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

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

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.

Study Overview

• Status: Enrolling by invitation
• Conditions: Inflammation; HIV; HIV-1-infection; HIV Disease Progression
• Intervention / Treatment: Procedure: Venipuncture; Procedure: Targeted physical medical exam; Procedure: Urine pregnancy test; Procedure: Anoscopy; Procedure: Esophagogastroduodenoscopy (EGD)

Detailed Description

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.

• Study Type: Observational
• Enrollment (Estimated): 40

Contacts and Locations

Study Locations

• United States
  • Washington
    • Seattle, Washington, United States, 98104
      • University of Washington Positive Research and the Gastroenterology Clinic at Harborview Medical Center

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 14 years to 61 years (Adult, Older Adult)
• Accepts Healthy Volunteers: No

• Sampling Method: Non-Probability Sample

Study Population

People living with HIV (PWH) on stable use of antiretroviral therapy (ART). They will be between 18 and 65 years of age. Children less than 18 years of age are excluded. Women of childbearing potential should not be pregnant to participate.

Description

Inclusion Criteria:

1. Confirmed HIV infection, by two different positive antibody tests and/or detectable plasma HIV RNA on two different dates
2. ≥18 and ≤65 years of age
3. Stable use of ART medication for ≥ 1 year
4. No switch of ART regimen within the past 180 days
5. CD4 > 350/mm3 within the past 180 days
6. HIV RNA <40 copies / mL on ≥ 2 occasions during continuous ART of ≥ 1 years, with no blip of >1000 HIV RNA copies / mL
7. Karnofsky score ≥80
8. Willingness and ability to provide informed consent for study participation
9. Willingness to undergo all required study procedures

Exclusion Criteria:

1. Active malignancy including myelodysplastic syndrome, or myeloproliferative disease within 24 weeks prior to study entry
2. Prior organ or bone marrow transplantation
3. Diagnosed autoimmune disease
4. Medical need for ongoing treatment with an immunosuppressive drug
5. Diagnosis of AIDS (defined as any AIDS-defining opportunistic infection or cancer, or a history of blood CD4+ T cell count < 200/μL)
6. Active opportunistic infection
7. Vomiting or diarrhea which prohibits consistent use of ART
8. Pregnant or breastfeeding
9. Excessive ingestion of ethanol determined by an AUDIT score of >8
10. Substance abuse
11. History of medical non-compliance

12. 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

13. Using disallowed medications:

    • Systemic corticosteroids
    • Other immunosuppressive medications (e.g., cyclosporine, sirolimus, tacrolimus, pimecrolimus, tofacitinib)
14. BMI > 40
15. Pulmonary dysfunction.
16. Use of narcotics.

Study Plan

How is the study designed?

Design Details

• Observational Models: Case-Control
• Time Perspectives: Cross-Sectional

Number of groups / cohorts

2

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
Tenofovir-containing ART; 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; Draw of peripheral blood (about 20 ml and 40 ml, during the screening and procedure visits, respectively).; Other Names: Peripheral blood draw by phlebotomy; Procedure: Targeted physical medical exam; 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.; Procedure: Urine pregnancy test; 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.; Procedure: Anoscopy; 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.; Procedure: Esophagogastroduodenoscopy (EGD); 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.; Other Names: Upper endoscopy
NRTI-sparing ART; Cohort 2: NRTI-sparing ART (specifically: rilpivirine PLUS dolutegravir OR rilpivirine PLUS cabotegravir) prescribed for daily use by participants' primary care providers.	Procedure: Venipuncture; Draw of peripheral blood (about 20 ml and 40 ml, during the screening and procedure visits, respectively).; Other Names: Peripheral blood draw by phlebotomy; Procedure: Targeted physical medical exam; 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.; Procedure: Urine pregnancy test; 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.; Procedure: Anoscopy; 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.; Procedure: Esophagogastroduodenoscopy (EGD); 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.; Other Names: Upper endoscopy

What is the study measuring?

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.

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
Determination of proteins that are secreted in the rectum and duodenum	We will conduct an unbiased proteomics approach to characterize the proteins that are secreted in both duodenum and rectum.	Cytobrush samples to assess secreted proteins will be collected during surgery. The assessment of this outcome (the determination of the proteome) will be done around within one year of completing sample collection.
Determination of the global transcriptome	We will do global transcriptomics analysis using sequencing of biopsy and blood mRNA	Blood and biopsy samples will be collected during surgery. The assessment of this outcome (the characterization of the transcriptome will be done around within one year of completing sample collection.

Collaborators and Investigators

• Sponsor: University of Washington
• Collaborators: National Institute of Allergy and Infectious Diseases (NIAID); National Center for Advancing Translational Sciences (NCATS)
• Investigators: Principal Investigator: German G Gornalusse, PhD, MSc, University of Washington; Principal Investigator: Florian Hladik, MD, PhD, University of Washington; Principal Investigator: Romel D Mackelprang, PhD, MSc, University of Washington

Publications and helpful links

General Publications

• Hughes SM, Levy CN, Calienes FL, Stekler JD, Pandey U, Vojtech L, Berard AR, Birse K, Noel-Romas L, Richardson B, Golden JB, Cartwright M, Collier AC, Stevens CE, Curlin ME, Holtz TH, Mugo N, Irungu E, Katabira E, Muwonge T, Lama JR, Baeten JM, Burgener A, Lingappa JR, McElrath MJ, Mackelprang R, McGowan I, Cranston RD, Cameron MJ, Hladik F. Treatment with Commonly Used Antiretroviral Drugs Induces a Type I/III Interferon Signature in the Gut in the Absence of HIV Infection. Cell Rep Med. 2020 Sep 22;1(6):100096. doi: 10.1016/j.xcrm.2020.100096.

Study record dates

Study Major Dates

• Study Start (Actual): September 1, 2022
• Primary Completion (Estimated): July 30, 2026
• Study Completion (Estimated): August 28, 2026

Study Registration Dates

• First Submitted: October 5, 2022
• First Submitted That Met QC Criteria: October 14, 2022
• First Posted (Actual): October 18, 2022

Study Record Updates

• Last Update Posted (Actual): April 29, 2026
• Last Update Submitted That Met QC Criteria: April 27, 2026
• Last Verified: April 1, 2026

More Information

Terms related to this study

• Keywords: Tenofovir; Blood; Interferon; NRTI; Rectum; HIV reservoir; Duodenum; Interferon Stimulated Genes
• Additional Relevant MeSH Terms: Pathologic Processes; Pathological Conditions, Signs and Symptoms; Inflammation; Investigative Techniques; Therapeutics; Specimen Handling; Clinical Laboratory Techniques; Diagnostic Techniques and Procedures; Diagnosis; Punctures; Surgical Procedures, Operative; Minimally Invasive Surgical Procedures; Diagnostic Techniques, Surgical; Endoscopy, Gastrointestinal; Diagnostic Techniques, Digestive System; Endoscopy; Digestive System Surgical Procedures; Blood Specimen Collection; Phlebotomy; Proctoscopy; Endoscopy, Digestive System; Gastroscopy
• Other Study ID Numbers: STUDY00011699; KL2TR002317 (U.S. NIH Grant/Contract); R01AI134293 (U.S. NIH Grant/Contract)

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: YES
• IPD Plan Description: We may share information encompassing global transcriptome analysis (e.g. RNA-Seq data) derived from biopsy or peripheral blood mononuclear cells (PBMC) mRNA, DNA sequencing of the bacterial 16S RNA gene from cytobrush material (to determine microbial composition in duodenum or rectum) and HIV proviral sequencing (to characterize the HIV reservoir). HIV status and the size/structure of the HIV reservoir will be data that will be shared. Direct identifiers (e.g., names, telephone numbers, emails, social security numbers, medical record numbers, etc.) will not be shared. Data will be submitted to the National Institute of Health (NIH) Genomic Data repository, with unrestricted access (i.e. data will be made accessible to anyone via a public website). The data will be coded or anonymized prior to submission to NIH such that the identities of subjects cannot be readily ascertained or otherwise associated with the data by the repository staff or secondary data users.
• IPD Sharing Time Frame: Data will be shared at the time of submission of the scientific paper(s).
• IPD Sharing Access Criteria: The information from this study will be stored in a public unrestricted data bank that anyone can use. The National Institutes of Health (NIH) has developed data banks that collect genomic study data. The NIH will store the de-identified information in these data banks for other researchers to use in future studies on any topic. This will include de-identified information about the participants' HIV status, mRNA sequences or genomic allelic variants (e.g. single nucleotide polymorphisms and copy number variants). The researchers could be from government, academic, or commercial institutions. We have a Extramural Institutional Certification dated 01/07/2022, Genomic Program Administrator: Chris Marcus from the National Institute of Allergy and Infectious Diseases (NIAID), NIH, HHS.
• IPD Sharing Supporting Information Type: STUDY_PROTOCOL; ICF; ANALYTIC_CODE

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No