Renata Grifantini

Previously unrecognized vaccine candidates against group B meningococcus identified by DNA microarrays

We have used DNA microarrays to follow Neisseria meningitidis serogroup B (MenB) gene regulation during interaction with human epithelial cells. Host-cell contact induced changes in the expression of 347 genes, more than 30% of which encode proteins with unknown function. The upregulated genes included transporters of iron, chloride, amino acids, and sulfate, many virulence factors, and the entire pathway of sulfur-containing amino acids. Approximately 40% of the 189 upregulated genes coded for peripherally located proteins, suggesting that cell contact promoted a substantial reorganization of the cell membrane. This was confirmed by fluorescence activated cell sorting (FACS) analysis on adhering bacteria using mouse sera against twelve adhesion-induced proteins. Of the 12 adhesion-induced surface antigens, 5 were able to induce bactericidal antibodies in mice, demonstrating that microarray technology is a valid approach for identifying new vaccine candidates and nicely complements other genome mining strategies used for vaccine discovery.

Identification of iron-activated and -repressed Fur-dependent genes by transcriptome analysis of Neisseria meningitidis group B

Iron is limiting in the human host, and bacterial pathogens respond to this environment by activating genes required for bacterial virulence. Transcriptional regulation in response to iron in Gram-negative bacteria is largely mediated by the ferric uptake regulator protein Fur, which in the presence of iron binds to a specific sequence in the promoter regions of genes under its control and acts as a repressor. Here we describe DNA microarray, computational and in vitro studies to define the Fur regulon in the human pathogen Neisseria meningitidis group B (strain MC58). After iron addition to an iron-depleted bacterial culture, 153 genes were up-regulated and 80 were down-regulated. Only 50% of the iron-regulated genes were found to contain Fur-binding consensus sequences in their promoter regions. Forty-two promoter regions were amplified and 32 of these were shown to bind Fur by gel-shift analysis. Among these genes, many of which had never been described before to be Fur-regulated, 10 were up-regulated on iron addition, demonstrating that Fur can also act as a transcriptional activator. Sequence alignment of the Fur-binding regions revealed that the N. meningitidis Fur-box encompasses the highly conserved (NATWAT)3 motif. Cluster analysis was effective in predicting Fur-regulated genes even if computer prediction failed to identify Fur-box-like sequences in their promoter regions. Microarray-generated gene expression profiling appears to be a very effective approach to define new regulons and regulatory pathways in pathogenic bacteria.

Gene Expression Profile in Neisseria meningitidis and Neisseria lactamica upon Host-Cell Contact

Abstract: Differential gene regulation in the human pathogen Neisseria meningitidis group B (MenB) and in Neisseria lactamica, a human commensal species, was studied by whole genome microarray after bacterial interaction with epithelial cells. Host‐cell contact induced changes in the expression of 347 and 285 genes in MenB and N. lactamica, respectively. Of these, only 167 were common to MenB and N. lactamica, suggesting that a different subset of genes is activated by pathogens and commensals. Change in gene expression was stable over time in N. lactamica, but short‐lived in MenB. A large part (greater than 30%) of the regulated genes encoded proteins with unknown function. Among the known genes, those coding for pili, capsule, protein synthesis, nucleotide synthesis, cell wall metabolism, ATP synthesis, and protein folding were down‐regulated in MenB. Transporters for iron, chloride and sulfate, some known virulence factors, GAPDH and the entire pathway of selenocysteine biosynthesis were upregulated. Gene expression profiling indicates that approximately 40% of the regulated genes encode putative surface‐associated proteins, suggesting that upon cell contact Neisseria undergoes substantial surface remodeling. This was confirmed by FACS analysis of adhering bacteria using mouse sera against a subset of recombinant proteins. Finally, a few surface‐located, adhesion‐activated antigens were capable of inducing bactericidal antibodies, indicating that microarray technology can be exploited for the identification of new vaccine candidates.

Approach to discover T- and B-cell antigens of intracellular pathogens applied to the design of Chlamydia trachomatis vaccines

Natural immunity against obligate and/or facultative intracellular pathogens is usually mediated by both humoral and cellular immunity. The identification of those antigens stimulating both arms of the immune system is instrumental for vaccine discovery. Although high-throughput technologies have been applied for the discovery of antibody-inducing antigens, few examples of their application for T-cell antigens have been reported. We describe how the compilation of the immunome, here defined as the pool of immunogenic antigens inducing T- and B-cell responses in vivo, can lead to vaccine candidates against Chlamydia trachomatis. We selected 120 C. trachomatis proteins and assessed their immunogenicity using two parallel high-throughput approaches. Protein arrays were generated and screened with sera from C. trachomatis-infected patients to identify antibody-inducing antigens. Splenocytes from C. trachomatis-infected mice were stimulated with 79 proteins, and the frequency of antigen-specific CD4+/IFN-γ+ T cells was analyzed by flow cytometry. We identified 21 antibody-inducing antigens, 16 CD4+/IFN-γ+–inducing antigens, and five antigens eliciting both types of responses. Assessment of their protective activity in a mouse model of Chlamydia muridarum lung infection led to the identification of seven antigens conferring partial protection when administered with LTK63/CpG adjuvant. Protection was largely the result of cellular immunity as assessed by CD4+ T-cell depletion. The seven antigens provided robust additive protection when combined in four-antigen combinations. This study paves the way for the development of an effective anti-Chlamydia vaccine and provides a general approach for the discovery of vaccines against other intracellular pathogens.

Multi High-Throughput Approach for Highly Selective Identification of Vaccine Candidates: the Group A Streptococcus Case

We propose an experimental strategy for highly accurate selection of candidates for bacterial vaccines without using in vitro and/or in vivo protection assays. Starting from the observation that efficacious vaccines are constituted by conserved, surface-associated and/or secreted components, the strategy contemplates the parallel application of three high throughput technologies, i.e. mass spectrometry-based proteomics, protein array, and flow-cytometry analysis, to identify this category of proteins, and is based on the assumption that the antigens identified by all three technologies are the protective ones. When we tested this strategy for Group A Streptococcus, we selected a total of 40 proteins, of which only six identified by all three approaches. When the 40 proteins were tested in a mouse model, only six were found to be protective and five of these belonged to the group of antigens in common to the three technologies. Finally, a combination of three protective antigens conferred broad protection against a panel of four different Group A Streptococcus strains. This approach may find general application as an accelerated and highly accurate path to bacterial vaccine discovery.

Mg 2+ signalling defines the group A streptococcal CsrRS (CovRS) regulon

CsrRS (or CovRS) is a two‐component system implicated in the control of multiple virulence determinants in the important human pathogen, group A Streptococcus (GAS). Earlier studies suggested that extracellular Mg2+ signalled through the presumed sensor histidine kinase, CsrS. We now confirm those findings, as complementation of a csrS mutant restored Mg2+‐dependent gene regulation. Moreover, we present strong evidence that Mg2+ signals through CsrS to regulate an extensive and previously undefined repertoire of GAS genes. The effect of Mg2+ on regulation of global gene expression was evaluated using genomic microarrays in an M‐type 3 strain of GAS and in an isogenic csrS mutant. Unexpectedly, of the 72 genes identified in the Mg2+‐stimulated CsrRS regulon, 42 were absent from the CsrR regulon (the latter being defined by comparison of wild‐type and CsrR mutant transcriptomes at low Mg2+). We observed CsrS‐dependent regulation of 72 of the 73 genes whose expression changed in response to elevated extracellular Mg2+ in wild‐type bacteria, a result that identifies CsrS as the principal, if not exclusive, sensor for extracellular Mg2+ in GAS. To our knowledge, this study is the first to characterize global gene regulation by a GAS two‐component system in response to a specific environmental stimulus.

Effect of Neisseria meningitidis Fur Mutations on Global Control of Gene Transcription

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.

Protein array profiling of tic patient sera reveals a broad range and enhanced immune response against Group A Streptococcus antigens

The human pathogen Group A Streptococcus (Streptococcus pyogenes, GAS) is widely recognized as a major cause of common pharyngitis as well as of severe invasive diseases and non-suppurative sequelae associated with the existence of GAS antigens eliciting host autoantibodies. It has been proposed that a subset of paediatric disorders characterized by tics and obsessive-compulsive symptoms would exacerbate in association with relapses of GAS-associated pharyngitis. This hypothesis is however still controversial. In the attempt to shed light on the contribution of GAS infections to the onset of neuropsychiatric or behavioral disorders affecting as many as 3% of children and adolescents, we tested the antibody response of tic patient sera to a representative panel of GAS antigens. In particular, 102 recombinant proteins were spotted on nitrocellulose-coated glass slides and probed against 61 sera collected from young patients with typical tic neuropsychiatric symptoms but with no overt GAS infection. Sera from 35 children with neither tic disorder nor overt GAS infection were also analyzed. The protein recognition patterns of these two sera groups were compared with those obtained using 239 sera from children with GAS-associated pharyngitis. This comparative analysis identified 25 antigens recognized by sera of the three patient groups and 21 antigens recognized by tic and pharyngitis sera, but poorly or not recognized by sera from children without tic. Interestingly, these antigens appeared to be, in quantitative terms, more immunogenic in tic than in pharyngitis patients. Additionally, a third group of antigens appeared to be preferentially and specifically recognized by tic sera. These findings provide the first evidence that tic patient sera exhibit immunological profiles typical of individuals who elicited a broad, specific and strong immune response against GAS. This may be relevant in the context of one of the hypothesis proposing that GAS antigen-dependent induction of autoantibodies in susceptible individuals may be involved the occurrence of tic disorders.

Multi-plasmid DNA vaccination avoids antigenic competition and enhances immunogenicity of a poorly immunogenic plasmid

DNA immunization is a very promising approach to the formulation of multivalent vaccines. However, little information is currently available on the immunogenicity of multi‐plasmid formulations. To address this issue, we immunized mice with a combination of four plasmids encoding malarial antigens and we compared antibody responses with those obtained with single‐plasmid injections. We found that when four plasmids encoding Plasmodium falciparum circumsporozoite protein, thrombospondin‐related anonymous protein, major merozoite surface protein (MSP)1 and Pfs25 are co‐injected into mice, Ab responses against each antigen are elicited at levels at least as high as the level obtained with single‐plasmid injection. The quality of antibody production, as determined by isotype analysis, was similar when single‐and multi‐plasmid administrations were compared, indicating the priming of the same cytokine profile for CD4+ T helper cells. The sera from mice immunized with the four‐plasmid formulation specifically recognized sporozoites, blood stage schizonts and gametes, indicating that DNA immunization induced antibody responses relevant to the native conformation. Finally and of particular interest, in the case of MSP1, the antibody response appears to be strongly potentiated by the presence of additional plasmids, indicating an adjuvant effect of DNA.

Role of FNR and FNR-regulated, sugar fermentation genes in Neisseria meningitidis infection

While it is generally accepted that anaerobic metabolism is required during infection, supporting experimental data have only been described in a limited number of studies. To provide additional evidence on the role of anaerobic metabolism in bacterial pathogens while invading mammalian hosts, we analysed the effect of the inactivation of FNR, the major regulatory protein involved in the adaptation to oxygen restrictive conditions, and of two of the FNR‐regulated genes on the survival of Neisseria meningitidis serogroup B (MenB) in vivo. We found that fnr deletion resulted in more than 1 log reduction in the meningococcal capacity to proliferate both in infant rats and in mice. To identify which of the FNR‐regulated genes were responsible for this attenuated phenotype, we defined the FNR regulon by combining DNA microarray analysis and FNR–DNA binding studies. Under oxygen‐restricted conditions, FNR positively controlled the transcription of nine transcriptional units, the most upregulated of which were the two operons NMB0388‐galM and mapA‐pgmβ implicated in sugar metabolism and fermentation. When galM and mapA were knocked out, the mutants were attenuated by 2 and 3 logs respectively. As the operons are controlled by FNR, from these data we conclude that MenB survival in the host anatomical sites where oxygen is limiting is supported by sugar fermentation.