Comparing Immune Activation and Latent HIV Reservoir Size Between People Living With HIV on Tenofovir-containing Versus NRTI-free ART

Study Overview
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Brief Summary
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The goal of the project is to determine the difference in immune activation and HIV reservoir size between People living with HIV (PWH) on tenofovir-containing antiretroviral therapy (ART) versus PWH on nucleoside reverse transcriptase inhibitor (NRTI)-sparing ART. Tenofovir (TFV), a phosphonated nucleoside reverse transcriptase inhibitor (NRTI), is being used for oral pre-exposure prophylaxis (PrEP). The investigators will test this hypothesis: tenofovir, and perhaps NRTIs in general, stimulate a type I/III interferon also in PWH who take these drugs. Because chronic interferon stimulation may promote the survival and proliferation of cells with integrated provirus, the investigators also hypothesize that these drugs antagonize decay of the HIV latent reservoir in PWH on ART. Consequently, the researchers hypothesize that PWH who have switched from NRTI-containing ART to NRTI-sparing ART exhibit lower type I/III interferon pathway activation and lower latent HIV reservoir size. The investigators also hypothesize that independently of treatment, the extent of type I/III interferon activation correlates with latent HIV reservoir size. Thus, the proposed study seeks to answer these two questions. Can the gastrointestinal epithelium be impacted by ART, and contribute to chronic immune activation and expansion of the HIV-1 reservoir? If so, what therapeutic approaches can the investigators implement to reduce the HIV-1 proviral load? The data will reveal pathways that can be targeted therapeutically to treat chronic immune activation in PWH. The findings of this study will immediately translate to optimize the standard of care in PWH.

Detailed Description
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The study will be open-label, cross-sectional, two-cohort study (20 participants per cohort will be recruited). Cohort 1: Tenofovir-containing ART (tenofovir disoproxil fumarate [TDF] OR tenofovir alafenamide [TAF] PLUS any other ART drugs) prescribed for daily use by participants' primary care providers. Cohort 2: NRTI-sparing ART (specifically: rilpivirine PLUS dolutegravir OR rilpivirine PLUS cabotegravir) prescribed for daily use by participants' primary care providers. The investigators plan to test type I/III IFN (interferon) pathway activation using Crystal digital PCR (Crystal-dPCR) to quantify mRNA copy numbers of ISG15 (ISG15 ubiquitin-like modifier), MX1 (MX dynamin like GTPase 1) and IFI6 (interferon alpha inducible protein 6). RNA will be isolated from the rectal and duodenal biopsies stored in RNALater using the RNeasy Fibrous Tissue Mini Kit (Qiagen) and from the PBMC using the RNeasy Plus Mini Kit (Qiagen). Crystal-dPCR will be performed on a 6-color Naica Crystal digital PCR instrument (Stilla Technologies). The latent HIV reservoir will be measured in DNA derived from peripheral blood mononuclear cells (PBMCs) using a novel intact/defective proviral HIV DNA Crystal-dPCR assay developed jointly by the Hladik and Jerome groups in Seattle.

Official Title
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Comparing Immune Activation and Latent HIV Reservoir Size Between People Living With HIV (PWH) on Tenofovir-containing Versus NRTI-sparing ART

Conditions
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HIV-1-infection, HIV Disease Progression, Inflammation, HIV

Intervention / Treatment
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* Procedure: Venipuncture
* Procedure: Targeted physical medical exam
* Procedure: Urine pregnancy test
* Procedure: Anoscopy
* Procedure: Esophagogastroduodenoscopy (EGD)


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Confirmed HIV infection, by two different positive antibody tests and/or detectable plasma HIV RNA on two different dates ≥18 and ≤65 years of age Stable use of ART medication for ≥ 1 year No switch of ART regimen within the past 180 days CD4 > 350/mm3 within the past 180 days HIV RNA <40 copies / mL on ≥ 2 occasions during continuous ART of ≥ 1 years, with no blip of >1000 HIV RNA copies / mL Karnofsky score ≥80 Willingness and ability to provide informed consent for study participation Willingness to undergo all required study procedures Exclusion Criteria: Active malignancy including myelodysplastic syndrome, or myeloproliferative disease within 24 weeks prior to study entry Prior organ or bone marrow transplantation Diagnosed autoimmune disease Medical need for ongoing treatment with an immunosuppressive drug Diagnosis of AIDS (defined as any AIDS-defining opportunistic infection or cancer, or a history of blood CD4+ T cell count < 200/μL) Active opportunistic infection Vomiting or diarrhea which prohibits consistent use of ART Pregnant or breastfeeding Excessive ingestion of ethanol determined by an AUDIT score of >8 Substance abuse History of medical non-compliance The following laboratory values (< 30 days before enrollment): Hemoglobin < 8.5 mg/dL Platelet count < 100,000/μL Coagulation (PT/PTT) tests above the normal reference Creatinine clearance < 60 mL/min Using disallowed medications: Systemic corticosteroids Other immunosuppressive medications (e.g., cyclosporine, sirolimus, tacrolimus, pimecrolimus, tofacitinib) BMI > 40 Pulmonary dysfunction. Use of narcotics.

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 65 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Tenofovir-containing ART<br>Cohort 1: Tenofovir-containing ART (tenofovir disoproxil fumarate [TDF] OR tenofovir alafenamide [TAF] PLUS any other ART drugs) prescribed for daily use by participants' primary care providers. | Procedure: Venipuncture<br>* Draw of peripheral blood (about 20 ml and 40 ml, during the screening and procedure visits, respectively).<br>* Other names: Peripheral blood draw by phlebotomy;Procedure: Targeted physical medical exam<br>* Study participants will be undergoing a physical exam where vitals will be recorded (e.g. temperature, blood pressure, heart rate). The registered nurse (RN) will also perform auscultation of heart and lungs. Study participants will be also asked to fill out a survey with questions related to his/her medical history, current use of medications, sexual history and substance abuse.<br>Procedure: Urine pregnancy test<br>* Women of childbearing potential will be asked to run an urine pregnancy test during the second (procedure) visit. Pregnant women will not be allowed to participate in the study.<br>Procedure: Anoscopy<br>* The anoscopy is an examination using a small, rigid, tubular instrument called anoscope (also called an anal speculum). This is inserted a few inches into the rectum in order to collect some small samples of mucosal tissue. We will collect 5 rectum biopsy samples and one cytobrush.<br>Procedure: Esophagogastroduodenoscopy (EGD)<br>* The EGD involves looking at the esophagus, stomach, and first and second portion of the duodenum. This procedure involves the use of an endoscope to remove small tissue samples. This procedure uses conscious sedation drugs given by a vein in the arm. The procedure takes about 1½-2 hours, including time for recovery. We will collect 5 duodenal biopsies and one cytobrush.<br>* Other names: Upper endoscopy;|
| NRTI-sparing ART<br>Cohort 2: NRTI-sparing ART (specifically: rilpivirine PLUS dolutegravir OR rilpivirine PLUS cabotegravir) prescribed for daily use by participants' primary care providers. | Procedure: Venipuncture<br>* Draw of peripheral blood (about 20 ml and 40 ml, during the screening and procedure visits, respectively).<br>* Other names: Peripheral blood draw by phlebotomy;Procedure: Targeted physical medical exam<br>* Study participants will be undergoing a physical exam where vitals will be recorded (e.g. temperature, blood pressure, heart rate). The registered nurse (RN) will also perform auscultation of heart and lungs. Study participants will be also asked to fill out a survey with questions related to his/her medical history, current use of medications, sexual history and substance abuse.<br>Procedure: Urine pregnancy test<br>* Women of childbearing potential will be asked to run an urine pregnancy test during the second (procedure) visit. Pregnant women will not be allowed to participate in the study.<br>Procedure: Anoscopy<br>* The anoscopy is an examination using a small, rigid, tubular instrument called anoscope (also called an anal speculum). This is inserted a few inches into the rectum in order to collect some small samples of mucosal tissue. We will collect 5 rectum biopsy samples and one cytobrush.<br>Procedure: Esophagogastroduodenoscopy (EGD)<br>* The EGD involves looking at the esophagus, stomach, and first and second portion of the duodenum. This procedure involves the use of an endoscope to remove small tissue samples. This procedure uses conscious sedation drugs given by a vein in the arm. The procedure takes about 1½-2 hours, including time for recovery. We will collect 5 duodenal biopsies and one cytobrush.<br>* Other names: Upper endoscopy;|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Type I/III Interferon pathway activation | Quantification of the mRNA copy number of the following Interferon-Stimulated Genes (ISGs): ISG15 (ISG15 ubiquitin-like modifier), MX1 (MX dynamin-like GTPase 1) and IFI6 (interferon alpha inducible protein 6). Copy numbers will be determined by Crystal digital PCR (dPCR), using the levels of UBC (Ubiquitin C) mRNA copies as normalizers. | Biopsy samples to assess interferon pathway activation will be collected during surgery. The assessment of this outcome (the determination of mRNA copy number) will be done around within one year of completing sample collection. | 
| Size of the latent intact proviral HIV reservoir in cell-associated HIV DNA (Ca-DNA) | Determination of size (copy number) of the HIV proviral intact reservoir using a recently published assay the Hladik's lab conjointly with Jerome's lab developed (reference: Levy et al., 2021, Cell Reports Medicine 2, 100243). This outcome will be expressed as intact HIV copy numbers (found in cell-associated HIV DNA) per 10^6 T cells. We will use a multiplexed digital PCR (dPCR) assay that simultaneously quantifies likely intact HIV-1 proviruses and T lymphocytes. We designed two triplex droplet digital PCR assays, each with 2 unique targets and 1 in common, and normalize the results to PCR-based T cell counts. Both HIV assays are specific, sensitive, and reproducible. Together, they estimate the number of proviruses containing all five primer-probe regions. | Blood samples to assess the latent HIV reservoir will be collected during surgery. The assessment of this outcome (the determination of HIV copy number) will be done around within one year of completing sample collection. | 
| Size of the latent defective proviral HIV reservoir in cell-associated HIV DNA (Ca-DNA) | Determination of size (copy number) of the HIV proviral defective reservoir using a recently published assay the Hladik's lab conjointly with Jerome's lab developed (reference: Levy et al., 2021, Cell Reports Medicine 2, 100243). This outcome will be expressed as defective HIV copy numbers (found in cell-associated HIV DNA) per 10^6 T cells. | Blood samples to assess the latent HIV reservoir will be collected during surgery. The assessment of this outcome (the determination of HIV copy number) will be done around within one year of completing sample collection. | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Composition of gastrointestinal microbiota | We will do 16s RNA sequencing in rectum and duodenum cytobrush samples to identify, classify and quantify gut microbiota. | Cytobrush samples to assess gut microbiota will be collected during surgery. The assessment of this outcome (the determination of 16s repertoire) will be done around within one year of completing sample collection. | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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HIV reservoir, Interferon Stimulated Genes, Tenofovir, Interferon, Duodenum, Blood, Rectum, NRTI