One of the major barriers to achieving HIV remission through combination antiretroviral therapy (cART) is the persistence of virus in reservoirs and tissue sanctuaries. B cell follicles and germinal centres (GCs) within lymph nodes (LNs) are immune privileged sites with the potential to support low level virus replication. However, the persistence of replicating virus within this tissue compartment during cART is controversial. Furthermore, the phenotype and localization of cell subsets that support virus replication in LNs in the presence of cART is unknown. Here, we used excised LNs and paired peripheral blood samples from 17 HIV infected subjects who initiated cART in Fiebig stage I from the well-characterized FRESH cohort to investigate the persistence of virus replication and to identify cell subsets that harbor residual virus during cART. Eight chronic untreated and eight HIV negative individuals were also included in the study as controls.
The phenotype and localization of GC cell subsets were defined using flow cytometry and immunohistochemistry (IHC) respectively. RNAscope; an in-situ RNA hybridization assay was used in combination with IHC to visualize the cellular localization of HIV RNA in LN sections. HIV RNA was further quantified ex-vivo in GC Tfh subsets using digital droplet PCR (ddPCR).
From IHC results, there was a positive correlation between the magnitude of GCs and HIV plasma viral load (r=0.6, p=0.07) in untreated individuals. Interestingly, the excessive GC T follicular helper (GCTfh) cells’ expansion observed in chronic HIV was significantly attenuated by early treatment (p=0.01). HIV Gag p24 antigen was detected almost exclusively in the GCs even after one year of cART mediated viral suppression. Specifically, follicular dendritic cells and GC Tfh cells harbored most of the detectable Gag p24. Furthermore, RNAscope for Gag-Pol multiplexed with BCL-6 confirmed GC localization of HIV RNA during early cART.
Taken together, our results demonstrate the persistence of low level viral replication in the lymph nodes of early treated HIV infected individuals. This study highlights the need for future interventions directed at eliminating residual virus replication in tissue sanctuaries during cART.