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.

Immunization with the Chlamydia trachomatis major outer membrane protein, using adjuvants developed for human vaccines, can induce partial protection in a mouse model against a genital challenge.

To test several vaccines for Chlamydia trachomatis we vaccinated BALB/c and C3H/HeN female mice with a purified preparation of the native mouse pneumonitis (MoPn) major outer membrane protein (MOMP). The MOMP was formulated with anyone of three different adjuvants MF59, LT-K63 or LT-R72. As a negative control the animals were immunized with ovalbumin. Positive controls were inoculated intranasally (i.n.) with 10(4) inclusion-forming units (IFU) of C. trachomatis MoPn. High levels of Chlamydia-specific antibodies were detected in the serum and vaginal washes of the mice immunized with MOMP. Using a lymphoproliferative assay (LPA) a significant response was obtained in splenocytes from most of the groups of mice vaccinated with MOMP. Two weeks after the last immunization the mice were challenged in the left ovarian bursa with 10(5) IFU of C. trachomatis MoPn and vaginal cultures were collected for a period of 6 weeks. Overall, BALB/c and C3H/HeN mice immunized with MOMP showed a decrease in the severity and length of the infection but the difference with the controls was not statistically significant. Following mating the percentage of mice with bilateral fertility was not significantly different between mice vaccinated with MOMP and their respective ovalbumin-immunized controls. However, the C3H/HeN mice immunized with MOMP using MF59 or LTR72 as adjuvants had significantly more embryos per mouse than the control groups. In conclusion, mice immunized with native MOMP and adjuvants developed for human vaccines showed significant Chlamydia-specific immune response and a limited protection against infection and long-term sequelae.

Identification of new potential vaccine candidates against Chlamydia pneumoniae by multiple screenings.

Chlamydia are intracellular bacteria associated to serious human disease. A vaccine has proved difficult to obtain so far, and current opinions agree that multi-antigen combinations may be required to induce optimal protective responses. In order to identify new potential vaccine candidates, we recently screened the Chlamydia pneumoniae (Cpn) genome and described 53 recombinant proteins which elicited antibodies binding to purified Cpn cells. We now report that six proteins in this group can also induce in vitro neutralizing antibodies. Antibody specificity for the corresponding antigens was assessed by immunoblot analysis of 2DE Cpn protein maps. Furthermore, four of the six in vitro neutralizing antigens (Pmp2, Pmp10, OmpH-like and enolase) could inhibit Cpn dissemination in a hamster model. The results show that these Cpn proteins are immunoaccessible in infectious EBs, and recommend further investigation on their value as vaccine components.

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.

Experimental infection by Chlamydia pneumoniae in the hamster.

We report that intraperitoneal injection of Chlamydia pneumoniae purified elementary bodies (EBs) in the hamster causes a systemic infection allowing the isolation of viable chlamydiae from several organs for several days post-infection (p.i.). In particular, spleen infection occurred up to Day 7 p.i. in 100% of animals. Systemic infection probably occurs via macrophages as intraperitoneally injected chlamydiae which are taken up by the hamster macrophages remain viable and can infect in vitro cell cultures. Immunization of 18 hamsters with heat-inactivated purified EBs, completely protected the spleens of 16 animals and substantially reduced infection levels in the remaining two. This model, therefore, provides a robust screening tool for the assessment of the protective activity of potential vaccine candidates. In a pilot study on five recombinant antigens recently described as EB surface proteins, three gave results undistinguishable from non-immunized, or mock-immunized controls; however two antigens, derived, respectively, from the product of the lcrE gene (a component of the putative TTSS of C. pneumoniae) and the product of Cpn0498 open reading frame, proved to be capable of inducing protective immune responses.

Proteome analysis of Neisseria meningitidis serogroup A.

Neisseria meningitidis is an encapsulated Gram-negative bacterium responsible for significant morbidity and mortality worldwide. Meningococci are opportunistic pathogens, carried in the nasopharynx of approximately 10% of asymptomatic adults. Occasionally they enter the bloodstream to cause septicaemia and meningitis. Meningococci are classified into serogroups on the basis of polysaccharide capsule diversity, and serogroup A strains have caused major epidemics mainly in the developing world. Here we describe a two-dimensional gel electrophoresis protein map of the serogroup A strain Z4970, a clinical isolate classified as ancestral to several pandemic waves. To our knowledge this is the first systematically annotated proteomic map for N. meningitidis. Total protein samples from bacteria grown on GC-agar were electrophoretically separated and protein species were identified by matrix-assisted laser desorption/ionization time of flight spectrometry. We identified the products of 273 genes, covering several functional classes, including 94 proteins so far considered as hypothetical. We also describe several protein species encoded by genes reported by DNA microarray studies as being regulated in physiological conditions which are relevant to natural meningococcal pathogenicity. Since menA differs from other serogroups by having a fairly stable clonal population structure (i.e. with a low degree of variability), we envisaged comparative mapping as a useful tool for microevolution studies, in conjunction with established genotyping methods. As a proof of principle, we performed a comparative analysis on the B subunit of the meningococcal transferrin receptor, a vaccine candidate encoded by the tbpB gene, and a known marker of population diversity in meningococci. The results show that TbpB spot pattern variation observed in the maps of nine clinical isolates from diverse epidemic spreads, fits previous analyses based on allelic variations of the tbpB gene.

Genomic approach for analysis of surface proteins in Chlamydia pneumoniae.

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.