Modified Alphavirus-Vesiculovirus Hybrid Vaccine Vectors for Homologous Prime-Boost Immunotherapy of Chronic Hepatitis B.

Abstract： Virus-like vesicles (VLV) are hybrid vectors based on an evolved Semliki Forest virus (SFV) RNA replicon and the envelope glycoprotein (G) from vesicular stomatitis virus (VSV) [...].

Virus-like Vesicles Expressing Multiple Antigens for Immunotherapy of Chronic Hepatitis B.

Infections with hepatitis B virus (HBV) can initiate chronic hepatitis and liver injury, causing more than 600,000 deaths each year worldwide. Current treatments for chronic hepatitis B are inadequate and leave an unmet need for immunotherapeutic approaches. We designed virus-like vesicles (VLV) as self-amplifying RNA replicons expressing three HBV antigens (polymerase, core, and middle surface) from a single vector (HBV-VLV) to break immune exhaustion despite persistent HBV replication. The HBV-VLV induces HBV-specific T cells in naive mice and renders them resistant to acute challenge with HBV. Using a chronic model of HBV infection, we demonstrate efficacy of HBV-VLV priming in combination with DNA booster immunization, as 40% of treated mice showed a decline of serum HBV surface antigen below the detection limit and marked reduction in liver HBV RNA accompanied by induction of HBsAg-specific CD8 T cells. These results warrant further evaluation of HBV-VLV for immunotherapy of chronic hepatitis B.

REH2C Helicase and GRBC Subcomplexes May Base Pair through mRNA and Small Guide RNA in Kinetoplastid Editosomes.

Mitochondrial mRNAs in Trypanosoma brucei undergo extensive insertion and deletion of uridylates that are catalyzed by the RNA editing core complex (RECC) and directed by hundreds of small guide RNAs (gRNAs) that base pair with mRNA. RECC is largely RNA-free, and accessory mitochondrial RNA-binding complex 1 (MRB1) variants serve as scaffolds for the assembly of mRNA-gRNA hybrids and RECC. However, the molecular steps that create higher-order holoenzymes ("editosomes") are unknown. Previously, we identified an RNA editing helicase 2-associated subcomplex (REH2C) and showed that REH2 binds RNA. Here we showed that REH2C is an mRNA-associated ribonucleoprotein (mRNP) subcomplex with editing substrates, intermediates, and products. We isolated this mRNP from mitochondria lacking gRNA-bound RNP (gRNP) subcomplexes and identified REH2-associated cofactors 1 and 2 ((H2)F1 and (H2)F2). (H2)F1 is an octa-zinc finger protein required for mRNP-gRNP docking, pre-mRNA and RECC loading, and RNP formation with a short synthetic RNA duplex. REH2 and other eukaryotic DEAH/RHA-type helicases share a conserved regulatory C-terminal domain cluster that includes an oligonucleotide-binding fold. Recombinant REH2 and (H2)F1 constructs associate in a purified complex in vitro. We propose a model of stepwise editosome assembly that entails controlled docking of mRNP and gRNP modules via specific base pairing between their respective mRNA and gRNA cargo and regulatory REH2 and (H2)F1 subunits of the novel mRNP that may control specificity checkpoints in the editing pathway.

Native Variants of the MRB1 Complex Exhibit Specialized Functions in Kinetoplastid RNA Editing.

Adaptation and survival of Trypanosoma brucei requires editing of mitochondrial mRNA by uridylate (U) insertion and deletion. Hundreds of small guide RNAs (gRNAs) direct the mRNA editing at over 3,000 sites. RNA editing is controlled during the life cycle but the regulation of substrate and stage specificity remains unknown. Editing progresses in the 3' to 5' direction along the pre-mRNA in blocks, each targeted by a unique gRNA. A critical editing factor is the mitochondrial RNA binding complex 1 (MRB1) that binds gRNA and transiently interacts with the catalytic RNA editing core complex (RECC). MRB1 is a large and dynamic complex that appears to be comprised of distinct but related subcomplexes (termed here MRBs). MRBs seem to share a 'core' complex of proteins but differ in the composition of the 'variable' proteins. Since some proteins associate transiently the MRBs remain imprecisely defined. MRB1 controls editing by unknown mechanisms, and the functional relevance of the different MRBs is unclear. We previously identified two distinct MRBs, and showed that they carry mRNAs that undergo editing. We proposed that editing takes place in the MRBs because MRBs stably associate with mRNA and gRNA but only transiently interact with RECC, which is RNA free. Here, we identify the first specialized functions in MRBs: 1) 3010-MRB is a major scaffold for RNA editing, and 2) REH2-MRB contains a critical trans-acting RNA helicase (REH2) that affects multiple steps of editing function in 3010-MRB. These trans effects of the REH2 include loading of unedited mRNA and editing in the first block and in subsequent blocks as editing progresses. REH2 binds its own MRB via RNA, and conserved domains in REH2 were critical for REH2 to associate with the RNA and protein components of its MRB. Importantly, REH2 associates with a ~30 kDa RNA-binding protein in a novel ~15S subcomplex in RNA-depleted mitochondria. We use these new results to update our model of MRB function and organization.

Native mitochondrial RNA-binding complexes in kinetoplastid RNA editing differ in guide RNA composition.

Mitochondrial mRNAs in kinetoplastids require extensive U-insertion/deletion editing that progresses 3'-to-5' in small blocks, each directed by a guide RNA (gRNA), and exhibits substrate and developmental stage-specificity by unsolved mechanisms. Here, we address compositionally related factors, collectively known as the mitochondrial RNA-binding complex 1 (MRB1) or gRNA-binding complex (GRBC), that contain gRNA, have a dynamic protein composition, and transiently associate with several mitochondrial factors including RNA editing core complexes (RECC) and ribosomes. MRB1 controls editing by still unknown mechanisms. We performed the first next-generation sequencing study of native subcomplexes of MRB1, immunoselected via either RNA helicase 2 (REH2), that binds RNA and associates with unwinding activity, or MRB3010, that affects an early editing step. The particles contain either REH2 or MRB3010 but share the core GAP1 and other proteins detected by RNA photo-crosslinking. Analyses of the first editing blocks indicate an enrichment of several initiating gRNAs in the MRB3010-purified complex. Our data also indicate fast evolution of mRNA 3' ends and strain-specific alternative 3' editing within 3' UTR or C-terminal protein-coding sequence that could impact mitochondrial physiology. Moreover, we found robust specific copurification of edited and pre-edited mRNAs, suggesting that these particles may bind both mRNA and gRNA editing substrates. We propose that multiple subcomplexes of MRB1 with different RNA/protein composition serve as a scaffold for specific assembly of editing substrates and RECC, thereby forming the editing holoenzyme. The MRB3010-subcomplex may promote early editing through its preferential recruitment of initiating gRNAs.