An adjuvant strategy enabled by modulation of the physical properties of microbial ligands expands antigen immunogenicity.

Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate lasting adaptive immunity. PRRs detect unique chemical patterns associated with invading microorganisms, but whether and how the physical properties of PRR ligands influence the development of the immune response remains unknown. Through the study of fungal mannans, we show that the physical form of PRR ligands dictates the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both the periphery and the dLN. When combined with viral glycoprotein antigens, this mannan formulation broadens epitope recognition, elicits potent antigen-specific neutralizing antibodies, and confers protection against viral infections of the lung. Thus, the physical properties of microbial ligands determine the outcome of the immune response and can be harnessed for vaccine development.

Restricted Clonality and Limited Germinal Center Reentry Characterize Memory B Cell Reactivation by Boosting.

Repeated exposure to pathogens or their antigens triggers anamnestic antibody responses that are higher in magnitude and affinity than the primary response. These involve reengagement of memory B cell (MBC) clones, the diversity and specificity of which determine the breadth and effectiveness of the ensuing antibody response. Using prime-boost models in mice, we find that secondary responses are characterized by a clonality bottleneck that restricts the engagement of the large diversity of MBC clones generated by priming. Rediversification of mutated MBCs is infrequent within secondary germinal centers (GCs), which instead consist predominantly of B cells without prior GC experience or detectable clonal expansion. Few MBC clones, generally derived from higher-affinity germline precursors, account for the majority of secondary antibody responses, while most primary-derived clonal diversity is not reengaged detectably by boosting. Understanding how to counter this bottleneck may improve our ability to elicit antibodies to non-immunodominant epitopes by vaccination.

Caspase-6 Is a Key Regulator of Innate Immunity, Inflammasome Activation, and Host Defense.

Caspases regulate cell death, immune responses, and homeostasis. Caspase-6 is categorized as an executioner caspase but shows key differences from the other executioners. Overall, little is known about the functions of caspase-6 in biological processes apart from apoptosis. Here, we show that caspase-6 mediates innate immunity and inflammasome activation. Furthermore, we demonstrate that caspase-6 promotes the activation of programmed cell death pathways including pyroptosis, apoptosis, and necroptosis (PANoptosis) and plays an essential role in host defense against influenza A virus (IAV) infection. In addition, caspase-6 promoted the differentiation of alternatively activated macrophages (AAMs). Caspase-6 facilitated the RIP homotypic interaction motif (RHIM)-dependent binding of RIPK3 to ZBP1 via its interaction with RIPK3. Altogether, our findings reveal a vital role for caspase-6 in facilitating ZBP1-mediated inflammasome activation, cell death, and host defense during IAV infection, opening additional avenues for treatment of infectious and autoinflammatory diseases and cancer.

A Site of Vulnerability on the Influenza Virus Hemagglutinin Head Domain Trimer Interface.

Here, we describe the discovery of a naturally occurring human antibody (Ab), FluA-20, that recognizes a new site of vulnerability on the hemagglutinin (HA) head domain and reacts with most influenza A viruses. Structural characterization of FluA-20 with H1 and H3 head domains revealed a novel epitope in the HA trimer interface, suggesting previously unrecognized dynamic features of the trimeric HA protein. The critical HA residues recognized by FluA-20 remain conserved across most subtypes of influenza A viruses, which explains the Ab's extraordinary breadth. The Ab rapidly disrupted the integrity of HA protein trimers, inhibited cell-to-cell spread of virus in culture, and protected mice against challenge with viruses of H1N1, H3N2, H5N1, or H7N9 subtypes when used as prophylaxis or therapy. The FluA-20 Ab has uncovered an exceedingly conserved protective determinant in the influenza HA head domain trimer interface that is an unexpected new target for anti-influenza therapeutics and vaccines.

Transcription Elongation Can Affect Genome 3D Structure.

How transcription affects genome 3D organization is not well understood. We found that during influenza A (IAV) infection, rampant transcription rapidly reorganizes host cell chromatin interactions. These changes occur at the ends of highly transcribed genes, where global inhibition of transcription termination by IAV NS1 protein causes readthrough transcription for hundreds of kilobases. In these readthrough regions, elongating RNA polymerase II disrupts chromatin interactions by inducing cohesin displacement from CTCF sites, leading to locus decompaction. Readthrough transcription into heterochromatin regions switches them from the inert (B) to the permissive (A) chromatin compartment and enables transcription factor binding. Data from non-viral transcription stimuli show that transcription similarly affects cohesin-mediated chromatin contacts within gene bodies. Conversely, inhibition of transcription elongation allows cohesin to accumulate at previously transcribed intragenic CTCF sites and to mediate chromatin looping and compaction. Our data indicate that transcription elongation by RNA polymerase II remodels genome 3D architecture.

Adenosine-to-inosine editing of endogenous Z-form RNA by the deaminase ADAR1 prevents spontaneous MAVS-dependent type I interferon responses.

Nucleic acids are powerful triggers of innate immunity and can adopt the Z-conformation, an unusual left-handed double helix. Here, we studied the biological function(s) of Z-RNA recognition by the adenosine deaminase ADAR1, mutations in which cause Aicardi-Goutières syndrome. Adar1mZα/mZα mice, bearing two point mutations in the Z-nucleic acid binding (Zα) domain that abolish Z-RNA binding, displayed spontaneous induction of type I interferons (IFNs) in multiple organs, including in the lung, where both stromal and hematopoietic cells showed IFN-stimulated gene (ISG) induction. Lung neutrophils expressed ISGs induced by the transcription factor IRF3, indicating an initiating role for neutrophils in this IFN response. The IFN response in Adar1mZα/mZα mice required the adaptor MAVS, implicating cytosolic RNA sensing. Adenosine-to-inosine changes were enriched in transposable elements and revealed a specific requirement of ADAR1's Zα domain in editing of a subset of RNAs. Thus, endogenous RNAs in Z-conformation have immunostimulatory potential curtailed by ADAR1, with relevance to autoinflammatory disease in humans.

Jamaican fruit bats mount a stable and highly neutralizing antibody response after bat influenza virus infection.

Antibodies are an essential component of the antiviral response in many species, but to date, there is no compelling evidence that bats are capable of eliciting a robust humoral immunity, including neutralizing antibodies. Here, we report that infection of Jamaican fruit bats with the bat influenza A virus H18N11 elicits a rapid and stable humoral immune response with a strong neutralizing capacity, associated with no detectable viral shedding after repeat challenge infection. Thus, the neutralizing antibody response of bats might play an important role in the bat immunity.

Dichotomous Expression of TNF Superfamily Ligands on Antigen-Presenting Cells Controls Post-priming Anti-viral CD4+ T Cell Immunity.

T cell antigen-presenting cell (APC) interactions early during chronic viral infection are crucial for determining viral set point and disease outcome, but how and when different APC subtypes contribute to these outcomes is unclear. The TNF receptor superfamily (TNFRSF) member GITR is important for CD4+ T cell accumulation and control of chronic lymphocytic choriomeningitis virus (LCMV). We found that type I interferon (IFN-I) induced TNFSF ligands GITRL, 4-1BBL, OX40L, and CD70 predominantly on monocyte-derived APCs and CD80 and CD86 predominantly on classical dendritic cells (cDCs). Mice with hypofunctional GITRL in Lyz2+ cells had decreased LCMV-specific CD4+ T cell accumulation and increased viral load. GITR signals in CD4+ T cells occurred after priming to upregulate OX40, CD25, and chemokine receptor CX3CR1. Thus IFN-I (signal 3) induced a post-priming checkpoint (signal 4) for CD4+ T cell accumulation, revealing a division of labor between cDCs and monocyte-derived APCs in regulating T cell expansion.

NS2 induces an influenza A RNA polymerase hexamer and acts as a transcription to replication switch.

Genome transcription and replication of influenza A virus (FluA), catalyzed by viral RNA polymerase (FluAPol), are delicately controlled across the virus life cycle. A switch from transcription to replication occurring at later stage of an infection is critical for progeny virion production and viral non-structural protein NS2 has been implicated in regulating the switch. However, the underlying regulatory mechanisms and the structure of NS2 remained elusive for years. Here, we determine the cryo-EM structure of the FluAPol-NS2 complex at ~3.0 Å resolution. Surprisingly, three domain-swapped NS2 dimers arrange three symmetrical FluPol dimers into a highly ordered barrel-like hexamer. Further structural and functional analyses demonstrate that NS2 binding not only hampers the interaction between FluAPol and the Pol II CTD because of steric conflicts, but also impairs FluAPol transcriptase activity by stalling it in the replicase conformation. Moreover, this is the first visualization of the full-length NS2 structure. Our findings uncover key molecular mechanisms of the FluA transcription-replication switch and have implications for the development of antivirals.

Profiling Cullin4-E3 ligases interactomes and their rewiring in influenza A virus infection.

Understanding the integrated regulation of cellular processes during viral infection is crucial for developing host-targeted approaches. We have previously reported that an optimal in vitro infection by influenza A (IAV) requires three components of Cullin 4-RING E3 ubiquitin ligases (CRL4) complexes, namely the DDB1 adaptor and two Substrate Recognition Factors (SRF), DCAF11 and DCAF12L1, which mediate non-degradative poly-ubiquitination of the PB2 subunit of the viral polymerase. However, the impact of IAV infection on the CRL4 interactome remains elusive. Here, using Affinity Purification coupled with Mass Spectrometry (AP-MS) approaches, we identified cellular proteins interacting with these CRL4 components in IAV-infected and non-infected contexts. IAV infection induces significant modulations in protein interactions, resulting in a global loss of DDB1 and DCAF11 interactions, and an increase in DCAF12L1-associated proteins. The distinct rewiring of CRL4's associations upon infection impacted cellular proteins involved in protein folding, ubiquitination, translation, splicing, and stress responses. Using a split-nanoluciferase-based assay, we identified direct partners of CRL4 components and via siRNA-mediated silencing validated their role in IAV infection, representing potential substrates or regulators of CRL4 complexes. Our findings unravel the dynamic remodeling of the proteomic landscape of CRL4's E3 ubiquitin ligases during IAV infection, likely involved in shaping a cellular environment conducive to viral replication and offer potential for the exploration of future host-targeted antiviral therapeutic strategies.

H3N2 influenza A virus gradually adapts to human-type receptor binding and entry specificity after the start of the 1968 pandemic.

To become established upon zoonotic transfer, influenza A viruses (IAV) need to switch binding from "avian-type" α2-3-linked sialic acid receptors (2-3Sia) to "human-type" Siaα2-6-linked sialic acid receptors (2-6Sia). For the 1968 H3N2 pandemic virus, this was accomplished by two canonical amino acid substitutions in its hemagglutinin (HA) although a full specificity shift had not occurred. The receptor repertoire on epithelial cells is highly diverse and simultaneous interaction of a virus particle with a range of low- to very low-affinity receptors results in tight heteromultivalent binding. How this range of affinities determines binding selectivity and virus motility remains largely unknown as the analysis of low-affinity monovalent HA-receptor interactions is technically challenging. Here, a biolayer interferometry assay enabled a comprehensive analysis of receptor-binding kinetics evolution upon host-switching. Virus-binding kinetics of H3N2 virus isolates slowly evolved from 1968 to 1979 from mixed 2-3/2-6Sia specificity to high 2-6Sia specificity, surprisingly followed by a decline in selectivity after 1992. By using genetically tuned HEK293 cells, presenting either a simplified 2-3Sia- or 2-6Sia-specific receptor repertoire, receptor-specific binding was shown to correlate strongly with receptor-specific entry. In conclusion, the slow and continuous evolution of entry and receptor-binding specificity of seasonal H3N2 viruses contrasts with the paradigm that human IAVs need to rapidly acquire and maintain a high specificity for 2-6Sia. Analysis of the kinetic parameters of receptor binding provides a basis for understanding virus-binding specificity, motility, and HA/neuraminidase balance at the molecular level.

Influenza A virus exploits transferrin receptor recycling to enter host cells.

Influenza A virus (IAV) enters host cells mostly through clathrin-dependent receptor-mediated endocytosis. A single bona fide entry receptor protein supporting this entry mechanism remains elusive. Here we performed proximity ligation of biotin to host cell surface proteins in the vicinity of attached trimeric hemagglutinin-HRP and characterized biotinylated targets using mass spectrometry. This approach identified transferrin receptor 1 (TfR1) as a candidate entry protein. Genetic gain-of-function and loss-of-function experiments, as well as in vitro and in vivo chemical inhibition, confirmed the functional involvement of TfR1 in IAV entry. Recycling deficient mutants of TfR1 do not support entry, indicating that TfR1 recycling is essential for this function. The binding of virions to TfR1 via sialic acids confirmed its role as a directly acting entry factor, but unexpectedly even headless TfR1 promoted IAV particle uptake in trans. TIRF microscopy localized the entering virus-like particles in the vicinity of TfR1. Our data identify TfR1 recycling as a revolving door mechanism exploited by IAV to enter host cells.

Epistasis reduces fitness costs of influenza A virus escape from stem-binding antibodies.

The hemagglutinin (HA) stem region is a major target of universal influenza vaccine efforts owing to the presence of highly conserved epitopes across multiple influenza A virus (IAV) strains and subtypes. To explore the potential impact of vaccine-induced immunity targeting the HA stem, we examined the fitness effects of viral escape from stem-binding broadly neutralizing antibodies (stem-bnAbs). Recombinant viruses containing each individual antibody escape substitution showed diminished replication compared to wild-type virus, indicating that stem-bnAb escape incurred fitness costs. A second-site mutation in the HA head domain (N129D; H1 numbering) reduced the fitness effects observed in primary cell cultures and likely enabled the selection of escape mutations. Functionally, this putative permissive mutation increased HA avidity for its receptor. These results suggest a mechanism of epistasis in IAV, wherein modulating the efficiency of attachment eases evolutionary constraints imposed by the requirement for membrane fusion. Taken together, the data indicate that viral escape from stem-bnAbs is costly but highlights the potential for epistatic interactions to enable evolution within the functionally constrained HA stem domain.

Getting on the right track: Interactions between viruses and the cytoskeletal motor proteins.

The cytoskeleton is an essential component of the cell and it is involved in multiple physiological functions, including intracellular organization and transport. It is composed of three main families of proteinaceous filaments; microtubules, actin filaments and intermediate filaments and their accessory proteins. Motor proteins, which comprise the dynein, kinesin and myosin superfamilies, are a remarkable group of accessory proteins that mainly mediate the intracellular transport of cargoes along with the cytoskeleton. Like other cellular structures and pathways, viruses can exploit the cytoskeleton to promote different steps of their life cycle through associations with motor proteins. The complexity of the cytoskeleton and the differences among viruses, however, has led to a wide diversity of interactions, which in most cases remain poorly understood. Unveiling the details of these interactions is necessary not only for a better comprehension of specific infections, but may also reveal new potential drug targets to fight dreadful diseases such as rabies disease and acquired immunodeficiency syndrome (AIDS). In this review, we describe a few examples of the mechanisms that some human viruses, that is, rabies virus, adenovirus, herpes simplex virus, human immunodeficiency virus, influenza A virus and papillomavirus, have developed to hijack dyneins, kinesins and myosins.

Bakuchiol targets mitochondrial proteins, prohibitins and voltage-dependent anion channels: New insights into developing antiviral agents.

We previously reported that bakuchiol, a phenolic isoprenoid anticancer compound, and its analogs exert anti-influenza activity. However, the proteins targeted by bakuchiol remain unclear. Here, we investigated the chemical structures responsible for the anti-influenza activity of bakuchiol and found that all functional groups and C6 chirality of bakuchiol were required for its anti-influenza activity. Based on these results, we synthesized a molecular probe containing a biotin tag bound to the C1 position of bakuchiol. With this probe, we performed a pulldown assay for Madin-Darby canine kidney cell lysates and purified the specific bakuchiol-binding proteins with SDS-PAGE. Using nanoLC-MS/MS analysis, we identified prohibitin (PHB) 2, voltage-dependent anion channel (VDAC) 1, and VDAC2 as binding proteins of bakuchiol. We confirmed the binding of bakuchiol to PHB1, PHB2, and VDAC2 in vitro using Western blot analysis. Immunofluorescence analysis showed that bakuchiol was bound to PHBs and VDAC2 in cells and colocalized in the mitochondria. The knockdown of PHBs or VDAC2 by transfection with specific siRNAs, along with bakuchiol cotreatment, led to significantly reduced influenza nucleoprotein expression levels and viral titers in the conditioned medium of virus-infected Madin-Darby canine kidney cells, compared to the levels observed with transfection or treatment alone. These findings indicate that reducing PHBs or VDAC2 protein, combined with bakuchiol treatment, additively suppressed the growth of influenza virus. Our findings indicate that bakuchiol exerts anti-influenza activity via a novel mechanism involving these mitochondrial proteins, providing new insight for developing anti-influenza agents.

Genome-wide CRISPR activation screen identifies JADE3 as an antiviral activator of NF-kB-dependent IFITM3 expression.

The innate immune system features a web of interacting pathways that require exquisite regulation. To identify novel nodes in this immune landscape, we conducted a gain-of-function, genome-wide CRISPR activation screen with influenza A virus. We identified both appreciated and novel antiviral genes, including Jade family PHD zinc finger 3 (JADE3) a protein involved in directing the histone acetyltransferase histone acetyltransferase binding to ORC1 complex to modify chromatin and regulate transcription. JADE3 is both necessary and sufficient to restrict influenza A virus infection. Our results suggest a distinct function for JADE3 as expression of the closely related paralogs JADE1 and JADE2 does not confer resistance to influenza A virus infection. JADE3 is required for both constitutive and inducible expression of the well-characterized antiviral gene interferon-induced transmembrane protein 3 (IFITM3). Furthermore, we find JADE3 activates the NF-kB signaling pathway, which is required for the promotion of IFITM3 expression by JADE3. Therefore, we propose JADE3 activates an antiviral genetic program involving NF-kB-dependent IFITM3 expression to restrict influenza A virus infection.

Cellular NS1-BP Protein Interacts with the mRNA Export Receptor NXF1 to Mediate Nuclear Export of Influenza Virus M mRNAs.

Influenza A viruses have eight genomic RNAs that are transcribed in the host cell nucleus. Two of the viral mRNAs undergo alternative splicing. The M1 mRNA encodes the matrix protein 1 (M1) and is also spliced into M2 mRNA, which encodes the proton channel matrix protein 2 (M2). Our previous studies have shown that the cellular NS1-binding protein (NS1-BP) interacts with the viral non-structural protein 1 (NS1) and M1 mRNA to promote M1 to M2 splicing. Another pool of NS1 protein binds the mRNA export receptor NXF1 (nuclear RNA export factor-1), leading to nuclear retention of cellular mRNAs. Here we show a series of biochemical and cell biological findings that suggest a model for nuclear export of M1 and M2 mRNAs despite the mRNA nuclear export inhibition imposed by the viral NS1 protein. NS1-BP competes with NS1 for NXF1 binding, allowing the recruitment of NXF1 to the M mRNAs after splicing. NXF1 then binds GANP (Germinal-center Associated Nuclear Protein), a member of the TRanscription and EXport complex (TREX)-2. Although both NS1 and NS1-BP remain in complex with GANP-NXF1, they dissociate once this complex docks at the nuclear pore complex (NPC), and the M mRNAs are translocated to the cytoplasm. Since this mRNA nuclear export pathway is key for expression of M1 and M2 proteins that function in viral intracellular trafficking and budding, these viral-host interactions are critical for influenza virus replication.

Transmission Bottleneck Size Estimation from De Novo Viral Genetic Variation.

Sequencing of viral infections has become increasingly common over the last decade. Deep sequencing data in particular have proven useful in characterizing the roles that genetic drift and natural selection play in shaping within-host viral populations. They have also been used to estimate transmission bottleneck sizes from identified donor-recipient pairs. These bottleneck sizes quantify the number of viral particles that establish genetic lineages in the recipient host and are important to estimate due to their impact on viral evolution. Current approaches for estimating bottleneck sizes exclusively consider the subset of viral sites that are observed as polymorphic in the donor individual. However, these approaches have the potential to substantially underestimate true transmission bottleneck sizes. Here, we present a new statistical approach for instead estimating bottleneck sizes using patterns of viral genetic variation that arise de novo within a recipient individual. Specifically, our approach makes use of the number of clonal viral variants observed in a transmission pair, defined as the number of viral sites that are monomorphic in both the donor and the recipient but carry different alleles. We first test our approach on a simulated dataset and then apply it to both influenza A virus sequence data and SARS-CoV-2 sequence data from identified transmission pairs. Our results confirm the existence of extremely tight transmission bottlenecks for these 2 respiratory viruses.

STAT6-induced production of mucus and resistin-like molecules in lung Club cells does not protect against helminth or influenza A virus infection.

Airway epithelial cells contribute to a variety of lung diseases including allergic asthma, where IL-4 and IL-13 promote activation of the transcription factor STAT6. This leads to goblet cell hyperplasia and the secretion of effector molecules by epithelial cells. However, the specific effect of activated STAT6 in lung epithelial cells is only partially understood. Here, we created a mouse strain to selectively investigate the role of constitutively active STAT6 in Club cells, a subpopulation of airway epithelial cells. CCSP-Cre_STAT6vt mice and bronchiolar organoids derived from these show an enhanced expression of the chitinase-like protein Chil4 (Ym2) and resistin-like molecules (Relm-α, -ß, -γ). In addition, goblet cells of these mice spontaneously secrete mucus into the bronchi. However, the activated epithelium resulted neither in impaired lung function nor conferred a protective effect against the migrating helminth Nippostrongylus brasiliensis. Moreover, CCSP-Cre_STAT6vt mice showed similar allergic airway inflammation induced by live conidia of the fungus Aspergillus fumigatus and similar recovery after influenza A virus infection compared to control mice. Together these results highlight that STAT6 signaling in Club cells induces the secretion of Relm proteins and mucus without impairing lung function, but this is not sufficient to confer protection against helminth or viral infections.

Headless hemagglutinin-containing influenza viral particles direct immune responses toward more conserved epitopes.

Seasonal influenza vaccines provide mostly strain-specific protection due to the elicitation of antibody responses focused on evolutionarily plastic antigenic sites in the hemagglutinin head domain. To direct the humoral response toward more conserved epitopes, we generated an influenza virus particle where the full-length hemagglutinin protein was replaced with a membrane-anchored, "headless" variant while retaining the normal complement of other viral structural proteins such as the neuraminidase as well as viral RNAs. We found that a single administration of a headless virus particle-based vaccine elicited high titers of antibodies that recognized more conserved epitopes on the major viral glycoproteins. Furthermore, the vaccine could elicit these responses even in the presence of pre-existing, hemagglutinin (HA) head-focused influenza immunity. Importantly, these antibody responses mediated protective, but non-neutralizing functions such as neuraminidase inhibition and antibody-dependent cellular cytotoxicity. Additionally, we show the vaccine can provide protection from homologous and heterologous challenges in mouse models of severe influenza without any measurable HA head-directed antibody responses. Thus, headless hemagglutinin containing viral particles may represent a tool to drive the types of antibody responses predicted to increase influenza vaccine breadth and durability.IMPORTANCECurrent seasonal influenza vaccines provide incomplete protection from disease. This is partially the result of the antibody response being directed toward parts of the virus that are tolerant of mutations. Redirecting the immune response to more conserved regions of the virus has been a central strategy of next-generation vaccine designs and approaches. Here, we develop and test a vaccine based on a modified influenza virus particle that expresses a partially deleted hemagglutinin protein along with the other viral structural proteins. We demonstrate this vaccine elicits antibodies that recognize the more conserved viral epitopes of the hemagglutinin stalk and neuraminidase protein to facilitate protection against influenza viruses despite a lack of classical viral neutralization activity.