Atractylenolide I improves behaviors in mice with depression-like phenotype by modulating neurotransmitter balance via 5-HT2A.

To explore the antidepressant effects and targets of atractylenolide I (ATR) through a network pharmacological approach. Relevant targets of ATR and depression analyzed by network pharmacology were scored (identifying 5-HT2A targets). Through elevated plus maze, open field, tail suspension, and forced swimming tests, the behavioral changes of mice with depression (chronic unpredictable mild stress [CUMS]) were examined, and the levels of neurotransmitters including serotonin, dopamine, and norepinephrine (5-HT, DA, and NE) were determined. The binding of ATR to 5-HT2A was verified by small molecular-protein docking. ATR improved the behaviors of CUMS mice, elevated their levels of neurotransmitters 5-HT, DA, and NE, and exerted a protective effect on their nerve cell injury. After 5-HT2A knockout, ATR failed to further improve the CUMS behaviors. According to the results of small molecular-protein docking and network pharmacological analysis, ATR acted as an inhibitor by binding to 5-HT2A. ATR can improve the behaviors and modulate the neurotransmitters of CUMS mice by targeting 5-HT2A.

Gastrodin improves nerve cell injury and behaviors of depressed mice through Caspase-3-mediated apoptosis.

AIM: We investigated the effects and target of gastrodin (GAS) for treating depression through network pharmacology combined with experimentation. METHODS: The therapeutic target and signal of GAS for depression were analyzed by network pharmacology. Depression in mice was mimicked with a chronic unpredictable mouse stress (CUMS) model. Through open field, elevated plus maze, forced swimming, and tail suspension tests, the effects of GAS on the CUMS mice behaviors were examined, and the levels of neurotransmitters were detected. The histopathological changes were assayed by H&E and IHC staining, and the protein expressions were detected by Western blotting. Small molecule-protein docking and molecular dynamics experiments were conducted to simulate the binding mode between GAS and Caspase-3. RESULTS: Network pharmacological analysis revealed that Caspase-3 was the action target of GAS. GAS could improve depression-like behaviors in CUMS mice, elevate their neurotransmitter levels, ameliorate their nerve cell injury, and inhibit their Caspase-3 expression. After knocking out Caspase-3, the effects of GAS were inhibited. Molecular dynamics simulation and small molecule-protein docking found that GAS bound to Caspase-3 at SER25, inhibiting the maturation and activation of Caspase-3. CONCLUSION: We find that GAS can act as a Caspase-3 inhibitor, which improves depression-like behaviors and nerve cell injury in CUMS mice by inhibiting Caspase-3-mediated apoptosis.

Dihydromyricetin suppresses inflammatory injury in microglial cells to improve neurological behaviors of Alzheimer's disease mice via the TLR4/MD2 signal.

AIM: We analyzed the role and mechanism of dihydromyricetin (DHM) in suppressing inflammatory injury in microglial cells via targeting MD2. METHODS: In vitro, BV2 cells were used as the objects of study to induce inflammatory injury with LPS + ATP, then the cell apoptosis level was identified, inflammatory factor levels were measured by ELISA, TLR4 and MD2 were stained with fluorescence staining, and protein expression was determined using Western-blot (WB) assay. Afterwards, MD2 expression was knocked down n BV2 cells to construct the BV2-MD2-/- cell line, so as to detect the role of DHM on BV2-MD2-/-. Moreover, the binding of DHM to MD2 was analyzed via mall molecule-protein docking and pull-down assays. In-vivo, wild-type (WT) C67BL/6 mice and APP/PS1 (AD) mice were used as the objects of study, which were intervened with DHM to detect the changes in mouse cognition. In addition, the pathological changes of brain tissues were analyzed with H&E staining. In addition, the inflammatory factor and protein levels in brain tissues were also detected. RESULTS: DHM suppressed inflammatory injury in BV2 cells, reduced the cell apoptosis rate and inflammatory factor levels, and suppressed the level of TLR4 and MD2. After MD2 knockdown, DHM was unable to further suppress BV2 cell injury. Results of small molecule-protein docking and pull-down assays suggested that DHM bound to MD2 to suppress the formation of TLR4 complex. In AD mice, DHM improved the cognitive disorder in mice, suppressed inflammatory injury in brain tissues and lowered the expression of TLR4 protein. CONCLUSION: DHM targeted MD2 to suppress the formation of TLR4 protein complex, thereby suppressing inflammatory injury in microglial cells and improving the cognition in AD mice.

Therapeutic equine hyperimmune antibodies with high and broad-spectrum neutralizing activity protect rodents against SARS-CoV-2 infection.

The emergence of SARS-CoV-2 variants stresses the continued need for broad-spectrum therapeutic antibodies. Several therapeutic monoclonal antibodies or cocktails have been introduced for clinical use. However, unremitting emerging SARS-CoV-2 variants showed reduced neutralizing efficacy by vaccine induced polyclonal antibodies or therapeutic monoclonal antibodies. In our study, polyclonal antibodies and F(ab')2 fragments with strong affinity produced after equine immunization with RBD proteins produced strong affinity. Notably, specific equine IgG and F(ab')2 have broad and high neutralizing activity against parental virus, all SARS-CoV-2 variants of concern (VOCs), including B.1.1,7, B.1.351, B.1.617.2, P.1, B.1.1.529 and BA.2, and all variants of interest (VOIs) including B.1.429, P.2, B.1.525, P.3, B.1.526, B.1.617.1, C.37 and B.1.621. Although some variants weaken the neutralizing ability of equine IgG and F(ab')2 fragments, they still exhibited superior neutralization ability against mutants compared to some reported monoclonal antibodies. Furthermore, we tested the pre-exposure and post-exposure protective efficacy of the equine immunoglobulin IgG and F(ab')2 fragments in lethal mouse and susceptible golden hamster models. Equine immunoglobulin IgG and F(ab')2 fragments effectively neutralized SARS-CoV-2 in vitro, fully protected BALB/c mice from the lethal challenge, and reduced golden hamster's lung pathological change. Therefore, equine pAbs are an adequate, broad coverage, affordable and scalable potential clinical immunotherapy for COVID-19, particularly for SARS-CoV-2 VOCs or VOIs.

PEGylation Prolongs the Half-Life of Equine Anti-SARS-CoV-2 Specific F(ab')2.

Therapeutic antibodies-F(ab')2 obtained from hyperimmune equine plasma could treat emerging infectious diseases rapidly because of their high neutralization activity and high output. However, the small-sized F(ab')2 is rapidly eliminated by blood circulation. This study explored PEGylation strategies to maximize the half-life of equine anti-SARS-CoV-2 specific F(ab')2. Equine anti-SARS-CoV-2 specific F(ab')2 were combined with 10 KDa MAL-PEG-MAL in optimum conditions. Specifically, there were two strategies: Fab-PEG and Fab-PEG-Fab, F(ab')2 bind to a PEG or two PEG, respectively. A single ion exchange chromatography step accomplished the purification of the products. Finally, the affinity and neutralizing activity was evaluated by ELISA and pseudovirus neutralization assay, and ELISA detected the pharmacokinetic parameters. The results displayed that equine anti-SARS-CoV-2 specific F(ab')2 has high specificity. Furthermore, PEGylation F(ab')2-Fab-PEG-Fab had a longer half-life than specific F(ab')2. The serum half-life of Fab-PEG-Fab, Fab-PEG, and specific F(ab')2 were 71.41 h, 26.73 h, and 38.32 h, respectively. The half-life of Fab-PEG-Fab was approximately two times as long as the specific F(ab')2. Thus far, PEGylated F(ab')2 has been prepared with high safety, high specificity, and a longer half-life, which could be used as a potential treatment for COVID-19.

A rabies virus-vectored vaccine expressing two copies of the Marburg virus glycoprotein gene induced neutralizing antibodies against Marburg virus in humanized mice.

Marburg virus disease (MVD) is a lethal viral haemorrhagic fever caused by Marburg virus (MARV) with a case fatality rate as high as 88%. There is currently no vaccine or antiviral therapy approved for MVD. Due to high variation among MARV isolates, vaccines developed against one strain fail to protect against other strains. Here we report that three recombinant rabies virus (RABV) vector vaccines encoding two copies of GPs covering both MARV lineages induced pseudovirus neutralizing antibodies in BALB/c mice. Furthermore, high-affinity human neutralizing antibodies were isolated from a humanized mouse model. The three vaccines produced a Th1-biased serological response similar to that of human patients. Adequate sequential immunization enhanced the production of neutralizing antibodies. Virtual docking suggested that neutralizing antibodies induced by the Angola strain seemed to be able to hydrogen bond to the receptor-binding site (RBS) in the GP of the Ravn strain through hypervariable regions 2 (CDR2) and CDR3 of the VH region. These findings demonstrate that three inactivated vaccines are promising candidates against different strains of MARV, and a novel fully humanized neutralizing antibody against MARV was isolated.

Discovery and characterization of SARS-CoV-2 reactive and neutralizing antibodies from humanized CAMouseHG mice through rapid hybridoma screening and high-throughput single-cell V(D)J sequencing.

The coronavirus disease 2019 pandemic has caused more than 532 million infections and 6.3 million deaths to date. The reactive and neutralizing fully human antibodies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are effective detection tools and therapeutic measures. During SARS-CoV-2 infection, a large number of SARS-CoV-2 reactive and neutralizing antibodies will be produced. Most SARS-CoV-2 reactive and neutralizing fully human antibodies are isolated from human and frequently encoded by convergent heavy-chain variable genes. However, SARS-CoV-2 viruses can mutate rapidly during replication and the resistant variants of neutralizing antibodies easily survive and evade the immune response, especially in the face of such focused antibody responses in humans. Therefore, additional tools are needed to develop different kinds of fully human antibodies to compensate for current deficiency. In this study, we utilized antibody humanized CAMouseHG mice to develop a rapid antibody discovery method and examine the antibody repertoire of SARS-CoV-2 RBD-reactive hybridoma cells derived from CAMouseHG mice by using high-throughput single-cell V(D)J sequencing analysis. CAMouseHG mice were immunized by 28-day rapid immunization method. After electrofusion and semi-solid medium screening on day 12 post-electrofusion, 171 hybridoma clones were generated based on the results of SARS-CoV-2 RBD binding activity assay. A rather obvious preferential usage of IGHV6-1 family was found in these hybridoma clones derived from CAMouseHG mice, which was significantly different from the antibodies found in patients with COVID-19. After further virus neutralization screening and antibody competition assays, we generated a noncompeting two-antibody cocktail, which showed a potent prophylactic protective efficacy against SARS-CoV-2 in cynomolgus macaques. These results indicate that humanized CAMouseHG mice not only provide a valuable platform to obtain fully human reactive and neutralizing antibodies but also have a different antibody repertoire from humans. Thus, humanized CAMouseHG mice can be used as a good complementary tool in discovery of fully human therapeutic and diagnostic antibodies.

Construction of Recombinant Rabies Virus Vectors Expressing H or F Protein of Peste des Petits Ruminants Virus.

Peste des petits ruminants (PPR) is one of the most contagious and fatal diseases of small ruminants in the world and is classified as a category A epidemic disease. It is the target of a global eradication campaign led by the Office International des Epizooties (OIE) and Food and Agriculture Organization of the United Nations (FAO). The PPR live attenuated vaccine is currently the most widely used and approved vaccine, but the use of this vaccine interferes with the serological testing of the PPR elimination program, and there is a potential safety risk. Viral vector vaccines are one of the most promising methods to solve this dilemma. In this study, the full-length infectious clone plasmid of rabies virus (RABV), pD-SRV9-PM-LASV, was used as the backbone, and the envelope glycoprotein H (hemagglutinin protein) or F (fusion protein) gene of PPRV was inserted into the backbone plasmid to construct the infectious clones pD-SRV9-PM-PPRV-H and pD-SRV9-PM-PPRV-F, which express the PPRV H and PPRV F genes, respectively. The correct construction of these infectious clones was verified after sequencing and double digestion. The infectious clones were transfected with a helper plasmid into BSR/T7 cells, and recombinant viruses were successfully rescued by direct immunofluorescence, indirect immunofluorescence, Western blotting, and transmission electron microscopy and named rSRV9-H and rSRV9-F. The results of growth kinetics studies indicated that the inserted gene did not affect virus proliferation. Stability studies revealed that the inserted target gene was stably expressed in recombinant RABV for at least 15 generations. In this study, the recombinant viruses rSRV9-H and rSRV9-F were successfully rescued. The constructed viruses had good proliferative activity and stability and provided potential bivalent inactivated vaccine candidate strains for the prevention of PPR and livestock rabies.

Substantially Improved Electrofusion Efficiency of Hybridoma Cells: Based on the Combination of Nanosecond and Microsecond Pulses.

As the initial antibody technology, the preparation of hybridoma cells has been widely used in discovering antibody drugs and is still in use. Various antibody drugs obtained through this technology have been approved for treating human diseases. However, the key to producing hybridoma cells is efficient cell fusion. High-voltage microsecond pulsed electric fields (µsHVPEFs) are currently one of the most common methods used for cell electrofusion. Nevertheless, the membrane potential induced by the external microsecond pulse is proportional to the diameter of the cell, making it difficult to fuse cells of different sizes. Although nanosecond pulsed electric fields (nsPEFs) can achieve the fusion of cells of different sizes, due to the limitation of pore size, deoxyribonucleic acid (DNA) cannot efficiently pass through the cell pores produced by nsPEFs. This directly causes the significant loss of the target gene and reduces the proportion of positive cells after fusion. To achieve an electric field environment independent of cell size and enable efficient cell fusion, we propose a combination of nanosecond pulsed electric fields and low-voltage microsecond pulsed electric fields (ns/µsLVPEFs) to balance the advantages and disadvantages of the two techniques. The results of fluorescence experiments and hybridoma culture experiments showed that after lymphocytes and myeloma cells were stimulated by a pulse (ns/µsLVPEF, µsHVPEF, and control), compared with µsHVPEF, applying ns/µsLVPEF at the same energy could increase the cell fusion efficiency by 1.5-3.0 times. Thus far, we have combined nanosecond and microsecond pulses and provided a practical solution that can significantly increase cell fusion efficiency. This efficient cell fusion method may contribute to the further development of hybridoma technology in electrofusion.

GEM-PA-Based Subunit Vaccines of Crimean Congo Hemorrhagic Fever Induces Systemic Immune Responses in Mice.

The Crimean Congo Hemorrhagic Fever Virus (CCHFV) is a tick-borne bunyavirus of the Narovirus genus, which is the causative agent of Crimean Congo Hemorrhagic Fever (CCHF). CCHF is endemic in Africa, the Middle East, Eastern Europe and Asia, with a high case-fatality rate of up to 50% in humans. Currently, there are no approved vaccines or effective therapies available for CCHF. The GEM-PA is a safe, versatile and effective carrier system, which offers a cost-efficient, high-throughput platform for recovery and purification of subunit proteins for vaccines. In the present study, based on a GEM-PA surface display system, a GEM-PA based vaccine expressing three subunit vaccine candidates (G-GP, including G-eGN, G-eGC and G-NAb) of CCHFV was developed, displaying the ectodomains of the structural glycoproteins eGN, eGC and NAb, respectively. According to the immunological assays including indirect-ELISA, a micro-neutralization test of pseudo-virus and ELISpot, 5 µg GPBLP3 combined with Montanide ISA 201VG plus Poly (I:C) adjuvant (A-G-GP-5 µg) elicited GP-specific humoral and cellular immunity in BALB/c mice after three vaccinations via subcutaneous injection (s.c.). The consistent data between IgG subtype and cytokine detection, ELISpot and cytokine detection indicated balanced Th1 and Th2 responses, of which G-eGN vaccines could elicit a stronger T-cell response post-vaccination, respectively. Moreover, all three vaccine candidates elicited high TNF-α, IL-6, and IL-10 cytokine levels in the supernatant of stimulated splenocytes in vitro. However, the neutralizing antibody (nAb) was only detected in A-G-eGC and A-G-eGC vaccination groups with the highest neutralizing titer of 128, suggesting that G-eGC could elicit a stronger humoral immune response. In conclusion, the GEM-PA surface display system could provide an efficient and convenient purification method for CCHFV subunit antigens, and the G-GP subunit vaccine candidates will be promising against CCHFV infections with excellent immunogenicity.

A Novel and Secure Pseudovirus Reporter System Based Assay for Neutralizing and Enhancing Antibody Assay Against Marburg Virus.

Marburg virus (MARV) is one of the principal members of the filovirus family, which can cause fatal hemorrhagic fever in humans. There are currently no prophylactic and therapeutic drugs on the market, and the high pathogenicity and infectivity of MARV make its research highly dependent on biosafety level 4 conditions, severely hindering the development of vaccines and therapies. Therefore, the development of medicines, such as MARV serological diagnosis, vaccines, and therapeutic antibody drugs, urgently needs a safe, convenient, and biosafety level 2 detection method to measure the neutralizing activity of MARV antibodies. To this end, we report a neutralization assay relying on a Rabies virus (RABV) reverse genetic operating system. We constructed infectious clones carrying the eGFP reporter gene and the full length of the original unmodified MARV GP gene. Based on the critical parameters of phylogenetic analysis, recombinant viruses targeting representative strains in the two major MARV lineages were successfully rescued. These pseudoviruses are safe in mice, and their inability to infect cells after being neutralized by antibodies can be visualized under a fluorescence microscope. We tested the system using the neutralizing antibody MR191. MR191 can significantly block the infection of BSR cells with pseudovirus. We compared it with the traditional lentivirus-type pseudovirus system to verify the system's credibility and obtained the same results as reported in the literature. In general, we have established a safe and visualized method for evaluating the neutralizing activity of MARV antibodies. Compared with traditional methods, it has the advantages of convenient operation, short cycle, and low cost. It is a candidate method that can replace actual viruses for a neutralization assay.

Nucleic acid visualization assay for Middle East Respiratory Syndrome Coronavirus (MERS-CoV) by targeting the UpE and N gene.

Since its first emergence in 2012, cases of infection with Middle East respiratory syndrome coronavirus (MERS-CoV) have continued to occur. At the end of January 2020, 2519 laboratory confirmed cases with a case-fatality rate of 34.3% have been reported. Approximately 84% of human cases have been reported in the tropical region of Saudi Arabia. The emergence of MERS-CoV has highlighted need for a rapid and accurate assay to triage patients with a suspected infection in a timely manner because of the lack of an approved vaccine or an effective treatment for MERS-CoV to prevent and control potential outbreaks. In this study, we present two rapid and visual nucleic acid assays that target the MERS-CoV UpE and N genes as a panel that combines reverse transcription recombinase polymerase amplification with a closed vertical flow visualization strip (RT-RPA-VF). This test panel was designed to improve the diagnostic accuracy through dual-target screening after referencing laboratory testing guidance for MERS-CoV. The limit of detection was 1.2×101 copies/µl viral RNA for the UpE assay and 1.2 copies/µl viral RNA for the N assay, with almost consistent with the sensitivity of the RT-qPCR assays. The two assays exhibited no cross-reactivity with multiple CoVs, including the bat severe acute respiratory syndrome related coronavirus (SARSr-CoV), the bat coronavirus HKU4, and the human coronaviruses 229E, OC43, HKU1 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Furthermore, the panel does not require sophisticated equipment and provides rapid detection within 30 min. This panel displays good sensitivity and specificity and may be useful to rapidly detect MERS-CoV early during an outbreak and for disease surveillance.

Development of a Visible Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for the Detection of Rift Valley Fever Virus.

Rift Valley fever (RVF) is a severe infectious disease, which can through mosquito bites, direct contact and aerosol transmission infect sheep, goats, people, camels, cattle, buffaloes, and so on. In this paper, a conserved region of the S RNA segment of Rift Valley fever virus (RVFV) ZH501 strain was used as target sequence. The RVFV RT-LAMP-VF assay was successfully established combined reverse transcription-loop-mediated isothermal amplification with a vertical flow visualization strip. The detection limit is up to 1.94 × 100 copies/µl of synthesized RVFV-RNA. RNA extracted from cell culture of an inactivated RVFV-BJ01 strain was also used as templates, and the detection limit is 1.83 × 103 copies/µl. In addition, there was no cross-reactivity with other viruses that can cause similar fever symptoms. The RVFV-LAMP-VF assay exhibited very high levels of diagnostic sensitivity, which had 100-fold more sensitive than RVFV real-time RT-PCR assay. Accordingly, the RVFV RT-LAMP-VF assay developed in this study is suitable for the rapid and sensitive diagnosis of RVFV without specialized equipment and can rapidly complete detection within 60 min, and the results are visible by vertical flow visualization strip within 5 min.

Molecular Phylogeny of an Avipoxvirus Isolated from Red-Flanked Blue Robin in China.

We first report avipoxvirus (APV) infection and an isolate named APV/03/2016 from a red-flanked blue robin (Tarsiger cyanurus) captured at Songhua Lake Scenic Area in Jilin City (Jilin Province, China) on March 24, 2016. The partial sequence of the 4b core protein gene and DNA polymerase gene of APV/03/2016 suggests that the virus belongs to the subclade B1 cluster of clade B (canarypox virus). The BLAST results showed the highest similarity of the two genes with the Pacific shearwater-isolated strain SWPV-2 (KX857215), canarypox virus strain D98-11133 (GQ487567), canarypox virus strain ATCC VR-111 (AY318871), avipoxvirus Mississippi isolate P89 (KC018048), and avipoxvirus Wisconsin isolate P92 (KC018051). The results indicate that APV/03/2016 is a canarypox-like virus. These findings demonstrate the continuous emergence of new APV hosts such as red-flanked blue robins and suggest that monitoring of APV circulation and evolution should be strengthened for T. cyanurus conservation.

Filogenia molecular de un Avipoxvirus aislado de ruiseñor coliazul en China. Se reporta por primera vez la infección por poxvirus aviar (APV) y un aislamiento denominado APV/03/2016 obtenido de un ruiseñor coliazul (Tarsiger cyanurus) capturado en el área escénica del Lago Songhua en la ciudad de Jilin (provincia de Jilin, China) el 24 de marzo de 2016. La secuencia parcial del gene de la proteína central 4b y el gene de la polimerasa de ADN del virus APV/03/2016 sugiere que el virus pertenece al subclado B1 del clado B (virus de la viruela del canario). Los resultados de la búsqueda mediante BLAST mostraron la mayor similitud de los dos genes con la cepa aislada de aves pelágicas del Pacífico (KX857215), virus de la viruela del canario cepa de virus D98-11133 (GQ487567), cepa de virus de la viruela del canario ATCC VR-111 (AY318871), aislamiento de avipoxvirus de Mississippi P89 (KC018048), y el aislamiento P89 de avipoxvirus en Wisconsin (KC018051). Los resultados indican que el virus APV/03/2016 es un virus similar al de la viruela del canario. Estos hallazgos demuestran la aparición continua de nuevos hospedadores de poxvirus aviares como el ruiseñor coliazul y sugieren que el monitoreo de la circulación y evolución de poxvirus aviares debería fortalecerse para la conservación del T. cyanurus.