Morphological changes and functional circRNAs screening of rabbit skeletal muscle development.

BACKGROUND: The temporal expression pattern of circular RNAs (circRNAs) across developmental stages is essential for skeletal muscle growth and functional analysis. However, there are few analyses on the potential functions of circRNAs in rabbit skeletal muscle development. RESULTS: Initially, the paraffin sections showed extremely significant differences in the diameter, number, area and density of skeletal muscle fibers of the fetus, child, adult rabbit hind legs (P < 0.01). Then, RNA-seq libraries of these three stages were constructed. A total of 481 differentially expressed circRNAs (DE-circRNAs) and 5,658 differentially expressed genes (DEGs) were identified. Subsequently, DE-circRNAs, whose host genes were DEGs or non-DEGs, were analyzed by GO respectively. In the fetus vs. child group, up-regulated DE-circRNAs (whose host genes were DEGs) were related to muscle fiber structure, and down-regulated ones were related to mitosis. The up-regulated DE-circRNAs (whose host genes were non-DEGs) were involved in enzyme activity, methylation and glycosylation, and the down-regulated ones were involved in mitosis and catabolism. In the fetus vs. adult group, the up-regulated DE-circRNAs (whose host genes were DEGs) were related to skeletal muscle basic structure, and the down-regulated ones were also associated with cell proliferation. But the up-regulated DE-circRNAs (whose host genes were non-DEGs) were connected with regulation of histone ubiquitination, chromatin and organelles. The down-regulated DE-circRNAs were connected with the catabolism processes. In addition, novel_circ_0022663 and novel_circ_0005489, which might have coding potential, and novel_circ_0004210 and novel_circ_0001669, which might have miRNA sponge capability, were screened out. CONCLUSIONS: In this study, hind leg muscles of fetus, child and adult rabbits were collected for paraffin section and RNA-seq to observe the structural changes of skeletal muscle and obtain circRNA expression profiles at different stages. These data provided a catalog of circRNAs related to muscle development in New Zealand rabbits, allowing us to better understand the functional transitions in mammalian muscle development.

Low-affinity but high-avidity interactions may offer an explanation for IgE-mediated allergen cross-reactivity.

BACKGROUND: Allergy is a global disease with overall frequencies of >20%. Symptoms vary from irritating local itching to life-threatening systemic anaphylaxis. Even though allergies are allergen-specific, there is a wide range of cross-reactivities (eg apple and latex) that remain largely unexplained. Given the abilities of low-affinity IgG antibodies to inhibit mast cells activation, here we elucidate the minimal affinity of IgE antibodies to induce type I hypersensitivity. METHODS: Three mature (high-affinity) IgE antibodies recognizing three distinct epitopes on Fel d 1, the major cat allergen, were back-mutated to germline conformation, resulting in binding to Fel d 1 with low affinity. The ability of these IgE antibodies to activate mast cells in vitro and in vivo was tested. RESULTS: We demonstrate that affinities as low as 10-7  M are sufficient to activate mast cells in vitro and drive allergic reactions in vivo. Low-affinity IgE antibodies are able to do so, since they bind allergens bivalently on the surface of mast cells, leading to high-avidity interactions. CONCLUSIONS: These results suggest that the underlying mechanism of allergen cross-reactivity may be low-affinity but high-avidity binding between IgE antibodies and cross-reactive allergen.

Major findings and recent advances in virus-like particle (VLP)-based vaccines.

Virus-like particles (VLPs) have made giant strides in the field of vaccinology over the last three decades. VLPs constitute versatile tools in vaccine development due to their favourable immunological characteristics such as their size, repetitive surface geometry, ability to induce both innate and adaptive immune responses as well as being safe templates with favourable economics. Several VLP-based vaccines are commercially available including vaccines against Human Papilloma Virus (HPV) such as Cervarix®, Gardasil® & Gardasil9® and Hepatitis B Virus (HBV) including the 3rd generation Sci-B-Vac™. In addition, the first licensed malaria-VLP-based vaccine Mosquirix™ has been recently approved by the European regulators. Several other VLP-based vaccines are currently undergoing preclinical and clinical development. This review summarizes some of the major findings and recent advances in VLP-based vaccine development and technologies and outlines general principles that may be harnessed for induction of targeted immune responses.

Vaccine against peanut allergy based on engineered virus-like particles displaying single major peanut allergens.

BACKGROUND: Peanut allergy is a severe and increasingly frequent disease with high medical, psychosocial, and economic burden for affected patients and wider society. A causal, safe, and effective therapy is not yet available. OBJECTIVE: We sought to develop an immunogenic, protective, and nonreactogenic vaccine candidate against peanut allergy based on virus-like particles (VLPs) coupled to single peanut allergens. METHODS: To generate vaccine candidates, extracts of roasted peanut (Ara R) or the single allergens Ara h 1 or Ara h 2 were coupled to immunologically optimized Cucumber Mosaic Virus-derived VLPs (CuMVtt). BALB/c mice were sensitized intraperitoneally with peanut extract absorbed to alum. Immunotherapy consisted of a single subcutaneous injection of CuMVtt coupled to Ara R, Ara h 1, or Ara h 2. RESULTS: The vaccines CuMVtt-Ara R, CuMVtt-Ara h 1, and CuMVtt-Ara h 2 protected peanut-sensitized mice against anaphylaxis after intravenous challenge with the whole peanut extract. Vaccines did not cause allergic reactions in sensitized mice. CuMVtt-Ara h 1 was able to induce specific IgG antibodies, diminished local reactions after skin prick tests, and reduced the infiltration of the gastrointestinal tract by eosinophils and mast cells after oral challenge with peanut. The ability of CuMVtt-Ara h 1 to protect against challenge with the whole extract was mediated by IgG, as shown via passive IgG transfer. FcγRIIb was required for protection, indicating that immune complexes with single allergens were able to block the allergic response against the whole extract, consisting of a complex allergen mixture. CONCLUSIONS: Our data suggest that vaccination using single peanut allergens displayed on CuMVtt may represent a novel therapy against peanut allergy with a favorable safety profile.

A Single Monoclonal Antibody against the Peanut Allergen Ara h 2 Protects against Systemic and Local Peanut Allergy.

BACKGROUND: Peanut allergy is the most prevalent and dangerous food allergy. Peanuts consist of a large number of different allergens and peanut-allergic patients are frequently sensitized to multiple allergens. Hence, conventional desensitization approaches aim at targeting as many allergens as possible. METHODS: The monoclonal anti-Ara h 2 antibody (mAb) was produced by hybridoma cells derived from WT BALB/c mice after immunization with a vaccine based on virus-like particles coupled to Ara h 2. BALB/c mice were sensitized intraperitoneally with peanut extract absorbed to alum and mAbs were applied i.v. Challenge was performed the next day with the whole peanut extract intravenously and via skin prick test. RESULTS: Here we show in peanut-allergic mice that a single high-affinity mAb specific for Ara h 2 is able to block systemic and local allergic reactions induced by the complex peanut extract. We confirm in vitro binding of the mAb to the inhibitory low-affinity FcγRIIb receptor using a sensitive biosensor and demonstrate in vivo that protection was dependent on FcγRIIb. CONCLUSION: A single mAb specific for Ara h 2 is able to improve local and systemic allergic symptoms induced by the whole allergen mixture.

Controlled re-activation of epigenetically silenced Tet promoter-driven transgene expression by targeted demethylation.

Faithful expression of transgenes in cell cultures and mice is often challenged by locus dependent epigenetic silencing. We investigated silencing of Tet-controlled expression cassettes within the mouse ROSA26 locus. We observed pronounced DNA methylation of the Tet promoter concomitant with loss of expression in mES cells as well as in differentiated cells and transgenic animals. Strikingly, the ROSA26 promoter remains active and methylation free indicating that this silencing mechanism specifically affects the transgene, but does not spread to the host's chromosomal neighborhood. To reactivate Tet cassettes a synthetic fusion protein was constructed and expressed in silenced cells. This protein includes the enzymatic domains of ten eleven translocation methylcytosine dioxygenase 1 (TET-1) as well as the Tet repressor DNA binding domain. Expression of the synthetic fusion protein and Doxycycline treatment allowed targeted demethylation of the Tet promoter in the ROSA26 locus and in another genomic site, rescuing transgene expression in cells and transgenic mice. Thus, inducible, reversible and site-specific epigenetic modulation is a promising strategy for reactivation of silenced transgene expression, independent of the integration site.

An unexpected protective role of low-affinity allergen-specific IgG through the inhibitory receptor FcγRIIb.

BACKGROUND: Induction of allergen-specific IgG antibodies is a critical parameter for successful allergen-specific immunotherapy. IgG antibodies can inhibit IgE-mediated mast cell activation through direct allergen neutralization or through the inhibitory receptor FcγRIIb. The affinity of IgE antibodies to the allergen has been shown to be critical for cellular activation. OBJECTIVE: Here we addressed the question of affinity thresholds of allergen-specific IgG antibodies for inhibition of mast cell activation using 2 different mAbs against the major cat allergen Fel d 1 both in vitro and in vivo in mice. METHODS: Sequences of the 2 high-affinity mAbs were back-mutated to germline, resulting in low-affinity (10-7 mol/L) antibodies of the exact same specificity. RESULTS: Using these newly generated recombinant antibodies, we demonstrate that low-affinity antibodies are still able to inhibit mast cell activation through FcγRIIb but do not neutralize the allergen. CONCLUSION: Antibody affinity dictates the mechanism of mast cell inhibition, and IgG antibodies triggering the inhibitory FcγRIIb pathway can show a broader cross-reactivity pattern than previously thought. This indicates that allergen-specific immunotherapy generates a larger protective umbrella of inhibitory IgG antibodies than previously appreciated.

Shape Self-Adaptive Liquid Embolic Agent for Ultrafast and Durable Vascular Embolization.

Minimally invasive embolization greatly decreases the mortality resulting from vascular injuries while still suffering from a high risk of recanalization and systematic thrombosis due to the intrinsic hydrophobicity and poor adhesion of the clinically used liquid embolic agent of Lipiodol. In this study, a shape self-adaptive liquid embolic agent was developed by mixing biocompatible poly(acrylic acid) (PAA), two-dimensional magnesium-aluminum layered double hydroxide (LDH), and poly(ethylene glycol)200 (PEG200). Upon contact with blood, the injectable PAA-LDH@PEG200 would quickly absorb water to form an adhesive and mechanically strong PAA-LDH thin hydrogel within 5 s, which could firmly adhere to the blood vessel wall for ultrafast and durable embolization. In addition, benefiting from the "positively charged nucleic center effect" of LDH nanosheets, the liquid PAA-LDH@PEG200 could avoid vascular distension by PAA overexpansion and possess high shock-resistant mechanical strength from the blood flow. Furthermore, both in vitro and in vivo embolization experiments demonstrated the complete embolic capacity of liquid PAA-LDH@PEG200 without the occurrence of recanalization for 28 days and also the great potential to act as a platform to couple with chemotherapeutic drugs for the minimized transcatheter arterial chemoembolization (TACE) treatment of VX2 tumors without recurrence for 18 days. Thus, liquid PAA-LDH@PEG200 developed here possesses great potential to act as a shape self-adaptive liquid embolic agent for ultrafast and durable vascular embolization.

Synthetic nanoparticles for the delivery of CRISPR/Cas9 gene editing system: classification and biomedical applications.

Gene editing has great potential in biomedical research including disease diagnosis and treatment. Clustered regularly interspaced short palindromic repeats (CRISPR) is the most straightforward and cost-effective method. The efficient and precise delivery of CRISPR can impact the specificity and efficacy of gene editing. In recent years, synthetic nanoparticles have been discovered as effective CRISPR/Cas9 delivery vehicles. We categorized synthetic nanoparticles for CRISPR/Cas9 delivery and discribed their advantages and disadvantages. Further, the building blocks of different kinds of nanoparticles and their applications in cells/tissues, cancer and other diseases were described in detail. Finally, the challenges encountered in the clinical application of CRISPR/Cas9 delivery materials were discussed, and potential solutions were provided regarding efficiency and biosafety issues.

Localization of Chicken Rab22a in Cells and Its Relationship to BF or Ii Molecules and Genes.

Rab22a is an important small GTPase protein the molecule that is involved in intracellular transportation and regulation of proteins. It also plays an important role in antigens uptake, transportation, regulation of endosome morphology, and also regulates the transport of antigens to MHC (Major Histocompatibility Complex) molecules. To investigate the role of Rab22a, the intracellular co-localization of chicken Rab22a (cRab22a) molecule and its relationship to BF and chicken invariant chain (cIi) molecules was studied. A 3D protein structure of Rab22a was constructed by using informatics tools (DNASTAR 4.0 and DNAMAN). Based on the model, the corresponding recombinant eukaryotic plasmids were constructed by point mutations in the protein's structural domains. HEK 293T cells were co-transfected with plasmids pEGFP-C1-cIi to observe the intracellular co-localization. Secondly, the DC2.4 Mouse Dendritic Cell and Murine RAW 264.7 cells were transfected with recombinant plasmids of pmCherry-cRab22a and pmCherry-mRab22a respectively. Subsequently, the intracellular localization of cRab22a in early and late endosomes was observed with specific antibodies against EEA1 and LAMP1 respectively. For gene expression-based studies, the cRab22a gene was down-regulated and up-regulated in HD11 cells, following the detection of transcription levels of the BFa (MHCIa) and cIi genes by real-time quantitative PCR (RT-qPCR). The interactions of the cRab22a gene with BFa and cIi were detected by co-immunoprecipitation (Co-IP) and Western blot. The results showed that the protein structures of chicken and mouse Rab22a were highly homologous (95.4%), and both localize to the early and late endosomes. Ser41 and Tyr74 are key amino acids in the Switch regions of Rab22a which maintain its intracellular localization. The down-regulation of cRab22a gene expression significantly reduced (p < 0.01) the transcription of BFa (MHCIa) and cIi in HD11 cells. However, when the expression of the cRab22a gene was increased 55 times as compared to control cells, the expression of the BFa (MHCIa) gene was increased 1.7 times compared to the control cells (p < 0.01), while the expression of the cIi gene did not significantly differ from control (p > 0.05). Western blot results showed that cRab22a could not directly bind to BFa and cIi. So, cRab22a can regulate BFa and cIi protein molecules indirectly. It is concluded that cRab22a was localized with cIi in the endosome. The Switch regions of cRab22a are the key domains that affect intracellular localization and colocalization of the cIi molecule.

Renal Clearable Mo-Based Polyoxometalate Nanoclusters: A Promising Radioprotectant against Ionizing Irradiation.

In response to diffused ionizing radiation damage throughout the body caused by nuclear leaks and inaccurate radiotherapy, radioprotectants with considerable free radical scavenging capacities, along with negligible adverse effects, are highly regarded. Herein, unlike being performed as toxic chemotherapeutic drug candidates, molybdenum-based polyoxometalate nanoclusters (Mo-POM NCs) were developed as a non-toxic potent radioprotectant with impressive free radical scavenging capacities for ionizing radiation protection. In comparison to the clinically used radioprotectant drug amifostine (AM), the as-prepared Mo-POM NCs exhibited effective shielding capacity by virtue of their antioxidant properties resulting from a valence shift of molybdenum ions, alleviating not only ionizing radiation-induced DNA damage but also disruption of the radiation-sensitive hematopoietic system. More encouragingly, without trouble with long-term retention in the body, ultra-small sized Mo-POM NCs prepared by the mimetic Folin-Ciocalteu assay can be removed from the body through the renal-urinary pathway and the hepato-enteral excretory system after completing the mission of radiation protection. This work broadened the biological applications of metal-based POM chemotherapeutic drugs to act as a neozoic radioprotectant.

Transcriptome analysis reveals critical factors for survival after adenovirus serotype 4 infection.

Fowl adenovirus serotype-4 (FAdV-4) is highly lethal to poultry, making it one of the leading causes of economic losses in the poultry industry. However, a small proportion of poultry can survive after FAdV-4 infection. It is unclear whether there are genetic factors that protect chickens from FAdV-4 infection. Therefore, the livers from chickens uninfected with FAdV-4 (Normal), dead after FAdV-4 infection (Dead) or surviving after FAdV-4 infection (Survivor) were collected for RNA-seq, and 2,649 differentially expressed genes (DEGs) were identified. Among these, many immune-related cytokines and chemokines were significantly upregulated in the Dead group compared with the Survivor group, which might indicate that death is related to an excessive inflammatory immune response (cytokine storm). Subsequently, the KEGG results for DEGs specifically expressed in each comparison group indicated that cell cycle and apoptosis-related DEGs were upregulated and metabolism-related DEGs were downregulated in the Dead group, which also validated the reliability of the samples. Furthermore, GO and KEGG results showed DEGs expressed in all three groups were mainly associated with cell cycle. Among them, BRCA1, CDK1, ODC1, and MCM3 were screened as factors that might influence FAdV-4 infection. The qPCR results demonstrated that these 4 factors were not only upregulated in the Dead group but also significantly upregulated in the LMH cells after 24 h infection by FAdV-4. Moreover, interfering with BRCA1, CDK1, ODC1, and MCM3 significantly attenuated viral replication of FAdV-4. And interfering of BRCA1, CDK1, and MCM3 had more substantial hindering effects. These results provided novel insights into the molecular changes following FAdV-4 infection but also shed light on potential factors driving the survival of FAdV-4 infection in chickens.

Screening and characterization of inhibitory vNAR targeting nanodisc-assembled influenza M2 proteins.

Influenza A virus poses a constant challenge to human health. The highly conserved influenza matrix-2 (M2) protein is an attractive target for the development of a universal antibody-based drug. However, screening using antigens with subphysiological conformation in a nonmembrane environment significantly reduces the generation of efficient antibodies. Here, M2(1-46) was incorporated into nanodiscs (M2-nanodiscs) with M2 in a membrane-embedded tetrameric conformation, closely resembling its natural physiological state in the influenza viral envelope. M2-nanodisc generation, an antigen, was followed by Chiloscyllium plagiosum immunization. The functional vNARs were selected by phage display panning strategy from the shark immune library. One of the isolated vNARs, AM2H10, could specifically bind to tetrameric M2 instead of monomeric M2e (the ectodomain of M2 protein). Furthermore, AM2H10 blocked ion influx through amantadine-sensitive and resistant M2 channels. Our findings indicated the possibility of developing functional shark nanobodies against various influenza viruses by targeting the M2 protein.

Defective interferon amplification and impaired host responses against influenza virus in obese mice.

OBJECTIVE: Obesity is a major risk factor that increases morbidity and mortality upon infection. Although type I and type III interferon (IFN)-induced innate immune responses represent the first line of defense against viral infections, their functionality in the context of metabolic disorders remains largely obscure. This study aimed to investigate IFN responses upon respiratory viral infection in obese mice. METHODS: The activation of IFNs as well as IFN regulatory factors (IRFs) upon H3N2 influenza infection in mice upon high-fat-diet feeding was investigated. RESULTS: Influenza infection of obese mice was characterized by higher mortalities. In-depth analysis revealed impaired induction of both type I and type III IFNs as well as markedly reduced IFN responses. Notably, it was found that IRF7 gene expression in obese animals was reduced in homeostasis, and its induction by the virus was strongly attenuated. CONCLUSIONS: The results suggest that the attenuated IRF7 expression and induction are responsible for the reduced expression levels of type I and III IFNs and, thus, for the higher susceptibility and severity of respiratory infections in obese mice.

A Novel Double Mosaic Virus-like Particle-Based Vaccine against SARS-CoV-2 Incorporates Both Receptor Binding Motif (RBM) and Fusion Domain.

COVID-19 has emerged, and has rapidly become a major health problem worldwide, causing millions of mortalities. Vaccination against COVID-19 is the most efficient way to stop the pandemic. The goal of vaccines is to induce neutralizing antibodies against SARS-CoV-2 virus. Here, we present a novel double mosaic virus-like particle (VLP) displaying two independent neutralizing epitopes, namely the receptor binding motif (RBM) located in S1 and the fusion peptide (AA 817-855) located in S2. CuMVTT virus-like particles were used as VLP scaffold and both domains were genetically fused in the middle of CuMVTT subunits, which co-assembled into double mosaic particles (CuMVTT-DF). A single fusion mosaic particle (CuMVTT-FP) containing the fusion peptide only was used for comparison. The vaccines were produced in E. coli, and electron microscopy and dynamic light scattering confirmed their integrity and homogeneity. In addition, the CuMVTT-DF vaccine was well recognized by ACE2 receptor, indicating that the RBM was in native conformation. Both CuMVTT-FP and CuMVTT-DF vaccines induced high levels of high avidity IgG antibodies as well as IgA recognizing spike and RBD in the case of CuMVTT-DF. Both vaccine candidates induced virus-neutralizing antibodies indicating that the fusion peptide can independently induce virus-neutralizing antibodies. In contrast, CuMVTT-DF containing both RBM and fusion peptide induced a higher level of neutralizing antibodies suggesting that the new double mosaic vaccine candidate CuMVTT-DF consisting of two antigens in one VLP maybe an attractive candidate for scale-up in a bacterial fermentation process for clinical development.

The impact of size on particle drainage dynamics and antibody response.

Vaccine-induced immune response can be greatly enhanced by mimicking pathogen properties. The size and the repetitive geometric shape of virus-like particles (VLPs) influence their immunogenicity by facilitating drainage to secondary lymphoid organs and enhancing interaction with and activation of B cells and innate humoral immune components. VLPs derived from the plant Bromovirus genus, specifically cowpea chlorotic mottle virus (CCMV), are T = 3 icosahedral particles. (T) is the triangulation number that refers to the number and arrangements of the subunits (pentamers and hexamers) of the VLPs. CCMV-VLPs can be easily expressed in an E. coli host system and package ssRNA during the expression process. Recently, we have engineered CCMV-VLPs by incorporating the universal tetanus toxin (TT) epitope at the N-terminus. The modified CCMVTT-VLPs successfully form icosahedral particles T = 3, with a diameter of ~30 nm analogous to the parental VLPs. Interestingly, incorporating TT epitope at the C-terminus of CCMVTT-VLPs results in the formation of Rod-shaped VLPs, ~1 µm in length and ~ 30 nm in width. In this study, we have investigated the draining kinetics and immunogenicity of both engineered forms (termed as Round-shaped CCMVTT-VLPs and Rod-shaped CCMVTT-VLPs) as potential B cell immunogens using different in vitro and in vivo assays. Our results reveal that Round-shaped CCMVTT-VLPs are more efficient in draining to secondary lymphoid organs to charge professional antigen-presenting cells as well as B cells. Furthermore, compared to Rod-shaped CCMVTT-VLPs, Round-shaped CCMVTT-VLPs led to more than 100-fold increased systemic IgG and IgA responses accompanied by prominent formation of splenic germinal centers. Round-shaped CCMVTT-VLPs could also polarize the induced T cell response toward Th1. To our knowledge, this is the first study investigating and comparing the draining kinetics and immunogenicity of one and the same VLP monomer forming nano-sized icosahedra or rods in the micrometer size.

TLR7 Signaling Shapes and Maintains Antibody Diversity Upon Virus-Like Particle Immunization.

Virus-like particles (VLPs) are used in different marketed vaccines and are able to induce potent antibody responses. The innate pattern recognition receptors TLR7/8 recognize single stranded (ss) RNA naturally packaged into some VLPs and have been shown to enhance the production of IgG antibodies upon immunization. Here we demonstrate that, upon immunization with RNA-loaded bacteriophage-derived VLP Qß, TLR7 signaling accelerates germinal center formation, promotes affinity/avidity maturation of VLP-specific IgG and isotype switching to IgG2b/2c. These findings extrapolated to antigens displayed on Qß; as Fel d 1, the major cat allergen, chemically attached to Qß also induced higher affinity/avidity IgG2b/2c antibodies in a TLR7-dependent fashion. Chimeric mice lacking TLR7-expression exclusively in B cells demonstrated that the enhanced IgG responses were driven by a B cell intrinsic mechanism. Importantly, deep sequencing of the BCR repertoire of antigen-specific B cells demonstrated higher diversity in mice with TLR7 signaling in B cells, suggesting that TLR7-signaling drives BCR repertoire development and diversity. Furthermore, the current data demonstrate that high levels of clonal diversity are reached early in the response and maintained by TLR7 signaling. In conclusion, TLR7 signaling enhances levels and quality of IgG antibodies, and this finding has major implications for vaccine design.

The fowl adenovirus serotype 4 (FAdV-4) induce cellular pathway in chickens to produce interferon and antigen-presented molecules (MHCI/II).

FAdV-4 is the major strain of adenovirus that responsible for hydro-pericardial syndrome (HPS) in poultry. In this study, the virus's specific gene fragments were isolated from clinically suspected cases and amplified by PCR. Finally, after a viral infection to investigate the immune response of the host, the gene expression of MHC (major histo-compatible) molecules (MHCIα, MHCIIß), Ii (Invariant Chain) gene, inflammatory cytokines (IFN-ß, IFN-γ, and IL-1ß), and transcription factors (MDA5, STING, IRF7, and NF-kB) were detected by real-time PCR (fluorescence technology). The results of sequence comparison showed that the clinically isolated virus was 100% homologous to a virulent strain of avian adenovirus group C serotype 4 (FAdV-4), which were named AH-FAdV-4. The TCID50 and pathogenicity of the virus were determined that was 106.52/0.1 mL with a mortality rate of 100% in chickens and 0% in ducks. Furthermore, results showed that the expression level of MHCIα, MHCIIß, and Ii genes in chicken embryo kidney cells significantly (P < 0.01) upregulated (increased) after infection, which was 43, 5.2, and 2.5 times higher than the control group. With the addition of PDTC, an inhibitor of NF-kB, then the expression level of MHCIα, MHCIIß, and Ii was decreased significantly (P < 0.01) than the control group. The transcription levels of these genes were decreased 0.64, 0.27, and 0.26 respectively. Simultaneously, the expression levels of IFN-ß, IFN-γ, and IL-1ß were also significantly (P < 0.01) up-regulated (increased) 7.8, 22.7, and 5 times higher than the control group. It was found that up-regulation of STING and NF-κB pathways are directly involved in the regulation of inflammatory cytokines (IFN-ß, IFN-γ, and IL-1ß), MHC molecules (MHCIα, MHCIIß), and Ii gene. The results also showed that the gene regulation pathways consecutively increased the expression levels of MDA5, STING, IRF7, and NF-kB. It is conducted that the expression levels of cytokines, MHC molecules, and li gene were increased by STING and NF-kB pathways.

Development of a Vaccine against SARS-CoV-2 Based on the Receptor-Binding Domain Displayed on Virus-Like Particles.

The ongoing coronavirus disease (COVID-19) pandemic is caused by a new coronavirus (severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2)) first reported in Wuhan City, China. From there, it has been rapidly spreading to many cities inside and outside China. Nowadays, more than 110 million cases with deaths surpassing 2 million have been recorded worldwide, thus representing a major health and economic issues. Rapid development of a protective vaccine against COVID-19 is therefore of paramount importance. Here, we demonstrated that the recombinantly expressed receptor-binding domain (RBD) of the spike protein can be coupled to immunologically optimized virus-like particles derived from cucumber mosaic virus (CuMVTT). The RBD displayed CuMVTT bound to ACE2, the viral receptor, demonstrating proper folding of RBD. Furthermore, a highly repetitive display of the RBD on CuMVTT resulted in a vaccine candidate that induced high levels of specific antibodies in mice, which were able to block binding of the spike protein to ACE2 and potently neutralize SARS-CoV-2 virus in vitro.

Vaccination with nanoparticles combined with micro-adjuvants protects against cancer.

BACKGROUND: Induction of strong T cell responses, in particular cytotoxic T cells, is a key for the generation of efficacious therapeutic cancer vaccines which yet, remains a major challenge for the vaccine developing world. Here we demonstrate that it is possible to harness the physiological properties of the lymphatic system to optimize the induction of a protective T cell response. Indeed, the lymphatic system sharply distinguishes between nanoscale and microscale particles. The former reaches the fenestrated lymphatic system via diffusion, while the latter either need to be transported by dendritic cells or form a local depot. METHODS: Our previously developed cucumber-mosaic virus-derived nanoparticles termed (CuMVTT-VLPs) incorporating a universal Tetanus toxoid epitope TT830-843 were assessed for their draining kinetics using stereomicroscopic imaging. A nano-vaccine has been generated by coupling p33 epitope as a model antigen to CuMVTT-VLPs using bio-orthogonal Cu-free click chemistry. The CuMVTT-p33 nano-sized vaccine has been next formulated with the micron-sized microcrystalline tyrosine (MCT) adjuvant and the formed depot effect was studied using confocal microscopy and trafficking experiments. The immunogenicity of the nanoparticles combined with the micron-sized adjuvant was next assessed in an aggressive transplanted murine melanoma model. The obtained results were compared to other commonly used adjuvants such as B type CpGs and Alum. RESULTS: Our results showed that CuMVTT-VLPs can efficiently and rapidly drain into the lymphatic system due to their nano-size of ~ 30 nm. However, formulating the nanoparticles with the micron-sized MCT adjuvant of ~ 5 µM resulted in a local depot for the nanoparticles and a longer exposure time for the immune system. The preclinical nano-vaccine CuMVTT-p33 formulated with the micron-sized MCT adjuvant has enhanced the specific T cell response in the stringent B16F10p33 murine melanoma model. Furthermore, the micron-sized MCT adjuvant was as potent as B type CpGs and clearly superior to the commonly used Alum adjuvant when total CD8+, specific p33 T cell response or tumour protection were assessed. CONCLUSION: The combination of nano- and micro-particles may optimally harness the physiological properties of the lymphatic system. Since the nanoparticles are well defined virus-like particles and the micron-sized adjuvant MCT has been used for decades in allergen-specific desensitization, this approach may readily be translated to the clinic.