Antibody and T cell responses following vaccination with OVA targeted to MHC class II molecules or CCR1/3/5.

<p>(a,b) Supernatants of 293E cells transfected with the indicated plasmids were tested for secreted proteins in ELISA (a) and examined by Western blotting with anti-OVA mAb under reducing (+ME) or non-reducing (-ME) conditions (b). Vaccine proteins are indicated below lanes. (c-f) Mice were immunized once i.d. with 25 µg DNA/EP, as indicated. (c-e) Sera were assayed for total IgG (c), IgG1 (d) or IgG2a (e) against OVA. (f) Splenocytes collected at day 14 post immunization were stimulated <i>in vitro</i> with OVA protein or controls as indicated, and analyzed by an IFNγ EliSpot. *indicates p<0.008 and **p<0.002.</p

Characterization of fusion vaccine proteins.

<p>a) Schematic overview of homodimeric vaccine proteins. The fusion proteins consists of HA antigen connected to a targeting unit via a shortened Ig hinge and a dimerizing human γ3 CH3 domain and Ig hinge. As targeting units we used a scFv directed against the MHC class II molecule I-E^d (αMHCII-HA), or the mouse chemokine MIP-1α (MIP-1α-HA). For non-targeted controls, a scFv directed against the hapten NIP (αNIP-HA), or a mutated MIP-1α (MIP-1α(C11S)-HA), replaced functional targeting units. b) Supernatants of transfected 293E cells were examined by Western blotting with anti-HA mAb under reducing (-ME) or non-reducing (+ME) conditions. Vaccine proteins are indicated below lanes, and MW by arrows. c) Binding of vaccine proteins to anti-C_H3 mAb in Sandwich ELISA, followed by detection with an anti-HA mAb. d) Binding of vaccine proteins to MHCII I-E^d-transfected L cell fibroblasts. Vaccine proteins were detected by anti-HA mAb. e) Supernatants of 293E cells transfected with MIP-1α-HA or the mutated counterpart (C11S) were examined for chemotaxis. Recombinant human MIP-1α(rLD78β) was included as positive control. Chemotactic index is shown. f, g) Binding of vaccine proteins to CD11b⁺ BALB/c splenocytes.</p

Antibodies in sera after intradermal DNA vaccination.

<p>(a-g) Mice were immunized once i.d. with 25 µg DNA/EP as indicated (n = 6/group), and assayed for total IgG (a), IgG1 (b), IgG2a (c), IgG2b (d) and IgG3 (e) against PR8 in ELISA (mean+/-SEM). f) Hemagglutination-inhibition (HI) titers (mean+/-SEM) in sera. g) Sera from day 14 were assayed in a micro neutralization assay (PR8 virus). Dotted line indicates threshold for positive neutralization (50%). (h,i) Mice were immunized twice i.d. at days 0 and 50 as indicated by arrows (↑), and sera assayed in ELISA against PR8 for induced IgG1 (h) and IgG2a (i) (mean +/- SEM).</p

Targeted DNA fusion vaccines enhance immune responses after intramuscular delivery.

<p>Mice were vaccinated once i.m. with 25 µg DNA/electroporation (EP) as indicated (n = 6/group). (a-c) Serum samples were assayed for total IgG (a), IgG1 (b) and IgG2a (c) against PR8 in ELISA (mean+/-SEM). (d-f) Three weeks after vaccination, splenocytes were harvested and stimulated <i>in vitro</i> with either class II restricted HA peptides [d, (SVSSFERFEIFPK) or e, (HNTNGVTAACSHEG)], a class I restricted HA peptide [f, (IYSTVASSL)], or a control peptide (GYKDGNEYI). Frequencies of IFNγ-producing cells were evaluated by EliSpot. The control peptide did not elicit responses beyond that observed for NaCl. Horizontal lines indicate sample means.</p