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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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Related in: MedlinePlus

Virus stock titers. DNA extracted from the purified virus of AAV/IE1 was used as the template of PCR. The DNA from 1000 μl, 500 μl and 250 μl purified virus was tested, respectively. We used three blank wells, with water, as negative controls. EG = encapsulated genomes.
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Figure 2: Virus stock titers. DNA extracted from the purified virus of AAV/IE1 was used as the template of PCR. The DNA from 1000 μl, 500 μl and 250 μl purified virus was tested, respectively. We used three blank wells, with water, as negative controls. EG = encapsulated genomes.

Mentions: Virus stock titers were determined by real-time PCR (Figure 2). We assessed the linearity of the real-time PCR by using a dilution row of the AAV/IE1 plasmid that would serve as standard curve in all further experiments. The obtained fragments corresponded to the expected size and no additional bands could be detected by gel electrophoresis, showing the specificity and selectivity of the PCR. We did not observe signals from the template sample in either the amplification plot or the agarose gel photograph (data not shown).


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)

Virus stock titers. DNA extracted from the purified virus of AAV/IE1 was used as the template of PCR. The DNA from 1000 μl, 500 μl and 250 μl purified virus was tested, respectively. We used three blank wells, with water, as negative controls. EG = encapsulated genomes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Virus stock titers. DNA extracted from the purified virus of AAV/IE1 was used as the template of PCR. The DNA from 1000 μl, 500 μl and 250 μl purified virus was tested, respectively. We used three blank wells, with water, as negative controls. EG = encapsulated genomes.
Mentions: Virus stock titers were determined by real-time PCR (Figure 2). We assessed the linearity of the real-time PCR by using a dilution row of the AAV/IE1 plasmid that would serve as standard curve in all further experiments. The obtained fragments corresponded to the expected size and no additional bands could be detected by gel electrophoresis, showing the specificity and selectivity of the PCR. We did not observe signals from the template sample in either the amplification plot or the agarose gel photograph (data not shown).

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