Streamlining the detection of latent HIV and investigating the causes of ineffective reactivation

Lead Research Organisation: University of Cambridge
Department Name: Medicine

Abstract

HIV incorporates its genetic material into that of an infected cell in a process called integration. Some of the integrated viruses can remain inactive, or become 'latent', but remain capable of reactivating later. These viruses periodically reactivate meaning that, if treatment is stopped they can rekindle the infection, rendering HIV incurable. One experimental approach to curing HIV is to reactivate these latent viruses whilst administering antiretroviral treatment at the same time, thereby flushing out the latent reservoir. Thus, it is crucial to be able to reactivate latent HIV effectively and measure the latent viral load efficiently.

The current gold standard measure of the latent HIV viral load is complex and labour intensive. It involves isolating the type of cells that harbour latent HIV, seeding them in defined small numbers in tissue culture wells, activating them, detecting proteins from reactivated viruses and working back to calculate the number of latently infected cells. This process takes many days to yield a result. HIV is genetically diverse and my pilot data show that each latent virus is genetically distinct. Activating a sample will add reactivated viruses to the circulating population. Thus one expects an increase in the number of distinct viral sequences after latent viruses have been reactivated. I will use modern sequencing methods and recently developed bioinformatics techniques to measure the number of viral sequences and correlate it with the latent viral load. This will reduce the amount of laboratory manipulation required for the assay.

Latent viruses are reactivated by activation of the cells containing them. However, even with the powerful stimuli currently available, only a minority can be reactivated. I will generate cultures containing latent viruses. Some cultures will have only reactivatable viruses, some only non-reactivatable viruses. I will study why viral reactivation is ineffective, focusing on areas that have been overlooked in previous studies.

There is evidence that some cells can be induced to produce viral RNA, but not virus particles. It is possible that there are additional blocks in the way the cell processes viral RNA and produces viruses. I will characterise these viral RNAs, sequence them and look for consistent mutations in apparently intact viral sequences that may account for their inability to produce virus. We can potentially uncover novel viral encoded mechanisms through which RNA processing and production are controlled.

Most integrated viruses are defective or non-reactivatable. Reactivatable viruses constitute a small minority. It is possible that there are special features in the sites into which some viruses are integrated that make them susceptible to reactivation. Data from cell line models are inconsistent, and existing data from patient is derived from unselected cells and non-reactivable infected cells, both contain large proportions of defective viruses. I will characterise the integration sites of reactivatable integrated viruses and identify features rendering the virus susceptible to reactivation.

Technical Summary

I am currently working to confirm that viruses from each latently infected cell are genetically distinguishable from each other. I am also working with my collaborators to define and reduce the errors introduced by virus outgrowth, cDNA and amplicon generation and next generation sequencing. After gathering the pilot data, I will proceed to characterise the repertoire of reactivated viruses in patient blood samples by next generation sequencing. I will look for an increase in the number of distinct viral sequences in a sample after viral reactivation and correlate it with the reactivatable viral load determined by virus outgrowth assay.

I will use the cultures generated in virus outgrowth assay to study the causes of ineffective reactivation. I will harvest intracellular RNA from cultures which viruses have not been reactivated, characterise and sequence any viral transcripts detected. I will look for mutations consistently present in viral intracellular RNAs from non-reactivatable cells.

I will examine the integration sites of the viruses which have been successfully reactivated. To facilitate this, I will expand the copies of genome available for analysis using low dose IL-7, which causes cellular proliferation without disrupting viral latency. To obtain the integration sites, I will modify a published method that involves DNA digestion, linker ligation, PCR amplification and sequencing.

I will optimize bisulphite genomic sequencing using a cell line model of HIV latency. Once optimized, I will perform bisulphite genomic sequencing on 5'LTR and Env in ex vivo cultures from virus outgrowth assay, correlating base modification with reactivability of the virus. I will also correlate DNA base modification with viral transcription activity. I aim to seek further collaboration to study the role of different base modifications such as CpG methylation and hydroxymethylation in HIV gene expression at single cell level.

Planned Impact

This project addresses some of the most important research questions surround HIV cure today.

One of the aims of the project is to develop a less labour intensive approach to measure the latent HIV viral load. This is a key area of research in HIV cure. The definitive virus outgrowth assay is labour intensive, limiting its widespread use. Our laboratory has since streamlined this assay and this project aims to reduce its labour requirement further. This can in turn facilitate clinical studies on latency reversal in the pursuit of HIV cure. The approach can also be used to study HIV latency ex vivo, as example to assess the effectiveness of different pharmacological agents in reactivating HIV. This part of the project is thus important for HIV cure research from a pragmatic perspective.

Effective reversal of HIV latency is crucial in the pursuit of the 'shock and kill' strategy of HIV cure. This project aims to identify the causes of ineffective viral reactivation. It has the potential to be a landmark study. One potential outcome is the identification of previously unrecognised defects in viral RNA that account for the apparent 'intact', non reactivatable latently infected cell. It will thus redefine the size of the latent reservoir. It will also identify new viral regulatory elements and thus enhance our understanding of the virus. By dissecting the genomic features and epigenetic changes rendering a virus susceptible or resistant to reactivation, this project will generate the data required to design more effective reactivation strategy. It also has the potential to push the boundary on the field of epigenetics, particular as we are studying a region in Env and our understanding on how intragenic epigenetic changes affect transcription is limited.

This project will generate data on the sequences of reactivated latent HIV. These can answer the question on whether clonal proliferation of infected cells plays a role in the maintenance of HIV latency.

In a wider context, having this work conducted in the UK will preserve expertise of HIV cure research in the UK. This will facilitate further research work in this area. HIV infected patients in the UK will thus have ready access to such expertise and potentially be amongst the first globally to benefit from the fruits of such research.
 
Description BHIVA National Conference 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Poster presentation of research results in National Conference, attended by researchers, clinicians and patient advocates
Year(s) Of Engagement Activity 2017
 
Description CHERUB Workshop 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Oral presentation in UK Collaborative HIV Latency Research Workshop in London, UK. Titled:-
Factors intrinsic to the virus significantly influence HIV silencing and inducibility.
Year(s) Of Engagement Activity 2018
 
Description Frontiers of Retrovirology 2018 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact poster presentation in Frontiers of Retrovirology Meeting, Leuven, Belgium. Poster titled:
A novel sensitive indicator cell line to quantify the reactivatable latent viral load.
Year(s) Of Engagement Activity 2018
 
Description Frontiers of Retrovirology 2018 - poster presentation 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Poster presentation in Leuven, Belgium - titled:-
Factors intrinsic to the virus significantly influence HIV silencing and inducibility.
Best poster Award
Year(s) Of Engagement Activity 2018
 
Description Grand round - 13 Feb 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Grand round - presentation of a patient involved in HIV cure, generated huge ongoing interest
Year(s) Of Engagement Activity 2019
 
Description Poster presentation - Blizard Institute HIV Symposium, London. 23 June 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Poster presentation in a national meeting
Year(s) Of Engagement Activity 2016
 
Description oral presentation - 10th annual meeting of Cambridge retrovirologists 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact meeting of researchers in the field locally
Year(s) Of Engagement Activity 2017
 
Description oral presentation - CHERUB workshop. London. December 2016 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Meeting of researchers in the CHERUB collaboration
Year(s) Of Engagement Activity 2016
 
Description poster presentation - Frontiers of Retrovirology Meeting, Erlangen, Germany. 12-14 September 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact poster presentation in an international conference
Year(s) Of Engagement Activity 2016