CHAPTER 26 Vaccination against Toxoplasmosis: Current Status and Future Prospects Craig W. Roberts*, Rima McLeod y , Fiona L. Henriquez**, James Alexander* *University of Strathclyde, Glasgow, Scotland, United Kingdom y The University of Chicago, Chicago, Illinois, USA **Institute of Biomedical and Environmental Health Research, School of Science, University of the West of Scotland, Paisley, Scotland, United Kingdom OUTLINE 26.1 Introduction 996 26.2 Scope of Problem and Potential Benefits of Vaccination 996 26.2.1 Toxoplasmosis in Animals and the Potential Benefits of Vaccination 996 26.2.1.1 Toxoplasmosis in Sheep and Goats 996 26.2.1.2 Toxoplasmosis in Pigs 997 26.2.1.3 Toxoplasmosis in Cattle 998 26.2.1.4 Toxoplasmosis in Chickens 998 26.2.1.5 Toxoplasmosis in Cats 998 26.2.2 Benefits of Animal Vaccination 998 26.2.3 Toxoplasmosis in Humans and the Potential Benefits of Vaccination 999 26.3 Current Status of Vaccines for Intermediate Hosts 1001 26.3.1 Vaccination Using Extracts or Killed Parasites 1001 26.3.2 Vaccination Using Live, Attenuated Parasites 1005 26.3.2.1 A Commercial Vaccine (S48) Against Toxoplasmosis in Sheep and Goats 1006 26.3.3 Vaccination Using Gene Deletion Attenuated Parasites 1007 26.3.4 Vaccination Using Viral Vectors 1010 26.3.5 Vaccination Using Bacterial Vectors 1011 26.3.6 DNA Vaccines 1012 26.3.7 Sub-Unit Vaccines 1021 26.3.8 Genomics and Immunosense 1030 26.4 The Rodent as a Model to Study Congenital Disease and Vaccination 1031 Toxoplasma gondii, second edition http://dx.doi.org/10.1016/B978-0-12-396481-6.00026-X Copyright Ó 2014 Elsevier Ltd. All rights reserved. 995
Transcript
C H A
P T E R
26
Vaccination against Toxoplasmosis:Current Status and Future ProspectsCraig W. Roberts*, Rima McLeody, Fiona L. Henriquez**,
James Alexander**University of Strathclyde, Glasgow, Scotland, United Kingdom yThe University of Chicago, Chicago,
Illinois, USA **Institute of Biomedical and Environmental Health Research, School of Science,University of the West of Scotland, Paisley, Scotland, United Kingdom
Th
O U T L I N E
26.1 Introduction 99
6
26.2 Scope of Problem and PotentialBenefits of Vaccination 996
26.2.1 Toxoplasmosis in Animals and the
Potential Benefits of Vaccination
996 26.2.1.1 Toxoplasmosis in Sheep
and Goats
996 26.2.1.2 Toxoplasmosis in Pigs 997 26.2.1.3 Toxoplasmosis in Cattle 998 26.2.1.4 Toxoplasmosis in
Chickens
998 26.2.1.5 Toxoplasmosis in Cats 998
26.2.2 Benefits of Animal Vaccination
998 26.2.3 Toxoplasmosis in Humans and
the Potential Benefits ofVaccination
999
26.3 Current Status of Vaccines forIntermediate Hosts 1001
oxoplasma gondii, second editionttp://dx.doi.org/10.1016/B978-0-12-396481-6.00026-X 995
26.3.1 Vaccination Using Extracts orKilled Parasites
1001
26.3.2 Vaccination Using Live,Attenuated Parasites
1005 26.3.2.1 A Commercial Vaccine
(S48) AgainstToxoplasmosis in Sheepand Goats
1006
26.3.3 Vaccination Using Gene DeletionAttenuated Parasites
1007
26.3.4 Vaccination Using Viral Vectors
1010 26.3.5 Vaccination Using Bacterial
Vectors
1011 26.3.6 DNA Vaccines 1012 26.3.7 Sub-Unit Vaccines 1021 26.3.8 Genomics and Immunosense 1030
26.4 The Rodent as a Model to StudyCongenital Disease and Vaccination 1031
Copyright � 2014 Elsevier Ltd. All rights reserved.
26. VACCINATION AGAINST TOXOPLASMOSIS: CURRENT STATUS AND FUTURE PROSPECTS996
26.5 Review of Vaccines for DefinitiveHost (CATS) 103
3
26.6 Future Strategies to Design NewVaccines for Coccidial Parasites in
General and Toxoplasma gondii inParticular 103
5
References 1037
26.1 INTRODUCTION
Toxoplasma gondii is a zoonotic disease and assuch a successful vaccinewould have both benefi-cial medical and veterinary impacts. An effectivevaccine for use in humans, while serving, in thefirst instance, to reduce mortality and morbidityassociated with infection, would also haveeconomic benefits as it would reduce the financialburden of lifelong care needed for those withsevere chronic disease. The ideal vaccine for veter-inary use would have the dual advantages ofincreasing livestock productivity while reducingthe public health risk associatedwith eatingmeat.
26.2 SCOPE OF PROBLEM ANDPOTENTIAL BENEFITS OF
VACCINATION
26.2.1 Toxoplasmosis in Animals andthe Potential Benefits of Vaccination
T. gondii is a widely disseminated parasite,which is capable of infecting all warm bloodedvertebrates to different degrees of severity,dependingonthespecies infected.Prior toadiscus-sion on the benefits of vaccination, we shall definethe problems associated with toxoplasmosis indifferent economically important livestockanimals such as sheep, goats, pigs, cattle andchickens and in the definitive host species, the cat.
26.2.1.1 Toxoplasmosis in Sheep and Goats
The main problem associated with T. gondiiinfections in sheep and goats is foetal death. In
countries with a high prevalence of T. gondii,such as the UK and Spain, the parasite isresponsible for 25% of all abortions (Buxton,1998; Pereira-Bueno et al., 2004). Sheep becomeinfected with oocysts derived from contaminatedfeed, pasture orwater. Sincemost sheep and goatsare kept outdoors (with the exception of milkgoats) they are all at risk. In non-pregnant sheepa T. gondii infection normally goes undetectedand is associated with mild flu-like symptoms,which coincide with the presence of tachyzoitesin the circulation. The induction of protectiveimmunity results in reduced parasitaemia andconversion of tachyzoites to bradyzoites, followedby a persistent lifelong infection with tissue cysts.Most evidence indicates that after a primary infec-tion, sheep become immune to reinfection and thisimmunity prevents foetal infections duringsubsequent pregnancies (reviewed in Buxtonand Innes, 1995; Munday, 1972; Frenkel, 1990;McColgan et al., 1988). However, a study byWilliams et al. (2005) would suggest that there isa high rate (54%) of transplacental transmissionof Toxoplasma in sheep, which was proposed tobe due to reactivation of chronic infections. Thesedata conflicted with older reports (Hartley, 1966;Munday, 1972) and led tomuch discussion,whichquickly resulted in another independent studythat could not confirm the results from Williams(Rodger et al., 2005). Thus, although pregnant,immune ewes may incidentally transmit Toxo-plasma to their offspring, it seems to remain morethe exception than the rule.
Sheep and goats that contract a primary infec-tion during gestation have a high risk (greaterthan 80%) that tachyzoites will infect the
26.2 SCOPE OF PROBLEM AND POTENTIAL BENEFITS OF VACCINATION 997
placenta and traverse to the foetus, leading toresorption, abortion or stillbirth (Buxton, 1998).The clinical outcome depends on when duringthe 145 day gestation period a pregnant ewebecomes infected. Infection early in pregnancyis likely to cause foetal resorption, whereas infec-tion late in pregnancy (after day 120) usuallyresults in the birth of an apparently normallamb, which may in fact be infected. Suchinfected newly born lambs usually have devel-oped a protective immune response to T. gondii,therefore few congenital defects are observed(Buxton, 1998). Infection in midterm (days 50e120) will cause foetal death, mummificationand abortion; the time from infection to abortionbeing about 40 days. T. gondii specific antibodiesmay be detected in the foetal circulation 30 daysafter initial infection of the ewe (reviewed inBuxton, 1998).
The impact and prevalence of T. gondii infec-tion in sheep and goats in the USA has recentlybeen reviewed in detail (Hill and Dubey, 2012).In one study cited, approximately 27% of lambsbutchered and on sale to the public harboured T.gondii infection. Types II and III lineages predo-minated in this sample, although some atypicaland mixed infections were recorded. In a sepa-rate study, approximately 53% of goats werefound to be seropositive for T. gondii. Approxi-mately 26% of commercially available goats’meat (heart) was found to contain viable T. gon-dii and Types II and III lineages predominatedwith some atypical and mixed infections noted(Hill and Dubey, 2012). These recent insights,specifically the identification of atypical lineages,highlight the potential risk of consuming thesemeats and the potential benefits of vaccination.
26.2.1.2 Toxoplasmosis in Pigs
Foetal T. gondii infections in pigs can lead toabortion and stillbirth, similar to sheep (Dubeyand Urban, 1990). Transplacental infection inpigs is less common than postnatal infections(reviewed in Dubey, 1986, 2009). In particular,young nursing pigs are susceptible to
toxoplasmosis, showing fever, coughing, weak-ness and wasting. In adults, toxoplasmosis ismostly sub-clinical, but infection is persistentwith tissue cysts being present in many differenttissues (reviewed in Dubey, 1986, 2009). As such,the contamination of pork meat with tissue cystsfor human consumption defines the majorproblem in pigs.
The prevalence of T. gondii positive pigs iscomplicated by the different farm facilities andfarm managements that are used and, further-more, depends on the age of the animal. Thereis an extremely high incidence of toxoplasmosisin pigs reared outdoors. In the late 1960s, whenpigs were kept outdoors, 75% of pigs wereinfected with T. gondii (Tenter et al., 2000). Theintroduction of indoor farming facilities hasdramatically reduced infection rates to as lowas 1% (Davies et al., 1998). In Argentina seropre-valence was only 4% in indoor reared pigs,whereas in some farms outdoor reared sowswere 100% positive (Venturini et al., 2004).A study in the Netherlands showed that pigsreared indoors were completely free of T. gondiiinfections while 3% of animal welfare-friendlyreared pigs were seropositive for T. gondii (Kijl-stra et al., 2004). Thus, T. gondii infections, atpresent, are mainly an issue for the minority ofpigs that are reared outdoors. Currently withinthe EU there is broad concern about animalwelfare and an increasing trend to purchaseanimal welfare-friendly products (Kyprianou,2005), which will undoubtedly lead to morefree ranging pigs within the EU and is likely tocoincide with a re-emergence of infected pigsand pork meat. Indeed, to highlight the likeli-hood of this happening a recent study in theUSA has demonstrated for the first time a highprevalence of T. gondii in ‘organic’ pigs withType II and Type III clonal lineages being identi-fied (Dubey et al., 2012). Throughout Europe andthe USA, Type II and III strains predominate(Dubey, 2009), although in China and SouthAmerica distinct genotypes may be the norm(Bezerra et al., 2012; Zhou et al., 2010).
26. VACCINATION AGAINST TOXOPLASMOSIS: CURRENT STATUS AND FUTURE PROSPECTS998
26.2.1.3 Toxoplasmosis in Cattle
Cattle do not get clinically ill from T. gondiiinfection and the only substantive concern iswhether or not beef can be infective toconsumers. Various surveys could not identifycattle that had been infected with T. gondii inthe field, and controlled infections with T. gondiidisplayed only transient infections, which werequickly eliminated (Dubey, 1990). Experimentalinfection followed by feeding of edible tissue tomice and cats demonstrated that T. gondii couldnot be isolated in significant quantities fromthese tissues; mice fed with homogenized organsremained negative and cats shed oocysts afterfeeding on heart and tongue (Dubey et al.,1993). A recent study has indicated that althoughinfrequent congenital infection can occurfollowing natural infection (Costa et al., 2011).Abortions in cattle reported as being due to T.gondii in the past were probably a result of Neo-spora caninum infection, which was first recog-nized in 1989 (Dubey and Lindsay, 1996).Overall, beef are only transiently T. gondii posi-tive generally, and this was thought to poseonly a small risk for human health. A quantita-tive risk assessment study from the Netherlandsto determine the relative contribution of sheep,pork and cattle to human infection, taking intoaccount prevalence, meat processing techniquesand consumption, demonstrated that, evenwith a low prevalence of infection in cattle,consumption of beef remained not only animportant source of infection for humans butprobably the major source (Opsteegh et al., 2011).
26.2.1.4 Toxoplasmosis in Chickens
Chickens can be infected with T. gondii, whichresults in the development of tissue cysts inmultiple organs and generation of specific anti-bodies, but not clinical toxoplasmosis (Dubeyet al., 1993). The parasite is present in free rangingchickens often at high levels and in theUSAplaysa major role in the epidemiology of the disease inthe rural environment (Hill and Dubey, 2012).
However, chicken meat is not considered a riskfactor for humans, because most chickens arereared indoors and poultry products are usuallyfrozen for storage that suffices to kill the para-sites, as well as being thoroughly cooked to avoidinfections with other pathogens.
26.2.1.5 Toxoplasmosis in Cats
Cats, as the definitive hosts, normally becomeinfected by ingestion of tissue cysts and, althoughadult cats (apart froma few isolated reports (Hen-riksen et al., 1994; Dubey, 1995)) usually remainclinically healthy, kittens sometimes die of acutetoxoplasmosis (Dubey andFrenkel, 1972).A largeproportion of all cats are seropositive, as evi-denced in a recent study from Ohio, where 48%of analysed cats were seropositive (Dubey et al.,2002). Cats that become infected shed around 20million oocysts over a short period of about twoweeks, before thedevelopment of a strongprotec-tive immune responsewhich limits further oocystshedding (Dubey, 1995). However, someimmune cats can re-shed oocysts if they are chal-lenged after a long period (six years) with a heter-ologous strain (Dubey, 1995). The shedding ofhighly infectious oocysts, which remain viablefor more than one year, into the environment bycats poses a major risk for both humans and live-stock. Oocysts can contaminate food and therebycreate an effective route of infection for bothhumans and livestock. Faeces from cats andwild felids can contaminate drinking water,thereby causing outbreaks of toxoplasmosis inhumans, as shown inPanama, theUSAandBrazil(Benenson et al., 1982; Bowie et al., 1997; Bahia-Oliveira et al., 2003). Finally, in contrast to contactwith cat litter, petting of cats that had recentlyshed oocysts was shown to pose a minor risk ofinfection for humans (Dubey, 1995).
26.2.2 Benefits of Animal Vaccination
In the case of the domestic cat, successfulvaccination would limit oocyst shedding andultimately reduce the incidence of toxoplasmosis
26.2 SCOPE OF PROBLEM AND POTENTIAL BENEFITS OF VACCINATION 999
generally. Otherwise, vaccination shouldimprove livestock productivity by reducingfoetal damage and hopefully also the incidenceof human disease by limiting contamination ofmeat products with tissue cysts.
As discussed above, clinical toxoplasmosisresulting in foetal damage occurs mostly insheep and goats. For these animals a commerciallive tachyzoite vaccine already exists, Toxovax(derived from the S48 ‘incomplete’ strain). Toxo-vax does efficiently reduce foetal deaths and asa non-persistent strain has a good safety record(further discussed in Section 26.3.2.1). It isunknown if Toxovax also aids in the reductionof contaminated lamb meat with tissue cystsand future vaccine studies on sheep and goatsare needed to address this question.
While the vast majority of pigs are rearedindoors, toxoplasmosis in this species remainsa minor problem and the market for a vaccine isperhaps too limited to be of commercial impor-tance. However, there is an increasing publicperception in the developed world that ‘organic’farming and ‘free range’ produce is superior tothat intensely reared indoors, both in terms ofquality and public health, not to mentionimproved animal welfare considerations. Conse-quently, free ranging pigs are becoming morecommon with the subsequent increasing humanhealth risk associatedwith an undoubted increasein the incidence of toxoplasmosis in pigs. There-fore, vaccination to protect both young nursingpigs, as well as to block cyst formation, is likelyto become necessary for a free ranging populationthat will increase in the future.
Since cattle display no clinical signs of toxo-plasmosis and as Toxoplasma infections do notpersist in these animals, it was thought previ-ously that they did not require vaccination.However, the recent quantitative risk assessmentstudy undertaken in the Netherlands suggeststhat when all parameters, such as consumption,processing and incidence, are taken into accountbeefwas found to be the likeliest conduit in infect-inghumans (Opsteegh et al., 2011). Consequently,
given the relative resistance of bovines to naturalinfection, they may offer an excellent target forsuccessful vaccination that would have realimpact on the incidence in humans. Chickens,also; although they can have persistent infectionwith tissue cysts, they do not require a vaccine,as they display no clinical manifestations andchicken meat is processed in such a way thattissue cysts are unlikely to survive.
Finally, as the definitive hosts, cats are a majorrisk responsible for contaminating food, pasturesand drinking water with oocysts. Cats area particular risk around farms and vaccinatingfarm cats with an experimental live bradyzoitevaccine (T-263) not only demonstrated it waspossible to neutralize oocyst shedding, butwithin a few years local mice were found to beToxoplasma seronegative and the incidence ofseropositivity in finishing pigs had decreased(Mateus-Pinilla et al., 1999). This trial clearlydemonstrates the feasibility and the potentialbenefits of vaccinating cats. Reduced oocystshedding from farm cats would not only limitthe incidence of toxoplasmosis in domestic live-stock, but also in humans via contaminated foodand water (Bahia-Oliveira et al., 2003). Similarly,vaccinating household cats would also reducethe likelihood of oocyst initiated infection tohumans and, in particular, to pregnant women.
In conclusion, a veterinary vaccine againsttoxoplasmosis already exists for sheep and goatsthat limits the incidence of abortion. The ques-tion that remains to be addressed is whether italso limits cyst burdens. Obviously, an idealvaccine would also have this outcome. If newveterinary Toxoplasma vaccines are developed,successful vaccination of cats would be expectedto have the biggest impact on reducing infectionin both livestock and humans.
26.2.3 Toxoplasmosis in Humans andthe Potential Benefits of Vaccination
Essentially all humans (approximately 6.5billion) are at risk of T. gondii infection and all
26. VACCINATION AGAINST TOXOPLASMOSIS: CURRENT STATUS AND FUTURE PROSPECTS1000
could arguably benefit from vaccination againstthis parasite. Current treatments are inadequateas they only control the proliferative tachyzoitestage of the life cycle, but do not eliminate thecyst stages associated with chronic infection(reviewed in Roberts et al., 2002). There area number of groups where the consequences ofinfection could be particularly severe and wherethe potential of vaccination would be great.T. gondii is a major cause of congenital diseasewith potentially severe sequelae. Pregnantwomen are more likely to acquire the infection(Avelino et al., 2004; Gilbert and Gras, 2003).Consequently, vaccination of women beforethey reach childbearing age may be a reasonablestrategy to reduce or eliminate this risk. The bene-fits of such a programme would vary accordingto country and clearly would have greatestimpact where the incidence of congenital infec-tion is highest. For example, the incidence ofcongenital toxoplasmosis is estimated to be 10per 1000 births in Paris, France (Desmonts andCouvreur, 1974), 0.5 per 1000 births in the UK(Williams et al., 1981) and one to 10 per 10,000births in the USA (Lopez et al., 2000). The finan-cial cost of such a programmewould be consider-able, butwould be offset by a reduction in the costof caring for those congenitally infected. In theUSA, the estimated total medical costs and lossof productivity as a consequence of human toxo-plasmosis, excluding AIDS patients, is approxi-mately $3 billion per annum and an annual lossof 11,000 quality adjusted years (Batz et al.,2012; Hoffmann et al., 2012).
At one time it was a widely held assumptionthat ocular toxoplasmosis only occurred in theimmune competent following congenital infec-tion, but recent evidence has found that it occursin a significant number of adult acquired infec-tions (reviewed in Roberts and McLeod, 1999).It has been estimated to occur in 2% to 3% ofadult acquired infections (Perkins, 1973) and 49cases were identified over a 13 year study inFrance (Couvreur and Thulliez, 1996). The inci-dence of adult acquired ocular disease varies
considerably by geographical region. In certainpopulations in Brazil, seropositivity was foundto be over 80% in adults over 25 years of age.The incidence of ocular disease within this pop-ulation was as high as 14%, although the contri-bution of congenital infections to this figure isdifficult to estimate (Petersen et al., 2001). Certainatypical strains of T. gondii have increased asso-ciation with ocular disease and may accountfor geographical differences in incidence ofocular disease (Grigg et al., 2001). Consequentlythe whole human population is at risk of adultacquired ocular disease and there is a good argu-ment for vaccinating the entire population. Thismay be especially important in regions whereatypical strains are more abundant.
The vast majority of congenitally infected chil-dren appear asymptomatic at birth. However, inone USA based study, around 20% of apparentlyasymptomatic children had ocular involvementat birth. Ocular disease had risen to over 80%in the subjects of this study by adolescence(reviewed in Roberts and McLeod, 1999). It hasbeen suggested that therapeutic vaccination inchildhood may be useful in reducing the inci-dence of ocular disease amongst asymptomatic,congenitally infected individuals (Wilson et al.,1980; Koppe et al., 1986). Such a vaccine wouldhave to overcome the mechanism that preventsthis patient group from developing solid immu-nity that is normally exhibited by those whohave adult acquired infection. Although immu-nological tolerance could play a role, the precisemechanism is currently unknown. Therefore,rational design of such a vaccine may provechallenging.
Other possible groups that might significantlybenefit from a therapeutic vaccine are those withactive disease. This would include those withactive adult acquired disease or the immunosup-pressed. There would be inherent problems invaccinating people with ongoing disease asthe immune response to the vaccine may beinfluenced in a detrimental manner by thenatural infection. Successful vaccination of
26.3 CURRENT STATUS OF VACCINES FOR INTERMEDIATE HOSTS 1001
immunosuppressed people as an alternative toantimicrobial therapy would also prove chal-lenging due to the very fact that they have poorlyfunctioning immune systems.
26.3 CURRENT STATUS OFVACCINES FOR INTERMEDIATE
HOSTS
26.3.1 Vaccination Using Extracts orKilled Parasites
The earliest studies to test the vaccine poten-tial of killed or crude antigen extracts againsttoxoplasmosis were carried out in 1956 (Cutch-ins and Warren, 1956; Jacobs, 1956). A summaryof more recent studies carried out since the early1970s is listed in Table 26.1. The vaccine potentialof whole fixed tachyzoites has been examined, aswell as whole tachyzoite lysates, soluble frac-tions, particulate fractions, excretory/secretoryproducts, detergent extracts, cysts, soluble cystfractions and crude whole rhoptry extracts.Numerous adjuvants have also been employedas part of the vaccine formulation includingFreund’s Complete (FCA) and Freund’s Incom-plete Adjuvant (FIA), lipid vesicles, ISCOMs(Immuno Stimulating Complexes), BCG, choleratoxin, PLG microspheres, exosomes derivedfrom an infected DC cell line, and CpG for vacci-nation. Guinea pigs, mice, rats, sheep and pigshave all been used as models and challengeinfections have utilized both virulent and aviru-lent tachyzoites, cysts and bradyzoites.
Overall, while some vaccines increasedsurvival following challenge infection (Krahen-buhl et al., 1972; Eissa et al., 2012), others did little(Waldeland and Frenkel, 1983; Saavedra et al.,2004). The fact that the immune system tendsto recognize life cycle stage-specific antigens(Kasper, 1989) in some respect may account forsome discrepancies. In addition, although nota hard and fast rule, lipid vesicles such as lipo-somes, non-ionic surfactant vesicles and
ISCOMs may be better adjuvants than FCAand FIA; excretory/secretory antigens mayinduce stronger protection than tachyzoitelysates, while the addition of cyst antigens totachyzoite preparations has improved efficacy(Elsaid et al., 2001). Again, while some vaccinesreduced cyst burden following challenge (Alex-ander et al., 1996; El-Malky et al., 2005), othersfailed to do so (McLeod et al., 1985; Lundenet al., 1993); however, in murine and rodentmodels of congenital toxoplasmosis vaccinationwith crude tachyzoite lysate (Roberts et al.,1994; Elsaid et al., 2001; Beauvillain et al., 2009),tachyzoite and cyst lysate (Elsaid et al., 2001) orexcretory secretory factors (Zenner et al., 1999)proved extremely effective at limiting bothmaternalefoetal transmission and foetal death.Nevertheless, killed whole tachyzoites alone(Beverley et al., 1971) or in FIA (Wilkins andO’Connell, 1992) could not protect sheep fromaborting.
More recently, tachyzoite sonicates wereformulated in QuilA-containing vesicles‘ISCOMs’ and used to vaccinate pregnant sheep.One vaccination given four weeks prior tomating was followed by two injections in thefirst 10 weeks of gestation. A challenge wasgiven at day 91 of pregnancy using oocysts ofthe M1 strain (Buxton et al., 1989). Ewes werenot protected against the acute phase of theinfection as was apparent from their febrileresponse comparable to non-vaccinated controls.However, fewer abortions were induced in thevaccinated ewes and the gestational time wascomparable to non-infected controls; probablydue to low numbers these differences were notsignificant. Foetal infection could not be pre-vented using this vaccine. Using a similarvaccine formulation, vaccination of pigs withrhoptry proteins in ISCOMs had little effect onthe febrile response following oral oocyst infec-tion, although the cyst burden was slightlyreduced (Garcia et al., 2005).
A similar approach was applied by Stanleyet al. (2004) using tachyzoite extracts formulated
TABLE 26.1 Killed and Crude Antigen Vaccine Studies and Their Outcomes in Animal Models
þþþFoetal deathAg w NISVee Foetal deathAg wo NISVþþ Maternal-foetaltransmissionAg w NISV
(Alexander et al.,1996)
Cysts þtachyzoitelysate
FCA/FIA s.c. BALB/K Beverleycysts (oral)
e Tachyzoitesþþ Cystsþþþ Tachyzoitecysts
(Zenner et al.,1999)
Excretory/secretory
FIA s.c. Fischer rats Antibodies 76K cysts(oral)
þþmaternalfoetaltransmission
(Elsaid et al., 1999) SolubletachyzoiteSoluble cystsSoluble cystsplus tachyzoite
LiposomesFCA
s.c. Swiss mice Antibodies P strain (oral) þþ wliposomes�FCA
þ w liposomes�FCA
(Elsaid et al., 2001) SolubletachyzoiteSoluble cystsSoluble cystsplus tachyzoite
LiposomesFCA
s.c. BALB/c AntibodiesLymphopro-liferation
P strain cysts(oral)
þþ Foetal Deathþ maternalfoetaltransmissionAg plusliposomes�Ag plus FCA
(Continued)
26.3CURREN
TST
ATUSOFVACCIN
ESFO
RIN
TER
MED
IATEHOST
S1003
TABLE 26.1 Killed and Crude Antigen Vaccine Studies and Their Outcomes in Animal Models (cont'd)
Reference Antigen
AdjuvantsorCarrier
Routeof Vac.
AnimalModel Immunology Challenge Survival
ParasiteBurden Other
(Daryani et al.,2003)
Tachyzoiteexcretory/secretory,lysate
FCA/FIA s.c. BALB/cmice
DTHLymphoproliferation
RHtachyzoites(s.c.)
þþ/þESA > TCA
(Garcia et al.,2005)
Rhoptryproteins
ISCOM s.c. Pigs Antibodies VEG oocysts(oral)
þþe Febrileresponse
(Beauvillain et al.,2007)
Tachyzoitelysate
SRDCderivedExosomes
s.c. CBA/J;C57BL/6
AntibodiesIFN-g, IL-4,IL-5, IL-10,IL-2
76 K cystsOral
þþþ CBA/Jþ C57BL/6
(Hedhli et al.,2009)
Tag Eimeriaprofiling-likeantigen
i.p. CBA/J Antibodies,IFN-g, IL-10,IL-2
70 76k cysts 62% lesscystburden
(Beauvillain et al.,2009)
Tachyzoitelysate
SRDCderivedExosomes
s.c. PregnantCBA/J
Antibodies,IFN-g, IL-4,IL-5, IL-10,IL-2
76K cystsOral
Pups þþþ
(Eissa et al., 2012) AutoclavedTachyzoitelysate
BCG i.d. Swiss albino Splenic CD8þ RHi.p.
þþ
Key for Survival: ee decreased survival: � no difference in survival; þ moderate (�50% increased survival); þþ significant (�50% increased survival); þþþ highly significant (�90%increased survival), compared with control groups.Key for Parasite Burden: ee increased parasite burden: e no difference in parasite burden; þ moderate (�50% decrease in parasite burden);þþsignificant (�50% decrease in parasite burden); þþþ highly significant (�90% decrease in parasite burden), compared with control groups.
26.VACCIN
ATIO
NAGAIN
STTOXOPLA
SMOSIS:
CURREN
TST
ATUSAND
FUTUREPR
OSPEC
TS
1004
26.3 CURRENT STATUS OF VACCINES FOR INTERMEDIATE HOSTS 1005
in cholera-toxin containing microspheres (Stan-ley et al., 2004). Vaccination of sheep was per-formed intranasally and induced mucosal andserum IgA. Since the oral route is the naturalroute of infection in sheep, mucosal immunitymay contribute to reducing the numbers ofinfectious parasites as was shown previouslyin mice (Bourguin et al., 1993), and reviewedin Kasper et al. (2004). Stanley and co-workersvaccinated non-pregnant sheep three times,but the effect on the acute infection measuredas a febrile response was only marginal.Antigenic differences between sporozoites, thestages invading the mucosal lining and the laterdeveloping tachyzoites, which cause thefebrile response, may account for theseobservations.
Thus, while vaccines based on killed or lysedtachyzoite antigens could induce protectiveimmunity in mice and limit maternalefoetaltransmission, results from a more practicalmodel employing outbred sheep were less prom-ising and could not compare to the effectsinduced by the incomplete S48 strain. However,relatively few studies have been documented insheep and further improvement could beachieved by applying different immunizationschedules or better adjuvants. The success ofthe killed Neospora vaccine which protectsagainst abortion in cattle warrants furtherstudies using antigen preparations to enhanceprotective immunity against Toxoplasma inducedabortion in sheep.
Although no studies are documented onvaccination of humans, it is known that chroni-cally infected women can protect their foetusfrom congenital infection. From the studiesdescribed above it is clear that sterile immunitycannot be achieved using killed or sub-unitvaccines in any of the infection models used.Since tachyzoites are very efficient in reachingthe foetus and since prevention of congenitalinfection is a prime target for vaccination ofhumans, a killed vaccine will probably not besufficiently efficacious.
26.3.2 Vaccination Using Live,Attenuated Parasites
The ubiquitous RH strain is a type I strain andinfection with only a few tachyzoites is sufficientto kill a mouse. This strain is, however, signifi-cantly less pathogenic in other animals, such aspigs. Moreover, inoculated tachyzoites did notcause a persistent infection in these animals,making this strain useful as a vaccine strain.Pigs, which can harbour a huge cyst burden, areconsidered a major source of infection forhumans. Vaccination of pigs using the RH strainprotected against challenge with a persistentstrain and reduced the presence of tissue cystsin the meat. When challenge occurred at 220days post-vaccination all vaccinated pigs werenegative for tissue cysts in a mouse bio-assay,whereas all control challenged animalswere positive (Dubey et al., 1994). Attenuatedlines were derived from this RH strain bychemical mutation using N-methyl-N0-nitro-N-nitrosoguanidine. Temperature-sensitivemutantswere isolated, of which the TS-4 strain demon-strated the most favourable phenotype ofretarded growth at 37�C while maintainingimmunogenicity in mammals (Pfefferkorn andPfefferkorn, 1976). Mice vaccinated with tachy-zoites of TS-4were protected against a lethal chal-lenge infectionwith RH.While vaccination couldalso reduce congenital transmission in a preg-nancy, model tissue cyst formation was not pre-vented after vaccination with TS-4 andchallenge with an avirulent strain (McLeodet al., 1988).
Frenkel et al. (1991)usedsimilarmutationmeth-odology and selected a straindeficient in the cocci-dial cycle of the parasite (the sexual replication inthe intestine of the cat) (Frenkel et al., 1991). Incontrast to RH and TS-4, this T-263 strain didproduce tissue cysts in the intermediate host. Itlost, however, the capacity to form oocysts incats that had ingested tissue cysts. The deficiencywas associatedwith infertilemicrogametes. Vacci-nating young cats with T-263 bradyzoites could
26. VACCINATION AGAINST TOXOPLASMOSIS: CURRENT STATUS AND FUTURE PROSPECTS1006
prevent oocyst shedding in 80% of recipientswhen challenged with an oocyst-forming strainof T. gondii. Such effects could not be induced byvaccination with live tachyzoites only (Freyreet al., 1993). This data confirms that bradyzoitesand tachyzoites carry different antigens andimmunity differs between intermediate anddefin-itive hosts relating to the interfaces where theinfection is occurring. Although commercializa-tion of the T-263 strain as a vaccine for cats wasconsidered, a product was never released.
Attenuation, while preserving immunoge-nicity, can also be achieved by gamma-irradiation as has been shown for many differentorganisms, including parasites, such as ina commercial lungworm vaccine (McKeand,2000). Dosages of less than 1000 Gy resulted intachyzoites that could invade cells, and althoughthey could not replicate they were still able toinduce cell mediated immune responses andsome protection (Seah and Hucal, 1975).However, the effectiveness of different irradiationdoses differed from study to study (summarizedin Dubey, 1996). Oocysts of the VEG strain failedto induce persistent infections, when irradiatedwith doses higher than 200 Gy (Dubey, 1996).Oocysts irradiated with 200 Gy could inducepartial protection in mice against oral challengeas measured by extended survival and fewerbrain cysts. This was once again confirmed ina more recent experiment (Hiramoto et al.,2002). Tachyzoites (107) irradiated with 200 Gyextended the survival time of mice by roughlyfour days when challenged with 1000 RH tachy-zoites. More importantly, it reduced tissue cystdevelopment in the brain by more than 10-foldwhen mice were challenged orally with 25 ME-49 tissue cysts. Since irradiated tachyzoites donot persist and are able to reduce the number oftissue cysts, such an approach may have implica-tions in the design of a therapeutic vaccine.
In addition to inducing an attenuated pheno-type by chemical mutation or irradiation, serialpassage of a type II strain has also been shownto evoke changes that lead to an attenuated
phenotype. This methodology was used todevelop the S48 strain, which is now commer-cially applied as an effective vaccine againstabortion in sheep (Buxton, 1993). This strainwas originally isolated from the cotyledon ofan aborted lamb and passaged twice weeklythereafter through mice for more than 30 years.The strain consequently lost its capacity toform tissue cysts in any animal challenged, butkept its immunogenic characteristics. S48 isdescribed in greater detail below.
In conclusion, a T. gondii infection is generallyable to induce a statusof lifelongprotection,whichwould be ideal if the primary infection did nothave the potential to damage neural tissues orinterfere with the outcome of pregnancy. Attenu-ated vaccines may have the potential to inducesimilar protection to natural primary infection inthe absence of such pathology. The preferredattenuation should be stable and, thus, irradiationis not a particularly good option. Selection of suffi-ciently attenuated strains after mutagenic actionsmay have the potential to deliver stable vaccinestrains. However, the use of such strains inimmune compromised animals or humans shouldbe restricted. New reverse genetic techniques willallow specific genes to be deleted such that thereversion to virulence can be excluded (discussedbelow).
26.3.2.1 A Commercial Vaccine (S48)Against Toxoplasmosis in Sheep and Goats
T. gondii normally causes disease when infec-tion occurs for the first time while the animal isin gestation, allowing the parasite to invade thenon-immune foetus. However, an animal that isalready immune prior to gestation is normallysufficiently protected to prevent foetal infection(Frenkel, 1990; McColgan et al., 1988). Indeed,while a natural infection with T. gondii in sheepinduced protective immunity (McColgan et al.,1988), vaccination with inactivated Toxoplasmatachyzoites could not protect pregnant sheepfrom infection (Beverley, 1971; Wilkins et al.,1987). This illustrated that vaccination with live
26.3 CURRENT STATUS OF VACCINES FOR INTERMEDIATE HOSTS 1007
T. gondii, but perhaps not a killed preparation,prior to gestation, couldprevent subsequent foetaldeath. Accordingly, the ‘incomplete’ S48 strainwas developed into a safe, live vaccine (Wilkinset al., 1988). S48 was isolated in 1956 from anaborted lamb in New Zealand. Since this timepre-dates the era of cryopreservation and tissueculturing, it was maintained by passaging thetachyzoites throughmicetwiceweeklyforaperiodof 30 years. During this time S48 lost its pathoge-nicity for sheep, as well as its ability to form tissuecysts (Wilkins and O’Connell, 1992). Not only hasS48 lost its ability to form tissue cysts, it also doesnot generate oocysts in cats (Intervet, unpublishedobservations). The safety of the strain was provenby its absence in any animal from fourweeks post-vaccination (Buxton, 1993). In 1988 it was intro-duced as Toxovax, a live vaccine, inNewZealand.Subsequently, it was registered for use in the UKand Ireland in 1992 and is now sold by Intervetas Ovilis� Toxovax in many European countries.
Ovilis� Toxovax is a live tissue culture grownvaccine that is supplied as a frozen product andis distributed to end users at 4�C. The vaccine isapplied either intramuscularly or subcutane-ously no later than three weeks prior to mating.To demonstrate the efficacy of the vaccine, theMoredun Research Institute together with Inter-vet performed a series of vaccination-challengeexperiments (Buxton et al., 1991; Buxton andInnes, 1995). Using 2000 sporulated oocysts ofthe M3 strain they induced a severe infection inpregnant S48-vaccinated and control sheep.While less than 18% of the lambs from controlsheep were born alive and viable, 80% of lambssurvived in the vaccinated group and theirweights were comparable with lambs fromnon-challenged ewes. Toxovax induced an IgGantibody response, although this responsewaned by 20 months post-vaccination, incontrast to persistent infections that maintainhigh antibody titres in the blood throughoutlife. S48 vaccination also induced specific CD4þ
and CD8þ T-cells that produced IFNg as docu-mented for normal live tachyzoite infections in
sheep (Buxton and Innes, 1995). Further studiesdemonstrated that animals were still immuneafter 18 months post-vaccination despite theirantibody levels having waned, highlighting theability of S48 to induce a potent adaptive type Icell mediated response (Buxton, 1993). Fieldtrials in the UK have shown the economic profit-ability of vaccination against T. gondii inducedabortion in sheep (Bos and Smith, 1993). Vacci-nation with S48 has also shown moderate effi-cacy in goats (Chartier and Mallereau, 2001).
26.3.3 Vaccination Using GeneDeletion Attenuated Parasites
Increased knowledge of T. gondii at the molec-ular level, greatly facilitated by the completion ofthe genomeproject and in combinationwith rapidprogress in the development of genetic tools tomanipulate the parasite, has generated opportuni-ties to create new attenuated vaccines. Such tar-geted approaches have been used to createparasites with incomplete life cycle stages withreduced proliferative capacity or with reducedvirulence. Mutant parasites have been generatedeither with an irreversible gene deletion or morerecently by conditionally inhibiting or activatingexpression of an essential gene. Genetically ‘crip-pled’ parasites have been analysed in vitro and invivo to characterize their mutant phenotypes andto determine whether a gene is possibly redun-dant. At present with the exception of a Mic1-3deletion mutant (Mevelec et al., 2010), the infec-tivity of all Toxoplasma mutants that have beengenerated by reverse genetics has only been ana-lysed in mice and not in larger animals. Whethervaccine potential or its absence demonstrated inmice can be translated into success or failure inlarger animals is a matter of some conjecture. Forexample, while both the RH strain and the incom-plete S48 strain are highly lethal tomice, RH is notpersistent in pigs and induces protective immu-nity upon challenge with oocysts (Dubey et al.,1994). Similarly, the incomplete S48 strain doesnot persist in any animal so far examined, and is
26. VACCINATION AGAINST TOXOPLASMOSIS: CURRENT STATUS AND FUTURE PROSPECTS1008
commercially used as a live vaccine. Thus, thepotential of a livemutant parasite cannot be deter-mined inmice only, but will ultimately have to beestablished in a larger animal.
In recent years, various Toxoplasma gene dele-tion mutants have been generated. Although theobjective was usually to gain further insightinto the function of a particular gene rather thanto generate a vaccine, it is likely that any
TABLE 26.2 Toxoplasma Mutant, Deficient and Condition
ReferenceTargeted Geneor Mutant Name
ParentalStrain Dosage
(McLeod et al., 1988) Ts-4 RH
(Lindsay et al., 1993) Ts-4 RH 5 � 105
(Pinckney et al., 1994) Ts-4 RH 3 � 105
(Fox and Bzik, 2002) CPSII RH Up to 1
(Rachinel et al. 2004) SAG1 RH 104 tach
(Dzierszinski et al., 2000) SAG3 RH 20 tach
(Soldati et al., 1995) ROP1 RH 50 tach
(Mercier et al., 1998) GRA2 RH 10 tach
(Bohne et al., 1998) BAG1 PLK** 104 tach
(Zhang et al., 1999) BAG1 PLK** 105 tach
(Moire et al., 2009) MIC1e3 RH Up to 1
(Lu et al., 2005) Ts-4 RH 105 tach
(Lu et al., 2009) Ts-4 RH 2 � 104
(Fox and Bzik et al., 2010) DOMPDC RH 105e10
(Hutson et al., 2010) DRPS13 RH 105 tach
(Zorgi et al., 2011) Irradiated RH VEG, M
*) applied via surgical injection in the intestines.**) PLK is a clonal derivative of ME49.C57BL/6 is a susceptible mouse, BALB/c is a relative resistant mouse and CD1
biologically important gene will contribute eitherto the fitness and/or virulence of the parasiteand consequently the vaccine potential of thesemutants has been of significant interest (see Table26.2).
The first genetically engineered mutant to beextensively studied was the RH strain witha disruption of carbamoyl phosphate synthetaseII (CPSII) (Fox and Bzik, 2002). CPSII is the first
26.3 CURRENT STATUS OF VACCINES FOR INTERMEDIATE HOSTS 1009
enzyme in the metabolic pathway for de novopyrimidine synthesis (generating the buildingblocks for RNA and DNA) and disrupting thisenzyme made Toxoplasma dependent on exter-nally supplied uracil, which it can salvage.Disruption of CPSII thus created a uracil auxo-troph, which only grew in vitro when host cellswere supplemented with uracil. In the absenceof uracil, CPSII knock-out parasites invadedhost cells normally but failed to replicate. Nogrowth was observed (without added uracil) invitro and in vivo. Injection of mice with CPSIIknock-out parasites did not kill BALB/c mice,and mice infected with CPSII knock-out para-sites 40 days previously were resistant to a lethalchallenge with 200 pfu of RH strain tachyzoites.
Surface antigens (SAGs) of Toxoplasma havealso been targeted for deletion. SAGs are thoughtto be involved in host cell attachment and the acti-vation of a host immune response. The majortachyzoite surface antigen is SAG1 and two typesof SAG1 mutants have been generated. One wasmade by chemical mutagenesis and the otherwas recently genetically engineered (DSAG1).Both attach to, enter and proliferate at approxi-mately normal rates within host cells (Mineoand Kasper, 1994; Kasper and Khan, 1993;Rachinel et al., 2004). DSAG1 tachyzoites werelethal in susceptible C57BL/6 mice (althoughsurvival was slightly prolonged compared withwild-type infected mice), but deletion of SAG1prevented an acute ileitis when tachyzoites weredirectly injected into the intestine.
SAG3 deletion mutants showed morepronouncedeffects thanDSAG1 tachyzoites; thesehad significantly reduced adherence to host cellsin vitro and mortality was reduced by 80% uponinfection in BALB/c mice compared with wild-type organisms (Dzierszinski et al., 2000).
Recently, a genomic cluster containing fourbradyzoite specific SAGs (SAG2c, SAG2d,SAG2x and SAG2y) was deleted in one knock-out. Deleting these four SAGs (DSAG2cdxy)yielded viable tachyzoites that could still differ-entiate into bradyzoites in vitro. In contrast,
preliminary studies in vivo showed that nineout of 10 DSAG2cdxy infected mice werenegative for brain cysts when assayed threemonths after infection (J. Saeij and J. Boothroyd,personal communication). Consequently,DSAG2cdxy appears promising as a vaccinecandidate because although it is still infectiveand can transform into bradyzoites, it persistspoorly if at all.
Two secretory vesicle proteins, ROP1 andGRA2, gene deletion mutants, have also beengenerated. Disruption of either ROP1 or GRA2resulted in no difference in growth rates orhost cell invasiveness in vitro, although DGRA2was less virulent in mice (Soldati et al., 1995;Mercier et al., 1998).
Disruption of BAG1, a bradyzoite specificheat shock protein, was thought to interferewith the formation or viability of tissue cysts.However, in one study (Bohne et al., 1998)disruption of BAG1 had no effect on tissue cystformation, while in a second study (Zhanget al., 1999) disruption of BAG1 could onlyreduce the number of tissue cysts in mousebrains by roughly five-fold. In the latter study,the lethal dose with DBAG1 did increase from2 � 106 to 5 � 107 compared with the parentalPLK strain (a clonal line derived from ME49).Nevertheless, tissue cysts were still being formedand were completely normal, proving that BAG1is not essential for bradyzoites. The authors sug-gested that BAG1 homologous genes may existin Toxoplasma, generating some redundancy.
Recently a Mic1e3 deficient RH strain ofT. gondii was developed. This strain is capableof conferring a degree of protection in miceagainst congenital infection. Fewer mice in vacci-nated groups were found to be infected than incontrol groups and those infected had fewercysts in their brains compared with those bornto unvaccinated control groups (Ismael et al.,2006). Similarly, this vaccine was found to confera degree of protection against congenital trans-mission when the pregnant ewes were chal-lenged with oocysts (Mévélec, 2010).
26. VACCINATION AGAINST TOXOPLASMOSIS: CURRENT STATUS AND FUTURE PROSPECTS1010
Attenuated parasites can also be generated bytargeting expression of essential genes. Since dele-tion of such genes will immediately result innon-viable parasites, targeting the expression ofessential genes should occur in a conditionalway. A number of systems have been developedthat achieve this goal (Meissner et al., 2001, 2002b;van Poppel et al., 2006; Hutson et al., 2010). Anumber of conditional knock-out parasites havebeen generated and one could envisage a vaccineapplication using these conditional knock-outs. Aconditional T. gondii knock-out for the small sub-unit ribosomal protein 13 (Hutson et al., 2010) hasbeen generated using the tetracycline repressorsystem (Hutson et al., 2010). In the absence ofanhydrotetracycline the conditional knock-outparasite exits the cell cycle in G1, but is arrestedin the G0 phase. Although able to persist a sus-tained time in vitro in this stage, it appears to becleared by the immune system of mice (Hutsonet al., 2010). Notably, mice vaccinated with theseparasites are protected against challenge witheither type 1 or type 2 strains of T. gondii.
In summary, reverse genetic techniques haveenabled the creation of mutant attenuated Toxo-plasma strains with vaccine potential. The uracilauxotrophic mutant generated by disruption ofCPSII and theRPS13 conditional knock-out strainsare particularly promising. It will be particularlyimportant to demonstrate the safety of themutants, before such genetically modified organ-isms (GMO) can be tested and used in the field.
26.3.4 Vaccination Using Viral Vectors
Viral delivery of immunogenic antigens hasbeen tested widely against various cancers andinfectious diseases including malaria, althoughonly a few studies have used this technique toexpress Toxoplasma antigens Table 26.5. Multipleviral vectors are available that are consideredsafe and are being tested in animals and humans.Poxviruses, adenoviruses and herpesviruses aremostly used as vectors. Some poxviruses suchas vaccinia can be a minor human pathogen
whereas others, such as fowlpox and MVA(modified vaccinia Ankara) cannot replicatein mammalian cells and are non-pathogenic(Paoletti, 1996). Likewise, replication defectiveadenoviruses, such as Ad5, are being used(Graham et al., 1977). Due to their safety, replica-tion defective viruses may also be considered fora human Toxoplasma vaccine.
If we consider the use of viral vectors asvaccine carriers for parasitic diseases, thenmost work has focused on malaria. For example,vaccination of mice with a Plasmodium yoelii cir-cumsporozoite protein, expressed by eithera replication defective Ad5 adenovirus ora combination of Ad5 with a vaccinia, inducedsterile and long lasting immunity (Rodrigueset al., 1997; Bruna-Romero et al., 2001). In anotherstudy, the multi-epitope vaccine fused to TRAP(MEeTRAP) induced sterile immunity in somehuman subjects (Webster et al., 2005). MEeTRAP was delivered with a prime and boostcombination of a recombinant fowlpox vectorfollowed by an MVA vector. These examplesillustrate the potential of such vectors.
A few infectious viral vaccine vectors havebeen tested against T. gondii including a felineherpesvirus (FHV1) tested in cats (Mishimaet al., 2002). FHV1was selected as carrier becauseit spreads amongst cats contagiously, therebyhopefully disseminating the vaccine to neigh-bouring domestic and stray cats. An attenuatedstrain was generated by deleting thymidinekinase and inserting Toxoplasma ROP2. FHVeROP2 induced specific antibodies and, upon bra-dyzoite challenge of cats, the number of braintissue cysts was reduced. However, it did notreduce oocyst secretion, an essential prerequisiteof a cat vaccine. In another study, the vaccinepotential of ROP2 was tested with MVA inmice (Roque-Resendiz et al., 2004). High dosagesof MVAeROP2 did induce specific antibodytitres and delayed the time of death (by twodays), but could not protect mice against a chal-lenge with 300 RH tachyzoites. Vaccination withMVAeROP2 also failed to reduce the formation
26.3 CURRENT STATUS OF VACCINES FOR INTERMEDIATE HOSTS 1011
of brain cysts if mice were orally challenged with20 ME49 cysts. Gazzinelli recently reportedpreliminary studies with three recombinantadenoviruses, expressing SAG1, SAG2 or SAG3(Gazzinelli et al., 2005; Caetano, 2006). Immuni-zation of BALB/c mice with these virusesinduced both antibodies and IFNg responses.However, upon challenge with a lethal dose ofRH parasites these mice were not protected.Conversely, challenge with a P-BR strain didshow a reduction of tissue cysts in the brain.Further studies have utilized baculovirus (Fanget al., 2009; 2012), vaccinia (Zhang et al., 2007a),pseudorabies (Liu et al. 2008; Shang et al., 2009;Nie et al., 2011) and influenza (Machado et al.,2010) as expression systems and additional anti-gens examined include AMA1 (Yu et al., 2012),GRA4 (Zhang et al., 2007) and MIC3 (Nie et al.,2011; Fang et al., 2012). Generally vaccines incor-porating some antigens are more effective thanothers (Mendes et al., 2011) and those expressingmore than one antigen are more effective thanthose expressing a single antigen (Qu et al.,2009; Nie et al., 2011; Fang et al., 2012).
Thus, recombinant viruses do have potentialas vaccine vectors, but undoubtedly the rightmix of Toxoplasma antigens expressed and thevector system utilized to yield the highest levelof protective immunity have yet to be found(see Table 26.5).
26.3.5 Vaccination Using BacterialVectors
Live bacterial vaccine vectors have beenextensively used to deliver and express heterolo-gous vaccine antigens to protect against cancerand various infectious agents, including AIDS(reviewed in Drabner and Guzman, 2001). Livebacterial vaccines have the advantage that theycan express multiple antigens, are easily massproduced, can be orally or intranasally appliedand induce strong immune responses. However,relatively few studies have tested whether heter-ologous expression of parasitic antigens with
bacterial vaccine vector strains can lead toprotective immunity Table 26.5.
Invasive bacteria such as Salmonella, Listeria,Yersinia, Shigella and Mycobacterium bovis BCGhave been used as vaccine vectors, capable ofmounting potent humoral and cellular immuneresponses. Since these are pathogenic bacteriathey were attenuated to generate suitable non-pathogenic vaccine strains. Many attenuatedstrains have been reported that are non-pathogenic and have limited proliferativecapacity in vivo. Attenuation can, however,lead to reduced immune stimulation. Moreover,overexpression of heterologous genes can resultin a rapid selection for low or non-expressers.To circumvent this potential obstacle, differentapproaches have been used to obtain stableexpression with bacterial vaccine strains for invivo use. For example, inducible promoterswere used, such as the Salmonella nirB promoter,which becomes activated in vivo under anaerobicconditions (Chatfield et al., 1992). Alternatively,a mixed population approach was testedwhereby expressing bacteria are constantlyderived from non-expressing carrier cells (Yanand Meyer, 1996). Finally, bacterial vaccinestrains have been successfully used to delivereukaryotic expression plasmids. In oneconvincing example, eukaryotic expression plas-mids containing Listeria antigens were success-fully delivered with Salmonella typhimurium,protecting mice from a lethal challenge with Lis-teria monocytogenes (Darji et al., 1997).
The earliest use of a bacterial vector as a vaccineagainstT. gondiiwasoral immunizationwitha liveattenuated Salmonella typhimurium vaccine strain(Cong et al., 2005). SAG1andSAG2weredeliveredwith a eukaryotic expression plasmid which alsocontained cholera toxin sub-units A2 and B.Cholera toxin (CT) is known to have an adjuvanteffect and indeed the addition of CT sub-unitsA2 and B induced a strong cellular immuneresponse, as measured by induced specificIgG2A titres, splenocyte proliferation and IFNgproduction. Upon challenge with 1000 RH
26. VACCINATION AGAINST TOXOPLASMOSIS: CURRENT STATUS AND FUTURE PROSPECTS1012
tachyzoites the vaccinated mice survived longerand 40% of the mice survived the whole trialperiod. There have been three further studiesusing bacterial vectors delivered orally in themouse model, two using attenuated Salmonellaincorporating either SAG1 (Qu et al., 2008) orSAG1 and MIC3 alone or in combination (Quet al., 2009) and one using BCG expressing ROP2(Wang et al., 2007). While all formulations deliv-ered a level of protection, vaccines comprisingmore than one antigen were generally more effec-tive (Qu et al., 2008) and were improved by incor-porating an adjuvant (Cong et al., 2005).
In conclusion, vaccination with live bacterialvectors can induce both strong humoral andcellular immunity and as they are delivered orallyshould also induce protection at the mucosallevel. However, as all studies to date have utilizedi.p. challenge with RH and their ability to protectagainst natural infection with cyst forming line-ages remains to be ascertained (see Table 26.5).
26.3.6 DNA Vaccines
It is the general consensus of opinion thata type 1 response, particularly associated withCD8þ T-cells producing IFNg, is the major medi-ator of immunity against T. gondii infection.Nevertheless, numerous vaccine and immuno-logical studies have also demonstrated thata broad spectrum of immune response requiringelements of type 2 immunity with antibodiesprovides the best overall protection against infec-tion. Consequently, as DNA vaccines are knownto induce CD8þ T-cell responses in addition tobroad spectrum immunity, there has been inrecent years a substantial effort to determine theireffectiveness against toxoplasmosis. While themajority of studies have concentrated on SAG1(Table 26.3), the vaccine potential of numerousother molecules GRA1, GRA2, GRA4, GRA6,GRA7, ROP1, ROP2, ROP16, ROP 18, HSP70,HSP30, MIC1, MIC2, MIC3, MIC4, MIC6,MIC8, M2AP, MAG1, AMA1, ROM1, BAG1,IMP1, Perforin-like-1 EFG and binding lectin
domains have also been investigated, either aloneor in combination, with varying degrees ofsuccess. Vaccination with SAG1 has been foundto be particularly effective at limiting mortalityagainst both virulent and avirulent challenge(Nielsen et al., 1999; Angus et al., 2000; Chenet al., 2002; Couper et al., 2003; Liu et al., 2010a).In addition, the effectiveness of SAG1 DNAvaccines was generally enhanced by utilizingcocktail vaccines comprising other antigenssuch as ROP2 (Fachado et al., 2003c), GRA4(Mevelec et al., 2005), MIC2 and MIC3 (Rosen-burg et al., 2009) and GRA2 (Zhou et al., 2012).The incorporation of adjuvants into the vaccines,particularly pGM-CSF (Desolme et al., 2000;Ismael et al., 2003; Mevelec et al., 2005), choleratoxin (Wang et al., 2009) and pIL-18 (Liu et al.,2010b; Yan et al., 2012b), also enhanced generalefficacy irrespective of the antigen under investi-gation. Surprisingly, while a number of studieshave indicated the potential of pIL-12 as adjuvant(Xue et al., 2008a, 2008b; Cui et al., 2008) a morerecent study suggested an adverse effect for thiscytokine (Khosroshahi et al., 2012) with regardto protection. These studies generally usedsimilar antigens, SAG1 and ROP2, the BALB/cmouse model and RH challenge and conse-quently it is difficult to draw a conclusion.However, the general consensus would favourmulti-epitoped, adjuvanted vaccines being moreefficacious than those comprising single antigensor non-adjuvanted preparations. Consequently,while a SAG1 DNA vaccine failed to limitmaternalefoetal transmission (Couper et al.,2003) a SAG1/GRA4 vaccine adjuvanted withpGM-CSF did increase pup survival if damswere infected during pregnancy, but againwithout preventing vertical transmission (Meve-lec et al., 2005). However, the former studyutilized BALB/c mice while the latter used SwissOF1 mice, which could also provide an explana-tion for relative success and failure. Indeed, it hasbeen shown that the degree of protectionafforded by a particular DNA vaccine can bedependent on the animal model used and
TABLE 26.3 DNA Vaccine Studies and Their Outcomes in Animal Models
(Min et al., 2012) GRA7 FCA, IFA i.m. BAL B/c Antibodies,IFN-g,
RH þþ DNA prime,protein boost
þ
(Hiszczynsk-Sawicka et al.,2012)
MIC3 Liposomes i.m. (neckdorsal)
Coopworthewes
Antibodies,IFN-g
*1 Vaccination did not prevent congenital toxoplasmosis in BALB/c mice*2 Vaccination did not limit congenital transmission in SWISS OF1 mice but increased survivalKey for Survival: �� decreased survival; � no difference in survival; þmoderate (�50% increased survival); þþ significant (�50% increased survival); þþþ highly significant(�90% increased survival), compared with control groups.Key for Parasite Burden: �� increased parasite burden; � no difference in parasite burden; þ moderate (�50% decrease in parasite burden);þþsignificant (�50% decrease in parasite burden); þþþ highly significant (�90% decrease in parasite burden), compared with control groups
26.3CURREN
TST
ATUSOFVACCIN
ESFO
RIN
TER
MED
IATEHOST
S1019
26. VACCINATION AGAINST TOXOPLASMOSIS: CURRENT STATUS AND FUTURE PROSPECTS1020
whereas GRA1 protected C3H mice both withregard to survival and cyst burden it did notprotect BALB/c or C57BL/6 mice (Vercammenet al., 2000). Conversely, ROP2 improved survivalof BALB/c mice following infection with RHtachyzoites but not survival of C57BL/6 orCBA/J mice (Leyva et al., 2001). Similarly,GRA6 protected BALB/c mice but not Kunmingsagainst IP challenge with RH strain (Sun et al.,2011). In addition to adjuvanting the vaccines,efficacy may also be enhanced by changing theroute of vaccination as in one study in whichintradermal inoculation proved more effectivethan the intramuscular or intraperitoneal routes(Mohamed et al., 2003). Many of the studies todate utilizing nucleic acid vaccines can be viewedas incomplete as they merely measure immuneresponses after vaccination; this is particularlytrue of numerous recent studies utilizing Coop-worth ewes. While many studies utilizing rodentmodels domeasure survival after challenge infec-tion, relatively few then monitor parasiteburdens and to date there are only two studiesthat investigate maternalefoetal transmission.
The normal portal of entry of T. gondii is via thegut mucosa. Furthermore, intraepithelial IFNgproducing CD8þ T-cells cytolytic for parasitizedenterocytes have been shown to be generatedfollowing infection (Chardes and Bout, 1993;Chardes et al., 1994) while IgA may protectmucosal surfaces from parasite invasion (Mineoet al., 1993). Unfortunately, conventional parenter-ally administered vaccines do not generallyinduce mucosal immune responses and the mostsuccessful method to induce this type of responsein addition to systemic immunity has been toadminister vaccines orally (Gallichan and Rosen-thal, 1996). Current evidence would suggest thatentrapment of such vaccines in lipid vesicleswould enhance the immune response generatedby both protecting the DNA from degradationand targeting theDNAdirectly toAPC (Gregoria-dis et al., 2002). Furthermore, it has been demon-strated that DNA vaccines can influence bothmucosal and systemic immunity by the oral route
if suitably encapsulated (Chen and Langer, 1998).However, only one study to date has tested thisroute using chitosan microparticle entrappedpGRA1 (Bivas-Benita et al., 2003). Unfortunately,mucosal immunitywasnotmeasuredandnochal-lenge infections were undertaken in this study.Nevertheless, a few recent studies that utilizedthe intranasal route have highlighted the potentialof specifically targeting the mucosal immunesystem (Dimier-Poisson et al., 2006; Wang et al.,2009). C57BL/6 mice receiving three intranasaldoses of tachyzoite mRNA developed systemicand mucosal humoral immunity as well assystemic and mucosal cell mediated immunity.Furthermore, survival rates were significantlyimproved and a partial reduction in brain cystburdens noted following normally lethal or suble-thal oral challenge with brain cysts of the 76Kstrain (Dimier-Poisson et al., 2006). Cholera toxin(pCTA2/B) may improve the efficacy of vaccina-tion by the oral route (Wang et al., 2009)
While overall DNA vaccine formulationsbased on the above antigens have resulted inreduced mortality, in those studies where braincyst burdens have also been quantified onlya reduction has been achieved with no sterileimmunity observed. What was thought signifi-cant initially was that the antigens utilizedtended to be either tachyzoite specific, e.g.SAG1, or shared, e.g. GRA1, 2 and 4, whilenone were primarily bradyzoite specific. Similarincomplete protection has also resulted whenattenuated or ‘crude’ tachyzoite antigen prepara-tions have constituted the vaccine (Roberts et al.,1994; Buxton, 1993). What could be of signifi-cance in this respect is that serological studiesin T. gondii infected humans (Kasper, 1989;Zhang et al., 1995; Lunden et al., 1993), as wellas mice, have indicated that recognition is gener-ally of immunodominant stage specific antigenswith little recognition of shared antigens. Thiswould imply a requirement for additional anti-gens to those expressed in tachyzoites forcomplete protection following vaccinationwhich can be concluded from some experimental
26.3 CURRENT STATUS OF VACCINES FOR INTERMEDIATE HOSTS 1021
studies (Alexander et al., 1996; Freyre et al., 1993).To date, the bradyzoite antigens BAG1 (Nielsenet al., 2006; Dautu et al., 2007) and MAG1 (Niel-sen et al., 2006; Hiszczy�nska-Sawicka et al.,2010b) have been utilized in DNA vaccinestudies with some limited success in reducingmortality (Dautu et al., 2007) and reducing cystburdens (Nielsen et al., 2006). However, theseantigens were not combined with thoseprimarily associated with tachyzoite stages.Thus, the ideal vaccine to induce completeimmunity against T. gondii may be one thatwould promote protection against more thanone life cycle stage, induce mucosal as well assystemic immunity and, in addition, a strongCD8 response. A DNA vaccine delivered bythe oral or other appropriate mucosal routewould offer a rational solution.
26.3.7 Sub-Unit Vaccines
While various crude antigen preparations havebeen tested for their vaccinepotential against toxo-plasmosis for almost 50 years, studies on the effi-cacy of purified sub-units or recombinantproducts is relatively new, with the earliest inves-tigations taking place around 20 and 10 years ago,respectively (Table 26.4). Sub-unit vaccines havethe advantage that specific immunogenic antigensare presented without adding antigens that are atbest irrelevant and at worst capable of inducingfebrile or disease exacerbating immune responses.In addition, antigens that induce little immunity inthe context of the parasite can be boosted to bemore immunogenic if applied in a non-naturalcontext. Thus, sub-unit vaccines are focused intheir immune objectives and safe but tend to lackimmunological potency and, therefore, requireformulation with appropriate adjuvants toenhance their effectiveness. Typically, SAG1, theimmunodominant tachyzoite life cycle specificsurface antigen, has been the most extensivelystudied product both as a purified sub-unit andas a recombinant antigen (Table 26.4). Additionalvaccine candidates that have been investigated
include ROP1, ROP2, ROP4, GRA1, GRA2,GRA4, GRA5, GRA6, HF10 (a GRA6 deriveddecapeptide), GRA7, GRA720-28 peptide, MIC1,MIC4, HSP70, SAG2, SAG3, SRS1, TgPI-1, ActinDepolymerization Factor, TCP, NTPasesII, P54andP24aswell asuncharacterized antigens recog-nized by monoclonal antibodies such as F3G3(Brinkmann et al., 1993) or SDSePAGE purifiedproteins associated with cell invasion (Azzouzet al., 2012). Collectively, these have been studiedboth individually and as ‘cocktail’ vaccinesadministered by a variety of routes and usinga variety of adjuvants. Recombinant sub-unitvaccines have also utilized live infectious expres-sion vectors such as BCG, Salmonella, feline herpesvirus and vaccinia virus (Supply et al., 1999;Mishima et al., 2002; Roque-Resendiz et al., 2004;Cong et al., 2005). In addition to tachyzoite lifecycle stage specific antigens, some success hasbeen achieved with antigens such as MAG1known to have shared expression in both tachy-zoite and bradyzoite stages (Parmley et al., 2002).
The choice of adjuvant can be crucial andprofoundly influence vaccine efficacy. Thus,a purified sub-unit vaccin which is protective asmeasured by vaccine survival (Bulow and Boot-hroyd, 1991; Khan et al., 1991) and cyst burdenfollowing infection when entrapped in liposomesor formulatedwith saponinQuilA is disease exac-erbatory if adjuvanted with FCA (Kasper et al.,1985). Adjuvants so far utilized for subcutaneousadministration include: FCA, FIA, liposomes,saponin QuilA, ISCOMs, SBAS1, Monophos-phoryl lipid A, lipopeptide, PADRE, GLA-SEand IL-12; for intraperitoneal administration:FCA, FIA, liposomes, VetL-10, Lactobacillus,FMA and aluminium hydroxide (ALUM); forintramuscular administration: ALUM, FIA andCpG; for oral administration: cholera toxin, chito-san microparticles; for intranasal administration:salbutamol, cholera toxin and enterotoxin.
Overall, SAG1, adjuvant withstanding, hasinduced comparatively good protection interms of decreasing mortality (e.g. Bulow andBoothroyd, 1991), cyst burden (Khan et al.,
TABLE 26.4 Sub-Unit Vaccine Studies and Their Outcomes in Animal Models
AS15 BMDCs f.p. C57BL/6 IFN-g, T cellproliferation
Prutachyzoitesi.p.
þþ þþ
Key for Survival: �� decreased survival; � no difference in survival; þ moderate (�50% increased survival); þþ significant (�50% increased survival); þþþ highly significant (�90%increased survival), compared with control groupsKey for Parasite Burden: �� increased parasite burden; � no difference in parasite burden; þ moderate (� 50% decrease in parasite burden);þþsignificant (�50% decrease in parasite burden); þþþ highly significant (�90% decrease in parasite burden), compared with control groups
26.3CURREN
TST
ATUSOFVACCIN
ESFO
RIN
TER
MED
IATEHOST
S1027
TABLE 26.5 Studies on Toxoplasma Nucleic Acid Incorporated into Live Infectious Vectors
26. VACCINATION AGAINST TOXOPLASMOSIS: CURRENT STATUS AND FUTURE PROSPECTS1030
1991) and limiting maternalefoetal transmission(Letscher-Bru et al., 2003). Variation in outcomesfollowing vaccination while, in part, dependenton adjuvant employed and, undoubtedly, routeof administration is often also dependent onstrain and life cycle stage of the parasite usedand route of challenge infection. The animalmodel (species/strain) utilized is also crucial tosuccess. Thus, subcutaneous SAG1 vaccinationprotected guinea pigs (Haumont et al., 2000)and BALB/c mice (Letscher-Bru et al., 2003)against maternalefoetal transmission but failedto protect CBA/J mice (Letscher-Bru et al.,2003). Subcutaneous vaccination of Fischer ratswith GRA2 and GRA5 adjuvanted with FIAalso inhibited maternalefoetal transmission(Zenner et al., 1999), but whether these sub-units would also be effective in other murinemodels or elsewhere requires further investiga-tion. Interestingly, vaccination with the Th2inducing adjuvant ALUM, incorporating SAG1(Petersen et al., 1998) or various combinationsof ROP2 and GRA4 (Martin et al., 2004), TgPI-1(Cuppari et al., 2008) or NTPase II (Tan et al.,2011), was able to promote survival of BALB/cmice infected with virulent parasites (Petersenet al., 1998; Tan et al., 2011) along with reducedparasite burdens (Tan et al., 2011) and increasesurvival in C3H mice (Cuppari et al., 2008) andreduce parasite burdens of C57BL/6 and C3Hmice infected with Me49 cysts (Martin et al.,2004; Cuppari et al., 2008). In these studies mixedType 1/Type 2 responses were inducedalthough the bias was antigen dependent:SAG1, ROP2 and TgPI-1 induced primarilya Th2 response and GRA4 and NTPase IIprimarily a Type 1 response. ALUM (as one ofa few licensed human adjuvants) has been usedin humans for over 60 years and althougha type 2 adjuvant if it is formulated with type 1inducers such as CpG (Stacey and Blackwell,1999) or IL-12 (Pollock et al., 2003) a strongtype 1 response can also be induced. In thiscontext Toxoplasma immunogens such as cyclo-philin (Aliberti et al., 2003) may be ideal
co-stimulators in an ALUM formulated vaccineto enhance a potent type 1 response.
It has also been suggested that multivalentvaccines may be more successful than thosecomprising a single antigen (Martin et al., 2004).Using various combinations of SAG1, ROP2,ROP4 and GRA4 adjuvanted with FIA thisappears to have been confirmed by Dziadeket al. (2011). Nevertheless, a recent study sug-gested that a combined ROP2 þ GRA4 vaccineadjuvanted with CpG was not as effective aseither antigen individually (Sánchez et al., 2011).However, in addition to other experimentaldifferences, the former study utilized an i.p. chal-lenge infection while in the latter study infectionwas by the oral route. More importantly, target-ing more than one life cycle stage immunodomi-nant antigen may further enhance vaccineefficacy. Bradyzoite specific sub-unit antigens orantigens shared between tachyzoites could havepotential for vaccination. In this respect, MAG1,originally reported as a bradyzoite specificantigen, was later demonstrated to be secretedfrom tachyzoites stages (Parmley et al., 2002).Significantly, vaccinationwithMAG1 adjuvantedwith QuilA not only reduced the cyst burden inthe brain but also reduced inflammation. AsSAG1 is also effective by this route using QuilA,the outcome of vaccination with a combinedSAG1/MAG1 vaccine would be extremely inter-esting. Indeed, as bradyzoites initiate infectionin the intestine the outcome of mucosal immuni-zation with this combination linked to choleratoxin, for example, would be intriguing.
26.3.8 Genomics and Immunosense
The sequencing of the three major lineages anda number of atypical strains will soon provide analmost complete supragenome (data available athttp://www.toxodb.org). This, with the appro-priate predictive algorithms, should, in theory,allow the identification of T. gondii, MHC1binding peptides for any host species. Recentwork has taken this ‘immunosense’ approach to
26.4 THE RODENT AS A MODEL TO STUDY CONGENITAL DISEASE AND VACCINATION 1031
epitope identification as previously successfullyused for other vaccines. This approach predictsHLA binding nonamer peptides from pathogenproteins predicted or proven to have vaccinepotential. Importantly, these algorithms can beused for multiple HLA class I supermotif haplo-types, HLA-B*0702, HLA-A03 and HLA-A*0201,at least one of which has been found to be presentin w80%e90% of the human population. Thesepeptides are then tested in vitro for their abilityto elicit IFNg production from PBLs isolatedfrom humans of the matching HLA with chronicT. gondii infection. Peptides that are stronginducers of IFNg are then tested in HLA trans-genic mice for their ability to elicit CD8 IFNgproduction and protection. Thus far, using thisapproach a number of candidate peptides capableof eliciting IFNg from human PBMCs expressingone of the HLA-B*0702, HLA-A03 and HLA-A*0201 supertypes and capable of protectingmice against challenge with a type II strain of T.gondii have been identified. In spite of the use ofdiverse adjuvanting systems, including conjuga-tion to lipopeptides, or administration witha new TLR-4 binding adjuvant derived fromSalmonella produced by IDRI/IDC, protectionwas impressive but not equivalent to thatobserved following vaccination with the RPS13conditional knock-out parasites (Cong et al.,2010, 2011, 2012). The ability of a single MHC1binding peptide to protect mice from challengehas been demonstrated as a proof of principlewith the GRA6 HF10 peptide in BALB/c mice,albeit using non-translational in vitro peptidepulsed dendritic cells as the delivery mechanism(Blanchard et al., 2008).
26.4 THE RODENT AS A MODEL TOSTUDY CONGENITAL DISEASE
AND VACCINATION
The success of an attenuated vaccine forpreventing Toxoplasma induced abortion insheep is reported in detail above. However,
experimental studies are extremely expensiveand comparatively difficult to perform andcoordinate in domestic animals. Consequently,much effort has been expended in elucidatingthe immunology of congenital toxoplasmosisin the rodent model and exploring the efficacyof prophylactic therapies. Very early studieson the mouse model indicated that verticaltransmission through successive generationswas the normal situation in mice (Beverley,1959) unlike either humans (Cook, 1990) orovids (Beverley and Watson, 1971) where onlya primary infection during pregnancy resultedin congenital infection. These differences wouldsuggest that mice would make poor substitutesfor studying immune prophylaxis of human orovine congenital disease. More recently, verticaldisease transmission has also been observed tooccur occasionally in humans in the apparentabsence of reinfection (Vogel et al., 1996; Kodji-kian et al., 2004). Conversely, the authors andothers have now demonstrated that verticaldisease transmission in mice in the absence ofprimary infection during pregnancy is, in fact,mouse strain dependent and that the BALB/cmouse is an excellent model of the humandisease (Roberts and Alexander, 1992; Robertset al., 1994; Alexander et al., 1998; Thouveninet al., 1997; Elsaid et al., 2001; Couper et al.,2003; Letscher-Bru et al., 2003; Abou-Bacaret al., 2004a, 2004b). BALB/c dams previouslyinfected with and recovered from cyst formingstrains of T. gondii, unlike mice infected for thefirst time during pregnancy, produced healthylitters even if infected for a second time by theoral route during pregnancy. Similarly, congen-ital transmission is significantly inhibitedfollowing oral challenge with cysts in Swissoutbred OF1 mice dams previously vaccinatedwith Mic1e3 deficient parasites (Ismael et al.,2006). Thus the mouse, if the appropriate strainis utilized, provides an appropriate modelto study the immunology of vertical diseasetransmission and design appropriate vaccinestrategies.
26. VACCINATION AGAINST TOXOPLASMOSIS: CURRENT STATUS AND FUTURE PROSPECTS1032
The physiological/immunological environ-ment associated with pregnancy, which favoursa type 2 immune response (reviewed in Robertset al., 1996, 2001), probably promotes a permis-sive environment for vertical transmission tooccur. Indeed, a type 2 response bias in themurine placenta has been demonstrated to facil-itate successful implantation, maintenance ofearly pregnancy and suppression of inflamma-tion: a switch towards a type 1 response canresult in foetal death (Krishnan et al., 1996a,1996b). It is well recorded that humans, as wellas mice, develop more severe primary infectionsduring pregnancy (Luft and Remington, 1982;Shirahata et al., 1992, 1993). This is associatedwith reduced IFNg levels (Shirahata et al., 1992)and administration of recombinant IFNgpromotes the resistance of pregnant mice againsttoxoplasmosis. Similarly, the Th1 cytokine IL-2also promotes resistance against lethal challengein pregnant mice (Shirahata et al., 1993). Further-more, IL-4 deficient mice that should have a type1 bias are more resistant to T. gondii infectionduring gestation than their wild-type counter-parts (Alexander et al., 1998; Thouvenin et al.,1997). In addition, although vertical diseasetransmission from infected dams to pups wasnot impaired in IL-4 deficient B6/129 (Alexanderet al., 1998), there was a 50% decrease in congen-ital infections in BALB/c IL-4 deficient micecompared with wild-type controls (Thouveninet al., 1997). Neutralization of IFNg and deple-tion of CD8þ T-cells increased the incidence ofmaternalefoetal transmission in BALB/c mice(Abou-Bacar et al., 2004a) although studies usingRAG2�/� mice which lack B- and T-cells alsoindicated a protective role for NK-cells, althoughin this study, paradoxically, neutralizing IFNginhibited transmission to the foetus in this model(Abou-Bacar et al., 2004b).
As previously infected BALB/c dams are ableto totally prevent vertical disease transmission totheir pups even when re-infected during preg-nancy this model offers a gold standard for test-ing putative vaccines. Consequently, vaccination
of dams with crude soluble tachyzoite antigens,with or without cyst antigens, entrapped in lipidvesicles prior to pregnancy has been demon-strated to limit vertical disease transmissionand pup mortality following infection with cystsorally on day 11 of pregnancy associated withenhanced IFNg production (Roberts et al., 1994;Elsaid et al., 2001). Indeed, a recombinant SAG1vaccine, although not a SAG1 DNA vaccine(Couper et al., 2003), has been demonstrated tobe sufficient to inhibit vertical transmission inBALB/c mice associated with CD8þ T-cells andIFNg production (Letscher-Bru et al., 2003).While rSAG1, though adjuvanted with a type 1promoter, also was protective in a congenitalguinea pig infection model (Haumont et al.,2000), rSAG1 actually promoted vertical diseasetransmission in CBA/J mice (Letscher-Bru et al.,2003). Thus the success of a vaccine may bemouse strain or species dependent but how thevaccine is adjuvanted is also crucial to success.For example while lipid vesicle entrappedSTAg (Roberts et al., 1994) successfully vacci-nated against maternalefoetal transmission,unadjuvanted STAg and FCA adjuvantedSTAg increased rates of foetal death, abortionand vertical transmission. FCA, reputably thegold standard type 1 adjuvant, had previouslybeen shown to be counterprotective in a SAG1vaccine (Kasper et al., 1985), promoting increaseddeath and parasite burdens following challengeinfection. However, while vesicular adjuvantssuch as lipid vesicles and ISCOMS have beendemonstrated to induce CD8þ T-cell responses(Debrick et al., 1991; Zhou et al., 1992) there islittle evidence that emulsion systems such asFCA are capable of inducing a similar response(Roberts et al., 1994).
These observations, however, do not explainwhy rSAG1, but not a SAG1 DNA vaccine,induces sufficient protective immunity to preventmaternalefoetal transmission in BALB/c mice asboth vaccines induce or should induce CD8þ
T-cell responses and IFNg production (Couperet al., 2003; Letscher-Bru et al., 2003). This could
26.5 REVIEW OF VACCINES FOR DEFINITIVE HOST (CATS) 1033
perhaps be a result of the utilization of thedifferent parasite strains, Beverley (Couperet al., 2003) and Me49 (Letscher-Bru et al., 2003),used for challenge although both these are type2 strains. The protection afforded by SAG1 inthe guinea pig study required inclusion ofa type 1 inducing immune response adjuvant(Haumont et al., 2000), SABS1, and the challengeinvolved a type 3 strain which is less virulentthan type 2 strains and not often associatedwith the human disease. However, using theBeverley strain, Roberts et al. (1994) did demon-strate significant protection against maternalefoetal transmission with a vaccine comprisinga cocktail of soluble tachyzoite antigens suggest-ing that an approach utilizing more than onevaccine candidate may be a more effective one(Roberts et al., 1994). This has been confirmedby Mevelec et al. (2005) who found thata combined SAG1, GRA4 DNA vaccine adju-vanted with plasmid GMeCSF was more effec-tive than vaccines expressing single antigens(Mevelec et al., 2005). Consequently, vaccinationof outbred Swiss OF1 dams with this ‘cocktail’reduced parasite induced foetal death duringpregnancy although it did not limit maternalefoetal transfer. Overall, these reports demon-strate that a type 1 response induced by anappropriately adjuvanted multivalent vaccinewould provide the most effective protectiveimmunity against murine congenital toxoplas-mosis. The protective activity of vaccines mayalso be enhanced firstly by introducing brady-zoite antigens to the cocktail (Elsaid et al., 2001)and secondly by inducing mucosal, as well assystemic, responses. Thus, McLeod et al. (1988)found that intraintestinal immunization of micewith the temperature sensitive mutant TS-4, butnot subcutaneous immunization, was effectivein reducing the incidence of congenital disease(McLeod et al., 1988).
The need to immunize using different lifecycle stage specific antigens of T. gondii hasbeen recently highlighted in the rat model ofcongenital transmission (Freyre et al., 2006).
Previous studies using Fischer (Zenner et al.,1993) and Wistar and Holtzman (Paulino andVitor, 1999) rats had indicated that rats recov-ered from a primary infection with T. gondiiand produced healthy non-infected pups evenif dams were re-infected during the gestationperiod. However, Freyre and colleagues (2006),using SpragueeDawley rats, have demonstratedthat immunization with RH tachyzoites inducedonly low rates of protection against cyst oroocyst challenge. Furthermore, immunizationwith cysts provided incomplete protectionagainst oocyst challenge even if the same para-site strain was used. Indeed, complete protectionwas only demonstrated in cyst immunized ratschallenged with cysts of the same strain andcomplete protection was rarely achievedfollowing challenge with different Toxoplasmastrains. Conversely, Zenner et al. (1999) foundthat full protection against vertical transmissionwas irrespective of which Toxoplasma strainwas used for immunization and which for chal-lenge (Zenner et al., 1999). Differences betweenthese two studies perhaps reflect the differentrat strains used in each. However, in the latterstudy, vaccinating dams with a vaccinecomprising excretory/secretory tachyzoite anti-gens did significantly protect against congenitalinfection, highlighting the potential importanceof the rat model. The hamster may providea further model to study the effectiveness ofvaccination, as it has recently been shown thatprevious infection almost completely protectsagainst congenital toxoplasmosis initiated byinoculation with cysts or oocysts (Freyre et al.,2012).
26.5 REVIEW OF VACCINES FORDEFINITIVE HOST (CATS)
Whether or not a cat vaccine is required isa matter of debate. Although cats are the defini-tive host for Toxoplasma they seldom developdisease. A future cat vaccine should therefore
26. VACCINATION AGAINST TOXOPLASMOSIS: CURRENT STATUS AND FUTURE PROSPECTS1034
not be aimed at protecting the cat from illness,but should prevent oocyst shedding, therebyreducing oocyst contamination of the environ-ment and risk to livestock and/or humans.Cats shed high numbers of oocysts after a firstinfection and most (but not all) cats remainimmune afterwards (Dubey, 1995). Kittensfrequently become infected soon after weaningwhen they start eating prey. Vaccinations shouldthus be applied as early as possible in kittens ofoutdoor-roaming cats. Ideally, domestic andstray cats should be vaccinated if one wouldlike to prevent oocyst contamination of food,water and soil. It is clear that this can onlybe accomplished if nationwide vaccinationcampaigns are organized, including the vaccina-tion of stray cats. Vaccination of stray cats couldbe accomplished by a similar approach as withrabies. Foxes were orally immunized by distrib-uting vaccine baits using a vacciniaerabiesglycoprotein recombinant virus, which success-fully controlled the disease in Europe and theUSA (Pastoret, 2002). Clearly, such a radicalvaccination approach for Toxoplasma would bemost beneficial to areas with high incidences oftoxoplasmosis, being mainly countries withpoor hygiene conditions, as exemplified inBrazil, where it was shown that contaminateddrinking water caused a high incidence ofhuman toxoplasmosis (Bahia-Oliveira et al.,2003). Although postnatal acquired infectionsare mostly asymptomatic, it has been demon-strated to cause ocular disease (Vallochi et al.,2002; Burnett et al., 1998), making a strong caseto control Toxoplasma. Apart from extensivenationwide vaccination campaigns, one canalso envisage a cat vaccine for domestic catowners to prevent or reduce the incidence ofToxoplasma infections during pregnancy, whichmay lead to congenitally infected babies orabortion.
A cat vaccine should thus prevent oocystshedding, which can be induced in cats uponinfection with each of the three infectious formsof Toxoplasma, being tachyzoites, bradyzoites or
sporozoites (Dubey, 1998). The natural andmost efficient route of infection of cats is via tissuecysts that are present in prey. Released brady-zoites can generate tachyzoites, but also directlyinitiate the enteroepithelial life cycle (Dubeyand Frenkel, 1972). A vaccine should be focusedon inhibiting this enteroepithelial cycle toprevent the formation of oocysts and shouldnot be limited to tachyzoites. Currently, noenteroepithelial antigens have been defined thatare specific to schizonts, gametocytes or zygotes.A few Toxoplasma antigens (including HXGPRT,HSP70 and a 14-3-3 homologue) have beencloned from enteroepithelial stages, but theseare not specific to these stages (Koyama et al.,2000). No antigens from enteroepithelial stageshave currently been tested as vaccines candi-dates. However, it may be questioned if a sub-unit vaccine will induce strong enough immunityto prevent or dramatically reduce oocyst shed-ding. Eimeria can serve as an example, wherevarious sub-unit vaccines against coccidiosis inchickens were tested and were shown to reduceoocyst shedding by only 50% (Vermeulen,1998). This demonstrates the difficulty of makingan effective sub-unit vaccine to significantlyreduce oocyst shedding. Eimeria is a related cocci-dial parasite with only enteroepithelial stages,and currently all commercial Eimeria vaccinescontain live sporulated oocysts. These vaccinescan reduce oocyst shedding by more than 90%and frequently even 100% (Vermeulen et al.,2001). It is therefore most likely that a live atten-uated Toxoplasma vaccine will be required toprevent oocyst shedding.
The T-263 mutant Toxoplasma strain has beenextensively tested as a live vaccine for cats (Fren-kel et al., 1991). The T-263 strain is unable toproduce oocysts in cats (due to a defect in thesexual stages), but can be propagated as tachy-zoites and bradyzoites. Oral vaccination withT-263 bradyzoites prevented oocyst sheddingin 84% of kittens following a single dose andwas 100% effective following a secondary vacci-nation (Freyre et al., 1993). Importantly, live
26.6 FUTURE STRATEGIES TO DESIGN NEW VACCINES FOR COCCIDIAL PARASITES IN GENERAL 1035
tachyzoites from the T-263 strain could notprotect cats from oocyst shedding upon chal-lenge (Freyre et al., 1993), indicating that livetachyzoite vaccines are not an option for cats.The effectiveness of the T-263 strain as a catvaccine was subsequently tested in a field trialon eight commercial swine farms in the USA(Mateus-Pinilla et al., 1999). Over a three yearperiod, during which time cats around the farmswere trapped and vaccinated, the number ofoocyst shedding cats was reduced and thenumber of seropositive pigs was reduced. Trap-ped mice were also found to be seronegative forToxoplasma. Despite this success, T-263 has neverbeen commercialized. A serious drawback forsuch a vaccine is the production and shelf life.Bradyzoites or tissue cysts are most efficientlyproduced in vivo and cannot be frozen withoutconsiderable loss of viability. On the otherhand, it is unknown how long tissue cystsremain viable at room temperature and/or at4�C. Currently, no alternatives to T-263 are avail-able, since attenuated strains such as S48 do notdevelop into bradyzoites and the TS4 mutantmay be too limited to develop in vivo (apartfrom the safety issue for TS4). Interestingly,a T-263 vaccine may be easily integrated intobait for wild animals, by simply infecting baitanimals prior to their deployment. As a precau-tion it should be realized that the use of baitwith live Toxoplasma parasites can be lethal tosome highly sensitive animal species, such asNewWorld monkeys and Australian marsupials(Hill et al., 2005).
More recent studies have used crude rhoptrypreparations administered either through thenasal or rectal route with QuilA as an adjuvant(Garcia et al., 2007; Zulpo et al., 2012). Intranasalimmunization was able to prevent oocyst shed-ding in two out of three cats challenged withT. gondii (VEG strain) oocysts (Garcia et al.,2007). In a subsequent study that challengedwith Me49 cysts, intranasal administration witha similar preparation was found to be superiorto intrarectal administration and was also
demonstrated to reduce the prepatent period(Zulpo et al., 2012).
In conclusion, the impact of reduced oocystburden on the risk of transmission to animalsand humans justifies the vaccination of the felinedefinitive host. Although such a target is techni-cally achievable, this would require large invest-ments from authorities and health organizationsmaking vaccination of cats compulsory. It is asyet not expected that this would be given suchpriority. Until then, hygiene measures remainthe only tools to reduce the risk of Toxoplasmainfection.
26.6 FUTURE STRATEGIES TODESIGN NEW VACCINES FORCOCCIDIAL PARASITES IN
GENERAL AND TOXOPLASMAGONDII IN PARTICULAR
It is generally believed that adult acquiredinfection of humans results in lifelong immunityto T. gondii. The evidence would suggest thatimmunity prevents reactivation of disease fromthe bradyzoite stage and reinfection by otherstrains of T. gondii. Furthermore, immunity nor-mally prevents congenital transmission fromchronically infected individuals even if thoseindividuals are re-exposed to infection. Thiswould suggest that a vaccine is feasible andthis should be achieved by mimicking theimmune response that occurs during a naturalinfection.
Immunity to T. gondii is complex and involvesmany facets of the immune system. The innateimmune system is important in containingparasite proliferation during the early stages ofinfection and drives the adaptive immuneresponse (Denkers and Gazzinelli, 1998). CD4þ
T-lymphocytes play an important role in shapingthe immune responses and provide IL-2 for thedevelopment of CD8þ T lymphocytes whichproduce IFNg and would appear to be the effec-tors of long term immunity. Although CD8þ
26. VACCINATION AGAINST TOXOPLASMOSIS: CURRENT STATUS AND FUTURE PROSPECTS1036
T-lymphocytes from experimentally vaccinatedmice are capable of killing T. gondii infected cellsinanMHCrestricted, perforindependentmanner,IFNgwould appear to be themajor effectormech-anism of long term immunity in vivo (Denkerset al., 1997; Wang et al., 2004). Thus, if a vaccine isto mimic natural immunity, it should comprisethe proteins or peptides thereof that are capableof being presented on MHC class I. These shouldbe administered in such a manner that facilitatesMHC class I processing and development ofCD8þ T-lymphocytes. In addition to the immu-nogen, an appropriate adjuvant is likely to berequired. Live attenuated parasites would appearto fulfil many of these requirements. Assumingthey maintain their fitness to invade cells, theyeffectively deliver the complete set of proteins tothe class II and class I processing pathways asoccurs during the course of natural infection. Inaddition, T. gondii has a number of endogenousadjuvants including cyclophilin 18, which hasthe ability to bind CCR-5 receptor and elicit IL-12production (Aliberti et al., 2003), profilin which isa ligand for murine TLR-11 (not present in allmammals) (Yarovinsky et al., 2005) and HSP70which induces dendritic cell maturation (Kanget al., 2004). Sub-unit vaccines are likely to be crit-icallydependent on adjuvant for effective deliveryto the endogenous class I processing pathway.DNA vaccination or the use of viral vectors mayrepresent alternative means of achieving this end.
Not surprisingly, live attenuated vaccines thatmimic a natural infection have been extremelysuccessful in inducing protection in murinemodels of infection. The use of live attenuatedparasites for human and coccidial veterinaryvaccines is still the best option if solid immunityis required. However, a number of concernswould have to be overcome for a live attenuatedToxoplasma vaccine. These would include thepotential of the parasite to revert to a virulentphenotype, the ability of these parasites to infectcells of the central nervous system, the possi-bility of persistence and the potential to causecongenital infection or abortion. However,
a live attenuated vaccine (Toxovax) has beenused in livestock for some time and has beensuccessful in limiting abortion in sheep. Thenature of the defect in this parasite that preventsit from completing a full life cycle is unknown.Consequently, the appeal of a rationally engi-neered, highly attenuated vaccine with multipledeleted genes conferring multiple auxotrophy ormultiple developmental disabilities is obvious.
The completion of the T. gondii genome hasprovided the amino acid sequence for all poten-tially immunogenic components of this path-ogen. As algorithms are refined it shall bepossible to make predictions of which predictedpeptides are likely to have MHC class I and classII epitopes not only for humans, but also for otherspecies. These peptide predictions will also needto take into account the polymorphisms in MHCmolecules as outlined in Section 26.3.8 forhumans and the various animal species that itmay be desirable to vaccinate. To enable thisinformation to be fully exploited for a sub-unitvaccine, adjuvants will be of key importance.
Alternatively, genome information can be usedin combination with mass spectrometry to iden-tify relevant sets of proteins (Mann et al., 2001).For example, surface antigens from differentToxoplasma life cycle stages could be isolated, ana-lysedwithMALDI-TOF and compared to peptidemasses from a database for identification. Thismay yield interesting new vaccine candidates, inparticular from bradyzoite stages and gastroin-testinal stages.
Another approach to identify novel vaccineantigens would be to use the full theoretical setof genes from Toxoplasma to select a subgroupof promising candidates and have all of themexpressed and assessed for their protectivecapacity. Such a major task has been undertakenwith the genome data from Meningococcus(serogroup B), from which 570 surface antigenswere identified and tested, resulting in sevennew vaccine candidates (Pizza et al., 2000). SinceT. gondii has a larger genome with 6927 genescurrently annotated (http://www.toxodb.org
(02/06)) this approach cannot be performed bya single group but would require a joint effortby multiple teams.
An ideal vaccine would be able to protectagainst all strains of T. gondii. This in theoryshould not be challenging as T. gondii is remark-ably clonal with most strains examined, fallinginto one of five types (Type IeV). However,a number of naturally occurring recombinantstrains have been identified in the USA anda number of exotic strains isolated in SouthAmerica. Natural infection has generally beenthought to protect from a secondary infection.However, there is now some evidence that in atleast some circumstances secondary infectionscan occur with separate strains in mice (Araujoet al., 1997; Dao et al., 2001). In addition, it hasbeen demonstrated in a murine system that cyto-toxic CD8þ lymphocytes can be parasite strainspecific (Johnson et al., 2002). This raises thepossibility that a vaccine might not be protectiveagainst all strains of T. gondii.
Most infections in humans are initiated by bra-dyzoites or sporozoites, but these stages onlypersist for a short length of time before givingrise to the tachyzoite form. The tachyzoite multi-plies extensively for around 14 days before trans-forming into bradyzoites that reside inside cyststructures. Each of these stages have stage specifi-cally expressed surface proteins (e.g. Kim andBoothroyd, 2005; Lyons et al., 2002) and secretedproteins (e.g. Reichmann et al., 2002; Meissneret al., 2002a).There is a clear advantage in targetinga vaccine against sporozoite or bradyzoite anti-gens as this might prevent infection entirely.However, such a strategy may necessitate twocomponents, one targeting sporozoites and onetargeting bradyzoites. Most vaccine studies haveto date used tachyzoite derived fractions (and insomecases specific components),which, in theory,could be protective in infections initiated by eitherof the infective stages. However, the ability of thetachyzoite todifferentiate into the bradyzoite formwith a different antigenic profile provides ameansof escaping an immune response as it develops
against the tachyzoite stage. Targeting the brady-zoite stage in a vaccine may prevent this meansof escape and result in sterile immunity. Sucha strategy might not be necessary for a vaccineaimed at preventing congenital transmission asthe tachyzoite stage would appear to be respon-sible for transplacental transmission. Notably,the live attenuated vaccine (S48) used in sheep toprevent congenital transmission consists of tachy-zoites. Thus, the choice of antigensmaybeaffectedby the intended use of the vaccine, but in realitya vaccine may require multiple components frommultiple life cycle stages.
To summarize, there is currently only a com-mercially available Toxoplasma vaccine for sheepand goats, but a vaccine for pigs, cats and humansis still lacking. In recent years, tremendous pro-gress has been made not only in cell and molec-ular biology of T. gondii, but also incharacterizing the immunobiology of the parasitein host species. This information allied to currentbiochemical, molecular and immunological prog-ress must make us optimistic about the likelihoodof developing new, safe and successful vaccinesfor both clinical and veterinary medicine.
ReferencesAbou-Bacar, A., Pfaff, A.W., Georges, S., Letscher-Bru, V., Filisetti, D., Villard, O.,
Antoni, E., Klein, J.P., Candolfi, E., 2004a. Role of NK cells and gammainterferon in transplacental passage of Toxoplasma gondii in a mouse modelof primary infection. Infect. Immun. 72, 1397e1401.
Abou-Bacar, A., Pfaff, A.W., Letscher-Bru, V., Filisetti, D., Rajapakse, R.,Antoni, E., Villard, O., Klein, J.P., Candolfi, E., 2004b. Role of gammainterferon and T-cells in congenital Toxoplasma transmission. ParasiteImmunol. 26, 315e318.
Alexander, J., Jebbari, H., Bluethmann, H., Brombacher, F., Roberts, C.W., 1998.The role of IL-4 in adult acquired and congenital toxoplasmosis. Int. J.Parasitol. 28, 113e120.
Alexander, J., Jebbari, H., Bluethmann, H., Satoskar, A., Roberts, C.W., 1996.Immunological control of Toxoplasma gondii and appropriate vaccine design.Curr. Top. Microbiol. Immunol. 219, 183e195.
Aliberti, J., Valenzuela, J.G., Carruthers, V.B., Hieny, S., Andersen, J., Charest, H.,Reis E Sousa, C., Fairlamb, A., Ribeiro, J.M., Sher, A., 2003. Molecularmimicry of a CCR5 binding-domain in the microbial activation of dendriticcells. Nat. Immunol. 4, 485e490.
Aosai, F., Mun, H.S., Norose, K., Chen, M., Hata, H., Kobayashi, M., Kiuchi, M.,Stauss, H.J., Yano, A., 1999. Protective immunity induced by vaccinationwith SAG1 gene-transfected cells against Toxoplasma gondii-infection in mice.Microbiol. Immunol. 43, 87e91.
Angus, C.W., Klivington-Evans, D., Dubey, J.P., Kovacs, J.A., 2000. Immuniza-tion with a DNA plasmid encoding the SAG1 (P30) protein of Toxoplasmagondii is immunogenic and protective in rodents. J. Infect. Dis. 181, 317e324.
26. VACCINATION AGAINST TOXOPLASMOSIS: CURRENT STATUS AND FUTURE PROSPECTS1038
Araujo, F., Slifer, T., Kim, S., 1997. Chronic infection with Toxoplasma gondii doesnot prevent acute disease or colonization of the brain with tissue cystsfollowing reinfection with different strains of the parasite. J. Parasitol. 83,521e522.
Araujo, F.G., Remington, J.S., 1974. Protection against Toxoplasma gondii in miceimmunized with Toxoplasma cell fractions, RNA and synthetic poly-ribonucleotides. Immunology 27, 711e721.
Avelino, M.M., Campos Jr., D., Parada, J.B., Castro, A.M., 2004. Risk factors forToxoplasma gondii infection in women of childbearing age. Braz. J. Infect.Dis. 8, 164e174.
Azzouz, S., Maache, M.O., Suna, A., Lawton, P., Pétavy, A.F., 2012. Toxoplasmagondii: identification and immune response against a group of proteinsinvolved in cellular invasion. Exp. Parasitol. 1, 63e68.
Bahia-Oliveira, L.M., Jones, J.L., Azevedo-Silva, J., Alves, C.C., Orefice, F.,Addiss, D.G., 2003. Highly endemic, waterborne toxoplasmosis in north Riode Janeiro state. Brazil. Emerg. Infect. Dis. 9, 55.
Batz, M.B., Hoffmann, S., Morris Jr., J.G., 2012. Ranking the disease burden of14 pathogens in food sources in the united states using attribution datafrom outbreak investigations and expert elicitation. J. Food Prot. 75,1278e1291.
Beauvillain, C., Juste, M.O., Dion, S., Pierre, J., Dimier-Poisson, I., 2009. Exosomesare an effective vaccine against congenital toxoplasmosis in mice. Vaccine27, 1750e1757.
Beauvillain, C., Ruiz, S., Guiton, R., Bout, D., Dimier-Poisson, I., 2007. A vaccinebased on exosomes secreted by a dendritic cell line confers protectionagainst T. gondii infection in syngeneic and allogeneic mice. Microbes Infect.14-15, 1614e1622.
Beghetto, E., Nielsen, H.V., Del Porto, P., Buffolano, W., Guglietta, S., Felici, F.,Petersen, E., Gargano, N., 2005. A combination of antigenic regions ofToxoplasma gondii microneme proteins induces protective immunity againstoral infection with parasite cysts. J. Infect. Dis. 191, 637e645.
Benenson, M.W., Takafuji, E.T., Lemon, S.M., Greenup, R.L., Sulzer, A.J., 1982.Oocyst-transmitted toxoplasmosis associated with ingestion of contami-nated water. N. Engl. J. Med. 307, 666.
Beverley, J.K., 1959. Congenital transmission of toxoplasmosis through succes-sive generations of mice. Nature 183, 1348e1349.
Beverley, J.K., 1971. [Clinical aspects, pathogenesis and prevention of animaltoxoplasmosis (with special reference to toxoplasmosis abortion in sheep)].Monatsh. Veterinarmed. 26, 893.
Beverley, J.K., Archer, J.F., Watson, W.A., Fawcett, A.R., 1971. Trial of a killedvaccine in the prevention of ovine abortion due to toxoplasmosis. Br. Vet. J.127, 529e535.
Beverley, J.K., Watson, W.A., 1971. Prevention of experimental and of naturallyoccurring ovine abortion due to toxoplasmosis. Vet. Rec. 88, 39e41.
Bezerra, R.A., Carvalho, F.S., Guimarães, L.A., Rocha, D.S., Maciel, B.M.,Wenceslau, A.A., Lopes, C.W., Albuquerque, G.R., 2012. Genetic charac-terization of Toxoplasma gondii isolates from pigs intended for humanconsumption in Brazil. Vet. Parasitol. 189, 153e161.
Bivas-Benita, M., Laloup, M., Versteyhe, S., Dewit, J., De Braekeleer, J.,Jongert, E., Borchard, G., 2003. Generation of Toxoplasma gondii GRA1protein and DNA vaccine loaded chitosan particles: preparation, charac-terization, and preliminary in vivo studies. Int. J. Pharm. 266, 17e27.
Blanchard, N., Gonzalez, F., Schaeffer, M., Joncker, N.T., Cheng, T., Shastri, A.J.,Robey, E.A., Shastri, N., 2008. Immunodominant, protective response to theparasite Toxoplasma gondii requires antigen processing in the endoplasmicreticulum. Nat. Immunol. 8, 937e944.
Bonenfant, C., Dimier-Poisson, I., Velge-Roussel, F., Buzoni-Gatel, D., DelGiudice, G., Rappuoli, R., Bout, D., 2001. Intranasal immunization withSAG1 and nontoxic mutant heat-labile enterotoxins protects mice againstToxoplasma gondii. Infect. Immun. 69, 1605e1612.
Bohne, W., Hunter, C.A., White, M.W., Ferguson, D.J., Gross, U., Roos, D.S.,1998. Targeted disruption of the bradyzoite-specific gene BAG1 does notprevent tissue cyst formation in Toxoplasma gondii. Mol. Biochem. Parasitol.92, 291.
Bos, H.J., Smith, J.E., 1993. Development of a live vaccine against ovine toxo-plasmosis. In the series analytic: Toxoplasmosis. Proceedings of a workshopheld June 28eJuly 2, 1992, Fontevraud, France.
Bourguin, I., Chardes, T., Bout, D., 1993. Oral immunization with Toxoplasmagondii antigens in association with cholera toxin induces enhanced protectiveand cell-mediated immunity in C57BL/6 mice. Infect. Immun. 61, 2082.
Bowie, W.R., King, A.S., Werker, D.H., Isaac-Renton, J.L., Bell, A., Eng, S.B.,Marion, S.A., 1997. Outbreak of toxoplasmosis associated with municipaldrinking water. The BC Toxoplasma Investigation Team. Lancet 350, 173.
Brinkmann, V., Remington, J.S., Sharma, S.D., 1993. Vaccination of mice with theprotective F3G3 antigen of Toxoplasma gondii activates CD4+ but not CD8+ T-cells and induces Toxoplasma specific IgG antibody. Mol. Immunol. 30, 353e358.
Bruna-Romero, O., Gonzalez-Aseguinolaza, G., Hafalla, J.C., Tsuji, M.,Nussenzweig, R.S., 2001. Complete, long-lasting protection against malariaof mice primed and boosted with two distinct viral vectors expressing thesame plasmodial antigen. Proc. Natl. Acad. Sci. U.S.A. 98, 11491.
Bulow, R., Boothroyd, J.C., 1991. Protection of mice from fatal Toxoplasma gondiiinfection by immunization with p30 antigen in liposomes. J. Immunol. 147,3496e3500.
Burnett, A.J., Shortt, S.G., Isaac-Renton, J., King, A., Werker, D., Bowie, W.R.,1998. Multiple cases of acquired toxoplasmosis retinitis presenting in anoutbreak. Ophthalmology 105, 1032.
Buxton, D., 1993. Toxoplasmosis: the first commercial vaccine. Parasitol. Today9, 335.
Buxton, D., 1998. Protozoan infections (Toxoplasma gondii, Neospora caninum andSarcocystis spp.) in sheep and goats: recent advances. Vet. Res. 29, 289.
Buxton, D., Innes, E.A., 1995. A commercial vaccine for ovine toxoplasmosis.Parasitology 110 (Suppl), S11.
Buxton, D., Thomson, K., Maley, S., Wright, S., Bos, H.J., 1991. Vaccination ofsheep with a live incomplete strain (S48) of Toxoplasma gondii and theirimmunity to challenge when pregnant. Vet. Rec. 129, 89.
Buxton, D., Uggla, A., Lovgren, K., Thomson, K., Lunden, A., Morein, B.,Blewett, D.A., 1989. Trial of a novel experimental Toxoplasma iscom vaccinein pregnant sheep. Br. Vet. J. 145, 451.
Caetano, B.C., Bruña-Romero, O., Fux, B., Mendes, E.A., Penido, M.L.,Gazzinelli, R.T., 2006. Vaccination with replication-deficient recombinantadenoviruses encoding the main surface antigens of Toxoplasma gondiiinduces immune response and protection against infection in mice. Hum.Gene. Ther. 17, 415e426.
Chardes, T., Bout, D., 1993. Mucosal immune response in toxoplasmosis. Res.Immunol. 144, 57e60.
Chardes, T., Buzoni-Gatel, D., Lepage, A., Bernard, F., Bout, D., 1994. Toxoplasmagondii oral infection induces specific cytotoxic CD8 alpha/beta+ Thy-1+ gutintraepithelial lymphocytes, lytic for parasite-infected enterocytes. J.Immunol. 153, 4596e4603.
Chartier, C., Mallereau, M.P., 2001. Vaccinal efficacy of Toxoplasma gondii S48strain tested in an experimental trial in goats. Ann. MÇd. VÇt. 145, 202.
Chatfield, S.N., Charles, I.G., Makoff, A.J., Oxer, M.D., Dougan, G., Pickard, D.,Slater, D., Fairweather, N.F., 1992. Use of the nirB promoter to direct the stableexpression of heterologous antigens in Salmonella oral vaccine strains: devel-opment of a single-dose oral tetanus vaccine. Biotechnology (N.Y.) 10, 888.
Chen, G., Chen, H., Guo, H., Zheng, H., 2002. Protective effect of DNA-mediatedimmunization with a combination of SAG1 and IL-2 gene adjuvant againstinfection of Toxoplasma gondii in mice. Chin. Med. J. (Engl.) 115, 1448e1452.
Chen, H., Langer, R., 1998. Oral particulate delivery: status and future trends.Adv. Drug Deliv. Rev. 34, 339e350.
Chen, H., Chen, G., Zheng, H., Guo, H., 2003. Induction of immune responses inmice by vaccination with Liposome-entrapped DNA complexes encodingToxoplasma gondii SAG1 and ROP1 genes. Chin. Med. J. 116, 1561e1566.
Chen, G., Guo, H., Lu, F., Zheng, H., 2001. Construction of a recombinantplasmid harbouring the rhoptry protein 1 gene of Toxoplasma gondii andpreliminary observations on DNA immunity. Chin. Med. J. 114, 837e840.
Chen, R., Lu, S.H., Tong, Q.B., Lou, D., Shi, D.Y., Jia, B.B., Huang, G.P.,Wang, J.F., 2009. Protective effect of DNA-mediated immunization withliposome-encapsulated GRA4 against infection of Toxoplasma gondii. J.Zhejiang Univ. Sci. B. 7, 512e521.
Cong, H., Gu, Q.M., Yin, H.E., Wang, J.W., Zhao, Q.L., Zhou, H.Y., Li, Y.,Zhang, J.Q., 2008. Multi-epitope DNA vaccine linked to the A2/B subunit ofcholera toxin protect mice against Toxoplasma gondii. Vaccine 26 (31),3913e21.
REFERENCES 1039
Cong, H., Mui, E.J., Witola, W.H., Sidney, J., Alexander, J., Sette, A.,Maewal, A., El Bissati, K., Zhou, Y., Suzuki, Y., Lee, D., Woods, S.,Sommerville, C., Henriquez, F.L., Roberts, C.W., McLeod, R., 2012. Toxo-plasma gondii HLA-B*0702-restricted GRA7(20-28) peptide with adjuvantsand a universal helper T-cell epitope elicits CD8(+) T-cells producinginterferon-g and reduces parasite burden in HLA-B*0702 mice. Hum.Immunol. 73, 1e10.
Cong, H., Mui, E.J., Witola, W.H., Sidney, J., Alexander, J., Sette, A., Maewal, A.,McLeod, R., 2011. Towards an immunosense vaccine to prevent toxoplas-mosis: protective Toxoplasma gondii epitopes restricted by HLA-A*0201.Vaccine 29, 754e762.
Cong, H., Mui, E.J., Witola, W.H., Sidney, J., Alexander, J., Sette, A., Maewal, A.,Mcleod, R., 2010. Human immunome, bioinformatic analyses using HLAsupermotifs and the parasite genome, binding assays, studies of humanT-cell responses, and immunization of HLA-A*1101 transgenic miceincluding novel adjuvants provide a foundation for HLA-A03 restrictedCD8+T cell epitope based, adjuvanted vaccine protective against Toxoplasmagondii. Immunome. Res. 6, 12.
Cong,H.,Gu,Q.M., Jiang, Y.,He, S.Y., Zhou,H.Y.,Yang,T.T., Li, Y.,Zhao,Q.L., 2005.Oral immunization with a live recombinant attenuated Salmonella typhimuriumprotects mice against Toxoplasma gondii. Parasite Immunol. 27, 29e35.
Cook, G.C., 1990. Toxoplasma gondii infection: a potential danger to the unbornfoetus and AIDS sufferer. Q. J. Med. 74, 3e19.
Costa, G.H., Da Costa, A.J., Lopes, W.D., Bresciani, K.D., Dos Santos, T.R.,Esper, C.R., Santana, A.E., 2011. Toxoplasma gondii: infection naturalcongenital in cattle and an experimental inoculation of gestating cows withoocysts. Exp. Parasitol. 127, 277e281.
Couper, K.N., Nielsen, H.V., Petersen, E., Roberts, F., Roberts, C.W.,Alexander, J., 2003. DNA vaccination with the immunodominant tachyzoitesurface antigen (SAG-1) protects against adult acquired Toxoplasma gondiiinfection but does not prevent maternalefoetal transmission. Vaccine 21,2813e2820.
Couvreur, J., Thulliez, P., 1996. Acquired toxoplasmosis of ocular or neurologicsite: 49 cases. Presse. Med. 25, 438e442.
Cutchins, E.C., Warren, J., 1956. Immunity patterns in the guinea pig followingToxoplasma infection and vaccination with killed Toxoplasma. Am. J. Trop.Med. Hyg. 5, 197e209.
Cui, X., Lei, T., Yang, D., Hao, P., Li, B., Liu, Q., 2012. Toxoplasma gondii immunemapped protein-1 (TgIMP1) is a novel vaccine candidate against toxoplas-mosis. Vaccine 30, 2282e2287.
Cui, Y.L., He, S.Y., Xue, M.F., Zhang, J., Wang, H.X., Yao, Y., 2008. Protectiveeffect of a multiantigenic DNA vaccine against Toxoplasma gondii with co-delivery of IL-12 in mice. Parasite Immunol. 30, 309e313.
Cuppari, A.F., Sanchez, V., Ledesma, B., Frank, F.M., Goldman, A., Angel, S.O.,Martin, V., 2008. Toxoplasma gondii protease inhibitor-1 (TgPI-1) is a novelvaccine candidate against toxoplasmosis. Vaccine 26, 5040e5045.
Da Cunha, I.A., Zulpo, D.L., Bogado, A.L., De Barros, L.D., Taroda, A.,Igarashi, M., Navarro, I.T., Garcia, J.L., 2012. Humoral and cellular immuneresponses in pigs immunized intranasally with crude rhoptry proteins ofToxoplasma gondii plus QuIL-A. Vet. Parasitol. 186, 216e221.
Daryani, A., Hosseini, A.Z., Dalimi, A., 2003. Immune responses againstexcreted/secreted antigens of Toxoplasma gondii tachyzoites in the murinemodel. Vet Parasitol 113 (2), 123e134.
Dimier-Poisson, I., Aline, F., Bout, D., Mévélec, M.N., 2006. Induction ofprotective immunity against toxoplasmosis in mice by immunization withToxoplasma gondii RNA. Vaccine 24, 1705e1709.
Dziadek, B., Gatkowska, J., Grzybowski, M., Dziadek, J., Dzitko, K.,Dlugonska, H., 2012. Toxoplasma gondii: the vaccine potential of threetrivalent antigen-cocktails composed of recombinant ROP2, ROP4, GRA4and SAG1 proteins against chronic toxoplasmosis in BALB/c mice. Exp.Parasitol. 131, 133e138.
Dziadek, B., Gatkowska, J., Brzostek, A., Dziadek, J., Dzitko, K., Grzybowski, M.,Dlugonska, H., 2011. Evaluation of three recombinant multi-antigenicvaccines composed of surface and secretory antigens of Toxoplasma gondii inmurine models of experimental toxoplasmosis. Vaccine 29, 821e830.
Dziadek, B., Gatkowska, J., Brzostek, A., Dziadek, J., Dzitko, K., Dlugonska, H.,2009. Toxoplasma gondii: the immunogenic and protective efficacy of
recombinant ROP2 and ROP4 rhoptry proteins in murine experimentaltoxoplasmosis. Exp. Parasitol. 123, 81e89.
Dao, A., Fortier, B., Soete, M., Plenat, F., Dubremetz, J.F., 2001. Successful rein-fection of chronically infected mice by a different Toxoplasma gondii geno-type. Int. J. Parasitol. 31, 63e65.
Dautu, G., Munyaka, B., Carmen, G., Zhang, G., Omata, Y., Xuenan, X.,Igarashi, M., 2007. Toxoplasma gondii: DNA vaccination with genes encodingantigens MIC2, M2ap, AMA1 and BAG1 and evaluation of their immuno-genic potential. Exp. Parasitol. Jul. 116 (3), 273e282.
Darcy, F., Maes, P., Gras-Masse, H., Auriault, C., Bossus, M., Deslee, D.,Godard, I., Cesbron, M.F., Tartar, A., Capron, A., 1992. Protection of miceand nude rats against toxoplasmosis by a multiple antigenic peptideconstruction derived from Toxoplasma gondii P30 antigen. J. Immunol. 149,3636e3641.
Davies, P.R., Morrow, W.E., Deen, J., Gamble, H.R., Patton, S., 1998. Seropre-valence of Toxoplasma gondii and Trichinella spiralis in finishing swine raisedin different production systems in North Carolina, USA. Prev. Vet. Med.36, 67.
Debard, N., Buzoni-Gatel, D., Bout, D., 1996. Intranasal immunization withSAG1 protein of Toxoplasma gondii in association with cholera toxindramatically reduces development of cerebral cysts after oral infection.Infect. Immun. 64, 2158e2166.
Debrick, J.E., Campbell, P.A., Staerz, U.D., 1991. Macrophages as accessory cellsfor class I MHC-restricted immune responses. J. Immunol. 147, 2846e2851.
Denkers, E.Y., Gazzinelli, R.T., 1998. Regulation and function of T-cell-mediatedimmunity during Toxoplasma gondii infection. Clin. Microbiol. Rev. 11,569e588.
Denkers, E.Y., Yap, G., Scharton-Kersten, T., Charest, H., Butcher, B.A., Caspar, P.,Heiny, S., Sher, A., 1997. Perforin-mediated cytolysis plays a limited role inhost resistance to Toxoplasma gondii. J. Immunol. 159, 1903e1908.
Desmonts, G., Couvreur, J., 1974. Toxoplasmosis in pregnancy and its trans-mission to the foetus. Bull. N. Y. Acad. Med. 50, 146e159.
Desolme, B., Mevelec, M.N., Buzoni-Gatel, D., Bout, D., 2000. Induction ofprotective immunity against toxoplasmosis in mice by DNA immunizationwith a plasmid encoding Toxoplasma gondii GRA4 gene. Vaccine 18,2512e2521.
Dimier-Poisson, I., Aline, F., Bout, D., Mevelec, M.N., 2006. Induction ofprotective immunity against toxoplasmosis in mice by immunization withToxoplasma gondii RNA. Vaccine 18, 1705e1709.
Drabner, B., Guzman, C.A., 2001. Elicitation of predictable immune responses byusing live bacterial vectors. Biomol. Eng. 17, 75.
Dubey, J.P., 1986. A review of toxoplasmosis in pigs. Vet. Parasitol. 19, 181.Dubey, J.P., 2009. Toxoplasmosis in pigs e The last 20 years. Vet. Parasitol.
164, 89.Dubey, J.P., 1990a. Status of toxoplasmosis in cattle in the United States. J. Am.
Vet. Med. Assoc. 196, 257.Dubey, J.P., 1995. Duration of immunity to shedding of Toxoplasma gondii oocysts
by cats. J. Parasitol. 81, 410.Dubey, J.P., 1996. Strategies to reduce transmission of Toxoplasma gondii to
animals and humans. Vet. Parasitol. 64, 65.Dubey, J.P., 1998. Advances in the life cycle of Toxoplasma gondii. Int. J. Parasitol.
of immunity to toxoplasmosis in pigs vaccinated with a nonpersistent strainof Toxoplasma gondii. Am. J. Vet. Res. 55, 982.
Dubey, J.P., Frenkel, J.K., 1972. Cyst-induced toxoplasmosis in cats. J. Protozool.19, 155.
Dubey, J.P., Lindsay, D.S., Saville, W.J., Reed, S.M., Granstrom, D.E., Speer, C.A.,2001. A review of Sarcocystis neurona and equine protozoal myeloencepha-litis (EPM). Vet. Parasitol. 95, 89.
Dubey, J.P., Ruff, M.D., Camargo, M.E., Shen, S.K., Wilkins, G.L., Kwok, O.C.,Thulliez, P., 1993. Serologic and parasitologic responses of domesticchickens after oral inoculation with Toxoplasma gondii oocysts. Am. J. Vet.Res. 54, 1668.
26. VACCINATION AGAINST TOXOPLASMOSIS: CURRENT STATUS AND FUTURE PROSPECTS1040
Dubey, J.P., Saville, W.J., Stanek, J.F., Reed, S.M., 2002. Prevalence of Toxoplasmagondii antibodies in domestic cats from rural Ohio. J. Parasitol. 88, 802.
Dubey, J.P., Urban Jr., J.F., 1990. Diagnosis of transplacentally induced toxo-plasmosis in pigs. Am. J. Vet. Res. 51, 1295e1299.
Dubey, J.P., Hill, D.E., Rozeboom, D.W., Rajendran, C., Choudhary, S.,Ferreira, L.R., Kwok, O.C., Su, C., 2012. High prevalence and genotypes ofToxoplasma gondii isolated from organic pigs in northern USA. Vet. Para-sitol. 2012 (188), 14e28.
Duquesne, V., Auriault, C., Darcy, F., Decavel, J.P., Capron, A., 1990. Protectionof nude rats against Toxoplasma infection by excreted-secreted antigen-specific helper T-cells. Infect. Immun. 58, 2120e2126.
Duquesne, V., Auriault, C., Gras-Masse, H., Boutillon, C., Darcy, F., Cesbron-Delauw, M.F., Tartar, A., Capron, A., 1991. Identification of T-cell epitopeswithin a 23-kD antigen (P24) of Toxoplasma gondii. Clin. Exp. Immunol. 84,527e534.
Dzierszinski, F., Mortuaire, M., Cesbron-Delauw, M.F., Tomavo, S., 2000. Tar-geted disruption of the glycosylphosphatidylinositol-anchored surfaceantigen SAG3 gene in Toxoplasma gondii decreases host cell adhesion anddrastically reduces virulence in mice. Mol. Microbiol. 37, 574.
Echeverria, P.C., DeMiguel,N., Costas,M., Angel, S.O., 2006. Potent antigen-specificimmunity to Toxoplasma gondii in adjuvant-free vaccination system using Rop2-Leishmania infantumHsp83 fusion protein. Vaccine 24, 4102e4110.
Eissa, M., El-Azzouni, M.Z., Mady, R.F., Fathy, F.M., Baddour, N.M., 2012. Initialcharacterization of an autoclaved Toxoplasma vaccine in mice. Exp. Parasitol.131, 310e316.
El-Malky, M., Shaohong, L., Kumagai, T., Yabu, Y., Noureldin, M.S., Saudy, N.,Maruyama, H., Ohta, N., 2005. Protective effect of vaccination with Toxo-plasma lysate antigen and CpG as an adjuvant against Toxoplasma gondii insusceptible C57BL/6 mice. Microbiol. Immunol. 49, 639e646.
Elsaid, M.M., Vitor, R.W., Frézard, F.J., Martins, M.S., 1999. Protection againsttoxoplasmosis in mice immunized with different antigens of Toxoplasmagondii incorporated into liposomes. Mem. Inst. Oswaldo Cruz. 94, 485e490.
Elsaid, M.M., Martins, M.S., Frezard, F., Braga, E.M., Vitor, R.W., 2001. Verticaltoxoplasmosis in a murine model. Protection after immunization withantigens of Toxoplasma gondii incorporated into liposomes. Mem. Inst.Oswaldo Cruz. 96, 99.
Fachado, A., Rodriguez, A., Angel, S.O., Pinto, D.C., Vila, I., Acosta, A.,Amendoeira, R.R., Lannes-Vieira, J., 2003. Protective effect of a naked DNAvaccine cocktail against lethal toxoplasmosis in mice. Vaccine 21, 1327e1335.
Fachado, A., Rodriguez, A., Molina, J., Silverio, J.C., Marino, A.P., Pinto, L.M.,Angel, S.O., Infante, J.F., Traub-Cseko, Y., Amendoeira, R.R., Lannes-Vieira, J., 2003. Long-term protective immune response elicited by vaccina-tion with an expression genomic library of Toxoplasma gondii. Infect. Immun.71, 5407e5411.
Fachado, A., Rodriguez, A., Angel, S.O., Pinto, D.C., Vila, I., Acosta, A.,Amendoeira, R.R., Lannes-Vieira, J., 2003b. Protective effect of a naked DNAvaccine cocktail against lethal toxoplasmosis in mice. Vaccine 21, 1327e1333.
Fang, R., Feng, H., Hu, M., Khan, M.K., Wang, L., Zhou, Y., Zhao, J., 2012.Evaluation of immune responses induced by SAG1 and MIC3 vaccinecocktails against Toxoplasma gondii. Vet. Parasitol. 187, 140e146.
Fang, R., Feng, H., Nie, H., Wang, L., Tu, P., Song, Q., Zhou, Y., Zhao, J., 2010.Construction and immunogenicity of pseudotype baculovirus expressingToxoplasma gondii SAG1 protein in BALB/c mice model. Vaccine 28,1803e1807.
Fang, R., Nie, H., Wang, Z., Tu, P., Zhou, D., Wang, L., He, L., Zhou, Y., Zhao, J.,2009. Protective immune response in BALB/c mice induced by a suicidalDNA vaccine of the MIC3 gene of Toxoplasma gondii. Vet. Parasitol. 164,134e140.
Fermin, Z., Bout, D., Ricciardi-Castagnoli, P., Hoebeke, J., 1999. Salbutamol as anadjuvant for nasal vaccination. Vaccine 17, 1936e1941.
Fox, B.A., Bzik, D.J., 2002. De novo pyrimidine biosynthesis is required forvirulence of Toxoplasma gondii. Nature 415, 926.
Frenkel, J.K., 1990. Transmission of toxoplasmosis and the role of immunity inlimiting transmission and illness. J. Am. Vet. Med. Assoc. 196, 233.
Frenkel, J.K., Pfefferkorn, E.R., Smith, D.D., Fishback, J.L., 1991. Prospectivevaccine prepared from a new mutant of Toxoplasma gondii for use in cats.Am. J. Vet. Res. 52, 759.
Freyre, A., Choromanski, L., Fishback, J.L., Popiel, I., 1993. Immunization of catswith tissue cysts, bradyzoites, and tachyzoites of the T-263 strain of Toxo-plasma gondii. J. Parasitol. 79, 716.
Freyre, A., Falcon, J., Mendez, J., Rodriguez, A., Correa, L., Gonzalez, M., 2006.Toxoplasma gondii: partial cross-protection among several strains of theparasite against congenital transmission in a rat model. Exp. Parasitol. 112,8e12.
Freyre, A., Araujo, F.A., Fialho, C.G., Bigatti, L.E., Falcón, J.D., 2012. Protection ina hamster model of congenital toxoplasmosis. Vet. Parasitol. 183, 359e363.
Gallichan, W.S., Rosenthal, K.L., 1996. Long-lived cytotoxic T lymphocytememory in mucosal tissues after mucosal but not systemic immunization.J. Exp. Med. 184, 1879e1890.
Garcia, J.L., Navarro, I.T., Biazzono, L., Freire, R.L., Da Silva Guimarães Junior, J.,Cryssafidis, A.L., Bugni, F.M., Da Cunha, I.A., Hamada, F.N., Dias, R.C.,2007. Protective activity against oocyst shedding in cats vaccinated withcrude rhoptry proteins of the Toxoplasma gondii by the intranasal route. Vet.Parasitol. 145, 197e206.
Gatkowska, J., Gasior, A., Kur, J., Dlugonska, H., 2008. Toxoplasma gondii:chimeric Dr fimbriae as a recombinant vaccine against toxoplasmosis. Exp.Parasitol. 118, 266e270.
Gazzinelli, R.T., Caetano, B., Fux, B., Garcia, M., Machado, A., Ferreira, A.M.,Vitor, R.W.A., Melo, M.N., Bruna-Romero, O., 2005. Protective immuneresponses involved in host resistance to Brazilian isolates of Toxoplasmagondii: implications on vaccine development employing recombinant viralvectors. Proceedings of the IXth International Coccidiosis Conference,84e90.
Gilbert, R., Gras, L., 2003. Effect of timing and type of treatment on the risk ofmother to child transmission of Toxoplasma gondii. Bjog 110, 112e120.
Golkar, M., Shokrgozar, M.A., Rafati, S., Musset, K., Assmar, M., Sadaie, R.,Cesbron-Delauw, M.F., Mercier, C., 2007. Evaluation of protective effect ofrecombinant dense granule antigens GRA2 and GRA6 formulated inmonophosphoryl lipid A (MPL) adjuvant against Toxoplasma chronic infec-tion in mice. Vaccine 25, 4301e4311.
Graham, F.L., Smiley, J., Russell, W.C., Nairn, R., 1977. Characteristics ofa human cell line transformed by DNA from human adenovirus type 5.J. Gen. Virol. 36, 59.
Gregoriadis, G., Bacon, A., Caparros-Wanderley, W., McCormack, B., 2002. Arole for liposomes in genetic vaccination. Vaccine 20 (Suppl. 5), B1eB9.
Grigg, M.E., Ganatra, J., Boothroyd, J.C., Margolis, T.P., 2001. Unusual abun-dance of atypical strains associated with human ocular toxoplasmosis.J. Infect. Dis. 184, 633e639.
Hartley, W.J., 1966. Some investigations into the epidemiology of ovine toxo-plasmosis. N. Z. Vet. J. 14, 106e107.
Haumont, M., Delhaye, L., Garcia, L., Jurado, M., Mazzu, P., Daminet, V.,Verlant, V., Bollen, A., Biemans, R., Jacquet, A., 2000. Protective immunityagainst congenital toxoplasmosis with recombinant SAG1 protein ina guinea pig model. Infect. Immun. 68, 4948e4953.
Hedhli, D., Dimier-Poisson, I., Judge, J.W., Rosenberg, B., Mévélec, M.N., 2009.Protective immunity against Toxoplasma challenge in mice by coadminis-tration of T. gondii antigens and Eimeria profilin-like protein as an adjuvant.Vaccine 27, 2274e2281.
Henriksen, P., Dietz, H.H., Henriksen, S.A., 1994. Fatal toxoplasmosis in five cats.Vet. Parasitol. 55, 15.
Hill, D.E., Chirukandoth, S., Dubey, J.P., 2005. Biology and epidemiology ofToxoplasma gondii in man and animals. Anim. Health Res. Rev. 6, 41.
Hill, D.E., Dubey, J.P., 2012. Toxoplasma gondii prevalence in farm animals in theUnited States. Int. J. Parasitol. (in press).
REFERENCES 1041
Hiramoto, R.M., Galisteo, A.J., Do, N.N., De Jr., A.H.F., 2002. 200 Gy sterilisedToxoplasma gondii tachyzoites maintain metabolic functions and mammaliancell invasion, eliciting cellular immunity and cytokine response similar tonatural infection in mice. Vaccine 20, 2072.
Hiszczy�nska-Sawicka, E., Li, H., Boy, U., Xu, J., Akhtar, M., Holec-Gasior, L.,Kur, J., Bickerstaffe, R., Stankiewicz, M., 2012. Induction of immuneresponses in sheep by vaccination with liposome-entrapped DNAcomplexes encoding Toxoplasma gondii MIC3 gene. Pol. J. Vet. Sci. 15, 3e9.
Hiszczy�nska-Sawicka, E., Oledzka, G., Holec-Gasior, L., Li, H., Xu, J.B.,Sedcole, R., Kur, J., Bickerstaffe, R., Stankiewicz, M., 2011a. Evaluation ofimmune responses in sheep induced by DNA immunization with genesencoding GRA1, GRA4, GRA6 and GRA7 antigens of Toxoplasma gondii. Vet.Parasitol. 177, 281e289.
Hiszczy�nska-Sawicka, E., Li, H., Xu, J.B., Holec-Gasior, L., Kur, J., Sedcole, R.,Bickerstaffe, R., Stankiewicz, M., 2011b. Modulation of immune response toToxoplasma gondii in sheep by immunization with a DNA vaccine encodingROP1 antigen as a fusion protein with ovine CD154. Vet. Parasitol.183, 72e78.
Hiszczy�nska-Sawicka, E., Akhtar, M., Kay, G.W., Holec-Gasior, L.,Bickerstaffe, R., Kur, J., Stankiewicz, M., 2010a. The immune responses ofsheep after DNA immunization with, Toxoplasma gondii MAG1 antigen-withand without co-expression of ovine interleukin 6. Vet. Immunol. Immuno-pathol. 136, 324e329.
Hiszczy�nska-Sawicka, E., Li, H., Xu, J.B., Oledzka, G., Kur, J., Bickerstaffe, R.,Stankiewicz, M., 2010b. Comparison of immune response in sheep immu-nized with DNA vaccine encoding Toxoplasma gondii GRA7 antigen indifferent adjuvant formulations. Exp. Parasitol. 124, 365e372.
Hoffmann, S., Batz, M.B., Morris Jr., J.G., 2012. Annual cost of illness and quality-adjusted life year losses in the United States due to 14 foodborne pathogens.J. Food Prot. 75, 1292e1302.
Hoseinian-Khosroshahi, K., Ghaffarifar, F., D'souza, S., Sharifi, Z., Dalimi, A.,2011. Evaluation of the immune response induced by DNA vaccine cocktailexpressing complete SAG1 and ROP2 genes against toxoplasmosis. Vaccine29, 778e783.
Huang, X., Li, J., Zhang, G., Gong, P., Yang, J., Zhang, X., 2012. Toxoplasma gondii:protective immunity against toxoplasmosis with recombinant actin depo-lymerizing factor protein in BALB/c mice. Exp. Parasitol. 130, 218e222.
Hutson, S.L., Mui, E., Kinsley, K., Witola, W.H., Behnke, M.S., El Bissati, K.,Muench, S.P., Rohrman, B., Liu, S.R., Wollmann, R., Ogata, Y., Sarkeshik, A.,Yates Jr, 3rd, McLeod, R., 2010. T. gondii RP promoters and knockdownreveal molecular pathways associated with proliferation and cell-cyclearrest. PLoS One 5, e14057.
Igarashi, M., Zulpo, D.L., Cunha, I.A., Barros, L.D., Pereira, V.F., Taroda, A.,Navarro, I.T., Vidotto, O., Vidotto, M.C., Jenkins, M.C., Garcia, J.L., 2010.Toxoplasma gondii: humoral and cellular immune response of BALB/c miceimmunized via intranasal route with rTgROP2. Rev. Bras. Parasitol. Vet. 19,210e216.
Igarashi, M., Kano, F., Tamekuni, K., Machado, R.Z., Navarro, I.T., Vidotto, O.,Vidotto, M.C., Garcia, J.L., 2008. Toxoplasma gondii: evaluation of an intra-nasal vaccine using recombinant proteins against brain cyst formation inBALB/c mice. Exp. Parasitol. 118, 386e392.
Ismael, A.B., Sekkai, D., Collin, C., Bout, D., Mevelec, M.N., 2003. The MIC3 geneof Toxoplasma gondii is a novel potent vaccine candidate against toxoplas-mosis. Infect. Immun. 71, 6222e6228.
Ismael, A.B., Hedhli, D., Cérède, O., Lebrun, M., Dimier-Poisson, I.,Mévélec, M.N., 2009. Further analysis of protection induced by the MIC3DNA vaccine against T. gondii: CD4 and CD8 T-cells are the major effectorsof the MIC3 DNA vaccine-induced protection, both Lectin-like and EGF-likedomains of MIC3 conferred protection. Vaccine 27, 2959e2966.
Ismael, A.B., Dimier-Poisson, I., Lebrun, M., Dubremetz, J.F., Bout, D.,Mevelec, M.N., 2006. Mic1-3 knock-out of Toxoplasma gondii is a successfulvaccine against chronic and congenital toxoplasmosis in mice. J. Infect. Dis.194, 1176e1183.
Jacobs, L., 1956. Propogation, morphology and biology of Toxoplasma. Annal ofthe New York Academy of Science 64, 154e159.
Johnson, J.J., Roberts, C.W., Pope, C., Roberts, F., Kirisits, M.J., Estes, R., Mui, E.,Krieger, T., Brown, C.R., Forman, J., McLeod, R., 2002. In vitro correlates of
Ld-restricted resistance to toxoplasmic encephalitis and their criticaldependence on parasite strain. J. Immunol. 169, 966e973.
Jongert, E., Verhelst, D., Abady, M., Petersen, E., Gargano, N., 2008a. ProtectiveTh1 immune responses against chronic toxoplasmosis induced by a protein-protein vaccine combination but not by its DNA-protein counterpart.Vaccine 26, 5289e5295.
Jongert, E., Melkebeek, V., De Craeye, S., Dewit, J., Verhelst, D., Cox, E., 2008b.An enhanced GRA1-GRA7 cocktail DNA vaccine primes anti-Toxoplasmaimmune responses in pigs. Vaccine 26, 1025e1031.
Jongert, E., De Craeye, S., Dewit, J., Huygen, K., 2007. GRA7 provides protectiveimmunity in cocktail DNA vaccines against Toxoplasma gondii. ParasiteImmunol. 29, 445e453.
Kang, H.K., Lee, H.Y., Lee, Y.N., Jo, E.J., Kim, J.I., Aosai, F., Yano, A., Kwak, J.Y.,Bae, Y.S., 2004. Toxoplasma gondii-derived heat shock protein 70 stimulatesthe maturation of human monocyte-derived dendritic cells. Biochem. Bio-phys. Res. Commun. 322, 899e904.
Kasper, L.H., 1989. Identification of stage-specific antigens of Toxoplasma gondii.Infect. Immun. 57, 668e672.
Kasper, L.H., Currie, K.M., Bradley, M.S., 1985. An unexpected response tovaccination with a purified major membrane tachyzoite antigen (P30) ofToxoplasma gondii. J. Immunol. 134, 3426e3431.
Kasper, L.H., Khan, I.A., 1993. Role of P30 in host immunity and pathogenesis ofT. gondii infection. Res. Immunol. 144, 45.
Khan, I.A., Ely, K.H., Kasper, L.H., 1991. A purified parasite antigen (p30)mediates CD8+ T-cell immunity against fatal Toxoplasma gondii infection inmice. J. Immunol. 147, 3501.
Khosroshahi, K.H., Ghaffarifar, F., Sharifi, Z., D’Souza, S., Dalimi, A.,Hassan, Z.M., Khoshzaban, F., 2012. Comparing the effect of IL-12 geneticadjuvant and alum non-genetic adjuvant on the efficiency of the cocktailDNA vaccine containing plasmids encoding SAG-1 and ROP-2 of Toxo-plasma gondii. Parasitol. Res. 111, 403e411.
Kijlstra, A., Eissen, O.A., Cornelissen, J., Munniksma, K., Eijck, I., Kortbeek, T.,2004. Toxoplasma gondii infection in animal-friendly pig production systems.Invest Ophthalmol. Vis. Sci. 45, 3165.
Kim, S.K., Boothroyd, J.C., 2005. Stage-specific expression of surface antigens byToxoplasma gondii as a mechanism to facilitate parasite persistence. J.Immunol. 174, 8038e8048.
Kikumura, A., Fang, H., Mun, H.S., Uemura, N., Makino, M., Sayama, Y.,Norose, K., Aosai, F., 2010. Protective immunity against lethal anaphylacticreaction in Toxoplasma gondii-infected mice by DNA vaccination with T.gondii-derived heat shock protein 70 gene. Parasitol. Int. 59, 105e111.
Kodjikian, L., Hoigne, I., Adam, O., Jacquier, P., Aebi-Ochsner, C., Aebi, C.,Garweg, J.G., 2004. Vertical transmission of toxoplasmosis from a chroni-cally infected immunocompetent woman. Pediatr. Infect. Dis. J. 23, 272e274.
Koppe, J.G., Loewer-Sieger, D.H., De Roever-Bonnet, H., 1986. Results of 20-yearfollow-up of congenital toxoplasmosis. Lancet 1, 254e256.
Koyama, T., Shimada, S., Ohsawa, T., Omata, Y., Xuan, X., Inoue, N., Mikami, T.,Saito, A., 2000. Antigens expressed in feline enteroepithelial-stages parasitesof Toxoplasma gondii. J. Vet. Med. Sci. 62, 1089.
Krahenbuhl, J.L., Ruskin, J., Remington, J.S., 1972. The use of killed vaccines inimmunization against an intracellular parasite: Toxoplasma gondii. J. Immu-nol. 108, 425.
Krishnan, L., Guilbert, L.J., Russell, A.S., Wegmann, T.G., Mosmann, T.R.,Belosevic, M., 1996a. Pregnancy impairs resistance of C57BL/6 mice toLeishmania major infection and causes decreased antigen-specific IFNgammaresponse and increased production of T helper 2 cytokines. J. Immunol. 156,644e652.
Krishnan, L., Guilbert, L.J., Wegmann, T.G., Belosevic, M., Mosmann, T.R.,1996b. T helper 1 response against Leishmania major in pregnant C57BL/6mice increases implantation failure and foetal resorptions. Correlation withincreased IFNgamma and TNF and reduced IL-10 production by placentalcells. J. Immunol. 156, 653e662.
Kyprianou, M., 2005. Speech to the Animal Welfare Intergroup of the EuropeanParliament. SPEECH/05/335. European Commission.
26. VACCINATION AGAINST TOXOPLASMOSIS: CURRENT STATUS AND FUTURE PROSPECTS1042
Laguía-Becher, M., Martín, V., Kraemer, M., Corigliano, M., Yacono, M.L.,Goldman, A., Clemente, M., 2010. Effect of codon optimization andsubcellular targeting on Toxoplasma gondii antigen SAG1 expression intobacco leaves to use in subcutaneous and oral immunization in mice. BMCBiotechnol. 10, 52.
Lau, Y.L., Thiruvengadam, G., Lee, W.W., Fong, M.Y., 2011. Immunogeniccharacterization of the chimeric surface antigen 1 and 2 (SAG1/2) of Toxo-plasma gondii expressed in the yeast Pichia pastoris. Parasitol. Res. 109,871e878.
Letscher-Bru, V., Pfaff, A.W., Abou-Bacar, A., Filisetti, D., Antoni, E., Villard, O.,Klein, J.P., Candolfi, E., 2003. Vaccination with Toxoplasma gondii SAG-1protein is protective against congenital toxoplasmosis in BALB/c mice butnot in CBA/J mice. Infect. Immun. 71, 6615e6619.
Leyva, R., Herion, P., Saavedra, R., 2001. Genetic immunization with plasmidDNA coding for the ROP2 protein of Toxoplasma gondii. Parasitol. Res. 87,70e79.
Li, B., Oledzka, G., McFarlane, R.G., Spellerberg, M.B., Smith, S.M., Gelder, F.B.,Kur, J., Stankiewicz, M., 2010. Immunological response of sheep to injectionsof plasmids encoding Toxoplasma gondii SAG1 and ROP1 genes. ParasiteImmunol. 32, 671e683.
Li, J., Han, Q., Gong, P., Yang, T., Ren, B., Li, S., Zhang, X., 2012. Toxoplasmagondii rhomboid protein 1 (TgROM1) is a potential vaccine candidate againsttoxoplasmosis. Vet. Parasitol. 184, 154e160.
Li, W.S., Chen, Q.X., Ye, J.X., Xie, Z.X., Chen, J., Zhang, L.F., 2011. Comparativeevaluation of immunization with recombinant protein and plasmid DNAvaccines of fusion antigen ROP2 and SAG1 from Toxoplasma gondii in mice:cellular and humoral immune responses. Parasitol. Res. 109, 637e644.
Lindsay, D.S., Blagburn, B.L., Dubey, J.P., 1993. Safety and results of challenge ofweaned pigs given a temperature-sensitive mutant of Toxoplasma gondii. J.Parasitol. 79, 71e76.
Luft, B.J., Remington, J.S., 1982. Effect of pregnancy on resistance to Listeriamonocytogenes and Toxoplasma gondii infections in mice. Infect. Immun. 38,1164e1171.
Lunden, A., Lovgren, K., Uggla, A., Araujo, F.G., 1993. Immune responses andresistance to Toxoplasma gondii in mice immunized with antigens of theparasite incorporated into immunostimulating complexes. Infect. Immun.61, 2639e2643.
Lunden, A., Parmley, S.F., Bengtsson, K.L., Araujo, F.G., 1997. Use of a recombi-nant antigen, SAG2, expressed as a glutathione-S-transferase fusion protein toimmunize mice against Toxoplasma gondii. Parasitol. Res. 83, 6e9.
Mann, M., Hendrickson, R.C., Pandey, A., 2001. Analysis of proteins and pro-teomes by mass spectrometry. Annu. Rev. Biochem. 70, 437e473.
Makino, M., Uemura, N., Moroda, M., Kikumura, A., Piao, L.X., Mohamed, R.M.,Aosai, F., 2011. Innate immunity in DNA vaccine with Toxoplasma gondii-heat shock protein 70 gene that induces DC activation and Th1 polarization.Vaccine 29, 1899e1905.
Mateus-Pinilla, N.E., Dubey, J.P., Choromanski, L., Weigel, R.M., 1999. A fieldtrial of the effectiveness of a feline Toxoplasma gondii vaccine in reducing T.gondii exposure for swine. J. Parasitol. 85, 855.
Martin, V., Supanitsky, A., Echeverria, P.C., Litwin, S., Tanos, T., De Roodt, A.R.,Guarnera, E.A., Angel, S.O., 2004. Recombinant GRA4 or ROP2 proteincombined with alum or the gra4 gene provides partial protection in chronicmurine models of toxoplasmosis. Clin. Diagn. Lab. Immunol. 11, 704e710.
Martínez-Gómez, F., García-González, L.F., Mondragón-Flores, R., Bautista-Garfias, C.R., 2009. Protection against Toxoplasma gondii brain cyst formationin mice immunized with Toxoplasma gondii cytoskeleton proteins andLactobacillus casei as adjuvant. Vet. Parasitol. 160, 311e315.
McColgan, C., Buxton, D., Blewett, D.A., 1988. Titration of Toxoplasma gondiioocysts in non-pregnant sheep and the effects of subsequent challengeduring pregnancy. Vet. Rec. 123, 467.
McKeand, J.B., 2000. Vaccine development and diagnostics of Dictyocaulusviviparus. Parasitology 120 (Suppl), S17eS23.
McLeod, R., Estes, R.G., Mack, D.G., 1985. Effects of adjuvants and Toxoplasmagondii antigens on immune response and outcome of peroral T. gondiichallenge. Trans. R. Soc. Trop. Med. Hyg. 79, 800e804.
McLeod, R., Frenkel, J.K., Estes, R.G., Mack, D.G., Eisenhauer, P.B., Gibori, G.,1988. Subcutaneous and intestinal vaccination with tachyzoites ofToxoplasma gondii and acquisition of immunity to peroral and congenitaltoxoplasma challenge. J. Immunol. 140, 1632.
Mendes, E.A., Caetano, B.C., Penido, M.L., Bruna-Romero, O., Gazzinelli, R.T.,2011. MyD88-dependent protective immunity elicited by adenovirus 5expressing the surface antigen 1 from Toxoplasma gondii is mediated byCD8(+) T lymphocytes. Vaccine 29, 4476e4484.
Mévélec, M.N., Bout, D., Desolme, B., Marchand, H., Magné, R., Bruneel, O.,Buzoni-Gatel, D., 2005. Evaluation of protective effect of DNA vaccinationwith genes encoding antigens GRA4 and SAG1 associated with GM-CSFplasmid, against acute, chronical and congenital toxoplasmosis in mice.Vaccine 23, 4489e4499.
Mévélec, M.N., Ducournau, C., Bassuny Ismael, A., Olivier, M., Sèche, E.,Lebrun, M., Bout, D., Dimier-Poisson, I., 2010. Mic1-3 Knock-out Toxo-plasma gondii is a good candidate for a vaccine against T. gondii-inducedabortion in sheep. Vet. Res. 41 (4), 49.
, M.N., Buzoni-Gatel, D., Bourguin, I., Chardes, T., Dubremetz, J.F., Bout, D.,1998. Mapping of B epitopes in GRA4, a dense granule antigen of Toxoplasmagondii and protection studies using recombinant proteins administered bythe oral route. Parasite Immunol. 20, 183e195.
Meissner, M., Brecht, S., Bujard, H., Soldati, D., 2001. Modulation of myosin Aexpression by a newly established tetracycline repressor-based induciblesystem in Toxoplasma gondii. Nucleic. Acids Res. 29, E115.
Meissner, M., Reiss, M., Viebig, N., Carruthers, V.B., Toursel, C., Tomavo, S.,Ajioka, J.W., Soldati, D., 2002a. A family of transmembrane micronemeproteins of Toxoplasma gondii contain EGF-like domains and function asescorters. J. Cell Sci. 115, 563e574.
Meissner, M., Schluter, D., Soldati, D., 2002b. Role of Toxoplasma gondii myosin Ain powering parasite gliding and host cell invasion. Science 298, 837.
Mercier, C., Howe, D.K., Mordue, D., Lingnau, M., Sibley, L.D., 1998. Targeteddisruption of the GRA2 locus in Toxoplasma gondii decreases acute virulencein mice. Infect. Immun. 66, 4176.
Min, J., Qu, D., Li, C., Song, X., Zhao, Q., Li, X.A., Yang, Y., Liu, Q., He, S.,Zhou, H., 2012. Enhancement of protective immune responses induced byToxoplasma gondii dense granule antigen 7 (GRA7) against toxoplasmosis inmice using a prime-boost vaccination strategy. Vaccine 30, 5631e5636.
REFERENCES 1043
Mineo, J.R., Kasper, L.H., 1994. Attachment of Toxoplasma gondii to host cellsinvolves major surface protein, SAG-1 (P30). Exp. Parasitol. 79, 11.
Mineo, J.R., McLeod, R., Mack, D., Smith, J., Khan, I.A., Ely, K.H., Kasper, L.H.,1993. Antibodies to Toxoplasma gondii major surface protein (SAG-1, P30)inhibit infection of host cells and are produced in murine intestine afterperoral infection. J. Immunol. 150, 3951e3964.
Mishima, M., Xuan, X., Yokoyama, N., Igarashi, I., Fujisaki, K., Nagasawa, H.,Mikami, T., 2002. Recombinant feline herpesvirus type 1 expressing Toxo-plasma gondi ROP2 antigen inducible protective immunity in cats. Parasitol.Res. 88, 144.
Mishima, M., Xuan, X., Shioda, A., Omata, Y., Fujisaki, K., Nagasawa, H.,Mikami, T., 2001. Modified protection against Toxoplasma gondii lethalinfection and brain cyst formation by vaccination with SAG2 and SRS1.J. Vet. Med. Sci. 63, 433e438.
Mital, J., Meissner, M., Soldati, D., Ward, G.E., 2005. Conditional expression ofToxoplasma gondii apical membrane antigen-1 (TgAMA1) demonstratesthat TgAMA1 plays a critical role in host cell invasion. Mol. Biol. Cell 16,4341.
Mohamed, R.M., Aosai, F., Chen, M., Mun, H.S., Norose, K., Belal, U.S., Piao, L.X.,Yano, A., 2003. Induction of protective immunity by DNA vaccination withToxoplasma gondii HSP70, HSP30 and SAG1 genes. Vaccine 21,2852e2861.
Moiré, N., Dion, S., Lebrun, M., Dubremetz, J.F., Dimier-Poisson, I., 2009. Mic1-3KO tachyzoite a live attenuated vaccine candidate against toxoplasmosisderived from a type I strain shows features of type II strain. Exp. Parasitol.23, 111e117.
Mun, H.S., Aosai, F., Yano, A., 1999. Role of Toxoplasma gondii HSP70 andToxoplasma gondii HSP30/bag1 in antibody formation and prophylacticimmunity in mice experimentally infected with Toxoplasma gondii. Microbiol.Immunol. 43, 471e479.
Munday, B.L., 1972. Transmission of Toxoplasma infection from chronicallyinfected ewes to their lambs. Br. Vet. J. 128, lxxi.
Nie, H., Fang, R., Xiong, B.Q., Wang, L.X., Hu, M., Zhou, Y.Q., Zhao, J.L., 2011.Immunogenicity and protective efficacy of two recombinant pseudorabiesviruses expressing Toxoplasma gondii SAG1 and MIC3 proteins. Vet. Para-sitol. 2011 (181), 215e221.
Nielsen, H.V., Di Cristina, M., Beghetto, E., Spadoni, A., Petersen, E.,Gargano, N., 2006. Toxoplasma gondii: DNA vaccination with bradyzoiteantigens induces protective immunity in mice against oral infection withparasite cysts. Exp. Parasitol. 12, 274e279.
Nielsen, H.V., Lauemoller, S.L., Christiansen, L., Buus, S., Fomsgaard, A.,Petersen, E., 1999. Complete protection against lethal Toxoplasma gondiiinfection in mice immunized with a plasmid encoding the SAG1 gene. Infect.Immun. 67, 6358e6363.
Opsteegh, M., Prickaerts, S., Frankena, K., Evers, E.G., 2011. A quantitativemicrobial risk assessment for meatborne Toxoplasma gondii infection in TheNetherlands. Int. J. Food Microbiol. 150, 103e114.
Overnes, G., Nesse, L.L., Waldeland, H., Lovgren, K., Gudding, R., 1991.Immune response after immunization with an experimental Toxoplasmagondii ISCOM vaccine. Vaccine 9, 25e28.
Paoletti, E., 1996. Applications of pox virus vectors to vaccination: an update.Proc. Natl. Acad. Sci. U.S.A. 93, 11349.
Parmley, S., Slifer, T., Araujo, F., 2002. Protective effects of immunization witha recombinant cyst antigen in mouse models of infection with Toxoplasmagondii tissue cysts. J. Infect. Dis. 185 (Suppl. 1), S90eS95.
He, Y., Lin, R.Q., Zhu, X.Q., 2009. Toxoplasma gondii microneme protein 6(MIC6) is a potential vaccine candidate against toxoplasmosis in mice.Vaccine 27, 6570e6574.
Pereira-Bueno, J., Quintanilla-Gozalo, A., Perez-Perez, V., Alvarez-Garcia, G.,Collantes-Fernandez, E., Ortega-Mora, L.M., 2004. Evaluation of ovineabortion associated with Toxoplasma gondii in Spain by different diagnostictechniques. Vet. Parasitol. 121, 33.
Perkins, E.S., 1973. Ocular toxoplasmosis. Br. J. Ophthalmol. 57, 1e17.
Petersen, E., Nielsen, H.V., Christiansen, L., Spenter, J., 1998. Immunization withE. coli produced recombinant T. gondii SAG1 with alum as adjuvant protectmice against lethal infection with Toxoplasma gondii. Vaccine 16,1283e1289.
Petersen, E., Pollak, A., Reiter-Owona, I., 2001. Recent trends in research oncongenital toxoplasmosis. Int. J. Parasitol. 31, 115e144.
Pinckney, R.D., Lindsay, D.S., Blagburn, B.L., Boosinger, T.R., Mclaughlin, S.A.,Dubey, J.P., 1994. Evaluation of the safety and efficacy of vaccination ofnursing pigs with living tachyzoites of two strains of Toxoplasma gondii.J. Parasitol. 80, 438e448.
Pizza, M., Scarlato, V., Masignani, V., Giuliani, M.M., Arico, B., Comanducci, M.,Jennings, G.T., Baldi, L., Bartolini, E., Capecchi, B., Galeotti, C.L., Luzzi, E.,Manetti, R., Marchetti, E., Mora, M., Nuti, S., Ratti, G., Santini, L., Savino, S.,Scarselli, M., Storni, E., Zuo, P., Broeker, M., Hundt, E., Knapp, B., Blair, E.,Mason, T., Tettelin, H., Hood, D.W., Jeffries, A.C., Saunders, N.J.,Granoff, D.M., Venter, J.C., Moxon, E.R., Grandi, G., Rappuoli, R., 2000.Identification of vaccine candidates against serogroup B meningococcus bywhole-genome sequencing. Science 287, 1816e1820.
Qu, D., Yu, H., Wang, S., Cai, W., Du, A., 2009. Induction of protective immunityby multiantigenic DNA vaccine delivered in attenuated Salmonella typhi-murium against Toxoplasma gondii infection in mice. Vet. Parasitol. 166,220e227.
Qu, D., Wang, S., Cai, W., Du, A., 2008. Protective effect of a DNA vaccinedelivered inattenuated Salmonella typhimurium against Toxoplasma gondiiinfection in mice. Vaccine 26, 4541e4548.
Quan, J.H., Chu, J.Q., Ismail, H.A., Zhou, W., Jo, E.K., Cha, G.H., Lee, Y.H., 2012.Induction of protective immune responses by a multiantigenic DNA vaccineencoding GRA7 and ROP1 of Toxoplasma gondii. Clin. Vaccine Immunol. 19,666e674.
Rachinel, N., Buzoni-Gatel, D., Dutta, C., Mennechet, F.J., Luangsay, S., Minns, L.A.,Grigg, M.E., Tomavo, S., Boothroyd, J.C., Kasper, L.H., 2004. The induction ofacute ileitis by a single microbial antigen of Toxoplasma gondii.J. Immunol. 173, 2725.
Rashid, I., Hedhli, D., Moiré, N., Pierre, J., Debierre-Grockiego, F., Dimier-Poisson, I., Mévélec, M.N., 2011. Immunological responses induced bya DNA vaccine expressing RON4 and by immunogenic recombinant proteinRON4 failed to protect mice against chronic toxoplasmosis. Vaccine 29,8838e8846.
Reichmann, G., Dlugonska, H., Fischer, H.G., 2002. Characterization of TgROP9(p36), a novel rhoptry protein of Toxoplasma gondii tachyzoites identified byT-cell clone. Mol. Biochem. Parasitol. 119, 43e54.
Roberts, C.W., Alexander, J., 1992. Studies on a murine model of congenitaltoxoplasmosis: vertical disease transmission only occurs in BALB/cmice infected for the first time during pregnancy. Parasitology 104(Pt 1), 19.
Roberts, C.W., Brewer, J.M., Alexander, J., 1994. Congenital toxoplasmosis in theBALB/c mouse: prevention of vertical disease transmission and foetal deathby vaccination. Vaccine 12, 1389.
Roberts, C.W., Roberts, F., Lyons, R.E., Kirisits, M.J., Mui, E.J., Finnerty, J.,Johnson, J.J., Ferguson, D.J., Coggins, J.R., Krell, T., Coombs, G.H.,Milhous, W.K., Kyle, D.E., Tzipori, S., Barnwell, J., Dame, J.B., Carlton, J.,McLeod, R., 2002. The shikimate pathway and its branches in apicomplexanparasites. J. Infect. Dis. 185 (Suppl. 1), S25eS36.
Roberts, C.W., Satoskar, A., Alexander, J., 1996. Sex steroids, pregnancy-associ-ated hormones and immunity to parasitic infection. Parasitol. Today 12,382e388.
Roberts, C.W., Walker, W., Alexander, J., 2001. Sex-associated hormones andimmunity to protozoan parasites. Clin. Microbiol. Rev. 14, 476e488.
Roberts, F., McLeod, R., 1999. Pathogenesis of toxoplasmic retinochoroiditis.Parasitol. Today 15, 51e57.
26. VACCINATION AGAINST TOXOPLASMOSIS: CURRENT STATUS AND FUTURE PROSPECTS1044
Rodger, S.M., Maley, S.W., Wright, S.E., MacKellar, A., Sales, J., Buxton, D., 2005.Toxoplasmosis in sheep; the possibility of endogenous transplacentaltransmission. Wiadomosci. Parazytologiczne 51, s15es18.
Rodrigues, E.G., Zavala, F., Eichinger, D., Wilson, J.M., Tsuji, M., 1997.Single immunizing dose of recombinant adenovirus efficiently inducesCD8+ T-cell-mediated protective immunity against malaria. J. Immunol.158, 1268.
Roque-Resendiz, J.L., Rosales, R., Herion, P., 2004. MVA ROP2 vaccinia virusrecombinant as a vaccine candidate for toxoplasmosis. Parasitology 128, 397.
Rosenberg, C., De Craeye, S., Jongert, E., Gargano, N., Beghetto, E., DelPorto, P., Vorup-Jensen, T., Petersen, E., 2009. Induction of partialprotection against infection with Toxoplasma gondii genotype II by DNAvaccination with recombinant chimeric tachyzoite antigens. Vaccine 27,2489e2498.
Saavedra, R., Leyva, R., Tenorio, E.P., Haces, M.L., Rodriguez-Sosa, M.,Terrazas, L.I., Herion, P., 2004. CpG-containing ODN has a limited role inthe protection against Toxoplasma gondii. Parasite Immunol. 26, 67e73.
Sánchez, V.R., Pitkowski, M.N., Fernández Cuppari, A.V., Rodríguez, F.M.,Fenoy, I.M., Frank, F.M., Goldman, A., Corral, R.S., Martin, V., 2011.Combination of CpG-oligodeoxynucleotides with recombinant ROP2 orGRA4 proteins induces protective immunity against Toxoplasma gondiiinfection. Exp. Parasitol. 128 (4), 448e453.
Scorza, T., D’Souza, S., Laloup, M., Dewit, J., De Braekeleer, J., Verschueren, H.,Vercammen, M., Huygen, K., Jongert, E., 2003. A GRA1 DNA vaccineprimes cytolytic CD8(+) T-cells to control acute Toxoplasma gondii infection.Infect. Immun. 71, 309e316.
Seah, S.K., Hucal, G., 1975. The use of irradiated vaccine in immunization againstexperimental murine toxoplasmosis. Can. J. Microbiol. 21, 1379.
Shang, L., Liu, Q., Liu, W., Men, J., Gao, S., Jiang, L., Wang, Z., Zhai, Y., Jin, H.,Lian, H., Chen, C., Xia, Z., Yuan, Z., Zhu, X.Q., 2009. Protection in miceimmunized with a heterologous prime-boost regime using DNA andrecombinant pseudorabies expressing TgSAG1 against Toxoplasma gondiichallenge. Vaccine 27, 2741e2745.
Shirahata, T., Muroya, N., Ohta, C., Goto, H., Nakane, A., 1992. Correlationbetween increased susceptibility to primary Toxoplasma gondii infection anddepressed production of gamma interferon in pregnant mice. Microbiol.Immunol. 36, 81e91.
Shirahata, T., Muroya, N., Ohta, C., Goto, H., Nakane, A., 1993. Enhancement byrecombinant human interleukin 2 of host resistance to Toxoplasma gondiiinfection in pregnant mice. Microbiol. Immunol. 37, 583e590.
Siachoque, H., Guzman, F., Burgos, J., Patarroyo, M.E., Gomez Marin, J.E., 2006.Toxoplasma gondii: immunogenicity and protection by P30 peptides ina murine model. Exp. Parasitol. 114, 62e65.
Soldati, D., Kim, K., Kampmeier, J., Dubremetz, J.F., Boothroyd, J.C., 1995.Complementation of a Toxoplasma gondii ROP1 knock-out mutant usingphleomycin selection. Mol. Biochem. Parasitol. 74, 87.
Stacey, K.J., Blackwell, J.M., 1999. Immunostimulatory DNA as an adjuvant invaccination against Leishmania major. Infect. Immun. 67, 3719e3726.
Stanley, A.C., Buxton, D., Innes, E.A., Huntley, J.F., 2004. Intranasal immuniza-tion with Toxoplasma gondii tachyzoite antigen encapsulated into PLGmicrospheres induces humoral and cell-mediated immunity in sheep.Vaccine 22, 3929.
Sun, X.M., Zou, J., A A, E.S., Yan, W.C., Liu, X.Y., Suo, X., Wang, H., Chen, Q.J.,2011. DNA vaccination with a gene encoding Toxoplasma gondii GRA6induces partial protection against toxoplasmosis in BALB/c mice. Parasit.Vectors 9 (4), 213.
Supply, P., Sutton, P., Coughlan, S.N., Bilo, K., Saman, E., Trees, A.J., CesbronDelauw, M.F., Locht, C., 1999. Immunogenicity of recombinant BCGproducing the GRA1 antigen from Toxoplasma gondii. Vaccine 17, 705e714.
Tan, F., Hu, X., Luo, F.J., Pan, C.W., Chen, X.G., 2011. Induction of protective Th1immune responses in mice by vaccination with recombinant Toxoplasma gondiinucleoside triphosphate hydrolase-II. Vaccine 29, 2742e2748.
Tan, T.G., Mui, E., Cong, H., Witola, W.H., Montpetit, A., Muench, S.P.,Sidney, J., Alexander, J., Sette, A., Grigg, M.E., Maewal, A., Mcleod, R.,2010. Identification of T. gondii epitopes, adjuvants, and host geneticfactors that influence protection of mice and humans. Vaccine 28,3977e3989.
Tenter, A.M., Heckeroth, A.R., Weiss, L.M., 2000. Toxoplasma gondii: from animalsto humans. Int. J. Parasitol. 30, 1217.
Thouvenin, M., Candolfi, E., Villard, O., Klein, J.P., Kien, T., 1997. Immuneresponse in a murine model of congenital toxoplasmosis: increasedsusceptibility of pregnant mice and transplacental passage of Toxoplasmagondii are type 2-dependent. Parassitologia 39, 279e283.
Vallochi, A.L., Nakamura, M.V., Schlesinger, D., Martins, M.C., Silveira, C.,Belfort Jr., R., Rizzo, L.V., 2002. Ocular toxoplasmosis: more than just whatmeets the eye. Scand. J. Immunol. 55, 324.
Van Poppel, N.F.J., Welagen, J., Duisters, R.F.J.J., Vermeulen, A.N., Schaap, D.,2006. Tight control of transcription in Toxoplasma gondii using an alternativetet repressor. Int. J. Parasitol. 36, 443e452.
Velge-Roussel, F., Moretto, M., Buzoni-Gatel, D., Dimier-Poisson, I., Ferrer, M.,Hoebeke, J., Bout, D., 1997. Differences in immunological response to aT. gondii protein (SAG1) derived peptide between two strains ofmice: effect on protection in T. gondii infection. Mol. Immunol. 34, 1045e1053.
Venturini, M.C., Bacigalupe, D., Venturini, L., Rambeaud, M., Basso, W.,Unzaga, J.M., Perfumo, C.J., 2004. Seroprevalence of Toxoplasma gondii insows from slaughterhouses and in pigs from an indoor and an outdoor farmin Argentina. Vet. Parasitol. 124, 161.
Vercammen, M., Scorza, T., Huygen, K., De Braekeleer, J., Diet, R., Jacobs, D.,Saman, E., Verschueren, H., 2000. DNA vaccination with genes encodingToxoplasma gondii antigens GRA1, GRA7, and ROP2 induces partiallyprotective immunity against lethal challenge in mice. Infect. Immun. 68,38e45.
Vermeulen, A.N., 1998. Progress in recombinant vaccine development againstcoccidiosis. A review and prospects into the next millennium. Int. J. Para-sitol. 28, 1121.
Vermeulen, A.N., Schaap, D.C., Schetters, T.P., 2001. Control of coccidiosis inchickens by vaccination. Vet. Parasitol. 100, 13.
Vogel, N., Kirisits, M., Michael, E., Bach, H., Hostetter, M., Boyer, K., Simpson, R.,Holfels, E., Hopkins, J., Mack, D., Mets, M.B., Swisher, C.N., Patel, D.,Roizen, N., Stein, L., Stein, M., Withers, S., Mui, E., Egwuagu, C.,Remington, J., Dorfman, R., McLeod, R., 1996. Congenital toxoplasmosistransmitted from an immunologically competent mother infected beforeconception. Clin. Infect. Dis. 23, 1055e1060.
Waldeland, H., Frenkel, J.K., 1983. Live and killed vaccines against toxoplas-mosis in mice. J. Parasitol. 69, 60.
Wang, X., Kang, H., Kikuchi, T., Suzuki, Y., 2004. Gamma interferon production,but not perforin-mediated cytolytic activity, of T-cells is required forprevention of toxoplasmic encephalitis in BALB/c mice genetically resistantto the disease. Infect. Immun. 72, 4432e4438.
Wang, H., He, S., Yao, Y., Cong, H., Zhao, H., Li, T., Zhu, X.Q., 2009. Toxoplasmagondii: protective effect of an intranasal SAG1 and MIC4 DNA vaccine inmice. Exp. Parasitol. 122, 226e232.
Wang, H., Liu, Q., Liu, K., Zhong, W., Gao, S., Jiang, L., An, N., 2007. Immuneresponse induced by recombinant Mycobacterium bovis BCG expressingROP2 gene of Toxoplasma gondii. Parasitol. Int. 56, 263e268.
Wang, Y., Wang, M., Wang, G., Pang, A., Fu, B., Yin, H., Zhang, D., 2011.Increased survival time in mice vaccinated with a branched lysine multipleantigenic peptide containing B- and T-cell epitopes from T. gondii antigens.Vaccine 29, 8619e8623.
Webster, D.P., Dunachie, S., Vuola, J.M., Berthoud, T., Keating, S., Laidlaw, S.M.,McConkey, S.J., Poulton, I., Andrews, L., Andersen, R.F., Bejon, P.,Butcher, G., Sinden, R., Skinner, M.A., Gilbert, S.C., Hill, A.V., 2005.Enhanced T-cell-mediated protection against malaria in human challengesby using the recombinant poxviruses FP9 and modified vaccinia virusAnkara. Proc. Natl. Acad. Sci. U.S.A. 102, 4836.
Wilkins, M.F., O’Connell, E., 1992. Vaccination of sheep against Toxoplasmaabortion. Surveillance 19, 20.
Wilkins, M.F., O’Connell, E., Te Punga, W.A., 1987. Toxoplasmosis in sheep. I.Effect of a killed vaccine on lambing losses caused by experimental chal-lenge with Toxoplasma gondii. N. Z. Vet. J. 35, 31.
Wilkins, M.F., O'Connell, E., Te Punga, W.A., 1988. Toxoplasmosis in sheep III.Further evaluation of the ability of a live Toxoplasma gondii vaccine toprevent lamb losses and reduce congenital infection following experimentaloral challenge. N. Z. Vet. J. 36, 86.
REFERENCES 1045
Williams, K.A., Scott, J.M., MacFarlane, D.E., Williamson, J.M., Elias-Jones, T.F.,Williams, H., 1981. Congenital toxoplasmosis: a prospective survey in theWest of Scotland. J. Infect. 3, 219e229.
Williams, R.H., Morley, E.K., Hughes, J.M., Duncanson, P., Terry, R.S., Smith, J.E.,Hide, G., 2005. High levels of congenital transmission of Toxoplasma gondii inlongitudinal and cross-sectional studies on sheep farms provides evidence ofvertical transmission in ovine hosts. Parasitology 130, 301e307.
Wilson, W.B., Sharpe, J.A., Deck, J.H., 1980. Cerebral blindness and oculomotornerve palsies in toxoplasmosis. Am. J. Ophthalmol. 89, 714e718.
Xiang, W., Qiong, Z., Li-Peng, L., Kui, T., Jian-Wu, G., Heng-Ping, S., 2009. Thelocation of invasion-related protein MIC3 of Toxoplasma gondii and protec-tive effect of its DNA vaccine in mice. Vet. Parasitol. 166, 1e7.
Xue, M., He, S., Zhang, J., Cui, Y., Yao, Y., Wang, H., 2008a. Comparison ofcholera toxin A2/B and murine interleukin-12 as adjuvants of Toxoplasmamulti-antigenic SAG1-ROP2 DNA vaccine. Exp. Parasitol. 119, 352e357.
Xue, M., He, S., Cui, Y., Yao, Y., Wang, H., 2008b. Evaluation of the immuneresponse elicited by multi-antigenic DNA vaccine expressing SAG1, ROP2and GRA2 against Toxoplasma gondii. Parasitol. Int. 57, 424e429.
Yan, Z.X., Meyer, T.F., 1996. Mixed population approach for vaccination withlive recombinant Salmonella strains. J. Biotechnol. 44, 197.
Yan, H., Yan, H., Tao, Y., Chen, H., Li, G., Gong, W., Jiao, H., Tian, F., Ji, M.,2012a. Application and expression of Toxoplasma gondii surface antigen 2(SAG2) and rhoptry protein 2 (ROP2) from recombinant Escherichia colistrain. Trans. R. Soc. Trop. Med. Hyg. 106, 356e362.
Yan, H.K., Yuan, Z.G., Song, H.Q., Petersen, E., Zhou, Y., Ren, D., Zhou, D.H., Li, H.X., Lin, R.Q., Yang, G.L., Zhu, X.Q., 2012b. Vaccination with a DNA vaccinecoding for perforin-like protein 1 and MIC6 induces significant protectiveimmunity against Toxoplasma gondii. Clin. Vaccine. Immunol. 19, 684e689.
Yan, H.K., Yuan, Z.G., Petersen, E., Zhang, X.X., Zhou, D.H., Liu, Q., He, Y.,Lin, R.Q., Xu, M.J., Chen, X.L., Zhong, X.L., Zhu, X.Q., 2011. Toxoplasma gondii:protective immunity against experimental toxoplasmosis induced by a DNAvaccine encoding the perforin-like protein 1. Exp. Parasitol. 128, 38e43.
Yang, C.D., Chang, G.N., Chao, D., 2004. Protective immunity against Toxoplasmagondii in mice induced by a chimeric protein rSAG1/2. Parasitol. Res. 92,58e64.
Yao, Y., He, S.Y., Wang, H.X., Zhou, H.Y., Zhao, H., Li, T., Xue, M.F., Zhu, X.Q.,2010. Protective immunity induced in mice by multiantigenic DNA vaccinewith genes encoding SAG1 and MIC8 of Toxoplasma gondii. Zhongguo JiSheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 28, 81e88.
Yarovinsky, F., Zhang, D., Andersen, J.F., Bannenberg, G.L., Serhan, C.N.,Hayden, M.S., Hieny, S., Sutterwala, F.S., Flavell, R.A., Ghosh, S., Sher, A.,2005. TLR11 activation of dendritic cells by a protozoan profilin-like protein.Science 308, 1626e1629.
Yu, L., Yamagishi, J., Zhang, S., Jin, C., Aboge, G.O., Zhang, H., Zhang, G.,Tanaka, T., Fujisaki, K., Nishikawa, Y., Xuan, X., 2012. Protective effect ofa prime-boost strategy with plasmid DNA followed by recombinantadenovirus expressing TgAMA1 as vaccines against Toxoplasma gondiiinfection in mice. Parasitol. Int. 61, 481e486.
Yuan, Z.G., Zhang, X.X., Lin, R.Q., Petersen, E., He, S., Yu, M., He, X.H., Zhou, D.H., He, Y., Li, H.X., Liao, M., Zhu, X.Q., 2011a. Protective effect againsttoxoplasmosis in mice induced by DNA immunization with gene encodingToxoplasma gondii ROP18. Vaccine 29, 6614e6619.
Zenner, L., Darcy, F., Cesbron-Delauw, M.F., Capron, A., 1993. Rat model ofcongenital toxoplasmosis: rate of transmission of three Toxoplasma gondiistrains to foetuses and protective effect of a chronic infection. Infect. Immun.61, 360e363.
Zenner, L., Estaquier, J., Darcy, F., Maes, P., Capron, A., Cesbron-Delauw, M.F.,1999. Protective immunity in the rat model of congenital toxoplasmosis andthe potential of excreted-secreted antigens as vaccine components. ParasiteImmunol. 21, 261.
Zhang, Y.W., Fraser, A., Balfour, A.H., Wreghitt, T.G., Gray, J.J., Smith, J.E., 1995.Serological reactivity against cyst and tachyzoite antigens of Toxoplasmagondii determined by FAST-ELISA. J. Clin. Pathol. 48, 908e911.
Zhang, Y.W., Kim, K., Ma, Y.F., Wittner, M., Tanowitz, H.B., Weiss, L.M., 1999.Disruption of the Toxoplasma gondii bradyzoite-specific gene BAG1 decreasesin vivo cyst formation. Mol. Microbiol. 31, 691.
Zhang, G., Huang, X., Boldbaatar, D., Battur, B., Battsetseg, B., Zhang, H., Yu, L.,Li, Y., Luo, Y., Cao, S., Goo, Y.K., Yamagishi, J., Zhou, J., Zhang, S.,Suzuki, H., Igarashi, I., Mikami, T., Nishikawa, Y., Xuan, X., 2010.Construction of Neospora caninum stably expressing TgSAG1 and evaluationof its protective effects against Toxoplasma gondii infection in mice. Vaccine28, 7243e7247.
Zhang, G., Huong, V.T., Battur, B., Zhou, J., Zhang, H., Liao, M., Kawase, O.,Lee, E.G., Dautu, G., Igarashi, M., Nishikawa, Y., Xuan, X., 2007a. Aheterologous prime-boost vaccination regime using DNA and a vacciniavirus, both expressing GRA4, induced protective immunity against Toxo-plasma gondii infection in mice. Parasitology 134, 1339e1346.
Zhang, J., He, S., Jiang, H., Yang, T., Cong, H., Zhou, H., Zhang, J., Gu, Q., Li, Y.,Zhao, Q., 2007b. Evaluation of the immune response induced by multi-antigenic DNA vaccine encoding SAG1 and ROP2 of Toxoplasma gondii andthe adjuvant properties of murine interleukin-12 plasmid in BALB/c mice.Parasitol. Res. 101, 331e338.
Zhou, X., Berg, L., Motal, U.M., Jondal, M., 1992. In vivo primary induction ofvirus-specific CTL by immunization with 9-mer synthetic peptides.J. Immunol. Methods 153, 193e200.
Zhou, D.H., Liang, R., Yin, C.C., Zhao, F.R., Yuan, Z.G., Lin, R.Q., Song, H.Q.,Zhu, X.Q., 2010. Seroprevalence of Toxoplasma gondii in pigs from southernChina. J. Parasitol. 96, 673e674.
Zhou, H., Min, J., Zhao, Q., Gu, Q., Cong, H., Li, Y., He, S., 2012. Protectiveimmune response against Toxoplasma gondii elicited by a recombinant DNAvaccine with a novel genetic adjuvant. Vaccine 30, 1800e1806.
Zorgi, N.E., Costa, A., Galisteo Jr., A.J., Do Nascimento, N., De Andrade Jr., H.F.,2011. Humoral responses and immune protection in mice immunized withirradiated T. gondii tachyzoites and challenged with three genetically distinctstrains of T. gondii. Immunol. Lett. 138, 187e196.
Zulpo, D.L., Headley, S.A., Biazzono, L., Da Cunha, I.A., Igarashi, M., DeBarros, L.D., Taroda, A., Cardim, S.T., Bogado, A.L., Navarro, I.T.,Garcia, J.L., 2012. Oocyst shedding in cats vaccinated by the nasal and rectalroutes with crude rhoptry proteins of Toxoplasma gondii. Exp. Parasitol. 131,223e230.
