Antigenic and immunogenic properties of recombinant proteins consisting of two immunodominant African swine fever virus proteins fused with bacterial lipoprotein OprI.

BACKGROUND: African swine fever (ASF) is a highly fatal swine disease, which threatens the global pig industry. There is no commercially available vaccine against ASF and effective subunit vaccines would represent a real breakthrough. METHODS: In this study, we expressed and purified two recombinant fusion proteins, OPM (OprI-p30-modified p54) and OPMT (OprI-p30-modified p54-T cell epitope), which combine the bacterial lipoprotein OprI with ASF virus proteins p30 and p54. Purified recombinant p30 and modified p54 expressed alone or fused served as controls. The activation of dendritic cells (DCs) by these proteins was first assessed. Then, humoral and cellular immunity induced by the proteins were evaluated in mice. RESULTS: Both OPM and OPMT activated DCs with elevated expression of relevant surface molecules and proinflammatory cytokines. Furthermore, OPMT elicited the highest levels of antigen-specific IgG responses, cytokines including interleukin-2, interferon-γ, and tumor necrosis factor-α, and proliferation of lymphocytes. Importantly, the sera from mice vaccinated with OPM or OPMT neutralized more than 86% of ASF virus in vitro. CONCLUSIONS: Our results suggest that OPMT has good immunostimulatory activities and immunogenicity in mice, and might be an appropriate candidate to elicit immune responses in swine. Our study provides valuable information on further development of a subunit vaccine against ASF.

Development of a competitive chemiluminescence immunoassay using a monoclonal antibody recognizing 3B of foot-and-mouth disease virus for the rapid detection of antibodies induced by FMDV infection.

BACKGROUND: Foot-and-mouth disease (FMD) is a devastating animal disease. Anti-non-structural protein (NSP) antibody detection is very important for confirming suspected cases, evaluating the prevalence of infection, certifying animals for trade and controlling the disease. METHODS: In this study, a competitive chemiluminescence immunoassay (3B-cCLIA) was developed for the rapid detection of antibodies against NSPs in different species of livestock animals using the monoclonal antibody (mAb) 9E2 as a competitive antibody that recognizes NSP 3B. RESULTS: The cut-off value (50%), diagnostic sensitivity (Dsn) (97.20%, 95.71%, and 96.15%) and diagnostic specificity (Dsp) (99.51%, 99.43%, and 98.36) of the assay were estimated by testing a panel of known-background sera from swine, cattle and sheep, respectively. The accuracy rate of the 3B-cCLIA was further validated and subsequently compared with that of two commercial diagnostic kits. The early diagnostic results showed that antibodies recognizing NSPs developed later (approximately 1-2 days) than antibodies recognizing structural proteins. Furthermore, anti-NSP antibody presence in animals vaccinated multiple times (false positives), especially cattle and sheep, was confirmed, and the false-positive rate increased with the number of vaccinations. CONCLUSIONS: These results indicate that the 3B-cCLIA is suitable for the rapid detection of antibodies against FMDV NSP 3B in a wide range of species.

Development of an indirect ELISA to specifically detect antibodies against African swine fever virus: bioinformatics approaches.

BACKGROUND: African swine fever (ASF), characterized by acute, severe, and fast-spreading, is a highly lethal swine infectious disease caused by the African swine fever virus (ASFV), which has caused substantial economic losses to the pig industry worldwide in the past 100 years. METHODS: This study started with bioinformatics methods and verified the epitope fusion protein method's reliability that does not rely on traditional epitope identification. Meanwhile, it will also express and purify the constructed genes through prokaryotic expression and establish antibody detection methods. RESULTS: The results indicated that the protein had good reactivity and did not cross-react with other swine diseases. The receiver-operating characteristic analysis was performed to verify the determination. The area under the receiver-operating characteristic curve was 0.9991 (95% confidence interval 0.9973 to 1.001). CONCLUSIONS: It was proved that the recombinant protein is feasible as a diagnostic antigen to distinguish ASFV and provides a new idea for ASFV antibody detection.

Development of an Indirect Chemiluminescence Immunoassay Using a Multiepitope Recombinant Protein To Specifically Detect Antibodies against Foot-and-Mouth Disease Virus Serotype O in Swine.

Foot-and-mouth disease virus (FMDV) has led to serious losses in animal husbandry worldwide. Seromonitoring of FMDV postvaccination is important for the control and eradication of foot-and-mouth disease (FMD) in regions and countries where vaccination is widespread. However, many commercial kits present high false-positive rates. In this study, a multiepitope-based indirect chemiluminescence immunoassay (ME-CLIA) was developed for specifically detecting antibodies against FMDV serotype O in swine sera. The developed method presented high diagnostic sensitivity and excellent diagnostic specificity, and it could detect a broad spectrum of antibodies against FMDV serotype O. The diagnostic performance, accuracy rate, and analytical sensitivity of ME-CLIA were compared with those of three commercial kits. The immune protection value of multiple-epitope recombinant vaccine detected using ME-CLIA was preliminarily determined by observation of clinical symptoms postimmunization challenge, the results of which indicated that the ME-CLIA can be employed as a matching detection method for evaluating multiple-epitope recombinant vaccine. The percent positive values of ME-CLIA determined using swine vaccinated with inactivated vaccine were significantly positively correlated with the titers of liquid-phase-blocking enzyme-linked immunosorbent assay (ELISA) (LBPE) (r = 0.8361; P < 0.0001). These results indicated that ME-CLIA is suitable for detection of antibodies against FMDV serotype O in swine and for potency evaluation of multiple-epitope and inactivated vaccines.