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Development of novel molecular and microfluidics tools for identification and characterization of latent HIV-1 reservoir
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH). Karolinska institutet.
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The existence of latent HIV-1 reservoir (LR) in all HIV-1 infected patients serves as a major obstacle to completely cure HIV-1 infection. However, up to now there is still no available assay that provides an accurate measurement of the reservoir size. This thesis aims to address this challenge from different aspects with several novel technologies, using both molecular and microfluidics-based tools. To find a proper tool to identify the latent HIV-1 reservoir, in Paper I and II, LIPS assay, RNAflow, and RNAscope assay were optimized and evaluated for indirect and direct detection of latent HIV-1 reservoir. The results indicated the LIPS method might not be sufficient for latent HIV-1 reservoir detection, although it has been proposed to quantify the latent HIV-1 reservoir indirectly. Furthermore, the optimized RNAscope technique performed better than RNAflow for transcription and translation competent latent HIV-1 reservoir identification. The RNAscope was also found to be independent of the HIV-1 subtype and can be applied to patient samples at single cell level. As there are currently no available surface biomarkers for latent HIV-1 reservoir, in Paper III, transcriptomics and proteomics-based analysis method for high-throughput selection of potential biomarker were established and applied to different patient groups. Twelve membrane protein-coding genes were identified as downregulated in the patient group who were hypothesized to have lower latent reservoir. These proteins might have the potential to be used as surface biomarkers for latent HIV-1 reservoir. CD4+ T cells, monocyte/macrophages, and natural killer cells are believed to be the primary source for HIV-1 reservoirs in peripheral blood. In paper IV, a microfluidic chip was developed to simultaneously isolate these three mononuclear leukocyte cell types directly from whole blood. The microfluidic method reduces the sample volume requirement and is a promising tool for latent HIV-1 reservoir study. Together, though further improvement and clinical verification are necessary, the work in this thesis has contributed to the advancement of latent HIV-1 reservoir characterization and may facilitate future development of the latent HIV-1 reservoir targeting and clearance methods with the ultimate goal – to cure HIV-1 infection.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2019. , p. 65
Series
TRITA-CBH-FOU ; 2019:8
Keywords [en]
HIV-1, Latent HIV-1 reservoir, HIV-1 characterization, Microfludics, Molecular detection
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Medical Technology
Identifiers
URN: urn:nbn:se:kth:diva-242235ISBN: 978-91-7873-089-6 (print)OAI: oai:DiVA.org:kth-242235DiVA, id: diva2:1283311
Public defence
2019-03-01, Air&Fire, Science for life lab, Tomtebodavägen 23a, 171 65 Solna, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

QC 20190129

Available from: 2019-01-29 Created: 2019-01-28 Last updated: 2019-04-12Bibliographically approved
List of papers
1. Characterization of Inducible Transcription and Translation-Competent HIV-1 Using the RNAscope ISH Technology at a Single-Cell Resolution
Open this publication in new window or tab >>Characterization of Inducible Transcription and Translation-Competent HIV-1 Using the RNAscope ISH Technology at a Single-Cell Resolution
2018 (English)In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 9, article id 2358Article in journal (Refereed) Published
Abstract [en]

Identifying the source and dynamics of persistent HIV-1 at single-cell resolution during cART is crucial for the design of strategies to eliminate the latent HIV-1 reservoir. An assay to measure latent HIV-1 that can distinguish inducible from defective proviruses with high precision is essential to evaluate the efficacy of HIV-1 cure efforts but is presently lacking. The primary aim of this study was therefore to identify transcription and translation competent latently infected cells through detection of biomolecules that are dependent on transcriptional activation of the provirus. We investigated the applicability of two commercially available assays; PrimeFlow (TM) RNA Assay (RNAflow) and RNAscope (R) ISH (RNAscope) for evaluation of the efficacy of latency reversal agents (LRAs) to reactivate the HIV-1 latent reservoir. The J-Lat cell model (clones 6.3, 9.3, and 10.6) and four LRAs was used to evaluate the sensitivity, specificity, and lower detection limit of the RNAflow and RNAscope assays for the detection and description of the translation-competent HIV-1 reservoir. We also checked for HIV-1 subtype specificity of the RNAscope assay using patient-derived subtype A1, B, C, and CRFOLAE recombinant plasmids following transfection in 293T cells and the applicability of the method in patient-derived peripheral blood mononuclear cells (PBMCs). The lower detection limit of RNAflow was 575 HIV-1 infected cells/million and 45 cells/million for RNAscope. The RNAscope probes, designed for HIV-1B, also detected other subtypes (A1, B, C, and CRF<b>01 _AE). RNAscope was applicable for the detection of in patient-derived PBMCs following LRA activation. In conclusion, our study showed that RNAscope can be used to quantify the number of directly observed individual cells expressing HIV-1 mRNA following LRA activation. Therefore, it can be a useful tool for characterization of translation-competent HIV-1 in latently infected cell at single-cell resolution in the fields of HIV-1 pathogenesis and viral persistence.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2018
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-235995 (URN)10.3389/fmicb.2018.02358 (DOI)000446068700001 ()2-s2.0-85055209198 (Scopus ID)
Funder
Swedish Research Council, 2017-01330Swedish Research Council, 2016-01675Stockholm County Council, ALF 20160074The Karolinska Institutet's Research Foundation, 2016-00221
Note

QC 20181015

Available from: 2018-10-15 Created: 2018-10-15 Last updated: 2019-01-29Bibliographically approved
2. Quantitative humoral profiling of the HIV-1 proteome in elite controllers and patients with very long-term efficient antiretroviral therapy
Open this publication in new window or tab >>Quantitative humoral profiling of the HIV-1 proteome in elite controllers and patients with very long-term efficient antiretroviral therapy
Show others...
2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 666Article in journal (Refereed) Published
Abstract [en]

A major challenge in evaluating the success of HIV eradication approaches is the need for accurate measurement of persistent HIV during effective antiretroviral therapy (ART). Previous studies have reported that the anti-HIV antibody assay "luciferase immuno-precipitation systems (LIPS)"can distinguish HIV-infected individuals harboring different sizes of the viral reservoirs. We performed antibody profiling of HIV-1 proteomes using LIPS in viremic progressors (n = 38), elite controllers (ECs; n = 19) and patients with fully suppressive long-term antiretroviral therapy (ART) (n = 19) (mean 17 years). IgG was quantified against six HIV-1 fusion proteins: p24, gp41, RT, Tat, integrase and protease. Lower antibody levels to all six-fusion proteins were observed in long-term ART patients compared to viremics (p < 0.05). In contrast ECs had lower antibody levels only against Tat and Integrase (p < 0.05). Principal component analysis and cluster-network analysis identified that 68% (13/19) of the long-term ART patients clustered together with 26% (5/19) ECs. The remaining ECs clustered together with the viremics indicating non-homogeneity among the ECs. The low anti-HIV levels in the long-term treated patients may indicate a restricted remaining viral replication. In contrast, the higher levels in ECs suggest a continuous viral expression with a limited concomitant release of extracellular virus.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2017
National Category
Nano Technology
Identifiers
urn:nbn:se:kth:diva-206253 (URN)10.1038/s41598-017-00759-8 (DOI)000398545400010 ()28386076 (PubMedID)2-s2.0-85018367653 (Scopus ID)
Note

QC 20170512

Available from: 2017-05-12 Created: 2017-05-12 Last updated: 2019-01-29Bibliographically approved
3. Transcriptomics and Targeted Proteomics Analysis to Gain Insights Into the Immune-control Mechanisms of HIV-1 Infected Elite Controllers
Open this publication in new window or tab >>Transcriptomics and Targeted Proteomics Analysis to Gain Insights Into the Immune-control Mechanisms of HIV-1 Infected Elite Controllers
Show others...
2018 (English)In: EBioMedicine, E-ISSN 2352-3964, Vol. 27, p. 40-50Article in journal (Refereed) Published
Abstract [en]

A small subset of HIV-1 infected individuals, the "Elite Controllers" (EC), can control viral replication and restrain progression to immunodeficiency without antiretroviral therapy (ART). In this study, a cross-sectional transcriptomics and targeted proteomics analysis were performed in a well-defined Swedish cohort of untreated EC (n = 19), treatment naive patients with viremia (VP, n = 32) and HIV-1-negative healthy controls (HC, n = 23). The blood transcriptome identified 151 protein-coding genes that were differentially expressed (DE) in VP compared to EC. Genes like CXCR6 and SIGLEC1were downregulated in EC compared to VP. A definite distinction in gene expression between males and females among all patient-groups were observed. The gene expression profile between female EC and the healthy females was similar but did differ between male EC and healthy males. At targeted proteomics analysis, 90% (29/32) of VPs clustered together while EC and HC clustered separately from VP. Among the soluble factors, 33 were distinctive to be statistically significant (False discovery rate = 0.02). Cell surface receptor signaling pathway, programmed cell death, response to cytokine and cytokine-mediated signaling seem to synergistically play an essential role in HIV-1 control in EC.

Place, publisher, year, edition, pages
ELSEVIER SCIENCE BV, 2018
Keywords
HIV-1 Elite Controllers, Transcriptome, Proteome
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:kth:diva-224058 (URN)10.1016/j.ebiom.2017.11.031 (DOI)000425875400009 ()29269040 (PubMedID)2-s2.0-85038835075 (Scopus ID)
Note

QC 20180316

Available from: 2018-03-16 Created: 2018-03-16 Last updated: 2019-01-29Bibliographically approved
4. Microfluidic based immunoaffinity mononuclear leukocytes isolation from whole blood
Open this publication in new window or tab >>Microfluidic based immunoaffinity mononuclear leukocytes isolation from whole blood
(English)Manuscript (preprint) (Other academic)
Abstract [en]

CD4+ T cells, monocyte/macrophages and natural killer cells are believed to be the main source for HIV-1 reservoirs in peripheral blood. However, despite the potential these subsets of providing a wealth of new information about immune function and host pathology, current HIV latency studies are often based on PBMCs or only CD4+ T cells, mainly due to the lack of appropriate cell subset isolation methods. We present here a microfluidic chip-based method to capture and enrich the three mononuclear cells sub-population peripheral leukocyte sub-populations: CD4+ lymphocytes, natural killer cells and monocytes; using a single source of whole blood (volume < 200 μL) on a single integrated platform, within a time frame of 20 min. The single step isolation method can be used for downstream proteomics and genomics analysis to study the aberrations in these cell types’ functions in critical diseases such as HIV.

Keywords
microfluidic, mononuclear leukocytes isolation, HIV-1 reservoir
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-242234 (URN)
Note

QC 20190131

Available from: 2019-01-28 Created: 2019-01-28 Last updated: 2019-02-14Bibliographically approved

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