A Novel System for Rapid and Cost-Effective Production of Detection and Diagnostic Reagents of West Nile Virus in Plants

The threat of West Nile virus (WNV) epidemics necessitates the development of a technology platform that can produce reagents to support detection and diagnosis rapidly and inexpensively. A plant expression system is attractive for protein production due to its low-cost and high-scalability nature and its ability to make appropriate posttranslational modifications. Here, we investigated the feasibility of using plants to produce two WNV detection and diagnostic reagents to address the current cost and scalability issues. We demonstrated that WNV DIII antigen and E16 monoclonal antibody are rapidly produced at high levels in two plant species and are easily purified. Furthermore, they are effective in identifying WNV and in detecting human IgM response to WNV infection. E16 mAb does not cross-react with other flaviviruses, therefore, is valuable for improving diagnostic accuracy. This study provides a proof of principle for using plants as a robust and economical system to produce diagnostic reagents for arboviruses

A Plant-Produced Vaccine Protects Mice Against Lethal West Nile Virus Infection Without Enhancing Zika or Dengue Virus Infectivity

West Nile virus (WNV) has caused multiple global outbreaks with increased frequency of neuroinvasive disease in recent years. Despite many years of research, there are no licensed therapeutics or vaccines available for human use. One of the major impediments of vaccine development against WNV is the potential enhancement of infection by related flaviviruses in vaccinated subjects through the mechanism of antibody-dependent enhancement of infection (ADE). For instance, the recent finding of enhancement of Zika virus (ZIKV) infection by pre-exposure to WNV further complicates the development of WNV vaccines. Epidemics of WNV and the potential risk of ADE by current vaccine candidates demand the development of effective and safe vaccines. We have previously reported that the domain III (DIII) of the WNV envelope protein can be readily expressed in Nicotiana benthamiana leaves, purified to homogeneity, and promote antigen-specific antibody response in mice. Herein, we further investigated the in vivo potency of a plant-made DIII (plant-DIII) in providing protective immunity against WNV infection. Furthermore, we examined if vaccination with plant-DIII would enhance the risk of a subsequent infection by ZIKV and Dengue virus (DENV). Plant-DIII vaccination evoked antigen-specific cellular immune responses as well as humoral responses. DIII-specific antibodies were neutralizing and the neutralization titersmet the threshold correlated with protective immunity by vaccines against multiple flaviviruses. Furthermore, passive administration of anti-plant DIII mouse serum provided full protection against a lethal challenge of WNV infection in mice. Notably, plant DIII-induced antibodies did not enhance ZIKV and DENV infection in Fc gamma receptor-expressing cells, addressing the concern of WNV vaccines in inducing cross-reactive antibodies and sensitizing subjects to subsequent infection by heterologous flavivirus. This study provides the first report of a WNV subunit vaccine that induces protective immunity, while circumventing induction of antibodies with enhancing activity for ZIKV and DENV infection

Antibody-Dependent Enhancement Activity of a Plant-Made Vaccine Against West Nile Virus

West Nile virus (WNV) causes annual outbreaks globally and is the leading cause of mosquito-borne disease in Unite States. In the absence of licensed therapeutics, there is an urgent need to develop effective and safe human vaccines against WNV. One of the major safety concerns for WNV vaccine development is the risk of increasing infection by related flaviviruses in vaccinated subjects via antibody-dependent enhancement of infection (ADE). Herein, we report the development of a plant-based vaccine candidate that provides protective immunity against a lethal WNV challenge mice, while minimizes the risk of ADE for infection by Zika (ZIKV) and dengue (DENV) virus. Specifically, a plant-produced virus-like particle (VLP) that displays the WNV Envelope protein domain III (wDIII) elicited both high neutralizing antibody titers and antigen-specific cellular immune responses in mice. Passive transfer of serum from VLP-vaccinated mice protected recipient mice from a lethal challenge of WNV infection. Notably, VLP-induced antibodies did not enhance the infection of Fc gamma receptor-expressing K562 cells by ZIKV or DENV through ADE. Thus, a plant-made wDIII-displaying VLP presents a promising WNV vaccine candidate that induces protective immunity and minimizes the concern of inducing ADE-prone antibodies to predispose vaccinees to severe infection by DENV or ZIKV

Impairment of Autophagy After Spinal Cord Injury Potentiates Neuroinflammation and Motor Function Deficit in Mice

Autophagy is a catabolic process that degrades cytoplasmic constituents and organelles in the lysosome, thus serving an important role in cellular homeostasis and protection against insults. We previously reported that defects in autophagy contribute to neuronal cell damage in traumatic spinal cord injury (SCI). Recent data from other inflammatory models implicate autophagy in regulation of immune and inflammatory responses, with low levels of autophagic flux associated with pro-inflammatory phenotypes. In the present study, we examined the effects of genetically or pharmacologically manipulating autophagy on posttraumatic neuroinflammation and motor function after SCI in mice. Methods: Young adult male C57BL/6, CX3CR1-GFP, autophagy hypomorph Becn1+/- mice, and their wildtype (WT) littermates were subjected to moderate thoracic spinal cord contusion. Neuroinflammation and autophagic flux in the injured spinal cord were assessed using flow cytometry, immunohistochemistry, and NanoString gene expression analysis. Motor function was evaluated with the Basso Mouse Scale and horizontal ladder test. Lesion volume and spared white matter were evaluated by unbiased stereology. To stimulate autophagy, disaccharide trehalose, or sucrose control, was administered in the drinking water immediately after injury and for up to 6 weeks after SCI. Results: Flow cytometry demonstrated dysregulation of autophagic function in both microglia and infiltrating myeloid cells from the injured spinal cord at 3 days post-injury. Transgenic CX3CR1-GFP mice revealed increased autophagosome formation and inhibition of autophagic flux specifically in activated microglia/macrophages. NanoString analysis using the neuroinflammation panel demonstrated increased expression of proinflammatory genes and decreased expression of genes related to neuroprotection in Becn1+/- mice as compared to WT controls at 3 days post-SCI. These findings were further validated by qPCR, wherein we observed significantly higher expression of proinflammatory cytokines. Western blot analysis confirmed higher protein expression of the microglia/macrophage marker IBA-1, inflammasome marker, NLRP3, and innate immune response markers cGAS and STING in Becn1+/- mice at 3 day after SCI. Flow cytometry demonstrated that autophagy deficit did not affect either microglial or myeloid counts at 3 days post-injury, instead resulting in increased microglial production of proinflammatory cytokines. Finally, locomotor function showed significantly worse impairments in Becn1+/- mice up to 6 weeks after SCI, which was accompanied by worsening tissue damage. Conversely, treatment with a naturally occurring autophagy inducer trehalose, reduced protein levels of p62, an adaptor protein targeting cargo to autophagosomes as well as the NLRP3, STING, and IBA-1 at 3 days post-injury. Six weeks of trehalose treatment after SCI led to improved motor function recovery as compared to control group, which was accompanied by reduced tissue damage. Conclusions: Our data indicate that inhibition of autophagy after SCI potentiates pro-inflammatory activation in microglia and is associated with worse functional outcomes. Conversely, increasing autophagy with trehalose, decreased inflammation and improved outcomes. These findings highlight the importance of autophagy in spinal cord microglia and its role in secondary injury after SCI

Brain Injury Accelerates the Onset of a Reversible Age-Related Microglial Phenotype Associated With Inflammatory Neurodegeneration

Lipofuscin is an autofluorescent (AF) pigment formed by lipids and misfolded proteins, which accumulates in postmitotic cells with advanced age. Here, we immunophenotyped microglia in the brain of old C57BL/6 mice (\u3e18 months old) and demonstrate that in comparison to young mice, one-third of old microglia are AF, characterized by profound changes in lipid and iron content, phagocytic activity, and oxidative stress. Pharmacological depletion of microglia in old mice eliminated the AF microglia following repopulation and reversed microglial dysfunction. Age-related neurological deficits and neurodegeneration after traumatic brain injury (TBI) were attenuated in old mice lacking AF microglia. Furthermore, increased phagocytic activity, lysosomal burden, and lipid accumulation in microglia persisted for up to 1 year after TBI, were modified b

Compaction Quality Inspection Method of Soil-Rock Filled Embankment Based on Continuous Compaction Control Technology

Affected by the site construction conditions, the measurement passes of the Taihang Expressway K8 + 105 ∼ K8 + 341 (K8 worksite) in the Taihang Expressway did not meet the requirements of data analysis, and the quantity of the control points was insufficient so that the linear correlation between the dynamic deformation modulus () and the vibratory compaction value (VCV) was not strong. Therefore, the target value of VCV cannot be used to diagnose the Ev d compaction quality of soil-rock filler. This paper analyzes the roller measurement VCV value and in situ measurements Ev d value separately. Results reveal the difference between the VCV mean measured in the last two passes and the standard deviation of the measured VCV mean in the last pass are used as the main basis for the actual compaction quality. In addition, the Ev d mean in the last rolling can be used as an auxiliary judgment basis for the quality control of the compaction

Influence of Maximum Particle Diameter on the Mechanical Behavior of Soil-Rock Mixtures

With the steady development of the development of the western region in China, the construction of mountain highways has developed rapidly, and the soil-rock mixed filler, as an excellent filler, is widely used in the subgrade filling of mountain highways. Unlike ordinary fine-grained soil, the source of the soil-rock mixtures (S-RMs) is not unique, and the particle size difference is large and the water content is not uniform, resulting in very complicated mechanical properties. But the current highway embankment codes are still mainly established on the fine-grained soil. It is not fully applicable to soil-rock filled embankment. Based on soil-rock filled embankment engineering practice, this research uses a large-scale direct shear test to research the mechanical characteristics of the S-RMs with different maximum particle diameters. According to the large-scale direct shear test of S-RMs with different maximum particle diameters, the shear displacement vs shear stress curve, shear dilation, and strength characteristics with maximum particle diameter were analyzed. Results demonstrate that whether secondary hardening occurs mainly depends on the normal stress and the maximum particle diameter of the filler. At different maximum particle diameters, the horizontal displacement vs vertical displacement curves of the S-RMs can be roughly divided into continuous shearing and beginning of shearing and quick dilation. And the shear strength increases with the increase of the maximum particle diameter. Moreover, the cohesion decreases first and then increases with the increase of the maximum particle diameter, and the internal friction angle increases with the increase of the maximum particle diameter. Therefore, some RBs with large particle diameter added to filler can effectively improve the shear strength of the S-RMs, which may be valuable for realistic engineering

Vibration Response Characteristics and Application of Existing Railway Subgrade

The existing conventional methods of subgrade disease assessment are not suitable for the existing lines. There are many research studies on the vibration response and attenuation law of the railway subgrade, but few research studies focus on the vibration response and attenuation law caused by the weak subgrade. In this study, vibration response tests were carried out at different positions and depths of the subgrade before and after reinforcement improvement. The results show that vibration response near the ballast is obvious, and it attenuates with the increase of the horizontal distance from the rail; the vibration acceleration response of the subgrade after reinforcement changes greatly; the vibration response curve of the reinforced section is spindle shaped, and the vertical vibration acceleration response attenuates obviously at the depth of 6.5 m, only about 10% to 30% of the surface; the vibration acceleration of the subgrade with reinforcement at the depth of 4.5 m attenuates to 60% of the surface; the vibration acceleration of the subgrade without reinforcement at the depth of 4.5 m attenuates to 50%–60% of the surface

Plant-Produced Antigen Displaying Virus-Like Particles Evokes Potent Antibody Responses against West Nile Virus in Mice

In this study, we developed a hepatitis B core antigen (HBcAg)-based virus-like particle (VLP) that displays the West Nile virus (WNV) Envelope protein domain III (wDIII) as a vaccine candidate for WNV. The HBcAg-wDIII fusion protein was quickly produced in Nicotiana benthamiana plants and reached a high expression level of approximately 1.2 mg of fusion protein per gram of leaf fresh weight within six days post gene infiltration. Electron microscopy and gradient centrifugation analysis indicated that the introduction of wDIII did not interfere with VLP formation and HBcAg-wDIII successfully assembled into VLPs. HBcAg-wDIII VLPs can be easily purified in large quantities from Nicotiana benthamiana leaves to >95% homogeneity. Further analysis revealed that the wDIII was displayed properly and demonstrated specific binding to an anti-wDIII monoclonal antibody that recognizes a conformational epitope of wDIII. Notably, HBcAg-wDIII VLPs were shown to be highly immunogenic and elicited potent humoral responses in mice with antigen-specific IgG titers equivalent to that of protective wDIII antigens in previous studies. Thus, our wDIII-based VLP vaccine offers an attractive option for developing effective, safe, and low-cost vaccines against WNV