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Статті в журналах з теми "HIV, phenotypic assay, cell based assay"
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.
Повний текст джерелаАнотація:
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.
Повний текст джерела
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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.
Повний текст джерелаАнотація:
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.
Повний текст джерелаАнотація:
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.
Повний текст джерелаАнотація:
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.
Повний текст джерелаАнотація:
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.
Повний текст джерелаАнотація:
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.
Повний текст джерелаАнотація:
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.
Повний текст джерелаАнотація:
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.
Дисертації з теми "HIV, phenotypic assay, cell based assay"
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Giannini, Alessia. "HIV drug discovery and resistance testing through cell based assays." Doctoral thesis, Università di Siena, 2019. http://hdl.handle.net/11365/1069117.
Повний текст джерелаАнотація:
Anti-HIV therapy has dramatically progressed from palliative care to high effectiveness, with virus replication and disease progression halted in most patients yet not eradicated. However, the need for lifelong therapy has kept anti-HIV drug development at high pace and novel strategies, drugs and drug classes have been regularly introduced over time. Issues to be tackled during prolonged antiretroviral therapy include adherence, toxicity, drug-drug interactions and the development of drug resistance. During my PhD course at the HIV Monitoring Laboratory (HML) of the Department of Medical Biotechnology at the University of Siena, I have been given the opportunity to focus my efforts in this area along two different lines.
The first activity has been done in the field of drug discovery within the EU FP7 funded THINPAD project. THINPAD is the acronym of “Targeting the HIV-1 Nucleocapsid Protein to fight Antiretroviral Drug Resistance”, thus the objective of this project was to discover and develop a novel class of anti-HIV agents targeting NCp7, a small nucleocapsid protein that plays a number of different roles in HIV replication. The project used a funnel strategy from virtual screening to biochemical testing, then cell based assays and finally humanized mice as the animal model to validate the candidate NCp7 inhibitors (NCIs). My unit measured the antiviral activity of the candidate NCIs through cell based assays developed at the HML. The compounds with the highest selectivity indexes were next demonstrated to retain comparable efficacy against wild-type virus and virus resistant to licensed drug classes, thus confirming NCp7 as a specific target suitable to block the replication of currently circulating drug resistant viruses. The same compounds were used to perform experiments aiming at the definition of the mechanism of action through the measurement of viral nucleic acids intermediates produced in infected cells in presence of inhibitory concentration of NCIs at different time points. The results of this analysis were in agreement with antiviral activity exerted at the early and/or late steps of viral replication, suggesting that NCIs are able to interfere with variable impact on the different functions of NCp7 during HIV life cycle. While preliminary efficacy tests in the humanized mouse model were not successful, in vitro data support further NCI development.
The second activity has investigated the role of natural HIV-1 variability or specific HIV-1 mutants in the susceptibility or genetic barrier to resistance to licensed or upcoming antiretrovirals. Completed studies of this kind have shown (i) a role for the natural polymorphisms E138A in the reverse transcriptase in lowering the genetic barrier to resistance to the non-nucleoside reverse transcriptase inhibitor etravirine, (ii) a minimal impact of the integrase E157Q polymorphism in resistance to integrase inhibitors and (iii) the infrequent occurrence of natural resistance to the novel entry inhibitor fostemsavir in the HIV-1 CRF02_AG, an evolutionary lineage of HIV-1 originated in Africa and substantially represented in Italy. Although different, the three topics shared the need to clarify uncertain areas and derive indications for optimal drug use in the clinic.
Both activities have been using cell based systems developed at the HML and further refined for the specific application. Phenotypic drug susceptibility testing is a fairly complex task, usually assigned to specialized companies in collaborative research projects and not used at all in the clinic. Among academic systems proposed over time, our assay has been uniquely validated through comparison with the de facto reference commercially available Phenosense assay from Monogram Biosciences. Overall, contributing to expanding and using the laboratory portfolio of systems required for advanced investigation of anti-HIV drug was perfectly in line with my expectations as a biotechnologist.
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Giammarino, Federica. "PHENOTYPIC CHARACTERIZATION OF NOVEL ANTIVIRALS FOR THE TREATMENT OF MULTIDRUG RESISTANT HIV-1 AND EMERGING VIRUSES." Doctoral thesis, Università di Siena, 2023. https://hdl.handle.net/11365/1224634.
Повний текст джерелаАнотація:
Abstract
Phenotypic characterization of novel antivirals for the treatment of multidrug resistant HIV-1 and emerging viruses
Doctoral Research School of Medical Biotechnologies – Cycle XXXV
Supervisor: Maurizio Zazzi; Candidate: Federica Giammarino
Background
The need for new antiviral drugs has increased overtime due to the worldwide circulation of different viruses together with the increased frequency and diversity of new outbreaks. The ideal option for a prompt response against both emerging and re-emerging viruses is represented by the use and the development of direct acting antiviral agents. During my PhD I was involved in several projects focused on the evaluation of the antiviral activity of licensed and investigational antiviral drugs against Human Immunodeficiency (HIV-1), West Nile (WNV), Dengue (DENV) and SARS-CoV-2 viruses.
Results and discussion
Doravirine
The antiviral activity of the NNRTI doravirine was evaluated against viruses harbouring different patterns of NNRTI resistance mutations in two studies. Globally, our data confirmed that the antiviral activity of doravirine may be compromised by the presence of multiple NNRTI resistance mutations, even in the absence of specific doravirine mutations.
A third study was focused on the role of the natural polymorphism of the reverse transcriptase V106I. Our results indicate that it minimally affects the susceptibility to doravirine in clinical isolates and that it does not impact the genetic barrier to resistance as compared to reference wild-type virus, while viruses including the NNRTI resistant mutation V106A or V106M rapidly showed viral breakthrough under doravirine pressure due to the reduced susceptibility.
Islatravir
Our study confirmed the decrease of susceptibility of the investigational NRTTI islatravir due to the presence of M184V mutation. The clinical impact of NRTI mutations in the activity of islatravir has still to be defined and the threshold of fold-change values associated to reduced activity in vivo remains to be established.
Ibalizumab
The combinatorial activity of ibalizumab together with other antivirals, both approved and investigational, was evaluated through a newly developed cell-based assay consisting in the infection of the MOLT4-R5 cell line with the wild-type strains NL4-3 and AD8, and by the analysis of the results using the innovative software SynergyFinderPlus. Our data suggest that ibalizumab positively interacts with other antivirals with possible synergistic effects in select cases. Further studies are needed to determine the impact of Env variability and viral tropism in combination with other entry inhibitors.
Development of a Cell-Based Immunodetection Assay for Simultaneous Screening of Antiviral Compounds Inhibiting Zika and Dengue Virus Replication
An easy-to-perform and fast flavivirus immunodetection assay (IA) was developed to determine antiviral activity of promising compounds against ZIKV and DENV. The system, validated with references compounds against both viruses, was able to distinguish between the inhibitory effect of molecules targeting the early and the post-budding phase of viral replication cycle.
Evaluation of sofosbuvir activity and resistance profile against West Nile virus in vitro
Since the activity of sofosbuvir has been documented against different flaviviruses, we investigated whether it may exert an activity also against WNV. In both cell-based and enzymatic assays sofosbuvir was able to inhibit WNV replication in the low micromolar range. Moreover, in vitro selection and molecular docking experiments indicated that HCV and WNV share a similar sofosbuvir resistance pattern.
ORIGINALE CHEMIAE in Antiviral Strategy - Origin and Modernization of Multi-Component Chemistry as a Source of Innovative Broad Spectrum Antiviral Strategy
The “ORIGINALE CHEMIAE in Antiviral Strategy” project aims to identify promising broad-spectrum antivirals by taking advantage of the Multi-Component Chemistry strategy. Following the synthetization of molecules, their antiviral activity was determined in in vitro standardized virus-cell systems against DENV, WNV, HIV-1 and SARS-CoV-2. We identified eight molecules able to inhibit at least one of the viruses tested. However, their low selectivity indexes indicate the need to further improve the design of these molecules to increase the antiviral activity and/or reduce the cell toxicity in order to identify candidates for preclinical testing in animal models.
Monoclonal antibodies and antivirals vs. SARS-CoV-2
After the development of a quantitative live-virus microneutralization assay, we evaluated the efficacy of licensed monoclonal Antibodies (mAbs) and the antiviral drugs remdesivir, nirmaltrevir and molnupiravir against different circulating SARS-CoV-2 variants. Our results showed that these drugs, contrary to the mAbs, retained activity against all tested variants.
Conclusions
A continuous challenge for public health is represented by the control of viral infections. Both vaccines and antiviral drugs may synergistically help to reduce the spread and the fatality of acute viral diseases and chronic infections. All the studies described in this thesis emphasize the role of the laboratory of virology within all the steps of the in vitro investigation of antiviral drugs, from the identification of molecules endowed with antiviral activity to the definition of the mechanism of action.
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Dragoni, Filippo. "Antiviral drug development for treatment of acute and chronic viral infections." Doctoral thesis, Università di Siena, 2021. http://hdl.handle.net/11365/1127988.
Повний текст джерелаАнотація:
Background
Viruses are aetiologic agents of several human infectious diseases, which can be distinguished into acute and chronic. Among the agents causing acute infections, arboviruses, such as Dengue (DENV), West Nile (WNV) and Zika (ZIKV) Viruses, have increasingly spread worldwide. Moreover, the newly discovered Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), causing the COVID-19 disease, is responsible for an incredibly quantity of infections and deaths. Conversely, the Human Immunodeficiency Virus (HIV-1) is a well-known pathogen able to cause chronic latent infection, but it still represents a global public health concern.
The main strategy for combating viral infections is a combination of vaccination and antiviral drugs. However, vaccines are available only for a minority of viral pathogens, thus the demand for new antiviral strategies has significantly increased. Anyway, the screening of candidate compounds requires the assessment of their antiviral effects in vitro.
Results and discussion
An easy-to-perform and fast flavivirus immunodetection assay (IA) was developed to determine antiviral activity of promising compounds against ZIKV and DENV, able to distinguish between the inhibitory effect of molecules targeting the early and post-budding phases of viral replication cycle.
Considering that the activity of sofosbuvir, a nucleotide analog licensed for HCV infection, has been documented against different flaviviruses, we investigated whether it may exert an activity also against WNV. We determined for the first time the antiviral activity of sofosbuvir against WNV in both cell-based and enzymatic assays. Moreover, in vitro selection and molecular docking experiments indicated that HCV and WNV share a similar sofosbuvir resistance pattern.
The “ORIGINALE CHEMIAE in Antiviral Strategy” project aims to identify promising broad-spectrum antivirals by taking advantage of the Multi-Component Chemistry strategy. Antiviral activity of molecules was determined in vitro against DENV, WNV, HIV-1 and SARS-CoV-2. Two compounds were able to inhibit viral replication against two different families of viruses (DENV/WNV and SARS-CoV-2), and two compounds were able to inhibit viral replication against viruses with remarkable differences in their replication cycle (DENV/WNV and HIV-1).
At the beginning of the pandemic, an Italian network named SCIRE (SARS-CoV-2 Italian Research Enterprise) was created in order to trace SARS-COV-2 evolution. By NGS whole genome sequencing, it was defined that the initial outbreak in Italy was mainly attributable to the SARS-CoV-2 lineage B.1 and to its uncontrolled circulation for an estimated period of 4 weeks.
Developing strategies to eradicate chronic HIV-1 infection are a high priority. Recently, it was hypothesized that maraviroc (MVC), may exert a latency reversing effect in addition to its antiviral activity. Thus, the MVC-mediated HIV-1 induction was explored. An increased HIV-1 expression was detected at the highest MVC concentration in two of the three cell line models tested. In ex vivo CD4 T cells, MVC-mediated induction was detected sparsely on individual samples. Such evidences suggest the role of MVC as a weak latency reversing agent of the HIV-1 provirus induction.
Conclusions
Control of viral infections is a continuous challenge for public health. The development of effective antiviral compounds could be reasonably the key to halt the spread or to mitigate severe clinical manifestations of acute viral diseases. Moreover, antiviral therapy is essential to the maintenance of undetectable viral loads in chronic infection. Furthermore, developing of compounds able to exert a reversal latency effect may be a remarkable benefit in exacerbate chronic viral infections.
In conclusion, the key concept of this thesis is to underline the strong need, and partially participate, in enhancing antiviral therapies strategies to counteract the burden of both acute and chronic viral diseases.
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Lai, Ming Chih, and 賴銘志. "Cloning of HIV-1 Integrase into a Moloney Murine Leukemia Virus Based Vector to Establish a Cell Line for In Vivo Integration Assay of HIV-1 Integrase." Thesis, 1994. http://ndltd.ncl.edu.tw/handle/48848916245915300130.
Повний текст джерелаЧастини книг з теми "HIV, phenotypic assay, cell based assay"
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Rajakuberan, Chitra, Brett J. Hilton, and Roland Wolkowicz. "Protocol for a Mammalian Cell-Based Assay for Monitoring the HIV-1 Protease Activity." In Methods in Molecular Biology, 393–405. Totowa, NJ: Humana Press, 2012. http://dx.doi.org/10.1007/978-1-61779-937-2_27.
Повний текст джерела
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Liu, Zhaoping, Andrea Gomez-Donart, Caroline Weldon, Nina Senutovitch, and John O’Rourke. "Developing a Novel Multiplexed Immune Assay Platform to Screen Kinase Modulators of T Cell Activation." In High-Throughput Screening for Drug Discovery [Working Title]. IntechOpen, 2021. http://dx.doi.org/10.5772/intechopen.97304.
Повний текст джерелаАнотація:
T cell activation plays a central role in inflammation, autoimmune diseases and cancer. Cancer immunotherapies, such as immune checkpoint inhibitor, bi-specific antibody, chimeric antigen receptor T (CAR T) cell, and adoptive tumor-infiltrating lymphocyte (TIL) therapies require the characterization and monitoring of T cell activation. Here we describe a novel, multiplex immune assay platform based on high-throughput flow cytometry technology and advanced computational algorithms for data analysis. The assay simultaneously measures T cell dynamics including phenotype, time-dependent expression of activation markers, secreted effector cytokines, and proliferation. The assay screened a kinase chemogenomic library and identified 25 kinase inhibitors with distinct inhibition profiles on early (CD69) and late (CD25) activation markers and the cytokines IFNγ and TNFα. We identified 5 kinase inhibitors with dissimilar effects on CD69 and CD25 expression, and a cluster of total 4 MEK1//2 inhibitors with similar activation profiles. The screening revealed 3 kinase inhibitors for PKC, IKK2, and MEK1/2 respectively, all with a phenotypic signature similar to ruxolitinib, a Jak1/2 inhibitor used to treat myelofibrosis disease. These results suggest this multiplexed assay platform, combined with a chemogenomic library screening, may be used as primary screen for phenotypic or target-based drug discovery, target identification, and potential drug repositioning.
Тези доповідей конференцій з теми "HIV, phenotypic assay, cell based assay"
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Gerckens, Michael, Katharina Heinzelmann, Oliver Eickelberg, and Gerald Burgstaller. "A phenotypic cell-based extracellular matrix deposition assay for target validation and drug discovery." In ERS International Congress 2017 abstracts. European Respiratory Society, 2017. http://dx.doi.org/10.1183/1393003.congress-2017.pa912.
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Chowdhury, Arnab Roy, Debabani Roy Chowdhury, Manoj Pandre, Samrat Roy, Sundarajan Kannan, and John W. Ellingboe. "Abstract 2868: A cell based phenotypic assay platform for cancer metastasis drug discovery and diagnostics." In Proceedings: AACR Annual Meeting 2021; April 10-15, 2021 and May 17-21, 2021; Philadelphia, PA. American Association for Cancer Research, 2021. http://dx.doi.org/10.1158/1538-7445.am2021-2868.
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Chan, Grace Ka-Yan, Tracy Kleinheinz, Matthew Martinson, Hok-Sum Cheung, and John G. Moffat. "Abstract 740: Phenotypic profiling and selectivity optimization of ERK inhibitors using a high-throughput imaged-based cell cycle assay." In Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA. American Association for Cancer Research, 2014. http://dx.doi.org/10.1158/1538-7445.am2014-740.
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Wehrman, Tom S., Erika A. O'Donnell, M. Jared Lumpe, Benjamin E. Osetek, and Peter O. Krutzik. "Abstract 5385: A novel cell-based kinase assay panel paired with high content phenotypic profiling to correlate kinase inhibitor specificity and cellular activity." In Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA. American Association for Cancer Research, 2014. http://dx.doi.org/10.1158/1538-7445.am2014-5385.
Повний текст джерелаЗвіти організацій з теми "HIV, phenotypic assay, cell based assay"
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Weil, Clifford F., Anne B. Britt, and Avraham Levy. Nonhomologous DNA End-Joining in Plants: Genes and Mechanisms. United States Department of Agriculture, July 2001. http://dx.doi.org/10.32747/2001.7585194.bard.
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Repair of DNA breaks is an essential function in plant cells as well as a crucial step in addition of modified DNA to plant cells. In addition, our inability to introduce modified DNA to its appropriate locus in the plant genome remains an important hurdle in genetically engineering crop species.We have taken a combined forward and reverse genetics approach to examining DNA double strand break repair in plants, focusing primarily on nonhomologous DNA end-joining. The forward approach utilizes a gamma-plantlet assay (miniature plants that are metabolically active but do not undergo cell division, due to cell cycle arrest) and has resulted in identification of five Arabidopsis mutants, including a new one defective in the homolog of the yeast RAD10 gene. The reverse genetics approach has identified knockouts of the Arabidopsis homologs for Ku80, DNA ligase 4 and Rad54 (one gene in what proves to be a gene family involved in DNA repair as well as chromatin remodeling and gene silencing)). All these mutants have phenotypic defects in DNA repair but are otherwise healthy and fertile. Additional PCR based screens are in progress to find knockouts of Ku70, Rad50, and Mre11, among others. Two DNA end-joining assays have been developed to further our screens and our ability to test candidate genes. One of these involves recovering linearized plasmids that have been added to and then rejoined in plant cells; plasmids are either recovered directly or transformed into E. coli and recovered. The products recovered from various mutant lines are then compared. The other assay involves using plant transposon excision to create DNA breaks in yeast cells and then uses the yeast cell as a system to examine those genes involved in the repair and to screen plant genes that might be involved as well. This award supported three graduate students, one in Israel and two in the U.S., as well as a technician in the U.S., and is ultimately expected to result directly in five publications and one Masters thesis.