Figure 1.
Unlike other antigens, βGal gene gun vaccines induced a balanced Th1/2 antibody response.
(A) Ratio of antigen-specific IgG2a:IgG1 in B6 and BALB/c mice (n = 3 to 8) two weeks after two gene gun immunizations with plasmid vaccines encoding different antigens administered at 2-week intervals. With each shot, 1 µg of plasmid was administered for all antigens; pCI-βGal was additionally tested with a dose of 100 ng/shot. *(p<0,05) and **(p<0,01) indicate groups that elicited significantly less IgG1 than IgG2a. (B) anti-βGal IgG1 and IgG2a in individual B6 (circles) or BALB/c (triangles) mice after gene gun immunization with pCI-βGal. Cumulative data from 8 independent experiments. (C) anti-βGal serum IgG1 and IgG2a in B6 mice (n = 5) 2 weeks after one (1×GG) or two gene gun immunizations (2×GG), and 2 or 8 weeks after a third immunization (3×GG). (D) IgG isotype titers and (E) cytotoxic activity against βGal in cytokine knockouts on BALB/c background (groups of n = 5, each). (F) anti-βGal serum IgG1 and IgG2a 2 weeks after 2 gene gun shots in Tlr4-deficient B10ScCR and in B6 wt mice. *, p<0,05 vs. WT.
Figure 2.
Gene gun vaccines encoding individual domains elicited predominately Th2-associated IgG1 antibodies.
(A) Sera from B6 mice (n = 5) gene gun-immunized with pCI-βGal twice at a 14 d interval, tested by ELISA on recombinant βGal domains or, for comparison, full length βGal (FL). (B) Sera from B6 mice (n = 4–5) gene gun-immunized with plasmids encoding individual βGal domains D1–D5, or full length βGal (FL), respectively, tested on full length βGal-coated ELISA plate wells. Mice were immunized twice at a 14 d interval and sera were collected 14 d after the boost. Diagrams present means +/− s.d. of log(10) isotype ratios of IgG2a:IgG1, i.e. positive values indicate predominating IgG2a and, hence, Th1-biased reactions.
Figure 3.
Disruption of the tetrameric structure of βGal reduced immunogenicity and abrogated IgG2a production.
(A) Size exclusion chromatography and Western Blot (inset) of lysates of BHK21 cells (ATCC CCL-10) transfected with, either, wild type (wt, dashed line) or N-terminally truncated ΔβGal (ΔN, solid line); fractions analyzed for βGal by ELISA. (B) Enzymatic βGal activity of serially diluted lysates of transfected cells (from samples shown in fig. 3A inset) as determined by luminescence and expressed as kilo-photon counts (kpc) per second. (C) in-vivo CTL activity in B6 mice (n = 5) 14 d after 2 gene gun immunizations with pCI-ΔβGal or the full length wild type sequence (WT βGal). (D,E) Serum IgG isotypes of individual mice, 2 weeks after two gene gun immunizations with pCI-βGal (D) or pCI-ΔβGal (E).
Figure 4.
Comparison of recombinant 6×HIS-tagged full length wild type βGal and N-terminally truncated ΔβGal.
(A) Circular dichroism spectra recorded at 20°C (left) or at 95°C (right). (B) Dynamic light scattering analysis of full length (WT) and truncated 6×HIS-tagged ΔβGal. (C) Size exclusion chromatography of the truncated ΔβGal protein. (D) Time course of microsomal degradation of full length (WT) βGal and truncated 6×HIS-tagged ΔβGal, calculated from densitometric analysis of SDS-PAGE samples drawn at the indicated time points (inset). (E) Serum IgG elicited by full length βGal (WT) or truncated 6×HIS-tagged ΔβGal. 10 µg were injected i.d. without adjuvant in groups of B6 mice (n = 5) twice at a 14 d interval. Sera were collected 2 weeks after the boost.
Figure 5.
Loss of enzymatic activity of βGal did not influence the type of immune response.
(A) Enzymatic activity of wild type (WT) βGal and the E537A mutant in transfected BHK21 cells, measured by luminogenic substrate hydrolysis and expressed as relative light units (RLU). Inset: Western blot of cell lysates (left: E537A, right: wild type βGal). (B) in vivo CTL assay and (C) Serum IgG in B6 mice (n = 5) 2 weeks after the second of 2 gene gun immunizations with, either, pCI-βGal (WT) or pCI-E537A-βGal. *, p<0.05 vs. WT. (D) Frequency of IFNγ-producing spleen cells of mice shown in (B, C) after restimulation in-vitro with either recombinant βGal protein or CTL-peptide.
Figure 6.
Gene gun immunization with a βGal-OVA fusion construct elicited IgG2a against OVA.
(A) Enzymatic βGal activity in BHK21 cells transfected with, either, βGal or 3 different fusion constructs: “cytoplasmic” OVA with a deletion of AA 20–145 fused to the N-terminus of βGal (cOVA-βGal), full length OVA fused to, either, the N-terminus (OVA-βGal) or the C-terminus of βGal (βGal-OVA). Inset: Western blot of BHK21 cells transfected with the indicated plasmids and developed with anti-βGal antiserum. (B) IFNγ production by spleen cells from B6 mice (n = 5) gene gun-immunized 3× at 2 week intervals with the fusion construct pCI-βGal-OVA, measured by cytokine ELISA of culture supernatants after 48 hrs of restimulation in-vitro with OVA (left) or βGal (right). Mice immunized with pCI-OVA (left) or pCI-βGal (right) were included for comparison. IFNγ in non-stimulated medium controls were below detection limits (not shown). (C) βGal-specific and (D) OVA-specific IgG isotypes in sera of mice shown in (B).
Figure 7.
βGal is not a Th1 adjuvant for other antigens.
(A) OVA-specific and (B) βGal-specific IgG in B6 mice (n = 5) gene gun-immunized with the fusion construct pCI-βGal-OVA or a mixture of pCI-OVA and pCI-βGal plasmids co-precipitated onto gold particles. For reference, groups of mice immunized with pCI-OVA (A) or pCI-βGal (B) were included. (C) OVA-specific IgG isotypes in B6 wild type mice or in mice constitutively expressing βGal (ROSA26), 2 weeks after 2 gene gun immunizations with pCI-OVA administered 2 weeks apart. (D) Serum IgG isotypes in B6 mice (n = 5) 2 weeks after two rounds of gene gun immunization, separated by a 2 week interval, with plasmid constructs in that secretion of OVA was prohibited by fusion to the C-terminus of, either, GFP, mCherry or βGal.