Structural integrity of the antigen is a determinant for the induction of T-helper type-1 immunity in mice by gene gun vaccines against E. coli beta-galactosidase.

The type of immune response is critical for successful protection and typically determined by pathogen-associated danger molecules. In contrast, protein antigens are usually regarded as passive target structures. Here, we provide evidence that the structure of the antigen can profoundly influence the type of response that is elicited under else identical conditions. In mice, gene gun vaccines induce predominantly Th2-biased immune reactions against most antigens. One exception is E. coli beta-galactosidase (βGal) that induces a balanced Th1/Th2 response. Because both, the delivered material (plasmid DNA-coated gold particles) as well as the procedure (biolistic delivery to the skin surface) is the same as for other antigens we hypothesized that Th1 induction could be a function of βGal protein expressed in transfected cells. To test this we examined gene gun vaccines encoding structural or functional variants of the antigen. Employing a series of gene gun vaccines encoding individual structural domains of βGal, we found that neither of them induced IgG2a antibodies. Even disruption of the homo-tetramer association of the native protein by deletion of a few N-terminal amino acids was sufficient to abrogate IgG2a production. However, enzymatically inactive βGal with only one point mutation in the catalytic center retained the ability to induce Th1 reactions. Thus, structural but not functional integrity of the antigen must be retained for Th1 induction. βGal is not a Th1 adjuvant in the classical sense because neither were βGal-transgenic ROSA26 mice particularly Th1-biased nor did co-administration of a βGal-encoding plasmid induce IgG2a against other antigens. Despite this, gene gun vaccines elicited Th1 reactions to antigens fused to the open reading frame of βGal. We interpret these findings as evidence that different skin-borne antigens may be differentially handled by the immune system and that the three-dimensional structure of an antigen is an important determinant for this

Gene gun immunization with a βGal-OVA fusion construct elicited IgG2a against OVA.

<p>(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).</p

Gene gun vaccines encoding individual domains elicited predominately Th2-associated IgG1 antibodies.

<p>(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.</p

Disruption of the tetrameric structure of βGal reduced immunogenicity and abrogated IgG2a production.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102280#pone-0102280-g003" target="_blank">fig. 3A</a> 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).</p

Loss of enzymatic activity of βGal did not influence the type of immune response.

<p>(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.</p