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Vaccination concepts against Toxoplasma gondii Expert Rev. Vaccines 8(2), 215–225 (2009)

João Luis Garcia Department of Preventive Veterinary Medicine, Londrina State University, Campus Universitário, Rodovia Celso Garcia Cid, Pr 445 Km 380, Cx. Postal 6001, Londrina, PR. 86051-990, Brazil Tel.: +55 433 371 5871 Fax: +55 433 371 4485 [email protected]

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Toxoplasma gondii is a parasite that infects animals and humans worldwide. Despite the current knowledge of immunology, pathology and genetics related to the parasite, a safe vaccine for prevention of the infection in both humans and animals does not exist. Here, we review some aspects concerning vaccination against T. gondii. Keywords : immunization • Toxoplasma gondii • toxoplasmosis • vaccine

Toxoplasma gondii is an intracellular obligate protozoan that can infect humans and warmblooded animals. Animals from the Felidae family are the only definitive hosts; the cat is of central importance in the transmission of toxoplasmosis [1] . After primary infection with T. gondii, cats may shed millions of oocysts into the soil [2] . In the soil, oocysts sporulate within 1–5 days and can persist as infection sources for months. Consuming sporulated oocysts located in either water or foodstuff can infect animals. Raw or undercooked meat from these animals that contain tissue cysts are important sources of T. gondii infections for humans. The risk of infection from meat has been described in many regions [3,4] . Navarro et al. demonstrated that more than 20% of pork samples were infected with tissue cysts in Brazil [5] . The estimated rate of congenital toxoplasmosis depends on where screening was carried out and can vary between 1.1 and 2.6 per 1000, one per 3000 and 2.1 per 10,000 live births in the USA, Brazil and Denmark, respectively [6–8] . The cost of human illness due to congenital toxoplasmosis is very high and was estimated at US$0.4–8.8 billion annually in the USA alone [6] . Additionally, in spite of highly active antiretroviral therapy, there is an estimated incidence of toxoplasmic encephalitis (TE) of 15.9% in HIV-infected patients [9] . Before highly active antiretroviral therapy, encephalitis induced by T. gondii was the most frequent cause of infection complications in AIDS [10] . Interpretations of the host immune responses to T. gondii associated with both appropriated parasite molecules and adjuvants will be necessary for effective vaccine production. However, only a few vaccines for veterinary parasitic diseases have been produced, such as live parasites 10.1586/14760584.8.2.215

(T. gondii and Eimeria species), killed vaccines (Neospora caninum and Anaplasma marginale) and subunit vaccines (Babesia canis) [11] , and none, so far, for human use. Life cycle

T. gondii was first described in 1908 concomitantly by Nicolle and Manceaux, and by Splendore. Long after their discovery, in the early 1970s, members of the Felidae family were described as definitive hosts [12,13] . This parasite is capable of infecting different nucleated cells from many hosts, including humans and endothermic animals (intermediate hosts). There are three infectious stages in their life cycle: sporozoites (inside sporulated oocyst), tachyzoites (multiplicative form during the acute phase) and bradyzoites (within-tissue cysts in the chronic phase). The hosts can be infected in three primary ways: tissue-cyst ingestion (carnivorism), oocyst ingestion (fecal–oral) and congenitally (transplacental infection). When the intermediate host becomes infected with either bradyzoites or sporozoites (this might occur with cats), they are transformed into tachyzoites that multiply rapidly by endodyogeny, and disrupt the cells and invade other host cells. The natural site of infection for T. gondii is the cells of the intestine, followed by the mesenteric lymph nodes and consequent dissemination to distant organs via the lymphatic and blood vessels. This intracellular growth causes tissue necrosis in many organs. After the host has developed immunity, the parasites are retained within tissue cysts and transformed to bradyzoites, which slowly multiply into tissue cysts. In humans and sheep, these cysts can persist throughout the life of the host [14] . These tissue cysts are frequently observed within

© 2009 Expert Reviews Ltd

ISSN 1476-0584

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the nervous system, eyes, cavitary organs and muscles. When the definitive host becomes infected, there is shedding of unsporulated oocysts after a sexual phase within intestinal cells. Within the environment, unsporulated oocysts begin the sporogony phase, are sporulated (two sporocysts with four sporozoites each) and become infectious within 1–5 days [15] . Cell invasion & immunity for T. gondii

The mechanism of host-cell invasion by tachyzoites is calcium dependent and regulated by several factors, such as conoid movements, motility and the secretion of organelles (micronemes, rhoptries and dense granules) [16–20] . This invasion is extremely rapid (15–40/s) being directly related to the sequential secretion of proteins by organelles, a complex process that is very important for parasitic survival within the cell [21,22] . The parasitophorous vacuole is formed after cellular invasion and has a membrane that is hybrid; this indicates that secreted/excreted proteins from the parasite will be part of this membrane and the parasitophorous vacuole is not fused with either endosomes or lysosomes [21,23] . Infections by T. gondii rarely produce clinical signs in the affected hosts and disease severity is related to species, age, nutritional and immunological status of the host, sex hormones, pregnancy, strain, parasite stages and concomitant infections [24–29] . The mechanisms involved in protection against infection are components of the innate and adaptive immune response. The first line of host defense against T. gondii occurs at the mucosa of the intestinal tract. Cytokines and chemokines secreted by intestinal epithelial cells are responsible for the migration of inflammatory cells, such as macrophages, neutrophils, lymphocytes and dendritic cells (DCs). Neutrophils produce chemokines that are important for the attraction and migration of DCs. DCs may act as a link between the innate and adaptive immune response. The immune response against T. gondii was thoroughly reviewed recently by Miller et al. [30] , Buzoni-Gatel and Kasper [31] , and Roberts et al. [32] . In the early stage of infection, lethal effects resulted from extremely elevated levels of IFN-γ and IL-18, whereas nonlethal infection was due to moderate levels of these cytokines. TNF-α has an important role in the resistance of toxoplasmosis; however, high levels of this cytokine might contribute towards pathogenesis. Perhaps during infection, elevated levels of IL-18, IFN-γ, IL-12 and TNF-α induce an increase in vascular permeability that could lead to multiple organ failure and consequent death [33] . CD4 + Th1 cells and CD8 + cytolytic T lymphocytes are vital for protective immunity and long-term survival during chronic infection; this characteristic is due to the ability to produce IFN-γ, a proinflammatory cytokine that is known as the major mediator of resistance to T. gondii [34] . Depletion of this cytokine during the chronic phase of infection demonstrated that continued IFN-γ production is necessary for long-term survival [35] . Tachyzoites located extracellularly might be destroyed due to the presence of specific antibodies and complement. Anti-T. gondii antibodies can prevent entry of the parasite into the cell, but not into phagocytes [16] . In sheep, high titers of antibodies against 216

T. gondii were observed after secondary infection with tachyzoites, and this coincided with the disappearance of viable tachyzoites from the site of infection [36] . There are many questions surrounding the immuno­regulation of T. gondii infection. Most of the studies that evaluated immunity against this parasite were performed in murine models. However, one important observation is that there is a clear difference between host susceptibilities. Mice are very susceptible to T. gondii infection while other species, such as humans, sheep and pigs, are not; therefore, it is unclear how the results from those murine studies can be extrapolated to these species  [14] . Susceptibility might be related to differences in the host immune system. This has been demonstrated in the development of human and mouse TE. In humans, TE is associated with a loss of T-cell function; in the mouse it is a defect in the CD8 + T-cell response, which is genetically based [37] . In addition, the immunity data obtained from mice should not be extrapolated to pigs, owing to some differences in immune-response mechanisms, such as the lack of an active nitric oxide pathway [38,39] . Proinflammatory cytokine levels were increased in Toxoplasma acute infection in the pig model [39] . Both IFN-γ production and CD8 + cells participate in the swine response to an acute T. gondii infection; however, the participation of IL-12 could not be important in the early stage of infection. The authors suggested that IL-10 in pigs becomes more prominent late in infection. These results regarding IL-12 and IL-10 indicated differences between pig and mouse immune responses. The magnitude of changes in proinflammatory gene expression of IFN-γ, IL-15, iNOS and TNF-α in experimentally infected pigs with T. gondii oocysts was higher at 7 days post infection (dpi) than at 14 dpi; this supported the clinical signs in the animals, indicating a partial resolution of infection at 14 dpi [40] . These authors observed a decrease in IL-12 in the gut-associated lymphoid tissues, which could reflect the enteritis and necrosis observed at 7 dpi, and this contributed to the immune suppression. Additionally, IL-15 was significantly induced in several tissues and IL-18 was only in the liver at 7 dpi. In sheep, both CD4 + and CD8 + cells play a significant role in the control of Toxoplasma acute infection; this phenotype co­incided with the time at which T. gondii were no longer detectable in the efferent lymph [41] . Innes described that the most important host factor to determine the susceptibility to T. gondii was how fast the immune system can produce IFN-γ [14] . Vaccines against T. gondii

The first generation of vaccines was elaborated with live, liveattenuated or killed parasites, followed by subunit vaccines and, finally, the genetic ones [42] . The live and killed vaccines are considered nonself by the immune system; they activate a number of reactive lymphocytes and induce antibody production that can block infection [43] . Vaccine studies in cats

Cats are considered key in T. gondii control, owing to the fact that the contaminated feces from these animals are responsible for oocyst spread within the environment. Therefore, Expert Rev. Vaccines 8(2), (2009)

Vaccination concepts against Toxoplasma gondii

a vaccine for this species must be protective against oocyst shedding. Although cats are important for the transmission of T. gondii, there are few studies using vaccines to control oocyst shedding. A live T. gondii mutant strain named T-263 was produced and evaluated in kittens to prevent oocyst shedding; a prevention rate greater than 84% was observed [44,45] . The use of this vaccine strain in cats from farms has reduced the exposure of pigs to T. gondii [46] . However, T-263 has its disadvantages: it uses live T. gondii bradyzoites, is expensive and suffers from cryopreservation problems [47] . Omata et al. tested a 60Co-irradiated tachyzoites vaccine in cats, and only three out of 14 did not shed oocysts [48] . Additionally, a DNA vaccine expressing recombinant ROP2 protein was tested in cats and also did not reduce oocyst shedding [49] . Garcia et al. used crude T. gondii rhoptry proteins plus Quil-A by the intranasal route to evaluate the control of the shedding of oocysts [50] . Two out of three cats did not shed oocysts after being immunized with three doses of the vaccine, and vaccinated cats had an estimated protection of 65%. During this study, the intestinal mucosa was the principal immunization target and, because of this, cats were immunized by the intranasal route. Currently, our group is examining the utilization of recombinant and crude proteins by the rectal and nasal routes to evaluate protection against oocyst shedding in cats. Vaccine studies in pigs

Tissue cysts in pork can persist for more than 2 years and are one of the most important sources of T.  gondii infections in humans  [51,52] . Consequently, a vaccine for pigs needs to avoid the formation of tissue cysts. Live vaccines (RH and S48) have shown protection against toxoplasmosis [27,51] , but these carry the risk of reverting to virulence [53] . Therefore, it is necessary to produce a killed vaccine against T. gondii in this species. Studies using live RH strain have shown restricted protection against tissue cyst formation [27,51,54,55] , but these results were not adequate to function as a live vaccine in pigs, owing to patho­ genicity differences among RH strains to pigs [54] . The vaccine does not remain in pig tissues at 64 dpi [51] . During a recent study in our laboratory, the RH strain was unable to produce tissue cysts at 69 dpi. The RH strain is the most widely studied and used T. gondii strain. This strain was isolated from a child with TE in 1939 and, since then, has been maintained in mice and cellular culture [56] . There is a genetic polymorphism between RH strains from different laboratories [24] ; however, this could not be due to changing behavior of RH strain in pigs. A vaccine study using crude T. gondii antigens incorporated in the immunostimulating complex (ISCOM) by the subcutaneous route in pigs did not isolate, by mouse bioassay, tissue cysts from vaccinated animals [57] . Garcia et al. used rhoptry proteins incorporated in ISCOM to prevent tissue cyst formation in pigs challenged with sporulated oocysts of the VEG strain [55] . These results have indicated that rhoptry vaccine conferred partial protection during the chronic phase of the disease. www.expert-reviews.com

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More recently, pigs were immunized intradermally with a cocktail DNA vaccine encoding GRA1 and seven dense granule proteins [58] . The authors described that this vaccine was able to elicit a strong humoral and type 1 cellular immune response in animals. Unfortunately, the results relative to the evaluation of tissue cyst burden were inconsistent; however, this study was important because an elicited immune response was observed in pigs by a DNA vaccine. Vaccine studies in sheep & goats

T. gondii is considered one of the main causes of abortion in goat and sheep herds of North America and Scotland [59,60] . These animals can act as an infection source for humans via the consumption of raw or undercooked meat containing tissue cysts [61] . Moreover, they act as a source of infection for humans, since transmission can occur via the consumption of in natura milk or meat that has not been cooked properly [61–63] . The main target for a vaccine strategy in sheep and goats would be protection against congenital toxoplasmosis and tissue cysts formation [64] . To the best of my knowledge, the only two studies to prevent toxoplasmosis in goats used the heterologous parasite Hammondia as immunization [65,66] . Killed and live vaccines have been tested in ewes [67–71] . While there was no beneficial effect on fertility by use of the killed vaccine, the authors observed that ewes vaccinated with a live incomplete strain had significantly lower maternofetal transmission than unvaccinated ewes. From these studies, a commercial vaccine was developed for use in ewes to avoid congenital toxoplasmosis (Toxovax, Intervet BV), which contained live-incomplete T. gondii tachyzoites (S48 strain) [72] . However, this vaccine is expensive, causes adverse effects, has a short shelf-life, might revert to a pathogenic strain and is potentially hazardous to users [73,74] . A nasal immunization study for sheep stimulated both humoral and cell-mediated responses and demonstrated the potential for the control of toxoplasmosis [74] . The authors tested soluble and particulate antigens of the T. gondii plus poly(d,l-lactide-co-glycolide) particles and cholera toxin by the nasal route in sheep. The particulate preparations induced more elevated local IgA responses than soluble antigen; however, these immunizations were not adequate to protect against infection with sporulated oocysts. Vaccine studies in rodents

Mice and rats are the main biological models for toxoplasmosis. While mice are relatively susceptible, rats, as with humans, are resistant to T. gondii. The use of these animals in experiments is very important for experimental toxoplasmosis, such as congenital and ocular toxoplasmosis and immunosuppressed host studies. The use of rodents is very frequent, either by their placenta, which is histologically similar to that of humans, or the use of nude rats and mice genetically, chemically or surgically immunosuppressed. Murine susceptibility to T. gondii is due to multigenetic control, with at least one of the genes linked to the MHC (H2 locus) [75] . Suzuki et al. described that the susceptibilities of mice for acute and chronic infection were not correlated [76] . BALB/c mice were 217

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more susceptible to the acute infection than CBA/Ca mice. In addition, the reduction in the number of brain cysts and encephalitis is mediated by the Ld gene in the D region of the H2 complex in mice. Mice (CBA/J, C3H/HeJ and C57BL6) with a b or k allele developed severe encephalitis during the chronic stage of Toxoplasma infection, whereas those with the d allele (BALB/c and A/J) did not [77] . There is a difference between the sexes: males demonstrated more resistance to acute and chronic infection than female mice, as was indicated by higher mortality and tissue cyst burden in female compared with male mice [78,79] . Apparently, the failure of females to respond quickly in terms of T cells (IFN-γ and IL-12) is responsible for this difference. There is a necessity for testing vaccines for congenital toxo­ plasmosis and the aforementioned murine models are useful for this. The use of rats is more relevant than the mouse model [80] . Mice are usually very susceptible to T. gondii infection and rats are more resistant (similar to humans). Vertical transmission of T. gondii through successive generations may occur in mouse models. This is different from women, where only a primary infection during pregnancy resulted in congenital infection; it is in fact mousestrain associated [81] . BALB/c has been described as a good model for congenital toxoplasmosis, as vertical transmission only occurs in this animal for the first time during pregnancy [82] . Thus, rat and BALB/c models are attractive for congenital and immunological studies and vaccine design. Female rats chronically infected with T. gondii were protected against congenital toxoplasmosis after challenge either with the same parasite strain or with a different strain [80] . Freyre et al. observed protection against congenital transmission in rats with homologous challenge; however, this was not reached with heterologous challenge [83] . The divergence observed in this study was a result of the excessive dose challenge. Swiss mice were immunized with ts4 strain by subcutaneous and intraintestinal routes to evaluate protection against mortality, tissue cyst burden and congenital transmission [84] . Animals that were immunized by the subcutaneous route had fewer tissue cysts and higher survival rates than mice immunized intraintestinally. The vaccination did not protect against congenital transmission; however, a small number of fetuses were protected by intra­intestinal immunization. The use of soluble tachyzoite antigens protected against fetal death in BALB/c mice [85]. This study demonstrated for the first time that a killed vaccine could protect against congenital infection. The intranasal route requires fewer antigens than the oral route because there is much less proteolytic activity within the nasal cavity [86] . This route promotes the production of both systemic and mucosal immune responses to an antigen [87] . There are many studies that have used mice for vaccine evaluations. The comparison of these results is rather difficult owing to the fact that the authors used different methods of immunization, challenge routes (with different zoites) and mouse strains. Table 1 lists some articles that have evaluated T. gondii vaccines for mice. There are some main antigens that were described as T. gondii vaccine candidates: these antigens are SAG1, ROP2, GRA1, 4, 5 and 7, 218

RA5 and TgPI-1 (Table 1) . They were used extensively, either as crude antigen, recombinant protein or DNA vaccine. All of these studies reached partial protection in mice against parasite challenge. Studies with mice must considerer the mouse strain, the route of immunization and stage of the parasite to be used in the challenge. Mouse susceptibility is associated with the T. gondii strain and how much heavier the inocula is [88] . T. gondii has been defined as virulent, intermediate-virulent or avirulent for mice [89] . Type I strains are usually fatal – even a single infectious parasite is able to cause acute disease – while types II and III have lethal doses higher than 103 organisms. RH is a type I strain (this strain lost the capacity to form tissue cysts), while ME49 and 76K belong to type II and VEG is type III. These strains are the most used in T. gondii studies. Recently, five virulence loci of T. gondii were identified, two of them associated with alleles in loci rop16 and rop18 [90,91] . Approaches to T. gondii vaccine production

Information obtained so far indicates that future studies for T. gondii vaccine development will have to use antigens that stimulate the Th1 immune response, expressed in all stages of the parasite and associated with an adequate immunization route [55] . The oral route is the natural site of infection of T. gondii in the sporozoite and bradyzoite stages. These stages express different antigens when compared with tachyzoites. Considering that the natural site of infection for T. gondii is the mucosal surface of the intestine, in developing a vaccine against T. gondii, it must be desirable to stimulate mucosal immunity. The tachyzoites, after being spread by host organisms, infect many tissues, including the eyes, brain and fetus. Thus, a combination of proteins from these three stages will be very desirable for producing a T. gondii vaccine. In conclusion, host vaccination will be one of the most important strategies for reducing T. gondii infection. This vaccination must prevent oocyst shedding in cats, which will be the key to controlling the spread of T. gondii, reducing fetal damage and reducing the number of tissue cysts in animals that represent a reservoir of the parasite for humans. However, only one live vaccine is commercially available for use in sheep and goats. Additionally, there is no vaccine against T. gondii for humans and, for the future, the development of a killed vaccine will be desirable. Expert commentary

It is evident that a vaccine against T. gondii will be required, not just due to infection costs worldwide, but also as a result of the serious consequences associated with humans, such as abortions, chorio­retinitis, mental retardation and deaths. Its utilization could improve sheep, goat and pig production, as T. gondii infection is also a reproductive problem of these animals. Another aspect is that the reduction of oocyst shedding in cats would be very important for disease control, so it may be necessary to consider these animals as the key to toxoplasmosis dissemination. There are immense publications relative to T. gondii, which make the parasite one of the most studied in parasitology and public health; however, to date, there is still a lot to be known regarding this parasite. For example, why does this parasite perform its sexual cycle only in the enteroepithelial cells from felids? Another question is: why aren’t Expert Rev. Vaccines 8(2), (2009)

Purified P30 (SAG1) plus ip. liposomes

SAG1 purified by HPLC, sc. MAP constructs and ESA

TSo (RH strain) plus CT

Swiss-Webster (6–8 weeks)

Darcy et al. (1992) OF1 (8–10 weeks)

C57BL/6 (8–10 weeks)

BALB/c (6–8 weeks)

CBA/J (8–10 weeks)

Swiss-webster

NMRI (8 weeks)

OF1 (7–8 weeks) BCG plus GRA1

Bourguin et al. (1993)

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Roberts et al. (1994)

Debard et al. (1996)

Dubey et al. (1996)

Petersen et al. (1998)

Supply et al. (1999)

p.o. 1000 cysts 76K strain

sc. 100 cysts SSI119 strain

p.o. decreasing dose of oocysts VEG strain

p.o. 100 cysts 76K strain

p.o. 20 cysts RRA strain

p.o. 90 cysts 76K strain 18 d.a.c.

p.o. 1200 cysts 76K strain

ip. 105 tachyzoites C strain

p.o. 200 cysts Me49 strain

Results

The percentage of survival was greater in the group that received TSo plus CT

Monomeric SAG1 did not protect against challenge and 40% of mice vaccinated with MAP survived up to 75 d.a.i.

Only 7% of the mice died compared with 73% of the control group

Mortality

Mortality and brain cyst burden

Mortality and brain cyst burden

Brain cyst burden

There was a slight delay in the mortality of vaccinated animals

The survival rate was higher in the mice immunized; however, there was no difference in cyst burden

Partial protection in the mortality and tissue cyst formation

Strong resistance to brain cyst formation

Congenital protection All pups from vaccinated dams survived into maturity

Brain cyst burden

Mortality

Mortality

Congenital protection Neonates from sc.-vaccinated dams were not protected. ii.-immunized mice had partial protection against congenital infection (36%)

Aim

[53]

[97]

[96]

[95]

[85]

[94]

[93]

[92]

[84]

Ref.

BCG: Bacille Calmette-Guérin; CLN: Cervical lymph node; CT: Cholera toxin; d.a.c.: Days after challenge; d.a.i.: Days after infection; EC2: Chimeric protein encoding fragments of MIC2, MIC3 and SAG1; ESA: Excreted–secreted antigens; Exo-Tag: Exosomes secreted by dendritic cells pulsed in vitro with Toxoplasma-derived antigens; FCA: Freund’s complete adjuvant; FIA: Freund’s incomplete adjuvant; GERBU: Adjuvant based on cationic lipid solid nanoparticles and glycopeptides derived from Lactobacillus bulgaricus cell wall; GRA: Dense granule antigen; ii.: Intraintestinal; im.: Intramuscular; in.: Intranasal; ip.: Intraperitoneal; iv.: Intravenous; LT: Heat-labile enterotoxin (LTR72 and LTK63 mutants); MAP: Multiple antigenic peptide; MIC: Microneme protein; MLN: Mesenteric lymph nodes; NISV: Nonionic surfactant vesicle; OF1: Oncins France 1; pIL-12: Plasmid-encoding IL-12; p.o.: Per oral; ROP2: Rhoptry 2 protein; SAG: Surface antigen; sc.: Subcutaneous; STAg: Soluble tachyzoites antigen; TSo: Tachyzoites sonicated.

ip.

sc.

p.o.

α-irradiated oocysts

Recombinant SAG1

in.

sc.

SAG1 purified plus CT

STAg plus NISV

p.o.

sc. or ii.

Bulow and Boothroyd (1991)

ts4 (temperaturesensitive mutant)

Swiss-Webster

Route of Challenge immunization

McLeod et al. (1988)

Antigen and adjuvant

Animal

Study (year)

Table 1. Toxoplasma gondii vaccine studies in mouse models.

Vaccination concepts against Toxoplasma gondii

Review

219

220

Swiss-Webster

C57BL/6, C3H

Roque-Reséndiz et al. (2004)

Martin et al. (2004)

im.

Plasmids plus MIC2a, MIC2b, MIC3, MIC4, M2AP and AMA1

im.

rROP2, rGRA4, mix im. rROP2 and rGRA4 plus alum plasmid plus GRA4

Vaccinia virus strain plus ROP2

sc.

in.

im. and ip.

p.o. 30 cysts SSI119

p.o. 20–100 cysts Me49 strain

ip. 300 tachyzoites RH strain p.o. 20 cysts Me49 strain

sc. 2 × 103 tachyzoites RH strain

p.o. 70 cysts 76K strain

sc. 6 × 103 tachyzoites RH strain

p.o. 80 cysts 76K strain

p.o. 40 cysts 76K strain

p.o. 10–200 cysts IPB-G and 76K strains

Brain cyst burden

Immunization reduced 84% of brain cyst burden compared with the control group

Plasmid and rGRA4 plus alum showed a similar protective level. rROP2 only conferred protection to C3H mice

The vaccine increased the lifeexpectancy; however, it did not reduce the number of parasites in the brain

Mortality and brain cyst burden

Mortality and brain cyst burden

Significant protection of mice immunized with ESAs

A significant smaller cysts were observed in immunized animals (LTR72: 77; LTK63: 78; LT: 75; and CT: 85% protection)

Mice immunized with plasmid did not resist to challenging while mice vaccinated with ts4 resisted. Death was delayed in the BALB/c group

Mice immunized exhibited 50% (MLN) and 60% (CLN) fewer cysts than control mice

Immunization resulted in 62% of survival. All control mice died after challenge

C3H mice showed partial protection against lethal challenge and lower brain cysts. DNA vaccination did not protect BALB/c and C57BL/6 mice

Results

Mortality

Brain cyst burden

Mortality

Brain cyst burden

Mortality and brain cyst burden

Mortality and brain cyst burden

Aim

[105]

[104]

[103]

[102]

[101]

[100]

[86]

[99]

[98]

Ref.

BCG: Bacille Calmette-Guérin; CLN: Cervical lymph node; CT: Cholera toxin; d.a.c.: Days after challenge; d.a.i.: Days after infection; EC2: Chimeric protein encoding fragments of MIC2, MIC3 and SAG1; ESA: Excreted–secreted antigens; Exo-Tag: Exosomes secreted by dendritic cells pulsed in vitro with Toxoplasma-derived antigens; FCA: Freund’s complete adjuvant; FIA: Freund’s incomplete adjuvant; GERBU: Adjuvant based on cationic lipid solid nanoparticles and glycopeptides derived from Lactobacillus bulgaricus cell wall; GRA: Dense granule antigen; ii.: Intraintestinal; im.: Intramuscular; in.: Intranasal; ip.: Intraperitoneal; iv.: Intravenous; LT: Heat-labile enterotoxin (LTR72 and LTK63 mutants); MAP: Multiple antigenic peptide; MIC: Microneme protein; MLN: Mesenteric lymph nodes; NISV: Nonionic surfactant vesicle; OF1: Oncins France 1; pIL-12: Plasmid-encoding IL-12; p.o.: Per oral; ROP2: Rhoptry 2 protein; SAG: Surface antigen; sc.: Subcutaneous; STAg: Soluble tachyzoites antigen; TSo: Tachyzoites sonicated.

BALB/c

BALB/c (8–10 weeks)

Daryani et al. (2003)

ESAs plus FCA and FIA

SAG1 plus CT, LT, LTR72 or LTK63

Beguetto et al. (2005)

im.

im.

Route of Challenge immunization

Passive transfer of CLN iv. or MLN lymphoid cells from mice in. vaccinated with SAG1 plus CT

CBA/J

CBA/J

Velge-Roussel et al. (2000)

Plasmid plus GRA4

Bonenfant et al. (2001)

C57BL/6 (6–8 weeks)

Desolme et al. (2000)

Plasmid plus GRA1, GRA7 and ROP2

Plasmid plus ROP2 and ts4

C57BL/6, BALB/c, C3H (6 weeks)

Vercammen et al. (2000)

Antigen and adjuvant

Leyva et al. (2001) BALB/C, C57BL/6, CBA/J

Animal

Study (year)

Table 1. Toxoplasma gondii vaccine studies in mouse models.

Review Garcia

Expert Rev. Vaccines 8(2), (2009)

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BALB/c

C3H/HeN

Swiss

ICR mice

Igarashi et al. (2008)

Cuppari et al. (2008)

Jongert et al. (2008)

Qu et al. (2008) p.o.

im.

im.

in.

im.

im.

iv.

ip. 500 tachyzoites RH strain

p.o. 20 cysts 76K strain

p.o. 20–50 cysts ME49 strain

p.o. 50 cysts VEG strain

p.o. 20–100 cysts 76K strain

ip. 104 tachyzoites RH strain

p.o. 30–80 cysts 76K strain

Mortality

Evaluate protein– protein and protein– DNA vaccine

Mortality and brain cyst burden

Brain cyst burden

Tested immunity conferred by cocktail of DNA vaccines

Mortality

Mortality and brain cyst burden

Mortality and brain cyst burden

Aim

Orally vaccinated animals were better protected than unvaccinated animals

Protein–protein vaccine conferred better protection than protein–DNA vaccine

Partial protection

Mice vaccinated had a partial protection against cyst formation (58%)

Animals that received pGRA7 had optimal protection

Vaccinated mice showed higher survival rates plus pIL-12

CBA/J mice and C57BL/6 mice showed fewer cysts and survival rates than controls, respectively

Vaccinated mice were protected in both lethal and sublethal challenge

Results

[113]

[112]

[111]

[110]

[109]

[108]

[107]

[106]

Ref.

BCG: Bacille Calmette-Guérin; CLN: Cervical lymph node; CT: Cholera toxin; d.a.c.: Days after challenge; d.a.i.: Days after infection; EC2: Chimeric protein encoding fragments of MIC2, MIC3 and SAG1; ESA: Excreted–secreted antigens; Exo-Tag: Exosomes secreted by dendritic cells pulsed in vitro with Toxoplasma-derived antigens; FCA: Freund’s complete adjuvant; FIA: Freund’s incomplete adjuvant; GERBU: Adjuvant based on cationic lipid solid nanoparticles and glycopeptides derived from Lactobacillus bulgaricus cell wall; GRA: Dense granule antigen; ii.: Intraintestinal; im.: Intramuscular; in.: Intranasal; ip.: Intraperitoneal; iv.: Intravenous; LT: Heat-labile enterotoxin (LTR72 and LTK63 mutants); MAP: Multiple antigenic peptide; MIC: Microneme protein; MLN: Mesenteric lymph nodes; NISV: Nonionic surfactant vesicle; OF1: Oncins France 1; pIL-12: Plasmid-encoding IL-12; p.o.: Per oral; ROP2: Rhoptry 2 protein; SAG: Surface antigen; sc.: Subcutaneous; STAg: Soluble tachyzoites antigen; TSo: Tachyzoites sonicated.

DNA vaccine delivery in attenuated Salmonella typhimurium (pSAG1)

rEC2 + rGRA7 plus GERBU; rEC2 + pGRA7 plus GERBU

RTgPI-1 plus Al2O3

rROP2, rGRA5 and rGRA7 plus CT

Cocktail DNA vaccine (pGRA1, pGRA7 and pROP2)

C3H/HeN

Exo-TAg

Jongert et al. (2007)

CBA/J, C57BL/6

Beauvillain et al. (2007)

p.o. 15–40 cysts 76K strain

in.

Toxoplasma gondii RNA

Multiantigenic DNA vaccine (pSAG1-ROP2)

C57BL/6 8–10 weeks)

Dimier-Poisson et al. (2006)

Route of Challenge immunization

Antigen and adjuvant

Zhang et al. (2007) BALB/c

Animal

Study (year)

Table 1. Toxoplasma gondii vaccine studies in mouse models.

Vaccination concepts against Toxoplasma gondii

Review

221

Review

Garcia

there any efficient killed vaccines for T. gondii? Answering these questions is not an easy task. The mechanisms of host cell invasion are very complex and involve a series of events and secreted– excreted proteins, and our understanding of the cell penetration process remains incomplete. Long-lasting protective immunity for T. gondii is associated with a cellular immune response, which is not easy to reach with killed vaccines, and this parasite has a wide range of important proteins for its survival inside cells. Our understanding of these proteins is also poor.

DNA and recombinant protein vaccines. Meanwhile, this arsenal of weapons was not sufficient to be used effectively as a production vaccine. A vaccine for toxoplasmosis must have different antigens expressed in all stages of the parasite associated with the adequate adjuvant and immunization route. Vaccination against bacterial and viral diseases is widespread, routine and successful, but only a few vaccines for veterinary protozoan diseases have been obtained, but none so far for human use. Financial & competing interests disclosure

Five-year view

The first and only commercial vaccine for toxoplasmosis that exists was launched in 1992 and consists of live tachyzoites of the S48 T. gondii strain to be used in sheep. However, it is infective for humans, expensive and has a short shelf-life. In the past, there have been technological improvements to produce vaccines, mainly

The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Key issues • A useful Toxoplasma gondii vaccine for pregnant females needs to be adequate for use without infecting the fetuses and should protect against transplacental infection. • A T. gondii vaccine for nonpregnant young women, who are negative for T. gondii and for people at high risk of acquiring ocular toxoplasmosis (e.g., people from south Brazil) must be safe (even if with live vaccine) and have a reasonable shelf-life. • A vaccine for improving human health needs to avoid oocyst shedding by cats and tissue cyst formation in animals. • Immunization of cats with crude rhoptry proteins has resulted in reduced oocyst shedding. • The use of recombinant proteins and DNA vaccines will be the future for vaccine production. • Live vaccines can be used for animal protection; however, they pose a risk of infecting handlers and this type of vaccine has a relatively reduced shelf-life. • It will be important to produce a subunit vaccine where antigens can be differentiated from natural infection.

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Affiliation •

João Luis Garcia, MVD, MSc, PhD Professor of Parasitology, Department of Preventive Veterinary Medicine, Londrina State University, Campus Universitário, Rodovia Celso Garcia Cid, Pr 445 Km 380, Cx. Postal 6001, Londrina, PR 86051-990, Brazil Tel.: +55 433 371 5871 Fax: +55 433 371 4485 [email protected]

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