Oral Presentation Australasian Cytometry Society 44th Annual Conference and Workshop

High titre neutralizing antibodies in response to SARS-CoV-2 infection require RBD-specific CD4 T cells that include proliferative memory cells (24538)

John Zaunders 1 , Chansavath Phetsouphanh 2 , Weng Hua Khoo 3 , Katherine Jackson 3 , Vera Klemm 2 , Annett Howe 2 , Anupriya Aggarwal 2 , Anouschka Akerman 2 , Vanessa Milogiannakis 2 , Alberto O Stella 2 , Romain Rouet 3 , Peter Schofield 3 , Megan L Faulks 3 , Hannah Law 2 , Thidarat Danwilai 1 , Mitchell Starr 1 , Cynthia Mee Ling Munier 2 , Daniel Christ 3 , Manu Singh 3 , Peter Croucher 3 , Fabienne Brilot 4 , Stuart Turville 2 , Tri Giang Phan 3 , Greg J Dore 2 , David Darley 5 , Philip Cunningham 1 , Gail V Matthews 2 , Anthony D Kelleher 2
  1. Centre for Applied Medical Research, St Vincent's Hospital, Darlinghurst, NSW, Australia
  2. Kirby Institute, UNSW , Sydney
  3. Garvan Institute of Medical Research, Darlinghurst, NSW, Australia
  4. Children's Hospital at Westmead, Sydney, NSW, Australia
  5. St Vincent's Hospital, Sydney, Darlinghurst, NSW, Australia

Background: Long-term immunity to SARS-CoV-2 infection, including neutralizing antibodies (nAb) and T cell-mediated immunity, is required to reduce ongoing disease burden. We investigated the association between variability of memory CD4 and CD8 T cells and levels of neutralizing antibodies in convalescent COVID-19 subjects.

Methods: Participants (n=49) were recruited in the ADAPT observational study at St Vincent’s Hospital, Sydney. SARS-CoV-2 specific CD4 T cells in PBMC, collected at 3 and 8 months post-infection, were measured by upregulation of CD25/CD134 at 48 hrs and by cell trace violet proliferation assay at day 7. Antibodies to SARS-CoV-2 spike protein were measured by a flow-based assay of binding to cells expressing variant spike proteins, and by live virus neutralization assays. Single cell RNA-seq of antigen-specific CD4 T cells was done using 10x Genomics 5’v2 Gene expression and immune profiling kit.

Results: Higher titres of nAb were associated with significantly higher levels of receptor binding domain (RBD)-specific CD4 T cells, including specific memory cells that proliferated vigorously in vitro. Conversely, low nAb titres in about half of convalescent individuals were found together with a lack of RBD-specific memory CD4 T cells. These low nAb subjects had other, non-RBD, spike-specific CD4 T cells, but they had more of an inhibitory Foxp3+ and CTLA-4+ cell phenotype. In contrast, high nAb subjects had effector T-bet+/granzymes+/perforin+ cytotoxic RBD-specific proliferative memory CD4 T cells. Single cell transcriptomics of RBD-specific CD4+ T cells from high nAb subjects revealed heterogeneity that included central and transitional memory, Tregs, and cytotoxic cell clusters containing diverse TCR repertoires. Nevertheless, vaccination of low nAb individuals led to slight but significant improvements in RBD-specific memory CD4 T cells and nAb titres.

Conclusions: Our results suggest that targeting CD4 T cell epitopes proximal to and within the RBD-region should be considered in booster vaccines.