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1、 Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright The American Society of Gene TherapyS210?557.Co-Administration of an AdenovirusEncoding the B Cell Stimulating Factor BAFF withHeat-Inactivated Pseudomonas aeruginosa Leadsto Increased Anti-pseudomonal Humoral ImmunityChristine Tertilt,1
2、Stefan Worgall,1 Michael C. Rivara,1 Ronald G.Crystal.11Weill Medical College of Cornell University, New York, NY.Pseudomonas aeruginosa is an important pathogen in conditionssuch as severe burns and cystic fibrosis, causing significant morbidityand mortality. Despite many efforts, no clinically eff
3、ective vaccineagainst P. aeruginosa is available to date especially in CF patients.In order to optimize an antipseudomonal vaccine and enhancemucosal immunity, we focused our study on the enhancement of theB cell response. Stimulating B cells through B cell activating moleculesduring immunization ag
4、ainst P. aeruginosa may increase theefficiency of a vaccine. B cell activating factor (BAFF), a 34kDsingle chain TNF-family member, is produced by activated antigenpresenting cells and stimulates B cells. Similar to other members ofthe TNF family, BAFF is secreted as a trimer by cleavage of theprote
5、in from the membrane. Following binding to its receptors on Bcells BAFF prolongs the life-span of activated B cells by preventingapoptosis and induces T cell independent immunoglobulin classswitch in vitro and is crucial for B cell development and for thegeneration of humoral immune responses in viv
6、o. Based on thesefindings, we hypothesized that the strong B cell stimulatoryproperties of BAFF can be exploited for the induction of immunityagainst P. aeruginosa and that the transient overexpression of BAFFduring immunization will favor the development of a rapid, stronghumoral immune response. T
7、o evaluate this concept, AdmBAFF,an E1-E3- adenovirus expressing full-length murine BAFF undercontrol of a CMV promotor, was constructed. After infection ofA549 cells with AdmBAFF, expression of full-length BAFF couldbe seen in the cell lysate by Western analysis, and a cleaved form ofBAFF was detec
8、ted in the supernatant, demonstrating that theoverexpressed BAFF was properly shed from the membrane. Toassess the potency of AdmBAFF to induce humoral immunityagainst P. aeruginosa in vivo, C57Bl/6 mice were injectedsubcutaneously with 7.5x1010 particle units of AdmBAFF togetherwith 105 cfu of heat
9、-inactivated P. aeruginosa strain PAO1. Miceinjected with AdNull or PBS plus PAO1 served as controls. Serumbinding antibodies against PAO1 were evaluated by ELISA 1, 2, 3,and 4 wk following immunization. Mice injected with PAO1 +AdmBAFF showed higher levels of PAO1-specific IgM titers 1 and2 wk afte
10、r immunization (1175 506 and 304 118, respectively)compared to mice immunized with PAO1 + AdNull (280 105 and73 48) or PAO1 + PBS (305 91 and 61 17, p0.02 for bothcomparisons at both timepoints). Similarly, higher PAO1-specifictotal IgG levels were observed 2, 3 and 4 wk following immunizationwith P
11、AO1 + AdmBAFF (373206, 15835 and 16499,respectively) compared to mice immunized with PAO1 + AdNull(7970, 47 45 and 38 32) or PAO1 + PBS (63 41, 30 9 and40 15, p0.3 all pairwise comparisons). At a dose of 1011 pu, anti-SARS-CoV neutralizing titers were also measurable (700160, intravenous;13020 intra
12、venous; 450210 subcutaneous). In contrast, naivemice and mice immunized with AdNull (an Ad with no transgene)had no detectable neutralizing antibody titers at any time point.Cellular immunity stimulated by immunization was evaluated inBALB/c mice injected intravenously with AdnS or AdNull at a doseo
13、f 1011 pu. After 2 wk, splenic CD8+ T cells were isolated andexposed to syngeneic target cells stably expressing S. Accumulationof intracellular IFN was observed in 8% of CD8+ cells as opposedto 2.5% of cells exposed to syngeneic cells not expressing S. Similarresults were observed in C57Bl/6 mice,
14、with 11.5% of CD8+ cellsaccumulating intracellular IFN in response to syngeneic cellsexpressing S, as opposed to 3.3% of CD8+ cells exposed to syngeneiccell lines not expressing S. The AdNull vaccinated controls did notstimulate antigen-specific IFN accumulation in the splenic CD8+cells. To determin
15、e the location of the major cellular epitopes in thespike glycoprotein, Ad vectors and target syngeneic cell linesexpressing the N-terminal domain (S1) or the C-terminal domain(S2) of spike were constructed. The specific accumulation of IFNin immunized BALB/c mice at 2 wk post-administration was mor
16、edependent on the S2 domain than the S1 domain (19% specificintracellular IFN accumulation using the S2 domain vs 6.3% withS1). We conclude that immunization with an Ad vaccine vectorexpressing the SARS-CoV S elicits high titers of SARS-CoV-neutralizing antibodies and that the S2 domain of spike contains theimmunodominant CD8+ T-cell epitopes.
