Anti-Toxin Responses to Natural Enterotoxigenic Escherichia coli (ETEC) Infection in Adults and Children in Bangladesh.

A sero-epidemiology study was conducted in Dhaka, Bangladesh between January 2020 and February 2021 to assess the immune responses to ETEC infection in adults and children. (1) Background: Enterotoxigenic Escherichia coli infection is a main cause of diarrheal disease in endemic countries. The characterization of the immune responses evoked by natural infection can guide vaccine development efforts. (2) Methods: A total of 617 adult and 480 pediatric diarrheal patients were screened, and 43 adults and 46 children (below 5 years of age) with an acute ETEC infection completed the study. The plasma samples were analyzed for antibody responses against the ETEC toxins. (3) Results: Heat-stable toxin (ST)-positive ETEC is the main cause of ETEC infection in adults, unlike in children in an endemic setting. We detected very low levels of anti-ST antibodies, and no ST-neutralizing activity. However, infection with ETEC strains expressing the heat-labile toxin (LT) induced systemic antibody responses in less than 25% of subjects. The antibody levels against LTA and LTB, as well as cholera toxin (CT), correlated well. The anti-LT antibodies were shown to have LT- and CT- neutralizing activity. The antibody reactivity against linear LT epitopes did not correlate with toxin-neutralizing activity. (4) Conclusions: Unlike LT, ST is a poor antigen and even adults have low anti-ST antibody levels that do not allow for the detection of toxin-neutralizing activity.

Evaluation of the Safety, Tolerability and Immunogenicity of ShigETEC, an Oral Live Attenuated Shigella-ETEC Vaccine in Placebo-Controlled Randomized Phase 1 Trial.

BACKGROUND: Shigella spp. and enterotoxigenic Escherichia coli (ETEC) cause high morbidity and mortality worldwide, yet no licensed vaccines are available to prevent corresponding infections. A live attenuated non-invasive Shigella vaccine strain lacking LPS O-antigen and expressing the ETEC toxoids, named ShigETEC was characterized previously in non-clinical studies. METHODS: ShigETEC was evaluated in a two-staged, randomized, double-blind and placebo-controlled Phase I clinical trial. A single dose of increasing amounts of the vaccine was given to determine the maximum tolerated dose and increasing number of immunizations were administered with an interval based on the duration of shedding observed. RESULTS: Oral immunization with ShigETEC was well tolerated and safe up to 4-time dosing with 5 × 1010 colony forming units. ShigETEC induced robust systemic immune responses against the Shigella vaccine strain, with IgA serum antibody dominance, as well as mucosal antibody responses evidenced by specific IgA in stool samples and in ALS (Antibodies in Lymphocyte Supernatant). Anti- ETEC toxin responses were detected primarily in the 4-times immunized cohort and for the heat-labile toxin correlated with neutralizing capacity. CONCLUSION: ShigETEC is a promising vaccine candidate that is scheduled for further testing in controlled human challenge studies for efficacy as well as in children in endemic setting for safety and immunogenicity.

Characterization of ShigETEC, a Novel Live Attenuated Combined Vaccine against Shigellae and ETEC.

Background: Shigella spp. and enterotoxigenic Escherichia coli (ETEC) remain the two leading bacterial causes of diarrheal diseases worldwide. Attempts to develop preventive vaccines against Shigella and ETEC have not yet been successful. The major challenge for a broad Shigella vaccine is the serotype-specific immune response to the otherwise protective LPS O-antigen. ETEC vaccines mainly rely on the heat-labile enterotoxin (LT), while heat-stable toxin (ST) has also been shown to be an important virulence factor. Methods: We constructed a combined Shigella and ETEC vaccine (ShigETEC) based on a live attenuated Shigella strain rendered rough and non-invasive with heterologous expression of two ETEC antigens, LTB and a detoxified version of ST (STN12S). This new vaccine strain was characterized and tested for immunogenicity in relevant animal models. Results: Immunization with ShigETEC resulted in serotype independent protection in the mouse lung shigellosis model and induced high titer IgG and IgA antibodies against bacterial lysates, and anti-ETEC toxin antibodies with neutralizing capacity. Conclusions: ShigETEC is a promising oral vaccine candidate against Shigella and ETEC infections and currently in Phase 1 testing.

Cationic amphipathic peptides accumulate sialylated proteins and lipids in the plasma membrane of eukaryotic host cells.

Cationic antimicrobial peptides (CAMPs) selectively target bacterial membranes by electrostatic interactions with negatively charged lipids. It turned out that for inhibition of microbial growth a high CAMP membrane concentration is required, which can be realized by the incorporation of hydrophobic groups within the peptide. Increasing hydrophobicity, however, reduces the CAMP selectivity for bacterial over eukaryotic host membranes, thereby causing the risk of detrimental side-effects. In this study we addressed how cationic amphipathic peptides-in particular a CAMP with Lysine-Leucine-Lysine repeats (termed KLK)-affect the localization and dynamics of molecules in eukaryotic membranes. We found KLK to selectively inhibit the endocytosis of a subgroup of membrane proteins and lipids by electrostatically interacting with negatively charged sialic acid moieties. Ultrastructural characterization revealed the formation of membrane invaginations representing fission or fusion intermediates, in which the sialylated proteins and lipids were immobilized. Experiments on structurally different cationic amphipathic peptides (KLK, 6-MO-LF11-322 and NK14-2) indicated a cooperation of electrostatic and hydrophobic forces that selectively arrest sialylated membrane constituents.

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.

Antimicrobial and immunostimulatory peptide, KLK, induces an increase in cytosolic Ca²(+) concentration by mobilizing Ca²(+) from intracellular stores.

The cationic antimicrobial immunomodulatory peptide, KLK (KLKL5KLK), exerts profound membrane interacting properties, impacting on ultrastructure and fluidity. KLK-membrane interactions that lead to these alterations require the ability of the peptide to move into an α-helical conformation. We show that KLK induces an increase of the intracellular Ca²(+) concentration in human T24 cells. The effect of KLK is buffer-sensitive, as it is detected when HBSS buffer is used, but not with PBS. This, together with the lack of effect of the middle leucine-to-proline-substituted peptide derivative [KPK (KLKLLPLLKLK)], indicates that it is the conformational propensity rather than the net positive charge that contributes to the effect of KLK on intracellular Ca²(+) level of T24 cells. We show that, although KLK slightly stimulates Ca²(+) influx into the cell, the bulk increase of Ca²(+) levels is due to KLK-induced depletion of intracellular Ca²(+) stores. Finally, we demonstrate a KLK-induced switch of PS (phosphatidylserine) from the inner to the outer plasma membrane leaflet that contributes to the onset of early apoptotic changes in these cells.

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.

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.

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.

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.

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.

RNA delivery by heat shock protein-70 into mammalian cells: a preliminary study.

Heat shock proteins (Hsps) are ubiquitous molecular chaperones with indispensable roles in assisting protein folding and giving protection from proteotoxic environmental harm. Members of the 70-kDa heat shock protein family have been demonstrated to recognize and bind with distinguished RNA sequences, which function as determinants of eukaryotic mRNA stability. We have earlier identified the molecular domains involved in RNA-binding and characterized in detail the specificity, affinity and some regulatory aspects of this molecular interaction using various deletion mutants and homologues of Hsp70. We have shown that wild type, but not any of the tested truncated mutants of Hsp70, is efficiently taken up by P388 mouse macrophage cells. Here we addressed the question of whether Hsp70 is capable of delivering bound RNA into mammalian cells. Employing fluorescence and confocal microscopy, we demonstrated that full length Hsp70 facilitates the uptake of RNA molecules into the cytoplasm of mammalian cells. We propose that further optimization of this system might enable the development of a valuable tool to deliver RNA molecules, such as siRNA, dsRNA or other regulatory RNA sequences to probe or influence various regulatory processes in eukaryotic cells.

[Antigenome technology: rapid and comprehensive identification and validation of antigens as putative components in vaccines against bacterial pathogens]. / Antigenom technológia: antigének gyors meghatározása és validálása bakteriális kórokozók elleni vakcinákban történo alkalmazáshoz.

The resolution of complete genome sequences of many pathogens has dramatically expanded our perspectives in developing novel vaccines by increasing both the speed and scale of target identification. Genomics-based technologies with their significant advantages can be applied to most pathogens and have exploited genome sequence information in alliance with adjunct technologies, including bioinformatics, expression analyses (microarrays, in vivo expression technologies), random mutagenesis or protein/peptide based selection methods (proteomics and immunoselection using expression libraries). A novel approach that combines the advantages of full genome coverage and serological antigen identification has been developed in the laboratory of the authors for the identification of protein antigens from bacterial pathogens. This technology has been applied to several bacterial pathogens and, as a result, defined the most relevant antigenic proteins that are targeted by the human immune system, including their antibody binding sites (epitopes). This catalogue of antigens is termed the antigenome and the novel technology offers an integrated approach for antigen validation in order to select the most promising candidates for the development of subunit vaccines against the targeted bacterial diseases. Using the antigenome technology, novel protective antigens had been discovered from several important human pathogens.

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.

Antigenome technology: a novel approach for the selection of bacterial vaccine candidate antigens.

A novel approach for the identification of protein antigens from bacterial pathogens was previously developed in our laboratory that combines the advantages of full genome coverage and serological antigen identification. We have applied this technology to several bacterial pathogens of the genera Staphylococcus and Streptococcus and have, as a result, defined the "antigenome" of these pathogens. This catalogue defines the most relevant antigenic proteins that are targeted by the human immune system, including their antibody binding sites. The antigenome technology offers an integrated approach for antigen validation in order to select the most promising candidates for the development of subunit vaccines against the targeted bacterial diseases. Using this technology, novel protective antigens were discovered from several important human pathogens.

Bacterial genomes pave the way to novel vaccines.

The availability of complete genome sequences of pathogens has dramatically changed the scope for developing improved and novel vaccines by increasing the speed of target identification. Genomics-based technologies have many advantages, compared to conventional approaches, which are time-consuming and usually identify only abundant antigens that are expressible under in vitro culture conditions. This review focuses on recent reports of genomics-based strategies that can be applied to most pathogens and that exploit genome sequence information in alliance with adjunct technologies, including bioinformatics, expression analyses, random mutagenesis or protein/peptide-based selection methods. Despite the caveats that are associated with the individual approaches, these technologies have already made major contributions to the identification and selection of novel vaccine candidates to combat bacterial infections.

Functional selection of vaccine candidate peptides from Staphylococcus aureus whole-genome expression libraries in vitro.

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.

Extending the 'stressy' edge: molecular chaperones flirting with RNA.

A number of intriguing observations have emerged during the past years indicating that certain classes of the evolutionarily highly conserved heat shock or stress proteins extend their molecular partnerships beyond the originally recognized protein world. In this review, following a brief introduction to the 70-kDa family of stress proteins, we summarize the main aspects of RNA recognition and binding by this class of molecules. By highlighting some biochemical features of both the protein and RNA partners, we attempt to embed the central parts of this interaction in the context of potential physiological relevance. As perhaps true for many newly recognized molecular interactions, the phenomenon of RNA recognition and binding by molecular chaperones discussed in this review calls for a place of ever-growing importance in the functional genomic era, where an expanding number of previously unsuspected molecular partnerships are uncovered by virtue of powerful high throughput methodologies. We suggest that integration of this new knowledge into the long-outlined 'classical' network of cellular metabolism at both the biochemical and architectural level is pivotal to the emerging 'synthesis era' of today's cell biology.

Surface binding and uptake of heat shock protein 70 by antigen-presenting cells require all 3 domains of the molecule.

The surprisingly efficient uptake of peptide-loaded heat shock proteins (Hsps) by antigen-presenting cells (APCs) has been recently associated with a specific receptor-ligand-based mechanism, and the identity of at least 1 receptor has been determined. In this study, we tested how the domain composition of the stress protein affected its surface association and internalization by APCs, and this was facilitated by the availability of the 70-kDa human heat shock protein (Hsp70) and its various deletion mutants. We show that both these processes strictly depend on the presence of all 3 domains of Hsp70. We propose that the previously described interdomain interactions as a determinant of a favorable conformational status might also govern a sterical adaptation of Hsps to components of the internalization machinery.

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.