Vedolizumab Use and the Associations Between Α4β7 Expression and HIV Reservoir in the Gut During Treated Primary HIV Infection --Manuscript Draft

AIDS

Vedolizumab use and the associations between α4β7 expression and HIV reservoir in the gut during treated primary HIV infection --Manuscript Draft--

Manuscript Number: AIDS-D-19-00507 Full Title: Vedolizumab use and the associations between α4β7 expression and HIV reservoir in the gut during treated primary HIV infection Article Type: Correspondence Section/Category: Keywords: HIV; HIV Reservoir; Vedolizumab; α4β7 integrin; GALT Corresponding Author: John Patrick Thornhill, Ph.D., M.B. B.Ch Imperial College London London, UNITED KINGDOM Corresponding Author Secondary Information: Corresponding Author's Institution: Imperial College London Corresponding Author's Secondary Institution: First Author: John Thornhill, PhD MB BCh First Author Secondary Information: Order of Authors: John Thornhill, PhD MB BCh Kate D Lynch, DPhil MBBS Jessica K Skelton, PhD Marcus Dorner, PhD Maryam Khan, BSc Genevieve E E Martin, DPhil MBBS Jonathan Hoare, PhD MBBS Simon Peake, MBBS John Frater, PhD MBBS Sarah Fidler, PhD MBBS Order of Authors Secondary Information:

Dr John Thornhill PhD, MB BCh BAO MRC Clinical Research Fellow, St Marys Hospital Campus Imperial College London W2 1NY Tel: +44 (0)1865 271288 Fax: +44 (0)1865 281890 Email: [email protected]

19th June 2019

To the Editor, JA Levy, AIDS

Re: Vedolizumab use and the associations between 47 expression and HIV reservoir in the gut during treated primary infection _ A report in Science by Byrareddy, et al. (Sustained virologic control in SIV+ macaques after antiretroviral and 47 antibody therapy. (Science 354, 197-202 (2016)) garnered much interest, suggesting that 47 antibody therapy may be a scalable intervention for use in HIV remission studies.

Given the recent interest in this novel intervention, we submit this report characterising 7 expression on CD4 T cells in gut tissue of HIV-infected participants. We observed a correlation between 7 on CD4 T cells and HIV reservoir in both the terminal ileum and rectum. Furthermore, we present the case of an individual treated with 47 antibody therapy during primary HIV infection, showing reduced 7 expression in the gut but no evidence of HIV remission (using a humanised mouse model). Our findings are consistent with an as yet published clinical study (NCT02788175).

I confirm that all authors have seen and approved the manuscript, and that all authors have significantly contributed to the work. Also, I can confirm that this work has not been previously published and it is not being considered for publication elsewhere.

We believe that our data are of interest to the readership of AIDS. We look forward to hearing from you.

With my best wishes

Dr John Thornhill, and on behalf of my co-authors Article

Vedolizumab use and the associations between 47 expression and HIV

reservoir in the gut during treated primary HIV infection

John P THORNHILL1,2,3, Kate D LYNCH2, Jessica K SKELTON1, Marcus DORNER1,

†, Maryam KHAN1, Genevieve E MARTIN2, Jonathan HOARE1, Simon PEAKE1,

John FRATER2,4.5, Sarah FIDLER1,3.

1. Division of Medicine, Wright Fleming Institute, Imperial College, London, UK

2. Peter Medawar Building for Pathogen Research, Nuffield Department of Medicine,

University of Oxford, Oxford, UK

3. Imperial College NIHR Biomedical Research Centre

4 National Institute of Health Research Biomedical Research Centre, Oxford, UK

5 Oxford NIHR Biomedical Research Centre

† deceased

Keywords: HIV; HIV Reservoir; Vedolizumab; α4β7 integrin; GALT

Running Head: 47 expression & HIV reservoir in gut

Word Count: 743

Corresponding author: John Thornhill, Imperial College London

([email protected])

Funding: This work was supported by an Medical Research Council Fellowship

(grant MR/N001265/1 to JT) and a British HIV Association Research Award.

Abstract

Latent HIV infection in gut CD4+ cells is a barrier to HIV eradication. α4β7 integrin is a gut homing marker expressed on CD4+ cells. Monoclonal antibodies against α4β are being trialled to induce HIV remission. Using gut biopsy samples taken from a cohort of HIV+ individuals treated during primary HIV infection, we characterised β7 expression and HIV DNA in terminal ileum and rectum. We demonstrate an association between HIV DNA and β7 expression in gut, and present the case of a

HIV+ individual treated with an α4β7 antibody; finding no evidence of HIV remission by humanized murine viral outgrowth assay.

A cure for human immunodeficiency virus (HIV) infection remains elusive with two cases of HIV cure to date[1, 2], both resulting from haematopoietic stem-cell transplantation (HSCT). Whilst interesting as proof of concept, HSCT is associated with high morbidity and mortality and is not scalable; therefore, alternative strategies towards a HIV cure are being explored. Antiretroviral therapy (ART) confers a significant survival benefit but cannot cure HIV infection - a consequence of latently infected CD4+ T-cells, particularly within tissue sites. Gut-associated lymphoid tissue

(GALT) serves as one of the main barriers to HIV eradication[3]. α4β7 integrin is a gut homing marker expressed on CD4+ T-cells which allows trafficking of CD4+ T-cells to gut tissue[4]. Furthermore, HIV preferentially infects 47+CD4+ T-cells[5]. Antibodies targeting α4β7 are used to treat inflammatory bowel disease[6]. In SIV-infected primates initial reports suggested treatment with a mononclonal antibody against α4β7 integrin and ART conferred viral control after stopping ART following administration in early infection[7].

We characterized β7 expression and total HIV DNA levels from gut lamina propria mononuclear cells (LPMC) in HEATHER: a UK multicenter prospective cohort of patients with PHI who commenced on ART within three months of PHI diagnosis[8]. In addition, we present the case of an individual from the HEATHER cohort, with ART- treated PHI who also received treatment with a licensed anti-α4β7 integrin monoclonal antibody, vedolizumab, for Crohn’s disease.

Matched peripheral blood mononuclear cell (PBMC) and gut LPMC isolated from the terminal ileum and rectum from twenty-four individuals in HEATHER were available.

β7 expression - the proportion of memory (CD45RA-CD4+) T cells which were β7

3 positive - was determined by a fluorescence minus one control using flow cytometry.

Total HIV DNA was quantified from collagenase digested LPMCs by qPCR.

One of the twenty-four individuals was diagnosed with Crohn’s disease five months prior to HIV acquisition (Figure 1a). This 31-year-old MSM tested negative for HIV at his Crohn’s diagnosis and again 2 months later. His Crohn’s disease was initially managed with prednisolone induction and subsequent azathioprine maintenance.

Eight months following the diagnosis of Crohn’s disease, he was diagnosed with PHI

(with a CD4 T cell count of 475 cells/mm3 and a HIV viral load of 44,038 copies/ml).

ART was commenced 28 days after PHI diagnosis with Truvada and raltegravir.

Raltegravir was switched to darunavir and ritonavir one month later based on HIV genotype testing. HIV viral load was <20 copies RNA/ml within five months of ART initiation. Twelve months after the diagnosis of Crohn’s disease and three months after

PHI, colonoscopy confirmed active ileitis and vedolizumab was commenced. Follow- up biopsy, four months after commencing vedolizumab, showed gross resolution of ileal inflammation with only mild residual chronic active granulomatous colitis on histopathology. Research gut biopsies were taken at this timepoint.

β7 expression on memory CD3+CD4+ LPMCs from the overall HEATHER cohort

(n=24) correlated with total HIV DNA in the terminal ileum (r=0.67 p=0.0007; Figure

1b) and rectum (r=0.46 p=0.03; Figure 1c). The vedolizumab–treated individual had the lowest β7 expression on CD3+CD4+ LPMCs measured in the terminal ileum

(5.2%) and relatively lower expression in the rectum (8.1%) when compared to other

HEATHER participants, as well as the highest measured β7 expression on PBMC- derived memory CD4 T cells (24%). Median β7 expression for the HEATHER cohort

4 was 27.1%, 18.3% & 11.0% in the terminal ileum, rectum & PBMC respectively (Figure

1d). Correspondingly, HIV DNA levels in gut LPMCs for the vedolizumab–treated participant were at or just below the median, while his PBMC HIV DNA was the highest measured in the overall cohort (Figure 1e). To confirm the presence of replication competent viral reservoirs in this patient, PBMCs obtained from the vedolizumab- treated participant were subsequently analysed by humanized murine viral outgrowth assay[9]. For this, two timepoints after vedolizumab treatment were evaluated and resulted in detectable serum HIV RNA in mice at days 14 and 28.

Together these results suggest that vedolizumab reduced trafficking of β7 expressing

CD4 T cells to the gut and support recent work from Uzzan et al. who reported significant attenuation of lymphoid aggregates, most notably in the terminal ileum with anti-α4β7 therapy[10]. These data also demonstrate an association between β7 expression and HIV DNA measured in gut LPMCs. Despite this, and consistent with other work in humanized mice[11] and the first unpublished human study using vedolizumab in HIV+ individuals[12]; our data suggests that while vedoluzimab treatment may confer lower expression of β7 on CD4 T cells in gut LPMCs, vedoluzimab is unlikely to induce sustained viral remission.

Authors’ contributions.

The study experiments were conceived and designed by JT, KL, GM, JH, SP, JF and

SF. Recruitment of the trial samples were performed by, JT, JF, SP, JH & SF. Gut biopsy processing, HIV DNA quantification and flow cytometry experiments were performed by JT, KL and MK. The humanized mouse model experiment was

5 performed by JS and MD. Data was analysed by JT, JS & MD. The paper was written by JT with input from all authors.

Acknowledgments: We thank the participants of HEATHER. The HEATHER study is conducted as part of the CHERUB (Collaborative HIV Eradication of Reservoirs: UK

BRC) collaboration. (CHERUB Steering Committee: Andrew Lever (University of

Cambridge), Mark Wills (University of Cambridge), Jonathan Weber (Imperial College,

London), Sarah Fidler (Imperial College, London), John Frater (University of Oxford),

Lucy Dorrell (University of Oxford), Mike Malim (King’s College, London), Julie Fox

(King’s College London), Ravi Gupta (University College London), Clare Jolly

(University College London

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Figure 1.

The clinical course of the HEATHER participant who received vedolizumab in primary

HIV infection is shown in (a). The correlation of the expression of β7 on memory CD4

T cells and total HIV DNA from individuals in the HEATHER cohort from the (b) terminal ileal and (c) rectal GALT is shown. (d) shows the β7 expression on memory

CD4 T cells across anatomical sites for the HEATHER cohort; data from the individual who received vedolizumab treatment is indicated by the solid shapes. (e) shows the total HIV DNA measured from CD4 T cells across anatomical sites for the HEATHER cohort; again, data from the individual who received vedolizumab treatment is indicated by the solid shapes.

Figure 1 Click here to download Figure Figure 1.jpg