Immunization with Live-Attenuated RHΔhad2a Strain Confers Partial Protective Immunity against Acute and Chronic Infection of Toxoplasma gondii in Mice

Toxoplasmosis caused by Toxoplasma gondii is an important zoonosis of human and animal health significance. Current chemical therapeutics have side effects, and no commercially available vaccine is licensed for the prevention of toxoplasmosis in humans and most animals. Developing a safe and effective vaccine with long-term protection against T. gondii infection is necessary to control toxoplasmosis. HAD2a is a key member of the haloacid dehalogenase (HAD) phosphatase family, which is essential for T. gondii daughter budding. However, the role of HAD2a in T. gondii virulence remains unknown. In this study, we successfully constructed the had2a gene knockout strain in the T. gondii-type I RH strain (RHΔhad2a) and determined its role in virulence and vaccination. These results demonstrate that HAD2a played an important role in parasite daughter budding and in vitro replication. Knockout of the had2a gene attenuated the virulence of the T. gondii-type I RH strain. Vaccination with RHΔhad2a tachyzoites induced a Th1-biased immune response, provided partial protection against acute T. gondii infection in mice by highly virulent tachyzoites of RH and PYS (ToxoDB#9, Chinese I) strains, and conferred strong protection against challenge infection by cysts and oocysts of the less virulent type II Pru strain. These results demonstrate that T. gondii had2a is important for its in vitro proliferation and virulence in mice and that RHΔhad2a may be used as a candidate strain to generate a multiple gene knockout live-attenuated strain or be collaboratively applied with other live-attenuated strains to confer more effective protection against T. gondii infection.


Introduction
Toxoplasma gondii is an obligate intracellular protozoan that infects almost all warmblooded animals and approximately 25 to 30% of the world's human population [1][2][3].Foodborne transmission is the primary route of T. gondii infection in humans [4,5].The consumption of undercooked or raw meat products contaminated with cysts or ingestion of vegetables or water contaminated with oocysts may result in T. gondii infection [1][2][3][4][5].Infection with T. gondii severely threatens the health of pregnant women and immunocompromised patients (such as HIV and organ transplant patients) [5][6][7].Primary T. gondii infection during pregnancy may cause premature delivery, stillbirth, congenital abnormalities, developmental disabilities, growth retardation and meningitis in fetuses [5,6,8,9].Although significant advances have been achieved in studies of T. gondii biology and epidemiology, current toxoplasmosis treatment, mainly pharmacotherapy, can only manage acute and reactivated infections without the clearing of T. gondii tissue cysts [2,5,8].
Pathogens 2024, 13, 121 2 of 12 Preventing T. gondii infection or clearing latent infection may be achieved by vaccination.Toxovax, a live-attenuated vaccine derived from T. gondii S48 tachyzoites and licensed in a few countries, has been used in sheep against toxoplasmosis, especially preventing miscarriage in pregnant sheep [10].However, this licensed vaccine has some application defects, such as a relatively short shelf life, uncomplete block to congenital infection and a possible reversion to a virulent phenotype [8].Significant toxoplasmosis vaccine development has been explored in the last few decades, such as live-attenuated strains, DNA vaccines, epitope vaccines, exosome-based, carbohydrate-based and nanoparticlebased vaccination [8,11,12].With the application of the CRISPR-Cas9 gene-editing tool in T. gondii research, several knockout strains of important T. gondii genes have been tested in vaccination studies in mice [8,13,14].These virulence-attenuated vaccine strains include RH∆tkl1 [13], RH∆gra17∆npt1 [15], RH∆ompdc∆uprt [16], ME49∆cdpk3 [17] and Pru∆cdpk2 [18], which have provided efficient protection against acute, chronic, and congenital infection.HAD2a (TGGT1_289910), which possesses a haloacid dehalogenase (HAD) phosphatase domain, is essential for T. gondii daughter budding [19].Conditional knockout of the had2a gene leads to incomplete cytokinesis, conjoined daughters, and disrupted proliferation [19].However, the role of HAD2a in T. gondii virulence remains unknown.The in vitro replication defect caused by the conditional knockout of the had2a gene indicates that the had2a gene mutant strain might have replication defect and attenuated virulence, suggesting a potential role as a live-attenuated vaccine.Here, we successfully constructed the had2a gene knockout strain RH∆had2a and characterized the virulence of RH∆had2a in mice.The immunization results revealed that RH∆had2a vaccination significantly induced specific anti-T.gondii antibodies and conferred partial protection against T. gondii acute and chronic infection.Our findings suggest that HAD2a is important for T. gondii virulence and RH∆had2a can be used as a parental strain to generate an attenuated multiple gene knockout vaccine strain.

Animals and Ethics Statement
The female Kunming mice (6-8 weeks old) used in the experiment were purchased from the Laboratory Animal Center of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Lanzhou, China).To reduce the impact of stress response on the experiment, mice were placed in a temperature-controlled room with a 12 h light/dark cycle and provided with sufficient sterile water and food for free intake.The study was approved by Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Approval no.2020-022).Every effort was made to reduce any suffering of the animals.

Construction of had2a Gene Knockout Strain
The CRISPR-Cas9 mediated homologous gene recombination was used to delete the had2a gene in the wild-type RH strain [23].The CRISPR plasmid pSAG1::CAS9-U6::Sg HAD2a and homologous dihydrofolate reductase (DHFR) drug-selective plasmid were constructed.To generate the had2a gene knockout CRISPR plasmid pSAG1::CAS9-U6::Sg HAD2a, the SgRNA of had2a gene was used to replace the SgRNA of the uracil phosphoribosyl transferase (uprt) gene in the pSAG1::CAS9-U6::SgUPRT template plasmid [24].The 5 ′ and 3 ′ homologous arms of the had2a gene (1000~1300 bp) were fused to the DHFR fragment and the pUC19 backbone fragment to construct the homologous DHFR-selective plasmid [25].The sequence-validated plasmid was used as a template to amplify the homologous DHFR fragment.To construct the had2a gene knockout strain (RH∆had2a), the purified homologous drug-selective cassette and sequencing-validated CRISPR plasmid were co-transfected into the wild-type RH tachyzoites.After pyrimethamine selection, single clones of RH∆had2a obtained by modified limiting dilution were confirmed by designed PCRs [20].The primers used are listed in Table 1.To visualize the effect of had2a gene deletion on parasite daughter budding, the purified RH and RH∆had2a tachyzoites were used to infect the HFF monolayers.After fixed with 4% paraformaldehyde for 20 min, the samples were permeabilized with 0.2% Triton X-100 for 15 min and incubated with polyclonal rabbit anti-IMC1 (1:500, available in our lab) [20] and goat anti-rabbit IgG (H+L) conjugated with Alexa Fluor 488 (1:500, Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at 37 • C, each of which was followed by five washes with PBS [26].A Leica confocal microscope system (TCS SP8, Leica, Wetzlar, Germany) was used to image the samples.

Plaque Assay
Confluent HFF monolayers grown in 12-well plates (Thermo Fisher Scientific, USA) were infected with 300 freshly egressed tachyzoites of wild-type RH or mutant RH∆had2a strains for 7 days.After the culture medium was removed, samples were washed with PBS, fixed with 4% paraformaldehyde for 20 min and stained with 0.2% crystal violet for 20 min, which was followed by two washes with PBS [25].Three independent replicates were performed.The plaque size and number were analyzed with the ImageJ software version 1.53a.

Intracellular Replication Assay
Freshly egressed tachyzoites (1 × 10 5 ) of RH∆had2a or RH strain were used to infect the confluent HFF monolayers cultured in 12-well plates for 1 h prior to washing the uninvaded tachyzoites.The number of tachyzoites in at least 100 parasitophorous vacuoles of samples was counted using IFA after 24 h incubation, in which mouse anti-SAG1 (1:500, Thermo Fisher Scientific, USA) and goat-anti mouse conjugated with Alexa Fluor 488 were used to visualize intracellular parasites [27].

Antibodies Assessment in Immunized Mice
Kunming mice were intraperitoneally immunized with 5 × 10 4 RH∆had2a tachyzoites, which was diluted in 200 µL PBS and quantified by plaque assay to ensure its number and viability used in vaccination.Naive mice were intraperitoneally injected with 200 µL PBS.Serum samples of the naive and immunized mice (six mice per group) were collected at 30 days post-immunization.To detect whether anti-T.gondii antibodies were induced, subclass antibodies IgG1 and IgG2a along with total IgG were determined using ELISA [13,15].

Protection of RH∆had2a Immunization against T. gondii Infection
Thirty days post-vaccination with 5 × 10 4 RH∆had2a tachyzoites, 1 × 10 3 T. gondii tachyzoites of the wild-type RH strain or PYS strain were used to intraperitoneally infect each of the immunized mice and naive mice (ten mice per group).For chronic infection, the naive and immunized mice (ten mice per group) were orally administrated with 20 Pru cysts or 50 Pru oocysts.The Pru cysts were collected and purified from the brain tissues of Kunming mice infected with cysts according to previous descriptions [13,29], whereas the Pru oocysts were collected and purified from feces of kittens orally infected with cysts [13,30,31].The survival time and toxoplasmosis clinical signs of all infected mice group were observed twice daily.Mice were euthanized immediately when reaching the humane endpoint.Brain cysts of all chronically survived mice were counted as previously [20,25].For the detection of brain cyst burden, the brains of the mice that survived for 30 days post-chronic challenge were collected, homogenized, and used to count brain cyst number, as described previously [15].

Statistical Analysis
All data obtained from three independent replicates were represented as means ± standard deviations (SD).Group difference was analyzed by a two-tailed, unpaired Student's t test using GraphPad Prism version 9.0.Difference was considered significant when the p value < 0.05.

Successful Construction of RH∆had2a
To investigate the role of HAD2a in T. gondii virulence, the had2a gene was knocked out in the T. gondii-type I RH strain by replacing the had2a gene with a homologous DHFR fragment using CRISPR-Cas9 (Figure 1A).The successful construction of gene knockout strain RH∆had2a was confirmed by PCRs.In PCR1 and PCR3, recombinant 5 ′ and 3 ′ homologous fragments (~1300 bp) were amplified in the RH∆had2a strain but not in the RH strain (Figure 1B), validating the successful insertion of 5 ′ and 3 ′ homologous fragments in RH∆had2a.The small had2a coding gene fragment was amplified in PCR2 of the RH strain but not in PCR2 of the RH∆had2a strain (Figure 1B), confirming the disruption of the had2a gen in RH∆had2a.These results demonstrated that had2a gene knockout strain RH∆had2a was successfully generated by CRISPR-Cas9 mediated homologous recombination.
the humane endpoint.Brain cysts of all chronically survived mice were counted as previously [20,25].For the detection of brain cyst burden, the brains of the mice that survived for 30 days post-chronic challenge were collected, homogenized, and used to count brain cyst number, as described previously [15].

Statistical Analysis
All data obtained from three independent replicates were represented as means ± standard deviations (SD).Group difference was analyzed by a two-tailed, unpaired Student's t test using GraphPad Prism version 9.0.Difference was considered significant when the p value < 0.05.

Successful Construction of RHΔhad2a
To investigate the role of HAD2a in T. gondii virulence, the had2a gene was knocked out in the T. gondii-type I RH strain by replacing the had2a gene with a homologous DHFR fragment using CRISPR-Cas9 (Figure 1A).The successful construction of gene knockout strain RHΔhad2a was confirmed by PCRs.In PCR1 and PCR3, recombinant 5′ and 3′ homologous fragments (~1300 bp) were amplified in the RHΔhad2a strain but not in the RH strain (Figure 1B), validating the successful insertion of 5′ and 3′ homologous fragments in RHΔhad2a.The small had2a coding gene fragment was amplified in PCR2 of the RH strain but not in PCR2 of the RHΔhad2a strain (Figure 1B), confirming the disruption of the had2a gen in RHΔhad2a.These results demonstrated that had2a gene knockout strain RHΔhad2a was successfully generated by CRISPR-Cas9 mediated homologous recombination.

Knockout of had2a Gene Severely Attenuated T. gondii Virulence in Mice
To assess the effect of had2a disruption on the replication and virulence of T. gondii, we performed IFA, in vitro plaque and in vivo virulence assays using RHΔhad2a and wildtype RH strains.In IFA, a division defect was observed in RHΔhad2a (Figure 2A).Some intravacuolar tachyzoites of RHΔhad2a were enlarged without daughter budding or the separation of dividing tachyzoites, lacking the rosette appearance of wild-type RH parasites.These results confirmed the role of HAD2a in parasite daughter budding as previously described [19].In an in vitro plaque assay, the same dose of RHΔhad2a or RH tachyzoites were used to infect the HFF monolayer.After 7 days of incubation, the HFF samples were stained with crystal violet.The results showed that knockout of the had2a gene caused a significant defect in the plaque-forming ability (Figure 2B,C).The in vitro

Knockout of had2a Gene Severely Attenuated T. gondii Virulence in Mice
To assess the effect of had2a disruption on the replication and virulence of T. gondii, we performed IFA, in vitro plaque and in vivo virulence assays using RH∆had2a and wildtype RH strains.In IFA, a division defect was observed in RH∆had2a (Figure 2A).Some intravacuolar tachyzoites of RH∆had2a were enlarged without daughter budding or the separation of dividing tachyzoites, lacking the rosette appearance of wild-type RH parasites.These results confirmed the role of HAD2a in parasite daughter budding as previously described [19].In an in vitro plaque assay, the same dose of RH∆had2a or RH tachyzoites were used to infect the HFF monolayer.After 7 days of incubation, the HFF samples were stained with crystal violet.The results showed that knockout of the had2a gene caused a significant defect in the plaque-forming ability (Figure 2B,C).The in vitro parasite replication rates were also monitored 24 h post infection.The deletion of had2a led to a significant decrease in the replication of T. gondii type I strain (Figure 2D).The significant defect of RH∆had2a in plaque-forming ability and replication efficacy demonstrates that HAD2a plays an important role in the in vitro proliferation of T. gondii.
RHΔhad2a or RH tachyzoites were used to infect Kunming mice.All mice infected with different doses (10 2 , 10 3 , 10 4 , 10 5 , 10 6 ) of RHΔhad2a tachyzoites survived without any clinical symptoms of toxoplasmosis, whereas mice infected with 10 2 tachyzoites of the wildtype RH strain were sacrificed within 10 days post-infection (Figure 2E).These results suggest that HAD2a is important for T. gondii virulence and that RHΔhad2a may be a potential candidate for developing anti-T.gondii live-attenuated vaccine.

RHΔhad2a Vaccination Induces a Th1-Dominated Immune Response
To examine whether RHΔhad2a vaccination induces the production of anti-T.gondiispecific IgG, Kunming mice were immunized with 5 × 10 4 RHΔhad2a tachyzoites (Figure 3A) [25].The sera of RHΔhad2a-vaccinated and unvaccinated mice were isolated at 30 days post-infection.The total IgG, subclass antibodies IgG1 and IgG2a were determined by ELISA (Figure 3B).These results showed that the sera obtained from RHΔhad2a-vaccinated mice had significantly higher levels of IgG, IgG1 and IgG2a compared with the sera of non-vaccinated mice (Figure 4A-C).The IgG2a level was higher than the IgG1 level in the sera of RHΔhad2a-vaccinated mice (Figure 4D).These results illuminated that RHΔhad2a vaccination induced a Th1-dominated immune response and that RHΔhad2a has the potential to be a live-attenuated vaccine candidate.To test whether HAD2a is important for T. gondii virulence, different doses of RH∆had2a or RH tachyzoites were used to infect Kunming mice.All mice infected with different doses (10 2 , 10 3 , 10 4 , 10 5 , 10 6 ) of RH∆had2a tachyzoites survived without any clinical symptoms of toxoplasmosis, whereas mice infected with 10 2 tachyzoites of the wild-type RH strain were sacrificed within 10 days post-infection (Figure 2E).These results suggest that HAD2a is important for T. gondii virulence and that RH∆had2a may be a potential candidate for developing anti-T.gondii live-attenuated vaccine.

RH∆had2a Vaccination Induces a Th1-Dominated Immune Response
To examine whether RH∆had2a vaccination induces the production of anti-T.gondiispecific IgG, Kunming mice were immunized with 5 × 10 4 RH∆had2a tachyzoites (Figure 3A) [25].The sera of RH∆had2a-vaccinated and unvaccinated mice were isolated at 30 days post-infection.The total IgG, subclass antibodies IgG1 and IgG2a were determined by ELISA (Figure 3B).These results showed that the sera obtained from RH∆had2avaccinated mice had significantly higher levels of IgG, IgG1 and IgG2a compared with the sera of non-vaccinated mice (Figure 4A-C).The IgG2a level was higher than the IgG1 level in the sera of RH∆had2a-vaccinated mice (Figure 4D).These results illuminated that RH∆had2a vaccination induced a Th1-dominated immune response and that RH∆had2a has the potential to be a live-attenuated vaccine candidate.

Vaccination of RHΔhad2a Provides Partial Protection in Mice against Acute Infection of Virulent Strains
To assess the protective efficacy of RHΔhad2a vaccination against tachyzoite infection, Kunming mice were immunized with 5 × 10 4 RHΔhad2a tachyzoites (Figure 3A) [25].After

Vaccination of RHΔhad2a Provides Partial Protection in Mice against Acute Infection of Virulent Strains
To assess the protective efficacy of RHΔhad2a vaccination against tachyzoite infection, Kunming mice were immunized with 5 × 10 4 RHΔhad2a tachyzoites (Figure 3A) [25].After

Vaccination of RH∆had2a Provides Partial Protection in Mice against Acute Infection of Virulent Strains
To assess the protective efficacy of RH∆had2a vaccination against tachyzoite infection, Kunming mice were immunized with 5 × 10 4 RH∆had2a tachyzoites (Figure 3A) [25].After 30 days post-immunization, the vaccinated mice and unvaccinated mice were infected by 10 3 wild-type RH or PYS tachyzoites (Figure 3C).The results showed that all unvaccinated mice were sacrificed within 10 days when challenged with RH or PYS tachyzoites (Figure 5A,B).Although the vaccinated mice were also sacrificed, the survival time was significantly prolonged in the vaccinated group (sacrificed within 18 days) (Figure 5A,B).These results indicate that immunization of the live-attenuated RH∆had2a confers partial protection against acute T. gondii infection by virulent strains.
Pathogens 2024, 13, x FOR PEER REVIEW 8 of 12 30 days post-immunization, the vaccinated mice and unvaccinated mice were infected by 10 3 wild-type RH or PYS tachyzoites (Figure 3C).The results showed that all unvaccinated mice were sacrificed within 10 days when challenged with RH or PYS tachyzoites (Figure 5A,B).Although the vaccinated mice were also sacrificed, the survival time was significantly prolonged in the vaccinated group (sacrificed within 18 days) (Figure 5A,B).These results indicate that immunization of the live-attenuated RHΔhad2a confers partial protection against acute T. gondii infection by virulent strains.

RHΔhad2a Vaccination Confers Partial Protection against Chronic Infection
To evaluate the protective efficacy of RHΔhad2a vaccination against chronic infection, mice were orally challenged with 20 Pru cysts or 50 Pru oocysts after 30 days post-vaccination (Figure 3D).The results showed that all unimmunized mice were sacrificed during the chronic stage of challenge infection by cysts or oocysts of the Pru strain (Figure 6A,B), whereas 80% of the vaccinated mice in the cysts-challenged group and 100% of the vaccinated mice in the oocysts-challenged group survived (Figure 6A,B).At 30 days postchallenge, the brain cyst numbers of the surviving mice were determined.In immunized groups, two out of ten mice in the Pru cysts-challenged group were sacrificed, and brain cysts were detected in only three out of the eight surviving mice (median = 45 ± 32, data not shown).In the oocysts-challenged group, only three out of ten surviving mice had brain cysts (median = 20 ± 22, data not shown).The numbers of brain cysts in the surviving vaccinated mice were significantly lower than that previously reported [15,25].These results suggest that RHΔhad2a vaccination induced partial protection against chronic T. gondii infection.

RH∆had2a Vaccination Confers Partial Protection against Chronic Infection
To evaluate the protective efficacy of RH∆had2a vaccination against chronic infection, mice were orally challenged with 20 Pru cysts or 50 Pru oocysts after 30 days post-vaccination (Figure 3D).The results showed that all unimmunized mice were sacrificed during the chronic stage of challenge infection by cysts or oocysts of the Pru strain (Figure 6A,B), whereas 80% of the vaccinated mice in the cysts-challenged group and 100% of the vaccinated mice in the oocysts-challenged group survived (Figure 6A,B).At 30 days post-challenge, the brain cyst numbers of the surviving mice were determined.In immunized groups, two out of ten mice in the Pru cysts-challenged group were sacrificed, and brain cysts were detected in only three out of the eight surviving mice (median = 45 ± 32).In the oocysts-challenged group, only three out of ten surviving mice had brain cysts (median = 20 ± 22).The numbers of brain cysts in the surviving vaccinated mice were significantly lower than that previously reported [15,25].These results suggest that RH∆had2a vaccination induced partial protection against chronic T. gondii infection.
cysts were detected in only three out of the eight surviving mice (median = 45 ± 32, data not shown).In the oocysts-challenged group, only three out of ten surviving mice had brain cysts (median = 20 ± 22, data not shown).The numbers of brain cysts in the surviving vaccinated mice were significantly lower than that previously reported [15,25].These results suggest that RHΔhad2a vaccination induced partial protection against chronic T. gondii infection.

Discussion
Toxoplasmosis caused by T. gondii infection is a significant public health threat to humans and animals [5,32].Due to its elaborate immune evasion system and complex life cycle of T. gondii, it is difficult to prevent and treat toxoplasmosis [5,32].Although the combined use of pyrimethamine and sulfadiazine is the primary therapeutic regimen, failure of treatment happened quite often [8].Side effects of current therapeutics may occur in treated patients [33].Vaccination of humans and animals with effective vaccines would reduce chemical therapeutic consequences and reliance, and it offers a better alternative for the efficient and long-term control of zoonotic toxoplasmosis [8,34].
HAD2a is essential for T. gondii daughter budding, which is an important part of the parasite lytic cycle [19].In this study, we successfully knocked out the had2a gene in the RH strain and constructed an RH∆had2a mutant strain.These results of IFA, plaque assay and intravacuolar replication assay demonstrated the important role of HAD2a in daughter budding, the separation of dividing tachyzoites and intracellular proliferation, which was consistent with a previous study [19].We used 10 2 , 10 3 , 10 4 , 10 5 , and 10 6 RH∆had2a tachyzoites and 10 2 RH tachyzoites to infect Kunming mice and monitored the survival rates of the mice.All infectious doses of RH∆had2a tachyzoites were not lethal to mice and caused no clinical symptoms of toxoplasmosis, suggesting that the deletion of had2a attenuated the virulence of the RH strain, indicating the possible application of RH∆had2a as a toxoplasmosis vaccine candidate.Infection of mice with the wild-type RH strain can cause histopathological changes in the liver, spleen, brain and lung of the infected mice [35].Thus, future studies should examine the histopathological changes in the mice infected with RH∆had2a tachyzoites to evaluate its safety in mice.
Toxoplasma gondii infection induces a complex protective immunity, including the innate immune response, adaptive immune response, and humoral immunity [36,37].In this study, we found that RH∆had2a vaccination induced a significant increase in anti-T.gondii specific antibodies, with significantly higher IgG2a levels than IgG1 levels, indicating that the vaccination of mice with RH∆had2a provokes a Th1-biased immunity.The increased IgG induced by RH∆had2a immunization may opsonize T. gondii by phagocytosis, inhibiting the attachment of the parasites to host cells and blocking the parasite invasion to exert their protective effect against T. gondii infection [38].The host immunity is primarily dependent on T helper 1 (Th1) cell-mediated immunity [8,36,37].High levels of interleukin-12 (IL-12) and interferon-γ (IFN-γ) are critical for the clearance of tachyzoites during acute infection, and it is essential for the formation of tissue cysts and sustainment of latent infection [8,36,37,39].Whether RH∆had2a vaccination induces the production of specific cytokines, such as IFN-γ, TNF-a, IL-12 or MCP-1, requires further studies.In addition, the determination of cytokines and antiparasitic factors, such as reactive nitrogen species and oxygen species, in mice infected with different doses of tachyzoites may help to understand whether the possible protective effect of a live-attenuated strain is dependent on the inoculated dose.
Tachyzoites and bradyzoites are the main infectious stages of T. gondii's asexual reproductive cycle.Rapidly dividing tachyzoites can disseminate to enormous and distant host tissues and provoke significant immune responses [5].Host immune defense and drug application can limit tachyzoite growth; however, some tachyzoites overcoming these challenges transform into slowly replicating bradyzoites [5].Cyst walls protect slowly dividing bradyzoites within tissue cysts to remain dormant in hosts by evading host immune responses, facilitating the establishment of long-term persistent infection [5,40].Toxoplasma gondii infection commonly occurs in humans via the oral consumption of water, vegetables, fruits contaminated with T. gondii oocysts or undercooked meat containing T. gondii tissue cysts [41].Thus, challenge infection with tachyzoites, cysts and oocysts after the immunization of mice with live-attenuated T. gondii vaccine strains is important in anti-T.gondii vaccine research.
In this study, we found that the survival rate was significantly higher in vaccinated mice challenged with Pru cysts compared with that of unvaccinated mice.Remarkably, RH∆had2a vaccination protected all mice from Pru oocyst infection.The brain cyst number of vaccinated mice challenged with Pru cysts and oocysts was significantly decreased comparing to previous data [15,25].However, T. gondii might be detected in other organs of the infected mice, in which no brain cyst was detected [17].In addition, the T. gondii load in vaccinated mice should be assessed before and after re-infection, in future studies, to rule out the residual infection and to verify that parasites are effectively eliminated by immunization.
Comparing to the naive group, mice vaccinated with RH∆had2a tachyzoites had a significantly longer survival time when challenged with tachyzoites of the wild-type I RH strain and PYS strain (ToxoDB#9).These results demonstrate that vaccination with the RH∆had2a strain conferred partial protection against acute infection and partially prevented the establishment of parasite infection in mice challenged with the Pru strain.Unlike RH∆tkl1 [13], ME49∆α-amy [14] and RH∆gra17∆npt1 [15], RH∆had2a did not fully protect mice from the virulent RH and PYS strains.Thus, RH∆had2a may not be individually applied as an effective live-attenuated vaccine.However, RH∆had2a may be applied to generate a multiple-attenuated knockout vaccine strain in further studies.One limitation of RH∆had2a constructed in this study is that it exhibits resistance to pyrimethamine, which is one of the primary drugs for treating toxoplasmosis.Thus, RH∆had2a should not be used for humans.

Conclusions
The findings of the present study demonstrates that the deletion of had2a suppressed the daughter budding and in vitro growth, and it attenuated the virulence of the T. gondiitype I RH strain.A single vaccination intraperitoneally of 5 × 10 4 RH∆had2a tachyzoites induces a Th1-skewed immune response in mice.Immunization of the RH∆had2a mutant strain provides a significantly longer survival time for mice against acute infection by highly virulent tachyzoites compared to the naive mice, partial protection against chronic infection by Pru cysts, and strong protection against Pru oocysts.These findings show that RH∆had2a could be a promising parent strain to generate a double-or triple-gene knockout mutant strain, or it could be cooperatively applied with another live-attenuated strain as live-attenuated vaccines.

Figure 1 .
Figure 1.Construction and validation of RHΔhad2a.(A) Schematic illustration of had2a gene deletion by CRISPR-Cas9 mediated homologous gene replacement.(B) PCRs of RHΔhad2a.Recombinant homologous fragments of 5′ and 3′ in RHΔhad2a were detected by PCR1 and PCR3.Deletion of had2a gene was detected by PCR2.

Figure 2 .
Figure 2. Knockout of had2a affected T. gondii daughter budding, replication, and virulence.(A) Division defect of intracellular RHΔhad2a dividing parasites.Wild-type RH parasites show normal daughter parasite budding (white arrows), while some RHΔhad2a parasites show the aberrant morphology of intercellular parasites and improper segregation of daughter cells (red arrows).(B-D) Replication defect of RHΔhad2a parasites in vitro.Plaques comparison of wild-type RH and RHΔhad2a parasites (B).Relative size and number of plaques were significantly affected by had2a gene deletion (**** p < 0.0001) (C).(D) Intracellular replication of RH and RHΔhad2a parasites at 24 h post infection of HFFs determined in three independent experiments.RHΔhad2a parasites had a slower replication compared to wild-type RH strain (*** p < 0.001).(E) Attenuated virulence of RHΔhad2a parasites.All mice infected with different doses of RHΔhad2a tachyzoites survived, while all mice infected with wild-type RH tachyzoites were sacrificed (**** p < 0.0001).

Figure 2 .
Figure 2. Knockout of had2a affected T. gondii daughter budding, replication, and virulence.(A) Division defect of intracellular RH∆had2a dividing parasites.Wild-type RH parasites show normal daughter parasite budding (white arrows), while some RH∆had2a parasites show the aberrant morphology of intercellular parasites and improper segregation of daughter cells (red arrows).(B-D) Replication defect of RH∆had2a parasites in vitro.Plaques comparison of wild-type RH and RH∆had2a parasites (B).Relative size and number of plaques were significantly affected by had2a gene deletion (**** p < 0.0001) (C).(D) Intracellular replication of RH and RH∆had2a parasites at 24 h post infection of HFFs determined in three independent experiments.RH∆had2a parasites had a slower replication compared to wild-type RH strain (*** p < 0.001).(E) Attenuated virulence of RH∆had2a parasites.All mice infected with different doses of RH∆had2a tachyzoites survived, while all mice infected with wild-type RH tachyzoites were sacrificed (**** p < 0.0001).

Figure 3 .
Figure 3. Schematic illustration of the study design of RHΔhad2a vaccination.Experimental overview of Kunming mice vaccination with 5 × 10 4 RHΔhad2a tachyzoites (A), and determination of anti-T.gondii antibodies of vaccinated mice prior to challenge (B) at acute (C) and chronic stages of infection (D).

Figure 3 . 12 Figure 3 .
Figure 3. Schematic illustration of the study design of RH∆had2a vaccination.Experimental overview of Kunming mice vaccination with 5 × 10 4 RH∆had2a tachyzoites (A), and determination of anti-T.gondii antibodies of vaccinated mice prior to challenge (B) at acute (C) and chronic stages of infection (D).

Figure 5 .
Figure 5. RHΔhad2a immunization significantly prolonged the survival time of mice with acute T. gondii infection.The naive and immunized mice were challenged with 1 × 10 3 tachyzoites of type I RH strain (A) or PYS (ToxoDB#9, Chinese I) strain (B) 30 days after immunization (*** p < 0.001, **** p < 0.0001).The survival time of the vaccinated mice was significantly longer compared to that of the naive mice.

Figure 5 .
Figure 5. RH∆had2a immunization significantly prolonged the survival time of mice with acute T. gondii infection.The naive and immunized mice were challenged with 1 × 10 3 tachyzoites of type I RH strain (A) or PYS (ToxoDB#9, Chinese I) strain (B) 30 days after immunization (*** p < 0.001, **** p < 0.0001).The survival time of the vaccinated mice was significantly longer compared to that of the naive mice.

Figure 6 .
Figure 6.Vaccination with RH∆had2a protected mice from chronic T. gondii infection.Survival time of the naive and immunized mice orally infected with 20 cysts (A) or 50 oocysts (B) of type II T. gondii Pru strain, 30 days after immunization with RH∆had2a tachyzoites (*** p < 0.001).Survival percentages of the vaccinated mice were significantly higher than that of the naive mice.

Table 1 .
Primers used in this study.