Do schistosome vaccine trials in mice have an intrinsic flaw that generates spurious protection data?
Wilson et al. Parasites & Vectors
Do schistosome vaccine trials in mice have an intrinsic flaw that generates spurious protection data?
R. Alan Wilson 0
Xiao-Hong Li 2
William Castro-Borges 1
0 Centre for Immunology and Infection, Department of Biology, University of York , Heslington, York YO10 5DD , UK
1 Departamento de Ciências Biológicas, Universidade Federal de Ouro Preto , Campus Morro do Cruzeiro, Ouro Preto, Minas Gerais , Brasil
2 National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention , Shanghai 200025 , People's Republic of China
The laboratory mouse has been widely used to test the efficacy of schistosome vaccines and a long list of candidates has emerged from this work, many of them abundant internal proteins. These antigens do not have an additive effect when co-administered, or delivered as SWAP homogenate, a quarter of which comprises multiple candidates; the observed protection has an apparent ceiling of 40-50 %. We contend that the low level of maturation of penetrating cercariae (~32 % for Schistosoma mansoni) is a major limitation of the model since 68/100 parasites fail to mature in naïve mice due to natural causes. The pulmonary capillary bed is the obstacle encountered by schistosomula en route to the portal system. The fragility of pulmonary capillaries and their susceptibility to a cytokine-induced vascular leak syndrome have been documented. During lung transit schistosomula burst into the alveolar spaces, and possess only a limited capacity to re-enter tissues. The acquired immunity elicited by the radiation-attenuated (RA) cercarial vaccine relies on a pulmonary inflammatory response, involving cytokines such as IFNγ and TNFα, to deflect additional parasites into the alveoli. A principal difference between antigen vaccine protocols and the RA vaccine is the short interval between the last antigen boost and cercarial challenge of mice (often two weeks). Thus, after antigen vaccination, challenge parasites will reach the lungs when both activated T cells and cytokine levels are maximal in the circulation. We propose that “protection” in this situation is the result of physiological effects on the pulmonary blood vessels, increasing the proportion of parasites that enter the alveoli. This hypothesis will explain why internal antigens, which are unlikely to interact with the immune response in a living schistosomulum, plus a variety of heterologous proteins, can reduce the level of maturation in a non-antigen-specific way. These proteins are “successful” precisely because they have not been selected for immunological silence. The same arguments apply to vaccine experiments with S. japonicum in the mouse model; this schistosome species seems a more robust parasite, even harder to eliminate by acquired immune responses. We propose a number of ways in which our conclusions may be tested.
Schistosoma mansoni; Schistosoma japonicum; Radiation attenuated vaccine; Antigen vaccine; Mouse; Hamster; Primate; Intravascular migration; Maturation; Pulmonary capillary; Inflammation
The question posed in the title of this review was
prompted by two recent sets of observations. Firstly, in
2014, publications from three Brasilian groups reported
protection against schistosome challenge after
vaccination of mice using what are unquestionably intracellular
proteins. The four proteins were Syntenin [
light chains DLC12 and DLC13 [
], and Y box protein
]. Secondly, we undertook a quantitative shotgun
proteomic analysis of the ubiquitous antigen preparation
SWAP, the soluble cytosolic extract of adult worms [
We found that about one quarter of the protein mass
comprised putative vaccine candidates identified by
other researchers. In this situation why does SWAP,
packed with multiple vaccine candidates, not have a
dramatic protective effect? Instead it performs no better
than individual candidates administered alone, and
sometimes worse. Do these antigens all trigger the same
mechanism, which has a ceiling of approximately 40–
50 % protection, whether one or many are used?
In this review we ask whether the protective outcome
of antigen vaccination is an artefact of the mouse
model and not a measure of acquired immunity. This is
not the same as saying that there are no protective
antigens but simply that the mouse model may not be
capable of discriminating between acquired immunity
induced by antigens and an effect of vaccination on
host physiology that diminishes schistosome
maturation in some non-specific way.
The mouse as an animal model for schistosomes
Whilst the laboratory mouse is a convenient host for
schistosomes, used in hundreds of vaccine testing
studies over the last 40 years, it does have a major drawback.
It is described as a permissive host but in reality the
actual numbers of penetrant Schistosoma mansoni
parasites that migrate from the skin to the portal system and
mature into adult worms is quite low. The statistic can
be obtained by counting the percentage of challenge
control cercariae that reach maturity in mice exposed to
the radiation-attenuated (RA) cercarial vaccine but
receiving no adjuvant or other treatment. In a sample of
26 experiments from the York group over a decade,
using the Puerto Rican isolate, the maturation in C57Bl/
6 mice was 32.3 % [
]. The maturation of cercariae of
the same isolate in CBA mice in seven experiments by
the London School of Hygiene group was an almost
identical 32.5 % [
]. The precise values may differ with
isolate and mouse strain but the stark fact from these
data is that of 100 cercariae penetrating the skin of a
naive mouse, 68 will fail to reach and mature in the site
of parasitisation, the portal tract. Vaccine-induced
protection is defined as the reduction in burden between
vaccinated and control groups and the low maturation
in mice is dealt with by using the formula: %
Protection = (Control burden - Test burden)/Control burden ×
100. For example with a ~40 % level of protection, in
the control group 68 parasites will fail to mature, while
vaccination of the test group eliminates only a further
13 parasites. Thus, of the 81 parasites that did not
mature in the test mouse, the vaccine treatment only
accounted for (13/81 × 100 =) ~16 %; the rest died of
natural causes. In truth, the way of calculating
protection in the mouse very effectively disguises only a small
achievement in worm elimination.
A larger number of penetrant S. mansoni cercariae
mature in the golden (Syrian) hamster with a mean of
56 % recorded in eight experiments [
], and values
as high as 76 % in single experiments . In
nonhuman primates, the hosts evolutionarily closest to
humans, there is comparable data available from
challenge control animals in vaccine experiments with RA
cercariae. A maturation of 82 % was recorded in a single
vervet experiment (Cercopithecus aethiops [
]) and a
mean of 80.5 % in three baboon experiments (Papio
]). In contrast, the laboratory rat presents an
even greater obstacle to migration than the mouse, with
22–27 % of juvenile worms detected in the liver between
days 11 and 21 [
]. We conclude that the laboratory
mouse, viewed solely in the context of parasite
maturation, does not seem the best choice for vaccine
experiments, and rats are even worse.
Exactly what are the “natural causes” that limit
It is important to establish at the outset that the 68 % of
non-maturing S. mansoni penetrants are not eliminated
by immunological processes as their protracted
migration and slow development (~5 weeks) allow ample time
for immune intervention. The best evidence is provided
by experiments in which mice were exposed to whole
body irradiation a few days before infection. The
treatment severely depresses immunological
responsiveness without administration of chemicals or alteration
of genotype. Most parameters decline by ~90 %, yet
parasite migration and maturation are not enhanced
]. This strongly supports a physical explanation
for the low maturation.
As a result of autoradiographic tracking experiments
on 75Seleno-methionine-labelled parasites we now have
explicit data on the route taken by schistosomula from
the skin to the portal system, the kinetics of the
process, and their fate along the way (Fig. 1; reviewed
]). The idea that most non-maturing
penetrants died in the skin was quickly dispelled and the
mincing and incubation of tissues to extract the larval
parasite burden was discredited as a quantitative
technique . A careful balance sheet of parasites in all
mouse organs up to 35 days post-infection revealed that
the vast majority exited the skin (Fig. 1a) and transited
to the lungs, where peak numbers of > 60 % penetrants
were detected (Fig. 1c) [
]. Note that the first larvae
arriving in the lungs pass through the vascular beds
and exit before the last larvae arrive from the skin, so
the maximum numbers in the lungs never equal the
skin total. Schistosomula begin to leave the lungs
around 5 days post-infection via the venous circulation
to the left side of the heart and are distributed in
arterial blood to all organs of the body (Fig. 1d). The
fractional distribution of cardiac output in the mouse is
approximately 0.33:0.66 splanchnic:systemic organs.
The one third of schistosomula entering the splanchnic
arteries (coeliac, superior and inferior mesenteric) pass
through the gastrointestinal capillary beds to reach the
portal system (Fig. 1e); the remaining two thirds
negotiate the capillary beds of systemic organs to return to
Fig 1 Balance sheet showing the profile of schistosome migration through all organs of the mouse. The numbers of schistosomula detected by
compressed organ autoradiography after exposure of mice to 75Se-Methionine-labelled cercariae (reproduced with permission from [
circles and solid lines are normal mice, closed circles and dashed lines are mice vaccinated five weeks previously with 500 RA cercariae. a and c
The vast majority of schistosomula leave the skin and travel to the lungs. d and e The impact of vaccination is revealed by the reduced parasite
numbers detected in the systemic organs and accumulating in the portal system, compared to normal mice. b The decline in parasite numbers
only begins from day 15 at which time < 10% remain in the skin. The lungs are the principal obstacle to maturation in normal mice, and more so
in vaccinated animals
the lungs. The principal feature revealed by the balance
sheet is that no parasites are eliminated before 15 days
post-infection (Fig. 1b), at which time < 10 % can be
detected in the skin; this is the clearest possible
demonstration that parasite death in the skin is minimal. At
15 days the population is distributed around all organs
of the mouse body, with the largest proportion in the
lungs. The question is ‘what happens from 15 days
When migrating schistosomula, recovered from the
pulmonary vasculature of donor mice, are injected via a
vein back to the lungs of naïve recipients, ~50 % reach
]. However, > 80 % of the same batch,
injected into the portal vessels, are recovered as mature
worms four weeks later. Such experiments confirm that
the mouse lungs are the real barrier on the migratory
route. The reason why less than 100 % of injected
schistosomula develop to maturity in the portal tract is that
as many as 25–30 % actually negotiate the hepatic
sinusoids back to the venous circulation and lungs .
They must then traverse the lung obstacle again, as must
those schistosomula returning from systemic organs.
The magnitude of the lung barrier is underlined by
experiments to determine the kinetics of larval migration
through different vascular beds [
22, 23, 28
]. The mean
transit time of day 7 schistosomula through the lungs is
30–35 h. This contrasts with values of 16 h for systemic
organs and 6.5 h for intestinal capillary beds [
(estimates from an independent experiment with fewer time
points gave values of 11.7 and 9.8 h, respectively [
Clearly negotiating the lung capillaries is hard work. It
must be emphasised that schistosomula leaving the skin
are short and stubby (100 to 190 μm long, Fig. 2b; [
and completely occlude pre-capillary arterioles of the
lungs 10 to 20 μm in diameter, when they arrive [
They must then undergo a phase of development,
becoming longer (> 400 μm), thinner (~8 μm) and losing
mid body spines (Fig. 2c; [
]) to facilitate transit
along capillaries only 7 μm wide. For the earliest arrivals,
the first transit through the lungs may take 78 h. In
addition, the percentage maturation for Day 3
schistosomula delivered i.v. to the lungs is 30 % and for Day 4
schistosomula 41 %, compared with ~50 % for Day 7
larvae . These data suggest that the first transit is the
most difficult and protracted; indeed, it is quite possible
that some schistosomula arriving from the skin never
progress further [
]. Conversely, the percentage
maturation of Day 12 and Day 17 schistosomula delivered to
Fig 2 The morphology of pulmonary migration. a Cast showing the complex meshwork of short capillaries that surround each alveolar
space, in contrast to more normal capillaries at the periphery (arrow). BV, blood vessel. The mean diameter of mouse alveoli ranges from 36
to 45 μm, depending on strain while the diameter of an alveolar capillary is ~ 6 μm. Image from Guntheroth et al. [
] with permission. b Dark-field
image of a Day 2 schistosomulum prior to exit from the skin via a blood vessel. c Dark-field image of a fully extended Day 6 lung schistosomulum,
approximately 400 μm in length. When crossing the vascular bed, the parasite body must extend through the capillary labyrinth of several alveoli
(b and c from [
]). d Transmission electron micrograph of a part of a lung schistosomulum lying within the lumen of an alveolar capillary. The tight
squeeze is confirmed by the bulge in the body where it lies adjacent to another capillary (arrowed) (from [
]). e Diagrammatic representation of the
way that a schistosomulum uses its anterior and posterior body spines to move along the capillary lumen, alternately anchoring one set and then the
other (from [
]). Scale-bars in a–c: 50 μm
the lungs i.v. is not significantly different from that of
Day 7 larvae [
]. We should therefore consider the Day
7 lung schistosomulum as fully adapted to intravascular
A combination of light and electron microscopy
revealed that at day 7 post-infection all lung schistosomula
were located in blood vessels of naïve mice [
]. At the
next sampling time, day 11, a proportion of them were
entirely or partially intra-alveolar, and this reached 80 %
at day 20 and later. No cellular reactions were evident
around intravascular parasites but at later sampling
times alveolar parasites were associated with large
inflammatory foci, probably elicited as a response to
nonspecific tissue damage when they burst out. In spite of
this inflammation, no damage to the schistosomula was
observed. These observations were confirmed in an
autoradiographic study which concluded that the
alveolar larvae were unharmed and were likely to be coughed
up, swallowed and digested [
]. Administration of
schistosomula via the trachea to the pulmonary airspace
revealed that a small number (~15 %) were able to
reenter blood vessels, migrate and mature in the liver, but
this proportion diminished as the parasites aged [
Exit into alveoli appears to occur accidentally as
parasites attempt to traverse pulmonary blood vessels,
and once there it becomes difficult to continue
We can estimate the proportion of schistosomula
becoming trapped and diverted to the airspaces on
each circuit using a simple spreadsheet calculation
(Additional file 1: Table S1A). An average P value of
0.36 for a schistosomulum getting stuck on each
passage through the lung vascular bed (including the first)
creates a realistic migration profile. Only a few passages
around the circulation, each taking about two days, are
sufficient to generate the cumulative portal population.
The remaining parasites blunder into the airspaces of the
lungs or get stuck in other locations (Fig. 1). This
physical explanation of worm demise, with the average P value
of 0.36, accounts almost precisely for the ~32 %
maturation in naïve mice. However, the maturation data for
Day 3 and Day 4 schistosomula indicate that they have
P values for getting stuck of P = 0.415 and 0.28,
respectively, while the 50 % maturation of day 7 (or older)
larvae delivered i.v. to the lungs is achieved by P = 0.203
(Additional file 1: Table S1B). So what feature of the
lungs, especially mouse lungs, presents a special
obstacle to onward migration?
The first and most obvious aspect is that the
pulmonary capillaries have very thin walls separated from
the airspaces only by alveolar basement membrane and
epithelium. The total thickness of the blood-gas barrier
is ~0.3 μm with the alveolar epithelial cell in some
parts having a thickness of 0.1 μm [
]. Thus the
pulmonary capillary is only separated from the alveolar
space by a layer of extracellular matrix and an
epithelial cytoplasm, each 0.1 μm thick. No other capillary in
the body is protected by such a thin layer of tissue,
making the pulmonary capillaries fragile and vulnerable
to failure [
]. The mechanical behaviour of alveolar
capillaries is determined by the extracellular matrix
layer, with type IV collagen the most important
constituent determining the strength of the blood-gas
barrier. The combined thickness of the three components
of the blood-gas barrier correlates with the
experimental pressures required to damage to the pulmonary
capillary wall in different species, the larger the animal,
the thicker the blood-gas barrier (horse > dog > rabbit)
]. Such measurements are not available for the
mouse but it is plausible that the alveolar capillaries of
this species may be particularly fragile due to its small
size. Indeed, it is easy to appreciate how the vigorous
and rhythmic extensions and contractions displayed by
elongating schistosomula [
], especially the younger
larvae, could provide the motive power to rupture
alveolar capillaries in the mouse lung.
A second difference is the nature of the pulmonary
capillaries themselves. Those enveloping the alveoli are
very short (~10 μm) forming a maze of vessels [
through which the schistosomulum must crawl (Fig. 2a).
This contrasts with the network of longer bifurcating
tubules that comprise the capillary bed of other organs
and non-alveolar parts of the lungs (Fig. 2a, arrowed).
Juxtaposing an image of an extended Day 6 lung
schistosomulum (Fig. 2c) next to that of the alveolar capillaries
brings the task it faces in traversing the pulmonary
vascular bed into sharp focus. Unsurprisingly it must take a very
convoluted path through the maze of short tubes with
many options, as revealed by in situ electron
microscopy (Fig. 2d; [
]). Adding in the fact that it progresses
along vessels in inchworm fashion using its anterior
and posterior spines (Fig. 2e; [
]) everything converges
to make pulmonary vascular transit a difficult
proposition, with a significant probability of bursting into an
The lungs are also subject to another potentially
relevant phenomenon, the vascular leak syndrome (VLS).
VLS was first reported following cytokine administration
(Interleukin 2) to patients to treat certain cancers [
and has been explored using the mouse as a model. It is
characterised by leakage of vascular fluids into tissues,
accompanied by hypotension; in the lungs this results in
pulmonary oedema. There is a general consensus that
IL-2-induced VLS is caused by secondary release of
inflammatory cytokines such as IFNγ and TNFα [
which may in turn increase production of vascular
mediators such a nitric oxide (NO). It has even been
suggested that VLS may be accompanied by modifications
in the extracellular matrix , which could weaken the
alveolar blood-air barrier to increase capillary fragility.
The relevance of VLS to schistosome vaccines is that its
causative agents are precisely those cytokines and
mediators involved in the protective effector mechanism
elicited by the RA vaccine (see below).
The radiation-attenuated (RA) cercarial vaccine in mice
The best studied model of acquired immunity to
schistosomes is that induced by exposure of rodents and
primates to a dose of RA cercariae. We have a wealth of
immunological and parasitological information about
the actions of attenuated parasites in generating
protection and the fate of a normal challenge in vaccinated
hosts (reviewed in [
]). In mice, optimally
attenuated parasites undergo a truncated migration as far as
the lungs, priming the immune response via
skindraining lymph nodes. These attenuated larvae also
“arm” the lungs by stimulating recruitment of effector
T cells to the interstitial spaces and airways [
Multiple vaccinations have an additive effect and almost
sterile immunity can be achieved by co-administration
of attenuated larvae and the cytokine IL-12 [
However, we now know that subcutaneous
administration of IL-12 can produce adverse systemic effects [
Initial priming has a strong Th1 component but
subsequent exposures to RA cercariae amplify Th2 cells and
enhance antibody involvement. A striking feature is that a
period of five weeks is left after application of the
attenuated parasites, before challenge with normal cercariae.
This allows any non-specific inflammatory events to
subside before challenge parasite entry; vaccination and
challenge are normally performed on separate sites
such the abdominal skin and the tail, to avoid residual
inflammation left by the initial exposure.
What happens to challenge parasites when they
penetrate the skin of previously vaccinated mice? They are a
little slower to leave but the population still travels to
the lungs where the discrepancy with migration in a
naïve mouse becomes apparent (Fig. 1). Fewer larvae
transit the lungs to reach the systemic and splanchnic
organs, as evidenced by the lower peak numbers in the
first (Fig. 1d) and the slower build up to a lower plateau
in the second (Fig. 1e). Total numbers detected begin
to decline a little earlier in the vaccinated hosts and
proceed to a lower end point but the rates of parasite
elimination are identical (Fig. 1b). Even without knowledge of
immunological events or mechanisms, it is clear that the
major site of parasite loss is the lungs. This organ, difficult
to negotiate in naïve mice, becomes more so in vaccinated
animals. Increasing the chance of getting stuck on each
circuit from P = 0.36 to P = 0.53 predicts the 40 %
reduction in worm burden (Additional file 1: Table S1), as an
additional 13 parasites fail to mature beyond the 68 in
naïve animals (Fig. 3). The corresponding values for a
70 % reduction in burden are P = 0.72 with an additional
22 parasites prevented from maturing, still less than a
quarter of the total.
What is the evidence that the protection induced by
the RA vaccine has an immunological basis? Adoptive
transfer of the immunity elicited by the RA vaccine has
been achieved using a parabiotic union between mice
]. Subsequent parabiotic experiments where the naïve
and vaccinated partners were separated before challenge
demonstrated two key facts about the model [
recruitment of effector cells to the lungs was a crucial
pre-arming component of the protective response.
Secondly, an anamnestic response was elicited by
percutaneous challenge and could act against migrating larvae in
the lungs. However, when the challenge was provided by
day seven schistosomula administered directly to the lung
vasculature, no protection was evident. Passive transfer of
protection with homologous serum from vaccinated mice
proved difficult to demonstrate [
]. However, it was
eventually achieved using serum from multiply vaccinated
], especially high-titre serum from
]; in both studies the serum could be
administered as late as day 4 or 7 post-challenge, revealing
that the target was the lung, not the skin schistosomulum.
To our knowledge, the passive or adoptive transfer of
protection to naïve mice has not been achieved using serum
or cells respectively, from any donors reportedly
exhibiting antigen-induced acquired immunity.
How are the extra challenge schistosomula arrested
in the lungs? A focal inflammatory response develops
Fig 3 Diagrammatic representation of the fate of migrating schistosomula in normal and vaccinated mice. a Cumulative parasite deaths in the
lungs. b Cumulative parasite survival and development in the portal system. The diagrams are based on the spreadsheet calculation in Additional
file 1: Table S1. For simplicity, the model assumes that parasites end their migration either in the lungs or the portal system. After an initial
elongation phase of about three days in the lungs, the time-base of a circuit around the body in the bloodstream is approximately 48 h. An
average P = 0.36 of getting stuck in the lungs of a naïve mouse on each passage produces an adult worm portal population equal to 32 % of
penetrating cercariae (red line). A “protection” of 40 % induced by a vaccine needs to increase the P of getting stuck to 0.53, with 13 more parasites
trapped (blue line). A “protection” of 70 % needs a P of 0.72, with 22 more worms trapped. This is still only one quarter of the total losses
to each larva over several days [
]. The mass of cells
around the parasite hinders onward migration and
increases the probability of deflection into an alveolus.
IFNγ is a key cytokine [
] and production of TNFα
also appears to be central to protection; mice lacking
the TNF receptor 1 (TNFRI-/-) were not protected by
exposure to the RA vaccine . However, pulmonary
inflammation alone is not sufficient since the cell
infiltration around migrating larvae in the lungs of
IFNγR-/and TNFRI-/- mice is actually greater, but is ineffective
at increasing the proportion of schistosomula deflected
into the alveoli; this implies that there are other
downstream mediators. Strikingly, the protection does not
involve direct immune damage to schistosomula.
Almost no evidence of cytological damage inflicted by the
enveloping inflammation was observed in trapped
challenge larvae in situ [
] and lung schistosomula were
not susceptible to ADCC mechanisms in vitro [
Most telling, transfer experiments with challenge
schistosomula trapped in the lungs (which would not
mature if left in situ) showed that they could migrate and
develop normally if introduced into the pulmonary or
portal circulation of naïve animals [
]. This durability
is a testament to the anti-oxidant capabilities of
migrating larvae [
]. In summary, the inflammatory effector
mechanism of the RA vaccine apparently alters the
pulmonary environment to decrease the probability that a
schistosomulum can successfully traverse the vascular
beds, but it does not cause it direct harm.
Although the effector mechanism in RA vaccinated
mice operates in the lungs, the effector T cells were
generated in the lymph nodes draining the skin
vaccination site. They exited via the efferent lymphatics to
join the circulation from where they were available for
recruitment to the lungs by the attenuated vaccinating
]. Such circulating effector T cells can
also be recruited to other sites like the footpad or pinna
by an inflammatory stimulus and this forms the basis of
a delayed-type hypersensitivity (DTH) assay to estimate
their level in the circulation. In mice exposed to the RA
vaccine, DTH T cells are high by ten days
postexposure, peak at day 17, are in decline by day 21 and
almost down to background by day 35 when the
cercarial challenge is administered (Fig. 4 [
]). Exposure to
the challenge rapidly stimulates a recall response with a
surge of DTH T cells into the circulation, peaking at
day 7, declining by day 14 and well towards background
by day 21. These data provide the clear justification for
the five-week interval between vaccination and
challenge, and also key evidence for an anamnestic
response to challenge larvae. The same kind of peripheral
responsiveness has been observed in chimpanzees
exposed to repeated doses of the RA vaccine. There was a
progressively increasing reactivity of peripheral blood
mononuclear cells to schistosome antigens with each
successive vaccination; reactive cells were still
circulating four weeks after challenge [
Vaccine antigen testing in the mouse
How does it differ from RA vaccine experiments?
In testing of vaccine antigens in the mouse a common
design is employed. A recombinant protein, less
frequently a parasite fraction or a purified protein, is
formulated with an adjuvant for subcutaneous or
intraperitoneal administration. Freund’s complete adjuvant
is a frequent choice for priming followed by two doses
of Freund’s incomplete adjuvant as the booster, spaced
two or three weeks apart. Mice that will serve as
Fig 4 Activated T cells in the circulation detected by footpad Delayed-Type Hypersensitivity (DTH) assay. a Reactivity after vaccination with 500
RA cercariae. Two identical experiments are plotted showing the Th1 cells reach a peak at approximately 17 days and have declined almost to
background levels before challenge at 35 days. b Reactivity of vaccinated mice (V + C) and naïve controls (CC) challenged at 5 weeks with 200 normal
cercariae. A sharp increase in circulating T cells is observed 7 days after challenge of the vaccinated animals, coincident with peak schistosomula numbers
in the lungs, and then a gradual decline. The reactivity of the control mice is muted in comparison, both with respect to the previously vaccinated animals
and to the naïve mice in part A exposed to RA cercariae. The gradually rising DTH response of the CC group up to day 20 is most likely a
response to worm vomitus released by the blood feeding worms accumulating in the liver. Replotted from [
challenge controls receive the adjuvant alone
formulated with saline; these are not equivalent. An
irrelevant antigen of similar immunogenicity would be a
better control; very seldom is a no-adjuvant group
included. (Vaccination with DNA constructs follows a
similar pattern, but without extraneous adjuvants).
The mice are all challenged with the same pool of
cercariae at an interval after the last boost, and then
perfused 5–8 weeks later to recover and count adult
A major, and we suggest significant, difference from
the RA vaccine is the much shorter interval between the
last boost and the day of challenge. The preferred time
appears to be around 14–15 days but it may be as short
as 10 days and less commonly three or occasionally four
weeks. Seldom is the interval five weeks as is standard
with the RA vaccine in mice, and we can find few
instances where it is longer than that. Recent experiments
with two tegument vaccine candidates appear to support
our contention. When C57Bl/6 mice were vaccinated
with Sm22.6 and challenged 15 days after the last boost,
they showed a mean 34.5 % protection [
]. In contrast,
when the antigen was administered to Balb/c mice that
were challenged 30 days after the last boost, the
protection elicited was 0 and 18 % [
]. A similar pattern was
observed with Sm29 where 51 % protection was
observed after a challenge of C57Bl/6 mice at 15 days after
the last boost [
] and 0 % protection after a challenge
of Balb/c mice at 30 days [
]. Of course it can be
argued that mouse strain was the key determinant of
protection, not the interval between boost and challenge, a
point that can only be resolved by further experiments.
A key feature of our hypothesis is that the short time
interval between boost and challenge may provide an
explanation for the apparent immunity elicited by some
very unlikely vaccine candidates. At the end of the
antigen vaccine schedule, is the level of inflammatory
cytokines in the circulation sufficient alone to modify
pulmonary vascular physiology? Alternatively, do
migrating challenge schistosomula elicit an inflammatory
stimulus in the lungs that recruits vaccine-activated cells
from the periphery, unrelated to their own antigen
specificity? Remember that only a small additional number
of larvae, above and beyond those that will not make it
anyway, need to be arrested by the lung obstacle for 40
or 50 % protection to be generated. It is not 40 or 50
out of 100 penetrants, it is 13 to 16 extra larvae. We
suggest that only a small additional effect on the
pulmonary capillaries would be needed to impede this extra
fraction of schistosomula.
Are the vaccine antigens plausible candidates?
Over the last three decades quite a long list of
schistosome candidates has been put forward, primarily based
on the results of protection experiments in mice. The
candidates divide into two categories, internal and
surface exposed/secreted. The first is larger and well
represented in SWAP [
]. It includes TPI, GST, Sm14 fatty
acid binding protein (FABP), Aldolase, GAPDH,
Calponin, Sm20.8, Sm22.6, paramyosin, myosin heavy chain,
14-4-3 chaperone, and stomatin. The shorter list of
exposed proteins, present in small amounts in SWAP,
includes: the tetraspanins Sm23 and TSP-2, tegument
Sm29 and calpain, and gut-secreted Cathepsin B.
The list encompasses the six WHO candidates that
have acquired almost mythical status. Two of them are
localised in muscle (paramyosin and myosin heavy
chain) and three are cytosolic (TPI, GST, FABP). They
did not perform well in independent murine trials but
acquired a life of their own such that today they can still
be described as the most promising candidates [
goal of 40 % protection for a usable vaccine was set by
WHO in the early 1990s so it is evident that, in spite of
much work with different adjuvants and live vectors over
the intervening 20+ years, a real and perhaps
insuperable obstacle to improvement remains. Multiple
exposures of mice to irradiated parasites drive protection
towards 100 %, whereas co-administration of two
vaccine antigens [
] or multiple epitope constructs [
] do not markedly increment protection. The real test
in this respect is the administration of SWAP with its
multiplicity of candidates, where protection seldom
reaches or exceeds 50 % [
]. There has always been
a conceptual problem with the cytosolic and cytoskeletal
constituents as vaccine antigens. How could an immune
effector mechanism “recognise” these internal proteins
in a live parasite and interact with them to cause its
demise? It is a question that has never received a
satisfactory answer from the internal candidate enthusiasts.
Live BCG as an adjuvant
For one candidate, paramyosin, there a comprehensive
body of relevant immunological data. However, the
purified protein was only effective when administered with
live BCG as an adjuvant. The production of IFNγ and
the activation of macrophages were shown to be key
components of the model [
] so the parallels with the
RA vaccine are obvious. Furthermore, strains with a
tendency to generate Th1 type responses were more readily
protected than e.g., Balb/c mice, or P strain mice with a
defect in macrophage activation [
]. Parasite tracking
experiments were mentioned as ‘in progress’ [
the site of challenge elimination was never identified. It
is noteworthy that both mouse monoclonal and rabbit
polyclonal anti-paramyosin antibodies failed to confer
any protection when administered -1 and +5 days after
]. A complicating factor in the paramyosin
experiments was that i.v. administration of BCG was
already known to reduce worm burden non-specifically
by generating pulmonary inflammation to interfere with
]. In the paramyosin experiments the BCG
was given intradermally (i.d.) with the assumption that it
would not enter the circulation and be disseminated,
although this does seem not to have been formally tested.
However, i.d. administration of BCG, with subsequent
detection in internal tissues does occur [
] so it may
not be possible to disentangle vaccine antigen and BCG
effects, making pulmonary involvement more plausible.
What can we deduce from the properties of the vaccine
It is a reasonable assumption that the closer the
identity of a schistosome protein to its nearest ortholog in
the murine host, the lower the probability that a
unique epitope will exist. The candidates range in
percentage homology between 29 and 74 % (mean
50 %; Table 1). Bepipred
(http://tools.immuneepitope.org/bcell/), the B cell epitope predictor indicates
that the larger schistosome proteins have higher
numbers of predicted epitopes, with myosin, paramyosin
and calpain the winners. The likely immunogenicity of
each candidate can be gauged using a simple formula
where the number of epitopes is divided by the
proportional identity of sequence times the Mw in kDa. On that
basis, the leaders are GST-28, Sm20.8 EF hand Ca2+
binding protein, Sm14 FABP and calpain (mean 36 %
homology). The least immunogenic are Enolase, 14-3-3,
Stomatin and Aldolase (mean 66 % homology). (Indeed
enolase is only included in the list to make the point that
when we cloned and expressed it for vaccine experiments
we could achieve no protection at all.) Our contention is
that in the mouse model pure immunogenicity is likely to
be the best predictor of an effect, simply because it will
provide the strongest stimulation to the immune system.
A different way of examining the vaccine potential of a
protein is to determine the effects that evolutionary
pressures might exert to alter the nucleotide sequence of the
encoding gene. This can be estimated by analysing the rates
of non-synonymous to synonymous substitutions (dN/dS)
between orthologs from different schistosome species. Such
a study was recently performed with the micro-exon
(MEG) and venom allergen-like (VAL) genes of
]. The proteins they encode are associated with
the glands and secretions of different life cycle stages [
], positioned at the parasite-host interface where they will
be exposed to selection pressure from the immune system.
The two classes of genes revealed significantly higher dN/
dS values when compared with a set of control genes
coding for secreted proteins, and for other proteins previously
localized to the tegument. Analyses of paralog genes
indicated that exposure of the protein to the definitive host
immune system was indeed a determining factor leading to
the higher dN/dS values. In addition, two other proteins
(Sm29 and TSP-2) exposed at the tegument surface, and
# epitopes > 4AAa
apredicted by Bepipred (http://tools.immuneepitope.org/bcell/)
several lipid-processing proteins present in the worm
vomitus displayed dN/dS values similar to those observed for
MEGs and VAL genes. This provides further evidence of an
additional selective pressure from the immune system on
exposed proteins rather than a phenomenon specific for
MEG and VAL genes. In complete contrast, almost all
previously proposed vaccine candidates display very low
rates of non-synonymous changes (Table 2). This is
entirely consistent with their internal location and
inaccessibility to immune effector responses in the live parasite. It
also explains their strong immunogenicity when finally
presented to the immune system upon release from the
damaged or dead parasite. They have not been selected to
be immunologically silent. This makes them the ideal
agents to elicit a strong acquired response when
administered multiple times with an adjuvant. Furthermore, when
cercarial challenge is given only 10 to 15 days after the last
boost, associated host responses to these reactive proteins
will be maximal in the circulation, and so well placed to
interfere non-specifically with an already fraught parasite
Sma, Schistosoma mansoni; Sha, S. haematobium; Sja, S. japonicum
aValue for the portion of the gene encoding the exposed
hydrophilic loop utilised in vaccine trials
What can protection experiments with heterologous antigens tell us?
Our hypothesis is that protection in the mouse model may
be a bystander effect caused by high levels of circulating
cytokines or the presence of activated T cells, macrophages
and other leucocytes in the circulation, coincident with
arrival of challenge larvae in the lungs. A corollary is that
vaccination with irrelevant antigens should produce the same
effect. The extensive experiments with live BCG are
evidence for an “irrelevant” antigen effect on parasite
] but other heterologous proteins have been tested.
Crude Fasciola hepatica extracts protected mice against
schistosome challenge [
] with the major activity attributed
to an abundant and immunogenic 12 kDa FABP [
Independently the S. mansoni homologue of this Fasciola protein
was cloned, shown to elicit protection in mice and proposed
as a dual Fasciola/Schistosoma vaccine [
]. Does the
cross-protection result from shared epitopes between the
two proteins? The sequence homology is only 49 %, spread
evenly throughout the polypeptide, and BepiPred does not
identify stretches of amino acids in common that might
serve as shared epitopes. This evolutionary distance is
confirmed by a comparison with S. bovis FABP which
has also been the subject of Fasciola cross-protection
studies . It differs from its S. mansoni equivalent
at only two amino acid positions but is again only
49 % identical to the F. hepatica protein. Only epitope
mapping of the respective fatty acid binding proteins
with specific antisera can resolve this point.
Protection experiments have also been performed
using heterologous proteases as adjuvants to boost
protective responses to vaccine candidates such as GAPDH
and 14-4-3 via a Th2-mediated immune response. It is
remarkable that papain alone, from the plant Carica
papaya, could induce a > 50 % reduction in worm burden
when mice were challenged 14 days after a second
administration of the enzyme subcutaneously [
logic of this approach was that papain can activate
basophils to secrete Th2-type cytokines in the absence of
antigen-specific IgE [
]. These cells normally
comprise ~1 % of circulating leucocytes and it would be
pertinent to discover if they had any effect on circulating
cytokines or pulmonary inflammation in papain-treated
mice. In a similar way, functionally active F. hepatica
cathepsin L1, which shares 47 % identity with its S.
mansoni orthologue, is capable alone of inducing up to 50 %
reduction in worm burden when injected 14 days before
cercarial challenge [
]. When co-administered with
schistosome proteins, a reduction in burden of up to 73 %
may ensue. (Remember that this is 73 % of 32 %, i.e., 23
worms stopped via the immune response against 68 from
natural causes, so still only a quarter.) A plausible
alternative to an adjuvant role for these proteases, and the
reactivity of F. hepatica FABP is that we are witnessing a
bystander effect in the circulation impacting on the lungs,
not specific acquired immunity.
Do vaccine antigens identified in mice protect non-human primates?
A perplexing feature of the candidates identified in mouse
vaccine trials is that they do not translate well to a
permissive primate host like the baboon (Papio anubis). Note that
since maturation in baboons can be > 80 % of penetrant
cercariae, an acquired immune response has much more
work to do than in a mouse to achieve a high level of
protection. Nevertheless, > 80 % protection has been induced
in primates like the baboon by multiple doses of the RA
]. The > 80 % level of maturation means that
only 20 out of 100 parasites die of “natural causes” during
migration and a further 64 die due to immune effector
mechanisms, the exact reverse of the mouse situation.
The protection elicited in the baboon by the RA vaccine is
also additive, in proportion to the number of exposures to
attenuated cercariae, and a clear saw-tooth pattern of
boost and decline in specific antibody production is
observed with each successive vaccination [
]. The duration
of protection has also been tested; it declines from 72 to
53 % when the interval between last boost and challenge
is extended for three weeks to three months. Thus we
contend that the baboon provides a robust test of vaccine
potential. How well have vaccination experiments
performed using the candidate antigens first trialled in mice?
A trial with SmGST28 produced contradictory results
], depending on the adjuvant used with -26 and 38 %
protection achieved. Another trial with myosin heavy
chain (IrV5) gave 25 and 26 % protection in two
different adjuvant formulations [
]. More recently, the
tegument antigen Sm29 proved to be immunogenic but
failed to induce protection (Kariuki, personal
communication) while three anti-oxidant enzymes (two
superoxide dismutases and glutathione peroxidase) in a DNA
vaccine formulation all failed to reduce worm burden
significantly, but did depress faecal egg excretion [
Only one vaccine candidate, Sm-p80 calpain has
achieved a good level of protection in baboon: 48 % with
a DNA construct [
], then 52–58 % with a recombinant
] (there was a 4 week gap between last boost
and challenge). Overall, it appears harder to elicit
protection with single antigen formulations in baboons than
in mice against a S. mansoni cercarial challenge: only
calpain passes the primate test but persistence of
protection induced does not yet appear to have been tested.
Are S. japonicum vaccine experiments in mice subject to the same constraints?
In comparison with S. mansoni there is much less detailed
information available about the mouse model infected
with S. japonicum. However, the percentage maturation in
naïve mice appears to be higher at ~50 % ([
strain), while that in hamsters (~53 %) is about the same
], and in rabbits it is 53.5 % [
]. The pattern of
schistosomulum migration and the site of elimination of
nonmaturing parasites are less well understood but an early
study concluded that migration was entirely in the
bloodstream, with passive transport between organs and active
crawling through capillary barriers [
]. A more rapid
migration of S. japonicum was noted with peak numbers in
the lungs at 3–4 days and the first arrivals in the portal
vein from 3.5 days. This qualitative pattern was later
confirmed by mincing and incubation of tissues to recover
migrating parasites [
]. The single quantitative
autoradiographic tracking study reported that the skin was not
a site of attrition after primary infection but that parasite
elimination occurred after migration to the lungs and
continued up to the liver stage [
]. The presence of large
numbers of petechial haemorrhages on the lung surface
has been recorded in several studies [
97, 99, 100
]; they are
seldom observed in S. mansoni-infected hosts in spite of
numerous schistosomula entering the alveoli. Most
striking, petechiae have been recorded on other organs like the
kidney, and on the walls of the stomach [
observations confirm that migration occurs through systemic
organs and that schistosomula arrive in the portal system
via the capillary beds of splanchnic organs. Based on
minimum recorded length, S. japonicum schistosomula
are apparently one third larger than those of S. mansoni
] so may cause greater vascular damage. In addition,
given the > 50 % maturation rate, they may also have a
greater capacity to re-enter the tissues to continue
Protection can be induced by exposure of mammalian
hosts to RA cercariae of S. japonicum, often attenuated
with UV light (reviewed in [
multiple vaccinations enhance that protection, compared to
single vaccinations, as observed in mice, [
] and cattle [
]. The immunological basis of
protection has been confirmed by passive transfer of serum
from 5× vaccinated mice to naive recipients [
exactly as with S. mansoni [
]. The most common
immunization protocol for antigens is three
immunizations, the first in complete Freund’s adjuvant, followed
by two boosts with incomplete Freund’s adjuvant. Two
weeks after immunization, mice are challenged with
cercaria, usually 40, but sometimes as few as 20, due to
the greater pathogenicity of S. japonicum. In contrast
to the RA vaccine, antigens including the six most
promising candidates selected by WHO, usually
induced protection ranging from 20 to 40 %, if any
(reviewed in [
]). Some researchers have reported that
two antigens administered together can induce higher
], while others reported the opposite,
compared to single antigens . S. japonicum SWAP,
presumably containing multiple candidates, has been
reported to elicit > 40 % protection [
]. Some unexpected
antigens also induced protection; for example,
conferred over 40 % protection against challenge [
mitochondrial succinic dehydrogenase was also reported
to induce significant protection [
]. The S. japonicum
counterparts of surface proteins reported to elicit good
protection against S. mansoni, (SjTSP2 [
], Sj29 [
were not as effective when used to vaccinate mice before
challenge with S. japonicum cercariae. Finally, protection
has been induced in mice against S. japonicum challenge
using heterologous antigens as diverse as Lumbricus
terrestris (earthworm; [
]) and Trichinella spiralis extracts
]. Although the evidence is more fragmentary, we
suggest that our bystander hypothesis is equally applicable to
S. japonicum vaccine experiments in mice.
In the title of this review we ask if there is a flaw in the
mouse model. We have built a case that indeed there is,
namely the fragility of the pulmonary capillaries. The
consequence is that during lung transit in a naïve
mouse, schistosomula burst into alveoli and have only a
limited capability to continue migration thereafter; this
is very much the dominant determinant of parasite
maturation. There is good evidence that the RA vaccine
makes pulmonary migration more difficult via a specific
acquired response to surface and secreted antigens of
the schistosomulum; additional worms are deflected but
they are not harmed by the inflammatory responses.
Conversely, our hypothesis is that for many of the
proposed vaccine antigens the vaccination protocol
increases the difficulty of pulmonary migration in a
nonantigen-specific way. The kinetics of T cell production
after vaccination with an antigen will likely mirror
those after exposure to the RA vaccine. As the interval
between last boost and challenge is usually short,
activated cells will still be available in the circulation for
recruitment to the lungs when the challenge parasites
arrive. Note, such recruitment is not antigen-specific,
but activation-specific. It is also likely that the level of
pro-inflammatory cytokines in the circulation will be
high, perhaps sufficient to modify the pulmonary
vessels. This would be independent of the accessibility of
the vaccine antigen in the live parasite. Our explanation
also encompasses the protective effects of the various
heterologous antigens - they are simply very
How can we probe the mouse model to test the assertions made in this review?
A major point of this overview of vaccine testing in
the mouse was to provide a series of pointers for
experiments to bring clarity to the situation. This is a
plea to stop treating the mouse as a black-box test bed
and make immunological measurements strictly in the
parasitological context of challenge parasite migration
and elimination. The onus is on vaccine researchers to
show that their specific antigen model has a solid
knowledge base in acquired immunity. Some key
(i) What happens to the level of protection if the
interval between the last boost and cercarial
challenge is extended at least to five weeks and
preferably longer? The very short interval of 10–15
days is a severe criticism of many antigen vaccine
(ii) What is the profile of activated T cells and of
cytokines in the circulation after vaccination? Has
it declined to background levels before cercarial
challenge? The emphasis here is on circulation, not
spleen or lymph nodes. Proliferation of peripheral
blood mononuclear cells, cytokine production with
and without antigen restimulation, detection of a
DTH response by footpad or pinna swelling, are all
appropriate assays. Key signatures would be IFNγ,
TNFα or nitric oxide production, or the presence
of activated monocytes/macrophages.
(iii) Does percutaneous cercarial challenge at 5 weeks
or later elicit a detectable secondary response to
the target antigen in the circulation of the
antigen-vaccinated mice in the days immediately
after challenge, measured as above? This is vital
missing evidence that the living challenge larvae
can trigger a recall response to vaccine antigens,
especially internal ones.
(iv) Where are challenge parasites eliminated in
antigen-vaccinated animals? Skin, lungs or later, we
simply do not know. This was crucial to
understanding how the RA vaccine operated. Although
75Se-Methionine is no longer commercially available,
35S Methionine and Cysteine provide an intensity
of radioactive label that is still sufficient to allow
detection of parasites in the skin and lungs by
autoradiography so the question of elimination in those
sites can be explored.
(v) Continuing the possibility of lung involvement, is
there evidence for cell recruitment to the lungs
after challenge that might interfere with migration
in a non-antigen specific way? This is testable by
broncho-alveolar lavage, and flow cytometric
phenotyping of recovered populations.
(vi) Are there physiological or pharmacological
interventions that might alter the migratory profile
of schistosomula through the lung vasculature,
independent of vaccination experiments? Does i.v.
administration of pro-inflammatory cytokines such
as IFNγ or TNFα diminish schistosome maturation
as implied by the RA vaccine? Alternatively, can
we emulate the protection achieved with the RA
vaccine using pharmacological interventions?
Molecules that disrupt pulmonary hemodynamics
could in theory modify the barrier to parasite
migration. In this context, would nitric
oxidegenerating molecules, by promoting vasodilation,
significantly alter vascular tonus in the lungs? This
would be difficult to test due to the short-lived
nature of these molecules; perhaps their effects
could be sustained by the use of phosphodiesterase
inhibitors. Other molecules capable of altering lung
fluid homeostasis (e.g., leukotriene D4,
plateletactivating factor, and thromboxane A2 mimetics)
could represent potential drugs to be tested.
A recent issue of Frontiers in Immunology was entitled
“The Schistosomiasis Vaccine - It is Time to Stand up”. We
doubt on present evidence that many claims of efficacy are
plausible. The reservations we raise about the mouse as a
test-bed for schistosome vaccine antigens require thorough
scrutiny. It is our contention that due to the poor level of
maturation of S. mansoni parasites, almost any laboratory
host would be a better option (only the rat is worse). The
possibility of bystander effects drastically altering migration
and maturation should diminish in hosts where a greater
proportion of penetrants mature. On that basis, the
hamster would be a better choice for large scale tests with S.
mansoni and the rabbit with S. japonicum. The baboon
provides the ultimate choice as a permissive primate. Its
body mass, typically 6–10 kg, coupled with a high
percentage maturation of penetrant cercariae, point to a robust
pulmonary blood/air barrier keeping accidental parasite loss
to the alveoli to a minimum. The baboon’s ability to tolerate
a large cercarial challenge without developing severe of
lethal pathology and its phylogenetic proximity to Homo
sapiens are further positive attributes. We suggest that the
results of antigen trials in baboons should be in place
before the expensive scale-up to human trials is ever
Additional file 1: Spreadsheet calculations documenting the
predicted migration profile of schistosomula when the probablility
of getting stuck in the lungs on each circuit of the vascular
system is modified. a Cercarial challenge, maturation in the naive
mouse = 32 %. b Intravenous injection of day 7 schistosomula to the
pulmonary vasculature, maturation = 50 %. c Cercarial challenge of an
antigen-vaccinated mouse, 40 % protection. d Ditto, 70 % protection.
(XLSX 31.2 kb)
i.v.: intravenous; IFNγ: interferon gamma; TNFα: tumour necrosis factor alpha.
The authors declare that they have no competing interests.
This review is the result of tripartite discussions between the three authors.
The section on Schistosoma japonicum is primarily the work of XHL. All
authors read and approved the final version of the manuscript.
This work was supported by Special Visiting Researcher Program (Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior - CAPES) grant number 170/
2012, Ministry of Education, Brazilian Federal Government to WCB that allowed
RAW to work in his laboratory. XHL is supported by grant number 15ZR1444330
from Natural Science Foundation of Shanghai, China.
1. Figueiredo BC , Assis NR , Morais SB , Ricci ND , Pinheiro CS , Martins VP , et al. Schistosome syntenin partially protects vaccinated mice against Schistosoma mansoni infection . PLoS Negl Trop Dis . 2014 ; 8 ( 8 ), e3107 .
2. Diniz PP , Nakajima E , Miyasato PA , Nakano E , de Oliveira RM , Martins EA . Two SmDLC antigens as potential vaccines against schistosomiasis . Acta Trop . 2014 ; 140 : 193 - 201 .
3. Dias SR , Boroni M , Rocha EA , Dias TL , de Laet SD , Oliveira FM , et al. Evaluation of the Schistosoma mansoni Y-box-binding protein (SMYB1) potential as a vaccine candidate against schistosomiasis . Front Genet . 2014 ; 5 : 174 .
4. Neves LX , Sanson AL , Wilson RA , Castro-Borges W. What 's in SWAP? Abundance of the principal constituents in a soluble extract of Schistosoma mansoni revealed by shotgun proteomics . Parasit Vectors . 2015 ; 8 : 337 .
5. Aitken R , Coulson PS , Wilson RA. Pulmonary leukocytic responses are linked to the acquired immunity of mice vaccinated with irradiated cercariae of Schistosoma mansoni . J Immunol . 1988 ; 140 ( 10 ): 3573 - 9 .
6. Mountford AP , Coulson PS , Saunders N , Wilson RA . Characteristics of protective immunity in mice induced by drug-attenuated larvae of Schistosoma mansoni. Antigen localization and antibody responses . J Immunol . 1989 ; 143 ( 3 ): 989 - 95 .
7. Smythies LE , Pemberton RM , Coulson PS , Mountford AP , Wilson RA. T cellderived cytokines associated with pulmonary immune mechanisms in mice vaccinated with irradiated cercariae of Schistosoma mansoni . J Immunol . 1992 ; 148 ( 5 ): 1512 - 8 .
8. Street M , Coulson PS , Sadler C , Warnock LJ , McLaughlin D , Bluethmann H , et al. TNF is essential for the cell-mediated protective immunity induced by the radiation-attenuated schistosome vaccine . J Immunol . 1999 ; 163 ( 8 ): 4489 - 94 .
9. Coulson PS , Wilson RA. Recruitment of lymphocytes to the lung through vaccination enhances the immunity of mice exposed to irradiated schistosomes . Infect Immun . 1997 ; 65 ( 1 ): 42 - 8 .
10. Long E , Harrison R , Bickle Q , Bain J , Nelson G , Doenhoff M. Factors affecting the acquisition of resistance against Schistosoma mansoni in the mouse. The effect of varying the route and the number of primary infections, and the correlation between the size of the primary infection and the degree of resistance that is acquired . Parasitology . 1980 ; 81 ( 2 ): 355 - 71 .
11. El Ridi R , Tallima H , Salah M , Aboueldahab M , Fahmy OM , Al-Halbosiy MF , et al. Efficacy and mechanism of action of arachidonic acid in the treatment of hamsters infected with Schistosoma mansoni or Schistosoma haematobium . Int J Antimicrob Agents . 2012 ; 39 ( 3 ): 232 - 9 .
12. Xiao SH , Keiser J , Chollet J , Utzinger J , Dong Y , Endriss Y , et al. In vitro and in vivo activities of synthetic trioxolanes against major human schistosome species . Antimicrob Agents Chemother . 2007 ; 51 ( 4 ): 1440 - 5 .
13. Warren KS , Peters PA . Quantitative aspects of exposure time and cercarial dispersion on penetration and maturation of Schistosoma mansoni in mice . Ann Trop Med Parasitol . 1967 ; 61 ( 3 ): 294 - 301 .
14. Xiao SH , Chollet J , Weiss NA , Bergquist RN , Tanner M. Preventive effect of artemether in experimental animals infected with Schistosoma mansoni . Parasitol Int . 2000 ; 49 ( 1 ): 19 - 24 .
15. Miller P , Wilson RA . Migration of the schistosomula of Schistosoma mansoni from the lungs to the hepatic portal system . Parasitology . 1980 ; 80 ( 2 ): 267 - 88 .
16. Yole DS , Reid GD , Wilson RA . Protection against Schistosoma mansoni and associated immune responses induced in the vervet monkey Cercopithecus aethiops by the irradiated cercaria vaccine . Am J Trop Med Hyg . 1996 ; 54 ( 3 ): 265 - 70 .
17. Yole DS , Pemberton R , Reid GD , Wilson RA . Protective immunity to Schistosoma mansoni induced in the olive baboon Papio anubis by the irradiated cercaria vaccine . Parasitology . 1996 ; 112 (Pt 1): 37 - 46 .
18. Knopf PM , Cioli D , Mangold BL , Dean DA . Migration of Schistosoma mansoni in normal and passively immunized laboratory rats . Am J Trop Med Hyg . 1986 ; 35 ( 6 ): 1173 - 84 .
19. Cheever AW , Duvall RH . Variable maturation and oviposition by female Schistosoma japonicum in mice: the effects of irradiation of the host prior to infection . Am J Trop Med Hyg . 1987 ; 37 ( 3 ): 562 - 9 .
20. Vignali DA , Bickle QD , Taylor MG . Studies on immunity to Schistosoma mansoni in vivo: whole-body irradiation has no effect on vaccine-induced resistance in mice . Parasitology . 1988 ; 96 (Pt 1): 49 - 61 .
21. Aitken R , Wilson RA. The growth and development of Schistosoma mansoni in mice exposed to sublethal doses of radiation . J Parasitol . 1989 ; 75 ( 6 ): 958 - 63 .
22. Wilson RA . The saga of schistosome migration and attrition . Parasitology . 2009 ; 136 ( 12 ): 1581 - 92 .
23. Wilson RA. Cercariae to liver worms: development and migration in the mammalian host . In: Rollinson D , Simpson AJG , editors. The biology of schistosomes: from genes to latrines . London: Academic; 1987 . p. 115 - 46 .
24. Dean DA , Mangold BL , Georgi JR , Jacobson RH . Comparison of Schistosoma mansoni migration patterns in normal and irradiated cercaria-immunized mice by means of autoradiographic analysis. Evidence that worm elimination occurs after the skin phase in immunized mice . Am J Trop Med Hyg . 1984 ; 33 ( 1 ): 89 - 96 .
25. Wilson RA , Coulson PS , Dixon B. Migration of the schistosomula of Schistosoma mansoni in mice vaccinated with radiation-attenuated cercariae, and normal mice: an attempt to identify the timing and site of parasite death . Parasitology . 1986 ; 92 (Pt 1): 101 - 16 .
26. Mangold BL , Dean DA , Coulson PS , Wilson RA. Site requirements and kinetics of immune-dependent elimination of intravascularly administered lung stage schistosomula in mice immunized with highly irradiated cercariae of Schistosoma mansoni . Am J Trop Med Hyg . 1986 ; 35 ( 2 ): 332 - 44 .
27. Coulson PS , Wilson RA. Examination of the mechanisms of pulmonary phase resistance to Schistosoma mansoni in vaccinated mice . Am J Trop Med Hyg . 1988 ; 38 ( 3 ): 529 - 39 .
28. Wilson RA , Coulson PS . Schistosoma mansoni: dynamics of migration through the vascular system of the mouse . Parasitology . 1986 ; 92 (Pt 1): 83 - 100 .
29. Wilson RA , Draskau T , Miller P , Lawson JR . Schistosoma mansoni: the activity and development of the schistosomulum during migration from the skin to the hepatic portal system . Parasitology . 1978 ; 77 ( 1 ): 57 - 73 .
30. Crabtree JE , Wilson RA . Schistosoma mansoni: an ultrastructural examination of pulmonary migration . Parasitology . 1986 ; 92 (Pt 2): 343 - 54 .
31. Crabtree JE , Wilson RA . The role of pulmonary cellular reactions in the resistance of vaccinated mice to Schistosoma mansoni . Parasite Immunol . 1986 ; 8 ( 3 ): 265 - 85 .
32. Dean DA , Mangold BL . Evidence that both normal and immune elimination of Schistosoma mansoni take place at the lung stage of migration prior to parasite death . Am J Trop Med Hyg . 1992 ; 47 ( 2 ): 238 - 48 .
33. West JB . Fragility of pulmonary capillaries . J Appl Physiol ( 1985 ). 2013 ; 115 ( 1 ): 1 - 15 .
34. Birks EK , Mathieu-Costello O , Fu Z , Tyler WS , West JB . Comparative aspects of the strength of pulmonary capillaries in rabbit, dog, and horse . Respir Physiol . 1994 ; 97 ( 2 ): 235 - 46 .
35. Guntheroth WG , Luchtel DL , Kawabori I. Pulmonary microcirculation: tubules rather than sheet and post . J Appl Physiol Respir Environ Exerc Physiol . 1982 ; 53 ( 2 ): 510 - 5 .
36. Crabtree JE , Wilson RA . Schistosoma mansoni: a scanning electron microscope study of the developing schistosomulum . Parasitology . 1980 ; 81 (Pt 3): 553 - 64 .
37. Baluna R , Vitetta ES . Vascular leak syndrome: a side effect of immunotherapy . Immunopharmacology . 1997 ; 37 ( 2-3 ): 117 - 32 .
38. Dubinett SM , Huang M , Lichtenstein A , McBride WH , Wang J , Markovitz G , et al. Tumor necrosis factor-alpha plays a central role in interleukin-2- induced pulmonary vascular leak and lymphocyte accumulation . Cell Immunol . 1994 ; 157 ( 1 ): 170 - 80 .
39. Park KY , Kim SJ , Oh E , Heo TH . Induction of vascular leak syndrome by tumor necrosis factor-alpha alone . Biomed Pharmacother . 2015 ; 70 : 213 - 6 .
40. Coulson PS . The radiation-attenuated vaccine against schistosomes in animal models: paradigm for a human vaccine? Adv Parasitol . 1997 ; 39 : 271 - 336 .
41. Hewitson JP , Hamblin PA , Mountford AP . Immunity induced by the radiationattenuated schistosome vaccine . Parasite Immunol . 2005 ; 27 ( 7-8 ): 271 - 80 .
42. Bickle QD . Radiation-attenuated schistosome vaccination-a brief historical perspective . Parasitology . 2009 ; 136 ( 12 ): 1621 - 32 .
43. Coulson PS , Wilson RA. Pulmonary T helper lymphocytes are CD44hi, CD45RB- effector/memory cells in mice vaccinated with attenuated cercariae of Schistosoma mansoni . J Immunol . 1993 ; 151 ( 7 ): 3663 - 71 .
44. Wynn TA , Jankovic D , Hieny S , Cheever AW , Sher A . IL -12 enhances vaccineinduced immunity to Schistosoma mansoni in mice and decreases T helper 2 cytokine expression , IgE production, and tissue eosinophilia . J Immunol . 1995 ; 154 ( 9 ): 4701 - 9 .
45. Anderson S , Shires VL , Wilson RA , Mountford AP . In the absence of IL-12, the induction of Th1-mediated protective immunity by the attenuated schistosome vaccine is impaired, revealing an alternative pathway with Th2- type characteristics . Eur J Immunol . 1998 ; 28 ( 9 ): 2827 - 38 .
46. Portielje JE , Kruit WH , Eerenberg AJ , Schuler M , Sparreboom A , Lamers CH , et al. Subcutaneous injection of interleukin 12 induces systemic inflammatory responses in humans: implications for the use of IL-12 as vaccine adjuvant . Cancer Immunol Immunother . 2005 ; 54 ( 1 ): 37 - 43 .
47. Dean DA , Bukowski MA , Clark SS . Attempts to transfer the resistance of Schistosoma mansoni-infected and irradiated cercaria-immunized mice by means of parabiosis . Am J Trop Med Hyg . 1981 ; 30 ( 1 ): 113 - 20 .
48. Bickle QD , Andrews BJ , Doenhoff MJ , Ford MJ , Taylor MG . Resistance against Schistosoma mansoni induced by highly irradiated infections: studies on species specificity of immunization and attempts to transfer resistance . Parasitology . 1985 ; 90 (Pt 2): 301 - 12 .
49. Mangold BL , Dean DA . Passive transfer with serum and IgG antibodies of irradiated cercaria-induced resistance against Schistosoma mansoni in mice . J Immunol . 1986 ; 136 ( 7 ): 2644 - 8 .
50. Wilson RA , Coulson PS , Mountford AP . Immune responses to the radiationattenuated schistosome vaccine: what can we learn from knock-out mice? Immunol Lett . 1999 ; 65 ( 1-2 ): 117 - 23 .
51. Smythies LE , Betts C , Coulson PS , Dowling MA , Wilson RA. Kinetics and mechanism of effector focus formation in the lungs of mice vaccinated with irradiated cercariae of Schistosoma mansoni . Parasite Immunol . 1996 ; 18 ( 7 ): 359 - 69 .
52. Smythies LE , Coulson PS , Wilson RA. Monoclonal antibody to IFN-gamma modifies pulmonary inflammatory responses and abrogates immunity to Schistosoma mansoni in mice vaccinated with attenuated cercariae . J Immunol . 1992 ; 149 ( 11 ): 3654 - 8 .
53. Wilson RA , Coulson PS , Betts C , Dowling MA , Smythies LE . Impaired immunity and altered pulmonary responses in mice with a disrupted interferon-gamma receptor gene exposed to the irradiated Schistosoma mansoni vaccine . Immunology . 1996 ; 87 ( 2 ): 275 - 82 .
54. Dessein A , Samuelson JC , Butterworth AE , Hogan M , Sherry BA , Vadas MA , et al. Immune evasion by Schistosoma mansoni: loss of susceptibility to antibody or complement-dependent eosinophil attack by schistosomula cultured in medium free of macromolecules . Parasitology . 1981 ; 82 (Pt 3): 357 - 74 .
55. Nare B , Smith JM , Prichard RK . Schistosoma mansoni: levels of antioxidants and resistance to oxidants increase during development . Exp Parasitol . 1990 ; 70 ( 4 ): 389 - 97 .
56. Mountford AP , Coulson PS , Pemberton RM , Smythies LE , Wilson RA . The generation of interferon-gamma-producing T lymphocytes in skin-draining lymph nodes, and their recruitment to the lungs, is associated with protective immunity to Schistosoma mansoni . Immunology . 1992 ; 75 ( 2 ): 250 - 6 .
57. Ratcliffe EC , Wilson RA . The magnitude and kinetics of delayed-type hypersensitivity responses in mice vaccinated with irradiated cercariae of Schistosoma mansoni . Parasitology . 1991 ; 103 (Pt 1): 65 - 75 .
58. Eberl M , Langermans JA , Frost PA , Vervenne RA , van Dam GJ , Deelder AM , et al. Cellular and humoral immune responses and protection against schistosomes induced by a radiation-attenuated vaccine in chimpanzees . Infect Immun . 2001 ; 69 ( 9 ): 5352 - 62 .
59. Pacifico LG , Fonseca CT , Chiari L , Oliveira SC . Immunization with Schistosoma mansoni 22.6 kDa antigen induces partial protection against experimental infection in a recombinant protein form but not as DNA vaccine . Immunobiology . 2006 ; 211 ( 1-2 ): 97 - 104 .
60. Alves CC , Araujo N , dos Santos VC , Couto FB , Assis NR , Morais SB , et al. Sm29, but not Sm22 . 6 retains its ability to induce a protective immune response in mice previously exposed to a Schistosoma mansoni infection . PLoS Negl Trop Dis . 2015 ; 9 ( 2 ): e0003537 .
61. Cardoso FC , Macedo GC , Gava E , Kitten GT , Mati VL , de Melo AL , et al. Schistosoma mansoni tegument protein Sm29 is able to induce a Th1-type of immune response and protection against parasite infection . PLoS Negl Trop Dis . 2008 ; 2 ( 10 ), e308 .
62. Stephenson R , You H , McManus DP , Toth I. Schistosome vaccine adjuvants in preclinical and clinical research . Vaccines (Basel) . 2014 ; 2 ( 3 ): 654 - 85 .
63. Ewaisha RE , Bahey-El-Din M , Mossallam SF , Amer EI , Aboushleib HM , Khalil AM . Combination of the two schistosomal antigens Sm14 and Sm29 elicits significant protection against experimental Schistosoma mansoni infection . Exp Parasitol . 2014 ; 145 : 51 - 60 .
64. Pinheiro CS , Ribeiro AP , Cardoso FC , Martins VP , Figueiredo BC , Assis NR , et al. A multivalent chimeric vaccine composed of Schistosoma mansoni SmTSP-2 and Sm29 was able to induce protection against infection in mice . Parasite Immunol . 2014 ; 36 ( 7 ): 303 - 12 .
65. Argiro L , Henri S , Dessein H , Kouriba B , Dessein AJ , Bourgois A . Induction of a protection against S. mansoni with a MAP containing epitopes of Sm37- GAPDH and Sm10-DLC. Effect of coadsorption with GM-CSF on alum . Vaccine . 2000 ; 18 ( 19 ): 2033 - 8 .
66. Yang W , Jackson DC , Zeng Q , McManus DP . Multi-epitope schistosome vaccine candidates tested for protective immunogenicity in mice . Vaccine . 2000 ; 19 ( 1 ): 103 - 13 .
67. Murrell KD , Dean DA , Stafford EE . Resistance to infection with Schistosoma mansoni after immunization with worm extracts or live cercariae: role of cytotoxic antibody in mice and guinea pigs . Am J Trop Med Hyg . 1975 ; 24 ( 6 Pt 1 ): 955 - 62 .
68. James SL , Pearce EJ . The influence of adjuvant on induction of protective immunity by a non-living vaccine against schistosomiasis . J Immunol . 1988 ; 140 ( 8 ): 2753 - 9 .
69. James SL . Activated macrophages as effector cells of protective immunity to schistosomiasis . Immunol Res . 1986 ; 5 ( 2 ): 139 - 48 .
70. James SL , DeBlois LA . Induction of protective immunity against Schistosoma mansoni by a nonliving vaccine. II. Response of mouse strains with selective immune defects . J Immunol . 1986 ; 136 ( 10 ): 3864 - 71 .
71. James SL . Induction of protective immunity against Schistosoma mansoni by a non-living vaccine . V. Effects of varying the immunization and infection schedule and site . Parasite Immunol . 1987 ; 9 ( 5 ): 531 - 41 .
72. Pearce EJ , James SL , Hieny S , Lanar DE , Sher A . Induction of protective immunity against Schistosoma mansoni by vaccination with schistosome paramyosin (Sm97), a nonsurface parasite antigen . Proc Natl Acad Sci U S A . 1988 ; 85 ( 15 ): 5678 - 82 .
73. Civil RH , Warren KS , Mahmoud AA . Conditions for bacille Calmette-Guerininduced resistance to infection with Schistosoma mansoni in mice . J Infect Dis . 1978 ; 137 ( 5 ): 550 - 5 .
74. Waeckerle-Men Y , Bruffaerts N , Liang Y , Jurion F , Sander P , Kundig TM , et al. Lymph node targeting of BCG vaccines amplifies CD4 and CD8 T-cell responses and protection against Mycobacterium tuberculosis . Vaccine . 2013 ; 31 ( 7 ): 1057 - 64 .
75. Philippsen GS , Wilson RA , DeMarco R. Accelerated evolution of schistosome genes coding for proteins located at the host-parasite interface . Genome Biol Evol . 2015 ; 7 ( 2 ): 431 - 43 .
76. DeMarco R , Mathieson W , Manuel SJ , Dillon GP , Curwen RS , Ashton PD , et al. Protein variation in blood-dwelling schistosome worms generated by differential splicing of micro-exon gene transcripts . Genome Res . 2010 ; 20 ( 8 ): 1112 - 21 .
77. Parker-Manuel SJ , Ivens AC , Dillon GP , Wilson RA . Gene expression patterns in larval Schistosoma mansoni associated with infection of the mammalian host . PLoS Negl Trop Dis . 2011 ; 5 ( 8 ), e1274 .
78. Li XH , de Castro-Borges W , Parker-Manuel S , Vance GM , Demarco R , Neves LX , et al. The schistosome oesophageal gland: initiator of blood processing . PLoS Negl Trop Dis . 2013 ; 7 ( 7 ), e2337 .
79. Chalmers IW , McArdle AJ , Coulson RM , Wagner MA , Schmid R , Hirai H , et al. Developmentally regulated expression, alternative splicing and distinct subgroupings in members of the Schistosoma mansoni venom allergen-like (SmVAL) gene family . BMC Genomics . 2008 ; 9 : 89 .
80. Hillyer GV , del Llano de Diaz A , Reyes CN . Schistosoma mansoni: acquired immunity in mice and hamsters using antigens of Fasciola hepatica . Exp Parasitol . 1977 ; 42 ( 2 ): 348 - 55 .
81. Hillyer GV , Garcia Rosa MI , Alicea H , Hernandez A . Successful vaccination against murine Schistosoma mansoni infection with a purified 12 Kd Fasciola hepatica cross-reactive antigen . Am J Trop Med Hyg . 1988 ; 38 ( 1 ): 103 - 10 .
82. Moser D , Tendler M , Griffiths G , Klinkert MQ . A 14-kDa Schistosoma mansoni polypeptide is homologous to a gene family of fatty acid binding proteins . J Biol Chem . 1991 ; 266 ( 13 ): 8447 - 54 .
83. Tendler M , Brito CA , Vilar MM , Serra-Freire N , Diogo CM , Almeida MS , et al. A Schistosoma mansoni fatty acid-binding protein, Sm14, is the potential basis of a dual-purpose anti-helminth vaccine . Proc Natl Acad Sci U S A . 1996 ; 93 ( 1 ): 269 - 73 .
84. Abane JL , Oleaga A , Ramajo V , Casanueva P , Arellano JL , Hillyer GV , et al. Vaccination of mice against Schistosoma bovis with a recombinant fatty acid binding protein from Fasciola hepatica . Vet Parasitol . 2000 ; 91 ( 1-2 ): 33 - 42 .
85. El Ridi R , Tallima H . Vaccine-induced protection against murine schistosomiasis mansoni with larval excretory-secretory antigens and papain or type-2 cytokines . J Parasitol . 2013 ; 99 ( 2 ): 194 - 202 .
86. Sokol CL , Medzhitov R . Role of basophils in the initiation of Th2 responses . Curr Opin Immunol . 2010 ; 22 ( 1 ): 73 - 7 .
87. El Ridi R , Tallima H , Selim S , Donnelly S , Cotton S , Gonzales Santana B , et al. Cysteine peptidases as schistosomiasis vaccines with inbuilt adjuvanticity . PLoS One . 2014 ; 9 ( 1 ), e85401 .
88. Kariuki TM , Farah IO , Yole DS , Mwenda JM , Van Dam GJ , Deelder AM , et al. Parameters of the attenuated schistosome vaccine evaluated in the olive baboon . Infect Immun . 2004 ; 72 ( 9 ): 5526 - 9 .
89. Boulanger D , Reid GD , Sturrock RF , Wolowczuk I , Balloul JM , Grezel D , et al. Immunization of mice and baboons with the recombinant Sm28GST affects both worm viability and fecundity after experimental infection with Schistosoma mansoni . Parasite Immunol . 1991 ; 13 ( 5 ): 473 - 90 .
90. Soisson LA , Reid GD , Farah IO , Nyindo M , Strand M. Protective immunity in baboons vaccinated with a recombinant antigen or radiation-attenuated cercariae of Schistosoma mansoni is antibody-dependent . J Immunol . 1993 ; 151 ( 9 ): 4782 - 9 .
91. Carvalho-Queiroz C , Nyakundi R , Ogongo P , Rikoi H , Egilmez NK , Farah IO , et al. Protective potential of antioxidant enzymes as vaccines for schistosomiasis in a non-human primate model . Front Immunol . 2015 ; 6 : 273 .
92. Zhang W , Ahmad G , Torben W , Noor Z , Le L , Damian RT , et al. Sm-p80- based DNA vaccine provides baboons with levels of protection against Schistosoma mansoni infection comparable to those achieved by the irradiated cercarial vaccine . J Infect Dis . 2010 ; 201 ( 7 ): 1105 - 12 .
93. Ahmad G , Zhang W , Torben W , Ahrorov A , Damian RT , Wolf RF , et al. Preclinical prophylactic efficacy testing of Sm-p80-based vaccine in a nonhuman primate model of Schistosoma mansoni infection and immunoglobulin G and E responses to Sm-p80 in human serum samples from an area where schistosomiasis is endemic . J Infect Dis . 2011 ; 204 ( 9 ): 1437 - 49 .
94. Ho YH . On th host specificity of Schistosoma japonicum . Chin Med J. 1963 ; 82 ( 7 ): 403 - 14 .
95. Xiao SH , Mei JY , Jiao PY . Schistosoma japonicum-infected hamsters (Mesocricetus auratus) used as a model in experimental chemotherapy with praziquantel, artemether, and OZ compounds . Parasitol Res . 2011 ; 108 ( 2 ): 431 - 7 .
96. Xiao SH , Jiqing Y , Jinying M , Huifang G , Peiying J , Tanner M. Effect of praziquantel together with artemether on Schistosoma japonicum parasites of different ages in rabbits . Parasitol Int . 2000 ; 49 ( 1 ): 25 - 30 .
97. Tang CC , Tang CT , Tang C . Studies on the migratory route of Schistosoma japonicum in its final host . Acta Zool Sin . 1973 ; 19 : 323 - 36 .
98. Gui M , Kusel JR , Shi YE , Ruppel A . Schistosoma japonicum and S. mansoni: comparison of larval migration patterns in mice . J Helminthol . 1995 ; 69 ( 1 ): 19 - 25 .
99. Laxer MJ , Tuazon CU . Migration of 75Se-methionine-labeled Schistosoma japonicum in normal and immunized mice . J Infect Dis . 1992 ; 166 ( 5 ): 1133 - 8 .
100. Moloney NA , Webbe G. The host-parasite relationship of Schistosoma japonicum in CBA mice . Parasitology . 1983 ; 87 (Pt 2): 327 - 42 .
101. Li XH , Liu SX . Progress on attenuated vaccines against schistosomiasis . Int J Parasit Dis . 2003 ; 30 ( 3 ): 97 - 102 .
102. Dunne DW , Jones FM , Cook L , Moloney NA . Passively transferable protection against Schistosoma japonicum induced in the mouse by multiple vaccination with attenuated larvae: the development of immunity, antibody isotype responses and antigen recognition . Parasite Immunol . 1994 ; 16 ( 12 ): 655 - 68 .
103. Lin D , Tian F , Wu H , Gao Y , Wu J , Zhang D , et al. Multiple vaccinations with UV- attenuated cercariae in pig enhance protective immunity against Schistosoma japonicum infection as compared to single vaccination . Parasit Vectors . 2011 ; 4 : 103 .
104. Hsu SY , Hsu HF , Xu ST , Shi FH , He YX , Clarke WR , et al. Vaccination against bovine schistosomiasis japonica with highly X-irradiated schistosomula . Am J Trop Med Hyg . 1983 ; 32 ( 2 ): 367 - 70 .
105. Li XH , Cao JP , Liu SX . Recent progess on candidate antigens for a vaccine against schistosomiasis japonicum in China . Chinese J Zoonoses . 2005 ; 21 ( 10 ): 901 - 5 .
106. Zhu YC , Ren JG , Si J , Harn DA , Yu CX , Liang YS , et al. Protective immunity with co-immunization of Sjc23kDa vaccine and SjcTPI DNA vaccine in Schistosoma japonicum infection . Chinese J Schistosomiasis Control . 2002 ; 14 ( 2 ): 84 - 7 .
107. Hu XM , Zhang ZS , Wu HW , Li CL , Su C , Ji MJ , et al. Studies on immunoprotection in mice after immunization with the epitopes of mithochondria-related protein rSj33.8 and rSj22.6 of Schistosoma japonicum . Chinese J Schistosomiasis Control . 2002 ; 14 ( 2 ): 88 - 91 .
108. Zhao W , Su C , Wu HW , Hu XM , Shen L , Ji MJ , et al. Studies on immunoprotection in mice after immunization with Schistosoma japonicum fatty acid binding protein (Sj-FABPc) recombinant protein . Chinese J Zoonoses . 2004 ; 18 ( 3 ): 42 - 4 .
109. Jiang MS , Chen JQ , Mei BS , Yang MX , Hong JL , Ni YH , et al. Induction of protective immunity against Schistosoma japonicum by soluble adult worm antigens . Chinese J Parasitic Dis Control . 1993 ; 6 ( 4 ): 269 - 72 .
110. Yu JL , Wang SP , He Z , Dai K , Xu SR , Liu XQ . Construction of the eukaryotic recombinant plasmid for hypoxanthine-guanine phosphoribosyltransferase of Schistosoma japonicum Chinese strain and its immunoprotection in mice . Chin J Microbiol Immunol . 2007 ; 27 ( 6 ): 565 - 9 .
111. Yu J , Wang S , Li W , Dai G , Xu S , He Z , et al. Cloning, expression and protective immunity evaluation of the full-length cDNA encoding succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum . Sci China C Life Sci . 2007 ; 50 ( 2 ): 221 - 7 .
112. Yu CX , Yin XR , Li J , Wu YD , Hua WQ , Liang YS , et al. Protective effect of recombinant TSP2 hydrophilic domain (TSP2HD) of Schistosoma japonicum in immunized mice . Chinese J Schistosomiasis Control . 2009 ; 21 ( 1 ): 6 - 10 .
113. Chen H , Fu ZQ , Chen L , Qiu CH , Fu GW , Li Y , et al. Immune protection of tegument protein rSj29 against Schistosoma japonicum in mice . Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi . 2009 ; 27 ( 6 ): 476 - 82 .
114. Wisnewski AV , Kresina TF . Induction of protective immunity to schistosomiasis with immunologically cross-reactive Lumbricus molecules . Int J Parasitol . 1995 ; 25 ( 4 ): 503 - 10 .
115. Tian M , Yi X , Zeng X , Zeng Q , Peng X . Cross-protection against Schistosoma japonicum infection in mice immunized with Trichinella spiralis muscle larva antigen . Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi . 1998 ; 16 ( 6 ): 411 - 4 .