Alexander von Gabain

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.

Tamm-Horsfall glycoprotein links innate immune cell activation with adaptive immunity via a Toll-like receptor-4–dependent mechanism

Tamm-Horsfall glycoprotein (THP) is expressed exclusively in the kidney and constitutes the most abundant protein in mammalian urine. A critical role for THP in antibacterial host defense and inflammatory disorders of the urogenital tract has been suggested. We demonstrate that THP activates myeloid DCs via Toll-like receptor-4 (TLR4) to acquire a fully mature DC phenotype. THP triggers typical TLR signaling, culminating in activation of NF-kappaB. Bone marrow-derived macrophages from TLR4- and MyD88-deficient mice were nonresponsive to THP in contrast to those from TLR2- and TLR9-deficient mice. In vivo THP-driven TNF-alpha production was evident in WT but not in Tlr4-/- mice. Importantly, generation of THP-specific Abs consistently detectable in urinary tract inflammation was completely blunted in Tlr4-/- mice. These data show that THP is a regulatory factor of innate and adaptive immunity and therefore could have significant impact on host immunity in the urinary tract.

Identification of a novel iron regulated staphylococcal surface protein with haptoglobin‐haemoglobin binding activity

Staphylococcus aureus is an extremely adaptable pathogen causing a wide variety of infections. Staphylococcal surface proteins that directly interact with host extracellular proteins greatly contribute to virulence and are involved in adhesion, immune escape and nutrient acquisition. In our extensive search for highly immunogenic, in vivo‐expressed, staphylococcal proteins, previously, we identified a novel member of the family of Gram‐positive anchor motif proteins with a predicted 895 amino acid long sequence. In order to determine the ligand for this novel LPXTG cell wall protein, we employed affinity purification of human plasma using the recombinant form of the protein. Two‐dimensional electrophoresis of eluted plasma proteins identified haptoglobin as a specific binding partner. Importantly, we also observed this specific ligand binding when living S. aureus cells were exposed to biotin‐labelled haptoglobin (Hp) in a FACS‐based assay. Targeted deletion of the gene eliminated Hp‐binding, a function that has not been attributed to S. aureus before. Based on these data we specified the protein as the staphylococcal haptoglobin receptor A (HarA). Similarly to other haptoglobin receptors identified in Gram‐negative pathogens, HarA binds not only Hp, but also haptoglobin‐haemoglobin complexes with an even higher affinity, as demonstrated in in vitro binding assays. Employing specific deletion mutants, ligand binding was localized to two homologous regions with about 145 amino acid residues located within the N‐terminal part of the protein. In addition, we demonstrated that expression of HarA was strictly controlled by iron through the iron‐dependent transcriptional regulator Fur. Based on these data we propose that HarA can be added to the list of staphylococcal virulence factors with a most likely function related to iron acquisition.

Therapy of Experimental Pseudomonas Infections with a Nonreplicating Genetically Modified Phage

ABSTRACT Bacteriophage therapy of bacterial infections has received renewed attention owing to the increasing prevalence of antibiotic-resistant pathogens. A side effect of many antibiotics as well as of phage therapy with lytic phage is the release of cell wall components, e.g., endotoxins of gram-negative bacteria, which mediate the general pathological aspects of septicemia. Here we explored an alternative strategy by using genetically engineered nonreplicating, nonlytic phage to combat an experimental Pseudomonas aeruginosa infection. An export protein gene of the P. aeruginosa filamentous phage Pf3 was replaced with a restriction endonuclease gene. This rendered the Pf3 variant (Pf3R) nonreplicative and concomitantly prevented the release of the therapeutic agent from the target cell. The Pf3R phage efficiently killed a wild-type host in vitro, while endotoxin release was kept to a minimum. Treatment of P. aeruginosa infections of mice with Pf3R or with a replicating lytic phage resulted in comparable survival rates upon challenge with a minimal lethal dose of 3. However, the survival rate after phage therapy with Pf3R was significantly higher than that with the lytic phage upon challenge with a minimal lethal dose of 5. This higher survival rate correlated with a reduced inflammatory response elicited by Pf3R treatment relative to that with the lytic phage. Therefore, this study suggests that the increased survival rate of Pf3R-treated mice could result from reduced endotoxin release. Thus, the use of a nonreplicating modified phage for the delivery of genes encoding proteins toxic to bacterial pathogens may open up a new avenue in antimicrobial therapy.

Mammalian Hsp70 and Hsp110 Proteins Bind to RNA Motifs Involved in mRNA Stability

In this study, in vitro RNA binding by members of the mammalian 70-kDa heat shock protein (Hsp) family was examined. We show that Hsp/Hsc70 and Hsp110 proteins preferentially bound AU-rich RNA in vitro. Inhibition of RNA binding by ATP suggested the involvement of the N-terminal ATP-binding domain. By using deletion mutants of Hsp110 protein, a diverged Hsp70 family member, RNA binding was localized to the N-terminal ATP-binding domain of the molecule. The C-terminal peptide-binding domain did not bind RNA, but its engagement by a peptide substrate abrogated RNA binding by the N terminus of the protein. Interestingly, removal of the C-terminal α-helical structure or the α-loop domain unique to Hsp110 immediately downstream of the peptide-binding domain, but not both, resulted in considerably increased RNA binding as compared with the wild type protein. Finally, a 70-kDa activity was immunoprecipitated from RNA-protein complexes formed in vitro between cytoplasmic proteins of human lymphocytes and AU-rich RNA. These findings support the idea that certain heat shock proteins may act as RNA-binding entities in vivo to guide the appropriate folding of RNA substrates for subsequent regulatory processes such as mRNA degradation and/or translation.

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.

The novel adjuvant IC31® strongly improves influenza vaccine-specific cellular and humoral immune responses in young adult and aged mice

The compromised immune responses in the elderly as well as the threat of pandemic influenza necessitate the development of improved influenza vaccines. This study provides evidence that IC31, a two-component synthetic adjuvant signalling through TLR-9, augments humoral and cellular immune responses to seasonal influenza vaccines. Experiments performed in young adult mice showed increased HI titres and higher levels of IgG2a antibodies that were accompanied by the induction of IFN-gamma producing CD4(+) T cells after single vaccination with reduced doses of vaccine antigens, even 200 days after single immunisation. Importantly, similar effects were seen in aged mice, although most pronounced upon booster immunisation. Thus, IC31 fulfils important criteria of novel influenza vaccine adjuvants.

IC31® and IC30, novel types of vaccine adjuvant based on peptide delivery systems

Toll-like receptor (TLR) agonists have a proven potential to become the adjuvants of the next generation when admixed and formulated with all kinds of vaccine compositions. The quality and magnitude of a vaccine-induced immune response is often strongly facilitated by TLR agonists, with the result that protection is increased and expanded toward type 1-driven immunity. DNA oligodeoxynucleotides bind to TLR9 and have been tested in a variety of vaccine settings with encouraging results. Combining oligodeoxynucleotides with poly-L-arginine (IC30) or certain artificial antimicrobial peptides dramatically improves and synergizes with the adjuvant action of TLR9 agonists, a notion that has prompted the development of IC31®, an adjuvant with a promising profile in both preclinical and clinical trials.