Toxoplasma gondii tkl1 Deletion Mutant Is a Promising Vaccine against Acute, Chronic, and Congenital Toxoplasmosis in Mice

Toxoplasmosis, caused by the obligate intracellular parasite Toxoplasma gondii, can affect nearly all warm-blooded animals and humans (1). Humans acquire infection through ingestion of raw or undercooked meat containing tissue cysts, food or water contaminated with oocysts, or congenitally via the placenta (2). Toxoplasmosis is a major cause of morbidity and mortality worldwide, especially in immune-compromised individuals and the unborn fetus, and causes significant economic losses to the livestock industry (3–5). Current treatment relies on the use of antiparasitic drugs, the efficacies of which have been compromised by side effects, the inability of eliminating tissue cysts, and the development of drug-resistant parasite strains (6). Therefore, development of a vaccine to protect against toxoplasmosis has been a priority, given the global burden of disease in at-risk immunocompromised individuals and pregnant women.

Vaccine is a promising approach for efficient long-term disease control and prevention while reducing the side effects of and reliance on chemotherapeutics (7). Several studies have employed various vaccination strategies, including inactivated vaccines, DNA vaccines, epitope vaccines, protein vaccines, nanovaccines, live vector-based vaccines, and live attenuated vaccines, with the ultimate goal to develop effective vaccines against toxoplasmosis (7). Among these, vaccination with a live attenuated strain is the most efficient vaccination strategy to confer a protective immune protection (7). A live attenuated vaccine (Toxovax) derived from S48 T. gondii tachyzoites is licensed in several countries for veterinary use to protect sheep against toxoplasmosis (8). However, this naturally attenuated vaccine has a relatively short shelf life and cannot fully block vertical transmission. Additionally, there is a possibility of reversion to a virulent phenotype, which may pose a risk to vulnerable hosts (8). In contrast, genetic deletions that modify the infectious potential of parasites may prevent reversion to the pathogenic status compared with the naturally attenuated strain (9). In Plasmodium, genetic engineering of attenuated malaria parasites has been widely used to protect against Plasmodium infection in animal models and humans, and vaccination-induced–sterilizing immunity has been reported in animal models and humans (10–12). However, to date, only a few gene-deleted attenuated strains have been constructed and have offered good protection against T. gondii infection (13–16). For example, the RHΔgra17 and PruΔcdpk2 strains have shown promising potentials as vaccines against acute, chronic, and congenital T. gondii infection in mice (13, 14).

T. gondii lacking tyrosine kinase–like 1 (tkl1) has limited ability to attach to the host cell, which, in turn, impairs parasite progression through the lytic cycle in vitro and results in a loss of virulence in mice, suggesting that tkl1 is an essential virulence factor with a potential immunogenicity (17). In this study, we sought to investigate the immune protective potential of a T. gondii strain lacking the tkl1 gene in a mouse model of T. gondii infection. Kunming mice were infected orally (i.e., the natural infection route) with oocysts or tissue cysts (i.e., the main infective stages of T. gondii). A significant level of protection was offered by vaccination using 1 × 10⁶ T. gondii RHΔtkl1 tachyzoites against acute, chronic, and congenital toxoplasmosis.

Kunming mice (Beijing strain) between 7 and 9 wk of age were obtained from the Center of Laboratory Animals of Lanzhou Veterinary Research Institute. All procedures were approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Kunming mice were selected because of their ability to produce more pups per litter, thus facilitating the evaluation of the vaccine-induced protection against congenital infection compared to C57BL/6 mice (18). Mice were housed under pathogen-free conditions in a controlled room under stable conditions (12/12–h dark/light cycle, 50–60% humidity, and 22°C temperature). Mice had free access to sterilized water and food ad libitum. Mice were acclimated for 1 wk before use in the experiment. Tachyzoites of T. gondii wild-type (WT) (RH, TgC7, PYS) and mutant (RHΔtkl1) strains were cultured in human foreskin fibroblast monolayers, and tissue cysts of the Prugniuad (Pru) strain were prepared as previously described (13, 14).

Infective oocysts of type 2 T. gondii Pru strain were obtained and purified according to a previously described method (19). Briefly, T. gondii Pru cysts were obtained from the brains of orally infected Kunming mice and maintained by monthly passage. After verification of the tissue cyst status by histological examination, a 10-wk-old specific pathogen–free kitten was fed with ∼100 freshly isolated tissue cysts. The cat feces were collected and examined daily for the presence of oocysts. Once detected, Pru oocysts were collected from the feces using a discontinuous cesium chloride gradient method, as described previously (20). To induce sporulation, the purified oocysts were suspended in 2% sulfuric acid and aerated on a shaker for 5–8 d at ambient temperature. Sporulated oocysts were washed twice with 0.85% saline and stored in 2% sulfuric acid at 4°C until use.

The tkl1 gene was deleted using clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 method, as previously described (13, 14). Briefly, single-guide RNA (sgRNA) of tkl1 was engineered into pSAG1::CAS9-U6::sgUPRT by PCR using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs), and the DHFR* (resistance cassettes) were amplified from the plasmid pUPRT-DHFR-D. Approximately 10 μg of purified DHFR* amplicons and 50 μg of positive plasmids were cotransfected into freshly harvested RH tachyzoites by electroporation. Positive clone was selected with pyrimethamine and examined by PCR analysis. All sgRNA, primers, and plasmids used in this study are listed in Table I.

Different doses (1 × 10², 1 × 10³, 1 × 10⁴, 1 × 10⁵, and 1 × 10⁶) of freshly egressed tachyzoites of RHΔtkl1 or WT RH were injected i.p. into Kunming mice (six mice per dose). The infected mice were observed at least once daily for the clinical signs of toxoplasmosis, and mortality was recorded for 30 d.

Kunming mice were vaccinated with 1 × 10⁶ tachyzoites of RHΔtkl1 or mock-vaccinated in a total volume of 200 μl of PBS by i.p. injection (Fig. 1). In vitro plaque assays were used to confirm the viability of the RHΔtkl1 prior to infection of the mice. Serum samples obtained at 2 mo after vaccination (1:25 diluted with PBS) were used to evaluate the levels of anti–T. gondii IgG, IgG1, and IgG2a Abs using ELISA, as previously described (13, 14). The level of secreted cytokines IL-2, IL-4, IL-5, IL-10, IL-12, and IFN-γ in mouse splenocyte supernatants were measured, as previously described (13, 14).

To assess the protective efficacy of RHΔtkl1 against acute infection, mice were infected i.p. with 1 × 10³ type 1 RH, TgC7, or PYS strains, at 2 mo after vaccination. Both TgC7 and PYS belong to ToxoDB#9 genotype, which is the predominant genotype in China with a similar virulence to type 1 RH (21). The ability of RHΔtkl1 to confer protection against challenge using cysts or oocysts were also evaluated by orally infecting mice using 10 or 100 tissue cysts or infective oocysts of type 2 Pru strain, respectively. These animals were observed daily for the development of clinical signs of toxoplasmosis, and mortality was recorded for 30 d. For protection against chronic infection, mice that survived at day 30 postinfection were euthanized, and their brain tissue cyst burdens were determined as previously described (13, 14).

Two months after vaccination, two female and one male mice per cage were housed overnight for mating, and the presence of a vaginal plug in the females was considered as day 1 of gestation. On day 5 of gestation, mice were orally administrated with 10 oocysts of Pru strain. Six mock-vaccinated uninfected mice and six mock-vaccinated mice orally administrated with 10 oocysts served as negative and positive controls, respectively. The Pru strain was used because the majority of human toxoplasmosis cases have been reported to be associated with type 2 strains (22). The protective efficacy of RHΔtkl1 strain against congenital T. gondii infection was assessed by analyzing litter size and survival rate of delivered pups at birth and at 30 d old. The body weight and brain tissue cyst burden of pups at 30 d old was also examined. In addition, the brain cyst burden of the dams on day 30 after delivery was determined.

All procedures were performed 2 mo after RHΔtkl1 immunization. Depletion of CD8⁺ and CD4⁺ T cells was achieved by i.p. inoculation of mice with 500 μg of anti-CD8 (YST-169.4) and 500 μg of anti-CD4 (GK1.5) mAbs (Bio X Cell, West Lebanon, NH), respectively. B cells were depleted by i.p. inoculation of mice with a combination of 300 μg of anti-B220 (RA3.3A1/6.1) and 300 μg of anti-CD19 (1D3) (Bio X Cell), as previously described (23). Mice were treated with these Abs on days −3, −2, −1, and 0, then every other day up to 3 wk. Parasite challenge (i.p. inoculation with 1 × 10³ tachyzoites of T. gondii RH strain) was performed 3 d after cell depletion. Control mice were treated with rat IgG. All mice were observed daily for signs of mortality.

For passive serum transfer experiments, RHΔtkl1-immune serum or naive serum from uninfected mice (300 μl) was transferred to recipient mice by i.v. route in the tail on the day of challenge (i.p. infection with 1 × 10³ RH tachyzoites) and daily for 5 d. Also, splenocytes were obtained from vaccinated mice at 2 mo after vaccination and were used for adoptive transfer into naive recipient mice. The splenic B cells, CD4⁺ and CD8⁺ T cells, were isolated from total splenocytes obtained from vaccinated mice using the EasySep Mouse Positive Selection Kit II (STEMCELL Technologies), as described previously (16). Naive mice received splenocytes (5 × 10⁸), CD19⁺ B cells (5 × 10⁷), CD4⁺ T cells (5 × 10⁷), CD8⁺ T cells (5 × 10⁷) from RHΔtkl1-vaccinated mice, or splenocytes (5 × 10⁸) from naive mice via tail i.v. injection. Twenty-four hours later, mice were i.p. inoculated with 1 × 10³ RH tachyzoites (24).

All statistical analyses were performed using GraphPad Prism version 5 (GraphPad Software, San Diego, CA). Levels of significance of the differences between groups were determined using two-tailed, unpaired Student t test (for comparing means between two groups) and one-way ANOVA (for comparing means between ≥3 groups). The log-rank (Mantel–Cox) test was used to determine significant differences in survival among the vaccinated and control groups. A p value < 0.05 was designated statistically significant.

CRISPR–Cas9 system was used to successfully disrupt the tkl1 gene in the RH strain by insertion of DHFR* into the guide RNA–targeted coding sequence region through nonhomologous end joining (Figs. 1, 2A). A single, stable RHΔtkl1 clone was generated and verified by specific PCR assay (Fig. 2B). In this study, we show that deletion of tkl1 (RHΔtkl1 mutant) leads to a complete attenuation of the virulence of WT T. gondii in mice. Virulence assays revealed that mice infected with up to 1 × 10⁶ RHΔtkl1 tachyzoites survived and exhibited no clinical signs of toxoplasmosis over the course of the entire 30-d experiment, whereas mice infected with 100 WT RH tachyzoites died within 8 d postinfection (dpi) (Fig. 2C). Thus, in all vaccination studies, mice were vaccinated with 1 × 10⁶ RHΔtkl1 tachyzoites because this dose achieved the required balance between safety and immunogenicity (Table I).

The sera from vaccinated mice were used to determine T. gondii–specific Ab response. A high level of anti–T. gondii IgG was observed in the serum of the vaccinated mice, suggesting that RHΔtkl1 mutant induced a strong humoral response. To characterize whether a Th1 and/or Th2 immune response was induced in the vaccinated mice, the IgG subclasses (IgG1 and IgG2a) were measured. Compared with mock-vaccinated mice, the levels of IgG1 and IgG2a were significantly higher in RHΔtkl1-vaccinated mice, and the levels of IgG2a were statistically higher than IgG1, suggesting that vaccination with RHΔtkl1 in mice elicits a mixed Th1/Th2 immune response with a predominant Th1 response 2 mo after vaccination (Fig. 3). Cytokine production by soluble T. gondii Ag–stimulated splenocytes were measured by ELISA at day 60 after vaccination. Supernatants from soluble T. gondii Ag–stimulated splenocyte cultures from vaccinated mice contained more Th1-type cytokines (IL-2, IL-12, and IFN-γ) and Th2-type cytokine (IL-10) than supernatants from mock-vaccinated mice, whereas the levels of other Th2-type cytokines (IL-4 and IL-5) were not increased in the vaccinated mice (Fig. 4).

Two months after vaccination, six mice were challenged with 1 × 10³ tachyzoites of type 1 strain RH or ToxoDB#9 strains (TgC7 or PYS). The survival of the mice was recorded for 30 d. Vaccination with RHΔtkl1 tachyzoites produced remarkable protection against subsequent T. gondii infection, regardless of the challenging strain used. All mock-vaccinated mice challenged with RH, TgC7, or PYS strain died within 10 dpi (Fig. 5). These results indicate that vaccination with RHΔtkl1 tachyzoites can protect against lethal acute challenge with WT T. gondii tachyzoites.

To investigate the immune protection induced by RHΔtkl1 vaccination against infection with cysts or oocysts, mice were orally administrated with cysts or oocysts of Pru strain 2 mo after RHΔtkl1 vaccination. All mock-vaccinated mice infected with a high (100) dose of cysts or oocysts appeared moribund, and the survival rates of mock-vaccinated mice that were orally administrated with a low (10) dose of cysts or oocysts were 75 and 62.5%, respectively. In contrast, all vaccinated mice completely survived both doses of challenge regardless of the type of parasite infective stage used (Fig. 6). One month later, the brain cyst burden in the mice that survived was determined. Remarkably, mice vaccinated with RHΔtkl1 and infected with a low dose of cysts or oocysts contained fewer cysts per brain (49 ± 27 [cyst-infected group] and 46 ± 42 [oocyst-infected group]) compared with mock-vaccinated mice (3215 ± 242 [cyst-infected group] and 8991 ± 1206 [oocyst-infected group]), respectively. Interestingly, vaccinated mice infected with a lethally high cyst dose (100 cysts) or oocyst dose (100 oocysts) exhibited a decrease in brain cyst burden similar to that observed in mice challenged with the low dose. These results indicate that vaccination with RHΔtkl1 provides protective immunity against chronic toxoplasmosis regardless of the parasite infective stage used.

Pregnant mice were orally infected with 10 oocysts of Pru strain on day 5 of gestation. Mice were monitored daily for abortion, and the litter size and body weight of the pups were recorded. No abortions were observed in the dams from RHΔtkl1 vaccinated plus oocysts-infected and mock-vaccinated plus uninfected groups. In both groups, the dams successfully delivered all their pups. The litter size and body weight of pups of RHΔtkl1 vaccinated plus oocysts-infected dams were similar to that of (control) mock-vaccinated plus uninfected dams (Fig. 7A, 7B). By contrast, two dams from the mock-vaccinated plus oocysts-infected group were unable to eat or drink and thus were humanely euthanized on days 14 and 17 postinfection, and their uteri showed severe pathologic lesions (Fig. 7C). In addition, abortions were observed (i.e., no neonates were delivered) in four dams, including one dam that was unable to reach water or food and was euthanized at 21 dpi. On day 30 after birth, pups born to RHΔtkl1 vaccinated plus oocyst-infected dams were humanely euthanized and their brain tissue cyst burden was determined. Few brain cysts (43 ± 29) were detected in 14.3% (11 of 77) of the pups born to RHΔtkl1 vaccinated plus oocyst-infected dams, and no brain tissue cysts were detected in the rest (66 of 77) of the pups. Also, brain tissue cyst burden in the surviving dams was determined at day 30 postpartum. The average brain cyst number was significantly higher in mock-vaccinated plus oocyst-infected dams (11,139 ± 1,197 cysts/brain) than in the RHΔtkl1 vaccinated plus oocyst-infected dams (52 ± 29 cysts/brain).

As mentioned above, high levels of specific anti–T. gondii IgG were produced in RHΔtkl1-vaccinated mice. To determine the roles of vaccine-elicited immune serum in inhibiting T. gondii infection, naive mice were infected i.p. with 1 × 10³ RH tachyzoites and received serum from the RHΔtkl1-vaccinated or naive mice for 5 d. Results showed that adoptive transfer of the sera of vaccinated mice enhanced the survival time of infected mice compared with mice transferred with sera of naive mice (Fig. 8A). However, adoptive transfer of the sera of vaccinated mice did not completely protect mice against T. gondii infection.

Although infection by live attenuated RHΔtkl1 strain provided strong protection from challenge with pathogenic strains in mice, little is known about which immune effectors are necessary for protection. Hence, we evaluated whether depletion of vaccinated mice of CD4⁺ T, CD8⁺ T, or B cells could increase their susceptibility to infection. We observed that selective depletion of CD4⁺ T or B cells did not render mice susceptible to infection with pathogenic T. gondii RH strain. All of the anti-CD4⁺– and rat IgG isotype control-treated mice survived, whereas 83.3% of B cell–depleted mice and only 16.7% of CD8⁺ T cell–depleted mice survived the RH infection (Fig. 8B). The depletion of CD8⁺ T cells rendered mice vulnerable to infection, causing significantly faster lethality when compared with the other mouse groups depleted of CD4⁺ T cells or B cells (Fig. 8B). These results confirm the protective role of CD8⁺ T cells in T. gondii infection and reveal the fundamental role of CD8⁺ T cells in the primary response to a lethal T. gondii infection.

To confirm that CD8⁺ T cells are the major lymphocyte subpopulation involved in the protective immune response to T. gondii infection, total splenocytes from naive mice or purified CD19⁺ B cells, CD4⁺ T cells, CD8⁺ T cells, or total splenocytes from RHΔtkl1-vaccinated mice were adoptively transferred into naive recipients, and the survival of adoptively transferred mice was monitored postinfection with 1 × 10³ RH tachyzoites. All naive mice treated with RHΔtkl1-vaccinated splenocytes or purified CD8⁺ T cells survived challenge infection. By contrast, mice receiving RHΔtkl1-vaccinated CD19⁺ B cells, CD4⁺ T cells, or naive splenocytes succumbed to illness by day 9 after RH infection (Fig. 8C).

Considerable work has gone into the development of a protective vaccine against toxoplasmosis (7). In previous studies, i.p. challenge with T. gondii tachyzoites, which does not mimic the natural infection route, has been frequently used in vaccination experiments (7). Also, although oral challenge has been used to mimic natural infection, most studies have focused on oral infection with parasite cysts, with few studies using cat-derived oocysts (7). In the current study, we immunized Kunming mice with RHΔtkl1 tachyzoites and evaluated the vaccine’s protective efficacy against acute and chronic toxoplasmosis induced by oral administration of tissue cysts or oocysts, respectively. We also tested the efficacy of the RHΔtkl1 vaccine against congenital toxoplasmosis, by establishing a mouse model based on challenging the dams orally with T. gondii Pru oocysts. This model provided advantages, such as mimicking the natural route of infection of dams and their offspring, offering the opportunity to examine long-term protective efficacies against vertical transmission.

In the current study, immunity induced by vaccination with RHΔtkl1 completely protected all mice against a lethal challenge with type 1 or ToxoDB#9 tachyzoites. Likewise, vaccine-induced immune response protected mice against chronic toxoplasmosis, as indicated by the significant reduction in the brain tissue cyst burden compared with mock-vaccinated mice. This protection offered by RHΔtkl1 vaccination seems to be mediated by enhanced cell- and humoral-mediated immune responses. In addition to the demonstrated effect of CD8⁺ T cells in controlling T. gondii infection, a contribution of this cell population to the reduction of the cyst burden in the mouse brain during chronic T. gondii infection has been also shown previously (25). A requirement for humoral- and cell-mediated immunity, has been reported for mice challenged with virulent T. gondii parasites after vaccination with attenuated tachyzoites, suggesting that Ab-mediated immunity also contributes to the protection against T. gondii (26). Consistent with other live attenuated toxoplasmosis vaccines, vaccination with RHΔtkl1 elicited a high level of anti–T. gondii IgG Abs (13–16). These specific IgG Abs can opsonize parasites by phagocytosis, hinder the parasite invasion and attachment to host cells, and activate the classical complement pathway (26). Although serum from RHΔtkl1-vaccinated mice have opsonizing Ab that can limit T. gondii propagation, protection offered by immune serum was limited, as shown by modest increase in the survival of naive mice treated with adoptively transferred serum from vaccinated mice compared with mice that received serum from naive mice (Fig. 8A).

Mice vaccinated with RHΔtkl1 developed a predominant Th1 type immune response, as indicated by a higher IgG2a to IgG1 ratio. These patterns of IgG subclasses were supported by the cytokines measured in the supernatants of splenocyte cultures, in which significantly higher levels of Th1-type cytokines (IL-2, IL-12, and IFN-γ) were detected in RHΔtkl1-vaccinated mice compared with mock-vaccinated mice. Previous studies have highlighted that elevated proinflammatory Th1-type cytokines, in particular, IL-12 and IFN-γ, are necessary for limiting T. gondii infection (27) and that Th1-biased immune response elicited by T. gondii infection leads to primarily CD8⁺ T cell–mediated immunity and control of parasite infection (28, 29). Mechanistically, lack of CD8⁺ T cells impairs the production of IFN-γ and IL-12, which is a major mediator of resistance against T. gondii infection (30). IL-12 cytokine has been shown to play a critical role in eliciting IFN-γ production by CD8⁺ T cells during acute toxoplasmosis (31).

Lower levels of Th2-type cytokines, IL-4 and IL-5, were detected in RHΔtkl1-vaccinated mice compared with Th1-type cytokines. IL-4 is a Th2-type cytokine, produced in response to receptor activation by CD4⁺ T cells, basophils, and mast cells, and possesses B cell–stimulatory and Th2-promoting properties (32). IL-4 functions are generally antagonistic to those of IFN-γ, which is in line with the increased IFN-γ and low IL-4 levels seen in our study. Elevated IFN-γ production, together with a low level of IL-4, was also detected in mice immunized with Mic1-3KO (15). IL-4 also promotes isotype switching in murine B cells to IgG1 and IgE but inhibits switching to IgG2a, IgG2b, and IgG3. This is also in agreement with the low IL-4 and a high IgG2a to IgG1 ratio observed in our study, providing more evidence of a biased Th1 type immune response.

Cell depletion and adoptive cell transfer approaches have demonstrated a protective role of CD4⁺ T and CD8⁺ T cells in mice infected with T. gondii (33). In the current study, using cell depletion and adoptive cell transfer, we demonstrated a critical role for CD8⁺ T cells from RHΔtkl1-vaccinated mice on the resistance of naive mice to a lethal T. gondii challenge in a mechanism that required IFN-γ production and CD8⁺ T cells in the recipient mice. The depletion or adoptive transfer of B cells and CD4⁺ T cells had no significant influence on T. gondii infection. These findings are consistent with the results reported in previous studies using various T. gondii–attenuated vaccine models, which suggest a crucial role of CD8⁺ T cells in the protective immune response to T. gondii (15, 16).

To evaluate protection against congenital toxoplasmosis, pregnant mice were orally administrated with 10 oocysts on day 5 of gestation, which corresponds to T. gondii infection in the first trimester. Consistent with the fact that infection during the first trimester causes abortion (34), all pregnant mice from the mock-vaccinated plus oocyst-infected group aborted, and no new-born mouse survived. In contrast, all pregnant mice from the RHΔtkl1-vaccinated plus oocyst-infected group delivered their pups successfully, and no abortion was observed. Moreover, the survival rate and body weight of the pups at day 30 were similar to that of pups born to mock-vaccinated plus uninfected dams. During pregnancy the maternal immune response is biased toward a Th2 humoral type to accommodate the growing fetus and support the pregnancy (35); however, this can increase susceptibility to infection. In contrast, Th1 cellular response is required to prevent congenital transmission and to protect pregnant mice against infection (36, 37). For example, a higher rate of maternal–fetal transmission of T. gondii was observed in IFN-γ–deficient C57BL/6 mice compared with WT mice (38). However, excessive inflammatory response can be detrimental to pregnancy and deleterious to the fetus. The pregnancy outcome of C57BL/6 mice infected with T. gondii was improved by treatment with rIL-10 and deteriorated in IL-10–deficient mice compared with T. gondii–infected naive mice (39). Therefore, a balanced pro/anti-inflammatory immune response is essential to achieve a successful pregnancy. In our study, mice vaccinated with 1 × 10⁶ RHΔtkl1 tachyzoites mounted high levels of Th1-type proinflammatory cytokines (IL-2, IL-12, and IFN-γ), which were balanced with increased production of the anti-inflammatory IL-10 cytokine. This immune balance is essential for minimizing host damage caused by excessive inflammatory responses (40–42) and has probably contributed to better pregnancy outcomes in vaccinated mice compared with mock-vaccinated plus infected mice.

In conclusion, data from our studies show that transfers of immune spleen cells into naive mice conferred significant protection against lethal T. gondii infections, and transfers of purified lymphocyte subsets demonstrated that this effect required immune responses involving CD8⁺ T cells. In addition, passive immunization experiments demonstrated that Abs alone did not prevent infection but slightly improved the survival rate of infected mice. These results suggest that, in combination with a Th1-skewed immune response, CD8⁺ T cells correlate with RHΔtkl1-mediated protection from T. gondii challenge in mice. Considering complete attenuation of its virulence, the high level of immune response mounted against T. gondii challenge, and the significant survival advantage in inoculated mice when used as a vaccine, the RHΔtkl1 strain seems to be a promising vaccine candidate. These findings may have implications for toxoplasmosis vaccine design in general.

The authors have no financial conflicts of interest.