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

-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