Immunoproteomic Identification of Polyvalent Vaccine Candidates from Vibrio parahaemolyticus Outer Membrane Proteins Hui Li,†,§ Ming-Zhi Ye,†,§ Bo Peng,‡ Hong-Kai Wu,† Chang-Xin Xu,‡ Xiao-Peng Xiong,† Chao Wang,† San-Ying Wang,‡ and Xuan-Xian Peng*,† Center for Proteomics, State Key Laboratory of Bio-Control, School of Life Sciences, Sun Yat-sen University, University City, Guangzhou 510006, People’s Republic of China, and School of Life Sciences, Xiamen University, Xiamen 361005, People’s Republic of China Received January 13, 2010
Bacterium is still a major cause of many infectious diseases and a global threat to human health, aquaculture, and animal feeding. Prevention by vaccination is the most efficient and economical way of fighting bacterial diseases, but one of the persistent challenges to prevent bacterial infections and disease transmissions is the existence of multiple bacterial species, families, and genera and the lack of efficient polyvalent vaccines against them. The information on candidate immunogens for polyvalent vaccine development is elusive, as well. For the development of broad cross-protective vaccines, we have employed heterogeneous antiserum-based immunoproteomics approaches to identify antigenically similar outer membrane (OM) proteins that could be used as potential polyvalent vaccine candidates against Vibrio parahaemolyticus, V. alginolyticus, V. fluvialis, Aeromonas hydrophila, and A. sobria infections. VPA1435, VP0764, VPA1186, VP1061, and VP2850 could be recognized by at least three antisera and demonstrated significantly passive and active immune protection against V. parahaemolyticus infection in a crucian carp model. VP1061 and VP2850 induced higher immune and protective abilities than the other three OM proteins. Furthermore, the abilities of VP1061 and VP2850 in the generation of broad cross-protective immune reaction against the infections of V. alginolyticus, A. hydrophila, and Pseudomonas fluorescens were also investigated in fish and mouse models. Our results suggested that VP1061 and VP2850 could potentially be used as polyvalent vaccine candidates for the development of novel polyvalent vaccines against V. parahaemolyticus and other Gram-negative pathogens. On the basis of these results, characteristics of OM proteins as polyvalent vaccine candidates have been addressed. Keywords: polyvalent vaccine • V. parahaemolyticus • immunoproteomics • cross-protective immunogens • outer membrane proteins • VP1061 • VP2850
Introduction Bacterium is still a major cause of many infectious diseases, which cause severe health problems in humans, and great losses in aquaculture and animal feeding.1-3 Although vaccines are very efficient in preventing human and animal hosts from pathogen’s infections, their roles in protecting human health and aquaculture improvement and animal feeding are limited because currently available vaccines are usually serotype- or species-specific.4 The efficacy of these serotype-specific vaccines is usually decreased by the narrow range of bacterial serotypes that epidemically cause a variety of diseases in geographical distribution.4 Any species-specific vaccine can prevent the infections caused by this type of specific bacterium only but not other bacterial species. However, bacterial pathogens have a variety of serotypes or species which belong to * To whom correspondence should be addressed. Phone: +86-20-31452846. Fax: +86-20-8403-6215. E-mail:
[email protected]. † Sun Yat-sen University. ‡ Xiamen University. § These authors contributed equally. 10.1021/pr1000219
2010 American Chemical Society
different genera and families and are classified into primary (obligate) or opportunistic pathogens. Therefore, development of cross-protective vaccines and antisera that can fight against as many pathogens as possible is in an urgent need. Crossprotective reactivity against different bacterial infections, including those against heterogeneous serotypes and species of pathogenic bacteria, has recently been reported.4-9 Vulnivaccine, a licensed vaccine against Vibrio vulnificus, could protect eels from vibriosis for at least 6 months after vaccination by triple prolonged immersion at the glass eel stage.6,7 The crossreactivity of outer membrane (OM) proteins in Gram-negative bacteria suggested that OM proteins could be served as promising polyvalent immunogens.8 Polyvalent vaccines, crossserotype and -species, even genus- and family-specific vaccines have recently become significant scientific issues.4,10-12 However, broad cross-protective immunogens remain to be investigated. Immunoproteomic methodologies are powerful tools for identification of dominant immunogens. Conventional immunoproteomics is based on homogeneous antisera and has been Journal of Proteome Research 2010, 9, 2573–2583 2573 Published on Web 02/11/2010
research articles utilized for identification of immunogens of several bacterial species.13-19 However, this approach cannot be used for identification of cross and protective immunogens. To solve the problem, we developed a novel immunoproteomic approach by combining the traditional approach with a bacterial immunization challenging method. With the developed approach, we successfully identified two cross-protective immunogens from A. hydrophila OM proteins against A. hydrophila and A. sobria infections.18 We also developed heterogeneous antiserum-based immunoproteomic methodologies for the identification of broad cross-protective vaccine candidates based on a heterophilic recognition principal.12 These approaches paved some new ways for identification of heterophilic antigens. The heterophilic antigens are the substances that occur in unrelated species of organisms but have similar serologic properties. The interactions between the heterophilic antigens and the heterophilic antibodies have been widely reported, and some of them were utilized in a pathogenesis study and diagnosis of clinical diseases.20,21 In summary, the combination of homogeneous and heterogeneous antiserumbased immunoproteomic methodologies with the bacterial immunization challenging method was successfully utilized to identify broad cross-protective immunogens of Vibrio parahaemolyticus OM proteins in the present study. VP2850 and VP1061 as potential broad cross-protective immunogens and polyvalent vaccine candidates against V. parahaemolyticus and other Gram-negative bacteria have been identified and characterized in fish and mouse models. Furthermore, the potential and characteristics of OM proteins as broad cross-protective vaccine candidates were also addressed in this study.
Materials and Methods Bacterial Strains and Animals. V. parahaemolyticus, V. alginolyticus, V. fluvialis, P. fluorescens, A. hydrophila, and A. sobria were the bacterial collections in our laboratory and were grown in LB medium at 28 °C. Approximately 20 g of crucian carps (Carassfus auriatus) was purchased from Zhujiang aquaculture farm in Guangzhou, China, and held in aquaria at 28 °C. Then, 179 Kunming mice with the average weight of 20 g, obtained from Animal Center of Sun Yat-sen University, were fostered in cages accommodated with sterile water and dry pellet diet. These animals were randomly divided into experimental and control groups and acclimated for 7-10 days for the investigation of the protective ability of polyvalent vaccine candidates. This study was approved by the Institutional Review Board of Sun Yat-sen University. Isolation and Analysis of OM Proteins. Extraction of V. parahaemolyticus OM proteins was prepared according to the procedure described previously.22 Briefly, the bacterial cells were harvested by centrifugation at 4000g for 15 min at 4 °C. The cells were then washed in 40 mL of saline solution three times and then resuspended in 5 mL of 50 mM Tris-HCl (pH 7.4). Cells were disrupted by intermittent sonic oscillation. Unbroken cells and cellular debris were removed by centrifugation at 5000g for 20 min, and supernatant was further centrifuged at 100 000g for 40 min at 4 °C. The pellet was resuspended in 10 mL of 2% w/v sodium lauryl sarcosinate (Sigma) and incubated at room temperature for 30 min. The solution was centrifuged at 100 000g for 40 min at 4 °C. The resulting pellet was resuspended in 50 mM Tris-HCl (pH 7.4) and then stored at -80 °C. The concentration of the OM proteins in the final preparation was determined using the Bradford method. 2574
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Li et al. 2-DE-Based Immunoproteomics. 2-DE-based immunoproteomics was performed according to the procedure described previously.22,23 Briefly, OM protein extracts containing 15 µg proteins were dissolved in solution (8 M urea, 2 M thiourea, 4% CHAPS, and 80 mM DTT). IEF was carried out using pH 3-10 carrier ampholyte for 8000 V. After equilibration for 15 min, the gels were transferred to the second dimension electrophoresis using 12% acrylamide gel. The gels were stained with Coomassie blue-R250 and then scanned in ImageScan (Amersham Bioscineces, Sweden). Protein spots of interest were cut from the gel for mass spectrometric analysis. The sample solution (30-100 ppm) with equivalent HCCA matrix solution was applied onto the MALDI-TOF target. Mass spectra were recorded in positive ion reflector mode on a Bruker ReFlex III MALDI-TOF instrument. The instrument was calibrated before each run using PeptideCalibStandard II (Bruker), and individual MS spectra were internally calibrated using tryptic autolytic peaks (if present). MS data were analyzed and peak lists generated using Flexanalysis version 2.0 (Bruker). MS peaks were selected between 800 and 3000 m/z. Search parameters allowed for one missed tryptic cleavage site, the carbamidomethylation of cysteine, and the possible oxidation of methionine; precursor ion mass tolerance was 100 ppm. Peptide masses were searched using the program Protein Prospector MS-FIT (http://prospector.ucsf.edu). For immunoblotting, proteins in gels were transferred to nitrocellulose (NC) membranes by electrophoresis, followed by Western blotting using rabbit antisera against relevant bacteria as the primary antibodies and peroxidase-conjugated goat antirabbit IgG (Bosheng Biotech Corp, Xiamen, China) as the secondary antibody. Antibodytagged protein spots were detected by DAB. Gene Cloning, Protein Purification, and Antiserum Preparing. Standard PCR and molecular biology protocols were used to amplify VPA1435, VP0425, VP0764, VPA1186, VP2850, and VP1061 genes of V. paraheamolyticus. Primes of these six genes and their restriction sites were designed, as shown in Table 1. PCR fragments were detected by agarose electrophoresis and directionally cloned into plasmid pET-32a or pET-28a. Recombinant plasmids were checked by restriction endonuclease digestion and sequencing and then transformed into Escherichia coli BL21. Overnight cultures of E. coli BL21 (DE3) harboring recombinant plasmids were diluted 1:100 (v/v) in fresh LB with ampicilin (60 µg/mL) or kanamycin (50 µg/mL), then incubated at 37 °C until the absorbent optical density reached 0.6 of 600 nm (OD600). Protein expressions were induced with 1 mM isopropyl-β-D-thiogalactoside (IPTG, from BBI, Canada) for 5 h at 37 °C after the optimization of expression conditions. Bacterial cells were harvested by centrifugation at 4000g for 20 min at 4 °C, washed with saline solution, resuspended in 50 mM sodium phosphate buffer (pH 8.0) containing 8 M urea, then incubated 30 min in an ice bath. The cell suspension was disrupted by sonication an in ice bath (350 W, 10 min × 3), followed by centrifugation at 12 000g for 20 min at 4 °C. The cell debris was discarded, and the clarified supernatant was loaded into a column packed with Ni2+nitriloacetate (NTA) super flow resin, which was charged with 50 mM NiSO4, and purified by affinity chromatography according to the manufacturer’s instructions (Qiagen, Germany). Concentrations of proteins were determined by the Bradford method, and the protein samples were stored at -80 °C until use. Antisera against the purified recombinant proteins were separately raised by immunizing New Zealand rabbits with 500 µg of purified protein emulsified with Freund’s complete
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Polyvalent Vaccine Candidates Table 1. Primers and Restricted Digestion Sites Used in This Study target gene
primers
VPA1435
sense primer: 5′-GCAGGATCCATGACGAAAAATAACTT-3′ antisense primer: 5′-GTAGAATTCGTTCAATTACCAGCGG-3′ sense primer: 5′-ATAGGATCCATGAAAAAACTAGCAGCGGT-3′ antisense primer: 5′-CGCAAGCTTTTATTGCTGAACTTGGTA-3′ sense primer: 5′-ATAGGATCCATGAACAAAGTAGCAAT-3′ antisense primer: 5′-GTGGAATTCGTGTGAACATTCAGAT-3′ sense primer: 5′-GCGGAATTCTTACTGCTTACCATCAT-3′ antisense primer: 5′-AGTCTCGAGGAACATCGCGGGCTTTTTG-3′ sense primer: 5′-ACAGGATCCAAGATGCAACTGAACAA-3′ antisense primer: 5′-CCGAAGCTTTTCCTCGAATTAGTAT-3′ sense primer: 5′-TGCAAGCTTACGTTATTTCGCTGGTT-3′ antisense primer: 5′-GCGGATCCATGAAAAAATTGCTTCCACT-3′
VP0764 VPA1186 VP2850 VP1061 VP0425
adjuvant. The first injection was followed by three other injections with Freund’s incomplete adjuvant at intervals of 2 weeks. The resulting antisera were collected and stored at -80 °C for later use. Passive and Active Immunization and Immune Challenge. Crucian carps and mice were randomly divided into 22 and 9 groups, respectively. For passive immunization and challenge, antisera against the recombinant proteins were adjusted into the same agglutinating titer and used for passive immunization. These animals were challenged using five times their lethal dose50 (LD50) of V. parahaemolyticus 2.5 h after the passive immunization. After 72 h observation, the relative percent survivals (RPS) of the experimental subjects were then measured. To ensure active immunity and challenge, the crucian carps and mice were injected with the immunogens at an interval of 2 weeks. Purified proteins emulsified with Freund’s complete adjuvant or with Freund’s incomplete adjuvant were utilized in the primary (50 µg for a carp and 100 µg for a mouse) and boost immunizations (25 µg for a carp and 100 µg for a mouse each), respectively. The immunized animals were randomly selected for measurement of agglutinating titers and challenged by bacteria with concentrations of five times their LD50 1 week following the boost. The negative control group was injected with 100 µL of saline solution. These animals were observed for 72 h to measure their RPS. Effect of Naı¨ve Sera and Antisera on the Expression of VP1061 and VP2850. V. parahaemolyticus was cultured in LB medium and harvested at 1.0 of OD600 and then divided into groups containing 900 µL of bacterial cells each. Some groups of bacterial cells were separately added into 0 (control), 50, 100, 200, and 400 µL of normal naı¨ve crucian carp plasma and then sterile saline solution added to a final volume of 1.3 mL. The other groups were added into 100 µL of 1:500, 1:5000, and 1:50000 of hyper-immune rabbit antisera against VP1061 or VP2850 and 100 µL of sterile saline solution (control). These mixtures of bacteria with fish plasma, rabbit sera, or saline solution were incubated at 30 °C for 15 min and then harvested by centrifugation at 10 000g for 20 min at 4 °C. The resulting cells were washed with saline solution and were solubilized at 100 °C for 5 min in sample buffer containing 2% (w/v) SDS and 5% (v/v) β-mercaptoethanol before being subjected to SDSPAGE and Western blotting using the primary antibodies rabbit antisera toward VP1061 or VP2850 and the secondary antibody peroxidase-conjugated goat antirabbit IgG. Then the membranes were reacted with substrate DAB until optimum color developed. The experiments were repeated three times. Database Searching and Bioinformatics Analysis. Tepitope prediction was performed using the software SYFPEITHI (http://
restriction digest site
BamHI-EcoRI BamHI-HIndIII BamHI-EcoRI EcoRI-XhoI BamHI-HIndIII BamHI-HIndIII
www.syfpeithi.de/Scripts/MHCServer.dll/EpitopePrediction.htm) and Propred http://www.imtech.res.in/raghava/propred/page3.html. B epitope prediction was done using ANTIGENIC from EMBOSS (http://mobyle.pasteur.fr/cgi-bin/MobylePortal/portal.py? form)antigenic. Amino acid sequence homologues were analyzed by Boxshade 3.21 (http://www.ch.embnet.org/software/ BOX_form.html and http://blast.ncbi.nlm.nih.gov/Blast.cgi). The distance and neighbor-joining (NJ) method was used to reconstruct phylogenetic unrooted trees for evolutionary distance data. The bootstrap was 100 times. Statistical Analysis. Differences between experimental and control groups were tested for significance using the statistic software SPSS and two significance levels (0.05 and 0.01).
Results Two-Dimensional Maps of V. parahaemolyticus OM Proteins. OM protein extracts obtained from V. parahaemolyticus cells were separated by 2-DE. Protein spots were visualized by Coomasie R-250 staining. Figure 1A shows a representative map of the OM proteins separated on a 2-DE gel. Approximately 44 spots were visualized after Coomassie R-250 staining. Thirtysix unique proteins were identified. The isoforms of three proteins were detected, and these three proteins were identified at multiple positions of the gel. These proteins were VPA1186 (spots 7, 32, 35, 36), VP2770 (spots 17, 19), and VPA1644 (spots 25, 26). Out of the 36 unique proteins identified, 29 (80.6%) of these proteins were predicted to be localized at OM (Table 2). Identification of Broad Cross-Immunogenic OM Proteins. Proteins in the 2-DE gels were transferred into the NC membrane and incubated with homogeneous and heterogeneous antisera. The heterogeneous sera included anti-V. fluvialis, anti-A. hydrophila, anti-V. alginolyticus, and anti-A. sobria. Thirteen spots representing 11 unique proteins were recognized by these antisera. These proteins were VPA0882 (spot 4), VPA1435 (spot 6), VPA0554 (spot 9), VPA1549 (spot 12), VP0425 (spot 16), VPA0248 (spot 28), VP0764 (spot 29), VPA1186 (spots 32, 35, and 36), VP2850 (spot 38), VP2310 (spot 40), VP1061 (spot 41). Out of them, VPA1435, VPA0554, and VP2850 were recognized by three antisera, and VP0425, VP0764, VPA1186, and VP1061 were reacted with all antisera used (Figure 1B-F, Table 3), indicating that these proteins were broad cross-immunogenic antigens. Interestingly, VPA0882, VPA1549, VP2850, and VP2310 were not recognized by the homogeneous antiserum but recognized by some heterogeneous antisera. These results may suggest that these proteins are not dominant antigens in V. parahaemolyticus, but their homologues are dominant ones in the corresponding bacteria. From 2-DE analysis, four different spots were identified as the Journal of Proteome Research • Vol. 9, No. 5, 2010 2575
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Figure 1. 2-DE analysis and Western blotting for identification of broad cross-reactive immunogens using V. parahaemolyticus OM proteins as antigens and homogeneous and heterogeneous antisera as the primary antibodies. (A) 2-DE; (B-F) immunoproteomics using homogeneous anti-Vibrio parahaemolyticus (B) and heterogeneous antisera of anti-V. fluvialis (C), anti-Aeromonas hydrophila (D), anti-V. alginolyticus (E), anti-A. sobria (F).
same protein VPA1186, but not all of these spots reacted with the antisera described above. Only spot 35 was recognized by all antisera used. Spots 32 and 36 reacted only with anti-V. parahaemolyticus and anti-V. alginolyticus sera, and spot 7 was not detected by any of these antisera. Thus, isoforms of proteins could be differentially recognized by the homogeneous and heterogeneous antisera. Our previous studies showed that the strongest immune response was not from the highest concentration of proteins in a pool of immunogens.12,18 In the present study, spot 35 had the highest percentage of spot volume in the 2-D gel, but spots 41 and 29 had the highest staining intensity when antiVibrio and anti-Aeromonas were used, respectively. These results showed that the volume percentage of a protein spot in 2-DE was not consistent with that of staining intensity in 2-DE Western blotting. Equally importantly, this study further revealed that the staining intensity was related to the species of antisera in reaction with a same antigen. The highest and second highest ratios came from spot 41 and spot 38 when the antisera against the three strains of Vibrio were used, whereas the highest and second highest ratios were generated from spots 29 and 4 or 16 and 29, respectively, when anti-A. 2576
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hydrophila and anti-A. sobria were used (Figure 2). Thus, the host response to dominant immunogens is associated with bacterial species. Investigation of Protective Ability in the Fish Model Using Passive and Active Immunization. The genes encoding VPA1435, VP0764, VPA1186, VP2850, VP1061, and VP0425 were cloned into pET-32a or pET-28a and expressed in E. coli BL21 (Figure 3). All of the recombinant and fused proteins except VP2850 were expressed at their predicted molecular mass. The recombinant VP2850 seemed to be expressed in the absence of the fused protein tag. The validity of the clone was also confirmed by DNA sequencing, and the identity of expressed VP2850 was validated using MALDI-TOF/MS (data not shown). These recombinant proteins were purified further for preparation of antisera (Figure 3). The prepared rabbit antisera were injected into crucian carps, and the passively immunized crucian carps were challenged using V. parahaemolyticus 2.5 h later. Five out of the six antisera, anti-VPA1435, VP0764, VPA1186, VP2850, and VP1061, showed significant immune protection from the bacterial infection. Their RPS was 50.0 (P < 0.01), 44.5 (P < 0.01), 33.4 (P < 0.05), 72.3 (P < 0.01), and 66.7% (P < 0.01), respectively (Table 4). The protective ability of the
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Polyvalent Vaccine Candidates
Table 2. Identification of V. parahaemolyticus OM Proteins Using PMF Searching and Subcellular Location Program PSORTb Version 2.0a spot no. 1 2 3 4 5 6 7 8 9 10 11 12 13 16 17 18 19 20 21 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
subcellular location
protein description OtnA protein ATP synthase F1, alpha subunit ferric vibrioferrin receptor heme transport protein HutA putative lipoprotein putative iron(III) compound receptor outer membrane protein OmpA putaive Fe-regulated protein B precursor methyl-accepting chemotaxis protein putative exported protein oligopeptide ABC transporter, periplasmic oligopeptide-binding protein hypothetical protein VPA1549 iron-regulated outer membrane virulence protein homologue outer membrane protein TolC elongation factor TU agglutination protein elongation factor TU putative outer membrane protein TolC polar flagellin C4-dicarboxylate-binding periplasmic protein hypothetical protein VP1475 maltose-inducible porin maltose-inducible porin uridine phosphorylase putative outer membrane protein OmpA outer membrane protein OmpA putative outer membrane protein OmpV long-chain fatty acid transport protein outer membrane protein OmpA long-chain fatty acid transport protein putative chitoporin outer membrane protein OmpA outer membrane protein OmpA outer membrane protein ompK precursor conserved hypothetical protein outer membrane protein OmpW surface antigen peptidoglycan-associated lipoprotein hypothetical protein VP1243 putative outer membrane lipoprotein Pcp lipoprotein-related protein
NCBI accession no.
no. of peptides matched
no. of peptides unmatched
Mr
MOWSE score
cover %
8
OM cytoplasmic OM OM OM OM OM OM CM OM periplasmic
28896994 28899845 14717832 28900737 28897223 28901290 28901041 28900519 28900409 28897576 28898865
17 18 9 11 5 11 13 5 6 9 7
30 9 20 18 24 23 31 37 28 36 26
98406/5.4 56637/5.1 78857/4.9 77341/4.7 67557/4.9 77060/4.6 36014/4.3 74206/4.5 50154/5.3 51780/4.6 62382/5.2
3.805 × 10 4.195 × 109 2.273 × 106 2.268 × 107 21 4.898 × 105 1.775 × 106 158 1508 9.867 × 105 1.609 × 104
25 37 20 32 13 24 44 14 18 24 19
unknown OM
28901404 28899376
3 7
31 25
11050/7.8 71948/4.8
47.4 9.673 × 104
10 23
OM cytoplasmic OM cytoplasmic OM extracellular periplasmic OM OM OM cytoplasmic OM OM OM OM OM OM OM OM OM OM unknown OM OM OM unknown OM unknown
28897199 28899544 28898405 28899544 28898772 551512 28897684 28898249 28901499 28901499 28897734 28900103 28897538 28900173 28898986 28901041 28900715 28897534 28901041 28901041 1709464 28899624 28899951 28899084 28897835 28898017 28897966 28897724
14 5 11 8 5 6 6 7 8 3 12 8 9 6 14 13 5 17 13 11 6 6 7 21 5 7 4 4
9 39 15 21 26 21 11 22 50 20 8 5 10 8 6 13 17 0 11 13 23 9 6 21 11 22 57 26
47983/4.7 43152/4.8 50705/5.3 43152/4.8 46573/5.3 40158/4.9 37133/5.9 32929/5.1 46963/4.6 46963/4.6 26936/5.4 35554/4.4 34073/4.4 28148/5.1 45759/4.7 36014/4.3 44961/4.5 40790/4.3 36014/4.3 36014/4.3 29467/5.1 20227/7.8 23468/5.0 90054/4.7 18713/4.6 19781/4.9 16121/8.9 15781/5.6
2.708 × 108 2787 2.571 × 106 1.287 × 105 6219 1.132 × 104 7211 1.267 × 105 8766 8766 5.687 × 108 6.393 × 104 6.720 × 104 5.499 × 105 9.762 × 106 9.427 × 104 7298 1.314 × 107 3.613 × 105 9.427 × 104 4190 2.836 × 104 4.689 × 104 3.185 × 1011 25.4 3.053 × 104 1410 1.780 × 104
34 19 39 33 24 23 25 44 32 12 59 40 36 35 60 45 22 64 49 37 45 34 25 33 31 48 41 48
a PMF (peptide mass fingerprinting) searching using the program Protein Prospector MS-Fit (http://prospector.ucsf.edu), in which V. parahaemolyticus protein database was defined as a matching species.
Table 3. Identification of the Immunoreactivity and Cross-Reactivity of V. parahaemolyticus OM Proteins Using Homogenous and Heterogeneous Antisera (-, no; +, yes) spot
4 6 9 12 16 28 29 32 35 36 38 40 41
protein description/name
anti-V. parahaemolyticus
anti-V. fluvialis
anti-V. alginolyticus
anti-A. hydrophila
anti-A. sobria
HutA/VPA0882 putative iron(III) compound receptor/ VPA1435 methyl-accepting chemotaxis protein/ VPA0554 hypothetical protein/VPA1549 TolC/VP0425 OmpA/VPA0248 OmpA/VP0764 OmpA/VPA1186 OmpA/VPA1186 OmpA/VPA1186 conserved hypothetical protein/VP2850 surface antigen/VP2310 PAL/VP1061
+
+
-
+ +
-
+
+
+
-
-
+ + + + + + +
+ + + + + + +
+ + + + + + +
+ + + + + + +
+ + + +
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Figure 2. Volumes and ratios of the spots from 2-D and 2-D Western blotting. (1) Volume of scanned dots from 2-D. (2) Volume of scanned dots from 2-D Western blotting. (3) Ratios of the corresponding scanned dots between 1 and 2.
five recombinant proteins was further investigated using an active immunization approach. Significantly protective rates were found in the animals immunized using VPA1435 (52.9%, P < 0.01), VP0764 (41.2%, P < 0.05), VPA1186 (41.2%, P < 0.05), VP2850 (82.4%, P < 0.01), and VP1061 (64.8%, P < 0.01) (Table 5). Investigation of Broad Cross-Protective Ability in Fish and Mouse Active Immunity Models. We further investigated the broad cross-protective ability of VP2850 and VP1061. Fish and mice were actively immunized and then challenged separately with V. alginolyticus, A. hydrophila, or P. fluorescens. Our results showed that VP2850 and VP1061 elicited significant protection of crucian carps from the infections of these three pathogens as compared with the control. The protective ability was ranked as follows: P. fluorescens > V. alginolyticus > A. hydrophila for VP2850 and P. fluorescens > A. hydrophila > V. alginolyticus for VP1061 (all P < 0.01) (Table 6). Significant difference was also found in the mouse challenging study. VP2850 and VP1061 elicited protective abilities against the three bacteria, and their abilities were ranked from high to low: V. alginolyticus > A. hydrophila > P. fluorescens for VP2850 and A. hydrophila > V. alginolyticus > P. fluorescens for VP1061 (all P < 0.01) (Table 6). With these results together, it may indicate that VP2850 and VP1061 are broad cross-protective immunogens. Investigation of the Cross-Reactive Mechanism. To understand the mechanism of the broad cross-reaction, the following three assays were performed. First, we investigated the evolutionary relationships of VP1061 and VP2850 among different bacterial species using distance and neighbor-joining methods. Some homologous genes of VP1061 and VP2850 were found, respectively, in more than 80 and 15 species of bacteria. The unrooted trees constructed from 17 representative VP1061 deduced protein sequences and the 15 VP2850 deduced protein sequences showed that these sequences were highly conserved in Gram-negative bacteria. VP1061 was more universal than VP2850 (Figure 4A,B). Importantly, these homologues of VP2850 were all putative proteins. In addition, an unrooted tree was constructed to investigate the conserved OmpA domain, as shown in Figure 4C because, significant protective activity of two OmpA domains (VP0764, VPA1186) was observed. The results showed that the conserved OmpA domain was found not only in these Gram-negative bacteria but also in Leptospira and Rickettsial. Second, the epitopes of VP1061 and VP2850 2578
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were predicted. There were 5 and 4 epitopes with 2 and 2 helices in VP1061 and VP2850, respectively (Table 7). These helix epitopes were utilized as primers for blast analysis in all species of bacteria because helical epitopes showed better immunogens than other epitopes.24 The two helical epitopes LLIALPVMA and MLAAHASYLSKN of VP1061 were shared by different families of bacteria including Enterobacteriaceae, Vibrionaceae, Deinococcaceae, Pseudomonadaceae, and Vibrionaceae, Pseudomonadaceae, Bacillaceae, respectively. The epitope LLIALPVMA was only recognized by T cells, and the epitope MLAAHASYLSKN was recognized by both B and T cells. The two helical epitopes NFGLFFGAAKV and IKMKKLMTS of VP2850 were recognized by B and T cells and shared by Vibrionaceae, Neisseria, Lactobacillus, and Vibrionaceae, Shewanella, Lactobacillus, respectively (Figure 4D-G). Third, rabbit antisera toward VP1061 and VP2850 were utilized to recognize the respective proteins in V. parahaemolyticus and their homologous proteins in V. alginolyticus, A. hydrophila and P. fluorescens. Immunoreactivity was detected in all four bacteria using antiserum against VP1061 but only in V. parahaemolyticus and V. alginolyticus using antiserum against VP2850 (data not shown). Thus, these shared epitopes recognized by B cells may provide sites for reactions with heterogeneous antibodies in the detection of VP1061, whereas other nonlinear epitopes may be involved in the detection of VP2850. Investigation of Host Regulation of VP1061 and VP2850. To investigate whether the expression of VP1061 and VP2850 were regulated by host products, we examined the effect of naı¨ve fish plasma or hyper-immune sera on the expressions of the two proteins using Western blotting. Up-regulation of VP2850 and VP1061 was detected in a dose-dependent fashion in response to the increase of naı¨ve fish plasma and decrease of hyper-immune rabbit sera (Figure 5). Thus, the regulation of the two vaccine candidate OM proteins by host sera may suggest the importance of the interactions between host and bacteria.25
Discussion Using the novel heterogeneous antiserum-based immunoproteomics developed in our laboratory and the bacterial immunization challenging method,12 we investigated the broad cross-protective activities of V. parahaemolyticus OM proteins
Polyvalent Vaccine Candidates
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Figure 3. Gene cloning and expression, purification of recombinant proteins, and validation of the polyclonal antibody specificity. (A1-A5) PCR products of OM proteins in 0.8% agarose gel. (B1-B6) Identification of recombinant plasmids by restriction enzyme analysis. (A,B) lane 1, VPA1435; lane 2, VP0425; lane 3, VP0764; lane 4, VPA1186; lane 5, VP2850; lane 6, VP1061; M, DNA molecular marker (DL2000). (C1-C6) SDS-PAGE of pET-omp expression in Escherichia coli BL21 (DE3); c, control. (D1-D6) SDS-PAGE analysis of purified recombinant OM proteins; a, expression strains; b, purified proteins. (C,D) 1, VPA1435; 2, VP0425; 3, VP0764; 4, VPA1186; 5, VP2850; 6, VP1061. (E) Validation of antibody specificity. Fraction of V. parahaemolyticus cells was isolated in 1-DE gels and transferred onto the NC membrane. Anti-VPA1435, VP0425, VP0764, VPA1186, VP2850, and VP1061 were used as the primary antibodies.
against infections caused by Gram-negative bacteria. The combined approach was used not only in the identification of antigenically similar protein spots among different bacterial
species using heterophilic antibodies in vitro but also in the investigation of broad cross-protective activities of these shared proteins in vivo. The specificity of the immunological recogniJournal of Proteome Research • Vol. 9, No. 5, 2010 2579
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Table 4. Passive Immune Protection from Bacterial Challenge in Crucian Carp Model V. parahaemolyticus immunogen
nos.
ADRa (%)
RPSa (%)
VPA1435 VP0425 VP0764 VPA1186 VP2850 VP1061 control
20 20 20 20 20 20 20
45 70 50 60 25 30 90
50.0b 22.3 44.5b 33.4c 72.3b 66.7b
a ADR, accumulating death rates; RPS, relative percent survival. RPS was calculated as RPS ) 1 - (% mortality of vaccinated group/% mortality of control group) × 100. b P < 0.01. c P < 0.05.
Table 5. Active Immune Protection from Bacterial Challenge in Crucian Carp Model V. parahaemolyticus immunogen
agglutinative titer
nos.
ADRa (%)
RPSa (%)
VPA1435 VP0764 VPA1186 VP2850 VP1061 control
1:400 1:200 1:200 1:200 1:200 1:8
20 20 20 20 20 20
40.0 50.0 50.0 15.0 30.0 85.0
52.9b 41.2b 41.2b 82.4c 64.8c
a ADR, accumulating death rates; RPS, relative percent survival. RPS was calculated as RPS ) 1 - (% mortality of vaccinated group/% mortality of control group) × 100. b P < 0.05. c P < 0.01.
tion was verified in this study. Differential detection of the four spots of VPA1186 by homologous and heterogeneous antisera suggested the diversified reactivity of the protein isoforms with these antibodies and the accuracy of antibody recognition to the isoforms of the antigen, as well. As a result, VP1061 (Pal) and VP2850 were identified to be cross-reactive OM proteins and showed significant protective abilities in fish and mouse models. Thus, these two OM proteins could be polyvalent vaccine candidates against Gram-negative bacterial infections. In addition to identification of the broad cross-reactive immunogens, we are also interested in the characteristic features of the broad cross-reactive immunogens. The potential reactive activity of cross-recognition was predicated using bioinformatics analysis. The bioinformatics results demonstrated the importance of homologous proteins as broad crossreactive immunogens. In our previous study, V. alginolyticus VA0764 (OmpA) and VA1061 (Pal) were found to have broad cross-protective activity.12 In the present study, we character-
ized the broad cross-protective features of V. parahaemolyticus VP1061 and VP2850. All of these three OM proteins, VP0764/ VA0764, VP1061/VA1061, and VP2850, are highly conserved in Gram-negative bacteria. On the other hand, homologous epitopes were found in both B and T cells in our bioinformatics analysis. Thus, broad cross-protective vaccine candidates may be highly conserved proteins and cell immunity may contribute to their broad cross-protective activity. VP1061 and VP2850 are annotated as OM proteins in the NCBI database. Information regarding the role of V. parahaemolyticus VP1061 and VP2850 is not available. However, the studies on the function and immunity of other bacterial Pal and their homologues have been reported. These reports indicated that Pal was essential for bacterial survival and pathogenesis, although its role in virulence had not been clearly defined.26 E. coli K12 CH202 (pRC2) Pal released into the blood contributed to bacterial virulence and inflammation27 and induced cardiomyocyte dysfunction via the TLR2/MyD88 signaling cascade in Gram-negative sepsis.28 This protein and its homologues were potential vaccine candidates of V. alginolyticus Haemophilus influenzae, and Legionella pneumophila.2,29-32 Unlike VP1061, little is known about the possible role of VP2850 in bacterial immunization. To our knowledge, the present study is the first report of possible roles of this protein as a vaccine candidate. Interestingly, both VP2850 and VP1061 could be regulated by host products. Like VP0764 and VPA1186, OmpA could induce significantly protective action against V. parahaemolyticus infection. A line of evidence has indicated that OmpA is also essential for bacterial survival and pathogenesis.33,34 In addition, OmpA has demonstrated its functional potential as a broad cross-protective and species-specific vaccine candidate.12,35-38 Pasteurella multocida recombinant OmpA induced strong but nonprotective and deleterious Th2-type immune response in mice.39 In summary, these broad crossprotective vaccine candidates may play important roles in the control of bacterial survival and pathogenesis. V. parahaemolyticus has four OmpA orthologs, VP0764, VPA1186, VP0636, and VPA0248. These proteins have no distinct homology based on full ORF (open reading frame) amino acid sequences. Their sequence similarities are 60% (VPA0248 vs VPA1186), 43.4% (VPA1186 vsVP0764), 42.4% (VPA0248 vsVP0764), 16.8% (VP0636 vs VP0764), 15.2% (VP0636 vs VPA0248), and 14.7% (VPA1186 vs VP0636). They also have no significant homologous relation with Pasteurella multocida and Klebsiella pneumoniae. However, their OmpA domains are highly conserved, and the protein is antigenically cross-reactive among Gram-negative bacteria. For example, the antiserum toward E.
Table 6. Cross Immune Protection from Bacterial Challenge in Crucian Carp and Mouse Models V. alginolyticus a
A. hydrophila a
a
P. fluorescens a
RPS (%)
a
ADR (%)
RPSa (%)
immunogen
nos.
ADR (%)
VP2850 VP1061 control
15 25 20
13.3 20.0 80.0
in crucian carp model 83.4b 33.3 75.0b 12.0 100.0
66.7b 88.0b
6.7 8.0 95.0
92.9b 91.6b
VP2850 VP1061 control
20 19 20
15 31.5 75.0
in mouse model 80.0b 20 58.0b 26.3 75.0
73.4b 64.9b
30 42.1 95.0
68.4b 55.7b
RPS (%)
ADR (%)
a ADR, accumulating death rates; RPS, relative percent survival. RPS was calculated as RPS ) 1 - (% mortality of vaccinated group/% mortality of control group) × 100. b P < 0.01.
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Polyvalent Vaccine Candidates
Figure 4. Construction of phylogenetic trees and blast analysis of helix epitopes. A phylogenetic tree with bootstrap values based on the full-length protein sequences of VP1061 (A) and VP2850 (B), and the conserved OmpA domain (C). The tree was constructed using a neighbor-joining method. Blast analysis of helix epitopes of VA1061 positions 10-18 (D) and 88-99 (E), and VP2850 positions 81-91 (F) and 3-11 (G) as primers. Table 7. Bioinformatic Analysis of VP1061 and VP2805 Epitopes
VP1061
VP2850
positions
amino acid sequence
structure
score
predicted type
10-18 133-141 88-99 153-166 43-51 165-176 140-149 81-91 3-11
LLIALPVMA YLQALGVQA MLAAHASYLSKN KPLLLGQSDEVYAK ETTVATPID IDGVQIGLLNCA MADVGLASIS NFGLFFGAAKV IKMKKLMTS
helix coil helix coil coil coil/strand strand strand/helix helix
30 27 1.103/34 1.102 1.087 1.114 1.107 1.079 33
T cell T cell B/T cells B cell B cell B cell B cell B cell T cell
coli K12 OmpA could react with the OmpA of other bacterial pathogens, and the antiserum against V. cholerae OmpA could
recognize E. coli K12 OmpA.40,41 These results suggest that the sequences besides the conserved OmpA domain play a role in Journal of Proteome Research • Vol. 9, No. 5, 2010 2581
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Figure 5. Effects of naı¨ve crucian carp plasma and hyper-immune rabbit sera on the expression of VP1061 and VP2850. V. parahaemolyticus was separately incubated with these sera for 15 min, and then the expression levels of proteins VP1061 and VP2850 were detected using Western blotting. (A,B) Naı¨ve crucian carp; (C,D) hyper-immune rabbit sera.
immune protection to some extent. Our previous report indicated that V. alginolyticus VA0764 was a potential vaccine candidate but not VPA1186 and VPA0248.12 Here we demonstrated the vaccination potential of VP0764 and VPA1186, but the protective ability of the two proteins was weaker than that of VP2850 and VP1061. In addition, differential recognitions to VPA1186 (OmpA) by different species of bacterial antisera were also observed in the present study, which was consistent with the previous report that E. coli OmpA showed a diversified behavior in a 1-DE study.42 Thus, broad cross-protective vaccine candidates should be comparatively selected from these homologous proteins. Indeed, a parallel investigation of the four OmpA orthologs and their homologues is required for a better understanding of their characteristic features. In summary, the antigenically similar proteins that could potentially serve as broad cross-protective vaccine candidates show different protective abilities. The present study indicates that the host response of these bacterial strains to the heterophilic antigens is largely related to the type of bacterial species. This can be attributed to the competence of these heterophilic antigens in stimulating the host. An immunogen is dominant in a bacterium, but its antigenically similar protein in another bacterium may only elicit weak or no response. In this regard, the dominant immunogens obtained from heterogeneous antiserum-based immunoproteomics using a bacterium as antigen do not include all antigenically similar proteins existent in other bacteria. For example, VP2850 was not recognized by homologous antiserum. The cross-immunogenicity proteins from V. alginolyticus screened by heterogeneous antisera in our previous report are not equal to those from V. parahaemolyticus determined in the present study.12 Thus, heterogeneous antiserum-based immunoproteomics analysis of different serotypes, species, genera, and families of bacteria could be used for the establishment of diversity maps between these different bacteria and antisera. The diversity maps would provide valuable information for the identification of broad cross2582
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protective vaccine candidates. In summary, not all broad crossprotective immunogens are dominant antigens in the bacteria tested. The modified immunoproteomics approaches developed and utilized in this study could also be used to identify differentiation biomarkers of bacteria.43 Homogeneous antiserum-based immunoproteomics may identify bacterial immunogenic proteins, but it cannot differentiate cross-reactive immunogens from immunogenic proteins. Thus, only parallel investigations of homologous and heterogeneous antiserumbased immunoproteomics can effectively identify bacterial serotype-, species-, genus-, and family-specific biomarkers from antigenically similar proteins of the bacteria.
Acknowledgment. This work was sponsored by grants from “973” project (2006CB101807), NSFC projects (30530610, 40876076), and the foundation of Guangdong for Natural Sciences (7117645). References (1) von Bernuth, H.; Picard, C.; Jin, Z. B.; Pankla, R.; Xiao, H. Pyogenic bacterial infections in humans with MyD88 deficiency. Science 2008, 321, 691–696. (2) Tian, Y.; Wang, Q. Y.; Liu, Q.; Ma, Y.; Cao, X. D.; Zhang, Y. X. Role of RpoS in stress survival, synthesis of extracellular autoinducer 2, and virulence in Vibrio alginolyticus. Arch Microbiol. 2008, 190, 585–594. (3) Jagusztyn-Krynicka, E. K.; Laniewski, P.; Wyszynska, A. Update on Campylobacter jejuni vaccine development for preventing human campylobacteriosis. Expert Rev. Vaccines 2009, 8, 625–645. (4) Maione, D.; Margarit, I.; Rinaudo, C. D.; Masignani, V.; Mora, M.; et al. Identification of a universal group B Streptococcus vaccine by multiple genome screen. Science 2005, 309, 148–150. (5) Troncoso, G.; Sanchez, S.; Moreda, M.; Criado, M. T.; Ferreiros, C. M. Antigenic cross-reactivity between outer membrane proteins of Neisseria meningitidis and commensal Neisseria species. FEMS Immunol. Med. Microbiol. 2000, 27, 103–109. (6) Collado, R.; Fouz, B.; Sanjuan, E.; Amaro, C. Effectiveness of different vaccine formulations against vibriosis caused by Vibrio vulnificus serovar E (biotype 2) in European eels Anguilla anguilla. Dis. Aquat. Org. 2000, 43, 91–101.
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