Lentivirus-induced immune dysregulation

FIV/HIV infections are associated with an early robust humoral and cellular anti-viral immune response followed by a progressive immune suppression that eventually results in AIDS. Several mechanisms responsible for this immune dysfunction have been proposed including cytokine dysregulation, immunologic anergy and apoptosis, and inappropriate activation of immune regulatory cells. Studies on FIV infection provide evidence for all three. Cytokine alterations include decreases in IL2 and IL12 production and increases in IFNγ and IL10 in FIV⁺ cats compared to normal cats. The elevated IL10:IL12 ratio is associated with the inability of FIV⁺ cats to mount a successful immune response to secondary pathogens. Additionally, chronic antigenic (FIV) stimulation results in an increase in the percent of activated T cells expressing B7 and CTLA4 co-stimulatory molecules in infected cats. The expression of these molecules is associated with T cells that are undergoing apoptosis in the lymph nodes. As ligation of CTLA4 by B7 transduces a signal for induction of anergy, one can speculate that the activated T cells are capable of T cell–T cell interactions resulting in anergy and apoptosis. The inability of CD4⁺ cells from FIV⁺ cats to produce IL2 in response to recall antigens and the gradual loss of CD4⁺ cell numbers could be due to B7–CTLA4 interactions. The chronic antigenemia may also lead to activation of CD4⁺CD25⁺ T regulatory cells. Treg cells from FIV⁺ cats are chronically activated and inhibit the mitogen-induced proliferative response of CD4⁺CD25⁻ by down-regulating IL2 production. Although Treg cell activation can be antigen-specific, the suppressor function is not, and thus activated Treg cells would suppress responses to secondary pathogens as well as to FIV. Concomitant with the well-known virus-induced immune suppression is a progressive immune hyper-activation. Evidence for immune hyper-activation includes polyclonal B cell responses, gradual replacement of naïve CD4⁺ and CD8⁺ T cell phenotypes with activation phenotypes (CD62L⁻, B7⁺, CTLA4⁺), and the chronic activation of CD4⁺CD25⁺ Treg cells. Thus lentivirus infections lead to severe immune dysregulation manifested as both chronic immune suppression and chronic immune activation. FIV infection of cats provides a number of advantages over other lentivirus infections as a model to study this immune dysregulation. It is a natural infection that has existed in balance with the cat's immune system for thousands of years. As such, the natural history and pathogenesis provides an excellent model to study the long-term relationships between AIDS lentivirus and host immune system function/dysregulation.     

板蓝根多糖对缺乳仔鼠免疫器官发育及T淋巴细胞亚群的影响

试验应用流式细胞术检测缺乳仔鼠CD3⁺、CD4⁺和CD8⁺ T淋巴细胞含量来研究添加不同剂量的板蓝根多糖(RIP)对缺乳仔鼠免疫器官及T淋巴细胞亚群的影响。结果表明:①RIP可增加缺乳仔鼠胸腺指数和脾脏指数。对胸腺指数和脾脏指数效果最好的RIP剂量随日龄增大而减小。②RIP可以增加缺乳仔鼠外周血CD3⁺、 CD4⁺ 、CD8⁺ T淋巴细胞数量。7~28日龄时初乳组(A组)、缺乳+中RIP组(C组)和缺乳+高RIP组(D组)3组CD3⁺ T淋巴细胞含量与缺乳组(B组)相比差异显著(P<0.05)。7~21日龄时初乳组(A组)、缺乳+中RIP组(C组)和缺乳+高RIP组(D组)3组CD4⁺ T淋巴细胞含量与缺乳组(B组)相比差异显著(P<0.05)。各处理组CD3⁺、 CD4⁺、CD8⁺ T淋巴细胞含量及CD4⁺/CD8⁺比值随着日龄的增加有所下降。适宜剂量的RIP可促进缺乳仔鼠免疫器官发育,提高T淋巴细胞亚群的水平。     

The Localization Changes of CD4+ and CD8+ T Lymphocytes in Chicken Lung at Different Ages

The aim of this study was to investigate the immune state of chicken lung in different periods.With lung tissue of Hy-line White chicken at different ages,the distribution and quantity changes of CD4⁺ and CD8⁺T lymphocytes in lung were studied using immunohistochemistry staining.The results showed that CD8⁺T lymphocytes appeared firstly in embryonic at 18 d,while CD4⁺T lymphocytes appeared at 1-day-old chicken.At 4-day-old,there were aggregates of lymphocytes at the junction of the primary and secondary bronchi,which formed obvious broncho-associated lymphoid tissue (BALT).CD4⁺T lymphocytes of each age mainly occupied the central area of BALT,while CD8⁺T lymphocytes mainly surrounded the periphery.Since 56 days old,CD8⁺T lymphocytes are distributed in the inner wall of third-order bronchial airway,atrial septum,gas exchange area and interlobular connective tissue,and are distributed throughout the lung.In terms of quantity change,with the growth of daily age,the number of CD4⁺T lymphocytes and CD8⁺T lymphocytes gradually increased,and the number of CD4⁺ T lymphocytes was more than that of CD8⁺ T lymphocytes before 35 days of age,while the number of CD8⁺ T lymphocytes was significantly more than that of CD4⁺ T lymphocytes at the same age thereafter.The results showed that the distribution and number of CD4⁺ and CD8⁺T lymphocytes in the lungs of chickens were correlated with the age,and the changes could reflect that the lungs before the age of 35 days were dominated by humoral immunity,while the lungs after that tended to be cellular immunity.     

黄牛呼吸道CD3+淋巴细胞分布的研究

CD3⁺是T细胞群的重要表面标志,呼吸道黏膜下分布的CD3⁺淋巴细胞作为抗感染黏膜免疫的基础,在保护机体抵抗呼吸道感染中发挥重要作用。为了解黄牛呼吸道CD3⁺淋巴细胞和淋巴组织的分布,本研究运用HE染色法和免疫组织学方法对5头7岁健康婆罗门黄牛的鼻黏膜、气管、肺内支气管及肺脏组织的CD3⁺淋巴细胞和淋巴组织的分布进行了研究。结果显示,黄牛的鼻黏膜、气管、肺内支气管和肺脏组织中均分布有CD3⁺淋巴细胞,且主要分布于黏膜上皮间及其下方固有层中及腺体周围;在鼻黏膜和肺脏组织中分布有CD3⁺淋巴细胞形成的弥散淋巴组织。牛呼吸道中CD3⁺淋巴细胞数量在鼻黏膜和肺脏组织中分布最多,气管黏膜、肺内支气管黏膜次之。本试验结果明确了CD3⁺淋巴细胞在黄牛呼吸道中的分布谱,表明黄牛呼吸道具备引发局部黏膜免疫的基础条件,为牛呼吸道黏膜免疫及呼吸道疾病防治研究提供了数据支撑。     

伯氏疟原虫ANKA株感染小鼠的T细胞、NK细胞及细胞因子变化

旨在系统分析伯氏疟原虫感染引起宿主T细胞、NK细胞、Tim-3表达及细胞因子的变化。选取64只雌性C57BL/6小鼠随机分为8组,每组8只,分别于感染后0、4、7、9、11、13、16和19 d获取小鼠脾及外周血免疫细胞,利用流式细胞术检测小鼠主要免疫细胞亚群及免疫检查点分子Tim-3表达水平的变化;同时检测血清中细胞因子的变化。结果表明,感染疟原虫后,小鼠脾CD3⁺CD4⁺ T细胞、CD3⁺CD8⁺ T细胞及NK细胞的比例均逐渐降低（P<0.01）,且伴随着3种细胞Tim-3表达量的升高（P<0.01）。小鼠外周血CD3⁺CD4⁺ T细胞的比例呈先降低后升高趋势,CD3⁺CD8⁺ T细胞的比例呈先升高后降低趋势（P<0.05）;小鼠外周血CD3^-NK1.1⁺细胞的比例呈先降低后升高趋势,但感染末期,其比例仍低于未感染组（P<0.05）。Tim-3分子在外周血CD3⁺CD4⁺ T细胞、CD3⁺CD8⁺ T细胞及CD3^-NK1.1⁺细胞的表达均显著升高（P<0.05）。感染疟原虫后,小鼠血清中促炎细胞因子IL-2的分泌量均显著高于未感染组（P<0.05）;促炎细胞因子IFN-γ、TNF-α和IL-6在血清中分泌均呈先升高后降低趋势（P<0.05）;具有免疫抑制作用的细胞因子IL-10呈逐渐升高趋势,且感染后期急剧升高（P<0.001）。以上结果表明,感染疟原虫后,小鼠的特异性免疫反应发挥了一定的杀伤作用,但由于Tim-3免疫检查点分子及一些发挥免疫抑制作用的细胞因子（IL-10）的过度表达,有利于疟原虫逃避宿主的免疫捕杀作用。该研究提示了从宿主免疫抑制角度研究疟原虫感染的重要性。     

免疫细胞在牦牛附睾和输精管的分布规律

旨在研究免疫细胞在健康雄性牦牛附睾和输精管的分布。采用免疫组织化学和实时荧光定量(qRT-PCR)方法对幼龄(5~6月龄)及成年(3~4岁)牦牛附睾(头、体、尾)和输精管中CD68⁺巨噬细胞、CD3⁺ T淋巴细胞、CD79α⁺ B淋巴细胞、IgA⁺和IgG⁺浆细胞的分布特征及其表面标志分子的表达水平进行研究。结果显示：CD68⁺巨噬细胞、CD3⁺ T淋巴细胞、CD79α⁺ B淋巴细胞、IgA⁺和IgG⁺浆细胞主要分布在附睾管和输精管的上皮和间质;另外,CD68和CD3 mRNA和蛋白水平在各年龄组牦牛附睾头和附睾体显著高于附睾尾和输精管(P < 0.05),而CD79α、IgA和IgG mRNA和蛋白水平在附睾尾和输精管显著高于附睾头和附睾体(P < 0.05);此外,在成年牦牛附睾和输精管CD3、CD79α、IgA、IgG、CD68 mRNA和蛋白水平均显著高于幼龄牦牛(P < 0.05)。综上提示,牦牛附睾头可能主要是细胞免疫发生的位点,而附睾尾和输精管则主要进行体液免疫应答,此外,成年牦牛附睾和输精管的局部免疫可能更完善,以上数据为进一步研究高原牦牛局部生殖免疫和病理提供了形态学资料。     

The immune response of sheep infected with larvae of the sheep blowfly Lucilia cuprina monitored via efferent lymph

Changes in lymphocyte traffic in efferent lymph from the prescapular lymph node of sheep were monitored during local primary and secondary infection with blowfly, Lucilia cuprina. During primary infections the response was characterised by an increase in the output of CD4⁺ T cells over CD8⁺ T cells for the first 48 h after wound initiation. By 72 h the output of CD8⁺ T cells exceeded that of CD4⁺ T cells. During secondary infections the increased output of CD8⁺ T cells was more pronounced and occurred earlier at approximately 48 h. The percentage of B lymphocytes as measured by sIg, CD45R and MHC class II expression increased at approximately 96–120 h after both primary and secondary infections, with the secondary response being greater than the primary. This increase in B cells corresponded with peak antibody titres recorded in the efferent lymph to a first instar antigen preparation as measured by ELISA. An increase in IFN-γ and soluble IL-2 receptor was recorded after both primary and secondary infections, with the response after secondary infection being greater than that recorded after primary larval infections.     

表达猪附红细胞体ENO基因的质粒DNA的构建及免疫效果研究

试验旨在构建表达猪附红细胞体ENO基因的质粒DNA,并测定其免疫效果。将猪附红细胞体ENO基因克隆到PVAX1真核表达载体上,然后转染到Vero细胞中进行表达并测定其免疫效果。将18只BALB/c小鼠（雌雄各半）随机分为3组（PVAX1-ENO质粒DNA免疫组、PVAX1空载体对照组及PBS对照组）,每组6只,每组小鼠对应免疫接种PVAX1-ENO质粒DNA、PVAX1空质粒和PBS。分离血清并通过ELISA方法测定血清中ENO蛋白抗体效价、IgG₁和IgG_2a抗体水平及IFN-γ和IL-4细胞因子水平。三免2周后每组选取3只小鼠检测CD4⁺和CD8⁺含量。结果显示,本试验成功构建了PVAX1-ENO质粒DNA,经PCR和酶切鉴定正确,并能在Vero细胞中成功表达。PVAX1-ENO质粒DNA免疫组ENO蛋白抗体水平、IgG₁和IgG_2a抗体水平、IFN-γ和IL-4细胞因子水平及CD4⁺和CD8⁺含量均显著高于PVAX1空载体对照组及PBS对照组（P<0.05）。结果表明,PVAX1-ENO质粒DNA可显著提高BALB/c小鼠的体液免疫水平,并在一定程度上刺激BALB/c小鼠细胞免疫。     

Killing of Brucella antigen-sensitized macrophages by T lymphocytes in bovine brucellosis

The present study was an investigation into the role of T lymphocytes in the killing of antigen-sensitized macrophages (MΦ) in bovine brucellosis. Following confirmation of bovine T lymphocyte cell lines derived from Brucella abortus Strain 19 vaccinated steers as antigen-specific in proliferation studies using various antigens, we adapted an apoptosis assay for evaluation of cytotoxicity by these bovine T cells against autologous monocyte-derived macrophages (MDMΦ) as target cells. Various B. abortus antigen preparations were tested including whole γ-irradiated B. abortus bacteria (γBA), a soluble cytosolic protein fraction and a membrane-associated protein fraction. Both polyclonal and cloned T lymphocyte cell lines exhibited cytotoxicity against MDMΦ targets in an antigen-specific fashion. Polyclonal and cloned T lymphocyte cell lines demonstrated cytotoxic responses to varying degrees against B. abortus antigens regardless of whether the antigen used was whole nonviable bacteria, a soluble protein extract or a membrane-associated fraction of extracted bacteria. To further develop correlation of these responses to an in vivo host defense mechanism, cytotoxicity was evaluated using target cells that had been infected with live B. abortus S19 or B. abortus Strain 2308. Cytotoxic responses were also demonstrated consistently against infected targets with either strain of B. abortus although in most cases, cytotoxicity was higher against target cells sensitized with γBA compared to those infected with live bacteria. Cloned T lymphocyte cell lines were all CD4⁺, CD8⁻ cells indicating that the observed cytotoxic responses were most likely due to an inflammatory T_h1 response and may represent an important host defense mechanism induced by vaccination with live attenuated strains of B. abortus in cattle.     

The Influence for Chickens' T Lymphocyte Subsets in Peripheral Blood after Inoculated with the Recombinant Chicken Interferon-α/Chicken Interleukin-2 Complex Protein

To explore the influence of recombinant chicken interferon-α/chicken interleukin-2 fusion protein (rChIFN-α-Linker-ChIL-2 protein, recombinant fusion protein) on the percentage content of peripheral T lymphocyte subsets (PBLC) of SPF chicken,the flow cytometry was used to detected the percentage contents of CD3⁺CD4⁺, CD3⁺CD8⁺ and the CD4⁺/CD8⁺ ratio in PBLC at different days post injection in the 14-day-old SPF chicken injected with rChIFN-α-Linker-ChIL-2 protein (group 2) and recombinant chicken interferon α (rChIFN-α) protein (group 3). The results indicated that the recombinant fusion protein and rChIFN-α protein could increase the percentage content of CD3⁺CD4⁺ T lymphocyte, decrease the percentage content of CD3⁺CD8⁺ T lymphocyte, improve CD4⁺ /CD8⁺ T lymphocyte ratio during 3 to 14 d post injection. The percentage contents of CD3⁺CD4⁺, CD3⁺CD8⁺ and CD4⁺/CD8⁺ ratio in chickens of group 2 were significantly different from the PBS control group (group 1) (P<0.01), and were notably different from group 3 during 3 to 7 d post injection (P<0.05). These results showed that both the rChIFN-α-Linker-ChIL-2 protein and rChIFN-α protein could affect the percentage contents of lymphocyte subsets, improve CD4⁺/CD8⁺ ratio, enhance the cellular immune function in chicken. The influences of rChIFN-α-Linker-ChIL-2 protein on the percentage contents of CD3⁺CD4⁺ and CD3⁺CD8⁺ T lymphocyte, and CD4⁺/CD8⁺ value were notable higher than rChIFN-α, which suggested that recombinant fusion protein play a synergistic role of interferon alpha and interleukin-2 on the cellular immune function by this pathway in chicken.     

Perforin expression can define CD8 positive lymphocyte subsets in pigs allowing phenotypic and functional analysis of natural killer, cytotoxic T, natural killer T and MHC un-restricted cytotoxic T-cells

In this study we have used the expression of perforin to characterize subsets of porcine cytotoxic lymphocytes. Perforin positive lymphocytes expressed both CD2 and CD8, most were small dense lymphocytes (SDL) and up to 90% were CD3 negative. However, the numbers of perforin positive T-cells increased with the age of the animal and their populations increased after specific antigen stimulation in vitro. The remaining perforin positive lymphocytes were large and granular and contained more CD3⁺CD5⁺CD6⁺ T-cells (−40%) of which a substantial proportion also co-expressed CD4. Perforin was expressed in subpopulations of both CD8 and CD8β lymphocytes, but was not expressed in γδ T-cells or monocyte/macrophages. The perforin positive CD3⁻ subset was phenotypically homogeneous and defined as CD5⁻CD6⁻CD8β⁻CD16⁺CD11b⁺. This population had NK activity and expressed mRNA for the NK receptor NKG2D, and adaptors DAP10 and DAP12. Perforin positive T-cells (CD3⁺) could be divided into at least three subsets. The first subset was CD4⁻CD5⁺CD6⁺CD11b⁻CD16⁻ most were small dense lymphocytes with cytotoxic T-cell activity but not all expressed CD8β. The second subset was mainly observed in the large granular lymphocytes. Their phenotype was CD4⁺CD5⁺CD6⁺CD8β⁺CD16⁻CD11b⁻ and also showed functional CTL activity. Thus not all of double positive T-cells are memory helper T-cells. The third subset did not express the T-cell co-receptor CD6, but up to half of them expressed another T-cell co-receptor CD5. The majority of this subset expressed CD11b and CD16, thus the third perforin positive T-cell subset was CD3⁺CD4⁻CD5⁺CD6⁻CD8β^±CD11b⁺CD16⁺, and possessed MHC-unrestricted cytotoxicity and LAK activity.     

Prediction,Identification and Preliminary Study on Immune Efficacy of S1 Protein Epitope-polypeptides of Avian Infectious Bronchitis Virus

For the sake of verifying the immunogenicity of candidate epitope-polypeptide, the B and T cell epitopes of S1 protein of avian infectious bronchitis virus (IBV) H120 strain were predicted and the corresponding epitope-polypeptides were synthesized, and then were used to immunize mice, the immune effect was analyzed. Five epitope-polypeptides against S1 protein of IBV H120 strain were selected by epitope prediction software and acquired by chemical synthesis, then were immunized to mice. The specific antibodies, neutralizing antibodies and T lymphocyte subsets induced by each epitope-polypeptides were analyzed by indirect ELISA, neutralization test and flow cytometry. The ELISA results showed that the five epitope-polypeptides had good reactivity. The antibody titers of antisera induced by the five epitope-polypeptides sorted from high to low as follows: Pep76-106, Pep240-257, Pep511-537, Pep403-421, Pep135-172. The neutralization test results showed that the neutralization titers of antisera induced by the five epitope-polypeptides groups in mice were higher than that of the blank control group, and the order of neutralization titers was Pep240-257 = Pep403-421 = Pep511-537 > Pep76-106 = Pep135-172. The flow cytometry results showed that the percentages of CD3⁺, CD4⁺CD8^- and CD8⁺CD4^- T lymphocytes in all the five epitope-polypeptides groups were significantly higher (P<0.01) than those in the blank control group. The number of the CD3⁺ and CD4⁺CD8^- T lymphocytes sorted from large to small as follows: Pep403-421, Pep240-257, Pep76-106, Pep511-537 and Pep135-172. The number of the CD8⁺CD4^- T lymphocytes sorted from large to small as follows: Pep403-421, Pep76-106, Pep511-537, Pep240-257 and Pep135-172. In conclusion, Pep240-257, Pep76-106 and Pep403-421 could induce humoral immunity among the five epitope-polypeptides, while Pep403-421 could induce cellular immunity. Thus, peptide of Pep403-421 could induce cellular immunity and humoral immunity. This study laid a foundation for further understanding the immunological characteristics of the S1 protein and the development of diagnostic reagents and effective epitope vaccines.     

鸡传染性支气管炎病毒S1蛋白抗原表位多肽的预测、鉴定及免疫效果的初步研究

旨在对鸡传染性支气管炎病毒（IBV）H120株S1蛋白的B细胞及T细胞可能的表位进行预测并合成相应的多肽,然后免疫小鼠,并分析其免疫效果,以验证候选表位多肽的免疫原性。利用表位预测软件筛选并化学合成针对IBV H120株S1蛋白的5条表位多肽,然后免疫小鼠,通过间接ELISA、中和试验和流式细胞技术检测各表位多肽诱导的特异性抗体、中和抗体和外周血T细胞亚群。ELISA检测结果显示,5条表位多肽均具有良好的反应原性,免疫小鼠的血清效价高低顺序依次为Pep76-106 > Pep240-257 > Pep511-537 > Pep403-421 > Pep135-172;中和试验结果表明,5条多肽免疫小鼠的血清中和滴度均高于空白对照组,其高低顺序依次为Pep240-257=Pep403-421=Pep511-537>Pep76-106=Pep135-172;流式检测结果表明,5条多肽免疫小鼠在CD3⁺、CD4⁺CD8^-、CD8⁺CD4^- T淋巴细胞水平上均极显著高于空白对照组（P<0.01）,CD3⁺T及CD4⁺CD8^-T淋巴细胞数大小顺序依次为Pep403-421 > Pep240-257 > Pep76-106 > Pep511-537 > Pep135-172,CD8⁺CD4^-T淋巴细胞数大小顺序依次为Pep403-421 > Pep76-106 > Pep511-537 > Pep240-257 > Pep135-172。合成的5条表位多肽中,Pep240-257、Pep76-106和Pep403-421可以诱导体液免疫,Pep403-421可以诱导细胞免疫,其中,Pep403-421可以同时诱导细胞免疫及体液免疫。本研究结果为深入了解S1蛋白的免疫学特性以及研发诊断试剂和有效表位疫苗奠定了基础。     

禽网状内皮组织增生病病毒感染对SPF雏鸡血液和局部淋巴组织CD4+/CD8+细胞及相关细胞因子表达的影响

旨在探讨禽网状内皮组织增生病病毒（reticuloendotheliosis virus,REV）对SPF雏鸡血液和局部淋巴组织中T淋巴细胞数量以及相关细胞因子表达的影响。将96只1日龄SPF雏鸡随机分为REV感染组和对照组,应用流式细胞术、酸性α-醋酸萘酯酶染色（acid α-naphthyl acetate esterase,ANAE）、荧光定量PCR等方法对上述指标进行检测。试验数据表明,与对照组相比,感染组雏鸡血液CD4⁺T淋巴细胞数量在第7~35天显著降低、CD8⁺T淋巴细胞数量在第7~28天显著升高,CD4⁺/CD8⁺比值在第7~28天显著降低（均P<0.05或P<0.01）;局部淋巴组织哈德尔腺（Hader’s gland,HG）、派伊尔结（Peyer’s patch,PP）和盲肠扁桃体（caecal tonsil,CT）中ANAE⁺T淋巴细胞数量均显著降低（均P<0.05或P<0.01）;GH、PP和CT中细胞因子IL-2、IFN-γ和TNF-α 转录量都有不同程度升高。本研究表明,REV感染引起雏鸡血液中CD4⁺ T淋巴细胞数量降低、CD4⁺/CD8⁺T淋巴细胞比例失衡、局部淋巴组织中T淋巴细胞数量相对减少及细胞因子TNF-α转录持续升高与REV造成感染雏鸡细胞免疫功能显著降低密切相关。     

Antigen presentation in vaccine development

A variety of microorganisms, nutrients or toxins are generally intrude our body through mucosal tissues or skin, where equipment for both preventing their invasions and catching their information to activate internal immune systems for adapting surroundings is arranged. Among the equipment, cells in charge of innate immunity, particularly dendritic cells (DCs), having an excellent capacity for prompt recognition of invaded pathogens via toll-like receptors (TLRs) to alert B and T cells for establishing aquired/adaptive immunity by presenting their processed antigenic fragments, have been paid great attention. These TLR-activated, antigen captured DCs are divided into two groups; one is pathogen-retaining unit and the other is pathogen-controlling unit. The latter DCs present processed antigenic molecules from the pathogens to competent β T cells together with special containers, such as class I, class II MHC and CD1 to generate specific cellular immunity. The former two MHC molecules can present processed peptide antigens, whereas the last CD1 molecule can present glycolipid/lipid antigens. In contrast, B lymphocytes that captured antigens via their specific immunoglobulin (Ig) receptors present digested peptide fragments with their class II MHC to stimulate suitable CD4⁺ helper T cells which in turn secrete various cytokines to efficiently expand and maintain antibody production from that partner B cells to establish humoral immunity. These β T cells and antibodies, recognize either processed antigenic peptide or glycolipid fragments, and thus, identification of these epitopes enables us to generate artificial pathogen-specific vaccines. Based on the recent findings about precise mechanisms of antigen processing and presentation orchestrated at the surface compartment, future development of vaccines against various pathogens are discussed.     

柔嫩艾美耳球虫对藏鸡和隐性白羽鸡致病性差异分析

为进一步明确柔嫩艾美耳球虫对球虫易感性差异鸡种的致病性,本研究以1×10⁵个柔嫩艾美耳球虫孢子化卵囊的剂量分别感染对球虫具有抗性的藏鸡和易感的隐性白羽鸡,接种后观察记录各组鸡的临床表征、血便记分、死亡率、增重、盲肠病变记分、卵囊产量,并于感染前和感染后3、6和8 d每个品种分别随机选择5只鸡采集抗凝血,应用流式细胞仪（FCAS）检测外周血CD4⁺T和CD8⁺T淋巴细胞亚群数量。结果显示,柔嫩艾美耳球虫感染后藏鸡相对增重率高于隐性白羽鸡,死亡率、血便记分和盲肠病变记分均低于隐性白羽鸡,但卵囊产量高于隐性白羽鸡。外周血T淋巴细胞亚群变化方面,感染前,藏鸡CD4⁺T淋巴细胞数及CD4⁺/CD8⁺比值均高于隐性白羽鸡。感染后第3天,藏鸡CD4⁺、CD8⁺ T淋巴细胞数及CD4⁺/CD8⁺比值下降,隐性白羽鸡CD8⁺ T淋巴细胞数略有下降,CD4⁺T淋巴细胞数及CD4⁺/CD8⁺比值上升,但CD4⁺/CD8⁺比值仍显著低于藏鸡（P<0.05）。感染后第6天,2个品种鸡CD4⁺ T淋巴细胞数及CD4⁺/CD8⁺比值均下降,其中藏鸡表现为显著下降（P<0.05）,而隐性白羽鸡仅CD4⁺/CD8⁺比值显著降低（P<0.05）,隐性白羽鸡CD8⁺ T淋巴细胞数显著升高（P<0.05）。感染后第8天,2个品种鸡CD4⁺/CD8⁺比值显著下降（P<0.05）,藏鸡CD8⁺ T淋巴细胞数显著高于隐性白羽鸡（P<0.05）,但CD4⁺/CD8⁺比值显著低于隐性白羽鸡（P<0.05）。结果表明,球虫对藏鸡和隐性白羽鸡的致病性存在差异,这种差异与T淋巴细胞介导的免疫应答反应密切相关。     

Beta-glucan enhancement of T cell IFNgamma response in swine

Beta-glucan has been shown to enhance anti-tumor and anti-infection functions in animals. Pigs at 4 months of age were infected with porcine reproductive and respiratory syndrome virus (PRRSV), and peripheral blood monocytes (PBMC) were isolated for the detection of interferon gamma (IFNgamma)-producing cells. We found that soluble high molecular weight beta-glucan could increase IFNgamma-producing cell frequency in a dose-dependent manner in the enzyme-linked immunospot assay (ELISPOT) in the absence of antigenic restimulation. A concentration as low as 1.6 microg/ml gave a significant increase and a similarly high enhancement was achieved at concentrations from 3.2 to 100 microg/ml. In PRRSV-specific IFNgamma ELISPOT, soluble beta-glucan elicited increased PRRSV-specific responses at concentrations from 3.2 to 50 microg/ml, but not at 100 microg/ml, whereas insoluble beta-glucan had no effect. Soluble beta-glucan augmented the porcine cellular immune response in an antigen-independent fashion, whereas insoluble beta-glucan had no activity. This finding suggests that soluble beta-glucan may enhance innate antiviral immunity against PRRSV.     

Analysis of Immune Response Induced by Recombinant Lactobacillus reuteri Expressing Cap Protein of Porcine Circovirus Type 2 in Mice

The purpose of this study was to construct a recombinant Lactobacillus reuteri (L. reuteri) expressing the cap protein of porcine circovirus type 2 (PCV2) and evaluate its effect on immune response in mice. The cap protein gene of PCV2b strain isolated and stored in the laboratory was amplified by PCR. A recombinant strain pPG-T7 g10-PPT-cap / L. reuteri expressing the cap protein was constructed using L. reuteri of pig origin as the host strain and explored the immune effect of BALB/c mice with recombinant bacteria orogastrically. Indirect ELISA was used to determine the level of antigen-specific IgG antibodies in the serum of mice after immunization, the levels of antigen-specific sIgA antibodies in stool, nasal wash, reproductive tract wash, and intestinal mucus, and the levels of various cytokines in mouse serum; MTT method was used to detect mouse spleen lymphocyte proliferation levels; flow cytometry (FCM) was used to detect the levels of CD4⁺ T cells and CD8⁺ T cells in mouse spleen lymphocytes; fluorescence quantitative PCR was used to detect the viral load of organs in challenged mice after immunization. The results showed that the serum levels of IgG antibodies in the mice of the oral immune recombinant strain group (OIG) were significantly higher than those in the control group (P<0.01); the levels of sIgA antibodies in the stool, nasal wash, reproductive tract wash, and intestinal mucus of the mice in OIG were significantly higher than those in the control group (P<0.01); Compared with the control group, the levels of cytokines in the serum of OIG were as follows: The levels of IFN-γ, IL-2, IL-4, IL-12 increased, the levels of IL-10 decreased, and the levels of IFN-α did not change significantly; Incubation of PCV2 and mouse spleen lymphocytes in vitro showed that the proliferation stimulating index of spleen lymphocytes in OIG was significantly higher than that of the control group (P<0.01); FCM results showed that CD4⁺ T cells and CD8⁺ T cells were higher than those of the control group; the results of fluorescent quantitative PCR showed that compared with the control group, the viral load in the OIG was significantly lower than that of the control group. In summary, the recombinant L. reuteri expressing the PCV2 cap protein were successfully constructed, and the constructed recombinant L. reuteri can stimulate mice to produce humoral and cellular immune responses after oragastrical immunization, and can exert a certain immune protection effect.     

表达猪圆环病毒2型cap蛋白的重组罗伊氏乳酸杆菌在小鼠诱导的免疫应答分析

旨在构建表达猪圆环病毒2型（PCV2）cap蛋白的重组罗伊氏乳酸杆菌（Lactobacillus reuteri,L.reuteri）,并评价其在小鼠体内诱导的免疫应答效果。利用PCR扩增实验室分离保存的PCV2b型毒株的cap蛋白基因,以猪源L.reuteri为宿主菌,构建表达cap蛋白的重组菌株pPG-T7 g10-PPT-cap/L.reuteri,通过口服免疫BALB/c小鼠。采用间接ELISA方法测定免疫后小鼠血清中抗原特异性IgG抗体水平,粪便、鼻腔洗液、生殖道洗液、肠黏液中抗原特异性sIgA抗体水平,小鼠血清中各细胞因子水平;MTT法检测小鼠脾淋巴细胞增殖水平;流式细胞技术检测小鼠脾淋巴细胞中CD4⁺T细胞、CD8⁺T细胞的水平;荧光定量PCR检测免疫后攻毒的小鼠体内器官的病毒载量。结果显示,口服免疫重组乳酸菌组小鼠血清IgG抗体水平显著高于对照组（P<0.01）;小鼠粪便、鼻腔洗液、生殖道洗液、肠黏液中sIgA抗体水平显著高于对照组（P<0.01）;小鼠血清中细胞因子水平和对照组相比,IFN-γ、IL-2、IL-4、IL-12水平升高,IL-10水平降低,IFN-α无显著变化;体外孵育PCV2和小鼠脾淋巴细胞结果表明,重组乳酸菌组小鼠脾淋巴细胞增殖刺激指数显著高于对照组（P<0.01）;流式细胞技术检测结果显示,口服免疫重组乳酸菌组小鼠脾细胞中CD4⁺T细胞、CD8⁺T细胞含量高于对照组;荧光定量PCR结果显示,相比于对照组,口服免疫重组乳酸菌组小鼠体内的病毒载量明显低于对照组。综上所述,本研究成功构建了表达PCV2 cap蛋白的重组罗伊氏乳酸杆菌,经口服途径免疫动物,构建的重组乳酸杆菌能够刺激小鼠产生体液免疫和细胞免疫应答,且具有一定的免疫保护效果。     

Immunological properties of recombinant classical swine fever virus NS3 protein in vitro and in vivo

Classical swine fever (CSF) is a highly contagious and often fatal disease of pigs characterised by fever, severe leukopenia and haemorrhages. With vaccines having an importance in disease control, studies are seeking improved protein-based subunit vaccine against the virus (CSFV). In this respect, recombinant viral NS3 protein was analysed for its immunopotentiating capacity, particularly in terms of cytotoxic immune responses. NS3 was effective at inducing in vitro responses, quantified by lymphoproliferation, IFN-gamma ELISPOT, flow cytometric detection of activated T cell subsets, and cytotoxic T cell assays. Peripheral blood mononuclear cells from CSFV-immune pigs could be stimulated, but not cells from na?ve animals. In addition to the IFN-gamma responses, induction of both CD4+ T helper cell and CD8+ cytotoxic T cells (CTL) were discernible--activation of the latter was confirmed in a virus-specific cytolytic assay. Attempts were made to translate this to the in vivo situation, by vaccinating pigs with an E2/NS3-based vaccine compared with an E2 subunit vaccine. Both vaccines were similar in their abilities to stimulate specific immune responses and protect pigs against lethal CSFV infection. Although the E2/NS3 vaccine appeared to have an advantage in terms of antibody induction, this was not statistically significant when group studies were performed. It was also difficult to visualise the NS3 capacity to promote T-cell responses in vivo. These results show that NS3 has potential for promoting cytotoxic defences, but the formulation of the vaccine requires optimisation for ensuring that NS3 is correctly delivered to antigen presenting cells for efficient activation of CTL.