SARS-CoV-2 is an RNA enveloped virus responsible for the COVID-19 pandemic that conducted in 6 million deaths worldwide so far. SARS-CoV-2 particles are mainly composed of the 4 main structural proteins M, N, E and S to form 100 nm diameter viral particles. Based on productive assays, we propose an optimal transfected plasmid ratio mimicking the viral RNA ratio in infected cells. This allows SARS-CoV-2 Virus-Like Particle (VLPs) formation composed of the viral structural proteins M, N, E and mature S. Furthermore, fluorescent or photoconvertible VLPs were generated by adding a fluorescent protein tag on N or M mixing with unlabeled viral proteins and characterized by western blots, atomic force microscopy coupled to fluorescence and immuno-spotting. Thanks to live fluorescence and super-resolution microscopies, we quantified VLPs size and concentration. SARS-CoV-2 VLPs present a diameter of 110 and 140 nm respectively for MNE-VLPs and MNES-VLPs with a concentration of 10e12 VLP/ml. In this condition, we were able to establish the incorporation of the Spike in the fluorescent VLPs. Finally, the Spike functionality was assessed by monitoring fluorescent MNES-VLPs docking and internalization in human pulmonary cells expressing or not the receptor hACE2. Results show a preferential maturation of S on N(GFP) labeled VLPs and an hACE2-dependent VLP internalization and a potential fusion in host cells. This work provides new insights on the use of non-fluorescent and fluorescent VLPs to study and visualize the SARS-CoV-2 viral life cycle in a safe environment (BSL-2 instead of BSL-3). Moreover, optimized SARS-CoV-2 VLP production can be further adapted to vaccine design strategies. This respiratory virus emerged in China during the month of December 2019 and spread all around the world, leading to the ongoing pandemic declared in March 2020 by the World Health Organization (WHO). Pathogenicity of coronaviruses was previously observed during SARS-CoV and MERS outbreaks, which respectively occurred in 2002 and 2012. Concerning the current pandemic, COVID-19 has affected up to 460 million cases and caused 6 million deaths (WHO). It’s demonstrating the importance of gathering knowledge on SARS-CoV-2 and developing vaccine design strategies. Among the possible strategies, Virus-Like Particles (VLPs) are high potential as they can be efficient and cheap vaccine candidates1. VLPs are multi-protein-lipid structures that mimic the organization and conformation of authentic native viruses without containing the viral genome.The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is an emerging virus belonging to the 2, Kerviel and collaborators showed that the Influenza minimal system requires at least four proteins for VLPs production3. SARS-CoV-2 is an enveloped virus containing one RNA molecule and 29 proteins, including four structural proteins: membrane protein M, nucleoprotein N, envelope protein E and spike protein S4. Thus, determining the optimal combination (and proportion) of these structural viral proteins remains to be tested to obtain a minimal system for SARS-CoV-2 VLPs production.To date, non-infectious VLPs have already been optimized using HIV-1 or Influenza structural proteins, allowing to study the entry and the assembly of these viruses in BSL-2 facilities. While the minimal requirement for HIV-1 VLPs uses only one viral protein, i.e. Gag11 exhibiting differences and contradictory results regarding the required structural viral proteins. Hence, different combinations regarding M, N, E and S were reported: the minimal system for VLPs production could require both M and E5 or M, E and S7 or M and E with N which plays a crucial role for VLPs formation10 as previously reported for SARS-CoV12. A combination of M with N or S was also suggested6. Here, based on years of experiences in the production of HIV-1 Gag VLPs and their characterization using advanced microscopies16, our work aims to revisit the minimal system for SARS-CoV-2 VLPs production. To optimize VLPs production from DNA plasmid ratio of M, N, E or S, we based our approach on viral RNA ratios obtained from transcriptomic analysis of the SARS-CoV-2 RNA genome in infected cells18. We also optimized the production of fluorescent VLPs (mono- or bi-color), made with tagged structural SARS-CoV-2 proteins, to create a valuable tool to image and study SARS-CoV-2 assembly and entry. We characterize these VLPs thanks to advanced microscopy methods such as Total Internal Reflection Fluorescence Microscopy (TIRF-M), Fluctuation Correlation Spectroscopy (FCS), correlative fluorescence-Atomic Force Microscopy (AFM) and Photo-Activable Localization Microscopy (PALM). These techniques allow us to determine the size, numbers and morphology of VLPs that we could compare to wild-type SARS-CoV-2 particles20. Finally, these fluorescent VLPs, made with tagged structural proteins (M or N) allowed us to evaluate the incorporation of the Spike and to follow viral particle internalization into host pulmonary cells, with or without the hACE2 receptor, using quantitative confocal microscopy.Recently, research teams establish several minimal systems for SARS-CoV-2 VLPs production2.The HEK293T human embryonic kidney cell line was obtained from ECACC  and maintained cultured in Dulbecco’s modified essential medium  supplemented with 10% heat inactivated fetal calf serum , 50 U/mL of penicillin , 50 µg/mL of streptomycin , 1 mM sodium-pyruvate  and 25 mM of HEPES , at 37 °C with 5% CO21 were obtained from ECACC  and cultured in Roswell Park Memorial Institute medium (RPMI) from Gibco supplemented with 10% heat-inactivated fetal calf serum , 50 U/mL of penicillin , 50 µg/mL of streptomycin , 1 mM sodium-pyruvate  and 25 mM HEPES , at 37 °C with 5% CO2. The human pulmonary alveolar A549-hACE2 and A549-hACE2mScarlet stable cell lines were engineered using 2 different lentiviral vectors. For A549-hACE2, A549 cells were transduced with VSVg pseudotyped particles derived from a lentiviral vector from Flash Therapeutics  to express the human receptor ACE2. Cells were then sorted by flow cytometry (BD FACSAria™ III Cell Sorter) to obtain an A549-hACE2 population stably expressing hACE2. To detect hACE2, cells were washed twice in PBS and incubated for 1 h at 4 °C with primary goat antibody anti-human ACE2 protein (R&D AF933) (1:100). After washing in PBS, cells were stained 45 min at 4 °C with secondary antibody AF488-labeled donkey-anti-goat (Life technologies A11055) (1:3000) for 45 min. h-ACE2 expression was monitored in comparison with isotype control using NovoCyte Flow Cytometer (Agilent) : GGGGSGGGGSGGGGSGGGGD to generate both M-Flex-mCherry and M-Flex-GFP. The plasmids were generated by the synthesis of Flex-mCherry and Flex-eGFP and inserted into humanized M plasmids described in7 using EZcloning from Genscript. The SARS-CoV-2 N protein was tagged at the N-terminus. The GFP and mEOS2 tags were amplified by PCR from peGFP and pmEOS2 using Pfu Polymerase , dNTP mix  and primers designed to carry NheI and KpnI restriction sites (Sigma Aldrich). PCR amplified inserts were added at the N-terminus of N protein in pcDNA3.1 + plasmid, and purified with the NucleoSpin Gel and PCR Clean-up kit (Macherey–Nagel). The plasmid N and inserts were digested and purified. Ligation between these digested products was performed using T4 DNA ligase . 50 µL of competent bacteria were next transformed with 5 µL of the ligation reaction product. Plasmids were extracted from various colonies with the NucleoSpin plasmid mini-kit . Validated plasmids were then sequenced by Eurofins.The plasmid expressing Wuhan SARS-CoV-2 M protein, N protein, E protein and S protein were humanized and put under the control of the cytomegalovirus (CMV) promoter driven mammalian expression vectors as described in Swann et al.2/HBS2X  (v/v) into HEK293T cells. A total of 1.4 µg of mixed plasmids was applied for the transfection of a well (for 6-well plate) and 0.7 µg/well for individual plasmid. Co-transfection of double, triple or quadruple plasmids was conducted with M, N, E and S with plasmid ratio 3:3:3:5 respectively (0.3 µg:0.3 µg:0.3 µg:0.5 µg per well to 6-well plate for example) in the first assay as shown in Fig. 18, corresponding, for an example, to a total of 2,2 µg/well of 6 well plate (with M:N:E:S corresponding to 0.3 µg:1.2 µg:0.2 µg:0.5 µg of transfected plasmid) as shown in Fig. HEK293T cells were seeded into a 6-well plate or 10 mL dishes 24 h before transfection. Plasmids expressing viral proteins were transfected with CaClVLPs were obtained from culture medium of transfected cells by clarification at 5000 rpm for 5 min at 4 °C. Then the VLPs were collected from clarified supernatants after ultracentrifugation, through a 25% sucrose cushion in TNE buffer , at 28,000 rpm or 30,000 rpm for 3 h at 4 °C in Beckman SW41Ti or SW55Ti rotors respectively depending on the volume. VLPs from ultracentrifugation were resuspended overnight at 4 °C in TNE buffer. The purified VLPs were stored at 4 °C.Immunoblotting were performed by using the following antibodies: rabbit anti-M 1:1000 ; rabbit anti-N 1:3000 ; mouse anti-S 1:1000 ; anti-GAPDH antibody coupled to HRP 1:25,000 . For immuno-spotting, rabbit anti-Spike neutralizing Antibody 1:100 , mouse anti-CD81 1:100 , fluorescent rabbit Alexa555 and mouse Alexa647-conjugated secondary antibodies 1:2000 (Invitrogen) were used in this study.Proteins from cell lysates  or VLPs (volume to volume) were loaded and separated on 8% and 12% SDS-PAGE gel and transferred onto a polyvinylidene difluoride transfer membrane (Thermo Fisher). Immunoblotting was performed by using the corresponding antibodies. Horse Radish Peroxidase signals were revealed by using Amersham ECL Prime (Sigma Aldrich). Images were acquired using the Chemidoc Imaging system (Bio-Rad). Each band intensity on the immunoblot was quantified using the ImageJ software. Quantification was established by the following formula for each viral protein: VLP/(VLP + cell lysate) relative to GAPDH as a loading control.l-lysine (Sigma) for 30 min at room temperature and rinsed three times with 1 mL of PBS (Thermo Fisher Scientific). Purified MNES, MNE, M(GFP)NES and M(GFP)NE VLPs were diluted 10 times in 100µL TNE buffer and were deposited for 30 min at room temperature on the coated dishes for adsorption. Dishes were next filled with 2 mL PBS 1X. AFM imaging was performed at room temperature on a NanoWizard IV atomic force microscope  mounted on an inverted microscope  equipped with a standard monochrome CCD camera . Fluorescence imaging was performed by wide-field illumination  with a 100X objective  and the appropriate filter cube for GFP fluorescence . A software module  was used to calibrate the tip position with the optical image. AFM topographic images were obtained in TNE using the quantitative imaging (QI) mode with BL-AC40TS cantilevers . Before each set of acquisitions, the sensitivity and spring constant of the cantilever were calibrated . The applied force was kept at 200 pN with a constant approach/retract speed of 25 µm/s (z-range of 100 nm). Using the JPK SPM-data processing software, images were flattened with a polynomial/histogram line fit. Low-pass Gaussian and/or median filtering was applied to remove minor noise from the images. The Z-color scale in all images is given as relative after processing. Particle height analysis, based on the height (measured) channel of the QI mode, was performed using the cross-section tool of the analysis software to calculate the maximal central height of each particle.Glass-bottom FluoroDish Cell Culture dishes (WPI) were coated with 0.01% poly-l-lysine (Sigma) for 30 min at room temperature and rinsed three times with 1 mL of PBS (Thermo Fisher Scientific). Purified M(GFP)NES and M(GFP)NE VLPs were diluted 10 times in 100µL TNE buffer and were deposited for 30 min at room temperature on the coated dishes for adsorption. Experiments were conducted on a Zeiss LSM780 confocal microscope. The 488 nm excitation laser beam is focused onto the sample using a water immersion objective  resulting in a 210 nm laser waist (2/s). When considering a Gaussian profile for illumination  is immediately obtained by Eq. 2\documenD diffusion time, obtained by fitting the experimental correlograms with Eq. (The VLPs diameters (d) were calculated using the following expression:mentclass2pt{miniml-lysine (Sigma-Aldrich) for 30 min at room temperature and rinsed three times with 1 mL of PBS (Thermo Fisher Scientific). Purified MN(mEOS2)E VLPs and N(mEOS2) particles were diluted 10 times in 100µL TNE buffer and were deposited for 30 min at room temperature on the coated dishes for adsorption. Images were acquired in TIRF mode on an inverted microscope  using a NA 1.49 SR-100 × TIRF objective at room temperature. A 561 nm laser was used to excite the 405 nm photo-convertible mEOS-2 protein. The fluorescence is collected through a dedicated cube containing a dichroic mirror and an emission bandpass filter centered at 617 ± 73 nm. 20,000 images were captured by an iXon U897 camera from Andor at a rate of 50 Hz. PALM acquisitions were analyzed using the ThunderSTORM plugin in Fiji23. The module DBSCAN of the super-resolution quantification software SR Tesseler24 was used to quantify cluster diameters.Glass coverslips were coated with 0.01% poly-l-lysine (Sigma) and rinsed three times with PBS. Purified fluorescent SARS-CoV-2 VLPs were diluted 10 times in TNE buffer and 100 µL were deposited 30 min at room temperature on the poly-l-lysine coated dish for adsorption. PFA 4% was deposited 15 min at room temperature for fixation. NH4Cl was added for 5 min at room temperature. Dishes were rinsed once with BSA 3%PBS and were blocked with BSA 3%PBS for 15 min at room temperature. Immuno-spotting was performed by using corresponding antibodies. After incubation, dishes were rinsed three times with PBS and stored at 4 °C. Then, images of fluorescent VLP were acquired using the TIRF-Microscopy (Nikon) with the 100 × objective. 13 Images corresponding to 250–300 fluorescent VLPs were analyzed by ImageJ/Fiji software and ComDet Plugin was used for quantifying the number of fluorescent VLPs and the double GFP/mCherry colocalization. The image background was estimated at 10% of the fluorescent intensity signal of VLPs and was removed from images before colocalization quantification.FluoroDish cell culture Petri Dishes  were coated overnight at room temperature with 0.01% poly-VLPs internalization assays were performed using fluorescent M(GFP)NE and M(GFP)NES particles or MN(GFP)ES VLPs. Human pulmonary A549 or A549-hACE2 cells were harvested live in cold PBS 1x (after 24 h in culture on glass coverslip). M(GFP)NE or M(GFP)NES or MN(GFP)ES VLPs were diluted (1:20) in transparent L15 Solution (Gibco) and put into contact with the live cells. The cells were then incubated either at 37 °C for 15 min or at 4 °C for 1 h. Unbound VLPs were removed by washing cells 3 times with cold PBS 1x. The cells were then fixed with 4% PFA and mounted with Prolong Gold antifade reagent with DAPI (Invitrogen) for confocal imaging. Confocal fluorescence images were generated using a CD7 confocal laser-scanning microscope  equipped with a 50 × 0.5 ×, 1.2 NA oil objective or with a LSM980 confocal laser-scanning microscope  equipped with a 63 × 1.4 NA oil objective. The cells were imaged as Z stack with 0.3 µm sections and deconvoluted using Deconvolution lab plugin in Fiji with a theoretical PSF. Fiji was also used to count the number of MN(GFP)ES VLP (ie. Fluorescent dots) per cell.To measure VLP distance from cell center, using Icy, we first performed a Z-projection of stacks, then segmented the cells to establish their center position and subsequently performed cell center/M(GFP)-VLP distance measurements.Statistical analyses were performed using Origin 8.5 and Graph Pad prism softwares. ANOVA and t-tests were used for the statistical comparison of data.Graph Pad was used for performing graphs.10. At 48 h post-transfection, VLPs released in cell supernatants were purified by ultracentrifugation over a 25% sucrose cushion in TNE buffer  coding for the 4 main structural proteins. For that, the expression of M, N, E and S is under the same CMV promotor, with a respective 3:12:2:5 ratio. This transfected plasmid ratio was compared with the arbitrary MNES ratio 3:3:3:5 , transfected individually, by pairs or with a plasmid ratio of 3:3:3:5. Transfections were performed on the highly productive HEK293T cell lines, widely used in VLPs production19. MNE and MNES VLPs appeared as spherical particles, with shapes similar to those of WT-SARS-CoV-2 particles. As seen previously reported with the wild-type virus, AFM in liquid in standard scanning mode did not resolved the S proteins at the surface of the MNES VLPs, which has been attributed to the fast dynamics of S in the envelope. The mean height of the VLP was 112 ± 48 nm for MNE and 126 ± 17 nm for MNES, showing a ~ 25% increase in size as compared with wild-type SARS-CoV-2 particles analyzed by AFM25 and electron microscopy (EM)27. Recent literature on SARS-CoV-2 VLPs reported a size around 100-200 nm in diameter either using AFM7 or EM9.The purified MNE and MNES VLPs were next imaged by AFM in the buffer for size and shape characterization Fig. D, in theWe next aimed at producing fluorescent SARS-CoV-2 VLPs from transfected HEK293T cells, by co-transfecting a M(Cter-GFP) fusion protein together with M, N and E, with a ratio M/M(GFP) of 2:2, supplemented or not with S. That way, M(GFP) incorporation into VLPs was possible and the protein incorporation was verified by Western Blot in both MNE and MNES conditions Fig. D; theref12 to 7.1012 particles/ml were found respectively for M(GFP)NES and M(GFP)NE VLPs suggesting a very efficient production considering that not all the VLPs are labeled with GFP. Interestingly, we were able to differentiate VLPs sizes, measuring diameters of 120 ± 10 nm (mean ± sd) for VLPs without S and 140 ± 20 nm for VLP with S NES and M(GFP)NE VLPs size and approximate concentration using Fluorescence Correlative Microscopy (FCS) Fig. D. GFP(+)15. We directly compared these sizes with the particles released into the cell supernatant from cells only transfected with N(mEOS2). The results are shown in Fig. 16. Dual color fluorescent labeled VLPs were next tried to challenge the limit of fluorescent tags incorporation into the VLPs. Dual color VLPs were produced by co-expression of M(Cter-mCherry) and N(Nter-GFP) with M, N and E supplemented or not with S in transfected HEK293T cells  protein. These photoconvertible VLPs were used to perform Photo-Activated Localization Microscopy (PALM) to provide another estimation of their sizeFurthermore, to estimate the Spike incorporation on fluorescent VLPs, we used the M(GFP)NES fluorescent VLPs to quantify the percentage of GFP labeled VLP incorporating S by immuno-spotting coupled to TIRF microscopy, using an anti-S neutralizing antibody and an AlexaFluor555 secondary antibody Fig. A. Non-spIn order to estimate the incorporation of the other viral proteins N and M with the N(GFP) and M(GFP) tagged proteins, respectively, we performed the same immuno-spotting assay with anti-N and anti-M antibodies on MN(GFP)NES and M(GFP)MNES VLPs Fig. C and qua1,d2,…dn) of VLPs, identified as GFP bright dots, to the center of the cell , established for each cell. As these radii represent the mean longest (r1) and shorter (r2) distances between the cell border and the cell center, it suggests that every VLP distance that will be found in between these two radii corresponds to a VLP located at the border of the cell. On the opposite, every cell center-VLP distance found below r2 indicates that the VLP is inside the cell. We then plot the distribution of the distances (n > 100) for each different condition and plot the average value of the major axis, r1 = 17.6 ± 3.7 µm (mean ± sd) and the minor axis, r2 = 11.6 ± 3.6 µm (mean ± sd) of the different cells (n = 16)  and at 37 °C (d = 13.5 ± 3.5 µm), these mean values being not statistically different . For MNES VLP-M(GFP), at 4 °C, we observed a slight but statistically significant (0.01 < p < 0.04) decrease in the mean VLP-cell center distance (d = 11.8 ± 5 µm) when compared to M(GFP)NE VLP. From Fig. 2, suggesting entry of these VLPs. This is confirmed when observing the M(GFP)NES VLP-cell center distance distribution at 37 °C. In this case the mean distance value is approximately divided by two (d = 6.1 ± 3.0 µm) and is statistically strongly different from all the other distributions (10–33 < p < 2.10–15) we established here. This clearly suggests that the presence of S on the surface of the VLPs induces a preferential endocytosis in A549-hACE2, cells carrying the hACE2 viral receptor.In order to study the functionality of the Spike incorporated in the fluorescent VLPs, we performed an internalization assay of the SARS-CoV-2 M(GFP) or N(GFP) tagged VLPs. To do so, M(GFP)MNE VLPs versus M(GFP)MNES VLPs were incubated with pulmonary A549 cells expressing the human receptor hACE2 NES VLPs internalization for 15 min at 37 °C on live cells. After fixation, z-projection imaging of M(GFP)NES VLPs in A549-hACE2mScarlet1 cells showed the internalization of the M(GFP)NES VLPs on the receptor ACE2. Z-projection imaging at 4 °C showed reduced internalization of the M(GFP)NES VLPs decorating the cell surface on the hACE2mScarlet1 receptor -VLP to mimic viral entry up to the fusion step of the VLP upon cell internalization. In order to explore that question, we used the fluorescent (N-GFP)-VLP internalization assay in A549 pulmonary cells with or without hACE2 receptor . We also observed that the addition of S reduces the incorporation of N and, to a lesser extent M . In this study, the addition of S is not changing the proportion of M or N, which differs from Xu et al.5. Finally, other teams concluded that SARS-CoV-2 VLP production was sufficient when VLPs are based on M, E, and S combination in mammalian cells  or in insect cells . Our study shows that MN is required and that the addition of E makes the yield of VLPs production 2.5 fold better. A combination of M, N, and E was recently also reported for producing SARS-CoV-2 VLPs in plants  and previously in mammalian cells in the case of SARS-CoV28. Overall, it seems that the minimal optimal combination remains MNE/S to form SARS-CoV-2 VLPs. In addition, the formation of these VLPs did not required the presence of a packageable genomic RNA since all these VLPs were produced without. However, it is possible to introduce a pseudo-viral RNA inside the MNES VLPs as recently reported29.As the SARS-CoV-2 pandemic persists around the world, it is necessary to further gather knowledge on this virus, especially in developing tools for studying virus assembly and virus entry in a safe BSL-2 environment (outside a BSL-3 and with non-infectious particles). Therefore, Virus-Like Particle production and characterization, if correctly mimicking the wild-type virus, would be essential to apprehend SARS-CoV-2 assembly mechanisms, and virus entry, in virology or to be used for the development of VLP-based vaccines in immunology. In this study, we established a minimal system for SARS-CoV-2 based VLPs production, requiring MNE/S with an optimized transfected plasmid ratio based on the RNAseq ratio measured in infected cells17 and in human pulmonary A549hACE2 cells18. Consequently, RNAseq data analysis allowed us to establish a plasmid ratio of 3:12:2:5 for M:N:E:S. This ratio increases VLPs production by 1.5 fold for MNE and MNES, as compared with the 3:3:3:5 ratio, with more S incorporation and more mature S2 on the VLP  by liquid AFM imaging. To confirm the size of these particles and to allow the study of SARS-CoV-2 using fluorescence and single molecule localization microscopy, we produced VLP incorporating M or N proteins in fusion with fluorescent or photoconvertible proteins. Using FCS and PALM, we found a 120-140 nm particle size range NES VLPs size in solution NES VLPs incorporated the spike. This number might be underestimated due to the deposit of the MNES VLPs on the glass surface and to the affinity of the S antibody to the Spike, but it tends to support the observation that not all the VLPs produced by the mammalian cells are incorporating the spike. It would be interesting to vary the ratio of Spike expression in the productive cells to see if it changes the level of incorporation in the VLPs, and to try different anti-S neutralizing antibodies. Our results also suggest that the injection of MNES VLP mimicking the virus to animal models should provide adequate antibody responses without being infectious.33 is demonstrating that this type of VLP can package viral RNAs to transduce and express genes in target cells, which reinforces the strong potential of SARS-CoV2 VLPs in vaccine development.Finally, our study has shown that in the presence of overexpressed hACE2 receptors at the cell surface, M(GFP)NES VLPs can recognize the receptor and be endocytosed, confirming that the Spikes incorporated in the VLPs are functional in recognizing the receptor ACE2. Interestingly, a recent articleOverall, we report that the minimal system required for VLPs formation is M, N, and E structural proteins incorporating S and that VLPs can be optimized in production and maturation by mimicking SARS-CoV-2 mRNA ratio of infected cells. Tagging VLPs with fluorescent proteins was possible and could be very useful to image SARS-CoV-2-hACE2 internalization in host cells using single particle fluorescence quantitative microscopy. SARS-CoV-2 VLPs could be useful tools to study the viral life cycle of SARS-CoV-2 in a BSL-2 or as vaccine prototypes.Supplementary Information.


Realgar, a poisonous traditional Chinese medicine, has been shown to cause liver injury when used for long periods or overdoses. However, the underlying molecular mechanisms and therapeutic targets have not been fully elucidated. The aim of this study is to explore the role of autophagy in sub-chronic realgar exposure-induced liver injury. Here, the liver injury model was established by continuously administrating mice with 1.35 g/kg realgar for 8 weeks. 3-methyladenine (3-MA) and rapamycin (RAPA) were used to regulate autophagy. The results showed that realgar induced abnormal changes in liver function, pathological morphology, expression of inflammatory cytokines, and upregulated NLRP3 inflammasome pathway in mouse livers. RAPA treatment (an inducer of autophagy) significantly improved realgar-induced liver injury and NLRP3 inflammasome activation, while 3-MA (an inhibitor of autophagy) aggravated the realgar-induced liver injury and NLRP3 inflammasome activation. Furthermore, we found that realgar-induced NLRP3 inflammasome activation in mouse livers is mediated by ROS. RAPA eliminates excessive ROS, inhibits NF-κB nuclear translocation and down-regulates the TXNIP/NLRP3 axis, consequently suppressing ROS-mediated NLRP3 inflammasome activation, which may be the underlying mechanism of the protective effect of autophagy on realgar-induced liver injury. In conclusion, the results of this study suggest that autophagy alleviates realgar-induced liver injury by inhibiting ROS-mediated NLRP3 inflammasome activation. Autophagy may represent a therapeutic target in modulating realgar-induced liver injury. In the clinic, it has been used for the treatment of acute promyelocytic leukemia (APL) and other cancers . Realgar2S2) . Althoug2S2) . The liv2S2) ,6,7. HowThe nucleotide-binding oligomerization domain (NOD)-like pyrin domain-containing protein 3 (NLRP3) inflammasome, which consists of NLRP3, adaptor apoptosis-associated speck-like protein (ASC), and pro-caspase-1 is deemed as a vital driving factor of various liver diseases and chemical/toxin-induced liver injury . NLRP3 rAutophagy is a process that eliminates damaged organelles and proteins through cytoplasmic degradation . IncreasTherefore, in this study, 3-methyladenine  or rapamycin  was used to regulate autophagy in realgar- induced liver injury mice model to explore the function of autophagy in realgar-induced liver injury and elucidate the relationship between autophagy and NLRP3 inflammasome activation in liver tissues. We aimed to provide insightful viewpoints into the mechanism and therapeutic target of realgar-induced liver injury.® Premix Ex Taq™ II (RR047A) were purchased from TaKaRa Biotechnology Company . Antibodies against NLRP3 (#19771-1-AP), Caspase-1 (#22915-1-AP), thioredoxin , p62 (#18420-1-AP), LC3 (#14600-1-AP), vacuolar protein sorting 34 , the mammalian target of rapamycin , nuclear receptor-κB p65  and Lamin B1 (#12987-1-AP) were purchased from Proteintech . Antibodies against ASC (#67824) and thioredoxin-interacting protein  were obtained from Cell Signaling Technology . Antibodies against IL-1β (#ab254360), IL-18 (#ab207323) and GSDMD (#ab209845) were obtained from Abcam . Pure Proteome Protein A/G Mix Magnetic Beads (#LSKMAGAG02) were purchased from Merck Millipore .Water-processed realgar was purchased from Sanmenxia Yuhuangshan Pharmaceutical Corporation . N-acetylcysteine  was purchased from Sigma . 3-MA  and Rapa  were purchased from TargetMol . Alanine aminotransferase , aspartate aminotransferase , alkaline phosphatase , total bilirubin , lactate lactic dehydrogenase , reactive oxygen species  kits were purchased from Nanjing Jiancheng Bioengineering Institute . Assay kits for urea  and albumin  were obtained from Shenyang BoKe Biological Technology Co. Itd and Shenyang Bolande Trading Co., Ltd. . Mouse γ- glutamyl transferase  ELISA Kits were provided by JIANGSU JINGMEI Biotechnology Co., Ltd. . Mouse serum creatinine  ELISA kits were purchased from Mode Biological Technology Co. Itd . IL-1β (#EK0394) ELISA Array Kits were purchased from Boster Biological Technology Co. Itd . Monodansylcadaverine  was purchased from solarbio . PrimeScript™ RT reagent Kit with gDNA Eraser (#RR820A), TB GreenSix-week-old healthy male Kunming (KM) mice (weighing 20~22 g) were obtained from the Laboratory Animal Center of China Medical University . The experimental mice were housed under SPF conditions under a 12 h light-dark cycle (8:00am–8:00pm) in an air-conditioned room with a room temperature of (25 ± 2) °C and (55 ± 10)% humidity and providing free access to food and water. The experimental protocol was processed according to the National Institute of Health Guide for the Care and Use of laboratory Animals  and approved by the Animal Ethics Committee of China Medical University. All efforts were exerted to minimize the number of mice used and their suffering.After one week of adaptive feeding, all mice were randomly divided into the following eight groups: (1) Control group; (2) Realgar group; (3) NAC group; (4) NAC+Realgar group; (5) 3-MA group; (6) 3-MA+Realgar group; (7) RAPA group, and (8) RAPA+Realgar group. Mice in the Control, NAC, 3-MA and RAPA groups received 0.5% CMC-Na; mice in the Realgar, NAC+Realgar, 3-MA+Realgar and RAPA+Realgar groups received 1.35 g/kg realgar by gavage once daily for 8 consecutive weeks. For the intervention studies, NAC, 3-MA, and RAPA were dissolved in 0.5% dimethylsulfoxide (DMSO). Mice in the NAC and NAC+Realgar groups were injected intraperitoneally with NAC at a dose of 20 mg/kg; mice in the RAPA and RAPA+Realgar groups were injected intraperitoneally with RAPA at a dose of 2 mg/kg; mice in the 3-MA and 3-MA+Realgar groups were injected intraperitoneally with 3-MA at a dose of 15 mg/kg. Mice in the Control and Realgar groups were injected intraperitoneally with 0.5% DMSO. All injections were given every two days. NAC was given for eight weeks, while 3-MA and RAPA were given for three weeks before isoflurane anaesthesia.The liver was removed carefully and rinsed with phosphate buffer. Then, the liver weights were measured after the liver tissues were dried with filter paper to remove the excess solution. The liver index was calculated as follows: liver index% = liver mass (g)/body mass (g) × 100%. The plasma ALT, AST, ALP, γ-GGT, TBIL, ALB, UN, Cr, LDH and IL-1β levels were detected with commercial kits on the basis of the manufacturer’s instructions.w/v) glutaraldehyde buffer. After perfusion for about 20 min, the liver was excised. Cross-sections (5 µm) of the tissue were cut and stained with hematoxylin and eosin (H&E) and then assessed under a microscope . For transmission electron microscopy (TEM) observation, the liver specimen was cut into 1 mm3 in size and fixed in 1% osmium tetroxide. Next, the samples were dehydrated through a graded series of ethanol and acetone and then embedded in the epoxy resin. After being sliced into 1-mm-thick sections and stained with lead citrate, the samples were detected by Hitachi  transmission electron microscope.The heart perfusion-fix technique was used to prepare the specimen for histopathological examination. The PBS was perfused initially to clear the blood from the liver followed by 4% paraformaldehyde and 2.5%  with an excitation wavelength of 502 nm and emission wavelength of 530 nm.The intracellular ROS was determined by detecting the fluorescent intensity of 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). Each liver tissue sample was homogenized in ice-cooled Tris-HCl buffer . The homogenate 190 μL was mixed with 10 μL DCFH-DA working solution (1 mmol/L), and then incubated in the dark at 37 °C for 30 min. Finally, the fluorescence intensity of DCF was assessed using a FLUOstar Omega6 cells/mL. The single-cell suspension was incubated with MDC (50 μM) for 45 min at room temperature avoiding light, followed by measuring with the help of a microplate reader  at the excitation wavelength of 335 nm, and the emission wavelength of 518 nm.The intensity of autophagic vacuoles (AVOs) was measured with MDC. The fresh liver tissue was minced in PBS and digested in 1 mL of trypsin solution at 37 °C for 30 min. Afterwards, 5 mL of DEME was added to terminate digestion. After filtration with sieves, the samples were washed two times using cold PBS and resuspended in binding buffer. The number of viable cells was counted and adjusted to 1 × 10Gapdh, analyzed by the 2−ΔΔCt method and presented as a ratio compared with the control. The primers of target genes were designed by Sangon Biotech  and the sequences are shown in For RNA extraction, total RNAs from mouse liver tissue were extracted by Trizol Reagent  according to the manufacturer’s instructions and the concentration of RNA was calculated. Approximately 1 μg of total RNA from each sample was reverse transcribed to cDNA by the PrimeScript RT reagent kit . The levels of mRNA expression were quantified using SYBR Green PCR Master Mix and an ABI prim 7500 Sequence Detection System . The PCR program included 95 °C for 5 s and then 95 °C for 30 s and 60 °C for 30 s for 40 cycles. Relative expression of target genes was normalized to After treatment, RIPA lysis buffer supplemented with protease and phosphatase inhibitors was used to extract the liver protein samples of mice according to the standard procedure. A BCA protein detection kit  was used for the quantification of protein content. After SDS-PAGE electrophoresis, the protein was transferred onto the PVDF membrane. The membranes were blocked using 5% skim milk for 1 h at room temperature. Then, the membrane was hatched by a specific primary antibody at 4 °C overnight. After washing three times with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies  for 1 h. The following primary antibodies were used: NLRP3 (1:1000), ASC (1:1000), Caspase-1 (1:1000), IL-1β (1:1000), GSDMD (1:1000), NF-κB (1:1000), TRX (1:1000), TXNIP (1:1000), IL-18 (1:1000), P62 (1:1000), LC3B (1:1500), VPS34 (1:1000), mTOR (1:5000), GAPDH (1:3000), and Lambin B1 (1:2500). GAPDH was used as an invariant control for the target proteins. The expressions of nuclear NF-κB proteins were normalized to Lambin B1 as a reference.In order to detect the protein-protein interactions, a Co-IP assay was performed. RIPA lysis of 1 mL was added to 0.05 g of the liver sample, and then homogenized and centrifuged at 12,000 rpm at 4 °C for 10 min. The supernatant was collected and incubated with mouse monoclonal TXNIP antibody or IgG control antibody for 4 h at 4 °C. Then 20 μL of protein A/G beads  were added to the protein-antibody complex and incubated for 2 h at 4 °C. Then, the protein-bead mixture was centrifuged and the supernatant was removed. The beads were washed three times with PBST buffer followed by denaturation with 1 × SDS-containing loading buffer at 100 °C for 5 min. Then, the expressions of captured proteins were assessed by Western blot. TXNIP-binding proteins from protein extract were immunoprecipitated with TXNIP antibodies. NLRP3 and TRX levels in pulled-down proteins were measured using Western blot analysis with NLRP3 and TRX antibody.t-test was used for statistical analysis of two groups. p < 0.05 was considered statistically significant.Data are presented as the mean ± standard error (SEM). IBM SPSS 24.0 software  was used for data analysis. The graphs were made with GraphPad Prism 5.0 software. The Student’s Atg5 mRNA and VPS34, LC3II/I, and P62 proteins were observed in the Realgar group as compared with controls , and the protein expressions mTOR and P62 were significantly lower, while the ratio of LC3II and LC3I was distinctly higher than that of the Realgar group (p < 0.05). In mice treated with 3-MA+Realgar, opposite results were obtained from the RAPA+Realgar group. The mRNA expression of Atg5 was decreased (p < 0.05), and the protein expression of VPS34, the ratio of LC3II and LC3I were down-regulated, whereas the protein expression of P62 was up-regulated (p < 0.05) as compared with the Realgar group. These results have demonstrated that RAPA and 3-MA can efficiently regulate autophagy in realgar-exposed mouse livers.In order to investigate the role of autophagy in realgar-induced liver injury, RAPA and 3-MA, the classical autophagic agonist and antagonist, were used to regulate the autophagy in vivo . In compp < 0.05), and the plasma AST activity of mice exposed to realgar showed an increasing trend (p > 0.05). The ALT, AST, ALP activities and TBIL, UN, Cr, and LDH levels in the plasma of mice in the RAPA+Realgar group were significantly lower than those of the Realgar group . The ALP, γ-GGT activities and TBIL, Cr levels in the plasma of mice in the 3-MA+Realgar group were further increased in comparison to the Realgar group . The level of ALB in mouse plasma was not obviously changed in any group. The results of H&E staining showed that the morphology of hepatocytes in the Realgar group revealed obvious damage . Compared with the control group, the mRNA expression of Tnf-α in the liver tissue of realgar exposed mice tended to increase  treatment (p < 0.05).The mRNA expression of increase B. Howevear group C. The exp < 0.05).The plasma IL-1β level in realgar exposed mice was 4.09 times higher than those of controls, while the IL-1β level apparently dropped in the RAPA+Realgar group  , which suggesting that autophagy enhancement can retard realgar induced NLRP3 inflammasome activation in mouse livers. The changes of NLRP3 inflammasome-related proteins and genes in the 3-MA+Realgar group were opposite to those of the RAPA+Realgar group. The protein expressions of ASC, Caspase-1, IL-1β, GSDMD, N-GSDMD  in the 3-MA+Realgar treatment group, suggesting that inhibition of autophagy aggravated NLRP3 inflammasome activation induced by realgar.The protein expressions of NLRP3, ASC, Caspase-1, IL-1β, GSDMD, N-GSDMD and mRNA expressions of controls A–C, whiccontrols A and therp3, Asc C were al N-GSDMD B and theaspase-1 C were reAs shown in p < 0.05), which indicates that autophagy can eliminate realgar-induced excessive ROS. Several studies reveal that autophagy can indirectly suppress inflammasome activation by removing ROS . In thisp < 0.05). Compared with the Realgar group, the expressions of NF-κBp65 in liver tissues and nuclear in the RAPA+Realgar group were significantly decreased (p < 0.05). The result suggested that enhanced autophagy can inhibit the entry of NF-κBp65 into the nucleus.NF-κB p65 is a vital factor in the initiation of NLRP3 inflammasome activation. Compared with the control group, the total and nuclear protein expression NF-κBp65 in mouse livers were significantly increased (p < 0.05), and reduced interaction between TXNIP and NLRP3 in realgar-induced liver injury mice . As the main organ of arsenic metabolism and accumulation, the liver is easily affected by arsenic toxicity. Epidemiology studies have indicated a close link between chronic arsenic exposure and abnormal liver function, hepatomegaly, hepatoportal sclerosis, liver fibrosis, cirrhosis and liver cancer in humans . MedicinNiuhuang Jiedu tablet, Angong Niuhuang Wan, etc. These realgar-containing TCMs have shown beneficial effects on detoxication, insecticidal, dampness elimination and dispelling phlegm [Many herbal medicines also contain arsenic, such as Salviae Miltiorrhizae, Safflower, Gentiana, Flower of Silktree Albizzia, etc. . Realgarg phlegm . Studiesg phlegm . Excessig phlegm . Autophag phlegm . To dateIn previous studies, we found that mouse exposure to realgar at 1.35 g/kg for 8 weeks exhibits obvious liver injury ,22,23. HRAPA and 3-MA were used to regulate the level of autophagy in the livers of realgar-exposed mice. 3-MA is the most commonly used autophagy inhibitor, and it functions by inhibiting VPS34. There is a report that pointed out that the generation of phosphatidyl-inositol-3-phosphate (PI3P) by VPS34 and the recruitment of PI3P-binding proteins is a critical step for phagophores nucleation and elongation . TherefoNext, we investigated the effect of autophagy on realgar-induced liver injury. It was found that after 8-week realgar exposure, the plasma ALT, ALP, γ-GGT activities and TBIL, UN, Cr, LDH levels are all apparently increased. In particular, the ALP, γ-GGT activities and TBIL levels are far higher than those in the controls, which may indicate that realgar may induce cholestatic liver injury in mouse livers . The resTo elucidate the mechanism by which autophagy inhibits the activation of NLRP3 inflammasome in realgar-induced liver injury mice, we focus on the role of ROS. As we know, ROS is one of the major endogenous danger-associated molecular patterns (DAMPs) that leads to the activation of NLRP3 . In ordeAutophagy may inhibit NLRP3 inflammasome activation by removing excess ROS. In this experiment, RAPA treatment apparently restored the increased ROS levels induced by realgar in mouse livers.Nlrp3, Asc and Il-1β. Therefore, we hypothesized that autophagy may down-regulate the realgar-induced NLRP3 inflammasome activation pathway by inhibiting NF-κB nuclear translocation, thereby alleviating realgar-induced inflammatory injury in mouse livers.NF-κB plays an important role in the priming stage of NLRP3 inflammasome activation. The NF-κB enters into the nuclear and upregulates the expression of inflammasome component NLRP3 and pro-IL-1β. It has been reported that autophagy can directly regulate NF-κB to regulate inflammatory responses . Our preGrowing evidence has shown that TXNIP is an active regulator of NLRP3 . It can Taken together, this study suggests that autophagy may exert a protective effect on realgar-induced liver injury by eliminating excessive ROS, inhibiting NF-κB nuclear translocation and down-regulating TXNIP/NLRP3 axis, consequently inhibiting NLRP3 inflammasome activation in mouse livers.In conclusion, in this study, we demonstrated that autophagy can alleviate realgar-induced liver injury via eliminating excessive ROS, inhibiting NF-κB nuclear translocation and down-regulating the TXNIP/NLRP3 axis, consequently inhibiting ROS-mediated NLRP3 inflammasome activation. The regulation of autophagy would be a potential strategy for preventing and curing realgar-induced liver toxicity, which will be worthy of further study.This report has some limitations. First, the RNA and proteins were extracted from whole livers in this experiment, thus, the analysis cannot discriminate between toxicity effects happening in hepatocytes versus other liver cells. In future studies, purified hepatocytes from the livers should be used to avoid confounding effects resulting from other cell types. Second, the suggested autophagy may exert a protective effect on realgar-induced liver injury via down-regulating TXNIP/NLRP3 axis, which needs confirmation by knocking down TXNIP.


A strong association between chemoreflex hypersensitivity, disordered breathing, and elevated sympathetic activity has been shown in experimental and human heart failure (HF). The contribution of chemoreflex hypersensitivity in HF pathophysiology is incompletely understood. There is ample evidence that increased peripheral chemoreflex drive in HF with reduced ejection fraction  leads to pathophysiological changes in autonomic and cardio-respiratory control, but less is known about the neural mechanisms mediating cardio-respiratory disturbances in HF with preserved EF . Importantly, it has been shown that activation of the central chemoreflex worsens autonomic dysfunction in experimental HFpEF, an effect mediated in part by the activation of C1 catecholaminergic neurons neighboring the retrotrapezoid nucleus (RTN), an important region for central chemoreflex control of respiratory and autonomic function. Accordingly, the main purpose of this brief review is to discuss the possible role played by activation of central chemoreflex pathways on autonomic function and its potential role in precipitating disordered breathing in HFpEF. Improving understanding of the contribution of the central chemoreflex to the pathophysiology of HFpEF may help in development of novel interventions intended to improve cardio-respiratory outcomes in HFpEF. Heart failure (HF) is a complex and heterogeneous clinical condition that affects an estimated 64 million people worldwide . HF in iAlthough, while HFrEF patients had benefit from complex pharmacological schemes and electronic-assisted devices for outcome improvements, these approaches showed low salutary potential when used in patients with HFpEF. Indeed, the complexity and heterogeneity of HFpEF may be one key reason for the poor track record of HFpEF clinical trials aiming to improve breathing disturbances in these patients. Thus, a more robust classification of the wide HFpEF phenotypic spectrum, particularly with a thoroughly characterization of chemoreflex drive, may help not only to improve HFpEF clinical management but also may shed lights into future therapeutic developments. In addition, there is an urgent need for new experimental HFpEF models that better reflects some of the specific pathological hallmarks of each (sub)type of human HFpEF for deeper understanding of the pathophysiological mechanisms involved in disease progression/maintenance .While there is conclusive evidence that demonstrates activation of the sympathetic nervous system (SNS) in HFrEF related with a chronically potentiated chemoreflex activity, there is limited information regarding chronic SNS activation in HFpEF . HoweverIn addition to autonomic dysfunction, sleep disordered breathing (SDB)  is highly prevalent in HFpEF patients and is associated with poor prognosis and increased mortality . SDB refImportantly, both cardiac autonomic imbalance and disordered breathing patterns in HF have been linked to altered chemoreflex function . Indeed,HFpEF is characterized in part by left ventricular diastolic dysfunction. Ventricular filling is progressively impaired as a result of slow left ventricular relaxation and increased left ventricular stiffness . This reThe presence of disordered breathing  is associated with increased mortality in HF patients . Most stChronic activation of the SNS and inhibition of the parasympathetic nervous system (PNS) are associated with poor outcomes in HFpEF . While aThe carotid body (CB) chemoreceptors are the main peripheral arterial chemoreceptor in humans, sensing a wide variety of blood borne stimuli . Upon activation, the CB chemoreflex drives a highly coordinated cardiorespiratory response characterized by increased ventilation and sympathetic nerve activity . In expeKristen et al. showed exaggerated increases in renal sympathetic nerve activity in response to hypercapnic stimulation in non-ischemic HF rats 2/pH homeostasis  and a proton-modulated potassium channel (TASK-2) (The RTN is defined as a set of neurons that: express the isotype 2 of vesicular glutamate transporter (VGLUT2), express the transcription factor Phox2b, lack catecholaminergic biosynthetic enzymes, and express the Neurokinin 1 receptor (NK1), and Neuromedin B . RTN che(TASK-2) . During (TASK-2) .2 fluctuations. Indeed, it has been shown that CB stimulation/inhibition modulates central chemoreceptors sensitivity to CO2/H+   . In addipopneas) . Importapopneas) . Furtherpopneas) . Therefo2/H+ via inhibition of a pH-sensitive inwardly rectifying K+ channel and release one or several putative molecules, which can regulate RTN neuron activity  elicited a two-fold increase in lumbar sympathetic nerve discharge and this was associated with a ∼70% increase in RVLM activity (i.e. sympatho-excitatory neurons)  . Yet theneurons) , and stineurons) . These oneurons) . Interesneurons) , but theIn HFpEF rats, partial lesion of RVLM-C1 neurons reduces the deleterious effects of central chemoreflex activation on cardiac autonomic imbalance, breathing disorders and cardiac function . Also, RAs mentioned previously, central chemoreflex sensitivity is enhanced in HFpEF and is associated with sympatho-excitation and cardiorespiratory dysfunction. While the precise mechanism(s) responsible for central chemoreflex sensitization, autonomic imbalance, and respiratory disorders in HFpEF are not yet clearly established, many studies suggest that neuroinflammation and oxidative stress observed in autonomic brain regions play a crucial role in cardiac pathophysiology in HF independent of its etiology (HFrEF vs. HFpEF). Persistent/chronic neuroinflammation and oxidative stress create a complex positive feedback loop that induces and maintains elevated sympathetic activity in HFpEF mediated in part by central chemoreflex hypersensitivity. Indeed, high levels of reactive oxygen species (ROS) have been linked to sympatho-excitation in HF  and our 2/H+ induces astrocyte-mediated release of pro-inflammatory cytokines in brainstem areas related to respiratory/sympathetic regulation which may contribute to enhanced SNS outflow and de-stabilize respiratory control. Understanding the complex interplay of the mechanisms involved in central chemoreflex hypersensitivity, increased sympathetic activity, and disordered breathing is an important step toward the development of novel interventions and improved clinical outcomes in HFpEF.Finally, as mentioned earlier, astrocytes contribute to ventilatory drive under hypercapnia through purinergic signaling . ImportaThe incidence and prevalence of HFpEF relative to HFrEF is rising at a rate of 10% every 10 years and will become the most common form of HF within the next decade . There a


Toxoplasma gondii virus-like particles displaying AMA1 of T. gondii and evaluated their diagnostic potential. We found that AMA1 VLPs were highly sensitive and reacted with the sera acquired from mice infected with either T. gondii ME49 or RH strains. The overall IgG and IgM antibody responses elicited by AMA1 VLPs were substantially higher than those induced by the conventionally used T. gondii lysate antigen (TLA). Importantly, AMA1 VLPs were capable of detecting parasitic infection with T. gondii RH and ME49 as early as 1 week post-infection, even when mice were exposed to low infectious doses . AMA1 VLPs also did not cross-react with the immune sera acquired from Plasmodium berghei-infected mice. Compared to TLA, stronger antibody responses were induced by AMA1 VLPs when tested using T. gondii-infected human sera. The sensitivities and specificities of the two antigens were substantially different, with AMA1 VLPs demonstrating over 90% sensitivity and specificity, whereas these values were in the 70% range for the TLA. These results indicated that AMA1 VLPs can detect infections of both T. gondii ME49 and RH at an early stage of infection caused by very low infection doses in mice, and these could be used for serological diagnosis of human toxoplasmosis.Toxoplasmosis diagnosis predominantly relies on serology testing via enzyme-linked immunosorbent assay (ELISA), but these results are highly variable. Consequently, various antigens are being evaluated to improve the sensitivity and specificity of toxoplasmosis serological diagnosis. Here, we generated  Toxoplasma gondii is an intracellular parasite and the etiologic agent causing the zoonotic disease toxoplasmosis, which can be transmitted to a wide array of warm-blooded vertebrates by ingestion of cyst-contaminated water, raw meat, or other food products [T. gondii infection in humans tends to result in non-specific symptoms in the immunocompetent population and the disease manifestation was also reported to be associated with the parasite’s clonal lineage [T. gondii infection can lead to transmission of the parasite to the fetus which ultimately results in miscarriage or stillbirth. Other symptoms associated with congenital toxoplasmosis include cerebral calcification, retinal choroiditis, hydrocephalus, and others [products . Currentproducts . T. gond lineage . Howeverd others . Due to d others . For theT. gondii infection, but they are frequently used as initial screening steps to identify the location of the affected area and the extent of the damage done [T. gondii diagnosis are increasing in popularity. For example, real-time PCR is a highly sensitive method that can detect extremely low concentrations of parasitic nucleic acid. One major advantage of molecular testing methods over conventional serology-based assays is that this method can confirm the presence of parasites in fetuses [To date, a wide array of toxoplasmosis diagnostic methods have been developed. Imaging techniques such as magnetic resonance imaging or ultrasonography are not specific for age done . Molecul fetuses .T. gondii-specific IgG and IgM were used as the basis for commercially manufactured diagnostic kits. However, there are issues with the serological testing methods. Due to the fact that IgM levels persist for several months or even years after infection, this can result in the false-positive classification of chronic infections as acute infections [T. gondii tachyzoite lysate antigen (TLA), but detection is usually possible after 2 to 3 months [Serology-based assays are the most commonly used toxoplasmosis diagnostic method which relies on measuring the serum IgM and IgG antibody responses. Notable examples of these methods include the Sabin and Feldman dye test (DT), enzyme-linked immunosorbent assays (ELISA), chemiluminescence assays (CIA), indirect fluorescent antibody test (IFAT), immunosorbent agglutination assays (ISAGA), the latex agglutination test (LAT), the serum IgG avidity test, and western blotting (WB) ,8. In pafections . Parasit3 months . In sero3 months ,12,13.T. gondii apical membrane antigen 1 (AMA1) expressed in Escherichia coli has been explored [T. gondii than prokaryote-derived proteins. Furthermore, baculovirus expression systems can be easily scaled to industrial levels at low costs, as well as being inherently safe for humans [T. gondii AMA1 in insect cells and evaluated the feasibility of this approach. Specifically, we assessed the murine and human serum response to the T. gondii AMA1 VLPs and compared the findings to conventional TLA-based ELISA.Although limited in number, several studies have explored the use of virus-like particles (VLPs) as a diagnostic tool. VLP-based ELISA was able to differentiate between West Nile virus and St. Louis encephalitis virus infections . The diaexplored . Howeverexplored . Baculovexplored  thus morr humans . To furtAll animal experiments in this study were carried out under the guidelines set out by Kyung Hee University IACUC. All experimental protocol involving animals was reviewed and approved by the IACUC (permit number: KHSASP-20-165). Blood collection was performed under mild anesthesia, which was induced and maintained with ketamine hydrochloride and xylazine. All efforts were made to minimize the number of animals used in the experiment as well as their suffering. Human subject research approval was obtained from the Kyung Hee University Institutional Review Board (KHSIRB-20-178(EA)). The need for consent was waived by the ethics committee.Toxoplasma gondii RH, ME49, and P. berghei ANKA strains were maintained in seven-week-old female BALB/c mice obtained from NARA Biotech  following the methods described previously [Spodoptera frugiperda Sf9 insect cells were cultured in serum-free SF900-II medium  and subsequently used to generate recombinant baculovirus (rBV) and virus-like particles (VLPs). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, IgA, IgM, and goat anti-human IgG antibodies were purchased from Southern Biotech  and Invitrogen . Influenza-infected sera were acquired by intranasally infecting mice with 50 plaque-forming units (pfu) of the H3N2 influenza virus (A/Hong Kong/1968). Sera were collected 4 weeks post-infection. Monoclonal influenza M1 antibody was purchased from Abcam . Toxoplasmosis patient sera (n = 17) were confirmed and provided by Chungnam National University  and Seoul National University .eviously ,22. SpodT. gondii apical membrane antigen 1 (AMA1) was conducted as previously described [EcoRI and XhoI restriction enzyme sites were PCR-amplified and transformed into pFastBac plasmid. After subsequent cloning into DH10Bac competent cell, bacmid DNA was acquired for rBV and VLP production. The baculoviruses and VLPs expressing AMA1 and influenza M1 were prepared as previously described [Cloning of escribed . Brieflyescribed . In brieT. gondii ME49 , RH , or P. berghei (5.0 × 103 per 100 μL in PBS). The ME49 and RH strains of T. gondii were inoculated using oral gavage, while P. berghei were inoculated through intraperitoneal (IP) injection. Blood samples were collected through retro-orbital plexus puncture. From RH-infected mice, sera were collected at 1 and 2 weeks post-infection (wpi). For ME49 infection, sera were acquired at 1, 2, 4, and 8 wpi. Mice were infected twice with P. berghei at 2-week intervals, and blood samples were drawn 1 week after the second infection. Sera were stored at −20 °C until use.Female BALB/c mice were orally infected with T. gondii RH lysate antigen (TLA) at concentrations of 4 μg/mL in carbonate coating buffer. After overnight coating at 4 °C, plates were blocked for 1 h at 37 °C using 0.2% gelatin in PBS with 0.05% Tween-20. A total of twenty human sera samples were acquired, with three from naïve individuals and seventeen from toxoplasmosis-positive patients. Human (n = 20) or mice sera (n = 32) were diluted (1:100 dilution in PBS) and inoculated into respective wells. M1 monoclonal antibodies were diluted 1:2000 in PBS. After incubating the plates for 2 h at 37 °C, 100 HRP-conjugated anti-mouse IgG, IgM, and IgA or anti-human IgG secondary antibodies were inoculated into respective wells (1:2000 dilution in PBS). Following 1 h incubation at 37 °C, o-phenylenediamine  was dissolved in a citrate substrate buffer with H2O2. Reactions were stopped with H2SO4 and optical density readings at 492 nm were measured using a microplate reader . ELISA cut-off value for the serum IgG, IgM, and IgA antibody responses was determined to be a mean OD + 3 standard deviation of negative sera. TLA used in the present study was validated by comparing its mouse serum ELISA results to that of commercialized TLA . For human sera, the relative sensitivity and specificity of both TLA and AMA1 VLPs were determined using the formula previously described elsewhere [Serum antibody responses were evaluated using enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well immunoplates were coated with either AMA1 VLPs or lsewhere . Brieflypost hoc test or Student’s t-test using GraphPad Prism version 6.0 . p-values (* p < 0.05) were considered statistically significant.All parameters were recorded for individuals within all groups. All data were presented as mean ± SD and statistical significances between groups were analyzed by one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison T. gondii were generated. The structural components of the VLPs were depicted using an illustration, with AMA1 antigen spikes protruding through the lipid bilayer (T. gondii (ME49)-infected mouse sera , irrespective of the infection dose. The overall inductions were strikingly higher than the ME49 positive control sera.To confirm that AMA1 VLPs could be used against other clonal lineages, as 1 wpi A–C. SpecT. gondii-specific IgM responses against TLA and AMA1 VLPs were determined using the sera acquired from ME49-infected mice. Antigen-specific IgM levels against TLA were consistent throughout the 8-week infection period. Contrary to this, IgM responses against AMA1 were significantly greater than those elicited against TLA at all time points irrespective of infection dosage . Overall, the IgM antibody responses to AMA1 VLPs were consistently maintained at higher levels throughout the 8-week infection period than those elicited against the TLA . Interestingly, there were several cases of false-negative results when ELISA was performed using TLA. Specifically, five patient samples had IgG OD values resembling that of naïve human sera results. Contrary to this, false-negative cases were not detected when sera were reacted with AMA1 VLPs. As indicated in To confirm whether the findings observed from murine sera were concordant with humans, toxoplasmosis-positive human sera (n = 17) were acquired and ELISA was performed. As expected, sera acquired from individuals with previous MA1 VLPs . ConsistSensitivity and specificity of ELISA test for serodiagnosis of toxoplasmosis using TLA and AMA1 VLPs. The sensitivity and specificity of each antigen indicated in the table were calculated as described in the Material section and determined using human sera by ELISA.T. gondii AMA1 VLPs can sufficiently interact with toxoplasmosis-positive sera, thus revealing an alternative to the TLA or other recombinant antigens. Our findings revealed that potent IgM and IgG responses to both types I and II T. gondii clonal lineages could be elicited using AMA1 VLPs. Moreover, the responses were highly parasite-specific as cross-reaction with the rodent malaria sera was not observed. Human sera ELISA results were reflective of the data acquired from mice sera and confirmed the possibility of developing AMA1 VLPs as a serodiagnostic tool.VLPs have been applied as serodiagnostic tools for several infectious diseases of viral origin ,25,26. TEscherichia coli. Due to the fact that mammalian-like glycosylation and other PTMs are lacking in E. coli, this may have strong implications when it comes to an accurate diagnosis. For example, in the case of a Strongyloides stercoralis infection diagnosis, removing the carbohydrate moieties from antigens prior to ELISA-based serodiagnosis resulted in reduced seroreactivity, reduced optical density reading values, and increased the number of cross-reaction which contributed to erroneous results [E. coli, VLPs produced in insect cells possess PTMs that are much closer to mammals, and therefore, carbohydrate-associated shortcomings would not affect serodiagnosis. This feature of the AMA1 VLPs may have contributed to the high specificity and sensitivity reported in our study. As with other organisms, a plethora of PTMs occurs in T. gondii. For example, both N- and O-linked glycosylations were observed in numerous T. gondii proteins including the rhoptries, micronemal proteins, surface antigens, and others [T. gondii is beyond the scope of this study, we speculate that these cellular processes are similar between the two. This notion is corroborated by the result of our previous T. gondii VLP vaccine studies [T. gondii in mice.The major drawback of using TLA-based ELISA is the lack of specificity and sensitivity resulting from its preparation method. Given the inter-laboratory heterogeneity in the TLA preparation method, the antigen lysates are prone to contain various forms of contaminants originating from eukaryotic cells, culture media, or others. This in turn renders standardizing the TLA-based ELISA somewhat arduous. Resultantly, recombinant antigens have emerged to replace the conventional TLA as they are much easier to standardize . To date results . Unlike d others . Glycosy studies ,32,33, wT. gondii IgM titers were reported to span for months after infection [T. gondii-specific IgM responses against ME49 strains were detected even after 8 wpi. Similar results were detected for IgG as these were detected as early as 1 wpi and were induced to high levels. The specificity and sensitivity of the AMA1 VLPs closely resembled those of other recombinant antigens reported in the aforementioned studies [T. gondii and other parasites, such as Neospora caninum, Hamondia spp., and Eimeria spp. [T. gondii and Plasmodium spp. Share a highly conserved arrangement of 16 cysteine residues, which has a large influence on cross-reactivity [T. gondii and P. berghei may occur to an extent. NCBI Protein BLAST analysis revealed 71% amino acid homology between AMA1 antigens of T. gondii (AF AF010264.1) and P. berghei (XM_034564734.1). Surprisingly, cross-reaction between the two was not detected at all and P. berghei immune sera failed to interact with T. gondii AMA1 VLPs. Meanwhile, all of the toxoplasmosis-positive sera reacted with the T. gondii AMA1 VLPs, thus signifying its high species-specificity. One of the noticeable differences in AMA1 of T. gondii and Plasmodium spp. is the PTM, especially palmitoylation. In the AMA1, a total of 18 cysteine residues are present: 16 conserved regions that likely form disulfide bridges, 1 in the signal peptide region, and the last cysteine residue located at position 504 near the cytoplasmic tail. However, the last cysteine residue where palmitoylation occurs in T. gondii is not conserved across multiple members of the Plasmodium spp., including P. berghei [T. gondii ME49 cysts, while human samples were acquired after natural infection with an unknown infection dose. Due to the fact that mice were infected with a higher dose of T. gondii than they would acquire upon natural exposure to the parasite, the antibody’s induction may have occurred to a greater extent. Consistent with this notion, a positive correlation between T. gondii ME49 infection dose and parasite-specific antibody induction in mice was reported in our previous study [T. gondii parasite-specific antibodies circulating in hosts most probably waned over time, thereby resulting in low antibody responses.Multiepitope-based recombinant antigen serodiagnosis is also emerging . Here, Dnfection . In line studies ,28,29. Sria spp. ,37. AMA1activity ,39. Base berghei . Investi berghei ,42, it wus study . The durT. gondii strain used for infection, the results of Ferra et al. are similar to the findings observed in our study, thus further validating the diagnostic potential of AMA1 VLPs. AMA1 VLPs were highly T. gondii-specific and were capable of detecting multiple strains of T. gondii clonal lineages. Infections with RH and ME49, belonging to T. gondii type I and II clonal lineages respectively, were successfully detected using the AMA1 VLPs. However, clonal lineage type III and other exotic strains were not tested and this remains to be evaluated in the future. Additionally, given the small human toxoplasmosis serum samples, further studies need to be conducted with a greater sample size to increase statistical power and identify potential outliers.Recombinant AMA1 has been evaluated as a serodiagnostic biomarker for toxoplasmosis. Recently, in a study by Ferra et al., the authors reported that the full-length AMA1 antigen demonstrated high specificity and sensitivity while serving as a better diagnostic indicator of toxoplasmosis than the N- or C-terminal AMA1 fragments . Apart fT. gondii infection incurred by as low as ten cysts (T. gondii-specific IgG response were observed as early as 1 wpi. This has important implications as it was reported from numerous human toxoplasmosis outbreaks that the parasite incubation period ranged from seven to thirty days [T. gondii AMA1 VLPs antigen possesses higher specificity and sensitivity than TLA, and can also detect T. gondii infections at low doses. Further development and standardization of this antigen could enable rapid and accurate diagnosis of toxoplasmosis.One major limitation of ELISA and antibody-based serology testing is the fact that the exact time of infection cannot be accurately discerned. As with other serology-based assays, AMA1 VLPs reported in our study could not address whether the infection is at an acute or chronic stage, as strong IgM and IgG responses were elicited at both infection time points. However, there are several advantages to using AMA1 VLPs over TLA. First, AMA1 VLPs were capable of detecting en cysts A. Althourty days . Based oT. gondii can detect infections of both T. gondii ME49 and RH, even at an early stage of infection caused by very low infection doses in mice. High sensitivity and specificity were found using T. gondii-infected human sera, indicating AMA1 VLPs could be used for serological diagnosis of human toxoplasmosis.Virus-like particles displaying AMA1 of


Virus-like particles (VLPs) are nanoplatforms comprised of one or more viral proteins with the capacity to self-assemble without viral genetic material. VLPs arise as promising nanoparticles (NPs) that can be exploited as vaccines, as drug delivery vehicles or as carriers of imaging agents. Engineered antibody constructs, namely single-chain variable fragments (scFv), have been explored as relevant molecules to direct NPs to their target. A vector containing the scFv of an antibody, aimed at the human epidermal growth factor receptor 2 (HER2) and fused to the human immunodeficiency virus (HIV) protein gp41, was previously constructed. The work herein describes the early results concerning the production and the characterization of HIV-1-based VLPs expressing this protein, which could function as potential non-toxic tools for transporting drugs and/or imaging agents. Virus-like particles (VLPs) are widely recognized for their immunogenic features but their flexibility allows them to be employed for a myriad of applications . Lately,Within the wide range of VLPs that are currently under development, HIV-1-based VLPs have been gaining relevance due to their pertinence as vaccine candidates ,10. The In previous work, we successfully constructed a plamid harboring a scFv of trastuzumab, a well-characterized anti-human epidermal growth factor receptor 2 (HER2) antibody, as a targeting moiety fused to the HIV viral protein gp41 and tagged with hemagglutinin (HA) . The val® Stable Competent Escherichia coli, and the cells were transformed by electroporation, in accordance with the protocol described by Lessard -1,3-benzene disulfonate) assay. SK-BR-3 and MDA-MB-231 cells were plated at a density of 1.0 × 106 cells/plate on 10 cm plates, transiently transfected with pX1665 and pMDLg/pRRE at a 1:1 ratio and the VLP collection occurred at 24 h and 52 h post-transfection, as per the manufacturer’s protocol for lentiviral production [rev, was added to the transfection protocol due to the relevance of the Rev protein in the transcription of structural genes [6 cells/well into 6-well plates, transient transfection with pX1665, pMDLg/pRRE and pRSV-REV at 1:1:1, 2:1:1, 5:1:1 and 10:1:1 ratios with the VLP collection taking place at 48 h post-transfection  was attained via crude harvested supernatants using a ratio of 1:1:1 with 1.0 × 106 cells/well plated into the 6-well plate with collection at 48 h post-transfection (The validation of the constructed VLPs and the determination of p24 concentration was accomplished by ELISA. Optimization of the production of the HIV-1-based VLPs addressed the ratio of the total DNA, transfected cell conditions and the time of the supernatant recovery. The yields obtained through the different protocols were compared, and the highest number of VLPs (1.41 × 10sfection . This prFollowing the production and the optimization of HER2-specific HIV-1-based VLPs, the Western Blot method was performed to determine whether the anti-HER2 scFv is expressed in the constructed VLPs. The molecular weight of the fusion protein is about 49 kDa B displayLastly, a cytotoxicity assay was conducted to assess the antiproliferative properties of the constructed VLPs in HER2-negative and HER2-positive cancer cell lines . Two breThe growing necessity to develop efficient therapies for diseases, such as cancer has spurred the design of novel nanoplatforms aimed at targeted therapy. VLPs are nanoscale platforms comprised of assembled viral proteins, which lack the viral genome, have emerged as an alternative to conventional nanoparticles because of their biocompatibility, stability, and their ability to target certain cells and tissues in order to deliver their cargo in a precise manner ,3,4. VLPAmong the different types of VLPs, HIV-1-based VLPs are particularly promising because self-assembly can be achieved with just one polyprotein . The abiIn this work, we aimed to design HIV-1-based VLPs that are directed at HER2 because of its well-established role in cancers, such as breast cancer  and gastE. coli, HEK-293T cells were seeded at different cell densities and transiently transfected with different plasmid ratios; the VLPs were then harvested at different times post-transfection for optimization. It was demonstrated that a density of 1.0 × 106 cells/well in 6-well plates with a 1:1:1 plasmid ratio and a VLP collection at 48 h post-transfection were the optimal conditions to obtain the highest HER2-specific HIV-1-based VLP yields in vitro. The technology herein described serves as a starting point to produce these VLPs and prompts further studies, which are focused on both scalability and cost-effectiveness.We used a previously constructed vector containing the scFv of an anti-HER2 antibody, trastuzumab, fused to the HIV viral protein gp41  for co-tAfter establishing the protocol for VLP production, we proceeded to the characterization of the constructed VLPs to assess whether the produced VLPs were correctly assembled and displayed the expected characteristics. We started by performing a Western Blot to evaluate the presence of the protein of interest. The expected molecular weight of the fusion protein, 47.1 kDa, was previously determined , and theThe goal of this study was accomplished as the innovative VLP was successfully constructed. The research of VLPs in precise medicine is growing exponentially. Therefore, we believe that this fast and systematic approach to the production of retargeted VLPs can have a positive impact in the scientific community.