The luminal part of the DC-Lamp protein is not required for induction of antigen specific T cell responses by means of antigen-DCLamp mRNA electroporated dendritic cells

Brenda De Keersmaecker; Carlo Heirman; Sabine Allard; Aude Bonehill; Jurgen Corthals; Kris Thielemans; Joeri Aerts

Abstract

-Previous studies showed that therapeutic immunization of HIV-1 infected patients by means of vaccination with autologous DC electroporated with mRNA encoding HIV antigens can induce antigen-specific T cell responses. Linking the viral antigen to a HLA class II-targeting sequence, such as DC-LAMP (Lysosome Associated Membrane Protein), in the mRNA construct results in presentation of antigenic peptides in both HLA class I and class II molecules and therefore enhances the induced T-cell responses.
In this study, we compared fusion constructs with or without the lumenal part of the DC-LAMP protein as described by Marques et al*. A human codon optimized consensus Gag sequence and of a chimeric cDNA sequence encompassing Tat, Rev and Nef codons (TaReNef) were cloned into a vector containing the DC-LAMP sequence with or without its lumenal domain: pSig-Gag-DC-LAMPLumTMCy versus pSig-Gag-DC-LAMPTMCy versus pGag, and pSig-TaReNef-DC-LAMPLumTMCy versus pSig-TaReNef-DC-LAMPTMCy.
The expression of Gag, after DC electroporation with the in vitro synthesized mRNA was analyzed by FACS staining. For all Gag constructs, >90% positivity was observed. Both in overnight ELISPOT assays and after in vitro stimulation during one week, T cell responses induced by this Gag protein lacking the DC-LAMP-derived sequence (pGag) did not exceed background values. DC electroporated with Sig-Antigen-DC-LAMPLumTMCy and Sig-Antigen-DC-LAMPTMCy were able to elicit similar levels of antigen-specific T cell responses for both Gag (n=4) and TaReNef (n=3).
We conclude that DC-LAMP mediated antigen targeting is absolutely required for optimal T cell stimulation, but that in our experimental setup, the lumenal part of DC-LAMP does not improve the overall induction of antigen specific T cell responses.

* Marques, E.T.A, et al., HIV-1 p55Gag encoded in the lysosome-associated membrane protein-1 as a DNA plasmid vaccine chimera is highly expressed, traffics to the major histocompatibility class II compartment, and elicits enhanced immune responses, J. Biol. Chem., 2003

Original language	English
Title of host publication	25 years of HIV, Institut Pasteur, Paris, France
Publication status	Published - 2008
Event	Finds and Results from the Swedish Cyprus Expedition: A Gender Perspective at the Medelhavsmuseet - Stockholm, Sweden Duration: 21 Sept 2009 → 25 Sept 2009

Conference

Conference	Finds and Results from the Swedish Cyprus Expedition: A Gender Perspective at the Medelhavsmuseet
Country/Territory	Sweden
City	Stockholm
Period	21/09/09 → 25/09/09

Keywords

dendritic cells
T-cell response
electroporation

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Cite this

@inbook{12ad6ac275514e55b910499c31ac2a1b,

title = "The luminal part of the DC-Lamp protein is not required for induction of antigen specific T cell responses by means of antigen-DCLamp mRNA electroporated dendritic cells",

abstract = "-Previous studies showed that therapeutic immunization of HIV-1 infected patients by means of vaccination with autologous DC electroporated with mRNA encoding HIV antigens can induce antigen-specific T cell responses. Linking the viral antigen to a HLA class II-targeting sequence, such as DC-LAMP (Lysosome Associated Membrane Protein), in the mRNA construct results in presentation of antigenic peptides in both HLA class I and class II molecules and therefore enhances the induced T-cell responses. In this study, we compared fusion constructs with or without the lumenal part of the DC-LAMP protein as described by Marques et al*. A human codon optimized consensus Gag sequence and of a chimeric cDNA sequence encompassing Tat, Rev and Nef codons (TaReNef) were cloned into a vector containing the DC-LAMP sequence with or without its lumenal domain: pSig-Gag-DC-LAMPLumTMCy versus pSig-Gag-DC-LAMPTMCy versus pGag, and pSig-TaReNef-DC-LAMPLumTMCy versus pSig-TaReNef-DC-LAMPTMCy. The expression of Gag, after DC electroporation with the in vitro synthesized mRNA was analyzed by FACS staining. For all Gag constructs, >90% positivity was observed. Both in overnight ELISPOT assays and after in vitro stimulation during one week, T cell responses induced by this Gag protein lacking the DC-LAMP-derived sequence (pGag) did not exceed background values. DC electroporated with Sig-Antigen-DC-LAMPLumTMCy and Sig-Antigen-DC-LAMPTMCy were able to elicit similar levels of antigen-specific T cell responses for both Gag (n=4) and TaReNef (n=3). We conclude that DC-LAMP mediated antigen targeting is absolutely required for optimal T cell stimulation, but that in our experimental setup, the lumenal part of DC-LAMP does not improve the overall induction of antigen specific T cell responses. * Marques, E.T.A, et al., HIV-1 p55Gag encoded in the lysosome-associated membrane protein-1 as a DNA plasmid vaccine chimera is highly expressed, traffics to the major histocompatibility class II compartment, and elicits enhanced immune responses, J. Biol. Chem., 2003",

keywords = "dendritic cells, T-cell response, electroporation",

author = "{De Keersmaecker}, Brenda and Carlo Heirman and Sabine Allard and Aude Bonehill and Jurgen Corthals and Kris Thielemans and Joeri Aerts",

year = "2008",

language = "English",

booktitle = "25 years of HIV, Institut Pasteur, Paris, France",

note = "Finds and Results from the Swedish Cyprus Expedition: A Gender Perspective at the Medelhavsmuseet ; Conference date: 21-09-2009 Through 25-09-2009",

}

De Keersmaecker, B, Heirman, C, Allard, S, Bonehill, A, Corthals, J, Thielemans, K & Aerts, J 2008, The luminal part of the DC-Lamp protein is not required for induction of antigen specific T cell responses by means of antigen-DCLamp mRNA electroporated dendritic cells. in 25 years of HIV, Institut Pasteur, Paris, France. Finds and Results from the Swedish Cyprus Expedition: A Gender Perspective at the Medelhavsmuseet, Stockholm, Sweden, 21/09/09.

The luminal part of the DC-Lamp protein is not required for induction of antigen specific T cell responses by means of antigen-DCLamp mRNA electroporated dendritic cells. / De Keersmaecker, Brenda; Heirman, Carlo; Allard, Sabine et al.
25 years of HIV, Institut Pasteur, Paris, France. 2008.

Research output: Chapter in Book/Report/Conference proceeding › Meeting abstract (Book)

TY - CHAP

T1 - The luminal part of the DC-Lamp protein is not required for induction of antigen specific T cell responses by means of antigen-DCLamp mRNA electroporated dendritic cells

AU - De Keersmaecker, Brenda

AU - Heirman, Carlo

AU - Allard, Sabine

AU - Bonehill, Aude

AU - Corthals, Jurgen

AU - Thielemans, Kris

AU - Aerts, Joeri

PY - 2008

Y1 - 2008

N2 - -Previous studies showed that therapeutic immunization of HIV-1 infected patients by means of vaccination with autologous DC electroporated with mRNA encoding HIV antigens can induce antigen-specific T cell responses. Linking the viral antigen to a HLA class II-targeting sequence, such as DC-LAMP (Lysosome Associated Membrane Protein), in the mRNA construct results in presentation of antigenic peptides in both HLA class I and class II molecules and therefore enhances the induced T-cell responses. In this study, we compared fusion constructs with or without the lumenal part of the DC-LAMP protein as described by Marques et al*. A human codon optimized consensus Gag sequence and of a chimeric cDNA sequence encompassing Tat, Rev and Nef codons (TaReNef) were cloned into a vector containing the DC-LAMP sequence with or without its lumenal domain: pSig-Gag-DC-LAMPLumTMCy versus pSig-Gag-DC-LAMPTMCy versus pGag, and pSig-TaReNef-DC-LAMPLumTMCy versus pSig-TaReNef-DC-LAMPTMCy. The expression of Gag, after DC electroporation with the in vitro synthesized mRNA was analyzed by FACS staining. For all Gag constructs, >90% positivity was observed. Both in overnight ELISPOT assays and after in vitro stimulation during one week, T cell responses induced by this Gag protein lacking the DC-LAMP-derived sequence (pGag) did not exceed background values. DC electroporated with Sig-Antigen-DC-LAMPLumTMCy and Sig-Antigen-DC-LAMPTMCy were able to elicit similar levels of antigen-specific T cell responses for both Gag (n=4) and TaReNef (n=3). We conclude that DC-LAMP mediated antigen targeting is absolutely required for optimal T cell stimulation, but that in our experimental setup, the lumenal part of DC-LAMP does not improve the overall induction of antigen specific T cell responses. * Marques, E.T.A, et al., HIV-1 p55Gag encoded in the lysosome-associated membrane protein-1 as a DNA plasmid vaccine chimera is highly expressed, traffics to the major histocompatibility class II compartment, and elicits enhanced immune responses, J. Biol. Chem., 2003

AB - -Previous studies showed that therapeutic immunization of HIV-1 infected patients by means of vaccination with autologous DC electroporated with mRNA encoding HIV antigens can induce antigen-specific T cell responses. Linking the viral antigen to a HLA class II-targeting sequence, such as DC-LAMP (Lysosome Associated Membrane Protein), in the mRNA construct results in presentation of antigenic peptides in both HLA class I and class II molecules and therefore enhances the induced T-cell responses. In this study, we compared fusion constructs with or without the lumenal part of the DC-LAMP protein as described by Marques et al*. A human codon optimized consensus Gag sequence and of a chimeric cDNA sequence encompassing Tat, Rev and Nef codons (TaReNef) were cloned into a vector containing the DC-LAMP sequence with or without its lumenal domain: pSig-Gag-DC-LAMPLumTMCy versus pSig-Gag-DC-LAMPTMCy versus pGag, and pSig-TaReNef-DC-LAMPLumTMCy versus pSig-TaReNef-DC-LAMPTMCy. The expression of Gag, after DC electroporation with the in vitro synthesized mRNA was analyzed by FACS staining. For all Gag constructs, >90% positivity was observed. Both in overnight ELISPOT assays and after in vitro stimulation during one week, T cell responses induced by this Gag protein lacking the DC-LAMP-derived sequence (pGag) did not exceed background values. DC electroporated with Sig-Antigen-DC-LAMPLumTMCy and Sig-Antigen-DC-LAMPTMCy were able to elicit similar levels of antigen-specific T cell responses for both Gag (n=4) and TaReNef (n=3). We conclude that DC-LAMP mediated antigen targeting is absolutely required for optimal T cell stimulation, but that in our experimental setup, the lumenal part of DC-LAMP does not improve the overall induction of antigen specific T cell responses. * Marques, E.T.A, et al., HIV-1 p55Gag encoded in the lysosome-associated membrane protein-1 as a DNA plasmid vaccine chimera is highly expressed, traffics to the major histocompatibility class II compartment, and elicits enhanced immune responses, J. Biol. Chem., 2003

KW - dendritic cells

KW - T-cell response

KW - electroporation

M3 - Meeting abstract (Book)

BT - 25 years of HIV, Institut Pasteur, Paris, France

T2 - Finds and Results from the Swedish Cyprus Expedition: A Gender Perspective at the Medelhavsmuseet

Y2 - 21 September 2009 through 25 September 2009