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Lyssaviruses cause rabies, which is an acute neurological disease responsible for more than 59,000 human deaths annually and has no available effective treatments. The phosphoprotein (P) of lyssaviruses (lyssavirus-P) plays multiple roles in virus replication and immune evasion. Lyssavirus-P has been identified as the major type I interferon (IFN-I) antagonist, while the precise site and precise molecular mechanism remain unclear. Herein, we found that substitution of site 179 of lyssavirus-P from serine (Ser) to proline (Pro) impairs its antagonism function of IFN-I by sequence alignment and site mutations. Subsequent studies demonstrated that lyssavirus-P containing S179 specifically interacted with I-kappa B kinase ε (IKKε). Specifically, lyssavirus-P containing S179 interacted simultaneously with the kinase domain (KD) and scaffold dimerization domain (SDD) of IKKε, competing with TNF receptor-associated factor 3 (TRAF3) and IFN regulatory factor 3 (IRF3) for binding with IKKε, leading to the inhibition of IFN production. Furthermore, S179 was involved in the viral pathogenicity of the typical lyssavirus rabies virus in a mouse model. Interestingly, we found that S179 is conserved among most lyssavirus-P and functional for IFN antagonism. Collectively, we identified S179 of lyssavirus-P is essential for IFN-I inhibition, which provides deep insight into the immune evasion strategies of lyssaviruses. IMPORTANCE Interferon (IFN) and the IFN-induced cellular antiviral response constitute the first line of defense against viral invasion. Evading host innate immunity, especially IFN signaling, is the key step required for lyssaviruses to establish infection. In this study, S179 of lyssavirus phosphoprotein (lyssavirus-P) was identified as the key site for antagonizing IFN-I production. Mechanistically, lyssavirus-P containing S179 specifically targets the key kinase IKKε and disrupts its interaction with TRAF3 and IRF3. S179P mutation in the P protein of the typical lyssavirus rabies virus (RABV) attenuated its pathogenicity in a mouse model. Our findings provide deep insight into the immune evasion strategies of lyssaviruses, which is helpful for the development of effective antiviral therapeutics.

Rabies, caused by rabies virus (RABV), is fatal to both humans and animals around the world. Effective clinical therapy for rabies has not been achieved, and vaccination is the most effective means of preventing and controlling rabies. Although different vaccines, such as live attenuated and inactivated vaccines, can induce different immune responses, different expressions of pattern recognition receptors (PRRs) also cause diverse immune responses. Toll-like receptor 4 (TLR4) is a pivotal PRR that induces cytokine production and bridges innate and adaptive immunity. Importantly, TLR4 recognizes various virus-derived pathogen-associated molecular patterns (PAMPs) and virus-induced damage-associated molecular patterns (DAMPs), usually leading to the activation of immune cells. However, the role of TLR4 in the humoral immune response induced by RABV has not yet been revealed. Based on TLR4-deficient (TLR4(−/−)) and wild-type (WT) mouse models, we report that TLR4-dependent recruitment of the conventional type 2 dendritic cells (CD8α(−) CD11b(+) cDC2) into secondary lymph organs (SLOs) is critical for antigen presentation. cDC2-initiated differentiation of follicular helper T (Tfh) cells promotes the proliferation of germinal center (GC) B cells, the formation of GCs, and the production of plasma cells (PCs), all of which contribute to the production of RABV-specific IgG and virus-neutralizing antibodies (VNAs). Collectively, our work demonstrates that TLR4 is necessary for the recruitment of cDC2 and for the induction of RABV-induced humoral immunity, which is regulated by the cDC2-Tfh-GC B axis. IMPORTANCE Vaccination is the most efficient method to prevent rabies. TLR4, a well-known immune sensor, plays a critical role in initiating innate immune response. Here, we found that TLR4-deficient (TLR4(−/−)) mice suppressed the induction of humoral immune response after immunization with rabies virus (RABV), including reduced production of VNAs and RABV-specific IgG compared to that occurred in wild-type (WT) mice. As a consequence, TLR4(−/−) mice exhibited higher mortality than that of WT mice after challenge with virulent RABV. Importantly, further investigation found that TLR4 signaling promoted the recruitment of cDC2 (CD8α(+) CD11b(−)), a subset of cDCs known to induce CD4(+) T-cell immunity through their MHC-II presentation machinery. Our results imply that TLR4 is indispensable for an efficient humoral response to rabies vaccine, which provides new insight into the development of novel rabies vaccines.

Rabies is a zoonotic disease caused by the rabies virus (RABV). RABV can lead to fatal encephalitis and is still a serious threat in most parts of the world. Interferon regulatory factor 7 (IRF7) is the main transcriptional regulator of type I IFN, and it is crucial for the induction of IFNα/β and the type I IFN-dependent immune response. In this study, we focused on the role of IRF7 in the pathogenicity and immunogenicity of RABV using an IRF7-/- mouse model. The results showed that the absence of IRF7 made mice more susceptible to RABV, because IRF7 restricted the replication of RABV in the early stage of infection. IRF7 deficiency affected the recruitment of plasmacytoid dendritic cells to the draining lymph nodes (dLNs), reduced the production of type I IFN and expression of IFN-stimulated genes. Furthermore, we found that the ability to produce specific RABV-neutralizing antibody was impaired in IRF7-/- mice. Consistently, IRF7 deficiency affected the recruitment of germinal-centre B cells to dLNs, and the generation of plasma cells and RABV-specific antibody secreting cells. Moreover, the absence of IRF7 downregulated the induction of IFN-γ and reduced type 1 T helper cell (Th1)-dependent antibody production. Collectively, our findings demonstrate that IRF7 promotes humoral immune responses and compromises the pathogenicity of RABV in a mouse model.

Rabies is a fatal zoonosis that causes encephalitis in mammals, and vaccination is the most effective method to control and eliminate rabies. Virus-like vesicles (VLVs), which are characterized as infectious, self-propagating membrane-enveloped particles composed of only Semliki Forest virus (SFV) replicase and vesicular stomatitis virus glycoprotein (VSV-G), have been proven safe and efficient as vaccine candidates. However, previous studies showed that VLVs containing rabies virus glycoprotein (RABV-G) grew at relatively low titers in cells, impeding their potential use as a rabies vaccine. In this study, we constructed novel VLVs by transfection of a mutant SFV RNA replicon encoding RABV-G. We found that these VLVs could self-propagate efficiently in cell culture and could evolve to high titers (approximately 10(8) focus-forming units [FFU]/ml) by extensive passaging 25 times in BHK-21 cells. Furthermore, we found that the evolved amino acid changes in SFV nonstructural protein 1 (nsP1) at positions 470 and 482 was critical for this high-titer phenotype. Remarkably, VLVs could induce robust type I interferon (IFN) expression in BV2 cells and were highly sensitive to IFN-α. We found that direct inoculation of VLVs into the mouse brain caused reduced body weight loss, mortality, and neuroinflammation compared with the RABV vaccine strain. Finally, it could induce increased generation of germinal center (GC) B cells, plasma cells (PCs), and virus-neutralizing antibodies (VNAs), as well as provide protection against virulent RABV challenge in immunized mice. This study demonstrated that VLVs containing RABV-G could proliferate in cells and were highly evolvable, revealing the feasibility of developing an economic, safe, and efficacious rabies vaccine. IMPORTANCE VLVs have been shown to represent a more versatile and superior vaccine platform. In previous studies, VLVs containing the Semliki Forest virus replicase (SFV nsP1 to nsP4) and rabies virus glycoprotein (RABV-G) grew to relatively low titers in cells. In our study, we not only succeeded in generating VLVs that proliferate in cells and stably express RABV-G, but the VLVs that evolved grew to higher titers, reaching 10(8) FFU/ml. We also found that nucleic acid changes at positions 470 and 482 in nsP1 were vital for this high-titer phenotype. Moreover, the VLVs that evolved in our studies were highly attenuated in mice, induced potent immunity, and protected mice from lethal RABV infection. Collectively, our study showed that high titers of VLVs containing RABV-G were achieved, demonstrating that these VLVs could be an economical, safe, and efficacious rabies vaccine candidate.

Rabies, caused by rabies virus (RABV), remains a serious threat to public health in most countries worldwide. At present, the administration of rabies vaccines has been the most effective strategy to control rabies. Herein, we evaluate the effect of colloidal manganese salt (Mn jelly [MnJ]) as an adjuvant of rabies vaccine in mice, cats, and dogs. The results showed that MnJ promoted type I interferon (IFN-I) and cytokine production in vitro and the maturation of dendritic cells (DCs) in vitro and in vivo. Besides, MnJ serving as an adjuvant for rabies vaccines could significantly facilitate the generation of T follicular helper (Tfh) cells, germinal center (GC) B cells, plasma cells (PCs), and RABV-specific antibody-secreting cells (ASCs), consequently improve the immunogenicity of rabies vaccines, and provide better protection against virulent RABV challenge. Similarly, MnJ enhanced the humoral immune response in cats and dogs as well. Collectively, our results suggest that MnJ can facilitate the maturation of DCs during rabies vaccination, which can be a promising adjuvant candidate for rabies vaccines. IMPORTANCE Extending the humoral immune response by using adjuvants is an important strategy for vaccine development. In this study, a novel adjuvant, MnJ, supplemented in rabies vaccines was evaluated in mice, cats, and dogs. Our results in the mouse model revealed that MnJ increased the numbers of mature DCs, Tfh cells, GC B cells, PCs, and RABV-specific ASCs, resulting in enhanced immunogenicity and protection rate of rabies vaccines. We further found that MnJ had the same stimulative effect in cats and dogs. Our study provides the first evidence that MnJ serving as a novel adjuvant of rabies vaccines can boost the immune response in both a mouse and pet model.

Cholesterol-25-hydroxylase (CH25H) is a reticulum-associated membrane protein that catalyzes the oxidation of cholesterol to 25-hydroxycholesterol (25HC). Recent studies have revealed that CH25H is an interferon-stimulated gene (ISG) that suppresses infection by several viruses. In the present study, we found that overexpression of both human and murine CH25H inhibited rabies virus (RABV) infection in HEK-293T (293T) cells. In contrast, silencing of CH25H enhanced RABV replication in 293T cells, and a catalytic mutant of CH25H lost its ability to inhibit RABV infection. Treatment with the oxysterol 25-hydroxycholesterol (25HC), the product of CH25H, dramatically decreased RABV replication in 293T, BSR and N2a cells by inhibiting viral membrane penetration. These data provide insights into the antiviral function of CH25H against RABV infection, which can potentially be used as a therapeutic agent for rabies.

Rabies, as one of the most threatening zoonoses in the world, causes a fatal central nervous system (CNS) disease. So far, vaccination with rabies vaccines has been the most effective measure to prevent and control this disease. At present, inactivated rabies vaccines are widely used in humans and domestic animals. However, humoral immune responses induced by inactivated rabies vaccines are relatively low and multiple shots are required to achieve protective immunity. Supplementation with an adjuvant is a practical way to improve the immunogenicity of inactivated rabies vaccines. In this study, we found that monophosphoryl-lipid A (MPLA), a well-known TLR4 agonist, could significantly promote the maturation of bone marrow-derived dendritic cells (BMDC) through a TLR4-dependent pathway in vitro and the maturation of conventional DCs (cDCs) in vivo. We also found that MPLA, serving as an adjuvant for inactivated rabies vaccines, could significantly facilitate the generation of T follicular helper (Tfh) cells, germinal center (GC) B cells, and plasma cells (PCs), consequently enhancing the production of RABV-specific total-IgG, IgG2a, IgG2b, and the virus-neutralizing antibodies (VNAs). Furthermore, MPLA could increase the survival ratio of mice challenged with virulent RABV. In conclusion, our results demonstrate that MPLA serving as an adjuvant enhances the intensity of humoral immune responses by activating the cDC&ndash, Tfh&ndash, GC B axis. Our findings will contribute to the improvement of the efficiency of traditional rabies vaccines.