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1
Trkola, Alexandra, Jamie Matthews, Cynthia Gordon, Tom Ketas, and John P. Moore. "A Cell Line-Based Neutralization Assay for Primary Human Immunodeficiency Virus Type 1 Isolates That Use either the CCR5 or the CXCR4 Coreceptor." Journal of Virology 73, no. 11 (November 1, 1999): 8966–74. http://dx.doi.org/10.1128/jvi.73.11.8966-8974.1999.
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ABSTRACT We describe here a cell line-based assay for the evaluation of human immunodeficiency virus type 1 (HIV-1) neutralization. The assay is based on CEM.NKR cells, transfected to express the HIV-1 coreceptor CCR5 to supplement the endogenous expression of CD4 and the CXCR4 coreceptor. The resulting CEM.NKR-CCR5 cells efficiently replicate primary HIV-1 isolates of both R5 and X4 phenotypes. A comparison of the CEM.NKR-CCR5 cells with mitogen-activated peripheral blood mononuclear cells (PBMC) in neutralization assays with sera from HIV-1-infected individuals or specific anti-HIV-1 monoclonal antibodies shows that the sensitivity of HIV-1 neutralization is similar in the two cell types. The CEM.NKR-CCR5 cell assay, however, is more convenient to perform and eliminates the donor-to-donor variation in HIV-1 replication efficiency, which is one of the principal drawbacks of the PBMC-based neutralization assay. We suggest that this new assay is suitable for the general measurement of HIV-1 neutralization by antibodies.
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Teeranaipong, Phairote, Noriaki Hosoya, Ai Kawana-Tachikawa, Takeshi Fujii, Tomohiko Koibuchi, Hitomi Nakamura, Michiko Koga, et al. "Development of a rapid cell-fusion-based phenotypic HIV-1 tropism assay." Journal of the International AIDS Society 16, no. 1 (January 2013): 18723. http://dx.doi.org/10.7448/ias.16.1.18723.
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Kalu, Amare Worku, Nigus Fikrie Telele, Shambhu G. Aralaguppe, Solomon Gebre-Selassie, Daniel Fekade, Gaetano Marrone, and Anders Sonnerborg. "Coreceptor Tropism and Maraviroc Sensitivity of Clonally Derived Ethiopian HIV-1C Strains Using an in-house Phenotypic Assay and Commonly Used Genotypic Methods." Current HIV Research 16, no. 2 (August 15, 2018): 113–20. http://dx.doi.org/10.2174/1570162x16666180515124836.
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Objectives:Genotypic Tropism Testing (GTT) tools are generally developed based on HIV-1 subtype B (HIV-1B) and used for HIV-1C as well but with a large discordance of prediction between different methods. We used an established phenotypic assay for comparison with GTT methods and for the determination of in vitro maraviroc sensitivity of pure R5-tropic and dual-tropic HIV-1C.Methods:Plasma was obtained from 58 HIV-1C infected Ethiopians. Envgp120 was cloned into a luciferase tagged NL4-3 plasmid. Phenotypic tropism was determined by in house method and the V3 sequences were analysed by five GTT methods. In vitro maraviroc sensitivity of R5-tropic and dual-tropic isolates were compared in the TZMbl cell-line.Results:The phenotypes were classified as R5 in 92.4% and dual tropic (R5X4) in 7.6% of 79 clones. The concordance between phenotype and genotype ranged from 64.7% to 84.3% depending on the GTT method. Only 46.9% of the R5 phenotypes were predicted as R5 by all GTT tools while R5X4 phenotypes were predicted as X4 by four methods, but not by Raymond’s method. All six tested phenotypic R5 clones, as well as five of six of dual tropic clones, showed a dose response to maraviroc.Conclusion:There is a high discordance between GTT methods, which underestimates the presence of R5 and overestimates X4 strains compared to a phenotypic assay. Currently available GTT algorithms should be further improved for tropism prediction in HIV-1C. Maraviroc has an in vitro activity against most HIV-1C viruses and could be considered as an alternative regimen in individuals infected with CCR5-tropic HIV-1C viruses.
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Nissley, Dwight V., Jessica Radzio, Zandrea Ambrose, Chih-Wei Sheen, Noureddine Hamamouch, Katie L. Moore, Gilda Tachedjian, and Nicolas Sluis-Cremer. "Characterization of novel non-nucleoside reverse transcriptase (RT) inhibitor resistance mutations at residues 132 and 135 in the 51 kDa subunit of HIV-1 RT." Biochemical Journal 404, no. 1 (April 26, 2007): 151–57. http://dx.doi.org/10.1042/bj20061814.
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Several rare and novel NNRTI [non-nucleoside reverse transcriptase (RT) inhibitor] resistance mutations were recently detected at codons 132 and 135 in RTs from clinical isolates using the yeast-based chimaeric TyHRT (Ty1/HIV-1 RT) phenotypic assay. Ile132 and Ile135 form part of the β7–β8 loop of HIV-1 RT (residues 132–140). To elucidate the contribution of these residues in RT structure–function and drug resistance, we constructed twelve recombinant enzymes harbouring mutations at codons 132 and 135–140. Several of the mutant enzymes exhibited reduced DNA polymerase activities. Using the yeast two-hybrid assay for HIV-1 RT dimerization we show that in some instances this decrease in enzyme activity could be attributed to the mutations, in the context of the 51 kDa subunit of HIV-1 RT, disrupting the subunit–subunit interactions of the enzyme. Drug resistance analyses using purified RT, the TyHRT assay and antiviral assays demonstrated that the I132M mutation conferred high-level resistance (>10-fold) to nevirapine and delavirdine and low-level resistance (∼2–3-fold) to efavirenz. The I135A and I135M mutations also conferred low level NNRTI resistance (∼2-fold). Subunit selective mutagenesis studies again demonstrated that resistance was conferred via the p51 subunit of HIV-1 RT. Taken together, our results highlight a specific role of residues 132 and 135 in NNRTI resistance and a general role for residues in the β7–β8 loop in the stability of HIV-1 RT.
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Hertogs, Kurt, Marie-Pierre de Béthune, Veronica Miller, Tania Ivens, Patricia Schel, Anja Van Cauwenberge, Christel Van den Eynde, et al. "A Rapid Method for Simultaneous Detection of Phenotypic Resistance to Inhibitors of Protease and Reverse Transcriptase in Recombinant Human Immunodeficiency Virus Type 1 Isolates from Patients Treated with Antiretroviral Drugs." Antimicrobial Agents and Chemotherapy 42, no. 2 (February 1, 1998): 269–76. http://dx.doi.org/10.1128/aac.42.2.269.
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ABSTRACT Combination therapy with protease (PR) and reverse transcriptase (RT) inhibitors can efficiently suppress human immunodeficiency virus (HIV) replication, but the emergence of drug-resistant variants correlates strongly with therapeutic failure. Here we describe a new method for high-throughput analysis of clinical samples that permits the simultaneous detection of HIV type 1 (HIV-1) phenotypic resistance to both RT and PR inhibitors by means of recombinant virus assay technology. HIV-1 RNA is extracted from plasma samples, and a 2.2-kb fragment containing the entire HIV-1 PR- and RT-coding sequence is amplified by nested reverse transcription-PCR. The pool of PR-RT-coding sequences is then cotransfected into CD4+ T lymphocytes (MT4) with the pGEMT3ΔPRT plasmid from which most of the PR (codons 10 to 99) and RT (codons 1 to 482) sequences are deleted. Homologous recombination leads to the generation of chimeric viruses containing PR- and RT-coding sequences derived from HIV-1 RNA in plasma. The susceptibilities of the chimeric viruses to all currently available RT and/or PR inhibitors is determined by an MT4 cell–3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based cell viability assay in an automated system that allows high sample throughput. The profile of resistance to all RT and PR inhibitors is displayed graphically in a single PR-RT-Antivirogram. This assay system facilitates the rapid large-scale phenotypic resistance determinations for all RT and PR inhibitors in one standardized assay.
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Buzon, Maria José, Itziar Erkizia, Christian Pou, Gerard Minuesa, Maria Carmen Puertas, Anna Esteve, Alfredo Castello, et al. "A non-infectious cell-based phenotypic assay for the assessment of HIV-1 susceptibility to protease inhibitors." Journal of Antimicrobial Chemotherapy 67, no. 1 (October 12, 2011): 32–38. http://dx.doi.org/10.1093/jac/dkr433.
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Carbonari, M., M. Cibati, M. Cherchi, D. Sbarigia, AM Pesce, L. Dell'Anna, A. Modica, and M. Fiorilli. "Detection and characterization of apoptotic peripheral blood lymphocytes in human immunodeficiency virus infection and cancer chemotherapy by a novel flow immunocytometric method." Blood 83, no. 5 (March 1, 1994): 1268–77. http://dx.doi.org/10.1182/blood.v83.5.1268.1268.
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Abstract We have developed a quantitative and sensitive flow cytometric method for the detection of human apoptotic lymphocytes that, unlike previously described assays, allows their identification in mixed populations of peripheral blood leukocytes as well as their immunophenotyping. Apoptotic lymphocytes are identified on the basis of peculiar light scatter changes, reflecting their smaller size and their modified nucleus/cytoplasm organization, and of the decreased expression of surface CD45 molecules. Based on these criteria, apoptotic lymphocytes generated by exposure to ionizing radiation can be easily distinguished from viable cells and from necrotic lymphocytes generated by treatment with antibody and complement. Using this assay, we reappraised the phenomenon of the in vitro apoptosis of lymphocytes from patients with human immunodeficiency virus (HIV) infection. Lymphocytes from HIV patients, unlike those from normal HIV-negative subjects, undergo apoptosis upon simple in vitro culture. We found that the percentages of lymphocytes undergoing apoptosis were significantly higher in patients with low CD4 cell counts (< 400/microL) than in patients at earlier stages (> 400 CD4 cells/microL). However, phenotypic analysis disclosed that apoptotic lymphocytes generated in these cultures were mostly CD8+ T cells and CD19+ B cells. Thus, in contrast to what has been previously suggested, the phenomenon of in vitro lymphocyte apoptosis might not be pathogenetically related to the depletion of CD4+ T cells in acquired immunodeficiency syndrome. Nevertheless, it might represent an useful marker of disease progression. Our assay allows the analysis of unfractionated peripheral blood leukocytes and thus the identification of apoptotic lymphocytes circulating in vivo. Apoptotic lymphocytes could indeed be detected in the circulation of a patient with cancer shortly after high-dose cytotoxic chemotherapy. By contrast, no apoptotic lymphocytes could be detected in vivo in patients with early or advanced HIV infection.
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Carbonari, M., M. Cibati, M. Cherchi, D. Sbarigia, AM Pesce, L. Dell'Anna, A. Modica, and M. Fiorilli. "Detection and characterization of apoptotic peripheral blood lymphocytes in human immunodeficiency virus infection and cancer chemotherapy by a novel flow immunocytometric method." Blood 83, no. 5 (March 1, 1994): 1268–77. http://dx.doi.org/10.1182/blood.v83.5.1268.bloodjournal8351268.
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We have developed a quantitative and sensitive flow cytometric method for the detection of human apoptotic lymphocytes that, unlike previously described assays, allows their identification in mixed populations of peripheral blood leukocytes as well as their immunophenotyping. Apoptotic lymphocytes are identified on the basis of peculiar light scatter changes, reflecting their smaller size and their modified nucleus/cytoplasm organization, and of the decreased expression of surface CD45 molecules. Based on these criteria, apoptotic lymphocytes generated by exposure to ionizing radiation can be easily distinguished from viable cells and from necrotic lymphocytes generated by treatment with antibody and complement. Using this assay, we reappraised the phenomenon of the in vitro apoptosis of lymphocytes from patients with human immunodeficiency virus (HIV) infection. Lymphocytes from HIV patients, unlike those from normal HIV-negative subjects, undergo apoptosis upon simple in vitro culture. We found that the percentages of lymphocytes undergoing apoptosis were significantly higher in patients with low CD4 cell counts (< 400/microL) than in patients at earlier stages (> 400 CD4 cells/microL). However, phenotypic analysis disclosed that apoptotic lymphocytes generated in these cultures were mostly CD8+ T cells and CD19+ B cells. Thus, in contrast to what has been previously suggested, the phenomenon of in vitro lymphocyte apoptosis might not be pathogenetically related to the depletion of CD4+ T cells in acquired immunodeficiency syndrome. Nevertheless, it might represent an useful marker of disease progression. Our assay allows the analysis of unfractionated peripheral blood leukocytes and thus the identification of apoptotic lymphocytes circulating in vivo. Apoptotic lymphocytes could indeed be detected in the circulation of a patient with cancer shortly after high-dose cytotoxic chemotherapy. By contrast, no apoptotic lymphocytes could be detected in vivo in patients with early or advanced HIV infection.
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Jørgensen, Louise Bruun, Terese L. Katzenstein, Jan Gerstoft, Lars R. Mathiesen, Court Pedersen, and Claus Nielsen. "Genotypic and Phenotypic Nevirapine Resistance Correlates with Virological Failure during Salvage Therapy Including Abacavir and Nevirapine." Antiviral Therapy 5, no. 3 (April 1, 1999): 187–94. http://dx.doi.org/10.1177/135965350000500302.
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Objective To study the development of resistance during 8 weeks of salvage therapy with abacavir and nevirapine in combination with other reverse transcriptase inhibitors (RTIs) and protease inhibitors (PIs). Methods Samples obtained at baseline and after 8 weeks of therapy from 16 heavily pretreated patients were analysed for genotypic and phenotypic resistance. Genotypic resistance was analysed in cell-associated DNA and plasma HIV-RNA using direct sequencing. Phenotypic resistance was analysed in a PBMC-based assay and in a recombinant virus assay. Plasma viral load was measured at baseline and after 2, 4 and 8 weeks of therapy. Results The majority of patients was genotypically and phenotypically resistant to lamivudine, abacavir, zidovudine and PIs, whereas 50% of the patients showed resistance to nevirapine at baseline in at least one of the methods used. After 8 weeks of salvage therapy, no additional development of resistance against nucleoside reverse transcriptase inhibitors and PIs could be detected. However, the amount of patients resistant to nevirapine increased to 83%. When the patients were divided into two groups according to baseline resistance against nevirapine, a significantly higher transient reduction in viral load was observed in patients with nevirapine-sensitive HIV at baseline compared to patients with resistant HIV at baseline. Conclusions The transient effect of salvage therapy including abacavir and nevirapine was due to the effect of nevirapine. The lack of effect of abacavir was most likely due to cross-resistance between abacavir and lamivudine/zidovudine used in previous treatment.
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Gasper, Melanie, David Sherman, and Donald Sodora. "Monocyte and NK cell dysfunctional responses to Mycobacteria in the context of chronic HIV-1 infection (P4377)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 183.24. http://dx.doi.org/10.4049/jimmunol.190.supp.183.24.
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Abstract Coinfection with M. tuberculosis (Mtb) and other Mycobacteria is a leading cause of morbity and mortality in HIV+ patients. While neither monocytes nor NK cells are directly infected with HIV, several of their phenotypic and functional properties become altered during chronic infection, and both cell types are important to the control of Mtb. We hypothesized that HIV-related NK cell dysfunction may result from impaired monocyte dysfunction and impaired crosstalk with NK cells, which we will evaluate in the context of Mycobacteria infection. In fact, we have observed a decrease in IL-12, an important cytokine in NK cell activation, by blood monocytes from HIV+ individuals following a 6h stimulation with M. Bovis BCG (opportunistic pathogen), compared to uninfected donors (p=0.017). In addition, while developing a flow-based functional assay to measure NK cell killing of autologous Mtb-infected monocytes, we have established that monocytes from HIV+ patients exhibit decreased phagocytosis of fluorescently-labeled BCG (p=0.016) but similar phagocytosis of Mtb compared to uninfected donors. Further, we found that NK cells from HIV+ donors exhibit decreased killing of target cells (p=0.04). Our data demonstrate a reduction in both monocyte and NK cell functionality in the same HIV+ patients and is suggestive of synergism in the dysfunction of two innate cell types with a potential for identifying novel therapeutic targets to enhance innate cellular function in HIV+ patients.
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Scully, Eileen P., Monica Gandhi, Rowena Johnston, Rebecca Hoh, Ainsley Lockhart, Curtis Dobrowolski, Amélie Pagliuzza, et al. "Sex-Based Differences in Human Immunodeficiency Virus Type 1 Reservoir Activity and Residual Immune Activation." Journal of Infectious Diseases 219, no. 7 (October 29, 2018): 1084–94. http://dx.doi.org/10.1093/infdis/jiy617.
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Abstract Plasma human immunodeficiency virus type 1 (HIV-1) RNA levels in women are lower early in untreated HIV-1 infection compared with those in men, but women have higher T-cell activation and faster disease progression when adjusted for viral load. It is not known whether these sex differences persist during effective antiretroviral therapy (ART), or whether they would be relevant for the evaluation and implementation of HIV-1 cure strategies. We prospectively enrolled a cohort of reproductive-aged women and matched men on suppressive ART and measured markers of HIV-1 persistence, residual virus activity, and immune activation. The frequency of CD4+ T cells harboring HIV-1 DNA was comparable between the sexes, but there was higher cell-associated HIV-1 RNA, higher plasma HIV-1 (single copy assay), and higher T-cell activation and PD-1 expression in men compared with women. These sex-related differences in immune phenotype and HIV-1 persistence on ART have significant implications for the design and measurement of curative interventions.
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Lin, P. F., H. Samanta, C. M. Bechtold, C. A. Deminie, A. K. Patick, M. Alam, K. Riccardi, R. E. Rose, R. J. White, and R. J. Colonno. "Characterization of siamycin I, a human immunodeficiency virus fusion inhibitor." Antimicrobial Agents and Chemotherapy 40, no. 1 (January 1996): 133–38. http://dx.doi.org/10.1128/aac.40.1.133.
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The human immunodeficiency virus (HIV) fusion inhibitor siamycin I, a 21-residue tricyclic peptide, was identified from a Streptomyces culture by using a cell fusion assay involving cocultivation of HeLa-CD4+ cells and monkey kidney (BSC-1) cells expressing the HIV envelope gp160. Siamycin I is effective against acute HIV type 1 (HIV-1) and HIV-2 infections, with 50% effective doses ranging from 0.05 to 5.7 microM, and the concentration resulting in a 50% decrease in cell viability in the absence of viral infection is 150 microM in CEM-SS cells. Siamycin I inhibits fusion between C8166 cells and CEM-SS cells chronically infected with HIV (50% effective dose of 0.08 microM) but has no effect on Sendai virus-induced fusion or murine myoblast fusion. Siamycin I does not inhibit gp120 binding to CD4 in either gp120- or CD4-based capture enzyme-linked immunosorbent assays. Inhibition of HIV-induced fusion by this compound is reversible, suggesting that siamycin I binds noncovalently. An HIV-1 resistant variant was selected by in vitro passage of virus in the presence of increasing concentrations of siamycin I. Drug susceptibility studies on a chimeric virus containing the envelope gene from the siamycin I-resistant variant indicate that resistance maps to the gp160 gene. Envelope-deficient HIV complemented with gp160 from siamycin I-resistant HIV also displayed a resistant phenotype upon infection of HeLa-CD4-LTR-beta-gal cells. A comparison of the DNA sequences of the envelope genes from the resistant and parent viruses revealed a total of six amino acid changes. Together these results indicate that siamycin I interacts with the HIV envelope protein.
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Pattacini, Laura, and Jennifer Lund. "Characterization of regulatory and conventional T cell phenotype and function in HIV exposed seronegative subjects. (170.29)." Journal of Immunology 188, no. 1_Supplement (May 1, 2012): 170.29. http://dx.doi.org/10.4049/jimmunol.188.supp.170.29.
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Abstract The role of regulatory T cells (Tregs) in preventing or facilitating HIV infection remains unclear. To address this question, we analyzed samples from 129 HIV-exposed seronegative (HESN) individuals, divided into high and low exposure groups based on partner viral load. The two groups had comparable frequencies of Tregs, but in the higher exposure group there was a trend for increased expression of the Treg suppressive markers CD39 and CTLA4. CD4+ and CD8+ T cell activation was similar in the two groups. To assess T cell function, we measured the proliferation of T cells in response to four HIV peptide pools by a CFSE-based assay. Both low and high exposure groups showed a comparable percentage of CD4+ (12.7 % versus 9.7% respectively) and CD8+T cell proliferation (3.2% versus 4.9%). To assess the Treg suppressive function, we compared the proliferation of peripheral blood mononuclear cells (PBMCs) and Treg-depleted PBMCs in response to the peptide pools. In 41% of the samples that showed proliferation in response to at least one peptide, T reg depletion induced an increased proliferation of CD4+ T cells. The suppression was similar in high and low exposure groups. In the remaining samples, Treg depletion led to a decrease in CD4+ T cell proliferation. In conclusion, our study suggests that HESN can mount an HIV-specific T cell response and further, Treg can either suppress or assist this response, though the mechanism for this difference remains under investigation.
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Rivière, Christel, Frédéric Subra, Karine Cohen-Solal, Véronique Cordette-Lagarde, Remi Letestu, Christian Auclair, William Vainchenker, and Fawzia Louache. "Phenotypic and Functional Evidence for the Expression of CXCR4 Receptor During Megakaryocytopoiesis." Blood 93, no. 5 (March 1, 1999): 1511–23. http://dx.doi.org/10.1182/blood.v93.5.1511.
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Abstract The identification of stromal cell–derived factor (SDF)-1 as a chemoattractant for human progenitor cells suggests that this chemokine and its receptor might represent critical determinants for the homing, retention, and exit of precursor cells from hematopoietic organs. In this study, we investigated the expression profile of CXCR4 receptor and the biological activity of SDF-1 during megakaryocytopoiesis. CD34+ cells from bone marrow and cord blood were purified and induced to differentiate toward the megakaryocyte lineage by a combination of stem-cell factor (SCF) and recombinant human pegylated megakaryocyte growth and development factor (PEG-rhuMGDF). After 6 days of culture, a time where mature and immature megakaryocytes were present, CD41+ cells were immunopurified and CXCR4mRNA expression was studied. High transcript levels were detected by a RNase protection assay in cultured megakaryocytes derived from cord blood CD34+ cells as well as in peripheral blood platelets. The transcript levels were about equivalent to that found in activated T cells. By flow cytometry, a large fraction (ranging from 30% to 100%) of CD41+cells showed high levels of CXCR4 antigen on their surface, its expression increasing in parallel with the CD41 antigen during megakaryocytic differentiation. CXCR4 protein was also detected on peripheral blood platelets. SDF-1 acts on megakaryocytes by inducing intracellular calcium mobilization and actin polymerization. In addition, in in vitro transmigration experiments, a significant proportion of megakaryocytes was observed to respond to this chemokine. This cell migration was inhibited by pertussis toxin, indicating coupling of this signal to heterotrimeric guanine nucleotide binding proteins. Although a close correlation between CD41a and CXCR4 expession was observed, cell surface markers as well as morphological criteria indicate a preferential attraction of immature megakaryocytes (low level of CD41a and CD42a), suggesting that SDF-1 is a potent attractant for immature megakaryocytic cells but is less active on fully mature megakaryocytes. This hypothesis was further supported by the observation that SDF-1 induced the migration of colony forming unit–megakaryocyte progenitors (CFU-MK) and the expression of activation-dependent P-selectin (CD62P) surface antigen on early megakaryocytes, although no effect was observed on mature megakaryocytes and platelets. These results indicate that CXCR4 is expressed by human megakaryocytes and platelets. Furthermore, based on the lower responses of mature megakaryocytes and platelets to SDF-1 as compared with early precursors, these data suggest a role for this chemokine in the maintenance and homing during early stages of megakaryocyte development. Moreover, because megakaryocytes are also reported to express CD4, it becomes important to reevaluate the role of direct infection of these cells by the human immunodeficiency virus (HIV)-1 in HIV-1–related thrombocytopenia.
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Rivière, Christel, Frédéric Subra, Karine Cohen-Solal, Véronique Cordette-Lagarde, Remi Letestu, Christian Auclair, William Vainchenker, and Fawzia Louache. "Phenotypic and Functional Evidence for the Expression of CXCR4 Receptor During Megakaryocytopoiesis." Blood 93, no. 5 (March 1, 1999): 1511–23. http://dx.doi.org/10.1182/blood.v93.5.1511.405k02_1511_1523.
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The identification of stromal cell–derived factor (SDF)-1 as a chemoattractant for human progenitor cells suggests that this chemokine and its receptor might represent critical determinants for the homing, retention, and exit of precursor cells from hematopoietic organs. In this study, we investigated the expression profile of CXCR4 receptor and the biological activity of SDF-1 during megakaryocytopoiesis. CD34+ cells from bone marrow and cord blood were purified and induced to differentiate toward the megakaryocyte lineage by a combination of stem-cell factor (SCF) and recombinant human pegylated megakaryocyte growth and development factor (PEG-rhuMGDF). After 6 days of culture, a time where mature and immature megakaryocytes were present, CD41+ cells were immunopurified and CXCR4mRNA expression was studied. High transcript levels were detected by a RNase protection assay in cultured megakaryocytes derived from cord blood CD34+ cells as well as in peripheral blood platelets. The transcript levels were about equivalent to that found in activated T cells. By flow cytometry, a large fraction (ranging from 30% to 100%) of CD41+cells showed high levels of CXCR4 antigen on their surface, its expression increasing in parallel with the CD41 antigen during megakaryocytic differentiation. CXCR4 protein was also detected on peripheral blood platelets. SDF-1 acts on megakaryocytes by inducing intracellular calcium mobilization and actin polymerization. In addition, in in vitro transmigration experiments, a significant proportion of megakaryocytes was observed to respond to this chemokine. This cell migration was inhibited by pertussis toxin, indicating coupling of this signal to heterotrimeric guanine nucleotide binding proteins. Although a close correlation between CD41a and CXCR4 expession was observed, cell surface markers as well as morphological criteria indicate a preferential attraction of immature megakaryocytes (low level of CD41a and CD42a), suggesting that SDF-1 is a potent attractant for immature megakaryocytic cells but is less active on fully mature megakaryocytes. This hypothesis was further supported by the observation that SDF-1 induced the migration of colony forming unit–megakaryocyte progenitors (CFU-MK) and the expression of activation-dependent P-selectin (CD62P) surface antigen on early megakaryocytes, although no effect was observed on mature megakaryocytes and platelets. These results indicate that CXCR4 is expressed by human megakaryocytes and platelets. Furthermore, based on the lower responses of mature megakaryocytes and platelets to SDF-1 as compared with early precursors, these data suggest a role for this chemokine in the maintenance and homing during early stages of megakaryocyte development. Moreover, because megakaryocytes are also reported to express CD4, it becomes important to reevaluate the role of direct infection of these cells by the human immunodeficiency virus (HIV)-1 in HIV-1–related thrombocytopenia.
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Ndhlovu, Zaza, Jacqueline Proudfoot, Daniel Kaufmann, and Bruce Walker. "Discordant immunodominant patterns of HIV-specific CD8 T cells defined by cytokine production or proliferative capacity in HIV elite controllers (39.4)." Journal of Immunology 184, no. 1_Supplement (April 1, 2010): 39.4. http://dx.doi.org/10.4049/jimmunol.184.supp.39.4.
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Abstract Several studies suggest that CTL proliferation is one of the functions specifically associated with elite control of HIV, but IFN-γ is generally used to define HIV epitope-specific CTL immunodominance. We performed multi-dimensional intra-donor analysis of HIV CTL-specific cytokine production and proliferation against panels of HLA-matched optimal epitopes. 9 elite controllers (EC) and 8 subjects with progressive infection (CP) were studied. Our analysis revealed two main immunodominance patterns defined by either predominant IFN-γ production or proliferative capacity with very different hierarchies of responses. In the EC, 40% of epitope-specific CTL that produced IFN-γ after 6 h stimulation did not proliferate whereas 47% of CTL with strong proliferative capacity did not produce IFN-γ after 6h stimulation. The contrasting patterns of immunodominance were associated with different phenotypes of memory and exhaustion markers. HIV CP usually had little HIV-specific proliferative capacity as compared to EC (21% vs 7% of tested epitopes, p=0.009). Our data indicate that immunodominance patterns of HIV-specific CD8 T cell responses differ markedly based on an IFN-γ read out assay compared to functional proliferation. The intra-individual heterogeneity of HIV-specific CTL responses shows that not all epitope specific responses are functionally similar. Assessing CTL functions at the single epitope level can facilitate identification of features critical for antiviral function.
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Lamorte, Louie, Steve Titolo, Christopher T. Lemke, Nathalie Goudreau, Jean-François Mercier, Elizabeth Wardrop, Vaibhav B. Shah, et al. "Discovery of Novel Small-Molecule HIV-1 Replication Inhibitors That Stabilize Capsid Complexes." Antimicrobial Agents and Chemotherapy 57, no. 10 (July 1, 2013): 4622–31. http://dx.doi.org/10.1128/aac.00985-13.
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ABSTRACTThe identification of novel antiretroviral agents is required to provide alternative treatment options for HIV-1-infected patients. The screening of a phenotypic cell-based viral replication assay led to the identification of a novel class of 4,5-dihydro-1H-pyrrolo[3,4-c]pyrazol-6-one (pyrrolopyrazolone) HIV-1 inhibitors, exemplified by two compounds: BI-1 and BI-2. These compounds inhibited early postentry stages of viral replication at a step(s) following reverse transcription but prior to 2 long terminal repeat (2-LTR) circle formation, suggesting that they may block nuclear targeting of the preintegration complex. Selection of viruses resistant to BI-2 revealed that substitutions at residues A105 and T107 within the capsid (CA) amino-terminal domain (CANTD) conferred high-level resistance to both compounds, implicating CA as the antiviral target. Direct binding of BI-1 and/or BI-2 to CANTDwas demonstrated using isothermal titration calorimetry and nuclear magnetic resonance (NMR) chemical shift titration analyses. A high-resolution crystal structure of the BI-1:CANTDcomplex revealed that the inhibitor bound within a recently identified inhibitor binding pocket (CANTDsite 2) between CA helices 4, 5, and 7, on the surface of the CANTD, that also corresponds to the binding site for the host factor CPSF-6. The functional consequences of BI-1 and BI-2 binding differ from previously characterized inhibitors that bind the same site since the BI compounds did not inhibit reverse transcription but stabilized preassembled CA complexes. Hence, this new class of antiviral compounds binds CA and may inhibit viral replication by stabilizing the viral capsid.
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Naumann, Nora, Suk See De Ravin, Uimook Choi, Morvarid Moayeri, Yasuhiro Ikeda, and Harry L. Malech. "Correction of Human X-Linked Chronic Granulomatous Disease (X-CGD) Following Transduction of X-CGD CD34+ Cells with a Modified RD114-Pseudotyped Simian Immunodeficiency Virus Lentiviral Vector in a NOD/SCID Mouse Xenograft Model." Blood 108, no. 11 (November 16, 2006): 3278. http://dx.doi.org/10.1182/blood.v108.11.3278.3278.
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Abstract X-linked chronic granulomatous disease (X-CGD) is an immune deficiency that results from mutations in the gp91phox subunit of the phagocyte NADPH-oxidase. We have developed a modified RD114-pseudotyped simian immunodeficiency virus (SIVmac) vector encoding human gp91phox and report here that this vector efficiently targets peripheral blood-mobilized CD34+ hematopoietic stem cells (PBSCs) from X-CGD patients, correcting the oxidase deficient phenotype. The majority of lentiviral gene transfer studies have relied on human immunodeficiency virus (HIV) 1-derived vectors pseudotyped with the vesicular stomatitis virus G protein (VSV-G) envelope. Because HIV is a human pathogen, there are theoretical benefits to considering other types of lentivectors engineered from viruses less pathogenic for humans such as SIV-based vectors. In addition, the VSV-G envelope has some toxicity for cells, limiting the titers of vector that can be used. RD114/TR-pseudotyped SIV gp91phox encoding vector particles were transiently produced following transfection of 293T cells and concentrated by high-speed centrifugation achieving stock titers of 1.5 × 10E7 infectious units/ml. PBSCs from two patients with X-CGD (P1 and P2) were cultured and transduced (multiplicity of infection 2–3) overnight four times before transplantation into sublethally irradiated NOD/SCID mice on day 5 of culture. Ex vivo transduction efficiency was 40.5% and 46%, respectively. Cells maintained in culture showed a progressive decrease in marking over the first 9 days, after which marking stabilized at 22.7% (P1) and 27.1% (P2) out to day 26 of culture with an averaged copy number of 2.2–2.4 per transduced cell at that time. Compared to healthy control, 18–19% of myeloid colonies derived from patient CD34+ cells stained positive in the nitroblue tetrazolium (NBT) test. Liquid cell cultures at 20 days were analyzed for production of reactive oxidative species using the flow cytometry dihydrorhodamine 123 (DHR) assay. Cells from P1 demonstrated 15.3% and from P2 9.1% of the activity of healthy cells. At 6 weeks after transplant, analysis of mouse bone marrow (mBM) samples revealed human cell engraftment levels of 36.8–74% (CD45+ cells). Compared to analysis of mice transplanted with healthy human PBSCs, 10.5% (P1) and 7.3% (P2) of the myeloid cells (CD13+) developing from X-CGD CD34+ cells in the chimeric mBM expressed gp91phox transgene. Human CD34+ cells were isolated from mBM and grown in liquid culture or plated for colony assays. 2.1% (P1) and 1.2% (P2) of cultured cells were gp91phox positive at day 14 with an averaged copy number of 2.3–5.2 per marked cell. 4.8% and 3.6% of colonies derived from the same cells were positive in the NBT test. DHR assay at day 21 of culture demonstrated 2.7% (P1) and 2.0% (P2) of the activity of healthy cells. Linear amplification-mediated PCR retrieved multiple unique vector insertion sites in CD34+ cells from both patients isolated from chimeric mBM, without predominance of any one clone suggesting polyclonal marking. In conclusion, these data suggest that modified RD114-pseudotyped SIVmac-based vectors may be suitable for hematopoietic gene therapy for CGD and represent an alternative to HIV-1 based vectors. Studies are ongoing to further improve vector production titers to achieve higher transduction levels of PBSCs.
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Maguire, Michael F., Rosario Guinea, Philip Griffin, Sarah Macmanus, Robert C. Elston, Josie Wolfram, Naomi Richards, et al. "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." Journal of Virology 76, no. 15 (August 1, 2002): 7398–406. http://dx.doi.org/10.1128/jvi.76.15.7398-7406.2002.
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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.
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Lee, Ah Ram, Ju-Yeon Cho, Jong Chul Kim, Mehrangiz Dezhbord, Soo Yeun Choo, Chang Hyun Ahn, Na Yeon Kim, et al. "Distinctive HBV Replication Capacity and Susceptibility to Tenofovir Induced by a Polymerase Point Mutation in Hepatoma Cell Lines and Primary Human Hepatocytes." International Journal of Molecular Sciences 22, no. 4 (February 5, 2021): 1606. http://dx.doi.org/10.3390/ijms22041606.
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Tenofovir disoproxil fumarate (TDF) has been regarded as the most potent drug for treating patients with chronic hepatitis B (CHB). However recently, viral mutations associated with tenofovir have been reported. Here, we found a CHB patient with suboptimal response after more than 4 years of TDF treatment. Clonal analysis of hepatitis B virus (HBV) isolated from sequential sera of this patient identified the seven previously reported TDF-resistant mutations (CYELMVI). Interestingly, a threonine to alanine mutation at the 301 amino acid position of the reverse-transcriptase (RT) domain, (rtT301A), was commonly accompanied with CYELMVI at a high rate (72.7%). Since the rtT301A mutation has not been reported yet, we investigated the role of this naturally occurring mutation on the viral replication and susceptibility to tenofovir in various liver cells (hepatoma cells as well as primary human hepatocytes). A cell-based phenotypic assay revealed that the rtT301A mutation dramatically impaired the replication ability with meaningful reduction in sensitivity to tenofovir in hepatoma cell lines. However, attenuated viral replication by the rtT301A mutation was significantly restored in primary human hepatocytes (PHHs). Our findings suggest that the replication capability and drug sensitivity of HBV is different between hepatoma cell lines and PHHs. Therefore, our study emphasizes that validation studies should be performed not only in the liver cancer cell lines but also in the PHHs to understand the exact viral fitness under antiviral pressure in patients.
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Miloud, Tewfik, Nathalie Dupas, and Felix Montero-Julian. "An 8-color panel for detection of human blood dendritic cells by flow cytometry (P3014)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 114.8. http://dx.doi.org/10.4049/jimmunol.190.supp.114.8.
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Abstract Dendritic cells (DCs) are antigen presenting cells capable of presenting antigen and priming a T cell response. They form a heterogeneous group of cells based on phenotype, location and function. In human blood, DCs represent less than 1% of white blood cells, and can be separated into 2 main cell subsets, namely the myeloid DCs (mDCs) and the plasmacytoid DCs (pDCs). Among the mDCs, 3 distinct cell subsets are identified: CD1c+mDCs, CD141+mDCs and CD16+mDCs. In blood, the frequency of DCs is affected in certain pathological conditions such as HIV, diabetes, asthma, chronic viral hepatitis, and graft-versus-host-disease. Thus, the detection and enumeration of different blood DC subsets is important to understand immune regulation in pathological conditions and to guide specific patient treatments. Due to the lack of specific markers for DC definition, the combination of several markers is required to allow their identification. Based on current knowledge in human DC biology, we have evaluated the expression and association of several DC markers to design an optimized 8-color panel for flow cytometry which allows for the detection of all DCs subsets in whole blood samples or peripheral blood mononuclear cells (PBMCs). This panel (CD1c/HLADR/Lineage/CD11c/CD16/Clec9A/CD123/CD45) provides an easy and robust assay to study the role of DCs in healthy donors and patient samples.
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Smith, Elizabeth B., Robert A. Ogert, David Pechter, Artjohn Villafania, Susan J. Abbondanzo, Karen Lin, Aida Rivera-Gines, Cheryl Rebsch-Mastykarz, and Frederick J. Monsma. "HIV Cell Fusion Assay." Journal of Biomolecular Screening 19, no. 1 (August 29, 2013): 108–18. http://dx.doi.org/10.1177/1087057113500074.
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The health and disease-related biology of the CXCR4 chemokine receptor presents the challenge of finding a small molecule that can bind CXCR4 and block T-cell tropic human immunodeficiency virus type 1 (HIV-1) cell entry, while preserving the ability of CXCR4 to respond to its native ligand, CXCL12. HIV entry into the host cell involves the interaction of the viral envelope glycoprotein gp120 binding to CD4, followed by a rearrangement in gp120, and subsequent interaction with the chemokine receptor CXCR4 or CCR5. These initial events can be re-created in a cell fusion assay that represents a surrogate system, mimicking the early stages of viral entry via these host cell receptors. In the current study, a T-tropic HIV cell fusion assay was established using U2OS cells expressing the envelope glycoprotein gp160 from the T-tropic HIV NL4-3 and HeLa cells expressing CD4 and CXCR4. Detection of the cell fusion event was based on a Gal4/VP16-activated β-lactamase signal and was measured by automated microscopy or laser scanning plate cytometry. Changes in morphology associated with cell fusion were combined with β-lactamase activity to generate results with robust assay statistics in both 384-well and 1536-well plates. Compounds were subsequently characterized by CXCR4 signaling assays to eliminate functional antagonists and allow the identification of a function-sparing HIV entry inhibitor.
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García Lerma, J. Gerardo, Raymond F. Schinazi, Amy S. Juodawlkis, Vincent Soriano, Yulin Lin, Kathleen Tatti, David Rimland, Thomas M. Folks, and Walid Heneine. "A Rapid Non-Culture-Based Assay for Clinical Monitoring of Phenotypic Resistance of Human Immunodeficiency Virus Type 1 to Lamivudine (3TC)." Antimicrobial Agents and Chemotherapy 43, no. 2 (February 1, 1999): 264–70. http://dx.doi.org/10.1128/aac.43.2.264.
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ABSTRACT Monitoring for lamivudine (3TC) resistance is important both for the clinical management of human immunodeficiency virus type 1 (HIV-1)-infected patients treated with 3TC and for surveillance of transmission of 3TC-resistant HIV-1. We developed a novel non-culture-based assay for the rapid analysis of phenotypic resistance to 3TC of HIV-1 in plasma. The assay measures the susceptibility of HIV-1 reverse transcriptase (RT) activity to 3TC triphosphate (3TC-TP) in plasma. RT detection was done by the Amp-RT assay, an ultrasensitive PCR-based RT assay. Under our assay conditions, we found that 5 μM 3TC-TP inhibited RT activity from wild-type (WT), zidovudine-resistant, or nevirapine-resistant HIV-1 but not from HIV-1 carrying either the M184V mutation or multidrug (MD) resistance mutations (77L/116Y/151M or 62V/75I/77L/116Y/151M). Mixing experiments showed a detection threshold of 10% 3TC-resistant virus (M184V) in a background of WT HIV-1. To validate the assay for the detection of phenotypic resistance of HIV-1 to 3TC in plasma samples, HIV-1 RT in 30 plasma specimens collected from 15 patients before and during therapy with 3TC was tested for evidence of phenotypic resistance by the Amp-RT assay. The results were compared with those of genotypic analysis. The RT in 12 samples was found to be 3TC sensitive, while the RT in 18 samples had evidence of phenotypic resistance. All 12 samples with 3TC-sensitive RT had WT genotypes at codon 184 and were retrieved before treatment with 3TC. In contrast, all 18 specimens with 3TC-resistant RT were posttherapy samples. This assay provides a simple, rapid, and reliable method for the detection of phenotypic resistance of HIV-1 to 3TC in plasma.
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Asin-Milan, Odalis, Yi Wei, Mohamed Sylla, Farida Vaisheva, Annie Chamberland, and Cécile L. Tremblay. "Performance of a clonal-based HIV-1 tropism phenotypic assay." Journal of Virological Methods 204 (August 2014): 53–61. http://dx.doi.org/10.1016/j.jviromet.2014.04.004.
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Guo, Stephanie, Adam Olm-Shipman, Andrew Walters, William R. Urciuoli, Stefanie Devito, Sergiy M. Nadtochiy, Andrew P. Wojtovich, and Paul S. Brookes. "A Cell-Based Phenotypic Assay to Identify Cardioprotective Agents." Circulation Research 110, no. 7 (March 30, 2012): 948–57. http://dx.doi.org/10.1161/circresaha.111.263715.
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Hachiya, Atsuko, Saori Aizawa-Matsuoka, Mari Tanaka, Yukiko Takahashi, Setsuko Ida, Hiroyuki Gatanaga, Yoshihiro Hirabayashi, Asato Kojima, Masashi Tatsumi, and Shinichi Oka. "Rapid and Simple Phenotypic Assay for Drug Susceptibility of Human Immunodeficiency Virus Type 1 Using CCR5-Expressing HeLa/CD4+ Cell Clone 1-10 (MAGIC-5)." Antimicrobial Agents and Chemotherapy 45, no. 2 (February 1, 2001): 495–501. http://dx.doi.org/10.1128/aac.45.2.495-501.2001.
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ABSTRACT We describe a rapid and simple novel phenotypic assay for drug susceptibility of human immunodeficiency virus type-1 (HIV-1) using a CCR5-expressing HeLa/CD4+ cell clone 1-10 (MAGIC-5). MAGIC-5 cells produced large amounts of HIV-1 in culture supernatants, which enabled us to perform the phenotypic resistance assay. Determination of HIV-1 susceptibility to various protease inhibitors (PI) and nucleoside reverse transcriptase inhibitors was completed within 15 days in T-cell-tropic (X4) and macrophage-tropic (R5) viruses using fresh plasma samples containing at least 104copies/ml. The nucleotide sequence of the envelope V3 region of HIV-1 in plasma was almost identical to that of the virus isolated by MAGIC-5 cells, suggesting a lack of selection bias in our assay. The assay variability was confined to within five-fold in all drugs examined. Accordingly, we used a 10-fold increase in the 50% inhibitory concentration as the cutoff value for viral resistance in the present assay. HIV-1 resistant to lamivudine, which was not detected by conventional genotypic assays, was isolated. In HIV-1 with PI-associated primary amino acid substitutions, our assay showed that drug resistance profiles correlated well with previously reported genotypic-assay data. Furthermore, our assay provided comprehensive results regarding PI resistance in the presence of multiple mutations. The novel assay successfully quantified the level of resistance of clinical HIV-1 isolates to a battery of anti-HIV drugs, indicating its clinical usefulness, particularly in patients who failed to respond to antiretroviral chemotherapy.
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Ayotte, Yann, François Bilodeau, Albert Descoteaux, and Steven R. LaPlante. "Fragment-Based Phenotypic Lead Discovery: Cell-Based Assay to Target Leishmaniasis." ChemMedChem 13, no. 14 (May 30, 2018): 1377–86. http://dx.doi.org/10.1002/cmdc.201800161.
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Su, Su, Dawn M. Betters, Muthalagu Ramanathan, Keyvan Keyvanfar, Aleah Smith, Xingmin Feng, Elissa Furutani, Mattias Carlsten, Andreas Lundqvist, and Richard Childs. "Optimizing Lentiviral Transduction of Human Natural Killer Cells." Blood 118, no. 21 (November 18, 2011): 4714. http://dx.doi.org/10.1182/blood.v118.21.4714.4714.
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Abstract Abstract 4714 The development of an efficient method to genetically modify natural killer (NK) cells could be used to characterize NK cell differentiation, acquisition of self-tolerance, tumor trafficking in vivo, as well as to manipulate NK cells to enhance their activity against infectious diseases and tumors. Although HIV-1 based lentiviral vectors (LVs) have been used to efficiently transfer genes into human T-cells, little data exists on LV transduction of either fresh or in vitro expanded human NK cells or its effects on NK cell phenotype and cytolytic function. In this study, we used an HIV-based LV expressing enhanced green fluorescence protein (EGFP) driven by a murine stem cell virus long terminal repeat (MSCV-LTR) promoter to transduce CD3− and CD56+ and/or CD16+ human NK cells that were either resting, IL-2 activated, or expanded in vitro using an irradiated EBV-LCL feeder cell line. We observed that resting NK cells were difficult to transduce with LVs, even at high multiplicities of infection (MOI), with transduction efficiencies (TE) in the range of only 3–14%. The efficiency of LV transduction improved when the NK cells were pre-stimulated in vitro with IL-2: TE improved to 21±0.2% in NK cells cultured for 24 hours in media containing IL-2 (200 U/mL) and 28.7±12.9% in NK cells that underwent in vitro expansion over 9 days prior to transduction using irradiated EBV-LCL feeder cells and media containing IL-2 (200U/mL). Subsequently, we evaluated incremental MOIs (3-200) to optimize LV transduction of expanded NK cells; optimal transduction was achieved using a spinoculation protocol at a MOI of 25 which resulted in the highest transduction efficiencies with the least amount of cell death. Increasing the MOI above this level resulted in a small increase in transduction, but was offset by an increase in NK cell apoptosis/death. Using a one-round, non-spinoculation protocol and an MOI of 30, we obtained a median transduction efficiency of 29% (range 16–41) with excellent retention of NK cell viability. This optimized protocol was used to transduce expanded NK cells with a LV vector encoding an shRNA targeting a region of the NK cell inhibitory receptor transcript NKG2A. Following transduction, surface expression of NKG2A decreased significantly on expanded NK cells compared to non-transduced expanded NK cells and “scramble transduced” LV controls; at a MOI of 10, the MFI of NKG2A on expanded human NK cells decreased 35% compared to non-transduced and LV transduced scramble controls (median MFI 428, 673, 659 in shRNA, non-transduced and scramble LV control transduced NK cells respectively). A comparison of transduction efficiencies using LVs expressing EGFP driven by MSCV-LTR, EF1a, and Ubi promoters showed MSCV-LTR mediated the highest level of gene expression in expanded NK cells. Transduced NK cells maintained stable EGFP transgene expression in vitro, which peaked 5 days following LV transduction and remained stable for an additional 9 days. The phenotype of lentiviral transduced NK cells was similar to non-transduced NK cells. Specifically, expression of CD56, CD16, granzyme A and B, perforin, the inhibitory receptors NKG2A, KIR3DL1, KIR3DL2, and KIR2DL1/DL2, and the activating receptors NKG2D, NCRs NKp46, and NKp30 were not altered in either fresh or expanded NK cells following LV transduction, although we did observe a significant reduction in NKp44 expression in LV transduced cells (22% compared to 50% on untransduced NK cells; 0.02). Furthermore, NK cell function, as assessed by cytokine production and cytotoxicity vs tumor targets was not altered in LV transduced NK cells. A 51Cr release cytotoxicity assay showed GFP+ NK cells, flow sorted following LV transduction of expanded NK cells, had similar cytotoxicity against K562 cells and human renal cell carcinoma cells (RCC) compared to non-transduced expanded NK cell controls (figures). In conclusion, we show that an HIV-1 based lentiviral vector driven by a MSCV-LTR, mediated efficient and stable gene transfer in IL-2 activated and in vitro expanded human NK cells. This study provides valuable insights for methods to optimize the long-term expression of LV transduced genes in human NK cells which could be used to improve their anti-tumor function in vivo. Target: K562 cells Target: RCC cell line Disclosures: No relevant conflicts of interest to declare.
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Reddy, Tejaswini P., Bijan Mahboubi, Roberto R. Rosato, Liliana Guzman-Rojas, Wei Qian, Jianying Zhou, Baek Kim, Stacy Moulder, Helen Piwnica-Worms, and Jenny C. Chang. "Abstract P5-17-04: Combined PI3K and NOS inhibition enhances efficacy of taxane-based chemotherapy in metaplastic breast cancer." Cancer Research 82, no. 4_Supplement (February 15, 2022): P5–17–04—P5–17–04. http://dx.doi.org/10.1158/1538-7445.sabcs21-p5-17-04.
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Abstract Background: Metaplastic breast cancer (MpBC) is a therapeutically chemoresistant, aggressive, and heterogeneous breast cancer variant accounting for <5% of all breast cancers. Most MpBCs harbor a triple-negative breast cancer (TNBC) phenotype, yet have a worse prognosis and decreased survival compared to TNBC. Despite its chemorefractory nature, the current mainstay of treatment for MpBC is surgery and systemic chemotherapy. Common molecular alterations found in MpBC associated with poor prognosis and worse overall survival include 1) hyperactivation of the phosphoinositide 3-kinase (PI3K) signaling pathway and 2) enhanced production of nitric oxide via inducible nitric oxide synthase (iNOS). In MpBC, both the PI3K and NOS signaling pathways may synergistically work together to enhance chemoresistance. We propose that combined inhibition of PI3K and iNOS will enhance the efficacy of taxane-based chemotherapy in MpBC. Methods: For in vitro and in vivo studies, we used MpBC cell lines (Hs578T and BT549) and TNBC/MpBC Patient-Derived Xenograft (PDX) models, respectively. For all studies, we used pan-NOS inhibitor NG-monomethyl-l-arginine (L-NMMA, L), PI3K inhibitor alpelisib (A), and docetaxel (D). Immunohistochemistry (IHC), Western Blotting (WB), Cell Proliferation Assays, Flow Cytometry (to evaluate cell death and cell cycle distribution analysis), and HIV reverse transcriptase-based dNTP assay to quantify dNTPs were performed. For in vivo studies, five MpBC PDX models were implanted into the mammary fat pad of NSG mice and they received single therapy (vehicle control, L, A, D), dual therapy (D+A, D+L), or triple combination therapy (D+A+L). Tumor volumes were recorded twice weekly. Results: 66% (4/6) MpBC and 33% (7/21) TNBC PDX models had double-positive IHC staining of both iNOS and p-Akt (Ser473), supporting the concept that both signaling pathways are typically activated in MpBC tumors, relative to non-metaplastic TNBC tumors. Apoptosis and cell proliferation analysis found that MpBC cell lines treated with triple-combination (D+A+L) had an increased number of apoptotic cells and decreased cell proliferation relative to MpBC cells treated with dual combination (L+A), or single treatment (vehicle, D, A, or L). Cell cycle distribution analysis of treated MpBC cells found that in a time-dependent manner, there was a substantial decrease in the % of MpBC cells in S-phase and an increase in the % cells in G2/M cell cycle arrest due to dual and triple combination. This result was supported by dNTP quantification analysis revealing that combined PI3K and NOS inhibition induced greater nucleotide depletion within 8 hours of treatment relative to single treatment in MpBC cells. WB analysis revealed that dual/triple combination therapy in MpBC cells resulted in an enhanced DNA damage response signaling relative to single treatment, as indicated with increased expression of γ-H2AX, p-Chk1, p-Chk2, p-P53 (Ser15 and 20), and p21. Pro-survival PI3K signaling pathway was activated in response to docetaxel treatment alone in MpBC cells, but its activation was significantly reduced when docetaxel was coupled with PI3K and NOS inhibition. In vivo studies revealed that triple combination therapy significantly reduced tumor volume and improved survival proportions compared to dual/single therapy and vehicle control. Conclusions: The present data suggest that combined PI3K and NOS inhibition enhances docetaxel-mediated DNA damage by depleting nucleotide pools, leading to enhanced DNA damage response, growth arrest, and apoptosis. Ongoing studies are investigating how docetaxel coupled with PI3K and NOS inhibition influences DNA repair signaling and MpBC metastatic capacity. The addition of PI3K and NOS inhibitors to taxane-based chemotherapy may be a novel therapeutic strategy for aggressive MpBCs. Citation Format: Tejaswini P Reddy, Bijan Mahboubi, Roberto R. Rosato, Liliana Guzman-Rojas, Wei Qian, Jianying Zhou, Baek Kim, Stacy Moulder, Helen Piwnica-Worms, Jenny C. Chang. Combined PI3K and NOS inhibition enhances efficacy of taxane-based chemotherapy in metaplastic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-17-04.
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Azegrouz, Hind, Gopal Karemore, Alberto Torres, Carlos M. Alaíz, Ana M. Gonzalez, Pedro Nevado, Alvaro Salmerón, et al. "Cell-Based Fuzzy Metrics Enhance High-Content Screening (HCS) Assay Robustness." Journal of Biomolecular Screening 18, no. 10 (September 17, 2013): 1270–83. http://dx.doi.org/10.1177/1087057113501554.
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High-content screening (HCS) allows the exploration of complex cellular phenotypes by automated microscopy and is increasingly being adopted for small interfering RNA genomic screening and phenotypic drug discovery. We introduce a series of cell-based evaluation metrics that have been implemented and validated in a mono-parametric HCS for regulators of the membrane trafficking protein caveolin 1 (CAV1) and have also proved useful for the development of a multiparametric phenotypic HCS for regulators of cytoskeletal reorganization. Imaging metrics evaluate imaging quality such as staining and focus, whereas cell biology metrics are fuzzy logic–based evaluators describing complex biological parameters such as sparseness, confluency, and spreading. The evaluation metrics were implemented in a data-mining pipeline, which first filters out cells that do not pass a quality criterion based on imaging metrics and then uses cell biology metrics to stratify cell samples to allow further analysis of homogeneous cell populations. Use of these metrics significantly improved the robustness of the monoparametric assay tested, as revealed by an increase in Z′ factor, Kolmogorov-Smirnov distance, and strict standard mean difference. Cell biology evaluation metrics were also implemented in a novel supervised learning classification method that combines them with phenotypic features in a statistical model that exceeded conventional classification methods, thus improving multiparametric phenotypic assay sensitivity.
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Willis, Clinton, Johanna Nyffeler, and Joshua Harrill. "Phenotypic Profiling of Reference Chemicals across Biologically Diverse Cell Types Using the Cell Painting Assay." SLAS DISCOVERY: Advancing the Science of Drug Discovery 25, no. 7 (June 17, 2020): 755–69. http://dx.doi.org/10.1177/2472555220928004.
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Cell Painting is a high-throughput phenotypic profiling assay that uses fluorescent cytochemistry to visualize a variety of organelles and high-content imaging to derive a large number of morphological features at the single-cell level. Most Cell Painting studies have used the U-2 OS cell line for chemical or functional genomics screening. The Cell Painting assay can be used with many other human-derived cell types, given that the assay is based on the use of fluoroprobes that label organelles that are present in most (if not all) human cells. Questions remain, however, regarding the optimization steps required and overall ease of deployment of the Cell Painting assay to novel cell types. Here, we used the Cell Painting assay to characterize the phenotypic effects of 14 phenotypic reference chemicals in concentration–response screening mode across six biologically diverse human-derived cell lines (U-2 OS, MCF7, HepG2, A549, HTB-9 and ARPE-19). All cell lines were labeled using the same cytochemistry protocol, and the same set of phenotypic features was calculated. We found it necessary to optimize image acquisition settings and cell segmentation parameters for each cell type, but did not adjust the cytochemistry protocol. For some reference chemicals, similar subsets of phenotypic features corresponding to a particular organelle were associated with the highest-effect magnitudes in each affected cell type. Overall, for certain chemicals, the Cell Painting assay yielded qualitatively similar biological activity profiles among a group of diverse, morphologically distinct human-derived cell lines without the requirement for cell type–specific optimization of cytochemistry protocols.
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Olivares-Ramírez, J. M., V. M. Ovando-Medina, A. Ortíz-Verdín, D. M. Amaya-Cruz, J. Coronel-Hernandez, A. Marroquín, and A. Dector. "Lateral flow assay HIV-based microfluidic blood fuel cell." Journal of Physics: Conference Series 1119 (November 2018): 012022. http://dx.doi.org/10.1088/1742-6596/1119/1/012022.
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Xu, Hong-Tao, Yudong Quan, Susan M. Schader, Maureen Oliveira, Tamara Bar-Magen, and Mark A. Wainberg. "The M230L Nonnucleoside Reverse Transcriptase Inhibitor Resistance Mutation in HIV-1 Reverse Transcriptase Impairs Enzymatic Function and Viral Replicative Capacity." Antimicrobial Agents and Chemotherapy 54, no. 6 (March 22, 2010): 2401–8. http://dx.doi.org/10.1128/aac.01795-09.
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ABSTRACT The M230L mutation in HIV-1 reverse transcriptase (RT) is associated with resistance to first-generation nonnucleoside reverse transcriptase inhibitors (NNRTIs). The present study was designed to determine the effects of M230L on enzyme function, viral replication capacity (RC), and the extent to which M230L might confer resistance to the second-generation NNRTI etravirine (ETR) as well as to the first-generation NNRTIs efavirenz (EFV) and nevirapine (NVP). Phenotyping assays with TZM-bl cells confirmed that M230L conferred various degrees of resistance to each of the NNRTIs tested. Recombinant viruses containing M230L displayed an 8-fold decrease in RC compared to that of the parental wild-type (WT) virus. Recombinant HIV-1 WT and M230L mutant RT enzymes were purified; and both biochemical and cell-based phenotypic assays confirmed that M230L conferred resistance to each of EFV, NVP, and ETR. RT that contained M230L was also deficient in regard to each of minus-strand DNA synthesis, both DNA- and RNA-dependent polymerase activities, processivity, and RNase H activity, suggesting that this mutation contributes to diminished viral replication kinetics.
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Kim, Sanggu, Robert E. Donahue, Gajendra Suryavanshi, Balamurugan Arumugam, Aylin C. Bonifacino, Yiming Xie, Otto Yang, Cynthia E. Dunbar, and Irvin Chen. "HSC Clonal Dynamics after T-Cell Depletion in a Nonhuman Primate Model of Lentiviral Gene Therapy." Blood 128, no. 22 (December 2, 2016): 3702. http://dx.doi.org/10.1182/blood.v128.22.3702.3702.
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Abstract Background: Hematopoietic stem cell (HSC) gene therapy is a new treatment paradigm that can potentially provide lifelong protection against HIV-1 infection. The basic principle is to genetically modify a patient's own HSC such that the progeny, including CD4+T cells and macrophages, are resistant to HIV-1. Given the expected low levels of the anti-HIV gene-marked cells in clinical studies, it is critical to understand how the vast number of HSC, each bearing a unique phenotype, and their mature progeny contribute to maintaining homeostatic regulation in HIV gene therapy settings. We have recently published novel and detailed insights about the long-term behavior patterns of individual HSC followed for 4 to 12 years post-transplant in our nonhuman primate (NHP) model of lentiviral gene therapy, revealing for the first time in primates the precise time point of HSC repopulation and the functional heterogeneity of HSCs (Kim, Cell Stem Cell, 2014; Goyal, BMC Biology, 2015). Consistent clonal behavior patterns have been observed in human gene therapy (Biasco, Cell Stem Cell, 2016), demonstrating the clinical relevance of our data. Here, in order to develop a systems-level understanding of HSC clonal dynamics in anti-HIV therapy settings requiring efficient immune recovery from T lymphopenia, we further analyzed HSC clonal repopulation after T-cell depletion in one of our NHP animals. Our study generated systems-level datasets useful for understanding the parameters of T-cell repopulation and homeostatic regulation in anti-HIV therapy, as well as other therapies requiring efficient immune recovery from disease- or treatment-induced T lymphopenia. Subjects and Methods: The HSC clonal behaviors in animal 95E132 have been well characterized for 16 years post-transplant. At the 16-year time point, this animal was treated with 8 doses (25 ug/kg/dose) of an anti-CD3e immunotoxin over 4 days. Hematopoietic recovery after T-cell depletion was monitored by a complete blood count, multi-color flow cytometry analysis, TCRv_ spectratyping, and a high-throughput lentiviral-tagging assay (Kim, Journal of Virology, 2010). HSC subtypes and their clonal behaviors were determined based on the clonal profiling of blood lineages, including CD4, CD8, CD20, CD14, and CD18 cells, over time. Results: The peripheral CD3+ T cells recovered in 2-3 months (Fig. 1B). Cytotoxic (CD8+) T-cell recovery was faster than that of T-helper cells. Effector and memory T cells expanded promptly after T-cell depletion, whereas the na•ve T cells had not recovered more than one year after CD3e-immunotoxin treatment. Clonal profiling analysis revealed a few dominant clones in the recovered T-cell compartment, while showing no notable clonal fluctuation in the CD18+ granulocytes over time (Fig. 1C-D). TCRv_ spectratyping showed a skewed T-cell receptor repertoire even a year after immunotoxin treatment. Conclusion: Our data can bolster our understanding of hematopoietic regulation in patients recovering from T lymphopenia. The data showed skewed a T-cell receptor repertoire and clonal dominance in the recovered T-cells after immunotoxin treatment in an aged animal, suggesting that T-cell recovery had occurred primarily as a result of peripheral T-cell expansion. A systems-level, clonal dynamics study of this extreme form of homeostatic regulation provides unique opportunities to identify and characterize the regenerative pathways, as well as the obstacles, that emerge in the process of restoring homeostasis after disease- or treatment-induced T-cell depletion. Figure 1 A. Hematopoietic recovery after CD3e-immunotoxin treatment (red arrow) was assessed at the clonal level. B. CD3+ T cells were effectively ablated by immunotoxin treatment. The T-cell percentage rebounded to the normal range by 2-3 months. C. A ternary diagram showing Myeloid-biased (black circles), Balanced (red circles), and Lymphoid-biased (green circles) HSC subtype clones. The relative position of a circle in the diagram indicates the lineage output potential toward G/M (granulocyte/monocyte), B-cell, and T-cell. The size of a circle indicates the relative frequency of a clone. D. The relative frequencies of the HSC clones in T-cell, B-cell, Granulocytes, and monocytes are shown at 1 month before (-1M), 1 month (1M) and 3 month (3M) immunotoxin treatment. The individual clones (circles) are located at the identical positions in C. Figure 1. A. Hematopoietic recovery after CD3e-immunotoxin treatment (red arrow) was assessed at the clonal level. B. CD3+ T cells were effectively ablated by immunotoxin treatment. The T-cell percentage rebounded to the normal range by 2-3 months. C. A ternary diagram showing Myeloid-biased (black circles), Balanced (red circles), and Lymphoid-biased (green circles) HSC subtype clones. The relative position of a circle in the diagram indicates the lineage output potential toward G/M (granulocyte/monocyte), B-cell, and T-cell. The size of a circle indicates the relative frequency of a clone. D. The relative frequencies of the HSC clones in T-cell, B-cell, Granulocytes, and monocytes are shown at 1 month before (-1M), 1 month (1M) and 3 month (3M) immunotoxin treatment. The individual clones (circles) are located at the identical positions in C. Disclosures Dunbar: GSK/Novartis: Research Funding. Chen:Calimmune: Membership on an entity's Board of Directors or advisory committees.
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Stacey, Peter, Anne Mai Wassermann, Laura Kammonen, Emma Impey, Anna Wilbrey, and Darren Cawkill. "Plate-Based Phenotypic Screening for Pain Using Human iPSC-Derived Sensory Neurons." SLAS DISCOVERY: Advancing the Science of Drug Discovery 23, no. 6 (March 16, 2018): 585–96. http://dx.doi.org/10.1177/2472555218764678.
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Screening against a disease-relevant phenotype to identify compounds that change the outcome of biological pathways, rather than just the activity of specific targets, offers an alternative approach to find modulators of disease characteristics. However, in pain research, use of in vitro phenotypic screens has been impeded by the challenge of sourcing relevant neuronal cell types in sufficient quantity and developing functional end-point measurements with a direct disease link. To overcome these hurdles, we have generated human induced pluripotent stem cell (hiPSC)–derived sensory neurons at a robust production scale using the concept of cryopreserved “near-assay-ready” cells to decouple complex cell production from assay development and screening. hiPSC sensory neurons have then been used for development of a 384-well veratridine-evoked calcium flux assay. This functional assay of neuronal excitability was validated for phenotypic relevance to pain and other hyperexcitability disorders through screening a small targeted validation compound subset. A 2700-compound chemogenomics screen was then conducted to profile the range of target-based mechanisms able to inhibit veratridine-evoked excitability. This report presents the assay development, validation, and screening data. We conclude that high-throughput-compatible pain-relevant phenotypic screening with hiPSC sensory neurons is feasible and ready for application for the identification of new targets, pathways, mechanisms of action, and compounds for modulating neuronal excitability.
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Weinheimer, S., L. Discotto, J. Friborg, H. Yang, and R. Colonno. "Atazanavir Signature I50L Resistance Substitution Accounts for Unique Phenotype of Increased Susceptibility to Other Protease Inhibitors in a Variety of Human Immunodeficiency Virus Type 1 Genetic Backbones." Antimicrobial Agents and Chemotherapy 49, no. 9 (September 2005): 3816–24. http://dx.doi.org/10.1128/aac.49.9.3816-3824.2005.
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ABSTRACT Substitution of leucine for isoleucine at residue 50 (I50L) of human immunodeficiency virus (HIV) protease is the signature substitution for atazanavir (ATV) resistance. A unique phenotypic profile has been associated with viruses containing the I50L substitution, which produces ATV-specific resistance and increased susceptibility to most other approved HIV protease inhibitors (PIs). The basis for this unique phenotype has not been clearly elucidated. In this report, a direct effect of I50L on the susceptibility to the PI class is described. Cell-based protease assays using wild-type and PI-resistant proteases from laboratory and clinical isolates and in vitro antiviral assays were used to demonstrate a strong concordance between changes in PI susceptibility at the level of protease inhibition and changes in susceptibility observed at the level of virus infection. The results show that the induction of ATV resistance and increased susceptibility to other PIs by the I50L substitution is likely determined at the level of protease inhibition. Moreover, the I50L substitution functions to increase PI susceptibility even in the presence of other primary and secondary PI resistance substitutions. These findings may have implications regarding the optimal sequencing of PI therapies necessary to preserve PI treatment options of patients with ATV-resistant HIV infections.
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Wu, Shouli, Pingping Yan, Yansheng Yan, Lijun Qiu, and Meirong Xie. "A single-loop recombinant pseudotyped-virus-based assay to detect HIV-1 phenotypic resistance." Archives of Virology 160, no. 6 (March 22, 2015): 1385–95. http://dx.doi.org/10.1007/s00705-015-2386-2.
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Xu, Hongtao, Yudong Quan, Bluma G. Brenner, Tamara Bar-Magen, Maureen Oliveira, Susan M. Schader, and Mark A. Wainberg. "Human Immunodeficiency Virus Type 1 Recombinant Reverse Transcriptase Enzymes Containing the G190A and Y181C Resistance Mutations Remain Sensitive to Etravirine." Antimicrobial Agents and Chemotherapy 53, no. 11 (August 24, 2009): 4667–72. http://dx.doi.org/10.1128/aac.00800-09.
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ABSTRACT Etravirine (ETR) is a second-generation nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) active against common human immunodeficiency virus type 1 (HIV-1) drug-resistant strains. This study was designed to determine the extent to which each of the Y181C or G190A mutations in RT might confer resistance to ETR and other members of the NNRTI family of drugs. Recombinant HIV-1 RT enzymes containing either the Y181C or the G190A mutation, or both mutations in tandem, were purified. Both RNA- and DNA-dependent DNA polymerase assays were performed in order to determine the extent to which each of these mutations might confer resistance in cell-free biochemical assays against each of ETR, efavirenz, and nevirapine. Both the biochemical and the cell-based phenotypic assays confirmed the susceptibility of G190A-containing enzymes and viruses to ETR. The results of this study indicate that the G190A mutation is not associated with resistance to ETR.
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Kint, Sam, Wim Van Criekinge, Linos Vandekerckhove, Winnok H. De Vos, Karol Bomsztyk, Diane S. Krause, and Oleg Denisenko. "Single cell epigenetic visualization assay." Nucleic Acids Research 49, no. 8 (January 28, 2021): e43-e43. http://dx.doi.org/10.1093/nar/gkab009.
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Abstract Characterization of the epigenetic status of individual cells remains a challenge. Current sequencing approaches have limited coverage, and it is difficult to assign an epigenetic status to the transcription state of individual gene alleles in the same cell. To address these limitations, a targeted microscopy-based epigenetic visualization assay (EVA) was developed for detection and quantification of epigenetic marks at genes of interest in single cells. The assay is based on an in situ biochemical reaction between an antibody-conjugated alkaline phosphatase bound to the epigenetic mark of interest, and a 5′-phosphorylated fluorophore-labeled DNA oligo tethered to a target gene by gene-specific oligonucleotides. When the epigenetic mark is present at the gene, phosphate group removal by the phosphatase protects the oligo from λ-exonuclease activity providing a quantitative fluorescent readout. We applied EVA to measure 5-methylcytosine (5mC) and H3K9Ac levels at different genes and the HIV-1 provirus in human cell lines. To link epigenetic marks to gene transcription, EVA was combined with RNA-FISH. Higher 5mC levels at the silenced compared to transcribed XIST gene alleles in female somatic cells validated this approach and demonstrated that EVA can be used to relate epigenetic marks to the transcription status of individual gene alleles.
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Rex, Elizabeth B., Suzie Kim, Jake Wiener, Navin L. Rao, Marcos E. Milla, and Daniel DiSepio. "Phenotypic Approaches to Identify Inhibitors of B Cell Activation." Journal of Biomolecular Screening 20, no. 7 (May 6, 2015): 876–86. http://dx.doi.org/10.1177/1087057115585724.
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An EPIC label-free phenotypic platform was developed to explore B cell receptor (BCR) and CD40R-mediated B cell activation. The phenotypic assay measured the association of RL non-Hodgkin’s lymphoma B cells expressing lymphocyte function-associated antigen 1 (LFA-1) to intercellular adhesion molecule 1 (ICAM-1)-coated EPIC plates. Anti-IgM (immunoglobulin M) mediated BCR activation elicited a response that was blocked by LFA-1/ICAM-1 specific inhibitors and a panel of Bruton’s tyrosine kinase (BTK) inhibitors. LFA-1/ICAM-1 association was further increased on coapplication of anti-IgM and mega CD40L when compared to individual application of either. Anti-IgM, mega CD40L, or the combination of both displayed distinct kinetic profiles that were inhibited by treatment with a BTK inhibitor. We also established a FLIPR-based assay to measure B cell activation in Ramos Burkitt’s lymphoma B cells and an RL cell line. Anti-IgM-mediated BCR activation elicited a robust calcium response that was inhibited by a panel of BTK inhibitors. Conversely, CD40R activation did not elicit a calcium response in the FLIPR assay. Compared to the FLIPR, the EPIC assay has the propensity to identify inhibitors of both BCR and CD40R-mediated B cell activation and may provide more pharmacological depth or novel mechanisms of action for inhibition of B cell activation.
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Pirounaki, Maria, Nancy A. Vander Heyden, Max Arens, and Lee Ratner. "Rapid phenotypic drug susceptibility assay for HIV-1 with a CCR5 expressing indicator cell line." Journal of Virological Methods 85, no. 1-2 (March 2000): 151–61. http://dx.doi.org/10.1016/s0166-0934(99)00163-9.
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Qari, Shoukat H., Mark Winters, Anne-Mieke Vandamme, Thomas Merigan, and Walid Heneine. "A Rapid Phenotypic Assay for Detecting Multiple Nucleoside Analogue Reverse Transcriptase Inhibitor-Resistant HIV-1 in Plasma." Antiviral Therapy 7, no. 2 (February 2002): 131–39. http://dx.doi.org/10.1177/135965350200700207.
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Zidovudine and other nucleoside analogue reverse transcriptase inhibitors (NRTIs), like zalcitabine and didanosine used for treatment of individuals infected with HIV-1, can select for viruses with Q151M and other associated mutations (for example, A62V, S68G, V75I, F77L, F116Y) in the reverse transcriptase (RT) enzyme. These mutations confer resistance to multiple nucleoside analogues, and thereby compromise the efficacy of this class of drugs. Presently available phenotypic assays for detection of multiple nucleoside analogue resistant (MNR) HIV-1 require testing for each NRTI individually. Here we report an enzymatic RT assay that uses resistance to zidovudine triphosphate (zidovudine-TP) as a diagnostic biochemical marker of MNR HIV-1. This assay exploits the different biochemical mechanisms for zidovudine-resistance conferred by either Q151M or T215Y/F mutations and the inability of conventional RT assays to detect T215Y/F-associated zidovudine resistance. The assay detects RT activity directly in plasma by using Amp-RT, an ultra-sensitive PCR-based RT assay. We show that enzymatic resistance to zidovudine-TP is specific to MNR RT and is distinguishable from both wild-type (WT) and RT containing classical zidovudine-resistant mutations (D67N, K70R, T215Y/F, K219Q). Compared to WT, MNR HIV-1 RT had 5- to 36-fold increases in the concentration of drug required to inhibit 50% (IC50) of RT activity, depending on the presence of Q151M alone or with additional MNR mutations. A screening assay utilizing 1 μM zidovudine-TP was developed and validated on 14 reference isolates, 37 plasma specimens, and seven patient-derived viruses. Twenty-three specimens were found to have reduced susceptibility to zidovudine-TP, and all had Q151M. In contrast, 21 specimens were sensitive to zidovudine-TP, of which 12 had WT genotypes, four had T215Y/F, and five had T69S-insertions along with T215Y/F mutations. This RT-based phenotypic assay provides a specific and rapid tool for the direct identification and monitoring of Q151M-associated MNR HIV-1 in plasma.
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Swamidass, S. Joshua, Constantino N. Schillebeeckx, Matthew Matlock, Mark R. Hurle, and Pankaj Agarwal. "Combined Analysis of Phenotypic and Target-Based Screening in Assay Networks." Journal of Biomolecular Screening 19, no. 5 (February 21, 2014): 782–90. http://dx.doi.org/10.1177/1087057114523068.
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Small-molecule screens are an integral part of drug discovery. Public domain data in PubChem alone represent more than 158 million measurements, 1.2 million molecules, and 4300 assays. We conducted a global analysis of these data, building a network of assays and connecting the assays if they shared nonpromiscuous active molecules. This network spans both phenotypic and target-based screens, recapitulates known biology, and identifies new polypharmacology. Phenotypic screens are extremely important for drug discovery, contributing to the discovery of a large proportion of new drugs. Connections between phenotypic and biochemical, target-based screens can suggest strategies for repurposing both small-molecule and biologic drugs. For example, a screen for molecules that prevent cell death from a mutated version of superoxide-dismutase is linked with ALOX15. This connection suggests a therapeutic role for ALOX15 inhibitors in amyotrophic lateral sclerosis. An interactive version of the network is available online ( http://swami.wustl.edu/flow/assay_network.html ).
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Kramer, Susanne, Peter Buontempo, Sony Agrawal, and Robert Ralston. "Imaging-Based Assay for Identification and Characterization of Inhibitors of CXCR4-Tropic HIV-1 Envelope-Dependent Cell-Cell Fusion." Journal of Biomolecular Screening 16, no. 6 (April 7, 2011): 668–75. http://dx.doi.org/10.1177/1087057111403480.
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Infection of certain cell types by HIV results in formation of syncytia. This process can be blocked by antibodies or compounds that prevent interaction of viral envelope protein with host cell receptors. Here the authors describe an automated imaging-based assay for inhibitors of cell-cell fusion mediated by interaction of HIV gp120 with CXCR4 coreceptor. The assay quantifies syncytia formation between U87MG astrocytoma cells constitutively expressing CD4/CXCR4 and morphologically distinct Jurkat T lymphoma cells inducibly expressing HIV env. Each cell type was differentially labeled with vital dyes. Fusion was quantified by measuring size, shape, and color of Jurkat cells and Jurkat-harboring cell syncytia. Dose–response experiments with reference inhibitors AMD 3100 and KRH-1636 yielded potencies consistent with those obtained using standard antiviral assays. This assay complements virus-based infectivity assays for identification of inhibitors of membrane fusion events triggered by interaction of HIV gp120 with host CXCR4.
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Honarnejad, Kamran, Achim K. Kirsch, Alexander Daschner, Aleksandra Szybinska, Jacek Kuznicki, and Jochen Herms. "FRET-Based Calcium Imaging." Journal of Biomolecular Screening 18, no. 10 (November 12, 2013): 1309–20. http://dx.doi.org/10.1177/1087057113502672.
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Perturbed intracellular store calcium homeostasis is suggested to play a major role in the pathophysiology of Alzheimer disease (AD). A number of mechanisms have been suggested to underlie the impairment of endoplasmic reticulum calcium homeostasis associated with familial AD-linked presenilin 1 mutations (FAD-PS1). Without aiming at specifically targeting any of those pathophysiological mechanisms in particular, we rather performed a high-throughput phenotypic screen to identify compounds that can reverse the exaggerated agonist-evoked endoplasmic reticulum calcium release phenotype in HEK293 cells expressing FAD-PS1. For that purpose, we developed a fully automated high-throughput calcium imaging assay using a fluorescence resonance energy transfer–based calcium indicator at single-cell resolution. This novel robust assay offers a number of advantages compared with the conventional calcium measurement screening technologies. The assay was employed in a large-scale screen with a library of diverse compounds comprising 20,000 low-molecular-weight molecules, which resulted in the identification of 52 primary hits and 4 lead structures. In a secondary assay, several hits were found to alter the amyloid β (Aβ) production. In view of the recent failure of AD drug candidates identified by target-based approaches, such a phenotypic drug discovery paradigm may present an attractive alternative for the identification of novel AD therapeutics.
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Sáez-Cirión, Asier, So Youn Shin, Pierre Versmisse, Françoise Barré-Sinoussi, and Gianfranco Pancino. "Ex vivo T cell–based HIV suppression assay to evaluate HIV-specific CD8+ T-cell responses." Nature Protocols 5, no. 6 (May 13, 2010): 1033–41. http://dx.doi.org/10.1038/nprot.2010.73.
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Tjaden, Amelie, Apirat Chaikuad, Eric Kowarz, Rolf Marschalek, Stefan Knapp, Martin Schröder, and Susanne Müller. "Image-Based Annotation of Chemogenomic Libraries for Phenotypic Screening." Molecules 27, no. 4 (February 21, 2022): 1439. http://dx.doi.org/10.3390/molecules27041439.
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Phenotypical screening is a widely used approach in drug discovery for the identification of small molecules with cellular activities. However, functional annotation of identified hits often poses a challenge. The development of small molecules with narrow or exclusive target selectivity such as chemical probes and chemogenomic (CG) libraries, greatly diminishes this challenge, but non-specific effects caused by compound toxicity or interference with basic cellular functions still pose a problem to associate phenotypic readouts with molecular targets. Hence, each compound should ideally be comprehensively characterized regarding its effects on general cell functions. Here, we report an optimized live-cell multiplexed assay that classifies cells based on nuclear morphology, presenting an excellent indicator for cellular responses such as early apoptosis and necrosis. This basic readout in combination with the detection of other general cell damaging activities of small molecules such as changes in cytoskeletal morphology, cell cycle and mitochondrial health provides a comprehensive time-dependent characterization of the effect of small molecules on cellular health in a single experiment. The developed high-content assay offers multi-dimensional comprehensive characterization that can be used to delineate generic effects regarding cell functions and cell viability, allowing an assessment of compound suitability for subsequent detailed phenotypic and mechanistic studies.
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Conway, Brian, and Francisco J. Diaz-Mitoma. "Virological and Immunological Aspects of AIDS Pathogenesis." Canadian Journal of Infectious Diseases 5, suppl e (1994): 13E—18E. http://dx.doi.org/10.1155/1994/157142.
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The most common and serious problem associated with long term antiretroviral therapy is waning efficacy over time. To date. a number of studies has suggested an association between drug resistance and clinical deterioration. However. a precise causal relationship has yet to be demonstrated. In a large American clinical trial. resistance to zidovudine (ZDV) was predictive of subsequent disease progression if this therapy was continued. Surprisingly. this was also predictive of deterioration if therapy was changed to didanosine (ddl). This suggests that other factors (perhaps virological and immunological) which may be present in addition to resistance. were as important (if not more so) in predicting clinical outcomes. It is likely that viral load. resistance. viral phenotype and alterations in immune function interact in this regard. Proper· studies may allow us to determine a “threshold” for a composite virological and immunological parameter beyond which disease progression will occur. As more antiretroviral agents become available. we will be in a position to intervene to “improve” laboratory markers and monitor them prospectively. potentially to maintain clinical latency for an indefinite period of time. In the authors' laboratories, a quantitative polymerase chain reaction assay for the evaluation of circulating proviral load has been developed. In an initial study of 70 patients. proviral load/ 106CD4 cells was clearly associated with the severity of immune disease. with up to 9.6% of cells being infected in subjects with CD4 cell counts below 200/µL. However. large variability in proviral load among individuals with comparable or dissimilar CD4 cell counts precludes the use of this measurement as an individual marker of the severity of immune disease. More recent work evaluated the combined use of proviral load (expressed as a dichotomous variable based on values above or below one copy/a03CD4 cells) and resistance in a prospective fashion. In five patients with high proviral loads and isolates resistant to their current therapy. a mean decrease of 72 CD4 cells/µL was observed over 12 months of observation. In contrast. in six patients with low proviral loads and sensitive isolates. there was a mean increase of 43 CD4 cells/µL. It appears that virological markers are associated with immune disease progression in this small cohort of patients. The association appears most marked when the two virological parameters are considered together rather than individually. The association is not always a tight one. and this may relate to a number of unmeasured factors. including viral phenotype. plasma viremia. and the immune response to HIV infection. Additional work incorporating these parameters into analysis is currently underway in the authors’ centre.
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Pires, Antonio, Jeffrey Pido-Lopez, Graeme Moyle, Brian Gazzard, Frances Gotch, and Nesrina Imami. "Enhanced T-Cell Maturation, Differentiation and Function in HIV-1-Infected Individuals after Growth Hormone and Highly Active Antiretroviral Therapy." Antiviral Therapy 9, no. 1 (January 2004): 67–75. http://dx.doi.org/10.1177/135965350400900110.
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Background Strong virus-specific helper and cytotoxic T-cell responses correlate with non-progression during HIV-1 infection. Administration of antiretroviral therapy (ART) during the chronic phases of HIV-1 infection fails to restore these responses in most patients. Design and methods We assessed the changes in immune function of 12 HIV-1-positive individuals treated with ART for over 4 years, who received 4 mg/day of recombinant human growth hormone (rhGH) for 12 weeks and were then randomized into groups receiving either placebo, twice weekly or alternate day dosing of rhGH. Peripheral blood was drawn for phenotypic analysis and functional assays at time points 0, 12 and 24 weeks. Results At week 12, we observed significant increases in naive CD4 T cells ( P<0.01) and effector CD8 T cells based on CD45RA and CCR7 expression ( P<0.02). In addition, we observed a rise in HIV-1 antigen-specific CD4 ( P<0.005) and CD8 ( P<0.05) T-cell responses. Twelve weeks post-randomization into placebo, alternate day or twice weekly dosing (24 weeks post-baseline), the phenotype and function of the virus-specific effector CD8 T cells seen at week 12 was maintained in most patients regardless of randomization arm and despite the disappearance of HIV-1-specific CD4 T-cell responses. Conclusions Concomitant administration of rhGH at 4 mg/day with highly active ART appears to partially reverse some of the defects exerted on the immune system by HIV-1. This combination may represent a valuable immunotherapeutic intervention aiding in the treatment of chronic HIV-1 infection.
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Ogasawara, Masahiro, Tomohiro Yamakawa, Yuki Katsura, Kanako Shima, Toshihiro Matsukawa, Minoru Kanaya, Koichiro Minauchi, et al. "Efficient Generation of HLA-A24-Restricted WT1-Specific Cytotoxic T Lymphocytes Using Gene-Engineered Artificial Antigen-Presenting Cells." Blood 116, no. 21 (November 19, 2010): 2101. http://dx.doi.org/10.1182/blood.v116.21.2101.2101.
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Abstract Abstract 2101 Background and Purpose: Recent success associated with adoptive transfer of antitumor T cells in lymphodepleted patients suggests the potential of adoptive immunotherapy to have a significant clinical impact. However, the widespread use of adoptive therapy has been hampered by the difficulty of consistently generating potent antitumor lymphocytes in a timely manner for every patient. To overcome this, we previously reported a culture system that can reproducibly generate antigen-specific cytotoxic T lymphocytes (CTL) from HLA-A2-positive melanoma patients by using K562-based artificial antigen-presenting cells (aAPCs). In the present study, we have applied the culture system to HLA-A24, one of the most common HLA class I antigen in the Asian population, and examined whether HLA-A2402-restricted WT1-specific CTL can be generated using aAPCs. Methods: HLA-A2402–positive peripheral blood mononuclear cells (PBMC) were obtained from healthy donors (n=4) and cancer patients (n=10). To establish antigen-specific T cells, CD8+ T cells were purified by positive selection using a magnetic beads method (Miltenyi Biotec). aAPCs were pulsed with an HLA-A2402 restricted, modified 9-mer WT1 immunodominant peptide (CYTWNQMNL). aAPCs were then irradiated with 200 Gy and added to purified CD8+ T cells at a ratio of 1:20 in 96-well plates in RPMI1640 supplemented with 10% human AB serum. Between stimulations, IL-2 (10 U/ml) and IL-15 (10ng/ml) (both from Peprotech) were added to the cultures. Results: Flow cytometry (FACS) analysis confirmed that aAPCs stably expressed transduced HLA class I, CD80 and CD83 molecules. aAPC did not express HLA class II molecules, CD40, CD154, or CD86. Following 3–4 rounds of weekly stimulation with peptide-pulsed aAPCs, WT1 peptide-specific CD8+ T cells were evaluated by a tetramer staining. The percentage of tetramer-positive cells was 0.082±0.0075% before stimulation. It increased to 0.865±0.528% (10.5 fold increase) and 1.89±1.12% (23.0 fold increase) following the third and the forth stimulation, respectively. There was no marked difference in magnitude of increase between healthy donors and cancer patients. However, when CD8+ T cells from patients vaccinated with WT1-peptide pulsed dendritic cells were stimulated with WT1-peptide-pulsed aAPCs, the percentage of tetramer-positive cells was significantly higher (11.72±6.46%) following the third stimulation. CD8+ T cells stimulated with WT1-peptide-pulsed aAPCs were negative for both A0201/WT1 and A2402/CMV tetramers, confirming HLA restriction and antigen specificity. FACS analysis revealed that WT1-specific CTL expanded with WT1-peptide-pulsed aAPCs expressed a memory phenotype. Furthermore, these CTL demonstrated cytotoxicity against CIR-A2402 target cells pulsed with a WT1 peptide in an LDH release assay. Unpulsed or HIV peptide-pulsed target cells were not lysed. Conclusions: These results demonstrated that HLA-A24-restricted WT1 specific CTLs with a memory phenotype can be generated ex vivo using peptide-pulsed gene-engineered aAPCs within a short period of time for clinical use. Disclosures: No relevant conflicts of interest to declare.