Efficient and productive virus infection often requires viral countermeasures that block innate immunity. The IFN-inducible 2′,5′-oligoadenylate (2-5A) synthetases (OASs) and ribonuclease (RNase) L are components of a potent host antiviral pathway. We previously showed that murine coronavirus (MHV) accessory protein ns2, a 2H phosphoesterase superfamily member, is a phosphodiesterase (PDE) that cleaves 2-5A, thereby preventing activation of RNase L. The PDE activity of ns2 is required for MHV replication in macrophages and for hepatitis. Here, we show that group A rotavirus (RVA), an important cause of acute gastroenteritis in children worldwide, encodes a similar PDE. The RVA PDE forms the carboxy-terminal domain of the minor core protein VP3 (VP3-CTD) and shares sequence and predicted structural homology with ns2, including two catalytic HxT/S motifs. Bacterially expressed VP3-CTD exhibited 2′,5′-PDE activity, which cleaved 2-5A in vitro. In addition, VP3-CTD expressed transiently in mammalian cells depleted 2-5A levels induced by OAS activation with poly(rI):poly(rC), preventing RNase L activation. In the context of recombinant chimeric MHV expressing inactive ns2, VP3-CTD restored the ability of the virus to replicate efficiently in macrophages or in the livers of infected mice, whereas mutant viruses expressing inactive VP3-CTD (H718A or H798R) were attenuated. In addition, chimeric viruses expressing either active ns2 or VP3-CTD, but not nonfunctional equivalents, were able to protect ribosomal RNA from RNase L–mediated degradation. Thus, VP3-CTD is a 2′,5′-PDE able to functionally substitute for ns2 in MHV infection. Remarkably, therefore, two disparate RNA viruses encode proteins with homologous 2′,5′-PDEs that antagonize activation of innate immunity.

As a neurotropic virus, human Enterovirus 71 (EV71) infection causes hand-foot-and-mouth disease (HFMD) and may develop severe neurological disorders in infants. Toll-like receptor 7 (TLR7) acts as an innate immune receptor and is also a death receptor in the central nervous system (CNS). However, the mechanisms underlying the regulation of TLR7-mediated brain pathogenesis upon EV71 infection remain largely elusive. Here we reveal a novel mechanism by which EV71 infects astrocytes in the brain and induces neural pathogenesis via TLR7 and interleukin-6 (IL-6) in C57BL/6 mice and in human astroglioma U251 cells. Upon EV71 infection, wild-type (WT) mice displayed more significant body weight loss, higher clinical scores, and lower survival rates as compared with TLR7^-/- mice. In the cerebral cortex of EV71-infected mice, neurofilament integrity was disrupted, and inflammatory cell infiltration and neurodegeneration were induced in WT mice, whereas these were largely absent in TLR7^-/- mice. Similarly, IL-6 production, Caspase-3 cleavage, and cell apoptosis were significantly higher in EV71-infected WT mice as compared with TLR7^-/- mice. Moreover, EV71 preferentially infected and induced IL-6 in astrocytes of mice brain. In U251 cells, EV71-induced IL-6 production and cell apoptosis were suppressed by shRNA-mediated knockdown of TLR7 (shTLR7). Moreover, in the cerebral cortex of EV71-infected mice, the blockade of IL-6 with anti-IL-6 antibody (IL-6-Ab) restored the body weight loss, attenuated clinical scores, improved survival rates, reduced the disruption of neurofilament integrity, decreased cell apoptotic induction, and lowered levels of Caspase-3 cleavage. Similarly, in EV71-infected U251 cells, IL-6-Ab blocked EV71-induced IL-6 production and cell apoptosis in response to viral infection. Collectively, it’s exhibited TLR7 upregulation, IL-6 induction and astrocytic cell apoptosis in EV71-infected human brain. Taken together, we propose that EV71 infects astrocytes of the cerebral cortex in mice and human and triggers TLR7 signaling and IL-6 release, subsequently inducing neural pathogenesis in the brain. Enterovirus 71 (EV71) infection causes aseptic meningitis, poliomyelitis-like paralysis and fatal encephalitis in infants. Besides an immune receptor, toll-like receptor 7 (TLR7) serves as a death receptor in central nervous system (CNS). However, the role of TLR7 in EV71-induced neural pathogenesis remains ambiguous. This study reveals a distinct mechanism by which EV71 induces neurodegeneration via TLR7 and interleukin-6 (IL-6). Upon EV71 infection, TLR7^-/- mice displayed less body weight loss, lower clinical score, and higher survival rate as compared with wild-type (WT) mice. Meanwhile, a severer histopathologic neurofilaments disruption, neurodegeneration and cell apoptosis were observed in brain of EV71-infected WT mice. IL-6 release, cell apoptosis, and Caspase-3 cleavage were attenuated by shRNA targeting TLR7 (shTLR7) in EV71-infected U251 cells. Moreover, anti-IL-6 antibody (IL-6-Ab) suppressed EV71-induced body weight loss, clinical score increase, and survival rate decrease as well as neurofilaments disruption and neurodegeneration in mice, and it also attenuated EV71-induced cell apoptosis and Caspase-3 cleavage in U251 cells. It’s retrospectively observed that TLR7 upregulation, IL-6 induction and astrocytic cell apoptosis in EV71-infected human brain. Therefore, TLR7 is required for neural pathogenesis by IL-6 induction upon EV71 infection.

Several recent attempts to improve the stability and immunogenicity of messenger RNA (mRNA) vaccines include the use of circular RNA (circRNA), targeted delivery, and lyophilization. However, these research directions have often been pursued independently, ignoring the impact of the modification of lipid nanoparticle-encapsulated mRNA vaccines on their targeted delivery after lyophilization and on their subsequent immunogenicity. Here, we develop a circRNA vaccine targeting lymph nodes that expresses rabies virus glycoprotein (G), termed circRNA-G. Mannose modification is introduced directly into the process of synthesizing PEG lipids, and the resulting PEG-mannose can be used in the preparation of mannose-LNPs (mLNPs) that target dendritic cells, thereby promoting the specific distribution of circRNA-G to lymph nodes (mLNP-circRNA-G). We demonstrated that mLNP-circRNA-G has continuous antigen availability that promotes the generation of T follicular helper cells, germinal center B cells, long-lived plasma cells, and memory B cells in mice. Importantly, the vaccine with this targeting modification remained stable for at least 24 weeks of storage at 4℃ after lyophilization, and its immunogenicity was also maintained. Notably, this strategy also enhances the antibody production of the SARS-CoV-2 trimeric receptor-binding domain circRNA vaccine and the stability of immunogenicity after lyophilization. In summary, this study provides a general platform for the design of lyophilized vaccines with targeted stability, demonstrating the potential of lymph node-targeting circRNAs as next-generation vaccines. IMPORTANCE: messenger RNA (mRNA) vaccines are a key technology in combating existing and emerging infectious diseases. However, the inherent instability of mRNA and the nonspecificity of lipid nanoparticle-encapsulated (LNP) delivery systems result in the need for cold storage and a relatively short-duration immune response to mRNA vaccines. Herein, we develop a novel vaccine in the form of circRNAs encapsulated in LNPs, and the circular structure of the circRNAs enhances their stability. Lyophilization is considered the most effective method for the long-term preservation of RNA vaccines. However, this process may result in irreversible damage to the nanoparticles, particularly the potential disruption of targeting modifications on LNPs. During the selection of lymph node-targeting ligands, we found that LNPs modified with mannose maintained their physical properties almost unchanged after lyophilization. Additionally, the targeting specificity and immunogenicity remained unaffected. In contrast, even with the addition of cryoprotectants such as sucrose, the physical properties of LNPs were impaired, leading to an obvious decrease in immunogenicity. This may be attributed to the protective role of mannose on the surface of LNPs during lyophilization. Freshly prepared and lyophilized mLNP-circRNA vaccines elicited comparable immune responses in both the rabies virus model and the SARS-CoV-2 model. Our data demonstrated that mLNP-circRNA vaccines elicit robust immune responses while improving stability after lyophilization, with no compromise in tissue targeting specificity. Therefore, mannose-modified LNP-circRNA vaccines represent a promising vaccine design strategy.

It was found previously that induction of innate immunity, particularly chemokines, is an important mechanism of rabies virus (RABV) attenuation. To evaluate the effect of overexpression of chemokines on RABV infection, chemokines macrophage inflammatory protein 1α (MIP-1α), RANTES, and IP-10 were individually cloned into the genome of attenuated RABV strain HEP-Flury. These recombinant RABVs were characterized in vitro for growth properties and expression of chemokines. It was found that all the recombinant viruses grew as well as the parent virus, and each of the viruses expressed the intended chemokine in a dose-dependent manner. When these viruses were evaluated for pathogenicity in the mouse model, it was found that overexpression of MIP-1α further decreased RABV pathogenicity by inducing a transient innate immune response. In contrast, overexpression of RANTES or IP-10 increased RABV pathogenicity by causing neurological diseases, which is due to persistent and high-level expression of chemokines, excessive infiltration and accumulation of inflammatory cells in the central nervous system, and severe enhancement of blood-brain barrier permeability. These studies indicate that overexpression of chemokines, although important in controlling virus infection, may not always be beneficial to the host.