Doing FLIPS: a new way to find latent HIV

Researchers have revealed a new way to understand the HIV that stays in the body during therapy. The group from Sydney’s Westmead Institute for Medical Research have developed a technique for detailing HIV at a molecular level. Bonnie Hiener and colleagues, led by Associate Professor Sarah Palmer, published the study in the journal Cell Reports in October 2017.

They call the technique Full-length Individual Proviral Sequencing or FLIPS. It detects single intact copies of HIV in cells, which can result in the production of new virus.

Why is this important?

The latent HIV reservoir is a barrier to cure. We need to understand where and how much virus remains in a person on therapy. This can help inform strategies to target and get rid of this remaining pool of HIV.

The problem is that finding HIV in people on treatment is hard! Only a tiny amount of virus stays around once therapy has begun, so infected cells are hard to find. Also, many cells contain virus that is defective and can’t replicate. The ‘gold-standard’ for detecting virus that can replicate (‘replication-competent’ virus) is a viral outgrowth assay. This test is difficult and time-consuming, and underestimates the amount of replication-competent HIV.

Detecting and analysing HIV DNA is an alternative. Most sequencing strategies focus on sub-regions of HIV. These strategies sensitively detect HIV, but can’t indicate whether the virus is able to replicate. Studying virus fragments also limits analysis of the full range of HIV diversity and evolution.

Hiener and colleagues take advantage of developments in sequencing technology to sequence nearly full-length HIV. Lead author, Sarah Palmer explains “FLIPS allows for in-depth genome-scale analyses of the HIV populations in cells sorted from the peripheral blood and anatomic tissue sites.”

What is the FLIPS assay?

The FLIPS assay creates copies of single HIV genomes and assesses their ability to make new virus. The primers (or starting points for the assay) pick up conserved regions that bookend the HIV genome. This enables analysis of the entire HIV coding region. After amplification, the researchers exclude sequences that would not be able to replicate. This includes those with missing segments, early stop sequences, additions that shift the code so that it no longer makes sense, and changes to critical packaging and processing sequences.

Using FLIPS to study HIV in people on long term therapy

The researchers used their new test to look at HIV in subsets of CD4 T cells from six men on HIV therapy. Three of these men had been on treatment since early infection, and three since chronic infection.

Hiener et al. looked at four separate subsets of CD4 T cells:

• Naïve T cells (T_N), which have never been involved in an immune response
• Central Memory T cells (T_CM), which have been involved in an immune response and remain, long lived, in lymph nodes and other sites
• Transitional Memory T cells (T_TM), which have been involved in an immune response and move away from lymph nodes into the rest of the body
• Effector Memory T cells (T_EM), which have been involved in an immune response, circulate in the body and are quick to respond again

Previous work has shown that HIV hides primarily in T_CM and T_TM cells.

What did they find?

The researchers were able to find over 500 HIV sequences in the various cell samples. Only 5% of them were intact, replication competent HIV. Of those that were not intact, most (68%) had large internal deletions.

The researchers found replication competent HIV mostly in the T_EM and T_TM cells. They did not find intact HIV in T_CM cells, which have previously been considered a key part of the HIV reservoir. Sarah Palmer says this is probably because the study only looked at a small number of people. She explains “T_CM are more abundant in the peripheral blood and these cells contain a lot of defective virus which was established prior to antiretroviral therapy. To date, we have not found any intact virus in T_CM which indicates that the frequency of intact virus is extremely low in this T cell subset within the peripheral blood”. This result highlights the importance of distinguishing any HIV from replication-competent HIV. It appears that T_CM do harbour a lot of HIV, but most of this virus is defective.

The FLIPS assay enables researchers to look at the relatedness of sequences within an individual. Identical sequences within a single cell type are thought to be a result of cell proliferation. The study found many identical sequences in some people. This adds to the evidence that the latent HIV reservoir is at least partially controlled by cell proliferation (rather than virus replication).

What comes next – what else can this assay reveal?

The FLIPS assay provides a new opportunity to understand the latent HIV reservoir.

It will be important to show that FLIPS correctly identifies HIV that is replication competent. Palmer explains “we plan to explore the replication competency of a subset of the genetically intact viruses which were identified by FLIPS. This would reconfirm that the genetically intact virus identified by FLIPS is replication competent”.

So far, only small numbers of people have had their reservoir assessed using FLIPS.  There is potential for the assay to be used on larger numbers of samples. Palmer says “although this is an expensive assay, it involves a relatively high throughput sequencing technique which could be applied to large numbers of samples from clinical trials.” Researchers could also batch samples and automate data analysis to reduce the assay cost and time.

The researchers are now preparing the assay for use on HIV in plasma. Most plasma virus is infectious and replication competent. Palmer explains “if we could sequence this virus and compare the HIV RNA from virions to the HIV DNA in the cells, we could identify which cells are contributing to the replication-competent virus which persists in the plasma. This would further identify the cells we need to target with new therapies in order to eliminate HIV from the body.”

The FLIPS assay is an important tool for understanding HIV latency. It opens new possibilities for targeting and eliminating latent HIV.

Listen to an interview with Sarah Palmer

antiretroviral therapy CD4 T Cells cure HIV latency research T cells viral reservoir