A Robust Quadruple Protein-Based Indirect ELISA for Detection of Antibodies to African Swine Fever Virus in Pigs.

African swine fever (ASF) emerged in domestic pigs and wild boars in China in 2018 and rapidly spread to neighboring Asian countries. Currently, no effective vaccine or diagnostic tests are available to prevent its spread. We developed a robust quadruple recombinant-protein-based indirect enzyme-linked immunosorbent assay (QrP-iELISA) using four antigenic proteins (CD2v, CAP80, p54, and p22) to detect ASF virus (ASFV) antibodies and compared it with a commercial kit (IDvet) using ASFV-positive and -negative serum samples. The maximum positive/negative value was 24.033 at a single antigen concentration of 0.25 µg/mL and quadruple ASFV antigen combination of 1 µg/mL at a 1:100 serum dilution. Among 70 ASFV-positive samples, 65, 67, 65, 70, 70, and 14 were positive above the cut-offs of 0.121, 0.121, 0.183, 0.065, 0.201, and 0.122, for CD2v, CAP80, p54, p22-iELISA, QrP-iELISA, and IDvet, respectively, with sensitivities of 92.9%, 95.7%, 92.9%, 100%, 100%, and 20%, respectively, all with 100% specificity. The antibody responses in QrP-iELISA and IDvet were similar in pigs infected with ASFV I. QrP-iELISA was more sensitive than IDvet for early antibody detection in pigs infected with ASFV II. These data provide a foundation for developing advanced ASF antibody detection kits critical for ASF surveillance and control.

Development of an indirect ELISA against African swine fever virus using two recombinant antigens, partial p22 and p30.

African swine fever (ASF) is a highly fatal viral disease affecting pigs. It is caused by the ASF virus (ASFV), and causes serious economic losses to the swine industry worldwide, including in Korea. Commercially available enzyme-linked immunosorbent assay (ELISA) kits for detecting anti-ASFV antibodies are used for the diagnosis and surveillance of ASF. In this study, an ELISA was developed to detect anti-ASFV antibodies using two recombinant proteins, p22 and p30, from genotype II ASFV. Recombinant transmembrane domain-deleted p22 (p22∆TM) and p30 were expressed in E.coli vector system pET32a and mixed for use as antigens in indirect ELISA. The p22∆TM/p30-based indirect ELISA was validated using 31 sera from genotype I ASFV-infected pigs and 1133 sera from uninfected pigs. Area under the curve of this test was 0.999 [95 % concentration interval 0.992-1.000], and sensitivity and specificity were 93.5 % and 99.8 %, respectively. The between run coefficient of variation for internal quality control serum was 6.61 %. In the seroconversion analysis, the p22∆TM/p30-based indirect ELISA showed equal or better ability to detect antibodies in pigs experimentally challenged with ASFV p72 genotypes I and II (p < 0.05). In conclusion, the p22∆TM/p30-based indirect ELISA is a reliable diagnostic method for detecting anti-ASFV antibodies.

Isolation and Genetic Characterization of African Swine Fever Virus from Domestic Pig Farms in South Korea, 2019.

On 17 September 2019, the first outbreak of African swine fever in a pig farm was confirmed in South Korea. By 9 October, 14 outbreaks of ASF in domestic pigs had been diagnosed in 4 cities/counties. We isolated viruses from all infected farms and performed genetic characterization. The phylogenetic analysis showed that all of fourteen ASFV isolates in South Korea belong to genotype II and serogroup 8. Additionally, all isolates had an intergenic region (IGR) II variant with additional tandem repeat sequences (TRSs) between the I73R and I329L genes and showed characteristics of central variable region (CVR) 1 of the B602L gene and IGR 1 of MGF 505 9R/10R genes. These are identical to the genetic characteristics of some European isolates and Chinese isolates.

Interleukin-32γ suppresses allergic airway inflammation in mouse models of asthma.

Asthma is a chronic airway inflammatory disease typically associated with T helper cell type 2 (Th2) cytokines. IL-32, first reported as an inducer of tumor necrosis factor (TNF)-α, is an inflammatory cytokine involved in various autoinflammatory diseases, viral infection, and cancer-related inflammation. However, the role of IL-32γ in asthma has not been clearly elucidated. In this study, the levels of IL-32γ in sputum from patients with asthma were measured by ELISA, and IL-32γ function was investigated in murine models of asthma with human IL-32γ-overexpressed transgenic (IL-32γ TG) mice. The therapeutic effect of recombinant IL-32γ (rIL-32γ) on allergic inflammation was also evaluated through bronchoalveolar lavage fluid analysis and histopathologic examinations. Sputum IL-32γ levels from patients with asthma were lower than those from healthy control subjects. In an acute mouse model of asthma, IL-32γ TG mice exhibited significantly reduced airway inflammation compared with that in wild-type mice. The production of Th1 cytokines, such as TNF-α and IFN-γ, and Th2 cytokines, such as IL-4, IL-5, and IL-13, was decreased in the lungs of IL-32γTG mice. On the contrary, the expression of IL-10 and IL-10-producing CD11b(+) monocytic cells was significantly increased in the lungs of ovalbumin-sensitized IL-32γ TG mice. In addition, rIL-32γ treatment revealed a suppressive effect on the airway inflammation in a chronic mouse model of asthma. The results of this study suggest that IL-32γ may have a preventive role in the development of allergic airway inflammation and could be a potential novel therapeutic target for bronchial asthma.

Engineering of a human kringle domain into agonistic and antagonistic binding proteins functioning in vitro and in vivo.

Here, we report the development of target-specific binding proteins based on the kringle domain (KD) ( approximately 80 residues), a ubiquitous modular structural unit occurring across eukaryotic species. By exploiting the highly conserved backbone folding by core residues, but using extensive sequence variations in the seven loop regions of naturally occurring human KDs, we generated a synthetic KD library on the yeast cell surface by randomizing 45 residues in the loops of a human KD template. We isolated KD variants that specifically bind to anticancer target proteins, such as human death receptor 4 (DR4) and/or DR5, and that function as agonists to induce apoptotic cell death in several cancer cell lines in vitro and inhibit tumor progression in mouse models. Combined treatments with KD variants possessing different recognition sites on the same target protein exerted synergisitic tumoricidal activities, compared to treatment with individual variants. In addition to the agonists, we isolated an antagonistic KD variant that binds human tumor necrosis factor-alpha (TNFalpha) and efficiently neutralizes TNFalpha-induced cytotoxicity in vitro and in vivo. The KD scaffold with seven flexible loops protruding from the central core was strongly sequence-tolerant to mutations in the loop regions, offering a potential advantage of distinct binding sites for target recognition on the single domain. Our results suggest that the KD scaffold can be used to develop target-specific binding proteins that function as agonists or antagonists toward given target molecules, indicative of their potential use as biotherapeutics.

Differences in circulating dendritic cell subtypes in pregnant women, cord blood and healthy adult women.

Different subtypes of dendritic cells (DC) influence the differentiation of naíve T lymphocytes into T helper type 1 (Th1) and Th2 effector cells. We evaluated the percentages of DC subtypes in peripheral blood from pregnant women (maternal blood) and their cord blood compared to the peripheral blood of healthy non pregnant women (control). Circulating DC were identified by flow cytometry as lineage (CD3, CD14, CD16, CD19, CD20, and CD56)-negative and HLA-DR-positive cells. Subtypes of DC were further characterized as myeloid DC (CD11c(+)/CD123(+/-)), lymphoid DC (CD11c(-)/CD123(+++)) and less differentiated DC (CD11c(-)/CD123(+/-)). The frequency of DC out of all nucleated cells was significantly lower in maternal blood than in control (P<0.001). The ratio of myeloid DC/lymphoid DC was significantly higher in maternal blood than in control (P<0.01). HLA-DR expressions of myeloid DC as mean fluorescence intensity (MFI) were significantly less in maternal blood and in cord blood than in control (P<0.001, respectively). The DC differentiation factors, TNF-alpha and GM-CSF, released from mononuclear cells after lipopolysaccharide stimulation were significantly lower in maternal blood than in control (P<0.01). The distribution of DC subtypes was different in maternal and cord blood from those of non-pregnant women. Their role during pregnancy remains to be determined.

Tumor necrosis factor alpha-induced interleukin-32 is positively regulated via the Syk/protein kinase Cdelta/JNK pathway in rheumatoid synovial fibroblasts.

OBJECTIVE: Interleukin-32 (IL-32) is a recently discovered cytokine that appears to play a critical role in human rheumatoid arthritis (RA). It is highly expressed in synovium and fibroblast-like synoviocytes (FLS) from RA patients, but not in patients with osteoarthritis (OA). This study was undertaken to assess IL-32 levels in RA synovial fluid (SF) and to investigate the secretion and regulation of IL-32 in RA FLS. METHODS: FLS and SF were obtained from the joints of RA patients. The secretion and expression of IL-32 and activation of signaling molecules were examined by enzyme-linked immunosorbent assay, immunoblotting, immunoprecipitation, reverse transcriptase-polymerase chain reaction, and small interfering RNA (siRNA) transfection. RESULTS: IL-32 levels were high in RA SF compared with OA SF. Furthermore, RA FLS expressed and secreted IL-32 when stimulated with tumor necrosis factor alpha (TNFalpha). TNFalpha-induced expression of IL-32 was significantly suppressed, in a dose-dependent manner, by inhibitors of Syk, protein kinase Cdelta (PKCdelta), and JNK and by knockdown of these kinases and c-Jun with siRNA. We also observed that PKCdelta mediated the activation of JNK and c-Jun, and experiments using specific inhibitors and siRNA demonstrated that Syk was the upstream kinase for the activation of PKCdelta. CONCLUSION: The present findings suggest that IL-32 may be a newly identified prognostic biomarker in RA, thereby adding valuable knowledge to the understanding of this disease. The results also demonstrate that the production of IL-32 in RA FLS is regulated by Syk/PKCdelta-mediated signaling events.

Identification of the most active interleukin-32 isoform.

Cytokines are crucial in host defence against pathogens such as bacteria, viruses, fungi and parasites. A newly described cytokine, interleukin-32 (IL-32), induces various proinflammatory cytokines (tumour necrosis factor-alpha, IL-1beta, IL-6) and chemokines in both human and mouse cells through the nuclear factor-kappaB and p38 mitogen-activated protein kinase inflammatory signal pathway. The IL-32 primarily acts on monocytic cells rather than T cells. In an attempt to isolate the IL-32 soluble receptor, we used an IL-32 ligand-affinity column to purify neutrophil proteinase 3, which is a serine proteinase involved in many inflammatory diseases. IL-32 has biological activity associated with Mycobacterium tuberculosis and chronic proinflammatory diseases such as rheumatoid arthritis. IL-32 is transcribed as six alternative splice variants and the biological activity of each individual isoform remains unknown. Here, we cloned the complementary DNA of the four IL-32 isoforms (alpha, beta, gamma and delta) that are the most representative IL-32 transcripts. To produce recombinant protein with a high yield, the amino acids of two cysteine residues were mutated to serine residues, because serine residues are not conserved among different species. The multi-step purified recombinant IL-32 isoform proteins were assessed for their biological activities with different cytokine assays. The gamma isoform of IL-32 was the most active, although all isoforms were biologically active. The present study will provide a specific target to neutralize endogenous IL-32, which may contribute to basic and clinical immunology.

Unique expression of a small IL-32 protein in the Jurkat leukemic T cell line.

Interleukin (IL)-32 was recently identified as a new cytokine which induces various proinflammatory cytokines in human monocytes and macrophages. Therefore, IL-32 has been primarily studied in inflammatory models such as rheumatoid arthritis and inflammatory bowel diseases. The regulation of endogenous IL-32 in other immune cells remains unknown. In the present study, we stimulated Jurkat T cells with phytohaemagglutinin (PHA) and phorbol myristate acetate (PMA) and examined IL-32 expression at both the mRNA and protein levels. All mRNAs of the four IL-32 isoforms and the 12-15 kDa IL-32 protein were independent of PHA and PMA stimulation, however a 9 kDa molecular weight IL-32 protein in the cell culture supernatant was induced by PHA and PMA after 16 h of stimulation. Compared to other human cell lines, the Jurkat cell line constitutively expressed a 12-15 kDa molecule of IL-32, which is smaller than the known IL-32 isoforms. We used IL-32 shRNA to examine the specificity of the 12-15 kDa molecule. Upon IL-32 shRNA transfection, the 12-15 kDa band was decreased specifically as compared to the control scrambled clone. Thus, the constitutive expression of IL-32 mRNA as well as the predominant production of a smaller sized IL-32 isoform in Jurkat cells may implicate a role for IL-32 in human T cell leukemia.