Elena Cartocci

Identification of protective and broadly conserved vaccine antigens from the genome of extraintestinal pathogenic Escherichia coli

Extraintestinal pathogenic Escherichia coli (ExPEC) are a common cause of disease in both mammals and birds. A vaccine to prevent such infections would be desirable given the increasing antibiotic resistance of these bacteria. We have determined the genome sequence of ExPEC IHE3034 (ST95) isolated from a case of neonatal meningitis and compared this to available genome sequences of other ExPEC strains and a few nonpathogenic E. coli. We found 19 genomic islands present in the genome of IHE3034, which are absent in the nonpathogenic E. coli isolates. By using subtractive reverse vaccinology we identified 230 antigens present in ExPEC but absent (or present with low similarity) in nonpathogenic strains. Nine antigens were protective in a mouse challenge model. Some of them were also present in other pathogenic non-ExPEC strains, suggesting that a broadly protective E. coli vaccine may be possible. The gene encoding the most protective antigen was detected in most of the E. coli isolates, highly conserved in sequence and found to be exported by a type II secretion system which seems to be nonfunctional in nonpathogenic strains.

The Region Comprising Amino Acids 100 to 255 of Neisseria meningitidis Lipoprotein GNA 1870 Elicits Bactericidal Antibodies

ABSTRACT GNA 1870 is a novel surface-exposed lipoprotein, identified by genome analysis of Neisseria meningitidis strain MC58, which induces bactericidal antibodies. Three sequence variants of the protein were shown to be sufficient to induce bactericidal antibodies against a panel of strains representative of the diversity of serogroup B meningococci. Here, we studied the antigenic and immunogenic properties of GNA 1870, which for convenience was divided into domains A, B, and C. The immune responses of mice immunized with each of the three variants were tested using overlapping peptides scanning the entire protein length and using recombinant fragments. We found that while most of the linear epitopes are located in the A domain, the bactericidal antibodies are directed against conformational epitopes located in the BC domain. This was also confirmed by the isolation of a bactericidal murine monoclonal antibody, which failed to recognize linear peptides on the A, B, and C domains separately but recognized a conformational epitope formed only by the combination of the B and C domains. Arginine in position 204 was identified as important for binding of the monoclonal antibody. The identification of the region containing bactericidal epitopes is an important step in the design of new vaccines against meningococci.

The RNA Chaperone Hfq Is Involved in Stress Response and Virulence in Neisseria meningitidis and Is a Pleiotropic Regulator of Protein Expression

ABSTRACT The well-conserved protein Hfq has emerged as the key modulator of riboregulation in bacteria. This protein is thought to function as an RNA chaperone and to facilitate base pairing between small regulatory RNA (sRNA) and mRNA targets, and many sRNAs are dependent on the Hfq protein for their regulatory functions. To address the possible role of Hfq in riboregulated circuits in Neisseria meningitidis, we generated an Hfq mutant of the MC58 strain, and the knockout mutant has pleiotropic phenotypes; it has a general growth phenotype in vitro in culture media, and it is sensitive to a wide range of stresses, including those that it may encounter in the host. Furthermore, the expression profile of a vast number of proteins is clearly altered in the mutant, and we have identified 27 proteins by proteomics. All of the phenotypes tested to date are also restored by complementation of Hfq expression in the mutant strain. Importantly, in ex vivo and in vivo models of infection the Hfq mutant is attenuated. These data indicate that Hfq plays a key role in stress response and virulence, and we propose a major role for Hfq in regulation of gene expression. Moreover, this study suggests that in meningococcus there is a large Hfq-mediated sRNA network which so far is largely unexplored.

Vaccine composition formulated with a novel TLR7-dependent adjuvant induces high and broad protection against Staphylococcus aureus.

Significance Staphylococcus aureus is a human pathogen causing life-threatening infections. The high incidence of methicillin-resistant S. aureus isolates resistant to all antibiotics makes the development of anti-S. aureus vaccines an urgent medical need. However, the unique ability of S. aureus to produce virulent factors, which counteract virtually all pathways of innate and adaptive immunity, has hampered all vaccine discovery efforts. Starting from the assumption that to be effective a vaccine should induce highly functional antibodies and potentiate the killing capacity of phagocytic cells, we selected a cocktail of five conserved antigens involved in different mechanisms of pathogenesis, and we formulated them with a potent adjuvant. This vaccine provides an unprecedented protective efficacy against S. aureus infection in animal models. Both active and passive immunization strategies against Staphylococcus aureus have thus far failed to show efficacy in humans. With the attempt to develop an effective S. aureus vaccine, we selected five conserved antigens known to have different roles in S. aureus pathogenesis. They include the secreted factors α-hemolysin (Hla), ess extracellular A (EsxA), and ess extracellular B (EsxB) and the two surface proteins ferric hydroxamate uptake D2 and conserved staphylococcal antigen 1A. The combined vaccine antigens formulated with aluminum hydroxide induced antibodies with opsonophagocytic and functional activities and provided consistent protection in four mouse models when challenged with a panel of epidemiologically relevant S. aureus strains. The importance of antibodies in protection was demonstrated by passive transfer experiments. Furthermore, when formulated with a toll-like receptor 7-dependent (TLR7) agonist recently designed and developed in our laboratories (SMIP.7–10) adsorbed to alum, the five antigens provided close to 100% protection against four different staphylococcal strains. The new formulation induced not only high antibody titers but also a Th1 skewed immune response as judged by antibody isotype and cytokine profiles. In addition, low frequencies of IL-17–secreting T cells were also observed. Altogether, our data demonstrate that the rational selection of mixtures of conserved antigens combined with Th1/Th17 adjuvants can lead to promising vaccine formulations against S. aureus.

Serum Response Factor and Protein-Mediated DNA Bending Contribute to Transcription of the Dystrophin Muscle-Specific Promoter

The minimal muscle-specific dystrophin promoter contains the consensus sequence CC(A/T)6GG, or the CArG element, which can be found in serum-inducible or muscle-specific promoters. The serum response factor (SRF), which mediates the transcriptional activation of the c-fos gene in response to serum stimulation, can bind to different CArG box elements, suggesting that it could be involved in muscle-constitutive transcription. Here we show that SRF binds to the dystrophin promoter and regulates its muscle-specific transcription. In transient transfections, an altered-binding-specificity SRF mutant restores the muscle-constitutive transcription of a dystrophin promoter with a mutation in its CArG box element. The muscle-constitutive transcription of the dystrophin promoter also requires the sequence GAAACC immediately downstream of the CArG box. This sequence is recognized by a novel DNA bending factor which was named dystrophin promoter-bending factor (DPBF). Mutations of the CArG flanking sequence abolish both DPBF binding and the promoter activity in muscle cells. Its replacement with a p62/ternary complex factor binding site changes the promoter specificity from muscle constitutive to serum responsive. These results show that, on the dystrophin promoter, the transcriptional activation induced by SRF requires the DNA bending induced by DPBF. The bending, next to the CArG box, could promote interactions between SRF and other proteins in the transcriptional complex.

Structure of the meningococcal vaccine antigen NadA and epitope mapping of a bactericidal antibody

Significance Serogroup B meningococcus (MenB) causes severe sepsis and invasive meningococcal disease, particularly affecting young children and adolescents. The genome-derived vaccine 4CMenB that targets MenB, has now been approved in over 30 countries worldwide. Here we report the crystal structure of the trimeric autotransporter Neisserial adhesin A (NadA), one of the three protein antigens included in 4CMenB, and the epitope mapping of a bactericidal mAb monoclonal antibody that targets the functional head domain of NadA. These results provide important insights into the structure and vaccine-induced immune response of this meningococcal antigen and may inform the engineering of improved immunogens by structure-based design. Serogroup B Neisseria meningitidis (MenB) is a major cause of severe sepsis and invasive meningococcal disease, which is associated with 5–15% mortality and devastating long-term sequelae. Neisserial adhesin A (NadA), a trimeric autotransporter adhesin (TAA) that acts in adhesion to and invasion of host epithelial cells, is one of the three antigens discovered by genome mining that are part of the MenB vaccine that recently was approved by the European Medicines Agency. Here we present the crystal structure of NadA variant 5 at 2 Å resolution and transmission electron microscopy data for NadA variant 3 that is present in the vaccine. The two variants show similar overall topology with a novel TAA fold predominantly composed of trimeric coiled-coils with three protruding wing-like structures that create an unusual N-terminal head domain. Detailed mapping of the binding site of a bactericidal antibody by hydrogen/deuterium exchange MS shows that a protective conformational epitope is located in the head of NadA. These results provide information that is important for elucidating the biological function and vaccine efficacy of NadA.

Expression of factor H binding protein in meningococcal strains can vary at least 15-fold and is genetically determined

Significance Complement is the main line of defense against bacterial pathogens; however, the molecular mechanisms triggering killing are largely unknown. Factor H binding protein (fHbp) is a component of two licensed vaccines against serogroup B meningococcus and a key target of complement-mediated bacterial killing. Selected reaction monitoring was used for the absolute quantification of fHbp on invasive meningococcal strains, showing that expression among strains can vary at least 15-fold and a minimum of 757 molecules separated by not more than 130 nm are required to engage C1q and kill the bacteria. Furthermore, the amount of fHbp is genetically determined by the sequence of the promoter region and correlated with the bactericidal activity. These findings increase the understanding of complement-mediated killing and vaccine protection. Factor H binding protein (fHbp) is a lipoprotein of Neisseria meningitidis important for the survival of the bacterium in human blood and a component of two recently licensed vaccines against serogroup B meningococcus (MenB). Based on 866 different amino acid sequences this protein is divided into three variants or two families. Quantification of the protein is done by immunoassays such as ELISA or FACS that are susceptible to the sequence variation and expression level of the protein. Here, selected reaction monitoring mass spectrometry was used for the absolute quantification of fHbp in a large panel of strains representative of the population diversity of MenB. The analysis revealed that the level of fHbp expression can vary at least 15-fold and that variant 1 strains express significantly more protein than variant 2 or variant 3 strains. The susceptibility to complement-mediated killing correlated with the amount of protein expressed by the different meningococcal strains and this could be predicted from the nucleotide sequence of the promoter region. Finally, the absolute quantification allowed the calculation of the number of fHbp molecules per cell and to propose a mechanistic model of the engagement of C1q, the recognition component of the complement cascade.

Neisseria Heparin Binding Antigen is targeted by the human alternative pathway C3-convertase

Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein specific for Neisseria and constitutes one of the three main protein antigens of the Bexsero vaccine. Meningococcal and human proteases, cleave NHBA protein upstream or downstream of a conserved Arg-rich region, respectively. The cleavage results in the release of the C-terminal portion of the protein. The C-terminal fragment originating from the processing of meningococcal proteases, referred to as C2 fragment, exerts a toxic effect on endothelial cells altering the endothelial permeability. In this work, we reported that recombinant C2 fragment has no influence on the integrity of human airway epithelial cell monolayers, consistent with previous findings showing that Neisseria meningitidis traverses the epithelial barrier without disrupting the junctional structures. We showed that epithelial cells constantly secrete proteases responsible for a rapid processing of C2 fragment, generating a new fragment that does not contain the Arg-rich region, a putative docking domain reported to be essential for C2-mediated toxic effect. Moreover, we found that the C3-convertase of the alternative complement pathway is one of the proteases responsible for this processing. Overall, our data provide new insights on the cleavage of NHBA protein during meningococcal infection. NHBA cleavage may occur at different stages of the infection, and it likely has a different role depending on the environment the bacterium is interacting with.