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

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

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

Comparison of recombinant 6×HIS-tagged full length wild type βGal and N-terminally truncated ΔβGal.

<p>(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.</p

βGal is not a Th1 adjuvant for other antigens.

<p>(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.</p

Immune Reactions against Gene Gun Vaccines Are Differentially Modulated by Distinct Dendritic Cell Subsets in the Skin

<div><p>The skin accommodates multiple dendritic cell (DC) subsets with remarkable functional diversity. Immune reactions are initiated and modulated by the triggering of DC by pathogen-associated or endogenous danger signals. In contrast to these processes, the influence of intrinsic features of protein antigens on the strength and type of immune responses is much less understood. Therefore, we investigated the involvement of distinct DC subsets in immune reactions against two structurally different model antigens, <i>E</i>. <i>coli</i> beta-galactosidase (betaGal) and chicken ovalbumin (OVA) under otherwise identical conditions. After epicutaneous administration of the respective DNA vaccines with a gene gun, wild type mice induced robust immune responses against both antigens. However, ablation of langerin⁺ DC almost abolished IgG1 and cytotoxic T lymphocytes against betaGal but enhanced T cell and antibody responses against OVA. We identified epidermal Langerhans cells (LC) as the subset responsible for the suppression of anti-OVA reactions and found regulatory T cells critically involved in this process. In contrast, reactions against betaGal were not affected by the selective elimination of LC, indicating that this antigen required a different langerin⁺ DC subset. The opposing findings obtained with OVA and betaGal vaccines were not due to immune-modulating activities of either the plasmid DNA or the antigen gene products, nor did the differential cellular localization, size or dose of the two proteins account for the opposite effects. Thus, skin-borne protein antigens may be differentially handled by distinct DC subsets, and, in this way, intrinsic features of the antigen can participate in immune modulation.</p></div

Epidermal LC are dispensable for βGal-specific immunity but down-regulate OVA-specific T cell functions.

<p>LangDTR or B6 WT mice were injected once with 1μg DT. Epidermal sheets were stained for langerin expression 3 wks later. (B) Repopulation of langerin⁺ DC in the dermis analyzed by FACS. LdDC from back skin were identified as CD11c⁺ EpCam^neg langerin⁺ cells. Groups of langDTR and B6 WT mice, treated once with 1μg DT were GG-immunized with either, pCI-OVA or pCI-βGal starting 1wk after DT treatment and boosted after 1wk. Splenocytes were in vitro restimulated with (C) CTL peptides DAPIYTNV for βGal- (left panel) or SIINFEKL for OVA-immunized mice (right panel) or with (E) βGal protein (left panel) or OVA protein (right panel) and analyzed for IFNγ secretion by ELISPOT technique. (D) Specific lysis of DAPIYTNV-pulsed (left panel) or SIINFEKL-pulsed (right panel) syngeneic target cells injected into GG-immunized langDTR and WT mice. (F) IgG1 antibody titers were analyzed by ELISA. Data represent means ± SD of groups of 4–5 mice and representative at least of two experiments.</p

Antigen-specific immune modulation is not due to differential cellular localization of the vaccine gene product.

<p>(A) Western blots of cell lysates (L) and supernatants (SN) of BHK cells transfected with the respective gene vaccines pCI-OVA or pCI-cherryOVA. LC-depleted langDTR and DT-treated B6 WT mice were GG-immunized with pCI-cherryOVA twice at a 1 wk interval, starting 1wk after a single DT injection. One week after the boost splenocytes were restimulated in vitro with (B) SIINFEKL or (<i>D</i>, <i>E</i>) OVA protein and analyzed for (B, D) IFNγ and (E) IL4 secretion by ELISPOT technique. (C) Specific lysis of SIINFEKL-pulsed syngeneic target cells injected into langDTR and WT mice immunized with pCI-cherryOVA. (F) Serum IgG1 titer from pCI-cherryOVA-immunized mice was analyzed by ELISA. Data represent means ± SD of groups of 5 mice. Data shown are representative of two independent experiments.</p