Protein microarray for profiling antibody responses to Yersinia pestis live vaccine.

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.

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INFECTION AND IMMUNITY, June 2005, p. 3734–3739
0019-9567/05/$08.00?0doi:10.1128/IAI.73.6.3734–3739.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 6
NOTES
Protein Microarray for Profiling Antibody Responses to
Yersinia pestis Live Vaccine
Bei Li,1† Lingxiao Jiang,2† Qifeng Song,3,4† Junxin Yang,1Zeliang Chen,1Zhaobiao Guo,1
Dongsheng Zhou,1Zongmin Du,1Yajun Song,1Jin Wang,1Hongxia Wang,1
Shouyi Yu,2Jian Wang,3and Ruifu Yang1*
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,
China1; The South Medical University, Guangzhou, China2; Beijing Genomics Institute, Chinese Academy
of Sciences, Beijing 100101, China3; and Graduate school of Chinese academy
of Sciences, Beijing 100039, China4
Received 20 October 2004/Returned for modification 24 November 2004/Accepted 6 January 2005
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.
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 re-
duced 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 be-
side 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 opportuni-
ties for vaccine design. The microarray immobilized with mul-
tiple antigens, using a simple fluorescence analysis, allows
high-throughput parallel detection and quantification of mul-
tiple specific antibodies in a miniaturized, low-sample-con-
sumption 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, pro-
viding 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 elec-
trophoresis analysis, 172 genes were successfully expressed in
Escherichia coli. The expressed proteins were subjected to pu-
rification in a 96-well format using Ni-NAT agarose (QIA-
GEN) 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 accept-
able for further analysis (26). After systematic optimization of
purification conditions, 149 purified proteins were obtained.
Western blotting was further conducted to examine 13 arbi-
trarily selected proteins, and the results confirmed that each
purified protein gave a band with the expected size. The con-
centration of these 13 proteins was determined using a BCA
protein assay reagent kit (Pierce) and fell in the range of 100
to 200 ?g/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.
* Corresponding author. Mailing address: 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. Phone: 86-10-66948594.
yangrf@nic.bmi.ac.cn.
† B.L, L.J., and Q.S. contributed equally to this work.
Fax:86-10-83820748. E-mail:
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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 antibod-
ies 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 ? 108EV76 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 ? 109booster 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 ?l 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 In-
struments). 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 nor-
malize the fluorescence signal of each spot. The negative val-
ues 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 con-
firmed in this study (data not shown), the averaged fluores-
cence intensity is considered as an indicator of the concentra-
tion 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 in-
duced in the immunized rabbits for the 149 Y. pestis proteins on
the protein chip. The immunized rabbits made antibody re-
sponses 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 ex-
pressed 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 pro-
teins 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 pro-
teins. Y. pestis strains typically carry three virulence plasmids,
i.e., pCD1, pPCP1, and pMT1. Seventy plasmid-encoding pro-
teins with presumed virulence-related functions were included
in the microarray analysis, and antibody responses to 26 pro-
teins 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 combi-
nation (1, 16). Our results confirm that even in the earlier
stages of plague infection, both F1 and V constitute the im-
munodominant 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 previ-
ous 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 ob-
served 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 recombi-
nant 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 data-
bases was found to induce antibody response. Detection of
antibody confirms that they are indeed accessible to the hu-
moral response system.
Evaluation of the immunogenicity of chromosomal proteins.
A total of 79 chromosomal proteins including the proven vir-
ulence factors, putative adhesins/invasins, outer membrane
VOL. 73, 2005NOTES3735

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FIG. 1. Profiles of serum antibodies against different proteins. The fluorescence values were normalized, logarithm transformed (base 2), and
viewed with TreeView. Genes are listed at right, while the time points of immunization and identification numbers of rabbits tested are listed at
the top. The color key shows the normalized absolute fluorescence values. The genes were designated with the CO92 gene definition (e.g.,
YPO0302).
3736 NOTESINFECT. IMMUN.

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proteins, insecticidal toxins, and genomic island-related pro-
teins 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 pre-
immunized 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 ad-
hesins/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 Enterobacteri-
aceae, and involved in bacterial virulence and growth (20).
OmpA appears as a new pathogen-associated molecular pat-
tern that interacts with antigen-presenting cells, suggesting that
TABLE 1. Proteins that induced antibody response in EV76-immunized rabbitsa
Protein
characteristic
Gene ID in
CO92
Protein FunctionCross-reaction
Plasmid encodingYPMT1.84?
YPCD1.31c?
YPCD1.51?
YPCD1.54
YPCD1.55
YPCD1.61
YPCD1.32c?
YPCD1.28?
YPCD1.48?
YPCD1.05c
YPMT1.12c
YPMT1.23c
YPMT1.24c
YPMT1.25Ac
YPMT1.34
YPMT1.42Ac
YPMT1.45c
YPMT1.48c
YPMT1.55c
YPMT1.75c
YPMT1.76
YPMT1.76A
YPMT1.86A
YPMT1.86c
YPCD1.08c
YPPCP1.06
Caf1
LcrV
YscB
YscE
YscF
YscL
LcrG
YopD
VirG
SycE
F1 capsule antigen
V antigen
Type III secretion apparatus component
Type III secretion apparatus component
Type III secretion apparatus component
Type III secretion apparatus component
Yop regulator
Yop negative regulation/targeting component
Targeting protein of the YscC complex
YopE chaperone
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Yes
No
Yes
No
Yes
Yes
Yes
No
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
No
Yes
Yes
No
Yes
ChromosomalYPO1303?
YPO0687
YPO1435?
YPO2394?
YPO3319?
YPO3382
YPO3674
YPO0388
YPO2090?
YPO2093
YPO2096
YPO2108
YPO2109
YPO2113?
YPO2118?
YPO2123
YPO2125
YPO2126
YPO2131
YPO1088
YPO1089
YPO1094
YPO1095
YPO2313
PsaA pH 6 antigen
Putative adherence protein
Outer membrane porin A protein
Major outer membrane lipoprotein
Catalase-peroxidase
Global stress requirement protein
Putative insecticidal toxin
Conserved hypothetical protein
Putative phage protein
Putative phage protein
Hypothetical phage protein
Hypothetical phage protein
Hypothetical phage protein
Hypothetical phage protein
Hypothetical phage protein
Putative phage minor tail protein
Putative phage-regulatory protein
Putative phage protein
Putative phage host specificity protein
Putative DNA-binding prophage protein
Putative regulatory prophage protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
No
No
Yes
No
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Yes
Yes
OmpA
MlpA
KatY
GsrA
TccC
a?, antibody response similar to or stronger than that to the known strongly immunogenic V antigen. The proteins, whose signal/noise ratio was ?3.0 (i.e., spot signal
against background signal) in the preimmune serum of at least three rabbits, were considered to have cross-reaction with the preimmune sera.
VOL. 73, 2005 NOTES3737

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the immune system has acquired the ability to recognize this
type of protein (20). MlpA is a major outer membrane lipopro-
tein 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 abun-
dance after growth in vitro at 37°C but not at 26°C. KatY
(antigen 5) with catalase-peroxidase activity is thought to me-
diate 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 se-
rodiagnostic markers. Unfortunately, there was cross-reaction
with the negative sera, although the three proteins encoded by
YPO0388, YPO2090, and YPO2093 were found to induce sig-
nificantantibodyresponses
YPO1087 to YPO1098 were present in only some Y. pseudo-
inthe immunizedrabbits.
tuberculosis strains but in all Y. pestis strains tested (6). Anti-
bodies to four proteins encoded by this locus (YPO1088,
YPO1089, YPO1094, and YPO1095) were detected in the im-
munized sera. Genes YPO2095 to YPO2135 constitute a 33-kb
chromosomal fragment that was absent from the biovar Micro-
tus 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, 109live cells of strain
91001 failed to cause any infectious symptoms in rabbits. The
most striking characteristic of strain 91001 is that 1.5 ? 107
cells challenging through the subcutaneous route caused nei-
ther 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 re-
sponse.
Sequence analysis predicted several genes, probably ac-
quired by horizontal gene transfer, to be homologues of insec-
ticidal 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 anti-
genic protein was expressed in the infected mammalian host.
Conclusions. Microarray profiling of the host immune re-
sponse to Y. pestis EV76 has identified immunodominant pro-
teins 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 anti-
TABLE 2. Evaluation of LCRS components as immunogenic and protective proteinsa
Protein Present in microarray
Immunogenic
Protective efficacy in bubonic/pneumonic model
(reference[s])
Previous study Present study
LcrV
YopD
YscF
VirG
YopN
YscO
YscP
TyeA
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
No
Bubonic and pneumonic—protective (22, 35)
Bubonic—partially protective (2)
Bubonic—not protective (17)
Bubonic—not protective (17)
Bubonic—not protective (2, 21)
Bubonic—not protective (17)
Bubonic—not protective (17)
Bubonic—not protective (17)
YopH
YopE
YopK
YopM
YpkA
YscJ
No
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
Yes
ND
ND
ND
ND
ND
ND
Bubonic—not protective (2)
Bubonic—not protective (2, 21)
Bubonic—not protective (2, 21)
Bubonic—not protective (2, 21)
Bubonic—delayed time to death (2)
Bubonic—not protective (17)
YscB
YscE
YscL
LcrG
SycE
Yes
Yes
Yes
Yes
Yes
ND
ND
ND
ND
ND
Yes
Yes
Yes
Yes
Yes
ND
ND
ND
ND
ND
aND, not determined. A total of 10 Yop proteins were actually subjected to cloning and expression. The genes yopB and yopM could not be successfully amplified
with Y. pestis 82009 DNA as a template. YopE, YopK, YopJ, and YopH were successfully expressed, but unfortunately, after being purified through 96-well format,
they could not pass the quality control process. Therefore, only YopD, YopN, and YopR were included in the final version of the protein microarrays.
3738NOTESINFECT. IMMUN.

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body 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.
We thank David A. Bastin for critically reading and carefully revis-
ing 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 Tech-
nology Research and Development Program of China (program 863,
no. 2004AA223110) and the National Natural Science Foundation of
China (no. 30430620).
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