RNA interference (RNAi) functions as a potent antiviral immunity in plants and invertebrates; however, whether RNAi plays antiviral roles in mammals remains unclear. Here, using human enterovirus 71 (HEV71) as a model, we showed HEV71 3A protein as an authentic viral suppressor of RNAi during viral infection. When the 3A-mediated RNAi suppression was impaired, the mutant HEV71 readily triggered the production of abundant HEV71-derived small RNAs with canonical siRNA properties in cells and mice. These virus-derived siRNAs were produced from viral dsRNA replicative intermediates in a Dicer-dependent manner and loaded into AGO, and they were fully active in degrading cognate viral RNAs. Recombinant HEV71 deficient in 3A-mediated RNAi suppression was significantly restricted in human somatic cells and mice, whereas Dicer deficiency rescued HEV71 infection independently of type I interferon response. Thus, RNAi can function as an antiviral immunity, which is induced and suppressed by a human virus, in mammals.

The re-emergence of Zika virus (ZIKV) in the Western Hemisphere has resulted in global public health crisis since 2015. ZIKV preferentially infects and targets human neural progenitor cells (hNPCs) and causes fetal microcephaly upon maternal infection. hNPCs not only play critical roles during fetal brain development, but also persist in adult brain throughout life. Yet the mechanism of innate antiviral immunity in hNPCs remains largely unknown. Here, we show that ZIKV infection triggers the abundant production of virus-derived small interfering RNAs in hNPCs, but not in the more differentiated progenies or somatic cells. Ablation of key RNAi machinery components significantly enhances ZIKV replication in hNPCs. Furthermore, enoxacin, a broad-spectrum antibiotic that is known as an RNAi enhancer, exerts potent anti-ZIKV activity in hNPCs and other RNAi-competent cells. Strikingly, enoxacin treatment completely prevents ZIKV infection and circumvents ZIKV-induced microcephalic phenotypes in brain organoid models that recapitulate human fetal brain development. Our findings highlight the physiological importance of RNAi-mediated antiviral immunity during the early stage of human brain development, uncovering a novel strategy to combat human congenital viral infections through enhancing RNAi.

Structural principles underlying the composition and synergistic mechanisms of protective monoclonal antibody cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic antibody cocktail against SARS-CoV-2. On the basis of our previously identified humanized cross-neutralizing antibody H014, we systematically analyzed a fully human naive antibody library and rationally identified a potent neutralizing antibody partner, P17, which confers effective protection in animal model. Cryo-EM studies dissected the nature of the P17 epitope, which is SARS-CoV-2 specific and distinctly different from that of H014. High-resolution structure of the SARS-CoV-2 spike in complex with H014 and P17, together with functional investigations revealed that in a two-antibody cocktail, synergistic neutralization was achieved by S1 shielding and conformational locking, thereby blocking receptor attachment and viral membrane fusion, conferring high potency as well as robustness against viral mutation escape. Furthermore, cluster analysis identified a hypothetical 3rd antibody partner for further reinforcing the cocktail as pan-SARS-CoVs therapeutics.

Mosquito-borne flaviviruses infect both mammals and mosquitoes. RNA interference (RNAi) has been demonstrated as an anti-flavivirus mechanism in mosquitoes; however, whether and how flaviviruses induce and antagonize RNAi-mediated antiviral immunity in mammals remains unknown. We show that the nonstructural protein NS2A of dengue virus-2 (DENV2) act as a viral suppressor of RNAi (VSR). When NS2A-mediated RNAi suppression was disabled, the resulting mutant DENV2 induced Dicer-dependent production of abundant DENV2-derived siRNAs in differentiated mammalian cells. VSR-disabled DENV2 showed severe replication defects in mosquito and mammalian cells and in mice that were rescued by RNAi deficiency. Moreover, NS2As of multiple flaviviruses act as VSRs in vitro and during viral infection in both organisms. Overall, our findings demonstrate that antiviral RNAi can be induced by flavivirus, while flavivirus uses NS2A as a bona fide VSR to evade RNAi in mammals and mosquitoes, highlighting the importance of RNAi in flaviviral vector-host life cycles.

Flaviviruses pose a significant threat to both animals and humans. Recently, a novel flavivirus, duck Tembusu virus (DTMUV), was identified to be the causative agent of a serious duck viral disease in Asia. Its rapid spread, expanding host range, and uncertain transmission routes have raised substantial concerns regarding its potential threats to nonavian hosts, including humans. Here, we demonstrate that DTMUV is not pathogenic for nonhuman primates and is highly sensitive to mammal type I interferon (IFN) signaling. In vitro assays demonstrated that DTMUV infected and replicated efficiently in various mammalian cell lines. Further tests in mice demonstrated high neurovirulence and the age-dependent neuroinvasiveness of the virus. In particular, the inoculation of DTMUV into rhesus monkeys did not result in either viremia or apparent clinical symptoms, although DTMUV-specific humoral immune responses were detected. Furthermore, we revealed that although avian IFN failed to inhibit DTMUV in avian cells, DTMUV was more sensitive to the antiviral effects of type I interferon than other known human-pathogenic flaviviruses. Knockout of the type I IFN receptor in mice caused apparent viremia, viscerotropic disease, and mortality, indicating a vital role of IFN signaling in protection against DTMUV infection. Collectively, we provide direct experimental evidence that this novel avian-origin DTMUV possesses a limited capability to establish infection in immunocompetent primates due to its decreased antagonistic activity in the mammal IFN system. Furthermore, our findings highlight the potential risk of DTMUV infection in immunocompromised individuals and warrant studies on the cross-species transmission and pathogenesis of this novel flavivirus. IMPORTANCEMosquito-borne flaviviruses comprise a large group of pathogenic and nonpathogenic members. The pathogenic flaviviruses include dengue, West Nile, and Japanese encephalitis viruses, and the nonpathogenic flaviviruses normally persist in a natural cycle and rarely cause disease in humans. A novel flavivirus, DTMUV (also known as duck egg drop syndrome flavivirus [DEDSV]) was identified in 2012 in ducks and then rapidly spread to several Asian countries. This new flavivirus was then shown to infect multiple avian species, resulting in neurological symptoms with unknown routes of transmission. There is public concern regarding its potential transmission from birds to humans and other nonavian hosts. Our present study shows that the mammalian IFN system can efficiently eliminate DTMUV infection and that the emergence of severe DTMUV-associated disease in mammals, especially humans, is unlikely. Currently, DTMUV infection mostly affects avian species.T he genus Flavivirus of the family Flaviviridae contains emerging and reemerging enveloped RNA viruses that cause serious diseases in humans and animals, including dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and tick-borne encephalitis virus (TBEV). A group ...

Japanese encephalitis (JE) caused by Japanese encephalitis virus (JEV) poses a serious threat to the world’s public health yet without a cure. Certain JEV-infected neural cells express a subset of previously identified intrinsic antiviral interferon stimulated genes (ISGs), indicating brain cells retain autonomous antiviral immunity. However, whether this happens in composited brain remains unclear. Human pluripotent stem cell (hPSC)-derived organoids can model disorders caused by human endemic pathogens such as Zika virus, which may potentially address this question and facilitate the discovery of a cure for JE. We thus generated telencephalon organoid and infected them with JEV. We found JEV infection caused significant decline of cell proliferation and increase of cell death in brain organoid, resulting in smaller organoid spheres. JEV tended to infect astrocytes and neural progenitors, especially the population representing outer radial glial cells (oRGCs) of developing human brain. In addition, we revealed variable antiviral immunity in brain organoids of different stages of culture. In organoids of longer culture (older than 8 weeks), but not of early ones (less than 4 weeks), JEV infection caused typical activation of interferon signaling pathway. Preferential infection of oRGCs and differential antiviral response at various stages might explain the much more severe outcomes of JEV infection in the younger, which also provide clues to develop effective therapeutics of such diseases.

Development of bio-therapeutics has exhibited exponential growth in China over the past decade. However, no biosimilar drug has been approved in China (CN) due to the lack of a national biosimilar regulatory guidance. HLX01, a rituximab biosimilar developed in China under European Medicines Agency biosimilar guidelines and requirements, was the first such drug submitted for regulatory review in China, and it is expected to receive approval there as a biosimilar product. To demonstrate the analytical similarities of HLX01, CN-rituximab (sourced in China but manufactured in Europe) and EUrituximab (sourced and manufactured in Europe), an extensive 3-way physicochemical and functional similarity assessment using a series of orthogonal and state-of-the-art techniques was conducted, following the similarity requirement guidelines recently published by China's Center for Drug Evaluation. The results of the similarity study showed an identical protein amino acid sequence and highly similar primary structures between HLX01 and the reference product (RP) MabThera®, along with high similarities in higher order structures, potency, integrity, purity and impurity profiles, biological and immunological binding functions, as well as degradation behaviors under stress conditions. In addition, HLX01 presented slightly lower aggregates and better photostability compared with the RP. Despite slight changes in relative abundance of glycan moieties and heavy chain C-terminal lysine modification, no differences in biological activities and immunological properties were observed between the RP and HLX01. In conclusion, HLX01 is highly similar to CN-and EU-sourced RP in terms of physicochemical properties and biological activities, suggesting similar product quality, efficacy, and safety. The regulatory requirements interpreted and applied towards the HLX01 marketing application sets a precedent for analytical similarity assessment of biosimilar products in China.

Background A biosimilar needs to demonstrate its similarity to the originator reference product (RP) in terms of structural and functional properties as well as nonclinical and clinical outcomes. Objectives The aim was to assess the analytical similarity between the trastuzumab biosimilar HLX02 and Europe-sourced Herceptin ® (EU-Herceptin ® ) and China-sourced Herceptin ® (CN-Herceptin ® ) following a quality-by-design (QbD) quality study and tier-based quality attribute evaluation. Methods A panel of highly sensitive and orthogonal methods, including a novel Fc gamma receptor IIIa (FcγRIIIa) affinity chromatography technique that enables quantitative comparison of glycan effects on effector function, was developed for the assessment. To ensure the full product variability was captured, ten batches of HLX02 were compared with 39 RP batches with expiry dates from August 2017 to March 2021. Results The extensive three-way similarity assessment demonstrated that HLX02 is highly similar to the RPs. Furthermore, the %afucose, %galactose, and FcγRIIIa affinity of the RPs were observed to first decrease and then return to the original level in relation to their expiry dates, and the RP batches can be subgrouped by their FcγRIIIa affinity chromatograms. HLX02 is demonstrated to be more similar to the RPs of the high FcγRIIIa affinity group. Conclusion Besides having an overall high analytical similarity to both EU-Herceptin ® and CN-Herceptin ® , HLX02 is more similar to Herceptin ® with high FcγRIIIa affinity, a result that demonstrates the power of the novel FcγRIIIa affinity chromatography technology in biosimilarity evaluation.