Signal sequence deletion and fusion to tetanus toxoid epitope augment antitumor immune responses to a human carcinoembryonic antigen (CEA) plasmid DNA vaccine in a murine test system

Abstract

Carcinoembryonic antigen (CEA, CEACAM5) is expressed on several human carcinomas including colon cancer. CEA contains signal peptides that target the protein through the endoplasmic reticulum and to the cell membrane. We constructed a plasmid DNA vaccine encoding a truncated CEA (DCEA), devoid of its signal peptides, and demonstrated that it was retained inside the cell, while full-length CEA (wtCEA) was expressed on the membrane. We hypothesized that intracellular retention of DCEA would enhance MHC class I presentation of CEA peptides, thus favoring cellular immune responses. In addition, a promiscuous T-helper epitope (Q830-L844 of tetanus toxoid) was fused to the N-terminal of the truncated CEA gene (tetDCEA). C57BL/6 mice immunized with DNA encoding wtCEA or tetDCEA developed both humoral and cellular immune responses to CEA. SCID mice transplanted with spleen cells from tetDCEA but not wtCEA-immunized C57BL/6 mice showed strong suppression of tumor growth after inoculation of human CEA-expressing colon carcinoma cells. Immune spleen cell populations depleted for either B, T or both B and T cells were active, indicating that effector cells might also reside in other populations. The present approach to manipulating antigen presentation may open new possibilities for immunotherapy against colon and other CEA-secreting carcinomas.

Proteoliposomal formulations of an HIV-1 gp41-based miniprotein elicit a lipid-dependent immunodominant response overlapping the 2F5 binding motif

“…This protein contains the C-terminal heptad repeat (HR2), the MPER and the transmembrane (TM) domains of gp41. In order to overcome potential limitations in MHC-II presentation of the processed antigen37, a TT promiscuous T-helper epitope 830 QYIKANSKFIGITEL 844 38 was fused in frame with the TM domain. A six-histidine tag was added at the C-terminal end to allow protein purification.…”

“…To overcome potential limitation in MHC-II presentation after antigen processing in C57 BL/6 mice and to increase immunogenicity, the original gp41-Min protein was engrafted in the C-terminal moiety with a tetanus toxoid (TT) promiscuous T-helper epitope3738. Furthermore, liposomes included several structural lipids overrepresented in the viral membrane (CHOL and SM)31 and lipids that may influence immune responses by promoting selective capture by antigen presenting cells (APC).…”

Mutations Conferring Drug Resistance Affect Eukaryotic Expression of HIV Type 1 Reverse Transcriptase

“…18 After 48 hr, cells were washed with phosphate-buffered saline (PBS), collected by centrifugation, resuspended (10 6 cells/ml), and lysed in the buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1-5 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethyleneglycoletetraacetic acid (EGTA), 1% NP-40 or 1% Triton X-100, 0.25% deoxycholate (DOC), 0.1% sodium dodecyl sulfate (SDS), and protease inhibitor cocktail for general use (Sigma, Deisenhofen, Germany). Cell debris was sedimented by centrifugation.…”

“…Immunofluorescence microscopy was carried out as described previously. 11,18 Pulse-chase analysis Cells were grown for 24 hr after transfection. Medium was withdrawn, and cells were incubated for another 45 min in the methionine-free/serum-free Dulbecco's modified Eagle medium (DMEM) (Sigma).…”

“…20 Mutations led to the loss of some sites (11)(12)(13)(14)(15) and acquisition of the new ones (12)(13)(14)(15)(16)(17)(18)(19)(20)(21). The balance was shifted toward a higher number of cleavage sites in RT mutants as compared to the wildtype protein.…”