Differential B-cell responses are induced by Mycobacterium tuberculosis PE antigens Rv1169c, Rv0978c, and Rv1818c.
ABSTRACT The multigene PE and PPE family represents about 10% of the genome of Mycobacterium tuberculosis. Here, we report that three members of the PE family, namely, Rv1169c, Rv0978c, and Rv1818c, elicit a strong, but differential, B-cell humoral response among different clinical categories of tuberculosis patients. The study population (n = 211) was comprised of different clinical groups of both adult and child patients: group 1 (n = 94) patients with pulmonary infection, group 2 (n = 30) patients with relapsed infection, group 3 (n = 31) patients with extrapulmonary infections, and clinically healthy donors (n = 56). Among the PE proteins studied, group 1 adult patient sera reacted to Rv1818c and Rv0978c, while Rv1169c elicited immunoreactivity in group 3 children. However, all three PE antigens studied as well as the 19-kDa antigen did not demonstrate humoral reactivity with sera from group 2 patients with relapsed infection. The current study shows that while responsiveness to all three PE antigens is a good marker for M. tuberculosis infection, a strong response to Rv0978c or to Rv1818c by group 1 adult patients with pulmonary infection or largely restricted reactivity to Rv1169c antigen in child patients with extrapulmonary infections offers the possibility of differential utility in the serodiagnosis of tuberculosis.
- SourceAvailable from: Stuti Arya[Show abstract] [Hide abstract]
ABSTRACT: The Mycobacterium tuberculosis has developed intricate strategies to evade the killing of microorganism and support its survival in phagocytes. The genome sequence of bacterium revealed the presence of several genes for lypolytic enzymes. Rv1923 gene, a member of Lip family in M. tuberculosis demonstrated the least sequence similarity with its counterpart in non-pathogenic strain M. smegmatis. The expression of Rv1923 gene (LipD) was not observed in in vitro growing cultures of M. tuberculosis H37Ra while an upregulation of transcription of Rv1923 gene was noticed in oxidative conditions. For detailed characterization of LipD enzyme the Rv1923 gene was cloned in pQE30-UA vector and expressed in E. coli M15 cells. LipD was purified from inclusion bodies and refolded with nearly 40 % protein yield. The specific activity of enzyme was calculated to be 16 U/mg with pNP-palmitate as a preferred substrate. Kinetic analysis showed K m 0.645 mM and V max 24.75 U/ml with pNP-palmitate. Ser-102, Asp-342, and His-369, predicted as the members of the catalytic triad, were confirmed by mutagenesis. Mutagenesis studies revealed that catalytic serine residues located in β-lactamase motifs (S-X-X-K) were responsible for lipolytic activity. Secondary structure analysis by CD spectroscopy demonstrated the presence of α helices and β sheets in the canonical structure of LipD. The enzyme was stable up to 50 °C and was active even at pH 6.0. The expression of enzyme under stress conditions and its activity and stability at high temperature and low pH suggested the possible role of LipD in the survival of mycobacterium in macrophage compartment.Current Microbiology 11/2013; · 1.36 Impact Factor
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ABSTRACT: Toll-like receptor 2 (TLR2), a member of pattern recognition receptors (PRRs) abundant on macrophages, dendritic cells (DCs) and respiratory epithelial cells lining the lung, plays critical role in host immune response against Mycobacterium tuberculosis (MTB) infection. TLR2-mediated elimination of MTB involves multiple pathways such as promoting DCs maturation, generating biased Th1, Th2, Th17 type response, regulating the macrophage activation and cytokine secretion. MTB can also hijack the TLR2 signaling to subvert the host immunity by dampening the macrophages response to IFN-γ, suppressing the processing and presentation of antigens. This review summarizes the intricate network of TLR2-mediated signaling and Mycobacteria effectors involved in MTB –host interaction with an aim to find better target for improved tuberculosis control, especially the host-derived therapy targets. TLR2 agonists with potential to be included in novel tuberculosis vaccines are also discussed.Biochimie 01/2014; · 3.12 Impact Factor
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ABSTRACT: The PE_PGRS33 protein is a member of the PE family, which encompasses the PE and the PE_PGRS subfamilies. Among PE_PGRS's, this protein is one of the most studied antigens and its immunomodulatory properties are influence by both PE and PGRS domains. However, the contribution of these domains to the host immune recognition of the PE_PGRS33 protein and their potential role in latent tuberculosis infection in humans is still unknown. In this study, the immunogenic properties of the complete PE_PGRS33 protein and each domain separately were evaluated in BALB/c mice and latent tuberculosis infected (LTBI) humans. In mice, PE_PGRS33 and its domains induced similar antibody production and secretion of IFN-γ. PE_PGRS33 and the PE domain stimulated higher CD4(+) and CD8(+) T-cell proliferation compared to the PGRS domain. This demonstrated that the principal difference in the immune recognition of the domains is the higher activation of T-cell subpopulations involved in the control of tuberculosis. In humans, the secretion of IFN-γ in response to PE_PGRS33 was detected in both LTBI and in non-infected vaccinated individuals. The same was observed for antibody response, which targets epitopes located in the PGRS domain but not in the PE domain. These observations suggest that T and B cell responses to PE_PGRS33 are induced by BCG vaccination and can be maintained for many years in non-infected individuals. This also indicates that the IFN-γ response detected might not be associated with latent tuberculosis infection. These results contribute to the elucidation of the role of the PE_PGRS33 protein and its PE and PGRS domains in the immune response against Mycobacterium tuberculosis.Frontiers in Immunology 01/2014; 5:236.
CLINICAL AND VACCINE IMMUNOLOGY, Oct. 2007, p. 1334–1341
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 14, No. 10
Differential B-Cell Responses Are Induced by Mycobacterium tuberculosis
PE Antigens Rv1169c, Rv0978c, and Rv1818c?
Yeddula Narayana,1Beenu Joshi,2V. M. Katoch,2Kanhu Charan Mishra,1and Kithiganahalli N. Balaji1*
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012,1and National Jalma Institute of
Leprosy and Other Mycobacterial Diseases, Tajganj, Agra 282-001,2India
Received 26 April 2007/Returned for modification 29 May 2007/Accepted 27 July 2007
The multigene PE and PPE family represents about 10% of the genome of Mycobacterium tuberculosis. Here,
we report that three members of the PE family, namely, Rv1169c, Rv0978c, and Rv1818c, elicit a strong, but
differential, B-cell humoral response among different clinical categories of tuberculosis patients. The study
population (n ? 211) was comprised of different clinical groups of both adult and child patients: group 1 (n ?
94) patients with pulmonary infection, group 2 (n ? 30) patients with relapsed infection, group 3 (n ? 31)
patients with extrapulmonary infections, and clinically healthy donors (n ? 56). Among the PE proteins
studied, group 1 adult patient sera reacted to Rv1818c and Rv0978c, while Rv1169c elicited immunoreactivity
in group 3 children. However, all three PE antigens studied as well as the 19-kDa antigen did not demonstrate
humoral reactivity with sera from group 2 patients with relapsed infection. The current study shows that while
responsiveness to all three PE antigens is a good marker for M. tuberculosis infection, a strong response to
Rv0978c or to Rv1818c by group 1 adult patients with pulmonary infection or largely restricted reactivity to
Rv1169c antigen in child patients with extrapulmonary infections offers the possibility of differential utility in
the serodiagnosis of tuberculosis.
Mycobacterium tuberculosis, the causative agent of pulmo-
nary tuberculosis (TB), infects one-third of the world’s popu-
lation (22). Despite the multiplicity of antimicrobial responses
mounted by its host, M. tuberculosis shows a remarkable ability
to survive either by evoking survival strategies or by interfering
with critical macrophage functions that are required to suc-
cessfully respond to infection (12, 21, 30). One such immune
evasion or survival strategy may be to express different sets of
proteins during various clinical stages of the disease in infected
macrophages of granulomas, which provides survival advan-
tages amid robust host immune responses. A group of genes
carried by M. tuberculosis that are expressed upon infection of
macrophages belongs to the PE family (1, 4, 9). This family is
comprised of about 100 genes, scattered throughout the ge-
nome, with highly homologous sequences corresponding to a
signature Pro-Glu (PE) amino acid sequence near the amino
terminus (1, 4, 9). In many proteins, the PE domain is often
linked to a unique domain of various lengths that is rich in
alanine and glycine amino acids, termed the PGRS domain
(PE_PGRS subfamily). It is generally believed that the PGRS
domain of PE family genes could be a source of antigenic
variability (5, 10, 13, 26, 29). The uniqueness of the PE genes
is further illustrated by the fact that these genes are restricted
to mycobacteria (4). However, despite their abundance in my-
cobacteria, very little is known regarding the expression or the
functions of PE family genes. Recent studies have provided
some insights into functional roles of selected PE family pro-
teins. It has been shown that Mycobacterium marinum ex-
presses a homologue of the M. tuberculosis PE (PE_PGRS)
Rv1651c gene in infected granulomas (27). Mutation studies
have shown that Rv1818c, a PE (PE_PGRS) gene product,
may play a role during growth in liquid medium as well as in
the infection of macrophages (5). In addition, aerosol infection
of mice with virulent M. tuberculosis strains generates a hu-
moral response to the Rv1818c protein (10). The involvement
of PE family genes in the virulence of the pathogen has also
been reported, and many members of PE family proteins, in-
cluding Rv1818c, are reported be localized on the surface of M.
tuberculosis bacilli (3, 11, 27). Additionally, it has been sug-
gested that the PE_PGRS subfamily of PE genes is enriched in
genes with a high probability of being essential for M. tuber-
culosis (19). Although those studies strongly support a role for
the PE family proteins in the biology, and possibly pathogen-
esis, of M. tuberculosis, an understanding of the function of PE
proteins will require extensive investigations.
In the current study, Rv1169c, Rv0978c, and Rv1818c were
selected based on the following observations. These PE family
proteins display a differential antigenic profile and are associ-
ated with pathological conditions, as evident from DNA mi-
croarray expression data (17, 26, 28, 31, 32). Furthermore,
Rv0978c and Rv1169c were upregulated in TB bacilli upon
infection of macrophages (28, 31). As mentioned above, a
mutation in Rv1818c affects the growth of bacilli as well as the
infection of macrophages (5). Recently published studies sug-
gested that the ectopic expression of Rv1818c in a nonpatho-
genic Mycobacterium smegmatis strain results in specific prop-
erties more typical of virulent mycobacteria, including
increased survival in macrophages and host tissues (13). In
addition, Rv1818c and Rv1169c have been detected in M. tu-
berculosis bacilli isolated from the granulomas of lungs of hu-
man pulmonary TB patients (26). Rv0978c was demonstrated
to be a member of a group of genes called in vivo-expressed
genomic island genes, which were shown to be upregulated in
* Corresponding author. Mailing address: Department of Microbi-
ology and Cell Biology, Indian Institute of Science, Bangalore 560012,
India. Phone: 91-80-22933223. Fax: 91-80-23602697. E-mail: balaji
?Published ahead of print on 8 August 2007.
M. tuberculosis bacilli during infection of mice (31). Rv0978c
was also shown to be upregulated, at least eightfold, in human
brain microvascular endothelial cell-associated M. tuberculosis,
suggesting a role for endothelial cell invasion and intracellular
survival (17). Furthermore, Rv0978c and Rv1169c are ex-
pressed upon infection by pathogenic species such as M. tuber-
culosis and Mycobacterium bovis only and not by environmental
mycobacteria like M. smegmatis or Mycobacterium avium. Sim-
ilarly, Rv1818c is constitutively expressed (5) by strains of M.
tuberculosis and M. bovis but not by environmental mycobac-
teria like M. smegmatis or M. avium. In the current investiga-
tion, we report that the above-mentioned antigens of the PE
family elicited differential humoral antibody reactivities in a
panel of human sera obtained from different clinical categories
of TB patients as measured by enzyme-linked immunosorbent
MATERIALS AND METHODS
Patients and control subjects. The study population (n ? 211) was comprised
of TB-infected patients reporting to the National Jalma Institute of Leprosy and
Other Mycobacterial Diseases in Agra, India. The patient population was cate-
gorized into different clinical groups as follows. Group 1 (n ? 94) patients were
diagnosed with pulmonary TB for the first time and had no history of chemo-
therapeutic intervention. Group 1 had 69 adults and 25 children. Group 2 (n ?
30) patients had relapsed infection, and all recruited patients were adults. Group
3 (n ? 31) patients with extrapulmonary TB infections consisted of 9 adults and
22 children. The patients were categorized according to guidelines of the Na-
tional TB Control Program, Central TB Division, Government of India. Pulmo-
nary TB in patients in groups 1 and 2 was confirmed by the presence of acid-fast
bacilli in at least two initial sputum smear examinations and growth of bacilli in
BACTEC cultures. Patients with active TB infection were also examined for
radiological abnormalities by chest X-ray. In the case of group 2 patients with
relapsed infection, all patients were adults and were diagnosed with pulmonary
TB. After diagnosis, these patients had a full course of antitubercular chemo-
therapeutic treatment but had a recurrence of the infection and disease symp-
toms after therapy. On the other hand, group 1 patients were patients with active
pulmonary TB at the onset of disease who were attending the Outpatient De-
partment at the National Jalma Institute of Leprosy and Other Mycobacterial
Diseases, Agra, India, and had not received any antitubercular chemotherapeutic
intervention previously. Members from group 3 had primarily abdominal TB
infection and tubercular meningitis. The diagnosis of extrapulmonary TB was
carried out by histological examination as well as with culture positivity of the
bacillus in specimens obtained from extrapulmonary sites. The age ranges of
adults and children were 18 to 60 years and 2 to 15 years, respectively. In the case
of pulmonary TB patients as well as patients belonging to other categories,
samples were obtained from active untreated patients (both adults and children)
at the time of diagnosis. Samples were taken prior to commencement of che-
motherapy. Relapsed patients were those patients who had a recurrence of
symptoms after taking a full course of antitubercular treatment. Sera were
separated from blood and kept at ?20°C until use. The samples were obtained
from all donors upon entry into the study, and samples from human immuno-
deficiency virus-positive subjects were excluded from the study. The healthy
donors (n ? 56) included in the study were recruited after radiological and
clinical examination to exclude individuals with active TB. Additionally, healthy
FIG. 1. Cloning, expression, and in silico analyses of Rv0978c, Rv1169c, and Rv1818c. In silico analysis of Rv0978c (A), Rv1169c (B), and
Rv1818c (C) reveals the regions with high antigenic indices for potential antigenic determinants. The boxed areas represent potential regions that
might elicit variable immune reposes among the above-mentioned three PE family proteins. (D, E, and F) The protein gels represent the expression
and purification of Rv0978c, Rv1169c, and Rv1818c, respectively. Lane 1, uninduced E. coli; lane 2, induced E. coli; lane 3, marker; lanes 4 and
5, purified protein. The arrow indicates the position of the recombinant protein.
VOL. 14, 2007 B-CELL RESPONSES TO M. TUBERCULOSIS PE ANTIGENS 1335
donors included in the study were age and gender matched to the different
clinical groups. The included subjects had given written consent, and the current
study was carried out after approval from the institutional bioethics committee.
Cloning, expression, and in silico analysis of Rv0978c, Rv1169c, and Rv1818c.
The selected PE genes were PCR amplified from genomic DNA using gene-
specific primers: 5?-GCGGATCCATGTCGTTTGTCAACGTGGC-3? (forward)
and 5?-CGCTCGAGAGCTGATTACCGACACCGTGT-3? (reverse) for Rv0978c,
5?-TGTCTTTTGTCACCACACGG-3? (forward) and 5?-GGTGGAGGTGCCC
GCGCGGTT-3? (reverse) for Rv1169c, and 5?-CCGGAATTCATGTCATTTG
TGGTCACG-3? (forward) and 5?-CGCGGATCCCGGTAACCCGTTCATCC
C-3? (reverse) for Rv1818c. The amplified PE gene PCR product was ligated into
the pGEMT-Easy vector (Promega Inc.), and the recombinant clones carrying
the appropriate PE gene insert were confirmed by DNA sequencing. The PE
gene inserts were subcloned into cloned pRSET series vectors for protein ex-
pression and purification. Escherichia coli BL21 cells carrying recombinant plas-
mids were induced with IPTG (isopropyl-?-D-thiogalactopyranoside), and His-
tagged recombinant proteins were purified with Ni-nitrilotriacetic acid columns
(QIAGEN). In addition, recombinant E. coli clones expressing Rv1818c and
19-kDa antigens were kind gifts from M. J. Brennan, FDA, and Neil Reiner,
University of British Columbia, Canada, respectively.
In silico analyses of Rv0978c, Rv1169c, and Rv1818c were carried out accord-
ing to a method described previously by Jameson and Wolf (18). The program
generates values for surface accessibility parameters and combines these values
with those obtained for regional backbone flexibility and predicted secondary
structure. Furthermore, the program offers a reliable prediction of potential
Serological characterization of Rv0978c, Rv1169c, and Rv1818c. ELISAs were
carried out with 96-well microtiter plates (Nunc) using the above-mentioned
recombinant PE proteins. The plates were incubated with recombinant PE pro-
teins (1.25 ?g/ml) overnight at 4°C, followed by three washes with phosphate-
buffered saline (PBS)–Tween 20 (0.05%) buffer. After being blocked with 3%
bovine serum albumin in PBS, wells were incubated with human sera (1:400
dilution in blocking buffer) for 1 h at 37°C, followed by washing with PBS-Tween
20 buffer. The above-mentioned serum dilution was selected after careful titra-
tion experiments with different dilutions of the patient sera were carried out. The
plates were further incubated with anti-human immunoglobulin (Ig)-horseradish
peroxidase, followed by development with o-phenylenediamine tetrahydrochlo-
ride. The absorbance values were measured at 492 nm by using an ELISA reader
Statistical analysis. Student’s t test was used for analysis of statistical signifi-
cance (P value). The data for serological reactivities of different categories of
patients were compared with data for healthy controls. Graphpad Quickcalcs
(online t test calculator [http://www.graphpad.com/quickcalcs/ttest1.cfm]) was
used for this purpose. Only P values that were less than 0.05 were considered to
be significant. Comparisons of immunoreactivities within clinical groups were
carried out using Bonferroni’s multiple-comparison test (Graphpad Prism).
Expression and purification of Rv0978c, Rv1169c, and
Rv1818c. The recombinant PE proteins Rv0978c, Rv1169c,
and Rv1818c were cloned, expressed, and purified as described
in Materials and Methods (Fig. 1). In silico analysis of PE
Rv0978c (331 amino acids [aa]), Rv1169c (100 aa), and
Rv1818c (498 aa) was performed according to a method de-
scribed previously by Jameson and Wolf (18), which predicts
the topological features of a protein directly from its primary
amino acid sequence. The output of this algorithm, the anti-
genic index, is used to create a linear surface contour profile of
FIG. 2. Differential humoral reactivity of PE proteins with sera from group 1 adult TB patients. The recombinant Rv0978c and Rv1818c PE
family proteins elicit a strong and differential antibody response in M. tuberculosis-infected patients (diagnosed for pulmonary TB) in group 1 as
opposed to healthy controls (HC). However, the antibody responses to Rv1169c (C) and the 19-kDa antigen (D) are not statistically significant
(P ? 0.5) compared to those to Rv0978c (P ? 0.04 [IgG]; P ? 0.001 [IgM]) (A) and Rv1818c (P ? 0.006 [IgG]; P ? 0.0001 [IgM]) (B).
1336 NARAYANA ET AL.CLIN. VACCINE IMMUNOL.
the protein. Since most of the antigenic sites are located within
surface-exposed regions of a protein, the program offers a
reliable means of predicting potential antigenic determinants.
The antigenic index is calculated by summing several weighted
measures of secondary structure. The hypothetical PE open
reading frames Rv0978c, Rv1169c, and Rv1818c have a high
antigenic profile score, as obtained by in silico analysis, reveal-
ing the regions with a high antigenic index for potential anti-
genic determinants (Fig. 1A to C). Among all the proteins
belonging to the PE family analyzed, the selected PE proteins
Rv1169c, Rv0978c, and Rv1818c exhibited very high antigenic
indices for potential antigenic determinants as mentioned in
the legend of Fig. 1A to C. Furthermore, only 8 PE proteins
out of 37 PE proteins and 23 PE_PGRS proteins out of 62
proteins exhibited similarly high antigenic indices. However,
according to recent reports in the literature (26, 32), Rv1169c,
Rv0978c, and Rv1818c are among nine PE family proteins that
were shown to be expressed in M. tuberculosis bacilli upon
infection of macrophages. As shown in Fig. 1, experiments
utilizing the peptides with boxed protein sequences for assess-
ing reactivity with humoral and cell-mediated immune re-
sponses of TB patients are under way.
Rv0978c, Rv1169c, and Rv1818c elicit differential B-cell re-
sponses during infection with TB. Humoral immune responses
of group 1 patients (those reporting pulmonary TB disease
symptoms for the first time) to the recombinant proteins
Rv0978c, Rv1169c, and Rv1818c and the 19-kDa antigen were
evaluated along with healthy controls as shown in Fig. 2A to D.
In the case of group 1 adult pulmonary TB patients, only
Rv0978c and Rv1818c showed statistically significant IgG and
IgM immunoreactivities, while Rv1169c or the 19-kDa antigen
did not react to the humoral response in either the IgG or IgM
subtype. However, in the case of children in group 1, only
Rv0978c, but not Rv1818c, Rv1169c, or the 19-kDa antigen,
elicited IgG antibody reactivity (Fig. 3). In contrast, all three
PE proteins studied as well as the 19-kDa antigen did not
demonstrate statistically significant reactivity to IgG and
IgM antibodies (Fig. 4 and data not shown, respectively) in
sera from adult patients with relapsed infection in group 2.
The sera derived from group 2 children were not available
for our studies.
In the case of group 3 patients who reported extrapulmonary
infections, only children and not adults elicited significant IgG
immunoreactivity (Fig. 5 and data not shown). Among the PE
antigens studied, only Rv0978c and Rv1169c showed strong
reactivity to IgG, but not to IgM, in sera from group 3 children
over healthy controls (Fig. 5 and Table 1). However, the lack of
reactivity to Rv1818c suggests the possibility of stage-specific
expression of Rv1818c in group 1 patients reporting pulmonary
TB infection. Similarly, the specific reactivity of Rv1169c with
sera from patients with extrapulmonary TB infection indicates
the likelihood of the stage-specific expression of some of the
above-mentioned PE antigens.
The specificity and sensitivity of IgG and IgM antibody re-
activity to Rv0978c, Rv1169c, and Rv1818c were calculated in
order to address the serodiagnostic efficacy of these antigens.
FIG. 3. Among studied PE proteins, only Rv0978c elicited reactiv-
ity in child pulmonary TB patients in group 1. The group 1 child
patients showed statistically significant humoral IgG antibody reactiv-
ity to Rv0978c (P ? 0.0348) and the 19-kDa antigen (P ? 0.0174) but
not to Rv1169c or Rv1818c (P ? 0.05). The elicited immune response
comprises only IgG but not the IgM class of humoral response. HC,
FIG. 4. The studied PE proteins did not react with sera from group
2 adult patients with relapsed TB infection. The selected PE antigen
Rv0978c, Rv1169c, or Rv1818c or the 19-kDa antigen (P ? 0.05) did
not demonstrate significant immunoreactivities in sera obtained from
adult patients with relapsed TB infections. The lack of serum immu-
noreactivities was observed in both the IgG and IgM classes of the
humoral responses, and data for the IgG class of antibodies are pre-
sented in this figure. HC, healthy controls.
FIG. 5. Rv1169c is recognized only by sera from patients with ex-
trapulmonary infections. The children reporting extrapulmonary infec-
tions belonging to group 3 mounted significant IgG responses to
Rv0978c (P ? 0.001), Rv1169c (P ? 0.0344), and the 19-kDa antigen
(P ? 0.0084) but not to the Rv1818c antigen (P ? 0.5). Among the
clinical categories studied, Rv1169c elicited immunoreactivity only
with group 3 children. The data represented here are comprised of IgG
antibody reactivities in sera of patients. HC, healthy controls.
VOL. 14, 2007B-CELL RESPONSES TO M. TUBERCULOSIS PE ANTIGENS1337
The specificities for Rv1818c and Rv0978c immunoreactivity in
the case of adult patients belonging to group 1 or group 3 were
93% and 94%, respectively, for IgG and 98% and 96%, re-
spectively, for IgM. However, Rv1169c did not elicit immuno-
reactivity in the case of group 1 or group 3 adult patients. The
specificity for Rv1169c and Rv0978c immunoreactivity in the
case of child patients belonging to group 3 was 84% for IgG,
and the immunoreactivity of IgM antibodies to Rv1169c or
Rv0978c was not statistically significant. In addition, Rv1818c
did not elicit IgG or IgM reactivity in the case of group 3
children. The sensitivity was generally over 85% for the above-
mentioned cases where specificity was over 80%.
To compare the serological sensitivities (both IgG and IgM)
to Rv0978c, Rv1169c, and Rv1818c, the data presented in Fig.
2 to 5 were recalculated as percentages of individuals showing
ELISA absorbance values above the means plus 2 standard
errors of the means for healthy controls (Table 1). Among all
patients, Rv0978c and Rv1818c elicited strong IgM antibody
responses in a very high percentage of adult group 1 individ-
uals (78% for Rv0978c and 90% for Rv1818c), while Rv1169c
elicited IgG responses (56%) more frequently than it elicited
IgM responses (41%) in group 3 children with extrapulmonary
infection. In addition, when the immunoreactivities to two
selected PE_PGRS antigens were compared within the clinical
group 1 adult pulmonary TB patients (Fig. 6A and B), while
Rv0978c elicited immunoreactivity in sera from adult and child
as in sera from children with extrapulmonary infection in group 3.
However, Rv1169c elicited specific reactivity in sera from group 3
FIG. 6. Immunoreactivities comprised of differential antibody responses to two selected PE_PGRS antigens, Rv1818c and Rv0978c. Shown is
a comparison of the immunoreactivities of adult patients to Rv1818c (A) and to Rv0978c (B) and of child patients to Rv1818c (C) and to Rv0978c
(D). The IgG antibody immunoreactivities are represented in the figure. HC, healthy controls.
TABLE 1. Immunoreactivities to the PE antigens studieda
OD ? 2
OD ? 2
OD ? 2
OD ? 2
OD ? 2
OD ? 2
Adults, group 1
0.29 ? 0.03669.50.79 ? 0.0878.20.78 ? 0.159.2 0.62 ? 0.06 89.80.55 ? 0.06 18.8 0.72 ? 0.0646.3
0.45 ? 0.06
0.61 ? 0.16
0.75 ? 0.16
0.98 ? 0.8
80.51 ? 0.14
0.56 ? 0.14
0.67 ? 0.16
0.8 ? 0.16
0.53 ? 0.16
0.50 ? 0.12
0.85 ? 0.16
0.85 ? 0.22
aOD, optical density. % of patients, percentage of patients with ELISA absorbance values greater than the means plus 2 standard errors of the means for healthy
1338 NARAYANA ET AL.CLIN. VACCINE IMMUNOL.
patients only (data not shown). The data presented in Fig. 6
represent the immunoreactivities of the IgG class of the humoral
These results indicate that while Rv0978c is recognized by
sera from pulmonary TB patients belonging to group 1 or by
sera from child patients belonging to group 3, Rv1818c and
Rv1169c elicit specific reactivity with adult group 1 patients
and group 3 patients, respectively. More importantly, the im-
munoreactivity elicited in patient sera demonstrated differen-
tial reactivities of the humoral antibodies to Rv1818c versus
Rv0978c (Fig. 6 and 7). The data presented in Fig. 6 and 7
represent the immunoreactivities of the IgG class of the hu-
moral response. The ratios of responses to Rv1818c and
Rv0978c are quite different in adult and child pulmonary TB
patients belonging to group 1. The ratio of responses to
Rv1818c and Rv0978c in the case of adult pulmonary TB
patients was anywhere between two- and ninefold, while the
ratio was much less than onefold in the case of child pulmonary
TB patients. The possible reasons for such a discrepancy in the
ratio of reactivity are described above, as Rv1818c clearly dem-
onstrated significant immunoreactivity only with adult, not
with the majority of child, group 1 patients. Overall, this dif-
ferential reactivity to Rv1818c and to Rv0978c clearly suggests
the degree of difference in recognition by patient sera even
though they share similar glycine-rich C-terminal regions.
M. tuberculosis is the etiologic agent of TB and a major cause
of morbidity and mortality worldwide, with one-third of the
world’s population estimated to be infected with this microor-
ganism (22). After acquiring M. tuberculosis by inhaling aero-
solized bacteria, the majority of healthy individuals develop a
cellular immune response and arrest the growth and spread of
the microorganism, without it progressing to clinical TB. This
protective cellular immune response to M. tuberculosis is initi-
ated in the lung and consists primarily of alveolar macrophages
and activated T cells. However, M. tuberculosis survives for
prolonged periods of time in the phagosomes of infected mac-
rophages in a hypoxic environment within the host (21) in an
asymptomatic latent state and can reactivate years later if the
host’s immune system wanes. In this context, M. tuberculosis
was reported to upregulate the expression of many genes that
might be important for its survival amid a hostile host re-
sponse. One such set of genes is the PE family of genes (9–12,
14). As mentioned above, some members of the PE family
are implicated in the replication and survival of bacilli in
granulomas as well as being the source of antigenic varia-
tions upon infection. However, the exact nature of the role
played by PE genes in providing survival benefits to macro-
phages harboring M. tuberculosis from surrounding T cells in
granulomas or during pathogenesis associated with infection
The principal objective of the current study is to compare
the selected PE proteins Rv1169c, Rv0978c, and Rv1818c to a
well-characterized antigen, the 19-kDa antigen, in terms of the
humoral immunoreactivities that they elicit in the sera of TB
patients as well as healthy individuals. The 19-kDa antigen is a
culture filtrate protein known to elicit strong humoral as well
as cell-mediated host immune responses (6, 15, 16, 20). Fur-
thermore, the 19-kDa antigen was suggested to exhibit several
immunomodulatory functions such as the inhibition of major
histocompatibility complex class II expression and antigen pro-
FIG. 7. Individual patients demonstrate varied antibody responses to Rv0978c versus Rv1818c. Differential humoral responses to Rv0978c and
Rv1818c from sera of selected TB adult (A) and child (B) group 1 patients (numbered) are shown. The figure represents the IgG antibody
responses to Rv0978c and Rv1818c that varied to a high degree in selected patients even though both PE proteins (PE_PGRS) share similar
glycine-rich C-terminal domains.
VOL. 14, 2007B-CELL RESPONSES TO M. TUBERCULOSIS PE ANTIGENS 1339
cessing and the inhibition of gamma interferon-induced im-
mune genes, etc. (23–25).
In the current investigation, Rv0978c, Rv1169c, and Rv1818c
elicited statistically significant yet differential immunoreactivi-
ties among three selected categories of TB patients. Impor-
tantly, not-so-significant or poorer reactivities were observed
against the three PE proteins in the sera of all healthy individ-
uals tested. Furthermore, the immunoreactivity data presented
in Fig. 2 to 6 suggest that Rv1818c or Rv1169c may be ex-
pressed during a specific stage of the disease. In contrast,
Rv0978c seems to be immunoreactive with sera derived from
either group 1 (pulmonary infection) or group 3 (extrapulmo-
nary) patients. In order to understand the molecular mecha-
nisms that underlie the humoral response elicited by PE anti-
gens, we would like to assess patient T-cell reactivity with
specific HLA class I and class II binding peptides derived from
the above-described PE family antigens, as the antibody re-
sponse in many cases is often regulated by initial T-cell re-
sponses during infection. However, we agree with the fact that
assessments of T-cell reactivity may not necessarily be a logical
step in the further establishment of the selected PE antigens as
The presence of antibodies to Rv0978c, Rv1169c, and
Rv1818c in sera of TB patients clearly suggests that these PE
proteins are expressed in vivo during active infection with M.
tuberculosis and that the above-described PE molecules are im-
munogenic. It has been observed that some of the PE_PGRS
proteins are antigenic, where antibodies raised against five
PE_PGRS proteins by DNA vaccination reacted with the re-
spective proteins expressed in epithelial cells or in reticulocyte
extracts (3, 5). Some of the antibodies showed cross-reactivity
with more than one PE_PGRS protein, suggesting the pres-
ence of common epitopes. However, the dot blot technique
used in the above-mentioned study is not a very reliable
method to ascertain either the size or the nature of the cross-
reactivity of antibodies raised against any given PE_PGRS
protein. In our studies, we have found that rabbit polyclonal
antibodies generated against Rv0978c do not cross-react with
recombinant Rv1818c (data not shown). In addition, Rv0978c
contains novel tandem repeats, called AB repeats, in the C
terminus, which are absent in most of the PE (or PE_PGRS
subfamily) genes, including Rv1818c (2).
Other studies have demonstrated that sera from TB patients
showed immunoreactivity to many of the PPE family proteins,
in addition to PE family proteins (7, 8). Many studies in the
literature clearly emphasized the observation of a lack of a
sufficient immune response in TB patients against many well-
characterized serodiagnostic antigens of TB bacilli. The above-
mentioned condition is more severe in newer cases of pulmo-
nary TB infection where the host immune system is not
sufficiently primed to elicit a strong humoral response against
most antigens of M. tuberculosis. Overall, our observations
suggest the possible serodiagnostic potential of Rv1818c,
Rv1169c, and Rv0978c during different stages of infection.
We are grateful to Kiran Katoch and D. S. Chauhan for the gener-
ous gift of TB patient sera. We are thankful to M. J. Brennan, FDA,
and to Neil Reiner, University of British Columbia, Canada, for the
kind gifts of recombinant expression clones for Rv1818c and 19-kDa
antigens. We thank Dipankar Nandi for critical comments and Naga-
suma Chandra, Rashmi Chaturvedi, and V. L. Suhas for their help
during the preparation of the manuscript.
The current study was funded by the Sir Dorabji Tata Center for
Research in Tropical Diseases to K.N.B. and V.M.K. as well as funds
from the Council for Scientific and Industrial Research, Department
of Biotechnology, and Department of Science and Technology, Gov-
ernment of India, to K.N.B. Y.N. and K.C.M. are supported by a
fellowship from the Council for Scientific and Industrial Research and
the University Grants Commission, Government of India, respectively.
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