A multivalent mRNA monkeypox virus vaccine (BNT166) protects mice and macaques from orthopoxvirus disease.

In response to the 2022 outbreak of mpox driven by unprecedented human-to-human monkeypox virus (MPXV) transmission, we designed BNT166, aiming to create a highly immunogenic, safe, accessible, and scalable next-generation vaccine against MPXV and related orthopoxviruses. To address the multiple viral forms and increase the breadth of immune response, two candidate multivalent mRNA vaccines were evaluated pre-clinically: a quadrivalent vaccine (BNT166a; encoding the MPXV antigens A35, B6, M1, H3) and a trivalent vaccine (BNT166c; without H3). Both candidates induced robust T cell responses and IgG antibodies in mice, including neutralizing antibodies to both MPXV and vaccinia virus. In challenge studies, BNT166a and BNT166c provided complete protection from vaccinia, clade I, and clade IIb MPXV. Furthermore, immunization with BNT166a was 100% effective at preventing death and at suppressing lesions in a lethal clade I MPXV challenge in cynomolgus macaques. These findings support the clinical evaluation of BNT166, now underway (NCT05988203).

LITAF protects against pore-forming protein-induced cell death by promoting membrane repair.

Pore-forming toxins (PFTs) are the largest class of bacterial toxins and contribute to virulence by triggering host cell death. Vertebrates also express endogenous pore-forming proteins that induce cell death as part of host defense. To mitigate damage and promote survival, cells mobilize membrane repair mechanisms to neutralize and counteract pores, but how these pathways are activated is poorly understood. Here, we use a transposon-based gene activation screen to discover pathways that counteract the cytotoxicity of the archetypal PFT Staphylococcus aureus α-toxin. We identify the endolysosomal protein LITAF as a mediator of cellular resistance to PFT-induced cell death that is active against both bacterial toxins and the endogenous pore, gasdermin D, a terminal effector of pyroptosis. Activation of the ubiquitin ligase NEDD4 by potassium efflux mobilizes LITAF to recruit the endosomal sorting complexes required for transport (ESCRT) machinery to repair damaged membrane. Cells lacking LITAF, or carrying naturally occurring disease-associated mutations of LITAF, are highly susceptible to pore-induced death. Notably, LITAF-mediated repair occurs at endosomal membranes, resulting in expulsion of damaged membranes as exosomes, rather than through direct excision of pores from the surface plasma membrane. These results identify LITAF as a key effector that links sensing of cellular damage to repair.

Innovative vaccine approaches-a Keystone Symposia report.

The rapid development of COVID-19 vaccines was the result of decades of research to establish flexible vaccine platforms and understand pathogens with pandemic potential, as well as several novel changes to the vaccine discovery and development processes that partnered industry and governments. And while vaccines offer the potential to drastically improve global health, low-and-middle-income countries around the world often experience reduced access to vaccines and reduced vaccine efficacy. Addressing these issues will require novel vaccine approaches and platforms, deeper insight how vaccines mediate protection, and innovative trial designs and models. On June 28-30, 2021, experts in vaccine research, development, manufacturing, and deployment met virtually for the Keystone eSymposium "Innovative Vaccine Approaches" to discuss advances in vaccine research and development.

MHC class II transactivator CIITA induces cell resistance to Ebola virus and SARS-like coronaviruses.

Recent outbreaks of Ebola virus (EBOV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have exposed our limited therapeutic options for such diseases and our poor understanding of the cellular mechanisms that block viral infections. Using a transposon-mediated gene-activation screen in human cells, we identify that the major histocompatibility complex (MHC) class II transactivator (CIITA) has antiviral activity against EBOV. CIITA induces resistance by activating expression of the p41 isoform of invariant chain CD74, which inhibits viral entry by blocking cathepsin-mediated processing of the Ebola glycoprotein. We further show that CD74 p41 can block the endosomal entry pathway of coronaviruses, including SARS-CoV-2. These data therefore implicate CIITA and CD74 in host defense against a range of viruses, and they identify an additional function of these proteins beyond their canonical roles in antigen presentation.

B Cell αv Integrins Regulate TLR-Driven Autoimmunity.

Systemic lupus erythematosus (SLE) is defined by loss of B cell tolerance, resulting in production of autoantibodies against nucleic acids and other cellular Ags. Aberrant activation of TLRs by self-derived RNA and DNA is strongly associated with SLE in patients and in mouse models, but the mechanism by which TLR signaling to self-ligands is regulated remains poorly understood. In this study, we show that αv integrin plays a critical role in regulating B cell TLR signaling to self-antigens in mice. We show that deletion of αv from B cells accelerates autoantibody production and autoimmune kidney disease in the Tlr7.1 transgenic mouse model of SLE. Increased autoimmunity was associated with specific expansion of transitional B cells, extrafollicular IgG2c-producing plasma cells, and activation of CD4 and CD8 T cells. Our data show that αv-mediated regulation of TLR signaling in B cells is critical for preventing autoimmunity and indicate that loss of αv promotes escape from tolerance. Thus, we identify a new regulatory pathway in autoimmunity and elucidate upstream signals that adjust B cell activation to prevent development of autoimmunity in a mouse model.

αv Integrins regulate germinal center B cell responses through noncanonical autophagy.

Germinal centers (GCs) are major sites of clonal B cell expansion and generation of long-lived, high-affinity antibody responses to pathogens. Signaling through TLRs on B cells promotes many aspects of GC B cell responses, including affinity maturation, class switching, and differentiation into long-lived memory and plasma cells. A major challenge for effective vaccination is identifying strategies to specifically promote GC B cell responses. Here, we have identified a mechanism of regulation of GC B cell TLR signaling, mediated by αv integrins and noncanonical autophagy. Using B cell-specific αv-KO mice, we show that loss of αv-mediated TLR regulation increased GC B cell expansion, somatic hypermutation, class switching, and generation of long-lived plasma cells after immunization with virus-like particles (VLPs) or antigens associated with TLR ligand adjuvants. Furthermore, targeting αv-mediated regulation increased the magnitude and breadth of antibody responses to influenza virus vaccination. These data therefore identify a mechanism of regulation of GC B cells that can be targeted to enhance antibody responses to vaccination.

GOP-1: Helping phagosomes pass the acid test.

Phagosomes form during engulfment of large particles and become increasingly acidic and proteolytic, ultimately fusing with lysosomes, in a process termed "phagosome maturation." In this issue, Yin et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201610001) identify GOP-1 as essential for the maturation of phagosomes containing apoptotic cells, through recruitment of the Rab GTPase UNC108.

ß8 Integrin Expression and Activation of TGF-ß by Intestinal Dendritic Cells Are Determined by Both Tissue Microenvironment and Cell Lineage.

Activation of TGF-ß by dendritic cells (DCs) expressing αvß8 integrin is essential for the generation of intestinal regulatory T cells (Tregs) that in turn promote tolerance to intestinal Ags. We have recently shown that αvß8 integrin is preferentially expressed by CD103(+) DCs and confers their ability to activate TGF-ß and generate Tregs. However, how these DCs become specialized for this vital function is unknown. In this study, we show that ß8 expression is controlled by a combination of factors that include DC lineage and signals derived from the tissue microenvironment and microbiota. Specifically, our data demonstrate that TGF-ß itself, along with retinoic acid and TLR signaling, drives expression of αvß8 in DCs. However, these signals only result in high levels of ß8 expression in cells of the cDC1 lineage, CD8α(+), or CD103(+)CD11b(-) DCs, and this is associated with epigenetic changes in the Itgb8 locus. Together, these data provide a key illustrative example of how microenvironmental factors and cell lineage drive the generation of regulatory αvß8-expressing DCs specialized for activation of TGF-ß to facilitate Treg generation.

αv Integrins combine with LC3 and atg5 to regulate Toll-like receptor signalling in B cells.

Integrin signalling triggers cytoskeletal rearrangements, including endocytosis and exocytosis of integrins and other membrane proteins. In addition to recycling integrins, this trafficking can also regulate intracellular signalling pathways. Here we describe a role for αv integrins in regulating Toll-like receptor (TLR) signalling by modulating intracellular trafficking. We show that deletion of αv or ß3 causes increased B-cell responses to TLR stimulation in vitro, and αv-conditional knockout mice have elevated antibody responses to TLR-ligand-associated antigens. αv regulates TLR signalling by promoting recruitment of the autophagy component LC3 (microtubule-associated proteins 1 light chain 3) to TLR-containing endosomes, which is essential for progression from NF-κB to IRF signalling, and ultimately for traffic to lysosomes where signalling is terminated. Disruption of LC3 recruitment leads to prolonged NF-κB signalling and increased B-cell proliferation and antibody production. This work identifies a previously unrecognized role for αv and the autophagy components LC3 and atg5 in regulating TLR signalling and B-cell immunity.

Tuberculosis vaccines: barriers and prospects on the quest for a transformative tool.

The road to a more efficacious vaccine that could be a truly transformative tool for decreasing tuberculosis morbidity and mortality, along with Mycobacterium tuberculosis transmission, is quite daunting. Despite this, there are reasons for optimism. Abetted by better conceptual clarity, clear acknowledgment of the degree of our current immunobiological ignorance, the availability of powerful new tools for dissecting the immunopathogenesis of human tuberculosis, the generation of more creative diversity in tuberculosis vaccine concepts, the development of better fit-for-purpose animal models, and the potential of more pragmatic approaches to the clinical testing of vaccine candidates, the field has promise for delivering novel tools for dealing with this worldwide scourge of poverty.

Innate host defense requires TFEB-mediated transcription of cytoprotective and antimicrobial genes.

Animal host defense against infection requires the expression of defense genes at the right place and the right time. Understanding such tight control of host defense requires the elucidation of the transcription factors involved. By using an unbiased approach in the model Caenorhabditis elegans, we discovered that HLH-30 (known as TFEB in mammals) is a key transcription factor for host defense. HLH-30 was activated shortly after Staphylococcus aureus infection, and drove the expression of close to 80% of the host response, including antimicrobial and autophagy genes that were essential for host tolerance of infection. TFEB was also rapidly activated in murine macrophages upon S. aureus infection and was required for proper transcriptional induction of several proinflammatory cytokines and chemokines. Thus, our data suggest that TFEB is a previously unappreciated, evolutionarily ancient transcription factor in the host response to infection.

Dectin-1-dependent LC3 recruitment to phagosomes enhances fungicidal activity in macrophages.

Autophagy has been postulated to play role in mammalian host defense against fungal pathogens, although the molecular details remain unclear. Here, we show that primary macrophages deficient in the autophagic factor LC3 demonstrate diminished fungicidal activity but increased cytokine production in response to Candida albicans stimulation. LC3 recruitment to fungal phagosomes requires activation of the fungal pattern receptor dectin-1. LC3 recruitment to the phagosome also requires Syk signaling but is independent of all activity by Toll-like receptors and does not require the presence of the adaptor protein Card9. We further demonstrate that reactive oxygen species generation by NADPH oxidase is required for LC3 recruitment to the fungal phagosome. These observations directly link LC3 to the inflammatory pathway against C. albicans in macrophages.

CD36 coordinates NLRP3 inflammasome activation by facilitating intracellular nucleation of soluble ligands into particulate ligands in sterile inflammation.

Particulate ligands, including cholesterol crystals and amyloid fibrils, induce production of interleukin 1ß (IL-1ß) dependent on the cytoplasmic sensor NLRP3 in atherosclerosis, Alzheimer's disease and diabetes. Soluble endogenous ligands, including oxidized low-density lipoprotein (LDL), amyloid-ß and amylin peptides, accumulate in such diseases. Here we identify an endocytic pathway mediated by the pattern-recognition receptor CD36 that coordinated the intracellular conversion of those soluble ligands into crystals or fibrils, which resulted in lysosomal disruption and activation of the NLRP3 inflammasome. Consequently, macrophages that lacked CD36 failed to elicit IL-1ß production in response to those ligands, and targeting CD36 in atherosclerotic mice resulted in lower serum concentrations of IL-1ß and accumulation of cholesterol crystals in plaques. Collectively, our findings highlight the importance of CD36 in the accrual and nucleation of NLRP3 ligands from within the macrophage and position CD36 as a central regulator of inflammasome activation in sterile inflammation.

Natural selection in a bangladeshi population from the cholera-endemic ganges river delta.

As an ancient disease with high fatality, cholera has likely exerted strong selective pressure on affected human populations. We performed a genome-wide study of natural selection in a population from the Ganges River Delta, the historic geographic epicenter of cholera. We identified 305 candidate selected regions using the composite of multiple signals (CMS) method. The regions were enriched for potassium channel genes involved in cyclic adenosine monophosphate-mediated chloride secretion and for components of the innate immune system involved in nuclear factor κB (NF-κB) signaling. We demonstrate that a number of these strongly selected genes are associated with cholera susceptibility in two separate cohorts. We further identify repeated examples of selection and association in an NF-κB/inflammasome-dependent pathway that is activated in vitro by Vibrio cholerae. Our findings shed light on the genetic basis of cholera resistance in a population from the Ganges River Delta and present a promising approach for identifying genetic factors influencing susceptibility to infectious diseases.

RhoGTPases--NODes for effector-triggered immunity in animals.

A recent study published in Nature by Keestra and colleagues addresses how the immune system detects the pathogenic potential of microbes and provides evidence that one strategy involves NOD1, which monitors the activation state of the RhoGTPases that are targeted by virulence effectors produced by pathogenic microbes. Interestingly, their findings reveal striking similarities with previous observations made in flies and plants, establishing the evolutionary conservation of this detection system in the innate immune arsenal in many taxa.

Activation of caspase-1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function.

Phagocytosis is a fundamental cellular process that is pivotal for immunity as it coordinates microbial killing, innate immune activation and antigen presentation. An essential step in this process is phagosome acidification, which regulates many functions of these organelles that allow phagosomes to participate in processes that are essential to both innate and adaptive immunity. Here we report that acidification of phagosomes containing Gram-positive bacteria is regulated by the NLRP3 inflammasome and caspase-1. Active caspase-1 accumulates on phagosomes and acts locally to control the pH by modulating buffering by the NADPH oxidase NOX2. These data provide insight into a mechanism by which innate immune signals can modify cellular defenses and establish a new function for the NLRP3 inflammasome and caspase-1 in host defense.

Dectin-1 activation controls maturation of ß-1,3-glucan-containing phagosomes.

Elimination of fungal pathogens by phagocytes requires phagosome maturation, a process that involves the recruitment and fusion of intracellular proteins. The role of Dectin-1, a ß-1,3-glucan receptor, critical for fungal recognition and triggering of Th17 responses, to phagosomal maturation has not been defined. We show that GFP-Dectin-1 translocates to the fungal phagosome, but its signal decays after 2 h. Inhibition of acidification results in retention of GFP-Dectin-1 to phagosome membranes highlighting the requirement for an acidic pH. Following ß-1,3-glucan recognition, GFP-Dectin-1 undergoes tyrosine phosphorylation by Src kinases with subsequent Syk activation. Our results demonstrate that Syk is activated independently of intraphagosomal pH. Inhibition of Src or Syk results in prolonged retention of GFP-Dectin-1 to the phagosome signifying a link between Syk and intraphagosomal pH. ß-1,3-glucan phagosomes expressing a signaling incompetent Dectin-1 failed to mature as demonstrated by prolonged Dectin-1 retention, presence of Rab5B, failure to acquire LAMP-1 and inability to acidify. Phagosomes containing Candida albicans also require Dectin-1-dependent Syk activation for phagosomal maturation. Taken together, these results support a model where Dectin-1 not only controls internalization of ß-1,3-glucan containing cargo and triggers proinflammatory cytokines, but also acts as a master regulator for subsequent phagolysosomal maturation through Syk activation.

Effector-triggered versus pattern-triggered immunity: how animals sense pathogens.

A fundamental question regarding any immune system is how it can discriminate between pathogens and non-pathogens. Here, we discuss how this discrimination can be mediated by a surveillance system distinct from pattern-recognition receptors that recognize conserved microbial patterns. It can be based instead on the ability of the host to sense perturbations in host cells induced by bacterial toxins or 'effectors' that are encoded by pathogenic microorganisms. Such 'effector-triggered immunity' was previously demonstrated mainly in plants, but recent data confirm that animals can also use this strategy.