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Table 4 Summary of key research assays

From: The RIO trial: rationale, design, and the role of community involvement in a randomised placebo-controlled trial of antiretroviral therapy plus dual long-acting HIV-specific broadly neutralising antibodies (bNAbs) in participants diagnosed with recent HIV infection—study protocol for a two-stage randomised phase II trial

Assay Sample Purpose Time-point
T cell immunology • Pre-ART where available
• On ART
• Pre-bNAb
• Fortnightly during ATI
• Following viral suppression
HIV-specific T cell intracellular cytokine staining for CD4 and CD8 phenotypic and functional responses PBMC Screen CD4 and CD8 T cell responses to HIV in response to bNAbs
HIV ELISpots: γ-interferon responses with peptides targeted according to ICS responses PBMC Higher resolution analyses of specific responses to peptide level and how they are impacted by intervention
CD8 tetramer responses targeted to specific peptides PBMC Further in-depth resolution of CD8 T cell functionality at cellular level
HLA Class I and II typing (4-digit) PBMC Profiling of HLA alleles to shape immune responses and confer advantage/disadvantages
T cell killing assays PBMC Functional killing assay to augment intracellular cytokine staining data
Natural Killer (NK) cell responses
Flow cytometry for NK cell phenotype and function PBMC Characterisation of NK function and profile in response to bNAbs
ADCC and NK cell killing PBMC Functional assessment of bNAbs using characterised killing protocols
NK-like population analyses (flow cytometry) PBMC High resolution characterisation of cells that fall between the innate and adaptive responses
Next-generation sequencing (NGS) single genome amplification (SGA) of HIV ENV: bNAb sensitivity PBMC Env sequencing to screen for bNAb sensitivity Screening
Full length HIV sequencing Plasma CD4 T cells Full length haplotype analysis of viral and proviral populations pre and post intervention to determine correlations with response • Pre-ART (virus) where available (HEATHER cohort)
• On ART, and pre-ATI (provirus)
• Rebound (virus)
• Post resuppression (provirus)
HIV Integration site analysis CD4 T cells Understanding of clonality of viral reservoir and relationship of integration sites to reactivation potential and susceptibility to inhibition Pre-ATI
HIV DNA and cell-associated RNA quantitation CD4 T cells Standard molecular analyses of proviral and HIV transcript quantitation, both surrogate markers of persistent infection and biomarkers of remission and rebound • Pre-ART where available (HEATHER cohort)
• On ART
• Pre-bNAb
• Pre-ATI
• Post resuppression
Host genomics
Transcriptome profiling*
*Initially bulk, but with single-cell 10× resolution dependent on results
CD4 and CD8 T cells
NK cells
Full host genomic analyses at bulk and single cell (likely tetramer driven for CD8 cells) to understand host determinants of outcomes • Pre-ART where available (HEATHER cohort)
• On ART
• Pre- ATI
Pharmacokinetics and anti-drug antibodies
bNAb pharmacokinetics
Quantitation of ‘anti-drug antibodies’ (ADA)
Plasma To determine bNAb plasma concentrations
To determine development of inhibitory antibodies against bNAbs
• Post bNAb infusion