旋毛虫Ts87抗原的免疫学特性及保护性的初步研究

应用Western btot和ELISA方法对纯化的重组蛋白PET—28a( )／Ts87进行免疫学特性鉴定及保护性研究。Western blot和ELISA结果显示,Ts87抗原可被人工感染旋毛虫的兔血清、病猪血清、病人血清及抗Ts87的兔血清所识别。Ts87抗原免疫BABL/c小鼠,较对照组减虫率为29％,说明Ts87抗原可作为旋毛虫免疫诊断和疫苗的候选抗原。     

Identification of oncofetal antigen/immature laminin receptor protein epitopes that activate BALB/c mouse OFA/iLRP-specific effector and regulatory T cell clones

During tumor development in mice and humans, oncofetal Ag/immature laminin receptor (OFA/iLRP)-specific Th1, CTL, and IL-10-secreting T (Ts) cells are induced. The presence of too many Ts or too few effector T cells appears to predict a poor prognosis. We established clones of OFA/iLRP-specific splenic Th1, CTL, and Ts cells from the OFA/iLRP+ MCA1315 fibrosarcoma-bearing BALB/c mice or from BALB/c mice vaccinated with 1 or 10 microg of rOFA/iLRP. The MCA1315 tumor cell-reactive T cell clones were characterized as to surface Ag phenotype, cytokine secretion profile, and specificity for OFA/iLRP presented by syngeneic splenic APC. OFA/iLRP-specific Th1 and Ts clones were established from all mice. OFA/iLRP-specific CTL could be established from all mice except for mice immunized with 10 microg of rOFA/iLRP. Analysis of the proliferation profile of the OFA/iLRP-specific clones to overlapping OFA/iLRP 12-mer peptides that spanned the OFA/iLRP protein sequence defined the epitopes to which the T cell clones responded. There was a similar spatial distribution of the epitopes to which the two types of CD8 T cell clones responded. The nonapeptide epitopes of the Ts clones were located between aa 36 and 147 of OFA/iLRP, while the epitopes of the CTL clones were located between aa 52 and 163. Even though the CTL and Ts epitopes shared part of the protein, all of the CD8 CTL epitopes were distinct and separable from those of CD8 Ts cells.     

Trichinella spiralis: macrophage activity and antibody response in chronic murine infection

The role of macrophages, their products, and the specific antibody response were examined during chronic Trichinella spiralis infection in BALB/c mice. Adult T. spiralis in intestines were detected from 5 to 20 dpi. Muscle larvae numbers peaked at 45 dpi and thereafter a reduction was noted. The highest numbers of macrophages in the peritoneal cavity of infected mice were obtained up to 30 dpi. The production of NO by macrophages in infected mice was suppressed at 5 dpi, and then NO release increased until 45 dpi. The levels of NO in plasma and urine were lower in infected mice during the entire experiment in comparison to control. The production of O(2)(-) in peritoneal macrophages was inhibited during the first two weeks after infection and then increased until 90 dpi. Circulating T. spiralis antigens in plasma and urine were detected from 5 to 30 dpi. Specific IgM and IgA in serum increased until 20 dpi. IgG, IgG(1), and IgG(2) levels in serum increased until 60 dpi.     

日本血吸虫新抗原基因Sj—Ts4的克隆、表达及免疫保护性

为探讨旋毛虫感染小鼠后抗血吸虫感染的分子机制 ,并为血吸虫病疫苗的研究提供新的抗原分子 ,用旋毛虫感染鼠血清筛选日本血吸虫成虫cDNA文库。经过 3轮筛选 ,获得 9个阳性克隆 ,其中新基因Sj Ts4有一完整的编码框 ,编码2 10个氨基酸。该蛋白质的理论分子量为 2 3kD ,等电点为 7.72。将Sj Ts4亚克隆于原核表达载体 pGEX 5X 3,以GST融合蛋白的形式在E .coliDH5α中表达。Western印迹分析显示 ,重组Sj Ts4蛋白 (rSj Ts4)能被慢性感染鼠血清识别。rSj Ts4或与弗氏完全佐剂混合后免疫小鼠 ,均可以诱导产生特异性的IgG抗体 ,抗体效价可高达 1∶2 5 6 0 0。两免疫组的减虫率分别为31.36 %和 36 .80 % ,与对照组相比都具有统计学意义     

Identification and characterization of a full-length cDNA encoding paramyosin of Trichinella spiralis

A full-length cDNA encoding Trichinella spiralis paramyosin (Ts-Pmy) was cloned by immunoscreening a cDNA library of the adult T. spiralis worm. Ts-Pmy cDNA consists of 2655 bp that encode 885 amino acids. The recombinant protein (rTs-Pmy) was expressed and purified by Ni-affinity chromatography. Western blot analysis showed that rTs-Pmy could be recognized by sera from T. spiralis-infected humans, swine, rabbits, and mice. Immunolocalization demonstrated that Ts-Pmy was abundant on the surface of T. spiralis larvae. BALB/c mice vaccinated with rTs-Pmy demonstrated 36.2% reduction in muscle larvae burden following T. spiralis larvae challenge. Vaccination of the mice with rTs-Pmy resulted in a high level of specific anti-Ts-Pmy IgG antibodies and generated a Th1/Th2 mixed type of immune response, with Th2 predominant. These studies showed that rTs-Pmy induced protective immunity in mice and could be considered as a potential vaccine candidate for trichinellosis.     

Clonal analysis of autoreactive B cells in a murine model of autoimmune hemolytic anemia

Hybridoma technology was used to examine the repertoire of autoantibody producing B cells in mice with autoimmune hemolytic anemia induced by injection of rat red blood cells (RBC). The results point to the importance of suppressor T cells (Ts) in both the induction as well as the maintenance of the self-specific B-cell repertoire at the clonal level. Thus when normal BALB/c mice were immunized to provoke a high autoantibody response, the hybrids generated were mainly (97%) cross-reactive with mouse RBC, whereas after immunization to elicit Ts and a low autoantibody response, hybrids were mainly (87%) rat RBC specific. In addition, when hybrids were generated from rat RBC immunized (CBA X B10A)F1 mice, in which Ts levels had been depleted during ontogeny, hybrids with "forbidden" purely anti-self (mouse RBC) specificity were detected.     

Regulation of the anti-alpha(1----3) dextran IgG antibody response of BALB/c mice by idiotype-specific T suppressor lymphocytes.

The immune response of BALB/c mice against the so-called thymus-independent bacterial Ag alpha(1----3) dextran (Dex) is restricted to the expression of few major idiotypes (Id). It is furthermore under the control of T lymphocytes which regulate the isotype expression in such a way that they prevent anti-Dex IgG antibody production upon immunization. At the same time these T cells are part of a regulatory system for Dex-specific B cell memory formation. The underlying Ts cell activity has previously been analyzed by using euthymic and athymic congenic animals. Now we have isolated CD4-positive Id-specific T cell lines and clones which by several criteria are representatives of the above Ts cells. They inhibit in vitro proliferation and antibody secretion of Dex-specific hybridoma B cells. They prevent Id-restricted in vivo IgG anti-Dex antibody formation in T cell-reconstituted BALB/c nu/nu mice. At the same time they enforce, again Id-specific, accumulation of Dex-specific B memory cells. As has been shown previously under the influence of splenic Ts cells, these B memory cells are arrested in the original host but can be expanded and activated for anti-Dex IgG antibody formation upon adoptive transfer into X-irradiated allotype congenic nonresponder BALB.Ighb mice. The data show that the regulatory influence of T cells on the anti-Dex response is Id specific. It can now be studied by means of cloned Ts cells.     

抗脐带间充质干细胞表面分子噬菌体ScFv抗体库的构建及初步鉴定

目的：利用噬菌体展示技术构建抗脐带间充质干细胞表面分子噬菌体ScFv抗体库。方法：收集P3代培养的UC-MSCs免疫BALB/c小鼠,提取其脾细胞总RNA,RT-PCR扩增全套VH和VL基因片段,将其先后克隆入噬菌粒pSEX81中,构建成完整的噬菌体ScFv抗体库。结果:构建的噬菌体ScFv抗体库的库容为2×107cfu,ScFv插入重组率为93％,BstN1酶切图谱呈不同多样性。ScFv抗体库经3轮初步筛选后插入重组率达100％,3个克隆出现了相同的酶切图谱,并且随着筛选次数的增加,输出/输入比明显提高,这说明抗体库得到了特异性富集。结论：成功地构建了抗脐带间充质干细胞表面分子噬菌体ScFv抗体库,这为将来筛选特异性抗体和进一步用于间充质干细胞表面特异性分子研究奠定了坚实的基础。     

Immunologic regulation of experimental cutaneous leishmaniasis. V. Characterization of effector and specific suppressor T cells

Resistant CBA mice infected with Leishmania tropica promastigotes develop concomitant and convalescent immunity against reinfection. This can be adoptively transferred by splenic and lymph node T cells with a threshold dosage of 1 to 2.5 x 10(7). The effector cells are of Thy-1+, Lyt-1+2- phenotype. The same immune cell population also adoptively transfers specific DTH to L. tropica, which is restricted by the major histocompatibility complex. On the other hand, highly susceptible BALB/c mice infected with L. tropica develop antigen-specific suppressor T (Ts) cells (previously shown to inhibit the induction and expression of DTH), which are capable, on transference, of reversing the healing of lesions induced by prior sublethal irradiation of BALB/c mice. As few as 10(6) of these T cells are effective in abrogating the potent prophylactic effect of 550 rad. The Ts cells are of Thy-1+, Lyt-1+2-, and I-J- phenotype. Sublethally irradiated and infected BALB/c mice produce antibody responses quantitatively and isotypically similar during the critical first 40 days after infection whether or not they are injected with 10(7) Ts cells (nonhealing vs healing). Thus, impairment of DTH and curative immune responses in BALB/c mice cannot be attributed to a helper function of these Thy-1+, Lyt-1+2- cells for the formation of suppressive antibody.     

利用噬菌体随机肽库筛选可结合志贺毒素B亚单位的短肽序列

以制备的重组志贺毒素B亚单位(StxB)为靶标,利用噬菌体展示亲和淘选技术,经4轮筛选,从随机十二肽库中筛选到与StxB结合的一批噬菌体克隆,对特异结合活性较高的27个噬菌体克隆的表面展示肽进行序列测定,其中A6序列出现16次,A9和A3序列分别出现2次和3次。为评价筛选克隆中和毒素毒性的能力,将展示肽出现频率最高的A6噬菌体克隆,体外与志贺毒素孵育进行动物试验,动物存活率达33.3%,表明毒素的毒性得到部分抑制,A6短肽可能发展成为志贺毒素的拮抗剂。     

Protection from murine cysticercosis by immunization with a parasite cysteine protease

Central nervous system infection by Taenia solium cysts causes neurocysticercosis, a common neurological infection in the Third World. We have previously isolated cysteine proteases from Taenia crassiceps and T. solium. In this study, we immunized BALB/c mice with the purified T. solium cysteine protease and challenged them with Taenia crassiceps. Immunized mice had a 72% reduction in parasite burden compared to mice that received no immunization. Immunized mice developed antigen specific lymphocyte proliferation. These data support further studies of the T. solium cysteine protease as a vaccine candidate.     

Generation and Characterization of a scFv Antibody Against T3SS Needle of Vibrio parahaemolyticus

Vibrio parahaemolyticus, a halophilic gram-negative bacterium, is a food-borne pathogen that largely inhabits marine and estuarine environments, and poses a serious threat to human and animal health all over the world. The hollow “needle” channel, a specific assemble of T3SS which exists in most of gram-negative bacteria, plays a key role in the transition of virulence effectors to host cells. In this study, needle protein VP1694 was successfully expressed and purified, and the fusion protein Trx-VP1694 was used to immunize Balb/c mice. Subsequently, a phage single-chain fragment variable antibody (scFv) library was constructed, and a specific scFv against VP1694 named scFv-FA7 was screened by phage display panning. To further identify the characters of scFv, the soluble expression vector pACYC-scFv-skp was constructed and the soluble scFv was purified by Ni²⁺ affinity chromatography. ELISA analysis showed that the scFv-FA7 was specific to VP1694 antigen, and its affinity constant was 1.07 × 10⁸ L/mol. These results offer a molecular basis to prevent and cure diseases by scFv, and also provide a new strategy for further research on virulence mechanism of T3SS in V. parahaemolyticus by scFv.     

Mimotope-Based Vaccines of Leishmania infantum Antigens and Their Protective Efficacy against Visceral Leishmaniasis

Background

The development of cost-effective prophylactic strategies to prevent leishmaniasis has become a high-priority. The present study has used the phage display technology to identify new immunogens, which were evaluated as vaccines in the murine model of visceral leishmaniasis (VL). Epitope-based immunogens, represented by phage-fused peptides that mimic Leishmania infantum antigens, were selected according to their affinity to antibodies from asymptomatic and symptomatic VL dogs'' sera.

Methodology/Main Findings

Twenty phage clones were selected after three selection cycles, and were evaluated by means of in vitro assays of the immune stimulation of spleen cells derived from naive and chronically infected with L. infantum BALB/c mice. Clones that were able to induce specific Th1 immune response, represented by high levels of IFN-γ and low levels of IL-4 were selected, and based on their selectivity and specificity, two clones, namely B10 and C01, were further employed in the vaccination protocols. BALB/c mice vaccinated with clones plus saponin showed both a high and specific production of IFN-γ, IL-12, and GM-CSF after in vitro stimulation with individual clones or L. infantum extracts. Additionally, these animals, when compared to control groups (saline, saponin, wild-type phage plus saponin, or non-relevant phage clone plus saponin), showed significant reductions in the parasite burden in the liver, spleen, bone marrow, and paws'' draining lymph nodes. Protection was associated with an IL-12-dependent production of IFN-γ, mainly by CD8⁺ T cells, against parasite proteins. These animals also presented decreased parasite-mediated IL-4 and IL-10 responses, and increased levels of parasite-specific IgG2a antibodies.

Conclusions/Significance

This study describes two phage clones that mimic L. infantum antigens, which were directly used as immunogens in vaccines and presented Th1-type immune responses, and that significantly reduced the parasite burden. This is the first study that describes phage-displayed peptides as successful immunogens in vaccine formulations against VL.     

Identification of antigenic proteins in Trichomonas vaginalis

Trichomoniasis is a sexually transmitted disease due to infection with Trichomonas vaginalis, and it can cause serious consequences for women's health. To study the virulence factors of this pathogen, T. vaginalis surface proteins were investigated using polyclonal antibodies specific to the membrane fractions of T. vaginalis. The T. vaginalis expression library was constructed by cloning the cDNA derived from mRNA of T. vaginalis into a phage λ Uni-ZAP XR vector, and then used for immunoscreening with the anti-membrane proteins of T. vaginalis antibodies. The immunoreactive proteins identified included adhesion protein AP65-1, α-actinin, kinesin-associated protein, teneurin, and 2 independent hypothetical proteins. Immunofluorescence assays showed that AP65-1, one of the identified immunogenic clones, is prevalent in the whole body of T. vaginalis. This study led us to identify T. vaginalis proteins which may stimulate immune responses by human cells.     

Protective Immunity against Trichinella spiralis Infection Induced by a Multi-Epitope Vaccine in a Murine Model

Trichinellosis is one of the most important food-borne parasitic zoonoses throughout the world. Because infected pigs are the major source of human infections, and China is becoming the largest international producer of pork, the development of a transmission-blocking vaccine to prevent swine from being infected is urgently needed for trichinellosis control in China. Our previous studies have demonstrated that specific Trichinella spiralis paramyosin (Ts-Pmy) and Ts-87 antigen could provide protective immunity against T. spiralis infection in immunized mice. Certain protective epitopes of Ts-Pmy and Ts-87 antigen have been identified. To identify more Ts-Pmy protective epitopes, a new monoclonal antibody, termed 8F12, was produced against the N-terminus of Ts-Pmy. This antibody elicited significant protective immunity in mice against T. spiralis infection by passive transfer and was subsequently used to screen a random phage display peptide library to identify recognized epitopes. Seven distinct positive phage clones were identified and their displayed peptides were sequenced. Synthesized epitope peptides conjugated to keyhole limpet hemocyanin were used to immunize mice, four of which exhibited larval reduction (from 18.7% to 26.3%, respectively) in vaccinated mice in comparison to the KLH control. To increase more effective protection, the epitope 8F7 that was found to induce the highest protection in this study was combined with two other previously identified epitopes (YX1 from Ts-Pmy and M7 from Ts-87) to formulate a multi-epitope vaccine. Mice immunized with this multi-epitope vaccine experienced a 35.0% reduction in muscle larvae burden after being challenged with T. spiralis larvae. This protection is significantly higher than that induced by individual-epitope peptides and is associated with high levels of subclasses IgG and IgG1. These results showed that a multi-epitope vaccine induced better protective immunity than an individual epitope and provided a feasible approach for developing a safer and more effective vaccine against trichinellosis.     

Expressed sequence tags of Trichinella spiralis muscle stage larvae

In order to obtain greater insight into the relevant genomic expression patterns of Trichinella spiralis, 992 expressed sequence tags (ESTs) were collected from a cDNA library of T. spiralis muscle stage larvae and assembled into 60 clusters and 385 singletons. Of them, 445 (44.7%) ESTs were annotated to their homologous genes, and small fractions were matched to known genes of nematodes. The annotated ESTs were classified into 25 eukaryotic orthologous groups (KOG). Cytochrome C oxidase (34 clones) was found to be most frequent species.     

Resistance to Toxoplasma gondii infection in mice treated with silk protein by enhanced immune responses

This study investigated whether elevated host immune capacity can inhibit T. gondii infection. For this purpose, we used silk protein extracted from Bombyx mori cocoons as a natural supplement to augment immune capacity. After silk protein administration to BALB/c mice for 6 weeks, ratios of T lymphocytes (CD4(+) and CD8(+) T-cells) and splenocyte proliferative capacities in response to Con A or T. gondii lysate antigen (TLA) were increased. Of various cytokines, which regulate immune systems, Th1 cytokines, such as IFN-γ, IL-2, and IL-12, were obviously increased in splenocyte primary cell cultures. Furthermore, the survival of T. gondii (RH strain)-infected mice increased from 2 days to 5 or more days. In a state of immunosuppression induced by methylprednisolone acetate, silk protein-administered mice were resistant to reduction in T-lymphocyte (CD4(+) and CD8(+) T-cells) numbers and the splenocyte proliferative capacity induced by Con A or TLA with a statistical significance. Taken together, our results suggest that silk protein augments immune capacity in mice and the increased cellular immunity by silk protein administration increases host protection against acute T. gondii infection.     

Suppressor T cells, distinct from "veto cells," are induced by alloantigen priming and mediate transferable suppression of cytotoxic T lymphocyte responses in vivo

Primary and secondary cytotoxic T lymphocyte responses to minor alloantigens can be suppressed by priming host mice with a high dose (10(8) cells) of alloantigenic donor spleen cells (SC). Such suppression is antigen specific and transferable into secondary hosts with T cells. One interpretation of this is that antigen-specific host suppressor T cells (Ts) are activated. Alternatively, donor Lyt-2+ T cells, introduced in the priming inoculum, may inactivate host CTL precursors (CTLp) that recognize the priming (donor) alloantigens. Donor cells that act in this way are termed veto T cells. The experiments described here exclude veto T cell participation in transferable alloantigen-specific suppression, and demonstrate the operation of an alloantigen-specific host-derived T suppressor (Ts) cell. The origin of the Ts has been studied directly by using Thy-1-disparate BALB/c mice. The cell responsible for the transfer of suppression of a secondary CTL response to B10 minors was of the host Thy-1 allotype, and so originated in the host spleen and was not introduced in the priming inoculum. Secondly, antigen-specific Ts generated in CBA female mice against B10 minors could act on CTL responses to an unequivocally non-cross-reactive-third party antigen (H-Y), provided the two antigens were expressed on the same cell membrane. Such third-party suppression is incompatible with the operation of veto T cells. Depletion of Thy-1.2+ or Lyt-2+ cells from the suppression-inducing donor SC inoculum did not abrogate suppression induction in BALB/c mice; instead, suppression was enhanced. The demonstration of veto cell activity in similarly primed mice by other groups of investigators indicates that both types of suppression may operate. However, our results show that only antigen-specific Ts can mediate the transferable suppression of CTL responses to alloantigens.