Antigenic Profiling of a Chlamydia trachomatis Gene-Expression Library
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MAJOR ARTICLE
Antigenic Profiling of a Chlamydia trachomatis
Gene-Expression Library
Frank Follmann,1 Anja Weinreich Olsen,1 Klaus Thorleif Jensen,1,a Paul Robert Hansen,2 Peter Andersen,1
and Michael Theisen1
1
Chlamydia Research, Department of Infectious Disease Immunology, Statens Serum Institut, and 2Bioorganic Chemistry Section, Department of
Natural Sciences, University of Copenhagen, Copenhagen, Denmark
The obligate intracellular bacterium Chlamydia trachomatis is the causative agent of sexually transmitted
chlamydia infections. A panel of 116 recombinant C. trachomatis proteins was evaluated comparatively to char-
acterize both cell-mediated and humoral immune responses in patients with confirmed C. trachomatis genital
infection. The antigens identified were categorized as being recognized exclusively by T cells (CT004, CT043,
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CT184, CT509, and CT611), B cells (CT082, CT089, CT322, CT396, and CT681), or both T cells and B cells (CT110
and CT443). This grouping of C. trachomatis antigens was correlated to their predicted cellular localization. The
comparative evaluation presented here indicates that T cell antigens are located in all bacterial compartments,
whereas antibody targets are mainly localized to the outer membrane (P ⴝ .0013). Overall, we have identified 5 T
cell antigens, 5 B cell antigens, and 2 T/B cell antigens that are potential components for a future chlamydia
vaccine.
Chlamydia trachomatis is an obligate intracellular bacte- [3]. Developing a vaccine against chlamydia highlights
ria and is the causative agent of the most common bac- the importance of knowing what constitutes a protective
terial sexually transmitted infection, with ⬎92 million immune response. Several studies in animal models
new cases per year worldwide [1]. The infection is often have all pointed to a central role for CD4⫹ Th1 cells and
asymptomatic, and the disease can persist over a pro- the cytokine interferon (IFN)–␥ [8 –10]. Studies in hu-
longed period of time [2, 3]. If left untreated, chlamydia mans have further emphasized the role played by CD4⫹
infection may lead to pelvic inflammatory disease, T cells. Female sex workers infected with HIV are more
which is a major cause of infertility among women [4]. susceptible to subsequent genital infection with C. tra-
C. trachomatis has also been implicated as a cofactor for chomatis than individuals not infected with HIV [5], and
HIV transmission and may confer an increased risk for IFN-␥ production by peripheral blood mononuclear
cervical squamous cell carcinoma [5–7]. On the basis of cells (PBMCs) stimulated with C. trachomatis Hsp60 is
serum reactivity, C. trachomatis can be divided into at associated with protection against reinfection [11]. The
least 18 serovars, of which D–K are responsible for the humoral branch of the immune system (IgG and IgA)
sexually transmitted disease. has a low effect on clearance of chlamydia in terms of
A chlamydia infection is effectively cured by antibiot- direct neutralization [12]. However, antibodies may as-
ics, but a vaccine-based prevention strategy is attractive sist cell-mediated responses through enhancing antigen
because of the high prevalence of asymptomatic cases uptake and presentation by antigen-presenting cells and
may also be involved in antibody-dependent cell cyto-
Received 10 August 2007; accepted 20 September 2007; electronically pub- toxicity [13]. Accordingly, antibodies have been shown
lished 20 February 2008. to play an important role in protection against reinfec-
Potential conflicts of interest: none reported.
a Present affiliation: Biotech Research and Innovation Center, University of tion [14, 15]. Thus, an optimal vaccine for chlamydia
Copenhagen, Copenhagen, Denmark. will probably combine antigenic targets for both cell-
Reprints or correspondence: Dr. Michael Theisen, Dept. of Infectious Disease
Immunology, Chlamydia Research, Statens Serum Institut, Artillerivej 5, DK-2300
mediated and humoral immunity.
Copenhagen S, Denmark (MTH@ssi.dk). Genome projects and the subsequent ability to inves-
The Journal of Infectious Diseases 2008; 197:897–905 tigate human immune responses by use of derived re-
© 2008 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2008/19706-0017$15.00
combinant proteins have prompted attempts at in silico
DOI: 10.1086/528378 prediction of T cell and B cell targets. In most studies, the
Antigenic Profiling of Chlamydia trachomatis ● JID 2008:197 (15 March) ● 897Table 1. Chlamydia trachomatis recombinant proteins tested in Table 1. (Continued)
this study.
Protein Protein
no.a Geneb Functionb Localizationc no.a Geneb Functionb Localizationc
1 CT002 Glu-tRNA Gln Cytoplasmic 35 CT110 Hsp60 Cytoplasmic
amidotransferase (C 36 CT111 10-kDa chaperonin Cytoplasmic
subunit)
37 CT112 Oligoendopeptidase Cytoplasmic
2 CT003 Glu-tRNA Gln Cytoplasmic
38 CT119 Inclusion membrane Outer membrane
amidotransferase (A
protein A
subunit)
39 CT123 Biotin carboxyl carrier Cytoplasmic
3 CT004 Glu-tRNA Gln Cytoplasmic
protein
amidotransferase (B
Subunit) 40 CT124 Biotin carboxylase Cytoplasmic
4 CT005 Hypothetical protein Outer membrane 41 CT125 L13 ribosomal protein Cytoplasmic
5 CT008 Ribonuclease HII Cytoplasmic 42 CT126 S9 ribosomal protein Cytoplasmic
6 CT009 HTH transcriptional Cytoplasmic 43 CT133 rRNA methylase Cytoplasmic
regulator 44 CT141 Protein translocase Cytoplasmic
7 CT015 ATPase Cytoplasmic 45 CT150 L33 ribosomal protein Cytoplasmic
8 CT016 Hypothetical protein Cytoplasmic 46 CT155 Phospholipase D Cytoplasmic
9 CT023 Peptide chain releasing Cytoplasmic endonuclease
superfamily
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factor (RF-1)
10 CT025 Signal recognition Cytoplasmic 47 CT168 Hypothetical protein Cytoplasmic
particle GTPase 48 CT172 Hypothetical protein Cytoplasmic
11 CT026 S16 ribosomal protein Cytoplasmic 49 CT175 Oligopeptide binding Cytoplasmic
12 CT027 tRNA (guanine N-1) Cytoplasmic protein permease
methyltransferase 50 CT184 YqgF family Inner membrane
13 CT028 L19 ribosomal protein Cytoplasmic 51 CT201 Oligopeptide transport Cytoplasmic
14 CT030 GMP kinase Cytoplasmic ATPase
15 CT032 Methionyl-tRNA Cytoplasmic 52 CT202 Oligopeptide transport Cytoplasmic
synthetase ATPase
16 CT035 Biotin protein ligase Cytoplasmic 53 CT212 Hypothetical protein Cytoplasmic
17 CT038 Hypothetical protein Cytoplasmic 54 CT213 Ribose-5-P isomerase A Cytoplasmic
18 CT040 Holliday junction Cytoplasmic 55 CT220 Phenylacrylate Cytoplasmic
helicase decarboxylase
19 CT043 Hypothetical protein Cytoplasmic 56 CT245 Pyruvate Cytoplasmic
dehydrogenase, ␣
20 CT048 SAM-dependent Cytoplasmic
methyltransferase 57 CT246 Pyruvate Cytoplasmic
21 CT052 Coproporphyrinogen III Cytoplasmic dehydrogenase, 
oxidase 58 CT250 Replication initiation Cytoplasmic
22 CT053 Hypothetical protein Cytoplasmic factor
23 CT055 Dihydrolipoamide Inner membrane 59 CT261 DNA Pol III epsilon chain Cytoplasmic
succinyltransferase 60 CT265 AcCoA Cytoplasmic
24 CT061 Sigma-28/WhiG family Cytoplasmic carboxylase/transferase,
␣
25 CT063 6-phosphogluconate Cytoplasmic
dehydrogenase 61 CT279 NADH (ubiquinone) Periplasmatic
oxidoreductase, ␥
26 CT067 Solute protein binding Cytoplasmic
62 CT288 Hypothetical protein Cytoplasmic
family
63 CT316 L7/L12 ribosomal protein Cytoplasmic
27 CT068 rRNA methylase Cytoplasmic
64 CT317 L10 ribosomal protein Cytoplasmic
28 CT071 Hypothetical protein Cytoplasmic
65 CT318 L1 ribosomal protein Cytoplasmic
29 CT078 Methylene Cytoplasmic
tetrahydrofolate 66 CT322 Elongation factor Tu Cytoplasmic
dehydrogenase 67 CT323 Initiation factor IF-1 Cytoplasmic
30 CT080 Hypothetical protein Cytoplasmic 68 CT324 Hypothetical protein Inner membrane
31 CT082 Hypothetical protein Outer membrane 69 CT325 Hypothetical protein Cytoplasmic
32 CT089 Low calcium response E Extracellular 70 CT336 PTS PEP Cytoplasmic
33 CT093 Riboflavin kinase/FAD Cytoplasmic phosphotransferase
synthase 71 CT341 Heat shock protein J Cytoplasmic
34 CT098 S1 ribosomal protein Cytoplasmic 72 CT342 S21 ribosomal protein Cytoplasmic
(continued) (continued)
898 ● JID 2008:197 (15 March) ● Follmann et al.Table 1. (Continued) focus has been on antibody [16 –18] and CD8⫹ T cell targets [19,
20]. Here, we present an antigen discovery strategy that focuses
on the identification of human T and B cell targets. We have
Protein
no.a Geneb Functionb Localizationc
selected a set of genes, comprising open reading frames (ORFs)
that are transcribed within the first 3 h of the intracellular life
73 CT357 Hypothetical protein Cytoplasmic
74 CT375 D-amino acid Cytoplasmic
cycle [21] as well as genes in the plasticity zone and flanking
dehydrogenase regions [22]. A total of 116 proteins were examined for their
75 CT376 Malate dehyrogenase Cytoplasmic ability to induce a cell-mediated immune response in a group of
76 CT395 Hsp70 cofactor Cytoplasmic 40 patients with confirmed genital C. trachomatis infection. Hu-
77 CT396 Hsp70 Cytoplasmic
moral responses were subsequently investigated in the same pa-
78 CT405 Riboflavin synthase Cytoplasmic
79 CT420 L21 ribosomal protein Cytoplasmic tients and led to a classification of the antigens as T cell, B cell, or
80 CT426 Fe-S oxidoreductase Cytoplasmic T/B cell antigens. This grouping of antigens was correlated with
81 CT428 Ubiquinone Cytoplasmic the predicted subcellular localization of the antigens [23]. To
methyltransferase
our knowledge, this work is the first comparative evaluation of
82 CT436 S10 ribosomal protein Cytoplasmic
83 CT437 Elongation factor G Cytoplasmic naturally occurring cell-mediated and humoral immune responses
84 CT439 S12 ribosomal protein Cytoplasmic in patients with diagnosed C. trachomatis genital infection.
85 CT443 60-kDa cysteine-rich Outer membrane
OMP MATERIALS AND METHODS
86 CT446 CHLPS Euo protein Cytoplasmic
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87 CT492 Phosphatase/kinase Cytoplasmic
Patients and samples. The study included 46 patients attend-
88 CT507 RNA polymerase, ␣ Cytoplasmic
89 CT509 S13 ribosomal protein Cytoplasmic ing the outpatient sexually transmitted disease clinic at Bispeb-
90 CT511 L15 ribosomal protein Cytoplasmic jerg Hospital, Copenhagen, who had tested positive for C. tra-
91 CT512 S5 ribosomal protein Cytoplasmic chomatis in a specific polymerase chain reaction assay and who
92 CT513 L18 ribosomal protein Cytoplasmic had provided informed consent; 23 patients were female (age
93 CT514 L6 ribosomal protein Cytoplasmic
range, 17–38 years; mean age, 24 years), and 23 were male (age
94 CT516 L5 ribosomal protein Cytoplasmic
95 CT520 L29 ribosomal protein Cytoplasmic range, 20 – 66 years; mean age, 30 years). PBMCs from 10 of
96 CT523 L22 ribosomal protein Cytoplasmic these patients were randomly selected from among the patients
97 CT526 L23 ribosomal protein Cytoplasmic responding positively to a whole-cell C. trachomatis serovar D
98 CT610 Hypothetical protein Cytoplasmic
extract. These 10 patients responded to the C. trachomatis ex-
99 CT611 Hypothetical protein Cytoplasmic
100 CT613 Dihydropteroate Cytoplasmic tract with a median level of IFN-␥ comparable to that in the
synthase overall group of 46 patients. Further testing for T cell responses
101 CT626 S4 ribosomal protein Cytoplasmic was done in 40 randomly selected patients, including 4 from the
102 CT630 HTH transcriptional Cytoplasmic original 10 patients. Antibody assay was performed on serum
regulatory protein
103 CT647 Hypothetical protein Inner membrane samples from all 46 patients. Blood donors with no history of
104 CT649 Formyltetrahydrofolate Cytoplasmic positive C. trachomatis testing were enrolled as control subjects
synthetase (n ⫽ 17; 11 female and 6 male). The study was approved by the
105 CT679 Elongation factor TS Cytoplasmic
Local Ethical Committee for Copenhagen (01– 008/03).
106 CT681 Major outer membrane Outer membrane
protein Generation of protein extract from serovar D elementary
107 CT725 Biotin synthetase Cytoplasmic body. Cultivation and harvesting of C. trachomatis serovar D was
108 CT734 Hypothetical protein Cytoplasmic done as described by Jensen et al. [24]. The purified bacteria was
109 CT747 Uroporphyrinogen Cytoplasmic sonicated, and the protein fraction was extracted with phenol and
decarboxylase
110 CT779 Hypothetical protein Cytoplasmic
ether, as described by Sauvé et al. [25]. Briefly, the lysate was mixed
111 CT801 S6 ribosomal protein Cytoplasmic 1:1 with phenol and vortexed for 20 s. After 5 min of centrifugation
112 CT803 L9 ribosomal protein Cytoplasmic at 12,000 g, the proteins in the phenol phase were precipitated with
113 CT833 Initiation factor 3 Cytoplasmic 2 volumes of ether and further centrifuged at 12,000 g for 5 min. The
114 CT835 L20 ribosomal protein Cytoplasmic
lower aqueous phase was dried and resuspended in an ammonia/3-
115 CT836 Phenylalanyl tRNA Cytoplasmic
synthetase, ␣ (cyclohexylamino)-1-propanesulfonic acid (CAPS) buffer (pH
116 CT845 Hypothetical protein Cytoplasmic 10.2). The EB protein extract was stored at ⫺20°C until use.
a
Protein no. assigned in this study. Serovar typing of clinical isolates from urine samples.
b
Gene annotation and predicted function are taken from GenBank Chlamydial chromosomal DNA was extracted, and DNA frag-
AE001273 via http://www.ncbi.nlm.nih.gov.
c
Subcellular localization as predicted by CELLO via the Web server at ments containing the gene and flanking regions of ompA were
http://cello.life.nctu.edu.tw. amplified essentially as described elsewhere [26].
Antigenic Profiling of Chlamydia trachomatis ● JID 2008:197 (15 March) ● 899Expression of C. trachomatis genes in Escherichia coli and Lymphocyte preparation and cell culture. PBMCs were
purification of recombinant proteins for T cell assay. The separated from 80 mL of whole blood by density gradient cen-
primary annotation for the C. trachomatis serovar D genome was trifugation using Lymphoprep (Nycomed) and frozen in liquid
used, as defined by Stephens et al. [22]. We selected 375 ORFs for nitrogen until use. For use in the T cell assay, PBMCs were
cloning; the list comprised early transcribed genes, the plasticity thawed and resuspended in RPMI 1640 supplemented with 1%
zone and its flanking regions, and ompA plus omcB. The full- penicillin/streptomycin, 1% nonessential amino acids, 1% glu-
length sequences of the specific C. trachomatis genes were cloned tamine, 1% sodium pyruvate, 1% HEPES, and 10% human AB
into pDEST17 (Invitrogen) in frame with a 6⫻His tag and ex- serum (local blood bank, Rigshospitalet, Copenhagen). The vi-
pressed recombinantly in E. coli. Recombinant proteins were ability and number of cells were determined by nigrosin stain-
initially purified by metal chelate affinity chromatography, es- ing. The cells were cultured in triplicate in round-bottom micro-
sentially as described elsewhere [27], followed by size fraction- titer plates (Nunc) at 1.25 ⫻ 10 5 cells/well in a total volume of
ation by SDS-PAGE, electroelution from SDS-PAGE gel pieces, 100 L. The antigen concentrations were 2 g/mL for C. tracho-
precipitation with 80%–95% acetone (high-performance liquid matis serovar D protein extract and 5 g/mL for recombinant
chromatography grade; Sigma-Aldrich) to remove SDS, washing antigens. Phytohemagglutinin (2 g/mL) was used as a positive
in 95% ethanol, and resuspension in 50 mmol/L Tris (pH 7.5), control, and cell cultures without antigen were included as neg-
150 mmol/L NaCl, and 8 mol/L urea. The buffer was changed on ative controls. The cells were incubated at 37°C in humidified air
a NAP-5 column (Amersham Biosciences) to 50 mmol/L Tris (5% CO2 and 95% air) for 5 days.
(pH 7.5), 150 mmol/L NaCl, and 20% glycerol plus 5% fetal calf IFN-␥ ELISA. The amount of IFN-␥ in the supernatants
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serum (Biochrom) and stored at ⫺20°C until use. Protein con- was determined by standard sandwich ELISA. The level of IFN-␥
centration was determined by densitometric scanning of SDS- was determined by using commercially available monoclonal
PAGE gels tested with the sample in multiple dilutions against a antibodies (Pierce), according to the manufacturer’s instruc-
standard of bovine serum albumin (GeneTools from Syngene). tions. Recombinant IFN-␥ was used as a standard (Pierce). The
Purification of recombinant C. trachomatis proteins for detection limit of the assay was 20 pg/mL, and the IFN-␥ release
antibody ELISA. Ninety-six DeepWell plates (Nunc) containing into unstimulated wells was below the detection level. The cutoff
1.4-mL E. coli cultures of individual recombinant proteins were in- level for each of the individual proteins was based on the upper
duced for 4 h at 37°C, and the bacterial pellet was collected by cen- 95% CI of the geometric mean for the control donors.
trifugation at 3000 g for 20 min. The pellet was resuspended in 200 Bioinformatical methods. Primary annotation and func-
tion was taken from C. trachomatis D/UW-3/CX (GenBank ac-
L of BugBuster (Novagen) with lysozyme (0.2 kU) and Benzonase
cession number AE001273). The subcellular localization of in-
(5 U) (Novagen) and left for 20 min on a shaker. Insoluble protein
dividual proteins in the entire genome were predicted by
aggregates were collected on Durapore DV membranes (Millipore).
CELLO, a subcellular localization predictive system [23]. Gram-
The membranes were washed in BugBuster, and inclusion bodies
negative bacteria have 5 subcellular sites, including cytoplasm,
were resuspended in 100 L of 50 mmol/L Tris (pH 7.5), 150
inner membrane, periplasm, outer membrane, and extracellular
mmol/L NaCl, and 8 mol/L urea. BugBuster filtrate and resus-
space. CELLO gives an output with probability for each location
pended inclusion bodies were analyzed by SDS-PAGE and stored
of each protein.
for further analysis at ⫺20°C.
Statistical methods. The Mann-Whitney rank-sum test was
Antibody screening of the recombinant proteins. Recom-
used to analyze differences between groups of data with a non-
binant proteins were coupled to nickel-chelated Immobilizer plates
Gaussian distribution. Fisher’s exact test was used to compare
(Nunc) at a concentration of 0.5 g/mL in 50 mmol/L NaHCO3
groups with categorical variables. Differences for which P ⬍ .05
buffer (pH 9) overnight. Serum samples from 10 patients and 5
were considered to be statistically significant.
control donors were preabsorbed with an E. coli extract (Promega)
to reduce background reactivity. Serum was diluted 1:200, and RESULTS
ELISA was performed essentially as described elsewhere [28]. Anti-
body binding was detected with peroxidase-conjugated rabbit anti– Library construction. A panel of C. trachomatis genes repre-
human immunoglobulin (Dako). The cutoff level in the initial senting ⬃42% (375 of 894 genes) of the annotated ORFs in the
screening with 10 patients was set to an optical density (OD) of 0.2. published serovar D genome [22] were cloned to create a full-
Antigens were selected for further analysis if they were recognized in length expression library. The selected ORFs comprise early
1 or more patients and showed no responses in the 5 control do- transcribed genes [21], genes in the plasticity zone and flanking
nors. In the larger screening using serum samples from 46 patients regions, and the ompA and omcB genes. The corresponding gene
and 17 control donors, we applied a cutoff value based on the upper products represent proteins with both known and unknown
95% confidence interval (CI) limit of the geometric mean for the functions and with different cellular localization and expression
control donors. profiles. The recombinant proteins were expressed in E. coli with
900 ● JID 2008:197 (15 March) ● Follmann et al.Downloaded from http://jid.oxfordjournals.org/ by guest on October 29, 2015
Figure 1. Reactivity of human T cells to 116 Chlamydia trachomatis recombinant proteins. Peripheral blood mononuclear cells isolated from 10
patients with C. trachomatis infection and 5 control donors were stimulated with the recombinant proteins at 5 g/mL. The release of interferon (IFN)–␥
was measured in the supernatants after 5 days. The no. of C. trachomatis patient samples that responded to the individual recombinant proteins was
plotted using a cutoff value based on the upper 95% confidence interval limit of the geometric mean for the control donors. Asterisks indicate proteins
inducing an IFN-␥ response in the patients with C. trachomatis infection that differed significantly from that in control donors (P ⬍ .05, Mann-Whitney
rank-sum test).
a protein expression efficiency of 64% (239 of the 375 genes
produced a recombinant protein as determined by SDS-PAGE).
In most cases, the dominant band migrated in accordance with
the expected molecular weight and was detected by a monoclo-
nal antibody against the His tag (data not shown). A total of 116
recombinant proteins (table 1) could be obtained at a purity
acceptable for testing with human PBMCs [29].
Identification of human T cell targets. To screen for spe-
cific T cell responses, PBMCs from 10 patients with a confirmed
C. trachomatis infection and from 5 control donors were stimu-
lated with 116 recombinant proteins (table 1). Proteins that met
the selection criteria of being significantly different from the
controls (P ⬍ .05, Mann-Whitney rank-sum test) and being
frequently recognized (by ⬎50% of the patients) were selected
for further analysis. Figure 1 summarizes the numbers of pa-
tients responding to each of the individual proteins. The pro- Figure 2. T cell reactivity against 14 selected antigens. Peripheral
teins inducing a significantly higher response in patients than in blood mononuclear cells isolated from 40 patients with Chlamydia tra-
control donors are marked by asterisks. Fourteen T cell targets chomatis infection (solid bars) and 17 control donors (open bars) were
met both selection criteria (CT004, CT015, CT035, CT043, stimulated with the recombinant proteins at 5 g/mL. The release of
interferon (IFN)–␥ was measured in the supernatants after 5 days. Each
CT110, CT111, CT168, CT184, CT342, CT396, CT443, CT492,
bar represents the median level of IFN-␥ release plus the upper limit of
CT509, and CT611). The 14 proteins were subsequently ana- the 95% confidence interval for the median. *P ⬍ .05, **P ⬍ .01, and
lyzed in more patients. Forty patients (20 female and 20 male) ***P ⬍ .001, for antigen response in patients vs. control donors (Mann-
were randomly selected from the patient group, and their IFN-␥ Whitney rank-sum test). Ct, C. trachomatis.
Antigenic Profiling of Chlamydia trachomatis ● JID 2008:197 (15 March) ● 901Table 2. Identity and characteristics of Chlamydia trachomatis T cell and B cell antigens.
Subcellular
Molecular localization Accession
Antigen Function weight, kDa by CELLOa Proteomicb no.c
T cell antigens
CT004 Gln amidotransferase 55 Cytoplasmic Yes O84007
CT043 Hypothetical protein 18 Cytoplasmic Yes O84047
CT184 YqgF family 16 Inner membrane No Q46370
CT509 S13 ribosomal protein 14 Cytoplasmic Yes Q46448
CT611 Hypothetical protein 32 Cytoplasmic Yes O84617
B cell antigens
CT082 Hypothetical protein 60 Extracellular Yes O84084
CT089 Low calcium response E 45 Extracellular Yes O84091
CT322 Elongation factor Tu 44 Cytoplasmic Yes P26622
CT396 Hsp70 71 Cytoplasmic Yes P17821
CT681 MOMP 42 Outer membrane Yes Q46409
T/B cell antigens
CT110 Hsp60 58 Cytoplasmic Yes P17203
CT443 Cysteine-rich OMP 59 Outer membrane Yes P18151
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NOTE. MOMP, major outer membrane protein; OMP, outer membrane protein.
a
CELLO is a subcellular localization predictive system [23].
b
Proteins identified in the literature as being expressed in either elementary body or reticulate body form.
c
Accession no. for the Swiss-Prot database.
responses were compared with those of 17 control donors with was relatively large donor-to-donor variation in the pattern of spe-
no previous history of a C. trachomatis infection (figure 2). Seven cific T cell responses, and no single donor recognized all antigens.
of the 14 proteins (CT004, CT043, CT110, CT184, CT443, Furthermore, no single T cell antigen was recognized as frequently
CT509, and CT611) induced significantly higher levels of IFN-␥ as the C. trachomatis protein extract, although the combination
secretion in patients than in control donors (P ⬍ .05, Mann- of T cell responses against CT004, CT110, and CT443 almost
Whitney rank-sum test) (figure 2 and table 2), and 6 of the 7 reached the same level (34 of 40 patients) as did responses to the
proteins (all except CT509) were recognized by ⬎50% of the C. trachomatis protein extract (38 of 40 patients). All antigens
patients, using the cutoff level for a positive response described were recognized equally well by PBMCs from patients infected
above. with the rare serovars G-K and those from patients infected with
Recognition profile of T cell antigens. To compare the pattern the frequent serovars D–F (P ⬍ .05, Fisher’s exact test). Thus,
of T cell responses in individual patient samples, the reactivity none of the antigens showed a tendency for serovar-restricted T
against the above-mentioned 7 antigens was displayed as a positive cell reactivity. Furthermore, patients with multiple infections
or negative signal in individual PBMCs (figure 3). In general, there did not show a broader pattern of specific T cell responses than
Figure 3. Recognition profile of T cell antigens. Black boxes represent positive responses (above the cutoff level), and gray boxes represent negative
responses. Each row shows the response to a single antigen in the 40 individual patients with Chlamydia trachomatis infection. The no. of patients
responding to each antigen is shown at the far right. The bottom 2 rows contain information on a confirmed previous infection (Pr. inf) with C.
trachomatis and serovariant typing of the clinical isolate, respectively; dashes indicate that it was not possible to amplify the ompA gene sequence
from chromosomal DNA extracted from urine. Each column summarizes data for 1 patient. Ct extr., C. trachomatis extract.
902 ● JID 2008:197 (15 March) ● Follmann et al.did patients with no history of a previous chlamydia infection Table 3. Summary of subcellular local-
(P ⬍ .05, Fisher’s exact test), and sex did not influence the re- ization of identified antigens.
sponse (data not shown).
Localization
Identification of antigens recognized by human antibodies.
Because it is generally accepted that both T and B cells play an Inside Outside
important role in defense against chlamydia, we continued by Antigen (n ⫽ 109) (n ⫽ 7) Pa
investigating the antibody reactivity against the large panel of T cell 5 0 NS
116 recombinant proteins (table 1). The proteins were screened B cell 2 3 .0013
by ELISA against serum from the same 10 patients with chla- T/B cell 1 1 NS
mydia infection used in the initial T cell screening. Twelve of the NOTE. NS, not significant.
a
116 proteins (CT028, CT082, CT089, CT110, CT123, CT279, For inside (cytoplasm, inner membrane, or
periplasmic) vs. outside (outer membrane or extra-
CT288, CT322, CT396, CT443, CT512, and CT681) were recog- cellular), by Fisher’s exact test.
nized (OD of ⬎0.2) by at least 1 patient and showed no response
in the 5 control donors tested (data not shown). These 12 pro-
rank-sum test). The majority of the 116 investigated proteins in
teins were further evaluated in the larger panel of 46 patients,
our study are predicted to be located inside the bacteria, which is
and CT082, CT089, CT322, CT396, CT681, CT443, and CT110
in agreement with the inclusion of early expressed genes in the
were identified as immunodominant antigens recognized by
gene library. Seven of the 116 proteins are characterized as outer
⬎50% of patients (figure 4). Among the 7 identified antibody
membrane or extracellular (table 1). To evaluate the predictive
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targets, 2 (CT443 and CT110) were also identified as T cell tar-
value of a given subcellular localization with a resulting immune
gets (figure 2 and table 2).
response, we analyzed the frequency of proteins eliciting a sig-
Subcellular localization of identified antigenic targets. A
nificant T cell response and a significant antibody response ac-
novel method for predicting the subcellular localization in
cording to their localization (table 3). This comparison showed
gram-negative bacteria, CELLO [23] was used to predict the cel-
significantly more outer membrane–associated proteins in the
lular localization of all published C. trachomatis ORFs: cyto-
group of B cell antigens (P ⫽ .0013, Fisher’s exact test) and in-
plasm (geometric mean molecular weight, 29 kDa [95% CI,
dicated that T cell targets were nonpredictive regarding their
28 –31 kDa]), inner membrane (34 kDa [31–38 kDa]), periplas-
localization. This observation is in agreement with the general
mic (23 kDa [20 –27 kDa]), outer membrane (50 kDa [44 –56
belief that proteins on the bacterial surface are more accessible
kDa]), and extracellular (49 kDa [35– 69 kDa]). For ease of com-
for antibodies than proteins in the cytoplasm.
parison, 2 groups were defined, with proteins predicted to be on
the inside (cytoplasm, inner membrane, or periplasmic) or out- DISCUSSION
side (outer membrane or extracellular) of the bacterium (table
3). Interestingly, these 2 groups differ significantly in their mo- The availability of genomic sequences from human pathogens
lecular weights, with geometric means of 30 kDa (95% CI, 29 –31 provides a rich source for discovering new vaccine candidates.
kDa) for proteins located inside and 50 kDa (95% CI, 44 –56 Considerable efforts have therefore been made to derive new
kDa) for proteins located outside (P ⬍ .0001, Mann-Whitney information that might potentially be used for the development
of a vaccine against sexually transmitted chlamydia [30, 31]. The
present approach has enabled us to evaluate specific immune
responses (T and B cell) against a large number of proteins in a
standardized manner.
Despite the importance of a T cell response in chlamydia in-
fection, only a few T cell antigens have been identified to date, as
reviewed by Brunham and Rey-Ladino [32]. In the present
study, we identified 5 antigens that were exclusively immuno-
dominant in human T cells: CT004 (Gln amidotransferase),
CT043 (hypothetical), CT184 (YqgF family), CT509 (S13), and
CT611 (hypothetical). Except for CT184, a predicted Holliday
Figure 4. Frequency of antibody responses. Serum samples from 46 junction resolvase, all of these proteins have been detected in
patients with Chlamydia trachomatis infection and 17 control donors their native form in in vitro grown C. trachomatis [16, 33, 34],
were tested by IgG ELISA at a 1:200 dilution. Each bar represents the
but only CT043 has previously been reported as a T cell antigen
percentage of patients responding above the cutoff level (upper 95%
confidence interval limit for the geometric mean of the control donors). [29]. CT004, CT184, and CT509 are all predicted to be part of
Antigens inducing an antibody responses in ⬎50% of the patients the Chlamydia transcriptional machinery and support the pre-
(dashed line) are considered immunodominant. vious finding that this group of proteins is presented to the im-
Antigenic Profiling of Chlamydia trachomatis ● JID 2008:197 (15 March) ● 903mune system during genital infection [29]. CT611 is a hypothet- protein is a major target of both T cells [43, 44] and B cells [45,
ical protein of unknown function, and the gene for CT611 is 46] and confirms the notion that heat-shock proteins are com-
situated adjacent to the gene for CT610, which has recently been mon targets of immune response to intracellular pathogens [47].
identified as a strong target of human T cell responses [26]. In- Together with CT681, CT443 is one of the most thoroughly in-
terestingly, the translation of these 2 genes may be coupled be- vestigated antigens in chlamydia [48 –50], and the identification
cause the stop codon of ct611 overlaps with the start codon of of CT443 here further emphasizes its immunological impor-
ct610. Thus, the close linkage of these proteins, the possibility of tance.
translational coupling, and their ability to induce a recall re- In conclusion, we have demonstrated that an unbiased com-
sponse in T cells from patients with a genital C. trachomatis in- parative evaluation of naturally occurring cell-mediated and hu-
fection may suggest that this region on the C. trachomatis ge- moral immune responses is feasible and has led to the identifi-
nome is an antigenic hot spot. cation of novel as well as previous described T and B cell
In view of the importance of a combined T and B cell response antigens. Importantly, we confirmed that prediction of subcel-
against C. trachomatis, we tested our panel for reactivity against lular localization is advantageous for identifying antibody tar-
serum IgG antibodies. We sought to identify novel antibody tar- gets.
gets and to describe an eventual overlap in T and B cell immune
responses. Five antigens—CT082 (hypothetical), CT089 (LcrE), Acknowledgments
CT322 (elongation factor Tu), CT396 (Hsp70), and CT681 (ma-
We thank Vita Skov, Lisbeth Abrahamsen, and Kathryn Wattam for ex-
jor outer membrane protein [MOMP])—were recognized ex- cellent technical assistance.
Downloaded from http://jid.oxfordjournals.org/ by guest on October 29, 2015
clusively by serum IgG antibodies. Of them, CT089, CT322,
CT396, and CT681 have been described previously as B cell tar-
gets [16, 35–38], whereas CT082 is a novel B cell target. CT082 is References
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