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Projects / Programmes source: ARIS

Evaluation of viral protein Nef induced extracellular vesicles as plasma biomarker of leaky HIV-1 reservoir in aviremic adults

Research activity

Code Science Field Subfield
3.01.00  Medical sciences  Microbiology and immunology   

Code Science Field
B510  Biomedical sciences  Infections 

Code Science Field
3.01  Medical and Health Sciences  Basic medicine 
Keywords
extracellular vesicles, HIV-1, viral reservoirs, Nef, biomarker, immuno-capture, ELISA, qPCR
Evaluation (rules)
source: COBISS
Researchers (13)
no. Code Name and surname Research area Role Period No. of publicationsNo. of publications
1.  33110  PhD Katja Goričar  Oncology  Researcher  2018 - 2021  287 
2.  33344  PhD Marija Holcar  Natural sciences and mathematics  Researcher  2018 - 2021  40 
3.  25780  PhD Samo Hudoklin  Neurobiology  Researcher  2018 - 2021  108 
4.  15666  PhD Marko Kreft  Neurobiology  Researcher  2018 - 2021  684 
5.  52660  Teja Lavrin  Biochemistry and molecular biology  Junior researcher  2019 - 2021  15 
6.  24288  PhD Metka Lenassi  Natural sciences and mathematics  Head  2018 - 2021  201 
7.  28393  PhD Nataša Resnik  Biochemistry and molecular biology  Researcher  2018 - 2021  88 
8.  27503  PhD Ana Rotter  Biotechnology  Researcher  2018 - 2021  328 
9.  31967  PhD Simona Sitar  Chemistry  Researcher  2018 - 2019  56 
10.  15467  PhD Matjaž Stenovec  Medical sciences  Researcher  2018 - 2021  202 
11.  17272  Mirjana Širca    Technical associate  2020 - 2021 
12.  08279  PhD Peter Veranič  Neurobiology  Researcher  2018 - 2021  359 
13.  12318  PhD Ema Žagar  Materials science and technology  Researcher  2018 - 2021  484 
Organisations (3)
no. Code Research organisation City Registration number No. of publicationsNo. of publications
1.  0104  National Institute of Chemistry  Ljubljana  5051592000  20,929 
2.  0105  National Institute of Biology  Ljubljana  5055784  13,240 
3.  0381  University of Ljubljana, Faculty of Medicine  Ljubljana  1627066  48,190 
Abstract
HIV remains a major global public health issue, with WHO estimating that around 36.7 million people were infected at the end of 2016. As HIV vaccine development turned out to be very complex, more focus is put into controlling the infection with antiretroviral therapy (ART) and improving the quality of life of infected individuals. Despite effective ART, the virus spreads and hides in small reservoirs distributed throughout the body of infected individuals, mostly consisting of CD4+ T cells. This persistence is one of the major obstacles to viral eradication, therefore much effort has been made towards identifying and measuring the latent, replication-competent HIV reservoir. Importantly, recent work has indicated the existence of translationally active reservoir, as viral protein Nef was detected in plasma of half of aviremic HIV-infected individuals. This leaky HIV reservoir could contribute to chronic inflammation and non-AIDS morbidity, but to what extent remains largely unknown. To tackle this, we first need a simple and accurate assay to quantify leaky HIV reservoir, and plasma Nef-extracellular vesicle (EV) level might serve as a good candidate biomarker. Nef is namely an early HIV transcript, which does not need other viral proteins for high expression in the host cell. It affects diverse processes inside the infected cell, but is also released extracellularly through incorporation into virions and importantly also into EVs. Nef expression in the parental cell affects lipid, protein and miRNA composition of released Nef-EVs, and also their size. Nef-EVs have a deleterious effect on surrounding cells since they cause apoptosis of resting T-lymphocytes, neurotoxicity and impair the blood-brain barrier. Thus we can assume Nef-EVs accumulate in plasma of aviremic individuals and differ significantly in characteristics from EVs released by uninfected cells. EVs are secreted by all types of cells in culture, and were also discovered in abundance in all body fluids, including blood. EVs protein, nucleic acid (miRNA) and lipid composition reflects the composition and the (patho)physiological state of the parent cell. Because of this and because they can be isolated from easy accessible body fluids, they are studied extensively as biomarkers for various diseases, but so far not in the context of HIV infection. Thus the objective of the proposed research is to evaluate viral protein Nef-EVs as plasma biomarker of leaky HIV reservoir in aviremic HIV-infected adults. Specifically, we will perform an in-depth characterization of Nef-EVs released from Nef-expressing primary human CD4+ T cells and immortalized human microglia, and identify: unique surface proteins, which we will use to develop a method for immunoisolation of Nef-EVs from diverse sources (aim 1); and unique cargo proteins and miRNAs, which we will use to develop a method for detection of Nef-EV levels and their cellular sources (aim 2). We will use these newly developed methods to quantify plasma Nef-EV levels in a cross-sectional study of ART- and spontaneously suppressed HIV-infected adults and examine Nef-EV association with subject’s HIV reservoir size and other clinical characteristics (aim 3). The hypothesis of this proposal is that Nef-EVs, released into the blood from leaky HIV reservoirs, reveal the extent and the cellular source of residual viral protein expression in suppressed HIV-infected individuals, and their presence is reflected in the subject’s clinical characteristics. Therefore plasma Nef-EVs are an appropriate biomarker of leaky HIV reservoir in aviremic adults. The proposal is a completely new approach to HIV diagnostics, and is the first step in developing a clinical assay for measuring leaky HIV reservoir that could be routinely used to monitor patient’s status. This could help adjust treatments and maybe even predict clinical outcomes, which would improve the life of many of HIV infected patients
Significance for science
The proposal is a completely new approach to HIV diagnostics, as it will explore plasma extracellular vesicles (EVs) as so far unexplored resource of HIV-infection related biomarkers, which would parallel similar approaches to cancer and neurodegenerative diseases. The approach is based on detecting Nef-EVs in plasma, where EVs released from near­by or remote infected cells accumulate, and are therefore indicative of the state of accessible and remote body HIV reservoirs. Importantly, we will access the SCOPE cohort (collaboration with prof. Steven G. Deeks, MD, bank co-director; UCSF, USA) of more than 1500 samples of HIV-1 infected volunteers with carefully characterized clinical data.   The study will also importantly contribute to the understanding of leaky HIV reservoirs, the concept that is attracting a lot of interest recently, as it questions the previously accepted model of latency (complete shutdown of viral expression). It suggests that low level of viral proteins are present in body fluids, which could contribute to chronic inflammation seen in virally suppressed, ART-treated individuals.   ART has transformed HIV infection from a progressive, typically fatal infection to a chronic disease, impacting healthy aging and burdening the health care system. Our project is the first step in developing a clinical assay for measuring the leaky HIV reservoir that could be routinely used to monitor patient’s status. Following the Nef-EV biomarker in the blood could help adjust treatments and maybe even predict clinical outcomes, which would improve the life of many of HIV infected patients and also benefit the health care systems. Importantly, the in-depth characterization of Nef-EVs described in the current proposal could in the future be used for development of therapeutics targeting these toxic Nef-EVs.   We will also establish methods for specific isolation and detection of plasma Nef-EVs, which could serve as an EV biomarker discovery platform for any disease. Our approach namely overcomes one of the weaknesses of EVs as biomarkers, that is the dilution of disease-related EVs in the pool of plasma EVs, which affects identification of unique, disease-related EV-molecules and later also the sensitivity of biomarker detection assay.   The novelty of the described research would also contribute importantly to the international recognition of Slovenian science in the newly developing research field of extracellular vesicles. The research will also contribute to the improvement of teaching and training of personnel, as most of the project co-workers are also involved in teaching at undergraduate and postgraduate levels and are mentoring Bachelor degree, master degree and PhD students. We are also very involved in promoting regional EV research through organizing yearly workshops, administrating the EV-focused monthly e-mailing list with 70 subscribers and organizing science outreach activities.
Significance for the country
The proposal is a completely new approach to HIV diagnostics, as it will explore plasma extracellular vesicles (EVs) as so far unexplored resource of HIV-infection related biomarkers, which would parallel similar approaches to cancer and neurodegenerative diseases. The approach is based on detecting Nef-EVs in plasma, where EVs released from near­by or remote infected cells accumulate, and are therefore indicative of the state of accessible and remote body HIV reservoirs. Importantly, we will access the SCOPE cohort (collaboration with prof. Steven G. Deeks, MD, bank co-director; UCSF, USA) of more than 1500 samples of HIV-1 infected volunteers with carefully characterized clinical data.   The study will also importantly contribute to the understanding of leaky HIV reservoirs, the concept that is attracting a lot of interest recently, as it questions the previously accepted model of latency (complete shutdown of viral expression). It suggests that low level of viral proteins are present in body fluids, which could contribute to chronic inflammation seen in virally suppressed, ART-treated individuals.   ART has transformed HIV infection from a progressive, typically fatal infection to a chronic disease, impacting healthy aging and burdening the health care system. Our project is the first step in developing a clinical assay for measuring the leaky HIV reservoir that could be routinely used to monitor patient’s status. Following the Nef-EV biomarker in the blood could help adjust treatments and maybe even predict clinical outcomes, which would improve the life of many of HIV infected patients and also benefit the health care systems. Importantly, the in-depth characterization of Nef-EVs described in the current proposal could in the future be used for development of therapeutics targeting these toxic Nef-EVs.   We will also establish methods for specific isolation and detection of plasma Nef-EVs, which could serve as an EV biomarker discovery platform for any disease. Our approach namely overcomes one of the weaknesses of EVs as biomarkers, that is the dilution of disease-related EVs in the pool of plasma EVs, which affects identification of unique, disease-related EV-molecules and later also the sensitivity of biomarker detection assay.   The novelty of the described research would also contribute importantly to the international recognition of Slovenian science in the newly developing research field of extracellular vesicles. The research will also contribute to the improvement of teaching and training of personnel, as most of the project co-workers are also involved in teaching at undergraduate and postgraduate levels and are mentoring Bachelor degree, master degree and PhD students. We are also very involved in promoting regional EV research through organizing yearly workshops, administrating the EV-focused monthly e-mailing list with 70 subscribers and organizing science outreach activities.
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