鼠疫耶尔森氏菌rV270抗原的克隆、表达及其免疫保护作用评价

目的：为研制鼠疫亚单位疫苗,克隆、表达并纯化去除产生免疫抑制作用序列后的鼠疫耶尔森氏菌LcrV抗原（rV270）。方法：依据已知的LcrV的核苷酸序列,避开其产生免疫抑制作用的区段设计引物,扩增rV270基因并克隆到pET24a载体中,在大肠杆菌BL21中表达His-rV270融合蛋白：表达产物先后经Co^2＋亲和层析和Sephacryl S-200HR凝胶柱纯化,并在纯化过程中应用凝血酶切除His标塔;氢氧化铝佐剂吸附重组抗原后免疫BALB／c小鼠,初次免疫后第21天加强免疫1次,第5周使用104CFU鼠疫菌141强毒株攻毒,测定其免疫保护作用。结果：rV270以可溶性方式表达;应用Co^2＋亲和层析柱和Sephacryl S-200HR凝胶柱结合凝血蛋白酶切除His标签的方法可得到不含标签的较高纯度的重组蛋白;攻毒实验中实验组小鼠全部存活,而对照组全部死亡。结论：获得了具有良好免疫保护作用的rV270蛋白,可用于鼠疫亚单位疫苗的研究。     

生物可降解微球作为乙型肝炎基因免疫佐剂的研究

探讨生物可降解微球对基因免疫的增强作用。采用有机溶剂蒸发法制备聚乳酸聚乙醇酸共聚 物（ＰＬＧＡ）微球,构建含有乙型肝炎病毒表面抗原Ｓ基因的ｐＲＣ－ＣＭＶ真核表达载体,用微球与基因 载体共孵育法制备其混合物。肌肉注射免疫Ｂａｌｂ／ｃ小鼠。结果表明：微球注射组的血清抗体滴度达到 ｌ：１６００,其效果与乙型肝炎病毒表面抗原加铝佐剂注射组相近,而裸ＤＮＡ注射组没有反应。说明了 生物可降解微球可显著的提高基因免疫的免疫反应。     

B型肉毒毒素重组Hc亚单位疫苗的免疫原性研究

目的:用大肠杆菌表达重组B型肉毒毒素受体结合区Hc抗原(BHc)作为亚单位疫苗,并研究其免疫原性。方法:构建pTIG-Trx-BHc原核表达载体,用大肠杆菌表达并纯化重组BHc抗原;免疫BALB/c小鼠以研究它的免疫原性。结果:大肠杆菌表达并纯化获得了重组BHc抗原,免疫小鼠后能刺激机体产生高效价的抗BHc抗体和保护性免疫反应。该重组BHc亚单位疫苗1μg剂量2次免疫小鼠即可产生对104LD50剂量B型肉毒毒素攻毒的完全保护,血清中和效价可达1.33 IU/m L。结论:大肠杆菌表达的重组BHc抗原具有良好的免疫原性,能够有效地保护小鼠抵抗B型肉毒毒素的攻击,该BHc抗原能够作为亚单位候选疫苗预防B型肉毒毒素中毒。     

F1-V重组亚单位疫苗对鼠疫耶尔森氏菌141强毒株皮下攻毒保护性的研究

在此实验中, 设计了一种包含2种成分的重组融合蛋白作为疫苗成分来防护鼠疫耶尔森氏菌(Yersinia)可能产生的生物威胁. 重组F1-V蛋白与铝佐剂结合, 分别以10, 20, 50 mg剂量免疫BALB/C小鼠, 周期为2个月. 检测小鼠血清抗体水平和T辅助细胞的亚型. 免疫后小鼠以25~600 LD₅₀剂量的鼠疫耶尔森氏菌141强毒株进行皮下攻毒实验. 结果证明, F1-V重组蛋白在 小鼠体内诱导产生足够保护的免疫应答. 血清IgG水平是产生最终保护力的一个重要因素. 20 μg的免疫剂量可以诱导血清抗体效价高达51200, 使小鼠对400 LD50的鼠疫耶尔森氏菌产生100%的保护. F1-V重组蛋白引发的抗体亚型主要为IgG1类, 说明抗体反应趋向Th2型反应. 流式细胞分析表明, 铝佐剂主要帮助F1-V重组融合蛋白诱导强烈的体液免疫而不是CTL细胞免疫应答.表明F1-V重组亚单位疫苗株有希望成为一种新型的鼠疫疫苗候选株.     

用电穿孔方法研究 H5N1 禽流感病毒DNA 疫苗对动物的免疫保护作用

为了研究 H5N1 DNA 疫苗对小鼠和鸡的保护效率,用 H5N1 禽流感病毒 HA DNA 疫苗免疫 BALB/c 小鼠和 SPF 鸡 . 小鼠和鸡分别经电穿孔和肌肉注射免疫两次,间隔为 3 周 . 二次免疫后,用致死量的同源病毒进行攻毒实验 . 空白对照组在攻毒后全部死亡,而经电穿孔免疫的小鼠和鸡均获得了完全的保护,并能有效地抑制病毒在小鼠肺脏和鸡泄殖腔的繁殖 . 同时,电穿孔免疫的小鼠和鸡均产生了高水平的特异性抗体 . 经电穿孔免疫的小鼠攻毒后 CTL 反应明显加强 . 这些结果表明, HA DNA 疫苗能有效地保护小鼠和鸡对禽流感病毒的感染,同时也表明电穿孔免疫是 DNA 疫苗免疫的有效途径之一 .     

PELA微球乙型肝炎疫苗的免疫原性研究

将疫苗包裹在可生物降解的微球中则一种极有潜力的新型疫苗载体系统。用聚－ＤＬ－乳酸－聚乙二醇共聚物（ＰＥＬＡ０为材料,包裹乙型肝炎表面抗原（ＨＢｓＡｇ０,缓释微球疫苗,以皮下注射或口服的方式免疫ＢＡＬＢ／ｃ小鼠,研究其免疫原笥。同时以乙肝常规铝佐剂疫苗免疫两剂作为对照。结果表明,皮下注射单剂微球疫苗后,在第１４周,小鼠血清ＩｇＧ滴度可达到与铝佐剂疫苗组相当的水平,维持较高的滴度;此外,口服微球疫苗组     

3种市售免疫调节剂作为流感病毒融合蛋白NM2e亚单位疫苗佐剂的研究

目的:观察具有免疫调节作用的市售左旋咪唑、西米替丁、伐地那非作为甲型流感病毒核蛋白(NP)与基质蛋白2胞外区多肽(M2e)融合蛋白NM2e亚单位疫苗佐剂的可能性。方法:将左旋咪唑、西米替丁、伐地那非单独或者分别与Al(OH)3配伍作为NM2e蛋白佐剂,经肌肉注射免疫BALB/c小鼠,通过体液和细胞免疫检测及病毒攻击实验研究这些免疫调节剂作为疫苗佐剂的可能性。结果:左旋咪唑对NM2e蛋白亚单位疫苗无明显的佐剂效应,但与铝佐剂合用可使攻毒后存活小鼠体重恢复加快;西米替丁可以明显提高NM2e蛋白诱发的特异性IgG2a滴度,提高攻毒后小鼠的存活率(30%),但保护效果明显低于铝佐剂(70%),与铝佐剂无明显的协同作用;伐地那非能明显增加NM2e蛋白诱发的特异性IgG1、IgG2a滴度,提高攻毒后小鼠的存活率(36%),但与铝佐剂合用有相互抑制作用。结论:左旋咪唑、西米替丁、伐地那非对NM2e蛋白均有一定的免疫调节作用,其中西米替丁和伐地那非能提高攻毒保护效果,但保护效果明显低于Al(OH)3,与Al(OH)3联合使用均无协同作用。     

新城疫壳聚糖微球疫苗免疫效果的研究

鸡新城疫是由新城疫病毒引起的鸡的一种急性、烈性、高度接触性传染病,是危害养禽业的最严重疫病之一。控制新城疫最根本的措施是进行有效的疫苗接种,目前常用的疫苗是弱毒活疫苗和灭活疫苗,但二者在实际应用中均存在一定的局限性。口服微球疫苗可以诱导较强的粘膜免疫;同时还能够诱导产生系统的体液免疫和细胞免疫,已成为ND疫苗研究的热点。以壳聚糖为囊材,新城疫La Sota抗原液为芯材,戊二醛为交联剂,制备出新城疫壳聚糖微球疫苗,通过了实验室安全检验和效力检验。将新城疫壳聚糖微球疫苗与LaSota活疫苗和新城疫油乳剂灭活苗分别免疫SPF鸡,利用MTT、血凝抑制法(HI)和ELISA等分别检测不同疫苗免疫后的细胞免疫、体液免疫和粘膜免疫抗体IgA,并在当免疫鸡HI抗体降到23的情况下进行了攻毒试验。结果表明,新城疫壳聚糖微球疫苗安全性好,免疫后可刺激机体产生较强的细胞免疫、体液免疫和局粘膜免疫,具有较好的保护作用。     

初步探索以“动物法则”评价鼠疫组分疫苗的临床有效性

目的依据"动物法则",探索被动免疫小鼠对鼠疫强毒菌攻击的有效性,为间接评价鼠疫组分疫苗临床有效性提供依据。方法以小鼠为观察对象,依据小鼠耐受正常人血清剂量和小鼠体内异源动物血清代谢动力学研究,确定小鼠耐受正常人血清剂量和攻击时间,基于此对小鼠进行鼠疫组分疫苗免疫人血清被动免疫,观察小鼠抵抗鼠疫强毒菌攻击的存活率和存活时间,判定小鼠被动保护鼠疫强毒菌攻击的有效性。结果 BALB/c小鼠对正常人血清耐受量为1.0 m L,3~4 h间抗体滴度最高。小鼠抵抗鼠疫强毒菌攻击试验显示,在14 d的观察期内,经2MLD、6 MLD和10 MLD鼠疫强毒菌攻击的被动免疫小鼠,存活率分别为57%、25%和42%,平均存活时间依次为11.2 d、8.7 d和9.8 d;1 MLD攻击非免对照小鼠全部死亡,平均存活时间5.3 d。结论初步证实利用被动免疫小鼠可间接评价鼠疫组分疫苗临床有效性的可行性。     

斑点叉尾鲖源海豚链球菌Srr蛋白海藻酸钠-壳聚糖口服疫苗的制备及其免疫效果研究

制备海藻酸钠-壳聚糖-海豚链球菌Srr蛋白微球疫苗, 并检测其对斑点叉尾鲙的免疫效果。采用乳化法利用海藻酸钠-壳聚糖包被Srr蛋白, 测定其包封率、载药率及包被蛋白的抗原性; 通过拌饲投喂免疫斑点叉尾鲙, 分为Srr组、Srr微球组、空微球组以及对照组, 间接ELISA法检测免疫后斑点叉尾鲙的血清抗体水平, 试剂盒检测多项血清非特异性指标; 于免疫后第4周利用海豚链球菌攻毒, 计算各组相对保护率, 并通过实时荧光定量PCR检测相关基因的表达量。结果显示, 通过乳化法制得形态为圆形或椭圆形、大小较为均一的微球疫苗, 粒径为(4.26±1.13) μm, 包封率为92.38%, 载药率为19.41%, Western-blot分析表明Srr蛋白微球具有较好的抗原性; Srr微球组的抗体效价峰值出现在第4周, 明显高于其他组, 血清总蛋白、T-SOD以及溶菌酶活力均显著或极显著高于其他实验组, 并获得60%的相对保护率。荧光定量分析结果显示, Srr微球组攻毒后24h和48h各免疫基因表达量均有所上调。Srr蛋白微球疫苗能够提高斑点叉尾鲙抵抗海豚链球菌的能力, 对海豚链球菌起到了一定的预防作用。     

Histopathological observation of immunized rhesus macaques with plague vaccines after subcutaneous infection of Yersinia pestis

In our previous study, complete protection was observed in Chinese-origin rhesus macaques immunized with SV1 (20 μg F1 and 10 μg rV270) and SV2 (200 μg F1 and 100 μg rV270) subunit vaccines and with EV76 live attenuated vaccine against subcutaneous challenge with 6×10(6) CFU of Y. pestis. In the present study, we investigated whether the vaccines can effectively protect immunized animals from any pathologic changes using histological and immunohistochemical techniques. In addition, the glomerular basement membranes (GBMs) of the immunized animals and control animals were checked by electron microscopy. The results show no signs of histopathological lesions in the lungs, livers, kidneys, lymph nodes, spleens and hearts of the immunized animals at Day 14 after the challenge, whereas pathological alterations were seen in the corresponding tissues of the control animals. Giemsa staining, ultrastructural examination, and immunohistochemical staining revealed bacteria in some of the organs of the control animals, whereas no bacterium was observed among the immunized animals. Ultrastructural observation revealed that no glomerular immune deposits on the GBM. These observations suggest that the vaccines can effectively protect animals from any pathologic changes and eliminate Y. pestis from the immunized animals. The control animals died from multi-organ lesions specifically caused by the Y. pestis infection. We also found that subcutaneous infection of animals with Y. pestis results in bubonic plague, followed by pneumonic and septicemic plagues. The histopathologic features of plague in rhesus macaques closely resemble those of rodent and human plagues. Thus, Chinese-origin rhesus macaques serve as useful models in studying Y. pestis pathogenesis, host response and the efficacy of new medical countermeasures against plague.     

The experimental validation of the advantages of combined emergency (fluoroquinolones) and specific (EV Nalr) prevention of plague versus their sequential use]

Mice immunization with reference vaccine at the early stage of plague infection provided animals survival and prolonged mean survival period up to 2-5 days. Ciprofloxacin, ofloxacin and pefloxacin prevents development of post vaccine immunity at white mice, immunized by reference vaccine strain EV. Nalidixic acid and norfloxacin effect on post vaccine immunity was lower. Use of immunogenic strain EV Nafr (resistant to nalidixic acid and fluoroquinolones) provided antiplague immunity formation at the background of fluoroquinolones prophylaxis. Ciprofloxacin, ofloxacin and pefloxacin used for plague prophylaxis at white mice infected with Yersinia pestis (about 1000 LD50) inhibited postinfective immunity development. Nalidixic acid and norfloxacin didn't demonstrate such effect. Urgent (fluoroquinolones) and specific (EV Nalr) combined prophylaxis was evaluated as more effective for a 5-day period and provided the development of antiplague immunity.     

Experimental study on the possibility of using live tularemia vaccine to increase resistance to heterologous infection disease

In experiments on guinea pigs immunized with Francisella tularensis 15, or live tularemia vaccine (LTV), the level of heterologous protective effect to dangerous infectious diseases caused by Yersinia pestis, Burkholderia pseudomallei, B. mallei, Mycobacterium tuberculosis was studied. The study revealed that during the first 4 weeks after the subcutaneous immunization with LTV the level of resistance of the immunized animals to heterologous infective agent reliably increased as indicated by the survival rate of the animals, as well as by the survival time of those killed by infection, in comparison with the controls. Later (on day 150 after immunization) differences in death rate between the groups perceptibly decreased. Nevertheless, the 1 1/2-fold increase of the survival time of the challenged immunized animals in comparison with the controls proved the possibility of using immunization with LTV for the urgent prophylaxis and treatment not only of tularemia, but also of plague, glanders, melioidosis and tuberculosis.     

Assessment of the effectiveness of different methoods of immunization with live plague vaccine EB in aerosol infections]

The work deals with the results of the comparative evaluation of the effectiveness of vaccines developed at the Sanitary Research Institute (Zagorsk) and the Mechnikov Research Institute for Vaccines and Sera (Moscow), as well as two methods of immunization against plague, by inhalation and subcutaneous injection, under the conditions of aerosol infection. The immunogenic effectiveness of both vaccines, when evaluated in terms of LD50, was shown to be approximately the same, but the animals immunized by the inhalation method with the vaccine developed at the Sanitary Research Institute proved to be less susceptible to infection than those immunized with the vaccine developed at the Mechnikov Research Institute for Vaccines and Sera in Moscow. After immunization by the inhalation method the vaccine developed at the Sanitary Research Institute rendered more effective protection (3- to 4-fold) against aerosol infection than after immunization by subcutaneous injection. The animals immunized by the inhalation method proved to be capable of surviving plague in the primary pneumonic form.     

鼠疫溶菌疫苗免疫小鼠的体液免疫应答

为选择以F1抗原为主要有效成分的鼠疫溶菌疫苗(Whole cell lysate of Yersinia pestis vaccine,WCLY)的免疫程序,设计了这组试验。在37℃培养鼠疫EV菌,通过超声波裂解法制备鼠疫溶菌疫苗。设计(0,2周)、(0,4周)、(0,2,4周)三种免疫程序,以每剂总蛋白量7.9μg、31.5μg和126.0μg三个剂量皮下接种NIH小鼠。分别在第一针免疫后2、4、8、12周采集血清,通过间接ELISA检测抗鼠疫菌F1抗原和总抗原抗体。结果显示:免疫后血清抗体上升很快,2周内即可测出;无论哪种免疫程序,至12周时抗体滴度仍保持高水平;加强免疫后,抗体水平在4周或8周达到较高,可与活疫苗免疫者相比;溶菌疫苗的接种剂量为7.9μg时,动物只出现轻度不良反应。提示鼠疫溶菌疫苗需要两剂免疫,最短可间隔2周,接种剂量应不超过7.9μg,疫苗中应富含F1抗原。     

Developing subunit immunogens using B and T cell epitopes and their constructs derived from the F1 antigen of Yersinia pestis using novel delivery vehicles

Yersinia pestis is the etiological agent of pneumonic and bubonic plague. As the currently licensed vaccines for plague have their own limitations, there is a need for a rational and more effective form of a subunit vaccine to combat both forms of the disease. Newer methods of antigen delivery coupled with adjuvant offer an alternative approach toward a plague vaccine. In order to develop a new generation vaccine against plague, we chose an immunodominant, outer membrane capsular protein, F1 of Y. pestis. The immunogenicity of the peptide sequences, predicted to possess B (three sequences, B1, B2 and B3) and T (two sequences, T1 and T2) cell determinants, was studied in a murine model with different genetic backgrounds, using alhydrogel and liposomes as delivery vehicles. All the peptide sequences are immunogenic in all mouse strains and showed primary and secondary immune response. B2 peptide was found to be most immunogenic, followed by B1 and B3 peptides. Chimeras made between B and T structures proved highly immunogenic and the antibody levels are comparable with native F1 antigen, thereby proving that T1 and T2 are helper sequences. Interestingly, the liposome mode of immunization was found to be more immunogenic and generated higher affinity antibodies than the alum-based preparation. Immunization using a mixture of all the peptides further proved B2 to be immunodominant. The IgG isotype profile showed predominance of IgG1, IgG2b followed by IgG2a for all the formulations irrespective of mode of antigen delivery. Lymphocyte proliferation of spleen cells primed in vivo with peptides, B-T conjugates and F1 antigen followed by in vitro stimulation with these antigens in soluble (medium) and particulate (liposome) form, showed dose-dependent stimulation of T cells, while B-T constructs showed a higher stimulation index, comparable to F1 antigen. The liposome mode of antigen presentation showed higher lymphoproliferation of spleen cells. Of all the peptides tested, T1 and T2 sequences showed the highest stimulation indices. The pattern of cytokine levels was in the following order: interferon-gamma>interleukin-2>interleukin-4. In vivo protective studies of the B-T conjugates revealed that B1T1 and a mixture of conjugates showed a survival rate of 10 days. Thus, the study highlights the importance of B and T cell epitopes as peptide-based immunogens, being a serious alternative for plague vaccine.     

Oral vaccination with salmonella simultaneously expressing Yersinia pestis F1 and V antigens protects against bubonic and pneumonic plague

The gut provides a large area for immunization enabling the development of mucosal and systemic Ab responses. To test whether the protective Ags to Yersinia pestis can be orally delivered, the Y. pestis caf1 operon, encoding the F1-Ag and virulence Ag (V-Ag) were cloned into attenuated Salmonella vaccine vectors. F1-Ag expression was controlled under a promoter from the caf1 operon; two different promoters (P), PtetA in pV3, PphoP in pV4, as well as a chimera of the two in pV55 were tested. F1-Ag was amply expressed; the chimera in the pV55 showed the best V-Ag expression. Oral immunization with Salmonella-F1 elicited elevated secretory (S)-IgA and serum IgG titers, and Salmonella-V-Ag(pV55) elicited much greater S-IgA and serum IgG Ab titers than Salmonella-V-Ag(pV3) or Salmonella-V-Ag(pV4). Hence, a new Salmonella vaccine, Salmonella-(F1+V)Ags, made with a single plasmid containing the caf1 operon and the chimeric promoter for V-Ag allowed the simultaneous expression of F1 capsule and V-Ag. Salmonella-(F1+V)Ags elicited elevated Ab titers similar to their monotypic derivatives. For bubonic plague, mice dosed with Salmonella-(F1+V)Ags and Salmonella-F1-Ag showed similar efficacy (>83% survival) against approximately 1000 LD(50) Y. pestis. For pneumonic plague, immunized mice required immunity to both F1- and V-Ags because the mice vaccinated with Salmonella-(F1+V)Ags protected against 100 LD(50) Y. pestis. These results show that a single Salmonella vaccine can deliver both F1- and V-Ags to effect both systemic and mucosal immune protection against Y. pestis.     

Prophylactic use of ceftriaxone in combination with F I antigen immunization in studies on uninbred albino mice infected by Yersinia pestis. Antiplague immunity development]

Administration of highly immunogenic (ED50 12.6 mcg/mouse) F I antigen (100 mcg/mouse) to albino mice 5 hours after their contamination approximately with 1000 LD50 of Yersinia pestis 231 provided 99-percent survival of same animals (17-50%) and 2-5-day prolongation of the life-span, that was indicative of the phenomenon analogous to the survival phenomenon observed in infected animals immunized by immunogenic strains of the plague microbe. The experiment on the mice confirmed high efficacy of ceftriaxone (100-percent survival) when used prophylactically for 5 days 5 hours after the contamination by Y. pestis 231 (approximately 1000 LD50) in the dose equivalent to the daily dose for humans. However, no antiplague immunity developed in the survivors: the immunity index (II) of 1.5x10. The use of ceftriaxone according to the same scheme simultaneously with single immunization by F I antigen in a dose of 100 mcg/mouse resulted not only in 100-percent survival of the animals but also in development of expressing antiplague immunity (II 2.2x10(5)). The protection level corresponded to the control with the same live-stock of the animals after a single immunization in the analogous dose of F I antigen (II 3.2x10(4)) and the ceftriaxone use (II 1.0x10(5)), as well as after immunization of the mice by 10(6) microbial cells of Y. pestis EV NIIEG (II 1.2x10(5)). The results of the study are indicative of the prospective use of subsingle vaccines of the new generation based on F I antigen for combined specific and urgent prophylaxis.     

Gold nanoparticle-adjuvanted S protein induces a strong antigen-specific IgG response against severe acute respiratory syndrome-related coronavirus infection,but fails to induce protective antibodies and limit eosinophilic infiltration in lungs

The spike (S) protein of coronavirus, which binds to cellular receptors and mediates membrane fusion for cell entry, is a candidate vaccine target for blocking coronavirus infection. However, some animal studies have suggested that inadequate immunization against severe acute respiratory syndrome coronavirus (SARS-CoV) induces a lung eosinophilic immunopathology upon infection. The present study evaluated two kinds of vaccine adjuvants for use with recombinant S protein: gold nanoparticles (AuNPs), which are expected to function as both an antigen carrier and an adjuvant in immunization; and Toll-like receptor (TLR) agonists, which have previously been shown to be an effective adjuvant in an ultraviolet-inactivated SARS-CoV vaccine. All the mice immunized with more than 0.5 µg S protein without adjuvant escaped from SARS after infection with mouse-adapted SARS-CoV; however, eosinophilic infiltrations were observed in the lungs of almost all the immunized mice. The AuNP-adjuvanted protein induced a strong IgG response but failed to improve vaccine efficacy or to reduce eosinophilic infiltration because of highly allergic inflammatory responses. Whereas similar virus titers were observed in the control animals and the animals immunized with S protein with or without AuNPs, Type 1 interferon and pro-inflammatory responses were moderate in the mice treated with S protein with and without AuNPs. On the other hand, the TLR agonist-adjuvanted vaccine induced highly protective antibodies without eosinophilic infiltrations, as well as Th1/17 cytokine responses. The findings of this study will support the development of vaccines against severe pneumonia-associated coronaviruses.