Genetically modified L3,7 and L2 lipooligosaccharides from Neisseria meningitidis serogroup B confer a broad cross-bactericidal response.

Currently available Neisseria meningitidis serogroup B (MenB) vaccines are based on outer membrane vesicles (OMVs) that are obtained from wild-type strains. They are purified with the aim of decreasing the lipooligosaccharide (LOS) content and hence reduce the reactogenicity of the vaccine even though LOS is a potential protective antigen. In <2-year-old children, these MenB vaccines confer protection only against strains expressing homologous PorA, a major and variable outer membrane protein. Our objective was to develop a safe LOS-based vaccine against MenB. To this end, we used modified porA knockout strains expressing genetically detoxified (msbB gene-deleted) L2 and L3,7 LOSs, allowing the production of LOS-enriched OMVs. The vaccine-induced antibodies were found to be bactericidal against nearly all invasive strains, irrespective of capsular serogroup. In addition, we have also demonstrated that LOS lacking the terminal galactose (with a lgtB mutation; truncated L3 LOS), but not LOS produced without the galE gene, induced a bactericidal antibody response in mice similar to that seen for LOS containing the full lacto-N-neotetraose (L3,7 LOS). In conclusion, a bivalent detoxified LOS OMV-based vaccine demonstrated the potential to afford a broad cross-protection against meningococcal disease.

Effects of adsorption of acellular pertussis antigens onto different aluminium salts on the protective activity in an intranasal murine model of Bordetella pertussis infection.

Adsorption of the pertussis antigens, pertussis toxoid (PT), filamentous hemagglutinin (FHA) and pertactin (PRN) onto aluminium phosphate rather than aluminium hydroxide leads to a lower humoral response and poorer protection against intranasal pertussis challenge in mice. These effects could be reversed by inclusion of fimbriae (FIM) 2 and 3 in the formulation. These data emphasis the importance of correct formulation for such vaccines.

Developing a nontypeable Haemophilus influenzae (NTHi) vaccine.

There is a current high demand for nontypable Haemophilus influenzae (NTHi) vaccines. Various options for the composition of such vaccines are possible. Decisions about the vaccine composition have to take into account the antigenic variability of NTHi, so even complex immunogens such as whole bacteria would preferentially have a tailor-made antigenic composition. We will present a summary of NTHi vaccine development, describing research efforts from SmithKline Beecham and other laboratories. Currently, major (P1, P2, P4, P5) and minor (P6, D15, TbpA/B, ellipsis) outer membrane proteins, LPS, adhesins (HMW, Hia, pili, P5) are being studied. Preclinical results with LPD, P5 (LB1) and OMP26 from our laboratories will be described including the use of animal models of otitis and lung infection.

Structure and function prediction of the Brucella abortus P39 protein by comparative modeling with marginal sequence similarities.

A methodology is proposed to solve a difficult modeling problem related to the recently sequenced P39 protein. This sequence shares no similarity with any known 3D structure, but a fold is proposed by several threading tools. The difficulty in aligning the target sequence on one of the proposed template structures is overcome by combining the results of several available prediction methods and by refining a rational consensus between them. In silico validation of the obtained model and a preliminary cross-check with experimental features allow us to state that this borderline prediction is at least reasonable. This model raises relevant hypotheses on the main structural features of the protein and allows the design of site-directed mutations. Knowing the genetic context of the P39 reading frame, we are now able to suggest a function for the P39 protein: it would act as a periplasmic substrate-binding protein.

Identification of the perosamine synthetase gene of Brucella melitensis 16M and involvement of lipopolysaccharide O side chain in Brucella survival in mice and in macrophages.

Brucella organisms are facultative intracellular bacteria that may infect many species of animals as well as humans. The smooth lipopolysaccharide (S-LPS) has been reported to be an important virulence factor of these organisms, but the genetic basis of expression of the S-LPS O antigen has not yet been described. Likewise, the role of the O side chain of S-LPS in the survival of Brucella has not been clearly defined. A mini-Tn5 transposon mutant library of Brucella melitensis 16M was screened by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies (MAbs) directed against the O side chain of Brucella. One mutant, designated B3B2, failed to express any O side chain as confirmed by ELISA, Western blot analysis, and colony coloration with crystal violet. Nucleotide sequence analysis demonstrated that the transposon disrupted an open reading frame with significant homology to the putative perosamine synthetase genes of Vibrio cholerae O1 and Escherichia coli O157:H7. The low G+C content of this DNA region suggests that this gene may have originated from a species other than a Brucella sp. The survival of B. melitensis mutant strain B3B2 in the mouse model and in bovine macrophages was examined. The results suggested that S-LPS or, more precisely, its O side chain is essential for survival in mice but not in macrophages.

Survival of a bacterioferritin deletion mutant of Brucella melitensis 16M in human monocyte-derived macrophages.

A bacterioferritin (BFR) deletion mutant of Brucella melitensis 16M was generated by gene replacement. The deletion was complemented with a broad-host-range vector carrying the wild-type bfr gene, pBBR-bfr. The survival and growth of the mutant, B. melitensis PAD 2-78, were similar to those of its parental strain in human monocyte-derived macrophages (MDM). These results suggest that BFR is not essential for the intracellular survival of B. melitensis in human MDM.

Identification of the major T-cell antigens present in the Brucella melitensis B115 protein preparation, Brucellergene OCB.

Brucellergene is a commercial allergen prepared from Brucella melitensis strain B115 and containing at least 20 cytoplasmic proteins. These proteins were separated by SDS-PAGE. The unstained gel was divided into 18 fractions and proteins were eluted from the gel fractions. The capacity of the separated proteins to elicit delayed-type hypersensitivity (DTH) in infected guinea-pigs or to induce the production of interferon-gamma (IFN-gamma) by blood cells from infected cattle was evaluated. The biological activity of the corresponding protein fractions blotted on to nitrocellulose was measured in a lymphocyte blastogenesis assay. Among the 18 fractions tested, two-spanning the mol. wt ranges 17-22 (fraction 8) and 35-42-kDa (fraction 17)-showed the maximum biological activity in the three tests. These fractions contain two antigens, the Brucella bacterioferritin (BFR) and P39 proteins. Both proteins are good candidates for the detection of cellular immunity to Brucella.

Humoral immune responses of Brucella-infected cattle, sheep, and goats to eight purified recombinant Brucella proteins in an indirect enzyme-linked immunosorbent assay.

Brucellosis research is currently focused on the identification of nonlipopolysaccharide (LPS) antigens which could potentially be useful for the specific serologic diagnosis of brucellosis as well as for vaccinal prophylaxis. On the basis of previous reports, we selected eight Brucella proteins (OMP36, OMP25, OMP19, OMP16, OMP10, p17, p15, and p39) as candidate antigens to be further evaluated. The genes encoding these proteins were cloned, sequenced, and overexpressed in Escherichia coli. The recombinant proteins were purified with a polyhistidine tag and metal chelate affinity chromatography and evaluated in an indirect enzyme-linked immunosorbent assay (iELISA). The specificity of the iELISA was determined with sera from healthy cattle, sheep, and goats and ranged from 95 to 99%, depending on the recombinant antigen and the species tested. Sera from experimentally infected, and from naturally infected, animals were used to evaluate the sensitivity of the iELISA. The antiprotein antibody response was often delayed when compared to the anti-smooth LPS (S-LPS) response and was limited to animals which developed an active brucellosis infection (experimentally infected pregnant animals and sheep and goats from areas where brucellosis is still endemic). Among the recombinant antigens, the three cytoplasmic proteins (p17, p15, and p39) gave the most useful results. More than 80% of the animals positive in S-LPS serology were also positive with one of these cytoplasmic proteins alone or a combination of two of them. None of the recombinant antigens detected experimentally infected nonpregnant cows and sheep or naturally infected cattle. This study is a first step towards the development of a multiprotein diagnostic reagent for brucellosis.

Characterization of smooth lipopolysaccharides and O polysaccharides of Brucella species by competition binding assays with monoclonal antibodies.

Previously, four epitope specificities on the O chain of Brucella species were reported: M, A, C, and C/Y. In this work, according to monoclonal antibody binding to smooth lipopolysaccharides of Yersinia enterocolitica 0:9, Brucella abortus W99 (A-dominant strain), and B. melitensis Rev1 (M-dominant strain), seven O-chain epitope specificities were defined: M, A, C (M > A), C (M = A), C/Y (M > A), C/Y (M = A) and C/Y (A > M). Competitive binding assays between these monoclonal antibodies suggested that these different epitopes are probably overlapping structures.

Characterization, occurrence, and molecular cloning of a 39-kilodalton Brucella abortus cytoplasmic protein immunodominant in cattle.

Monoclonal antibodies and polyclonal antisera recognizing a 39-kDa protein (P39) of brucellin, a cytoplasmic extract from Brucella melitensis rough strain B115, were produced. The P39 was purified by anion-exchange chromatography. Eleven of fourteen Brucella-infected cows whose infections had been detected by the delayed-type hypersensitivity (DTH) test with brucellergen also developed a DTH reaction when purified P39 was used as the trigger. The T-cell proliferative responses to P39 of peripheral blood lymphocytes from Brucella-infected cows were also positive. None of the animals infected with other bacterial species that are presumed to induce immunological cross-reactions with Brucella spp. reacted to P39, either in DTH tests or in lymphocyte proliferation assays. A lambda gt11 genomic library of Brucella abortus was screened with a monoclonal antibody specific for P39, and the gene coding for this protein was subsequently isolated. The nucleotide sequence of the P39 gene was determined, and the deduced amino acid sequence is in accordance with the sequence of an internal peptide isolated from P39.

Detection of Yersinia enterocolitica serogroup O:3 by a PCR method.

Yersinia enterocolitica is the etiologic agent of a range of clinical situations in humans, but only a small number of serotypes are involved. Among these, Y. enterocolitica O:3 is the most frequently implicated. A PCR method was developed to detect Y. enterocolitica O:3. For this purpose, two pairs of primers were designed to amplify two fragments of the rfb cluster of Y. enterocolitica O:3: a 253-bp fragment of the rfbB gene and a 405-bp fragment of the rfbC gene. A specific detection was obtained only with rfbC primers, which yielded a PCR product of the expected size exclusively with pathogenic Y. enterocolitica of serotype O:3. This pair of primers was combined with the ail, inv, and virF primers previously described (H. Nakajima, M. Inoue, T. Mori, K.-I. Itoh, E. Arakawa, and H. Watanabe, J. Clin. Microbiol. 30:2484-2486, 1992) to allow both the detection and the differentiation between Y. pseudotuberculosis, pathogenic Y. enterocolitica of serotype O:3 and other pathogenic Y. enterocolitica.

Characterization of a monoclonal antibody specific for Brucella smooth lipopolysaccharide and development of a competitive enzyme-linked immunosorbent assay to improve the serological diagnosis of brucellosis.

The reactivity of monoclonal antibody (MAb) 12G12 was analyzed in regard to the main biovars of Brucella species and some members of the families Enterobacteriaceae and Vibrionaceae which present serological cross-reactions with the smooth lipopolysaccharide (S-LPS) of Brucella species. This MAb was strictly directed against the common specific epitope of the Brucella S-LPS. It recognized all of the smooth Brucella strains and biovars except B. suis biovar 2. In order to improve the specificity of the serological diagnosis of brucellosis, a competitive enzyme-linked immunosorbent assay (cELISA) was developed with the horseradish peroxidase-conjugated MAbs 12G12 and S-LPS of B. melitensis Rev1. The specificity of the cELISA was analyzed with 936 serum samples from healthy cattle. The assay was evaluated with sera from heifers (n = 18) experimentally infected with B. abortus 544. After infection, the performance of the cELISA was in agreement with those of the complement fixation test and the rose Bengal plate test. Finally, the specificity of the assay was also evaluated in regard to false-positive serological reactions by using sera from heifers experimentally infected with Yersinia enterocolitica 0:9 (n = 4) and with field sera presenting false-positive reactions (n = 74). The specificity of the cELISA was greater than the specificities of the complement fixation test and the rose Bengal plate test. Indeed, the new assay detected only 31 of the 101 false-positive serum samples detected by at least one serological test.

Infection of cattle with Yersinia enterocolitica O:9 a cause of the false positive serological reactions in bovine brucellosis diagnostic tests.

During the last four years, an increasing number of cattle herds were classified positive by brucellosis screening tests in areas of Belgium and France free of the disease. No clinical symptom of brucellosis was reported in these animals and no Brucella abortus strains were isolated. After two years, no brucellosis outbreak was registered in all of the herds concerned. On this basis, all the serological reactions observed were classified as false positive. An ELISA using Yersinia Outer membrane Proteins (YOPs) as antigens was developed in order to discriminate between a Yersinia enterocolitica O:9 infection and a Brucella abortus infection. Antibodies against YOPs were detected in sera from Y. enterocolitica O:9 experimentally infected cattle (n = 4) but not in sera from B. abortus experimentally infected cattle (n = 4). In a field study, 66.7% of the 174 serum samples from cattle presenting false positive serological reactions showed anti-YOPs antibodies whereas only 10% of 454 sera, classified negative by the brucellosis screening tests, showed anti-YOPs antibodies. Our results suggest that infections with Y. enterocolitica O:9 may cause false positive reactions in brucellosis testing.

Cloning and sequencing of the bacterioferritin gene of Brucella melitensis 16M strain.

The 40 N-terminal amino acids of the 20 kDa antigen A2 from Brucella melitensis were sequenced and showed important similarities with 4 bacterioferrins. A monoclonal antibody raised against this antigen cross-reacted with Escherichia coli bacterioferritin. Hybridization of two sets of degenerate primers with B. melitensis HindIII-digested genomic DNA identified a 3.8 kb fragment. This fragment was shown to contain a bacterioferritin gene (bfr) encoding a 161-amino acid protein. The sequence of the Brucella bacterioferritin is 69% similar to that of E. coli, and many of the ferroxidase centre and haem-ligation residues are conserved.

Molecular cloning, nucleotide sequence, and occurrence of a 16.5-kilodalton outer membrane protein of Brucella abortus with similarity to pal lipoproteins.

Recombinant lambda gt11 phages were selected by screening a genomic library of Brucella abortus DNA with monoclonal antibodies specific for a 16.5-kDa Brucella outer membrane protein (Omp16). The corresponding gene, named pal, was subcloned on a 0.7-kb AluI fragment. Immunoblotting confirmed the expression of a recombinant Omp16 in the transformants. DNA sequence analysis revealed an open reading frame of 168 codons. The deduced amino acid sequence agrees with an internal peptide sequence of native Omp16 and contains a potential lipoprotein signal peptide cleavage site, giving rise to a predicted mature protein of 144 amino acids. The predicted sequence of Omp16 also shows a remarkable degree of similarity to the sequences of three peptidoglycan-associated bacterial lipoproteins. In immunoblotting with a monoclonal antibody specific for Omp16, we demonstrated that Omp16 was expressed in the 34 Brucella strains tested, representing all six species and known biovars.