基于载体减毒单增李斯特菌的弧菌疫苗菌株构建

目的：利用减毒单增李斯特菌作为疫苗载体的优势,拟构建基于载体减毒单增李斯特菌的弧菌疫苗菌株。方法：使用单增李斯特菌表达载体pERL3过表达弧菌中共同的靶标抗原Ompk;利用PCR和RT-PCR技术分别对过表达菌株鉴定和抗原基因的检测。结果：PCR结果显示成功构建3株Ompk过表达菌株Lm-Ompk（L-O）、Lm-Lmo0576-Ompk（L-L-O）和Lm-P-Ompk（L-P-O）;测序结果显示过表达菌株中外源基因的序列与NCBI 结果相似性为100%;RT-PCR结果显示Ompk基因在3株过表达菌株中的转录水平均极为显著（P<0.001）;在抗生素条件下,Ompk基因在各过表达菌株中的表达水平显著高于在非抗性条件下的转录水平（P<0.05）。结论：成功构建了3株基于载体减毒单增李斯特菌的弧菌疫苗菌株,弧菌中共同的靶标抗原Ompk在3株重组菌株中均可以稳定表达。     

Stable integration vector for nutrient broth-based selection of attenuated Listeria monocytogenes strains with recombinant antigen expression

Recombinant Listeria monocytogenes strains induce strong cellular immune responses and may prove useful for antigen delivery for the vaccination of humans. However, the genetic systems currently available for the stable expression of recombinant antigens by L. monocytogenes rely on the use of antibiotic resistance genes. We report on a derivative, pPL2dalGlnA, of the Listeria monocytogenes pPL2 integration vector that completely lacks drug resistance genes. The selectable markers in pPL2dalGlnA are glutamine synthetase (GlnA) and alanine racemase (Dal). This novel vector was stably maintained in auxotropic L. monocytogenes strains that normally require d-alanine. The pPL2dalGlnA vector also partially restored the ability of an L. monocytogenes Deltadal Deltadat strain to colonize the spleens and livers of infected mice. A novel, highly attenuated strain of L. monocytogenes with quadruple deletions was also engineered by deleting the L. monocytogenes actA and plcB virulence genes from a Deltadal Deltadat strain. Infection of mice with recombinants of this mutant strain that express the antigen from pPL2dalGlnA were shown to elicit CD8(+) T-cell responses to human immunodeficiency virus Tat. This vector system is thus useful for stable antigen expression and vaccination studies.     

Attenuated Listeria monocytogenes as a live vector for induction of CD8+ T cells in vivo: a study with the nucleoprotein of the lymphocytic choriomeningitis virus

Listeria monocytogenes spends most of its intracellular lifecycle in the cytosol of the infected eucaryotic cells. Withinthis cellular compartment originates the endogenous pathwayof antigen processing and presentation. We thus assumed thatrecombinant L. monocytogenes expressing an heterologous protein,the nucleoprotein of the lymphocytic choriomeningltis virus(LCMV), should be able to induce antigen-specific CD8⁺ T cellsin vivo. The LCMV nucleoprotein gene was inserted in phase withthe sequence coding for the putative signal sequence of thehemolysin of L. monocytogenes in order to target its secretioninto the cytosol of the infected cell. The ability of this recombinantbacterium to induce LCMV-reactive CD8⁺ T cells was then monitoredin BALB/c mice. The immune status of the immunized BALB/c micewas studied on the seventh day after a single i.v.injectionof a sublethal dose of the recombinant bacteria: (i) cytotoxicCD8⁺ T cells were detected in liver; (ii) using in vitro re-stimulationwith PMA and ionomycin, secondary cytotoxic CD8⁺ T cells weredetected in spleen; (iii) an early inflammatory reaction dependenton the presence of CD8⁺ T cells occurred in the footpad afterintraplantar inoculation of live LCMV; and (iv) mice were protectedagainst an otherwise lethal intracerebral LCMV challenge; theprotection was accompanied by elimination of the virus. Whenthe immune status of the immunized hosts was monitored for alonger period post-immunization, the balance between immuneprotectiosn and immunopathology described for the anti-LCMVimmune responses was observed; two phases of protection weredetected, flanking a transitory phase of exacerbation of thelymphocytic choriomeningitis disease (weeks 2–5). Takentogether, these results indicate the feasibility of using attenuatedL. monocytogenes as a model of a live vector to induce in vivoCD8-ependent immune responses against intracellular pathogens.     

Peptide immunization in humans: a combined CD8+/CD4+ T cell-targeted vaccine restimulates the memory CD4 T cell response but fails to induce cytotoxic T lymphocytes (CTL)

Immunization with short antigenic peptides represents a potential strategy to induce peptide-specific CTL in vivo. In this study, a synthetic vaccine consisting of an HIV-derived, HLA-A2.1-binding CTL epitope and a tetanus toxin-derived T helper epitope was evaluated for its capacity to induce peptide-specific CTL in humans. Thirteen volunteers were immunized and boosted twice with 100 μg of the CTL epitope plus 300 μg of the T helper peptide (p30). Peripheral blood mononuclear cells (PBMC) were regularly analysed for cytotoxic and proliferative responses before, between and after the immunizations, and the serum was tested for anti-peptide antibodies. No unequivocal induction of HIV peptide-specific CTL in any of the volunteers was observed. However, a wide pattern of mild and transient side reactions was observed, ranging from local redness at the injection site to generalized exanthema, myalgias, arthralgias and fever. The side-effects were related to the T helper epitope, as they were similar to the side-effects experienced after tetanus immunization, correlated to the magnitude of the p30-specific in vitro proliferative response, and occurred only if p30 was co-injected. No antibodies against the HIV-derived peptides nor against p30 were detectable in the serum after repeated immunizations. The data suggest that the CTL peptide, at the concentration used in this study, failed to induce a cytotoxic immune response in vivo, although the T helper peptide seems to be capable of restimulating the specific memory T cells.     

Attenuated Listeria monocytogenes as a cancer vaccine vector for the delivery of CD24, a biomarker for hepatic cancer stem cells

Attenuated Listeria monocytogenes （LM） is a promising candidate vector for the delivery of cancer vaccines. After phagocytosis by antigen-presenting cells, this bacterium stimulates the major histocompatibility complex （MHC）-I and MHC-II pathways and induces the proliferation of antigen-specific T lymphocytes. A new strategy involving genetic modification of the replication-deficient LM strain AdalAdat （Lmdd） to express and secrete human CD24 protein has been developed. CD24 is a hepatic cancer stem cell biomarker that is closely associated with apoptosis, metastasis and recurrence of hepatocellular carcinoma （HCC）. After intravenous administration in mice, Lmdd-CD24 was distributed primarily in the spleen and liver and did not cause severe organ injury. Lmdd-CD24 effectively increased the number of interferon （IFN）-7-producing CD8＋ T cells and IFN-7 secretion. Lmdd-CD24 also enhanced the number of IL-4- and IL-lO-producing T helper 2 cells. The efficacy of the Lmdd-CD24 vaccine was further investigated against Hepa1- 6-CD24 tumors, which were inguinally inoculated into mice. Lmdd-CD24 significantly reduced the tumor size in mice and increased their survival. Notably, a reduction of T regulatory cell （Treg） numbers and an enhancement of specific CD8＋ T-cell activity were observed in the tumor-infiltrating lymphocytes （TILs）. These results suggest a potential application of the Lmdd-CD24 vaccine against HCC.     

Heat-killed Listeria monocytogenes and L. monocytogenes soluble antigen induce clonable CD4+ T lymphocytes with protective and chemotactic activities in vivo.

In the present study we attempted to analyze the possibility to induce in mice a T-cell response using killed Listeria monocytogenes in adjuvant. Clearly, nonviable antigen is capable of inducing protective and granuloma-forming T cells in C57BL/6 mice when emulsified in complete Freund's adjuvant. These T cells were cloned in vitro by using antigen and irradiated splenocytes, as antigen-presenting cells, and the clones were characterized in vivo. Listeria-specific T-cell clones showed protective and chemotactic activity upon adoptive transfer, although some degree of functional heterogeneity among different clones was observed. The heterogeneous in vivo functions could not be correlated with the ability of the clones to produce gamma interferon or T-cell growth factor (interleukin-2 and/or interleukin-4). It was demonstrated that an in vivo relevant fraction of listeria-specific T lymphocytes can be induced by nonviable antigen in complete Freund's adjuvant. These results show that the low immunogenicity of heat-killed bacteria is not due to the expression of specific protective T-cell epitopes only by live bacteria.     

In vitro properties of a Listeria monocytogenes bacteriophage-resistant mutant predict its efficacy as a live oral vaccine strain

A Listeria monocytogenes glcV mutation precludes the binding of certain listerial phages and produces a profound attenuation characterized by the absence of detectable mutants in the livers and spleens of orally inoculated mice. In vitro, we found that the mutant formed plaques on mouse enterocyte monolayers as efficiently as the parent but the plaques formed were smaller. Intracellular growth rate determinations and examination of infected enterocytes by light and fluorescence microscopy established that the mutant was impaired not in intracellular growth rate but in cell-to-cell spreading. Because this property is shared by other immunogenic mutants (e.g., actA mutants), our glcV mutant was tested for vaccine efficacy. Oral immunization with the mutant and subsequent oral challenge (22 days postvaccination) with the parent revealed a ca. 10,000-fold increase in protection afforded by the mutant compared to sham-vaccinated controls. The glcV mutant did not stimulate innate immunity under the dose and route employed for vaccination, and an infectivity index time course experiment revealed pronounced mutant persistence in Peyer's patches. The immunogenicity of the glcV mutant compared to an isogenic actA mutant reference strain was next tested in an experiment with a challenge given 52 days postvaccination. Both mutant strains showed scant vital organ infectivity and high levels of protection similar to those seen using the glcV mutant in the 22-day postvaccination challenge. Our results indicate that oral administration of a profoundly attenuated listerial mutant can safely elicit solid protective immunity.     

The tumor recall response of antitumor immunity primed by a live, recombinant Listeria monocytogenes vaccine comprises multiple effector mechanisms

Listeria monocytogenes, a facultative intracellular bacterium, can induce a potent antitumor immune response if engineered to express a model tumor antigen also expressed by the tumor cells. The effectiveness of this approach is dependent on L. monocytogenes-induced tumor-specific CD4(+) and CD8(+) T-cells. CD8(+) T-cells may mediate tumor eradication largely through direct CTL activity, but the role of CD4(+) T-cells and other cells of the immune system is less clear. Here we investigate their role and the role of the cytokines they produce in the ability of L. monocytogenes-induced antitumor immunity to protect against tumor challenge. Our results suggest that a complex cytokine response, involving type 2 as well as type 1 cytokines, is responsible for the ability of Lm-NP-immunized mice to resist tumor challenge, potentially mediating tumor cell killing through multiple effector pathways.     

Induction of protective immunity to Listeria monocytogenes with dendritic cells retrovirally transduced with a cytotoxic T lymphocyte epitope minigene

In the present study, we developed a cytotoxic T lymphocyte (CTL) epitope minigene-transduced dendritic cell (DC)-based vaccine against Listeria monocytogenes. Murine bone marrow-derived DCs were retrovirally transduced with a minigene for listeriolysin O (LLO) 91-99, a dominant CTL epitope of L. monocytogenes, and were injected into BALB/c mice intravenously. We found that the DC vaccine was capable of generating peptide-specific CD8+ T cells exhibiting LLO 91-99-specific cytotoxic activity and gamma interferon production, leading to induction of protective immunity to the bacterium. Furthermore, we demonstrated that the retrovirally transduced DC vaccine was more effective than a CTL epitope peptide-pulsed DC vaccine and a minigene DNA vaccine for eliciting antilisterial immunity. These results provide an alternative strategy in which retrovirally transduced DCs are used to design vaccines against intracellular pathogens.     

Presentation of Listeria monocytogenes antigens by major histocompatibility complex class I molecules to CD8 cytotoxic T lymphocytes independent of listeriolysin secretion and virulence

Virulence and intracellular persistence of Listeria monocytogenes markedly depend on secretion of listeriolysin (Hly), which promotes invasion of the pathogen from the endosome into the cytosol. Recent studies have provided compelling evidence that Hly also facilitates recognition of listerial antigens, in association with major histocompatibility complex (MHC) class I molecules, by CD8 T lymphocytes. Data presented here confirm that the Hly-deficient strains, the prfA^? mutant L. monocytogenes SLCC53 and the transposon mutants L. monocytogenes M3 and M20 are avirulent for mice, and unable to replicate inside bone marrow-derived macrophages (BMMΦ). Furthermore, BMMΦ infected with M3, M20 or SLCC53 were as efficiently lysed as BMMΦ infected with the Hly-positive wild-type strain EGD by MHC class I-dependent CD8 cytotoxic T lymphocytes. Using the highly sensitive polymerase chain reaction method, hly mRNA was detectable in BMMΦ infected with L. monocytogenes EGD or SLCC53, but totally absent in M3-infected BMMΦ. In the case of M20, an excision of the transposon occurred, but the excision was not precise and the hly gene was approximately 400 base pairs shorter. These findings argue against a unique role for Hly in MHC class I presentation of listerial antigens, although Hly appears central to virulence and intracellular replication. Thus, virulence of L. monocytogenes is dissociable from MHC class I presentation of listerial antigens.     

Avidity maturation following immunization with two human cytomegalovirus (CMV) vaccines: a live attenuated vaccine (Towne) and a recombinant glycoprotein vaccine (gB/MF59)

Two human cytomegalovirus (CMV) vaccines have been previously evaluated for their immunogenicity: a recombinant gB/MF59 vaccine and an attenuated strain of CMV (Towne). In healthy adults, we measured the antibody avidity maturation indices that occurred after vaccination with each. For Towne, administered as a single dose, the rise in IgG antibody avidity to CMV glycoprotein gB occurred slowly and continued for 24 months post-immunization. For gB/MF59, administered as two priming doses and a booster dose given at 6 months, the booster rapidly induced IgG antibodies to gB whose avidity was maximal at 7 months after the initial priming dose. Both vaccines induced antibody levels and avidity maturation indices that equaled those induced by wild-type virus suggesting that both vaccines may be effective in controlling CMV infections.     

Simian immunodeficiency virus-specific cytotoxic T lymphocytes and protection against challenge in rhesus macaques immunized with a live attenuated simian immunodeficiency virus vaccine

In this study, we examined the role of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocytes (CTLs) in macaques immunized with an attenuated strain of simian immunodeficiency virus (SIVmac239Deltanef) in protection against pathogenic challenge with SIVmac251. Our results indicate that attenuated SIVmac239Deltanef can elicit specific CTL precursor cells (CTLp), but no correlation was observed between breadth or strength of CTLp response to structural proteins SIV-Env, -Gamg or -Pol (as measured by limiting dilution assay) and protection against infection. In one animal, we longitudinally followed the SIV-Gag-specific response to an MHC class I Mamu-A*01-restricted epitope p11C, C-M using a tetrameric MHC/peptide complex reagent. A low frequency of SIV p11C, C-M peptide-specific tetramer-reactive cells was present at the time of challenge but could be expanded in vitro. Surprisingly, the low level of Mamu-A*01/p11C, C-M-specific CTLs induced through attenuated SIVmac239Deltanef vaccination increased in the absence of detectable SIVmac251 or SIVmac239Deltanef proviral DNA. Overall, our results suggest that protection against infection in this model can be achieved through more than one mechanism, with SIV-specific CTLs being important in controlling SIVmac239Deltanef viral replication postchallenge.     

Oral and rectal immunization of adult female volunteers with a recombinant attenuated Salmonella typhi vaccine strain.

An attenuated strain of Salmonella typhi delta(cya) delta(crp-cdt) delta(asd) expressing a gene encoding a hepatitis B virus core-pre-S protein was tested in female adult volunteers for its ability to elicit a systemic and a mucosal immune response. Specifically, our purpose was to evaluate the potential of such a vaccine strain to induce specific secretory immunoglobulin A (sIgA) at genital and rectal surfaces. Oral and rectal routes of immunization were compared: oral immunization induced seroconversion against the bacterial lipopolysaccharide (LPS) in six out of seven volunteers, while after rectal immunization only one out of six volunteers seroconverted against LPS. To our disappointment, the latter volunteer was also the only one who seroconverted against the carried antigen (pre-S1), demonstrating the poor ability of this live vaccine to induce an immune response against the carried antigen. Anti-LPS sIgA was found in both the vaginal and cervical secretions of a volunteer who presented a strong seroconversion after oral immunization (16-fold increase in anti-LPS IgG). Smaller amounts of anti-LPS sIgA were found in the rectal secretions of one orally and one rectally immunized volunteer and in the saliva of three orally and one rectally immunized woman. Our data show for the first time that it is possible to induce specific sIgA in the genital and rectal tracts of women by using an S. typhi vaccine strain.     

Evidence for a significant role of CD4+ T cells in adoptive immunity to Listeria monocytogenes in the liver.

Although the ability of CD8+ T cells to adoptively immunize mice against Listeria monocytogenes in the spleen is well established, the role of different T-cell subsets in anti-bacterial protection in the liver, a major target of Listeria infection, remains unclear. Therefore, the ability of sorted CD4+ and CD8+ T cells to adoptively immunize mice against a L. monocytogenes infection in the liver was studied. The results show that positively sorted CD4+ T cells from day 7 Listeria-immune mice were as effective as sorted CD8+ cells in transferring significant anti-Listeria protection in the liver. Similar findings were obtained when CD4+ and CD8+ T cells, negatively selected by antibody-induced complement-mediated depletion in vitro, were used for adoptive transfer. CD8+ T cells, however, were more efficient than CD4+ T cells in transferring protection in the spleen. Taken together, the results show that CD4+ T cells are at least as protective as CD8+ T cells against a L. monocytogenes infection in the liver, thereby arguing against the view that CD4+ T cells are of limited importance in adoptive immunity against listeriosis.     

Immunity to experimental infection with Leishmania major: generation of protective L3T4+ T cell clones recognizing antigen(s) associated with live parasites

Exacerbation and resolution of lesions induced by Leishmania major promastigotes are, at least in part, the result of the activity of distinct parasite-specific L3T4+ T lymphocytes. The present report describes L. major-specific cloned L3T4+ T lymphocytes capable of transferring substantial protective immunity to normal highly susceptible BALB/c mice. The two protective T cell clones analyzed appear to recognize antigen associated only with live L. major parasites. Therefore, the pattern of antigen reactivity of these protective T cell clones is different from that of the previously described parasite-specific L3T4+ T cells which contribute to exacerbation of disease. The results presented in this report indicate that the two opposite effects of parasite-specific L3T4+ T cells on the disease process could be mediated by functionally similar L3T4+ T cells differing in their antigen specificity.     

儿童接种新甲型(H1N1)流感疫苗后特异性CD4+ T细胞亚群的表型特征

目的 探讨儿童接种新甲型(H1N1)流感疫苗接种后远期疫苗特异性CD4+记忆T细胞亚群的特征.方法 根据自愿原则,选择31例接种甲型H1N1流感疫苗47个月后的儿童,取静脉血并分离淋巴细胞,细胞培养中滴入甲型H1N1流感疫苗,同时设不加疫苗刺激的作为对照组,流式细胞仪检测CD4+记忆T细胞亚群的表型特征.结果 外周血单个核细胞(PBMC)中CD4+疫苗特异刺激组为29.85％,对照组为39.00％,实验组低于对照组;CD4+初始T细胞实验组为74.32％,对照组为70.08％(P＞0.05);CD4+记忆T细胞实验组为28.54％,与对照组25.52％比较,无统计学意义(P＞0.05);记忆T细胞分中央型与效应型记忆T细胞,检测结果:CCR7和CD62L单阳性记忆T细胞亚群实验组分别为72.52％、29.85％,对照组分别为84.0％、93.44％,实验组的明显低于对照组(P＜0.05).结论 实验儿童初始T细胞比例都较高;甲型H1N1流感疫苗能诱导抗原特异性记忆CD4+T细胞的产生,可是为数较少,当中主要是中央型记忆CD4+T细胞,其中CCR7+和CD62L+记忆T细胞数量较低.     

CD4+ T cell associated cytokine gene expression during experimental infection with Listeria monocytogenes: the mRNA phenotype of granuloma formation

In murine listeriosls, elimination of bacteria and immunityto re-infection critically depend on Thy-1⁺ CM^– cells,while cell-mediated inflammatory phenomena like delayed-typehypersensltlvlty and granuloma formation are mediated by CD4⁺T cells. In an attempt to correlate T cell phenotype and functionwith a particular set of cytokines produced in vivo, we examinedthe cytokine gene expression profile associated with the presenceor absence of CD4⁺ and/or CD8⁺ cells in the livers of mice duringexperimental infection with Usterta monocytogenes. T cell subsetdepletion was achieved by l.p. administration of saturatingamounts of the appropriate mAbs, and mRNA detection was carriedout using a qualitative and semi-quantitative polymerase chainreaction-based mRNA amplification protocol. In both primaryand secondary infection, the presence of CD4⁺ cells was a prerequisitefor granuloma formation, and was found to be closely associatedwith mRNA expression for IL-2, IL-3 and IL-4, a 5-fold increasein expression of tumor necrosis factor (TNF)- and granulocytemacrophage colony stimulating factor, and a 25-fold increasein expression of IFN- and TNF-ß mRNAs, suggestinga role for these cytokines in granuloma formation. In strikingcontrast, depletion of CD8⁺ cells did not result in reducedmRNA expression for any one of the cytokines studied, Implyingthat CD8⁺ T cell mediated cure and prevention of listerlosismay operate via qualitatively distinct mechanisms.