Bi-functional gold nanocages enhance specific immunological responses of foot-and-mouth disease virus-like particles vaccine as a carrier and adjuvant.

Virus-like particle (VLP) vaccines have become one of the dominant vaccine candidates for foot-and-mouth disease (FMD). To further enhance the immunogenicity of VLP vaccines, gold nanocages (AuNCs) were selected as an adjuvant for the vaccine. Our experiments demonstrated that AuNCs had little biotoxicity in vivo and in vitro and improved the uptake of VLP in BHK-21 and RAW264.7 cell lines. The VLP-AuNCs activated DCs mainly through toll-like receptor 4 (TLR4) and promoted the secretion of IL-6, IL-1ß, and TNF-α. The conjugation of VLP and AuNCs triggered a strong immune response against FMD virus (FMDV) in mice and guinea pigs. The VLP-AuNCs significantly enhanced the proliferation of CD8+ T cells (P < 0.05) and the secretion of cellular immune-related cytokines (IFN-γ, P < 0.05; IL-12p70, P < 0.01) compared with VLP. The present study demonstrated that AuNCs, as a great potential adjuvant for FMDV VLP vaccines, significantly enhance the immune response.

Enhanced protective immune response of foot-and-mouth disease vaccine through DNA-loaded virus-like particles.

Foot-and-mouth disease virus (FMDV) is the etiological agent of a highly contagious disease that affects cloven-hoofed animals. Virus-like particles (VLPs) can induce a robust immune response and deliver DNA and small molecules. In this study, a VLP-harboring pcDNA3.1/P12A3C plasmid was generated, and the protective immune response was characterized. Guinea pigs were injected with VLPs, naked DNA vaccine, DNA-loaded VLPs, or phosphate-buffered saline twice subcutaneously at four-week intervals. Results demonstrated that the VLPs protected the naked DNA from DNase degeneration and delivered the DNA into the cells in vitro. The DNA-loaded VLPs and the VLPs alone induced a similar level of specific antibodies (P > 0.05) except at 49 dpv (P < 0.05). The difference in interferon-γ was consistent with that in specific antibodies. The levels of neutralizing antibodies induced by the DNA-loaded VLPs were significantly higher than those of other samples (P < 0.01). Similarly, the lymphocyte proliferation by using DNA-loaded VLPs was significantly higher than those using other formulas after booster immunization. Vaccination with DNA-loaded VLPs provided higher protection (100%) against viral challenge compared with vaccination with VLPs (75%) and DNA vaccine (25%). This study suggested that VLPs can be used as a delivery carrier for DNA vaccine. In turn, the DNA vaccine can enhance the immune response and prolong the serological duration of the VLP vaccine. This phenomenon contributes in providing complete protection against the FMDV challenge in guinea pigs and can be valuable in exploring novel nonreplicating vaccines and controlling FMD in endemic countries worldwide.

Development of a novel SYBR green I-based quantitative RT-PCR assay for Senecavirus A detection in clinical samples of pigs.

Porcine vesicular disease caused by Senecavirus A (SVA) is a newly emerging disease in many countries. Based on clinical signs only, it is very challenging to distinguish SVA infection from other similar diseases, such as foot and mouth disease, swine vesicular disease, and vesicular stomatitis. Therefore, it is crucial to establish a detection assay for the clinical diagnosis of SVA infection. In this study, a pair of specific primers were designed based on the highly conserved L/VP4 gene sequence of SVA. The established SYBR green I-based quantitative reverse transcription polymerase chain reaction (qRT-PCR) method was used to detect SVA nucleic acids in clinical samples. The limit of detection SVA nucleic acids by qRT-PCR was 6.4 × 101 copies/µL, which was significantly more sensitive than that by gel electrophoresis of 6.4 × 103 copes/µL. This assay was specific and had no cross-reaction with other seven swine viruses. Using SYBR green I-based qRT-PCR, the SVA positive rates in experimental animal samples and field samples were 67.60% (96/142) and 80% (24/30) respectively. The results demonstrate that SYBR green I-based qRT-PCR is a rapid and specific method for the clinical diagnosis and epidemiological investigation of related vesicular diseases caused by SVA.

Foot-and-mouth disease virus-like particles as integrin-based drug delivery system achieve targeting anti-tumor efficacy.

The surface of foot-and-mouth disease virus (FMDV)-like particles (VLPs) contains a conserved arginine-glycine-aspartic acid (RGD) motif. Natural FMDV specifically attaches to overexpressed integrin receptors in several cancer cells. The FMDV VLPs produced in Escherichia coli were used for the first time as a delivery system of anti-tumor drug doxorubicin (DOX). The DOX-loaded VLPs exhibited a distinct release profile in different physiological conditions. The effects of FMDV-VLPs-DOX on cellular internalization and viability were evaluated in vitro by cell imaging, MTT assay and apoptosis, respectively. The anti-tumor efficacy in vivo was also determined in a nude mouse xenograft model based on tumor volume/weight and histological changes. The FMDV-VLPs-DOX complex significantly inhibited the proliferation of tumor and improved the pathological damage of DOX to non-targeting tissues. All results supported the potential of FMDV VLPs as a platform for specific targeted delivery of drugs or chemical reagents.

Exploring Hollow Mesoporous Silica Nanoparticles as a Nanocarrier in the Delivery of Foot-And-Mouth Disease Virus-like Particle Vaccines.

Virus-like particle (VLP) vaccine is considered to be the most promising candidate alternative to the traditional inactivated vaccine for foot-and-mouth disease (FMD). To elicit a desired immune response, hollow mesoporous silica nanoparticles (HMSNs) have been synthesized and utilized as a nanocarrier for FMD VLP vaccine delivery. The as-prepared HMSNs displayed a relatively small particle size (∼260 nm), large cavity (∼150 nm), and thin wall (∼55 nm). The inherent structural superiorities make them ideal nanocarriers for the FMD VLP vaccine, which exhibited good biocompatibility, great protein-loading capacity, high antibody-response level, and protective efficiency, even comparable to commercial adjuvant ISA 206. All the results suggested that HMSNs may be a valid nanocarrier in VLP-based vaccines.

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.

[Evaluation of the humoral immunity in mice induced by foot-and-mouth disease virus-like particles-ZIF-8 complexes with different sizes].

To further enhance the immune effect of the foot-and-mouth disease (FMD) virus-like particles (VLPs) vaccine, this study prepared FMDV VLPs-zeolitic imidazolate (framework-8, ZIF-8) complexes with different particle sizes. We used a biomimetic mineralization method with Zn2+ and 2-methylimidazole in different concentration ratios to investigate the effect of size on the immunization effect. The results showed that FMDV VLPs-ZIF-8 with three different sizes were successfully prepared, with an approximate size of 70 nm, 100 nm, and 1 000 nm, respectively. Cytotoxicity and animal toxicity tests showed that all three complexes exhibited excellent biological safety. Immunization tests in mice showed that all three complexes enhanced the titers of neutralizing and specific antibodies, and their immune effects improved as the size of the complexes decreased. This study showed that ZIF-8 encapsulation of FMDV VLPs significantly enhanced their immunogenic effect in a size-dependent manner.

Poly(I:C) and CpG improve the assembly of foot-and-mouth disease virus-like particles and immune response in mice.

Virus-like particles (VLPs) are extremely potent, safe, and serviceable vaccine platforms. Good assembly efficiency of VLPs is the key to reducing vaccine production costs and eliciting a robust immune response. This study adopted CpG and Poly (I:C) as scaffolds to facilitate the assembly of foot-and-mouth disease virus (FMDV) VLPs in vitro. The VLPs and the co-assembly products were characterized by particle size, zeta potential, gel retardation measurement, nuclease digestion experiments, size-exclusion chromatography, transmission electron microscopy and circular dichroism analysis. Our results indicated the successful encapsulation of CpG and Poly (I:C) inside VLPs without any effect on shape or size. Vaccination in mice also elicited a robust immune response. This study demonstrated that CpG and Poly (I:C) improved the efficiency of FMDV VLPs assembly and enhanced immune response, further proposing a new idea for improving the efficiency of VLPs assembly and enriching the in vitro VLPs assembly strategies.

Self-Assembling Nanovaccine Fused with Flagellin Enhances Protective Effect against Foot-and-Mouth Disease Virus.

Nanovaccines based on self-assembling nanoparticles (NPs) can show conformational epitopes of antigens and they have high immunogenicity. In addition, flagellin, as a biological immune enhancer, can be fused with an antigen to considerably enhance the immune effect of antigens. In improving the immunogenicity and stability of a foot-and-mouth disease virus (FMDV) antigen, novel FMDV NP antigens were prepared by covalently coupling the VP1 protein and truncated flagellin containing only N-terminus D0 and D1 (N-terminal aa 1-99, nFLiC) with self-assembling NPs (i301). The results showed that the fusion proteins VP1-i301 and VP1-i301-nFLiC can assemble into NPs with high thermal tolerance and stability, obtain high cell uptake efficiency, and upregulate marker molecules and immune-stimulating cytokines in vitro. In addition, compared with monomeric VP1 antigen, high-level cytokines were stimulated with VP1-i301 and VP1-i301-nFLiC nanovaccines in guinea pigs, to provide clinical protection against viral infection comparable to an inactivated vaccine. This study provides new insight for the development of a novel FMD vaccine.

Bio-mineralization of virus-like particles by metal-organic framework nanoparticles enhances the thermostability and immune responses of the vaccines.

Virus-like particle (VLPs) vaccines have been extensively studied due to their good immunogenicity and safety; however, they highly rely on cold-chain storage and transportation. Nanotechnology of bio-mineralization as a useful strategy has been employed to improve the thermal stability and immunogenicity of VLPs. A zeolitic imidazole framework (ZIF-8), a core-shell structured nanocomposite, was applied to encapsulate foot-and-mouth disease virus (FMDV) VLPs. It was found that the ZIF-8 shell enhanced the heat resistance of VLPs and promoted their ability to be taken up by cells and escape from lysosomes. The VLPs-ZIF-8 easily activated antigen-presenting cells (APCs), triggered higher secretion levels of cytokines, and elicited stronger immune responses than VLPs alone even after being treated at 37 °C for 7 days. This platform has good potential in the development of VLP-based vaccine products without transportation.

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.

Sec62 Regulates Endoplasmic Reticulum Stress and Autophagy Balance to Affect Foot-and-Mouth Disease Virus Replication.

Endoplasmic reticulum (ER) stress-induced autophagy is closely associated with viral infection and propagation. However, the intrinsic link between ER stress, autophagy, and viral replication during foot-and-mouth disease virus (FMDV) infection is not fully elucidated. Our previous studies demonstrated that FMDV infection activated the ER stress-associated UPR of the PERK-eIF2a and ATF6 signaling pathway, whereas the IRE1a signaling was suppressed. We found that the activated-ATF6 pathway participated in FMDV-induced autophagy and FMDV replication, while the IRE1α pathway only affected FMDV replication. Further studies indicated that Sec62 was greatly reduced in the later stages of FMDV infection and blocked the activation of the autophagy-related IRE1α-JNK pathway. Moreover, it was also found that Sec62 promoted IRE1a phosphorylation and negatively regulated FMDV proliferation. Importantly, Sec62 may interact with LC3 to regulate ER stress and autophagy balance and eventually contribute to FMDV clearance via fusing with lysosomes. Altogether, these results suggest that Sec62 is a critical molecule in maintaining and recovering ER homeostasis by activating the IRE1α-JNK pathway and delivering autophagosome into the lysosome, thus providing new insights on FMDV-host interactions and novel antiviral therapies.

Four Simple Biomimetic Mineralization Methods to Improve the Thermostability and Immunogenicity of Virus-like Particles as a Vaccine against Foot-and-Mouth Disease.

The need for a cold chain system during storage and transport substantially increases the cost of vaccines. Virus-like particles (VLPs) are among the best countermeasures against foot and mouth disease virus (FMDV). However, VLPs are composed of pure proteins, and thus, are susceptible to heat. To address this problem, four simple biomimetic mineralization methods with the use of calcium phosphate were developed to improve heat tolerance via biomineralization. The results showed that biomineralization can significantly improve the heat resistance of VLPs. The biomineralized VLPs can be stored at low as 25 °C for eight days, and 37 °C for four days. Animal experiments showed that biomineralization had no effect on the immunogenicity of VLPs or the expression of specific antibodies (Abs) and neutralizing Abs. Even after heat treatment at 37 °C for four days, the biomineralized VLPs remained immunogenic and produced highly specific and neutralizing Abs with a high rate of protection. These results suggest that these biomineralization approaches can promote the thermal stability of VLPs against and significantly reduce dependence on cold storage and delivery systems.

Enhanced immunogenicity of foot and mouth disease DNA vaccine delivered by PLGA nanoparticles combined with cytokine adjuvants.

Although the immunogenicity of DNA vaccines is nonideal, they are still considered as potential alternative vaccine candidates to conventional vaccines. Various DNA delivery systems, including nanoparticles, have been extensively explored and validated to further enhance the immunogenicity of DNA vaccines. DNA vaccines are considered as alternative vaccine candidates. Various DNA delivery systems, including nanoparticles, have been extensively explored to enhance the immunogenicity of DNA vaccines. In this study, positively charged Poly (D, l-lactide-co-glycolic acid) (PLGA) nanoparticles were generated and characterized as a delivery system for O-serotype foot-and-mouth DNA vaccine. A recombinant plasmid encoding swine interleukin (IL)-18, IL-2, or granulocyte-macrophage colony-stimulating factor (GM-CSF) gene was introduced into the DNA vaccine to further improve its immunogenicity, which was evaluated in a guinea pig model. PLGA-pVAX-VP013/IL-18 elicited significantly (P = 0.0149) higher FMDV-specific antibody levels than naked DNA before the challenge. The level of neutralizing antibodies induced by PLGA-pVAX-VP013/IL-18, PLGA-pVAX-VP013/IL-2, and PLGA-pVAX-VP013/GM-CSF significantly increased compared with that induced by naked DNA (P < 0.0001). The lymphocyte proliferation assay showed that cellular immunity induced by PLGA-pVAX-VP013/IL-18 and PLGA-pVAX-VP013/GM-CSF was dramatically enhanced compared with that induced by the inactivated vaccine. The protection by PLGA-pVAX-VP013/IL-18 was consistent with that by the inactivated vaccine post-challenge and was followed by PLGA-pVAX-VP013/GM-CSF. Therefore, cationic PLGA nanoparticles can deliver DNA vaccines and induce humoral and cellular immune responses. The co-administration of FMD DNA vaccine with IL-18 formulated with PLGA nanoparticles was the optimal strategy to improve the immunogenicity of FMD DNA vaccines.

Potent Protective Immune Responses to Senecavirus Induced by Virus-Like Particle Vaccine in Pigs.

Senecavirus A (SVA) is the pathogen that has recently caused porcine idiopathic vesicular disease (PIVD). The clinical symptoms of PIVD are similar to those of acute foot-and-mouth disease and also can result in the death of newborn piglets, thus entailing economic losses. Vaccine immunization is the most effective way to prevent and control SVA. Among all SVA vaccines reported, only the SVA inactivated vaccine has been successfully developed. However, to ensure the elimination of this pathogen, safer and more effective vaccines are urgently required. A virus-like particles (VLPs)-based vaccine is probably the best alternative to inactivated vaccine. To develop an SVA VLPs vaccine and evaluate its immune effect, a prokaryotic expression system was used to produce SVA capsid protein and assemble VLPs. The VLPs were characterized by affinity chromatography, sucrose density gradient centrifugation, ZetaSizer and transmission electron microscopy. Meanwhile, the SVA CH-HB-2017 strain was used to infect pigs and to determine infection routes and dose. Experimental pigs were then immunized with the SVA VLPs vaccine emulsified in an ISA 201 adjuvant. The results showed that the VLPs vaccine induced neutralizing and specific antibodies at similar levels as an inactivated SVA vaccine after immunization. The level of INF-γ induced by the VLPs vaccine gradually decreased-similar to that of inactivated vaccine. These results indicated that VLPs vaccine may simultaneously cause both cellular and humoral immune responses. Importantly, after the challenge, the VLPs vaccine provided similar levels of protection as the inactivated SVA vaccine. In this study, we successfully obtained novel SVA VLPs and confirmed their highly immunogenicity, thus providing a superior candidate vaccine for defense and elimination of SVA, compared to the inactivated vaccine.

Golden-star nanoparticles as adjuvant effectively promotes immune response to foot-and-mouth disease virus-like particles vaccine.

Virus-like particles (VLPs) have become a hot topic in modern vaccine research because of its safety, facile production, and immune properties. To further enhance the immune effect of VLPs, we synthesized and used gold-star nanoparticles (AuSNs) as adjuvant for vaccine. Foot-and-mouth disease (FMD) VLPs as target antigen were combined with AuSNs. The FMD VLPs-AuSNs complex was characterized through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, ultraviolet light absorption, and morphological measurement analyses. Result indicated that the FMD VLPs-AuSNs complex is non-toxic in different cell lines. AuSNs can effectively promote the entry of FMD VLPs into cells and improve macrophages activation when combined with FMD VLPs compared with FMD VLPs alone. Further animal vaccination and challenge tests revealed that the specific immune response and protection rate of AuSNs adjuvant group is higher than that of conventional mineral oil (ISA206) adjuvant group. AuSNs can effectively improve the immune protection effects of FMD VLPs vaccines, and exhibit potential as a new adjuvant for other vaccines.

Purification of foot-and-mouth disease virus by heparin as ligand for certain strains.

The goal of this project was to develop an easily operable and scalable process for the recovery and purification of foot-and-mouth disease virus (FMDV) from cell culture. Heparin resins HipTrap Heparin HP and AF-Heparin HC-650 were utilized to purify FMDV O/HN/CHA/93. Results showed that the purity of AF-Heparin HC-650 was ideal. Then, the O/HN/CHA/93, O/Tibet/CHA/99, Asia I/HN/06, and A/CHA/HB/2009 strains were purified by AF-Heparin HC-650. Their affinity/virus recoveries were approximately 51.2%/45.8%, 71.5%/70.9%, 96.4%/73.5, and 59.5%/42.1%, respectively. During a stepwise elution strategy, the viral particles were mainly eluted at 300mM ionic strength peaks. The heparin affinity chromatography process removed more than 94% of cellular and medium proteins. Anion exchange resin Capto Q captured four FMD virus particles; 40% of binding proteins and 80%-90% of viral particles were eluted at 450mM NaCl. Moreover, ionic strength varied from 30 to 450mM had no effect on the immunity to FMDV. The results revealed that heparin sulfate may be the main receptor for CHA/99 strain attachment-susceptible cells. Heparin affinity chromatography can reach perfect results, especially when used as a ligand of the virus. Anion exchange is useful only as previous step for further purification.