Min Cui

MicroRNA-19b-3p Modulates Japanese Encephalitis Virus-Mediated Inflammation via Targeting RNF11

ABSTRACT Japanese encephalitis virus (JEV) can invade the central nervous system and consequently induce neuroinflammation, which is characterized by profound neuronal cell damage accompanied by astrogliosis and microgliosis. Albeit microRNAs (miRNAs) have emerged as major regulatory noncoding RNAs with profound effects on inflammatory response, it is unknown how astrocytic miRNAs regulate JEV-induced inflammation. Here, we found the involvement of miR-19b-3p in regulating the JEV-induced inflammatory response in vitro and in vivo. The data demonstrated that miR-19b-3p is upregulated in cultured cells and mouse brain tissues during JEV infection. Overexpression of miR-19b-3p led to increased production of inflammatory cytokines, including tumor necrosis factor alpha, interleukin-6, interleukin-1β, and chemokine (C-C motif) ligand 5, after JEV infection, whereas knockdown of miR-19b-3p had completely opposite effects. Mechanistically, miR-19b-3p modulated the JEV-induced inflammatory response via targeting ring finger protein 11, a negative regulator of nuclear factor kappa B signaling. We also found that inhibition of ring finger protein 11 by miR-19b-3p resulted in accumulation of nuclear factor kappa B in the nucleus, which in turn led to higher production of inflammatory cytokines. In vivo silencing of miR-19b-3p by a specific antagomir reinvigorates the expression level of RNF11, which in turn reduces the production of inflammatory cytokines, abrogates gliosis and neuronal cell death, and eventually improves the survival rate in the mouse model. Collectively, our results demonstrate that miR-19b-3p positively regulates the JEV-induced inflammatory response. Thus, miR-19b-3p targeting may constitute a thought-provoking approach to rein in JEV-induced inflammation. IMPORTANCE Japanese encephalitis virus (JEV) is one of the major causes of acute encephalitis in humans worldwide. The pathological features of JEV-induced encephalitis are inflammatory reactions and neurological diseases resulting from glia activation. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally. Accumulating data indicate that miRNAs regulate a variety of cellular processes, including the host inflammatory response under pathological conditions. Recently, a few studies demonstrated the role of miRNAs in a JEV-induced inflammatory response in microglia; however, their role in an astrocyte-derived inflammatory response is largely unknown. The present study reveals that miR-19b-3p targets ring finger protein 11 in glia and promotes inflammatory cytokine production by enhancing nuclear factor kappa B activity in these cells. Moreover, administration of an miR-19b-3p-specific antagomir in JEV-infected mice reduces neuroinflammation and lethality. These findings suggest a new insight into the molecular mechanism of the JEV-induced inflammatory response and provide a possible therapeutic entry point for treating viral encephalitis.

Recombinant Rabies Viruses Expressing GM-CSF or Flagellin Are Effective Vaccines for Both Intramuscular and Oral Immunizations

Our previous studies indicated that recombinant rabies viruses (rRABV) expressing chemokines or cytokines (including GM-CSF) could enhance the immunogenicity by recruiting and/or activating dendritic cells (DC). In this study, bacterial flagellin was cloned into the RABV genome and recombinant virus LBNSE-Flagellin was rescued. To compare the immunogenicity of LBNSE-Flagellin with recombinant virus expressing GMCSF (LBNSE-GMCSF), mice were immunized with each of these rRABVs by intramuscular (i.m.) or oral route. The parent virus (LBNSE) without expression of any foreign molecules was included for comparison. The i.m.-immunized mice were bled at three weeks after the immunization for the measurement of virus neutralizing antibody (VNA) and then challenged with 50 LD50 challenge virus standard (CVS-24). Orally immunized mice were boosted after three weeks and then bled and challenged one week after the booster immunization. It was found that both LBNSE-GMCSF and LBNSE-Flagellin recruited/activated more DCs and B cells in the periphery, stimulated higher levels of adaptive immune responses (VNA), and protected more mice against challenge infection than the parent virus LBNSE in both the i.m. and the orally immunized groups. Together, these studies suggest that recombinant RABV expressing GM-CSF or flagellin are more immunogenic than the parent virus in both i.m. and oral immunizations.

Recombinant rabies virus expressing dog GM-CSF is an efficacious oral rabies vaccine for dogs

Developing efficacious oral rabies vaccines is an important step to increase immunization coverage for stray dogs, which are not accessible for parenteral vaccination. Our previous studies have demonstrated that recombinant rabies virus (RABV) expressing cytokines/chemokines induces robust protective immune responses after oral immunization in mice by recruiting and activating dendritic cells (DCs) and B cells. To develop an effective oral rabies vaccine for dogs, a recombinant attenuated RABV expressing dog GM-CSF, designated as LBNSE-dGM-CSF was constructed and used for oral vaccination in a dog model. Significantly more DCs or B cells were activated in the peripheral blood of dogs vaccinated orally with LBNSE-dGM-CSF than those vaccinated with the parent virus LBNSE, particularly at 3 days post immunization (dpi). As a result, significantly higher levels of virus neutralizing antibodies (VNAs) were detected in dogs immunized with LBNSE-dGM-CSF than with the parent virus. All the immunized dogs were protected against a lethal challenge with 4500 MICLD50 of wild-type RABV SXTYD01. LBNSE-dGM-CSF was found to replicate mainly in the tonsils after oral vaccination as detected by nested RT-PCR and immunohistochemistry. Taken together, our results indicate that LBNSE-dGM-CSF could be a promising oral rabies vaccine candidate for dogs.

Comparison of complete genome sequences of dog rabies viruses isolated from China and Mexico reveals key amino acid changes that may be associated with virus replication and virulence

Rabies is a global problem, but its impact and prevalence vary across different regions. In some areas, such as parts of Africa and Asia, the virus is prevalent in the domestic dog population, leading to epidemic waves and large numbers of human fatalities. In other regions, such as the Americas, the virus predominates in wildlife and bat populations, with sporadic spillover into domestic animals. In this work, we attempted to investigate whether these distinct environments led to selective pressures that result in measurable changes within the genome at the amino acid level. To this end, we collected and sequenced the full genome of two isolates from divergent environments. The first isolate (DRV-AH08) was from China, where the virus is present in the dog population and the country is experiencing a serious epidemic. The second isolate (DRV-Mexico) was taken from Mexico, where the virus is present in both wildlife and domestic dog populations, but at low levels as a consequence of an effective vaccination program. We then combined and compared these with other full genome sequences to identify distinct amino acid changes that might be associated with environment. Phylogenetic analysis identified strain DRV-AH08 as belonging to the China-I lineage, which has emerged to become the dominant lineage in the current epidemic. The Mexico strain was placed in the D11 Mexico lineage, associated with the West USA-Mexico border clade. Amino acid sequence analysis identified only 17 amino acid differences in the N, G and L proteins. These differences may be associated with virus replication and virulence–for example, the short incubation period observed in the current epidemic in China.

TLR7 Deficiency Leads to TLR8 Compensative Regulation of Immune Response against JEV in Mice

Japanese encephalitis virus (JEV) is a highly fatal pathogen to human beings. Toll-like receptor 7 (TLR7) plays a role as the first host defense against most single-stranded RNA flaviviruses. This study aims to investigate the role of TLR7 in inducing adaptive immune response in mice against JEV. In vitro and in vivo studies were conducted to examine the expression of toll-like receptors (TLRs) in mice. After JEV infection, physical parameters of mice (survival rate and body weight) were evaluated, and organs or cells were collected for further analysis. The expression of TLR7 was increased significantly as compare to other TLR molecules post-JEV infection. The expression of CD80, CD86, and CD273 on bone marrow-derived dendritic cells was increased significantly in TLR7−/− mice. Furthermore, viral load was also increased significantly in TLR7−/− mice as compare to C57BL/6 mice. But there was no significant difference among survival rate and body weight in TLR7−/− mice as compare to C57BL/6. Interestingly, we also found that TLR8 was upregulated in TLR7−/− mice. The study concluded that TLR8 was upregulated in TLR7-deficient mice, and it might play a compensatory role in the immune response in TLR7−/− mice.

A Novel Rabies Vaccine Expressing CXCL13 Enhances Humoral Immunity by Recruiting both T Follicular Helper and Germinal Center B Cells

ABSTRACT Rabies remains a public health threat in most parts of the world, and approximately 99% of the cases are transmitted by dogs. There is an urgent need to develop an efficacious and affordable vaccine to control canine-transmitted rabies in developing countries. Our previous studies demonstrate that overexpression of chemokines/cytokines such as CCL-3 (MIP-1α) and granulocyte-macrophage colony-stimulating factor (GM-CSF) can enhance the immunogenicity of rabies vaccines. In the present study, the chemokine CXCL13 was inserted into the genome of the recombinant rabies virus (rRABV) strain LBNSE, and the effect of the chemokine CXCL13 on the immunogenicity of RABV was investigated. It was found that LBNSE-CXCL13 recruited follicular helper T (Tfh) and germinal center (GC) B cells, promoted the formation of GCs, and increased the population of plasma cells in immunized mice. Further studies showed that mice immunized with LBNSE-CXCL13 produced more rabies virus-neutralizing antibodies (VNAs) and developed better protection than those immunized with the parent virus LBNSE or the GM-CSF-expressing RABV (LBNSE-GM-CSF). Collectively, these findings provide a better understanding of the role of CXCL13 expression in the immunogenicity of the RABV, which may help in designing more-efficacious rabies vaccines. IMPORTANCE Rabies is endemic in most parts of the world, and more effort is needed to develop affordable and effective vaccines to control or eliminate this disease. The chemokine CXCL13 recruits both Tfh and B cells, which is essential for the homing of Tfh cells and the development of B cell follicles. In this study, the effect of the overexpression of CXCL13 on the immunogenicity of the RABV was evaluated in a mouse model. We found that CXCL13 expression promoted humoral immunity by recruiting Tfh and GC B cells, facilitating the formation of GCs, and increasing the number of plasma cells. As expected, the overexpression of CXCL13 resulted in enhanced virus-neutralizing antibody (VNA) production and protection against a virulent RABV challenge. These findings provide a better understanding of the role of CXCL13 in RABV-induced immune responses, which will help in designing more efficacious rabies vaccines.

Myeloid-Derived Suppressor Cells Inhibit T Follicular Helper Cell Immune Response in Japanese Encephalitis Virus Infection

Resolution of viral infections requires activation of innate cells to initiate and maintain adaptive immune responses. In this study, we examined Japanese encephalitis virus (JEV) infection leading to acute encephalopathy depending on suppression of the adaptive immune responses mediated by innate cells. Infection with P3 strains of JEV enhanced myeloid-derived suppressor cell (MDSC) populations, and the survival rate of JEV-infected mice improved after MDSC depletion. Mechanically, P3-induced MDSCs suppressed CD4+ T cell immune responses, especially responses of T follicular helper (Tfh) cells, leading to decreased splenic B cells (CD19+) and blood plasma cells (CD19+CD138+) and reduced levels of total IgM and JEV-specific neutralizing Abs. Upon depleting P3-induced MDSCs in vivo, the Tfh cell population, B cells, plasma cells, and Ab production recovered. These findings provide unique insights regarding MDSC functions in mediating immune suppression via inhibiting Tfh cell responses and further impairing humoral immunity, which facilitate the progression of infection.

Recombinant rabies virus expressing IL-21 enhances immunogenicity through activation of T follicular helper cells and germinal centre B cells

Previous studies have demonstrated that the lack of interleukin-21 (IL-21) signalling could affect specific antibody induction after rabies vaccination. Here, to further investigate the over-expression of IL-21 on the immunogenicity of rabies virus (RABV), a recombinant RABV expressing murine IL-21, designated LBNSE-IL21, was constructed and evaluated in a mouse model. It was found that in mice immunized with LBNSE-IL21, there was a substantial increase in the number of T follicular helper cells and germinal centre B cells but no enhancement of dendritic cell activation. Furthermore, significantly higher rabies virus-neutralizing antibody (VNA) titres were produced in mice immunized with LBNSE-IL21 than in mice immunized with the parent virus LBNSE in the first six weeks, resulting in higher protection. Together, these results suggest that LBNSE-IL21 can induce a rapid and robust VNA titre, and it has the potential to be developed as a promising rabies vaccine.

Lab-Attenuated Rabies Virus Causes Abortive Infection and Induces Cytokine Expression in Astrocytes by Activating Mitochondrial Antiviral-Signaling Protein Signaling Pathway

Rabies is an ancient disease but remains endemic in most parts of the world and causes approximately 59,000 deaths annually. The mechanism through which the causative agent, rabies virus (RABV), evades the host immune response and infects the host central nervous system (CNS) has not been completely elucidated thus far. Our previous studies have shown that lab-attenuated, but not wild-type (wt), RABV activates the innate immune response in the mouse and dog models. In this present study, we demonstrate that lab-attenuated RABV causes abortive infection in astrocytes, the most abundant glial cells in the CNS. Furthermore, we found that lab-attenuated RABV produces more double-stranded RNA (dsRNA) than wt RABV, which is recognized by retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated protein 5 (MDA5). Activation of mitochondrial antiviral-signaling protein (MAVS), the common adaptor molecule for RIG-I and MDA5, results in the production of type I interferon (IFN) and the expression of hundreds of IFN-stimulated genes, which suppress RABV replication and spread in astrocytes. Notably, lab-attenuated RABV replicates in a manner identical to that of wt RABV in MAVS−/− astrocytes. It was also found that lab-attenuated, but not wt, RABV induces the expression of inflammatory cytokines via the MAVS- p38/NF-κB signaling pathway. These inflammatory cytokines increase the blood–brain barrier permeability and thus enable immune cells and antibodies infiltrate the CNS parenchyma, resulting in RABV control and elimination. In contrast, wt RABV restricts dsRNA production and thus evades innate recognition by RIG-I/MDA5 in astrocytes, which could be one of the mechanisms by which wt RABV evades the host immune response in resident CNS cells. Our findings suggest that astrocytes play a critical role in limiting the replication of lab-attenuated RABV in the CNS.

Recombinant rabies virus expressing IL-15 enhances immunogenicity through promoting the activation of dendritic cells in mice

Rabies remains a public health threat that kills approximately 59,000 people worldwide each year, most of which are from the developing countries of Africa and Asia where dog rabies are endemic. Therefore, developing an affordable and efficacious vaccine is crucial for rabies control in these countries. Interleukin (IL)-15, an immunoregulatory cytokine, is a pluripotent molecule with therapeutic potential, which targets many cell types and links the innate and adaptive immune system. In this study, IL-15 gene was cloned and inserted into the genome of a recombinant rabies virus (RABV) strain LBNSE (designated as LBNSE-IL15), and the effect of over-expression of IL-15 on the immunogenicity of RABV was investigated. It was found that mice vaccinated with LBNSE-IL15 could induce significantly higher level of virus-neutralizing antibody (VNA) than those immunized with LBNSE, resulting in the higher protection after challenge. Further investigation was performed to find out the possible role of IL-15 plays in the process of antibody induction, and it was found that LBNSE-IL15 could enhance the maturation of dendritic cells (DCs) in immunized mice. Furthermore, the mice immunized with LBNSE-IL15 could promote the TFH cells differentiation and the generation of germinal center B cells and plasma cells. Together, these data indicated that IL-15 could be a potential adjuvant in enhancing the immunogenicity of RABV, contributing to the development of more-efficacious rabies vaccines.