Dendritic cells require STAT-1 phosphorylated at its transactivating domain for the induction of peptide-specific CTL.

Phosphorylation of transcription factor STAT-1 on Y701 regulates subcellular localization whereas phosphorylation of the transactivating domain at S727 enhances transcriptional activity. In this study, we investigate the impact of STAT-1 and the importance of transactivating domain phosphorylation on the induction of peptide-specific CTL in presence of the TLR9-dependent immune adjuvant IC31. STAT-1 deficiency completely abolished CTL induction upon immunization, which was strongly reduced in animals carrying the mutation of the S727 phospho-acceptor site. A comparable reduction of CTL was found in mice lacking the type I IFN (IFN-I) receptor, whereas IFN-gamma-deficient mice behaved like wild-type controls. This finding suggests that S727-phosphorylated STAT-1 supports IFN-I-dependent induction of CTL. In adoptive transfer experiments, IFN-I- and S727-phosphorylated STAT-1 were critical for the activation and function of dendritic cells. Mice with a T cell-specific IFN-I receptor ablation did not show impaired CTL responses. Unlike the situation observed for CTL development S727-phosphorylated STAT-1 restrained proliferation of naive CD8(+) T cells both in vitro and following transfer into Rag-deficient mice. In summary, our data reveal a dual role of S727-phosphorylated STAT-1 for dendritic cell maturation as a prerequisite for the induction of CTL activity and for T cell autonomous control of activation-induced or homeostatic proliferation.

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.

Helices alpha2 and alpha3 of West Nile virus capsid protein are dispensable for assembly of infectious virions.

The internal hydrophobic sequence within the flaviviral capsid protein (protein C) plays an important role in the assembly of infectious virions. Here, this sequence was analyzed in a West Nile virus lineage I isolate (crow V76/1). An infectious cDNA clone was constructed and used to introduce deletions into the internal hydrophobic domain which comprises helix alpha2 and part of the loop intervening helices alpha2 and alpha3. In total, nine capsid deletion mutants (4 to 14 amino acids long) were constructed and tested for virus viability. Some of the short deletions did not significantly affect growth in cell culture, whereas larger deletions removing almost the entire hydrophobic region significantly impaired viral growth. Efficient growth of the majority of mutants could, however, be restored by the acquisition of second-site mutations. In most cases, these resuscitating mutations were point mutations within protein C changing individual amino acids into more hydrophobic residues, reminiscent of what had been observed previously for another flavivirus, tick-borne encephalitis virus. However, we also identified viable spontaneous pseudorevertants with more than one-third of the capsid protein removed, i.e., 36 or 37 of a total of 105 residues, including all of helix alpha3 and a hydrophilic segment connecting alpha3 and alpha4. These large deletions are predicted to induce formation of large, predominantly hydrophobic fusion helices which may substitute for the loss of the internal hydrophobic domain, underlining the unrivaled structural and functional flexibility of protein C.

Chemokine degradation by the Group A streptococcal serine proteinase ScpC can be reconstituted in vitro and requires two separate domains.

Streptococcus pyogenes is one of the most common human pathogens and possesses diverse mechanisms to evade the human immune defence. One example of its immune evasion is the degradation of the chemokine IL (interleukin)-8 by ScpC, a serine proteinase that prevents the recruitment of neutrophils to an infection site. By applying the ANTIGENome technology and using human serum antibodies, we identified Spy0416, annotated as ScpC, as a prominent antigen that induces protective immune responses in animals. We demonstrate here for the first time that the recombinant form of Spy0416 is capable of IL-8 degradation in vitro in a concentration- and time-dependent manner. Mutations in the conserved amino acid residues of the catalytic triad of Spy0416 completely abolished in vitro activity. However, the isolated predicted proteinase domain does not exhibit IL-8-degrading activity, but is dependent on the presence of the C-terminal region of Spy0416. Binding to IL-8 is mainly mediated by the catalytic domain. However, the C-terminal region modulates substrate binding, indicating that the proteolytic activity is amenable to regulation via the non-catalytic regions. The specificity for human substrates is not restricted to IL-8, since we also detected in vitro protease activity for another CXC chemokine GRO-alpha (growth-related oncogene alpha), but not for NAP-2 (neutrophil-activating protein 2), SDF (stromal-cell-derived factor)-1alpha, PF-4 (platelet factor 4), I-TAC (interferon-gamma-inducible T-cell alpha-chemoattractant), IP-10 (interferon-gamma-inducible protein 10) and MCP-1 (monocyte chemoattractant protein 1). The degradation of two human CXC chemokines in vitro, the high sequence conservation, the immunogenicity of the protein in humans and the shown protection in animal studies suggest that Spy0416 is a promising vaccine candidate for the prevention of infections by S. pyogenes.

Innovating vaccines: from gene expression to translational entrepreneurship.

Born 1950 in the small town of Coburg in post-war West Germany, I was surrounded by a strange mindset that was coded by a "Gemisch" of "Verdrängung" of the country's Nazi past and by a blind optimism that the divided country-in spite of the cruelties and the horrors of the war and the Nazi period-could be rebuilt and regain the respect of other nations. Luckily, I was raised in a family where the reality of destruction and Nazi crimes of the past were openly discussed and not suppressed; the exodus and deportation of Jewish citizens and concentration camps such as Auschwitz were not taboo topics.

The antigenome: from protein subunit vaccines to antibody treatments of bacterial infections?

New strategies are needed to master infectious diseases. The so-called "passive vaccination", i.e., prevention and treatment with specific antibodies, has a proven record and potential in the management of infections and entered the medical arena more than 100 years ago. Progress in the identification of specific antigens has become the hallmark in the development of novel subunit vaccines that often contain only a single immunogen, frequently proteins, derived from the microbe in order to induce protective immunity. On the other hand, the monoclonal antibody technology has enabled biotechnology to produce antibody species in unlimited quantities and at reasonable costs that are more or less identical to their human counterparts and bind with high affinity to only one specific site of a given antigen. Although, this technology has provided a robust platform for launching novel and successful treatments against a variety of devastating diseases, it is up till now only exceptionally employed in therapy of infectious diseases. Monoclonal antibodies engaged in the treatment of specific cancers seem to work by a dual mode; they mark the cancerous cells for decontamination by the immune system, but also block a function that intervenes with cell growth. The availability of the entire genome sequence of pathogens has strongly facilitated the identification of highly specific protein antigens that are suitable targets for neutralizing antibodies, but also often seem to play an important role in the microbe's life cycle. Thus, the growing repertoire of well-characterized protein antigens will open the perspective to develop monoclonal antibodies against bacterial infections, at least as last resort treatment, when vaccination and antibiotics are no options for prevention or therapy. In the following chapter we describe and compare various technologies regarding the identification of suitable target antigens and the foundation of cognate monoclonal antibodies and discuss their possible applications in the treatment of bacterial infections together with an overview of current efforts.

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.

Unique membrane-interacting properties of the immunostimulatory cationic peptide KLKL(5)KLK (KLK).

We have monitored the effects of KLKL(5)KLK (KLK), a derivative of a natural cationic antimicrobial peptide (CAP) on isolated membrane vesicles, and investigated the partition of the peptide within these structures. KLK readily interacted with fluorescent dyes entrapped in the vesicles without apparent pore formation. Fractionation of vesicles revealed KLK predominantly in the membrane. Peptide-treated vesicles appeared with generally disorganized bilayers. While KLK showed no effect on osmotic resistance of human erythrocytes, dramatic decrease in core and surface membrane fluidity was observed in peptide-treated erythrocyte ghosts as measured by fluorescence anisotropy. Finally, CD spectroscopy revealed lipid-induced random coil to beta-sheet and beta-sheet to alpha-helix conformational transitions of KLK. Together with the oligonucleotide oligo-d(IC)(13) [ODN1a], KLK functions as a novel adjuvant, termed IC31. Among other immunological effects, KLK appears to facilitate the uptake and delivery of ODN1a into cellular compartments, but the nature of KLK's interaction with the cell surface and other membrane-bordered compartments remains unknown. Our results suggest a profound membrane interacting property of KLK that might contribute to the immunostimulatory activities of IC31.

Sustained viral load reduction in treatment-naive HCV genotype 1 infected patients after therapeutic peptide vaccination.

BACKGROUND: Novel antivirals augment treatment efficacy in chronic HCV infection, to overcome limitations on safety profile alternative approaches are warranted. The effect of a therapeutic peptide vaccine on HCV viral load was investigated in treatment-naïve genotype 1 HCV patients. METHODS: Fifty patients received 8 intradermal IC41 vaccinations biweekly with topical application of the TLR7 agonist imiquimod (Group A). In Group B, 21 patients received a condensed schedule of 16 subcutaneous vaccinations weekly without imiquimod. RESULTS: At Week 16 Group A (n=44) showed a statistically significant (p=0.0013) HCV viral load decline of 0.21 log. 24 weeks after the last vaccination the viral load decreased by 0.47 log (p<0.0001) in 34 subjects. This effect was more pronounced in 17 patients with high baseline HCV (>2×10(6)IU/ml) with a 0.61 log decline, which was statistically significant (p<0.02) starting two weeks after the third vaccination. No apparent effect on HCV viral load was observed in Group B (n=21). In Group A eight patients (24%) showed a viral load response defined as a decline of >0.8 log. Overall, about 30-55% of patients showed T cell responses during the vaccination series and up to six months in both groups. No significant correlations between the HCV viral load decrease and T cell immune response were detected. CONCLUSIONS: This is the first report on a significant antiviral effect of a peptide vaccine in HCV infected patients. Response kinetics with increased HCV RNA decline 24 weeks after the last IC41 vaccination is encouraging.

Adjuvating the adjuvant: facilitated delivery of an immunomodulatory oligonucleotide to TLR9 by a cationic antimicrobial peptide in dendritic cells.

IC31(®) is a novel bi-component vaccine adjuvant consisting of the peptide KLKL(5)KLK (KLK) and the TLR9 agonist oligonucleotide d(IC)(13) (ODN1a). While membrane-interacting properties of KLK and immuno-modulating capabilities of ODN1a have been characterized in detail, little is known of how these two molecules function together and synergize in interacting with their primary target cells, dendritic cells (DCs). We have found that KLK-triggered aggregates entrapped ODN1a and these complexes readily associated with the DC cell surface. KLK stimulated the uptake and internalization of ODN1a via endocytosis, while the bulk of the peptide remained associated with the cell periphery. ODN1a co-localized with early and late endosomes as well as endoplasmic reticular structures. ODN1a co-localized with TLR9 positive compartments following KLK mediated uptake. These features did not depend on the expression of TLR-9. Our results reveal novel mechanisms that allow KLK to enhance the effects of the TLR-9 ligand ODN1a in immunomodulation.

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.

Type I interferons as mediators of immune adjuvants for T- and B cell-dependent acquired immunity.

Originally identified as antiviral substances produced by infected cells, type I interferons (IFN-I) are now known to have a wide range of additional activities within both the innate and adaptive immune response. Here we review properties of IFN-I contributing to their 'natural immune adjuvant' character, and their important role for the function of complete Freund's adjuvant (CFA) and the TLR9-dependent immune adjuvant IC31. We show data to demonstrate that treatment with IFN-I boosts the ability of vaccine/adjuvant combinations to induce peptide-specific CTL in both young and old mice. We view these findings in the perspective of previous clinical applications of IFN-I for vaccination.

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.

Anti-infective antibodies: a novel tool to prevent and treat nosocomial diseases.

The emergence of multidrug-resistant bacteria is a growing challenge for healthcare in the treatment of infectious diseases. In particular, nosocomial infections are getting out of control and reduce the likelihood to recover without, sometimes lethal, complications and long-term damage. Current antibiotics are unable to keep nosocomial infections in check and novel ones move only reluctantly forward and are expected to only delay the problem of multidrug resistance. Progress made in the identification of suitable pathogen targets, a better understanding of host-parasite interactions and the recent inclusion of monoclonal antibodies into the arsenal of novel therapies has provoked the interest to revitalize a historical concept of medicine to treat and prevent bacterial infections with antibodies.

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.

Protective anti-mycobacterial T cell responses through exquisite in vivo activation of vaccine-targeted dendritic cells.

Vaccine efficacy largely depends upon DC targeting and activation. The most potent TLR soluble ligands induce diffuse DC activation, which may be associated with marked pro-inflammatory responses and possibly adverse effects. This raises the concern that effective vaccine adjuvants may similarly rely on widespread DC activation. Using a promising candidate vaccine against tuberculosis (fusion protein of Ag85B and 6-kDa early secretory antigenic target (ESAT-6)) formulated in the potent IC31 adjuvant, DC targeting and activation was studied in vivo, following the fate of antigen and adjuvant in the draining lymph nodes, to define the magnitude of DC targeting/activation required in vivo to induce protective vaccine responses. Unexpectedly, protective IFN-gamma-mediated Ag85B-ESAT-6/IC31 responses were associated to the activation of a minute population (less than 0.3%) of CD11c(+) lymph node DC, without detectable systemic pro-inflammatory responses. This activated peripheral tissue-derived DC population, characterized by enhanced CD80, CD86, CD40 and IL-12p40 expression, was only identified when focusing on adjuvant- or antigen-labeled CD11c(+) DC, which were found to support T cell proliferation. Immunization with aluminum hydroxide adjuvant (Alum) resulted in a similar proportion of antigen-associated DC but without detectable enhancement of CD80, CD86, CD40 or IL-12p40 expression. Thus, potent protective IFN-gamma-producing responses may be elicited by the exquisite activation of a minute number of in vivo targeted DC.

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.