HIV-1CN54 DNA疫苗滴鼻免疫小鼠诱发系统和黏膜免疫应答

目的：探讨重组痘苗病毒rVVsyngp120或rVVmCN54gp120候选疫苗是否增强HIV-1CN54合成gp120基因(syngp120)DNA疫苗的免疫原性。方法：第0、7、14、21天用DNA疫苗滴鼻免疫小鼠,第28、35、42天再滴鼻接种rVVsyngp120或rVVmCN54gp120。体外测脾和肠系膜淋巴结(MLN)淋巴细胞增殖应答与CD8^ CTL应答。测血清和黏膜洗液特异的IgG和IgA,并测其是否中和实验室适应株HIV-1SF33。结果：单纯DNA免疫后,脾和MLN淋巴细胞在体外发生增殖应答和CTL应答,且测出血清特异的IgG和黏膜洗液特异的IgA。重组痘苗病毒末次免疫后第2周(第56天),发现rVVmCN54gp120增强MLN淋巴细胞增殖应答和CTL应答,脾CTL应答也增强。rVVsyngp120则增强MLN CTL应答。同时发现：2组重组痘苗病毒免疫的动物其血清中特异IgG抗体滴度均有所增高,但黏膜(粪便和阴道)洗液特异IgA抗体滴度却未增高,未测出血清特异IgA和黏膜洗液特异IgG。免疫血清可中和HIV-1SF33,而阴道洗液却不能。结论：单纯DNA疫苗滴鼻免疫可诱发较弱的系统和黏膜体液免疫与细胞免疫,但维持时间短。重组痘苗病毒主要增强局部黏膜的细胞免疫应答,且稍增强系统体液免疫应答,未增强黏膜的IgA应答。免疫血清有中和作用。     

小鼠对HIV-2 gp105核酸疫苗免疫应答的研究

目的: 探讨HIV- 2gp105基因核酸疫苗在小鼠体内的免疫应答, 为开发HIV- 2核酸疫苗提供实验依据。方法:将HIV- 2外膜蛋白 (gp105 )基因插入真核表达质粒载体pVAX1中, 构建pVAX1 gp105重组表达质粒。将其肌注免疫BALB/c小鼠, 用ELISA法检测小鼠血清抗HIV -2抗体, 用流式细胞仪测定CD4 、CD8 T细胞亚群数, 以乳酸脱氢霉释放法检测脾特异性CTL的杀伤活性。结果: 重组质粒pVAX1 -gp105免疫组小鼠的血清抗体滴度、脾T细胞亚群的数量及特异性CTL的杀伤活性, 均明显高于对照组, 分别为P<0. 01, P<0. 05和P<0. 01。结论: HIV -2gp105核酸疫苗能诱导小鼠产生特异性细胞和体液免疫。     

表达HIV-1 gag-gp120嵌合基因的DNA疫苗在小鼠体内的免疫应答

目的 :检测表达HIV 1gag gp12 0嵌合基因的DNA疫苗在小鼠体内的免疫应答效果。方法 :将真核表达质粒pVAXGE肌肉注射BALB C小鼠 ,观察免疫小鼠脾T淋巴细胞亚群的数量、特异性CTL杀伤率及小鼠免疫后不同时间点血清中IgG抗体滴度。结果 :重组质粒pVAXGE免疫组小鼠脾淋巴细胞进行了增殖 ,脾特异性CTL杀伤率显著高于对照组 (P <0 0 1) ;小鼠免疫后于第 8周血清抗体达到最高。结论 :表达HIV 1gag gp12 0嵌合基因的DNA疫苗质粒可诱导BALB C小鼠发生免疫应答     

改善HIV—1DNA疫苗免疫原性的方法

1　 HIV-1 DNA疫苗的特点自 1 981年首次发现艾滋病以来 ,艾滋病病毒 ( HIV)的感染在全世界迅速广泛流行。到2 0 0 0年底 ,我国在 HIV感染人数已达 60万。由于药物的昂贵和耐药株的产生影响艾滋病的治疗。科学家认为预防 HIV传播的最佳措施是研制和应用安全而有效的预防性疫苗 ,而且认为研制有效的 HIV疫苗是可行的。目前科研人员研究了多种类型疫苗 ,其中最引人关注的是基因疫苗。HIV-DNA疫苗之所以受到科学家的关注 ,是因为 :基因疫苗与传统疫苗、基因工程疫苗相比 ,有以下优点 :1直接接种 DNA疫苗 ,生产成本低。 2接种基因疫…     

IL-12的真核表达载体促进小鼠对DNA疫苗的免疫应答

慢性乙型肝炎及其病毒携带者细胞免疫功能明显低下。ＤＮＡ疫苗可被体细胞摄取并表达相应抗原 ,激发出保护性免疫应答 ,包括特异性ＣＴＬ应答及特异性抗体的产生〔1〕。ＩＬ 12是目前发现对体内免疫活性细胞诱导和调节作用最强、范围最广的细胞因子。我们将ＨＢＶＤＮＡ疫苗 (ｐＣＲ3.1 Ｓ)与编码鼠ＩＬ 12的真核表达载体 (ｐＷＲＧ316 9,简称ＩＬ 12 )共同免疫小鼠后 ,观察其对小鼠免疫应答的影响。材料与方法材料 :5～ 8周龄雌性ＢＡＬＢ/ｃ小鼠购自本校实验动物中心 ,体重 15～ 2 0ｇ ;Ｐ815小鼠肥大细胞瘤细胞系 ,编码ＨＢＶＳ抗原的真…     

DNA疫苗的免疫机制及其优化策略

DNA疫苗的研究成为疫苗研究领域的热点,被誉为疫苗研究的第三次革命,开创了人类与疾病作斗争的疫苗研究新篇章。DNA疫苗既可以诱导细胞免疫又可以诱导体液免疫应答,成为最有希望的新型疫苗之一,但其免疫机制还不是十分清楚,免疫原性相对较弱,限制了DNA疫苗的广泛应用。     

HCV核心-包膜E2抗原融合基因DNA疫苗的构建及对小鼠的免疫应答试验

目的：观察一种结构新颖的HCV融合抗原DNA疫苗在BALB/c小鼠的免疫效果，探讨其用于防治丙型肝炎的可行性。方法：用重叠延伸PCR拼接编码小鼠IgG kappa链信号肽和通用型辅助性T细胞表位PADRE的DNA片段，PCR分别扩增HCV核心抗原基因和包膜E2抗原基因，将3段基因插入真核表达载体pcDNA3.1，构成重组表达质粒pST-CE2t，转染COS7细胞，免疫组化检测HCV抗原的表达。将pST-CE2t和HCV核心抗DNA疫苗pcDNA3.1core分别肌肉注射接种BALB/c小鼠，检测小鼠的血清抗体、T细胞增殖和CTL反应。结果：pST-CE2t可在COS7细胞内表达HCV核心抗原和E2抗原，接种于BALB/c小鼠能有效诱导体液和细胞免疫应答，其中抗HCV核心抗原免疫应答的强度明显超过pcDNA3.1core，且更趋向于TH1型免疫应答。结论：pST-CE2t对于丙型肝炎的防治有潜在的应用价值。     

HIV-1CN54合成gag DNA疫苗鼻内免疫BALB/c小鼠诱发的免疫应答

目前 8 0 %的ＨＩＶ感染是通过粘膜表面 (主要是肠道和生殖道 )发生的。粘膜免疫保护粘膜表面不感染ＨＩＶ ,除抗体的作用之外 ,ＣＤ8+ＣＴＬ细胞应答也发挥重要作用。ＨＩＶ 1ＣＮ54 是Ｂ′ Ｃ重组毒株 ,中国与欧盟合作以其为代表毒株研制疫苗 ,以期预防和控制艾滋病。我们以前的研究表明用ＨＩＶ 1ＣＮ54 合成ｇａｇ(ｓｙｎｇａｇ)的ＤＮＡ疫苗进行肌肉注射免疫小鼠诱发比较强的体液和细胞免疫应答 ,本研究则采用鼻内接种ＤＮＡ疫苗的方法 ,观察是否诱发系统和粘膜的免疫应答。取雌性ＢＡＬＢ ｃ(6～ 8周 ,重 18～2 0ｇ) ,每笼 4只 ,任意…     

B7-2表达质粒对HBV DNA疫苗诱导的特异性免疫应答的影响

目的：探讨B7－2分子是否能够增强乙型肝炎病毒（HBV）DNA疫苗诱导的特异性免疫应答。方法：将B7－2表达质粒与HBV DNA疫苗共接种于小鼠腓肠肌内，检测细胞毒性T淋巴细胞（CTL）活性，迟发性超敏反应（DTH）及抗－HBs滴度。结果：B7－2表达质粒与HBV DNA疫苗共接种组的DTH反应和CTL活性，明显强于单独接种HBV DNA疫苗组（P＜0．01）。两组的抗－HBs滴度差异无显著性（P＞0．05）。结论：B7－2表达质粒与HBV DNA疫苗共接种可显著增强抗－HBV特异性细胞免疫应答（CMI）。     

HIV-1C亚型nef基因构建的DNA质粒诱导小鼠免疫反应的研究

目的构建表达中国流行株HIV-1C亚型调控nef基因的重组质粒pVAX-nef，并免疫BALB／c小鼠，观察其免疫效果，为探索新型HIV DNA疫苗提供数据。方法利用分子生物学技术，将nef基因克隆到pVAX，并在体外进行表达与鉴定。以纯化的Nef蛋白作为包被抗原，用ELISA检测其体液免疫反应，用ELISPOT检测其细胞免疫反应。结果重组质粒pVAX-nef成功构建。接种小鼠后2周，ELISPOT结果显示产生了针对HIV特异的CD4和CD8细胞抗原表位的免疫应答，且与免疫剂量存在一定的正相关性。ELISA实验诱导产生了抗HIV-1特异性抗体，其中40μg免疫组诱导的抗体水平最高。结论重组质粒pVAX-nef免疫小鼠后可有效地诱导机体产生细胞免疫和体液免疫反应。     

柯萨奇B3病毒结构和非结构蛋白DNA 疫苗诱导小鼠产生免疫应答的研究

目的 制备4种柯萨奇B3病毒（CVB3）结构和非结构蛋白重组质粒DNA疫苗,并探讨其诱导机体产生体液和细胞免疫应答的效果。方法 用基因重组技术构建4种CVB3结构和非结构蛋白重组质粒,将各重组质粒体外转染真核细胞,用Western blot检测表达产物;于BALB／c小鼠后腿胫骨前肌注射免疫,于0、4、8周共免疫3次,100μg/次。免疫后不同时间检测体液和细胞免疫应答指标。结果 4种重组质粒酶切出相应大小的片段,经测序证实为CVB3序列,Western blot证实能够在体外真核细胞中表达。pcDNA3/vp2、pcDNA3/VP1、pcDNA3/2A和pcDNA3/3D均可诱导小鼠产生相应的特异性抗体、细胞毒性T淋巴细胞（CTL）和淋巴细胞增殖反应、迟发型超敏反应（DTH）,并对致死量的CVB3m、CVB5和CVB2攻击具有保护作用,表现为病毒攻击后第3天血中病毒滴度降低,第10天心肌病理变化比对照组明显减轻,且小鼠生存率显著提高。其中以pcDNA/VP1和pcDNA3／3D组保护作用最明显。结论 CVB3结构蛋白VP1和非结构蛋白3D质粒DNA有可能用作CVB DNA疫苗的候选基因,值得进一步深入研究。     

微小隐孢子虫子孢子表面蛋白质粒DNA接种诱导Balb/c小鼠的免疫应答

目的　探讨微小隐孢子虫子孢子表面蛋白CP2 3重组质粒pCR3.1～ 2 3DNA疫苗诱导机体产生体液和细胞免疫应答的效果。方法　用重组的DNA疫苗于Balb/c小鼠后腿胫骨前肌注射免疫 ,于 0、3、6周共免疫 3次 ,10 0 μg/次。免疫后不同时间检测体液和细胞免疫应答指标。并用 1× 10 6卵囊进行攻虫试验。结果　pCR3.1～ 2 3可诱导机体产生相应的特异性抗体 ,对C .parvum卵囊攻击具有保护作用。结论　微小隐孢子虫子孢子表面蛋白CP2 3重组质粒pCR3.1～ 2 3有可能作为侯选的隐孢子虫DNA疫苗 ,值得进一步深入研究。     

人乳头瘤病毒18型L1-E6、E7嵌合基因DNA疫苗的体内免疫效应

目的：检测HPV18 L1-E6、E7嵌合基因DNA疫苗在小鼠体内的体液和细胞免疫效应。方法：将实验动物BALB/c小鼠54只随机分为9组，按不同免疫方式(肌肉接种或鼻内滴注)分别给予不同的重组质粒(pVAX1-L1-E6M3或pVAX1-L1-E7M3)和免疫佐剂(pLXHDmB7-2或LTB)。用免疫原免疫3次，末次免疫后取眼球后血进行ELISA抗体检测。末次免疫后进行小鼠足垫迟发型超敏反应(DTH)试验。断足进行小鼠足垫HE染色。取小鼠脾脏制成单细胞悬液，进行脾细胞增殖试验，并进行CD4^ /CD8^ T细胞中IFN-γ^ 或IL-4^ 细胞的FACS分析。结果：与对照组相比，各实验组均获得明显的免疫效果。实验组免疫后血清抗体4值均高于相应组别免疫前；肌肉注射组每次免疫后抗体水平较前次明显升高。实验组小鼠注射VLP抗原的左后足垫局部有红肿硬结，镜下观察可见大量单核细胞侵润。肌肉接种组的DTH反应强度、脾细胞增碹刺激指数(SI)和CD8^ IFN-γ^ 细胞数均高于鼻内滴注组；而鼻内滴注组CD4^ IL-4^ 细胞数高于单纯质粒肌肉接种组；加入pLXHDmB7-2的联合免疫组各项指标均高于单纯质粒组。结论-证实了重组pVAX1-HPV18L1/E6、E7嵌和基因DNA疫苗能诱导小鼠的体液免疫和细胞免疫效应：同时，证实B7-2分子能显著提高该质粒在小鼠体内的免疫反应效果.     

A single subcutaneous or intranasal immunization with adenovirus-based SARS-CoV-2 vaccine induces robust humoral and cellular immune responses in mice

Optimal vaccines are needed for sustained suppression of SARS-CoV-2 and other novel coronaviruses. Here, we developed a recombinant type 5 adenovirus vector encoding the gene for the SARS-CoV-2 S1 subunit antigen (Ad5.SARS-CoV-2-S1) for COVID-19 immunization and evaluated its immunogenicity in mice. A single immunization with Ad5.SARS-CoV-2-S1 via S.C. injection or I.N delivery induced robust antibody and cellular immune responses. Vaccination elicited significant S1-specific IgG, IgG1, and IgG2a endpoint titers as early as 2 weeks, and the induced antibodies were long lasting. I.N. and S.C. administration of Ad5.SARS-CoV-2-S1 produced S1-specific GC B cells in cervical and axillary LNs, respectively. Moreover, I.N. and S.C. immunization evoked significantly greater antigen-specific T-cell responses compared to unimmunized control groups with indications that S.C. injection was more effective than I.N. delivery in eliciting cellular immune responses. Mice vaccinated by either route demonstrated significantly increased virus-specific neutralization antibodies on weeks 8 and 12 compared to control groups, as well as BM antibody forming cells (AFC), indicative of long-term immunity. Thus, this Ad5-vectored SARS-CoV-2 vaccine candidate showed promising immunogenicity following delivery to mice by S.C. and I.N. routes of administration, supporting the further development of Ad-based vaccines against COVID-19 and other infectious diseases for sustainable global immunization programs.     

Cellular and humoral immune responses against cancer: implications for cancer vaccines.

The key issue in tumor immunology is to identify antigens as target structures for a cancer-selective immunological attack in the tumor-bearing host, resulting in tumor rejection. There is a growing detailed understanding of structural and regulatory gene alterations giving rise to candidate rejection antigens and peptides in tumor cells. As well as reviewing the development of new adjuvant and recombinant vector systems, new approaches are suggested for the construction of cancer vaccines.     

Structural characteristics correlate with immune responses induced by HIV envelope glycoprotein vaccines

HIV envelope glycoprotein (Env) is the target for inducing neutralizing antibodies. Env is present on the virus surface as a trimer, and, upon binding to CD4, a cascade of events leads to structural rearrangement exposing the co-receptor binding site and entry into the CD4+ host target cells. We have designed monomeric and trimeric Env constructs with and without deletion of the variable loop 2 (ΔV2) from SF162, a subtype B primary isolate, and performed biophysical, biochemical and immunological studies to establish a potential structure-functional relationship. We expressed these Envs in CHO cells, purified the proteins to homogeneity and performed biophysical studies to define the binding properties to CD4, structural characteristics and exposure of epitopes recognized by b12 and CD4i mAb (17B) on both full-length and mutant HIV Env proteins. Parameters evaluated include oligomerization state, number and affinity of CD4 binding sites, enthalpy and entropy of the Env-CD4 interaction and affinity for b12 and 17b mAbs. We observed one CD4 binding site per monomer and three active CD4 binding sites per trimer. A 40-fold difference in affinity of the gp120 monomer vs. the o-gp140 trimer towards CD4 was observed (Kd = 58 nM and 1.5 nM, respectively), whereas only a 2-fold difference was observed for the V2 deleted Envs (Kd of gp120ΔV2 = 19 nM, Kd of o-gp140DV2 = 9.3 nM). Monomers had 3-fold higher affinity to the mAb 17b and at least 3-fold weaker affinity to b12 compared to trimers, with gp120DV2 having the weakest affinity for b12 (Kd = 446 nM). Affinity of CD4 binding correlated with proportion of the antibodies induced against the conformational epitopes by the corresponding Envs, and changes in mAb binding correlated with the induction of antibodies directed against linear epitopes. Furthermore, biophysical analysis reveals that the V2 deletion has broad structural implications in the monomer not shared by the trimer, and these changes are reflected in the quality of the immune responses induced in rabbits. These data suggest that biophysical characteristics of HIV Env, such as affinity for CD4, and exposure of important neutralizing epitopes, such as those recognized by b12 mAb, may be important predictors of its in vivo efficacy and may serve as important surrogate markers for screening Env structures as potential vaccine candidates.     

Durability of humoral and cell-mediated immune response after SARS-CoV-2 mRNA vaccine administration

Vaccination against the SARS-Cov-2 virus is an effective way to protect against the disease and the severe course of COVID-19. Forty-nine fully vaccinated with mRNA vaccines (BNT162b2 or mRNA-1273) SARS-CoV-2 infection-naïve volunteers aged 33–89 were enrolled in the study. Evaluation of the cellular and humoral immune response was performed within 1 to 3 months (T1) and 6–9 months (T2) after the second injection, and within 2–3 months (T3) after a booster dose. Additionally, a comparative analysis of the specific immune status was made between two age groups—below 60 (n = 22) and over 60 (n = 27) years. SARS-CoV-2-specific T-cell response was evaluated by IFN-γ-producing spot forming cells (SFCs) using a standardized ELISPOT assay. Virus neutralizing antibodies (VNA) against SARS-CoV-2 were measured by a blocking ELISA test and spike protein specific IgG (S-IgG) and IgA (S-IgA) antibodies—by semiquantitative ELISA. IFN-γ-producing SFCs, S-IgG, S-IgA and VNA significantly decreased 6–9 months after the second dose. After the third injection S-IgG and S-IgA markedly increased compared to T2 and reached the levels at T1. Of note, the highest values of VNA were observed at T3. No differences in the tested immune parameters were found between the two age groups. Data obtained showed that for a long period—6–9 months after a full course of immunization with mRNA vaccine, immune reactivity is present, but both cellular and humoral immune responses gradually decrease. The administration of a third dose mainly restores the specific humoral immune response against the SARS-CoV-2 virus.     

沙眼衣原体主要外膜蛋白核酸疫苗诱导小鼠产生特异性细胞和体液免疫应答

目的构建沙眼衣原体主要外膜蛋白核酸疫苗,并观察其诱导小鼠产生的体液免疫和细胞免疫。方法将核酸疫苗(pcDNA3.1MOMP)或对照空质粒(pcDNA3.1)注射于4～6周龄小鼠后腿股四头肌,每次剂量为100mg。间隔2周加强免疫2次。末次免疫后,ELISA法测定脾淋巴细胞培养上清液中IFNγ及小鼠血清中抗MOMP水平;MTT法测定脾淋巴细胞特异性增殖反应。结果小鼠接种核酸疫苗后,能产生特异性抗体,第3次免疫后抗体最高滴度达1∶1024,培养上清液中IFNγ达(532.0±45.4)pg/mL;实验组小鼠脾淋巴细胞刺激指数为3.94±0.25,其抗原特异性反应明显高于对照组。结论沙眼衣原体主要外膜蛋白核酸疫苗能刺激机体产生特异的细胞免疫和体液免疫。