日本乙型脑炎病毒单克隆抗体介导ADCC效应的研究

以日本乙型脑炎病毒(JEV)感染的BHK_2细胞为靶细胞,小鼠脾细胞为效应细胞,测定了6株日本乙型脑炎病毒单克隆抗体(JEV-McAb)介导ADCC效应的活性。结果表明,2H_4、2F_2、nG_2、mG_9等种特异性及属特异性McAb无介导~(51)Cr释放的活性,而mC_3及2D_2两株亚组特异性McAb可介导ADCC效应;但mC_3和2D_2介导ADCC效应的活性也不相同。JEV-     

抗单纯疱疹病毒单克隆抗体的研究——Ⅱ、单克隆抗体介导ADCC效应的探讨

本文用HSV-1SM44株感染BHK细胞,经放射性同位素~(51)Cr标记制备靶细胞,以正常小鼠脾细胞为效应细胞,应用5株抗HSV McAb,建立了McAb介导ADCC-~(51)Cr释放试验的测定方法,对McAb介导的ADCC效应测定的有关试验条件做了选择,确定了最适工作条件。ADCC测定结果表明,5株抗HSV McAb介导ADCC的活性不同,McAb 1A12,2A8,1G8无ADCC活性;而1D10和2C5 2株McAb 1:10稀释时的~(51)Cr释放率分别为27.09和25.07％,进一步稀释至10~(-2)或10~(-3)时,这2株McAb仍有ADCC活性。结果提示,不同的HSV抗原决定簇诱导产生的抗体,在介导ADCC免疫保护作用上是有差异的。并为McAb用于临床治疗HSV感染的可能性提供了实验资料。     

单克隆抗体治疗日本乙型脑炎的实验研究

本文应用JEV-McAb对感染JEV的小白鼠及幼山羊进行实验性治疗,并对乙脑免疫机理作一初步探讨。结果表明,JEV-McAb对经不同途径感染各种JEV毒株的小白鼠都有较好的保护作用,其效果明显优于PcAb。小鼠感染后120小时,大部分已发病,个别已濒死,这时给予混合McAb仍可保护约半数存活。McAb对感染JEV的幼羊也有较好的保护作用。羊脑内注入JEV48小时后,出现发热不安等症状,这时由腹腔和椎管两种途径同时给予混合McAb,保护率达100％。但单一腹腔或椎管注射无效,可能与血脑屏障的作用有关。存活实验动物脑组织内未分离出病毒;而死亡实验动物病毒分离阳性,脑组织双PAP及HE染色证实JEV抗原阳性并伴有脑炎的病理学改变。中和活性是JEV-McAb的主要保护方式,中和效价高的McAb保护率也高。     

家鼠型肾综合征出血热病毒单克隆抗体的制备与鉴定

以家鼠型HFRS病毒R22株免疫BALB/c小鼠,取免疫鼠脾细胞与小鼠骨髓瘤细胞系Sp2/0-Ag14融合,获得了23株能稳定分泌HFRS病毒特异性McAb的杂交瘤细胞系。以18株不同型别的HFRS病毒株进行分析,结果表明上述McAb中有些为HFRS病毒组特异性的,有些为家鼠型特异性的。有2株McAb对家鼠型HFRS病毒株具有较高的中和活性,对HFRS病毒R22株感染乳鼠也有明显的保护作用。     

单克隆抗体治疗日本乙型脑炎的实验研究

本文应用JEV-McAb对感染JEV的小白鼠及幼山羊进行实验性治疗,并对乙脑免疫机理作一初步探讨。结果表明,JEV-McAb对经不同途径感染各种JEV毒株的小白鼠都有较好的保护作用,其效果明显优于PcAb。小鼠感染后120小时,大部分已发病,个别已濒死,这时给予混合McAb或其F(ab′)_2,仍可保护约半数存活。McAb对感染JEV的幼羊也有较好的     

抗日本脑炎病毒上山株单克隆抗体的免疫学性状的分析(文摘)

日本脑炎病毒(JEV)与黄病毒属中约60种病毒有广泛的交叉反应。历来均采取免疫血清进行血球凝集抑制试验(HIT)帮助诊断,但分析鉴定病毒则显得困难。本文以B淋巴细胞杂交瘤技术,建立15种分泌抗日本流行的JEV上山株种特异性单克隆抗体(McAb)细胞系(KAMIMA),结果发现只与JEV发生HI反应,而与墨累山谷(Muray Valley)脑炎病毒、西尼罗(West—nile)病毒、圣路易(St.Louis)脑炎病毒、苏联春夏(Russian Spring-Summer)脑炎病毒及登革1型(Dengue-1)病毒5种黄病     

小鼠流行性乙型脑炎疾病进展中反应性星形胶质细胞活化模式的变化及干预

目的 明确星形胶质细胞活化模式与流行性乙型脑炎疾病进展的关系。方法 首先通过足垫注射乙型脑炎病毒(JEV)构建流行性乙型脑炎小鼠模型,采用免疫荧光技术(IFA)检测JEV非结构蛋白3(NS3)在小鼠全脑的表达情况;进一步利用IFA、 RNA测序技术(RNA-seq)、实时定量PCR明确流行性乙型脑炎发病不同阶段星形胶质细胞活化模式的变化;最后,通过侧脑室注射鸢尾素(irisin)调控补体C3阳性A1型星形胶质细胞、 S100钙结合蛋白A10(S100A10)阳性的A2型星形胶质细胞活化比例,探索其是否可改善乙型脑炎模型小鼠的体质量、行为学评分及脑组织损伤情况。结果 流行性乙型脑炎模型组小鼠的M1、 M2等运动皮层脑区和海马组织中NS3蛋白显著增加。上述区域胶质原纤维酸性蛋白(GFAP)阳性星形胶质细胞的数目和体积较对照组也显著增加,其中JEV感染后与A1/A2型星形胶质细胞活化相关的基因均显著升高。侧脑室或者腹腔注射irisin虽不能改善流行性乙型脑炎预后,但可在一定程度上抑制A1型星形胶质细胞活化,减轻神经炎症。结论 JEV主要感染小鼠的运动皮质和海马神经元,且星形胶质细胞异常激活导...     

日本脑炎病毒单克隆抗体的研究进展

近年来,抗各种不同日本脑炎病毒(JEV)株的McAb不断产生,并已在JEV的研究中广泛应用,取得了前所末有的进展。在分子病毒学中,McAb为进一步弄清JEV的抗原位点,不同位点之间的关系,不同位点与不同生物学功能之间的关系,以及选择保护性位点,制备亚单位疫苗提供了有力的工具。在乙脑检验诊断中,McAb为检出JEV,阐明JEV不同毒林之间的关系提供了条件。在乙脑实验性治疗以及对临床患者探索性治疗中,McAb为乙脑的特异性防治提供了依据,是一种很有希望的制剂。     

乙型脑炎病毒prM蛋白单克隆抗体的制备

目的 克隆日本乙型脑炎病毒(Japanese encephalitis virus,JEV)前膜蛋白(prM)编码基因,原核表达、纯化prM蛋白,以纯化产物为免疫原制备单克隆抗体(mAb);方法从感染JEV病毒的鼠脑中克隆编码JEV prM蛋白的基因并将其克隆入原核表达载体pET32a,转化大肠埃希菌BL21(DE3)LvsS后以IPTG诱导表达.表达产物经Ni-NTA纯化后进行SDS-PAGE分析.用纯化的蛋白免疫BALB/c小鼠,经细胞融合和亚克隆后筛选出能分泌识别prM蛋白的mAb的杂交瘤细胞株.用Western Blot和免疫组化方法检测单克隆抗体的特异性.结果 从鼠脑中克隆出约500 bp的JEV prM基因,将其克隆入原核表达载体中,在大肠埃希菌中获得了较好表达.纯化的prM蛋白免疫BALB/c小鼠,经传统细胞融合及筛选方法制备出单克隆抗体,抗体滴度为105.ELISA、Western Blot和免疫组化检测结果证实该株单抗具有较好的特异性.结论 成功的表达和纯化了日本乙型脑炎病毒的prM蛋白,并完成了单克隆抗体的制备,为乙型脑炎病毒感染的早期诊断及预防的研究奠定了良好的基础.     

肾综合征出血热病毒血凝素单克隆抗体的研制与鉴定

本文报道,从肾综合征出血热病毒(HFRSV)感染乳鼠鼠脑中提取高滴度的血凝素(HAN),以该血凝素免疫BALB/c小鼠,取脾细胞与Sp2/0骨髓瘤细胞进行融合。融合率为60％和78％,阳性孔率为15％和44％。IFA筛选后共获得56株分泌抗HFRSV McAb的杂交瘤细胞系。对其中1A_3,2A_(11),1B_3,3D_8,2F_7,1G_4,1G_9,3G_1,3G_(11),2H_1十株细胞系用有限稀释法进行了3～4次克隆化,阳性克隆率达到100％。杂交瘤细胞系经连续传代培养巳6个多月,仍能稳定地分泌单克隆抗体。冻存的细胞系经复苏后仍能稳定地分泌McAb。应用血凝抑制(Hl)试验证明其中9株为特异性分泌抗HFRSVHHAN McAb 的杂交瘤细胞系,1株(2A_(11))是抗HFRSV McAb的杂交瘤细胞系,其中(1:81920)和3G_1(1:40960)具有高滴度的血凝抑制活性。应用微量细胞培养中和试验(NT)证明其中6抗1A_8,1B_3,3D_8,1G_4,3G_1,3G_(11))具有中和活性.尤其3D_8(1:5120)和3G_1(>1:10240)有较高的中和效价。IFA测定了10株杂交瘤细胞的McAb与HFRSV陈株抗原片反应的抗体效价,其中培养上清为1:40～1:320,杂交瘤腹水为1:1万～1:8万。10种单克隆抗体与JEV、HSV-I和正常Vero-E_6细胞抗原片均无交叉反应。但均能与不同疫区、不同宿主分离的HFRSV陈株81-14A,A9和R_(22)抗原片反应,说明HFRSV血凝素McAb具     

Suppressor T cells for delayed-type hypersensitivity to Japanese encephalitis virus.

The delayed-type hypersensitivity (DTH) to Japanese encephalitis virus (JEV) and the suppressor cells controlling it and the antibody-forming cells in inbred Swiss mice have been studied. JEV induces DTH, with a peak response at day 7 following infection which persists at low levels at least up to 119 days. Suppressor activity appeared on day 18. It was transferable by immune spleen cells. Treatment of spleen cells with anti-Thy-1.2 antisera and complement abrogated the suppressor activity. The homogenate of the spleen was equally effective in mediating suppression of DTH and the humoral response as measured by direct antibody plaque-forming cell (IgM-PFC) assay. The suppressor activity was antigen-specific both on DTH and T helper for antibody response as the immune responses against SRBC or Coxsackie B4 virus were not suppressed. The suppressor cells were sensitive to cyclophosphamide treatment when the drug was given 48 hr before their appearance. It is, therefore, concluded that in JEV infection of mice, antigen-specific suppressor T cells are generated, both for DTH and IgM antibody, which are cyclophosphamide-sensitive and mediate suppression through soluble product(s).     

Preparation and characterization of the monoclonal antibodies against Japanese encephalitis virus.

Six mouse monoclonal antibodies (MoAbs) against Japanese encephalitis virus (JEV) were prepared and analyzed with indirect immunofluorescence assay (IFA), enzyme linked immunosorbent assay (ELISA), haemagglutination inhibition test (HI), neutralization test (NT), antibody dependent cell mediated cytotoxicity (ADCC) assay, antigenic site specific analysis and relative affinity measurement. These MoAbs could be divided into three classes by indirect immunofluorescence cross reactivity among four flaviviruses, 2H4, 2F2, and nG2 were type specific; 2D2 and mC3 were subgroup specific; and mG9 was family specific. 2H4 and 2F2 had higher neutralization activity, 2D2 and mC3 had the function of inducing ADCC effect, mG9 had higher titer in HI. The six MoAbs recognized five antigenic sites on JEV envelope glycoprotein, 2H4 and 2F2 recognized the same or very similar antigenic site and their relative affinity was ranked as: nG2 > 2H4 > 2D2 > mG9 > 2F2 > mC3.     

Induction of suppressor cells in Japanese encephalitis virus infected mice.

Adoptive transfer of spleen cells obtained from mice primed with Japanese encephalitis virus (JEV) suppressed IgM antibody plaque forming cells (PFC) against JEV in the spleen. Similar suppression of PFC was also shown in vitro by adding primed spleen cells to JEV-stimulated spleen cell cultures. The suppressor activity appeared sharply in the third week after priming and persisted up to 6 weeks. By using various cell separation procedures it was found that the suppressor activity resided in the T cell enriched fraction and not in B cells or macrophages. Sensitivity of the cells to treatment with anti-Thy 1.2 antiserum and complement confirmed that suppressor cells were T lymphocytes. It was noted that the suppression was effective against dengue virus antigen also. Our findings thus show generation of suppressor T lymphocytes in JEV-infected mice.     

Induction of suppressor cells in Japanese encephalitis virus infected mice

Adoptive transfer of spleen cells obtained from mice primed with Japanese encephalitis virus (JEV) suppressed IgM antibody plaque forming cells (PFC) against JEV in the spleen. Similar suppression of PFC was also shown in vitro by adding primed spleen cells to JEV-stimulated spleen cell cultures. The suppressor activity appeared sharply in the third week after priming and persisted up to 6 weeks. By using various cell separation procedures it was found that the suppressor activity resided in the T cell enriched fraction and not in B cells or macrophages. Sensitivity of the cells to treatment with anti-Thy 1.2 antiserum and complement confirmed that suppressor cells were T lymphocytes. It was noted that the suppression was effective against dengue virus antigen also. Our findings thus show generation of suppressor T lymphocytes in JEV-infected mice.     

A tripeptide (NSK) inhibits Japanese encephalitis virus infection in vitro and in vivo

Japanese encephalitis virus (JEV) is a major pathogen that can cause acute viral encephalitis in both humans and animals. Domain III of the viral envelope protein (EDIII) is involved in binding to host cell receptor(s) to facilitate virus entry. Our previous study showed that the loop3 peptide of EDIII possesses antiviral activity against JEV infection. In this paper, we demonstrate that three residues (NSK) in loop3 are responsible for the antiviral activity of loop3 peptide. In vitro experiments showed that the tripeptide NSK could inhibit JEV infection in both BHK-21 and Neuro-2A cells by inhibiting attachment of JEV to the cells, with IC₅₀ values of 8 μM and 6.5 μM, respectively. In vivo experiments showed that the tripeptide could increase the survival of mice challenged with JEV to 75 % when administrated intracerebrally. Therefore, this tripeptide may serve as the basis for the development of novel antiviral agents against Japanese encephalitis virus infection.     

Effect of macrophage-derived factor on hypoferraemia induced by Japanese encephalitis virus in mice.

Depression of serum iron following Japanese encephalitis virus (JEV) infection was observed in mice. The hypoferraemia was associated with the accumulation of iron in reticulo-endothelial cells in the spleen. Splenectomy (compared with sham-operation) prevented the depression in serum iron concentration after JEV infection. It also prevented the rise in levels of liver iron. The effect of JEV-stimulated, splenic macrophage-derived factor (MDF) was evaluated in causing hypoferraemia. MDF produced a rapid reduction in the serum iron levels with accumulation of iron in spleen. These observations suggest that MDF plays a key role in the regulation of iron metabolism during JEV infection.     

Induction of secondary immune response by reactivated Japanese encephalitis virus in latently infected mice.

Development of secondary immune response has been studied following reactivation of latent Japanese encephalitis virus (JEV) infection in mice. The virus could be reactivated in 43% of the latently infected mice at 27 weeks p.i. by treatment with cyclophosphamide. The reactivated virus induced delayed-type hypersensitivity (DTH) and leucocyte migration inhibition (LMI) responses in mice, with peak activity on Day 5 post-reactivation (p.r.). The DTH persisted at low levels for long periods. Humoral immunity measured by haemagglutination-inhibiting antibody showed a four-fold rise in antibody titres. DTH was transferable by immune spleen cells for 5 days p.r. only. It is, therefore, concluded that JEV reactivation generates a quick and short-lived secondary immune response.     

Japanese encephalitis virus infection of mouse cell lines: ability to prime mice for generation of virus specific cytotoxic T lymphocytes and differences in CTL recognisable viral determinants

Summary Ten different mouse cell lines were examined for Japanese encephalitis virus (JEV) infection in vitro and then tested for their ability to generate virus specific cytotoxic T lymphocytes (CTL). Among all cell lines examined, Neuro 2a (a neuroblastoma) was readily infected with JEV as examined by immunofluorescence and viral replication. Among other cells, P388D1, RAW 264.7 (Macrophage origin), Sp2/0 (B-cell Hybridoma), YAC-1 (T-cell lymphoma), and L929 (Fibroblast) were semipermissive to JEV infection. The cytopathic effects caused by progressive JEV infection varied from cell line to cell line. In the case of YAC-1 cells long-term viral antigen expression was observed without significant alterations in cell viability. Intermediate degrees of cytopathicity are seen in RAW 264.7 and L929 cells while infection of PS, Neuro 2a, P388D1 and Sp2/0 caused major viability losses. All infected cell lines were able to prime adult BALB/c (H-2^d) mice for the generation of secondary JEV specific CTL. In contrast to YAC-1, the permissive neuroblastoma cell line Neuro 2a (H-2K^kD^d) was found to be least efficient in its ability to stimulate anti-viral CTL generation. Cold target competition studies demonstrated that both Neuro 2a and YAC-1 (H-2K^kD^d) cells expressed similar viral determinants that are recognised by CTL, suggesting that the reason for the lower ability of Neuro 2a to stimulate anti-viral CTL was not due to lack of viral CTL determinants. These findings demonstrate that a variety of mouse cell lines can be infected with Japanese encephalitis virus, and that these infected cells could be utilised to generate virus specific CTL in BALB/c mice.     

Effective inhibition of different Japanese encephalitis virus genotypes by RNA interference targeting two conserved viral gene sequences in vitro and in vivo

Japanese encephalitis is a zoonotic, mosquito-borne, infectious disease caused by Japanese encephalitis virus (JEV), which is prevalent in China. At present, there are no specific drugs or therapies for JEV infection, which can only be treated symptomatically. Lentivirus-mediated RNA interference (RNAi) is a highly efficient method to silence target genes. In this study, two lentiviral shRNA, LV-C and LV-NS5, targeting the conserved viral gene sequences were used to inhibit different JEV genotypes strains in BHK21 cells and mice. The results showed that LV-C significantly inhibited JEV genotype I and genotype III strains in cells and mice. Quantitative RT-PCR analysis showed that JEV mRNA were reduced by 83.2–90.9% in cells by LV-C and that flow cytometry analysis confirmed the inhibitory activity of LV-C. The viral titers were reduced by about 1000-fold in cells and the brains of suckling mice by LV-C, and the pretreatment of LV-C protected 60–80% of mice against JEV-induced lethality. The inhibitory activities of LV-NS5 in cells and mice were weaker than those of LV-C. These results indicate that RNAi targeting of the two conserved viral gene sequences had significantly suppressed the replication of different JEV genotypes strains in vitro and in vivo, highlighting the feasibility of RNAi targeting of conserved viral gene sequences for controlling JEV infection.     

Passive protection of mice, goats, and monkeys against Japanese encephalitis with monoclonal antibodies

Six monoclonal antibodies (McAbs) against Japanese encephalitis virus (JEV) were tested for passive protection in JEV-infected mice, goats, and rhesus monkeys. mG9 and nG2 had no protective effect; mG3 and 2D2 had some protective effect, but not sufficient to be of therapeutic significance; and 2H4 and 2F2 had excellent protective efficacy in mice even 120 hr after infection when most of the mice in the virus control group were sick. The mixture of 2H4, 2F2, mC3 (M-McAb), and their F(ab')2 fragments showed excellent protection in mice, goats, and monkeys and was safe. The protective effects of McAbs correlated with their neutralization titers, but cytotoxicity-mediated activities also played a role in protection.