 was not protective. Since TFV-DP blocks reverse transcription by competing with the natural dATP substrate, we measured dATP contents in peripheral lymphocytes, lymphoid tissue, and rectal mononuclear cells. Compared to those in circulating lymphocytes and lymphoid tissue, rectal lymphocytes had 100-fold higher dATP concentrations and dATP/TFV-DP ratios, likely reflecting the activated status of the cells and suggesting that TFV-DP may be less active at the rectal mucosa. Our results identify dATP/TFV-DP ratios as a possible correlate of protection by TFV and suggest that natural substrate concentrations at the mucosa will likely modulate the prophylactic efficacy of nucleotide reverse transcriptase inhibitors. |
Delayed maturation of antibody avidity but not seroconversion in rhesus macaques infected with SHIV during oral pre-exposure prophylaxis
Curtis KA , Kennedy MS , Luckay A , Cong ME , Youngpairoj AS , Zheng Q , Smith J , Hanson D , Heneine W , Owen SM , Garcia-Lerma JG . J Acquir Immune Defic Syndr 2011 57 (5) 355-62 Pre-exposure prophylaxis (PrEP) is a novel intervention strategy for the prevention HIV transmission. As several clinical trials are at various stages of completion, it is important to understand the impact of PrEP treatment on the development of the immune response to HIV, particularly in individuals that exhibit breakthrough infections despite PrEP. A model of HIV infection, using rhesus macaques and the simian/human immunodeficiency virus (SHIV), was employed to evaluate the effects of PrEP on the evolution of the humoral immune response. Time to seroconversion, neutralizing and binding antibody levels, and antibody avidity were measured in 12 rhesus macaques infected during daily or intermittent PrEP with FTC (emtricitabine) or Truvada (FTC/Tenofovir combination) and compared to 11 untreated, SHIV-infected controls. Macaques that became infected while receiving PrEP exhibited significantly lower peak virus loads during acute infection as compared to untreated animals. While the timing of seroconversion and SHIV binding and neutralizing antibody levels were not impacted by treatment, lower maturation rates of antibody avidity for anti-p27, gp120, gp160, and gp41 were observed. This study suggests that reduced virus loads associated with PrEP treatment has little impact on timing of seroconversion and neutralizing/binding antibody levels: however, maturation of antibody avidity was suppressed. |
Protection against rectal transmission of an emtricitabine (FTC)-resistant SHIV162p3M184V mutant by intermittent prophylaxis with Truvada
Cong ME , Youngpairoj AS , Zheng Q , Aung W , Mitchell J , Sweeney E , Hanson DL , Hendry RM , Dobard C , Heneine W , Garcia-Lerma JG . J Virol 2011 85 (15) 7933-6 Daily pre-exposure prophylaxis (PrEP) with Truvada (FTC and TDF) is a novel HIV prevention strategy recently found to reduce HIV incidence among men who have sex with men. We used a macaque model of HIV transmission to investigate if Truvada maintains prophylactic efficacy against an FTC-resistant isolate containing the M184V mutation. Five macaques received a dose of Truvada 3 days before exposing them rectally to SHIV162p3(M184V) followed by a second dose 2h afterwards. Five untreated animals were used as controls. Virus exposures were done weekly for up to 14 weeks. Despite the high (>100-fold) level of FTC resistance conferred by M184V, all five treated animals were protected from infection while the five untreated macaques were infected (p=0.0008). Our results show that Truvada maintains high prophylactic efficacy against an FTC-resistant isolate. Increased susceptibility to tenofovir due to M184V and other factors including residual antiviral activity by FTC and/or reduced virus fitness due to M184V may have all contributed to the observed protection. |
T cell chemo-vaccination effects after repeated mucosal SHIV exposures and oral pre-exposure prophylaxis
Kersh EN , Adams DR , Youngpairoj AS , Luo W , Zheng Q , Cong ME , Aung W , Mitchell J , Otten R , Hendry RM , Heneine W , McNicholl J , Garcia-Lerma JG . PLoS One 2011 6 (4) e19295 Pre-exposure prophylaxis (PrEP) with anti-viral drugs is currently in clinical trials for the prevention of HIV infection. Induction of adaptive immune responses to virus exposures during anti-viral drug administration, i.e., a "chemo-vaccination" effect, could contribute to PrEP efficacy. To study possible chemo-vaccination, we monitored humoral and cellular immune responses in nine rhesus macaques undergoing up to 14 weekly, low-dose SHIV(SF162P3) rectal exposures. Six macaques concurrently received PrEP with intermittent, oral Truvada; three were no-PrEP controls. PrEP protected 4 macaques from infection. Two of the four showed evidence of chemo-vaccination, because they developed anti-SHIV CD4(+) and CD8(+) T cells; SHIV-specific antibodies were not detected. Control macaques showed no anti-SHIV immune responses before infection. Chemo-vaccination-induced T cell responses were robust (up to 3,940 SFU/10(6) PBMCs), predominantly central memory cells, short-lived (≤22 weeks), and appeared intermittently and with changing specificities. The two chemo-vaccinated macaques were virus-challenged again after 28 weeks of rest, after T cell responses had waned. One macaque was not protected from infection. The other macaque concurrently received additional PrEP. It remained uninfected and T cell responses were boosted during the additional virus exposures. In summary, we document and characterize PrEP-induced T cell chemo-vaccination. Although not protective after subsiding in one macaque, chemo-vaccination-induced T cells warrant more comprehensive analysis during peak responses for their ability to prevent or to control infections after additional exposures. Our findings highlight the importance of monitoring these responses in clinical PrEP trials and suggest that a combination of vaccines and PrEP potentially might enhance efficacy. |
Generation and mucosal transmissibility of emtricitabine- and tenofovir-resistant SHIV162P3 mutants in macaques
Cong ME , Youngpairoj AS , Aung W , Sharma S , Mitchell J , Dobard C , Heneine W , Garcia-Lerma JG . Virology 2011 412 (2) 435-40 Transmission of drug-resistant HIV has been widely documented. We generated tenofovir (TFV)- and emtricitabine (FTC)-resistant SHIV162P3 mutants that can be used to investigate the transmission efficiency of drug-resistant viruses and their impact on the efficacy of pre-exposure prophylaxis. Both SHIV162p3(M184V) and SHIV162p3(K65R) replicated in vitro at high titers. Drug resistance profiles were similar to those seen in HIV. Virus infectivity to virion particle ratios were 4- and 10-fold lower in SHIV162p3(M184V) and SHIV162p3(K65R), compared to a concurrently generated WT SHIV162p3, respectively. Mucosal transmissibility studies using a repeat low-dose macaque model of rectal and vaginal transmission showed that both mutants were able to efficiently infect macaques only after the dose was increased to adjust for fitness reductions due to K65R and M184V. Our results in limited number of macaques suggest that the reduction in fitness due to M184V and K65R decreases virus transmissibility, and identify in vitro infectivity parameters that associate with mucosal transmissibility. |
Minority HIV mutation detection in dried blood spots indicates high specimen integrity and reveals hidden archived drug resistance.
Wei X , Youngpairoj AS , Garrido C , Zahonero N , Corral A , de Mendoza C , Heneine W , Johnson JA , Garcia-Lerma JG . J Clin Virol 2010 50 (2) 148-52 BACKGROUND: Dried blood spots (DBS) could serve as an attractive, cost-effective alternative to plasma for HIV drug resistance testing. OBJECTIVES: To assess the utility and potential gain in genotypic information with sensitive testing of DBS compared to conventional bulk plasma genotyping, and examine the correlation of majority and minority-level resistance mutations in DBS with treatment history. STUDY DESIGN: Evaluate nucleic acids from the DBS of 33 antiretroviral-experienced subtype B-infected subjects for minority M41L, K65R, K70R, K103N, Y181C, M184V, and T215Y/F mutations by real-time PCR. Compare minority resistance mutations in DBS with bulk genotypes from the same DBS cards and available plasma specimens. RESULTS: All but one (50/51, 98%) mutation from the original plasma bulk sequencing were still detectable in the DBS after three years of storage. The one mutation not identified in DBS was also no longer detectable by bulk sequencing. Furthermore, sensitive testing found 12 additional drug resistance mutations at minority levels in the DBS of 11 (33%) patients. Six minority mutations were in the RNA compartment and six were detected only in the DNA compartment. Resistance was detected in the DBS RNA compartment only in cases where the associated drug was in use within one year of sample collection. CONCLUSIONS: Our ability to identify majority and additional minority-level resistance mutations demonstrated that DBS, if stored properly, is a high-integrity specimen type for conventional and sensitive drug resistance testing. Our data further support the global utility of DBS for drug resistance surveillance and clinical monitoring. |
Intermittent prophylaxis with oral Truvada protects macaques from rectal SHIV infection
Garcia-Lerma JG , Cong ME , Mitchell J , Youngpairoj AS , Zheng Q , Masciotra S , Martin A , Kuklenyik Z , Holder A , Lipscomb J , Pau CP , Barr JR , Hanson DL , Otten R , Paxton L , Folks TM , Heneine W . Sci Transl Med 2010 2 (14) 14ra4 HIV continues to spread globally, mainly through sexual contact. Despite advances in treatment and care, preventing transmission with vaccines or microbicides has proven difficult. A promising strategy to avoid transmission is prophylactic treatment with antiretroviral drugs before exposure to HIV. Clinical trials evaluating the efficacy of daily treatment with the reverse transcriptase inhibitors tenofovir disoproxil fumarate (TDF) or Truvada (TDF plus emtricitabine) are under way. We hypothesized that intermittent prophylactic treatment with long-acting antiviral drugs would be as effective as daily dosing in blocking the earliest stages of viral replication and preventing mucosal transmission. We tested this hypothesis by intermittently giving prophylactic Truvada to macaque monkeys and then exposing them rectally to simian-human immunodeficiency virus (SHIV) once a week for 14 weeks. A simple regimen with an oral dose of Truvada given 1, 3, or 7 days before exposure followed by a second dose 2 hours after exposure was as protective as daily drug administration, possibly because of the long intracellular persistence of the drugs. In addition, a two-dose regimen initiated 2 hours before or after virus exposure was effective, and full protection was obtained by doubling the Truvada concentration in both doses. We saw no protection if the first dose was delayed until 24 hours after exposure, underscoring the importance of blocking initial replication in the mucosa. Our results show that intermittent prophylactic treatment with an antiviral drug can be highly effective in preventing SHIV infection, with a wide window of protection. They strengthen the possibility of developing feasible, cost-effective strategies to prevent HIV transmission in humans. |
On-line coupling of anion exchange and ion-pair chromatography for measurement of intracellular triphosphate metabolites of reverse transcriptase inhibitors
Kuklenyik Z , Martin A , Pau CP , Holder A , Youngpairoj AS , Zheng Q , Cong ME , Garcia-Lerma JG , Heneine W , Pirkle JL , Barr JR . J Chromatogr B Analyt Technol Biomed Life Sci 2009 877 (29) 3659-66 We developed an automated on-line weak anion exchange (WAX) solid-phase extraction (SPE) method coupled with ion-pair (IP) chromatography-tandem mass spectrometry (MS/MS) detection for quantitatively measuring triphosphorylated metabolites of three reverse transcriptase inhibitors (RTI). The administered pro-drugs were Tenofovir disoproxil fumarate (TDF), Emtricitabine (FTC) and Lamivudine (3TC). Their intracellular metabolites Tenofovir-diphosphate (TFV-DP), Emtricitabine-triphosphate (FTC-TP), and Lamivudine-triphosphate (3TC-TP) were measured in peripheral blood mononuclear cells (PBMC). We coupled the WAX and IP chromatography systems using a combination of 6-port and 10-port switching valves, and we mixed the WAX elute with 1,5-dimethyl-hexyl-amine before IP chromatography separation. Multiple waste outlets allowed for eliminating potential matrix components interfering with MS/MS detection. Limits of detection were 9, 200 and 75pg per sample for TFV-DP (448/176 m/z), FTC-TP (488/130 m/z) and 3TC-TP (468/119 m/z), respectively. |
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