Self-Assembling E2-Based Nanoparticles Improve Vaccine Thermostability and Protective Immunity against CSFV.

Classical swine fever virus (CSFV) is a highly contagious pathogen causing significant economic losses in the swine industry. Conventional inactivated or attenuated live vaccines for classical swine fever (CSF) are effective but face biosafety concerns and cannot distinguish vaccinated animals from those infected with the field virus, complicating CSF eradication efforts. It is noteworthy that nanoparticle (NP)-based vaccines resemble natural viruses in size and antigen structure, and offer an alternative tool to circumvent these limitations. In this study, we developed an innovative vaccine delivery scaffold utilizing self-assembled mi3 NPs, which form stable structures carrying the CSFV E2 glycoprotein. The expressed yeast E2-fused protein (E2-mi3 NPs) exhibited robust thermostability (25 to 70 °C) and long-term storage stability at room temperature (25 °C). Interestingly, E2-mi3 NPs made with this technology elicited enhanced antigen uptake by RAW264.7 cells. In a rabbit model, the E2-mi3 NP vaccine against CSFV markedly increased CSFV-specific neutralizing antibody titers. Importantly, it conferred complete protection in rabbits challenged with the C-strain of CSFV. Furthermore, we also found that the E2-mi3 NP vaccines triggered stronger cellular (T-lymphocyte proliferation, CD8+ T-lymphocytes, IFN-γ, IL-2, and IL-12p70) and humoral (CSFV-specific neutralizing antibodies, CD4+ T-lymphocytes, and IL-4) immune responses in pigs than the E2 vaccines. To sum up, these structure-based, self-assembled mi3 NPs provide valuable insights for novel antiviral strategies against the constantly infectious agents.

Virus-like particle-based multipathogen vaccine of FMD and SVA elicits balanced and broad protective efficacy in mice and pigs.

Inactivated vaccines lack the capability to serologically differentiate between infected and vaccinated animals, thereby impeding the effective eradication of pathogen. Conversely, vaccines based on virus-like particles (VLPs) emulate natural viruses in both size and antigenic structure, presenting a promising alternative to overcome these limitations. As the complexity of swine infectious diseases increases, the increase of vaccine types and doses may intensify the stress response. This exacerbation can lead to diminished productivity, failure of immunization, and elevated costs. Given the critical dynamics of co-infection and the clinically indistinguishable symptoms associated with foot-and-mouth disease virus (FMDV) and senecavirus A (SVA), there is a dire need for an efficacious intervention. To address these challenges, we developed a combined vaccine composed of three distinct VLPs, specifically designed to target SVA and FMDV serotypes O and A. Our research demonstrates that this trivalent VLP vaccine induces antigen-specific and robust serum antibody responses, comparable to those produced by the respective monovalent vaccines. Moreover, the immune sera from the combined VLP vaccine strongly neutralized FMDV type A and O, and SVA, with neutralization titers comparable to those of the individual vaccines, indicating a high level of immunogenic compatibility among the three VLP components. Importantly, the combined VLPs vaccines-immunized sera conferred efficient protection against single or mixed infections with FMDV type A and O, and SVA viruses in pigs. In contrast, individual vaccines could only protect pigs against homologous virus infections and not against heterologous challenges. This study presents a novel combined vaccines candidate against FMD and SVA, and provides new insights for the development of combination vaccines for other viral swine diseases.

Local and systemic immune responses induced by intranasal immunization with biomineralized foot-and-mouth disease virus-like particles.

Introduction: Foot-and-mouth disease virus (FMDV) infects the host by invading mucosal epithelial cells of the respiratory or digestive tract. Therefore, establishing a specific antiviral mucosal immune barrier can effectively block viral invasion. Methods: We evaluated local mucosal and systemic immune responses elicited by intranasal immunization of mice with foot-and-mouth disease (FMD) calcium phosphate mineralized virus-like particles (CaP-VLPs) and tested whether three commercial mucosal adjuvants enhanced the immunogenicity of the antigen. The biosafety of the vaccine was verified through gross observation and pathological analysis of the lungs. Results: CaP-VLPs effectively induced secretion of IgA (sIgA) from multiple sites in mouse mucosa and produced anti-FMD-specific IgG in the serum. Splenic lymphocytes specifically proliferated and secreted IFN-γ following antigen stimulation, indicating the vaccine can induce a certain level of cellular immune response. Finally, the pathological examination confirmed that CaP-VLPs did not cause substantial damage to the lungs of animals after immunization via mucosal administration. Notably, the vaccine mixed with S adjuvant increased the content of sIgA and serum IgG, and the high level of IgG in serum was maintained at least 7 weeks. Discussion: Overall, this study reveals that FMD CaP-VLPs can induce good local mucosal immune and systemic immune response through intranasal immunization, and the immune response was specifically enhanced by S adjuvant. These data support that CaP-VLPs-S as a candidate mucosal vaccine for the prevention of FMD vaccine infection.

Development and Efficacy Evaluation of a Novel Nano-Emulsion Adjuvant for a Foot-and-Mouth Disease Virus-like Particles Vaccine Based on Squalane.

The successful development of foot-and-mouth disease virus-like particles (FMD-VLPs) has opened a new direction for researching a novel subunit vaccine for foot-and-mouth disease (FMD). Therefore, it is urgent to develop an adjuvant that is highly effective and safe to facilitate a better immune response to be pair with the FMD-VLP vaccine. In this research, we prepared a new nano-emulsion adjuvant based on squalane (SNA) containing CpG using the pseudo-ternary phase diagram method and the phase transformation method. The SNA consisted of Span85, Tween60, squalane, polyethene glycol-400 (PEG400) and CpG aqueous solution. The average particle diameter of the SNA was about 95 nm, and it exhibited good resistance to centrifugation, thermal stability, and biocompatibility. Then, SNA was emulsified as an adjuvant to prepare foot-and-mouth disease virus-like particles vaccine, BALB/c mice and guinea pigs were immunized, and we evaluated the immunization effect. The immunization results in mice showed that the SNA-VLPs vaccine significantly increased specific antibody levels in mice within 4 weeks, including higher levels of IgG1 and IgG2a. In addition, it increased the levels of IFN-γ and IL-1ß in the immune serum of mice. Meanwhile, guinea pig-specific and neutralizing antibodies were considerably increased within 4 weeks when SNA was used as an adjuvant, thereby facilitating the proliferation of splenic lymphocytes. More importantly, in guinea pigs immunized with one dose of SNA-VLPs, challenged with FMDV 28 days after immunization, the protection rate can reach 83.3%, which is as high as in the ISA-206 control group. In conclusion, the novel squalane nano-emulsion adjuvant is an effective adjuvant for the FMD-VLPs vaccine, indicating a promising adjuvant for the future development of a novel FMD-VLPs vaccine.

Hepatic histopathology and apoptosis in diet-induced-obese mice under Escherichia coli pneumonia.

This research was to investigate the difference of hepatic histopathology and apoptosis between the diet-induced obesity (DIO) and normal (lean) mice after Escherichia coli (E. coli) pneumonia. A total of 128 ICR mice were selected to be challenged intranasally with phosphate-buffered saline (PBS) or 4×109CFUs/mL of E. coli, and the liver histopathology and apoptosis were examined pre- and post-infection. Results showed that the liver index, levels of lipid droplets, cytokines, adipocytokines, oxidative stress, apoptotic percentage, and apoptotic related factors in the E. coli-infected mice were generally higher than those in the uninfected mice, whereas the hepatic glycogen and Bcl-2 were the opposite. Interestingly, after E. coli infection, the DIO-E. coli mice exhibited decreased liver index and apoptotic percentages, and reduced levels of TNF-α, IL-6, resistin, MDA, GSH, CAT, Caspase-3, Caspase-9, Bax as well as Bax/Bcl-2 ratio in comparison to the lean-E. coli mice. Our results indicated that E. coli-induced pneumonia caused hepatic histopathological damage, increased hepatic apoptosis, oxidative damages, and higher levels of cytokines and adipocytokines. However, such changes showed less severely in the DIO mice than in the lean mice following E. coli pneumonia.

Histopathological Injuries, Ultrastructural Changes, and Depressed TLR Expression in the Small Intestine of Broiler Chickens with Aflatoxin B1.

To explore AFB1-induced damage of the small intestine, the changes in structure and expression of TLRs (Toll-like Receptors) in the small intestine of chickens were systematically investigated. Ninety healthy neonatal Cobb chickens were randomized into a control group (0 mg/kg AFB1) and an AFB1 group (0.6 mg/kg AFB1). The crypt depth of the small intestine in the AFB1 group was significantly increased in comparison to the control chickens, while the villus height and area were evidently decreased, as well as the villus:crypt ratio and epithelial thickness. The histopathological observations showed that the villi of the small intestine exposed to AFB1 were obviously shedding. Based on ultrastructural observation, the absorptive cells of small intestine in the AFB1 group exhibited fewer microvilli, mitochondrial vacuolation and the disappearance of mitochondrial cristae, and junctional complexes as well as terminal web. Moreover, the number of goblet cells in the small intestine in the AFB1 group significantly decreased. Also, AFB1 evidently decreased the mRNA expression of TLR2-2, TLR4, and TLR7 in the small intestine. Taken together, our study indicated that dietary 0.6 mg/kg AFB1 could induce histopathological injuries and ultrastructural changes, and depress levels of TLR mRNA in the chicken small intestine.

AMPKα pathway involved in hepatic triglyceride metabolism disorder in diet-induced obesity mice following Escherichia coli Infection.

To investigate the different effects of acute pulmonary infection induced by Escherichia coli (E. coli) on lipid metabolism between diet-induced obesity (DIO, fed with high-fat diet) mice and lean mice. A total of 180 ICR mice were selected to be challenged intranasally with phosphate-buffered saline or 109 CFUs/mL of E. coli, and the body character indexes, biochemical indexes and expressions of genes and proteins involved in lipid metabolism were examined pre- and post-infection. Results revealed that, before infection, DIO mice had significantly higher body weight, adipose and liver indexes, free fatty acid and triglyceride contents than lean mice. After infection, increased free fatty acid and triglyceride contents, increased expressions of resistin, SREBP-1c, ACC1, FAS and SCD-1, and declined PPARα, CPT-1α expressions and AMPKα phosphorylation were detected in the infected group, while the change rates were more serious in the lean mice than the DIO mice. The above-mentioned findings verified that, after being infected with E. coli, hepatic lipid metabolism disorder was aggravated by activating SREBP-1c related lipid synthesis pathway and inhibiting PPARα related fatty acid oxidation pathway. However, infection-induced lipid metabolic disorders was slighter in the DIO mice than the lean mice through AMPKα pathway.

The molecular mechanism of cell cycle arrest in the Bursa of Fabricius in chick exposed to Aflatoxin B 1.

Aflatoxin B1 shows potent hepatotoxic, carcinogenic, genotoxic, immunotoxic potential in humans and many species of animals. The aim of this study was to clarify the underlying mechanism of G0G1 phase and G2M phase arrest of cell cycle in the bursa of Fabricius in broilers exposed to dietary AFB1. 144 one-day-old healthy Cobb broilers were randomly divided into two groups and fed on control diet and 0.6 mg·Kg-1 AFB1 diet for 3 weeks. Histological observation showed that AFB1 induced the increase of nuclear debris and vacuoles in lymphoid follicle of BF. Results of flow cytometry studies showed that bursal cells arrested in G2M phase at 7 days of age and blocked in G0G1 phase at 14 and 21 days of age following exposure to AFB1. The qRT-PCR analysis indicated that cell cycle arrested in G2M phase via ATM-Chk2-cdc25-cyclin B/cdc2 pathway, and blocked in G0G1 phase through ATM-Chk2-cdc25-cyclin D/CDK6 pathway and ATM-Chk2-p21-cyclin D/CDK6 route. In a word, our results provided new insights that AFB1 diet induced G2M and G0G1 phase blockage of BF cells in different periods, and different pathways were activated in different arrested cell cycle phase.

A study on the expression of apoptotic molecules related to death receptor and endoplasmic reticulum pathways in the jejunum of AFB1-intoxicated chickens.

Aflatoxin B1 (AFB1) is a common contaminant of poultry feeds in tropical and subtropical climates. Early researches have well established the hepatotoxic, carcinogenic, and immunotoxic effects of AFB1 on humans and animals. Recently, it has been shown that AFB1 could cause the up- or down-alteration of mitochondrial pathway molecule expression. However, the information on the expression of death receptor and endoplasmic reticulum molecules in the jejunal apoptosis induced by AFB1 were unavailable. So the present study was conducted to explore the expression of apoptotic molecules related to death receptor and endoplasmic reticulum in the jejunal cells of chickens exposed to AFB1 diet for 3 weeks. Total of 144 one-day-old chickens was randomly divided into two groups, namely control group (containing 0 mg/kg AFB1) and AFB1 group (containing 0.6 mg/kg AFB1). Histopathological observation and microscopic quantitative analysis revealed morphological changes in the jejunum such as the shedding of the mucosal epithelial cells in the apical region of villi along with the decrease of villus height, villus area and villus/crypt ratio in the AFB1 group. Both TUNEL and flow cytometry assays showed that AFB1 intake induced excessive apoptosis of jejunal cells. Quantitative real-time PCR test displayed the general upregulation of death receptors (FAS, FASL, TNF-α and TNF-R1), endoplasmic reticulum signals (GRP78 and GRP94) as well as initiator and executioner caspases (CASPASE-10, CASPASE-8 and CASPASE-3) in the jejunum of AFB1-intoxicated chickens. It's the first study demonstrating that AFB1 induced apoptosis of chickens' jejunum accompanied by the alteration of death receptor and endoplasmic reticulum molecule expression.

Aflatoxin B1 affects apoptosis and expression of death receptor and endoplasmic reticulum molecules in chicken spleen.

Aflatoxin B1 (AFB1) is a natural product of the Aspergillus genus of molds, which grow on several foodstuffs stored in hot moist conditions, and is among the most potent hepatocarcinogens and immunosuppression presently known. The latter was related to the up-regulated apoptosis of immune organs. However, the effect of expression of death receptor and endoplasmic reticulum molecules in AFB1-induced apoptosis of chicken splenocytes was largely unknown. The objective of this study was to investigate this unknown field. One hundred and forty four one-day-old chickens were randomly divided into control group (0 mg/kg AFB1) and AFB1 group (0.6 mg/kg AFB1), respectively and fed with AFB1 for 21 days. Histological observation demonstrated that AFB1 caused slight congestion and lymphocytic depletion in the spleen. TUNEL and flow cytometry assays showed the excessive apoptosis of splenocytes provoked by AFB1. Moreover, quantitative real-time PCR analysis revealed that AFB1 induced the elevated mRNA expression of Fas, FasL, TNF-α, TNF-R1, Caspase-3, Caspase-8, Caspase-10, Grp78 and Grp94 in the spleen. These findings suggested that AFB1 could lead the excessive apoptosis and alter the expression of death receptor and endoplasmic reticulum molecules in chicken spleen.