Analysis Of The Function Of Toxoplasma Gondii Nucleoside Vaccine Triphosphate Hydrolase (NTPase) And Study Of The Possibility Of Candidate Target For Toxoplasmosis

Toxoplasma gondii is an obligate intracellular parasite and a significant pathogen of immunocompromised patients, notably persons with acquired immune deficiency syndromes (AIDS). Parasite replication and the strong inflammatory response result in massive tissue destruction and severe clinical manifestations. Although acute toxoplasmosis can be effectively treated with a variety of antibiotics, the drugs commonly cause side effects and treatment usually does not eradicate the infection. Therefore, new drugs, especially those against the proliferative stage of the parasite and with none or less side effect, are highly desirable for the treatment of toxoplasmosis.T. gondii is surrounded with parasitophorous vacuole membrane after invasion of host cells. The parasite could modify the vacuolar environment by the secretion of a large number of proteins; one of them with dithiol-activated enzymatic activity is the nucleoside triphosphate hydrolase (NTPase). The NTPase is released from dense granules and accumulates as a soluble protein in the vacuolar space. The enzyme functions as an apyrase and is capable of degrading ATP to ADP and ADP to AMP. In host cells, T. gondii lacks the enzymes necessary for de novo synthesis of purines and therefore must salvage purines from the host for survival and replication, and NTPase has been proved to function in the purine salvage pathway by participating in the generation of the preferred purine salvage substrate, adenosine. So that it is supposed to take a significant role in parasite survival and replication.There are two isoforms, NTPase-Ⅰand NTPase-Ⅱ, expressed in T. gondii. The genomic construction of the NTP genes has no intron and includes three tandemly repeated NTP genes:NTP1, NTP2 and NTP3, in which NTP1 encodes NTPase-Ⅱisoform, NTP3 encodes NTPase-Ⅰisoform and NTP2 does not encode protein. The gene encoding NTPase-Ⅱis found in all strains of T. gondii, while the gene encoding NTPase-Ⅰis confined only to virulent strains. As a result, the application of NTPase-Ⅱisoform may be more preferable as a potential vaccine candidate than NTPase-Ⅰisoform.In this study, the monoclonal antibodies against recombinant T. gondii NTPase-Ⅱ(rTgNTPase-Ⅱ) were developed firstly, and then we undertook a series of experiments to test whether these McAbs inhibited the enzyme activity and the growth of T. gondii, and finally illustrate the effect of NTPase-Ⅱon growth of Toxoplasma gondii tachyzoite. Finally, in order to find novel T. gondii recombinant antigens with protective values, we describe here the ability of a recombinant form of TgNTPase-II co-administered with alum, a safe adjuvant that can be used in humans, to induce protection against toxoplasmosis in a murine model.1. Clone and Expression of NTPase-Ⅱgene of Toxoplasma gondiiTo construct a pBAD-HisB-NTPase recombinant plasmid and express the His-tag fusion protein NTPase, and identify the purified protein.The NTPase gene was amplified by polymerase chain reaction (PCR) from RH strain of T.gondii and cloned into pGEM-T Easy vector. Positive clones were screened and identified by BglⅡ,HindⅢdigestion and sequenced. The homology of DNA sequence was 100% to that in the Genbank. The target gene was then subcloned into the prokaryotic expression vector pBAD-HisB and transformed into E.coli BL21 (DE3). So prokaryotic recombinant plasmids pBAD-HisB-NTPase were successfully constructed, SDS-PAGE showed that the recombinant NTPase proteins were over-expressed as inclusion body with molecular weight of 70 kDa. The expressed recombinant proteins were purified with Ni-NTA agarose and further analyzed by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western blotting. Western blotting and ELISA analysis testified that the purified recombinant protein could be specifically recognized by mouse serum immunized with T. gondii and mouse anti-recombinant protein sera, and indicated the protein had highly antigenicity and immunogenicity.2. Development and analysis of the monoclonal antibody against recombinant T. gondii NTPase-Ⅱ(rTgNTPase-II):To develop the monoclonal antibodies against recombinant T. gondii NTPase-II (rTgNTPase-Ⅱ), undertake a series of experiments to test whether these McAbs inhibited the enzyme activity and the growth of T. gondii, and finally illustrate the effect of NTPase-Ⅱon growth of Toxoplasma gondii tachyzoite.Syngeneic 6- to 8-week-old female BALB/c mice were injected subcutaneously with purified rTgNTPase-Ⅱantigen (50μg/100μl) mixed with an equal volume of Freund's complete adjuvant, and boosted 3 times subcutaneously with rTgNTPase-Ⅱ(50μg/100μl) mixed with Freund's incomplete adjuvant at 2 week intervals. Spleen cells obtained from immunized mice three days after the last immunization were fused with Sp2/0 myeloma cells by using polyethylene glycol. Supernatants from 534 hybridomas were screened respectively. Selection of positive hybridomas was made by ELISA using purified rTgNTPase-II as the antigen. Then, a positive hybridoma obtained was subcloned 3 times by limiting dilution. Finally, two McAb-producing hybridoma cell lines with the specific immunoactivity were identified and named as MNT1 and MNT2 respectively. Then, two McAb-producing hybridoma cell lines were transplanted intraperitoneally into BALB/c mice previously treated with pristane to collect ascites.2.1. Characterization of McAbs:At first, These McAbs isotypes were determined by Sigma kit and isotypes of both McAbs were IgG2a class. And titers of the culture supernatant and ascetic fluids of hybridoma cells were detected by indirect ELISA after serial dilutions. Results showed that the titers of MNT1 and MNT2 were 1:12800 and 1:6400 in culture supernatants; and 1:51200 and 1:12800 in ascitic fluids, respectively. Antibodies of cells were still positive after passage for three months and storage in liquid N2 for one month.Then, these McAbs were purified on a protein A-agarose column and identified using SDS-PAGE and Western blotting. The BL21 (DE3) cell lysate containing pBAD-HisB was used as the negative control and whole tachyzoite lysate was used as positive controls. MNT1 and MNT2 can bind with the protein sized around 70 kDa and the protein sized around 63 kDa, respectively, but not with BL21 (DE3) cell lysate containing pBAD-HisB. The result displayed that the developed McAbs, MNTl and MNT2, can recognize the recombined and native NTPase-Ⅱspecifically.Finally, in order to determine whether the two McAbs could react with NTPase-Ⅱprotein in tachyzoites, confocal laser microscopy was used for indirect fluorescence antibody test (IFAT) after immunostaining was carried out by treating tachyzoites with McAb-containing supernatants, both supernatants from Sp2/0 cultures and an isotype-matched mouse myeloma IgG2a as the negative control. Images showed that a specific reaction was observed on tachyzoites when T. gondii tachyzoites were analyzed with MNTl or MNT2 in IFAT.2.2. Examination of the protective effects of McAbs:COS-7 cells seeded in multiwell cell culture dishes were infected with tachyzoites (a parasite:cell ratio of 2:1). Prior to infection, tachyzoites were pre-incubated in 50μg/ml two McAbs for 1 h at 37℃, isotype-matched mouse myeloma IgG2a and medium as negative control. One hour after incubation, the monolayer was washed to remove extracellular parasites and re-incubated with medium for 2 h,8 h and 14 h, respectively. Cells on the coverslips were stained with Giemsa solution and examined by light microscopy. The number of infected cells per 100 cells and the number of tachyzoites per 100 infected cells were examined at 3 h after incubation for evaluation of the effects of McAbs on inhibition, and the number of tachyzoites per 100 infected cells was compared for evaluation of the effects of McAbs on replication at 3 h,9 h and 15 h after infection, respectively. The infective rates of MNT1, MNT2, IgG2a and medium treated tachyzoites were 12%,10%,12% and 13%, respectively and not significantly different among four groups(χ2= 0.458, P=0.928). However, there were significant differences in the number of intracellular parasites per infected cell between MNT1 or MNT2 treated group and IgG2a or medium treated group at 9h and 15h after infection. Pretreatment of tachyzoites with MNT1 or MNT2 significantly inhibited parasite replication in COS-7 compared to the IgG2a or medium control group at 9h (P=0.014, MNT1 versus medium; P=0.017, MNT1 versus IgG2a; P=0.037, MNT2 versus medium; P=0.040, MNT1 versus IgG2a) and 15h (P=0.000, MNT1 versus medium; P= 0.000, MNT1 versus IgG2a; P= 0.015, MNT2 versus medium; P=0.002, MNT1 versus IgG2a) after infection. Furthermore, the inhibition in tachyzoites pretreated with MNT1 McAb was significantly higher than those pretreated with MNT2 McAb (P= 0.003) at 15h after infection.In order to examine the inhibition of NTPase activity by two McAbs in vitro, tachyzoites were pre-incubated with MNT1 or MNT2, respectively. After incubation, 1mM DTT was added and incubated for 10 min. Then Triton X-100 was added to a final concentration of 1%, and tachyzoites were incubated for 5 min at room temperature. The detergent lysates of tachyzoites were assayed for enzymatic activity. An isotype-matched mouse myeloma IgG2a was used as a negative control. The concentration of Pi reached 23.0±5.4μM in control group (P= 0.002, MNT1 versus IgG2a; P=0.035 MNT2 versus IgG2a), but it was 6.8±2.6μM in MNT1 group and 14.9±1.9μM in MNT2 group (P=0.036, MNT1 group versus MNT2 group), indicating that both MNT1 and MNT2 can inhibit the ATP diphosphohydrolase activity of NTPase in vitro, which hinted that the reduction of tachyzoite replication might be owing to the inhibition of NTPase-II by the McAbs.To evaluate the protection of passive immunization, two groups of BALB/c mice, MNT1-1 and MNT1-2, were immunized intraperitoneally with 0.2 ml (500μg/ml) of purified MNT1 McAb per mouse. Group MNT1-1 was challenged with two hundred RH strain tachyzoites on the same day of MNT1 injection. Group MNT1-2 was challenged with the same number of tachyzoites 48 h later. Mice in group MNT2-1 and MNT2-2 were injected with MNT2. The negative control groups A1 and A2 were injected with mouse myeloma IgG2a. The survival periods were recorded daily until all mice were dead. The mice, received MNT1 or MNT2 McAbs and challenged on the same day, displayed significantly longer survival times compared with the control group (P=0.004, MNT1 versus control; P=0.018, MNT2 versus control). On the other hand, although the survival time of mice receiving MNT1 then challenged after 48 h was significantly prolonged compared to the control (P=0.030), there was not significantly different between MNT2 group and control group (P=0.189). These results indicated that mice passively immunized with MNT1 survived considerably longer than other groups after challenge infection.Taken together, we concluded that the McAbs against NTPase-Ⅱcan reduce the replication of T. gondii and have a crucial effect on the protection of host from T. gondii infection.3. Study of the immune protection and the mechanism of rTgNTPase-Ⅱon T. gondii infectionIn an effort to find novel T. gondii recombinant antigens with protective values, we describe here the ability of a recombinant form of TgNTPase-Ⅱco-administered with alum, a safe adjuvant that can be used in humans, to induce protection against toxoplasmosis in a murine model.The recombinant NTPase-Ⅱprotein (rTgNTPase-Ⅱ) was expressed and purified. Endotoxin was removed. After endotoxin removal, the lipopolysaccharide (LPS) content was measured. Less than 10pg/ml of LPS was detected in the final protein preparations. Before inoculation into mice or stimulation in vitro, rTgNTPase-II was dialyzed against PBS, filtered throughout a 0.2μm-pore membrane and stored at-70℃. The purified recombinant protein was quantified by the Bradford method.Then, female BALB/c mice 6-8 weeks old were divided into three groups with 20 mice per group. Mice were immunized with 10μg of rTgNTPase-Ⅱalone or adsorbed to 0.5mg of aluminum hydroxide gel (alum) by bilateral intramuscular injection into the quadriceps and boosted with the same dose three times per two-week interval. Another 20 mice received injection with PBS plus alum and were used as non-immunized controls. Two weeks after the last immunization, humoral and cellular immune responses were evaluated.3.1. Evaluation of humoral immune response:To determine the specific antibody titers, blood samples were obtained after the immunization schedule was completed, and assayed by an ELIS A with rTgNTPase-Ⅱas the bound target. The IgG antibodies against rTgNTPase-Ⅱwere significantly greater in sera of mice immunized with rTgNTPase-Ⅱ+alum (1.038±0.479) than those immunized with rTgNTPase-Ⅱalone (0.434±0.204) (P=0.000). As expected, mice immunized with PBS+alum did not present significant specific antibody titers (0.032±0.024).In order to characterize whether a Thl and/or Th2 response was elicited in immunized mice, the distribution of IgG subtypes against rTgNTPase-Ⅱwas analyzed. Both IgG1 and IgG2a were found in the sera of mice vaccinated with rTgNTPase-Ⅱalone or with rTgNTPase-Ⅱ+alum, which showed a mixed anti-rTgNTPase-ⅡIgG1/IgG2a profile. However, immunization by rTgNTPase-Ⅱalone gave a Th1 response (IgG2a/IgG1 ratio>1), by rTgNTPase-Ⅱwith alum adjuvant appeared predominantly Th2 response (IgG2a/IgG1 ratio<1). These results indicate that the presence of alum enhances the specific humoral response.3.2. Evaluation of cellular immune responseIn order to study the antigen-specific lymphocyte responses induced by the vaccination, an in vitro lymphocyte proliferation assay was performed. Spleens were removed from 5 mice per group two weeks after the last booster injection under aseptic conditions and single-cell preparations were obtained. The cells were then plated at a density of 3×105 cells and cultured in the presence of rTgNTPase-Ⅱ(10μg/ml) or Concanavalin A (ConA; 5μg/ml; positive control) or medium alone (negative control). The plates were incubated for 72h and pulsed with 10μl of CCK-8 reagent. The stimulation index (SI=the mean OD450 values from recombinant antigen-stimulated cultures/the mean OD450 values from non-stimulated cultures) of each group was calculated. Both splenocytes from the mice immunized with adjuvant rTgNTPase-Ⅱ(P=0.000) and rTgNTPase-Ⅱalone (P=0.002) elicited a significant lymphocyte proliferative response to the rTgNTPase-Ⅱantigen stimulation when compared to the control group. Control mice vaccinated with PBS-alum did not respond to rTgNTPase-II stimulation. However, splenocytes from all immunized and control groups proliferated to comparable levels in response to the mitogen ConA.To further characterize the immuno-modulation properties of this antigen, the concentration of cytokines in supernatants of spleen cells stimulated with rTgNTPase-II was analyzed. Spleen cells were obtained as described above and cultured. Supernatants from cultured splenocytes (5×106) were collected after 24,72 or 96 h of stimulation with rTgNTPase-Ⅱ(10μg/ml) and were analyzed for interleukin-2 (IL-2) and interleukin-4 (IL-4) activity at 24 h, for interleukin-10 (IL-10) activity at 72 h, and for gamma-interferon (IFN-γ) activity at 96 h. Both mice immunized with rTgNTPase-Ⅱ+alum and rTgNTPase-Ⅱalone produced a significant increase in the amount of secreted IFN-γ(P=0.000, rTgNTPase-Ⅱ+alum or alone group versus control) and IL-2 (P=0.002, rTgNTPase-Ⅱ+alum versus control; P=0.013, alone group versus control) when compared to the PBS+alum groups. Furthermore, only splenocytes of mice immunized by rTgNTPase-Ⅱ+alum produced specific amounts of IL-10 (P=0.021, rTgNTPase-Ⅱ+alum versus control; P=0.031, alone group versus control). In contrast, IL-4 was not detected in any group following stimulation with rTgNTPase-Ⅱantigen. In the meanwhile, Con A induced the highest cytokine release levels for these cytokines assayed in all experimental groups. These results show that immunization with rTgNTPase-Ⅱ+ alum induces a mixed Th1/Th2 response.For phenotypic analysis of splenocytes, cell surface staining was accomplished by using the following fluorochrome-conjugated McAbs:anti-mouse-CD4-FITC, anti-mouse-CD8a-PE, anti-mouse-CD3e-PE-cy5 and appropriate IgG isotype controls. A single cell suspension was prepared as described above, then cells were delivered to each well already containing of CD4-FITC, CD8a-PE or CD3e-PE-Cy5 or appropriate IgG isotype controls and incubated. Erythrocytes were then lysed once. After washing, incubated cells were fixed and immediately analyzed on flow cytometer collecting at least 10,000 cells per sample. Although the percentages of CD3+CD4+ T lymphocytes were slightly higher in rTgNTPase-Ⅱ+alum and rTgNTPase-Ⅱalone groups than those in control group, there were not statistically significant differences (P=0.597). However, the ratio of CD3+CD4+ T cells/ CD3+CD8+ T cells was significantly reduced in all groups except the control group because of the significant increase of percentages of CD3+CD8+ T lymphocytes, suggesting that rTgNTPase-Ⅱ-induced immunity is also CD8+ T cell mediated.3.3. Evaluation of protection against acute and chronic infectionTo evaluate whether rTgNTPase-Ⅱcould potentially provide protection against T. gondii acute infection, five mice of each group were intraperitoneally challenged with 103 tachyzoites of the virulent RH strain 2 weeks after last immunization. A significant increase in the survival rate was observed in the rTgNTPase-Ⅱ+alum (P =0.021) and rTgNTPase-Ⅱalone immunized group (P=0.020) compared to control group, indicating that rTgNTPase-Ⅱcould induce partial protection against challenge with the highly virulent RH strain of T. gondii.In addition to determining the protection against acute infection, rTgNTPase-Ⅱ-dependent resistance to T. gondii chronic infection was also evaluated. Ten mice of each group were inoculated orally with 20 PRU tissue cysts at two weeks after the last immunization. Mice were observed daily for mortality. Five weeks after the challenge, surviving mice were sacrificed and their brains were removed. The mean number of cysts per brain was determined by counting. Compared to the mice in control group, the average parasite burden was reduced significantly by 62.9% and 57.6% for rTgNTPase-Ⅱ+alum (P=0.008) and rTgNTPase-Ⅱalone vaccinated mice (P=0.014), respectively. These results demonstrated the protective effect of rTgNTPase-Ⅱagainst T. gondii chronic infection.