Sven Mostböck

Priming of CD8+ and CD4+ T Cells in Experimental Leishmaniasis Is Initiated by Different Dendritic Cell Subtypes

The biological role of Langerin+ dendritic cells (DCs) such as Langerhans cells and a subset of dermal DCs (dDCs) in adaptive immunity against cutaneous pathogens remains enigmatic. Thus, we analyzed the impact of Langerin+ DCs in adaptive T cell-mediated immunity toward Leishmania major parasites in a Lang-DTR mouse model that allows conditional diphtheria toxin (DT)-induced ablation of Langerin+ DCs in vivo. For the first time, infection experiments with DT-treated Lang-DTR mice revealed that proliferation of L. major-specific CD8+ T cells is significantly reduced during the early phase of the immune response following depletion of Langerin+ DCs. Consequently, the total number of activated CD8+ T cells within the draining lymph node and at the site of infection is diminished. Furthermore, we show that the impaired CD8+ T cell response is due to the absence of Langerin+ dDCs and not Langerhans cells. Nevertheless, the CD4+ T cell response is not altered and the infection is cleared as effectively in DT-treated Lang-DTR mice as in control mice. This clearly demonstrates that Langerin+ DCs are, in general, dispensable for an efficient adaptive immune response against L. major parasites. Thus, we propose a novel concept that, in the experimental model of leishmaniasis, priming of CD4+ T cells is mediated by Langerin− dDCs, whereas Langerin+ dDCs are involved in early priming of CD8+ T cells.

Genetic Vaccination against Malaria Infection by Intradermal and Epidermal Injections of a Plasmid Containing the Gene Encoding the Plasmodium berghei Circumsporozoite Protein

ABSTRACT The circumsporozoite protein (CSP) from the surface of sporozoite stage Plasmodium sp. malaria parasites is among the most important of the malaria vaccine candidates. Gene gun injection of genetic vaccines encoding Plasmodium berghei CSP induces a significant protective effect against sporozoite challenge; however, intramuscular injection does not. In the present study we compared the immune responses and protective effects induced by P. berghei CSP genetic vaccines delivered intradermally with a needle or epidermally with a gene gun. Mice were immunized three times at 4-week intervals and challenged by a single infectious mosquito bite. Although 50 times more DNA was administered by needle than by gene gun, the latter method induced significantly greater protection against infection. Intradermal injection of the CSP genetic vaccine induced a strong Th1-type immune response characterized by a dominant CSP-specific immunoglobulin G2a (IgG2a) humoral response and high levels of gamma interferon produced by splenic T cells. Gene gun injection induced a predominantly Th2-type immune response characterized by a high IgG1/IgG2a ratio and significant IgE production. Neither method generated measurable cytotoxic T lymphocyte activity. The results indicate that a gene gun-mediated CS-specific Th2-type response may be best for protecting against malarial sporozoite infection when the route of parasite entry is via mosquito bite.

Immunize and disappear—Safety-optimized mRNA vaccination with a panel of 29 allergens

BACKGROUND The spread of type I allergic diseases has reached epidemic dimensions. The success of therapeutic intervention is limited, and hence prophylactic vaccination is now seriously considered. However, immunization of healthy individuals requires safety standards far beyond those applicable for therapeutic approaches. mRNAs encoding allergen molecules represent an attractive tool for preventive vaccination because of the inherent safety features of this vaccine type. OBJECTIVE In the current study we investigated whether mRNA constructs would be capable of protecting against type I allergic reactions in a murine model using the grass pollen allergen Phl p 5 and 28 other major pollen, food, animal, mold, and latex allergens. METHODS BALB/c mice were immunized intradermally either with conventional or replicase-based mRNA constructs. Subsequently, animals were sensitized by means of subcutaneous injection of allergen/alum, followed by airway provocation. IgG1/IgG2a/IgE titers were determined by using ELISAs. Allergen-specific functional IgE levels were assessed by using the basophil release assay. Measurement of cytokines in splenocyte cultures and bronchoalveolar lavage fluids were performed by using enzyme-linked immunosorbent spot assays/sandwich ELISAs. Eosinophil and CD8(+) counts in bronchoalveolar lavage specimens were determined by means of flow cytometry. Airway hyperreactivity was assessed with whole-body plethysmography and invasive resistance/dynamic compliance measurement. RESULTS mRNA vaccination proved its antiallergic efficacy in terms of IgG subclass distribution, functional IgE suppression, reduction of IL-4 and IL-5 levels, induction of IFN-gamma-producing cells, and reduction of airway hyperreactivity and eosinophil counts in the lung. CONCLUSION Immunization with mRNA induces T(H)1-biased immune responses similar to those elicited through DNA-based vaccination but additionally offers the advantage of a superior safety profile.

Isoforms of the Major Allergen of Birch Pollen Induce Different Immune Responses after Genetic Immunization

Background: Recent publications indicate that immunization with plasmid DNA encoding allergens might represent a potential approach in allergen–specific immunotherapy. Objective: In the present study we have compared the immune responses induced by plasmid DNA encoding for two isoforms of Bet v 1, the major allergen of birch pollen. Methods: BALB/c mice were injected intradermally with plasmid DNA encoding for the genes of Bet v 1a (pCMV–Beta) and Bet v 1d (pCMV–Betd). In addition, the effect of immunostimulatory DNA sequences was investigated by appending and/or coinjecting CpG motifs. Antibody responses and IFN–γ and IL–4 levels were measured by ELISA. Allergen–specific proliferation was determined by incorporation of [3H]–thymidine. Results: The two isoforms induced a similar humoral response. The lack of any IgE production and the ratio of IgG1 to IgG2a clearly indicated a Th–1–type response. The antisera against both isoforms were highly cross–reactive, which was supported by the energy plot indicating similar folding of the two protein isoforms. However, determination of IFN–γ and IL–4 in the serum elicited a strikingly different cytokine profile during the course of the immune response. In contrast to pCMV–Beta, pCMV–Betd caused no significant allergen–specific proliferation and induced only marginal levels of the key cytokines. Conclusions: Based on the assumption that the induction of a strong Th–1 type response is a prerequisite for successful treatment of allergy, our results favor the use of isoform Bet v 1a in combination with CpG motifs for a novel type of allergen immunotherapy based on plasmid DNA immunization. Additionally, the data also confirm the assumption that the antigen itself can have a marked influence on the immune response after genetic immunization.

Removal of the circumsporozoite protein (CSP) glycosylphosphatidylinositol signal sequence from a CSP DNA vaccine enhances induction of CSP-specific Th2 type immune responses and improvesprotection against malaria infection

The C terminus of the circumsporozoite protein (CSP) is anchored to the parasite cell membrane by a glycosylphosphatidylinositol (GPI) glycolipid. This GPI signal sequence functions poorly in heterologous eukaryotic cells, causing CSP retention within internal cell organelles during genetic immunization. Cellular location of antigen has quantitative and qualitative effects on immune responses induced by genetic immunization. Removal of the GPI signal sequence had a profound effect on induction and efficacy of CSP‐specific immune response after genetic immunization of BALB/c mice with a gene gun. The CSP produced from the plasmid lacking the GPI anchor signal sequence (CSP‐A) was secreted and soluble, but that produced by the CSP+A plasmid was not. The CSP‐A plasmid induced a highly polarized Th2 type response, in which the CSP‐specific IgG antibody titer was three‐ to fourfold higher, and the protective effect was significantly greater than that induced by the CSP+A plasmid. Thus, these two physical forms of CSP induced quantitatively and qualitatively different immune responses that also differed in protective efficacy. Engineering plasmid constructs for proper cellular localization of gene products is a primary consideration for the preparation of optimally efficacious DNA vaccines.

A DNA vaccine encoding the outer surface protein C from Borrelia burgdorferi is able to induce protective immune responses.

The outer surface protein C (OspC) of Borrelia burgdorferi, the spirochete that causes Lyme disease, is a promising candidate for a vaccine against borreliosis. BALB/c and C3H/HeJ mice were immunized either with recombinant OspC protein or with plasmid DNA encoding OspC fused to the human tissue plasminogen activator leader sequence (pCMV-TPA/ZS7). The influence of the route of administering the DNA and the use of oligodeoxynucleotides containing CpG-motifs on the development of the immune response was investigated. In both mouse strains, protein as well as gene-gun immunization induced Th2 type responses, whereas needle injection of plasmid DNA resulted in Th1 type antibody production. Co-injection of CpG-motifs did not significantly modify the response type in any immunization group, as indicated by only marginal changes of antibody subclass distribution. The protection rate after challenge with 10(4) B. burgdorferi organisms per mouse was between 80% and 100% for all groups. These results demonstrate, for the first time, that a DNA vaccine encoding OspC of B. burgdorferi is suitable for inducing protection against Lyme borreliosis.

Myeloid suppressor cells require membrane TNFR2 expression for suppressive activity

TNF and TNF receptor type 2 (TNFR2) have been shown to be important for generation of myeloid‐derived suppressor cells (MDSC). In order to analyze whether and how TNFR2 passes the effect of TNF on, myeloid cells from TNFR2‐deficient mice were compared to respective cells from wild‐type mice. Primary TNFR2‐deficient myeloid cells showed reduced production of NO and IL‐6 which was attributable to CD11b+ CD11c− Ly6C+ Ly6G− immature monocytic MDSC. TNFR2‐deficient MDSC isolated from bone marrow were less suppressive for T cell proliferation compared to WT‐derived MDSC. These differences on myeloid cells between the two mouse lines were still observed after co‐culture of bone marrow cells from the two mouse lines together during myeloid cell differentiation, which demonstrated that the impaired functional capacity of TNFR2‐deficient cells was independent of soluble factors but required membrane expression of TNFR2. Similarly, adoptive transfer of TNFR2‐deficient bone marrow cells into wild‐type hosts did not rescue the TNFR2‐specific phenotype of bone marrow‐derived myeloid cells. Therefore, membrane TNFR2 expression determines generation and function of monocytic MDSC.

IL-2/Anti-IL-2 Antibody Complex Enhances Vaccine-Mediated Antigen-Specific CD8+ T Cell Responses and Increases the Ratio of Effector/Memory CD8+ T Cells to Regulatory T Cells

IL-2 is well described as a cytokine with two markedly distinct functionalities: as a necessary signal during CD4+ and CD8+ T cell activation/expansion and as an essential cytokine for the maintenance of CD4+CD25+FoxP3+ T cells (regulatory T (TREG) cells) during homeostasis. In this study we demonstrate for the first time that, compared with the use of IL-2 alone, a complex of IL-2 and anti-IL-2 Ab (IL-2 complex) enhances the effectiveness of a viral vaccine in a mouse model with known Ag specificity. IL-2 complex led to an increase in the number of Ag-specific effector/memory CD8+ T cells, cytokine production, and CTL lysis following Ag-specific restimulation in a vaccination setting. Our results further demonstrate that this effect is temporary and declines over the course of a few days after the IL-2 complex treatment cycle. Moreover, in contrast to the use of IL-2 alone, IL-2 complex greatly increased the ratio of effector/memory CD8+ T cells to TREG cells. This phenomenon can thus potentially be used in the enhancement of immune responses to vaccination.

Improvement of the immune response against plasmid DNA encoding OspC of Borrelia by an ER-targeting leader sequence.

The present study outlines the characterization of a DNA-based immune response against the OspC antigen, one of the most promising candidates for a Borrelia vaccine. Balb/c mice were injected intradermally with plasmid DNA encoding the OspC gene (lacking the natural leader sequence) under transcriptional control of the cytomegalovirus (CMV) promotor. Immunization with this construct elicited only a marginal response, which was drastically improved by a fusion construct containing the human tissue plasminogen activator (hTPA) signal sequence. The results indicate that for DNA-based immunization against OspC an ER-targeting signal may be necessary for both antibody production as well as cellular immune responses.

Sepsis leads to a reduced antigen-specific primary antibody response

Immunosuppression, impaired cytokine production and high susceptibility to secondary infections are characteristic for septic patients, and for mice after induction of polymicrobial septic peritonitis by sublethal cecal ligation and puncture (CLP). Here, we demonstrate that CLP markedly altered subsequent B‐cell responses. Total IgG and IgM levels, as well as the memory B‐cell response, were increased in septic mice, but antigen‐specific primary antibody production was strongly impaired. We found that two days after CLP, CD11b+ splenocytes were activated as demonstrated by the increased expression of activation markers, expression of arginase and production of NO by immature myeloid cells. The in vivo clearance of a bacterial infection was not impaired. DCs demonstrated reduced IL‐12 production and altered antigen presentation, resulting in decreased proliferation but enhanced IFN‐γ production by CD4+ cells. CD4+ T cells from mice immunized on day 2 after CLP showed reduced Th1 and Th2 cytokine production. In addition, there was an increase in Treg cells. Interestingly, levels of immature B cells decreased but levels of mature B cells increased two days after CLP. However, adoptive transfer of naïve CD4+ T cells, naïve B cells, or naïve DCs did not rescue the antigen‐specific antibody response.