Evaluation of humoral and cellular immune responses induced by a cocktail of recombinant African swine fever virus antigens fused with OprI in domestic pigs.

BACKGROUND: African swine fever (ASF) is a highly fatal disease in domestic pigs caused by ASF virus (ASFV), for which there is currently no commercial vaccine available. The genome of ASFV encodes more than 150 proteins, some of which have been included in subunit vaccines but only induce limited protection against ASFV challenge. METHODS: To enhance immune responses induced by ASFV proteins, we expressed and purified three fusion proteins with each consisting of bacterial lipoprotein OprI, 2 different ASFV proteins/epitopes and a universal CD4+ T cell epitope, namely OprI-p30-modified p54-TT, OprI-p72 epitopes-truncated pE248R-TT, and OprI-truncated CD2v-truncated pEP153R-TT. The immunostimulatory activity of these recombinant proteins was first assessed on dendritic cells. Then, humoral and cellular immunity induced by these three OprI-fused proteins cocktail formulated with ISA206 adjuvant (O-Ags-T formulation) were assessed in pigs. RESULTS: The OprI-fused proteins activated dendritic cells with elevated secretion of proinflammatory cytokines. Furthermore, the O-Ags-T formulation elicited a high level of antigen-specific IgG responses and interferon-γ-secreting CD4+ and CD8+ T cells after stimulation in vitro. Importantly, the sera and peripheral blood mononuclear cells from pigs vaccinated with the O-Ags-T formulation respectively reduced ASFV infection in vitro by 82.8% and 92.6%. CONCLUSIONS: Our results suggest that the OprI-fused proteins cocktail formulated with ISA206 adjuvant induces robust ASFV-specific humoral and cellular immune responses in pigs. Our study provides valuable information for the further development of subunit vaccines against ASF.

Antigenicity and immunogenicity of recombinant proteins comprising African swine fever virus proteins p30 and p54 fused to a cell-penetrating peptide.

African swine fever (ASF) is a highly fatal swine disease threatening the global pig industry. Currently, vaccine is not commercially available for ASF. Hence, it is desirable to develop effective subunit vaccines against ASF. Here, we expressed and purified two recombinant fusion proteins comprising ASFV proteins p30 and p54 fused to a novel cell-penetrating peptide Z12, which were labeled as ZPM (Z12-p30-modified p54) and ZPMT (Z12-p30-modified p54-T cell epitope). Purified recombinant p30 and modified p54 expressed alone or fused served as controls. The transduction capacity of these recombinant proteins was assessed in RAW264.7 cells. Both ZPM and ZPMT exhibited higher transduction efficiency than the other proteins. Subsequently, humoral and cellular immune responses elicited by these proteins were evaluated in mice. ZPMT elicited the highest levels of antigen-specific IgG responses, cytokines (interleukin-2, interferon-γ, and tumor necrosis factor-α) and lymphocyte proliferation. Importantly, sera from mice immunized with ZPM or ZPMT neutralized greater than 85% of ASFV in vitro. Our results indicate that ZPMT induces potent neutralizing antibody responses and cellular immunity in mice. Therefore, ZPMT may be a suitable candidate to elicit immune responses in swine, providing valuable information for the development of subunit vaccines against ASF.

Identification of a specific surface epitope of OmpC for Escherichia coli O157:H7 with protein topology facilitated affinity mass spectrometry.

The goal of this work was to identify the target protein and epitope of a previously reported Escherichia coli O157:H7 (ECO157)-specific monoclonal antibody (mAb) 2G12. mAb 2G12 has shown high specificity for the recovery and detection of ECO157. To achieve this goal, the target protein was first separated by two-dimensional gel electrophoresis (2-DE) and located by Western blot (WB). The protein spots were identified to be the outer membrane protein (Omp) C by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). After that, the target protein was purified by immunoaffinity chromatography (IAC) and subjected to in situ enzymatic cleavage of the vulnerable peptides. Eight eluted peptides of OmpC identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) were further mapped onto the homologous protein structure of E. coli OmpC (2IXX). The topology of OmpC showed that three peptides had extracellular loops. Epitope mapping with overlapping peptide library and sequence homology analysis revealed that the epitope consisted of a specific peptide, "LGVING," and an adjacent conservative peptide, "TQTYNATRVGSLG." Both peptides loop around the overall structure of the epitope. To test the availability of the epitope when ECO157 was grown under different osmolarity, pH, and nutrition levels, the binding efficacy of mAb 2G12 with ECO157 grown in these conditions was evaluated. Results further demonstrated the good stability of this epitope under potential stressful environmental conditions. In summary, this study revealed that mAb 2G12 targeted one specific and one conservative extracellular loop (peptide) of the OmpC present on ECO157, and the epitope was stable and accessible on ECO157 cells grown in different environment. KEY POINTS: • OmpC is the target of a recently identified ECO157-specific mAb 2G12. • Eight peptides were identified from the OmpC by using LC-MS/MS. • The specificity of mAb 2G12 is mainly determined by the "LGVING" peptide.

First report of the prevalence and genetic characterization of Giardia duodenalis and Cryptosporidium spp. in Yunling cattle in Yunnan Province, southwestern China.

Yunling cattle is an unique cattle breed distributed in Yunnan Province, southwestern China. It is yet to know whether Yunling cattle are infected with Giardia duodenalis and Cryptosporidium spp.. The objectives of the present study were to investigate the prevalence and characterize the assemblages of G. duodenalis and species of Cryptosporidium spp. in Yunling cattle in Yunnan province. The overall prevalence of G. duodenalis and Cryptosporidium spp. were 10.49% (41/391) and 0.77% (3/391), respectively. The age was considered as the risk factor for Yunling cattle infection with G. duodenalis (χ2 = 8.082, OR = 2.56, P = 0.004). Two assemblages of G. duodenalis, assemblage A (n = 1) and assemblage E (n = 40), were identified by amplification of the ß-giardin (bg) and glutamate dehydrogenase (gdh) gene loci using the nested PCR methods. Furthermore, Cryptosporidium andersoni (n = 1) and Cryptosporidium ryanae (n = 2) were detected by nested PCR targeting the small subunit (SSU) rRNA gene. This is the first report of G. duodenalis and Cryptosporidium spp. in Yunling cattle in China, which provided baseline date for further studies of the prevalence, genetic identity, and public health potential of these parasites in Yunling cattle.

Molecular characterization of Eimeria spp. and Blastocystis in rabbits in Shandong Province, China.

Eimeria spp. and Blastocystis are the common parasites that parasitize the intestinal tract of rabbits, which can seriously threaten the health of rabbits and lead to economic losses to the rabbit industry. However, information about the prevalence and transmission of these two parasites in rabbits is limited in China. The objective of this study was to survey the prevalence of Eimeria spp. and Blastocystis in rabbits in Shandong Province. A total of 616 rabbit fecal samples were collected from two cities (Rizhao and Weihai) in Shandong Province, eastern China, and Eimeria spp. and Blastocystis were identified by polymerase chain reaction based on species-specific markers. The prevalence of Eimeria spp. was 20% (123/616) and the Blastocystis prevalence was 0.97% (6/616). Five different Eimeria species (Eimeria intestinalis, E. perforans, E. magna, E. media, and E. irresidua) and the ST4 subtype of Blastocystis were identified in rabbits by sequence analysis. This is the first report of Blastocystis prevalence and subtype ST4 in rabbits in Shandong Province. The findings provide baseline data for the prevention and control of Eimeria spp. and Blastocystis in rabbits in Shandong Province, China.

Distance-dependent quenching and enhancing of electrochemiluminescence from tris(2, 2'-bipyridine) ruthenium (II)/tripropylamine system by gold nanoparticles and its sensing applications.

Understanding the role of gold nanoparticles (AuNPs) in electrochemiluminescence (ECL) processes of the Ru(bpy)32+ (bpy= 2, 2'-bipyridine)/tripropylamine (TPA) system would be beneficial to develop novel ECL sensors for a variety of applications. In this work, we found that the AuNPs on the surface of indium tin oxide (ITO) electrode could catalyze the electrochemical oxidation of TPA, greatly enhancing the ECL signal of Ru(bpy)32+/TPA, present in the solution. If physical separation of AuNPs away from electrode surface after hybridization with target ssDNA, ECL signal decreased dramatically due to the loss of electrochemical activity of AuNPs, based on which a sensitive and specific DNA sensor in a "switch-off" mode was constructed with a detection limit of 0.2 pM. In addition, a suppressing effect of the AuNPs on the ECL of Ru(bpy)32+ was experimentally confirmed by decreasing the electrocatalytic effect to overall ECL emission, including selection of oxalate as a coreactant instead of TPA, or introduction of gold electrode as substrate. Furthermore, when Ru(bpy)32+ and AuNPs were both immobilized on the ITO electrode at close proximity, the ECL quenching induced by energy/electron transfer was predominant. ECL emission of the Ru(bpy)32+/TPA system resulted from a competition between electrocatalytic enhancement and quenching effect. However, the quenched ECL signal would return in case of the AuNPs moving far away from ECL emitters after a hybridization reaction as before, and a separation distance dependent surface enhancement was observed as well. Based on the role change for AuNPs from quenching to enhancing ECL intensity of Ru(bpy)32+/TPA system, a novel ECL DNA sensing strategy in a "turn-on" mode was developed, enabling quantitative analysis of target ssDNA over the range of 0.05 pM to 0.5 nM with a detection limit of 12 fM. Overall, we demonstrated the existence of three effects of AuNPs on the ECL of Ru(bpy)32+/TPA system, and which played the leading role was dependent on the placement of AuNPs, Ru(bpy)32+, and their separation distance. The ECL sensors based on the role change for AuNPs showed both high sensitivity and excellent selectivity.