Evaluations of Alkyl hydroperoxide reductase B cell antigen epitope as a potential epitope vaccine against Campylobacter jejuni

ObjectiveThe present study aimed to screen and find alkyl hydroperoxide reductase (AhpC) B cell dominant epitope of Campylobacter jejuni (C. jejuni).Materials and methodsBio-informatic algorithms were used to predict B cell epitopes of AhpC. The AhpC protein and chemically synthesized antigenic epitopes of C. jejuni were considered as antigens, and the AhpC antibody was used as the primary antibody, ELISA and dot blot were used to analyze and screen the dominant epitope. The specific IgG of mice serum and IL-4 in splenocyte culture supernatant were detected by ELISA. The protective efficacy was evaluated by animal disease index and tissue histopathological staining of the jejunum.ResultsSeven epitopes of AhpC were predicted, one epitope (AhpC_4–16) was found to recognize the antibodies of AhpC and had strong antigenicity by ELISA and dot blot analysis. In epitope AhpC_4–16 immunized mice, specific IgG of serum and IL-4 in splenocyte culture supernatant were significantly higher. The illness index decreased significantly, the protective rate was 66.67%. Histopathology displayed that the jejunum morphology was better than the control group.ConclusionsThese findings suggested that epitope AhpC_4–16 showed effective protective role against C. jejuni and is a candidate epitope of vaccine against this pathogen.     

177 T-cell vaccine design for Streptococcus pneumoniae: an in silico approach

Streptococcus pneumoniae (pneumococcus) remains an important cause of meningitis, bacteremia, acute otitis media, community acquired pneumonia associated with significant morbidity, and mortality world wide. Conjugated polysaccharide, glycoconjugated, and capsular polysaccharide based vaccines were existent for pneumococcal disease but are still specific and restricted to serotypes of S. pneumoniae. Proteome of eight serotypes of S. pneumoniae was retrieved and identified in common proteins (Munikumar et al., 2012). 18 membrane proteins were distinguished from 1657 common proteins of eight serotypes of S. pneumoniae. Implementing comparative genomic approach and subtractive genomic approach, three membrane proteins were predicted as essential for bacterial survival and non-homologous to human (Munikumar et al., 2012; Umamaheswari et al., 2011). ProPred server was used to propose four promiscuous T-cell epitopes from three membrane proteins and validated through published positive control, SYFPEITHI and immune epitope database (Munikumar et al., in press). The four epitopes docked into peptide binding region of predominant HLA-DRB alleles with good binding affinity in Maestro v9.2. The T-cell epitope 89-VVYLLPILI-97 and HLA-DRB5^?0101 docking complex was with best XPG score (?13.143?kcal/mol). Further, the stability of the complex was checked through molecular dynamics simulations in Desmond v3.3. The simulation results had revealed that the complex was stable throughout 5000?ps (Munikumar et al., in press). Thus, the epitope would be the ideal candidate for T-cell driven subunit vaccine design against selected serotypes of S. pneumoniae.     

A specific DNA probe for the identification of Campylobacter jejuni

A 6.1 kb DNA probe for the human enteric pathogen Campylobacter jejuni has been isolated from a genomic library constructed in the plasmid vector pBR322 in Escherichia coli. The DNA sequence used as a probe was identified from recombinant plasmids following immunological screening of transformants using polyclonal antisera to whole cells and to membrane antigens of C. jejuni. Restriction endonuclease fragment mapping of C. jejuni DNA inserts from three of the recombinant plasmids showed an overlapping DNA fragment. One of these recombinant plasmids, when used as a DNA probe in Southern hybridization, specifically hybridized with chromosomal DNA from all of the C. jejuni strains tested. Hybridization was not detected at high stringency between the DNA probe and chromosomal DNA from any other Campylobacter species tested except weakly with the chromosomal DNA of strains of Campylobacter coli. Hybridization was also not detected with chromosomal DNA from a range of other enteric bacteria likely to be encountered in faecal material. The intensity of hybridization with C. coli could be increased by reducing the stringency of hybridization.     

Multi high-throughput approach for highly selective identification of vaccine candidates: the Group A Streptococcus case

We propose an experimental strategy for highly accurate selection of candidates for bacterial vaccines without using in vitro and/or in vivo protection assays. Starting from the observation that efficacious vaccines are constituted by conserved, surface-associated and/or secreted components, the strategy contemplates the parallel application of three high throughput technologies, i.e. mass spectrometry-based proteomics, protein array, and flow-cytometry analysis, to identify this category of proteins, and is based on the assumption that the antigens identified by all three technologies are the protective ones. When we tested this strategy for Group A Streptococcus, we selected a total of 40 proteins, of which only six identified by all three approaches. When the 40 proteins were tested in a mouse model, only six were found to be protective and five of these belonged to the group of antigens in common to the three technologies. Finally, a combination of three protective antigens conferred broad protection against a panel of four different Group A Streptococcus strains. This approach may find general application as an accelerated and highly accurate path to bacterial vaccine discovery.     

Evaluation of a commercial diagnostic PCR for the identification of Campylobacter jejuni and Campylobacter coli

AIMS: DuPont Qualicon recently developed a new PCR assay for the identification of Campylobacter jejuni and Campylobacter coli. We evaluated the selectivity and utility of this assay compared with a PCR method already in use in our laboratory. METHODS AND RESULTS: A group of 133 Campylobacter isolates from poultry carcass rinse samples were screened using the commercial PCR and standard PCR. Identical results were found for 89.5% (119/133) of the isolates. However, 10.5% (14/133) gave conflicting results suggesting mixed cultures. These 14 strains were retested by both PCR methods. Of these, 78.6% (11/14) showed identical results for both PCR methods after retesting; the results for the remaining 21.4% (3/14) again indicated mixed cultures. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF STUDY: The new multiplex PCR is a rapid and accurate alternative to more conventional PCR methods. The persistence of mixed Campylobacter cultures noted in this study suggests certain strains may be very difficult to isolate clonally by standard culture methods.     

Whole-genome analysis of Leptospira interrogans to identify potential vaccine candidates against leptospirosis

Leptospirosis is an important global human and veterinary health problem. Humans can be infected by exposure to chronically infected animals and their environment. An important focus of the current leptospiral research is the identification of outer membrane proteins (OMPs). Due to their location, leptospiral OMPs are likely to be relevant in host-pathogen interactions, hence their potential ability to stimulate heterologous immunity. The existing whole-genome sequence of Leptospira interrogans serovar Copenhageni offers a unique opportunity to search for cell surface proteins. Predicted genes encoding potential surface proteins were amplified from genomic DNA by PCR methodology and cloned into an Escherichia coli expression system. The partially purified recombinant proteins were probed by Western blotting with sera from human patients diagnosed with leptospirosis. Sixteen proteins, out of a hundred tested, were recognized by antibodies present in human sera. Four of these proteins were conserved among eight serovars of L. interrogans and absent in the non-pathogenic Leptospira biflexa. These proteins might be useful for the diagnosis of the disease as well as potential vaccine candidates.     

Some potential sources for transmission of Campylobacter jejuni to broiler chickens

AIMS: The aim of the study was to determine Campylobacter jejuni contamination and prevalence on fomites moving between broiler farms and the processing plant in the period after cleaning and before departure to harvest chickens. In addition, changes in the proportion of contaminated fomites in the course of a day were assessed. METHODS AND RESULTS: Pooled swab samples were obtained from pallets, crates, wheels of trucks, tractors and forklifts, truck beds, and from drivers' and catchers' boots. After enrichment in Bolton's broth Campylobacter were recovered on modified blood-free Campylobacter selective agar (mCCDA). Isolates were identified using tests for phenotypic and biochemical characteristics. Of the 209 samples collected, 53% were positive for C. jejuni, with all fomites positive except tractor wheels. Pallets had the highest contamination rate at 75%. More than 50% of catchers' boots, drivers' boots, crates and truck wheels were positive. Forty-seven per cent and 31% of truck beds and forklift wheels, respectively, were contaminated. The proportion of contaminated fomites did not change significantly during the day. CONCLUSIONS: This study has identified trucks, forklifts, pallets, crates, drivers' and catchers' boots as potential sources of C. jejuni for broilers. SIGNIFICANCE AND IMPACT OF THE STUDY: Campylobacter jejuni contamination of broiler processing plant fomites was found to be extensive ranging from 31% for truck beds to 75% for pallets. The proportion of contaminated fomites was observed to be similar throughout the day. The impact of contaminated fomites as sources of colonization of broilers with C. jejuni is discussed.     

New antigenic candidates in multiple sclerosis: identification by serological proteome analysis

Myelin antigen targets that are clearly associated with pathogenic events in multiple sclerosis (M.S.) patients remain to be defined. We recently demonstrated that the analysis of global IgG antibody response against human brain antigens using one-dimensional (1-D) immunoblotting, allowed us to discriminate M.S. patients from controls (both healthy subjects and patients with Sj?gren's syndrome). Additionally, this approach also differentiated the three clinical forms of M.S. Indeed, 42 brain antigenic bands (26 from healthy brain and 16 from the M.S. brain) showed the discriminant IgG immune responses. The aim of our study was to characterize the 26 discriminant antigenic bands detected in healthy brain. Protein identification was successively performed by 1-D and two-dimensional (2-D) immunoblottings using sera from 18 M.S. patients, followed by mass spectrometry (MS) analysis and a database search. One hundred and two antigenic spots were then detected on 2-D immunoblots, with M.S. sera against healthy brains. Sixty-four spots were successfully matched with 2-D Coomassie brillant blue stained gels, which were further selected for MS analysis and annotated leading to the identification of 14 of the 26 discriminant antigens. Thus, serological proteome analysis may provide a useful tool for the identification of potentially new M.S.-associated antigens, whose relevance to physiopathological events remains to be defined.     

Psoroptes ovis: identification of vaccine candidates by immunoscreening

Serum from successful vaccine trials against the sheep scab mite, Psoroptes ovis, was used to immunoscreen a cDNA library constructed from mixed-stage and gender P. ovis to identify potential recombinant vaccine candidates. Immunodominant recombinant proteins recognised by IgG in these sera were selected for further analysis. Two candidates were identified in this way; a catchin-like protein (CLP) and a novel mu class glutathione S-transferase (GST). Both candidates were expressed in bacteria as recombinant proteins, the GST as an active enzyme, and combined with four other recombinant allergens in a multi-component recombinant vaccine. Strong serum IgG responses were induced in sheep against each of the components of the recombinant vaccine, however, the protective efficacy of the vaccine could not be determined because of variability in the establishment of a challenge infection.     

Evaluation of different generic in silico methods for predicting HLA class I binding peptide vaccine candidates using a reverse approach

Since CD8⁺ T cell response is crucial to combat intracellular infections and cancer, identification of class I HLA binding peptides is of immense clinical value. The experimental identification of such peptides is protracted and laborious. Exploiting in silico tools to discover such peptides is an attractive alternative. However, this approach needs a thorough assessment before its elaborate application. We have adopted a reverse approach to evaluate the reliability of eight different servers (inclusive of 55 predictors) by exploiting experimentally proven data. A comprehensive data set of more than 960 peptides was employed to test the efficacy of the programs. We have validated commonly used strategies to predict peptides that bind to seven most prevalent HLA class I alleles. We conclude that four of the eight servers are more adept in predictions. Although the overall predictions for class I MHC binders were superior to class II MHC binders, individual predictors for different alleles belonging to the same program were highly variable in their efficiencies. We have also addressed whether a consensus approach can yield better prediction efficiency. We observed that combining the results from different in silico programs could not increase the efficiency significantly.     

Whole genome identification of Mycobacterium tuberculosis vaccine candidates by comprehensive data mining and bioinformatic analyses

Background

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), infects ~8 million annually culminating in ~2 million deaths. Moreover, about one third of the population is latently infected, 10% of which develop disease during lifetime. Current approved prophylactic TB vaccines (BCG and derivatives thereof) are of variable efficiency in adult protection against pulmonary TB (0%–80%), and directed essentially against early phase infection.

Methods

A genome-scale dataset was constructed by analyzing published data of: (1) global gene expression studies under conditions which simulate intra-macrophage stress, dormancy, persistence and/or reactivation; (2) cellular and humoral immunity, and vaccine potential. This information was compiled along with revised annotation/bioinformatic characterization of selected gene products and in silico mapping of T-cell epitopes. Protocols for scoring, ranking and prioritization of the antigens were developed and applied.

Results

Cross-matching of literature and in silico-derived data, in conjunction with the prioritization scheme and biological rationale, allowed for selection of 189 putative vaccine candidates from the entire genome. Within the 189 set, the relative distribution of antigens in 3 functional categories differs significantly from their distribution in the whole genome, with reduction in the Conserved hypothetical category (due to improved annotation) and enrichment in Lipid and in Virulence categories. Other prominent representatives in the 189 set are the PE/PPE proteins; iron sequestration, nitroreductases and proteases, all within the Intermediary metabolism and respiration category; ESX secretion systems, resuscitation promoting factors and lipoproteins, all within the Cell wall category. Application of a ranking scheme based on qualitative and quantitative scores, resulted in a list of 45 best-scoring antigens, of which: 74% belong to the dormancy/reactivation/resuscitation classes; 30% belong to the Cell wall category; 13% are classical vaccine candidates; 9% are categorized Conserved hypotheticals, all potentially very potent T-cell antigens.

Conclusion

The comprehensive literature and in silico-based analyses allowed for the selection of a repertoire of 189 vaccine candidates, out of the whole-genome 3989 ORF products. This repertoire, which was ranked to generate a list of 45 top-hits antigens, is a platform for selection of genes covering all stages of M. tuberculosis infection, to be incorporated in rBCG or subunit-based vaccines.     

In silico approach towards identification of potential inhibitors of Helicobacter pylori DapE

Helicobacter pylori is a gastric mucosal pathogen and is associated with diseases like peptic ulcer and gastric cancer. To combat H. pylori infection, there is an urgent need for new class of antibiotics due to the emergence of drug-resistant strains. Enzymes involved in bacterial lysine biosynthetic pathways may be potential targets for antibacterial drug development, since lysine is an essential component of the bacterial peptidoglycan cell wall. No pathway exists for lysine biosynthesis in humans; hence, the inhibitors targeting bacterial enzymes may have selective toxicity. dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) is a critical enzyme of this pathway and deletion of DapE gene is lethal to H. pylori, since the organism has no alternative pathway for lysine biosynthesis. In this study, we reported a 3D model structure of H. pylorie DapE, which consisted of a catalytic domain and a dimerization domain generated by MODELLER software. We also confirmed the stability of the modeled structure through 10 ns molecular dynamics simulation using GROMACS software. Next, to identify potential small molecule inhibitors of DapE, drug-like small molecule-screening library was generated. This was performed by Tanimoto-based similarity searching in the PubChem Database with DapE substrate L,L-SDAP as a query molecule, followed by fragment-based docking approach using GLIDE XP. This approach identified two potential substrate-competitive small molecule inhibitors of DapE. These new molecules may provide a starting point to search for novel therapeutics.     

An integrated in silico based subtractive genomics and reverse vaccinology approach for the identification of novel vaccine candidate and chimeric vaccine against XDR Salmonella typhi H58

Salmonella typhi is notorious for causing enteric fever which is also known as typhoid fever. It emerged as an extreme drug resistant strain that requires urgent attention to prevent its global spread. Statistically, about 11–17 million typhoid illnesses are reported worldwide annually. The only alternative approach for the control of this illness is proper vaccination. However, available typhoid vaccine has certain limitations such as poor long-term efficacy, and non-recommendation for below 6 years children, which opens the avenues for designing new vaccines to overcome such limitations. Computational-based reverse vaccinology along with subtractive genomics analysis is one of the robust approaches used for the prioritization of vaccine candidates through direct screening of genome sequence assemblies. In the current study, we have successfully designed a peptide-based novel antigen chimeric vaccine candidate against the XDR strain of S. typhi H58. The pipeline revealed four peptides from WP_001176621.1 i.e., peptidoglycan-associated lipoprotein Pal and two peptides from WP_000747548.1 i.e., OmpA family lipoprotein as promising target for the induction of immune response against S. typhi. The six epitopes from both proteins were found as immunogenic, antigenic, virulent, highly conserved, nontoxic, and non-allergenic among whole Salmonella H58 proteome. Furthermore, the binding interaction between a chimeric vaccine and human population alleles was unveiled through structure-based studies. So far, these proteins have never been characterized as vaccine targets against S. typhi. The current study proposed that construct V2 could be a significant vaccine candidate against S. typhi H58. However, to ascertain this, future experimental holistic studies are recommended as follow-up.     

MALDI-TOF-MS方法快速鉴定食品中空肠弯曲菌的技术研究

运用基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time of-flight mass spectrometry,MALDI-TOF-MS)技术快速鉴定食品中空肠弯曲菌.通过对该方法的样品前处理的选择、稳定性、特异性等方面进行研究,确定了方法...     

CSF proteome: a protein repository for potential biomarker identification

Proteomic analysis is not limited to the analysis of serum or tissues. Synovial, peritoneal, pericardial and cerebrospinal fluid represent unique proteomes for disease diagnosis and prognosis. In particular, cerebrospinal fluid serves as a rich source of putative biomarkers that are not solely limited to neurologic disorders. Peptides, proteolytic fragments and antibodies are capable of crossing the blood-brain barrier, thus providing a repository of pathologic information. Proteomic technologies such as immunoblotting, isoelectric focusing, 2D gel electrophoresis and mass spectrometry have proven useful for deciphering this unique proteome. Cerebrospinal fluid proteins are generally less abundant than their corresponding serum counterparts, necessitating the development and use of sensitive analytical techniques. This review highlights some of the promising areas of cerebrospinal fluid proteomic research and their clinical applications.     

Phase-Variable Surface Structures Are Required for Infection of Campylobacter jejuni by Bacteriophages

This study characterizes the interaction between Campylobacter jejuni and the 16 phages used in the United Kingdom typing scheme by screening spontaneous mutants of the phage-type strains and transposon mutants of the sequenced strain NCTC 11168. We show that the 16 typing phages fall into four groups based on their patterns of activity against spontaneous mutants. Screens of transposon and defined mutants indicate that the phage-bacterium interaction for one of these groups appears to involve the capsular polysaccharide (CPS), while two of the other three groups consist of flagellatropic phages. The expression of CPS and flagella is potentially phase variable in C. jejuni, and the implications of these findings for typing and intervention strategies are discussed.     

Yoghurt: an unlikely source of Campylobacter jejuni/coli

Knowledge of the relative insensitivity of Campylobacter jejuni to moderately acid environments prompted us to study its survival in different batches of yoghurt of pH range 4.2–5.3 and the role of organic or inorganic acid in the die-off of this pathogen. None of the 11 strains of C. jejuni or C. coli survived more than 25 min in yoghurt. Suspecting that this rapid die-off cannot be accounted for by the pH of the yoghurt we compared the survival rates of C. jejuni in milk, whose pH had been adjusted by lactic, propionic and hydrochloric acid respectively, with that of yoghurt. Even for an inoculum of 10⁵–10⁸ cfu/ml propionic acid was bactericidal in minutes. Lactic acid reduced the bacterial populations by 3–5 logs in 30 min. Strong inorganic acid HC1, by contrast, had little or no effect on the populations. Although lactic acid is quite bactericidal for C. jejuni , it is apparently not the only factor to which the prompt elimination of this pathogen from yoghurt could be attributed.     

Yoghurt: an unlikely source of Campylobacter jejuni/coli

Knowledge of the relative insensitivity of Campylobacter jejuni to moderately acid environments prompted us to study its survival in different batches of yoghurt of pH range 4.2-5.3 and the role of organic or inorganic acid in the die-off of this pathogen. None of the 11 strains of C. jejuni or C. coli survived more than 25 min in yoghurt. Suspecting that this rapid die-off cannot be accounted for by the pH of the yoghurt we compared the survival rates of C. jejuni in milk, whose pH had been adjusted by lactic, propionic and hydrochloric acid respectively, with that of yoghurt. Even for an inoculum of 10(5)-10(8) cfu/ml propionic acid was bactericidal in minutes. Lactic acid reduced the bacterial populations by 3-5 logs in 30 min. Strong inorganic acid HCl, by contrast, had little or no effect on the populations. Although lactic acid is quite bactericidal for C. jejuni, it is apparently not the only factor to which the prompt elimination of this pathogen from yoghurt could be attributed.