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Clostridioides difficile (C. difficile) is the most common cause of nosocomial antibiotic associated diarrhoea. The incidence of C. difficile infection (CDI) has been rising worldwide over the last 20 years with consequent rises in morbidity, mortality and healthcare costs, although the incidence has fallen in the UK over the last few years. Confirmation of diagnosis and early intervention are critical to the management of CDI. The standard treatment for CDI is the administration of antibiotics. However, vaccination has been recognized as the most cost-effective treatment for the prevention and possible long-term protection against CDI episode. There are several promising vaccine candidates in various stages of development. Many of these vaccines have displayed good efficacy for CDI under laboratory conditions or in clinical trials. With the emergence of vaccines against C. difficile, here we describe the development and verification of an Enzyme Linked Immunosorbent Assay (ELISA) that can be used for the quality control testing of candidate vaccines against C. difficile through the measurement of vaccine antigen content. Verification of the assay was performed by assessment of specificity, sensitivity, intermediate precision and relative accuracy. The ELISAs were specific for the toxoids being detected and the detection limit of the assay for toxoid A was 4.88 ng/mL and 3.91 ng/mL for toxoid B. The geometric coefficients of variation for intermediate precision did not exceed 25% and relative accuracy was within 77-130%. We therefore conclude that the ELISA described here is sufficiently sensitive, specific, precise and accurate for use for the quality control testing of candidate C. difficile vaccines., (Copyright © 2020. Published by Elsevier B.V.)

Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia, a chronic respiratory disease in swine resulting in enormous economic losses. To identify the components that contribute to virulence and unveil those biological processes potentially related to attenuation, we used isobaric tags for relative and absolute quantification technology (iTRAQ) to compare the protein profiles of the virulent M. hyopneumoniae strain 168 and its attenuated highly passaged strain 168L. We identified 489 proteins in total, 70 of which showing significant differences in level of expression between the two strains. Remarkably, proteins participating in inositol phosphate metabolism were significantly downregulated in the virulent strain, while some proteins involved in nucleoside metabolism were upregulated. We also mined a series of novel promising virulence-associated factors in our study compared with those in previous reports, such as some moonlighting adhesins, transporters, lipoate-protein ligase, and ribonuclease and several hypothetical proteins with conserved functional domains, deserving further research. Our survey constitutes an iTRAQ-based comparative proteomic analysis of a virulent M. hyopneumoniae strain and its attenuated strain originating from a single parent with a well-characterized genetic background and lays the groundwork for future work to mine for potential virulence factors and identify candidate vaccine proteins.

Fusobacterium nucleatum is a Gram-negative bacterium commonly found in the oral cavity and is often involved in periodontal diseases. Recent studies have shown increased F. nucleatum prevalence in colorectal cancer (CRC) tissues, and causal data has linked this bacterium to CRC tumorigenesis. Immune-based approaches to contain, reduce or eradicate its gut colonization may prevent CRC. Outer membrane vesicles (OMVs) are naturally produced by Gram-negative bacteria, typically contain multiple putative virulence factors and may elicit protective immune responses if used as vaccines. Here, OMVs were isolated from F. nucleatum cultures and purified using gradient centrifugation. Proteins contained within the OMVs were identified by nano LC/MS/MS analysis. Of 98 proteins consistently identified from duplicate analyses, 60 were predicted to localize to the outer membrane or periplasm via signal peptide driven translocation. Of these, six autotransporter proteins, which constitute the majority of protein mass of OMVs, were associated with Type V secretion system. In addition, other putative virulence factor proteins with functional domains, including FadA, MORN2 and YadA-like domain, were identified with multiple exposed epitope sites as determined by in silico analysis. Altogether, the non-replicative OMVs of F. nucleatum contain multiple antigenic virulence factors that may play important roles in the design and development of vaccines against F. nucleatum. SIGNIFICANCE: Fusobacterium nulceatum has been proved playing significant role in colorectal carcinogenesis. Outer membrane vesicles are nanoparticles that naturally secreted by Gram-negative bacterial containing various antigenic components, which provides new insight in vaccine development. Understanding the constituents of F. nucleatum OMVs will provide fundamental information and potential strategies for OMV-based F. nucleatum vaccines design. Based on our knowledge this is the first proteomic study of OMVs from F. nucleatum., (Copyright © 2018. Published by Elsevier B.V.)

The sexually transmitted disease gonorrhea (causative agent: Neisseria gonorrhoeae ) remains an urgent public health threat globally because of its reproductive health repercussions, high incidence, widespread antimicrobial resistance (AMR), and absence of a vaccine. To mine gonorrhea antigens and enhance our understanding of gonococcal AMR at the proteome level, we performed the first large-scale proteomic profiling of a diverse panel ( n = 15) of gonococcal strains, including the 2016 World Health Organization (WHO) reference strains. These strains show all existing AMR profiles - established through phenotypic characterization and reference genome publication - and are intended for quality assurance in laboratory investigations. Herein, these isolates were subjected to subcellular fractionation and labeling with tandem mass tags coupled to mass spectrometry and multi-combinatorial bioinformatics. Our analyses detected 904 and 723 common proteins in cell envelope and cytoplasmic subproteomes, respectively. We identified nine novel gonorrhea vaccine candidates. Expression and conservation of new and previously selected antigens were investigated. In addition, established gonococcal AMR determinants were evaluated for the first time using quantitative proteomics. Six new proteins, WHO_F_00238, WHO_F_00635c, WHO_F_00745, WHO_F_01139, WHO_F_01144c, and WHO_F_01126, were differentially expressed in all strains, suggesting that they represent global proteomic AMR markers, indicate a predisposition toward developing or compensating gonococcal AMR, and/or act as new antimicrobial targets. Finally, phenotypic clustering based on the isolates' defined antibiograms and common differentially expressed proteins yielded seven matching clusters between established and proteome-derived AMR signatures. Together, our investigations provide a reference proteomics data bank for gonococcal vaccine and AMR research endeavors, which enables microbiological, clinical, or epidemiological projects and enhances the utility of the WHO reference strains., (© 2019 El-Rami et al.)

The temperature-sensitive (ts+) Mycoplasma synoviae vaccine strain MS-H harbors a non-synonymous mutation which results in Glycine to Arginine substitution at position 123 in the highly conserved glycine-rich motif of Obg-fold in the GTP-binding protein Obg. In-silico analysis of the wild-type and mutant Obgs of M. synoviae has indicated that this amino acid substitution affects structure of the protein, potentially leading to abrogation of Obg function in vivo. Present study was conducted to develop the first expression vector for M. synoviae and to investigate the potential effect(s) of complementation of MS-H vaccine with the wild-type obg from 86079/7NS, the parent strain of MS-H. An oriC vector, pKS-VOTL, harboring the 86079/7NS obg gene, downstream of the variable lipoprotein haemagglutinin (vlhA) gene promoter, also cloned from 86079/7NS, was used to transform MS-H. The plasmid was localised at the chromosomal oriC locus of MS-H without any detectable integration at the chromosomal obg locus. Analysis of the MS-H transformants revealed abundant obg transcripts as well as Obg protein, when compared to the MS-H transformed with a similar vector, pMAS-LoriC, lacking obg coding sequence. The MS-H transformants complemented with wild-type Obg maintained their original temperature-sensitivity phenotype (consistent with MS-H vaccine) but, when compared to the MS-H transformed with pMAS-LoriC, had significantly higher (p < 0.05) growth rate and viability at the permissive (33°C) and non-permissive temperature (39.5°C), respectively. Analysis of Obg expression in MS-H and its wild-type parent strain revealed comparatively lower levels of Obg in MS-H. These results indicate that not only the mutation in Obg, but also the level of Obg expression, can confer functional abnormalities in the bacterial host. Furthermore, with the construction of first expression vector for M. synoviae, this study has set foundation for the development of recombinant vaccine(s) based on MS-H.

Background: An increasing resistance of bacteria to the commonly used antimicrobials forces to search for alternative or supportive ways to cure infections. Targeting virulence factors is one of such approaches. The bacterial HtrA proteins are strongly involved in virulence and the lack of functional HtrA in many cases impairs invasiveness of pathogens. HtrAs act by protecting the cells under stressful conditions as well as they take direct part in invasion of the host. The latter function is played predominantly by the recently identified extracellular fraction of HtrA. This review aims to evaluate HtrAs as therapeutic targets, including design of chemical inhibitors and vaccines., Methods: We undertook a thorough search of bibliographic databases for peer-reviewed scientific literature., Results: One hundred and sixty-four papers were included in the review. First, we briefly summarized key structural and functional properties of known HtrA proteins with the special focus on the extracellular HtrA fraction. Then we provided an overview of efforts and advancements to target HtrAs of pathogenic bacteria as a promising antimicrobial therapy. In some cases, encouraging results were obtained and application of HtrAspecific inhibitors protected tissues from damage and killed bacteria. Also promising reports concerning the use of HtrA as a protective antigen in several disease models have recently been published., Conclusion: The findings of this review suggest that the exported HtrA proteins are very attractive therapeutic targets due to their accessibility, significance in virulence and immunogenicity. However, further extensive studies are still needed to develop a safe antimicrobial treatment., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhea, is not preventable by vaccination and is rapidly developing resistance to antibiotics. However, the transferrin (Tf) receptor system, composed of TbpA and TbpB, is an ideal target for novel therapeutics and vaccine development. Using a three-dimensional structure of gonococcal TbpA, we investigated two hypotheses, i.e., that loop-derived antibodies can interrupt ligand-receptor interactions in the native bacterium and that the loop 3 helix is a critical functional domain. Preliminary loop-derived antibodies, as well as optimized second-generation antibodies, demonstrated similar modest ligand-blocking effects on the gonococcal surface but different effects in Escherichia coli. Mutagenesis of loop 3 helix residues was employed, generating 11 mutants. We separately analyzed the mutants' abilities to (i) bind Tf and (ii) internalize Tf-bound iron in the absence of the coreceptor TbpB. Single residue mutations resulted in up to 60% reductions in ligand binding and up to 85% reductions in iron utilization. All strains were capable of growing on Tf as the sole iron source. Interestingly, in the presence of TbpB, only a 30% reduction in Tf-iron utilization was observed, indicating that the coreceptor can compensate for TbpA impairment. Complete deletion of the loop 3 helix of TbpA eliminated the abilities to bind Tf, internalize iron, and grow with Tf as the sole iron source. Our studies demonstrate that while the loop 3 helix is a key functional domain, its function does not exclusively rely on any single residue., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Borrelia burgdorferi, the causative agent of Lyme disease, alters its gene expression in response to environmental signals unique to its tick vector or vertebrate hosts. B. burgdorferi carries one superoxide dismutase gene (sodA) capable of controlling intracellular superoxide levels. Previously, sodA was shown to be essential for infection of B. burgdorferi in the C3H/HeN model of Lyme disease. We employed two-dimensional electrophoresis (2-DE) and immunoblot analysis with antibodies specific to carbonylated proteins to identify targets that were differentially oxidized in the soluble fractions of the sodA mutant compared to its isogenic parental control strain following treatment with an endogenous superoxide generator, methyl viologen (MV, paraquat). HPLC-ESI-MS/MS analysis of oxidized proteins revealed that several proteins of the glycolytic pathway (BB0057, BB0020, BB0348) exhibited increased carbonylation in the sodA mutant treated with MV. Levels of ATP and NAD/NADH were reduced in the sodA mutant compared with the parental strain following treatment with MV and could be attributed to increased levels of oxidation of proteins of the glycolytic pathway. In addition, a chaperone, HtpG (BB0560), and outer surface protein A (OspA, BBA15) were also observed to be oxidized in the sodA mutant. Immunoblot analysis revealed reduced levels of Outer surface protein C (OspC), Decorin binding protein A (DbpA), fibronectin binding protein (BBK32), RpoS and BosR in the sodA mutant compared to the control strains. Viable sodA mutant spirochetes could not be recovered from both gp91/phox-⁄- and iNOS deficient mice while borrelial DNA was detected in multiple tissues samples from infected mice at significantly lower levels compared to the parental strain. Taken together, these observations indicate that the increased oxidation of select borrelial determinants and reduced levels of critical pathogenesis-associated lipoproteins contribute to the in vivo deficit of the sodA mutant in the mouse model of Lyme disease. This study, utilizing the sodA mutant, has provided insights into adaptive capabilities critical for survival of B. burgdorferi in its hosts.

Bacillus anthracis, B. thuringiensis and B. cereus are members of the B. cereus group. They share high genetic similarity. Whereas plcR (Phospholipase C regulator) usually encodes a functional pleiotropic activator protein in B. cereus and B. thuringiensis isolates, a characteristic nonsense mutation is found in all B. anthracis strains investigated, making the gene dysfunctional. To study the function of PlcR in B. anthracis, we used the B. cereus CMCC63301 genome as a template and constructed a recombinant expression plasmid pBE2A-plcR, and introduced it into the B. anthracis vaccine strain A16R, and then analyzed the activity of the hemolysin and sphingomyelinase. The results showed that transformation of B. anthracis with plasmid pBE2A-plcR carrying the native B. cereus plcR gene active the expression of sphingomyelinase gene, but did not activate expression of hemolysin genes of B. anthracis A16R.

The live vaccine based on the Francisella tularensis subsp. holarctica vaccine strain 15 NIIEG line is used in Russia against tularemia. This vaccine is highly effective, but fairly unstable. Therefore, development of stable live tularemia vaccine with minimal side effect is rather urgent. The method of allel removal in the F. tularensis vaccine strain was used to remove one copy of the iglC gene, which is required to provide intracellular production of the vaccine strain, as well as removal of the recA gene. The latter is crucial for homological recombination. pGM5 suicide vector based on pHV33 bireplicon plasmid was constructed to provide replacement of intact F. tularensis chromosome segments by modified segments. Modified chromosome segments contain F. Tularensis DNA fragment without iglC structural gene segment 545 p. b. (in pGMΔiglC plasmid), as well as DNA fragment containing no recA structural gene segment 1060 p.b. (pGMΔrecA plasmid). The constructed 15/23-1ΔrecA mutant, in contrast to the vaccine strain 15, was capable of reproducing in the macrophage-like cells J774A.1 line, whereas the efficiency of the reproduction was 8-10 times less. BALB/c mouse responded to immunization by the 15/23-1ΔrecA strain by smaller weight decrease (-2%) as compared to the strain 15 (-14%). Bacteria of the 15/23-1ΔrecA strain were virtually incapable of germinating from the BALB/c murine spleen 14 days after invasion, whereas bacteria of the strain 15 were found in the murine organs even after 21 days. The F. tularensis 15/23-1ΔrecA strain having smaller reaction ability can be used as a basis for construction of stable live safe tularemia vaccine.

Pathogenic Leptospira spp. causes leptospirosis in China and throughout the world. Here, we have sequenced two L. interrogans moderately virulent vaccine strains JDL03 (serovar Canicola) and JDL10 (serovar Hebdomadis) used in China. We selected a subproteomic approach to identify surface-exposed proteins including OMPs and extracellular proteins of these two strains plus a highly virulent vaccine strain 56601 (serovar Lai). Comparative surface-exposed proteome among the three strains indicated 81 cores, 61 dispensable and 122 unique surface-exposed proteins. Finally, the 10 highly conserved surface-exposed or subsurface proteins included two known cross-reactive antigens (LipL32 and LA&#95;3469) and another two novel antigens (LA&#95;0136 and LA&#95;0505) displaying conserved immunoreactivity among 15 Chinese epidemic serovars. Furthermore, many potential virulence factors were detected in these identified surface-exposed proteins, such as Loa22, LipL32, LenC, LenF and OmpL37. Interestingly, LipL45, ClpA and ClpB, exhibiting obvious amino acid mutations among str.56601, str.JDL03 and JDL10, might contribute to virulence differences observed among these strains. Additionally, specific surface-exposed proteins in virulent str.56601 were considered to be key virulence determinants, such as Zn-dependent protease, cholesterol oxidase precursor, and so on. In all, we had relatively complete surface-exposed subproteomes of L. interrogans, which will enhance our understanding of leptospiral pathogenesis and key virulence determinants., Biological Significance: The present work demonstrates the use of genomic sequencing and subproteomic studies for the identification of potential vaccine and diagnostic antigen candidates against leptospirosis. The data show the conserved surface-exposed proteins to be novel potentially vaccine/diagnostic candidates. Furthermore, the data also show that LipL45, ClpA, ClpB and a lipoprotein from these three strains plus another highly virulent strain Fiocruz L1-130 contain specific amino acid mutations in strains JDL03 and JDL10. The surface-exposed subproteome of pathogenic L. interrogans could provide valuable information to gain a more complete understanding of leptospiral pathogenesis and virulence determinants., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Clostridium difficile is a Gram-positive bacterium and is the most commonly diagnosed cause of hospital-associated and antimicrobial-associated diarrhea. Despite the emergence of epidemic C. difficile strains having led to an increase in the incidence of the disease, a vaccine against this pathogen is not currently available. C. difficile strains produce two main toxins (TcdA and TcdB) and express three highly complex cell-surface polysaccharides (PSI, PSII and PSIII). PSII is the more abundantly expressed by most C. difficile ribotypes offering the opportunity of the development of a carbohydrate-based vaccine. In this paper, we evaluate the efficacy, in naive mice model, of PSII glycoconjugates where recombinant toxins A and B fragments (TcdA&#95;B2 and TcdB&#95;GT respectively) have been used as carriers. Both glycoconjugates elicited IgG titers anti-PSII although only the TcdB&#95;GT conjugate induced a response comparable to that obtained with CRM197. Moreover, TcdA&#95;B2 and TcdB&#95;GT conjugated to PSII retained the ability to elicit IgG with neutralizing activity against the respective toxins. These results are a crucial proof of concept for the development of glycoconjugate vaccines against C. difficile infection (CDI) that combine different C. difficile antigens to potentially prevent bacterial colonization of the gut and neutralize toxin activity.

The Borrelia burgdorferi outer membrane (OM) contains numerous surface-exposed lipoproteins but a relatively low density of integral OM proteins (OMPs). Few membrane-spanning OMPs of B. burgdorferi have been definitively identified, and none are well characterized structurally. Here, we provide evidence that the borrelial OMP P66, a known adhesin with pore-forming activity, forms a β-barrel in the B. burgdorferi OM. Multiple computer-based algorithms predict that P66 forms a β-barrel with either 22 or 24 transmembrane domains. According to our predicted P66 topology, a lysine residue (K487) known to be sensitive to trypsin cleavage is located within a surface-exposed loop. When we aligned the mature P66 amino acid sequences from B. burgdorferi and B. garinii, we found that K487 was present only in the B. burgdorferi P66 protein sequence. When intact cells from each strain were treated with trypsin, only B. burgdorferi P66 was trypsin sensitive, indicating that K487 is surface exposed, as predicted. Consistent with this observation, when we inserted a c-Myc tag adjacent to K487 and utilized surface localization immunofluorescence, we detected the loop containing K487 on the surface of B. burgdorferi. P66 was examined by both Triton X-114 phase partitioning and circular dichroism, confirming that the protein is amphiphilic and contains extensive (48%) β-sheets, respectively. Moreover, P66 also was able to incorporate into liposomes and form channels in large unilamellar vesicles. Finally, blue native PAGE (BN-PAGE) revealed that under nondenaturing conditions, P66 is found in large complexes of ∼400 kDa and ∼600 kDa. Outer surface lipoprotein A (OspA) and OspB both coimmunoprecipitate with P66, demonstrating that P66 associates with OspA and OspB in B. burgdorferi. The combined computer-based structural analyses and supporting physicochemical properties of P66 provide a working model to further examine the porin and integrin-binding activities of this OMP as they relate to B. burgdorferi physiology and Lyme disease pathogenesis.

1. The single-copy domain of the N-terminal region of the vlhA gene of Mycoplasma synoviae was sequenced, analysed and verified and used to type Iranian field isolates of M. synoviae and the MS-H live vaccine strain. In addition, a restriction fragment length polymorphism (RFLP) method was developed to differentiate between field isolates of Iranian and MS-H vaccine strains. 2. All sequences were analysed and aligned; the percentage similarity of the DNA was calculated and dendrograms were constructed. Based on single nucleotide polymorphism (SNP) that existed in all field isolates in Iran, the PCR-RFLP method allowed the differentiation of all M. synoviae field isolates from the vaccine strain. 3. Using phylogenetic analysis, the isolates were assigned to 8 unique genotypes and, within each group, DNA had a high level of similarity. 4. DNA sequence analysis and PCR-RFLP of the amplicon based on percent similarity and evolutionary relationship appeared to be useful tools for strain differentiation whether M. synoviae clinical isolates from infected chickens were derived from the vaccine strain or wild-type strains. 5. This study confirms the potential value of strain typing for epidemiological purposes and suggests that phylogenetic studies are essential to understand the true relationships between strains.

Background: Campylobacter jejuni is an important food-borne and zoonotic pathogen with a worldwide distribution. Humans and chickens are hosts of this pathogen. At present, there is no ideal vaccine for controlling human campylobacteriosis or the carriage of C. jejuni by chickens. Bacterial in vivo-induced antigens are useful as potential vaccine candidates and biomarkers of virulence., Methods: In this study, we developed a novel systematic immunoproteomics approach to identify in vivo-induced antigens among the total cell proteins of C. jejuni using pre-adsorbed sera from patients infected with C. jejuni., Results: Overall, 14 immunoreactive spots were probed on a PVDF membrane using pre-adsorbed human sera against C. jejuni. Then, we excised these protein spots from a duplicate gel and identified using MALDI-TOF MS. In total, 14 in vivo-induced antigens were identified using PMF and BLAST analysis. The identified proteins include CadF (CadF-1 and CadF-2), CheW, TufB, DnaK, MetK, LpxB, HslU, DmsA, PorA, ProS, CJBH&#95;0976, CSU&#95;0396 and hypothetical protein cje135&#95;05017. Real-time RT-PCR was performed on 9 genes to compare their expression levels in vivo and in vitro. The data showed that 8 of the 9 analyzed genes were significantly upregulated in vivo relative to in vitro., Conclusion: We successfully developed a novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens by using pre-adsorbed sera from infected patients., General Significance: This new analysis method may prove to be useful for identifying in vivo-induced antigens within any host infected by bacteria and will contribute to the development of new subunit vaccines., (© 2013. Published by Elsevier B.V. All rights reserved.)

Moraxella catarrhalis causes significant health problems, including 15-20% of otitis media cases in children and ~10% of respiratory infections in adults with chronic obstructive pulmonary disease. The lack of an efficacious vaccine, the rapid emergence of antibiotic resistance in clinical isolates, and high carriage rates reported in children are cause for concern. In addition, the effectiveness of conjugate vaccines at reducing the incidence of otitis media caused by Streptococcus pneumoniae and nontypeable Haemophilus influenzae suggest that M. catarrhalis infections may become even more prevalent. Hence, M. catarrhalis is an important and emerging cause of infectious disease for which the development of a vaccine is highly desirable. Studying the pathogenesis of M. catarrhalis and the testing of vaccine candidates have both been hindered by the lack of an animal model that mimics human colonization and infection. To address this, we intranasally infected chinchilla with M. catarrhalis to investigate colonization and examine the efficacy of a protein-based vaccine. The data reveal that infected chinchillas produce antibodies against antigens known to be major targets of the immune response in humans, thus establishing immune parallels between chinchillas and humans during M. catarrhalis infection. Our data also demonstrate that a mutant lacking expression of the adherence proteins MhaB1 and MhaB2 is impaired in its ability to colonize the chinchilla nasopharynx, and that immunization with a polypeptide shared by MhaB1 and MhaB2 elicits antibodies interfering with colonization. These findings underscore the importance of adherence proteins in colonization and emphasize the relevance of the chinchilla model to study M. catarrhalis-host interactions.

Borrelia burgdorferi alternates between ticks and mammals, requiring variable gene expression and protein production to adapt to these diverse niches. These adaptations include shifting among the major outer surface lipoproteins OspA, OspC, and VlsE at different stages of the infectious cycle. We hypothesize that these proteins carry out a basic but essential function, and that OspC and VlsE fulfil this requirement during early and persistent stages of mammalian infection respectively. Previous work by other investigators suggested that several B. burgdorferi lipoproteins, including OspA and VlsE, could substitute for OspC at the initial stage of mouse infection, when OspC is transiently but absolutely required. In this study, we assessed whether vlsE and ospA could restore infectivity to an ospC mutant, and found that neither gene product effectively compensated for the absence of OspC during early infection. In contrast, we determined that OspC production was required by B. burgdorferi throughout SCID mouse infection if the vlsE gene were absent. Together, these results indicate that OspC can substitute for VlsE when antigenic variation is unnecessary, but that these two abundant lipoproteins are optimized for their related but specific roles during early and persistent mammalian infection by B. burgdorferi., (Published 2013. This article is a U.S. Government work and is in the public domain in the USA.)

Coxiella burnetii is the etiological agent of Q fever. To identify its major seroreactive proteins, a subgenomic protein array was developed. A total of 101 assumed virulence-associated recombinant proteins of C. burnetii were probed with sera from mice experimentally infected with C. burnetii and sera from Q fever patients. Sixteen proteins were recognized as major seroreactive antigens by the mouse sera. Seven of these 16 proteins reacted positively with at least 45% of Q fever patient sera. Notably, HspB had the highest fluorescence intensity value and positive frequency of all the proteins on the array when probed with both Q fever patient sera and mouse sera. These results suggest that these seven major seroreactive proteins, particularly HspB, are potential serodiagnostic and subunit vaccine antigens of Q fever.

Lactic acid bacteria (LAB), widely used in the food industry, are present in the intestine of most animals, including humans. The potential use of these bacteria as mucosal delivery vehicles for vaccinal, medical or technological use has been extensively investigated. Lactococcus lactis, a LAB species, is a potential candidate for the production of biologically useful proteins and for plasmid DNA delivery to eukaryotic cells. Several delivery systems have been developed to target heterologous proteins to a specific cell location (i.e., cytoplasm, cell wall or extracellular medium) and more recently to efficiently transfer DNA to eukaryotic cells. A promising application of L. lactis is its use for the development of live mucosal vaccines. Here, we have reviewed the expression of heterologous protein and the various delivery systems developed for L. lactis, as well as its use as an oral vaccine carrier., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Helicobacter pylori (H. pylori) has been identified as the main pathogenic factors of chronic gastritis and peptic ulcer, and the Class I carcinogen of gastric cancer by WHO. Vaccine has become the most effective measure to prevent and cure H. pylori infection. The UreB is the most effective and common immunogen of all strains of H. pylori and may stimulate the immunoresponse protecting the human body against the challenge of H. pylori. UreB antigen gene was cloned into the binary vector pBI121 which contains a seed-specific promoter Oleosin of peanut and a kanamycin resistance gene, and then UreB gene was transformed into peanut embryo leaflets by Agrobacter-mediated method. The putative transgenic plants were examined for the presence of UreB in the nuclear genome of peanut plants by PCR analysis. Expression of UreB gene in plants was identified by RT-PCR and Western blot analysis. These results suggest that the UreB transgenic peanut can be potentially used as an edible vaccine for controlling H. pylori.

Background: Leptospirosis, a zoonosis caused by Leptospira spp., is recognized as an emergent infectious disease. Due to the lack of adequate diagnostic tools, vaccines are an attractive intervention strategy. Recombinant proteins produced in Escherichia coli have demonstrated promising results, albeit with variable efficacy. Pichia pastoris is an alternative host with several advantages for the production of recombinant proteins., Results: The vaccine candidates LigANI and LipL32 were cloned and expressed in P. pastoris as secreted proteins. Large-scale expression resulted in a yield of 276 mg/L for LigANI and 285 mg/L for LipL32. The recombinant proteins were glycosylated and were recognized by antibodies present in the sera of patients with severe leptospirosis., Conclusions: The expression of LigANI and LipL32 in P. pastoris resulted in a significant increase in yield compared to expression in E. coli. In addition, the proteins were secreted, allowing for easy purification, and retained the antigenic characteristics of the native proteins, demonstrating their potential application as subunit vaccine candidates.

3-Deoxy-D-manno-octulosonic acid (Kdo) is an eight-carbon sugar ubiquitous in Gram-negative bacterial lipopolysaccharides (LPS). Although its biosynthesis is well described, no protein has yet been identified as a Kdo hydrolase. However, Kdo hydrolase enzymatic activity has been detected in membranes of Helicobacter pylori and Francisella tularensis and may be responsible for the removal of side-chain Kdo from the LPS core saccharides. We now report the identification of genes encoding a Kdo hydrolase in F. tularensis Schu S4 and live vaccine strain strains, in H. pylori 26695 strain and in Legionella pneumophila Philadelphia 1 strain. We have renamed the genes kdhA for keto-deoxyoctulosonate hydrolase A. Deletion of kdhA abolished Kdo hydrolase activity in membranes of F. tularensis live vaccine strain. The F. tularensis kdhA mutant synthesized a core oligosaccharide containing a Kdo disaccharide with one of the Kdo residues being a terminal side chain. This side-chain Kdo monosaccharide was absent in the wild-type core oligosaccharide. Expression in Escherichia coli of recombinant KdhA from F. tularensis, H. pylori, and L. pneumophila resulted in a reduction of membrane-associated side-chain Kdo. The identification of this previously faceless enzyme will accelerate study of the biosynthetic basis and biologic impact for postbiosynthetic LPS structural modification.

Objective: We evaluated the effects of surface protective antigen A (SpaA) and its N-teminal protective domain (rSpaA-N) against Erysipelothrix rhusiopathiae infection in mice., Methods: The SpaA was purified by electroelution from NaOH-extracted antigen of Erysipelothrix rhusiopathiae strain C43311. The rSpaA-N was expressed in E. coli BL21 as a soluble protein by IPTG inducing, and purified with GST affinity chromatography. Mice of each group were subcutaneously immunized three times with 50 microg or 100 microg of native SpaA, rSpaA-N or NaOH-extracted antigen with in complete or incomplete Freund adjuvant at 2-week intervals. Five mice of each group were challenged with 100 LD50 of Erysipelothrix rhusiopathiae virulent strain C43065 two weeks after the third immunization, and the specific antibody responses for SpaA was determined by indirect ELISA., Results: Mice immunized with 50 microg or 100 microg of native SpaA, rSpaA-N, or NaOH-extracted antigen were protected completely against the challenge with strain C43065. There was no significant difference (P < 0.05) between the antibody responses observed for native SpaA, rSpaA-N and NaOH-extracted antigen at any dosages. Western blot results indicated that the native SpaA and rSpaA-N were recognized specifically by an antiserum against the native SpaA of strain C43311., Conclusion: These results demonstrated that the rSpaA-N is a protective antigen of Erysipelothrix rhusiopathiae serotype 2 strains, and might be a useful vaccine candidate against swine erysipelas.

Background: S. Typhi, a human-restricted Salmonella enterica serovar, causes a systemic intracellular infection in humans (typhoid fever). In comparison, S. Typhimurium causes gastroenteritis in humans, but causes a systemic typhoidal illness in mice. The PhoP regulon is a well studied two component (PhoP/Q) coordinately regulated network of genes whose expression is required for intracellular survival of S. enterica., Methodology/principal Findings: Using high performance liquid chromatography mass spectrometry (HPLC-MS/MS), we examined the protein expression profiles of three sequenced S. enterica strains: S. Typhimurium LT2, S. Typhi CT18, and S. Typhi Ty2 in PhoP-inducing and non-inducing conditions in vitro and compared these results to profiles of phoP(-)/Q(-) mutants derived from S. Typhimurium LT2 and S. Typhi Ty2. Our analysis identified 53 proteins in S. Typhimurium LT2 and 56 proteins in S. Typhi that were regulated in a PhoP-dependent manner. As expected, many proteins identified in S. Typhi demonstrated concordant differential expression with a homologous protein in S. Typhimurium. However, three proteins (HlyE, STY1499, and CdtB) had no homolog in S. Typhimurium. HlyE is a pore-forming toxin. STY1499 encodes a stably expressed protein of unknown function transcribed in the same operon as HlyE. CdtB is a cytolethal distending toxin associated with DNA damage, cell cycle arrest, and cellular distension. Gene expression studies confirmed up-regulation of mRNA of HlyE, STY1499, and CdtB in S. Typhi in PhoP-inducing conditions., Conclusions/significance: This study is the first protein expression study of the PhoP virulence associated regulon using strains of Salmonella mutant in PhoP, has identified three Typhi-unique proteins (CdtB, HlyE and STY1499) that are not present in the genome of the wide host-range Typhimurium, and includes the first protein expression profiling of a live attenuated bacterial vaccine studied in humans (Ty800).

Molecular mechanisms underlying the reciprocal regulation of the two major surface lipoproteins and virulence factors of Borrelia burgdorferi, OspA and OspC, are not fully understood. Herein, we report that inactivation of the ospAB operon resulted in overproduction of OspC and many other lipoproteins via the constitutive activation of the Rrp2-RpoN-RpoS pathway. Complementing the ospAB mutant with a wild-type copy of ospA, but not an ospA variant that lacks the lipoprotein signal sequence, restored normal regulation of the Rrp2-RpoN-RpoS pathway; these results indicate that the phenotype was not caused by spurious mutations. Interestingly, while most of the ospAB mutant clones displayed a constitutive ospC expression phenotype, some ospAB mutant clones showed little or no ospC expression. Further analyses revealed that this OspC-negative phenotype was independent of abrogation of ospAB. While activation of the Rrp2-RpoN-RpoS pathway was recently shown to downregulate ospA, our findings suggest that reduction of OspA can also activate this pathway. We postulate that the activation of the Rrp2-RpoN-RpoS pathway and downregulation of OspA form a positive feedback loop that allows spirochaetes to produce and maintain a constant high level of OspC and other lipoproteins during tick feeding, a strategy that is critical for spirochaetal transmission and mammalian infection.

The surface lipoproteins of the Lyme disease spirochaete Borrelia burgdorferi directly interact with tissue microenvironments during mammalian infection, and thus potentially affect various aspects of infection. To investigate the influence of surface antigen synthesis on infectious behaviour, B. burgdorferi was modified to constitutively produce the well-characterized surface lipoproteins OspA and invariant VlsE. Although increasing OspA or VlsE production did not significantly affect synthesis of other surface lipoproteins or spirochaetal growth in vitro, overexpressing vlsE resulted in increased ospA but decreased ospC expression, and overexpressing ospA led to decreased ospC and vlsE expression in severe combined immunodeficient (SCID) mice. Increasing the expression of either ospA or vlsE did not alter the ID(50), but affected spirochaetal dissemination and significantly reduced tissue spirochaete loads in SCID mice. In immunocompetent mice, increased vlsE expression resulted in quick clearance of infection, while constitutive ospA expression led to a substantial ID(50) increase and severely impaired dissemination. Furthermore, B. burgdorferi with constitutive ospA expression persisted in the skin tissue but was cleared from both heart and joints of chronically infected immunocompetent mice. Taken together, the study indicates that increasing production of OspA or invariant VlsE influences lipoprotein gene expression in the murine host and alters the infectious behaviour of B. burgdorferi.

Heat shock protein B (HspB) is one of the dominant proteins recognized by most Helicobacter pylori-infected persons and is being considered as potential candidates for subunit vaccines. In the present study we describe the generation of an antibody against HspB and its use in immunohistochemical assays on gastric biopsies. We have demonstrated that our rabbit polyclonal antibody against HspB did not recognize any protein in lysates from a lung human epithelial cell (H1299) line and did not cross-react with the other members of human heat shock proteins. Secondly, we have observed that in gastric biopsies, HspB immunostaining was present inside the cytoplasm of human epithelial cells with a particular localization in the apical portion of gastric epithelial cells other than in the extracellular spaces among gastric cells of human stomach. Finally, we have demonstrated a cytoplasmic HspB immunostaining in groups of neoplastic cells of MALT lymphoma. In conclusion, our observations suggest a possible involvement of HspB in the pathogenesis of H. pylori-related pathologies such as gastritis, ulcer and gastric cancer., ((c) 2008 Wiley-Liss, Inc.)

Aim: To investigate the immunobiology of Rpf domain from Micrococcus luteus., Methods: BALB/c mice were immunized with Rpf domain three times at 2-week interval. ELISA was used to detect the title of the anti-Rpf domain antibody titer in the immunized mice sera. The spleen lymphocytes of the immunized mice were separated and the stimulation index (SI) was measured by MTT colorimetry. The levels of secreted IFN-gamma, IL-10 and IL-12 upon specific antigen stimulation was detected by ELISA. The Rpf domain immunized BALB/c mice were intravenously infected with 10(5) CFU MTB H37Rv. The number of CFU in the spleens was determined four weeks after final injection., Results: The titer of the specific antibody in sera of the immunized BALB/c mice was 1:128 000. The SI of Rpf domain immunized group (2.10+/-0.12) was significantly higher than that of saline immunized group (0.90+/-0.21). The lever of IFN-gamma, IL-10 and IL-12 levels in culture supernatant of spleen lymphocytes from the fusion protein immunized mice was (1 126+/-36) ng/L, (368+/-13) ng/L and (289+/-14) ng/L, respectively, which was markedly higher than that of saline immunized group (P<0.01). Compared with normal saline immunized mice (6.64+/-0.13) four weeks after final injection, dramatic reduction in MTB replication was observed in the spleen (5.03+/-0.11) from the BALB/c mice immunized with fusion proteins., Conclusion: Rpf domain can be used as a candidate for a new TB vaccine.

Many potential vaccine candidates for serogroup B Neisseria meningitidis (NMB) have been identified by reverse vaccinology, a genome-based approach. However, some candidates may be unseen owing to uncertain annotation or their peculiar properties. In this study, we describe the preparation and identification of a novel lipoprotein expressed in all meningococcal strains tested. mAb were first prepared from mice immunized with a meningococcal B strain isolated in Taiwan. Total proteins from the immunizing strain were separated by 2-DE in duplicate. Clone 4-7-3, which crossreacted to 174 tested meningococcal isolates, was used as the primary antibody for Western blotting. The immunoreactive spot was identified by LC-mass spectrometric analysis of the corresponding spot from the silver-stained gel and confirmed by molecular biology approach to be a novel lipoprotein encoded by the hypothetical NMB1468 gene. The potential use of this protein, designated Ag473/NMB1468, as a vaccine component was evaluated using the recombinant protein produced in Escherichia coli. Immunized mice were found to be protected from developing meningococcal disease after intraperitoneal inoculation with a lethal dose of meningococcal strain Nm22209, suggesting that Ag473/NMB1468 may be a promising vaccine candidate. This study also demonstrates the usefulness of the immunoproteomic approach in identification of novel vaccine candidates.

The spore forming bacterium Clostridium perfringens is a widely occurring pathogen. Vaccines against C. perfringens type B and C are currently manufactured using beta-toxin secreted by virulent C. perfringens strains. Large-scale production of vaccines from virulent strains requires stringent safety conditions and costly detoxification and control steps. Therefore, it would be beneficial to produce this toxin in a safe production host and in an immunogenic, but non-toxic form (toxoid). For high-level expression of beta-toxoid, we cloned the highly active ribosomal rpsF promoter of Bacillus subtilis in a broad host range multicopy plasmid. In B. subtilis, we obtained high intracellular production, up to 200 microg ml(-1) culture. However, the beta-toxoid was poorly secreted. The employed rpsF expression system allowed using the same expression plasmids in other heterologous hosts such as Lactococcus lactis and Streptococcus pneumoniae. In these organisms secretion of beta-toxoid was ten times higher compared to the best producing B. subtilis strain. These results show the usefulness of the rpsF based broad host range expression system.

Live attenuated Salmonella strains have been extensively explored as oral delivery systems for recombinant vaccine antigens and effector proteins with immunoadjuvant and immunomodulatory potential. The feasibility of this approach was demonstrated in human vaccination trials for various antigens. However, immunization efficiencies with live vaccines are generally significantly lower compared to those monitored in parenteral immunizations with the same vaccine antigen. This is, at least partly, due to the lack of secretory expression systems, enabling large-scale extracellular delivery of vaccine and effector proteins by these strains. Because of their low complexity and the terminal location of the secretion signal in the secreted protein, Type I (ATP-binding cassette) secretion systems appear to be particularly suited for development of such recombinant extracellular expression systems. So far, the Escherichia coli hemolysin system is the only Type I secretion system, which has been adapted to recombinant protein secretion in Salmonella. However, this system has a number of disadvantages, including low secretion capacity, complex genetic regulation, and structural restriction to the secreted protein, which eventually hinder high-level in vivo delivery of recombinant vaccines and effector proteins. Thus, the development of more efficient recombinant protein secretion systems, based on Type I exporters can help to improve efficacies of live recombinant Salmonella vaccines. Type I secretion systems, mediating secretion of bacterial surface layer proteins, such as RsaA in Caulobacter crescentus, are discussed as promising candidates for improved secretory delivery systems.

N-Formylated (N-f-met) peptides derived from proteins of the intracellular bacterium Listeria monocytogenes generate a protective, H2-M3-restricted CD8 T cell response in C57BL/6 mice. N-f-met peptide-specific CTL were generated in vitro when mice previously immunized with gp96 isolated from donor mice infected with L. monocytogenes were stimulated with these peptides. No significant peptide-specific CTL activity was observed in mice immunized with gp96 from uninfected animals. Masses corresponding to one N-f-met peptide were found by matrix-assisted laser desorption/ionization-mass spectrometry on gp96 isolated from C57BL/6 mice infected with L. monocytogenes, but not on gp96 from noninfected mice. Therefore, bacterial N-f-met peptides from intracellular bacteria can bind to gp96 in the infected host, and gp96 loaded with these peptides can generate N-f-met-peptide-specific CTL. We assume a unique role of gp96 in Ag processing through the H2-M3 pathway.