mRNA-LNP prime boost evolves precursors toward VRC01-like broadly neutralizing antibodies in preclinical humanized mouse models.

Germline-targeting (GT) protein immunogens to induce VRC01-class broadly neutralizing antibodies (bnAbs) to the CD4-binding site of the HIV envelope (Env) have shown promise in clinical trials. Here, we preclinically validated a lipid nanoparticle-encapsulated nucleoside mRNA (mRNA-LNP) encoding eOD-GT8 60mer as a soluble self-assembling nanoparticle in mouse models. In a model with three humanized B cell lineages bearing distinct VRC01-precursor B cell receptors (BCRs) with similar affinities for eOD-GT8, all lineages could be simultaneously primed and undergo diversification and affinity maturation without exclusionary competition. Boosts drove precursor B cell participation in germinal centers; the accumulation of somatic hypermutations, including in key VRC01-class positions; and affinity maturation to boost and native-like antigens in two of the three precursor lineages. We have preclinically validated a prime-boost regimen of soluble self-assembling nanoparticles encoded by mRNA-LNP, demonstrating that multiple lineages can be primed, boosted, and diversified along the bnAb pathway.

Potent TRIM21 and complement-dependent intracellular antiviral immunity requires the IgG3 hinge.

Humans have four IgG antibody subclasses that selectively or differentially engage immune effector molecules to protect against infections. Although IgG1 has been studied in detail and is the subclass of most approved antibody therapeutics, increasing evidence indicates that IgG3 is associated with enhanced protection against pathogens. Here, we report that IgG3 has superior capacity to mediate intracellular antiviral immunity compared with the other subclasses due to its uniquely extended and flexible hinge region, which facilitates improved recruitment of the cytosolic Fc receptor TRIM21, independently of Fc binding affinity. TRIM21 may also synergize with complement C1/C4-mediated lysosomal degradation via capsid inactivation. We demonstrate that this process is potentiated by IgG3 in a hinge-dependent manner. Our findings reveal differences in how the four IgG subclasses mediate intracellular immunity, knowledge that may guide IgG subclass selection and engineering of antiviral antibodies for prophylaxis and therapy.

Virus neutralisation by intracellular antibodies.

For decades antibodies were largely thought to provide protection in extracellular spaces alone, mediating their effector functions by mechanisms such as entry-blocking, complement activation and phagocyte recruitment. However, a wealth of research has shown that antibodies are also capable of neutralising numerous viruses inside cells. Efficacy has now been demonstrated at virtually all intracellular stages of the viral life cycle. Antibodies can neutralise viruses in endosomes by blocking uncoating, fusion mechanisms, or new particle egress. Neutralisation can also occur in the cytosol via recruitment of the intracellular antibody receptor TRIM21. In addition to these direct neutralisation effects, recent research has shown that antibodies can mediate virus control indirectly by promoting MHC class I presentation and thereby increasing the CD8 T cell response. This provides valuable new insight into how non-neutralising antibodies can mediate potent protection in vivo. Overall, the importance of understanding the mechanisms of intracellular neutralisation by antibodies is highlighted by the ongoing need to develop new methods to control viruses. Using or inducing antibodies to block virus replication inside cells is now an innovative approach used by several vaccination and therapeutic strategies.

TRIM21-From Intracellular Immunity to Therapy.

Tripartite motif containing-21 (TRIM21) is a cytosolic ubiquitin ligase and antibody receptor that provides a last line of defense against invading viruses. It does so by acting as a sensor that intercepts antibody-coated viruses that have evaded extracellular neutralization and breached the cell membrane. Upon engagement of the Fc of antibodies bound to viruses, TRIM21 triggers a coordinated effector and signaling response that prevents viral replication while at the same time inducing an anti-viral cellular state. This dual effector function is tightly regulated by auto-ubiquitination and phosphorylation. Therapeutically, TRIM21 has been shown to be detrimental in adenovirus based gene therapy, while it may be favorably utilized to prevent tau aggregation in neurodegenerative disorders. In addition, TRIM21 may synergize with the complement system to block viral replication as well as transgene expression. TRIM21 can also be utilized as a research tool to deplete specific proteins in cells and zebrafish embryos. Here, we review our current biological understanding of TRIM21 in light of its versatile functions.

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-1ß 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-1ß 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.

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.

Intracellular Antiviral Immunity.

Innate immunity is traditionally thought of as the first line of defense against pathogens that enter the body. It is typically characterized as a rather weak defense mechanism, designed to restrict pathogen replication until the adaptive immune response generates a tailored response and eliminates the infectious agent. However, intensive research in recent years has resulted in better understanding of innate immunity as well as the discovery of many effector proteins, revealing its numerous powerful mechanisms to defend the host. Furthermore, this research has demonstrated that it is simplistic to strictly separate adaptive and innate immune functions since these two systems often work synergistically rather than sequentially. Here, we provide a broad overview of innate pattern recognition receptors in antiviral defense, with a focus on the TRIM family, and discuss their signaling pathways and mechanisms of action with special emphasis on the intracellular antibody receptor TRIM21.

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.