Generation of the RH ΔompdcΔuprt Strains Using the CRISPR/Cas9 System
To determine whether the ompdc and uprt -deficient RH mutant could be employed as a live attenuated vaccine against toxoplasmosis, the ompdc and uprt genes were deleted through a CRISPR/cas9 editing system in RHΔku80Δhxgprt strain. The HXGPRT marker was inserted into the uprt-specific guide RNA-targeted sequence region (Fig. 1a). The single and stable RHΔku80Δuprt::HXGPRT clone was verified by PCR detection (Fig. 1b). Under the effects of sgRNA of hxgprt and enzyme Cas9, the HXGPRT marker was deleted (Fig. 1c) and PCR results indicated a single RHΔku80ΔuprtΔhxgprt strain was successfully selected (Fig. 1d). Subsequently, the ompdc gene was replaced by HXGPRT marker in RHΔku80ΔuprtΔhxgprt strain to yield the RHΔompdcΔuprt mutant (Fig. 1e), and the stable mutant clone was determined by PCR and qPCR assay (Fig. 1f-g). In summary, the double gene knockout RHΔku80ΔuprtΔhxgprt strain was successfully generated.
RH ΔompdcΔuprt mutant exhibited a reduced cellular replication ability
In order to explore the biological characteristics of the RHΔompdcΔuprt mutant, invasion, parasite replication, and plaque assays were carried out. Plaque assay results showed that in the presence of uracil and ump, the mutant could grow as normally as wild type RH strain to form plagues, while rarely plagues were observed when the uracil and ump were removed from the culture medium of RHΔompdcΔuprt mutant (Fig. 2a-c). In addition, the parasite replication assay showed that at 24 h and 48 h post infection, the number of RHΔompdcΔuprt mutant tachyzoites per parasitophorous vacuoles (PV) was notably lower than that of wide type RH tachyzoites in absence of uracil and ump, and most PV of this mutant consisted 1 or 2 tachyzoites indicating a nearly quiescent condition of cellular proliferation (Fig. 2d-e, 1S). However, the number of RHΔompdcΔuprt mutant tachyzoites per PV presented a similar level with that of wide type once supplied with uracil and ump, indicating the replication restore. Furthermore, the results of invasion assay showed limited difference between wide type strain and RHΔompdcΔuprt mutant no matter supplied with or without uracil and ump (Fig. 2S), suggesting that the deletion of ompdc and uprt had no effect on invasion. Taken together, these results suggested that the RHΔompdcΔuprt mutant remarkably reduced its cellular proliferation ability.
The virulence of RH ΔompdcΔuprt mutant was severely attenuated in mice
Mice were used to evaluate the virulence of RHΔompdcΔuprt. BALB/c mice and BALB/c-nu mice were intraperitoneally (i.p.) injected with different doses of RHΔompdcΔuprt mutant tachyzoites, in parallel, mice intraperitoneally injected with 1 × 102 tachyzoites of the parental RHΔku80 strain were treated as control. The survival percentage of mice was monitored daily, and the results showed that mice injected with wild-type strain all died within 9 days, while the survival rates of RHΔompdcΔuprt mutant-injected mice were 100%, even when challenged with infectious high dose of 106 tachyzoites (Fig. 3a-b), indicating the virulence of this mutant was significantly attenuated. Subsequently, ascites samples from different groups of mice were collected from 1 dpi to 5 dpi for parasite examination. The parasite number in wide type-infected mice increased remarkably day by day, while that of RHΔompdcΔuprt-injected mice notably decreased every day (Fig. 3c). Similarly, a large number of T. gondii tachyzoites were detected in the liver and lung tissues of RHΔku80-injected mice, while limited tachyzoites were detected by qPCR in these tissues from RHΔompdcΔuprt-injected mice on the 4th day post infection (Fig. 3d-e). These results demonstrated a significant reduce of parasite burden caused by RHΔompdcΔuprt mutant. Next, the histological section of liver, spleen, and lung tissues were subjected to HE staining for pathological examination. As shown in Fig. 3f, rarely changes were observed in tissue sections from RHΔompdcΔuprt-injected mice compared with naïve one, while in wild type infected mice, large amount of necrotic cells and absent lymphoid follicles were appeared in spleen tissue, obviously cellular separation was displayed in liver, and notably thicker alveolar walls were observed in lung tissues. These results indicated limited pathological change or harm would be induced by RHΔompdcΔuprt mutant to mice.
Robust humoral and cellular immune responses were elicited by vaccination of RH ΔompdcΔuprt mutant in mice
The previous results indicated the RHΔompdcΔuprt mutant could be used as a candidate vaccine, in order to furtherly illuminate the mechanism of immune response induced by the mutant, the vaccination procedure was performed as described in Fig. 4a, and the analysis of antibody and T cell subsets were carried out. Sera obtained from mice were used to determine the specific antibody response. Significantly high levels of anti-T. gondii IgG were detected in vaccinated mice, and the IgG titer was increased remarkably after every vaccination (Fig. 4b), indicating a robust humoral response was induced. The levels of IgG subclasses (IgG1 and IgG2a) were tested to characterize the immune response type. Both the levels of IgG1 and IgG2a were significant higher in RHΔompdcΔuprt –vaccinated mice compared with control mice (Fig. 4c). In addition, the level of IgG2a was notable higher than that of IgG1, indicating that the vaccination of RHΔompdcΔuprt in mice elicited a Th1/Th2 mixed and Th1-biased immune response.
In determining what T lymphocytes may be contributing to the development of protective immunity against T. gondii the flow cytometry was used to analyze based on differential cell populations. After immunization with RHΔompdcΔuprt mutant, a significant increase in percentages of CD3+CD4+ T cell by 2 weeks post vaccination were observed compared with controls (Fig. 4d-e). Additionally, the percentage of CD3+CD8+ T cell of vaccinated mice was increased to a much higher degree (Fig. 4f-g). In parallel, the rest of splenocytes from vaccinated and unvaccinated mice were cultured in vitro for further stimulation with STAg. The cytokine levels of splenocyte supernatant were then detected by flow cytometry. In consistent with the levels of IgG1 and IgG2a, the pro-inflammatory cytokines levels including Th1-type cytokines (IL-2, IL-12, IFN-γ) and Th2-type cytokines (IL-10, IL-4) of the immunized mice were notably higher than those of the control mice (Fig. 5a-e). The result also showed a quick and robust proliferation of splenocytes once stimulated by the STAg (Fig. 5f), indicating the efficient cellular immune response induced by the immunization of the mutant. Of note, IL-12 and IFN-γ which are the key factors in cellular immune clearance of tachyzoites were also found significantly increased in sera samples of immunized mice and lasting for nearly a week post vaccination (Fig. 3S). These results along with the above described IgG titer suggested that robust humoral and cellular immune responses were elicited by the vaccination of RHΔompdcΔuprt mutant for the further protection against T. gondii.
RH ΔompdcΔuprt immunization confers long lasting protection against infection with various types of T. gondii tachyzoites in mice
Now that the above results demonstrated the strong immune responses were successfully stimulated, therewith the protective efficacy provoked by RHΔompdcΔuprt mutant was evaluated. Two weeks after the third vaccination, lethal dose challenge of type I RH strains were subjected to naïve and vaccinated mice. All naive mice died within 10 days while the vaccinated ones 100% completely survived (Fig. 6a). In addition, when challenged with type II ME49 tachyzoites or Chinese locally isolated strain WH6, RHΔompdcΔuprt mutant-vaccinated mice presented 100% survival, while naïve mice all died within 13 or 14 days post infection (Fig. 6b-c). Subsequently, tissues including liver, spleen, and lung were collected for pathological examination after 6 days (RH) or 12 days (ME49) challenged with tachyzoites. The obviously changes were observed in unvaccinated mice, such as liver cell necrosis, spleen body destruction, and lung congestion as described above, while the tissue sections of vaccinated mice showed limited changes (Fig. 6d). In summary, these results indicated that vaccination with RHΔompdcΔuprt mutant can provide long lasting effective protection against challenge with various T. gondii tachyzoites without causing any pathological damage.
The long lasting immune protection induced by RH ΔompdcΔuprt vaccination could be adoptively transferred against acute infection in mice
The above results demonstrated that vaccination of RHΔompdcΔuprt elicited high levels of specific IgG antibody and T lymphocytes to active immune response against T. gondii infection. To confirm which immune cells are responsible for protection, purified CD19+ B cells, CD4+ T cells, CD8+ T cells, total splenocytes from RHΔompdcΔuprt-vaccinated mice or total splenocytes from naïve mice were adoptively transferred into naïve mice through the tail vein. All mice were challenged with lethal dose of RH strain and the survival were monitored daily. Mice received naïve splenocytes or RHΔompdcΔuprt-vaccinated CD4+ T cells succumbed to death by 9 dpi (Fig. 6e), in contrast, the passive immunization of purified CD19+ B cells, CD8+ T cells, or total splenocytes from RHΔompdcΔuprt-vaccinated mice survived a significantly longer time compared with naïve mice (Fig. 6e). These results indicated that the B cells and CD8+ T cells may be the effective cells for the development of protective immune response. Subsequently, sera from RHΔompdcΔuprt-vaccinated mice were transferred into naïve mice to determine the contribution in inhibiting T. gondii infections. At 0 to 4 days post infection with lethal dose of RH strain, positive sera of RHΔompdcΔuprt-vaccinated mice or negative sera were injected into naïve mice via the tail vein. The effect of passive immunization was evaluated by monitoring the survival rate and the parasite load in ascites fluid. The data showed a relative but significant longer survival rate in passively immunized mice compared with the control mice (Fig. 6f). In consistent with this result, a significant lower parasite load was observed in passively immunized mice (Fig. 6g). Altogether, the long lasting protective immune response including sera and effective B cells and CD8+ T cells elicited by RHΔompdcΔuprt mutant could be adoptively transferred into naïve mice against T. gondii infection to a certain extent.
Vaccination of the RH ΔompdcΔuprt mutant was determined safe in cats and induced robust immune response
Based on the above results that the RHΔompdcΔuprt mutant was safe and avirulent in the mouse model, we deduced that it may be used as a live attenuated vaccine for cats and performed a safety evaluation. Cats were then intramuscularly injected with RHΔompdcΔuprt to furtherly determine the virulence for felidae animals. Feacal samples of RHΔompdcΔuprt injected and PBS injected cats were collected for oocysts inspection from 1 dpi to 10 dpi, and none oocysts were found in all RHΔompdcΔuprt injected cats (Table 2S). The result suggests that RHΔompdcΔuprt has a good safety profile as a T. gondii vaccine candidate for cats. Then the immune response in cat was furtherly evaluated. After vaccination for 2 doses, serum obtained from cats were used to determine the specific antibody response. Significantly high levels of anti-T. gondii IgG were detected in vaccinated cats, and the IgG titer was increased remarkably after every vaccination (Fig. 7a), which indicating a robust humoral response was induced by the mutant.
RH ΔompdcΔuprt -vaccinated cats notably reduced the oocysts shed number and period
In order to evaluate the possibility of applying the vaccine in cats, we performed a challenge and tested the expulsion of oocysts. After immunization, cats were orally challenged with 200 cysts of ME49 strain. Subsequently, the numbers of oocysts in feces from each were counted from 1dpi to 15dpi. Oocysts were detectable at 3 dpi for all-infected cats, while RHΔompdcΔuprt-vaccinated cats notably reduced the shedding period (6 days) compared with that of naïve cats (11 days) as shown in Fig. 7b. Furthermore, the results showed a remarkable decrease in total number of oocysts (95.3%) in vaccinated cats than in naïve cats (Fig. 7c). These results suggested that inoculation of the RHΔompdcΔuprt-vaccinated strain in cats reflected the effective protection against toxoplasmosis.