Background
Generating robust virus-specific CD8+ T cell responses is the major objective of most HIV therapeutic vaccines. However, standard T cell assays fail to distinguish whether vaccine-expanded T cell populations are boosted pre-existing responses and/or newly-recruited clonotypes. Here, we utilized two single cell T cell receptor (TCR) sequencing approaches to characterize CD8+ T cell clonotypes expanded in the peripheral blood of five people living with HIV two weeks after receiving a multiclade consensus HIV gag/pol ± env DNA vaccine/IL-12 regimen (n=3) versus placebo (n=2) in the PENNVAX trial (NCT03606213).
Methods
First, we sequenced single paired TCR alpha and beta chains from enriched CD8+ T cells using the high-throughput Omniscope OS-T assay (per sample sequenced: median 1.3e6 [1.7e5-1.9e6] cells and 1.1e5 [6.1e4-1.6e5] unique clonotypes, defined as matching CDR3b amino acid [aa] sequence plus V/J usage). To investigate clonal dynamics between pre- and post-vaccination, expanded clones were defined as having a log2 fold change of ≥2 post- vs pre-vaccination (FDR adj. p-value <0.05, Fisher’s exact test). Clusters of clonotypes with similar CDR3 beta sequences were identified using TCRdist3. Additionally, we performed 10X single cell (sc)RNA/TCR sequencing from sorted MHC class I multimer+ HIV-specific CD8+ T cells from the same participants and timepoints.
Results
Among the three vaccinated individuals, we observed a significant expansion of 133 (15% newly detected), 415 (49% new), and 445 (12.5% new) CD8+ T cell clonotypes. Network graphs of the expanded clonotypes revealed hubs containing both boosted and newly detected clonotypes. The CDR3 beta aa sequence of 3.4% of boosted and 6.4% of newly detected clonotypes matched publicly available sequences reported to be HIV-specific. Expansion dynamics of clonotypes sequenced by 10X from multimer+ HIV-specific CD8+ T cells matched their expansion dynamics in the high-throughput assay.
Conclusions
By performing scTCRseq on approximately 1 million CD8+ T cells per sample, we successfully identified coordinated expansion following an HIV therapeutic vaccine of both boosted clonotypes as well as what appear to be newly expanded clonotypes with similar TCRs. Our data provide a benchmark for understanding how scTCRseq assays can be used to study vaccine-elicited HIV-specific T cells and lay the groundwork for future studies aimed at optimizing therapeutic strategies for HIV.
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