Early initiation of Combination Antiretroviral Therapy (cART) during acute HIV infection blunts peak viremia, reduces HIV viral reservoirs and preserves immune function, but treatment interruptions often results in rapid viral rebound. We studied persons identified and treated at the onset of plasma viremia, in many when viral load is less than 1000 RNA copies/ml to define the dynamics of HIV suppression in lymph node (LN) tissues. Additionally, we investigated the cell subset that remained persistently HIV infected.
We studied 16 hyperacute HIV-infected subjects who initiated therapy in Fiebig stage I, subdivided into three groups based on when the LN sample was obtained. Group 1 was sampled within 3 months, group 2 was sampled at one year and group 3 was sampled after two years on therapy. Immunofluorescence (IF) microscopy and RNAscope in situ hybridization (ISH) techniques were used to quantify Gag p24 protein and Gagpol RNA respectively. The Cobas AmpliPrep HIV-1 test was used to quantify LN and plasma viral loads and viral Gag, Nef and Envelope sequencing were conducted using ABI 3130xl sequencing platform. Digital droplet PCR was used to quantify HIV RNA levels in FACs sorted LN cell subsets and follicular CD4+ T cells harboring HIV antigens were extensively phenotyped by flow cytometry.
Despite rapid plasma viral suppression at a median of 16.5 days, Gag p24 antigen and quantifiable RNA were readily detectable in the LN in 12 out of the 16 donors sampled in all three experimental groups. Moreover, sequencing analysis revealed viral evolution in Gag, Nef and/or Envelope sequences in 4 out of 6 LNs sampled >3 months after therapy compared to the transmitter found virus sequences obtained just before cART initiation. There was no significant reduction in Gagp24 antigen in LN samples obtained after a year on cART compared to the samples obtained within 3 months on cART (p=0.4). RNA quantification of FACs sorted TFH subsets showed significantly higher levels of Gag p24 mRNA copies in CXCR3+ follicular CD4+ T cells compared to other TFH subsets (p=0.01).
Our results highlight the huge difference in viral load decay kinetics between peripheral blood and LN, despite very early cART initiation. Importantly, we identify that CXCR3+ TFH contribute significantly to viral persistence in the LN during therapy. These results underscore the need for future interventions directed at eliminating residual virus in tissue sanctuaries.