Rapid recall and de novo T cell responses during SARS-CoV-2 breakthrough infection

While the protective role of neutralising antibodies against COVID-19 is well-established, questions remain about the relative importance of cellular immunity. Using 6 pMHC-multimers in a cohort with early and frequent sampling we define the phenotype and kinetics of recalled and primary T cell responses following Delta or Omicron breakthrough infection. Recall of spike-specific CD4+ T cells was rapid, with cellular proliferation and extensive activation evident as early as 1 day post-symptom onset. Similarly, spike-specific CD8+ T cells were rapidly activated but showed variable levels of expansion. Strikingly, high levels of SARS-CoV-2-specific CD8+ T cell activation at baseline and peak were strongly correlated with reduced peak SARS-CoV-2 RNA levels in nasal swabs and accelerated clearance of virus. Our study demonstrates rapid and extensive recall of memory T cell populations occurs early after breakthrough infection and suggests that CD8+ T cells contribute to the control of viral replication in breakthrough SARS-CoV-2 infections.

SARS-CoV-2 mRNA vaccination elicits robust and persistent T follicular helper cell response in humans

SARS-CoV-2 mRNA vaccines induce robust anti-spike (S) antibody and CD4+ T cell responses. It is not yet clear whether vaccine-induced follicular helper CD4+ T (TFH) cell responses contribute to this outstanding immunogenicity. Using fine needle aspiration of draining axillary lymph nodes from individuals who received the BNT162b2 mRNA vaccine, we show that frequency of TFH correlates with that of S-binding germinal center B cells. Mining of the responding TFH T cell receptor repertoire revealed a strikingly immunodominant HLADPB1* 04-restricted response to S167-180 in individuals with this allele, which is among the most common HLA alleles in humans. Paired blood and lymph node specimens show that while circulating S-specific TFH cells peak one week after the second immunization, S-specific TFH persist at nearly constant frequencies for at least six months. Collectively, our results underscore the key role that robust TFH cell responses play in establishing long-term immunity by this efficacious human vaccine.

Suboptimal SARS-CoV-2-specific CD8+ T-cell response associated with the prominent HLA-A*02:01 phenotype

An improved understanding of human T-cell-mediated immunity in COVID-19 is important if we are to optimize therapeutic and vaccine strategies. Experience with influenza shows that infection primes CD8+ T-cell memory to shared peptides presented by common HLA types like HLA-A2. Following re-infection, cross-reactive CD8+ T-cells enhance recovery and diminish clinical severity. Stimulating peripheral blood mononuclear cells from COVID-19 convalescent patients with overlapping peptides from SARS-CoV-2 Spike, Nucleocapsid and Membrane proteins led to the clonal expansion of SARS-CoV-2-specific CD8+ and CD4+ T-cells in vitro, with CD4+ sets being typically robust. For CD8+ T-cells taken directly ex vivo, we identified two HLA-A*02:01-restricted SARS-CoV-2 epitopes, A2/S269-277 and A2/Orf1ab3183-3191. Using peptide-HLA tetramer enrichment, direct ex vivo assessment of the A2/S269+CD8+ and A2/Orf1ab3183+CD8+ populations indicated that the more prominent A2/S269+CD8+ set was detected at comparable frequency ([~]1.3x10-5) in acute and convalescent HLA-A*02:01+ patients. But, while the numbers were higher than those found in uninfected HLA-A*02:01+ donors ([~]2.5x10-6), they were low when compared with frequencies for influenza-specific (A2/M158) and EBV-specific (A2/BMLF1280) ([~]1.38x10-4) populations. Phenotypic analysis ex vivo of A2/S269+CD8+ T-cells from COVID-19 convalescents showed that A2/S269+CD8+ T-cells were predominantly negative for the CD38, HLA-DR, PD-1 and CD71 activation markers, although the majority of total CD8+ T-cells were granzyme and/or perforin-positive. Furthermore, the bias towards naive, stem cell memory and central memory A2/S269+CD8+ T-cells rather than effector memory populations suggests that SARS-CoV2 infection may be compromising CD8+ T-cell activation. Priming with an appropriate vaccine may thus have great value for optimizing protective CD8+ T-cell immunity in COVID-19.

Crystal structure of the SARS-CoV-2 non-structural protein 9, Nsp9

Many of the proteins produced by SARS-CoV-2 have related counterparts across the Severe Acute Respiratory Syndrome (SARS-CoV) family. One such protein is non-structural protein 9 (Nsp9), which is thought to mediate both viral replication and virulence. Current understanding suggests that Nsp9 is involved in viral genomic RNA reproduction. Nsp9 is thought to bind RNA via a fold that is unique to this class of betacoronoaviruses although the molecular basis for this remains ill-defined. We sought to better characterise the SARS-CoV-2 Nsp9 protein and subsequently solved its X-ray crystal structure, in an apo-form and, unexpectedly, in a peptide-bound form with a sequence originating from a rhinoviral 3C protease sequence (LEVL). The structure of the SARS-CoV-2 Nsp9 revealed the high level of structural conservation within the Nsp9 family. The exogenous peptide binding site is close to the dimer interface and impacted on the relative juxtaposition of the monomers within the homodimer. Together we have established a protocol for the production of SARS-CoV-2 Nsp9, determined its structure and identified a peptide-binding site that may warrant further study from the perspective of understanding Nsp9 function.