Interactions of porcine alveolar macrophages and bone marrow cells with African swine fever virus and virus-infected cells

Virus yields from porcine alveolar macrophages (AM) infected with African swine fever virus (ASFV) were greater and were achieved more rapidly, when inoculated at a high multiplicity of infection (MOI) than at low MOI. The difference was related to a lower percentage of cells becoming infected after low MOI inoculation. The reduced yields after low MOI were not caused by prolongation of the culture time, by bacterial endotoxins or by production of inhibitory substances by infected AM. Virus-infected AM were not susceptible to lysis in antibody-dependent cell mediated cytotoxicity (ADCC) assays and this was apparently due to a paucity of viral antigen expressed on the cell surface. Uninfected AM did not act as effectors in ADCC.Porcine bone marrow (PBM) cells were effective in mediation of ADCC and their activity was reduced after ASFV infection. Cells separated into adherent and non-adherent populations, depleted by carbonyl iron treatment or separated by Ficoll-Hypaque centrifugation, all showed effector activity in ADCC. The effector cells were not mature neutrophils or lymphocytes and were probably granulocytic precursors.     

In vitro immune serum-mediated protection of pig monocytes against African swine fever virus

Serum samples from pigs that had recovered from infection with a Dominican Republic isolate of African swine fever virus (ASFV) were mixed with dilutions of the virus, then assayed in microcultures of normal pig mononuclear leukocytes to determine whether the samples contained antibodies that protected monocytes against the virus. Protection was determined by the difference in titer (log10) between virus mixed with healthy pig serum and virus mixed with immune pig serum, using 50% cytopathogenic effect end points; protection was expressed as an immune serum-protection index. After addition of virus-serum mixtures to mononuclear leukocyte microcultures, a time-dependent decrease in protective index and production of infectious virus (determined by use of yield reduction assays) were observed. Protective effects were associated with the immunoglobulin fraction of serum, were rapidly lost on dilution, and were independent of complement. Antibody was most protective for the homologous Dominican Republic isolate of ASFV, with decreased protection against Lisbon '60 ASFV, and no protection against foot-and-mouth disease virus or bluetongue virus. Low concentrations of protective antibody were found during the acute viremic phase in infected pigs; antibody increased to maximal concentrations as the viremia decreased.     

Serum containing ovine IgG2 antibody specific for maedi visna virus envelope glycoprotein mediates antibody dependent cellular cytotoxicity

Antibody-dependent cell-mediated cytotoxicity (ADCC) specific for maedi visna virus (MVV) has never been described. The IgG antibody response to MVV is restricted to an IgG1 response whilst MVV specific IgG2 is never seen in persistently infected sheep. To determine whether the isotypic restriction of the antibody response is responsible for the lack of ADCC, an ADCC assay was developed using polyclonal serum raised to recombinant MVV ENV protein. Sheep immunised with a recombinant GST:SUenv fusion protein in complete Freund's adjuvant produced an antibody response which contained IgG1 and IgG2 antibodies. The activity of this serum in an ADCC assay was compared to serum from persistently infected sheep. Serum from immunised sheep mediated ADCC reactions whilst no activity was ever seen in persistently infected sheep serum. IgG2 may therefore be the possible effector isotype for ADCC reactions against MVV. Failure of the IgG2 dependent ADCC system in vivo may contribute to the persistence of MVV-infected macrophages in vivo.     

Antibody-dependent cell-mediated cytotoxicity in sheep

The ability of sheep leukocytes to mediate antibody — dependent cell-mediated cytotoxicity (ADCC) and that of sheep serum IgG₁ and IgG₂ to induce ADCC were investigated. Partial characterization of effector cells was attempted. These investigations revealed that ADCC occurs in sneep. With chicken erythrocytes (CRBC) as the target cells, polymorphonu-cleated cells (PMN), and monocytes, were the most effective leukocytes. Ovine peripheral blood lymphocytes (PBL) also mediated ADCC, and within the PBL population, T-cells were capable of mediating ADCC. The T-cells were obtained by nylon wool fractionation and selective agglutination by peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA). Both nylon wool adherent and non-adherent fractions were active in ADCC, although the former were more active, implying heterogeneity in nylon wool adherence among ovine K-cells. Depletion of B (SIg⁺) cells did not affect ADCC activity of the remaining cells. Depletion of Fc⁺ cells markedly reduced cytotoxic activity of PBL. Both sheep IgG₁ and IgG₂ anti-CRBC immunoglobulins were able to induce ADCC.     

单克隆抗体介导对细粒棘球蚴原头节细胞毒作用的体外试验

用抗体依赖性细胞介导的细胞毒（ADCC）试验在体外观察了单克隆抗体对细粒棘球蚴原头节的杀伤作用。原头节在1C2、2E3、3E6、3F10、3C2 5株单抗浓缩上清（1：5）及嗜酸性粒细胞存在情况下培养40小时，发育抑制率分别为100%，100%，100%，100%，57%；死亡率分别为100%，100%，100%，96%和14%。这一结果显示我们所获单抗中部分具有较强保护性，为寻求保护性抗原提供了线索，同时表明ADCC是宿主获得性抵抗力的重要组成部分。     

Antibodies to Aujeszky's disease virus in pigs immunized with purified virus glycoproteins

Antibodies to Aujeszky's disease virus (ADV) glycoproteins gII, gIII, and gp50 were compared using four in vitro tests. Antibodies generated by vaccination with a modified-live vaccine (MLV) were also compared. The serological assays employed were: serum neutralization test (SNT), complement facilitated serum neutralization test (C'SNT), complement-mediated cytolysis and antibody dependent cellular cytotoxicity (ADCC). Pigs were immunized with single glycoproteins twice 14 days apart, or once with the modified-live vaccine. Fourteen days after the second immunization, sera were collected. Virus neutralizing activity (SNT) was demonstrated in the sera from all pigs immunized with gp50 and in one out of three immunized with gIII. Sera from the MLV group all had neutralization titers higher than animals immunized with single glycoproteins. Addition of guinea pig complement to the serum neutralization test (i.e., C'SNT) produced an enhancement of antibody titers in all groups except the pigs immunized with gIII. The complement-mediated cytolysis test rendered antibody titers similar in magnitude for all pigs immunized with single glycoproteins, but slightly lower than values for MLV vaccinated pigs. ADCC activity was clearly displayed in sera from pigs immunized with gIII or vaccinated with MLV, whereas sera from pigs immunized with gII or gp50 had a minimal response. The results indicate that the relative efficiency of antibodies against ADV glycoproteins in protection should be considered for selecting or producing gene-deleted strains for use in vaccine production.     

抗非洲猪瘟病毒单链抗体的构建、表达及活性鉴定

制备抗非洲猪瘟病毒（ASFV）猪源单链抗体（ScFv）,并对其生物学活性进行鉴定,筛选出具有ASFV反应活性的猪源单链抗体（ScFv）,为ASFV的诊断提供新的材料。采集ASFV感染康复猪的外周血,分离淋巴细胞,提取淋巴细胞总RNA,以此为模板通过PCR扩增得到猪源IgG重链可变区与轻链可变区基因,利用SOE-PCR技术扩增拼接得到猪源ScFv基因;构建pET-30a-ScFv表达载体,进行蛋白的表达与纯化,用ELISA和IFA鉴定ScFv抗体的反应活性。结果显示,成功扩增出猪源ScFv（VH-VLκ、VH-VLλ）基因,鉴定到1株与ASFV存在反应活性的ScFv（VH-VLλ₁₁）抗体。结果表明,成功筛选到1株与ASFV存在反应活性的ScFv（VH-VLλ₁₁）抗体,为ASFV诊断和防控提供了新型原材料。     

非洲猪瘟病毒p37蛋白生物信息学分析及其多克隆抗体制备

【目的】获取ASFV p37蛋白,并制备抗ASFV p37蛋白的多克隆抗体,为ASFV p37蛋白结构和功能研究提供材料。【方法】应用生物信息学工具对ASFV HLJ/2018(GenBank登录号：MK333180.1)p37蛋白进行分析,设计合成p37基因,并构建pET32a-p37重组质粒。将重组质粒转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导表达后,通过SDS-PAGE对重组蛋白的可溶性进行分析。收集菌体进行超声破碎,分离沉淀并使用8 mol/L尿素变性后离心,用0.45μm滤膜过滤离心后的上清,使用镍亲和层析柱纯化蛋白,通过SDS-PAGE、Western blotting对其纯化效果及特异性进行验证。将纯化后的p37蛋白按照50μg/只免疫小鼠,制备抗ASFV p37蛋白多克隆抗体。利用间接ELISA方法测定制备的多克隆抗体效价;通过Western blotting、间接免疫荧光试验检测该多克隆抗体的特异性。【结果】生物信息学分析表明,p37蛋白为稳定亲水性蛋白,无跨膜区和信号肽。二级结构主要含有α-螺旋(45.28%)、延伸链(15.09%)、无规则卷曲(31...     

非洲猪瘟病毒360-12L蛋白的原核表达及其多克隆抗体制备

[目的] 获得抗原性好的非洲猪瘟病毒（African swine fever virus）12L蛋白用于ASFV血清学诊断技术研究。[方法] 根据GenBank ASFV参考序列（登录号：NC_044959.2）合成360-12L基因,并插入pET-28a（+）载体,构建原核表达质粒pET-28a-12L,将经测序鉴定正确的质粒转化大肠杆菌BL21（DE3）感受态细胞进行诱导培养,筛选最佳诱导温度,同时分析其可溶性。随后通过亲和层析纯化蛋白,并对所获得的12L重组蛋白进行SDS-PAGE分析。将获得的12L重组蛋白作为免疫原免疫BALB/c雌性小鼠,以获得鼠抗ASFV 12L蛋白的多克隆抗体,通过间接ELISA方法测定所制备的多克隆抗体效价,并用间接免疫荧光试验进行验证。[结果] 成功构建了ASFV 12L蛋白原核表达质粒,重组菌pET-28a-12L-BL21在37 ℃ 6 h时表达量较高,主要以包涵体形式表达,可在54.7 ku处出现明显条带。间接ELISA结果显示,制备的鼠抗ASFV 12L蛋白多克隆抗体效价>1：512 000,间接免疫荧光试验结果表明,所制备的鼠抗ASFV 12L蛋白多克隆抗体具有良好的特异性,可特异性识别12L重组蛋白。[结论] 原核表达的12L重组蛋白具有良好的免疫原性,制备的鼠抗ASFV 12L蛋白的多克隆抗体具有较高的反应性和特异性,为进一步研究12L蛋白的生物学功能、ASFV血清学诊断技术和疫苗研究提供生物材料。     

Effector mechanisms in the pig. Antibody-dependent cellular cytolysis of African swine fever virus infected cells

Antibody dependent cellular cytolysis (ADCC) against African swine fever virus infected nucleated cells was investigated in a porcine system. Of the peripheral blood components examined, only neutrophils acted as effectors. Lymph node derived cells displayed no ADCC activity. In vitro yield reduction assays suggested that neutrophil mediated ADCC may play a role in recovery from African swine fever virus infection.     

Immunostimulatory effects of human recombinant interleukin-12 on peripheral blood mononuclear cells from normal dogs

Interleukin-12 (IL-12) plays a pivotal role in regulating cellular immune responses involving autoimmunity, infectious disease, and cancer. Human recombinant (hr) IL-12 is being evaluated for therapy of human cancer. We investigated the potential of hrIL-12 to activate canine peripheral blood mononuclear cells (PBMC) using proliferation and cytotoxicity as readouts. Human rIL-12 caused increased proliferation of PBMC, and enhanced lysis of allogeneic canine tumor targets mediated by PBMC from normal dogs in vitro. In addition, antibody-dependent cellular cytotoxicity (ADCC) mediated by canine PBMC was enhanced by hrIL-12. These results indicate that hrIL-12 is recognized by canine immune cells, triggering a number of immune responses in canine PBMC, that may be important for immunotherapy of canine cancer. Information from this investigation provides impetus for evaluation of the effects of hrIL-12 on PBMC from tumor-bearing dogs and should be helpful in the development of hrIL-12 as an immune cell activator in vivo in the dog.     

非洲猪瘟病毒p22蛋白表达、多克隆抗体制备及间接ELISA方法的建立

【目的】试验旨在表达与纯化非洲猪瘟病毒(African swine fever virus, ASFV)的结构蛋白p22,将其作为包被抗原建立ASFV抗体的间接ELISA检测方法,用于诊断非洲猪瘟。【方法】将ASFV p22编码基因KP177R的截短体(24―145位氨基酸)克隆至原核表达载体pET-32a(+)中,将重组质粒pET-32a-p22转化大肠杆菌BL21(DE3)感受态细胞,经0.1 mmol/L IPTG诱导表达5 h,利用镍柱亲和纯化p22蛋白,并进行Western blotting鉴定。利用p22蛋白免疫BALB/c小鼠制备抗血清,并以含p22全长基因的真核表达质粒pCAGGS-EGFP-fp22转染HEK293T细胞为抗原基质,利用间接免疫荧光(IFA)鉴定抗血清的反应性。以重组p22蛋白为包被抗原,优化最佳抗原包被浓度、待检血清稀释度、封闭条件、抗原抗体反应时间、酶标二抗工作浓度等参数,建立ASFV抗体间接ELISA检测方法,并对临床猪血清样品进行检测。【结果】ASFV p22截短蛋白在大肠杆菌中高水平表达,蛋白产量为0.85 mg/100 g菌体;p22蛋白具...     

Comparative functional characterization of canine IgG subclasses

To date, very little is known about the functional characteristics of the four published canine IgG subclasses. It is not clear how each subclass engages the immune system via complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC), or how long each antibody may last in serum. Such information is critical for understanding canine immunology and for the discovery of canine therapeutic monoclonal antibodies. Through both in vitro and ex vivo experiments to evaluate canine Fc's for effector function, complement binding, FcRn binding, and ADCC, we are now able to categorize canine subclasses by function. The subclasses share functional properties with the four human IgG subclasses and are reported herein with their function-based human analog. Canine Fc fusions, canine chimeras, and caninized antibodies were characterized. Canine subclasses A and D appear effector-function negative while subclasses B and C bind canine Fc gamma receptors and are positive for ADCC. All canine subclasses bind the neonatal Fc receptor except subclass C. By understanding canine IgGs in this way, we can apply what is known of human immunology toward translational and veterinary medicine. Thus, this body of work lays the foundation for evaluating canine IgG subclasses for therapeutic antibody development and builds upon the fundamental scholarship of canine immunology.     

Influence of arachidonic acid metabolites and steroids on function of bovine polymorphonuclear neutrophils.

Polymorphonuclear neutrophils (PMN) from 4 ovariectomized healthy cows were incubated with 0 (control), 10(-8), 10(-7), and 10(-6) M arachidonic acid metabolites of the cyclo- and lipoxygenase pathways for 30 minutes, and with steroids for 2 hours. Immediately after incubation, PMN were subjected to the following function assays: chemotaxis against zymosan-activated serum, chemotaxis against arachidonic acid metabolite or steroid at the doses given (only control PMN were tested), random migration, ingestion of 125I-iododeoxyuridine-labeled Staphylococcus aureus (125I-IdUR-S aureus), iodination of proteins, cytochrome C reduction, antibody-independent and -dependent cell-mediated cytotoxicity (AICC and ADCC). Prostaglandin F2 alpha was chemoattractant and stimulated ingestion of 125I-IdUR-S aureus. Prostaglandin E2 stimulated cytochrome C reduction, whereas prostacyclin inhibited iodination of proteins. Thromboxane B2 stimulated ADCC. Leukotriene B4 was chemoattractant for bovine PMN and stimulated random migration and AICC. 5-Hydroxyeicosatetraenoic acid was also chemoattractant, but inhibited ingestion of 125I-IdUR-S aureus. 15-Hydroxyeicosatetraenoic acid was chemoattractant and decreased ADCC. Lipoxin A4 stimulated random migration, whereas lipoxin B4 inhibited chemotaxis against zymosan-activated serum, but was chemoattractant and stimulated cytochrome C reduction. 12-Hydroxyhepadecatrienoic acid and 12-hydroxyeicosatetraenoic acid did not influence any of the PMN functions tested. Of the steroids tested, cortisol increased ADCC, and progesterone stimulated cytochrome C reduction, but decreased ADCC. 17 beta-Estradiol and estrone were chemoattractant and stimulated cytochrome C reduction. In addition, estrone also stimulated random migration.(ABSTRACT TRUNCATED AT 250 WORDS)     

非洲猪瘟病毒p30蛋白单克隆抗体的制备及阻断ELISA抗体检测方法的建立

为建立检测非洲猪瘟病毒(ASFV)抗体的阻断ELISA方法,本研究利用原核表达的ASFV p30重组蛋白免疫BALB/c小鼠制备单克隆抗体。以重组p30蛋白作为包被抗原,以辣根过氧化物酶(HRP)标记的p30单克隆抗体作为检测抗体,经条件优化,建立了一种检测ASFV抗体的阻断ELISA方法。ROC曲线分析显示,该方法最佳阻断率临界值为16.63%。该方法与CSFV、FMDV-O/A、PRRSV、PEDV、SVA的阳性血清均无交叉反应;最低能检出1∶128稀释的阳性血清;批内和批间变异系数(CV)均＜10%。用本方法与商品化试剂盒平行检测208份血清样品,Kappa值为0.96,表明具有高度一致性。上述结果表明,本研究建立的阻断ELISA方法具有较高的特异性和敏感性,可用于血清ASFV抗体的检测,为ASFV流行病学调查及猪群疫情监控提供技术支持。     

Identification of feline monocytes and neutrophils as effector cells in antibody-dependent cellular cytotoxicity: sequential analysis, using light microscopy, histochemistry, and scanning electron microscopy

Feline monocytes and neutrophils functioned as effector cells in antibody-dependent cellular cytotoxicity (ADCC) against antibody-coated chicken erythrocytes. Using light microscopy, effector cell populations were identified in effector-target cell interactions, with further characterization of these identical individual effector cells by histochemical evaluations and scanning electron microscopy. Monocytes and neutrophils, but not lymphocytes, were observed attacking target cells. Carbonyl iron depletion of monocytes and neutrophils from peripheral blood leukocytes caused a marked reduction from a mean of 62% to 3.6% lysis in ADCC as measured by a 4-hour 51Cr release assay. Effector cells functioning in the ADCC reaction were visualized, using sequential analysis and light microscopy, histochemistry, and scanning electron microscopy.     

Immune responses of specific pathogen free foals to EHV-1 infection.

Four foals were raised under specific pathogen free (SPF) conditions. At 3 to 4 months of age, SPF foals and 1 other non-SPF foal were intranasally inoculated with equine herpes virus type 1 (EHV-1). Clinical signs included depression, fever, inappetence and intermittent coughing. Clinical recovery was complete by seven days but high titres of virus were detected in nasal mucus for at least 10 days after inoculation. Clinical illness was less severe in the non-SPF foal. Interferon was detected in the nasal mucus of all foals from 2 days post infection (dpi), persisting until 8 or 10 dpi. ELISA antibody was detected in serum from 6 dpi. Titres continued to rise throughout the period of observation, and were slightly stimulated by re-inoculation. EHV antibody, identified as belonging to the IgM class by the double sandwich ELISA, was detected from 6 dpi. Peak IgM titres were observed between day 10 and 18, declining to base levels by day 42. Virus neutralizing antibody was detectable in serum from day 14 and rises in titre were parallel to that of total ELISA antibody. Cellular immunity in EHV-1 infected SPF horses was examined by the antibody dependent cytotoxicity (ADCC) test and the specific lymphocyte transformation test. The ability of foal neutrophils to effect ADCC decreased significantly between 3 to 10 days after inoculation. Peripheral blood mononuclear cells (PBMC) displayed reactivity towards EHV-1 antigens from about day 14, with maximum stimulation indices being obtained between 28 and 42 dpi.     

Swine antibody-dependent cellular cytotoxicity against pseudorabies virus-infected cells

In pseudorabies virus (PRV) infection of pigs, antibody-dependent cellular cytotoxicity (ADCC) may be an early defense mechanism. Peripheral blood leukocytes (PBL) and pulmonary macrophages mediate ADCC activity. Antibody-dependent cellular cytotoxicity against PRV-infected target cells was assessed, and the effect of infection of cells having an ADCC-effector function was determined. Although pulmonary lavage cells (PLC) had ADCC activity, in vitro infection of PLC led to PRV replication, loss of cell viability, and loss of ADCC activity. In contrast, infection of PBL did not lead to replication, decreased cell viability, or reduced ADCC activity, compared with those in non-infected controls. Measuring ADCC activity in a longitudinal study revealed that PBL from neonates had lower ADCC activity than did PBL from pigs greater than 3.5 months old. Peripheral blood leukocytes and not PLC may have a greater role in control of PRV dissemination in the pig. The difference in activity between cells from neonates and older pigs might explain, in part, the age dependency in the severity of the disease.     

Limited efficacy of West Nile virus vaccines in large falcons (Falco spp.)

West Nile virus (WNV) can lead to fatal diseases in raptor species. Unfortunately, there is no vaccine which has been designed specifically for use in breeding stocks of falcons. Therefore the immunogenicity and protective capacity of two commercially available WNV vaccines, both approved for use in horses, were evaluated in large falcons. One vaccine contained adjuvanted inactivated WNV lineage 1 immunogens, while the second represented a canarypox recombinant live virus vector vaccine. The efficacy of different vaccination regimes for these two vaccines was assessed serologically and by challenging the falcons with a WNV strain of homologous lineage 1. Our studies show that the recombinant vaccine conveys a slightly better protection than the inactivated vaccine, but moderate (recombinant vaccine) or weak (inactivated vaccine) side effects were observed at the injection sites. Using the recommended 2-dose regimen, both vaccines elicited only sub-optimal antibody responses and gave only partial protection following WNV challenge. Better results were obtained for both vaccines after a third dose, i.e. alleviation of clinical signs, absence of fatalities and reduction of virus shedding and viraemia. Therefore the consequences of WNV infections in falcons can be clearly alleviated by vaccination, especially if the amended triple administration scheme is used, although side effects at the vaccination site must be accepted.     

The role of specific immunoglobulins in antibody-dependent cell-mediated cytotoxicity assays during Babesia bovis infection

A stabilate prepared from Babesia bovis-infected Boophilus microplus ticks was used to infect intact adult cattle. Whole sera and immunoglobulin fractions from representative sera were tested by complement fixation (CF), indirect fluorescent antibody (IFA), and antibody-dependent cell-mediated cytotoxicity (ADCC) assays. The last test utilized 51Cr-labeled chicken erythrocytes coated with Babesia bovis antigen as targets. Mononuclear cell preparations, obtained from peripheral blood of normal donors and consisting of lymphocytes with 2--6% large monocytes, were used as the source of effector cells. Antibody activity was detected by all tests between 14 and 16 days following infection. Specific IgM and IgG1 were reactive in both CF and IFA tests, although the development of high titers was attributable to IgG, alone. The ADCC activity was restricted to IgG1 fractions and was greater in those sera or fractions with greater CF activity. No activity was demonstrated in IgG2 fractions by any test used.