铝佐剂类型及吸附率对吸附无细胞百白破联合疫苗免疫原性的影响

目的研究铝佐剂类型及吸附能力对吸附无细胞百白破联合疫苗免疫原性的影响,筛选最佳佐剂。方法用磷酸铝(AP)、不同浓度磷酸盐处理的氢氧化铝(PTAH)以及氢氧化铝(AH)佐剂吸附百白破抗原,检测不同佐剂对各抗原的吸附率。制备不同佐剂类型和吸附率的制剂,腹腔免疫NIH小鼠,ELISA法测定免疫后各组小鼠血清抗体滴度。结果百日咳丝状血凝素(FHA)在所有佐剂中吸附率≥95%,百日咳类毒素(PT)、百日咳溶血素(PRN)、精制白喉类毒素(DT)和破伤风类毒素(TT)则随着铝佐剂中磷酸根增多吸附率降低。动物实验产生PT抗体水平大小关系:AP/AHPTAH/无佐剂组;产生FHA抗体水平大小关系:AP/PTAH-3/AHPTAH-2/PTAH-3无佐剂组;产生PRN抗体水平大小关系:PTAH/AP/AH无佐剂组;产生DT抗体水平大小关系:APPTAHAH无佐剂组;产生TT抗体水平大小关系:PTAHAH/AP/无佐剂组。结论铝佐剂吸附力不影响抗原(DT除外)免疫原性,磷酸铝更适合作吸附无细胞百白破联合疫苗的佐剂。     

用模式抗原OVA研究CTA1-DD／IgG佐剂的免疫调节作用

目的：进一步研究新型佐剂CTA1-DD/IgG的免疫调节作用。方法：小鼠经鼻内免疫抗原（鸡卵清白蛋白OVA）联合佐剂CTA1-DD或CTA1-DD/IgG复合物,用ELISA检测血清中OVA特异性IgG抗体生成和IgG抗体亚类,用酶联免疫斑点试验检测脾脏抗体分泌细胞。结果：CTA1-DD/IgG佐剂在小鼠中诱导血清特异性IgG抗体及脾脏特异性抗体分泌细胞的能力均高于CTA1-DD佐剂;CTA1-DD/IgG或CTA1-DD作为佐剂联合OVA免疫后IgG1、IgG2a、IgG2b 抗体皆提高,且IgG2a/IgG1＞1。结论：CTA1-DD/IgG佐剂的免疫调节作用强于CTA1-DD,且能产生混合IgG抗体亚型,促进平衡的免疫应答。     

新型升血小板因子的免疫原性实验研究

目的明确自行研制的新型升血小板因子是否具有免疫原性。方法分别用佐剂、新型升血小板因子＋佐剂、新型升血小板因子、鸡卵清蛋白＋佐剂免疫Balb／c小鼠,采用琼脂免疫扩散试验和酶联免疫吸附试验（EIJSA）检测小鼠血清中的抗体。结果ELISA法未检测到血清中的抗体;琼脂免疫扩散试验血清经72h扩散观察未出现沉淀线,没有形成抗原．抗体复合物。结论用新型升血小板因子免疫Balb／c小鼠,未发现能够刺激小鼠产生抗新型升血小板因子抗体。     

水痘带状疱疹病毒gE蛋白联合不同佐剂对小鼠免疫效果的比较

目的比较利用AS01和Al/CpG联合佐剂, 水痘带状疱疹病毒糖蛋白E(Varicella Zoster virus glycoprotein E, VZV gE)在BALB/c小鼠体内的免疫效果。方法 BALB/c小鼠分别于0和21天免疫, 小鼠血清抗体检测采用酶联免疫吸附实验;用VZV检测小鼠血清中和抗体滴度;酶联免疫吸附斑点实验检测细胞免疫反应。结果经过2次肌肉注射免疫, 实验组(Shingrix、gE+Al/CpG和gE+AS01)中小鼠均能产生高滴度的中和抗体, 增加分泌IFN-γ和IL-4的淋巴细胞数量。gE+Al/CpG免疫组中小鼠产生的中和抗体滴度最高(1943), 但是AS01佐剂组(Shingrix和gE+AS01)中小鼠产生的分泌IFN-γ和IL-4的淋巴细胞数量高于Al/CpG佐剂组(gE+Al/CpG)。相比于AS01佐剂, Al/CpG佐剂诱导小鼠产生了偏向Th2型的体液免疫应答。各实验组中小鼠CD4+T细胞、CD8+T细胞的比例均没有统计学差异。结论 Al/CpG佐剂联合gE蛋白诱导高滴度中和抗体, 但是诱导产生的细胞免疫应答强度低于AS01佐剂组。     

恶性疟疾疫苗M.RCAg-1与候选佐剂纳米乳配伍的免疫原性研究

为了评价纳米乳作为佐剂对恶性疟疾人工重组蛋白疫苗M.RCAg-1免疫原性的影响,将纳米乳佐剂与M.RCAg-1相配伍,于第0、14、28 d肌肉注射免疫小鼠,抗原量为20μg/只。第3次免疫后10 d,眼球取血并取脾细胞。采用ELISA法检测小鼠血清特异性Ig G抗体水平、抗体亚类及其对抗原单表位的识别,用间接免疫荧光反应(IFA)检测抗体对恶性疟原虫天然蛋白的识别,用ELISPOT法检测小鼠脾淋巴细胞免疫应答水平。结果显示疫苗佐剂组小鼠血清中抗M.RCAg-1的抗体水平可达1∶108;疫苗佐剂组产生的特异性Ig G抗体以Ig G1为主;疫苗佐剂组抗体对抗原单表位和天然虫体的识别效果显著。各表位和蛋白诱发免疫小鼠分泌IFN-γ的特异性淋巴细胞克隆数高于分泌IL-4的特异性淋巴细胞克隆数。结果提示纳米乳作为恶性疟疾疫苗M.RCAg-1的佐剂,能够显著提高机体的体液免疫和细胞免疫应答水平。     

BALB/C小鼠对不同佐剂SARS灭活疫苗的早期免疫反应

目的：研究含不同佐剂的SARS灭活疫苗免疫小鼠后，小鼠的早期细胞免疫和体液免疫反应。方法：灭活的SAPS冠状病毒F69株分别与弗氏佐剂、氢氧化铝、CpG佐剂配伍，制备灭活疫苗。接种6周龄SPF’级BALB／C小鼠。定时采血，分析小鼠外周血中CD4^ 、CD8^ T细胞亚群动态变化，同时测定鼠血清中特异性IgG抗体及抗体的病毒中和活性。结果：由3种佐剂制备的SARS灭活疫苗免疫小鼠后，CD4^ 、CD8^ T细胞百分比升高或无明显改变，CD4／CD8比值无显著性改变。其中，弗氏佐剂疫苗免疫小鼠的CD4^ T细胞百分比升高较明显，且在一段时间内，维持在一个较高的水平；铝佐剂疫苗免疫小鼠后，未观察到明显T细胞亚群改变；而CoG佐剂疫苗免疫小鼠的CD8^ T细胞百分比改变较其它佐剂疫苗改变更明显。与此相异的是，3种疫苗均可诱导小鼠产生较高滴度特异性IgG抗体，并且所产生的抗体具有中和活性。在初免后第34天，3种佐剂组的IgG抗体滴度分别为1：12800、1：3200和1：1600，中和效价分别为1：2560、1：960和1：320。结论：SARS灭活疫苗接种小鼠后，可诱导较强的体液免疫反应，同时轻度上调CD4^ 、CD8^ T细胞活性，程度因佐剂而异。     

FK506作为佐剂刺激Tfh细胞增强体液免疫水平的研究

目的:探讨他克莫司(FK506)作为佐剂通过刺激Tfh细胞增强疫苗体液免疫水平的机制。方法:利用FK506与模式蛋白(卵清白蛋白,OVA)皮下注射免疫BALB/c小鼠,免疫三次,利用ELISA方法检测抗体水平;利用抗体染色流式细胞仪检测Tfh细胞、B细胞表面分子IL-21R及记忆性B细胞标志分子CD27表达。结果:FK506作为OVA蛋白佐剂,能够显著提高小鼠OVA特异性的IgG水平,增强体液免疫水平;免疫后的小鼠Tfh细胞表达IL-21水平显著高于其他对照组。同时,B细胞表达IL-21R及记忆性B细胞标志分子CD27显著高于其他对照组。表明FK506作为佐剂刺激Tfh细胞表达IL-21,作用于B细胞增强抗体水平。结论:FK506能够作为蛋白疫苗佐剂刺激Tfh细胞表达IL-21;IL-21可能通过与B细胞表面IL-21R作用,增强抗体的分泌;并刺激记忆性B细胞的产生,进而增强体液免疫水平。     

单磷酸脂质A佐剂对无细胞百日咳疫苗的免疫保护效果研究

目的 探讨单磷酸脂质A(MPLA)佐剂对无细胞百日咳疫苗(aP)的免疫保护效果影响.方法 aP中添加MPLA佐剂,在Balb/c小鼠上进行免疫以及感染保护实验.通过检测小鼠百日咳特异性IgG抗体及其分型抗体IgG1和IgG2a水平、感染后白细胞变化以及气管和肺组织细菌定植来进行免疫效果的评估.结果 aP+MPLA免疫的...     

尿酸钠对BALB/c小鼠抗体应答及树突状细胞和迟发型超敏反应的影响

目的:探讨尿酸钠作为佐剂对BALB/c小鼠体液和细胞免疫应答的影响。方法:利用尿酸钠悬浮液为佐剂、天花粉蛋白(TCS)为免疫原对BALB/c小鼠进行免疫,以酶联免疫测定法检测特异性抗体IgG的效价。体外诱导小鼠树突状细胞(DC),流式细胞术分析DC表型,评价尿酸钠体外对DC成熟的效应。以二硝基氟苯建立迟发型超敏反应(DTH)模型,分析尿酸钠在体内对细胞免疫应答的影响。结果:传统弗氏佐剂可极大地增强小鼠对TCS的抗体应答,尿酸钠佐剂对抗体应答不但没有促进,与单独使用免疫原相比,抗体应答反而明显降低。流式细胞术分析显示,尿酸钠对DC表达CD11c和CD83没有影响,但可明显提高MHCII的表达水平。DTH模型中,尿酸钠增强致敏原引发的耳廓肿胀程度,并促进DTH小鼠淋巴细胞的体外增殖能力。结论:尿酸钠悬浮液作为佐剂,对细胞免疫有显著增强作用,而对体液免疫应答却有一定的抑制作用,提示该佐剂在疫苗研究中有潜在应用前景。     

弗氏佐剂与氢氧化铝佐剂对诱导小鼠获得性免疫应答作用的比较

为研究、比较不同佐剂对诱导小鼠产生获得性免疫应答的不同作用,以卵清白蛋白(OVA)为抗原,分别混合完全弗氏佐剂(CFA)或Al(OH)3佐剂,对C57BL/6小鼠进行常规免疫,采用流式细胞技术对细胞内细胞因子IFN-γ和IL-4进行检测;ELISA方法对特异性抗OVA抗体滴度及抗体亚型进行了检测。结果显示在免疫后CFA组产生以IFN-γ为主的细胞因子而Al(OH)3组产生以IL-4为主的细胞因子;两组中均产生特异性抗OVA IgG抗体,但CFA组以IgG2a亚型为主,而Al(OH)3组则以IgG1亚型为主,不产生IgG2a亚型抗体。实验表明,经CFA加抗原免疫后机体产生的免疫应答以Th1型细胞免疫为主,抗体类型为IgG2a;而Al(OH)3佐剂则诱导机体产生Th2型细胞免疫应答,抗体类型为IgG1。     

Adjuvant activity of purified peptidoglycan of Listeria monocytogenes in mice and guinea pigs.

The immunological properties of peptidoglycan (L-PG) purified from the cell wall skeleton (L-CWS) of Listeria monocytogenes strain EGD were investigated and compared with the properties of L-CWS. L-PG consisted of alanine, glutamic acid, alpha, epsilon-diaminopimelic acid, muramic acid, and glucosamine. L-PG showed potent adjuvant activities for circulating antibody formation and development of delayed-type hypersensitivity to bacterial alpha-amylase in vivo and for the primary immune response to sheep erythrocytes in vitro, as well as L-CWS. Both L-PG and L-CWS enhanced the generation of cell-mediated cytotoxicity in allogeneic mice and activated thioglycolate-elicited peritoneal macrophages and macrophage cell line RAW 264 to kill tumor target cells in vitro. We also found that L-PG acted on normal spleen cells as a mitogen. Both L-PG and L-CWS had tumor (Meth A)-suppressive and -regressive activities in syngeneic mice. Our results suggest that the L-PG moiety retains the adjuvant and antitumor activities of L-CWS.     

Oral adjuvants enhance IgA responses to Streptococcus mutans

The induction of immune responses to orally-administered trinitrophenyl (TNP)-haptenated Streptococcus mutans or its cell wall components and enhancement of immune responses with oral adjuvants has been studied in high IgA responsive C3H/HeJ mice and in gnotobiotic rats. Gastric intubation of TNP-S. mutans to LPS non-responsive C3H/HeJ or syngeneic, LPS responsive C3H/HeN mice induced IgA responses as determined by measuring splenic plaque-forming cell (PFC) responses and IgA anti-TNP antibodies in serum, saliva, and urine. Higher IgA responses always occurred in C3H/HeJ mice given oral S. mutans antigen than similarly treated C3H/HeN animals. Oral administration of the adjuvants concanavalin A or S. mutans cell wall peptidoglycan (PG) with antigen resulted in augmented IgA responses, especially in C3H/HeJ mice. On the other hand, oral administration of muramyl dipeptide (MDP) with antigen boosted anti-TNP responses in C3H/HeN, but not in C3H/HeJ, mice. Gnotobiotic rats given S. mutans whole cells (WC) or purified cell walls (CW) by the oral route exhibited a salivary IgA immune response which was potentiated greater than twofold when antigen was given with PG or MDP. In other studies, S. mutans WC or CW antigen in water-oil-water (W/O/W) emulsion or liposomes was administered by gastric intubation to rats. Significant salivary IgA responses were induced with these antigen-adjuvant preparations. Although rats given S. mutans WC or CW were protected from S. mutans challenge, the greatest degree of caries immunity was obtained in animals which received antigen and adjuvant and which exhibited significant salivary IgA antibody levels. In preliminary studies, it was observed that local injection of rats in the salivary gland region with a ribosomal preparation from S. mutans resulted in a significant salivary IgA response and caries immunity. The potential for soluble and lipid carrier adjuvants in oral vaccines for induction of protective antibodies to S. mutans is discussed.     

S-O_2-1细菌细胞壁骨架的制备及其对巨噬细胞的激活作用

本实验提取的S-O_2-1细菌细胞壁骨架(Cell Wall Skeleton,S-CWS)测出有16种氨基酸。 实验证明,经过S-CWS激活后的小鼠巨噬细胞的吞噬百分率和吞噬指数均有提高,酵母(YC)花环形成率达对照组的3.3倍。 巨噬细胞对肿瘤细胞的体外细胞阻滞实验结果证明,经S-CWS激活后的巨噬细胞对肿瘤细胞的阻滞效应(Cytostasis effect)显著提高。     

p185抗体可致小鼠流产的研究

目的通过体内实验进一步验证ｐ１８５蛋白的免疫原性，旨在研究该蛋白作为肿瘤疫苗的可能性。方法用ＳｅｐｈａｄｅｘＧ２００层析柱分离的ｐ１８５蛋白组分与佐剂混合免疫ＢＡＬＢ／ｃ小鼠，经ＥＬＩＳＡ验证血清中出现ｐ１８５抗体后与健康的同品系雄性鼠交配，以阴栓形成提示交配成功，观察雌鼠产仔率，子宫形态及免疫病理。结果免疫鼠产仔率明显低于仅用佐剂免疫的对照组（Ｐ＜０．０５），子宫内发现胚胎坏死，病检显示子宫组织有弥散性中性粒细胞浸润和局灶性坏死，并有免疫复合物沉积，提示ｐ１８５免疫能诱导小鼠流产。结论这一实验结果从体内进一步证实ｐ１８５的免疫原性。为该蛋白作为肿瘤疫苗提供实验资料，同时该模型可用于对胚胎性肿瘤抗原的免疫原性研究     

Adjuvant activity of mycobacterial fractions: adjuvant activity of synthetic N-acetylmuramyl-dipeptide and the related compounds.

Immunological activity of synthetic cell wall peptidoglycan subunits was examined in guinea pigs and mice. It was concluded that the minimal adjuvant-active subunit of cell wall peptidoglycan for the induction of delayed-type hypersensitivity to monoazobenzenearsonate-N-acetyl-L-tyrosine and for circulating-antibody formation to bacterial alpha-amylase and the thymus-independent antigen DNP-Ficoll was N-acetylmuramyldipeptide, MurNAc-L-Ala-D-isoGln. N-acetylmuramyldipeptide and 6-O-stearoyl-N-acetylmuramyldipeptide showed no adjuvant activity in the generation of cell-mediated cytotoxic effector cells in the spleens of C57Bl/6J mice after in vivo immunization with the allogeneic antigen mastocytoma P815-X2 cells, but N-acetylmuramyldipeptide showed adjuvant activity after in vitro sensitization of C57Bl/6J mouse spleen cells to the alloantigen mitomycin C-treated DBA/2 mouse spleen cells. It was also shown that 6-O-stearoylation of N-acetylmuramylpeptide could not potentiate the adjuvant activity of N-acetylmuramyldipeptide. Mitogenic and antitumor activities were not observed in either N-acetylmuramyldipeptide or 6-O-stearoyl-N-acetylmuramyldipeptide in mouse systems.     

A plaque assay for assessment of immune responses to Streptococcus mutans cell wall carbohydrate

A plaque-forming cell (PFC) assay has been developed for measurement of single cell responses to the serotype carbohydrate antigen of the Streptococcus mutans cell wall. Serotype g carbohydrate was purified from a mutanolysin (M1) enzyme digest (and designated M1g) of S. mutans 6715 cell walls by ion exchange and gel filtration chromatography. M1g carbohydrate was esterified by stearoylization (sM1g), and subsequently coated to sheep erythrocytes (sM1g-SRBC). This coating antigen was then used for enumeration of IgM anti-M1g PFC responses from spleens of either mice or rats immunized with S. mutans 6715 antigen. Good splenic IgM anti-M1g PFC responses were seen in either mice or rats given S. mutans whole cells or cell walls, while cell wall lysates or M1g were lowly immunogenic. Of interest was the finding that sM1g induced good IgM anti-M1g PFC responses in mice and in murine spleen cell cultures, in vitro. This study describes a method for assessment of individual antibody-producing cells to a major S. mutans cell wall determinant which should facilitate studies directed to determine mechanisms involved in the induction of immune responses to this important bacterium.     

Vaccination with pure Mycobacterium leprae proteins inhibits M. leprae multiplication in mouse footpads.

In this study, we evaluated vaccination with a number of purified, as well as recombinant, Mycobacterium leprae proteins for protective efficacy in mice. BALB/c mice were immunized intradermally with various native somatic (purified) or recombinant M. leprae proteins and their synthetic polypeptides emulsified in Freund's incomplete adjuvant. The protective efficacy of these preparations was assessed by enumeration of bacilli in the footpads of mice challenged with viable M. leprae 1 to 2 months following immunization. Protection was afforded by the purified and recombinant 10-kDa M. leprae cytoplasmic heat shock protein, the recombinant cell wall-associated 65-kDa M. leprae heat shock protein, and to a lesser extent, the purified 28-kDa M. leprae cytoplasmic protein (superoxide dismutase). Vaccination with either the purified or recombinant 35-kDa M. leprae cell membrane protein, the synthetic 27-amino-acid N-terminal peptide of the 10-kDa protein, the recombinant 18-kDa M. leprae protein, or the purified 22-kDa cell membrane protein was ineffective. When the interval between immunization and challenge was increased to 6 months, the purified 10-kDa M. leprae protein and the recombinant 65-kDa M. leprae protein lost vaccine efficacy, while a sodium dodecyl sulfate-soluble protein fraction of the M. leprae cell wall (soluble proteins), as had been found previously, continued to protect, suggesting that multiple M. leprae protein epitopes are critical for solid vaccine protection.     

用糖基化磷脂酰肌醇B7-1锚定肿瘤细胞膜进行免疫治疗

目的 研制抗肿瘤免疫治疗的新疫苗.方法 用蛋白转化法将B7-1锚定在肿瘤细胞膜上,免疫C57BL-6小鼠后,一组小鼠取脾细胞进行T细胞扩增和细胞毒T淋巴细胞(CTL)功能检测,另一组小鼠进行肿瘤细胞接种试验.结果 用GPI-B7-1修饰的肿瘤细胞膜免疫小鼠后诱发了肿瘤特异性T细胞扩增和CTL.且该疫苗有一定的保护小鼠免受肿瘤细胞侵袭的作用.结论 GPI蛋白转化法为人类抗肿瘤免疫治疗提供了新的、有效的修饰肿瘤细胞膜的方法.     

Xylaria hypoxylon Lectin as Adjuvant Elicited Tfh Cell Responses

Foot‐and‐mouth disease (FMD) caused by FMD virus (FMDV) is a major health and economic problem in the farming industry. Vaccination of livestock against this highly infectious viral disease is crucial, and inactivated FMD vaccine has been effective at controlling infection. However, accumulated data show that the inactivated vaccine generates weak immune responses and that the oil formulation results in undesirable side effects. Mushroom lectins have recently been shown to display adjuvant effects when incorporated into DNA vaccines. In this study, to enhance the cellular immune response of FMDV antigen (146S), C57BL/6 mice were immunized with 146S combined with Xylaria hypoxylon lectin (XHL). The oil formulation (146S/Oil) was served as control group. Strong humoral immune responses were elicited in mice immunized with 146S/XHL as shown by high 146S antigen‐specific IgG levels, and also in 146S/Oil group. Interestingly, XHL in conjunction with inactivated FMD vaccine activated strong Th1 and Tc1 cell responses, especially Tfh cell responses, in immunized mice. XHL stimulated dendritic cell maturation by upregulating expression of major histocompatibility complex II (MHCII) molecules and co‐stimulatory molecules CD40 and CD86 in immunized mice. No XHL‐specific IgG or inflammatory factors were detected indicating the safety of XHL as an adjuvant. Taken together, these results suggest the effectiveness of XHL at inducing cellular immune responses and therefore confirm its suitability as an adjuvant for inactivated FMD vaccine.     

Comparison of antitumor activity ofLactobacillus casei with other bacterial immunopotentiators

Antitumor activity ofLactobacillus casei YIT 9018 (LC9018) was demonstrated by intralesional (i. 1.) or intravenous (i. v.) administration into tumor-bearing mice which were inoculated with methylcholanthrene-induced fibrosarcoma (Meth A) or Kirsten murine sarcoma virus-transformed tumor (K234) cells. Its activity was significantly superior to the activity of two other species of lactobacilli but was nearly the same as that ofCorynebacterium parvum orMycobacterium bovis Bacille Calmette-Guérin (BCG). I. l. or i. v. administration of LC9018 into the tumor bearers caused local transient swelling or hepatosplenomegaly but did not cause other pronounced lesions. There was no significant difference in the degree of hepatosplenomegaly in LC9018 and that in other immunopotentiators. In mice whose tumors had regressed as a result of administration of LC9018 or the other immunopotentiators, the phytohemagglutinin P (PHA-P) response of the spleen cells was less than that of mice whose tumors progressed, and approached the normal level. The PHA-P response of popliteal lymph node cells proximal to the tumor lesion was fairly low compared with the splenic PHA-P response and there was no difference between the lymphocytes from mice whose tumors had regressed or progressed. Adjuvant activity of LC9018 in inducing tumor immunity was demonstrated by administering a mixture of LC9018 and Meth A cells to mice. This adjuvant activity was of the same efficiency as that ofC. parvum and BCG. The presence of the antitumor activity of LC9018 in cell wall components was deduced from fact that removal of its cell wall by endo-N-acetylmuramidase (M-1 enzyme) abolished the activity. The possible availability of LC9018 for immunotherapy of tumors is discussed.