Investigating the distribution & impact of HIV-specific broadly neutralising antibodies on mucosal immunity and the HIV reservoir in rectal tissue

Antiretroviral treatment (ART) has improved survival for people with HIV. But it cannot cure HIV as ART cannot remove HIV from cells containing virus in a resting state, known as the HIV reservoir. Current ART options usually requires daily oral medications, challenges include long-term side effects, treatment fatigue, drug resistance and expense. Broadly neutralising antibodies (bNAbs) are a new type of HIV treatment that may provide a safe, effective, and long-acting alternative to ART.
Early studies suggest bNAbs may 'train' the immune system leading to long lasting HIV-specific responses. Monkeys given bNAbs early in SHIV infection, demonstrated virus control for up to 4 years. Two individuals treated with bNAbs in a small human study have unexpectedly continued to maintain undetectable virus levels in blood even beyond 30 weeks. However these studies were not randomised.
The largest reservoir of HIV in the body is in the gut. It can be up to 5 times larger than in blood. It is uncertain if bNAbs enter gut tissue as well as in blood. Immune responses in the gut also differ from blood in many ways, such as slower recovery even after starting ART. Finally, we do not know how bNAbs affect the gut HIV reservoir.
The RIO trial is an ongoing funded study. It compares a single dose of two types of bNAbs against inactive drug (placebo). Participants then stop ART and will return for frequent viral load tests. When the virus is detectable in blood, they will restart ART. Participants receiving placebo will be given the bNAbs after restarting ART. The RIO trial provides a unique chance to study the impact of bNAbs on the gut immune system and HIV reservoir through rectal biopsies.
I will study rectal biopsies samples collected from RIO participants before and after they receive the study drugs (see Annex 1). These small 3mm biopsies are collected by an experienced clinician through a safe and painless procedure in clinic, using a short plastic tube in their rectum. This is less invasive than other common gut procedures such as colonoscopies and have minimal risks.
The project aims to answer 3 research questions:
1: Are the bNAbs levels in the gut comparable to blood?
Paired rectal tissue and blood bNAb levels will be measured using a highly sensitive test that can measure even single molecules of bNAbs present. If peak gut tissue bNAb concentrations are comparable to blood, these data will support further research of bNAbs for the eradication of HIV gut reservoir. This work will be done on an expected n = 10 participants who are randomised to receive the bNAbs in the main study.
2: Do bNAbs 'train' immune cells in the gut?
I will use fluorescent antibody 'dyes' to look for the presence of both bNAbs and immune cells in biopsy sections through an automatic microscope. If bNAbs can 'train' HIV-specific immune responses, we will expect to see bNAbs in areas of immune cells called germinal centres. Learning how bNAbs 'train' gut immune responses may help researchers design better treatment or vaccines.
3: Do bNAbs affect the HIV reservoir in the gut?
Studying resting HIV reservoir containing cells is challenging, as most contain defective HIV DNA and only about 1% contain intact HIV DNA. Cells containing intact HIV DNA are likely the source of rebound virus when ART is stopped. I will improve an existing intact HIV DNA test to sort intact from defective HIV DNA in the rectal tissue for analysis. This test will provide a more accurate, in-depth analysis of the HIV reservoir.
Using the improved test, I will be able to map how the resting HIV reservoir has changed after being exposed to bNAbs. By comparing samples from the same individual, I can test if the viral reservoir has grown during ART interruption, and if bNAbs has an impact on the HIV reservoir.
Findings from this project will provide insight into how the bNAbs work in the gut, and aid development of bNAbs and other strategies to eradicate the gut HIV reservoir.

Broadly neutralising antibodies (bNAbs) are a promising novel HIV therapeutic, potentially capable of inducing long-term immune responses even after treatment interruption. The RIO trial is a placebo-controlled randomised trial of single dose long-acting dual bNAbs and treatment interruption. I will establish a sub-study collecting 7 rectal biopsies per person, in 20 RIO participants to investigate the effect of bNAbs on gut tissue, the largest HIV reservoir site in the body.
The project aims to determine 1) if bNAbs in rectal tissue achieve concentrations comparable to blood; 2) immunohistochemistry analysis of bNAbs interaction with rectal lymphoid tissue; and 3) the effect of bNAbs on the rectal latent HIV proviral reservoir.
Methodology:
Homogenised rectal tissue bNAb concentrations will be measured using a single-molecule ELISA and compared with paired serum bNAb concentrations for pharmacokinetic analyses (n=10, 5-7 timepoints per participant).
Formalin fixed, paraffin embedded biopsy sections exposed to bNAbs will be used to optimise conjugated bNAb-specific fluorescent-labelled anti-idiotype and immune cellular markers antibodies. Once optimized, the sections will be screened with the high-throughput Vectra Polaris multiplex automated imaging system and the HALO multiples imaging analysis platform. Regions of interest comprising isolated lymphoid follicle structures and bNAbs (n=20, 3 timepoints per participant) will be identified using cellular markers (see CfS) and bNAb antibody stains.
The development of a novel IPDA using double emulsion droplet technology will improve on the existing IPDA, to allow sorting and sequencing of rare intact HIV DNA sequences in tissue and PBMCs in addition to intact reservoir size quantification. Phylogenetic analyses will compare the intact HIV reservoir sequences to determine the presence of changes in the HIV reservoir compared at baseline and post viral rebound and ART restart (n=20,2 timepoints per participant).