Identification of a novel linear B-cell epitope on the p30 protein of African swine fever virus using monoclonal antibodies.

The outbreak of African Swine Fever (ASF) has caused huge economic losses to the pig industry. There are no safe and effective vaccines or diagnostics available. The p30 protein serves as a key target for the detection of ASFV antibodies and is an essential antigenic protein for early serological diagnosis. Here, the p30 protein was purified after being expressed in E. coli and its immunogenicity was verified in sera from pigs naturally infected with ASFV. Furthermore, a monoclonal antibody (McAb) designated as McAb 1B4G2-4 (subtype IgG1/kappa-type) was produced and it was verified to specifically recognize the ASFV Pig/HLJ/2018/strain and eukaryotic recombinant ASFV p30 protein. The epitope identified by McAb 1B4G2-4, defining the unique B-cell epitope 164HNFIQTI170, was located using peptide scanning. Comparing amino acid (aa) sequence revealed that this epitope is conserved in all reference ASFV strains from different regions of China, including the highly pathogenic strain Georgia 2007/1 (NC_044959.2) that is widely distributed. It is also exposed to the surface of the p30 protein, suggesting that it could be an important B-cell epitope. Our study may serve as a basis for the development of serological diagnostic methods and subunit vaccines.

A novel conserved B-cell epitope in pB602L of African swine fever virus.

African swine fever virus (ASFV) is a complex DNA virus and the only member of the Asfarviridae family. It causes high mortality and severe economic losses in pigs. The ASFV pB602L protein plays a key role in virus assembly and functions as a molecular chaperone of the major capsid protein p72. In addition, pB602L is an important target for the development of diagnostic tools for African swine fever (ASF) because it is a highly immunogenic antigen against ASFV. In this study, we expressed and purified ASFV pB602L and validated its immunogenicity in serum from naturally infected pigs with ASFV. Furthermore, we successfully generated an IgG2a κ subclass monoclonal antibody (mAb 7E7) against pB602L using hybridoma technology. Using western blot and immunofluorescence assays, mAb 7E7 specifically recognized the ASFV Pig/HLJ/2018/strain and eukaryotic recombinant ASFV pB602L protein in vitro. The 474SKENLTPDE482 epitope in the ASFV pB602L C-terminus was identified as the minimal linear epitope for mAb 7E7 binding, with dozens of truncated pB602l fragments characterized by western blot assay. We also showed that this antigenic epitope sequence has a high conservation and antigenic index. Our study contributes to improved vaccine and antiviral development and provides new insights into the serologic diagnosis of ASF. KEY POINTS: • We developed a monoclonal antibody against ASFV pB602L, which can specifically recognize the ASFV Pig/HLJ/2018/ strain. • This study found one novel conserved B-cell epitope 474SKENLTPDE482. • In the 3D structure, 474SKENLTPDE482 is exposed on the surface of ASFV pB602L, forming a curved linear structure.

A candidate nanoparticle vaccine comprised of multiple epitopes of the African swine fever virus elicits a robust immune response.

The African swine fever (ASF) pandemics pose a significant threat to the global swine industry, and the development of safe and effective vaccines is a daunting but necessary challenge. The level and persistence of immunity are very important for the effectiveness of the vaccine. Targeting antigens to antigen presenting cells (APCs) can greatly enhance immunogenicity. In this study, we developed a self-assembled nano-ASFV vaccine candidate (NanoFVax) targeting DCs, by covalently coupling the self-assembled 24-mer ferritin with the dominant B and T cell epitopes of the highly immunogenic ASFV antigen (p72, CD2v, pB602L and p30) and fused with the chemokine receptor XCL1 (a DC targeting molecule) through the SpyTag/SpyCatcher protein ligase system. Compared to monomeric protein, the nanoparticle vaccines can induce a more robust T-cell response, and the high-level antibody response against ASFV can last for more than 231 days. Therefore, the NanoFVax is a novel and promising vaccine candidate for ASFV.

Establishment of a Dual-Antigen Indirect ELISA Based on p30 and pB602L to Detect Antibodies against African Swine Fever Virus.

African swine fever (ASF) is an acute, virulent, and highly fatal infectious disease caused by the African swine fever virus (ASFV). There is no effective vaccine or diagnostic method to prevent and control this disease currently, which highlights the significance of ASF early detection. In this study, we chose an early antigen and a late-expressed antigen to co-detect the target antibody, which not only helps in early detection but also improves accuracy and sensitivity. CP204L and B602L were successfully expressed as soluble proteins in an Escherichia coli vector system. By optimizing various conditions, a dual-antigen indirect ELISA for ASFV antibodies was established. The assay was non-cross-reactive with antibodies against the porcine reproductive and respiratory syndrome virus, classical swine fever virus, porcine circovirus type 2, and pseudorabies virus. The maximum serum dilution for detection of ASFV-positive sera was 1:1600. The intra-batch reproducibility coefficient of variation was <5% and the inter-batch reproducibility coefficient of variation was <10%. Compared with commercial kits, the dual-antigen indirect ELISA had good detection performance. In conclusion, we established a detection method with low cost, streamlined production process, and fewer instruments. It provides a new method for the serological diagnosis of ASF.

Development of an Effective Double Antigen Sandwich ELISA Based on p30 Protein to Detect Antibodies against African Swine Fever Virus.

African swine fever (ASF), the highly lethal swine infectious disease caused by the African swine fever virus (ASFV), is a great threat to the swine industry. There is no effective vaccine or diagnostic method to prevent and control this disease currently. The p30 protein of ASFV is an important target for serological diagnosis, expressed in the early stage of viral replication and has high immunogenicity and sequence conservatism. Here, the CP204L gene was cloned into the expression vector pET-30a (+), and the soluble p30 protein was successfully expressed in the E. coli prokaryotic expression system and then labeled with horseradish peroxidase (HRP) to be the enzyme-labeled antigen. Using the purified recombinant p30 protein, a double-antigen sandwich ELISA for ASFV antibody detection was developed. This method exhibits excellent specificity, sensitivity and reproducibility in clinical sample detection with lower cost and shorter production cycles. Taken together, this study provides technical support for antibody detection for ASFV.

Prevalence of multiple sclerosis in Guangzhou, China: A population-based case-finding prospective study.

BACKGROUND: Multiple sclerosis (MS) is rare in China, and the prevalence previously reported may be biased. Currently, few studies that have investigated the prevalence of MS in China based on the latest diagnostic criteria. METHODS: Through a population-based survey from August 8, 2021 to December 31, 2021, we calculated the prevalence of multiple sclerosis in 18,676,605 residents of Guangzhou, China. MS patients were identified through the health insurance system of the Guangzhou Health Insurance Bureau, and we surveyed 17 large tertiary hospitals using a case-finding approach. All MS patients were diagnosed according to the 2017 McDonald criteria. RESULTS: A total of 143 patients in the resident population of Guangzhou were diagnosed with MS, with a crude prevalence of 0.77 per 100,000 (95% confidence interval (CI): 0.65-0.90), and the prevalence was higher in in females (1.14/100,000) than in males (0.44/100,000). The age-adjusted prevalence was 0.92 per 100,000 (95% CI: 0.77-1.10). The prevalence peaked at the age of 25-29 years (2.86/100,000) for both males and females (1.44/100,000 and 4.42/100,000, respectively). CONCLUSIONS: This is the first study to report the prevalence of MS in Guangzhou, China, according to the criteria. Our study shows that the prevalence of MS in Guangzhou is lower than that in other cities in China.

ZIF-8 derived porous carbon to mitigate shuttle effect for high performance aqueous zinc-iodine batteries.

Rechargeable aqueous zinc-iodine batteries (ZIBs) with low environmental impacts and abundant natural reserves have emerged as promising electrochemical energy storage devices. However, the shuttle effect and low conductivity of the iodine species cause poor electrochemical performance and hinder their practical application. Herein, we propose a ZIF-8 derived porous carbon (ZPC) for iodine species immobilization in ZIBs. The rich porous structure and highly conductive framework of ZPC provide efficient iodine loading and allow the fast transmission of electrons. In addition, the presence of N, Zn and ZnO in the carbon framework can build chemical anchoring with the iodine species to mitigate the shuttle effect. Thus, the ZPC/I2 cathode exhibits a reversible capacity of 156 mAh g-1 after 100 cycles at 100 mA g-1 and a long-term stability of 1000 cycles at a high rate. This study will open a new paradigm for devolving highly reversible ZIBs.

Concurrent production of glycyrrhetic acid 3-O-mono-ß-d-glucuronide and lignocellulolytic enzymes by solid-state fermentation of a plant endophytic Chaetomium globosum.

Glycyrrhetic acid 3-O-mono-ß-d-glucuronide (GAMG) as an important derivative of glycyrrhizin (GL) shows stronger biological activities and higher sweetness than GL. The biotransformation process is considered as an efficient strategy for GAMG production, due to its mild reaction, high production efficiency and environmentally friendly status. In this study, licorice straw was used for the first time as a medium for GAMG and lignocellulosic enzyme production via solid-state fermentation (SSF) of endophytic fungus Chaetomium globosum DX-THS3. The fermentation conditions including particle size, temperature, seed age, inoculum size, and moisture of substrate were optimized. Furthermore, additional nitrogen sources and carbon sources were screened for GAMG production by C. globosum DX-THS3 of SSF. Under optimal fermentation conditions, the percent conversion of glycyrrhizin reached 90% in 15 days, whereas the control needed 35 days to achieve the same result. The productivity of optimization (P = 2.1 mg/g/day) was 2.33-fold that of non-optimization (P = 0.9 mg/g/day). Meanwhile, high activities of filter paper enzyme (FPase) (245.80 U/g), carboxymethyl cellulase (CMCase) (33.67 U/g), xylanase (83.44 U/g), and ß-glucuronidase activity (271.42 U/g) were obtained faster than those in the control during SSF. Our study provides a novel and efficient strategy for GAMG production and indicates C. globosum DX-THS3 as a potential producer of lignocellulolytic enzymes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40643-021-00441-y.

Rapid detection of Salmonella in milk by a nuclear magnetic resonance biosensor based on the streptavidin-biotin system and O-carboxymethyl chitosan target gadolinium probe.

Rapid and sensitive detection of foodborne pathogens is of great importance for food safety. Here, a set of nuclear magnetic resonance (NMR) biosensors based on a O-carboxymethyl chitosan target gadolinium (Gd) probe was developed to quickly detect Salmonella in milk by combining NMR technology and bioimmunotechnology with membrane filtration technology. First, O-carboxymethyl chitosan (O-CMC) was biotinylated to prepare biotinylated O-carboxymethyl chitosan (biotin-O-CMC) through amide reaction, and biotinylated magnetic complexes (biotin-O-CMC-Gd) were obtained by using O-CMC, which has strong chelating adsorption on Gd. The target probe was obtained by combining biotin-O-CMC-Gd with the biotinylated antibody (biotin-antibody) via streptavidin (SA) by introducing the SA-biotin system. Then, Salmonella was captured by the target probe through antigen-antibody interaction. Finally, NMR was used to measure the longitudinal relaxation time (T1) of the filtrate collected by membrane filtration. This NMR biosensor with good specificity and high efficiency can detect Salmonella with the sensitivity of 1.8 × 103 cfu/mL within 2 h; in addition, it can realize the detection of complex samples because of its strong anti-interference capability and may open up a new method for rapid detection of Salmonella, which has a great application potential.