Necroptosis blockade prevents lung injury in severe influenza.

Severe influenza A virus (IAV) infections can result in hyper-inflammation, lung injury and acute respiratory distress syndrome1-5 (ARDS), for which there are no effective pharmacological therapies. Necroptosis is an attractive entry point for therapeutic intervention in ARDS and related inflammatory conditions because it drives pathogenic lung inflammation and lethality during severe IAV infection6-8 and can potentially be targeted by receptor interacting protein kinase 3 (RIPK3) inhibitors. Here we show that a newly developed RIPK3 inhibitor, UH15-38, potently and selectively blocked IAV-triggered necroptosis in alveolar epithelial cells in vivo. UH15-38 ameliorated lung inflammation and prevented mortality following infection with laboratory-adapted and pandemic strains of IAV, without compromising antiviral adaptive immune responses or impeding viral clearance. UH15-38 displayed robust therapeutic efficacy even when administered late in the course of infection, suggesting that RIPK3 blockade may provide clinical benefit in patients with IAV-driven ARDS and other hyper-inflammatory pathologies.

rAAV expressing a COBRA-designed influenza hemagglutinin generates a protective and durable adaptive immune response with a single dose.

Influenza remains a worldwide public health threat. Although seasonal influenza vaccines are currently the best means of preventing severe disease, the standard-of-care vaccines require frequent updating due to antigenic drift and can have low efficacy, particularly in vulnerable populations. Here, we demonstrate that a single administration of a recombinant adenovirus-associated virus (rAAV) vector expressing a computationally optimized broadly reactive antigen (COBRA)-derived influenza H1 hemagglutinin (HA) induces strongly neutralizing and broadly protective antibodies in naïve mice and ferrets with pre-existing influenza immunity. Following a lethal viral challenge, the rAAV-COBRA vaccine allowed for significantly reduced viral loads in the upper and lower respiratory tracts and complete protection from morbidity and mortality that lasted for at least 5 months post-vaccination. We observed no signs of antibody waning during this study. CpG motif enrichment of the antigen can act as an internal adjuvant to further enhance the immune responses to allow for lower vaccine dosages with the induction of unique interferon-producing CD4+ and CD8+ T cells specific to HA head and stem peptide sequences. Our studies highlight the utility of rAAV as an effective platform to improve seasonal influenza vaccines. IMPORTANCE: Developing an improved seasonal influenza vaccine remains an ambitious goal of researchers and clinicians alike. With influenza routinely causing severe epidemics with the potential to rise to pandemic levels, it is critical to create an effective, broadly protective, and durable vaccine to improve public health worldwide. As a potential solution, we created a rAAV viral vector expressing a COBRA-optimized influenza hemagglutinin antigen with modestly enriched CpG motifs to evoke a robust and long-lasting immune response after a single intramuscular dose without needing boosts or adjuvants. Importantly, the rAAV vaccine boosted antibody breadth to future strains in ferrets with pre-existing influenza immunity. Together, our data support further investigation into the utility of viral vectors as a potential avenue to improve our seasonal influenza vaccines.

Multi-variate model of T cell clonotype competition and homeostasis.

Diversity of the naive T cell repertoire is maintained by competition for stimuli provided by self-peptides bound to major histocompatibility complexes (self-pMHCs). We extend an existing bi-variate competition model to a multi-variate model of the dynamics of multiple T cell clonotypes which share stimuli. In order to understand the late-time behaviour of the system, we analyse: (i) the dynamics until the extinction of the first clonotype, (ii) the time to the first extinction event, (iii) the probability of extinction of each clonotype, and (iv) the size of the surviving clonotypes when the first extinction event takes place. We also find the probability distribution of the number of cell divisions per clonotype before its extinction. The mean size of a new clonotype at quasi-steady state is an increasing function of the stimulus available to it, and a decreasing function of the fraction of stimuli it shares with other clonotypes. Thus, the probability of, and time to, extinction of a new clonotype entering the pool of T cell clonotypes is determined by the extent of competition for stimuli it experiences and by its initial number of cells.

Quantifying T Cell Cross-Reactivity: Influenza and Coronaviruses.

If viral strains are sufficiently similar in their immunodominant epitopes, then populations of cross-reactive T cells may be boosted by exposure to one strain and provide protection against infection by another at a later date. This type of pre-existing immunity may be important in the adaptive immune response to influenza and to coronaviruses. Patterns of recognition of epitopes by T cell clonotypes (a set of cells sharing the same T cell receptor) are represented as edges on a bipartite network. We describe different methods of constructing bipartite networks that exhibit cross-reactivity, and the dynamics of the T cell repertoire in conditions of homeostasis, infection and re-infection. Cross-reactivity may arise simply by chance, or because immunodominant epitopes of different strains are structurally similar. We introduce a circular space of epitopes, so that T cell cross-reactivity is a quantitative measure of the overlap between clonotypes that recognize similar (that is, close in epitope space) epitopes.

TCR meta-clonotypes for biomarker discovery with tcrdist3 enabled identification of public, HLA-restricted clusters of SARS-CoV-2 TCRs.

T-cell receptors (TCRs) encode clinically valuable information that reflects prior antigen exposure and potential future response. However, despite advances in deep repertoire sequencing, enormous TCR diversity complicates the use of TCR clonotypes as clinical biomarkers. We propose a new framework that leverages experimentally inferred antigen-associated TCRs to form meta-clonotypes - groups of biochemically similar TCRs - that can be used to robustly quantify functionally similar TCRs in bulk repertoires across individuals. We apply the framework to TCR data from COVID-19 patients, generating 1831 public TCR meta-clonotypes from the SARS-CoV-2 antigen-associated TCRs that have strong evidence of restriction to patients with a specific human leukocyte antigen (HLA) genotype. Applied to independent cohorts, meta-clonotypes targeting these specific epitopes were more frequently detected in bulk repertoires compared to exact amino acid matches, and 59.7% (1093/1831) were more abundant among COVID-19 patients that expressed the putative restricting HLA allele (false discovery rate [FDR]<0.01), demonstrating the potential utility of meta-clonotypes as antigen-specific features for biomarker development. To enable further applications, we developed an open-source software package, tcrdist3, that implements this framework and facilitates flexible workflows for distance-based TCR repertoire analysis.

TCR meta-clonotypes for biomarker discovery with tcrdist3: identification of public, HLA-restricted SARS-CoV-2 associated TCR features.

As the mechanistic basis of adaptive cellular antigen recognition, T cell receptors (TCRs) encode clinically valuable information that reflects prior antigen exposure and potential future response. However, despite advances in deep repertoire sequencing, enormous TCR diversity complicates the use of TCR clonotypes as clinical biomarkers. We propose a new framework that leverages antigen-enriched repertoires to form meta-clonotypes - groups of biochemically similar TCRs - that can be used to robustly identify and quantify functionally similar TCRs in bulk repertoires. We apply the framework to TCR data from COVID-19 patients, generating 1831 public TCR meta-clonotypes from the 17 SARS-CoV-2 antigen-enriched repertoires with the strongest evidence of HLA-restriction. Applied to independent cohorts, meta-clonotypes targeting these specific epitopes were more frequently detected in bulk repertoires compared to exact amino acid matches, and 59.7% (1093/1831) were more abundant among COVID-19 patients that expressed the putative restricting HLA allele (FDR < 0.01), demonstrating the potential utility of meta-clonotypes as antigen-specific features for biomarker development. To enable further applications, we developed an open-source software package, tcrdist3, that implements this framework and facilitates flexible workflows for distance-based TCR repertoire analysis.