阿尔茨海默病核酸疫苗的构建及诱导体液免疫反应的初步研究

近年,内源性蛋白主动免疫预防和治疗阿尔茨海默病(AD)的策略成为AD研究领域的热点,并获得迅猛发展。构建了以β-片层结构的淀粉样蛋白(AB)及其可溶性与病理性突变体为抗原基因的DNA疫苗用于免疫小鼠,经过对免疫方案的探索和调整,结果能够打破其自身耐受,在外周血中诱发出较高滴度的抗-AB抗体。初步探讨了诱导体液免疫学效应的规律。并在原代培养的海马神经元AB毒性模型中验证了免疫后血清的生物学活性。     

重组CHO细胞乙肝表面抗原诱导小鼠细胞免疫应答的研究

为了研究重组CHO细胞乙肝表面抗原(CHO-rHBsAg)在小鼠中诱导T细胞免疫应答的能力,全面评价疫苗的免疫原性,以CHO-rHBsAg免疫BALB/c小鼠,常规制备小鼠脾脏淋巴细胞并在体外以抗原或特异多肽刺激;采用ELISA法测定抗原特异性T淋巴细胞分泌的细胞因子,乳酸脱氢酶法(LDH)测定抗原特异性细胞毒T淋巴细胞(CTL)活性,酶联斑点法(ELISPOT)测定CTL频数(CTLp),应用流式细胞仪分析T淋巴细胞亚群。结果显示,rHBsAg可在小鼠中诱导Th1及Th2类细胞因子;加铝佐剂的rHBsAg较未加佐剂的抗原可诱导较高水平的IFN-γ、CTL克隆及较高百分比的CD8+T淋巴细胞亚群。重组CHO细胞来源的HBsAg可在BALB/c小鼠中诱导一定程度的细胞免疫应答。     

Leishmania major: immune response in BALB/c mice immunized with stress-inducible protein 1 encapsulated in liposomes

Protection against leishmaniasis is depending upon generation of a Th1 type of immune response. Field trials of first generation Leishmania vaccine showed a limited efficacy even with multiple doses mainly due to lack of an appropriate adjuvant. In this study, susceptible BALB/c mice were immunized with rLmSTI1 encapsulated in liposomes to explore the extent of protection induced by Leishmania antigen encapsulated in the liposomes against challenge with Leishmania major. The results showed that s.c. immunization of BALB/c mice with liposomal rLmSTI1 induced a significant protection against challenge and a significant lower parasite burden in spleen up to 14 weeks after challenge. The protected animals showed a significantly smaller footpad thickness after challenge, and a higher level of anti-SLA IgG antibodies before and after challenge with a predominant IgG2a titer. The data supports the possibility of using liposomal Leishmania antigens as a vaccine.     

Effects of a commercial malathion dip preparation on the cellular and humoral immune response of BALB/c mice

Because of the widespread use of malathion as a treatment for ectoparasitism, a study was undertaken to determine the effects of a malathion dip preparation on the BALB/c mouse immune system. Mice were treated with either 2% (recommended dosage) or 8% solutions of malathion or a water control. The cellular immune response was evaluated by in vitro exposure of lymphocytes to mitogens, and the humoral immune response was assessed by using an enzyme-linked immunosorbent assay (ELISA) to quantify antibody production against sheep red blood cells (SRBC). Responses to the mitogens and to the SRBC were not significantly different between 2% and 8% malathion treated and water treated mice. Results indicated that malathion did not affect these two aspects of the mouse immune system when used as a 2% or 8% dipping solution.     

Immunity induced by Staphylococcus aureus surface protein A was protective against lethal challenge of Staphylococcus aureus in BALB/c mice

Staphylococcus aureus is the most common cause of hospital-acquired bacteremia. Due to emergence of antibiotic-resistant strains, these infections present a serious public health threat. In this study, to develop a broadly protective vaccine, we tested whether immune responses induced by several proteins associated with S. aureus toxicity could protect mice from lethal challenge with human clinical S. aureus isolate USA300. We found that the surface protein A (SasA) of S. aureus could protect mice from lethal challenge of the bacteria.     

Interleukin-4 production in BALB/c mice immunized with Anisakis simplex.

We investigated the interleukin (IL-4) levels in BALB/c mice immunized with Anisakis extract in single or multiple doses and in mice orally infected with a larva. From animals immunized maximum responses were obtained with the multiple doses with an only IL-4 peak. Conversely, in the mice inoculated with a larva per os, the IL-4 levels showed two peaks of different rates.     

香菇多糖对急性弓形虫感染小鼠Th应答调节作用的实验研究

目的 探讨香菇多糖(Lentinan,Lent)对急性弓形虫感染小鼠BALB/c Th1/Th2免疫应答调节效应的影响.方法 对RH强毒株感染的BALB/c小鼠进行不同时间点的香菇多糖预处理,动态观察用药后各组感染小鼠的生存率;在感染后第0、3、5、8和10天提取小鼠的脾细胞,ELISA法检测脾细胞培养上清中IFN-γ和IL-10的分泌水平.结果 感染前6 d 1 mg/kg LNT用药组与药物未处理组相比显著提高了生存率;显著增强了Th1免疫应答中关键细胞因子IFN-γ和Th2免疫应答中关键细胞因子IL-10的分泌水平.结论 对急性弓形虫感染的BALB/c小鼠采用香菇多糖预处理之后能有效激发Th1/Th2型免疫应答的有效建立,对于抵抗弓形虫感染具有重要意义.     

Growth of sarcoma 180 in normal and splenectomized BALB/c mice immunized with BCG

An evaluation was made on the growth of Sarcoma 180 (S 180) in normal and splenectomized BALB/c mice which had been immunized 40 days before the tumor challenge with BCG, either intradermally or in diffusion chambers placed in the peritoneal cavity. Immunization with intradermal BCG did not modify the growth of subcutaneous S 180, whereas it provided significant protection when the tumor was inoculated with BCG. Previous treatment with BCG in diffusion chambers had no effect on the development of S 180, but significantly decreased the percentage of survival of mice inoculated with S 180 mixed with BCG, as compared to the homologous group immunized with intradermal BCG. Splenectomy performed before the challenge with S 180, enabled 56% of mice lacking previous immunization to survive and increased this percentage to 100% when the tumor was inoculated mixed with BCG. As for splenectomized mice immunized with BCG within diffusion chambers and challenged with S 180, there was 90% of survival and 69% in those challenged with S 180 mixed with BCG. These results would suggest that the action of BCG on the growth of S 180 differs according to whether the mycobacteria are in direct contact with the host or exert their effect by means of soluble antigens capable of passing through the millipore filter of the diffusion chambers. These effects would be conditioned by the immunological state of the host.     

Activation of suppressor cells of the delayed hypersensitivity response in BALB/c mice infected or immunized with Leishmania mexicana pifanoi

A T suppressor cell population that specifically shut down delayed hypersensitivity responses (DHR) to the parasite was found in both BALB/c mice chronically infected with Leishmania mexicana pifanoi and in naive mice which had received a single IV supraoptimal dose of killed parasites. At the early phase of infection mice exhibited a transitory state of cell-mediated immunity against the parasite that was abrogated when lesions reached their accelerated phase of growth. Results suggest that in both infected and high-dose immunized mice, the activation of T suppressor cells of DHR is related to antigen overload.     

Clustered epitopes within a new poly-epitopic HIV-1 DNA vaccine shows immunogenicity in BALB/c mice

Despite a huge number of studies towards vaccine development against human immunodeficiency virus-1, no effective vaccine has been approved yet. Thus, new vaccines should be provided with new formulations. Herein, a new DNA vaccine candidate encoding conserved and immunogenic epitopes from HIV-1 antigens of tat, pol, gag and env is designed and constructed. After bioinformatics analyses to find the best epitopes and their tandem, nucleotide sequence corresponding to the designed multiepitope was synthesized and cloned into pcDNA3.1+ vector. Expression of pcDNA3.1-tat/pol/gag/env plasmid was evaluated in HEK293T cells by RT-PCR and western-blotting. Seven groups of BALB/c mice were intramuscularly immunized three times either with 50, 100, 200 µg of plasmid in 2-week intervals or with similar doses of insert-free plasmid. Two weeks after the last injection, proliferation of T cells and secretion of IL4 and IFN-γ cytokines were evaluated using Brdu and ELISA methods, respectively. Results showed the proper expression of the plasmid in protein and mRNA levels. Moreover, the designed multiepitope plasmid was capable of induction of both proliferation responses as well as IFN-γ and IL-4 cytokine production in a considerable level compared to the control groups. Overall, our primary data warranted further detailed studies on the potency of this vaccine.     

Secondary immune response of mice immunized with a protein-cellulose complex

It has been previously established that an intravenous injection of a protein antigen solution into mice primed with the same antigen in the form of a protein-cellulose complex induces an intensive antibody production (up to 10,000 antibody-forming cells/10(6) splenocytes and up to 3 mg of antibodies/ml of serum). The present study has shown that secondary immune response can be considerably enhanced if large amounts of the antigen are administered intraperitoneally in a protein-cellulose complex during secondary immunization. In these experiments the mean number of antibody-forming cells was 50.000/10(6) splenocytes and the antibody serum level averaged 10 to 12 mg/ml. The effect persisted for a long time: as late as on day 80 the antibody concentration was 2 mg/ml of serum.     

The role of liposome charge on immune response generated in BALB/c mice immunized with recombinant major surface glycoprotein of Leishmania (rgp63)

Liposomes as a lipid-based system have been shown to be an effective adjuvant formulation. In this study, the role of liposome charge in induction of a Th1 type of immune response and protection against leishmaniasis in BALB/c mice was studied. Liposomes containing rgp63 were prepared by Dehydration-Rehydration Vesicle (DRV) method. Neutral liposomes consisted of dipalmitoylphosphatidylcholine and cholesterol. Positively and negatively charged liposomes were prepared by adding dimethyldioctadecylammonium bromide (DDAB) or dicetyl phosphate (DCP) to the neutral liposome formulation, respectively. Female BALB/c mice were immunized subcutaneously with negatively, positively charged or neutral liposomes encapsulated with rgp63, rgp63 in soluble form or PBS, three times in 3 week intervals. The extent of protection and type of immune response generated were studied in different groups of mice. The group of mice immunized with rgp63 encapsulated in neutral liposomes showed a significantly (P < 0.01) smaller footpad swelling upon challenge with Leishmania major compared with positively or negatively charged liposomes. The mice immunized with neutral liposomes also showed a significantly (P < 0.01) the lowest splenic parasite burden, the highest IgG2a/IgG1 ratio and IFN-γ production and the lowest IL-4 level compared to the other groups. The results indicated that a Th1 type of immune response was induced in mice immunized with neutral liposomes more efficiently than positively charged liposomes and conversely negatively charged liposomes induced a Th2 type of immune response.     

The role of T-lymphocytes in the development of a humoral immune response to the corpuscular antigen of Staphylococcus aureus

The dependence of humoral immune response and the formation of immunological memory to corpuscular staphylococcal antigen (CSA) on the T-system of immunity was studied in experiments on B-mice and on mice with the congenital absence of the thymus (nude). Primary and secondary immune response to CSA in athymic mice was found to be considerably less than in normal animals. After the repeated immunization of genetically athymic mice the pronounced secondary reaction of the formation of antibodies to CSA was observed. As shown in this investigation carried out with the use of adoptive transfer techniques, the induction of memory B-cells to CSA may occur in animals with congenital or experimentally induced T-immunodeficiency. The conclusion was made on the T-dependence of humoral immune response to CSA, the formation of immunological memory to this antigen being relatively T-independent.     

Evaluation of the contraceptive potential of brZPCp vaccine in BALB/c mice: their safety and efficacy

In this study we have examined the potential ability of Microtus branditi partial ZPC (brZPCp) cDNA sequence (436-1150 nt) as a target for immunocontraception. Immunogenicity studies and fertility trials were performed in BALB/c mice using recombinant construction pCR3.1-brZPC(p). ELISA outcome indicated that antibodies could be generated by immunized mice, and IgG titer was increased compared to the control. Immunohistochemistry outcome indicated that antibodies could recognize native ZP in vivo, which in turn, prevented the binding of sperm to ovulated eggs. Antibodies could also recognize recombinant protein expressed by BL21 in vitro, which was confirmed by SDS-PAGE and Western blot. Fertility rate was reduced by 45% compared to the control immunized with pCR3.1. Meanwhile, there was no incidence of significant ovarian pathology in treated mice. This experiment indicates that this vaccine can elicit the specific antibody which binds exactly to the corresponding ZPC. This construction is proved to be immunogenic, and can reduce fertility without obvious oophoritis. The result in this study suggests a potentially important method for controlling population for its safety and easy production.     

Leishmania infantum: mixed T-helper-1/T-helper-2 immune response in experimentally infected BALB/c mice

The main goal of the present study was to characterise the course of infection and immunological responses developed by Leishmania infantum infected BALB/c mice. Parasite load was determined by Real-time TaqMan PCR while cytokine and Immunoglobulin G (IgG) production were assessed by ELISA. Leishmania DNA was detected in spleen and liver as soon as day 1 post-inoculation (pi) and the parasitism was sustained until the end of the experiment. The cytokine kinetics in spleen and liver was generally associated with the oscillations of parasite load. Overall, it was not observed a distinct Th1 or Th2 pattern of cytokine production during the time of experiment. The infected mice developed a mixed immune response, with concomitant production of IFN-gamma, IL-4 and IL-10, both in spleen and liver, and both IgG isotypes. However, our results suggest that, compared to liver, the spleen is more susceptible to L. infantum infection.     

Modulated immune responsiveness associated with experimental Encephalitozoon cuniculi infection in BALB/c mice

Spleen cell blastogenesis to mitogens and antibody responses to sheep erythrocytes (sRBC) were tested in BALB/c mice with experimental E. cuniculi infections. Blastogenesis responses of spleen cells 1 week post-infection were significantly lower than normal to T-cell mitogens (Con A and PHA) and were unchanged in response to B-cell mitogens (LPS and PWM). After 2 weeks post-infection, the responses to T cell mitogens returned to normal. Mixing spleen cells from 1-week infected mice with cells from uninfected mice failed to reveal the presence of suppressor cells. Antibody responses to sRBC were significantly slower to develop in 1 week-infected mice compared with uninfected mice or mice infected 2 weeks earlier or at the same time as sRBC challenge. Infected mice displayed splenomegaly which was most pronounced 1 week post-infection and the differential spleen cell counts revealed the presence of lymphoblasts. Lymphohyperplasia appeared to cause the splenomegaly. No shifts in the proportion of Thy 1.2+ T cells, Ig+ B cells, or esterase-positive macrophages were detected. These results indicate that the immune system in BALB/c mice is depressed early during E. cuniculi infections.     

Temporal development of protective cell-mediated and humoral immunity in BALB/c mice infected with Brucella abortus

In BALB/c mice infected i.v. with attenuated strain 19 of Brucella abortus, the organism replicates to high numbers in the spleen and reaches peak concentrations at 2 wk postinfection (p.i.). The infection is then progressively cleared so that by 8 wk p.i. numbers of bacteria have decreased 10,000 fold or more. Passive transfer assays were performed with T cell-enriched spleen cells and serum of donor mice infected 2, 3, 4, 5, 6, or 8 wk previously. Antibodies conferred significant protection to recipients at and after 3 wk p.i., whereas protection by T cells was not evident until 4 wk p.i. The combined transfer of serum and cells enhanced protection over that provided by serum or cells alone when transfers were made before, but not after, challenge infection. Protection conferred by T cell-enriched spleen cells of 6-wk donors was unaffected by the presence of equal quantities of cells from 3-wk donors, but was abrogated by the removal of both CD4 and CD8 T cell subsets. Experiments with purified CD4 and CD8 subsets revealed that cell-mediated protection resided at equivalent levels in both subsets. Daily treatment of mice with Cyclosporin A for 4 wk after infection caused some increase in numbers of brucellae in spleens and livers. Although immune responses of treated animals were markedly suppressed, there was little effect of treatment on numbers of macrophages in the spleen, on enhanced killing of Listeria monocytogenes in the spleen, or on the nature and intensity of splenic and hepatic inflammatory responses. These data indicate that acquired resistance to infection with B. abortus in mice is the result of independent, and probably also interactive, effects of antibodies and T effector cells of both CD4 and CD8 phenotypes. The initial decline in bacterial numbers in the spleen, which occurred in the absence of detectable cell-mediated immunity in that organ, could probably be ascribed principally to effects of antibodies and to nonimmune stimuli responsible for increased formation, attraction, and activation of macrophages.