Explore the vast range of titles available.

T cell epitope candidates are commonly identified using computational prediction tools in order to enable applications such as vaccine design, cancer neoantigen identification, development of diagnostics and removal of unwanted immune responses against protein therapeutics. Most T cell epitope prediction tools are based on machine learning algorithms trained on MHC binding or naturally processed MHC ligand elution data. The ability of currently available tools to predict T cell epitopes has not been comprehensively evaluated. In this study, we used a recently published dataset that systematically defined T cell epitopes recognized in vaccinia virus (VACV) infected C57BL/6 mice (expressing H-2Db and H-2Kb), considering both peptides predicted to bind MHC or experimentally eluted from infected cells, making this the most comprehensive dataset of T cell epitopes mapped in a complex pathogen. We evaluated the performance of all currently publicly available computational T cell epitope prediction tools to identify these major epitopes from all peptides encoded in the VACV proteome. We found that all methods were able to improve epitope identification above random, with the best performance achieved by neural network-based predictions trained on both MHC binding and MHC ligand elution data (NetMHCPan-4.0 and MHCFlurry). Impressively, these methods were able to capture more than half of the major epitopes in the top N = 277 predictions within the N = 767,788 predictions made for distinct peptides of relevant lengths that can theoretically be encoded in the VACV proteome. These performance metrics provide guidance for immunologists as to which prediction methods to use, and what success rates are possible for epitope predictions when considering a highly controlled system of administered immunizations to inbred mice. In addition, this benchmark was implemented in an open and easy to reproduce format, providing developers with a framework for future comparisons against new tools.

Inflammasomes are important for host defence against pathogens and homeostasis with commensal microbes. Here, we show non-haemolytic enterotoxin (NHE) from the neglected human foodborne pathogen Bacillus cereus is an activator of the NLRP3 inflammasome and pyroptosis. NHE is a non-redundant toxin to haemolysin BL (HBL) despite having a similar mechanism of action. Via a putative transmembrane region, subunit C of NHE initiates binding to the plasma membrane, leading to the recruitment of subunit B and subunit A, thus forming a tripartite lytic pore that is permissive to efflux of potassium. NHE mediates killing of cells from multiple lineages and hosts, highlighting a versatile functional repertoire in different host species. These data indicate that NHE and HBL operate synergistically to induce inflammation and show that multiple virulence factors from the same pathogen with conserved function and mechanism of action can be exploited for sensing by a single inflammasome. The Bacillus haemolytic enterotoxin haemolysin BL has been shown to activate the NLRP3 inflammasome. Here the authors show that a non-haemolytic enterotoxin (NHE) from B. cereus can also activate the NLRP3 inflammasome with a similar mechanism of lytic pore formation.

The magnitude of T cell responses to infection is a function of the naïve T cell repertoire combined with the context and duration of antigen presentation. Using mass spectrometry, we identify and quantify 21 class 1 MHC-restricted influenza A virus (IAV)-peptides following either direct or cross-presentation. All these peptides, including seven novel epitopes, elicit T cell responses in infected C57BL/6 mice. Directly presented IAV epitopes maintain their relative abundance across distinct cell types and reveal a broad range of epitope abundances. In contrast, cross-presented epitopes are more uniform in abundance. We observe a clear disparity in the abundance of the two key immunodominant IAV antigens, wherein direct infection drives optimal nucleoprotein (NP) 366–374 presentation, while cross-presentation is optimal for acid polymerase (PA) 224–233 presentation. The study demonstrates how assessment of epitope abundance in both modes of antigen presentation is necessary to fully understand the immunogenicity and response magnitude to T cell epitopes. CTL responses are critical in protection against pathogens. Here, using mass spectrometry and flow cytometry, the authors characterize the kinetics of influenza A virus class I MHC epitopes cross-presented in professional antigen presenting cells and identify new epitopes that elicit T cell responses in infected mice.

Herpes simplex virus (HSV) has provided the prototype for viral latency with previously well-defined acute or lytic and latent phases. More recently, the deep quiescence of HSV latency has been questioned with evidence that lytic genes can be transcribed in this state. However, to date the only evidence that these transcripts might be translated has come from immunological studies that show activated T cells persist in the nervous system during latency. Here we use a highly sensitive Cre-marking model to show that lytic and latent phases are less clearly defined in two significant ways. First, around half of the HSV spread leading to latently infected sites occurred beyond the initial acute infection and second, we show direct evidence that lytic promoters can drive protein expression during latency.

Herpes simplex viruses (HSV) are significant human pathogens that provide one of the best-described examples of viral latency and reactivation. HSV latency occurs in sensory neurons, being characterized by the absence of virus replication and only fragmentary evidence of protein production. In mouse models, HSV latency is especially stable but the detection of some lytic gene transcription and the ongoing presence of activated immune cells in latent ganglia have been used to suggest that this state is not entirely quiescent. Alternatively, these findings can be interpreted as signs of a low, but constant level of abortive reactivation punctuating otherwise silent latency. Using single cell analysis of transcription in mouse dorsal root ganglia, we reveal that HSV-1 latency is highly dynamic in the majority of neurons. Specifically, transcription from areas of the HSV genome associated with at least one viral lytic gene occurs in nearly two thirds of latently-infected neurons and more than half of these have RNA from more than one lytic gene locus. Further, bioinformatics analyses of host transcription showed that progressive appearance of these lytic transcripts correlated with alterations in expression of cellular genes. These data show for the first time that transcription consistent with lytic gene expression is a frequent event, taking place in the majority of HSV latently-infected neurons. Furthermore, this transcription is of biological significance in that it influences host gene expression. We suggest that the maintenance of HSV latency involves an active host response to frequent viral activity.

Naïve CD8 + T cells need to undergo a complex and coordinated differentiation program to gain the capacity to control virus infections. This not only involves the acquisition of effector functions, but also regulates the development of a subset of effector CD8 + T cells into long-lived and protective memory cells. Microbiota-derived metabolites have recently gained interest for their influence on T cells, but much remains unclear about their role in CD8 + T cell differentiation. In this study, we investigated the role of the G protein-coupled receptors (GPR)41 and GPR43 that can bind microbiota-derived short chain fatty acids (SCFAs) in CD8 + T cell priming following epicutaneous herpes simplex virus type 1 (HSV-1) infection. We found that HSV-specific CD8 + T cells in GPR41/43-deficient mice were impaired in the antigen-elicited production of interferon-gamma (IFN-γ), tumour necrosis factor-alpha (TNF-α), granzyme B and perforin, and failed to differentiate effectively into memory precursors. The defect in controlling HSV-1 at the site of infection could be restored when GPR41 and GPR43 were expressed exclusively by HSV-specific CD8 + T cells. Our findings therefore highlight roles for GPR41 and GPR43 in CD8 + T cell differentiation, emphasising the importance of metabolite sensing in fine-tuning anti-viral CD8 + T cell priming.

Current knowledge about the dynamics of antigen presentation to T cells during viral infection is very poor despite being of fundamental importance to our understanding of anti-viral immunity. Here we use an advanced mass spectrometry method to simultaneously quantify the presentation of eight vaccinia virus peptide-MHC complexes (epitopes) on infected cells and the amounts of their source antigens at multiple times after infection. The results show a startling 1000-fold range in abundance as well as strikingly different kinetics across the epitopes monitored. The tight correlation between onset of protein expression and epitope display for most antigens provides the strongest support to date that antigen presentation is largely linked to translation and not later degradation of antigens. Finally, we show a complete disconnect between the epitope abundance and immunodominance hierarchy of these eight epitopes. This study highlights the complexity of viral antigen presentation by the host and demonstrates the weakness of simple models that assume total protein levels are directly linked to epitope presentation and immunogenicity.

The value of predictive algorithms for identifying CD8 + T (T CD8+ )-cell epitopes has not been adequately tested experimentally. Here we demonstrate that conventional bioinformatic methods predict the vast majority of T CD8+ -cell epitopes derived from vaccinia virus WR strain (VACV-WR) in the H-2 b mouse model. This approach reveals the breadth of T-cell responses to vaccinia, a widely studied murine viral infection model, and may provide a tool for developing comprehensive antigenic maps of any complex pathogen.

Mucosal-associated invariant T cells (MAIT cells) are “unconventional” immune cells that are becoming increasingly appreciated to play important roles in a variety of viral infections. Herpes simplex virus (HSV) causes significant human disease and is a master manipulator of multiple immune functions, but how this virus may control MAIT cells is poorly understood. We discovered that HSV can infect human MAIT cells and impair their functional capacity and also show that MAIT cells exposed to HSV, but which do not show evidence of infection, are similarly impaired. This study therefore identifies an additional immunomodulatory function of HSV.

In standard uses of CRISPR/Cas9 technology, the cutting of genomes and their efficient repair are considered to go hand-in-hand to achieve desired genetic changes. This includes the current approach for engineering genomes of large dsDNA viruses. However, for poxviruses we show that Cas9-guide RNA complexes cut viral genomes soon after their entry into cells, but repair of these breaks is inefficient. As a result, Cas9 targeting makes only modest, if any, improvements to basal rates of homologous recombination between repair constructs and poxvirus genomes. Instead, Cas9 cleavage leads to inhibition of poxvirus DNA replication thereby suppressing virus spread in culture. This unexpected outcome allows Cas9 to be used as a powerful tool for selecting conventionally generated poxvirus recombinants, which are otherwise impossible to separate from a large background of parental virus without the use of marker genes. This application of CRISPR/Cas9 greatly speeds up the generation of poxvirus-based vaccines, making this platform considerably more attractive in the context of personalised cancer vaccines and emerging disease outbreaks. Gowripalan, Smith et al. use CRISPR/Cas9 technology to rapidly select recombinant poxviruses without using selectable marker genes. They find that Cas9 cleavage inhibits poxvirus DNA replication, suppressing virus spread in culture. This application makes poxviruses more attractive vector platforms for fighting cancer and emerging disease outbreaks.