First-in-human immunoPET imaging of HIV-1 infection using 89Zr-labeled VRC01 broadly neutralizing antibody

A major obstacle to achieving long-term antiretroviral (ART) free remission or functional cure of HIV infection is the presence of persistently infected cells that establish a long-lived viral reservoir. HIV largely resides in anatomical regions that are inaccessible to routine sampling, however, and non-invasive methods to understand the longitudinal tissue-wide burden of HIV persistence are urgently needed. Positron emission tomography (PET) imaging is a promising strategy to identify and characterize the tissue-wide burden of HIV. Here, we assess the efficacy of using immunoPET imaging to characterize HIV reservoirs and identify anatomical foci of persistent viral transcriptional activity using a radiolabeled HIV Env-specific broadly neutralizing antibody, 89Zr-VRC01, in HIV-infected individuals with detectable viremia and on suppressive ART compared to uninfected controls (NCT03729752). We also assess the relationship between PET tracer uptake in tissues and timing of ART initiation and direct HIV protein expression in CD4 T cells obtained from lymph node biopsies. We observe significant increases in 89Zr-VRC01 uptake in various tissues (including lymph nodes and gut) in HIV-infected individuals with detectable viremia (N = 5) and on suppressive ART (N = 5) compared to uninfected controls (N = 5). Importantly, PET tracer uptake in inguinal lymph nodes in viremic and ART-suppressed participants significantly and positively correlates with HIV protein expression measured directly in tissue. Our strategy may allow non-invasive longitudinal characterization of residual HIV infection and lays the framework for the development of immunoPET imaging in a variety of other infectious diseases.


Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Field-collected samples This is an exploratory first-in human PET imaging study and incorporated three comparison groups. The sample size for this study was based on prior non-human primate imaging as described in the manuscript text, that observed differences between tissue PET uptake regions with fewer animals than included in this study (Santangelo PJ et al. Nat Methods, 2015;12(5):427-32. This study used 4 viremic macaques and 3 controls; significant differences in lymphoid and other tissues were identified using PET imaging with these numbers of animals.
Data were acquired from the planned tissue regions of interest that were determined prior to final study data analysis. ROIs were excluded if it was not possible to perform adequate tissue gating using the Osirix software and quality of PET-MR data. No other data was excluded.
Image analysis was performed for each imaging time point (ranging from 2-4) for each of the five individuals from each of the three comparison groups. For axillary and inguinal lymph node analyses, mean SUVs were calculated from two separate lymph nodes on contralateral sides of the body.
This was not a randomized clinical trial or case controlled study, but all attempts were made to include participants with similar demographic and clinical factors across the comparison groups. This data is explicitly detailed in the stud manuscript.
Raw image files were analyzed (ROI selection, tissue SUV calculation) blinded as to the participant information, including the comparison group. Regions of interest were selected without knowing the HIV status or other personal information. Given the nature of the imaging study, investigators were not blinded as to the selection of participants within each group as they were responsible for enrolling participants and scheduling imaging visits. p24 KC57-PE (Beckman Coulter, 6604667)· p24 28B7-APC (Medimabs, MM-0289-APC) p24 antibodies as above were titrated upon reception of each new batch acquired from the manufacturer. Lot numbers and expiration dates were recorded. Positive controls were used with each flow cytometry experiment.
Balb/C mice (5 females and 5 males). Mice were allowed natural sleep/wake cycles and were approximately 6-8 weeks old.
No wild animals were used in this study.
No field-collected samples were used in this study.
Fifteen participants 18 years of age or older were recruited for this imaging study, including HIV-infected participants on continuous suppressive ART with viral load < 40 RNA copies/mL prior to imaging and within 12 months of study entry or detectable plasma HIV RNA > 1,000 copies per/mL, and uninfected controls. Viremic individuals had plasma HIV-1 RNA ranging from 3,499 to 789,705 copies/mL) and ART-suppressed individuals had times on ART ranging from 105 to 8,509 days on ART). One viremic and one control participant were female. The mean ages of uninfected control, viremic and ARTsuppressed participants were 58, 52 and 53 years, respectively. In addition, HIV+ participants had HIV-1 envelope RNA or DNA consensus sequences from peripheral blood suggestive of VRC01 binding activity (HIV infected participants only) when able. Study procedures were conducted between 2017 and 2019.
Participants were identified from the UCSF SCOPE cohort and HIV clinics at Zuckerberg San Francisco General Hospital between 2017 and 2019. These are outpatient units that provide clinical care or research to people living with HIV in the greater San Francisco-Bay Area. Participants were co-enrolled in the HIV SCOPE study for additional collection of blood and tissue for measures of HIV persistence. To minimize self selection bias, investigators identified potential participants based on ART usage, time on ART and viral load levels (for viremic participants).
The University of California San Francisco Committee on Human Research approved this clinical study and all participants provided written informed consent and underwent MRI screening prior to imaging.
The full study protocol can be obtained from the corresponding authors upon request.
Participants were recruited at ZSFGH and PET-MR imaging and data acquisition was performed at the UCSF China Basin radiology/ clinical imaging center. Participants provided informed consent and peripheral blood collection at SFGH Research outpatient Ward 84 (SCOPE cohort); PET-MR imaging and tracer injection was performed at the outpatient UCSF Radiology Center at China Basin, San Francisco. Lymph node biopsies were performed at the ZSFG outpatient clinical research ward.
The primary outcome was to observe a difference in rSUV values in lymph node tissues (and other ROIs including gut tissue) between participants with HIV off ART and uninfected controls. Secondary outcomes were to observe differences between those with HIV on ART and uninfected controls. CD4+ T cells were isolated from LNMCs by negative magnetic selection (StemCell) and directly stained with the Aqua Live/ Dead staining kit and with antibodies against cell-surface molecules in PBS + 4% human serum for 30 min at 4°C. Cells were fixed/permeabilized with the FoxP3 Staining Buffer Set (eBioscience) for 45 min and stained with anti-p24 KC57 and anti-p24 28B7 antibodies for an additional 45 min in the permeabilization buffer. All antibodies are commercially available. Titrations were performed for all antibodies to determine optimal concentrations. Dilution used in this study are indicated below: CD3 UCHT-1 A700 (reference 557943, dilution 1/100), CD4 SK3 BUV496 (reference 564651, dilution 1/25), CD8 RPA-T8 BUV395 (reference 563795, dilution 1/100), CD45RA HI100 BV786 (reference 563870, dilution 1/100), CXCR5 RF8B2 BB515 (reference 564624, dilution 1/50), PD-1 EH12.1 BUV737 (reference 565299, dilution 1/100) were purchased from BD Biosciences. ICOS C398.4A BV421 (reference 313523, dilution 1/100) was obtained from Biolegend. p24 KC57-FITC (dilution 1/1000) was obtained from Beckman Coulter. The certificate of analysis can be found on the Beckman Coulter website. It is certified that each batch of p24 KC57 meets the requirements for flow cytometry experiments.