TRIM21 mediates antibody inhibition of adenovirus-based gene delivery and vaccination

Adenovirus has enormous potential as a gene-therapy vector, but preexisting immunity limits its widespread application. What is responsible for this immune block is unclear because antibodies potently inhibit transgene expression without impeding gene transfer into target cells. Here we show that antibody prevention of adenoviral gene delivery in vivo is mediated by the cytosolic antibody receptor TRIM21. Genetic KO of TRIM21 or a single-antibody point mutation is sufficient to restore transgene expression to near-naïve immune levels. TRIM21 is also responsible for blocking cytotoxic T cell induction by vaccine vectors, preventing a protective response against subsequent influenza infection and an engrafted tumor. Furthermore, adenoviral preexisting immunity can lead to an augmented immune response upon i.v. administration of the vector. Transcriptomic analysis of vector-transduced tissue reveals that TRIM21 is responsible for the specific up-regulation of hundreds of immune genes, the majority of which are components of the intrinsic or innate response. Together, these data define a major mechanism underlying the preimmune block to adenovirus gene therapy and demonstrate that TRIM21 efficiently blocks gene delivery in vivo while simultaneously inducing a rapid program of immune transcription

Corrigendum: Antibody-antigen kinetics constrain intracellular humoral immunity

This Article contains a typographical error in the Methods section under the subheading ‘Crystallization’, where the Protein Data Bank accession code ‘5LDN’ was incorrectly given as ‘5LDV’

Antibody-antigen kinetics constrain intracellular humoral immunity

During infection with non-enveloped viruses, antibodies stimulate immunity from inside cells by activating the cytosolic Fc receptor TRIM21. This intracellular humoral response relies on opsonized viral particles reaching the cytosol intact but the antigenic and kinetic constraints involved are unknown. We have solved the structure of a potent TRIM21-dependent neutralizing antibody in complex with human adenovirus 5 hexon and show how these properties influence immune activity. Structure-guided mutagenesis was used to generate antibodies with 20,000-fold variation in affinity, on-rates that differ by ~50-fold and off-rates by >175-fold. Characterization of these variants during infection revealed that TRIM21-dependent neutralization and NFκB activation was largely unaffected by on-rate kinetics. In contrast, TRIM21 antiviral activity was exquisitely dependent upon off-rate, with sub-μM affinity antibodies nevertheless unable to stimulate signaling because of fast dissociation kinetics. These results define the antibody properties required to elicit an efficient intracellular immune response during viral infection

Antibody and DNA sensing pathways converge to activate the inflammasome during primary human macrophage infection

Inflammasomes are potent innate immune signalling complexes that couple cytokine release with pro-inflammatory cell death. However, pathogens have evolved strategies to evade this cell autonomous system. Here, we show how antibodies combine with innate sensors in primary human macrophages to detect viral infection and activate the inflammasome. Our data demonstrate that antibody opsonisation of virions can activate macrophages in multiple ways. In the first, antibody binding of adenovirus causes lysosomal damage, activating NLRP3 to drive inflammasome formation and IL-1β release. Importantly, this mechanism enhances virion capture but not infection and is accompanied by cell death, denying the opportunity for viral replication. Unexpectedly, we also find that antibody-coated viruses, which successfully escape into the cytosol, trigger a second system of inflammasome activation. These viruses are intercepted by the cytosolic antibody receptor TRIM21 and the DNA sensor cGAS. Together, these sensors stimulate both NLRP3 inflammasome formation and NFκB activation, driving dose-dependent IL-1β and TNF secretion, without inducing cell death. Our data highlight the importance of cooperativity between multiple sensing networks to expose viruses to the inflammasome pathway, which is particularly important for how our innate immune system responds to infection in the presence of pre-existing immunity

Complement C4 prevents viral infection through capsid inactivation

The complement system is vital for anti-microbial defense. In the classical pathway, pathogen-bound antibody recruits the C1 complex (C1qC1r2C1s2) that initiates a cleavage cascade involving C2, C3, C4, and C5 and triggering microbial clearance. We demonstrate a C4-dependent antiviral mechanism that is independent of downstream complement components. C4 inhibits human adenovirus infection by directly inactivating the virus capsid. Rapid C4 activation and capsid deposition of cleaved C4b are catalyzed by antibodies via the classical pathway. Capsid-deposited C4b neutralizes infection independent of C2 and C3 but requires C1q antibody engagement. C4b inhibits capsid disassembly, preventing endosomal escape and cytosolic access. C4-deficient mice exhibit heightened viral burdens. Additionally, complement synergizes with the Fc receptor TRIM21 to block transduction by an adenovirus gene therapy vector but is partially restored by Fab virus shielding. These results suggest that the complement system could be altered to prevent virus infection and enhance virus gene therapy efficacy

Mitochondrial–cell cycle cross-talk drives endoreplication in heart disease

Endoreplication, duplication of the nuclear genome without cell division, occurs in disease to drive morphologic growth, cell fate, and function. Despite its criticality, the metabolic underpinnings of disease-induced endoreplication and its link to morphologic growth are unknown. Heart disease is characterized by endoreplication preceding cardiac hypertrophy. We identify ATP synthase as a central control node and determinant of cardiac endoreplication and hypertrophy by rechanneling free mitochondrial ADP to methylenetetrahydrofolate dehydrogenase 1 L (MTHFD1L), a mitochondrial localized rate-limiting enzyme of formate and de novo nucleotide biosynthesis. Concomitant activation of the adenosine monophosphate-activated protein kinase (AMPK)-retinoblastoma protein (Rb)-E2F axis co-opts metabolic products of MTHFD1L function to support DNA endoreplication and pathologic growth. Gain- and loss-of-function studies in genetic and surgical mouse heart disease models and correlation in individuals confirm direct coupling of deregulated energetics with endoreplication and pathologic overgrowth. Together, we identify cardiometabolic endoreplication as a hitherto unknown mechanism dictating pathologic growth progression in the failing myocardium