Antigenic and cross-protection studies of biotype 1 and biotype 2 isolates of Yersinia ruckeri in rainbow trout, Oncorhynchus mykiss (Walbaum)

Aims: The study investigated antigen characteristics of biotype (bt) 1 and bt 2 isolates of Yersinia ruckeri. Methods and Results: The cell surface characteristics of Y. ruckeri were compared for their antigenic characteristics using polyclonal antibodies that revealed that both biotypes had a homogenous whole‐cell protein antigenic profile. Notable differences in the antigenic properties were observed in the lipopolysaccharide profile of both biotypes. Two iron‐regulated outer membrane proteins (IROMP) of c. 90 and 100 kDa were shown to be major specific antigens. The results demonstrate for the first time differences in antigens between bt 1 and bt 2 isolates of serotype O1 isolates of Y. ruckeri. The protection induced in rainbow trout by a commercial monovalent, and bivalent inactivated vaccine was tested with the outcome that the ability of isolates to cause mortality in vaccinated fish varied with geographical location. In this context, vaccination studies suggested that the O antigen was the dominant immunogenic molecule involved in protection against the disease. Conclusions: The O antigen of Y. ruckeri was the dominant immunogenic molecule involved in the protection of rainbow trout against enteric redmouth disease. Significance and Impact of the Study: There are distinct phenotypic and antigenic differences in Y. ruckeri bt 1 and bt 2 with O antigen recognized as the dominant immunogenic molecule. The data have significance in explaining the lack of success of the earlier monovalent vaccine and demonstrate the effectiveness of the newer bivalent vaccine.     

Identification of vaccine candidate antigens of an ESBL producing Klebsiella pneumoniae clinical strain by immunoproteome analysis

Klebsiella pneumoniae is an opportunistic pathogen which causes pneumoniae, urinary tract infections and septicemia in immunocompromised patients. Hospital outbreaks of multidrug-resistant K. pneumoniae, especially those in neonatal wards, are often caused by strains producing the extended-spectrum-beta-lactamases (ESBLs). An immunoproteome based approach was developed to identify candidate antigens of K. pneumoniae for vaccine development. Sera from patients with acute K. pneumoniae infections (n = 55) and a control group of sera from healthy individuals (n = 15) were analyzed for reactivity by Western blot against ESBL K. pneumoniae outer membrane proteins separated by 2-DE. Twenty highly immunogenic protein spots were identified by immunoproteomic analysis. The immunogenic proteins that are most frequently recognized by positive K. pneumoniae sera were OmpA, OmpK36, FepA, OmpK17, OmpW, Colicin I receptor protein and three novel proteins. Two of the vaccine candidate genes, OmpA (Struve et al. Microbiology 2003, 149, 167-176) and FepA (Lai, Y. C. et al.. Infect Immun 2001, 69, 7140-7145), have recently been shown to be essential in colonization and infection in an in vivo mouse model. Hence, these two immunogenic proteins could serve as potential vaccine candidates.     

In vivo human immune response to transferrin-binding protein 2 and other iron-regulated proteins of Neisseria meningitidis

Abstract When grown under iron restriction, Neisseria meningitidis expresses new outer-membrane proteins, some of which are antigenic and potentially useful as vaccine components. This is particularly relevant to N. meningitidis serogroup B, against which neither polysaccharide nor conjugate vaccines are effective. We investigated recognition of N. meningitidis serogroup B outer-membrane antigens by three sera from patients recovered from meningitis. Recognition of antigens from the homologous strain provided information on in vivo expression during infection and immunogenicity, while cross-reactivity with outer membrane proteins from the other two strains and from another five strains in our collection allowed evaluation of antigenic heterogeneity. Our results demonstrate that transferrin-binding protein 2 (TBP2) is immunogenic in humans, to varying degrees depending on the strain, and that TBP2s (like the equivalent proteins of Haemophilus influenzae type b) are among the most important iron-regulated outer membrane antigens expressed during infection. Other immunogenic outer membrane proteins (some iron-regulated) are also expressed during infection; in a previous study in mouse, three of these proteins (with M _r of 50, 70 and 77 kDa) did not induce an immune response. Our cross-reactivity data provide some support for Robki et al.'s two-group classification of N. meningitidis strains, and provide evidence against the possibility that the antigenic domains shared by the TBP2s of all N . meningitidis strains induce immune responses in vivo.     

Intranasal Immunization with Nontypeable Haemophilus influenzae Outer Membrane Vesicles Induces Cross-Protective Immunity in Mice

Haemophilus influenzae is a Gram-negative human-restricted bacterium that can act as a commensal and a pathogen of the respiratory tract. Especially nontypeable H. influenzae (NTHi) is a major threat to public health and is responsible for several infectious diseases in humans, such as pneumonia, sinusitis, and otitis media. Additionally, NTHi strains are highly associated with exacerbations in patients suffering from chronic obstructive pulmonary disease. Currently, there is no licensed vaccine against NTHi commercially available. Thus, this study investigated the utilization of outer membrane vesicles (OMVs) as a potential vaccine candidate against NTHi infections. We analyzed the immunogenic and protective properties of OMVs derived from various NTHi strains by means of nasopharyngeal immunization and colonization studies with BALB/c mice. The results presented herein demonstrate that an intranasal immunization with NTHi OMVs results in a robust and complex humoral and mucosal immune response. Immunoprecipitation revealed the most important immunogenic proteins, such as the heme utilization protein, protective surface antigen D15, heme binding protein A, and the outer membrane proteins P1, P2, P5 and P6. The induced immune response conferred not only protection against colonization with a homologous NTHi strain, which served as an OMV donor for the immunization mixtures, but also against a heterologous NTHi strain, whose OMVs were not part of the immunization mixtures. These findings indicate that OMVs derived from NTHi strains have a high potential to act as a vaccine against NTHi infections.     

Identification and cloning of immunogenic Aliivibrio salmonicida Pal-like protein present in profiled outer membrane and secreted subproteome

Aliivibrio salmonicida is the aetiological agent of cold water vibriosis affecting farmed fish species, a disease that today is fully controlled by vaccination. However, the molecular mechanisms behind the successful vaccine are largely unknown. In order to gain insight into the possible mechanisms of A. salmonicida vaccines, we report here the profiles of both the outer membrane and secreted subproteomes of A. salmonicida LFI315. The 2 subproteomes were resolved by 2-dimensional electrophoresis that identified a total of 82 protein entries. Monoclonal antibodies specific to an unidentified protein antigen were utilized in the immunoproteomic analysis of both outer membrane proteins and extracellular proteins. The immunogenic protein was located in both subproteomes and identified as a 20 kDa peptidoglycan-associated lipoprotein (Pal). The identity of the antigen was verified by heterologous expression of the cloned A. salmonicida pal gene (VSAL_I1899). It is likely that the immunogenic Pal-like protein is among the constituents that act as a protective antigen in the successful vaccine used today. In view of this, it may be considered a potentially useful component in future vaccine development and pathogenicity studies.     

百日咳杆菌69KDa外膜蛋白的分离纯化及生物学特性研究

本文发展了一种从百日咳杆菌Ⅰ相菌株中纯化６９ＫＤａ外膜蛋白的简易方法,将细菌体经加热浸提、乙醇沉淀蛋白、ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ５０柱层析精制而成。用ＳＤＳ－ＰＡＧＥ、免疫印迹、光密度仪扫描分析,证明纯化制剂为均一的、特异性的６９ＫＤａ外膜蛋白,其收率为５４．２％,纯度达９９．２％,每微克６９ＫＤａ蛋白制剂中的内毒素含量低于０．８５ＥＵ;ＰＴ残留量小于０．１０５ｎｇ。抗６９ＫＤａ蛋白抗血清能     

FetA Antibodies Induced by an Outer Membrane Vesicle Vaccine Derived from a Serogroup B Meningococcal Isolate with Constitutive FetA Expression

Invasive meningococcal disease causes over 3500 cases each year in Europe, with particularly high incidence among young children. Among serogroup B meningococci, which cause most of the cases, high diversity in the outer membrane proteins (OMPs) is observed in endemic situations; however, comprehensive molecular epidemiological data are available for the diversity and distribution of the OMPs PorA and FetA and these can be used to rationally design a vaccine with high coverage of the case isolates. The aim of this study was to determine whether outer membrane vesicles (OMVs) derived from an isolate with constitutive FetA expression (MenPF-1 vaccine) could be used to induce antibodies against both the PorA and FetA antigens. The immunogenicity of various dose levels and number of doses was evaluated in mice and rabbits, and IgG antibody responses tested against OMVs and recombinant PorA and FetA proteins. A panel of four isogenic mutants was generated and used to evaluate the relative ability of the vaccine to induce serum bactericidal activity (SBA) against FetA and PorA. Sera from mice were tested in SBA against the four target strains. Results demonstrated that the MenPF-1 OMVs were immunogenic against PorA and FetA in both animal models. Furthermore, the murine antibodies induced were bactericidal against isogenic mutant strains, suggesting that antibodies to both PorA and FetA were functional. The data presented indicate that the MenPF-1 vaccine is a suitable formulation for presenting PorA and FetA OMPs in order to induce bactericidal antibodies, and that proceeding to a Phase I clinical trial with this vaccine candidate is justified.     

Characterization of immune response elicited by the recombinant outer membrane protein OmpF of Aeromonas hydrophila,a potential vaccine candidate in murine model

Porins, the outer membrane proteins of gram negative bacteria, perform vital roles in bacterial survival and virulence, such as nutrient transportation across the membrane as well as adhesion to host cells during infection. The outer membrane proteins, OmpF and OmpC, are part of a two-component regulatory system, essential for the maintenance of solute concentrations in the cytoplasmic milieu of bacteria, and are thus considered vital for bacterial survival. Exposed on the surface of gram-negative bacteria, these channel proteins are highly immunogenic and can thus be exploited as vaccine candidates. In the present study, we have cloned, characterized, and expressed outer membrane protein OmpF of Aeromonas hydrophila, a major fish pathogen and also known to cause severe infections in humans. The cloned ompF gene of A. hydrophila consisting of an open reading frame corresponding to mature OmpF was expressed and purified from the heterologous host, E. coli. High level of expression resulted in recovery of ~120 mg/L of the purified rOmpF at shake flask level. Polyclonal antisera raised against the recombinant OmpF showed a very high endpoint titer (>1:80,000) and were able to specifically agglutinate live A. hydrophila. Further, anti-OmpF antisera cross-reacted with the cell lysates of various Aeromonas isolates, suggesting that anti-rOmpF antibodies can be used to identify different A. hydrophila isolates in infected conditions. Antibody isotyping, cytokine ELISA, and ELISPOT assay indicated predominantly Th1 type of immune response. The recombinant OmpF reported in the present study thus has the potential to be used as a vaccine candidate against A. hydrophila.     

Polymorphism of the major surface epitope of the CopB outer membrane protein of Moraxella catarrhalis

The CopB outer membrane protein has been considered a vaccine candidate for the prevention of infections due to Moraxella catarrhalis. Monoclonal antibody 10F3 recognizes whole cells of about 70% of clinical isolates, suggesting that this epitope is reasonably conserved. To determine whether CopB has other surface epitopes, we analyzed M. catarrhalis isolates using polyclonal sera against recombinant CopB proteins from a 10F3 positive isolate and a 10F3 negative isolate, and polyclonal sera against synthetic peptides that contained the sequence corresponding to the 10F3 epitope region of three different isolates. Extensive cross-reactivity was observed with the anti-CopB sera towards purified recombinant CopB proteins in Western blot and antigen ELISA, implying that antigenic regions common to both proteins were present. However, anti-CopB sera resembled anti-CopB peptide sera in exhibiting similar binding specificity to whole cells, segregating M. catarrhalis isolates into four CopB groups. We subsequently cloned and sequenced the copB genes from representative isolates. The deduced CopB amino acid sequences and the degree of sequence identity also demonstrated the existence of the same four CopB groups. Each of the four groups had a unique sequence in the 10F3 epitope region and a fifth group had the epitope deleted. The polymorphism of the major surface epitope prompts further consideration regarding the utility of CopB as a vaccine component as well as the design of an efficacious CopB-based vaccine to achieve broad protection against Moraxella infection.     

Proteolytic digestion and labelling studies of the organization of the proteins in the outer membrane of Escherichia coli.

The arrangement of the proteins in the outer membrane of Escherichia coli was examined by treating intact cells and isolated membrane preparations with fluorescamine and with pronase. Intact wild-type cells, or those of a mutant in which the core region of the lipopolysaccharide was absent, were equally resistant to pronase treatment. The protein components of isolated outer membrane preparations varied in their rate of digestion and labelling with fluorescamine. The N-terminal portion of protein B was removed by pronase to yield a fragment (protein Bp) still embedded in the membrane. Protein Bp was not significantly enriched in nonpolar amino acids, suggesting that protein B may not be held in the membrane primarily by hydrophobic interactions. This was confirmed by reconstitution experiments in which protein B could be reassociated with itself, without lipopolysaccharide or phospholipid, in the presence of divalent cation such that pronase digestion of the reassociated material gave protein Bp.     

Subdominant Outer Membrane Antigens in Anaplasma marginale: Conservation,Antigenicity, and Protective Capacity Using Recombinant Protein

Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP) extracts or a defined surface protein complex reproducibly induce protective immunity. However, there are several knowledge gaps limiting progress in vaccine development. First, are these OMPs conserved among the diversity of A. marginale strains circulating in endemic regions? Second, are the most highly conserved outer membrane proteins in the immunogens recognized by immunized and protected animals? Lastly, can this subset of OMPs recognized by antibody from protected vaccinates and conserved among strains recapitulate the protection of outer membrane vaccines? To address the first goal, genes encoding OMPs AM202, AM368, AM854, AM936, AM1041, and AM1096, major subdominant components of the outer membrane, were cloned and sequenced from geographically diverse strains and isolates. AM202, AM936, AM854, and AM1096 share 99.9 to 100% amino acid identity. AM1041 has 97.1 to 100% and AM368 has 98.3 to 99.9% amino acid identity. While all four of the most highly conserved OMPs were recognized by IgG from animals immunized with outer membranes, linked surface protein complexes, or unlinked surface protein complexes and shown to be protected from challenge, the highest titers and consistent recognition among vaccinates were to AM854 and AM936. Consequently, animals were immunized with recombinant AM854 and AM936 and challenged. Recombinant vaccinates and purified outer membrane vaccinates had similar IgG and IgG2 responses to both proteins. However, the recombinant vaccinates developed higher bacteremia after challenge as compared to adjuvant-only controls and outer membrane vaccinates. These results provide the first evidence that vaccination with specific antigens may exacerbate disease. Progressing from the protective capacity of outer membrane formulations to recombinant vaccines requires testing of additional antigens, optimization of the vaccine formulation and a better understanding of the protective immune response.     

Progressive Decrease in the Potential Usefulness of Meningococcal Serogroup B Vaccine (4CMenB,Bexsero?) in Gipuzkoa,Northern Spain

The effectiveness of a vaccine is determined not only by the immunogenicity of its components, but especially by how widely it covers the disease-causing strains circulating in a given region. Because vaccine coverage varies over time, this study aimed to detect possible changes that could affect vaccine protection during a specific period in a southern European region. The 4CMenB vaccine is licensed for use in Europe, Canada, and Australia and is mainly directed against Neisseria meningitidis serogroup B. This vaccine contains four main immunogenic components: three recombinant proteins, FHbp, Nhba and NadA, and an outer membrane vesicle [PorA P1.4]. The allelic distribution of FHbp, Nhba, NadA, and PorA antigens in 82 invasive isolates (B and non-B serogroups) isolated from January 2008 to December 2013 were analyzed. 4CMenB was likely protective against 61.8% and 50% of serogroup B and non-B meningococci, respectively, in the entire period, but between 2012 and 2013, the predicted protection fell below 45% (42.1% for serogroup B isolates).The observed decreasing trend in the predicted protection during the 6 years of the study (Χ ² for trend  = 4.68, p = 0.03) coincided with a progressive decrease of several clonal complexes (e.g., cc11, cc32 and cc41/44), which had one or more antigens against which the vaccine would offer protection.     

Characterization of Francisella tularensis outer membrane proteins

Francisella tularensis is a gram-negative coccobacillus that is capable of causing severe, fatal disease in a number of mammalian species, including humans. Little is known about the proteins that are surface exposed on the outer membrane (OM) of F. tularensis, yet identification of such proteins is potentially fundamental to understanding the initial infection process, intracellular survival, virulence, immune evasion and, ultimately, vaccine development. To facilitate the identification of putative F. tularensis outer membrane proteins (OMPs), the genomes of both the type A strain (Schu S4) and type B strain (LVS) were subjected to six bioinformatic analyses for OMP signatures. Compilation of the bioinformatic predictions highlighted 16 putative OMPs, which were cloned and expressed for the generation of polyclonal antisera. Total membranes were extracted from both Schu S4 and LVS by spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation, which separated OMs from cytoplasmic (inner) membrane and other cellular compartments. Validation of OM separation and enrichment was confirmed by probing sucrose gradient fractions with antibodies to putative OMPs and inner membrane proteins. F. tularensis OMs typically migrated in sucrose gradients between densities of 1.17 and 1.20 g/ml, which differed from densities typically observed for other gram-negative bacteria (1.21 to 1.24 g/ml). Finally, the identities of immunogenic proteins were determined by separation on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analysis. This is the first report of a direct method for F. tularensis OM isolation that, in combination with computational predictions, offers a more comprehensive approach for the characterization of F. tularensis OMPs.     

The identification of outer membrane proteins and flagella of Campylobacter jejuni

The outer membrane proteins of five clinical isolates of Campylobacter jejuni were identified by 125I-surface labelling and SDS-PAGE of outer membrane preparations. All isolates expressed a major outer membrane protein of variable molecular weight (43 000-46 000: 43K-46K). Several constant surface proteins were also identified including a 27K protein which was surface-exposed and acid-extractable but was not present in the outer membrane preparations. Isolated flagella comprised a major 62K protein and a minor 87K protein. Both proteins were absent in an aflagellate variant. The 62K protein was immunoblotted and immunoprecipitated by rabbit anti-flagella antisera.     

Cloning, expression and immunogenicty analysis of five outer membrane proteins of Vibrio parahaemolyticus zj2003

Genes of five outer membrane proteins of Vibrio parahaemolyticus zj2003, including OmpW, OmpV, OmpK, OmpU and TolC, were cloned and expressed as N-terminal His(6)-tagged proteins in Escherichia coli. The recombinant fusion proteins were purified with nickel chelate affinity chromatography. To analyze the immunogenicity of these proteins, large yellow croaker (Pseudosciaena crocea) were immunized by intraperitoneal injection. Antibody response was assessed by method of enzyme-linked immunosorbent assay. Titres to all five recombinant proteins increased during 4 to 8 weeks post immunization, within the range of log 2 values of 5.75 to 10.8. Recorded relative survival percent (RPS) of the vaccinated groups varied from 80% to 90%, while 10 fish in control group all died. Western blot tests were undertaken with the serum of survival fish after experimental infection. Except for recombinant TolC, the other four recombinant proteins were recognized by the serum. It is indicated that four outer membrane proteins of V. parahaemolyticus zj2003, including OmpW, OmpV, OmpU and OmpK, are immunogenic during in vivo infection, which would be of some significance in developing efficient vaccine in aquaculture. This is the first report of successful vaccination against V. parahaemolyticus with purified recombinant outer membrane proteins.     

Comparative membrane proteome analysis of three Borrelia species

The versatility of the surface of Borrelia, the causative agent of Lyme borreliosis, is very important in host-pathogen interactions allowing bacteria to survive in ticks and to persist in a mammalian environment. To identify the surface proteome of Borrelia, we have performed a large comparative proteomic analysis on the three most important pathogenic Borrelia species, namely B. burgdorferi (strain B31), B. afzelii (strain K78), and B. garinii (strain PBi). Isolation of membrane proteins was performed by using three different approaches: (i) a detergent-based fractionation of outer membrane proteins; (ii) a trypsin-based partial shedding of outer cell surface proteins; (iii) biotinylation of membrane proteins and preparation of the biotin-labelled fraction using streptavidin. Proteins derived from the detergent-based fractionation were further sub-fractionated by heparin affinity chromatography since heparin-like molecules play an important role for microbial entry into human cells. All isolated proteins were analysed using either a gel-based liquid chromatography (LC)-MS/MS technique or by two-dimensional (2D)-LC-MS/MS resulting in the identification of 286 unique proteins. Ninety seven of these were found in all three Borrelia species, representing potential targets for a broad coverage vaccine for the prevention of Lyme borreliosis caused by the different Borrelia species.     

Profiling the Bordetella pertussis proteome during iron starvation

Regulation of gene expression in response to local iron concentration is commonly observed in bacterial pathogens that face this nutrient limitation during host infection. In this study, a proteomic approach was used to analyze the differential protein expression of Bordetella pertussis under iron limitation. Whole cell lysates (WCL) and outer membrane fractions of bacteria grown either under iron-starvation or iron-excess conditions were analyzed by two-dimensional (2-D) gel electrophoresis. Statistical analysis revealed 36 proteins displaying differential expression, 9 with higher expression under iron-excess and 27 with increased expression under iron-starvation. These proteins were subjected to tryptic digestion and MALDI-TOF MS. Apart from those previously reported, we identified new low-iron-induced proteins that might help to explain the increased virulence of this phenotype. Additionally, we found evidence that at least one of the identified proteins, solely expressed under iron starvation, is highly immunogenic in infected individuals.     

Genome-wide screening and identification of antigens for rickettsial vaccine development

The capacity to identify immunogens for vaccine development by genome-wide screening has been markedly enhanced by the availability of microbial genome sequences coupled to proteomic and bioinformatic analysis. Critical to this approach is in vivo testing in the context of a natural host–pathogen relationship, one that includes genetic diversity in the host as well as among pathogen strains. We aggregate the results of three independent genome-wide screens using in vivo immunization and protection against Anaplasma marginale as a model for discovery of vaccine antigens for rickettsial pathogens. In silico analysis identified 62 outer membrane proteins (Omp) from the 949 predicted proteins in the A. marginale genome. These 62 Omps were reduced to 10 vaccine candidates by two independent genome-wide screens using IgG2 from vaccinates protected from challenge following vaccination with outer membranes (screen 1) or bacterial surface complexes (screen 2). Omps with broadly conserved epitopes were identified by immunization with a live heterologous vaccine, A. marginale ssp. centrale (screen 3), reducing the candidates to three. The genome-wide screens identified Omps that have orthologs broadly conserved among rickettsial pathogens, highlighted the importance of identifying immunologically subdominant antigens, and supported the use of reverse vaccinology approaches in vaccine development for rickettsial diseases.     

Analysis of Borrelia burgdorferi membrane architecture by freeze-fracture electron microscopy.

Freeze-fracture electron microscopy was used to investigate the membrane architectures of high-passage Borrelia burgdorferi B31 and low- and high-passage isolates of B. burgdorferi N40. In all three organisms, fractures occurred almost exclusively through the outer membrane (OM), and the large majority of intramembranous particles were distributed randomly throughout the concave OM leaflet. The density of intramembranous particles in the concave OM leaflet of the high-passage N40 isolate was significantly greater than that in the corresponding leaflet of the low-passage N40 isolate. Also noted in the OMs of all three organisms were unusual structures, designated linear bodies, which typically were more or less perpendicular to the axis of the bacterium. A comparison of freeze-fractured B. burgdorferi and Treponema pallidum, the syphilis spirochete, revealed that the OM architectures of these two pathogens differed markedly. All large membrane blebs appeared to be bounded by a membrane identical to the OM of B. burgdorferi whole cells; in some blebs, the fracture plane also revealed a second bilayer closely resembling the B. burgdorferi cytoplasmic membrane. Aggregation of the lipoprotein immunogens outer surface protein A (OspA) and OspB on the bacterial surface by incubation of B. burgdorferi B31 with specific polyclonal antisera did not affect the distribution of OM particles, supporting the contention that lipoproteins do not form particles in freeze-fractured OMs. The expression of poorly immunogenic, surface-exposed proteins as virulence determinants may be part of the parasitic strategy used by B. burgdorferi to establish and maintain chronic infection in Lyme disease.