Giulio Ratti

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.

Genomic Approach for Analysis of Surface Proteins in Chlamydia pneumoniae

ABSTRACT Chlamydia pneumoniae, a human pathogen causing respiratory infections and probably contributing to the development of atherosclerosis and heart disease, is an obligate intracellular parasite which for replication needs to productively interact with and enter human cells. Because of the intrinsic difficulty in working with C. pneumoniae and in the absence of reliable tools for its genetic manipulation, the molecular definition of the chlamydial cell surface is still limited, thus leaving the mechanisms of chlamydial entry largely unknown. In an effort to define the surface protein organization of C. pneumoniae, we have adopted a combined genomic-proteomic approach based on (i) in silico prediction from the available genome sequences of peripherally located proteins, (ii) heterologous expression and purification of selected proteins, (iii) production of mouse immune sera against the recombinant proteins to be used in Western blotting and fluorescence-activated cell sorter (FACS) analyses for the identification of surface antigens, and (iv) mass spectrometry analysis of two-dimensional electrophoresis (2DE) maps of chlamydial protein extracts to confirm the presence of the FACS-positive antigens in the chlamydial cell. Of the 53 FACS-positive sera, 41 recognized a protein species with the expected size on Western blots, and 28 of the 53 antigens shown to be surface-exposed by FACS were identified on 2DE maps of elementary-body extracts. This work represents the first systematic attempt to define surface protein organization in C. pneumoniae.

Identification of immunoreactive proteins of Chlamydia trachomatis by Western blot analysis of a two‐dimensional electrophoresis map with patient sera

Western blots of two‐dimensional electrophoretic maps of proteins from Chlamydia trachomatis were probed with sera from 17 seropositive patients with genital inflammatory disease. Immunoblot patterns (comprising 28 to 2 spots, average 14.8) were different for each patient; however, antibodies against a spot‐cluster due to the chlamydia‐specific antigen outer membrane protein‐2 (OMP2) were observed in all sera. The next most frequent group of antibodies (15/17; 88%) recognized the hsp60 GroEL‐like protein, described as immunopathogenic in chlamydial infections. Reactivity to the major surface‐exposed and variable antigen major outer membrane protein (MOMP) was observed at a relatively lower frequency (13/17; 76%). The hsp70 DnaK‐like protein was also frequently recognized (11/17; 64.7%) in this patient group. Besides the above confirmatory findings, the study detected several new immunoreactive proteins, with frequencies ranging from 11/17 to 1/17. Some were characterized also by N‐terminal amino acid sequencing and homology searches. Amongst these were a novel outer membrane protein (OmpB) and, interestingly, five conserved bacterial proteins: four (23%) sera reacted with the RNA polymerase alpha‐subunit, five (29%) recognized the ribosomal protein S1, eight (47%) the protein elongation factor EF‐Tu, seven (41%) a putative stress‐induced protease of the HtrA family, and seven sera (41%) the ribosomal protein L7/L12. Homologs of the last two proteins were shown to confer protective immunity in other bacterial infections. The data show that immunological sensitization processes commonly thought to play a role in chlamydial pathogenicity may be sustained not only by the hsp60 GroEl‐like protein, but also by other conserved bacterial antigens, some of which may be also considered as potential vaccine candidates.

Diversity of the Chlamydia trachomatis common plasmid in biovars with different pathogenicity

The 7.5-kb plasmid of Chlamydia trachomatis (CT) is believed to encode essential genes and might have a role in CT pathogenicity. Accordingly, analysis of plasmid-linked mutations in isolates from biovars with different pathogenic properties should help in identifying which plasmid-encoded genes, if any, may be involved in modulating virulence. For this purpose, the plasmid present in a low-virulence isolate (trachoma biovar, serotype D) was cloned and sequenced. Nucleotide changes were experimentally checked against the sequence of the plasmid variant from the highly virulent strain L2/434/Bu (LGV biovar). By aligning our data with two published sequences of different trachoma and LGV variants a general consensus structure was determined, comprising eight major open reading frames (ORF) and a number of points where there is consensus only between isolates of the same biovar (biovar-specific mutations). The degree of variation between different isolates is less than 1%. In particular, comparison of serotype-D and -L2 plasmids shows mutations which are generally silent or lead to few (one to four), often conservative, amino acid changes in ORFs 1, 2, 4, 5, 6, and 7. The protein encoded by ORF8 is completely conserved. In contrast, the polypeptide variants encoded by ORF3 show nine amino acid changes, seven of which are due to biovar-specific mutations.

DNA immunization with pgp3 gene of Chlamydia trachomatis inhibits the spread of chlamydial infection from the lower to the upper genital tract in C3H/HeN mice

Chlamydia trachomatis pgp3 DNA immunized (no. 300) and non-immunized (no. 300) C3H/HeN mice were infected by vaginal inoculation with infectious C. trachomatis serotype D elementary bodies (EBs) and the spread of infection to the salpinges was assessed by cell culture isolation from tissue homogenates 7, 14, 21, 28, 35 and 42 days post-infection (p.i.). Overall, the pgp3-DNA immunization prevented salpinx infection in 94 (56%) mice, if compared with the 168 positive animals found among the non-immunized animals (P < 0.001). A group of negative control animals (i.e. mice immunized with plasmid DNA containing an irrelevant insert) was not protected, whereas all the mice of a positive immune control group (mice that had resolved a primary genital C. trachomatis infection) were resistant to re-infection. Pgp3 DNA immunization induced both humoral and mucosal anti-pgp3 antibodies.

The structure of a plasmid of Chlamydia trachomatis believed to be required for growth within mammalian cells.

Sequence analysis of a 7.5 kb DNA plasmid isolated from Chlamydia trachomatis shows 8 open reading frames (ORFs) regularly spaced along most of the sequence. One of these ORFs encodes a 451‐amino‐acid polypeptide highly homologous to the DnaB protein of Escherichia coli. A region between ORFs 6 and 7 contains a cluster of alternating ATs and a 22 bp sequence tandemly repeated 4 times, suggesting a replication control region. Several ORFs correspond to plasmid‐specific polypeptides that have been described. Codons ending with A or T are more frequent, as might be expected from the high A/T content (64%) of the plasmid, and codon usage is similar to that of the C. trachomatis chromosomal gene, omp1L2.

Transcriptional regulation in the Chlamydia trachomatis pCT plasmid

We have analyzed transcriptional regulation of the chlamydial plasmid pCT. Transcription of a full-length 2.9-kb ORF1-ORF2 mRNA is likely to be regulated by the sigma 66 transcription factor which recognizes the TATAAT and TNGNCA sequences at the -10 and -35 DNA regions, respectively. RNA synthesis starts 39 nucleotides (nt) upstream from the ATG start codon of ORF1 and terminates within the downstream ORF3 DNA region. A 2.8-kb transcript transverses the ORF3-6 DNA region, while two transcripts of 2.2 and 1.9 kb cover the ORF4-6 DNA region. These mRNAs overlap two abundant transcripts which regulate the expression of the ORF3 and ORF4 genes. The accumulation of transcripts associated with these ORFs is likely to be regulated at the level of RNA synthesis by an unknown sigma factor which could select the RTTTAAA and TTYTTR sequences located at the -10 and -35 DNA regions, respectively. This new promoter consensus sequence could be unique to the gene expression machinery of Chlamydiae.

Analysis of HLA DP, DQ, and DR allesles in adult Italian rheumatoid arthritis patients

We analyzed the distribution of DRB1, DQA1, DQB1, and DPB1 allelic variants in 48 rheumatoid arthritis (RA) patients, compared with 109 Italian random controls, using PCR amplification and hybridization with specific oligonucleotides. We confirm the previously reported increase of DR4 specificity, in comparison with healthy Italian individuals. In particular, we find a statistically significant positive association of DRB1*0401 and DRB1*0404 alleles with RA. However, when we compare the DR4+ groups, none of the DRB1*04 alleles is increased in the RA group. By sequence analysis, performed on 10 patients, we demonstrate that the DRB1*04 genes of RA show no difference from the DRB1*04 sequences previously published. From the molecular analysis of the other DRB1 polymorphic variants, we find a trend of positive association of DRB1*0101 in DR4-negative patients versus DR4-negative healthy controls and, in the group of DR4-negative and/or DR1-negative patients, a similar increase of DRB1*06. Also, we observe in RA patients a statistically significant increase of DQA1*0301 and DQB1*0302 accompanied by a significant decrease of DQA1*0201, DQA1*0501 and DQB1*0201. Finally, from the analysis of DPB1 gene, it can be assessed that the distribution of DPB1 alleles does not differ significantly between RA patients and healthy controls.

Development of transplantable ascites tumours which continuously produce polyclonal antibodies in pristane primed BALB/c mice immunized with bacterial antigens and complete Freund's adjuvant

Bacterial immunogens (whole cells of Borrelia burgdorferi, elementary bodies of Chlamydia trachomatis and purified proteins of 22 and 24 kDa of Borrelia hermsii) were emulsified with an excess of complete Freunds adjuvant and injected (i.p.) on days 0, 7, 14 and 21, into BALB/c mice treated with pristane on day 6. This procedure induced the development of antibody-producing ascites tumours which could be serially transplanted in pristane-conditioned mice. Ascites tumours continued to yield a consistent amount of specific polyclonal antibody after ten serial transplants. The method described appears to be particularly useful for the production of a large amount of antibody when only small amounts of immunogen are available.

Conformational changes in diphtheria toxoids: Analysis with monoclonal antibodies

Monoclonal antibodies (Mab) were raised against CRM197, a non‐toxic mutant of diphtheria toxin (DT). The ability of four Mabs to bind DT and the six functional mutants CRM197, CRM176, CRM228, CRM1001, CRM45 and CRM30 was assessed by immunoblotting and by a radioimmunoassay in which the protein antigen in solution competes with labeled CRM197 for the Mab binding site. The results show that the peptides recognized by Mab11.3, Mab53 and Mab23 are accessible in the mutant molecules in solution but not when they are part of the native DT structure, which could therefore be described for this purpose as ‘closed’ in contrast with an ‘open’ conformation of CRM197, CRM176 and CRM228. In particular, the behaviour of Mab53 indicates that the single amino acid substitutions in the A fragments of CRM197 and CRM176 also affect the conformation of their B fragments.