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Protective CD8+ T-cell responses to cytomegalovirus driven by rAAV/GFP/IE1 loading of dendritic cells.

Yu Y, Pilgrim P, Yan J, Zhou W, Jenkins M, Gagliano N, Bumm K, Cannon M, Milzani A, Dalle-Donne I, Kast WM, Cobos E, Chiriva-Internati M - J Transl Med (2008)

Bottom Line: As an extension of the versatility of the rAAV system, we incorporated immediate-early 1 (IE1), expressed in HCMV.CTLs were capable to lyse low doses of peptides pulsed into target cells.These data suggest that AAV-based antigen loading of DCs is highly effective for generating human CTL responses against HCMV antigens.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Hematology & Oncology, Texas Tech University Health Sciences Center and Southwest Cancer Treatment and Research Center, Lubbock, TX, USA. yuefei.yu@ttuhsc.edu

ABSTRACT

Background: Recent studies demonstrate that recombinant adeno-associated virus (rAAV)-based antigen loading of dendritic cells (DCs) generates in vitro, significant and rapid cytotoxic T-lymphocyte (CTL) responses against viral antigens.

Methods: We used the rAAV system to induce specific CTLs against CVM antigens for the development of cytomegalovirus HCMV) gene therapy. As an extension of the versatility of the rAAV system, we incorporated immediate-early 1 (IE1), expressed in HCMV. Our rAAV vector induced a strong stimulation of CTLs directed against the HCMV antigen IE1. We then investigated the efficiency of the CTLs in killing IE1 targeted cells.

Results: A significant MHC Class I-restricted, anti-IE1-specific CTL killing was demonstrated against IE1 positive peripheral blood mononuclear cells (PBMC) after one, in vitro, stimulation.

Conclusion: In summary, single PBMC stimulation with rAAV/IE1 pulsed DCs induces strong antigen specific-CTL generation. CTLs were capable to lyse low doses of peptides pulsed into target cells. These data suggest that AAV-based antigen loading of DCs is highly effective for generating human CTL responses against HCMV antigens.

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Immunofluorescence on HEK293 cells. Microphotographs show fluorescent labeling for AAV/IE1 (A, B) in HEK293 cells. A: original magnification: 20×; B: original magnification: 63×.
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Figure 1: Immunofluorescence on HEK293 cells. Microphotographs show fluorescent labeling for AAV/IE1 (A, B) in HEK293 cells. A: original magnification: 20×; B: original magnification: 63×.

Mentions: The goal of this study was to determine whether rAAV-based gene loading of IE1 genes into DCs could elicit a significant CTL response against IE1-positive target cell lines. This was the first time that the gene encoding IE1 was inserted into the AAV vector. First, the IE1 gene was amplified by PCR from plasmid pCGN-IE1. The IE1 cDNA obtained from pCGN-IE1 was inserted into the gutted AAV vector to generate AAV/IE1 as described in the materials and methods section. Figure 1A shows a structural map of the AAV/IE1 vector. In this vector, the IE1 gene was expressed from the AAV p5 promoter, which is known to be active in DCs [31]. After rAAV vector generation, we evaluated their ability to infect HEK293 cells. The rAAV-vector infected cells expressed the target antigens, as confirmed by immunofluorecence labeling, which showed the expression of IE1 transduced HEK293 cells. (Figure 1)


Protective CD8+ T-cell responses to cytomegalovirus driven by rAAV/GFP/IE1 loading of dendritic cells.

Yu Y, Pilgrim P, Yan J, Zhou W, Jenkins M, Gagliano N, Bumm K, Cannon M, Milzani A, Dalle-Donne I, Kast WM, Cobos E, Chiriva-Internati M - J Transl Med (2008)

Immunofluorescence on HEK293 cells. Microphotographs show fluorescent labeling for AAV/IE1 (A, B) in HEK293 cells. A: original magnification: 20×; B: original magnification: 63×.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2570669&req=5

Figure 1: Immunofluorescence on HEK293 cells. Microphotographs show fluorescent labeling for AAV/IE1 (A, B) in HEK293 cells. A: original magnification: 20×; B: original magnification: 63×.
Mentions: The goal of this study was to determine whether rAAV-based gene loading of IE1 genes into DCs could elicit a significant CTL response against IE1-positive target cell lines. This was the first time that the gene encoding IE1 was inserted into the AAV vector. First, the IE1 gene was amplified by PCR from plasmid pCGN-IE1. The IE1 cDNA obtained from pCGN-IE1 was inserted into the gutted AAV vector to generate AAV/IE1 as described in the materials and methods section. Figure 1A shows a structural map of the AAV/IE1 vector. In this vector, the IE1 gene was expressed from the AAV p5 promoter, which is known to be active in DCs [31]. After rAAV vector generation, we evaluated their ability to infect HEK293 cells. The rAAV-vector infected cells expressed the target antigens, as confirmed by immunofluorecence labeling, which showed the expression of IE1 transduced HEK293 cells. (Figure 1)

Bottom Line: As an extension of the versatility of the rAAV system, we incorporated immediate-early 1 (IE1), expressed in HCMV.CTLs were capable to lyse low doses of peptides pulsed into target cells.These data suggest that AAV-based antigen loading of DCs is highly effective for generating human CTL responses against HCMV antigens.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Hematology & Oncology, Texas Tech University Health Sciences Center and Southwest Cancer Treatment and Research Center, Lubbock, TX, USA. yuefei.yu@ttuhsc.edu

ABSTRACT

Background: Recent studies demonstrate that recombinant adeno-associated virus (rAAV)-based antigen loading of dendritic cells (DCs) generates in vitro, significant and rapid cytotoxic T-lymphocyte (CTL) responses against viral antigens.

Methods: We used the rAAV system to induce specific CTLs against CVM antigens for the development of cytomegalovirus HCMV) gene therapy. As an extension of the versatility of the rAAV system, we incorporated immediate-early 1 (IE1), expressed in HCMV. Our rAAV vector induced a strong stimulation of CTLs directed against the HCMV antigen IE1. We then investigated the efficiency of the CTLs in killing IE1 targeted cells.

Results: A significant MHC Class I-restricted, anti-IE1-specific CTL killing was demonstrated against IE1 positive peripheral blood mononuclear cells (PBMC) after one, in vitro, stimulation.

Conclusion: In summary, single PBMC stimulation with rAAV/IE1 pulsed DCs induces strong antigen specific-CTL generation. CTLs were capable to lyse low doses of peptides pulsed into target cells. These data suggest that AAV-based antigen loading of DCs is highly effective for generating human CTL responses against HCMV antigens.

Show MeSH
Related in: MedlinePlus