Vincent Weynants

Additive and Synergistic Bactericidal Activity of Antibodies Directed against Minor Outer Membrane Proteins of Neisseria meningitidis

ABSTRACT Neisseria meningitidis serogroup B is a major cause of bacterial meningitis in younger populations. The available vaccines are based on outer membrane vesicles obtained from wild-type strains. In children less than 2 years old they confer protection only against strains expressing homologous PorA, a major, variable outer membrane protein (OMP). We genetically modified a strain in order to eliminate PorA and to overproduce one or several minor and conserved OMPs. Using a mouse model mimicking childrens PorA-specific bactericidal activity, it was demonstrated that overproduction of more than one minor OMP is required to elicit antibodies able to induce complement-mediated killing of strains expressing heterologous PorA. It is concluded that a critical density of bactericidal antibodies needs to be reached at the surface of meningococci to induce complement-mediated killing. With minor OMPs, this threshold is reached when more than one antigen is targeted, and this allows cross-protection.

An Outer Membrane Receptor of Neisseria meningitidis Involved in Zinc Acquisition with Vaccine Potential

Since the concentration of free iron in the human host is low, efficient iron-acquisition mechanisms constitute important virulence factors for pathogenic bacteria. In Gram-negative bacteria, TonB-dependent outer membrane receptors are implicated in iron acquisition. It is far less clear how other metals that are also scarce in the human host are transported across the bacterial outer membrane. With the aim of identifying novel vaccine candidates, we characterized in this study a hitherto unknown receptor in Neisseria meningitidis. We demonstrate that this receptor, designated ZnuD, is produced under zinc limitation and that it is involved in the uptake of zinc. Upon immunization of mice, it was capable of inducing bactericidal antibodies and we could detect ZnuD-specific antibodies in human convalescent patient sera. ZnuD is highly conserved among N. meningitidis isolates and homologues of the protein are found in many other Gram-negative pathogens, particularly in those residing in the respiratory tract. We conclude that ZnuD constitutes a promising candidate for the development of a vaccine against meningococcal disease for which no effective universal vaccine is available. Furthermore, the results suggest that receptor-mediated zinc uptake represents a novel virulence mechanism that is particularly important for bacterial survival in the respiratory tract.

Antigenic Conservation of an Immunodominant Invariable Region of the VlsE Lipoprotein among European Pathogenic Genospecies of Borrelia burgdorferi SL

Lyme disease is caused by genetically divergent spirochetes, including 3 pathogenic genospecies: Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii. Serodiagnosis is complicated by this genetic diversity. A synthetic peptide (C(6)), based on the 26-mer invariable region (IR(6)) of the variable surface antigen of B. burgdorferi (VlsE), was used as ELISA antigen, to test serum samples collected from mice experimentally infected with the 3 genospecies and from European patients with Lyme disease. Regardless of the infecting strains, mice produced a strong antibody response to C(6), which indicates that IR(6) is antigenically conserved among the pathogenic genospecies. Twenty of 23 patients with culture-confirmed erythema migrans had a detectable antibody response to C(6). A sensitivity of 95.2% was achieved, with serum samples collected from patients with well-defined acrodermatitis chronica atrophicans. Fourteen of 20 patients with symptoms of late Lyme disease also had a positive anti-IR(6) ELISA. Thus, it is possible that C(6) may be used to serodiagnose Lyme disease universally.

Inhibition of Borrelia burgdorferi-Tick Interactions In Vivo by Outer Surface Protein A Antibody

Borrelia burgdorferi outer surface protein (Osp) A is preferentially expressed by spirochetes in the Ixodes scapularis gut and facilitates pathogen-vector adherence in vitro. Here we examined B. burgdorferi-tick interactions in vivo by using Abs directed against OspA from each of the three major B. burgdorferi sensu lato genospecies: B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii. Abs directed against B. burgdorferi sensu stricto (isolate N40) destroy the spirochete and can protect mice from infection. In contrast, antisera raised against OspA from B. afzelii (isolate ACA-1) and B. garinii (isolate ZQ-1) bind to B. burgdorferi N40 but are not borreliacidal against the N40 isolate. Our present studies assess whether these selected OspA Abs interfere with B. burgdorferi-tick attachment in a murine model of Lyme disease with I. scapularis. We examined engorged ticks that had fed on B. burgdorferi N40-infected scid mice previously treated with OspA (N40, ACA-1, ZQ-1, or mAb C3.78) or control Abs. OspA-N40 antisera or mAb C3.78 destroyed B. burgdorferi N40 within the engorged ticks. In contrast, treatment of mice with OspA-ACA-1 and OspA-ZQ-1 antisera did not kill B. burgdorferi N40 within the ticks but did effectively interfere with B. burgdorferi-I. scapularis adherence, thereby preventing efficient colonization of the vector. These studies show that nonborreliacidal OspA Abs can inhibit B. burgdorferi attachment to the tick gut, highlighting the importance of OspA in spirochete-arthropod interactions in vivo.

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.

Quantitative assessment by flow cytometry of T-lymphocytes producing antigen-specific γ-interferon in Brucella immune cattle

Peripheral blood mononuclear cells (PBMC) isolated from cattle infected with Brucella secreted gamma-interferon (IFN-gamma) after antigen-specific stimulation with Brucellergene, which is a mixture of cytoplasmic proteins of rough Brucella melitensis B115. Following the depletion of the monocyte-macrophages from the PBMC, the enriched lymphocyte populations stimulated with Brucellergene did not produce IFN-gamma. Two-colour immunofluorescence staining of intracellular IFN-gamma and bovine cell surface molecules identified the cells producing IFN-gamma among the PBMC stimulated with Brucellergene. Moreover, this method could be used to estimate the number of T-cells specifically producing IFN-gamma. For a given animal, there is a significant correlation (p < 0.05) between the production of IFN measured by an ELISA of the supernatant of whole blood stimulated with Brucellergene and the number of T-cells producing IFN-gamma after in vitro stimulation with Brucellergene. The development of the immunofluorescence staining technique provides a new tool for analysing and for measuring the T-cell immune response in cattle.

Immunogenicity and Safety of Three Doses of a Bivalent (B:4:P1.19,15 and B:4:P1.7-2,4) Meningococcal Outer Membrane Vesicle Vaccine in Healthy Adolescents

ABSTRACT An experimental bivalent meningococcal outer membrane vesicle (OMV) vaccine (B:4:P1.19,15 and B:4:P1.7-2,4) has been developed to provide wide vaccine coverage particularly of the circulating strains in Europe. A randomized, controlled phase II study (study identification number, 710158/002; ClinicalTrials.gov identifier number, NCT00137917) to evaluate the immunogenicity and safety of three doses of the OMV vaccine when given to healthy 12- to 18-year-olds on a 0-2-4 month (n = 162) or 0-1-6 month schedule (n = 159). A control group received two doses of hepatitis A and one of conjugated meningococcal serogroup C vaccine on a 0-1-6 month schedule (n = 157). Immune response, defined as a fourfold increase in serum bactericidal titer using a range of vaccine-homologous or PorA-related and heterologous strains, was determined for samples taken before and 1 month after vaccination; assays were performed at two laboratories. As measured at the GlaxoSmithKline (GSK) laboratory, the OMV vaccine induced an immune response against homologous or PorA-related strains (in at least 51% of subjects against strains of serosubtype P1.19,15 and at least 66% against strains of serosubtype P1.7-2,4) and against a set of three heterologous strains (in 28% to 46% of subjects). Both laboratories showed consistent results for immune response rates. The OMV vaccine had a similar reactogenicity profile for each schedule. Pain preventing normal activities occurred in approximately one-fifth of the subjects; this was significantly higher than in the control group. The immune responses induced by the bivalent OMV vaccine demonstrated the induction of bactericidal antibodies against the vaccine-homologous/PorA-related strains but also against heterologous strains, indicating the presence of protective antigens in OMVs and confirming the potential of clinical cross-protection.

Function of Neisserial Outer Membrane Phospholipase A in Autolysis and Assessment of Its Vaccine Potential

ABSTRACT Outer membrane phospholipase A (OMPLA) is an outer membrane-localized enzyme, present in many gram-negative bacterial species. It is implicated in the virulence of several pathogens. Here, we investigated the presence, function, and vaccine potential of OMPLA in the human pathogen Neisseria meningitidis. Immunoblot analysis showed the presence of OMPLA in 28 out of 33 meningococcal strains investigated. The OMPLA-negative strains all contained a pldA gene, but these alleles contained premature stop codons. All six Neisseria gonorrhoeae strains tested, but only two out of seven commensal neisserial strains investigated, expressed OMPLA, showing that OMPLA is expressed by, but not limited to, many pathogenic neisserial strains. The function of OMPLA was investigated by assessing the phenotypes of isogenic strains, expressing no OMPLA, expressing wild-type levels of OMPLA, or overexpressing OMPLA. OMPLA exhibited phospholipase activity against endogenous phospholipids. Furthermore, OMPLA was characterized as an autolysin that acted under specific conditions, such as prolonged growth of the bacteria. The vaccine potential of the protein was investigated by immunizing mice with in vitro refolded, recombinant OMPLA. High levels of antibody titers were obtained, but the murine sera were neither bactericidal nor protective. Also, convalescent patients and vaccinee sera did not contain detectable levels of anti-OMPLA antibodies, indicating that OMPLA may not be sufficiently immunogenic to be included in a meningococcal vaccine.

Genetically Modified L3,7 and L2 Lipooligosaccharides from Neisseria meningitidis Serogroup B Confer a Broad Cross-Bactericidal Response

ABSTRACT 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.

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.