Increasing the Vaccine Potential of Live M. bovis BCG by Coadministration with Plasmid DNA Encoding a Tuberculosis Prototype Antigen

The attenuated live M. bovis Bacille-Calmette-Guérin (BCG) is still the sole vaccine used against tuberculosis, but confers only variable efficacy against adult pulmonary tuberculosis (TB). Though no clear explanation for this limited efficacy has been given, different hypotheses have been advanced, such as the waning of memory T-cell responses, a reduced antigenic repertoire and the inability to induce effective CD8+ T-cell responses, which are known to be essential for latent tuberculosis control. In this study, a new BCG-based vaccination protocol was studied, in which BCG was formulated in combination with a plasmid DNA vaccine. As BCG is routinely administered to neonates, we have evaluated a more realistic approach of a simultaneous intradermal coadministration of BCG with pDNA encoding the prototype antigen, PPE44. Strongly increased T- and B-cell responses were observed with this protocol in C57BL/6 mice when compared to the administration of only BCG or in combination with an empty pDNA vector, as measured by Th1-type spleen cell cytokine secretion, specific IgG antibodies, as well as specific IFN-γ producing/cytolytic-CD8+ T-cells. Moreover, we observed a bystander activation induced by the coding plasmid, resulting in increased immune responses against other non-plasmid encoded, but BCG-expressed, antigens. In all, these results provide a proof of concept for a new TB vaccine, based on a BCG-plasmid DNA combination

Macrophages and Fibroblasts Differentially Contribute to Tattoo Stability

International audienceBackground: Little information is available about the complexity and function of skin cells contributing to the high stability of tattoos. It has been shown that dermal macrophages play an important role in the storage and maintenance of pigment particles. By contrast, the impact of dermal fibro-blasts, forming the connective tissue of the skin, on the stability of the tattoo is not known. Method: In this study, we compared the cell number and the particle load in dermal macrophages versus dermal fibroblasts, isolated from tail skin of tattooed mice. Results: Microscopic analysis revealed that both cell populations contained the tattoo particles, although in largely different amounts. A small number of mac-rophages with high side scatter intensity contained a large quantity of pigment particles, whereas a high number of der-mal fibroblasts harbored only a few pigment particles. Using the CD64 dtr mouse model that allows for selective, diphtheria toxin-mediated depletion of macrophages, we have previously shown that macrophages hold the tattoo in place by capture-release and recapture cycles. In the tattooed skin of macrophage-depleted mice, the content of pigment particles in fibroblasts did not change; however, the total number of fibroblasts carrying particles increased. Conclusion: The present study demonstrates that dermal macrophages and fibroblasts contribute in different ways to the tattoo stability and further improves our knowledge on tattoo persistence

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

Langerin(+) Dermal Dendritic Cells Are Critical for CD8(+) T Cell Activation and IgH gamma-1 Class Switching in Response to Gene Gun Vaccines

International audienceThe C-type lectin langerin/CD207 was originally discovered as a specific marker for epidermal Langerhans cells (LC). Recently, additional and distinct subsets of langerin(+) dendritic cells (DC) have been identified in lymph nodes and peripheral tissues of mice. Although the role of LC for immune activation or modulation is now being discussed controversially, other langerin(+) DC appear crucial for protective immunity in a growing set of infection and vaccination models. In knock-in mice that express the human diphtheria toxin receptor under control of the langerin promoter, injection of diphtheria toxin ablates LC for several weeks whereas other langerin(+) DC subsets are replenished within just a few days. Thus, by careful timing of diphtheria toxin injections selective states of deficiency in either LC only or all langerin(+) cells can be established. Taking advantage of this system, we found that, unlike selective LC deficiency, ablation of all langerin(+) DC abrogated the activation of IFN-γ-producing and cytolytic CD8(+) T cells after gene gun vaccination. Moreover, we identified migratory langerin(+) dermal DC as the subset that directly activated CD8(+) T cells in lymph nodes. Langerin(+) DC were also critical for IgG1 but not IgG2a Ab induction, suggesting differential polarization of CD4(+) T helper cells by langerin(+) or langerin-negative DC, respectively. In contrast, protein vaccines administered with various adjuvants induced IgG1 independently of langerin(+) DC. Taken together, these findings reflect a highly specialized division of labor between different DC subsets both with respect to Ag encounter as well as downstream processes of immune activation

Epidermal langerhans cells are dispensable for humoral and cell-mediated immunity elicited by gene gun immunization.

Gene gun immunization, i.e., bombardment of skin with DNA-coated particles, is an efficient method for the administration of DNA vaccines. Direct transfection of APC or cross-presentation of exogenous Ag acquired from transfected nonimmune cells enables MHC-I-restricted activation of CD8(+) T cells. Additionally, MHC-II-restricted presentation of exogenous Ag activates CD4(+) Th cells. Being the principal APC in the epidermis, Langerhans cells (LC) seem ideal candidates to accomplish these functions. However, the dependence on LC of gene gun-induced immune reactions has not yet been demonstrated directly. This was primarily hampered by difficulties to discriminate the contributions of LC from those of other dermal dendritic cells. To address this problem, we have used Langerin-diphtheria toxin receptor knockin mice that allow for selective inducible ablation of LC. LC deficiency, even over the entire duration of experiments, did not affect any of the gene gun-induced immune functions examined, including proliferation of CD4(+) and CD8(+) T cells, IFN-gamma secretion by spleen cells, Ab production, CTL activity, and development of protective antitumor immunity. Together, our data show that gene gun immunization is capable of inducing humoral and cell-mediated immune reactions independently of LC