Discovery of novel Schistosoma japonicum antigens using a targeted protein microarray approach
Parasites & Vectors
Discovery of novel Schistosoma japonicum antigens using a targeted protein microarray approach
Hamish EG McWilliam 0
Donald P McManus
Els NT Meeusen
0 Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity , Melbourne, Victoria , Australia
Background: Novel vaccine candidates against Schistosoma japonicum are required, and antigens present in the vulnerable larval developmental stage are attractive targets. Post-genomic technologies are now available which can contribute to such antigen discovery. Methods: A schistosome-specific protein microarray was probed using the local antibody response against migrating larvae. Antigens were assessed for their novelty and predicted larval expression and host-exposed features. One antigen was further characterised and its sequence and structure were analysed in silico. Real-time polymerase chain reaction was used to analyse transcript expression throughout development, and immunoblotting and enzyme-linked immunosorbent assays employed to determine antigen recognition by antibody samples. Results: Several known and novel antigens were discovered, two of which showed up-regulated transcription in schistosomula. One novel antigen, termed S. japonicum Ly-6-like protein 1 (Sj-L6L-1), was further characterised and shown to share structural and sequence features with the Ly-6 protein family. It was found to be present in the worm tegument and expressed in both the larval and adult worms, but was found to be antigenic only in the lungs that the larvae migrate to and traverse. Conclusions: This study represents a novel approach to vaccine antigen discovery and may contribute to schistosome vaccine development against this important group of human and veterinary pathogens.
Schistosoma japonicum; Vaccine development; Ly-6 proteins; Protein microarray; Immunomics
An effective vaccine against schistosomiasis would be a
major step towards eliminating this devastating and
widespread tropical parasitic disease, and has been the focus of
significant research effort for decades . Numerous
schistosome vaccine candidates have been discovered
showing varying levels of protection, and several
molecules are in development ; however, a commercial
vaccine still remains elusive. It has been suggested that more
effective vaccine candidates remain undiscovered,
indicating a need for novel approaches to target discovery [1,3].
The radiation-attenuated larval vaccine model provides
evidence that high levels of protection can be generated
against schistosome infection. When a range of animal
models are infected with radiation-attenuated cercariae,
the resulting immunity rejects up to 90% of the challenge
infection [4,5]. In this model the larvae do not progress to
adult worms, and the consensus is that the early
developing schistosomula represent a potent source of protective
antigens and are the target of this protective immune
response [4,5]. Furthermore, larval stages are also suspected
of being the target of naturally acquired immunity in
humans [6-8]. In contrast, the adult worms can live in the
host bloodstream for decades despite being surrounded by
specific components of the immune response [9,10].
Therefore larval-specific antigens, or molecules exposed
uniquely in larvae, have the potential to serve as
effective novel vaccine candidates. Despite this, the current
schistosome vaccine targets are largely adult antigens
and there is a lack of larval-specific antigens being
Recently the genomes of all three major schistosome
species have been sequenced [12-14], and several
postgenomic approaches and high throughput methods have
been developed to take advantage of this wealth of
information . One such approach is a schistosome-specific
protein microarray, which contains 232 unique antigens
displayed on nitrocellulose slides . These proteins
were selected from bioinformatic data using criteria biased
towards promising vaccine candidates i.e. up-regulated
expression in larvae, predicted/known parasite surface
expression, and limited similarity with mammalian
sequences . Many of these are novel molecules and the
majority are from Schistosoma japonicum, with the
remainder from S. mansoni. The arrayed proteins can be
probed with antibodies from immune hosts as a powerful
new technology for vaccine antigen discovery .
In the present study, this protein microarray was
screened with antibodies generated from lymph nodes
draining the sites of larval S. japonicum migration .
Referred to as antibody secreting cell-probes (ASC-probes),
these antibodies are specific to the migrating skin and lung
larvae , and were generated from S.
japonicum-infected rats, which is a well-studied model of
antibodymediated immunity against the migrating larvae during a
secondary infection [18-20]. Hence, these ASC-probes
have the potential to recognise protective larval antigens.
Using this approach we identified several novel antigens
expressed by schistosomula; one promising candidate was
further investigated indicating it may be an important
target for vaccine development.
The conducts and procedures involving animal
experiments were approved by the Monash University animal
ethics review board, and the Animals Ethics Committee
of the Queensland Institute of Medical Research (project
no. P288). This study was performed in accordance with
the recommendations of the Australian code of practice
for the care and use of animals for scientific purposes,
Protein microarray screening
A protein microarray consisting of 232 unique
schistosome proteins was prepared as described by Driguez et al.
. The microarray slides were hydrated using Blocking
Buffer (BB; Whatman) for 3060 min at RT, using 16-pad
incubation chambers and frames (Whatman) to separate
arrays. Antibodies used for screening the microarrays were
obtained from lymph nodes of challenged rats as detailed
in McWilliam et al. . Briefly, previously infected rats
were challenged with 350 cercariae and skin and lung
lymph nodes (LN) removed at 5 and 9 days after challenge
respectively. This protocol was designed based on
previous observations that rats show antibody-mediated
immunity against larvae during secondary infections [18,19].
LN cell suspensions were cultured in vitro for 5 days to
allow spontaneous secretion of antibodies by in vivo
induced antibody secreting cells (ASC) and the supernatant
collected and used as the antibody probe (=ASC-probe).
Considering ASC-probes from non-infected (NI) rats
contain little antibody , they are not ideal controls for
non-specific antibody binding; NI rat sera was therefore
used as the negative control. Four skin and four lung
ASC-probes (undiluted) or 15 NI serum samples (1:100 in
BB) were incubated on the arrays in a sealed box
overnight at 4C. Skin and lung ASC-probes were selected
based on those containing the highest antibody
concentration as documented in . Arrays were washed 3 times
with tris-buffered saline (TBS; 20 mM tris, 150 mM NaCl)
with 0.05% Tween-20 (TBST), followed by
biotin-conjugated anti-rat IgG (1:1000 in BB) for 1 h at RT. After 3
washes in TBST, streptavidin-conjugated Cy5 fluorophore
(Surelight P3, Columbia Biosciences; 1:200 in BB) was
incubated for 1 h at RT. After a final 3 washes in TBST the
array slides were separated from the chambers, washed in
distilled water and dried by centrifuging for 5 minutes at
500 g. The slides were stored in the dark until scanned.
Microarray scanning and antigen identification
Scanning was performed on a confocal laser microarray
scanner (Genepix 4300A, Molecular Devices), and the
signal intensity (SI) was quantified using image analysis
software (Genepix Pro 7, Molecular Devices) and transformed
and normalised using the vsn statistical package (http://
www.r-project.org). Finally the data were re-transformed
(inverse log2) to a normalised SI [21-23].
Antigens were considered positively recognised by an
ASC-probe sample when the SI was greater than two
standard deviations above the mean of NI rat sera. Amino
acid sequences of the identified antigens were then
analysed for features of potentially important vaccine
candidates i.e. developmental expression based on EST Profile
Viewer (http://www.ncbi.nlm.nih.gov/ unigene), presence
of a signal peptide using SignalP 4.1 (www.cbs.dtu.dk/
services/SignalP), and a predicted transmembrane domain
with TMpred (www.ch.embnet.org/software/TMPRED_
form.html). Also, literature searches were performed to
determine the novelty of each antigen, which were
considered novel when there were no reports of vaccine
efficacy against S. japonicum or significant characterisation.
RNA isolation from schistosome life-stages
Cercariae were obtained from freshly-shed infected
Oncomelania hupensis hupensis snails and transferred using a
sterile bacterial loop directly to Qiazol Lysis Reagent
(Qiagen). Two-day (2d) schistosomula were manually
transformed from cercariae using the syringe method as
described in McWilliam et al. , and cultured in vitro
for 2 days at 37C with 5% CO2. Three-day (3d)
schistosomula were obtained from infected mice lungs 3 days
post-infection (lung-stage schistosomula) as described by
Gobert et al. , and were lysed directly in Qiazol. Adult
worm pairs were obtained from freshly-perfused mice,
and either left as pairs or carefully separated, and washed
in PBS before homogenising in Qiazol. The RNA was then
extracted from each stage following the manufacturers
protocol and stored in water at 80C until required.
Developmental expression of novel antigens by
quantitative real-time PCR (qPCR)
After determining the RNA concentration using a
NanoDrop spectrophotometer (Thermo Scientific), total RNA
(300 ng) was converted to cDNA using the QuantiTect
Reverse Transcription Kit (Qiagen) including the genomic
DNA removal step. To perform the qPCR, primers were
designed based on the novel antigen sequences identified
in the protein microarray screening and using NADH
dehydrogenase ubiquinone flavoprotein 2 (NDUFV2) as
the reference gene . The primer sequences were:
AY815838 (forward: 5- CGTCGACATTCAAGTTGG
TC -3, reverse: 5- GGGGCATAATCTTCACTTTGA -3);
Ly-6-like-1 (forward: 5- TGAAAGTTTTGGGACTTTG
TATG -3, reverse: 5- CGAATGGATTCGGACAGT
CT -3); calponin-like (forward: 5- CATGTCATTCGGT
GCTCAAC -3, reverse: 5- TTCAGCAATATGACGTT
GATTACTA -3) and NDUFV2 (forward: 5- CGAG
GACCTAACAGCAGAGG -3, reverse: 5- TCCGAAC
GAACTTTGAATCC -3). Because calponin-like protein
has homology to another calponin homologue in S.
japonicum (accession #AAD11976), the primers were designed
in the most dissimilar region. The qPCR reactions
included SYBR Master Mix (Applied Biosystems) and the
above primers at 0.5 M and 5 l of 1:20 diluted cDNA,
and were run in technical triplicates on an Eppendorf
Realplex4 Mastercycler for 35 cycles, using an annealing
temperature of 55C. Melt curve analysis was performed
to ensure a single product was amplified. The relative
copy number of SYBR green for each gene was calculated
from a standard curve of serial dilutions of cDNA, and the
relative expression of the target gene was determined
relative to the reference gene copy number. Finally, the
expression of each gene was calculated relative to the
cercarial cDNA level to observe the change throughout
Sequence analysis of S. japonicum ly-6-like-1 (Sj-L6L-1)
The NCBI databases (http://blast.ncbi.nlm.nih.gov/Blast.
cgi) were used to identify Sj-L6L-1 related S. japonicum,
S. mansoni, and mammalian species (Mus musculus and
Homo sapiens). For other parasitic trematodes (S.
haematobium, Fasciola hepatica, F. gigantica, Opisthorchis viverrini
and Clonorchis sinensis), searches were performed using the
databases at www.gasserlab.org. Sequence alignments were
performed by ClustalW2 (http://www.ebi.ac.uk/Tools/msa/
clustalw2/). The presence of a signal peptide and omega site
(which denotes the position where the C-terminal
propeptide is cleaved off in the mature protein and is
replaced with a glycosylphosphatidylinositol (GPI)-anchor)
was predicted by SignalP 4.1 (www.cbs.dtu.dk/services/
SignalP/) and big-PI predictor (http://mendel.imp.ac.at/
gpi/gpi_server.html), respectively. Structural homology
searches were performed using the amino acid sequence
of mature Sj-L6L-1 without the predicted N-terminal
signal peptide and C-terminal propeptide (beginning at M20
and truncated at the predicted GPI-anchor attachment
site at N96) in the Phyre2 server (www.sbg.bio.ic.ac.uk/
phyre2) , and the protein structure was displayed
using PyMOL (v1.3; www.pymol.org).
Production and purification of recombinant Sj-L6L-1
The Sj-L6L-1 sequence was amplified from the pXi T7
vector used in the construction of the protein arrays .
Primers were designed to produce the mature Sj-L6L-1
protein (M20-N96) (forward primer: 5-ATGAAAAA
TAAAAAGGTCAAATG-3, reverse: 5-ATTACAATAAT
CTTCATCACAAC-3). Amplification of the 231 bp
SjL6L-1 fragment was performed using Phusion High Fidelity
DNA Polymerase (New England Biolabs). Next 3 adenine
overhangs were added by incubating the PCR product with
1 unit of Platinum Taq DNA Polymerase (Life
Technologies) and 0.2 mM dATP for 10 min at 72C. This was
purified and then cloned in-frame into the pBAD/TOPO
ThioFusion plasmid (Life Technologies) according to the
manufacturers directions. This resulted in a fusion protein
with thioredoxin (Escherichia coli) as a solubility tag and
6His purification tag at the amino- and carboxy- termini,
The plasmid was sequenced to confirm the correct
sequence, and then transformed into TOP10 E. coli cells
(Life Technologies). Expression was induced by adding
arabinose (0.005% w/v), and soluble rSj-L6L-1 present
in the bacterial lysate supernatant was purified on a
HisTrap HP column (GE Healthcare) according to the
manufacturers instructions. Fractions were analysed
by sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE) and the purest fraction was
dialysed into TBS (pH 8.0) and stored at 20C. The
thioredoxin fusion partner alone (with the 6His tag;
referred to as rTrx) was also produced using the empty
pBAD vector, and purified in the same way for use as a
Antiserum against rSj-L6L-1 and rTrx was generated
by injecting a rat with 50 g of rSj-L6L-1 or rTrx with
200 g Quil A in 0.1 ml PBS. A secondary immunisation
was administered 2 weeks later, after which a test bleed
indicated a specific antibody response to the
Schistosome protein extracts
Crude worm extracts were prepared by collecting each
developmental stage (as above), washing with TBS, and
homogenising in 1% (w/v) SDS in TBS. Samples were
centrifuged at 12,000 g for 15 min at RT. The protein
concentration of the supernatants was determined by
BCA assay (Pierce, Rockford). To determine whether
Sj-L6L-1 was soluble and present in the tegument, adult
worms were separated into different fractions. Tegument
was gently removed by a modified freeze/thaw/vortex
technique [27,28]. Briefly, washed and snap-frozen worm
pairs were thawed in TBS (pH 7.5) on ice for 5 min,
followed by vortexing for 5 1 sec to allow gentle
removal of tegument. The supernatant was removed and
centrifuged at 13,000 g for 10 min at 4C. The
tegument pellet was resuspended in lithium dodecyl sulphate
(LDS) sample buffer (Life Technologies) and heated to
95C for 10 min. The remaining worms were washed in
20 mM Tris (pH 7.4) and then homogenised in the same
buffer. After centrifugation, as before, the supernatant
was kept as the soluble fraction. The pellet was washed
twice with buffer followed by another centrifugation
step, and then solubilised in 1% SDS. After spinning
again, the supernatant was kept as the aqueous-insoluble
Western blotting for detection of native and recombinant
Purified rSj-L6L-1 or rTrx (1 g) in LDS sample buffer
with or without reducing agent (50 mM dithiothreitol;
DTT), were run on 10% NuPAGE Bis-Tris gels, along with
Novex Sharp Pre-stained Protein Standards (Life
Technologies) and stained with Coomassie blue. For western
blotting, rSj-L6L-1 (0.5 g) or worm extracts (10 g) were
separated by SDS-PAGE and transferred onto
nitrocellulose membranes, which were then blocked overnight at
4C in 5% w/v skim milk powder in PBST (SM-PBST).
After washing three times in PBST, the primary antibodies
were added: rSj-L6L-1 was probed with neat pooled rat
lung-LN ASC-probes from infected rats; worm extracts
were probed with either anti-rSj-L6L-1 or anti-rTrx
antiserum (1:500 in 1% SM-PBST). After washing, anti-rat Ig
(H + L):HRP (1:1000) (Life Technologies) was incubated
with the membranes for 1 h at RT, and the membranes
rewashed. Finally ECL substrate (GE Healthcare) was
applied and chemiluminescence detected on Super RX film
Recognition of rSj-L6L-1 by enzyme-linked immunosorbant
An ELISA was used to quantify the antigenicity of rSj-L6L-1
during schistosomiasis, using the ASC-probes and serum
samples from infected rats as described in McWilliam et al.
. Some samples (particularly NI serum) showed binding
to the Trx fusion partner alone (data not shown); therefore
binding was measured to both rSj-L6L-1 and rTrx. Wells
were coated with recombinant protein (3 g/ml) in
carbonate coating buffer (pH 9.6) overnight at 4C, and
blocked with 5% SM-PBST. The samples used for probing
consisted of NI rat serum at 1:200 (n = 3) or neat
ASCprobes from infected rat skin LN (n = 5); lung LN (n = 5);
liver LN (n = 5); and spleen (n = 4). Duplicate samples were
incubated in the wells for 2 h at 37C. After washing
antirat Ig (H + L):HRP (1:5000) was incubated for 1 h at 37C.
TMB solution (Life Technologies) was added to each well,
followed by 2 M H2SO4, and the optical density (OD) was
read at 450 nm. Statistical differences in binding of
ASCprobes to Sj-L6L-1 were assessed using a Kruskall-Wallis
one-way analysis of variance.
Protein array screening with ASC-probes for antigen
Screening of the 232 schistosome protein microarray with
antibodies obtained from lymph nodes (ASC-probes) of
S. japonicum challenged rats resulted in significant
recognition of 11 antigens (Table 1). Nine of these were
recognised by the lung ASC-probes, three by the skin
ASCprobes and one (tetraspanin-2; TSP-2) was recognised by
both skin and lung ASC-probes. Only 3 antigens were
consistently recognised by all four of the lung ASC-probes;
these were the novel hypothetical protein AY815838, and
the known vaccine candidates TSP-2 and 21.7 kDa antigen
(Sj21.7). Other known molecules that were recognised
by the ASC-probes included 22.6 kDa antigen (Sj22.6),
S. mansoni filamin, dynein light chain 1 (DLC1), S. mansoni
tetraspanin-3, and the 29 kDa antigen (Sj29). Because the
amount of protein in each spot is not standardised due to
the cell-free expression system used [29,30], it should be
noted, however, that the degree of antibody binding to
proteins on the array does not necessarily indicate antigenicity.
Therefore, even weakly recognised antigens, such as
Ly-6like protein 1 (Sj-L6L-1), which was only recognised by one
skin and lung ASC-probe sample, may also be considered
as relevant antigenic targets.
To select antigens for further characterisation, each of
the 11 sequences were investigated as to its novelty, its
potential for up-regulated larval expression, and predicted
exposure to the hosts immune system (indicated by the
presence of a signal peptide and/or transmembrane
domain). Five of the eleven proteins identified were novel
and recognised by lung ASC probes, and three of these
Table 1 S. japonicum protein array antigen recognition by rat ASC-probes
Recognition* by ASC-probes
21.7 kDa antigen (Sj21.7)
Zinc finger protein
29 kDa antigen (Sj29)
Tetraspanin-3 SmTSP3 (S. mansoni)
*Recognition level score: the average signal intensity of the individual(s) which were greater than the mean + 2SD of control serum. High (+++) >10000 SI; Moderate
(++) 5009999 SI; Low (+) 0499 SI; Negative () 0 SI. Number of ASC-probe samples (out of four) that recognised each antigen is indicated in parentheses. Novel:
defined as not having been tested as a vaccine candidate against S. japonicum and limited characterisation. #Expression profile: The stage(s) with the highest expression
is indicated, based on EST Profile Viewer, UniGene, NCBI: cercariae (C), schistosomula (S), adult worm (A), egg (E), sporocyst (Sp). Signal peptide determined by SignalP 4.1
(www.cbs.dtu.dk/services/SignalP/). Transmembrane prediction by TMpred (www.ch.embnet.org/software/TMPRED_form.html) ^Data on sequence not present in
UniGene. Three antigens possessing ideal characteristics as novel vaccine candidates are highlighted in bold.
had indications that they were highly expressed in the
schistosomula stage. Two of the novel antigens, AY815838
and Sj-L6L-1, were predicted to have signal peptides and
transmembrane domains. The third novel antigen
identified, calponin-like protein, had no predicted host-exposed
The AY815838 protein is currently unknown, but has
limited amino acid sequence identity to two surface
antigens of S. mansoni: Sm25 (accession #AAA29943; 34%
identity) and Sm13 (accession #AAC25419.1; 30%
identity). Sj-L6L-1 has significant homology to a S. mansoni
antigen that was briefly investigated in a DNA vaccine
study  and recently designated Sm-CD59.2 . Finally
calponin-like protein has a predicted size of 27 kDa and
has some homology with a 38 kDa S. japonicum calponin
homologue (accession #AAD11976; 63% identity)
previously investigated .
Developmental expression analysis of novel antigens
The developmental expression of the three novel antigen
transcripts was investigated by qPCR (Figure 1). The
AY815838 transcript was most highly expressed in the
2 day schistosomula, elevated to 65 times the cercarial
expression, which then reduced to 5 and 10 times cercarial
expression levels in 3-day schistosomula and adult pairs,
respectively. The calponin-like protein showed a steady
increase in expression throughout the development of the
intravascular stages, peaking in the adult worms at 41
times the cercarial expression. Finally, Sj-L6L-1 was very
highly expressed in the developing schistosomula; the
Figure 1 Developmental expression of novel S. japonicum antigens recognised by lymph node-derived ASC-probes. Expression levels of
AY815838 (A), calponin-like protein (B) and Sj-L6L-1 (C) in developmental stages of S. japonicum were determined by quantitative real-time PCR.
Stages examined were cercariae (C), 2 day- (2d) and 3 day-old (3d) schistosomula (S), adult pairs (A), adult males (M), adult females (F) and eggs
(E). Normalized fold expression of the genes relative to the expression in cercariae is presented, and bars represent standard error of the mean.
2-day in vitro cultured and the 3-day in vivo lung-stage
larvae had 23 and 27 times the cercarial expression
respectively, whereas adult males had just a 3-fold -increase
in transcript level. For all three genes, expression in adult
worms was predominantly, or restricted to, the males.
These data suggested AY815838 and Sj-L6L-1 were
promising novel targets with predominant larval expression.
The larval expression of Sj-L6L-1 was particularly
convincing since there was strong agreement in transcript levels in
samples from both in vitro and in vivo-generated larvae
(2- and 3-day schistosomula, respectively). AY815838 was
identified in a previous screening of the microarray and is
being currently investigated (P. Driguez and D. McManus,
personal communication); therefore Sj-L6L-1 was selected
for further detailed characterisation.
Sequence analysis of Sj-L6L-1
Database searching revealed Sj-L6L-1 has similarity with
the Lymphocyte Antigen 6 (Ly-6) family of proteins,
present in many eukaryotic species (Figure 2). The
membrane-anchored members of this family share several
features: a predicted N-terminal signal peptide, a
glycosylphosphatidylinositol (GPI)-anchor omega site where the
C-terminal propeptide is cleaved off in the mature protein,
Figure 2 Sequence and predicted structure of mature Sj-L6L-1. A: The Sj-L6L-1 sequence aligned with the highly similar orthologous sequences
from S. mansoni (Sm-CD59.2) and S. haematobium (Sh-L6L-1) and compared with Ly-6 family proteins from mice (Mm-Ly-6D) and humans (Hs-E48 and
Hs-CD59). The N-terminal signal peptide and the C-terminal propeptide are highlighted in grey, while structurally important cysteine residues are
highlighted in red. The predicted omega site (GPI-anchor site) is highlighted in blue. The predicted mature Sj-L6L-1 sequence is indicated within the
black triangles. B: The predicted structure of mature Sj-L6L-1 was modelled based on a Phyre2 structure homology search, revealing a structure with
the typical three-finger fold common to Ly-6 family proteins. Each finger is labelled with green roman numerals. C: The mature Sj-L6L-1 amino acid
sequence has the predicted disulfide bonding pattern, also characteristic of this family, including the GPI-anchor at the C-terminus. The disulfide bonds
are shown and numbered in magenta, with those predicted by the in silico analysis depicted by solid lines and those manually inferred by dotted
and the Ly-6/uPAR (LU) protein domain characterised by
810 conserved cysteine residues .
Highly related orthologues to Sj-L6L-1 were found in
S. mansoni (SmCD59.2; accession number XP_002570561)
and S. haematobium (Sh-L6L-1; B_00489), which have 78
and 79% amino acid identity, respectively. All three have
most of the classical Ly-6 family features: predicted signal
peptides, GPI-anchor omega site, and ten cysteines in
the mature region (eight of these align with conserved
mammalian residues but two are in differing positions)
(Figure 2A). However, domain searching using the amino
acid sequence did not predict the presence of an LU
domain in these schistosome orthologues, most likely due to
the altered positions of the 3rd and 4th cysteines (see
Figure 2A). These altered cysteines were common in all
trematode orthologues examined (Figure 2A and data not
shown). The S. mansoni orthologue, SmCD59.2, was
recently described along with other members of its family by
Farias et al. . This family was referred to as CD59-like
proteins because they have some similarity with the
mammalian Ly-6 family member, CD59. However, since the
schistosome proteins more closely resemble the family of
Ly-6 proteins rather than one particular member, we
propose to describe them as Ly-6-like proteins. S.
japonicum also has several orthologous sequences to
Sj-L6L-1, although the closest Sj-L6L-2 (accession
number AAW26563.1), has only 38% identity.
The full pre-protein sequence of Sj-L6L-1 is predicted
to be 14.1 kDa, while the mature protein after the signal
peptide and propeptide are cleaved is estimated to be
8.9 kDa. However, the addition of the GPI anchor is
estimated to add a similar amount to the cleaved propeptide
, so the native form would be approximately 12 kDa.
Other parasitic trematode sequences with low identity
(31-34%) to Sj-L6L-1 were identified in F. hepatica
(Fh_Contig6273), F. gigantica (Contig25430), C.sinensis
(CS1_c757) and O. viverrini (OV1_c8524). The closest
mammalian orthologues are the mouse Ly-6D (also known
as ThB; 28% identity; accession # EDL29445), the human
E48 protein (32% identity; accession # CAA73189).
Human CD59 in contrast has 25% identity (accession #
Despite not having a traditional LU domain by
sequence searching, structural homology searching of the
mature Sj-L6L-1 sequence predicted structural similarity
with Ly-6 proteins. The highest scoring template
following the Phyre2 server search was the Ly-6 protein
Lynx1, with 98.3% confidence. As shown in Figure 2B, in
silico modelling predicted a three-fingered structure
which is common to Ly-6-like proteins . This
structure was predicted to contain 3 disulfide bonds,
numbered 1, 4 and 5 in Figure 2B and C. Disulfide bonds 2
and 3 depicted in Figure 2 were manually added since
this is the same pattern seen in other Ly-6 proteins such
as human Lynx1  and CD59 [38,39], and because the
residues are in close proximity in the model (Figure 2B).
Recombinant Sj-L6L-1 is in the native antigenic
Recombinant Sj-L6L-1 was produced fused to thioredoxin
(Trx) as a solubility tag, and induction with arabinose
caused a significant expression of a soluble protein band
at approximately 27 kDa as observed by reducing
SDSPAGE (Figure 3). This was purified by the 6His tag using a
nickel column. The protein band was excised and the
sequence confirmed by LC-MS/MS using a HCT ULTRA
ion trap mass spectrometer (Bruker Daltonics; Monash
Biomedical Proteomics Facility). Under non-reducing
conditions the protein had a molecular weight of 25 kDa
(Figure 3A, lane 4). This reduction in size was likely due
to a change in the Sj-L6L-1 portion of the fusion protein,
since reducing or non-reducing conditions had no effect
on the size of rTrx which remained at 16 kDa (lanes 2 and
3). Finally, immunoblotting revealed rSj-L6L-1 was
recognised by the rat lung ASC-probes from infected rats, but
only in the non-reduced form (Figure 3A, lane 6). This
indicated that the recombinant protein shares
conformational epitopes with the native protein, and these are
abolished by treating with reducing agent, presumably by
disrupting the disulfide bonds between the
It was also noted that there was a ladder effect with
rSjL6L-1 in the non-reduced sample (Figure 3A, lane 4).
These corresponded to multimers of the protein, visible at
50 kDa (dimer) and 75 kDa (trimer) and then increasing
masses that were not distinguishable. In the reduced
sample (lane 5) only an additional 55 kDa band was present,
likely to be a dimer of the reduced form. These bands also
reacted with an anti-His tag antibody (data not shown),
indicating that they are aggregates of rSj-L6L-1, but none
of the multimers were recognised by the lung ASC-probes
compared to the 25 kDa monomer.
Recombinant Sj-L6L-1 is recognised by the local lung
antibody response in rats
Since lung ASC-probes were found to recognise
nonreduced rSj-L6L-1 (Figure 3A), an ELISA was used to
investigate the binding of the different rat ASC-probe
samples to rSj-L6L-1 (Figure 3B). To account for some
background binding to the fusion tag alone, the data are
presented as relative binding (RB) of rSj-L6L-1 to rTrx,
where RB > 1 means the sample bound to rSj-L6L-1
greater than the rTrx control. Figure 3B illustrates that
the non-infected (NI) rat sera had no recognition of
rSjL6L-1 with a mean of 0.8 RB. The only sample type to
have a statistically significant recognition of rSj-L6L-1
was the lung ASC-probes, with a mean of 4.8 RB
(p = 0.039). The skin ASC-probes had some slight
Figure 3 Recombinant Sj-L6L-1 (rSj-L6L-1) contains conformational epitopes. A: The purified fusion protein of rSj-L6L-1 with thioredoxin (Trx),
or rTrx alone, were analysed by SDS-PAGE under non-reducing conditions (lanes 2, 4, 6) or reducing conditions with dithiothreitol (DTT; lanes 3, 5, 7).
DTT had no effect on rTrx but slightly increased the size of rSj-L6L-1. Reduced and non-reduced rSj-L6L-1 was transferred to a nitrocellulose membrane
and probed with lung-LN ASC-probes (lanes 67). B: rSj-L6L-1 ELISA with rat ASC-probes obtained from skin-, lung- or liver-LN or spleen compared
with non-infected (NI) rat serum. Relative binding to rSj-L6L-1 is the ratio of the rSj-L6L-1 optical density to that of the fusion partner, rTrx, alone. Bars
represent the mean standard error.
recognition of rSj-L6L-1 at 1.6 RB, although this was not
significantly higher than the other groups.
Native Sj-L6L-1 is present in tegument extract
Schistosome extracts probed with antiserum to rSj-L6L-1
recognised a band of approximately 12 kDa in all
developmental stages examined (Figure 4A), with an additional
band of approximately 10 kDa observed only in the
schistosomula. Neither of these were recognised by antiserum
to rTrx (data not shown). The 12 kDa Sj-L6L-1 band was
similar in intensity comparing schistosomula and adult
worm pairs, and it was clear that the protein is more
abundant in male worms than females. Due to the scarcity
of larval material and the difficulty in removing the larval
tegument , tegument extracts were generated from
adult worms only. The same 12 kDa band was seen in the
worm tegument and insoluble fractions (Figure 4B), but
not in the aqueous-soluble fraction. This indicates
In this study, a novel post-genomic technique, a protein
microarray, was employed to identify schistosome vaccine
candidates. To focus on the identification of antigens
important in the vulnerable larval stages, rat lymph node
antibody samples (ASC-probes) generated previously from
the sites of larval infection , the skin and lungs, were
used to screen the arrays. The combination of these two
immunomic technologies the protein microarray,
providing multiple antigen binding data, and the ASC-probe
samples, which provide tissue-specific reactivity, resulted
in a list of targets which may contribute to a vaccine
targeting the migrating larvae . Several novel and also
known vaccine candidates were recognised; one of these,
named Sj-L6L-1, was further characterised and has several
Figure 4 Detection of native Sj-L6L-1 in S. japonicum extracts. A: Protein extracts from S. japonicum cercariae (C), schistosomula (S), adult
worm pairs (A), or separated male (M) or female (F) worms were analysed by SDS-PAGE and stained with coomassie (lanes 26) or subjected to
immunoblotting (lanes 711) with antisera to rSj-L6L-1. Molecular weight (MW) markers are shown in lane 1. B: Adult worms were fractionated
into tegument (teg), aqueous-soluble (sol) and -insoluble (ins) worm fractions, and analysed by immunoblotting with antisera to rSj-L6L-1.
features suggesting it is a promising vaccine candidate
relevant to the developing larvae.
The known targets included TSP-2, a tegumental
antigen for which the homologue in S. mansoni is currently
under further development as a vaccine candidate . The
present study suggests that TSP-2 is exposed to the hosts
immune system in both the skin and lung sites during rat
S. japonicum infection, since it was strongly recognised by
skin and lung ASC-probes. Other known vaccine
candidates recognised by the lung ASC-probes were: Sj22.6 and
Sj21.7, both tegument-associated proteins from the TAL
protein family ; S. mansoni filamin, a large structural
protein shown to confer some protection in a mouse
model ; and DLC1 which has been found associated
with the worm surface [43,44]. In addition, two further
molecules, S. mansoni TSP-3 and Sj29, were recognised
by the skin ASC-probes, indicating that they are antigenic
predominantly during skin invasion. These are both
purported to be surface molecules and Sj29 has been
investigated as a vaccine candidate . Gobert et al.  found
that in S. mansoni, the genes encoding both proteins had
significantly up-regulated transcript expression after
transformation from cercariae to larvae, and notably Sm29 (the
homologue of Sj29) had the highest expression in the
3 hour schistosomulum, the stage which develops shortly
after skin penetration.
A major criterion used in the present study to select
novel antigens for further characterisation was increased
larval expression. Initially, the NCBI EST database was
used as a preliminary measure of developmental
expression. This indicated that only one of the four novel
targets was not up-regulated in schistosomula, the zinc
finger protein (AY223099). This antigen also lacked a
signal peptide and transmembrane domain, and is likely
to be an intracellular protein and therefore may only be
exposed to the hosts immune system during parasite
damage. The developmental expression of the remaining
three antigens was confirmed by qPCR, and both
AY815838 and Sj-L6L-1 showed high larval gene
expression relative to cercariae. AY815838 was dramatically
up-regulated in the 2 day in vitro-cultured, but not the
3 day in vivo lung-isolated, schistosomula. This high
expression in the in vitro larvae could indicate an early
high expression that is then reduced after 3 days, or
could be an artefact of in vitro culture. Sj-L6L-1,
however, showed a more consistent larval expression, with
similar up-regulated levels in the 2 day and 3 day larvae
compared with other stages.
Based on these analyses, Sj-L6L-1 was selected for
further characterisation and was produced in recombinant
form. The E. coli-expressed rSj-L6L-1 fusion protein was
recognised by the lung ASC-probes indicating that it
was at least partly in the correct antigenic conformation.
By treating with reducing agent, the protein structure
was altered sufficiently to ablate recognition by these
antibodies, indicating that the schistosomiasis-induced
antibodies recognised only conformational epitopes on
the recombinant protein The disulfide bonds in Ly-6
proteins are known to be important for their structural
conformation, stabilising the typical three-finger motif;
for example, when CD59 is treated with reducing agent
it loses its ability to inhibit the complement system .
The Sj-L6L-1 protein was identified in all life-stages
examined. No increase in protein expression was seen in the
schistosomula despite the significant increase in
transcription. However, an additional band was recognised in the
schistosomula and this could represent either an
immature form of the protein or another variation unique to
this developmental stage. The Sj-L6L-1 protein was also
detected in the tegument extract from adult S. japonicum
worms, and the same band was found in the insoluble
fraction from the denuded adult worms. This indicates
that Sj-L6L-1 is at least associated with the outer
tegument, and is highly likely to be on the external surface as
is the case for its homologue in S. mansoni . It also
suggests that the protein is attached to the plasma
membrane, since it was only detected in the insoluble fraction;
hence, it is unlikely to be secreted like some Ly-6 proteins.
Another important observation was that local antibodies
obtained from rat lymph nodes draining the lung, the site
of larval killing, were specific for rSj-L6L-1. No specific
antibody was evident against the protein in lymph node
samples obtained from the liver, where more mature
worms reside, despite the fact that male adults do produce
the protein. This suggests that Sj-L6L-1 is uniquely
exposed during larval development and not in adult worms.
It is possible that this occurs after the transformation of
the schistosomula from cercariae, when the larvae rapidly
synthesise the new tegument  and before they acquire
host proteins which mask their own antigens .
While Sj-L6L-1 is novel for S. japonicum, the closest
homologue in S. mansoni (SmCD59.2) and its family have
been recently characterised . Previously, two of the
members of this family were identified in the adult S.
mansoni worm tegument using proteomics . Farias
et al.  analysing the S. mansoni transcriptome for
genes up-regulated from the cercariae to the
schistosomula, identified SmCD59.2 (which they refer to as dif 5)
and performed a limited DNA vaccine trial which resulted
in a slight (but non-significant) reduction (22%) in worm
The Ly-6 family of proteins was originally described in
mice, but Ly-6-like proteins have since been found in
many animal species from C. elegans to humans and
comprise the Ly-6 super gene family . These are
broadly grouped together based on the presence of 810
conserved cysteines which comprise the LU domain
. These conserved cysteines create 45 disulfide
bonds resulting in a three-finger structure, a motif also
common to the related snake venom toxins . Since
Sj-L6L-1 is related to this family by containing most of
these features, it is referred to here as Ly-6-like and part
of the Ly-6 super family. The Ly-6 family members and
Ly-6-like proteins appear to have extremely diverse
roles, although their precise functions are as yet unclear
. This family of proteins also exhibit limited
sequence identity between members , which makes
assigning a putative function to Sj-L6L-1 difficult. They
are generally thought to participate in development, cell
adhesion, and cell signalling, although how the latter
occurs is still unknown .
In summary, a novel protein microarray was combined
with a tissue-specific antibody source to investigate the
immunome of migrating S. japonicum larvae and to
identify novel antigens expressed by the schistosomula stage,
which is generally considered the likely target of an
antischistosome vaccine. Several novel and known proteins
were found to be antigenic in the regions of larval
migration and could form part of a multivalent vaccine
specifically targeting the schistosomula. Although these targets
were identified using rat samples, their vaccine efficacy
should ideally be tested in a final host such as the water
buffalo to account for differences in immune mechanisms
between species . Of the identified molecules, a novel
S. japonicum protein, Sj-L6L-1, was characterised and
found to be antigenic in the larval stage and present, but
not antigenic, in the worm tegument, and may provide a
valuable vaccine candidate against schistosomiasis.
PD probed the microarrays, HM & PD analysed the microarray data and
isolated parasite samples, HM synthesised and characterised Sj-L6L-1, and
drafted the manuscript. HM, PD, EM, DP and DPM interpreted results and
edited, drafted and approved the final manuscript.
We wish to acknowledge Mary Duke for her S. japonicum infection and
animal handling expertise, and financial support from the National Health
and Medical Research Council of Australia and the Australian Research
Council Centre of Excellence in Structural & Functional Microbial Genomics.
HM was supported by a NHMRC postgraduate scholarship (number 607203).
DPM is a NHMRC Senior Principal Research Fellow.
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