<Emphasis Type="Italic">Photobacterium damselae</Emphasis> subsp. <Emphasis Type="Italic">piscicida</Emphasis>: an integrated view of a bacterial fish pathogen

Pasteurellosis, or pseudotuberculosis, is a bacterial septicaemia caused by the halophilic bacterium Photobacterium damselae subsp. piscicida (formerly Pasteurella piscicida). Although this disease was first described in wild populations of white perch and striped bass, currently the natural hosts of the pathogen are a wide variety of marine fish. The disease has great economic impact both in Japan, where it affects mainly yellowtail cultures, and in the Mediterranean area, due to the losses it causes in seabream and seabass farms. This microorganism serves as a perfect model to study a bacterial fish pathogen, either at an applied level, to resolve or to mitigate the high economic losses of fish farmers, or at a basic level, for a better understanding of P. damselae subsp. piscicida biology. This article discusses the methods employed in our laboratory to study the causative agent of pasteurellosis. It reviews important aspects, from the diverse procedures for the detection and isolation of the pathogen to the latest molecular studies that have allowed its correct taxonomic allocation. Characterization of some virulence mechanisms and the available methods to prevent the disease are also presented. Electronic Publication     

Prime/boost immunotherapy of HPV16-induced tumors with E7 protein delivered by Bordetella adenylate cyclase and modified vaccinia virus Ankara

The Bordetella adenylate cyclase toxoid (CyaA) targets cells expressing the α_Mβ₂ integrin receptor CD11b/CD18 (CR3 or Mac-1) and can penetrate into cytosol of professional antigen-presenting cells, such as dendritic cells. This allows us to use CyaA for delivery of passenger antigens into the cytosolic pathway of processing and MHC class I-restricted presentation, which can promote induction of antigen-specific CD8⁺ cytotoxic T-lymphocyte immune responses. We show here that vaccination with a genetically detoxified CyaA336/E7 protein, carrying the full-length oncoprotein E7 of the human papilloma virus 16 inserted at position 336 of the cell-invasive AC domain of CyaA, induces an E7-specific CD8⁺ T-cell immune response and confers on mice protective, as well as therapeutic immunity against challenge with TC-1 tumor cells expressing the E7 oncoprotein. The therapeutic efficacy of priming with the CyaA336/E7 vaccine could further be enhanced by a heterologous booster immunization with a highly attenuated modified vaccinia virus Ankara (MVA) expressing the E7 protein fused to the lysosome-associated membrane protein (LAMP1). These results establish the potential of CyaA as a new antigen delivery tool for prime/boost immunotherapy of tumors. This paper won the poster prize at the conference “Progress in Vaccination against Cancer 4”, PIVAC 4, held in Freudenstadt-Lauterbad, Black Forest, Germany, from 22 to 25 September 2004. For further material on this conference, please see the series of Symposium Papers, published     

中国脊髓灰质炎病毒疫苗株基因特征及其神经毒力的初步观察

中国脊髓灰质炎病毒疫苗株普遍存在基因变异的现象,如重组,点突变等。我们选出１０株有代表性的变异株,在具有人脊灰病毒受体基因的转基因小鼠ＰＶＲ－Ｔｇ２１中做毒力分析,发现Ｓａｂｉｎ １基因型与野毒基因型的重组株显示很强的神经毒力,其ＰＤ５０ｉｎＴＣＩＤ５０值为４．５,而Ｓｂｉｎ１标准株的ＰＤ５０值大于８０。另外两株Ｉ型疫苗株只有关键性核苷酸位点发生突变,只在位点５２５－发生突变的一株,其ＰＤ５０值为     

禽流感抗原表位在沙门氏菌菌毛的表达

目的:经抗原表位预测和同源性比较,禽流感M1蛋白位于58-66序列的九肽是A型流感病毒中保守并具有很强免疫原性的T细胞表位,鉴于鼠伤寒沙门氏菌LT2的SEF17菌毛基因agfA作为疫苗载体的优势,在其上构建引起机体细胞免疫的沙门氏茵口服活体重组疫苗,以求在人类对抗禽流感过程中发挥作用。方法:利用两步重叠延伸PCR和基因置换,将外源表位插入LT2茵毛,并利用抗生素抗性,温度敏感质粒,及茵毛的刚果红吸附能力筛选菌毛上插有外源抗原表位的重组菌,并通过测序进一步验证外源基因的插入。结果:两步重叠延伸PCR产物AB,CD,AD长度与理论大小530bp,423bp,932bp一致。两次转化PCR鉴定,产物长度与理论大小932bp,634bp一致。刚果红吸附测定,菌毛上插入有外源肽的菌落因吸附刚果红能力减弱呈粉色,对筛选出的KmS型粉色茵落的PCR鉴定,产物长度与理论大小417bp一致,测序结果也显示agfA中外源表位基因的插入。结论:禽流感M1蛋白位于58-66序列的T细胞表位成功插入沙门氏茵SEF17菌毛基因agfA。     

Selective recognition of oncogene promoter G-quadruplexes by Mg2+

The epitope sequences within the hemagglutinin (HA) of influenza A virus H3N2 at amino acid residues 173-181 and 227-239 that forms anti-parallel β-sheet structure are similarly recognized by human monoclonal antibodies (HuMAbs), B-1 and D-1 that we recently obtained using the peripheral blood lymphocytes from two influenza-vaccinated volunteers. Both HuMAbs showed strong global neutralization of H3N2 strains. Here we show the significant conservation of the β-sheet region consisting of the above-mentioned two epitope regions in H3N2. In addition, we also identified the corresponding regions with similar structure in other subtypes such as H1N1 and H5N1. These two regions are similarly located underneath the receptor-binding sites of individual subtypes. Analysis of those regions using sequences available from the Influenza Virus Resource at the National Center for Biotechnology Information revealed that compared with those in the known neutralizing epitopes A-E, those sequences were fairly conserved in human H3N2 (n = 7955), swine H1N1 (n = 360) and swine H3N2 (n = 235); and highly conserved in human H1N1 (n = 2722), swine-origin pandemic H1N1 (n = 1474), human H5N1 (n = 319) and avian H5N1 (n = 2349). Phylogenetic tree for these regions formed clearly separable clusters for H1N1, H3N2 and H5N1, irrespective of different host origin. These data may suggest a possible significance of those regions for development of alternative vaccine that could induce neutralizing antibodies reactive against wide-range of influenza virus strains.     

Regulation of immunity to Plasmodium: Implications from mouse models for blood stage malaria vaccine design

Malaria, a disease caused by the protozoan parasite Plasmodium, remains a serious healthcare problem in developing countries worldwide. While the host-parasite relationship in humans has been difficult to determine, the pliability of murine malaria models has enabled valuable contributions to the understanding of the pathogenesis of disease. Although no single model reflects precisely malaria infection of the human, different models collectively provide important information on the mechanisms of protective immunity and immunopathogenesis. This review summarizes progress towards understanding the broad spectrum of immune responsiveness to the blood stages of the malaria parasite during experimental infections in mice and highlights how examination of murine malarias sheds light on the factors involved in the modulation of vaccine-potentiated immunity.     

Immunisation with non-integral OMPs promotes pulmonary clearance of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can cause fatal acute lung infections in critically ill individuals. Lung damage due to chronic infections in cystic fibrosis sufferers is the major cause of morbidity and mortality in this group. The bacterium produces various immunomodulatory products that enable it to survive in the lung. Innate and increasing resistance to antibiotic therapy shown by this organism heightens the need for development of a vaccine. This study reports the identification of six non-integral protein antigens; Pa13, azurin, acyl carrier protein (ACP), amidase, aminopeptidase and KatE, purified from a mucoid strain of P. aeruginosa. N-terminal amino acid sequencing was used to identify these proteins and, based on their ascribed functions, determined that their normal cellular location was cytosolic. A rat model of acute pulmonary infection was used to investigate the ability of these protein antigens to enhance pulmonary clearance of a live P. aeruginosa challenge. Mucosal immunisation with four of the six antigens significantly enhanced bacterial clearance from both the lavage fluid and lung tissue. The greatest level of clearance was demonstrated for the antigens; KatE, aminopeptidase and amidase. Enhanced bacterial clearance was maintained when the antigens amidase and aminopeptidase were produced in recombinant form. When delivered parenterally, aminopeptidase demonstrated its continued efficacy as a vaccine candidate. This study has demonstrated that non-integral outer membrane proteins are antigenic and protective and warrant further investigation as potential components of a vaccine.     

Immuno-informatics based approaches to identify CD8+ T cell epitopes within the Leishmania donovani 3-ectonucleotidase in cured visceral leishmaniasis subjects

Leishmaniases are vector-borne diseases for which no vaccine exists. These diseases are caused by the Leishmania species complex. Activation of the CD8⁺ T cell is crucial for protection against intracellular pathogens, and peptide antigens are attractive strategies for the precise activation of CD8⁺ T in vaccine development against intracellular infections. The traditional approach to mine the epitopes is an arduous task. However, with the advent of immunoinformatics, in silico epitope prediction tools are available to expedite epitope identification. In this study, we employ different immunoinformatics tools to predict CD8⁺ T cell specific 9 mer epitopes presented by HLA-A*02 and HLA-B40 within the highly conserved 3′-ectonucleotidase of Leishmania donovani. We identify five promiscuous epitopes, which have no homologs in humans, theoretically cover 85% of the world's population and are highly conserved (100%) among Leishmania species. Presentation of selected peptides was confirmed by T2 cell line based HLA-stabilization assay, and three of them were found to be strong binders. The in vitro peptide stimulation of peripheral blood mononuclear cells (PBMC) from cured HLA-A02⁺ visceral leishmaniasis (VL) subjects produced significantly higher IFN-γ, IL-2 and IL-12 compared to no peptide control healthy subjects. Further, CD8⁺ cells from treated VL subjects produced significantly higher intracellular IFN-γ, lymphocyte proliferation and cytotoxic activity against selected peptides from the PBMCs of treated HLA-A02⁺ VL subjects. Thus, the CD8⁺ T cell specific epitopes shown in this study will speed up the development of polytope vaccines for leishmaniasis.     

Development of Leptospira in vitro potency assays: EU/industry experience and perspectives

Nobivac^® Lepto (MSD Animal Health) is a non-adjuvanted canine leptospirosis vaccine containing inactivated whole cells of Leptospira interrogans serogroup Canicola serovar Portlandvere and L. interrogans serogroup Icterohaemorrhagiae serovar Copenhageni. The current standard in vivo potency test is a hamster challenge test associated with major drawbacks such as animal suffering and poor reproducibility. Here, the quantification of antigenic mass by ELISA as a new in vitro potency test is described, supporting the 3Rs concept (replacement, reduction, and refinement of animal tests) and in accordance with European Pharmacopoeia Monograph 0447 (Canine Leptospirosis Vaccine [Inactivated]). The two corresponding sandwich ELISAs are based on monoclonal antibodies specific for immunodominant leptospiral lipopolysaccharide epitopes. Protection in passive immunization experiments demonstrate that these monoclonal antibodies recognize key protective antigens in currently licensed human and veterinary whole cell Leptospira vaccines. The high precision and robustness renders the two ELISAs much more reliable correlates of potency in dogs than the hamster potency test. The recent approval of these assays for a new canine leptospirosis vaccine is an important contribution to the 3Rs in quality control testing of Leptospira vaccines.     

减毒鼠伤寒沙门氏菌全长hpaA基因工程菌的构建

为构建表达HpaA蛋白的重组减毒鼠伤寒沙门氏菌 ,并探讨以减毒鼠伤寒沙门氏菌为载体构建H .pylori疫苗株的意义 ,应用PCR法从H .pylori基因组DNA中扩增 783bp的hpaA基因 ,经酶切 连接反应将其克隆入原核表达质粒pTrc99A的NcoⅠ SalⅠ位点 ,并进行了核苷酸序列测定。重组质粒转化减毒鼠伤寒沙门氏菌SL3 2 6 1 ,提取重组菌质粒 ,PCR和酶切鉴定 ,筛选阳性克隆。用SDS PAGE电泳和Westernblot进行HpaA表达分析和鉴定 ,用薄层扫描分析HpaA含量。重组菌C5 7BL 6小鼠喂灌 ,分批两d和 1 0d后处死小鼠 ,取脾和末段回肠进行细菌培养 ,挑菌落提质粒鉴定。结果表明 ,经PCR和酶切证实 ,构建了含 783bphpaA基因的重组原核表达质粒 ,并将后者成功转化了减毒鼠伤寒沙门氏菌。重组菌能表达约3 0kDHpaA蛋白 ,重组HpaA量约占全菌体蛋白量的 3 8 9% ,Westernblot证实其有免疫反应性。小鼠重组菌喂灌两d或 1 0d后 ,脾和末段回肠均发现携目的基因的菌落。这些结果提示 ,构建了表达H .pyloriHpaA的重组减毒…