Figure 5

Selection of epitopes and production of epitope-specific IgG by a complex of peptide and Lipoplex(O). (a-c) Selection of B cell epitopes from hTM4SF5 protein by immunization with complexes consisting of each predicted hTM4SF5 peptide and Lipoplex(O). BALB/c mice (n = 3/group) were immunized with complexes consisting of each peptide (hTM4SF5R1, hTM4SF5R2-1, hTM4SF5R2-2, hTM4SF5R2-3, hTM4SF5R2-4, and hTM4SF5R2-5) and Lipoplex(O) (Lipoplex(O) + peptide). (a) Amounts of anti-each peptide-specific total IgG. (b) Amounts of anti-hTM4SF5R2-3 peptide-specific IgG isotypes. (c) Titers of anti-hTM4SF5R2-3 peptide-specific IgG isotypes. (d) The kinetics of IgG production. BALB/c mice (n = 3/group) were immunized with the complex of hTM4SF5R2-3 peptide and Lipoplex(O). BALB/c mice were injected i.p. with hTM4SF5R2-3 peptide and Lipoplex(O) complex. The sera were collected one day before each injection and 10 days after final injection, and an ELISA kit was used to assay amounts of the peptide-specific total IgG, IgG1, IgG2a and IgM were assayed with an ELISA kit. (e) Induction of DOPE:CHEMS encapsulation-dependent peptide-specific IgG production. BALB/c mice (n = 5/group) were immunized with 50 μg of hTM4SF5R2-3 peptide and 50 μg of MB-ODN 4531(O) coencapsulated in a DOPE:CHEMS (1:1 ratio), DSPC:Chol (45:55 ratio), DSPC:CHEMS:PEG-PE (6:4:0.3 ratio), Chol:DOPE:PEG-PE (4:6:0.06 ratio), or Dc-Chol:DOPE:PEG-PE (4:6:0.06 ratio) complex. The sera were collected, and amounts of anti-hTM4SF5R2-3 peptide-specific total IgG were assayed with an ELISA kit. (f) Effect of GC dinucleotide and phosphorothioate backbone modification. BALB/c mice (n = 5/group) were immunized with 50 μg of hTM4SF5R2-3 peptide and one of the following complexes (50 μg): Lipoplex(O), or LipoplexGC(O). The sera were collected, and amounts of peptide-specific total IgG were assayed with an ELISA kit. The results are expressed as a Mean ± SD of three or five mice. These experiments were performed 3 times with similar results. **P < 0.01 (vs PBS control).