Bio-mineralization has emerged as a promising strategy to enhance vaccine immunogenicity. This study optimized the calcium phosphate (CaP) mineralization process of foot-and-mouth disease virus-like particles (FMD VLPs) to achieve high mineralization efficiency and scalability. Key parameters, including concentrations of Ca²⁺, HPO₄^2-, NaCl, and VLPs, as well as stirring speed, were systematically optimized. Stability of the scaled-up reaction system and immunogenicity of the mineralized vaccine were evaluated. Optimal conditions [25.50 mmol/L Ca(NO₃)₂, 15 mmol/L Na₂HPO₄, 300 mmol/L NaCl, 0.75 mg/mL VLPs, and 1 500 r/min] yielded CaP-mineralized VLPs (VLPs-CaP) with high mineralization efficiency, uniform morphology, and a favorable particle size. Scaling up the reaction by 25 folds maintained consistent mineralization efficiency and particle characteristics. Immunization in mice demonstrated that VLPs-CaP induced higher titers of specific antibodies and neutralizing antibodies than unmineralized VLPs (P < 0.05). Higher IgG2a/IgG1 ratio and enhanced IFN-γ secretion (P < 0.05) further indicated robust cellular immune responses. We establish a stable and scalable protocol for VLPs-CaP, providing a theoretical and technical foundation for developing high-efficacy VLPs-CaP vaccines.
Vaccines, Virus-Like Particle/immunology* ; Immunogenicity, Vaccine ; Calcium Phosphates/chemistry* ; Foot-and-Mouth Disease Virus ; Biomineralization ; Particle Size ; Animals ; Mice ; Antibodies, Neutralizing/blood* ; Antibodies, Viral/blood* ; Immunity, Cellular

Vaccination is a crucial strategy for the prevention and control of infectious diseases. Virus-like particles (VLPs), composed of structural proteins, have garnered significant attention as a novel type of vaccine due to their excellent safety and immunogenicity. However, similar to most vaccine antigens, VLPs exhibit insufficient thermal stability, which not only restricts the widespread application of vaccines but also increases the risk of vaccine inactivation. This study aims to enhance the stability and shelf life of VLPs derived from type A foot-and-mouth disease virus (FMDV) by employing vacuum freeze-drying technology. The optimal lyoprotectant formulation was determined through single-factor and combinatorial screening. Subsequently, the correlation between the immunogenicity of the freeze-dried vaccine and the content of FMDV VLPs was evaluated via a mouse model. The stability of FMDV VLPs before and after freeze-drying was further assessed by storing them at 4, 25, and 37 ℃ for varying time periods. Results indicated that the lyoprotectant formulation No.1, composed of 7.5% trehalose, 0.1% Tween 80, 50 mmol/L glycine, 1% sodium glutamate, and 3% polyvinylpyrrolidone (PVP), effectively preserved the content of FMDV VLPs during the vacuum freeze-drying process. The immunization trial in mice revealed that the levels of specific antibodies, immunoglobulin G1 (IgG1), interleukin-4 (IL-4), and neutralizing antibodies induced by freeze-dried FMDV VLPs were comparable to those induced by non-freeze-dried FMDV VLPs. The heat treatment results showed that the storage periods of freeze-dried FMDV VLPs at 4, 25, and 37 ℃ were significantly longer than those of non-freeze-dried FMDV VLPs. In conclusion, the selected lyoprotectant formulation effectively improved the stability of FMDV VLPs vaccines. This study provides valuable insights for enhancing the stability of novel subunit vaccines.
Freeze Drying/methods* ; Animals ; Foot-and-Mouth Disease Virus/immunology* ; Mice ; Vaccines, Virus-Like Particle/chemistry* ; Foot-and-Mouth Disease/immunology* ; Vacuum ; Drug Stability ; Mice, Inbred BALB C ; Viral Vaccines/immunology*

Objective:To investigate the detection status and genotypes distribution characteristics of norovirus(NoV)in environmental sewage from three monitoring points in Fujian province, and to explore the significance of its application to NoV monitoring.Methods:Sewage samples were collected monthly at 5 sampling sites in representative monitoring cities, enriched and concentrated. Partial gene fragments of norovirus VP1 were amplified by reverse transcription-semi nested polymerase chain reaction (RT-snPCR), TA cloned and sequenced. Genotypes were identified based on the sequencing.Results:A total of 56 sewage samples were collected from July 2022 to June 2023. The detection rates of GⅠ and GⅡ were 89.29% (50/56) and 94.64% (53/56), respectively. A total of 7 NoV GⅠ genotypes and 13 GⅡgenotypes were identified. GⅠ.1, GⅠ.4, GⅡ.4 and GⅡ.17 were the dominant genotypes. NoV genotypes detected in different sampling sites were not exactly the same. The detection rate of NoV was low from August to November 2022, and the prevalence of the dominant genotypes was different in different seasons. GⅠ.1 and GⅡ.4 were highly prevalent from August to November 2022, but were replaced by GⅠ.4 and GⅡ.17 from December 2022 to June 2023, respectively. More NoV genotypes were detected in January-June 2023, comparing to the July-December 2022. The dominant genotype GII.17, has multiple clades and new variants have been discovered that are different from the 2014/2015 circulating strains.Conclusions:The detection rates of NoV in environmental sewage were very high, and genotypes were diverse. Environmental sewage surveillance could be an important complementary method for NoV cases surveillance.

The variable domain of heavy-chain antibody (VHH) has been developed widely in drug therapy, diagnosis, and research. Escherichia coli is the most popular expression system for VHH production, whereas low bioactivity occurs sometimes. Mammalian cells are one of the most ideal hosts for VHH expression at present. To improve the yield of VHH in Expi293F cells, we optimized the signal peptide (SP) and codons of VHH. Firstly, the fusion protein VHH1-Fc was used to screen SPs. The SP IFN-α2 showed the highest secretion as quantified by enzyme-linked immunosorbent assay (ELISA). Subsequently, codon optimization by improving GC3 and GC content doubled the yield of VHH1 and kept its binding activity to Senecavirus A (SVA). Finally, the mean yields of other 5 VHHs that fused with SP IFN-α2 and codon-optimized were over 191.6 mg/L, and these VHHs had high recovery and high purity in the culture supernatant. This study confirms that SP IFN-α2 and codon optimization could produce VHHs in Expi293F cells efficiently, which provides a reference for the large-scale production of VHHs.
Codon/genetics* ; Protein Sorting Signals/genetics* ; Escherichia coli/metabolism* ; Humans ; Recombinant Fusion Proteins/biosynthesis* ; Interferon-alpha/metabolism* ; Immunoglobulin Heavy Chains/immunology* ; Cell Line ; Immunoglobulin Variable Region/immunology*

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.
Animals ; Mice ; Foot-and-Mouth Disease/prevention & control* ; Foot-and-Mouth Disease Virus ; Antibodies, Neutralizing ; Immunity, Humoral ; Immunization ; Vaccines, Virus-Like Particle ; Antibodies, Viral ; Viral Vaccines

Transient expression is the major method to express foot-and-mouth disease virus (FMDV) capsid proteins in mammalian cells. To achieve stable expression of FMDV capsid proteins and efficient assembly of virus like particles (VLPs) in cells, the plasmids of piggyBac (PB) transposon-constitutive expression and PB transposon-tetracycline (Tet) inducible expression vectors were constructed. The function of the plasmids was tested by fluorescent proteins. By adding antibiotics, the constitutive cell pools (C-WT, C-L127P) expressing P12A3C (WT/L127P) genes and the inducible cell pools (I-WT, I-L127P) expressing P12A3C (WT/L127P) genes were generated. The genes of green fluorescent protein, 3C protease and reverse tetracycline transactivator (rtTA) were integrated into chromosome, which was confirmed by fluorescence observation and PCR testing. The cell pool I-L127P has a stronger production capacity of capsid proteins and VLPs, which was confirmed by Western blotting and enzyme linked immunosorbent assay (ELISA), respectively. In conclusion, inducing the chromosomal expression of FMDV capsid proteins was firstly reported, which may facilitate the technical process of mammalian production of FMDV VLPs vaccine and the construction of mammalian inducible expression systems for other proteins.
Animals ; Foot-and-Mouth Disease Virus/genetics* ; Capsid Proteins ; Viral Proteins/metabolism* ; Foot-and-Mouth Disease/prevention & control* ; Tetracyclines/metabolism* ; Viral Vaccines ; Antibodies, Viral ; Mammals/metabolism*

Objective:To investigate the regulation of intestinal microbiota by Roux-en-Y gastric bypass (RYGB) on patients with obesity or obesity combined with diabetes.Methods:The retrospective and descriptive study was conducted. The stool samples before and after surgery and clinical data of 20 patients with obesity, including 9 simple obesity cases and 11 obesity combined with diabetes cases, who underwent RYGB in the First Affiliated Hospital of Ji′nan University from July 2016 to August 2017 were collected. There were 11 males and 9 females, aged (33±11)years. Observation indicators: (1) changes in composition and structure of intestinal microflora; (2) changes of intestinal microflora in simple obesity patients after operation; (3) changes of intestinal microflora in obesity combined with diabetes patients after operation. Follow up was conducted using telephone interview or outpatient examinations to detect the body mass, the application of antimicrobial agent and the blood glucose control of patients. According to the unified training points, the stool samples were collected and stored into the DNA stabilizer, and then conducted to laboratory analysis within 45 hours. The follow up was up to November 2018. Measurement data with normal distribution were represented as Mean± SD, and independent-samples t test was used for inter-group comparison and paired-samples t test was used for intra-group comparison. Measurement data with skewed distribution were represented as M( Q1, Q3), and Wilcoxon signed rank test of two independent samples was used for inter-group comparison. Count data were described as absolute numbers, and the chi-square test, ANOSIM analysis, linear discriminant (LEfSe) analysis and the Metastats analysis were used for inter-group comparison. Results:(1) Changes in composition and structure of intestinal microflora. The Shannon index of α diversity of preoperative intestinal microflora in simple obesity patients and obesity combined with diabetes patients was 4.37±0.69 and 4.47±0.85, respectively, showing no significant difference between them ( t=0.28, P>0.05). Results of preoperative LEfSe analysis showed that there were differences in the bacterial abundance of Firmicutes and Bacteroidea between simple obesity patients and obesity combined with diabetes patients. The abundances of Parasutterella in simple obesity patients and obesity combined with diabetes patients was 0.000 113 0(0, 0.004 378 2) and 0.008 464 0(0.001 325 7, 0.034 983 1), respectively, showing a significant difference between them ( Z=2.12, P<0.05). Results of preoperative PCoA analysis showed that the contribution rates of principal component 1, principal component 2 and principal component 3 were 24.98%, 22.24% and 16.33% in simple obesity patients and obesity combined with diabetes patients and results of ANOSIM comparison showed that there was no significant difference in preoperative intestinal microflora between them ( r=?0.11, P>0.05). The Shannon index of α diversity of postoperative intestinal microflora in simple obesity patients and obesity combined with diabetes patients was 4.60±0.65 and 4.66±0.40, respectively, showing no significant difference between them ( t=0.24, P>0.05). Results of postoperative LEfSe analysis showed that there were differences in the bacterial abundance of Bacteroidea, Proteus and Firmicutes between simple obesity patients and obesity combined with diabetes patients. The abundances of Morganella and Coprococcus_2 in simple obesity patients and obesity combined with diabetes patients were 0.000 192 0(0.000 011 9,0.001 569 0), 0(0,0) and 0(0,0), 0.000 054 1(0,0.000 419 0), showing significant differences between them ( Z=2.70, 2.29, P<0.05). (2) Changes of intestinal microflora in simple obesity patients after operation. There were 10 genera of bacteria of intestinal bacteria changing after surgery, including 7 species of bacteria increasing in the Firmicutes and the Proteobacteria as Veillonella, Morganella, Granulicatella, Aeromonas, Streptococcus, Rothia and Megasphaera and the bacteria decreasing in the Firmicutes and the Actinobacteria as Ruminococcus_torques_group, Romboutsia and Erysipelo-trichaceae_UCG-003. Results of LEfSe analysis showed that the bacteria significantly enriched in simple obesity patients before surgery were Ruminococcus_torques_group, Romboutsia and Erysipelotri-chaceae_UCG-003, belonging to Firmicutes, and the bacteria significantly enriched in simple obesity patients after surgery were Rothia, Granulicatella, Enterococcus, Streptococcus, Megasphaera, Veillonella, A eromonas and Morganella, belonging to Actinobacteria, Firmicutes and Proteobacteria. (3) Changes of intestinal microflora in obesity combined with diabetes patients after operation. There were 16 bacteria of intestinal bacteria increasing after surgery, including Streptococcus, Veillonella, Haemophilus, Pluralibacter, Gemella, Lachnospiraceae_NC2004_group, Granulicatella,Aeromonas, uncultured_ bacterium_f_ Saccharimonadaceae, R uminiclostridium_9, Butyricicoccus, Fusobacterium, Anaerotruncus, Fusicateni-bacter, Klebsiella and E ubacterium_eligens_group, which belonged to the Firmicutes and the Proteo-bacteria. Results of LEfSe analysis showed that the bacteria significantly enriched in obesity combined with diabetes patients before surgery were Fusicatenibacter, Tyzzerella_3 and Butyricicoccus, belonging to the Firmicutes, and the bacteria significantly enriched in obesity combined with diabetes patients after surgery were Gemella, Granulicatella, Enterococcus, Streptococcus, Lachnospiraceae_NC2004_group, Eubacterium_eligens_group, Anaerotruncus, Ruminiclostridium_9, Anaeroglobus, Veillonella, Fusobacterium, uncultured_bacterium_f_Saccharimonadaceae, Aeromonas, Klebsiella, Pluralibacter, Proteus and Haemophilus, belonging to the Firmicutes and the Proteobacteria. Conclusions:RYGB can significantly increases the intestinal microflora abundance in simple obesity patients and obesity combined with diabetes patients. The two types of patients have specific changes in intestinal microflora at the genus level.

Animals ; Antibodies, Neutralizing ; Antibodies, Viral ; Foot-and-Mouth Disease Virus

The stability of virus-like particles (VLPs) is currently the main factor affecting the quality of foot-and-mouth disease VLPs vaccines. In order to further improve the quality of the VLPs vaccine of foot-and-mouth disease (FMD), three amino acid modification sites were designed and screened through kinetic analysis software, based on the three-dimensional structure of FMDV. The three mutant recombinant plasmids were successfully prepared by the point mutation kit, transformed into Escherichia coli strain BL21 and expressed in vitro. After purification by Ni ion chromatography column, SDS-PAGE proved that the three amino acid mutations did not affect the expression of the target protein. The results of the stability study of three FMD mutant VLPs obtained by in vitro assembly show that the introduction of internal hydrophobic side chain amino acids made the morphology of VLPs more uniform (N4017W), and their stability was significantly improved compared to the other two VLPs. The internal hydrophobic force of the capsid contributes to the formation of VLPs and helps to maintain the stability of the capsid, providing new experimental ideas for improving the quality of VLPs vaccines, and helping to promote the development of VLPs vaccines.
Amino Acids ; Animals ; Capsid Proteins/genetics* ; Foot-and-Mouth Disease/prevention & control* ; Foot-and-Mouth Disease Virus/genetics* ; Kinetics ; Vaccines, Virus-Like Particle/genetics* ; Viral Vaccines/genetics*

Purpose: This study was to investigate the work experience of newly recruited male nurses during the COVID-19 pandemic. Methods: With a phenomenological approach, this qualitative study was adopted semistructured interviews by phone or video calls. A total of 9 male nurses newly recruited for the COVID-19 wards in Chinese hospitals were interviewed for this study. And Colaizzi's method was applied for evaluation in the data analysis. Results: Based on our findings, three themes were extracted. First, the newly recruited male nurses showed negative emotions at the beginning of COVID-19 epidemic, which was caused by changes in working conditions and content, but also prompted the nurses to change the way of coping with the crisis. Second, they gradually mastered the working skills and psychological training to cope with COVID-19 and developed a positive attitude toward life and a high sense of professional responsibility. Finally, we learned about their needs to respond to public health emergencies such as the COVID-19 pandemic. Conclusion COVID-19 is a disaster for all of humanity. The newly recruited male nurses are an important force in emergency rescue. Although they suffered from short-term negative emotions, they quickly adapted to the crisis. In order to better prepare for future emergencies, the disaster response capacity of newly recruited male nurses needs to be further improved. In addition, newly recruited male nurses have a strong demand for timely and personalized career development guidance.