Protein Microarray for Profiling Antibody Responses to Yersinia pestis Live Vaccine
http://www.100md.com
感染与免疫杂志 2005年第6期
Laboratory of Analytical Microbiology, National Center for Biomedical Analysis, Army Center for Microbial Detection and Research, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China
The South Medical University, Guangzhou, China
Beijing Genomics Institute, Chinese Academy of Sciences, Beijing 100101, China
Graduate school of Chinese academy of Sciences, Beijing 100039, China
ABSTRACT
A protein microarray representing 149 Yersinia pestis proteins was developed to profile antibody responses in EV76-immunized rabbits. Antibodies to 50 proteins were detected. There are 11 proteins besides F1 and V antigens to which the predominant antibody response occurred, and these proteins show promise for further evaluation as candidates for subunit vaccines and/or diagnostic antigens.
TEXT
Plague, one of the most dangerous diseases, is caused by Yersinia pestis. The increasing possibility of antibiotic-resistant Y. pestis strains means that a vaccine effective against bubonic and pneumonic plague is urgently needed (12, 14, 18). The current interest is in developing plague vaccines that consist of purified protein subunits, with improved protection and reduced side effects (25, 31, 34). The F1 or V single-subunit vaccine and the F1 plus V combination vaccine have been shown to provide effective protection against bubonic and pneumonic plague in animal models (1, 31, 34). There may still exist other Y. pestis antigens that provide protection. These novel vaccine candidates in conjunction with F1 and V can be developed as multicomponent subunit vaccines (21). It may improve protection against F1- and/or V-antigen mutant but virulent strains. Therefore, evaluation of Y. pestis proteins beside F1 and V for their efficacy in inducing specific antibody in the infected animals or human patients is urgently needed for plague vaccine development.
Genomic sequences of Y. pestis CO92 (27), KIM (9), and 91001 (30) have been released in the past 3 years. Decoding of whole-genome sequence provides unprecedented opportunities for vaccine design. The microarray immobilized with multiple antigens, using a simple fluorescence analysis, allows high-throughput parallel detection and quantification of multiple specific antibodies in a miniaturized, low-sample-consumption format. In this study, a microarray representing 149 Y. pestis proteins was developed to profile the serum antibodies of rabbits that were immunized with live plague vaccine, providing an overall picture of the immunogenicity of the proteins tested.
Construction of an antigen microarray representing 149 Y. pestis proteins. In our present work, 202 genes were selected for cloning and expression (additional information is available at http://bioinflab.org/journals/ruifu/supplementary_TableS1.pdf/). The digested PCR products of specific genes were cloned into expression plasmid vector pET-32a (Novagen), and recombinant plasmids were transformed into BL21(DE3) cells. According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, 172 genes were successfully expressed in Escherichia coli. The expressed proteins were subjected to purification in a 96-well format using Ni-NAT agarose (QIAGEN) according to Brauns' method with a few modifications (5). For quality control, the purified proteins were printed onto silylated glass slides (CEL) and incubated with Cy5-labeled antibody specific for the six-His tag. Only the proteins giving a signal-to-background ratio of 3.0 were thought to be acceptable for further analysis (26). After systematic optimization of purification conditions, 149 purified proteins were obtained. Western blotting was further conducted to examine 13 arbitrarily selected proteins, and the results confirmed that each purified protein gave a band with the expected size. The concentration of these 13 proteins was determined using a BCA protein assay reagent kit (Pierce) and fell in the range of 100 to 200 μg/ml. Then, the 149 proteins were printed in triplicate on the slides to fabricate the final version of the microarrays. Rabbit immunoglobulin G (IgG) was printed as a positive control, while cell lysate of E. coli BL21 transformed with PET-32a was used as a negative control. The printed slides were deposited at room temperature for at least 1 h and then stored at 4°C.
Microarray profiling of serum antibody response in the EV76-immunized rabbits. The microarray was used as a tool to screen for the relative amounts of the corresponding antibodies present in the sera of EV76-immunized rabbits. Overnight cultures of the live vaccine EV76 were used for preparation of bacterial suspension with physiological saline. Four rabbits of 2 to 2.5 kg received a primary subcutaneous immunization of 2.5 x 108 EV76 adsorbed to the complete Freund's adjuvant and 2 weeks later a second immunization of the same quantity with the incomplete Freund's adjuvant. On days 29, 36, 43, and 50, each animal received a 1 x 109 booster intravenous dose. Sera were collected before immunization and 1 day before each booster since the second booster. Before being used for microarray profiling, all the sera were incubated with lysate of E. coli BL21 carrying pET-32a for 2 h so as to eliminate antibodies against E. coli in the tested sera.
Microarrays used for serum profiling were blocked using bovine serum albumin-0.01 mM phosphate-buffered saline (PBS), pH 7.5, for 1 h at room temperature. Then, 200 μl of diluted serum (1:200) was incubated with them for another 1 h. After washing one time with PBS-Tween 20 and two times with PBS, the protein chips were incubated with Cy5 dye-labeled goat antirabbit IgG (1:1,000 dilution) generated by using a Cy5 antibody labeling kit (Amersham Biosciences) for 1 h. Slides were washed as described above and imaged with an Axon 4100A scanner (Axon Instruments). Image and data analysis was performed using the GenePix pro 4.0 software (Axon Instruments). The fluorescence signal of each spot was calculated as the median fluorescence intensity subtracted from the local background median intensity. The spot signals for each protein in three replicated hybridizations were averaged. In order to reduce differences produced in the operation process, we used the fluorescence signal of rabbit IgG as the criterion to normalize the fluorescence signal of each spot. The negative values of spot intensity were set to zero to reflect that local background intensity is equal to spot signal. The normalized data sets were logarithm transformed (base 2) and displayed with TreeView software (10).
Since there was a good correlation between the fluorescence intensity and the IgG concentration captured by the printed antigen according to our previous study (7), which was confirmed in this study (data not shown), the averaged fluorescence intensity is considered as an indicator of the concentration of antibodies. Given that all the proteins were printed in roughly equal amounts, the technology afforded a screening strategy that was relatively unbiased in terms of the effect of protein concentration on sensitivity of detection. Figure 1 gives a schematic representation of the serum antibody profiles induced in the immunized rabbits for the 149 Y. pestis proteins on the protein chip. The immunized rabbits made antibody responses to 50 of the 149 proteins, and each antibody titer gave an increment tendency with the development of immunizing times (Fig. 1 and Table 1). However, 37 of these 50 proteins showed cross-reaction with the preimmunized sera. According to the fluorescence intensity-displaying relative concentration of specific antibody (IgG) presented in the immune sera, a group of 12 proteins (labeled with an asterisk in Table 1) to which antibody response appeared to be similar to or stronger than that to the known strongly immunogenic V antigen can be identified. Notwithstanding, our studies show that 12 proteins described in Table 1 are immunodominant when rabbits are immunized with live EV76 and suggest these proteins are expressed during the course of a plague infection.
The remaining proteins tested in our study were unable to evoke antibody signals for several reasons. First, some of these proteins are putative or hypothetical, and actually they may not be expressed by the bacterium. Second, some proteins are not expressed under our culturing and immunizing conditions. Third, the amount of proteins expressed may be too low to induce an immune response. Fourth, the immunogenicity of these proteins might be too weak to contact antigen-presenting cells to raise antibodies, or this breed of rabbit may lack the genetic ability to respond to certain epitopes. Finally, the proteins printed on slides are recombinant and therefore differ in conformation from their naive forms, so the corresponding antibodies cannot efficiently recognize them.
Evaluation of the immunogenicity of plasmid-encoding proteins. Y. pestis strains typically carry three virulence plasmids, i.e., pCD1, pPCP1, and pMT1. Seventy plasmid-encoding proteins with presumed virulence-related functions were included in the microarray analysis, and antibody responses to 26 proteins were recorded (Table 1). As show in Fig. 1, antibody response to F1 antigen was the most dramatic in terms of both speed and magnitude of the increment in antibody titers, while V antigen was assigned to a group of 13 proteins with very strong antigenicity (see above). F1 and V, when tested as vaccine antigens, provide a high level of protection against experimental plague (31, 34), especially when used in combination (1, 16). Our results confirm that even in the earlier stages of plague infection, both F1 and V constitute the immunodominant antigens in Y. pestis. As a Y. pestis-specific, strongly antigenic protein, F1 is the ideal candidate for plague serodiagnosis (29).
Plasmid pCD1 harbors a gene cluster named LCRS (low calcium response stimulon) that encodes a type III secretion system (4). Through the type III secretion system, Y. pestis injects the YOP effector proteins into the cytosol of eukaryotic cells when docking at the surface of the host cell, mediating resistance to phagocytosis (8). The antigenicity and protective efficacy of a portion of LCRS components have been tested in previous studies; beside V antigen, only YopD was found to provide partial protection against nonencapsulated Y. pestis subcutaneous challenge (2, 3, 21). Twenty-three of the 43 LCRS proteins were included in the microarray analysis. Table 2 shows the comparison of our results and those of the previous studies. Five new proteins (YscB, SycE, YscE, LcrG, and YscL) were found to be immunogenic in the current work. No antibody response to YopN, YscO, YscP, or TyeA was observed in our study, which is totally opposite to the previous results (17, 21). The discrepancy may be due to the fact that we used the live attenuated vaccine but not the purified recombinant proteins or the fully virulent strain for immunization.
Interestingly, a group of proteins that have no homology with any known or hypothetical proteins currently in the databases was found to induce antibody response. Detection of antibody confirms that they are indeed accessible to the humoral response system.
Evaluation of the immunogenicity of chromosomal proteins. A total of 79 chromosomal proteins including the proven virulence factors, putative adhesins/invasins, outer membrane proteins, insecticidal toxins, and genomic island-related proteins were included in the microarray analysis; 24 of them were found to induce an antibody response (Table 1). pH 6 antigen, whose expression is induced in infected macrophage in acidic environment (23), can bind to glycosphingolipids that can be found on a range of host cell types (28) and apolipoprotein B-containing lipoproteins in human plasma (24), and it was recently identified as an antiphagocytic factor (19). In all preimmunized animals, we could not detect the specific antibody to this protein, but in the EV76-immunized rabbits at 42 days after immunization, the fluorescence intensity is increased to the level even higher than that of V antigen at the same time point. The microarray still included seven other putative adhesins/invasins, while only antibody to the protein encoded by YPO0687 was detected in the immune sera.
Outer membrane protein A (OmpA) is highly represented in the bacterial cell wall, conserved among the Enterobacteriaceae, and involved in bacterial virulence and growth (20). OmpA appears as a new pathogen-associated molecular pattern that interacts with antigen-presenting cells, suggesting that the immune system has acquired the ability to recognize this type of protein (20). MlpA is a major outer membrane lipoprotein that contributes to the structural integrity of the outer membrane along with OmpA (32). Both OmpA and MlpA represent a new type of candidate for vaccine design. In our study, OmpA (YPO1435) and MlpA (YPO2394) induced a strong increment of antibody titers with a pattern similar to that of V antigen.
Both KatY (13) and HtrA (33) are produced in great abundance after growth in vitro at 37°C but not at 26°C. KatY (antigen 5) with catalase-peroxidase activity is thought to mediate resistance to killing by professional phagocytes (13). Our results showed that the EV76-immunized rabbits made a strong antibody response to KatY with a pattern similar to that with V antigen. In contrast to the wild-type strain, the htrA mutant fails to grow at an elevated temperature of 39°C but shows only a small increase in sensitivity to oxidative stress and is only partially attenuated in the animal model (33). Antibody to HtrA was detected in the immunized rabbits.
Horizontal gene transfer entails the direct integration of genetic elements into the bacterial genome to form "genomic islands" with functions of increasing bacterial fitness (15). Three genomic islands (YPO0387 to YPO0397, YPO2087 to YPO2135, and YPO1087 to YPO1098) were tested in our present work because they appear to be newly acquired. YPO0387 to YPO0397 and YPO2087 to YPO2094 are uniquely conserved in Y. pestis (6, 37). If strongly antigenic, proteins encoded by this locus will have huge promise as serodiagnostic markers. Unfortunately, there was cross-reaction with the negative sera, although the three proteins encoded by YPO0388, YPO2090, and YPO2093 were found to induce significant antibody responses in the immunized rabbits. YPO1087 to YPO1098 were present in only some Y. pseudotuberculosis strains but in all Y. pestis strains tested (6). Antibodies to four proteins encoded by this locus (YPO1088, YPO1089, YPO1094, and YPO1095) were detected in the immunized sera. Genes YPO2095 to YPO2135 constitute a 33-kb chromosomal fragment that was absent from the biovar Microtus strains (30, 38). Y. pestis 91001, a member of the biovar Microtus strains, has a 50% lethal dose of 23.2 for mice by subcutaneous challenge; meanwhile, 109 live cells of strain 91001 failed to cause any infectious symptoms in rabbits. The most striking characteristic of strain 91001 is that 1.5 x 107 cells challenging through the subcutaneous route caused neither bubonic plague nor pneumonic plague in a volunteer trial (11). Microtus strains are supposed to be avirulent to humans, although they are highly lethal to mice, so this fragment likely contributes the ability to infect humans in fully virulent strains. Twenty-nine putative proteins encoded by this fragment were tested, and nine of them were found to induce antibody response.
Sequence analysis predicted several genes, probably acquired by horizontal gene transfer, to be homologues of insecticidal toxins (TcaA, TcaB, TcaC, TccC, viral enhancin, etc.) of insect pathogens, and they were implicated in the adaptation of Y. pestis to the flea life cycle (27, 36). This study showed that at least the TccC (YPO3674) homologue could induce the host antibody response, which clearly demonstrated that this antigenic protein was expressed in the infected mammalian host.
Conclusions. Microarray profiling of the host immune response to Y. pestis EV76 has identified immunodominant proteins of this live plague vaccine strain. Our results revealed a set of Y. pestis proteins to which the predominant antibody response occurred in immunized rabbits, raising a wealth of new candidates for us to further test as vaccines and diagnostic antigens. For example, proteins that can induce strong antibody response during immunization—such as those encoded by YPO2394, YPO1435, YPO2090, YPO2118, YPO3382, YPO3319, and YPCD1.48—are currently being investigated in our laboratory for their protective potency against bubonic and pneumonic plague in animal models.
ACKNOWLEDGMENTS
We thank David A. Bastin for critically reading and carefully revising the manuscript. We are grateful to Heng Zhu for very fruitful discussions and suggestions and to Haipan Zeng and Guozheng Liu for their technical assistance in microarray printing.
Financial supports for this work came from the National High Technology Research and Development Program of China (program 863, no. 2004AA223110) and the National Natural Science Foundation of China (no. 30430620).
B.L, L.J., and Q.S. contributed equally to this work.
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The South Medical University, Guangzhou, China
Beijing Genomics Institute, Chinese Academy of Sciences, Beijing 100101, China
Graduate school of Chinese academy of Sciences, Beijing 100039, China
ABSTRACT
A protein microarray representing 149 Yersinia pestis proteins was developed to profile antibody responses in EV76-immunized rabbits. Antibodies to 50 proteins were detected. There are 11 proteins besides F1 and V antigens to which the predominant antibody response occurred, and these proteins show promise for further evaluation as candidates for subunit vaccines and/or diagnostic antigens.
TEXT
Plague, one of the most dangerous diseases, is caused by Yersinia pestis. The increasing possibility of antibiotic-resistant Y. pestis strains means that a vaccine effective against bubonic and pneumonic plague is urgently needed (12, 14, 18). The current interest is in developing plague vaccines that consist of purified protein subunits, with improved protection and reduced side effects (25, 31, 34). The F1 or V single-subunit vaccine and the F1 plus V combination vaccine have been shown to provide effective protection against bubonic and pneumonic plague in animal models (1, 31, 34). There may still exist other Y. pestis antigens that provide protection. These novel vaccine candidates in conjunction with F1 and V can be developed as multicomponent subunit vaccines (21). It may improve protection against F1- and/or V-antigen mutant but virulent strains. Therefore, evaluation of Y. pestis proteins beside F1 and V for their efficacy in inducing specific antibody in the infected animals or human patients is urgently needed for plague vaccine development.
Genomic sequences of Y. pestis CO92 (27), KIM (9), and 91001 (30) have been released in the past 3 years. Decoding of whole-genome sequence provides unprecedented opportunities for vaccine design. The microarray immobilized with multiple antigens, using a simple fluorescence analysis, allows high-throughput parallel detection and quantification of multiple specific antibodies in a miniaturized, low-sample-consumption format. In this study, a microarray representing 149 Y. pestis proteins was developed to profile the serum antibodies of rabbits that were immunized with live plague vaccine, providing an overall picture of the immunogenicity of the proteins tested.
Construction of an antigen microarray representing 149 Y. pestis proteins. In our present work, 202 genes were selected for cloning and expression (additional information is available at http://bioinflab.org/journals/ruifu/supplementary_TableS1.pdf/). The digested PCR products of specific genes were cloned into expression plasmid vector pET-32a (Novagen), and recombinant plasmids were transformed into BL21(DE3) cells. According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, 172 genes were successfully expressed in Escherichia coli. The expressed proteins were subjected to purification in a 96-well format using Ni-NAT agarose (QIAGEN) according to Brauns' method with a few modifications (5). For quality control, the purified proteins were printed onto silylated glass slides (CEL) and incubated with Cy5-labeled antibody specific for the six-His tag. Only the proteins giving a signal-to-background ratio of 3.0 were thought to be acceptable for further analysis (26). After systematic optimization of purification conditions, 149 purified proteins were obtained. Western blotting was further conducted to examine 13 arbitrarily selected proteins, and the results confirmed that each purified protein gave a band with the expected size. The concentration of these 13 proteins was determined using a BCA protein assay reagent kit (Pierce) and fell in the range of 100 to 200 μg/ml. Then, the 149 proteins were printed in triplicate on the slides to fabricate the final version of the microarrays. Rabbit immunoglobulin G (IgG) was printed as a positive control, while cell lysate of E. coli BL21 transformed with PET-32a was used as a negative control. The printed slides were deposited at room temperature for at least 1 h and then stored at 4°C.
Microarray profiling of serum antibody response in the EV76-immunized rabbits. The microarray was used as a tool to screen for the relative amounts of the corresponding antibodies present in the sera of EV76-immunized rabbits. Overnight cultures of the live vaccine EV76 were used for preparation of bacterial suspension with physiological saline. Four rabbits of 2 to 2.5 kg received a primary subcutaneous immunization of 2.5 x 108 EV76 adsorbed to the complete Freund's adjuvant and 2 weeks later a second immunization of the same quantity with the incomplete Freund's adjuvant. On days 29, 36, 43, and 50, each animal received a 1 x 109 booster intravenous dose. Sera were collected before immunization and 1 day before each booster since the second booster. Before being used for microarray profiling, all the sera were incubated with lysate of E. coli BL21 carrying pET-32a for 2 h so as to eliminate antibodies against E. coli in the tested sera.
Microarrays used for serum profiling were blocked using bovine serum albumin-0.01 mM phosphate-buffered saline (PBS), pH 7.5, for 1 h at room temperature. Then, 200 μl of diluted serum (1:200) was incubated with them for another 1 h. After washing one time with PBS-Tween 20 and two times with PBS, the protein chips were incubated with Cy5 dye-labeled goat antirabbit IgG (1:1,000 dilution) generated by using a Cy5 antibody labeling kit (Amersham Biosciences) for 1 h. Slides were washed as described above and imaged with an Axon 4100A scanner (Axon Instruments). Image and data analysis was performed using the GenePix pro 4.0 software (Axon Instruments). The fluorescence signal of each spot was calculated as the median fluorescence intensity subtracted from the local background median intensity. The spot signals for each protein in three replicated hybridizations were averaged. In order to reduce differences produced in the operation process, we used the fluorescence signal of rabbit IgG as the criterion to normalize the fluorescence signal of each spot. The negative values of spot intensity were set to zero to reflect that local background intensity is equal to spot signal. The normalized data sets were logarithm transformed (base 2) and displayed with TreeView software (10).
Since there was a good correlation between the fluorescence intensity and the IgG concentration captured by the printed antigen according to our previous study (7), which was confirmed in this study (data not shown), the averaged fluorescence intensity is considered as an indicator of the concentration of antibodies. Given that all the proteins were printed in roughly equal amounts, the technology afforded a screening strategy that was relatively unbiased in terms of the effect of protein concentration on sensitivity of detection. Figure 1 gives a schematic representation of the serum antibody profiles induced in the immunized rabbits for the 149 Y. pestis proteins on the protein chip. The immunized rabbits made antibody responses to 50 of the 149 proteins, and each antibody titer gave an increment tendency with the development of immunizing times (Fig. 1 and Table 1). However, 37 of these 50 proteins showed cross-reaction with the preimmunized sera. According to the fluorescence intensity-displaying relative concentration of specific antibody (IgG) presented in the immune sera, a group of 12 proteins (labeled with an asterisk in Table 1) to which antibody response appeared to be similar to or stronger than that to the known strongly immunogenic V antigen can be identified. Notwithstanding, our studies show that 12 proteins described in Table 1 are immunodominant when rabbits are immunized with live EV76 and suggest these proteins are expressed during the course of a plague infection.
The remaining proteins tested in our study were unable to evoke antibody signals for several reasons. First, some of these proteins are putative or hypothetical, and actually they may not be expressed by the bacterium. Second, some proteins are not expressed under our culturing and immunizing conditions. Third, the amount of proteins expressed may be too low to induce an immune response. Fourth, the immunogenicity of these proteins might be too weak to contact antigen-presenting cells to raise antibodies, or this breed of rabbit may lack the genetic ability to respond to certain epitopes. Finally, the proteins printed on slides are recombinant and therefore differ in conformation from their naive forms, so the corresponding antibodies cannot efficiently recognize them.
Evaluation of the immunogenicity of plasmid-encoding proteins. Y. pestis strains typically carry three virulence plasmids, i.e., pCD1, pPCP1, and pMT1. Seventy plasmid-encoding proteins with presumed virulence-related functions were included in the microarray analysis, and antibody responses to 26 proteins were recorded (Table 1). As show in Fig. 1, antibody response to F1 antigen was the most dramatic in terms of both speed and magnitude of the increment in antibody titers, while V antigen was assigned to a group of 13 proteins with very strong antigenicity (see above). F1 and V, when tested as vaccine antigens, provide a high level of protection against experimental plague (31, 34), especially when used in combination (1, 16). Our results confirm that even in the earlier stages of plague infection, both F1 and V constitute the immunodominant antigens in Y. pestis. As a Y. pestis-specific, strongly antigenic protein, F1 is the ideal candidate for plague serodiagnosis (29).
Plasmid pCD1 harbors a gene cluster named LCRS (low calcium response stimulon) that encodes a type III secretion system (4). Through the type III secretion system, Y. pestis injects the YOP effector proteins into the cytosol of eukaryotic cells when docking at the surface of the host cell, mediating resistance to phagocytosis (8). The antigenicity and protective efficacy of a portion of LCRS components have been tested in previous studies; beside V antigen, only YopD was found to provide partial protection against nonencapsulated Y. pestis subcutaneous challenge (2, 3, 21). Twenty-three of the 43 LCRS proteins were included in the microarray analysis. Table 2 shows the comparison of our results and those of the previous studies. Five new proteins (YscB, SycE, YscE, LcrG, and YscL) were found to be immunogenic in the current work. No antibody response to YopN, YscO, YscP, or TyeA was observed in our study, which is totally opposite to the previous results (17, 21). The discrepancy may be due to the fact that we used the live attenuated vaccine but not the purified recombinant proteins or the fully virulent strain for immunization.
Interestingly, a group of proteins that have no homology with any known or hypothetical proteins currently in the databases was found to induce antibody response. Detection of antibody confirms that they are indeed accessible to the humoral response system.
Evaluation of the immunogenicity of chromosomal proteins. A total of 79 chromosomal proteins including the proven virulence factors, putative adhesins/invasins, outer membrane proteins, insecticidal toxins, and genomic island-related proteins were included in the microarray analysis; 24 of them were found to induce an antibody response (Table 1). pH 6 antigen, whose expression is induced in infected macrophage in acidic environment (23), can bind to glycosphingolipids that can be found on a range of host cell types (28) and apolipoprotein B-containing lipoproteins in human plasma (24), and it was recently identified as an antiphagocytic factor (19). In all preimmunized animals, we could not detect the specific antibody to this protein, but in the EV76-immunized rabbits at 42 days after immunization, the fluorescence intensity is increased to the level even higher than that of V antigen at the same time point. The microarray still included seven other putative adhesins/invasins, while only antibody to the protein encoded by YPO0687 was detected in the immune sera.
Outer membrane protein A (OmpA) is highly represented in the bacterial cell wall, conserved among the Enterobacteriaceae, and involved in bacterial virulence and growth (20). OmpA appears as a new pathogen-associated molecular pattern that interacts with antigen-presenting cells, suggesting that the immune system has acquired the ability to recognize this type of protein (20). MlpA is a major outer membrane lipoprotein that contributes to the structural integrity of the outer membrane along with OmpA (32). Both OmpA and MlpA represent a new type of candidate for vaccine design. In our study, OmpA (YPO1435) and MlpA (YPO2394) induced a strong increment of antibody titers with a pattern similar to that of V antigen.
Both KatY (13) and HtrA (33) are produced in great abundance after growth in vitro at 37°C but not at 26°C. KatY (antigen 5) with catalase-peroxidase activity is thought to mediate resistance to killing by professional phagocytes (13). Our results showed that the EV76-immunized rabbits made a strong antibody response to KatY with a pattern similar to that with V antigen. In contrast to the wild-type strain, the htrA mutant fails to grow at an elevated temperature of 39°C but shows only a small increase in sensitivity to oxidative stress and is only partially attenuated in the animal model (33). Antibody to HtrA was detected in the immunized rabbits.
Horizontal gene transfer entails the direct integration of genetic elements into the bacterial genome to form "genomic islands" with functions of increasing bacterial fitness (15). Three genomic islands (YPO0387 to YPO0397, YPO2087 to YPO2135, and YPO1087 to YPO1098) were tested in our present work because they appear to be newly acquired. YPO0387 to YPO0397 and YPO2087 to YPO2094 are uniquely conserved in Y. pestis (6, 37). If strongly antigenic, proteins encoded by this locus will have huge promise as serodiagnostic markers. Unfortunately, there was cross-reaction with the negative sera, although the three proteins encoded by YPO0388, YPO2090, and YPO2093 were found to induce significant antibody responses in the immunized rabbits. YPO1087 to YPO1098 were present in only some Y. pseudotuberculosis strains but in all Y. pestis strains tested (6). Antibodies to four proteins encoded by this locus (YPO1088, YPO1089, YPO1094, and YPO1095) were detected in the immunized sera. Genes YPO2095 to YPO2135 constitute a 33-kb chromosomal fragment that was absent from the biovar Microtus strains (30, 38). Y. pestis 91001, a member of the biovar Microtus strains, has a 50% lethal dose of 23.2 for mice by subcutaneous challenge; meanwhile, 109 live cells of strain 91001 failed to cause any infectious symptoms in rabbits. The most striking characteristic of strain 91001 is that 1.5 x 107 cells challenging through the subcutaneous route caused neither bubonic plague nor pneumonic plague in a volunteer trial (11). Microtus strains are supposed to be avirulent to humans, although they are highly lethal to mice, so this fragment likely contributes the ability to infect humans in fully virulent strains. Twenty-nine putative proteins encoded by this fragment were tested, and nine of them were found to induce antibody response.
Sequence analysis predicted several genes, probably acquired by horizontal gene transfer, to be homologues of insecticidal toxins (TcaA, TcaB, TcaC, TccC, viral enhancin, etc.) of insect pathogens, and they were implicated in the adaptation of Y. pestis to the flea life cycle (27, 36). This study showed that at least the TccC (YPO3674) homologue could induce the host antibody response, which clearly demonstrated that this antigenic protein was expressed in the infected mammalian host.
Conclusions. Microarray profiling of the host immune response to Y. pestis EV76 has identified immunodominant proteins of this live plague vaccine strain. Our results revealed a set of Y. pestis proteins to which the predominant antibody response occurred in immunized rabbits, raising a wealth of new candidates for us to further test as vaccines and diagnostic antigens. For example, proteins that can induce strong antibody response during immunization—such as those encoded by YPO2394, YPO1435, YPO2090, YPO2118, YPO3382, YPO3319, and YPCD1.48—are currently being investigated in our laboratory for their protective potency against bubonic and pneumonic plague in animal models.
ACKNOWLEDGMENTS
We thank David A. Bastin for critically reading and carefully revising the manuscript. We are grateful to Heng Zhu for very fruitful discussions and suggestions and to Haipan Zeng and Guozheng Liu for their technical assistance in microarray printing.
Financial supports for this work came from the National High Technology Research and Development Program of China (program 863, no. 2004AA223110) and the National Natural Science Foundation of China (no. 30430620).
B.L, L.J., and Q.S. contributed equally to this work.
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