Abstract: The effects of immunodeficiency associated with chronic HIV infection on COVID-19 disease and viral persistence have not been directly addressed in a controlled setting. In this pilot study, we exposed two pigtail macaques (PTMs) chronically infected with SIVmac239, exhibiting from very low to no CD4 T cells across all compartments, to SARS-CoV-2. We monitored the disease progression, viral replication and evolution, and compared these outcomes with SIV-naïve PTMs infected with SARS-CoV-2. No overt signs of COVID-19 disease were observed in either animal, and the SARS-CoV-2 viral kinetics and evolution in the SIVmac239 PTMs were indistinguishable from those in the SIV-naïve PTMs in all sampled mucosal sites. However, the single-cell RNA sequencing of bronchoalveolar lavage cells revealed an infiltration of functionally inert monocytes after SARS-CoV-2 infection. Critically, neither of the SIV-infected PTMs mounted detectable anti-SARS-CoV-2 T-cell responses nor anti-SARS-CoV-2 binding or neutralizing antibodies. Thus, HIV-induced immunodeficiency alone may not be sufficient to drive the emergence of novel viral variants but may remove the ability of infected individuals to mount adaptive immune responses against SARS-CoV-2.
Overall design: Single-Cell RNA Sequencing (scRNAseq) of BAL Cells
For single-cell sequencing of bronchoalveolar lavage (BAL) cells, we collected samples before SARS-CoV-2 inoculation and on days 2, 7, 21, and 28 post-challenge. BAL samples were centrifuged at room temperature for 5 min at 1800 rpm, and the resulting cell pellets were resuspended in DMEM supplemented with 10% FBS and 1% Anti-Anti. We used the Parse Biosciences cell fixation kit, following the manufacturer’s instructions for PBMCs, to fix the cells (Parse Biosciences, Seattle, WA, USA). Specifically, we fixed 1 million cells per animal/timepoint in a 15 mL falcon tube. The fixed cells were stored at −20 °C until all samples were collected.
To enable multiplexing of samples, the Parse Single-Cell whole transcriptome kit, which utilizes a combinatorial barcoding approach (Evercode WT, Parse Biosciences, USA), was employed. This allowed us to barcode and multiplex 10 samples collected from the coinfected animals across five timepoints. For analysis of the processed cells, we conducted two separate runs: the first run included approximately 15,000 cells, while the second run consisted of approximately 42,000 cells. The sublibraries from each run were pooled and sequenced on an Illumina NextSeq 2000 platform, yielding an average depth of 27,165 reads per cell for the first batch and 29,088 reads per cell for the second.
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