Human immune response to epitopes on the meningococcal outer membrane class 5 protein following natural infection

Abstract Two monoclonal antibodies (mAbs) were produced against a serogroup B Neisseria meningitidis strain. These mAbs recognized two epitopes in the class 5 outer membrane proteins (OMP), designated P5.7 and P5.Bm, and were able to kill the homologous strain through complement activation. Both epitopes were surface exposed and 68% of group B meningococcal clinical isolates had one or both epitopes present in their class 5 OMP. Antibodies to one or both epitopes were demonstrated in 17 patients with meningococcal meningitis using an ELISA inhibition assay. Of the 17 paired sera, 41% and 29% of the acute-phase sera had antibodies to the P5.7 and P5.Bm epitopes, respectively. Immunoglobulin G to P5.Bm were found in all 17 convalescent-phase sera while specific antibodies against P5.7 were only found in 6 of these sera. These results demonstrate the potential importance of the P5.Bm and P5.7 epitopes on the class 5 OMP as candidates for vaccine composition.     

Human antibody response to outer membrane proteins and fimbriae of Haemophilus influenzae type b

We investigated the prevalence of antibodies in childrens' sera directed against outer membrane proteins (OMP) and fimbriae of Haemophilus influenzae type b. Invasive isolates of H. influenzae type b were enriched for fimbriae production; OMP and fimbriae were resolved by SDS-PAGE. After blotting to nitrocellulose, the proteins were incubated with homologous patient sera or with sera from healthy children. IgG antibodies bound to OMP were detected by immunoperoxidase staining. Immunoblotting was also performed using purified, nondenatured fimbriae as antigen. Nine of the 10 patients studied had antibodies in the acute serum directed against one or more of the OMP. Neither the acute nor the convalescent serum of the remaining patient contained antibodies against OMP. Antibodies against a greater number of OMP were present in the convalescent serum, in comparison to the acute serum, in 4 of the 10 patients. Five of 10 patients had antibodies against the purified fimbriae of an unrelated invasive isolate in either the acute or the convalescent serum. Acute sera from patients more frequently contained antibodies directed against OMP 60K (p less than or equal to 0.01) and OMP 51K (p less than or equal to 0.003) compared with the sera of healthy controls. In contrast, the sera of healthy children more frequently contained antibodies directed against OMP 40K (p less than or equal to 0.04). Sera from both patients and controls contained antibodies against commensal Haemophilus. We conclude that although antibodies against OMP are commonly present in healthy children, antibodies against certain OMP may be markers for susceptibility or protection.     

NMR dynamics and antibody recognition of the meningococcal lipidated outer membrane protein LP2086 in micellar solution

Neisseria meningitidis is a major cause of meningitis. Although protective vaccination is available against some pathogenic serogroups, serogroup B meningococci have been a challenge for vaccinologists. A family of outer membrane lipoproteins, LP2086 (or factor H binding proteins, fHbp), has been shown to elicit bactericidal antibodies and is currently part of a cocktail vaccine candidate. The NMR structure of the variant LP2086-B01 in micellar solution provided insights on the topology of this family of proteins on the biological membrane. Based on flow cytometry experiments on whole meningococcal cells, binding experiments with monoclonal antibodies, and the NMR structure in micellar solution, we previously proposed that LP2086-B01 anchors the outer bacterial membrane through its lipidated N-terminal cysteine, while a flexible 20 residue linker positions the protein above the layer of lipo-oligosaccharides that surrounds the bacteria. This topology was suggested to increase the antigen exposure to the immune system. In the present work, using micellar solution as a membrane mimicking system, we characterized the backbone dynamics of the variant LP2086-B01 in both its lipidated and unlipidated forms. In addition, binding experiments with a Fab fragment derived from the monoclonal MN86-1042-2 were also performed. Our data suggests that due to the length and flexibility of the N-terminal linker, the antigen is not in contact with the micelle, thus making both N- and C-domains highly available to the host immune system. This dynamic model, combined with the binding data obtained with MN86-1042-2, supports our previously proposed arrangement that LP2086-B01 exposes one face to the extracellular space. Binding of MN86-1042-2 antibody shows that the N-domain is the primary target of this monoclonal, providing further indication that this domain is immunologically important for this family of proteins.     

Conformation-dependent antibody response to Pseudomonas aeruginosa outer membrane proteins induced by immunization in humans

Outer membrane proteins (OMPs) of pathogenic bacteria have been used as protective antigens in developing bacterial vaccines. In the present study, we compared the antibody responses to a Pseudomonas aeruginosa OMP vaccine elicited in humans and rabbits by immunization. Immunization with the vaccine induced high titers of serum IgG antibody both in rabbits and humans but reactivities of the induced antibodies with the OMPs were different. The rabbit immune sera recognized most of the OMPs in the vaccine both in immunoblot and immunoprecipitation analyses. In contrast, a great variation in band pattern and intensity was observed among the human immune sera in immunoblot analysis, but not in immunoprecipitation analysis. Denaturation of the OMPs did not affect the binding activity of the rabbit immune sera as determined by ELISA, but substantially reduced those of the human immune sera and anti-OMP IgG purified from a pooled normal human plasma. These data suggest that antibody response to P. aeruginosa OMPs elicited by immunization in humans is mainly directed against discontinuous or conformation-dependent epitopes, which should be taken into account in developing vaccines, especially for OMP-derived synthetic peptides.     

Human serum and mucosal antibody responses to outer membrane protein G1b of Moraxella catarrhalis in chronic obstructive pulmonary disease

Moraxella catarrhalis is an important human pathogen that causes otitis media, sinusitis, and lower respiratory tract infections in adults with chronic obstructive pulmonary disease. Outer membrane protein G1b is a approximately 29-kDa protein that has a high degree of homology among strains, contains surface-exposed epitopes, and is a potential vaccine candidate. The ompG1b gene was cloned, expressed in Escherichia coli, and purified. To assess the expression of outer membrane protein G1b during human infection, paired serum and sputum supernatants from patients with chronic obstructive pulmonary disease followed prospectively were studied by enzyme-linked immunosorbent assays with recombinant outer membrane protein G1b to detect antibodies made specifically during carriage of M. catarrhalis. Overall, 39% of patients developed either a serum IgG (28.6%) or a sputum supernatant IgA (19.2%) response to outer membrane protein G1b following 100 episodes of acquisition and clearance of M. catarrhalis. A sputum supernatant IgA response was more likely following exacerbations compared with asymptomatic colonizations, whereas a serum IgG response occurred at similar rates. Serum IgG antibodies following natural infection were directed toward surface-exposed epitopes of outer membrane protein G1b. Overall, these studies show that outer membrane protein G1b is expressed during infection of the human respiratory tract and that human antibodies bind to outer membrane protein G1b epitopes on the bacterial surface. These observations indicate that outer membrane protein G1b should be evaluated further as a vaccine antigen.     

Immune response of Japanese flounder Paralichthys olivaceus to outer membrane protein of Edwardsiella tarda

The humoral and cellular immune responses of Japanese flounder, Paralichthys olivaceus, were investigated following intraperitioneal injection with outer membrane protein (OMP) of Edwardsiella tarda in Freund's incomplete adjuvant (FIA). The specific serum antibody titre against OMP of E. tarda were measured using ELISA for 14 weeks, and the total serum antibody concentrations were also determined according to the sandwich ELISA standard model constructed using purified IgM. Both of the specific and total antibodies had an increase and reached their peaks 4 weeks after immunization. Simultaneously, the percentages of sIg + lymphocytes in blood, spleen, pronephros and mesonephros were detected by flow cytometry. It was shown that the percentages of sIg + lymphocytes in all lymphoid organs reached their peak levels 4 weeks after immunization, and then decreased gradually. To investigate the protection against infection, three challenges were performed in the same way at day 14, 30 and 100 after immunization, fish challenged at day 30 showed a higher relative percentage survival (RPS) of 71 compared to the 14-day group (30) and 100-day group (53), which indicated a positive correlation between the survival and the levels of the antibody.     

Deoxyribonucleic acid and outer membrane: binding to outer membrane involves a specific protein.

The binding of deoxyribonucleic acid (DNA) to the outer membrane of Escherichia coli was examined. The amount of DNA found to be bound to outer membrane was low and was estimated to be about 0.4% of the total DNA. Treatment of cells with chloramphenicol or rifampin caused a disassociation of the apparent DNA-outer membrane complex. The results presented here suggest that the binding between membrane and DNA is specific and involves a membrane protein having a molecular weight of 13,000.     

Heterogeneous antibody response to polyvalent melanoma vaccines in syngeneic mice

In this study, a human melanoma vaccine induced antibody responses in mice that varied significantly from animal to animal. BALB/c mice were immunized to a xenogenic human polyvalent melanoma vaccine that has been used in phase II clinical trials in over 600 patients. Mice were bled biweekly for up to 6 weeks to measure antibody responses. IgG antibody responses to the melanoma vaccine components were detectable within 2 weeks but were much stronger at 4 and 6 weeks. When the pooled sera were further analyzed by Western blot, a complex pattern of antigens was detected. When individual sera from identically immunized mice were assayed by Western blot, a consistent, reproducible pattern of antigen recognition was not seen. Rather, we found significantly different antibody responses among the mice. Both the intensity of antibody responses and the pattern of antigens recognized varied from animal to animal. Although there appeared to be immunodominant antigens that produced antibody responses in most mice, no single antigen induced antibody responses in all mice. These results demonstrate that polyvalent vaccines induce heterogeneous antibody responses in mice treated identically. Analysis of the response of selected melanoma patients immunized to the same vaccine revealed similar antibody responses to the antigens in the melanoma vaccine. Heterogeneity may hamper interpretation of vaccine immunogenicity and relevant tumor antigens in humans.     

Bactericidal antibody response to Neisseria meningitidis serogroup B in patients with bacterial meningitis: effect of immunization with an outer membrane protein vaccine

We evaluated the bactericidal antibody response to Neisseria meningitidis serogroup B in convalescent patients (n=65) from bacterial meningitis. Patients infected with B meningococci were stratified according to their vaccination status (Cuban BC vaccine) into group 1 (immunized) (n=12) and group 2 (non-immunized) (n=15). The results suggested that antibody titers > or =2 (log(2)) indicate a specific immune response to N. meningitidis. In group 1, 64% of patients had a significant antibody titer (> or =2) in their acute sera against a B:4:P1.15 strain, compared to only 21% of group 2 patients. All patients from group 1 without bactericidal antibodies in their acute sera had a significant increase (at least 2-fold increase in log(2) titers) in antibody titers in their convalescent sera, in contrast, to only 27% of patients from group 2 (P=0.06). Using mutant strains lacking OMP1 or OMP5, it was shown that OMP1 was an important antigen recognized by immunized patients but not by non-immunized patients.     

Acinetobacter baumannii secretes cytotoxic outer membrane protein A via outer membrane vesicles

Acinetobacter baumannii is an important nosocomial pathogen that causes a high morbidity and mortality rate in infected patients, but pathogenic mechanisms of this microorganism regarding the secretion and delivery of virulence factors to host cells have not been characterized. Gram-negative bacteria naturally secrete outer membrane vesicles (OMVs) that play a role in the delivery of virulence factors to host cells. A. baumannii has been shown to secrete OMVs when cultured in vitro, but the role of OMVs in A. baumannii pathogenesis is not well elucidated. In the present study, we evaluated the secretion and delivery of virulence factors of A. baumannii to host cells via the OMVs and assessed the cytotoxic activity of outer membrane protein A (AbOmpA) packaged in the OMVs. A. baumannii ATCC 19606(T) secreted OMVs during in vivo infection as well as in vitro cultures. Potential virulence factors, including AbOmpA and tissue-degrading enzymes, were associated with A. baumannii OMVs. A. baumannii OMVs interacted with lipid rafts in the plasma membranes and then delivered virulence factors to host cells. The OMVs from A. baumannii ATCC 19606(T) induced apoptosis of host cells, whereas this effect was not detected in the OMVs from the ΔompA mutant, thereby reflecting AbOmpA-dependent host cell death. The N-terminal region of AbOmpA(22-170) was responsible for host cell death. In conclusion, the OMV-mediated delivery of virulence factors to host cells may well contribute to pathogenesis during A. baumannii infection.     

Backbone and side-chain assignment of the lipidated and non-lipidated forms of the meningococcal outer membrane protein LP2086

LP2086 is a lipidated outer membrane protein from Neisseria meningitidis that elicits bactericidal antibodies and represents a promising vaccine candidate against meningococcal infections. Here we report the backbone and side-chain assignment for two forms of LP2086: non-lipidated in aqueous buffer and the lipidated protein in micellar solution. Ethical standards  All experiments described comply with the current laws of the United States of America. The authors declare that they have no conflict of interest     

A common-immunogenic Vibrio outer membrane protein

Abstract The presence of antibodies in rabbit antisera to cell envelope proteins of Vibrio cholerae has been examined using a two-dimensional system, in which the cell envelopes are electrophoresed in sodium dodecyl sulfate (SDS) in polyacrylamide gels in the first dimension, and in agarose containing antibodies in the second. The results show that a 25-kDa protein is markedly immunogenic and appears to be common to Vibrio strains; it is not present in a number of other organisms. This 25-kDa protein is an outer membrane protein as judged by sucrose gradient centrifugation and it is accessible to iodination by lactoperoxidase, suggesting that it is exposed on the cell surface.     

Insights into outer membrane protein crystallization

Outer membrane proteins are structurally distinct from those that reside in the inner membrane and play important roles in bacterial pathogenicity and human metabolism. X-ray crystallography studies on >40 different outer membrane proteins have revealed that the transmembrane portion of these proteins can be constructed from either β-sheets or less commonly from α-helices. The most common architecture is the β-barrel, which can be formed from either a single anti-parallel sheet, fused at both ends to form a barrel or from multiple peptide chains. Outer membrane proteins exhibit considerable rigidity and stability, making their study through x-ray crystallography particularly tractable. As the number of structures of outer membrane proteins increases a more rational approach to their crystallization can be made. Herein we analyse the crystallization data from 53 outer membrane proteins and compare the results to those obtained for inner membrane proteins. A targeted sparse matrix screen for outer membrane protein crystallization is presented based on the present analysis.     

Gene polymorphism analysis of Yersinia enterocolitica outer membrane protein A and putative outer membrane protein A family protein

Background

Yersinia enterocolitica outer membrane protein A (OmpA) is one of the major outer membrane proteins with high immunogenicity. We performed the polymorphism analysis for the outer membrane protein A and putative outer membrane protein A (p-ompA) family protein gene of 318 Y. enterocolitica strains.

Results

The data showed all the pathogenic strains and biotype 1A strains harboring ystB gene carried both ompA and p-ompA genes; parts of the biotype 1A strains not harboring ystB gene carried either ompA or p-ompA gene. In non-pathogenic strains (biotype 1A), distribution of the two genes and ystB were highly correlated, showing genetic polymorphism. The pathogenic and non-pathogenic, highly and weakly pathogenic strains were divided into different groups based on sequence analysis of two genes. Although the variations of the sequences, the translated proteins and predicted secondary or tertiary structures of OmpA and P-OmpA were similar.

Conclusions

OmpA and p-ompA gene were highly conserved for pathogenic Y. enterocolitica. The distributions of two genes were correlated with ystB for biotype 1A strains. The polymorphism analysis results of the two genes probably due to different bio-serotypes of the strains, and reflected the dissemination of different bio-serotype clones of Y. enterocolitica.     

Acinetobacter baumannii outer membrane protein A modulates the biogenesis of outer membrane vesicles

Acinetobacter baumannii secretes outer membrane vesicles (OMVs) during both in vitro and in vivo growth, but the biogenesis mechanism by which A. baumannii produces OMVs remains undefined. Outer membrane protein A of A. baumannii (AbOmpA) is a major protein in the outer membrane and the C-terminus of AbOmpA interacts with diaminopimelate of peptidoglycan. This study investigated the role of AbOmpA in the biogenesis of A. baumannii OMVs. Quantitative and qualitative approaches were used to analyze OMV biogenesis in A. baumannii ATCC 19606T and an isogenic ΔAbOmpA mutant. OMV production was significantly increased in the ΔAbOmpA mutant compared to wild-type bacteria as demonstrated by quantitation of proteins and lipopolysaccharides (LPS) packaged in OMVs. LPS profiles prepared from OMVs from wild-type bacteria and the ΔAbOmpA mutant had identical patterns, but proteomic analysis showed different protein constituents in OMVs from wild-type bacteria compared to the ΔAbOmpA mutant. In conclusion, AbOmpA influences OMV biogenesis by controlling OMV production and protein composition.     

Quantification of lipopolysaccharides in outer membrane vesicle vaccines against meningococcal disease. High-performance liquid chromatographic determination of the constituent 3-hydroxy-lauric acid.

A high-performance liquid chromatographic (HPLC) assay for quantification of lipopolysaccharides (LPSs, endotoxins) in outer membrane vesicle vaccines against meningococcal disease has been developed. The LPS constituent, 3-hydroxy-lauric acid, served as marker substance for the quantification. LPS from the vaccine was precipitated by ethanol and the fatty acid constituents, including 3-hydroxy-lauric acid, were released by acidic hydrolysis, collected and purified by solid phase extraction on C18 disc-cartridges and converted into phenacyl esters for UV detection at 240 nm. Quantification of the derivatized 3-hydroxy-lauric acid was achieved by HPLC using a Brownlee RP-18 reversed phase column with acetonitrile/water (68:32, v/v) as mobile phase. The method was found to be linear over the range 3-49 microg LPS/ml with a sensitivity of 1.6 (microg/ml)(-1). The repeatability (within-day precision) of the method at three levels (3-49 microg LPS/ml) was 6-14% relative standard deviation and the intermediate (between-day) precision was 7% relative standard deviation (at level 15 microg LPS/ml). The method has been successfully used in the quality control of a meningococcal B outer membrane vesicle vaccine, containing 4-8% LPS relative to protein (w/w), in our laboratory for three years.     

Surface antigen analysis of group B Neisseria meningitidis outer membrane by monoclonal antibodies: Identification of bactericidal antibodies to class 5 protein

Twenty-four monoclonal antibodies (mAbs) against group B Neisseria meningitidis surface antigens were analyzed by immunoenzymatic assays and by a bactericidal test. Two mAbs were specific to polysaccharide B and one to lipopolysaccharide. The others were directed against outer membrane proteins ranging in molecular mass from 25 to 200 kDa. The outer membrane protein epitopes recognized by the mAbs were not conformational and were located on the outer surface of the microorganism. Linear epitopes on the class 5 protein, exposed on the surface of the membrane, were able to induce bactericidal antibodies to the homologous strain. The susceptibility of Neisseria meningitidis to these antibodies was unchanged when this organism was cultivated under conditions of iron depletion. These results demonstrate that peptides derived from class 5 proteins are potentially important in synthetic peptide or in recombinant protein vaccines containing linear bactericidal epitopes.