• Neutral Lipopolyplexes for In Vivo Delivery of Conventional and Replicative RNA Vaccine.

      Perche, Federico; Clemençon, Rudy; Schulze, Kai; Ebensen, Thomas; Guzmán, Carlos A; Pichon, Chantal; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier, 2019-09-06)
      Nucleic acid vaccination relies on injecting DNA or RNA coding antigen(s) to induce a protective immune response. RNA vaccination is being increasingly used in preclinical and clinical studies. However, few delivery systems have been reported for in vivo delivery of RNA of different sizes. Using a tripartite formulation with RNA, cationic polymer, and anionic liposomes, we were able to encapsulate RNA into neutral lipopolyplexes (LPPs). LPPs were stable in vitro and successfully delivered conventional RNA and replicative RNA to dendritic cells in cellulo. Their injection led to reporter gene expression in mice. Finally, administration of LPP-Replicon RNA (RepRNA) led to an adaptive immune response against the antigen coded by the RepRNA. Accordingly, LPPs may represent a universal formulation for RNA delivery.
    • Mucosal CD8+ T cell responses induced by an MCMV based vaccine vector confer protection against influenza challenge.

      Zheng, Xiaoyan; Oduro, Jennifer D; Boehme, Julia D; Borkner, Lisa; Ebensen, Thomas; Heise, Ulrike; Gereke, Marcus; Pils, Marina C; Krmpotic, Astrid; Guzmán, Carlos A; et al. (PLOS, 2019-09-01)
      Cytomegalovirus (CMV) is a ubiquitous β-herpesvirus that establishes life-long latent infection in a high percentage of the population worldwide. CMV induces the strongest and most durable CD8+ T cell response known in human clinical medicine. Due to its unique properties, the virus represents a promising candidate vaccine vector for the induction of persistent cellular immunity. To take advantage of this, we constructed a recombinant murine CMV (MCMV) expressing an MHC-I restricted epitope from influenza A virus (IAV) H1N1 within the immediate early 2 (ie2) gene. Only mice that were immunized intranasally (i.n.) were capable of controlling IAV infection, despite the greater potency of the intraperitoneally (i.p.) vaccination in inducing a systemic IAV-specific CD8+ T cell response. The protective capacity of the i.n. immunization was associated with its ability to induce IAV-specific tissue-resident memory CD8+ T (CD8TRM) cells in the lungs. Our data demonstrate that the protective effect exerted by the i.n. immunization was critically mediated by antigen-specific CD8+ T cells. CD8TRM cells promoted the induction of IFNγ and chemokines that facilitate the recruitment of antigen-specific CD8+ T cells to the lungs. Overall, our results showed that locally applied MCMV vectors could induce mucosal immunity at sites of entry, providing superior immune protection against respiratory infections.
    • The STING activator c-di-AMP exerts superior adjuvant properties than the formulation poly(I:C)/CpG after subcutaneous vaccination with soluble protein antigen or DEC-205-mediated antigen targeting to dendritic cells.

      Volckmar, Julia; Knop, Laura; Stegemann-Koniszewski, Sabine; Schulze, Kai; Ebensen, Thomas; Guzmán, Carlos A; Bruder, Dunja; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier, 2019-08-14)
      Vaccination is the most efficient strategy to protect from infectious diseases and the induction of a protective immune response not only depends on the nature of the antigen, but is also influenced by the vaccination strategy and the co-administration of adjuvants. Therefore, the precise monitoring of adjuvant candidates and their immune modulatory properties is a crucial step in vaccine development. Here, one central aspect is the induction of appropriate humoral and cellular effector mechanisms. In our study we performed a direct comparison of two promising candidates in adjuvant development, the STING activator bis-(3,5)-cyclic dimeric adenosine monophosphate (c-di-AMP) and the Toll-like receptor ligand formulation poly(I:C)/CpG. These were evaluated in C57BL/6 mice using the model antigen ovalbumin (OVA) in subcutaneous vaccination with soluble protein as well as in a dendritic cell (DC) targeting approach (αDEC-OVA). Strikingly, c-di-AMP as compared to poly(I:C)/CpG resulted in significantly higher antigen-specific IgG antibody levels when used in immunization with soluble OVA as well as in antigen targeting to DC. In vaccination with soluble OVA, c-di-AMP induced a significantly stronger CTL, Th1 and IFNγ-producing CD8+ memory T cell response than poly(I:C)/CpG. The response was CTL and Th1 cell dominated, a profile shared by both adjuvants. In the context of targeting OVA to DC, c-di-AMP induced significantly increased Th1 and Th2 cell responses as compared to poly(I:C)/CpG. Interestingly, the Th1 response dominated the overall T cell response only when c-di-AMP was used, indicating a distinct modulatory property of c-di-AMP when the DC targeting immunization approach was exploited. Taken together, we describe superior properties of c-di-AMP as compared to poly(I:C)/CpG in subcutaneous vaccination with soluble antigen as well as antigen targeting to DC. This indicates exceptionally effective adjuvant properties for c-di-AMP and provides compelling evidence of its potential for further adjuvant development, especially also when using DC targeting approaches.
    • In vivo Neutralization of Pro-inflammatory Cytokines During Secondary Streptococcus pneumoniae Infection Post Influenza A Virus Infection

      Sharma-Chawla, Niharika; Stegemann-Koniszewski, Sabine; Christen, Henrike; Boehme, Julia D.; Kershaw, Olivia; Schreiber, Jens; Guzmán, Carlos A.; Bruder, Dunja; Hernandez-Vargas, Esteban A.; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers Media SA, 2019-08-14)
      An overt pro-inflammatory immune response is a key factor contributing to lethal pneumococcal infection in an influenza pre-infected host and represents a potential target for therapeutic intervention. However, there is a paucity of knowledge about the level of contribution of individual cytokines. Based on the predictions of our previous mathematical modeling approach, the potential benefit of IFN-γ- and/or IL-6-specific antibody-mediated cytokine neutralization was explored in C57BL/6 mice infected with the influenza A/PR/8/34 strain, which were subsequently infected with the Streptococcus pneumoniae strain TIGR4 on day 7 post influenza. While single IL-6 neutralization had no effect on respiratory bacterial clearance, single IFN-γ neutralization enhanced local bacterial clearance in the lungs. Concomitant neutralization of IFN-γ and IL-6 significantly reduced the degree of pneumonia as well as bacteremia compared to the control group, indicating a positive effect for the host during secondary bacterial infection. The results of our model-driven experimental study reveal that the predicted therapeutic value of IFN-γ and IL-6 neutralization in secondary pneumococcal infection following influenza infection is tightly dependent on the experimental protocol while at the same time paving the way toward the development of effective immune therapies.
    • Systems Immunology Characterization of Novel Vaccine Formulations for Mycoplasma hyopneumoniae Bacterins.

      Matthijs, Anneleen M F; Auray, Gaël; Jakob, Virginie; García-Nicolás, Obdulio; Braun, Roman O; Keller, Irene; Bruggman, Rémy; Devriendt, Bert; Boyen, Filip; Guzman, Carlos A; et al. (Frontiers, 2019-01-01)
      We characterized five different vaccine candidates and a commercial vaccine in terms of safety, immunogenicity and using a systems vaccinology approach, with the aim to select novel vaccine candidates against Mycoplasma hyopneumoniae. Seven groups of six M. hyopneumoniae-free piglets were primo- and booster vaccinated with the different experimental bacterin formulations, the commercial vaccine Hyogen® as a positive control or PBS as a negative control. The experimental bacterin was formulated with cationic liposomes + c-di-AMP (Lipo_AMP), cationic liposomes + Toll-like receptor (TLR) 2/1, TLR7, and TLR9 ligands (TLR ligands; Lipo_TLR), micro-particles + TLR ligands (PLGA_TLR), squalene-in-water emulsion + TLR ligands (SWE_TLR), or DDA:TDB liposomes (Lipo_DDA:TDB). Lipo_DDA:TDB and Lipo_AMP were the most potent in terms of serum antibody induction, and Lipo_DDA:TDB, Lipo_AMP, and SWE_TLR significantly induced Th1 cytokine-secreting T-cells. Only PLGA_TLR appeared to induce Th17 cells, but was unable to induce serum antibodies. The transcriptomic analyses demonstrated that the induction of inflammatory and myeloid cell blood transcriptional modules (BTM) in the first 24 h after vaccination correlated well with serum antibodies, while negative correlations with the same modules were found 7 days post-vaccination. Furthermore, many cell cycle and T-cell BTM upregulated at day seven correlated positively with adaptive immune responses. When comparing the delivery of the identical TLR ligands with the three formulations, we found SWE_TLR to be more potent in the induction of an early innate immune response, while the liposomal formulation more strongly promoted late cell cycle and T-cell BTM. For the PLGA formulation we found signs of a delayed and weak perturbation of these BTM. Lipo_AMP was found to be the most potent vaccine at inducing a BTM profile similar to that correlating with adaptive immune response in this and other studies. Taken together, we identified four promising vaccine candidates able to induce M. hyopneumoniae-specific antibody and T-cell responses. In addition, we have adapted a systems vaccinology approach developed for human to pigs and demonstrated its capacity in identifying early immune signatures in the blood relating to adaptive immune responses. This approach represents an important step in a more rational design of efficacious vaccines for pigs.
    • The Combination Vaccine Adjuvant System Alum/c-di-AMP Results in Quantitative and Qualitative Enhanced Immune Responses Post Immunization.

      Ebensen, Thomas; Delandre, Simon; Prochnow, Blair; Guzmán, Carlos A; Schulze, Kai (2019-01-01)
    • Invariant NKT Cell-Mediated Modulation of ILC1s as a Tool for Mucosal Immune Intervention.

      Trittel, Stephanie; Vashist, Neha; Ebensen, Thomas; Chambers, Benedict J; Guzmán, Carlos A; Riese, Peggy; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2019-01-01)
      Non-NK group 1 innate lymphoid cells (ILC1s), mainly investigated in the mucosal areas of the intestine, are well-known to contribute to anti-parasitic and anti-bacterial immune responses. Recently, our group revealed that lung ILC1s become activated during murine influenza infection, thereby contributing to viral clearance. In this context, worldwide seasonal influenza infections often result in severe disease outbreaks leading to high morbidity and mortality. Therefore, new immune interventions are urgently needed. In contrast to NK cells, the potential of non-NK ILC1s to become functionally tailored by immune modulators to contribute to the combat against mucosal-transmitted viral pathogens has not yet been addressed. The present study aimed at assessing the potential of ILC1s to become modulated by iNKT cells activated through the CD1d agonist αGalCerMPEG. Our results demonstrate an improved functional responsiveness of murine lung and splenic ILC1s following iNKT cell stimulation by the mucosal route, as demonstrated by enhanced surface expression of TNF-related apoptosis-inducing ligand (TRAIL), CD49a and CD28, and increased secretion of IFNγ. Interestingly, iNKT cell stimulation also induced the expression of the immune checkpoint molecules GITR and CTLA-4, which represent crucial points of action for immune regulation. An in vivo influenza infection model revealed that intranasal activation of ILC1s by αGalCerMPEG contributed to increased viral clearance as shown by reduced viral loads in the lungs. The findings that ILC1s can become modulated by mucosally activated iNKT cells in a beneficial manner emphasize their up to now underestimated potential and renders them to be considered as targets for novel immune interventions.
    • Large-scale production of megakaryocytes in microcarrier-supported stirred suspension bioreactors.

      Eicke, Dorothee; Baigger, Anja; Schulze, Kai; Latham, Sharissa L; Halloin, Caroline; Zweigerdt, Robert; Guzman, Carlos A; Blasczyk, Rainer; Figueiredo, Constança; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-07-05)
      Megakaryocytes (MKs) are the precursors of platelets (PLTs) and may be used for PLT production in vivo or in vitro, as well as a source for PLT-derived growth factors. Induced pluripotent stem cells represent an unlimited cell source for the in vitro production of MKs. This study aimed at developing an effective, xeno-free and scalable system to produce high numbers of MKs. In particular, microcarrier beads-assisted stirred bioreactors were evaluated as a means of improving MK yields. This method resulted in the production of 18.7 × 10
    • Influenza-Activated ILC1s Contribute to Antiviral Immunity Partially Influenced by Differential GITR Expression.

      Vashist, Neha; Trittel, Stephanie; Ebensen, Thomas; Chambers, Benedict J; Guzmán, Carlos A; Riese, Peggy; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2018-01-01)
      Innate lymphoid cells (ILCs) represent diversified subsets of effector cells as well as immune regulators of mucosal immunity and are classified into group 1 ILCs, group 2 ILCs, and group 3 ILCs. Group 1 ILCs encompass natural killer (NK) cells and non-NK ILCs (ILC1s) and mediate their functionality via the rapid production of IFN-γ and TNF-α. The current knowledge of ILC1s mainly associates them to inflammatory processes. Much less is known about their regulation during infection and their capacity to interact with cells of the adaptive immune system. The present study dissected the role of ILC1s during early influenza A virus infection, thereby revealing their impact on the antiviral response. Exploiting in vitro and in vivo H1N1 infection systems, a cross-talk of ILC1s with cells of the innate and the adaptive immunity was demonstrated, which contributes to anti-influenza immunity. A novel association of ILC1 functionality and the expression of the glucocorticoid-induced TNFR-related protein (GITR) was observed, which hints toward a so far undescribed role of GITR in regulating ILC1 responsiveness. Overexpression of GITR inhibits IFN-γ production by ILC1s, whereas partial reduction of GITR expression can reverse this effect, thereby regulating ILC1 functionality. These new insights into ILC1 biology define potential intervention targets to modulate the functional properties of ILC1s, thus contributing toward the development of new immune interventions against influenza.
    • Signatures of T and B Cell Development, Functional Responses and PD-1 Upregulation After HCMV Latent Infections and Reactivations in Nod.Rag.Gamma Mice Humanized With Cord Blood CD34 Cells.

      Theobald, Sebastian J; Khailaie, Sahamoddin; Meyer-Hermann, Michael; Volk, Valery; Olbrich, Henning; Danisch, Simon; Gerasch, Laura; Schneider, Andreas; Sinzger, Christian; Schaudien, Dirk; et al. (Frontiers, 2018-01-01)
      uman cytomegalovirus (HCMV) latency is typically harmless but reactivation can be largely detrimental to immune compromised hosts. We modeled latency and reactivation using a traceable HCMV laboratory strain expressing the Gaussia luciferase reporter gene (HCMV/GLuc) in order to interrogate the viral modulatory effects on the human adaptive immunity. Humanized mice with long-term (more than 17 weeks) steady human T and B cell immune reconstitutions were infected with HCMV/GLuc and 7 weeks later were further treated with granulocyte-colony stimulating factor (G-CSF) to induce viral reactivations. Whole body bio-luminescence imaging analyses clearly differentiated mice with latent viral infections vs. reactivations. Foci of vigorous viral reactivations were detectable in liver, lymph nodes and salivary glands. The number of viral genome copies in various tissues increased upon reactivations and were detectable in sorted human CD14+, CD169+, and CD34+ cells. Compared with non-infected controls, mice after infections and reactivations showed higher thymopoiesis, systemic expansion of Th, CTL, Treg, and Tfh cells and functional antiviral T cell responses. Latent infections promoted vast development of memory CD4+ T cells while reactivations triggered a shift toward effector T cells expressing PD-1. Further, reactivations prompted a marked development of B cells, maturation of IgG+ plasma cells, and HCMV-specific antibody responses. Multivariate statistical methods were employed using T and B cell immune phenotypic profiles obtained with cells from several tissues of individual mice. The data was used to identify combinations of markers that could predict an HCMV infection vs. reactivation status. In spleen, but not in lymph nodes, higher frequencies of effector CD4+ T cells expressing PD-1 were among the factors most suited to distinguish HCMV reactivations from infections. These results suggest a shift from a T cell dominated immune response during latent infections toward an exhausted T cell phenotype and active humoral immune response upon reactivations. In sum, this novel in vivo humanized model combined with advanced analyses highlights a dynamic system clearly specifying the immunological spatial signatures of HCMV latency and reactivations. These signatures can be merged as predictive biomarker clusters that can be applied in the clinical translation of new therapies for the control of HCMV reactivation.
    • Superior immunogenicity of HCV envelope glycoproteins when adjuvanted with cyclic-di-AMP, a STING activator or archaeosomes.

      Landi, A; Law, J; Hockman, D; Logan, M; Crawford, K; Chen, C; Kundu, J; Ebensen, T; Guzman, C A; Deschatelets, L; et al. (2017-12-15)
      Three decades after the discovery, hepatitis C virus (HCV) is still the leading cause of liver transplantation and poses a major threat to global health. In spite of recent advances in the development of direct acting antivirals, there is still a need for a prophylactic vaccine to limit the virus spread and protect at-risk populations, especially in developing countries, where the cost of the new treatments may severely limit access. The use of recombinant HCV glycoproteins E1E2 (rE1E2) in combination with the MF59, an oil-in-water emulsion-based adjuvant, has previously been shown to reduce the rate of chronicity in chimpanzees and to induce production of cross-neutralizing antibodies and cellular immune responses in human volunteers. To further improve neutralizing antibody responses in recipients along with robust T cell responses, we have explored the immunogenicity of different adjuvants when formulated with the HCV rE1E2 vaccine in mice. Our data show that cyclic di-adenosine monophosphate (c-di-AMP) and archaeosomes elicit strong neutralizing antibodies similar to those elicited using aluminum hydroxide/monophosphoryl lipid A (Alum/monophos. /MPLA) and MF59. However, both c-di-AMP and archaeosomes induced a more robust cellular immune response, which was confirmed by the detection of vaccine-specific poly-functional CD4+ T cells. We conclude that these adjuvants may substantially boost the immunogenicity of our E1E2 vaccine. In addition, our data also indicates that use of a partial or exclusive intranasal immunization regimen may also be feasible using c-di-AMP as adjuvant.
    • Bivalent mucosal peptide vaccines administered using the LCP carrier system stimulate protective immune responses against Streptococcus pyogenes infection.

      Schulze, Kai; Ebensen, Thomas; Chandrudu, Saranya; Skwarczynski, Mariusz; Toth, Istvan; Olive, Colleen; Guzman, Carlos A; Helmholtz Centre for infection researchGmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-09-05)
      Despite the broad knowledge about the pathogenicity of Streptococcus pyogenes there is still a controversy about the correlate of protection in GAS infections. We aimed in further improving the immune responses stimulated against GAS comparing different vaccine formulations including bis-(3',5')-cyclic dimeric adenosine monophosphate (c-di-AMP) and BPPCysMPEG, a derivative of the macrophage-activating lipopeptide (MALP-2), as adjuvants, respectively, to be administered with and without the universal T helper cell epitope P25 along with the optimized B cell epitope J14 of the M protein and B and T cell epitopes of SfbI. Lipopeptide based nano carrier systems (LCP) were used for efficient antigen delivery across the mucosal barrier. The stimulated immune responses were efficient in protecting mice against a respiratory challenge with a lethal dose of a heterologous S. pyogenes strain. Moreover, combination of the LCP based peptide vaccine with c-di-AMP allowed reduction of antigen dose at the same time maintaining vaccine efficacy.
    • Roads to advanced vaccines: influenza case study.

      Riese, Peggy; Guzmán, Carlos A; Helmholz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-09)
      Vaccines represent a cornerstone to ensure healthy lives and promote well-being for all at all ages. However, there are many diseases for which vaccines are not available, are relatively ineffective or need to be adapted periodically. Advances in microbial biotechnology will contribute to overcoming these roadblocks by laying the groundwork for improving and creating new approaches for developing better vaccines, as illustrated here in the case of influenza.
    • Type I IFN and not TNF, is Essential for Cyclic Di-nucleotide-elicited CTL by a Cytosolic Cross-presentation Pathway.

      Lirussi, Darío; Ebensen, Thomas; Schulze, Kai; Trittel, Stephanie; Duran, Veronica; Liebich, Ines; Kalinke, Ulrich; Guzmán, Carlos Alberto; Helmholtz Centre for infection research, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017-07-19)
      Cyclic di-nucleotides (CDN) are potent stimulators of innate and adaptive immune responses. Cyclic di-AMP (CDA) is a promising adjuvant that generates humoral and cellular immunity. The strong STING-dependent stimulation of type I IFN represents a key feature of CDA. However, recent studies suggested that this is dispensable for adjuvanticity. Here we demonstrate that stimulation of IFN-γ-secreting CD8(+) cytotoxic T lymphocytes (CTL) is significantly decreased after vaccination in the absence of type I IFN signaling. The biological significance of this CTL response was confirmed by the stimulation of MHC class I-restricted protection against influenza virus challenge. We show here that type I IFN (and not TNF-α) is essential for CDA-mediated cross-presentation by a cathepsin independent, TAP and proteosome dependent cytosolic antigen processing pathway, which promotes effective cross-priming and further CTL induction. Our data clearly demonstrate that type I IFN signaling is critical for CDN-mediated cross-presentation.
    • Anti-nuclear autoantibodies in the general German population: prevalence and lack of association with selected cardiovascular and metabolic disorders-findings of a multicenter population-based study.

      Akmatov, Manas K; Röber, Nadja; Ahrens, Wolfgang; Flesch-Janys, Dieter; Fricke, Julia; Greiser, Halina; Günther, Kathrin; Kaaks, Rudolf; Kemmling, Yvonne; Krone, Bastian; et al. (2017-06-06)
      We determined the prevalence of anti-nuclear autoantibodies (ANAs) in the German adult population and examined the association between ANAs and cardiovascular and metabolic disorders.
    • Intranasal vaccination with an adjuvanted polyphosphazenes nanoparticle-based vaccine formulation stimulates protective immune responses in mice.

      Schulze, Kai; Ebensen, Thomas; Babiuk, Lorne A; Gerdts, Volker; Guzman, Carlos A.; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017-06-01)
      The most promising strategy to sustainably prevent infectious diseases is vaccination. However, emerging as well as re-emerging diseases still constitute a considerable threat. Furthermore, lack of compliance and logistic constrains often result in the failure of vaccination campaigns. To overcome these hurdles, novel vaccination strategies need to be developed, which fulfill maximal safety requirements, show maximal efficiency and are easy to administer. Mucosal vaccines constitute promising non-invasive approaches able to match these demands. Here we demonstrate that nanoparticle (polyphosphazenes)-based vaccine formulations including c-di-AMP as adjuvant, cationic innate defense regulator peptides (IDR) and ovalbumin (OVA) as model antigen were able to stimulate strong humoral and cellular immune responses, which conferred protection against the OVA expressing influenza strain A/WSN/OVAI (H1N1). The presented results confirm the potency of nanoparticle-based vaccine formulations to deliver antigens across the mucosal barrier, but also demonstrate the necessity to include adjuvants to stimulate efficient antigen-specific immune responses.
    • Targeted antigen delivery to dendritic cells elicits robust antiviral T cell-mediated immunity in the liver.

      Volckmar, Julia; Gereke, Marcus; Ebensen, Thomas; Riese, Peggy; Philipsen, Lars; Lienenklaus, Stefan; Wohlleber, Dirk; Klopfleisch, Robert; Stegemann-Koniszewski, Sabine; Müller, Andreas J; et al. (2017-03-07)
      Hepatotropic viruses such as hepatitis C virus cause life-threatening chronic liver infections in millions of people worldwide. Targeted in vivo antigen-delivery to cross-presenting dendritic cells (DCs) has proven to be extraordinarily efficient in stimulating antigen-specific T cell responses. To determine whether this approach would as well be suitable to induce local antiviral effector T cells in the liver we compared different vaccine formulations based on either the targeting of DEC-205 or TLR2/6 on cross-presenting DCs or formulations not involving in vivo DC targeting. As read-outs we used in vivo hepatotropic adenovirus challenge, histology and automated multidimensional fluorescence microscopy (MELC). We show that targeted in vivo antigen delivery to cross-presenting DCs is highly effective in inducing antiviral CTLs capable of eliminating virus-infected hepatocytes, while control vaccine formulation not involving DC targeting failed to induce immunity against hepatotropic virus. Moreover, we observed distinct patterns of CD8+ T cell interaction with virus-infected and apoptotic hepatocytes in the two DC-targeting groups suggesting that the different vaccine formulations may stimulate distinct types of effector functions. Our findings represent an important step toward the future development of vaccines against hepatotropic viruses and the treatment of patients with hepatic virus infection after liver transplantation to avoid reinfection.
    • Cell aggregation enhances bone formation by human mesenchymal stromal cells.

      Chatterjea, A; LaPointe, V L; Barradas, A; Garritsen, H; Yuan, H; Renard, A; van Blitterswijk, C A; de Beor, J; Hemholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Vraunschweig, Germany. (2017-02-15)
      The amount of bone generated using current tissue engineering approaches is insufficient for many clinical applications. Previous in vitro studies suggest that culturing cells as 3D aggregates can enhance their osteogenic potential, but the effect on bone formation in vivo is unknown. Here, we use agarose wells to generate uniformly sized mesenchymal stromal cell (MSC) aggregates. When combined with calcium phosphate ceramic particles and a gel prepared from human platelet-rich plasma, we generated a tissue engineered construct which significantly improved in vivo bone forming capacity as compared to the conventional system of using single cells seeded directly on the ceramic surface. Histology demonstrated the reproducibility of this system, which was tested using cells from four different donors. In vitro studies established that MSC aggregation results in an up-regulation of osteogenic transcripts. And finally, the in vivo performance of the constructs was significantly diminished when unaggregated cells were used, indicating that cell aggregation is a potent trigger of in vivo bone formation by MSCs. Cell aggregation could thus be used to improve bone tissue engineering strategies.
    • Exploitation of prokaryotic expression systems based on the salicylate-dependent control circuit encompassing nahR/P(sal)::xylS2 for biotechnological applications.

      Becker, Pablo D; Royo, Jose L; Guzman, Carlos A; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-02-15)
      Expression vectors appear to be an indispensable tool for both biological studies and biotechnological applications. Controlling gene overexpression becomes a critical issue when protein production is desired. In addition to several aspects regarding toxicity or plasmid instability, tight control of gene expression is an essential factor in biotechnological processes. Thus, the search for better-controlled circuits is an important issue among biotechnologists. Traditionally, expression systems involve a single regulatory protein operating over a target promoter. However, these circuits are limited on their induction ratios (e.g., by their restriction in the maximal expression capacity, by their leakiness under non-induced conditions). Due to these limitations, regulatory cascades, which are far more efficient, are necessary for biotechnological applications. Thus, regulatory circuits with two modules operating in cascade offer a significant advantage. In this review, we describe the regulatory cascade based on two salicylate-responsive transcriptional regulators of Pseudomonas putida (nahR/P(sal)::xylS2), its properties, and contribution to a tighter control over heterologous gene expression in different applications.Nowadays, heterologous expression has been proven to be an indispensable tool for tackling basic biological questions, as well as for developing biotechnological applications. As the nature of the protein of interest becomes more complex, biotechnologists find that a tight control of gene expression is a key factor which conditions the success of the downstream purification process, as well as the interpretation of the results in other type of studies. Fortunately, different expression systems can be found in the market, each of them with their own pros and cons. In this review we discuss the exploitation of prokaryotic expression systems based on a promising expression system, the salicylate-dependent control circuit encompassing nahR/P(sal)::xylS2, as well as some of the improvements that have been done on this system to exploit it more efficiently in the context of both biotechnological applications and basic research.
    • Immunization with Tc52 or its amino terminal domain adjuvanted with c-di-AMP induces Th17+Th1 specific immune responses and confers protection against Trypanosoma cruzi.

      Matos, Marina N; Cazorla, Silvia I; Schulze, Kai; Ebensen, Thomas; Guzmán, Carlos Alberto; Malchiodi, Emilio L; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-02)
      The development of new adjuvants enables fine modulation of the elicited immune responses. Ideally, the use of one or more adjuvants should result in the induction of a protective immune response against the specific pathogen. We have evaluated the immune response and protection against Trypanosoma cruzi infection in mice vaccinated with recombinant Tc52 or its N- and C-terminal domains (NTc52 and CTc52) adjuvanted either with the STING (Stimulator of Interferon Genes) agonist cyclic di-AMP (c-di-AMP), a pegylated derivative of α-galactosylceramide (αGC-PEG), or oligodeoxynucleotides containing unmethylated CpG motifs (ODN-CpG). All groups immunized with the recombinant proteins plus adjuvant: Tc52+c-di-AMP, NTc52+c-di-AMP, CTc52+c-di-AMP, NTc52+c-di-AMP+αGC-PEG, NTc52+CpG, developed significantly higher anti-Tc52 IgG titers than controls. Groups immunized with c-di-AMP and Tc52, NTc52 or CTc52 showed the highest Tc52-specific IgA titers in nasal lavages. All groups immunized with the recombinant proteins plus adjuvant developed a strong specific cellular immune response in splenocytes and lymph node cells with significant differences for groups immunized with c-di-AMP and Tc52, NTc52 or CTc52. These groups also showed high levels of Tc52-specific IL-17 and IFN-γ producing cells, while NTc52+CpG group only showed significant difference with control in IFN-γ producing cells. Groups immunized with c-di-AMP and Tc52, NTc52 or CTc52 developed predominantly a Th17 and Th1immune response, whereas for NTc52+CpG it was a dominant Th1 response. It was previously described that αGC-PEG inhibits Th17 differentiation by activating NKT cells. Thus, in this work we have also included a group immunized with both adjuvants (NTc52+c-di-AMP+αGC-PEG) with the aim to modulate the Th17 response induced by c-di-AMP. This group showed a significant reduction in the number of Tc52-specific IL-17 producing splenocytes, as compared to the group NTc52+c-di-AMP, which has in turn correlated with a reduction in protection against infection. These results suggest that the Th17 immune response developed after immunizing with NTc52+c-di-AMP could have a protective role against T. cruzi infection. Groups NTc52+c-di-AMP, Tc52+c-di-AMP and NTc52PB, were the ones that showed better protection against infection with lower parasitemia and weight loss, and higher survival.