Biomedical subjects
Publications and source records attributed to Rino Rappuoli.
Streptococcus pneumoniae is a major public health threat worldwide. The recent discovery that this pathogen possesses pili led us to investigate their protective abilities in a mouse model of intraperitoneal infection. Both active and passive immunization with recombinant pilus subunits afforded protection against lethal challenge with the S. pneumoniae serotype 4 strain TIGR4.
An ongoing public health challenge is to develop vaccines that are effective against infectious diseases that have global relevance. Vaccines against serotypes of group B Streptococcus (GBS) that are prevalent in the United States and Europe are not optimally efficacious against serotypes common to other parts of the world. New technologies and innovative approaches are being used to identify GBS antigens that overcome serotype-specificity and that could form the basis of a globally effective vaccine against this opportunistic pathogen. This Review highlights efforts towards this goal and describes a template that can be followed to develop vaccines against other bacterial pathogens.
We have examined the intranasal administration of a vaccine against Mycobacterium tuberculosis (M.tb) consisting of the mucosal adjuvant LTK63 and the Ag Ag85B-ESAT-6. Vaccination with LTK63/Ag85B-ESAT-6 gave a strong and sustained Th1 response mediated by IFN-gamma-secreting CD4 cells, which led to long-lasting protection against tuberculosis, equivalent to that observed with bacillus Calmette-Guérin (BCG) or Ag85B-ESAT-6 in dimethyldioctadecylammonium bromide/monophosphoryl lipid A. Because a crucial element of novel vaccine strategies is the ability to boost BCG-derived immunity, we also tested whether LTK63/Ag85B-ESAT-6 could act as a BCG booster vaccine in BCG-vaccinated mice. We found that vaccinating with LTK63/Ag85B-ESAT-6 strongly boosted prior BCG-stimulated immunity. Compared with BCG-vaccinated nonboosted mice, we observed that infection with M.tb led to a significant increase in anti-M.tb-specific CD4 T cells in the lungs of LTK63/Ag85B-ESAT-6-boosted animals. This correlated with a significant increase in the protection against M.tb in LTK63/Ag85B-ESAT-6-boosted mice, compared with BCG-vaccinated animals. Thus, LTK63/Ag85B-ESAT-6 represents an efficient preventive vaccine against tuberculosis with a strong ability to boost prior BCG immunity.
The advent of whole-genome sequencing of bacteria and advances in bioinformatics have revolutionized the study of bacterial pathogenesis, enabling the targeting of possible vaccine candidates starting from genomic information. Nowadays, the availability of hundreds of bacterial genomes enables identification of the genetic differences across several genomes from the same species. The unexpected degree of intra-species diversity suggests that a single genome sequence is not entirely representative and does not offer a complete picture of the genetic variability of a species. The practical consequence is that, in many cases, a universal vaccine is possible only by including a combination of antigens and this combination must take into account the pathogen population structure.
Meningitis and sepsis caused by serogroup B meningococcus are two severe diseases that still cause significant mortality. To date there is no universal vaccine that prevents these diseases. In this work, five antigens discovered by reverse vaccinology were expressed in a form suitable for large-scale manufacturing and formulated with adjuvants suitable for human use. The vaccine adjuvanted by aluminum hydroxide induced bactericidal antibodies in mice against 78% of a panel of 85 meningococcal strains representative of the global population diversity. The strain coverage could be increased to 90% and above by the addition of CpG oligonucleotides or by using MF59 as adjuvant. The vaccine has the potential to conquer one of the most devastating diseases of childhood.
NhhA, Neisseriahia/hsf homologue, or GNA0992, is an oligomeric outer membrane protein of Neisseria meningitidis, recently included in the family of trimeric autotransporter adhesins. In this study we present the structural and functional characterization of this protein. By expressing in Escherichia coli the full-length gene, deletion mutants and chimeric proteins of NhhA, we demonstrated that the last 72 C-terminal residues are able to allow trimerization and localization of the N-terminal protein domain to the bacterial surface. In addition, we investigated on the possible role of NhhA in bacterial-host interaction events. We assessed in vitro the ability of recombinant purified NhhA to bind human epithelial cells as well as laminin and heparan sulphate. Furthermore, we shown that E. coli strain expressing NhhA was able to adhere to epithelial cells, and observed a reduced adherence in a meningococcal isogenic MC58DeltaNhhA mutant. We concluded that this protein is a multifunctional adhesin, able to promote the bacterial adhesion to host cells and extracellular matrix components. Collectively, our results underline a putative role of NhhA in meningococcal pathogenesis and ascertain its structural and functional belonging to the emerging group of bacterial autotransporter adhesins with trimeric architecture.
For over a century, vaccines were developed according to Pasteur's principles of isolating, inactivating and injecting the causative agent of an infectious disease. The availability of a complete microbial genome sequence in 1995 marked the beginning of a genomic era that has allowed scientists to change the paradigm and approach vaccine development starting from genomic information, a process named reverse vaccinology. This can be considered as one of the most powerful examples of how genomic information can be used to develop therapeutic interventions, which were difficult or impossible to tackle with conventional approaches. As the genomic era progressed, it became apparent that multi-strain genome analysis is fundamental to the design of universal vaccines. In the post-genomic era, the next challenge of the vaccine biologist will be the merging of the vaccinology with structural biology.
Although currently available vaccines represent an outstanding success story in modern medicine and have had a dramatic effect on morbidity and mortality worldwide, it is clear that improvements in vaccine delivery are required. Vaccine delivery improvements may include the addition of novel injectable adjuvants, or the use of novel routes of delivery, including mucosal immunization. Mucosal delivery may be required to provide protection against pathogens that infect at mucosal sites, including sexually transmitted diseases. Alternatively, novel approaches to delivery, including mucosal administration, may be used to improve compliance for existing vaccines. Of particular interest for safer mass immunization are needle-free delivery devices, which would avoid problems due to needle re-use in many parts of the world, or needle-stick injuries.
To test whether inactivated influenza vaccines distributed during the 2003-2004 influenza season in the northern hemisphere were able to confer protection against the mismatched variant A/Fujian/411/2002 virus strain, we measured haemagglutination inhibiting (HI) antibodies in elderly subjects vaccinated with three inactivated vaccines against the homologous A/H3N2 vaccine strain (A/Panama) and against the mismatched A/Fujian strain. The results showed that, while 76 to 80% of elder people vaccinated with conventional vaccines had protected levels of antibodies against the A/Fujian heterovariant strain, those vaccinated with the MF59-adjuvanted vaccine have protective levels of antibodies in >98% of the cases. We conclude that MF59-adjuvanted vaccines confer protection also against influenza virus strains which are not fully matched with those included in the vaccine.
The recent influenza vaccine shortages have provided a timely reminder of the tenuous nature of the world's vaccine supply and the potential for manufacturing issues to severely disrupt vital access to important vaccines. The application of new technologies to the discovery, assessment, development and production of vaccines has the potential to prevent such occurrences and enable the introduction of new vaccines. Gene-based vaccines, virus-like particles, plant-derived vaccines and novel adjuvants and delivery systems represent promising approaches to creating safer, more potent vaccines. As a consequence, more people will have faster access to more effective vaccines against a broader spectrum of infectious diseases. However, the increased cost of producing new vaccines and regulatory uncertainty remain challenges for vaccine manufacturers.
Most bacterial pathogens have long filamentous structures known as pili or fimbriae extending from their surface. These structures are often involved in the initial adhesion of the bacteria to host tissues during colonization. In gram-negative bacteria, pili are typically formed by non-covalent interactions between pilin subunits. By contrast, the recently discovered pili in gram-positive pathogens are formed by covalent polymerization of adhesive pilin subunits. Evidence from studies of pili in the three principal streptococcal pathogens of humans indicates that the genes that encode the pilin subunits and the enzymes that are required for the assembly of these subunits into pili have been acquired en bloc by the horizontal transfer of a pathogenicity island.
To better understand Neisseria meningitidis genomes and virulence, microarray comparative genome hybridization (mCGH) data were collected from one Neisseria cinerea, two Neisseria lactamica, two Neisseria gonorrhoeae and 48 Neisseria meningitidis isolates. For N. meningitidis, these isolates are from diverse clonal complexes, invasive and carriage strains, and all major serogroups. The microarray platform represented N. meningitidis strains MC58, Z2491 and FAM18, and N. gonorrhoeae FA1090. By comparing hybridization data to genome sequences, the core N. meningitidis genome and insertions/deletions (e.g. capsule locus, type I secretion system) related to pathogenicity were identified, including further characterization of the capsule locus, bioinformatics analysis of a type I secretion system, and identification of some metabolic pathways associated with intracellular survival in pathogens. Hybridization data clustered meningococcal isolates from similar clonal complexes that were distinguished by the differential presence of six distinct islands of horizontal transfer. Several of these islands contained prophage or other mobile elements, including a novel prophage and a transposon carrying portions of a type I secretion system. Acquisition of some genetic islands appears to have occurred in multiple lineages, including transfer between N. lactamica and N. meningitidis. However, island acquisition occurs infrequently, such that the genomic-level relationship is not obscured within clonal complexes. The N. meningitidis genome is characterized by the horizontal acquisition of multiple genetic islands; the study of these islands reveals important sets of genes varying between isolates and likely to be related to pathogenicity.
Squalene is a naturally occurring oil which has been used in the development of vaccine adjuvants, such as the oil-in-water emulsion MF59. In past years, by use of noncontrolled and nonvalidated assays, a claim was made that antisqualene antibodies were detectable in the sera of individuals with the so-called Gulf War syndrome. Using a validated enzyme-linked immunosorbent assay for the quantitation of immunoglobulin G (IgG) and IgM antibodies against squalene, we demonstrated that antisqualene antibodies are frequently detectable at very low titers in the sera of subjects who were never immunized with vaccines containing squalene. More importantly, vaccination with a subunit influenza vaccine with the MF59 adjuvant neither induced antisqualene antibodies nor enhanced preexisting antisqualene antibody titers. In conclusion, antisqualene antibodies are not increased by immunization with vaccines with the MF59 adjuvant. These data extend the safety profile of the MF59 emulsion adjuvant.
Macrophages (Mphi) may play an important role in the pathogenesis of invasive meningococcal infection. Previously, we have shown that the class A Mphi scavenger receptor (SR-A) is a major nonopsonic receptor for Neisseria meningitidis on Mphi. SR-A contributes to host defense by binding proinflammatory polyanionic ligands such as lipopolysaccharide (LPS) and by the uptake and killing of live organisms. SR-A-deficient mouse Mphi display a substantial reduction in the number of meningococci ingested compared to wild-type Mphi, and SR-A is required for meningococcal phagocytosis but not for the release of tumor necrosis factor alpha. Although soluble lipid A and lipid(IV)A are reported as ligands for SR-A, we demonstrated that LPS and LPS expression were not essential for the uptake of whole meningococci. In the present study, we set out to discover protein ligand(s) for SR-A in N. meningitidis lysates and outer membrane vesicles. Using various microbial mutant strains, we determined that molecules comprising the membrane capsule and pili, as well as the abundant surface Opa proteins were not essential for SR-A recognition. We developed a binding assay to detect SR-A ligands and identified three candidate proteins expressed on intact organisms, namely, NMB1220, NMB0278, and NMB0667. Soluble forms of these ligands were shown to block the binding of meningococci to CHO cells stably transfected with SR-A. Furthermore, NMB1220 was endocytosed by SR-A on Mphi and prevented internalization of soluble acetylated low-density lipoprotein. Thus, we have identified novel, unmodified protein ligands for SR-A that are able to inhibit meningococcal interactions with macrophages in vitro.
Iron homeostasis is particularly important in pathogenic bacteria, which need to compete with the host for this essential cofactor. In Helicobacter pylori, a causative agent of several gastric pathologies, iron uptake and storage genes are regulated at the transcriptional level by the ferric uptake regulator Fur. The regulatory circuit of Fur has recently come under focus because of an intimate interlink with a broader regulatory network governing metal homeostasis, acidic response, and virulence. To dissect the Fur regulatory circuit and identify in vivo targets of regulation, we developed a genome-wide location analysis protocol which allowed the identification of 200 genomic loci bound by Fur as well as the investigation of the binding efficiency of the protein to these loci in response to iron. Comparative analysis with transcriptomes of wild-type and fur deletion mutant strains allowed the distinction between targets associated with Fur regulation and genes indirectly influenced by the fur mutation. The Fur regulon includes 59 genes, 25 of which appear to be positively regulated. A case study conducted by primer extension analysis of two oppositely regulated genes, hpn2 and flaB, suggests that negative regulation as well as positive regulation occurs at the transcriptional level. Furthermore, the results revealed the existence of 13 Fur targeted loci within polycistronic operons, which were associated with transcript deregulation in the fur mutant strain. This study provides a systematic insight of Fur regulation at the genome-wide level in H. pylori and points to regulatory functions extending beyond the classical Fur repression paradigm.
The ferric uptake regulator Fur is a well-known iron-responsive repressor of gene transcription, which is used by many bacteria to respond to the low-iron environment that pathogens encounter during infection. In this study we used comparative transcriptome analysis to define the role of the Fur protein in the global control of gene transcription and iron regulation in Neisseria meningitidis. By using the Fur-null mutant and its complemented derivative, we identified 83 genes whose transcription is controlled by Fur. We report that Fur may control differential expression of these genes by binding directly to their promoters or through indirect mechanisms. In addition, mutation of the fur gene resulted in the induction of the heat shock response, and transcription of these genes does not respond to iron limitation. Furthermore, analysis of the iron starvation stimulon in the Fur-null mutant provided evidences of iron-responsive regulation that is independent of Fur. We began to dissect the regulatory networks of Fur and the heat shock (stress) response in N. meningitidis, and the observed interlink between the two circuits is discussed.
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GNA1870, a 28-kDa surface-exposed lipoprotein of Neisseria meningitidis recently discovered by reverse vaccinology, is one of the most potent antigens of Meningococcus and a promising candidate for a universal vaccine against a devastating disease. Previous studies of epitope mapping and genetic characterization identified residues critical for bactericidal response within the C-terminal domain of the molecule. To elucidate the conformation of protective epitopes, we used NMR spectroscopy to obtain the solution structure of the immunodominant 18-kDa C-terminal portion of GNA1870. The structure consists of an eight-stranded antiparallel beta-barrel overlaid by a short alpha-helix with an unstructured N-terminal end. Residues previously shown to be important for antibody recognition were mapped on loops facing the same ridge of the molecule. The sequence similarity of GNA1870 with members of the bacterial transferrin receptor family allows one to predict the folding of this class of well known bacterial antigens, providing the basis for the rational engineering of high affinity B cell epitopes.