Andreas Meinke

Discovery of a novel class of highly conserved vaccine antigens using genomic scale antigenic fingerprinting of pneumococcus with human antibodies

Pneumococcus is one of the most important human pathogens that causes life-threatening invasive diseases, especially at the extremities of age. Capsular polysaccharides (CPSs) are known to induce protective antibodies; however, it is not feasible to develop CPS-based vaccines that cover all of the 90 disease-causing serotypes. We applied a genomic approach and described the antibody repertoire for pneumococcal proteins using display libraries expressing 15–150 amino acid fragments of the pathogens proteome. Serum antibodies of exposed, but not infected, individuals and convalescing patients identified the ANTIGENome of pneumococcus consisting of ∼140 antigens, many of them surface exposed. Based on several in vitro assays, 18 novel candidates were preselected for animal studies, and 4 of them showed significant protection against lethal sepsis. Two lead vaccine candidates, protein required for cell wall separation of group B streptococcus (PcsB) and serine/threonine protein kinase (StkP), were found to be exceptionally conserved among clinical isolates (>99.5% identity) and cross-protective against four different serotypes in lethal sepsis and pneumonia models, and have important nonredundant functions in bacterial multiplication based on gene deletion studies. We describe for the first time opsonophagocytic killing activity for pneumococcal protein antigens. A vaccine containing PcsB and StkP is intended for the prevention of infections caused by all serotypes of pneumococcus in the elderly and in children.

Identification of in vivo expressed vaccine candidate antigens from Staphylococcus aureus

For the design of potent subunit vaccines, it is of paramount importance to identify all antigens immunologically recognized by a patient population infected with a pathogen. We have developed a rapid and efficient procedure to identify such commonly recognized antigens, and here we provide a comprehensive in vivo antigenic profile of Staphylococcus aureus, an important human pathogen. S. aureus peptides were displayed on the surface of Escherichia coli via fusion to one of two outer membrane proteins (LamB and FhuA) and probed with sera selected for high Ab titer and opsonic activity. A total of 60 antigenic proteins were identified, most of which are located or predicted to be located on the surface of the bacterium or secreted. The identification of these antigens and their reactivity with individual sera from patients and healthy individuals greatly facilitate the selection of promising vaccine candidates for further evaluation. This approach, which makes use of whole genome sequence information, has the potential to greatly accelerate and facilitate the formulation of novel vaccines and is applicable to any pathogen that induces Abs in humans and/or experimental animals.

The fibrinogen receptor FbsA promotes adherence of Streptococcus agalactiae to human epithelial cells

ABSTRACT Streptococcus agalactiae is a major cause of bacterial pneumonia, sepsis, and meningitis in human neonates. During the course of infection, S. agalactiae adheres to a variety of epithelial cells but the underlying mechanisms are only poorly understood. The present report demonstrates the importance of the fibrinogen receptor FbsA for the streptococcal adherence and invasion of epithelial cells. Deletion of the fbsA gene in various S. agalactiae strains substantially reduced their binding of soluble fibrinogen and their adherence to and invasion of epithelial cells, indicating a role of FbsA in these different processes. The adherence and invasiveness of an fbsA deletion mutant were partially restored by reintroducing the fbsA gene on an expression vector. Heterologous expression of fbsA in Lactococcus lactis enabled this bacterium to adhere to but not to invade epithelial cells, suggesting that FbsA is a streptococcal adhesin. Flow cytometry experiments revealed a dose-dependent binding of FbsA to the surface of epithelial cells. Furthermore, tissue culture experiments exhibited an intimate contact of FbsA-coated latex beads with the surfaces of human epithelial cells. Finally, host cell adherence and invasion were significantly blocked in competition experiments with either purified FbsA protein or a monoclonal antibody directed against the fibrinogen-binding epitope of FbsA. Taken together, our studies demonstrate that FbsA promotes the adherence of S. agalactiae to epithelial cells but that FbsA does not mediate the bacterial invasion into host cells. Our results also indicate that fibrinogen-binding epitopes within FbsA are involved in the adherence of S. agalactiae to epithelial cells.

Functional Selection of Vaccine Candidate Peptides from Staphylococcus aureus Whole-Genome Expression Libraries In Vitro

ABSTRACT An in vitro protein selection method, ribosome display, has been applied to comprehensively identify and map the immunologically relevant proteins of the human pathogen Staphylococcus aureus. A library built up from genomic fragments of the virulent S. aureus COL strain (methicillin-resistant S. aureus) allowed us to screen all possible encoded peptides for immunoreactivity. As selective agents, human sera exhibiting a high antibody titer and opsonic activity against S. aureus were used, since these antibodies indicate the in vivo expression and immunoreactivity of the corresponding proteins. Identified clones cluster in distinct regions of 75 genes, most of them classifiable as secreted or surface-localized proteins, including previously identified virulence factors. In addition, 14 putative novel short open reading frames were identified and their immunoreactivity and in vivo mRNA expression were confirmed, underscoring the annotation-independent, true genomic nature of our approach. Evidence is provided that a large fraction of the identified peptides cannot be expressed in an in vivo-based surface display system. Thus, in vitro protein selection, not biased by the context of living entities, allows screening of genomic expression libraries with a large number of different ligands simultaneously. It is a powerful approach for fingerprinting the repertoire of immune reactive proteins serving as target candidates for active and passive vaccination against pathogens.

High-Affinity Binding of the Staphylococcal HarA Protein to Haptoglobin and Hemoglobin Involves a Domain with an Antiparallel Eight-Stranded β-Barrel Fold

Iron scavenging from the host is essential for the growth of pathogenic bacteria. In this study, we further characterized two staphylococcal cell wall proteins previously shown to bind hemoproteins. HarA and IsdB harbor homologous ligand binding domains, the so called NEAT domain (for “near transporter”) present in several surface proteins of gram-positive pathogens. Surface plasmon resonance measurements using glutathione S-transferase (GST)-tagged HarAD1, one of the ligand binding domains of HarA, and GST-tagged full-length IsdB proteins confirmed high-affinity binding to hemoglobin and haptoglobin-hemoglobin complexes with equilibrium dissociation constants (KD) of 5 to 50 nM. Haptoglobin binding could be detected only with HarA and was in the low micromolar range. In order to determine the fold of this evolutionarily conserved ligand binding domain, the untagged HarAD1 protein was subjected to nuclear magnetic resonance spectroscopy, which revealed an eight-stranded, purely antiparallel β-barrel with the strand order (-β1↓-β2↑-β3↓-β6↑-β5↓-β4↑-β7↓-β8↑), forming two Greek key motifs. Based on structural-homology searches, the topology of the HarAD1 domain resembles that of the immunoglobulin (Ig) fold family, whose members are involved in protein-protein interactions, but with distinct structural features. Therefore, we consider that the HarAD1/NEAT domain fold is a novel variant of the Ig fold that has not yet been observed in other proteins.

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.

Bacterial Phage Receptors, Versatile Tools for Display of Polypeptides on the Cell Surface

Four outer membrane proteins of Escherichia coli were examined for their capabilities and limitations in displaying heterologous peptide inserts on the bacterial cell surface. The T7 tag or multiple copies of the myc epitope were inserted into loops 4 and 5 of the ferrichrome and phage T5 receptor FhuA. Fluorescence-activated cell sorting analysis showed that peptides of up to 250 amino acids were efficiently displayed on the surface of E. coli as inserts within FhuA. Strains expressing FhuA fusion proteins behaved similarly to those expressing wild-type FhuA, as judged by phage infection and colicin sensitivity. The vitamin B(12) and phage BF23 receptor BtuB could display peptide inserts of at least 86 amino acids containing the T7 tag. In contrast, the receptors of the phages K3 and lambda, OmpA and LamB, accepted only insertions in their respective loop 4 of up to 40 amino acids containing the T7 tag. The insertion of larger fragments resulted in inefficient transport and/or assembly of OmpA and LamB fusion proteins into the outer membrane. Cells displaying a foreign peptide fused to any one of these outer membrane proteins were almost completely recovered by magnetic cell sorting from a large pool of cells expressing the relevant wild-type platform protein only. Thus, this approach offers a fast and simple screening procedure for cells displaying heterologous polypeptides. The combination of FhuA, along with with BtuB and LamB, should provide a comprehensive tool for displaying complex peptide libraries of various insert sizes on the surface of E. coli for diverse applications.

Jak2-Stat5 Interactions Analyzed in Yeast

Many cytokine receptors employ Janus protein tyrosine kinases (Jaks) and signal transducers and activators of transcription (Stats) for nuclear signaling. Here, we have established yeast strains in which an autoactivated Jak2 kinase induces tyrosine phosphorylation, dimerization, nuclear translocation, and DNA binding of a concomitantly expressed Stat5 protein. Transcriptional activity of Stat5 on a stably integrated, Stat-dependent reporter gene required the C-terminal fusion of the VP16 transactivation domain. In such yeast strains, the interaction between Jak2 and Stat5 was analyzed without interference by other mammalian proteins involved in regulating Jak-Stat signaling, and mutant versions of both proteins were analyzed for their ability to productively interact. Complexes between Jak2 and Stat5 were found to be stable under stringent co-immunoprecipitation conditions. Deletion of the Jak homology regions 2–7 (JH2–JH7) of Jak2, leaving only the kinase domain (JH1) intact, reduced the ability of the kinase to phosphorylate Stat5, whereas deletion of the JH2 domain caused an increased enzymatic activity. A site-directed R618K mutation in the Stat5 SH2 domain abolished the phosphorylation by Jak2, while deletion of the C terminus led to Stat5 hyperphosphorylation. A single phosphotyrosine-SH2 domain interaction was sufficient for the dimerization of Stat5, but such dimers bound to DNA very inefficiently. Together, our data show that yeast cells are appropriate tools for studying Jak-Stat or Stat-Stat interactions. Our mutational analysis suggests that the Stat5 SH2 domain is essential for the interaction with Jak2 and that the kinase domain of Jak2 is sufficient for Jak2-Stat5 interaction. Therefore, the Jak kinase domain may be all that is needed to cause Stat phosphorylation in situations where receptor docking is dispensable.

Colony-stimulating factors and interferon-γ activate a protein related to MGF-Stat 5 to cause formation of the differentiation-induced factor in myeloid cells

The Jak‐Stat pathway of intracellular signals is used by growth factor‐ and cytokine receptors to induce gene transcription. We have recently reported that differentiation of myeloid cells, induced by phorbol ester, interferon‐γ (IFN‐γ) or colony‐stimulating factor‐1 (CSF‐1) is accompanied by the activation of the differentiation‐induced factor (DIF). Activated DIF specifically associates with a subclass of gamma‐interferon activation site (GAS)‐like DNA elements. We now report that GM‐CSF, which like CSF‐1 promotes the generation of mature macrophages, activates DIF. No activation was observed after treatment with the granulocyte growth and differentiation factor G‐ CSF. Antibodies raised against a Stat family protein, designated mammary gland factor‐Stat 5 (MGF‐Stat 5), reacted with DIF induced by either CSF‐1, GM‐CSF or IFN‐γ. Antisera to other known Stats were without effect on the DIF complex in electrophoretic mobility shift assays (EMSA). A 112 kDa protein could be isolated from either GM‐CSF‐ or IFN‐γ‐treated cells by GAS oligonucleotide precipitation. This protein reacted with antibodies to both MGF‐Stat 5 and phosphotyrosine. MGF‐Stat 5 and closely related proteins thus define a subfamily of Stat transcription factors that are present in a variety of cell types and are required for the onset of immediate gene expression in response to differentiating stimuli.

Selection of peptide entry motifs by bacterial surface display

Surface display technologies have been established previously to select peptides and polypeptides that interact with purified immobilized ligands. In the present study, we designed and implemented a surface display-based technique to identify novel peptide motifs that mediate entry into eukaryotic cells. An Escherichia coli library expressing surface-displayed peptides was combined with eukaryotic cells and the gentamicin protection assay was performed to select recombinant E. coli, which were internalized into eukaryotic cells by virtue of the displayed peptides. To establish the proof of principle of this approach, the fibronectin-binding motifs of the fibronectin-binding protein A of Staphylococcus aureus were inserted into the E. coli FhuA protein. Surface expression of the fusion proteins was demonstrated by functional assays and by FACS analysis. The fibronectin-binding motifs were shown to mediate entry of the bacteria into non-phagocytic eukaryotic cells and brought about the preferential selection of these bacteria over E. coli expressing parental FhuA, with an enrichment of 100000-fold. Four entry sequences were selected and identified using an S. aureus library of peptides displayed in the FhuA protein on the surface of E. coli. These sequences included novel entry motifs as well as integrin-binding Arg-Gly-Asp (RGD) motifs and promoted a high degree of bacterial entry. Bacterial surface display is thus a powerful tool to effectively select and identify entry peptide motifs.