A bivalent Adenovirus-Vectored Vaccine induces a robust humoral response, but does not protect cynomolgus macaques against a lethal challenge with Sudan virus.

The most recent Sudan virus (SUDV) outbreak in Uganda was first detected in September 2022 and resulted in 164 laboratory-confirmed cases and 77 deaths. There are no approved vaccines against SUDV. Here, we investigated the protective efficacy of ChAdOx1-biEBOV in cynomolgus macaques using a prime or a prime-boost regimen. ChAdOx1-biEBOV is a replication-deficient simian adenovirus vector encoding SUDV and Ebola virus (EBOV) glycoproteins (GPs). Intramuscular vaccination induced SUDV and EBOV GP-specific IgG responses and neutralizing antibodies. Upon challenge with SUDV, vaccinated animals showed signs of disease like those observed in control animals, and no difference in survival outcomes were measured among all three groups. Viral load in blood samples and in tissue samples obtained after necropsy were not significantly different between groups. Overall, this study highlights the importance of evaluating vaccines in multiple animal models and demonstrates the importance of understanding protective efficacy in both animal models and human hosts.

Pteropus vampyrus TRIM40 Is an Interferon-Stimulated Gene That Antagonizes RIG-I-like Receptors.

Nipah virus (NiV; genus: Henipavirus; family: Paramyxoviridae) naturally infects Old World fruit bats (family Pteropodidae) without causing overt disease. Conversely, NiV infection in humans and other mammals can be lethal. Comparing bat antiviral responses with those of humans may illuminate the mechanisms that facilitate bats' tolerance. Tripartite motif proteins (TRIMs), a large family of E3-ubiquitin ligases, fine-tune innate antiviral immune responses, and two human TRIMs interact with Henipavirus proteins. We hypothesize that NiV infection induces the expression of an immunosuppressive TRIM in bat, but not human cells, to promote tolerance. Here, we show that TRIM40 is an interferon-stimulated gene (ISG) in pteropodid but not human cells. Knockdown of bat TRIM40 increases gene expression of IFNß, ISGs, and pro-inflammatory cytokines following poly(I:C) transfection. In Pteropus vampyrus, but not human cells, NiV induces TRIM40 expression within 16 h after infection, and knockdown of TRIM40 correlates with reduced NiV titers as compared to control cells. Bats may have evolved to express TRIM40 in response to viral infections to control immunopathogenesis.

The RNA helicase DHX16 recognizes specific viral RNA to trigger RIG-I-dependent innate antiviral immunity.

Type I interferons (IFN-I) are essential to establish antiviral innate immunity. Unanchored (or free) polyubiquitin (poly-Ub) has been shown to regulate IFN-I responses. However, few unanchored poly-Ub interactors are known. To identify factors regulated by unanchored poly-Ub in a physiological setting, we developed an approach to isolate unanchored poly-Ub from lung tissue. We identified the RNA helicase DHX16 as a potential pattern recognition receptor (PRR). Silencing of DHX16 in cells and in vivo diminished IFN-I responses against influenza virus. These effects extended to members of other virus families, including Zika and SARS-CoV-2. DHX16-dependent IFN-I production requires RIG-I and unanchored K48-poly-Ub synthesized by the E3-Ub ligase TRIM6. DHX16 recognizes a signal in influenza RNA segments that undergo splicing and requires its RNA helicase motif for direct, high-affinity interactions with specific viral RNAs. Our study establishes DHX16 as a PRR that partners with RIG-I for optimal activation of antiviral immunity requiring unanchored poly-Ub.

SARS-CoV-2 proteases PLpro and 3CLpro cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species.

The genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory responThe genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory response observed in COVID-19 patients. We demonstrate that in the mouse NLRP12 protein, one of the recognition site is not cleaved in our in-vitro assay. We pushed this comparative alignment of IRF-3 and NLRP12 homologs and show that the lack or presence of cognate cleavage motifs in IRF-3 and NLRP12 could contribute to the presentation of disease in cats and tigers, for example. Our findings provide an explanatory framework for indepth studies into the pathophysiology of COVID-19.

Envelope protein ubiquitination drives entry and pathogenesis of Zika virus.

Zika virus (ZIKV) belongs to the family Flaviviridae, and is related to other viruses that cause human diseases. Unlike other flaviviruses, ZIKV infection can cause congenital neurological disorders and replicates efficiently in reproductive tissues1-3. Here we show that the envelope protein (E) of ZIKV is polyubiquitinated by the E3 ubiquitin ligase TRIM7 through Lys63 (K63)-linked polyubiquitination. Accordingly, ZIKV replicates less efficiently in the brain and reproductive tissues of Trim7-/- mice. Ubiquitinated E is present on infectious virions of ZIKV when they are released from specific cell types, and enhances virus attachment and entry into cells. Specifically, K63-linked polyubiquitin chains directly interact with the TIM1 (also known as HAVCR1) receptor of host cells, which enhances virus entry in cells as well as in brain tissue in vivo. Recombinant ZIKV mutants that lack ubiquitination are attenuated in human cells and in wild-type mice, but not in live mosquitoes. Monoclonal antibodies against K63-linked polyubiquitin specifically neutralize ZIKV and reduce viraemia in mice. Our results demonstrate that the ubiquitination of ZIKV E is an important determinant of virus entry, tropism and pathogenesis.