Projects / Programmes
Identification and evaluation of novel biomarkers for residual active HIV reservoirs.
| Code |
Science |
Field |
Subfield |
| 3.01.00 |
Medical sciences |
Microbiology and immunology |
|
| Code |
Science |
Field |
| B007 |
Biomedical sciences |
Medicine (human and vertebrates) |
| Code |
Science |
Field |
| 3.01 |
Medical and Health Sciences |
Basic medicine |
HIV-1, Nef, TAR RNA, microRNA, exosomes, virus rezervoar, biomarkers
Researchers (1)
| no. |
Code |
Name and surname |
Research area |
Role |
Period |
No. of publicationsNo. of publications |
| 1. |
30767 |
PhD Jana Ferdin |
Natural sciences and mathematics |
Head |
2016 - 2017 |
53 |
Organisations (1)
Abstract
HIV remains a major global public health issue, with WHO estimating that around 35.0 million people were infected at the end of 2013. Despite the relative efficacy of anti-retroviral therapy (ART) in controlling HIV infection, the virus spreads and hides in small reservoirs distributed throughout the body of infected individuals, which can on the long run contribute to diverse health problems. Some assays to determine HIV reservoirs already exist, but have many limitations, the important one being that they only detect HIV at the level of DNA or RNA, which does not tell anything about the residual “activity” of the reservoir, e.g. are the reservoirs capable of limited translation of certain viral proteins. Nef is one of the earliest and most abundantly expressed viral proteins. It is a critical HIV pathogenic factor, known to disrupt cellular signaling and trafficking pathways and to alter the metabolism even in uninfected cells. Importantly, Nef is released from infected cells with exosomes and was detected in plasma of infected individuals. Exosomes are extracellular vesicles (40 - 100 nm in diameter) secreted by all types of cells in culture, and were also discovered in abundance in all body fluids, including blood (3 x 106 exosomes/ul), CSF, saliva, urine, breast milk and others. Importantly, they are capable to cross the blood-brain barrier. Exosome protein, nucleic acid and lipid composition reflects the composition and the physiological state of the parent cell. Because of the above characteristics, they are studied extensively as biomarkers for various diseases. Before exosomes, miRNAs in body fluids also attracted a lot of attention as novel class of biomarkers for diverse diseases. Recent studies support that most of miRNAs are packed in exosomes, where they are protected from degradation and can be specifically targeted to certain cells/tissues. HIV infection effects miRNA cellular profiles and one study also showing that HIV-1 TAR (trans-activation response element) miRNA is present in the exosomes isolated from the serum of ART-treated patients or elite controllers.
Thus the hypothesis of this proposal is that plasma Nef and/or TAR miRNA concentrations are good indicators for residual “active” HIV reservoirs in ART treated individuals and elite controllers. MicroRNA profiling and comparison between different patient groups will determine several novel putative biomarkers of HIV reservoirs for further evaluation. To evaluate our hypothesis, we will first obtain 140 plasma samples of well-characterized HIV-infected patients from the SCOPE collection (4 groups: HIV-uninfected subjects; non-controllers; ART suppressed individuals; elite controllers). After optimizing the commercially available Nef ELISA assay, we will determine Nef concentration in all plasma samples. Next, we will optimize extraction of miRNAs from plasma or from plasma exosomes, and determine TAR miRNA concentrations by RT-qPCR using previously published primers and commercially available SYBER qPCR kit. In the end, miRNA expression profiling will be performed on three plasma samples for each of the four different patient groups. Extracted miRNAs will be reverse transcribed and tested by miRNA ready to use PCR human panels with unique larger set of miRNAs and several controls. All obtained results will be statistically evaluated.
The findings of this proposal are the first step in a completely new approach to AIDS diagnostics based on molecular components of exosomes, like Nef and HIV-derived miRNAs, in blood plasma. Importantly, plasma Nef concentrations could in the future be tested as a prognostic biomarker of HIV-associated neurocognitive disorders, as Nef has a known role in HIV neuropathogenesis. The results of our study could also be the basis for identification of new targets for AIDS therapeutics.
Significance for science
The proposed project is the first step towards a novel diagnostic approach in AIDS, similar to exosomes-based approach in cancer (etc. glioblastoma). Diagnostics based on detecting exosomes in plasma is a better alternative to diagnostic procedures based on tissue sampling, since it is less invasive, dangerous, faster, simplified and more cost-effective analysis. The proposed approach based on determining Nef or miRNA (viral or human) circulating in plasma within exosomes released from bystander or distant HIV-infected cells. The level of Nef and plasma miRNAs are either indicators of the nearby events or events in more distant tissues. Thus, they have a great potential as biomarkers used for the evaluation of the HIV-reservoirs in the body. Some tests to determine HIV-reservoirs exist, but they have many limitations. Importantly, they detect the level of viral DNA or RNA, but tell us nothing about the "activity" of the viral reservoir, such as limited translation of viral proteins with known pathological effect. Our studies have shown that viral (Nef and TAR RNA) as well as changed miRNA profile expression in plasma could serve as potential biomarkers of HIV-reservoir, that could also be tested (individually or in combination) as a prognostic biomarker of HIV-associated neuro-cognitive disorders. Results of our study could present a basis for new therapeutic targets used in AIDS treatment and inhibition or prevention of negative effects of viral / human molecules in the body.
Significance for the country
Within the framework of the project Z3-7198 we have contributed to the following areas important for the development of Slovenia: 1-Important result of our study is the estimate (Nef concentrations and the presence of TAR RNA) of virus reservoir and the expression level of specific (miRNA) human biofluids as biomarkers to evaluate the size of HIV reservoirs and its remaining activity in treated HIV positive individuals. These findings contributed importantly to the development of Slovenian science, both in general and specifically at this newly developing field of HIV infection research involving extracellular vesicles. We presented our research results at numerous international congresses and in the form of lectures at local and foreign institutions, thus contributing to the recognition of Slovenian science in the international scientific environment. 2- On the long-term, we also significantly contributed to the improvement in treatment and thus the quality of life of HIV-1 infected individuals, in Slovenia and elsewhere in the world. We have shown that protein Nef is present in the plasma of half of HIV-infected individuals; in which effective antiretroviral therapy maintains the level of plasma HIV RNA below detectable levels. Nef is HIV pathogenic factor, with many known negative effects. It might also contribute to inflammatory and to non- AIDS-related disorders, observed in otherwise successfully treated individuals. As part of this project, we have optimized the assay to detect Nef in plasma, which can be used to identify those individuals who requiring an adjustment of their current therapy. We have also shown that Nef-exosomes could serve as a potential therapeutic target. 3- During the project, we also wrote a review article published in Medicinski rezgledi (in Slovene language), where we summarized the current knowledge of extracellular vesicles, with description of various types of extracellular vesicles, the methods for their isolation and analysis, already known biological roles and their clinical potential. With this publication, we presented the extracellular vesicles and their promising clinical potential in a simple and transparent way to clinicians and healthcare professionals, as well as to researchers who are new in this field of research. Additionally I have shared my knowledge about extracellular vesicles (practical and theoretical) as a working mentor in the student research thesis. This research focused on determining the presence of viral (TAR RNA) and / or the level of expressed human miRNA as potentially novel viral or human plasma biomarkers in HIV-1 infected individuals. This student thesis was also awarded with the Faculty Prešeren Award and nominated for the University Prešeren Award.
Most important scientific results
Interim report,
final report
Most important socioeconomically and culturally relevant results
Interim report,
final report
