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

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 pneumococcal eukaryotic-type serine/threonine protein kinase StkP co-localizes with the cell division apparatus and interacts with FtsZ in vitro.

The importance of serine/threonine phosphorylation in signalling and regulation of gene expression in prokaryotes has been widely recognized. Driven by our interest in StkP (the pneumococcal serine/threonine kinase homologue) for vaccine development, we studied its cellular localization. We found that the C-terminally located PASTA (penicillin-binding protein and serine/threonine kinase associated) domains, but not the N-terminal kinase domain of StkP, were located on the surface of live pneumococcal cells grown in vitro and were also accessible to antibodies during pneumococcal infection in mice and man. Most importantly, we discovered, by immunofluorescence microscopy, that StkP co-localized with the cell division apparatus. StkP and FtsZ, the prokaryotic tubulin homologue, co-localized at mid-cell in most cells. Formation and constriction of the ring-like structure of StkP followed the dynamic changes of FtsZ in dividing cells. This pattern resembles that of the 'late' divisome protein penicillin-binding protein 2X. The lack of StkP in gene deletion mutants did not disturb FtsZ ring formation, further suggesting that StkP joins the divisome after the FtsZ ring is assembled. We also present evidence that StkP binds and phosphorylates recombinant FtsZ in vitro; however, we could not detect changes in the phosphorylation of FtsZ in a stkP deletion strain relative to wild-type cells. Based on its cell-division-dependent localization and interaction with FtsZ, we propose that StkP plays a currently undefined role in cell division of pneumococcus.

Monoclonal antibodies targeting different cell wall antigens of group B streptococcus mediate protection in both Fc-dependent and independent manner.

Group B streptococcus remains an important neonatal pathogen in spite of widely adopted intrapartum antibiotic administration; therefore immune prophylaxis for GBS infections is highly warranted. In passive immunization and lethal challenge studies with multiple GBS strains, we characterized the protective effect of rabbit polyclonal and murine monoclonal antibodies specific for four multi-functional cell wall anchored proteins, FbsA, BibA, PilA and PilB. Single specificity rabbit sera or mAbs induced high level, but strain dependent protection, while their combinations resulted in superior and broad efficacy against all GBS strains tested. Polyclonal and monoclonal antibodies specific for the pilus proteins exerted very potent opsonophagocytic killing activity in vitro and required the Fc domain for protection in vivo. In contrast, FbsA and BibA specific antibodies failed to show OPK activity, but their Fab fragments fully protected animals, suggesting that blocking the function of these proteins was the major mode of action. These data are supportive for developing immune prophylaxis with human mAbs for prematurely born neonates who receive low levels of antibodies by maternofetal transport and are characterized by not fully developed phagocytic and complement activity.

Immunological fingerprinting of group B streptococci: from circulating human antibodies to protective antigens.

Group B streptococcus is one of the most important pathogens in neonates, and causes invasive infections in non-pregnant adults with underlying diseases. Applying a genomic approach that relies on human antibodies we identified antigenic GBS proteins, among them most of the previously published protective antigens. In vitro analyses allowed the selection of conserved candidate antigens that were further evaluated in murine lethal sepsis models using several GBS strains. In active and passive immunization models, we identified four protective GBS antigens, FbsA and BibA, as well as two hypothetical proteins, all shown to contribute to virulence based on gene deletion mutants. These protective antigens have the potential to be components of novel vaccines or targets for passive immune prophylaxis against GBS disease.

Composition of the ANTIGENome of Helicobacter pylori defined by human serum antibodies.

Helicobacter pylori is the most prevalent human pathogen and although, it remains silent in most individuals for lifetime, colonization may develop into severe gastric and duodenal conditions. Rapidly developing resistance to antibiotic treatment urgently calls for the development of effective vaccines. We determined the ANTIGENome of two clinical isolates of H. pylori, KTH-Ca1 and KTH-Du, derived from patients with gastric cancer and duodenal ulcer, respectively. Using disease-relevant human sera from well-characterized donors we identified 124 annotated ORFs and 54 non-annotated peptides as antigens. Through in vitro validation assays we selected the 20 most promising vaccine candidates. Importantly, two candidates represent proteins that were previously shown to provide protection in models of H. pylori infection. One of the most frequently selected and conserved protein, the siderophore-dependent transporter HP1341, was confirmed to show high reactivity with human serum IgGs. These analyses provide the means to identify novel antigens for the selection of vaccine candidates, as well as disease associated biomarkers.

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 pathogen's proteome. Serum antibodies of exposed, but not infected, individuals and convalescing patients identified the ANTIGENome of pneumococcus consisting of approximately 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.

High-affinity binding of the staphylococcal HarA protein to haptoglobin and hemoglobin involves a domain with an antiparallel eight-stranded beta-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 (K(D)) 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 beta-barrel with the strand order (-beta1 -beta2 -beta3 -beta6 -beta5 -beta4 -beta7 -beta8), 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.

Comparison of antibody repertoires against Staphylococcus aureus in healthy individuals and in acutely infected patients.

The management of staphylococcal diseases is increasingly difficult with present medical approaches. Preventive and therapeutic vaccination is considered to be a promising alternative; however, little is known about immune correlates of protection and disease susceptibility. To better understand the immune recognition of Staphylococcus aureus by the human host, we studied the antistaphylococcal humoral responses in healthy people in comparison to those of patients with invasive diseases. In a series of enzyme-linked immunosorbent assay analyses performed using 19 recombinant staphylococcal cell surface and secreted proteins, we measured a wide range of antibody levels, finding a pronounced heterogeneity among individuals in both donor groups. The analysis revealed marked differences in the antibody repertoires of healthy individuals with or without S. aureus carriage, as well as in those of patients in the acute phase of infection. Most importantly, we identified antigenic proteins for which specific antibodies were missing or underrepresented in infected patients. In contrast to the well-described transient nature of disease-induced antistaphylococcal immune response, it was demonstrated that high-titer antistaphylococcal antibodies are stable for years in healthy individuals. In addition, we provide evidence obtained on the basis of opsonophagocytic and neutralizing activity in vitro assays that circulating antistaphylococcal serum antibodies in healthy donors are functional. In light of these data we suggest that proper serological analysis comparing the preexisting antibody repertoires of hospitalized patients with different outcomes for nosocomial staphylococcal infections could be extremely useful for the evaluation of candidate vaccine antigens in addition to protection data generated with animal models.

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.

PIBF (progesterone induced blocking factor) is overexpressed in highly proliferating cells and associated with the centrosome.

PIBF was previously identified as a 34 kDa immunomodulatory molecule secreted by pregnancy lymphocytes and is thought to play a crucial role in preventing rejection of the embryo by the maternal immune response. Recent data suggested that PIBF protein was also expressed by the progesterone receptor (PR) positive MCF-7 breast tumor cell line. Therefore our study was designed to analyze the expression of PIBF in malignant cell lines and primary tumors both at the mRNA and protein levels. RNA expression analyses of several human cell lines with different tissue origin and paired human tumor/normal tissues, as well as of several PR+ and PR- breast tumors revealed that PIBF mRNA was overexpressed in highly proliferating cells independent of the presence of PR. In addition to the full-length PIBF mRNA encoding for a 90 kDa protein, several alternatively spliced species were detected, all resulting from perfect exon skipping. The most frequently identified splice variant is predicted to encode for an approximately 35 kDa protein. Immunofluorescence microscopy revealed a centrosomal localization for the full-length PIBF, while the 35 kDa form showed a diffuse cytoplasmic staining. These data, together with the identification of the PIBF gene in the chromosomal region associated with breast cancer susceptibility, reveal a strong parallel with known tumor suppressor proteins, such as BRCA1 and p53 having the same centrosomal localization. Given the notion that a number of proteins shown to be involved in tumorigenesis are associated with the centrosome and disturbed centrosome function causes unequal segregation of chromosomes, studies to evaluate whether or not PIBF that is highly expressed in tumors is directly involved in tumorigenesis are thus warranted.

Concomitant determination of absolute values of cellular protein amounts, synthesis rates, and turnover rates by quantitative proteome profiling.

Two-dimensional gel electrophoresis of protein fractions isolated from (35)S-radiolabeled cells provides qualitative information on intracellular amounts, (35)S incorporation rates, protein modifications, and subcellular localizations of up to thousands of individual proteins. In this study we extended proteome profiling to provide quantitative data on synthesis rates of individual proteins. We combined fluorescence detection of radiolabeled proteins with SYPRO ruby(TM) staining and subsequent autoradiography of the same gels, thereby quantifying protein amounts and (35)S incorporation. To calibrate calculation of absolute synthesis rates, we determined the amount and autoradiograph intensity of radiolabeled haptoglobin secreted by interleukin-6 pretreated HepG2 cells. This allowed us to obtain a standard calibration value for (35)S incorporation per autoradiograph intensity unit. This value was used to measure protein synthesis rates during time course experiments of heat-shocked U937 cells. We measured the increasing amounts of hsp70 and calculated it by integration of the determined hsp70 synthesis rates over time. Similar results were obtained by both methods, validating our standardization procedure. Based on the assumption that the synthesis rate of proteins in a steady state of cell metabolism would essentially compensate protein degradation, we calculated biological half-lives of proteins from protein amounts and synthesis rates determined from two-dimensional gels. Calculated protein half-lives were found close to those determined by pulse-chase experiments, thus validating this new method. In conclusion, we devised a method to assess quantitative proteome profiles covering determination of individual amounts, synthesis, and turnover rates of proteins.

Plasma from cancer patients featuring a characteristic protein composition mediates protection against apoptosis.

By comparative proteome analysis we searched for characteristic alterations of human plasma accompanying neoplastic disease. We identified protein alterations in plasma of prostate-, lung-, and breast-cancer patients in comparison to controls, comprising elevated levels of fibrinogen gamma-chain dimer, degradation products of antiplasmin and laminin gamma-chain, and elevated levels of acute phase proteins. The latter proteins and laminin fragments have been described as anti-apoptotic factors. We raised the question whether these alterations may have any relevance for the regulation of apoptosis. In contrast to plasma derived from healthy donors, samples from prostate-, lung-, and breast-cancer patients selectively inhibited Fas- and staurosporine-induced apoptosis in Jurkat cells but remained ineffective upon UV light-induced apoptosis. These data suggested that inhibition occurred by extracellular interference with apoptosis induction. Supporting this hypothesis, we found that formation of the CD95 death-inducing signal complex was strongly inhibited in the presence of plasma from cancer patients.