Kai Schulze

The missing link: Bordetella petrii is endowed with both the metabolic versatility of environmental bacteria and virulence traits of pathogenic Bordetellae

BackgroundBordetella petrii is the only environmental species hitherto found among the otherwise host-restricted and pathogenic members of the genus Bordetella. Phylogenetically, it connects the pathogenic Bordetellae and environmental bacteria of the genera Achromobacter and Alcaligenes, which are opportunistic pathogens. B. petrii strains have been isolated from very different environmental niches, including river sediment, polluted soil, marine sponges and a grass root. Recently, clinical isolates associated with bone degenerative disease or cystic fibrosis have also been described.ResultsIn this manuscript we present the results of the analysis of the completely annotated genome sequence of the B. petrii strain DSMZ12804. B. petrii has a mosaic genome of 5,287,950 bp harboring numerous mobile genetic elements, including seven large genomic islands. Four of them are highly related to the clc element of Pseudomonas knackmussii B13, which encodes genes involved in the degradation of aromatics. Though being an environmental isolate, the sequenced B. petrii strain also encodes proteins related to virulence factors of the pathogenic Bordetellae, including the filamentous hemagglutinin, which is a major colonization factor of B. pertussis, and the master virulence regulator BvgAS. However, it lacks all known toxins of the pathogenic Bordetellae.ConclusionThe genomic analysis suggests that B. petrii represents an evolutionary link between free-living environmental bacteria and the host-restricted obligate pathogenic Bordetellae. Its remarkable metabolic versatility may enable B. petrii to thrive in very different ecological niches.

Non-invasive delivery of nanoparticles to hair follicles: a perspective for transcutaneous immunization.

Transfollicular vaccination aims to reach the peri-follicular antigen presenting cells without impairing the stratum corneum (SC) barrier. This would be an optimal vaccination strategy under critical hygienic conditions. Nanoparticles (NPs) are the ideal vehicles for transfollicular delivery of vaccines as they are able to (i) penetrate deeper into the hair follicles than molecules in solution, (ii) can help to stabilize protein based antigen and (iii) improve and modulate the immune response. This study investigates the potential of transfollicular delivery of polymeric NPs using ovalbumin (OVA) as a model antigen. NPs were prepared by a double emulsion method from pharmaceutically well characterized biocompatible and biodegradable polymers poly(lactide-co-glycolide) (PLGA) or chitosan-coated PLGA (Chit-PLGA) using polyvinyl alcohol as stabilizer. The NP formulations are available as freeze dried product which can be re-constituted with water or cell culture medium before use to yield any desired OVA/NP concentration. OVA was protected from cleavage or aggregation inside the NPs and retained its biological activity to 74% (PLGA) and 64% (Chit-PLGA). Thus, when applying a typical dose of 8.5 μl/cm(2) NP formulation (50mg NPs/ml, 54.3±0.047 and 66.5±0.044 μg OVA/mg NPs for PLGA and Chit-PLGA NPs, respectively) an effective dose of 17 μg/cm(2) (PLGA) or 18 μg/cm(2) (Chit-PLGA) of active OVA is administered. In a cell culture assay encapsulated OVA stimulated the proliferation of CD4+ (PLGA and Chit-PLGA) and CD8+ T-cells (only Chit-PLGA) to a larger extent than OVA in solution. An adoptive transfer experiment demonstrated that the model antigen OVA can be delivered via the transfollicular route. This preliminary experiment is a proof of concept that by this transfollicular immunization approach it is possible to deliver antigens, thereby stimulating antigen-specific T cells. Both NP formulations improved the delivery efficiency of OVA into the hair follicles on excised pig ears by a factor of 2-3 compared to OVA solution. This delivery efficiency could further be increased by increasing the number of NPs applied per skin area by a factor of ≈2-2.4. Consequently formulation of OVA into PLGA and Chit-PLGA NPs may offer to reduce the dose which needs to be applied for transfollicular immunization.

Novel conserved group A streptococcal proteins identified by the antigenome technology as vaccine candidates for a non-M protein-based vaccine.

ABSTRACT Group A streptococci (GAS) can cause a wide variety of human infections ranging from asymptomatic colonization to life-threatening invasive diseases. Although antibiotic treatment is very effective, when left untreated, Streptococcus pyogenes infections can lead to poststreptococcal sequelae and severe disease causing significant morbidity and mortality worldwide. To aid the development of a non-M protein-based prophylactic vaccine for the prevention of group A streptococcal infections, we identified novel immunogenic proteins using genomic surface display libraries and human serum antibodies from donors exposed to or infected by S. pyogenes. Vaccine candidate antigens were further selected based on animal protection in murine lethal-sepsis models with intranasal or intravenous challenge with two different M serotype strains. The nine protective antigens identified are highly conserved; eight of them show more than 97% sequence identity in 13 published genomes as well as in approximately 50 clinical isolates tested. Since the functions of the selected vaccine candidates are largely unknown, we generated deletion mutants for three of the protective antigens and observed that deletion of the gene encoding Spy1536 drastically reduced binding of GAS cells to host extracellular matrix proteins, due to reduced surface expression of GAS proteins such as Spy0269 and M protein. The protective, highly conserved antigens identified in this study are promising candidates for the development of an M-type-independent, protein-based vaccine to prevent infection by S. pyogenes.

The Bacterial Second Messenger cdiGMP Exhibits Promising Activity as a Mucosal Adjuvant

ABSTRACT The development of mucosal adjuvants is still a critical need in vaccinology. In the present work, we show that bis(3′,5′)-cyclic dimeric GMP (cdiGMP), a second messenger that modulates cell surface properties of several microorganisms, exerts potent activity as a mucosal adjuvant. BALB/c mice were immunized intranasally with the model antigen β-galactosidase (β-Gal) coadministered with cdiGMP. Animals receiving cdiGMP as an adjuvant showed significantly higher anti-β-Gal immunoglobulin G (IgG) titers in sera than controls (i.e., 512-fold [P < 0.05]). Coadministration of cdiGMP also stimulated efficient β-Gal-specific secretory IgA production in the lung (P < 0.016) and vagina (P < 0.036). Cellular immune responses were observed in response to both the β-Gal protein and a peptide encompassing its major histocompatibility complex class I-restricted epitope. The IgG1-to-IgG2a ratio of anti-β-Gal antibodies and the observed profiles of secreted cytokines suggest that a dominant Th1 response pattern is promoted by mucosal coadministration of cdiGMP. Finally, the use of cdiGMP as a mucosal adjuvant also led to the stimulation of in vivo cytotoxic T-lymphocyte responses in C57BL/6 mice intranasally immunized with ovalbumin and cdiGMP (up to 30% of specific lysis). The results obtained indicate that cdiGMP is a promising tool for the development of mucosal vaccines.

A Pegylated Derivative of α-Galactosylceramide Exhibits Improved Biological Properties

The glycolipid α-galactosylceramide (αGalCer) has immunomodulatory properties, which have been exploited to combat cancer, chronic inflammatory diseases, and infections. However, its poor solubility makes αGalCer a suboptimal compound for in vivo applications. In this study, a pegylated derivative of αGalCer is characterized, which exhibits improved physical and biological properties. The new compound, αGalCerMPEG, is water-soluble and retains the specificity for the CD1d receptor of αGalCer. The in vitro stimulatory properties on immune cells (e.g., dendritic cells and splenocytes) are maintained intact, even when tested at a 33-fold lower concentration of the active moiety than αGalCer. NK cells isolated from mice treated with αGalCerMPEG also had stronger cytotoxic activity on YAC-1 cells than those obtained from animals receiving either αGalCer or CpG. Intranasal immunization studies performed in mice showed that αGalCerMPEG exerts stronger adjuvant activities than the parental compound αGalCer when tested at 0.35 vs 11.7 nM/dose. Coadministration of β-galactosidase with αGalCerMPEG resulted not only in high titers of Ag-specific Abs in serum (i.e., 1:512,000), but also in the stimulation of stronger Th2 and secretory IgA responses, both at local and remote mucosal effector sites (i.e., nose, lung, and vagina). The new synthetic derivative αGalCerMPEG represents a promising tool for the development of immune interventions against infectious and noninfectious diseases.

Characterization of the domain of fibronectin-binding protein I of Streptococcus pyogenes responsible for elicitation of a protective immune response.

ABSTRACT Fibronectin-binding protein I (SfbI) represents a major adhesin ofStreptococcus pyogenes. Mice were intranasally immunized with recombinant proteins spanning different portions of SfbI to identify the minimal fragment able to elicit a protective response against a lethal challenge with S. pyogenes. The strongest cellular responses and the highest levels of antigen-specific secretory immunoglobulin A (IgA) were detected in mice immunized with the fibronectin-binding region of SfbI. In contrast, animals vaccinated with a polypeptide spanning the aromatic and proline-rich regions showed the highest titers and fastest IgG response in serum. Vaccination with either SfbI without a membrane anchor and signal peptide or a polypeptide encompassing its fibronectin-binding regions resulted in efficient protection against heterologous challenge (60% and 80%, respectively), whereas the use of a polypeptide lacking this region conferred marginal protection (10%) with respect to the control group (0%). These results demonstrate that the fibronectin-binding region of SfbI is a promising candidate antigen for developing anti-S. pyogenes vaccines.

Modeling Influenza Virus Infection: A Roadmap for Influenza Research

Influenza A virus (IAV) infection represents a global threat causing seasonal outbreaks and pandemics. Additionally, secondary bacterial infections, caused mainly by Streptococcus pneumoniae, are one of the main complications and responsible for the enhanced morbidity and mortality associated with IAV infections. In spite of the significant advances in our knowledge of IAV infections, holistic comprehension of the interplay between IAV and the host immune response (IR) remains largely fragmented. During the last decade, mathematical modeling has been instrumental to explain and quantify IAV dynamics. In this paper, we review not only the state of the art of mathematical models of IAV infection but also the methodologies exploited for parameter estimation. We focus on the adaptive IR control of IAV infection and the possible mechanisms that could promote a secondary bacterial coinfection. To exemplify IAV dynamics and identifiability issues, a mathematical model to explain the interactions between adaptive IR and IAV infection is considered. Furthermore, in this paper we propose a roadmap for future influenza research. The development of a mathematical modeling framework with a secondary bacterial coinfection, immunosenescence, host genetic factors and responsiveness to vaccination will be pivotal to advance IAV infection understanding and treatment optimization.

Efficient immune responses against Intimin and EspB of enterohaemorragic Escherichia coli after intranasal vaccination using the TLR2/6 agonist MALP-2 as adjuvant

Mucosal vaccine formulations based on purified recombinant C280 gamma-Intimin and EspB (Escherichia coli secreted protein B) from enterohaemorragic E. coli co-administered with a pegylated derivative of the TLR2/6 agonist MALP-2 (macrophage-activating lipopeptide) as adjuvant were evaluated in BALB/c mice. After intranasal vaccination, strong humoral and cellular immune responses were observed against C280 gamma-Intimin and EspB. Sera of immunized mice inhibit bacterial haemolytic activity in vitro. Antigen-specific T-cell proliferation, IL-4, IL-2 and IFN-gamma producing cells, and secretory IgA were mostly detected in animals receiving MALP-2 as adjuvant. These results suggest that C280 gamma-Intimin and EspB are good candidate antigens to be incorporated into mucosal vaccines against this important pathogen.

Vacuum-assisted generation and control of atomic coherences at x-ray energies.

The control of light-matter interaction at the quantum level usually requires coherent laser fields. But already an exchange of virtual photons with the electromagnetic vacuum field alone can lead to quantum coherences, which subsequently suppress spontaneous emission. We demonstrate such spontaneously generated coherences (SGC) in a large ensemble of nuclei operating in the x-ray regime, resonantly coupled to a common cavity environment. The observed SGC originates from two fundamentally different mechanisms related to cooperative emission and magnetically controlled anisotropy of the cavity vacuum. This approach opens new perspectives for quantum control, quantum state engineering and simulation of quantum many-body physics in an essentially decoherence-free setting.

Flunarizine prevents hepatitis C virus membrane fusion in a genotype-dependent manner by targeting the potential fusion peptide within E1.

To explore mechanisms of hepatitis C viral (HCV) replication we screened a compound library including licensed drugs. Flunarizine, a diphenylmethylpiperazine used to treat migraine, inhibited HCV cell entry in vitro and in vivo in a genotype‐dependent fashion. Analysis of mosaic viruses between susceptible and resistant strains revealed that E1 and E2 glycoproteins confer susceptibility to flunarizine. Time of addition experiments and single particle tracking of HCV demonstrated that flunarizine specifically prevents membrane fusion. Related phenothiazines and pimozide also inhibited HCV infection and preferentially targeted HCV genotype 2 viruses. However, phenothiazines and pimozide exhibited improved genotype coverage including the difficult to treat genotype 3. Flunarizine‐resistant HCV carried mutations within the alleged fusion peptide and displayed cross‐resistance to these compounds, indicating that these drugs have a common mode of action. Conclusion: These observations reveal novel details about HCV membrane fusion; moreover, flunarizine and related compounds represent first‐in‐class HCV fusion inhibitors that merit consideration for repurposing as a cost‐effective component of HCV combination therapies. (Hepatology 2016;63:49–62)