Toxoplasma gondii is a species of parasitic protozoa in the genus Toxoplasma. The definitive host of T. gondii is the cat, but the parasite can be carried by many warm-blooded animals (birds or mammals). Toxoplasmosis, the disease of which T. gondii is the causative agent, is usually minor and self-limiting but can have serious or even fatal effects on a fetus whose mother first contracts the disease during pregnancy or on an immunocompromised human or cat (Wiki: T. gondii).
4. Microbial Pathogenesis
T. gondii is an intestinal coccidian of cat with man as one of the intermediate host. Humans are generally infected by ingesting oocysts released in cat faeces or by consuming meat from infected herd animals containing the long lived tissue cysts. Following ingestion, the outer wall of cysts or oocysts are disrupted by enzymatic degradation and bradyzoites and sporozoites are liberated into intestinal lumen. They rapidly invade and multiply within surrounding cells where they become tachyzoites. Thereafter, tachyzoites are disseminated via blood or lymphatic system to most of the organs of human body. At these sites, the tachyzoites infect host cells, replicate and invade the adjoining cell. In this fashion, the hallmarks of the infection develop: cell death and focal necrosis surrounded by an acute inflammatory response (Bhopale, 2003).
5. Host Ranges and Animal Models
The life cycle of T. gondii has two phases. The sexual part of the life cycle (coccidia like) takes place only in members of the Felidae family (domestic and wild cats), which makes these animals the parasite's primary host. The asexual part of the life cycle can take place in any warm-blooded animal, like other mammals (including felines) and birds (Wiki: T. gondii).
Molecule Role Annotation :
Oral immunization of mice with ZJ111/pcDNA3-SAG1 elicited specific humoral responses and stimulated proliferation of splenocytes (P<0.05). Mice immunized with ZJ111/pcDNA3-SAG1 displayed significant protection against an intraperitoneally challenge with 500 tachyzoite forms of T. gondii RH strain. Vaccination at 10^7 and 10^8 CFU per mice provided a 20% and 10% survival rate comparing 100% mortality of the non-immunized mice, exhibiting longer living time and better survival rate (Qu et al., 2008).
Protein Note :
encoded by transcript TGME49_227620; Signal peptide predicted by SignalP 2.0 HMM (probability 0.997) with cleavage site probability 0.917 at residue 23; GO_component: GO:0020003 - symbiont-containing vacuole;ev_code=EXP; GO_component: GO:0045177 - apical part of cell;ev_code=EXP
Protein Note :
encoded by transcript TGGT1_297880 (Gold et al., 2015); Signal peptide predicted by SignalP 2.0 HMM (probability 1.000) with cleavage site probability 0.976 at residue 30; Predicted trans-membrane domain (TMHMM2.0):12-35
Protein Note :
encoded by transcript TGME49_286450; Signal peptide predicted by SignalP 2.0 HMM (probability 0.923) with cleavage site probability 0.441 at residue 26; Predicted trans-membrane domain (TMHMM2.0):6-26:70-93; GO_component: GO:0020003 - symbiont-containing vacuole;ev_code=EXP; GO_component: GO:0045177 - apical part of cell;ev_code=EXP
Protein Note :
Micronemal proteins: The Toxoplasma lytic cycle begins when the parasite actively invades a target cell. In association with invasion, T. gondii sequentially discharges three sets of secretory organelles beginning with the micronemes, which contain...; cd01471
Molecule Role Annotation :
A mic2 mutant is attenuated in mice and induces protection from challenge with wild type T. gondii (Huynh and Carruthers, 2006).
>XP_002369833.2 EGF family domain-containing protein [Toxoplasma gondii ME49]
MRGGTSALLHALTFSGAVWMCTPAEALPIQKSVQLGSFDKVVPSREVVSESLAPSFAVTETHSSVQSPSK
QETQLCAISSEGKPCRNRQLHTDNGYFIGASCPKSACCSKTMCGPGGCGEFCSSNWIFCSSSLIYHPDKS
YGGDCSCEKQGHRCDKNAECVENLDAGGGVHCKCKDGFVGTGLTCSEDPCSKRGNAKCGPNGTCIVVDSV
SYTCTCGDGETLVNLPEGGQGCKRTGCHAFRENCSPGRCIDDASHENGYTCECPTRVLT
Molecule Role :
Protective antigen
Molecule Role Annotation :
Protective experiments of DNA vaccine pcDNA3-MIC3 (pMIC3i) in Kumming mice showed that the vaccine could significantly prolong the survival time of the mice challenged by virulent RH strains of T. gondii (Xiang et al., 2009).
Protein Note :
Calcium-binding EGF-like domain, present in a large number of membrane-bound and extracellular (mostly animal) proteins. Many of these proteins require calcium for their biological function and calcium-binding sites have been found to be located at the...; cl21504
Molecule Role Annotation :
The gene sequence encoding MIC8 was inserted into the eukaryotic expression vector pVAX I, and the pVAX-MIC8 expression plasmid was constructed, and the plasmid diluted with PBS to 100 mg/100 microl was injected into the Kunming mice muscularly. Levels of IgG antibody, gamma-interferon (IFN-gamma), interleukin-2 (IL-2), interleukin-4, and interleukin-10 were detected. The mice were challenged with tachyzoites of the virulent T. gondii RH strain at the 14th day after the last immunization to observe the survival time. The high level of IFN-gamma, IL-2, and IgG antibody indicated that mice vaccinated with recombinant pVAX-MIC8 plasmid could elicit strong cellular and humoral immune responses and showed a significantly increased survival time (10.3 +/- 0.9 days) compared with control mice which died within 5 days of challenge infection (Liu et al., 2010).
Molecule Role Annotation :
An OMPDC mutant, alone or in combination with an UP mutation, is attenuated in mice and induces significant protection from challenge with wild type T. gondii (Fox and Bzik, 2010).
Protein Name :
serine protease inhibitor 1 isoform beta
Protein pI :
4.61
Protein Weight :
30858.24
Protein Length :
373
Protein Note :
Kazal type serine protease inhibitors and follistatin-like domains. Kazal inhibitors inhibit serine proteases, such as, trypsin, chyomotrypsin, avian ovomucoids, and elastases. The inhibitory domain has one reactive site peptide bond, which serves the...; cd00104
Molecule Role Annotation :
A study to examine the immunostimulatory properties of Leishmania infantum Hsp83 (83) to elicit humoral and cellular response against the Toxoplasma gondii Rop2 protein in an adjuvant-free vaccination system was conducted. Mice immunized with fusion Rop2-83 elicited a stronger humoral and cellular response in comparison to mice immunized with Rop2 alone, or a mix of LiHsp83 and Rop2. The fusion protein induced a Th1 type response, with predominance of specific IgG2a/IgG2c isotype and IFN-gamma secretions, whereas Rop2 alone or mixed with LiHsp83 produced a Th1/Th2 mixed response. Vaccination with fusion protein conferred a remarkable resistance against oral infection with ME49 cysts in C57BL/6 and C3H mice, in comparison to mice immunized with Rop2 alone or the protein mixture. Following lethal challenge, a significant survival rate was observed in Rop2-83 immunized Balb/c and C57BL/6 mice in comparison to control groups (Echeverria et al., 2006).
Molecule Role Annotation :
Oral immunization of mice with ZJ111/pcDNA3-SAG1 elicited specific humoral responses and stimulated proliferation of splenocytes (P<0.05). Mice immunized with ZJ111/pcDNA3-SAG1 displayed significant protection against an intraperitoneally challenge with 500 tachyzoite forms of T. gondii RH strain. Vaccination at 10(7) and 10(8)CFU per mice provided a 20% and 10% survival rate comparing 100% mortality of the non-immunized mice, exhibiting longer living time and better survival rate (Qu et al., 2008).
Protein Note :
WD40 domain, found in a number of eukaryotic proteins that cover a wide variety of functions including adaptor/regulatory modules in signal transduction, pre-mRNA processing and cytoskeleton assembly; typically contains a GH dipeptide 11-24 residues from...; cd00200
Molecule Role Annotation :
A UP mutant, in combination with an OMPDC mutation, is attenuated in mice and induces significant protection from wild type T. gondii (Fox and Bzik, 2010).
Molecule Role Annotation :
IFN-gamma plays a critical role in Th1 type immune response. It is important for protection against infections by various viruses and intracellular bacteria.
Additional Molecule Role :
Vaximmutor
Additional Molecule Role Annotation :
The experimental data demonstrated that three time vaccinations with BCG in BALB/c mice induced strong TB Ag-specific IFN-gamma immune responses in splenocytes (Wang et al., 2009).
ROP16: Rhoptry protein 16: a specialised kinase released from rhoptries into the host cell during invasion, directly target the host cell nucleus and activate both the signal transducer and activator of transcription 3 and 6 (STAT3 and STAT6) signaling pathways. (Li et al., 2016)
f. Gene Engineering of
ROP16
Type:
Recombinant vector construction
Description:
pPolyII-CAV-â–³E3-ROP16 was constructed. The Kpn I fragment containing the E3 region of CAV-2 from pPolyII-CAV-2 was cloned to pVAX. RPO16 was amplified by PCR using a forward primer introducing Nhe I recognition sites and a reverse primer introducing Bgl II recognition sites. ROP16 was identified, purified, and then cloned to pVAX-E3 vector. The 6.7 kb fragment of Nru I and Sal I double-digested pVAX-ΔE3-ROP16, containing the ROP16 expression cassette flanked by residual E3 sequences, and was cloned back into pPolyII-CAV-2. The recombinant viruses was then generated in MDCK cells. (Li et al., 2016)
CAV-2 (Li et al., 2016): canine adenovirus type 2: a serotype attenuated strain, induce Infectious tracheobronchitis (ITB) which is characterized by respiratory symptoms. (Zhu et al., 2022)
h. Immunization Route
Intramuscular injection (i.m.)
i. Description
Recombinant canine adenovirus expressing the ROP16 gene of the RH strain of T. gondii (Li et al., 2016)
Vaccination Protocol:
The mice were randomly assigned to four experimental groups (23 mice per group). Group I was intramuscularly inoculated once with 0.1 ml CAV-2-ROP16 (108.0 PFU/ml); group II received 0.1 ml CAV-2 (108.25 PFU/ml) once intramuscularly; group III was inoculated once intramuscularly with 0.1 ml PBS; and group IV was not given any injection. Group 2, 3, and 4 are negative controls. (Li et al., 2016)
Immune Response:
Humoral: The antibody titres of ROP16 were drastically enhanced in CAV-2-ROP16 group compared to the controls. The ratio of IgG1 to IgG2a in CAV-2-ROP16 group was significantly higher than that of the controls (P < 0.05). (Li et al., 2016) Cellular: Values of IFN-γ and IL-2 in the CAV-2-ROP16 group were significantly higher than the control groups (P < 0.05). Low concentration of IL-4 showed a weak but obviously proliferative response in CAV-2-ROP16 group compared to the other control groups (P < 0.05). There were no statistically significant differences in the levels of IL-10 in all groups (P > 0.05). There was significant increase in IFN-γ and TNF-α production that was induced by CD4+ and CD8+ T cells, respectively in the mice vaccinated with CAV-2-ROP16 compared to the other three groups (P < 0.05). The percentage of CD3+/CD4+ and CD3+/CD8+ T-cells were significantly increased in mice vaccinated with CAV-2-ROP16 compared to the other three groups (P < 0.05).(Li et al., 2016)
Challenge Protocol:
Eight weeks after the prime immunisation, 20 mice in all groups were challenged intraperitoneally (i.p.) with 1 × 10^3 tachyzoites of the virulent T. gondii RH strain. The mice were observed daily for mortality. (Li et al., 2016)
Efficacy:
The mice vaccinated with a single dose of CAV-2-ROP16 displayed 25% protection 80 days after RH strain infection; the administration of either CAV-2 or PBS did not prevent mortality, that all mice received CAV-2 or PBS died within seven days. (Li et al., 2016)
2. CAV-2-ROP18
a. Type:
Recombinant vector vaccine
b. Status:
Research
c. Host Species for Licensed Use:
Human
d. Antigen
ROP18: Rhoptry protein 18: a polymorphic serine-threonine kinase which is secreted in the host cell during the invasion process, and its catalytic activity is required for the acute virulence phenotype. It is considered one of the key virulence factors in the pathogenesis of the T. gondii infection. (Li et al., 2015)
e. Gene Engineering of
ROP18
Type:
Recombinant vector construction
Description:
Recombinant plasmid pPolyII-CAV-â–³E3-ROP18 was constructed by in vitro ligation. The plasmid includes E3 (the E3 region of CAV-2); CMV (human cytomegalovirus (hCMV) immediate-early gene promoter), ROP18, and polyA (the SV40 early mRNA polyadenylation signal). The plasmid were digested with Asc I and Pme I to release the linear recombinant genome. The recombinant genome was used to transfect MDCK cells. (Li et al., 2015)
CAV-2 (Li et al., 2015): canine adenovirus type 2: a serotype attenuated strain, induce Infectious tracheobronchitis (ITB) which is characterized by respiratory symptoms. (Zhu et al., 2022)
g. Immunization Route
Intramuscular injection (i.m.)
h. Description
Recombinant canine adenovirus expressing the ROP18 gene of the RH strain of T. gondii (Li et al., 2015)
i.
Mouse Response
Host Strain:
specific-pathogen-free (SPF) grade inbred Kunming mice (Li et al., 2015)
Vaccination Protocol:
The mice were randomly assigned into four experimental groups (33 mice per group). Group I was intramuscularly inoculated once with 0.1 ml CAV-2-ROP18 (10 8.125 p.f.u. ml−1); group II received 0.1 ml CAV-2 (108.25 p.f.u. ml−1) once as a negative control; group III was inoculated intramuscularly with 0.1 ml PBS as control at weeks 0, 2 and 4; and group IV was not injected with anything as a negative control. (Li et al., 2015)
Immune Response:
Humoral: Antibody titers significantly increased in the recombinant virus CVA-2-ROP18 group at week 2, 4 and 6 after immunization, compared to CAV-2, PBS and blank control immunized group (P < 0.05). Both IgG1 and IgG2a levels were detected. There was no significant difference in IgG1 and IgG2a levels between the groups immunized CAV-2, PBS, and nothing (P > 0.05). HI antibodies against CAV-2 were detected in all mice vaccinated with CAV-2 and the recombinant CAV-2-ROP18 at 2 weeks post-primary immunization, reaching comparable titers throughout the test period. (Li et al., 2015) Cellular: Splenocytes from mice immunized with CAV-2-ROP18 showed a significant proliferative response to ROP18 (P < 0.05), which was significantly higher than proliferation by splenocytes from all other controls (P < 0.05). Splenocytes from all groups proliferated to comparable levels in response to the mitogen ConA. Significant CTL activity was tested in mice immunized with CAV-2-ROP18. The percentage of CD3+/CD4+ T cells and CD3+/CD8+ T cells were significantly increased in mice immunized with CAV-2-ROP18 compared to all controls. Similarly, CAV-2-ROP18 significantly altered CD4+ or CD8+ T cell profiles in terms of IFN-γ and TNF-α expression in comparison with all controls. IL-2 and IFN-γ values in CAV-2-ROP18 group are significantly higher than in the controls (P < 0.05). Low levels of IL-4 showed a slight but significantly production from CAV-2-ROP18 groups compared to the controls (P < 0.05). (Li et al., 2015)
Challenge Protocol:
Eight weeks after the immunization, 20 mice in each group were challenged intraperitoneally (i.p.) with 1 × 103 tachyzoites of the virulent T. gondii RH strain, and 10 other mice were inoculated intragastrically with 5 cysts of the PRU strain. All mice were observed daily for mortality. Two months after the challenge, the surviving mice were euthanized and their brains were removed. Each brain was homogenized in 2 ml of PBS. The mean number of cysts per brain was determined by counting in three samples of 25 μl aliquots of each homogenized brain under an optical microscope. (Li et al., 2015)
Efficacy:
RH: Mice immunized with only a single dose of CAV-2-ROP18 showed 40% protection until 60 days after challenge, while the administration of either CAV-2 or PBS did not prevent mortality (mice died within 7 days). (Li et al., 2015) PRU: Mice from the CAV-2-ROP18 group developed a significantly lower (P < 0.05) number of brain cysts (8000 ± 1414 cysts per brain) compared to mice from the other three control control groups (approximately 18000 cysts). (Li et al., 2015)
3. CTLVECNS
a. Type:
Inactivated or "killed" vaccine
b. Status:
Research
c. Host Species for Licensed Use:
Human
d. Antigen
Toxoplasma lysate: Tachyzoites were collected from previously infected mice and were sedimented by centrifugations and sonications. The supernatant material (the lysated antigen of Toxoplasma) was collected. (El et al., 2016)
Vaccination Protocol:
Mice were divided into two main groups, Group I control group (100 mice) which was further subdivided into five equal subgroups (20 mice): Ia: normal non infected; Ib: chitosan delivery control subgroup, received 20 µg of BCNS in 100 µl of PBS/mouse/dose; Ic: FIA control subgroup, received 100 µl of FIA/mouse/dose, Id & Ie: non vaccinated groups for further challenge. Group II experimental vaccinated group (100 mice) was further subdivided into two equal subgroups (50 mice). Subgroup IIa, received CTLVECNS suspended in PBS while subgroup IIb received CTLV + FIA. All vaccinated mice received three doses 2 weeks apart. (El et al., 2016)
Immune Response:
Higher ratio of IgG2a to IgG1; Higher level of IFN-γ and IL-2 (El et al., 2016)
Challenge Protocol:
RH: Group Id (20 mice) were infected with 2,500 tachyzoites of RH strain without immunization, serving as RH infected control group. 25 mice from group IIa and 25 mice from group IIb were challenged by 2,500 viable tachyzoites of virulent RH strain. 10 mice out of 25 were sacrificed 5 days post infection and the remaining mice were observed daily to record mortality. (El et al., 2016) Me49: Group Ie (20 mice) were perorally inoculated by 10 cysts of Me49 strain without immunization, serving as Me49 infected control group. The other 25 mice from group IIa and 25 mice from group IIb were challenged orally by 10 cysts of Me49 strain. 10 mice out of 25 were sacrificed 60 days post infection and the remaining were observed daily to record mortality. (El et al., 2016) Serum samples were collected from RH and Me49 infected control subgroups mice before being sacrificed simultaneously with their corresponding experimental subgroups. (El et al., 2016)
Efficacy:
RH: None of the mice of RH infected control subgroup remained alive beyond the seventh day post infection with a mean survival time of 5.9 ± 0.88 days. Mice vaccinated with CTLV either in chitosan nanospheres or in combination with FIA and challenged with RH strain showed significant increase in their survival time. (BCNS: maximum survival time of 80 days with a mean of 35.73 ± 31.82; FIA: 57 days with a mean of 33.67 ± 18.30 days). There was a statistically significant reduction in parasitic count in all studied organs in vaccinated animals. The reduction in the mean tachyzoites count was more evident in the subgroup receiving encapsulated chitosan vaccine in comparison to the subgroup receiving the vaccine in combination with FIA .(El et al., 2016) Me49: The mean survival time in the control group was 60.7 ± 8.11 days with only one mouse which lived for 120 days post infection. Only mice vaccinated with CTLV in combination with FIA showed a statistically significant increase in the survival time (a mean of 120.53 ± 33.13 days). There was a statistically significant difference between mice vaccinated with CTLV whether in chitosan nanospheres (a mean of 79.20 ± 17.91 days) or in combination with FIA. A statistically significant reduction in the mean cyst count of the brain was found in both experimental subgroups. The difference between vaccinated subgroups was statistically significant with the highest reduction in the subgroup receiving the vaccine in combination with FIA. (El et al., 2016)
Description:
The GRA7(20–28) peptide sequence was cloned in between the NsiI/BamHI restriction sites of the modified pPEP-T vector to yield the final LP amino acid sequence. The sequence is composed of the his-tag sequence (1-12aa), the CD8+ epitope (13-21aa), the pentameric coiled coil (22-60aa), a glycine-glycine linker (61-62aa), the trimeric coiled coil (63-107aa) and a solubility tag (108-128). The trimeric coiled coil contains a PADRE derivative as a CD4+ epitope (86-98aa). LP monomer was expressed in E. coil, purified, and eventually self-assembled to form nanoparticles (GRA7(20-28)SAPN). (El et al., 2014)
Self-assembling nanoparticles displaying the GRA7(20–28) in conjunction with PADRE. (El et al., 2014)
h.
Mouse Response
Host Strain:
HLA-B*0702 transgenic mice: express a chimeric HLA-B07/H2-Db MHC Class I Molecule and are on a C57BL/6 × Balb/C background backcrossed through many generations (El et al., 2014)
Vaccination Protocol:
Mice were inoculated subcutaneously with 50 μg GRA7(20–28) SAPN or P4c-RD control SAPN three times at two weeks intervals. (El et al., 2014)
Immune Response:
Increase of IFN-γ secretion (El et al., 2014)
Challenge Protocol:
RH: Mice (n = 5 per group) were challenged i.p. with 2000 RH T. gondii expressing stable YFP. Peritoneal fluid was collected 120 h post infection and parasite fluorescence and numbers were measured using a fluorometer and hemocytometer, respectively. (El et al., 2014) Me49: Mice (n = 6 for PBS(negative control) and GRA7(20–28)SAPN, n = 3 for ΔRPS13(positive control)) were challenged intraperitoneally 14 days post-immunization using 2000 Me49-FLUC tachyzoites and were imaged 21 days post-challenge. Mice were then euthanized, and the number of tissue cysts per brain were counted. (El et al., 2014)
Efficacy:
RH: Fluorescence from GRA7(20–28)SAPN immunized mice was significantly lower than the control. (El et al., 2014) Me49: The numbers of luciferase expressing parasites in mice immunized with GRA7(20–28)SAPN were significantly reduced compared to the mice immunized with control SAPN or PBS. This correlates with the reduction of the number of cysts per brain in GRA7(20–28)SAPN immunized mice. (El et al., 2014)
Description:
TgCST2 95-325aa was amplified and then constructed to the expression vector pET-30a. Escherichia coli strain BL-21 (DE3) system was used for protein expression. The recombinant protein was purified and the endotoxin was removed using His Bind® Resin Chromatography kit (Merck, Darmstadt, Germany) and Detoxi-Gel Affinity Pak Prepacked columns (Pierce, Rockford, USA). (Tian et al., 2022)
Vaccination Protocol:
A total of 60 BALB/c mice were randomized into three groups (20/group), 20 µg of rTgCST2 was immunized subcutaneously each mouse with an equal volume of ISA 201 (Seppic, France) and mice immunized ISA 201 alone or PBS only were enrolled as controls. All mice were vaccinated 3 times at 2-week intervals. (Tian et al., 2022)
Immune Response:
Humoral: Anti-rTgCST2 titers reached up to 1:10^4 for vaccinated mice. Serum IgG levels induced by rTgCST2 had a substantial increase compared with control groups (P < 0.001). Both IgG1 and IgG2a levels induced by rTgCST2 showed considerable rise after inoculation (P < 0.001), IgG1 was the predominant subtype. (Tian et al., 2022) Cellular: On 6 weeks post-vaccination, only the levels of IFN-γ and IL-10 in ISA201-rTgCST2 immunized groups were significantly higher (P < 0.05 and P < 0.001, respectively) than the control groups. (Tian et al., 2022)
Challenge Protocol:
Two weeks after the last vaccination, 10 immunized mice per group were challenged intraperitoneally with 1 × 10^2 tachyzoites of RH T. gondii strain and 10 additional mice were challenged intragastrically with 10 cysts of PRU strain. (Tian et al., 2022)
Efficacy:
Intraperitoneal inoculation of 10^2 RH strain tachyzoites induced all death of the mice within 9 days after challenge. There was no significant difference in survival time between two control groups (PBS and ISA 201, P > 0.05). The survival time of mice immunized with ISA 201-rTgCST2 was significantly longer than that of PBS group (P < 0.001). (Tian et al., 2022) After 2 months of observation, 10 of 10 mice infected orally with 10 cysts of PRU strain in ISA 201-rTgCST2 immunized group survived. However, only one animal in the ISA 201 group and two animals in the PBS group died throughout the experiment. Brain cyst burdens in ISA 201-rTgCST2 immunized group had decreased a bit compared with PBS control groups (reduction by 36.4%, P < 0.05). Meanwhile, compared with PBS control groups, there displayed a reduction in brain cyst diameter of ISA 201-rTgCST2 immunized group (P > 0.05, reduction by 16.7%). (Tian et al., 2022)
Description:
Total RNA of T. gondii tachyzoites was extracted. Complementary DNA (cDNA) was synthesized and Toxoplasma gondii IMC gene was amplified by PCR from cDNA with primers containing EcoRI and XhoI sites. The gene was cloned into pFastBac vector and was transfected and formed recombinant baculovirus (rBV). Sf9 insect cells was co-infected by rBVs expressing T. gondii IMC or influenza M1, and VLPs containing both T. gondii IMC and influenza M1 were released. (Lee et al., 2016)
Virus-like particle (VLP) vaccine consisting of the influenza M1 protein as a core protein together with IMC ISP3 of T. gondii. (Lee et al., 2016)
i.
Mouse Response
Vaccination Protocol:
Mice (6 per group) were intranasally immunized twice with 100 μg total VLP protein at 4-week intervals. (Lee et al., 2016)
Immune Response:
Higher levels of IgA and IgG, higher IgG2a than IgG1; higher levels of IFN-γ, IL-6 and IL-11 (Lee et al., 2016)
Challenge Protocol:
Naïve or immunized mice were infected with T. gondii ME49 intraperitoneally with 20 cysts in 100 μl PBS at 1 month after boosting. Body weight changes and survival were observed daily, and cysts in the brain were counted. (Lee et al., 2016)
Efficacy:
Significantly decreased cyst counts and cyst sizes in brain were detected in mice upon challenge infections compared to non-immunized mouse controls (Reduction rate of cyst count: 75%, **P < 0.01; Reduction rate of cyst size: 50%, *P < 0.05) Immunized mice gained body weight whereas control mice lost body weight or died upon challenge. All mice immunized survived whereas control mice showed 60% survival. (Lee et al., 2016)
Description:
TgROP18 complete open reading frame was amplified from the PRU strain RNA by RT-PCR with primers introducing BamHI and SalI sites.The gene was inserted into pET-30a(+) vectors.The recombinant plasmids were transformed into E. coli BL21-codon plus (DE3)-RIL competent cells (Stratagene). The cells were selected, grown, and harvested, releasing the TgROP18 protein. The protein was made into PLG microparticles by water-in-oil-in-water double emulsion solvent evaporation technique. (Xu et al., 2015)
Description:
TgROP38 complete open reading frame was amplified from the PRU strain genomic DNA with primers introducing BsmBI and BamHI sites. The gene was inserted into pSMK vectors. The recombinant plasmids were transformed into E. coli BL21-codon plus (DE3)-RIL competent cells (Stratagene). The cells were selected, grown, and harvested, releasing the TgROP38 protein.The protein was made into PLG microparticles by water-in-oil-in-water double emulsion solvent evaporation technique. (Xu et al., 2015)
Vaccination Protocol:
Mice were randomly divided into nine groups (19 mice each) and were injected subcutaneously at five different points at a 2-week interval. Each group were injected of different vaccines (with 10μg proteins): 1) 100μg PBS, 2) 100μg PLG, 3) 100μg rTgROP38, 4) 100μg rTgROP18, 5) 50μg rTgROP38 + 50μg rTgROP18, 6) 100μg PLG-rROP38, 7) 100μg PLG-rROP18, 8) 100μg PLG-rROP38-rROP18 (50μg rROP38 + 50μg rROP18), and 9) blank controls which were not treated until challenged. (Xu et al., 2015)
Challenge Protocol:
Eight weeks after the second immunization, 10 mice in all groups were challenged orally with 10 cysts of the PRU strain and were used to calculate the mean number of cysts per brain 1 month later. (Xu et al., 2015)
Efficacy:
Immunization with various protein vaccines significantly decreased the brain cyst formation in mice (P < 0.01), compared with all the control groups. The brain cyst number in the mice immunized with PLG-rROP38-rROP18 was the lowest, with a cyst reduction of 81.3 %. (Xu et al., 2015)
Following amplification of the T. gondii cyclophilin gene, the shuttle expression plasmid pMV261-TgCyP and integrative expression plasmid pMV361-TgCyP were constructed, and their expression was stimulated after transfection into BCG (Yu et al., 2013).
h. Immunization Route
Intramuscular injection (i.m.)
i.
Mouse Response
Vaccination Protocol:
rBCGpMV261-TgCyP and rBCGpMV361-TgCyP (106 CFU) were used to immunise the mice in two different manners (through an i.v. or oral route) (Yu et al., 2013).
Vaccine Immune Response Type:
VO_0003057
Challenge Protocol:
Two weeks after the third vaccination, mice were selected randomly from each group and challenged intraperitoneally (i.p.) with 10^3 highly virulent T. gondii RH strain tachyzoites (Yu et al., 2013).
Efficacy:
The levels of Th1-type IFN-γ, IL-2 and IL-12 were significantly increased following immunisation with the rBCG vaccines via the i.v. or oral route, which indicated that catalytic activity against T. gondii infection was generated in the mice. rBCGpMV361-TgCyP i.v. inoculation resulted in a higher protection efficiency, as demonstrated by the increased survival time and survival rate (17%) of BALB/c mice (Yu et al., 2013).
Description:
The ORF of TgCDPK6 were amplified with RT–PCR using primers containing Kpn I and Not I restriction sites. The RT–PCR products were inserted into the pET-30a(+) via restriction sites, forming pET-CDPK6. E. coli BL21(DE3) was transformed with pET-CDPK6. rTgCDPK6 was expressed, purified, and encapsulated in 50:50 PLG using the water-in-oil-in-water double emulsion solvent evaporation technique. (Zhang et al., 2016)
Description:
The ORF of TgROP18 were amplified with RT–PCR using primers containing BamH I and Sal I restriction sites. The RT–PCR products were inserted into the pET-30a(+) via restriction sites, forming pET-ROP18. E. coli BL21(DE3) was transformed with pET-ROP18. rTgROP18 was expressed, purified, and encapsulated in 50:50 PLG using the water-in-oil-in-water double emulsion solvent evaporation technique. (Zhang et al., 2016)
Vaccination Protocol:
A total of 13 groups of mice were injected subcutaneously with 10 μg of each protein. The vaccinated groups were immunized with rCDPK6, rROP18, rCDPK6 + rROP18, rCDPK6 + Montanide™ ISA 206 VG (206), rROP18 + 206, rCDPK6 + rROP18 + 206, rCDPK6 + PLG, rROP18 + PLG, or rCDPK6 + rROP18 + PLG. The control groups were immunized with PBS alone, PBS + 206, or PBS + PLG, or was not treated. (Zhang et al., 2016)
Immune Response:
Humoral: Higher levels of IgG in all protein vaccines. The levels of IgG increased sequentially with successive immunizations with the tested antigens, and reached their highest levels in week 8. Six weeks after the last vaccination, the levels of IgG antibodies in the mice immunized with rROP18 + PLG or rCDPK6 + rROP18 + PLG were significantly higher than those in the mice immunized with rROP18 or rCDPK6 + rROP18 (P < 0.01), but were not statistically different from those in the mice immunized with various proteins + 206 adjuvant (P > 0.05). (Zhang et al., 2016) Cellular: The splenocytes proliferated more strongly in the vaccinated mice than in the controls, and the highest proliferative response was detected in rCDPK6 + rROP18 + PLG mice (P < 0.001). Higher levels of T-cell subtypes were detected in mice immunized with protein vaccines. protein–PLG induced significantly higher levels of CD4+ (P < 0.001) and CD8+ T lymphocytes (P < 0.01) than the controls. rROP18 + 206 and rROP18 + PLG induced significantly higher levels of CD4+ than the controls (P < 0.05). Only mice from rROP18 + 206 group were showed significantly higher level of CD8+ compared to that in controls (P < 0.05). (Zhang et al., 2016)
Challenge Protocol:
Six weeks after the immunization, 10 mice in each group were challenged intraperitoneally with 10^3 tachyzoites of the T. gondii RH strain, and their survival was recorded daily until all the mice had died. Six mice in each group were inoculated orally with 10 PRU tissue cysts, and their brain cysts were determined 4 weeks after chronic infection was established. (Zhang et al., 2016)
Efficacy:
Acute: The average survival time of the mice immunized with the protein vaccines (8.56 days) was slightly longer than that in the controls (8 days). Immunization with rROP18 + PLG (10.9 days ± 2.58), rROP18 (10.1 days ± 1.52), and rCDPK6 + PLG (9.1 days ± 0.24) significantly prolong the average survival time in mice compared with that of the controls (P < 0.05). (Zhang et al., 2016) Chronic: The tissue cyst loadings in the brains of mice vaccinated with the proteins varied from 47.7 % to 73.6 %, and were significantly lower than those in the control groups (P < 0.001). The protein antigens plus PLG microparticles provided more effective protection to the mice than immunizations only with proteins, but the differences were not statistically significant (P > 0.05). (Zhang et al., 2016)
11. RH-AMA1KO
a. Type:
Live, attenuated vaccine
b. Status:
Research
c. Host Species for Licensed Use:
Human
d. Antigen
Live, attenuated T. gondii RH strain with AMA1 knockout (Lagal et al., 2015)
e. Immunization Route
Intraperitoneal injection (i.p.)
f. Description
T. gondii Live, attenuated vaccine using RH strain with AMA1 knockout (Lagal et al., 2015)
Vaccination Protocol:
Viable parasites (2.5 × 10^2 to 10^6) were injected i.p. or subcutaneously into mice in a 100-μl volume of phosphate-buffered saline (PBS). (Lagal et al., 2015)
Challenge Protocol:
RH: Naive and AMA1KO-injected mice received an i.p. injection of 10^3 (C57BL/6J) or 10^5 (BALB/c, CD-1) AMA1+ parasites 6.5 to 15 weeks after the first injection. (Lagal et al., 2015) Chronic: 10^3 Me49 tachyzoites were injected in BALB/c mice that had been inoculated with 10^5 AMA1KO tachyzoites 8 weeks earlier. (Lagal et al., 2015)
Efficacy:
BALB/c: When challenged with 10^5 AMA1+ type I parasites, BALB/c naive mice died within 6 days, while mice injected with either 10^5 or 10^6 AMA1KO parasites 8 to 10 weeks earlier survived over the assay period of 60 days. The protection went down to 18% when a lower AMA1KO dose (10^3 AMA1KO parasites) was first injected. Immunity lasting more than 8 months for BALB/c mice immunized with 10^5 AMA1KO tachyzoites, which resisted the challenge from 10^5 virulent tachyzoites. 60% of BALB/c mice immunized with 10^5 AMA1KO parasites and challenged 8 weeks later with 10^3 type II ME49 tachyzoites gave negative results by qPCR for the presence of parasites in their brain whereas 100% of the nonimmunized mice gave positive results. (Lagal et al., 2015) C57BL/6J: A rather long-lasting immunity was conferred to C57BL/6J mice immunized with 2.5 × 10^2 and 1.5 × 10^3 AMA1KO tachyzoites, which resisted challenge from 10^3 AMA1+ tachyzoites. (Lagal et al., 2015) CD-1: CD-1 mice immunized with 10^4 or 10^6 AMA1KO parasites survived challenge with 10^5 AMA1+ tachyzoites. (Lagal et al., 2015)
Description:
ORF of TgADF was amplified using PCR from the cDNA template with forward primer introducing BamHI site and reverse primer introducing XhoI site. The PCR products were cloned into the ppET30a(+) vector and transformed into E. coli DH5α competent cells. rTgADF was harvested from E. coil and was purified. (Liu et al., 2016)
Vaccination Protocol:
The mice were randomly divided into 5 groups (8 mice per group). Four groups were intranasally administered with 10, 20, 30, or 40 μg of rTgADF that was separately dissolved in 20 μL of phosphate-buffered saline (PBS). The control group was immunized with PBS. On days 0, 14, and 21, the nostrils of the mice were slowly instilled with rTgADF protein solution (10 μL per nostril). (Liu et al., 2016)
Immune Response:
Mucosal: Significantly higher levels of sIgA titers were observed in the nasal, intestinal, and vesical washes in mice immunized with 20, 30, or 40 μg of rTgADF than in the control groups (P < 0.05). The sIgA levels in three mucosal washes in the 30- and 40-μg groups were prominently higher than that in the 20-μg group. Moreover, an apparent predominance of the 30-μg group over the 40-μg group was observed on the basis of the vesical washes. (Liu et al., 2016) Humoral: High IgG titers were detected in the serum samples of all immunized mice (P < 0.05). The OD values for IgG were continuously increased following an increase in the immunization dosage, and the 40-μg group showed the highest titer among all the immunized groups (P < 0.01). (Liu et al., 2016) Cellular: Splenocytes from the 30-μg and 40-μg groups exhibited a significantly greater proliferative response to rTgADF than splenocytes from the control group. Splenocytes from all the immunized groups secreted significantly high levels of IFN-γ and IL-2 when compared with the control group, and the highest levels were elicited by 30 μg of rTgADF. In contrast, IL-4 and IL-10 levels displayed no significant changes between the immunized and control groups (P > 0.05). (Liu et al., 2016)
Challenge Protocol:
Two groups of BALB/c mice (30 mice per group) were immunized with PBS or 30 μg of rTgADF as mentioned in vaccination protocol. Twenty-two mice from each group were intragastric administrated with a dose of 4 × 10^4 tachyzoites for the acute assay. Survival times of the infected mice were recorded daily. The other 8 mice per group were challenged with 1 × 10^4 tachyzoites for the chronic model. Four weeks later, real-time PCR was used to quantify tachyzoite loads in the liver and brain for detecting the SAG1 gene. (Liu et al., 2016)
Efficacy:
Acute: Most of the mice in the control group were dead within 9 days post-infection, with the last mouse dying on the 23rd day. However, 36.36 % of the mice immunized with 30 μg of rTgADF remained alive 30 days post-infection. (Liu et al., 2016) Chronic: The tachyzoite load in the livers and brains was significantly lower in the immunized mice {13.89 ± 1.27 (105/g) and 6.33 ± 0.43 (105/g), respectively)} than in the control mice {43.09 ± 3.03 (105/g) and 12.92 ± 3.30 (105/g), respectively}, which showed a reduction in the tachyzoite load by 67.77 % in the liver and 51.01 % in the brain when compared with the control group. (Liu et al., 2016)
Description:
ORF of the TgPGAM 2 gene was amplified from the RH strain T. gondii tachyzoites and cloned into the pET-30a(+) vector. The rTgPGAM 2 protein was express in BL21 (DE3) cells, purified, and the endotoxin in rTgPGAM2 was removed. (Wang et al., 2016)
Vaccination Protocol:
The mice were divided into five groups (10 mice per group) and were intranasally immunised with 10, 20, 30 or 40 μg of rTgPGAM2 suspended in 20 μL of sterile PBS, or PBS only as control. The mice were immunised using the same protocol on days 0, 14 and 21. (Wang et al., 2016)
Challenge Protocol:
On the 15th day after the last immunisation, eight mice in each group were challenged orally with 1 × 10^4 T. gondii RH strain tachyzoites for the tachyzoite load assay, and 12 mice in each group were challenged orally with 4 × 10^4 tachyzoites for the survival assay. The numbers of tachyzoites in the brains and livers of the mice were measured to assess the results of the chronic challenge infection assay. The time to death and survival of the mice were recorded and assessed for one month after parasite challenge. (Wang et al., 2016)
Efficacy:
Chronic infection: The tachyzoite loads in the brain tissues were (7.64 ± 1.47) × 10^6/g in the control group and (2.92 ± 0.51) × 10^6/g in the rTgPGAM 2-vacinated group. The tachyzoite loads in the liver tissues were (10.39 ± 2.17) × 10^6/g in the control group and (3.85 ± 1.17) × 10^6/g in the rTgPGAM 2-vacinated group. Compared with the mice in the control group, the average parasite burden was reduced significantly by 56.9% and 69.2% in the brain and liver tissues, respectively. (Wang et al., 2016) Acute infection: A significant increase in the survival time and survival rate (70%) was observed in the rTgPGAM 2-immunised group compared with the control group. (Wang et al., 2016)
Description:
First strand cDNA was synthesised from the total RNA extracted from tachyzoites of T. gondii. TgRACK1 was amplified by PCR, which introduced EcoRI and NotI sites. The PCR product was digested and cloned into pGEX-6P-1 vector. The resulting pGEX-6P-1-TgRACK1 plasmid was transformed into E. coli BL21 (DE3) host cells. rTgRACK1 was harvested from E. coil and purified. (Wang et al., 2014)
Vaccination Protocol:
Fifty 6-week-old female BALB/c mice were randomly divided into five groups (10 per group) and were intranasally immunized with 15, 25, 35 or 45 μg of rTgRACK1 dissolved in 20 μl sterile phosphate-buffered saline (PBS) on days 0, 14, and 21. Control mice received PBS alone. (Wang et al., 2014)
Immune Response:
Mucosal: The sIgA antibody titers in the nasal washes of the rTgRACK1-treated groups were significantly higher than those of the PBS control, with the highest titer of sIgA antibody detected in the 35 μg rTgRACK group (P<0.01). sIgA levels from the intestinal and vesical washes were higher in 25, 35, 45 μg rTgRACK1 groups, and 35 μg rTgRACK1 could also elicit the highest sIgA levels in intestinal and vesical washes (P < 0.01). Enhanced production of both IFN-γ and IL-2 were detected in the 25 μg rTgRACK1 group (P < 0.05) and reached higher levels in the 35 and 45 μg rTgRACK1 groups (P < 0.01). (Wang et al., 2014) Humoral: Significantly increased levels of total IgG and IgA antibodies were observed in the 25, 35 and 45 μg rTgRACK1 groups compared to the control group (P < 0.05). The maximum IgG antibody response was detected in the 35 μg rTgRACK1-treated group. A mixed IgG1/IgG2a response with predominant IgG2a production was detected. (Wang et al., 2014) Cellular: Significant productions of IFN-γ, IL-2 and IL-4 were all evident at 25 μg rTgRACK1 treatment and reached a maximum level at 35 μg rTgRACK1. No significant changes in production of IL-10 were seen (P>0.05). (Wang et al., 2014)
Challenge Protocol:
Mice immunised with 35 μg of rTgRACK1 and PBS control mice (30 mice/group, 10 mice for chronic infection and 20 mice for lethal assay) were orally challenged with either 1 × 10^4 tachyzoites for the chronic infection or 4 × 10^4 tachyzoites for an acute infection on the fifteenth day after the last immunisation.The time to death of the challenged mice was monitored for the acute challenge. On the 31st day, the numbers of tachyzoite in the murine brains and livers of the chronic challenge mice were determined by real-time PCR assay. (Wang et al., 2014)
Efficacy:
Chronic: Significant reductions of tachyzoite were observed in both the brain and liver tissues of rTgRACK1-immunised mice (1.12 ± 0.17 × 10^6/g and 2.61 ± 0.28 × 10^6/g respectively) in comparison with those of PBS controls (2.09 ± 0.32 × 106/g and (5.63 ± 0.74) × 10^6/g, respectively), representing a 57.09% reduction in brain (P < 0.01) and 53.64% reduction in liver (P < 0.01) with rTgRACK1 vaccination. (Wang et al., 2014) Acute: The death time of mice immunised with 35 μg rTgRACK1 was between 8 and 21 days after the challenge, while all of the mice in the PBS control group died from 6 to 10 days after the challenge, representing significantly increase in the survival rate of vaccinated mice (approximately 45%) (P < 0.01). (Wang et al., 2014)
Description:
ORF of the TgROP17 gene was amplified by RT-PCR and was cloned into the pGEX-6P-1 vector. The recombinant plasmid was transferred into E. coli DH5a and were selected. The successful pGEX-6P-1/TgROP17 construct was transformed into E. coli Rosetta (DE3). The rTgROP17 protein was express in E. coli Rosetta (DE3) cells, purified, and the endotoxin in rTgROP17 was removed. (Wang et al., 2014)
Vaccination Protocol:
Mice were randomly divided into 5 groups (8 mice per group) and were immunized nasally with 20 µl PBS containing 15, 25, 35 or 45 µg of rTgROP17. The control mice were given PBS solution only. All animals were vaccinated three times on days 0, 14, and 21. (Wang et al., 2014)
Immune Response:
Humoral: The total IgG antibody productions of the mice immunized with 35 and 45 µg rTgROP17 were significantly higher than those of the control group (P<0.01) but not significantly different from each other (P>0.05). 25 µg but not 15 µg rTgROP17 elicited elevated IgG antibody levels compared to the control groups (P<0.05). Both IgG1 and IgG2a were detected in the sera of all the mice immunized with rTgROP17, and greater levels of IgG2a were detected than IgG1 in general. (Wang et al., 2014) Cellular: 35 µg and 45µg rTgROP17 significantly stimulated the production of IFN-γ(P<0.01), IL-2(P<0.01), and IL-4 (P<0.05) over the PBS control group. 25 µg rTgROP17 also stimulated the production of IFN-γ and IL-2 (P<0.05) but not IL-4 (P>0.05). 15 µg rTgROP17 did not stimulated the production of IFN-γ, IL-2, or IL-4 (P>0.05). No significant difference was observed for the levels of IL-5 between the rTgROP17-vaccinated and PBS control mice (P>0.05). The splenocyte stimulation indices (SIs) of the mice in 35 and 45 µg rTgROP17 groups were significantly greater than those in 15 µg rTgROP17 group or PBS control (P<0.01). The SI of the 25 µg group was greater than that of the PBS control (P<0.05). No significant difference was found for the SIs of the 15 µg group and PBS controls (P> 0.05). (Wang et al., 2014) Mucosal: Higher levels of SIgA were detected in the nasal, vaginal and intestinal washes of rTgROP17-immunized mice compared to those of their PBS controls (35 and 45 µg: P<0.01; 25 µg: P<0.05). (Wang et al., 2014)
Challenge Protocol:
Mice at 6 weeks of age were randomly divided into two groups (20 mice per group) and vaccinated intranasally with 35 µg of rTgROP17 or GST control in 20 µl volumes on days 0, 14, and 21. On day 14 after the final immunization, 8 mice from each group were orally challenged with 1×10^4 tachyzoites of the RH strain for chronic assay, and the other 12 mice from each group were challenged with 4×10^4 tachyzoites for acute infection. (Wang et al., 2014)
Efficacy:
Chronic infection: The tachyzoite loads in the brains and livers of the mice immunized with 35 µg rTgROP17 were significantly reduced to 59.17% (P<0.01) and 49.08% (P<0.05) of the loads found in the GST-treated control mice, respectively (Wang et al., 2014) Acute infection: The survival rates of the mice in rTgROP17 group were significantly increased (75%) on the 30th day after the challenge when compared to those in GST-treated control group (25%) (P<0.01). Survival of rTgROP17-immunized mice was three times over the GST-treated mice. The death time of the control mice ranged from 5 to 9 days, while the mice immunized with rTgROP17 died between days 10 and 14 after the challenge. (Wang et al., 2014)
Description:
To construct the pVAX-MIC8 expression plasmid, the coding sequence of the T. gondii MIC8 gene was amplified by polymerase chain reaction (PCR) from genomic DNA of T. gondii. The PCR product was cloned in pGEM-T easy vector (Promega, USA) and sequenced in both directions to ensure fidelity and generated pGEM-MIC8. The MIC8 fragment was inserted into the BamH I/Xba I sites of pVAX I by double digestion with BamH I/Xba I restriction enzymes, and ligation was finally done using T4 DNA ligase. The resulting plasmid was named pVAX-MIC8 (Liu et al., 2010).
Vaccination Protocol:
Mice (20 per group) received injections of 100 μl of pVAXMIC8 plasmid or empty pVAX I vector into each anterior tibial muscle (final plasmid concentration, 100 μg/100 μl). The injections were repeated using the same protocol 2 and 4 weeks after vaccination. Tail bleeds were performed on vaccinated mice on the day of first vaccination, second vaccination, and third vaccination and pre-challenge to ascertain the presence of specific anti-T. gondii antibodies (Liu et al., 2010).
Challenge Protocol:
Mice in all groups were intraperitoneally challenged with the virulent T. gondii RH strain (10^3 tachyzoites per mouse) 2 weeks after the last immunization (Liu et al., 2010).
Efficacy:
Immunization of mice with pVAXMIC8 dramatically increased the survival time (10.3± 0.9 days) compared with control mice which died within 5 days of challenge (Liu et al., 2010).
17. T. gondii DNA vaccine encoding multi-epitope ROP8
a. Type:
DNA vaccine
b. Status:
Research
c. Host Species for Licensed Use:
Human
d. Antigen
ROP8: expressed in tachyzoite and bradyzoite stages. Involved in the pathogenicity of the parasite as well as host cell modulation, critical for acute virulence of T. gondii. (Foroutan et al., 2020)
e. Gene Engineering of
ROP8
Type:
DNA vaccine construction
Description:
The linear B cell epitopes, major histocompatibility complex (MHC) class I and class II molecules for ROP8 protein were predicted using bioinformatic tools. MHC I, MHC II, and linear B-cell epitopes were linked together by the SAPGTP linker, and the Kozak sequence was added at N-terminal. Several physico-chemical parameters, post translational modifications, and the secondary and tertiary structure for the multi-epitope peptide were predicted. The final sequence of the peptide was sent for the construction and cloning into eukaryotic expression vector pcDNA3.1. (Foroutan et al., 2020)
Multi-epitope DNA vaccine encoding the potential B and T-cell epitopes from ROP8 protein using eukaryotic expression vector pcDNA3.1. (Foroutan et al., 2020)
Vaccination Protocol:
Three groups of BALB/c mice (8 per experimental group) were immunized on days 0, 21, and 42 with 100 μg of plasmid DNA suspended in 100 μl sterile endotoxin free PBS (100 μg/100 μl), including two control groups (PBS and empty pcDNA3.1 vector). (Foroutan et al., 2020)
Immune Response:
Humoral: Significantly high levels of IgG antibodies were detected post vaccination with multi-epitope ROP8 vaccine compared with PBS and empty pcDNA3.1 vector (P < 0.05). The multi-epitope vaccine elicited both IgG1 and IgG2a production with the predominance of IgG2a compared with control groups (P < 0.05). (Foroutan et al., 2020) Cellular: Spleen cells from mice vaccinated with multi-epitope ROP8 vaccine generated a significantly higher level of IFN-γ and IL-4 than the mice in the PBS and empty pcDNA3.1 vector groups (P < 0.05). The BALB/c mice vaccinated with multi-epitope vaccine have the greater splenocyte proliferative response compared with control groups (P < 0.05). (Foroutan et al., 2020)
Challenge Protocol:
Three weeks after the last immunization, five mice per group were intraperitoneally (i.p) challenged with 2 × 10^3 tachyzoite of virulent T. gondii RH strain. Their survival periods were monitored daily. (Foroutan et al., 2020)
Efficacy:
The BALB/c mice that received the multi-epitope ROP8 vaccine had prolonged survival time compared with PBS and pcDNA3 control groups (P < 0.05). (Foroutan et al., 2020)
Vaccination Protocol:
All studies were done with 6–8 weeks old Kunming mice. For experimental group, 100 μg pcDNA3-MIC3 were injected into mouse hind footpad three times, at week 0, 3 and 6, respectively. The mice of control groups were vaccinated with 100 μg pcDNA3 or normal saline (NS) three times (Xiang et al., 2009).
Challenge Protocol:
Immunized Kunming mice were challenged intraperitoneally with 500 tachyzoites of RH strain T. gondii 9 weeks after the first immunization (Xiang et al., 2009).
Efficacy:
The survival time of mice in pcDNA3-MIC3 group was significantly longer than those of in group pcDNA3 and NS (Xiang et al., 2009).
Description:
GRA6 ORF was PCR amplified and the PCR product was ligated into the pcDNA3.1 vectors to construct recombinant plasmid pcDNA3.1-HisGRA6. (Sun et al., 2011)
T. gondii DNA vaccine encoding GRA6 using pcDNA3.1 vector. (Sun et al., 2011)
i.
Mouse Response
Host Strain:
BALB/c mice carrying H-2Ld gene and Kunming mice without H-2Ld gene. (Sun et al., 2011)
Vaccination Protocol:
BALB/c mice received three injections of 100 μg of pcDNA3.1-HisGRA6 DNA at 2-week intervals in both tibialis anterior muscles. Kunming mice were immunized with pcDNA3.1-HisGRA6 and BALB/c with empty pcDNA3.1 as controls. One group of BALB/c mice was immunized with pcDNA3.1-HisGRA6 formulated in 1% LMS as the adjuvant. (Sun et al., 2011)
Challenge Protocol:
The immunized mice were challenged intraperitoneally with 1, 000 tachyzoites of T. gondii RH strain (Type I). One week after the challenge, brain, liver and spleen were removed and fixed in 10% formalin for histological examination. Two weeks after the last immunization, the animals were observed daily for mortality. (Sun et al., 2011)
Efficacy:
Immunization of BALB/c mice with pcDNA3.1-HisGRA6 significantly increased the survival rate of these mice (40% survival) (P < 0.05) in comparison with BALB/c immunized with the control vector or Kunming mice immunized with pcDNA3.1-HisGRA6 (0% survival) (P < 0.05). Survival was further increased in mice immunized with pcDNA3.1-HisGRA6 plus LMS (53.3% survival cf. 40% without LMS; P < 0.05). Mice alive at week 12 stayed alive for at least 2 months. No parasite was detected in the brain, liver or spleen of BALB/c mice immunized with pcDNA3.1-HisGRA6 alone or pcDNA3.1-HisGRA6 plus LMS. Parasites were clearly visible in the tissues of the control groups. (Sun et al., 2011)
Efficacy:
When challenged with Toxoplasma gondii RH strain, mice immunized with pIL-12 co-administration had significantly higher survival rates compared to co-administration with pCTA2/B or pcDNA3.1-SAG1-ROP2 alone (control) (Xue et al., 2008).
Description:
The α-chain of MIC11 gene was PCR amplified and was ligated into pcDNA3.1 vector forming pcDNA/MIC11 plasmid. The plasmid was cloned in E. coli (DH5α strain) and was confirmed by restriction digestion and sequencing analysis. The large scale plasmid extraction was performed using Endo-Free Plasmid Maxi Kit (OMEGA bio-tek, USA) by following the manufacturer's instructions. (Tao et al., 2013)
Vaccination Protocol:
Sixty BALB/c mice were randomly divided into three groups (20 in each group). 100 μg (for each injection) of pcDNA/MIC11 plasmid or the pcDNA3.1 empty vector were suspended in 100 μl sterile PBS and intramuscularly injected into different groups of mice, respectively. The mice injected with 100 μl sterile PBS only were served as the blank control group. All groups of mice were immunized three times at a 2-week interval using same dosages. (Tao et al., 2013)
Immune Response:
Humoral: higher anti-TLA IgG Cellular: higher splenocyte proliferation activity, higher levels of IFN-γ, IL-12, and IL-2 (Tao et al., 2013)
Challenge Protocol:
Ten days after the last immunization, twelve mice from each group were randomly selected and injected intraperitoneally with 1 × 10^3 tachyzoites of T. gondii RH strain. Tachyzoites were counted with a hemocytometer and adjusted to 5 × 10^3 parasites/ml with 0.1 M PBS (pH 7.4). The parasite solution (200 μl) was injected into each mouse, and mice survival was subsequently monitored daily for 15 days. (Tao et al., 2013)
Efficacy:
The mice immunized with pcDNA/MIC11 showed significantly prolonged survival. All mice in the control groups immunized with PBS or pcDNA3.1 died within 8 or 10 days post-infection, respectively. However, 50 % of the mice immunized with pcDNA/MIC11 were alive at day 10, and 17 % of them survived to day 15 after the parasite challenging. (Tao et al., 2013)
22. T. gondii DNA vaccine pDREP-TgNTPase-II
a. Type:
DNA vaccine
b. Status:
Research
c. Host Species for Licensed Use:
Human
d. Antigen
TgNTPase-II: Toxoplasma gondii nucleoside triphosphate hydrolase-II: facilitate the parasite to salvage purines from the host cell for survival and replication (Zheng et al., 2017)
e. Vector:
pDREP: a DNA-based alphaviral RNA replicon vector (suicidal DNA vector). Induce apoptotic cell death of the transfected cells (Zheng et al., 2017).
f. Immunization Route
Intramuscular injection (i.m.)
g. Description
T. gondii DNA vaccine encoding TgNTPase using pDREP I vector. (Zheng et al., 2017)
T. gondii DNA vaccine encoding GRA8 using pDsRed2-N1 vector. (Chu et al., 2018)
24. T. gondii DNA vaccine pEGFP-C1/GRA41
a. Type:
DNA vaccine
b. Status:
Research
c. Host Species for Licensed Use:
Human
d. Antigen
GRA41: dense granular protein 41. Induce the interface formation between host cells and parasite. (Zhou et al., 2019)
e. Gene Engineering of
GRA41
Type:
DNA vaccine construction
Description:
Amplified by PCR using a forward primer introducing HindIII recognition sites and a reverse primer introducing BamHI recognition sites. The amplified GRA41 was purified and was inserted into the pEGFP-C1 vector. (Zhou et al., 2019)
Vaccination Protocol:
Mice were assigned to three groups where there were 24 mice in each one. All the mice were vaccinated with 100 μL of PBS containing 100 μg pGRA41, 100 μg empty vectors, or 100 μg PBS respectively. There were three injections with 2 week interval. (Zhou et al., 2019)
Immune Response:
Humoral: The level of IgG in pEGFP-C1/GRA41 group increased continuously after the first injection. There was a reliable difference in the IgG quantity between the test group and the control groups in week 4 and week 6 (P<0.05). The difference was significant between IgG2a and IgG1 in pEGFPC1/GRA41 group (P<0.05). The quantity of the former is approximately two times than that of the latter. (Zhou et al., 2019) Cellular: For IFN-γ, there was a statistically signifcant difference between the pEGFPC1/GRA41 group and the two controlled groups. However, for IL-4 or IL-10, there was no statistically significant difference between all groups. (Zhou et al., 2019)
Challenge Protocol:
Two weeks after the fnal immunization, ten mice were challenged intraperitoneally with 1×10^4 tachyzoites of the high virulence T. gondii RH strain, and another 10 mice were challenged intragastrically with 20 T. gondii PRU strain cysts. (Zhou et al., 2019)
Efficacy:
RH: All mice in pEGFPC1/GRA41 group died on day 18, with an average survival duration of 13.3±3.37 days, compared to mice in the control groups which all died on day 7. For survival duration, there was remarkable difference between the pEGFPC1/GRA41 group and the control groups (P<0.05). (Zhou et al., 2019) PRU: The mean number of cysts in control groups (1520±278) was approximately two times higher than that of pEGFP-C1/GRA41 group (618±132), which was statistically signifcant (P<0.05). (Zhou et al., 2019)
Description:
Amplified by PCR using a forward primer introducing Sma I recognition sites and a reverse primer introducing Xba I recognition sites. (Yan et al., 2012) The amplified MIC6 was cloned into the pGEM-T Easy vector (Promega) and was sequenced. The ROP18 fragment was cleaved by SmaI/XbaI from pGEM-MIC6, purified, and then cloned into the SmaI/XbaI sites of pIRESneo, generating recombinant plasmid pIRESneo/MIC6. The purified TgPLP1 was cloned into pIRESneo/MIC6 and formed pIRESneo/MIC6/PLP1. (Yan et al., 2012)
Description:
Amplified by PCR using a forward primer introducing Cla I recognition sites and a reverse primer introducing BamH I recognition sites. (Yan et al., 2012) The amplified TgPLP1 was digested, purified, and cloned into pIRESneo/MIC6 and formed pIRESneo/MIC6/PLP1. (Yan et al., 2012)
T. gondii DNA vaccine encoding MIC6 and TgPLP1 as antigin using pIRESneo vector. pVAX I plasmids encoding IL-18 are codelivered as adjuvant. (Yan et al., 2012)
Vaccination Protocol:
Seven groups of mice (25 mice per group) were injected with 100 μg of plasmid DNA suspended in 100 μl sterile PBS. Group I mice were injected with PBS as a blank control; group II to group VI mice were injected with the empty vector pVAX I or pIRESneo, pVAX/IL-18, pVAX/TgPLP1, or pVAX/MIC6, respectively, also as controls; and groups VII and VIII were injected with pIRESneo/MIC6/TgPLP1 or pIRESneo/MIC6/TgPLP1+pVAX/IL-18 (100 μl each) respectively. Two booster injections were administered at 2 week interval. (Yan et al., 2012)
Immune Response:
Humoral: Mice injected with pIRESneo/MIC6/TgPLP1 or pIRESneo/MIC6/TgPLP1+pVAX/IL-18 had significantly high levels of anti-TgPLP1/MIC6 IgG antibodies, especially after the third immunization. Mice injected with pVAX/PLP1 or pVAX/MIC6 alone also generated anti-TgPLP1/MIC6 antibodies but at significantly lower levels (P < 0.05). Mice injected with PBS, pVAX I, pIRESneo, or pVAX/IL-18 alone did not generate anti-TgPLP1 antibodies, significantly different from the groups mentioned above (P < 0.05). (Yan et al., 2012) Cellular: Splenocytes from all groups proliferated to comparable levels. The level of splenocyte proliferation increased over those of the other groups when mice were coinjected with pIRESneo/MIC6/TgPLP1 + pVAX/IL-18 (P < 0.05). Mice immunized with pIRESneo/MIC6/TgPLP1 had higher lymphocyte responses than the controls (P < 0.05). Mice immunized with pVAX/TgPLP1, pVAX/MIC6, or pVAX/IL-18 alone had significantly higher lymphocyte responses than the rest of the controls (P < 0.05). Very large amounts of specific IFN-γ, IL-2, and IL-12 were produced in pIRESneo/MIC6/TgPLP1+pVAX/IL-18, pIRESneo/MIC6/TgPLP1, pVAX/IL-18, pVAX/PLP1, or pVAX/MIC6 groups. Specific amounts of IL-4 and IL-10 were synthesized in pIRESneo/MIC6/TgPLP1 or pIRESneo/MIC6/TgPLP1+pVAX/IL-18 groups. (Yan et al., 2012)
Challenge Protocol:
Five mice were randomly chosen from every group and challenged intragastrically with 80 cysts of strain PRU, and observed daily for mortality. Eight mice of each group were orally infected with 20 cysts of strain PRU at the 14th day after the last immunization. Cysts in the brain were counted 6 weeks after challenge. (Yan et al., 2012)
Efficacy:
Acute infection: All mice immunized with PBS, pIRESneo, or pVAX I died at day 25. Immunization with pIRESneo/MIC6/TgPLP1 dramatically increased the survival time (42.8 ± 2.9 days) (P < 0.05). Coimmunization with pIRESneo/MIC6/TgPLP1 and pVAX/IL-18 enhanced the survival time even further (45.0 ± 2.9 days), although the difference was not statistically significant (P > 0.05). (Yan et al., 2012) Chronic infection: The lowest brain cyst burdens were observed in mice immunized with pIRESneo/MIC6/PLP1+pVAX/IL-18 (1,085.67 ± 49.32 cysts per brain) or pIRESneo/MIC6/PLP1 (1,206.00 ± 10.02 cysts per brain) and were significantly lower than those in the PBS group (3,140.33 ± 96.72 cysts per brain) and the other five groups (P < 0.05). Brain cyst burdens were significantly lower (P < 0.05) in mice immunized with pVAX/PLP1 (1,759.00 ± 60.47) or pVAX/MIC6 (1,890.00 ± 46.36) than in the control animals.(Yan et al., 2012)
Description:
Amplified by PCR using a forward primer introducing Sma I recognition sites and a reverse primer introducing Xba I recognition sites. (Chen et al., 2018) The amplified ROP18 was cloned into the pGEM-T Easy vector (Promega) and was sequenced. The ROP18 fragment was cleaved by SmaI/XbaI from pGEM-ROP18, purified, and then cloned into the SmaI/XbaI sites of pIRESneo, generating recombinant plasmid pIRESneo/ROP18. The purified TgPLP1 was cloned into pIRESneo/ROP18 and formed pIRESneo/ROP18/PLP1. (Yan et al., 2012)
Description:
Amplified by PCR using a forward primer introducing Cla I recognition sites and a reverse primer introducing BamH I recognition sites. (Chen et al., 2018) The amplified TgPLP1 was digested, purified, and cloned into pIRESneo/ROP18 and formed pIRESneo/ROP18/PLP1. (Yan et al., 2012)
T. gondii DNA vaccine encoding ROP18 and TgPLP1 as antigin using pIRESneo vector. pVAX I plasmids encoding IL-18 are codelivered as adjuvant. (Chen et al., 2018)
j.
Mouse Response
Host Strain:
Specific-pathogen-free (SPF) Kunming mice (Chen et al., 2018)
Vaccination Protocol:
Eight groups of mice (30 mice/group) were vaccinated intramuscularly with 100 μg of plasmid dissolved in 100 μl of sterile PBS. The experimental groups received pIRESneo/ROP18/PLP1 + pVAX/IL-18, pIRESneo/ROP18/PLP1, pVAX/PLP1, or pVAX/ROP18 respectively. Injection of pVAX/IL-18, PBS, empty pIRESneo vector, or empty pVAX1 vector was performed as controls. All of the mice were boosted twice at 2-week intervals. (Chen et al., 2018)
Immune Response:
Humoral: Immunization with PBS, pVAXI, pIRESneo, or pVAX/IL-18 alone (four control groups) did not produce an antibody response, which was significantly different (P < 0.05) from the response observed in the four experimental groups. Significant differences were observed between the mice treated with pROP18/PLP1 or pROP18/PLP1 + pVAX/IL-18 and mice treated with pVAX/ROP18 (P < 0.05). (Chen et al., 2018) Cellular: The lymphocyte proliferative response in mice injected with pROP18/PLP1 or pROP18/PLP1 + pVAX/IL-18 was significantly higher than that in control mice (P < 0.05). Th1-type cytokines (IFN-γ, IL-2 and IL-12) were induced at significantly higher levels by ROP18/PLP1 fusion protein (P < 0.05) than by ROP18 or PLP1 alone. Compared to mice immunized with pROP18/PLP1 + pVAX/IL-18, mice immunized with pROP18/PLP1 had than significantly lower IL-2, IL-12 and IFN-γ levels (P < 0.05) but significantly higher IL-4 and IL-10 levels (P < 0.05). (Chen et al., 2018)
Challenge Protocol:
Two weeks after the last stimulation, 12 mice per group were randomly chosen for intragastric administration of 80 T. gondii PRU cysts, and survival was monitored daily. Another 8 mice per group were orally infected with 20 PRU tissue cysts, and brain cysts were counted at 6 weeks post-infection. (Chen et al., 2018)
Efficacy:
Acute infection: the survival time was significantly longer (P < 0.05) in mice vaccinated with pROP18/PLP1 (44 ± 2.9 days) or pROP18/PLP1 + pVAX/IL-18 (47 ± 2.9 days) than in control mice (≤36 days). (Chen et al., 2018) Chronic infection: brain cyst burden was significantly lower (P < 0.05) in mice immunized with pROP18/PLP1 + pVAX/IL-18 or pROP18/PLP1 than in control mice. (Chen et al., 2018)
27. T. gondii DNA vaccine pMIC3i encoding the complete MIC3-ORF (RH(ERP) strain)
Efficacy:
Mice immunized with pMIC3i displayed significant protection against an oral challenge with 76K T. gondii strain cysts, exhibiting fewer brain cysts than the control mice immunized with the empty plasmid pcDNA3. The co-administration of pGM-CSF enhanced this protection (Ismael et al., 2009).
Vaccination Protocol:
Six groups (14 per group) of mice were injected intramuscularly (50 µL in each thigh skeletal muscle) with 100 µg of plasmid DNA suspended in 100 µL sterile PBS. Group I was injected with PBS as control, group II with pcDNA3·1 vector alone as control, group III with pSAG2, group IV with pSAG1-ROP2, group V with pSAG1-ROP2-SAG2 and group VI with pSAG1-ROP2-SAG2 + pIL-12 (100 µg each). The mice were immunized on days 0, 14 and 28 with the same protocol. (Cui et al., 2008)
Vaccine Immune Response Type:
VO_0000286
Challenge Protocol:
On day 56, seven immunized mice per group were challenged with 10^4 tachyzoites of the T. gondii RH strain. The survival days of the mice was recorded. (Cui et al., 2008)
Efficacy:
Higher survival rates after lethal challenge were obtained in mice immunized with pSAG1-ROP2-SAG2 as compared to the mice immunized with PBS, expression vector alone or pSAG2, or pSAG1-ROP2. In addition, the protection induced by pSAG1-ROP2-SAG2 was remarkably enhanced by pIL-12 co-administration (Cui et al., 2008).
29. T. gondii DNA vaccine pSAG5B/SAG5C
a. Type:
DNA vaccine
b. Status:
Research
c. Host Species for Licensed Use:
Human
d. Antigen
SAG5B and SAG5C: sequence branches of surface antigen protein 5, transcribed in T. gondii tachyzoites and bradyzoites, play an important role in cyst persistence in the host. (Lu et al., 2017)
e. Gene Engineering of
SAG5B
Type:
DNA vaccine construction
Description:
Amplified by PCR. The PCR products from SAG5B and SAG5C genes were cloned into the pEASY-T1 vector and digested with restriction enzymes. The SAG5B and SAG5C fragments were purified and inserted into the mammalian expression vector pBudCE4.1 to produce pSAG5B and pSAG5C. The NotI/KpnI fragment encoding SAG5B was excised and cloned into the HindIII/XbaI sites of the pSAG5C to produce pSAG5B/SAG5C. (Lu et al., 2017)
Description:
Amplified by PCR. The PCR products from SAG5B and SAG5C genes were cloned into the pEASY-T1 vector and digested with restriction enzymes. The SAG5B and SAG5C fragments were purified and inserted into the mammalian expression vector pBudCE4.1 to produce pSAG5B and pSAG5C. The NotI/KpnI fragment encoding SAG5B was excised and cloned into the HindIII/XbaI sites of the pSAG5C to produce pSAG5B/SAG5C. (Lu et al., 2017)
Vaccination Protocol:
Mice were divided randomly into five groups (28 in each group) and were immunized four times on weeks 0, 2, 4, and 6. Three experimental groups were immunized with 100 μL of PBS containing 100 μg pSAG5B, 100 μg pSAG5C, or 100 μg pSAG5B/SAG5C . The other two mouse groups (the controls) were injected with the empty vector or with PBS. (Lu et al., 2017)
Immune Response:
Humoral: The IgG levels for the experimental mice increased gradually over time. Significant differences were found in the sera of mice vaccinated using pSAG5B, pSAG5C, or pSAG5B/SAG5C compared with the mice injected with PBS or pBudCE4.1 (P < 0.05). Moreover, IgG levels in the pSAG5B/SAG5C-immunized mice were significantly higher compared with the mice immunized with the single-gene (P < 0.05). A predominance of IgG2a over IgG1 was observed. (Lu et al., 2017) Cellular: The IFN-γ levels in mice immunized with pSAG5B, pSAG5C, or pSAG5B/SAG5C were significantly higher than those of the mice immunized with PBS or pBudCE4.1 (P < 0.05). The pSAG5B/SAG5C-immunized mice generated the highest IFN-γ level. The pSAG5C-immunized mice generated a higher level of IFN-γ than the pSAG5B-immunized mice, but no significant difference was found between these two groups (P > 0.05). The splenocyte supernatant IL-4 levels of all groups were similar (P > 0.05). (Lu et al., 2017)
Challenge Protocol:
Two weeks after the final inoculation (day 56), 12 mice from each group were challenged intraperitoneally with 1 × 10^4 tachyzoites of the highly virulent T. gondii RH strain, while another 12 mice from each group were infected intragastrically with 20 cysts of the T. gondii PRU strain. The mice challenged with the RH strain were observed and those that showed signs of illness were sacrificed immediately using CO2 gas. (Lu et al., 2017)
Efficacy:
RH: The survival time of the mice immunized with pSAG5B (8.5 ± 0.53), pSAG5C (7 ± 0.56) or pSAG5B/SAG5C (12.3 ± 0.68) was longer than that of the PBS-immunized mice (3.8 ± 0.24) or the pBudCE4.1-immunized mice (3.9 ± 0.26) (P < 0.05). Mice in the pSAG5B/SAG5C immunization group survived longer (17 days) than the mice immunized with the single gene (P < 0.05), while the number of survival days for the single gene groups were similar (P > 0.05). (Lu et al., 2017) PRU: Mice in the pSAG5B, pSAG5C, and pSAG5B/SAG5C groups showed significantly fewer brain cysts than the PBS- or pBudCE4.1-immunized mice (P < 0.05). Brain cyst numbers in the mice vaccinated with pSAG5B/SAG5C were much lower than those of the other mouse groups (P < 0.05). (Lu et al., 2017)
30. T. gondii DNA vaccine pVAX-CDPK1 with pVAX-IL21-IL15
a. Type:
DNA vaccine
b. Status:
Research
c. Host Species for Licensed Use:
Human
d. Antigen
CDPK1: calcium-dependent protein kinase 1: involved in the regulation of the parasite’s life cycle at stages dependent on microneme secretion, the key regulator of calcium dependent exocytosis, acts in calcium-dependent secretion of specialized organelles called micronemes, which play a critical role in direct parasite motility, host-cell invasion, and egress (Chen et al., 2014)
e. Gene Engineering of
CDPK1
Type:
DNA vaccine construction
Description:
CDPK1 was amplified by RT-PCR using a forward primer introducing Kpn I recognition sites and a reverse primer introducing Xba I recognition sites. The amplified CDPK1 was purified and was inserted into the pVAX I vector and generated plasmid pVAX-CDPK1. (Chen et al., 2014)
Vaccination Protocol:
Six groups (35 mice in each group) were intramuscularly injected twice at 2-week intervals in three immunizations (at weeks 0, 2 and 4) with 100 μg pVAX-CDPK1 DNA in 100 μl sterile PBS, 100 μg pVAX-CDPK1 + pVAX-IL-21-IL-15 DNA in 100 μl sterile PBS, 100 μg pVAX/IL-21/IL-15 DNA in 100 μl sterile PBS, 100 μg the empty vector pVAX, PBS (100 μl/each), respectively, and one group of mice was not inoculated to constitute blank control. (Chen et al., 2014)
Immune Response:
Humoral: A significant antibody responses corresponding to total antibodies including IgG, IgG1and IgG2a (P < 0.05) were induced in pVAX-CDPK1, pVAX-IL-21-IL-15 and pVAX-CDPK1 plus pVAX-IL-21-IL-15 groups compared to the controls. The highest antibody levels were observed in pVAX-IL-21-IL-15 and pVAX-CDPK1 co-injection group. There was a predominance of IgG2a over IgG1, and co-injection of pVAX-IL-21-IL-15 with pVAX-CDPK1 significantly increased the ratio (P < 0.05). (Chen et al., 2014) Cellular: Stimulation index in spleen cells from mice immunized with pVAX-CDPK1 or pVAX-IL-21-IL-15 alone was significantly higher compared to the controls, and the level of splenocyte proliferation was further increased (P < 0.05) in mice co-injected pVAX-IL-21-IL-15 and pVAX-CDPK1. The percentage of CD3 + CD8 + CD4-T cells and CD3 + CD4 + CD8-T cells were significantly increased in all immunized mice compared with the controls, and the most increase presented in pVAX-CDPK1 plus pVAX-IL-21-IL-15 co-injection group. IFN-γ and IL-2 significantly increased in all immunized mice, and the largest amounts of both two cytokines were produced in pVAX-CDPK1 and pVAX-IL-21-IL-15 co-injection group compared to the controls (P < 0.05). Small amounts of IL-4 and IL-10 were secreted from splenocytes stimulated by TLA in vitro in all immunized mice compared to the control groups (P < 0.05). (Chen et al., 2014)
Challenge Protocol:
Two weeks after the last immunization, 15 mice per group were challenged intraperitoneally (IP) with 1 × 10^3 tachyzoites of the RH strain. The survival time for each mouse and the percentages of mice survived were recorded until a fatal outcome for all animals. In addition, 10 mice per group were inoculated orally with 20 cysts of the PRU strain at day 14th after the third immunization, and observed mice daily for mortality. Four weeks after the PRU strain challenge, surviving mice were sacrificed. The mean number of cysts per brain was determined by counting three samples of 10 μl aliquots of each homogenized brain under an optical microscope. (Chen et al., 2014)
Efficacy:
RH: Mice immunized with pVAX-CDPK1 (17.3 ± 4.3 days) or pVAX/IL-21/IL-15 (12.0 ± 2.0 days) significantly prolonged survival time after challenge in comparison to mice in control groups. There was significant difference of survival time between the two groups (P < 0.05). Co-injection with pVAX-CDPK1 and pVAX/IL-21/IL-15 enhanced the survival time of the immunized mice (19.2 ± 5.1 days) in contrast to the group of pVAX-CDPK1 or pVAX/IL-21/IL-15 (P < 0.05). (Chen et al., 2014) PRU: The immunized groups challenged with PRU strain cysts showed a significant reduction in the number of cysts in the brain (P < 0.05) (Chen et al., 2014)
31. T. gondii DNA vaccine pVAX-CDPK1 with pVAX-IL7-IL15
a. Type:
DNA vaccine
b. Status:
Research
c. Host Species for Licensed Use:
Human
d. Antigen
CDPK1 (Chen et al., 2016): calcium-dependent protein kinase 1: involved in the regulation of the parasite’s life cycle at stages dependent on microneme secretion, the key regulator of calcium dependent exocytosis, acts in calcium-dependent secretion of specialized organelles called micronemes, which play a critical role in direct parasite motility, host-cell invasion, and egress (Chen et al., 2014)
e. Gene Engineering of
CDPK1
Type:
DNA vaccine construction
Description:
CDPK1 was amplified by RT-PCR using a forward primer introducing Kpn I recognition sites and a reverse primer introducing Xba I recognition sites. The amplified CDPK1 was purified and was inserted into the pVAX I vector and generated plasmid pVAX-CDPK1.(Chen et al., 2014)
Vaccination Protocol:
Mice were randomly divided into six groups (30 mice each). Three vaccination groups were immunized three times at 2-week intervals with 100 μg plasmid dissolved in 100 μl sterile PBS (pVAX-CDPK1, pVAX-IL-7-IL-15, or pVAX-CDPK1 + pVAX-IL-7-IL-15). Another two groups of mice were injected with empty pVAX I vector or PBS respectively as negative control, and one group of mice was not inoculated to constitute blank control. (Chen et al., 2016)
Immune Response:
Humoral: Specific total IgG antibodies were detected in the experimental group, with the highest antibody levels in the group of pVAX-CDPK1 + pVAX-IL-7-IL-15 (CDPK1.7.15) in contrast to PBS, pVAX I or blank control (p < 0.05), and the antibody levels increased with successive DNA immunizations. Immunization with pVAX-CDPK1 induced a higher IgG2a/IgG1 ratio compared to the pVAX-IL-7-IL-15 alone immunized animals, and co-injection of pVAX-CDPK1 with pVAX-IL-7-IL-15 induced the highest IgG2a/IgG1 ratio. (Chen et al., 2016) Cellular: Mice immunized with pVAX-CDPK1 + pVAX-IL-7-IL-15 generated significantly higher lymphocytes proliferative response (SI) and levels of IFN-γ and IL-2 compared with mice immunized with pVAX-CDPK1 or pVAX-IL-7-IL-15 alone (p < 0.05). IL-4 and IL-10 levels increased in pVAX-CDPK1, pVAX-IL-7-IL-15 and pVAX-CDPK1 + pVAX-IL-7-IL-15 groups compared to the control groups (p < 0.05). The percentages of CD3+CD8+CD4- T cells and CD3+CD4+CD8– T cells in pVAX-CDPK1, pVAX-IL-7-IL-15 and pVAX-CDPK1 + pVAX-IL-7-IL-15 groups were significantly increased compared to the controls. CD8+ and CD4+ T cell profiles were also significantly altered in theses groups in terms of IFN-γ expression compared to all controls (p < 0.05). There were no difference between changes in CD8+ and CD4+ T cell profiles in terms of IFN-γ secretion (p > 0.05). (Chen et al., 2016)
Challenge Protocol:
10 mice per group were challenged intraperitoneally with 1 × 10^3 tachyzoites of virulent T. gondii (RH strain). The survival periods were recorded until a fatal outcome for all animals. The other 10 mice per group were inoculated orally with 10 cysts of cyst T. gondii (PRU strain) 14 days after the last immunization. The cysts in their brains were counted 30 days post challenge. (Chen et al., 2016)
Efficacy:
RH: immunized mice showed a prolonged survival time compared to mice receiving the controls (all these mice died within 9 days, p > 0.05), with 14.13 ± 3.85 days in the group of pVAX-CDPK1, 11.73 ± 1.83 days in the group of pVAX-IL-7-IL-15 and 18.07 ± 5.43 days in the group of pVAX-CDPK1 + pVAX-IL-7-IL-15 (p < 0.05). (Chen et al., 2016) PRU:Immunization with pVAX-CDPK1, pVAX-IL-7-IL-15 and pVAX-CDPK1 + pVAX-IL-7-IL-15 induced reduction in average parasite burden significantly by 46.0%, 45.0% and 73.5% respectively (p < 0.05) in comparison with the control groups (p < 0.05). (Chen et al., 2016)
Description:
TgGRA16 gene was PCR amplified, and the PCR products were subcloned into the pVAX I and generated recombinant plasmid pVAX-GRA16. The recombinant plasmids pVAX-GRA16 were purified from transformed E. coli DH5α cells by anion exchange chromatography. (Hu et al., 2017)
T. gondii DNA vaccine encoding GRA16 as antigen using pVAX I vector. (Hu et al., 2017)
i.
Mouse Response
Vaccination Protocol:
A total of 4 groups were performed (28 mice per group). Mice in different groups were intramuscularly injected with pVAX-GRA16 plasmids, empty pVAX I, and PBS (100 μl/each) 3 times at a 2-week interval. The mice that received nothing were used as negative control. (Hu et al., 2017)
Challenge Protocol:
10 mice in each group were intraperitoneally (IP) challenged with 10^3 tachyzoites of the virulent T. gondii RH strain 2 weeks after the last immunization. The survival time for each mouse and the percentages of mice survived were recorded until a fatal outcome for all animals. Meanwhile, 6 mice in all groups were inoculated orally with 10 tissue cysts as experimental chronic toxoplasmosis. One month after infection, brains of mice from each group were homogenized in 1 ml PBS. The number of cysts per brain was determined. (Hu et al., 2017)
Efficacy:
The average survival time of immunized mice (8.4 ± 0.78 days) showed an extension tendency compared to that of the control groups (7.1 ± 0.30 days), but the differences were not significant (p > 0.05). Compared to pVAX I, PBS, and negative control groups, immunization with pVAX-GRA16 significantly reduced brain cyst numbers in the immunized mice (p < 0.001), with a cyst reduction of 43.89%. (Hu et al., 2017)
33. T. gondii DNA vaccine pVAX-GRA24 + pVAX-GRA25 + pVAX-MIC6
Description:
MIC6 was PCR amplified and was cloned in pGEM-T easy vector, generated pGEM-MIC6. The MIC6 fragment was cleaved by BamHI/XhoI from pGEM-MIC6 and cloned into the BamHI/XhoI sites of pVAXI. (Xu et al., 2019)
Description:
GRA24 was PCR amplified and was cloned in pMD-18 T vector, generated pMD-GRA24. The GRA24 fragment was cleaved by KpnI/XbaI from pMD-GRA24 and cloned into the KpnI/XbaI sites of pVAXI. (Xu et al., 2019)
Vaccination Protocol:
Eight groups (30 Kunming mice per group) were used for this study, consisting of five experimental and three control groups. Mice in the experimental groups were immunized three times (2-week intervals) with 100 μL (1 μg/μL) of pVAX-GRA24, pVAX-GRA25, pVAX-MIC6, pVAX-GRA24 + pVAX-GRA25 or pVAX-GRA24 + pVAX-GRA25 + pVAX-MIC6 plasmids by intramuscular injection into the quadriceps, respectively. Control groups included mice injected with 100 μL empty pVAX I vector (1 μg/μL), 1× PBS or blank control, respectively. (Xu et al., 2019)
Immune Response:
Humoral: Highest antibodies titer was observed in pVAX-GRA24 + pVAX-GRA25 + pVAX-MIC6 group. Also, boosting with pVAX-GRA24 and pVAX-GRA25 increased the IgG titer induced by pVAX-GRA24 or pVAX-GRA25. The levels of IgG titer in the pVAX-GRA24, pVAX-GRA25 or pVAX-MIC6 groups were significantly higher (p < 0.05) than those in the three control groups. Higher IgG2a to IgG1 ratio. Cellular: Highest levels of IL-2, IFN-γ, IL-12 and IL-23 in co-injected mice. The percentages of CD3+CD4+CD8− and CD3+CD8+CD4− T lymphocytes were significantly increased in all experimental groups. pVAX-GRA24 + pVAX-GRA25 showed higher percentage than single plasmid group, whereas pVAX-GRA24 + pVAX-GRA25 + pVAX-MIC6 group showed the highest percentage. Higher SI in all experimental groups. (Xu et al., 2019)
Challenge Protocol:
Two weeks after the third booster vaccine dose, 10 mice from all groups were challenged intraperitoneally with 1 × 10^3 tachyzoites of the virulent RH strain, and the survival periods were recorded daily until all mice were dead. Meanwhile, another 10 mice per group were challenged with a non-lethal dose of 20 cysts of the Pru strain. Then, 4 weeks after the challenge, the surviving mice were sacrificed via cervical dislocation, and the mean number of cysts per brain was determined by counting three samples of 10 μL aliquots of each homogenized brain under an optical microscope. (Xu et al., 2019)
Efficacy:
Mice immunized with pVAX-GRA24 (8.1 ± 0.5 days), pVAX-GRA25 (9.4 ± 0.7 days), pVAX-MIC6 (11.5 ± 0.8 days), pVAX-GRA24 + pVAX-GRA25 (13.8 ± 0.9 days) and with pVAX-GRA24 + pVAX-GRA25 + pVAX-MIC6 (18.7 ± 1.3 days) had a significantly longer survival time compared to the three control groups. The mice in the three control groups died within 6 days after challenge. (Xu et al., 2019) The number of cysts in the mouse brain was reduced significantly in the pVAX-GRA24 (29.03%), pVAX-GRA25 (40.88%), pVAX-MIC6 (37.70%), pVAX-GRA24 + pVAX-GRA25 (48.06%) and pVAX-GRA24 + pVAX-GRA25 + pVAX-MIC6 (55.37%) groups, compared to the control group (p < 0.05). There was no apparent reduction of brain cysts among the three control groups. (Xu et al., 2019)
34. T. gondii DNA vaccine pVAX-ROP38
a. Type:
DNA vaccine
b. Status:
Research
c. Host Species for Licensed Use:
Human
d. Antigen
ROP38: Rhoptry protein 38: an active rhoptry protein kinase (ROPK) that could have an inhibitory effect on host cell transcription by down-regulating the MAPK signaling track, observed inside rhoptries. (Xu et al., 2014)
e. Gene Engineering of
ROP38
Type:
DNA vaccine construction
Description:
Amplified by PCR using a forward primer introducing Kpn I recognition sites and a reverse primer introducing Xba I recognition sites. The amplified ROP38 was purified and was inserted into the pVAX I vector. (Xu et al., 2014)
Vaccination Protocol:
Three groups of mice (28 per group) were injected with 100 μl diluted pVAX-ROP38 DNA vaccine, PBS or empty pVAX I plasmid, respectively. Another group of 28 mice were kept as blank control without injection. Mice in the pVAX-ROP38 DNA vaccine, PBS and pVAX I groups were vaccinated three times with a two-week interval. (Xu et al., 2014)
Immune Response:
Humoral: Compared with the three control groups, a statistically significantly higher level of IgG antibody was detected in the sera of mice immunized with pVAX-ROP38 (P < 0.01), and the OD values of IgG were continuously increased with successive DNA immunization. The levels of IgG1 and IgG2a in the experimental group (immunized with TgROP38) were also the highest (P < 0.01). A significantly higher IgG2a value than IgG1 was detected. (Xu et al., 2014) Cellular: The percentage of CD3+ CD4+ CD8- and CD3+ CD8+ CD4- T lymphocytes in the pVAX-ROP38 group were significantly higher than those in the control groups, respectively (P < 0.01). There were no significantly differences in the ratio of CD8+/CD4+ between mice immunized with pVAX-ROP38 and in controls (P > 0.05). A significantly high level of both IFN-γ and IL-2 in the pVAX-ROP38 group was detected in pVAX-ROP38 group compared with that in the three control groups (P < 0.05). (Xu et al., 2014)
Challenge Protocol:
Two weeks after the last immunization, 10 mice in each group were challenged intraperitoneally with 1000 tachyzoites of the virulent T. gondii RH strain, and other 6 mice were inoculated with 10 cysts of the attenuated virulent PRU strain orally. Mice injected with RH strain were observed twice a day for mortality until a fatal outcome for all animals. The brain cysts were determined one month after the challenge infection. Each brain was homogenized in 2 ml PBS, and the mean number of cysts per brain was calculated. (Xu et al., 2014)
Efficacy:
RH: The average survival time of mice in the pVAX-ROP38 group was slightly longer than that of the three control groups, but the difference was not statistically significant (P > 0.05). (Xu et al., 2014) PRU: The number of brain cysts in mice from the pVAX-ROP38 group was significantly decreased (76.6%) compared to that of the three control groups (P <0.01). (Xu et al., 2014)
Description:
The coding sequences of GRA17 was amplified by RT-PCR from total RNA, and then were inserted into pMD18-T. The GRA1 fragment was cleaved from pMD-TgGRA17 and was sub-cloned into the corresponding sites of pVAX I, eventually formed pVAX-TgGRA17. (Zhu et al., 2017)
Vaccination Protocol:
A total of 78 mice were randomly divided into six groups (13 mice per group). For the experimental groups, mice were immunized with 100 μl (100 μg) of pVAX-TgGRA17, pVAX-TgGRA23, and pVAX-TgGRA17 + pVAX-TgGRA23, respectively, by intramuscular injections and given booster immunizations 2 and 4 weeks later. Mice injected with empty pVAX I vector or PBS were served as negative control groups, and the blank control group received nothing. (Zhu et al., 2017)
Challenge Protocol:
Two weeks after the last immunization, 10 mice per group were challenged intraperitoneally with 1 × 10^3 tachyzoites of the highly virulent T. gondii RH strain and the survival time of mice was monitored daily. (Zhu et al., 2017)
Efficacy:
The average survival time of mice immunized with pVAX-TgGRA17 (9.1 ± 0.32 days) was significantly longer than that of the three control groups (blank control, 7.11 ± 0.33 days; PBS control, 7.22 ± 0.44 days; pVAX I control, 7.11 ± 0.33 days) (P < 0.05). The difference in the average survival time of mice among the three control groups was not statistically significant, which all died within 8 days after challenge. (Zhu et al., 2017)
36. T. gondii DNA vaccine pVAX-TgGRA17 + pVAX-TgGRA23
Description:
The coding sequences of GRA17 was amplified by RT-PCR from total RNA, and then were inserted into pMD18-T. The GRA1 fragment was cleaved from pMD-TgGRA17 and was sub-cloned into the corresponding sites of pVAX I, eventually formed pVAX-TgGRA17. (Zhu et al., 2017)
Description:
The coding sequences of GRA23 was amplified by RT-PCR from total RNA, and then were inserted into pMD18-T. The GRA1 fragment was cleaved from pMD-TgGRA23 and was sub-cloned into the corresponding sites of pVAX I, eventually formed pVAX-TgGRA23. (Zhu et al., 2017)
Vaccination Protocol:
A total of 78 mice were randomly divided into six groups (13 mice per group). For the experimental groups, mice were immunized with 100 μl (100 μg) of pVAX-TgGRA17, pVAX-TgGRA23, and pVAX-TgGRA17 + pVAX-TgGRA23, respectively, by intramuscular injections and given booster immunizations 2 and 4 weeks later. Mice injected with empty pVAX I vector or PBS were served as negative control groups, and the blank control group received nothing. (Zhu et al., 2017)
Challenge Protocol:
Two weeks after the last immunization, 10 mice per group were challenged intraperitoneally with 1 × 10^3 tachyzoites of the highly virulent T. gondii RH strain and the survival time of mice was monitored daily. (Zhu et al., 2017)
Efficacy:
The average survival time of mice immunized with pVAX-TgGRA17 + pVAX-TgGRA23 (12.6 ± 2.55 days) was significantly longer than that of the three control groups (blank control, 7.11 ± 0.33 days; PBS control, 7.22 ± 0.44 days; pVAX I control, 7.11 ± 0.33 days) (P < 0.05). The difference in the average survival time of mice among the three control groups was not statistically significant, which all died within 8 days after challenge. (Zhu et al., 2017)
Description:
The coding sequences of GRA23 was amplified by RT-PCR from total RNA, and then were inserted into pMD18-T. The GRA1 fragment was cleaved from pMD-TgGRA23 and was sub-cloned into the corresponding sites of pVAX I, eventually formed pVAX-TgGRA23. (Zhu et al., 2017)
Vaccination Protocol:
A total of 78 mice were randomly divided into six groups (13 mice per group). For the experimental groups, mice were immunized with 100 μl (100 μg) of pVAX-TgGRA17, pVAX-TgGRA23, and pVAX-TgGRA17 + pVAX-TgGRA23, respectively, by intramuscular injections and given booster immunizations 2 and 4 weeks later. Mice injected with empty pVAX I vector or PBS were served as negative control groups, and the blank control group received nothing. (Zhu et al., 2017)
Challenge Protocol:
Two weeks after the last immunization, 10 mice per group were challenged intraperitoneally with 1 × 10^3 tachyzoites of the highly virulent T. gondii RH strain and the survival time of mice was monitored daily. (Zhu et al., 2017)
Efficacy:
The average survival time of mice immunized with pVAX-TgGRA23 (10.8 ± 0.79 days) was significantly longer than that of the three control groups (blank control, 7.11 ± 0.33 days; PBS control, 7.22 ± 0.44 days; pVAX I control, 7.11 ± 0.33 days) (P < 0.05). The difference in the average survival time of mice among the three control groups was not statistically significant, which all died within 8 days after challenge. (Zhu et al., 2017)
Immune Response:
Immunization with pVAX-TgMIC13 induced a strong immune responses demonstrated by significant lymphocyte proliferation, cytokine production and antibody responses (Yuan et al., 2013).
Challenge Protocol:
Acute: Subgroup A were challenged intraperitoneally with 1 x 10^3 tachyzoites of virulent T. gondii (RH strain) 14 day after the final immunization. The time of the death was recorded. Chronic: Subgroup B were challenged intragastrically with 10 cysts of cyst T. gondii (PRU strain) 14 day after the final immunization. The cysts in their brain were counted 30 days post challenge. (Yuan et al., 2013)
Efficacy:
Immunized mice showed increased survival time (21.3±11.3 days) and reduced number of cysts in brain of mice (57.14%) after challenge with tachyzoites of the virulent T. gondii RH strain and cysts of the T. gondii PRU strain, respectively (Yuan et al., 2013).
Description:
Full-length coding region of SAG1 were PCR amplified and were cloned into pTZ57R/T vectors. The recombinant construct pTZ-SAG1 was digested with BglII and NheI, and the ligations were subcloned into empty pVitro2-neo-mcs vectors, previously digested by corresponding restriction enzymes. (Mavi et al., 2019)
Description:
Full-length coding region of GRA7 were PCR amplified and were cloned into pTZ57R/T vectors. The recombinant construct pTZ-GRA7 was digested with BamHI and ClaI, and the ligations were subcloned into empty pVitro2-neo-mcs vectors, previously digested by corresponding restriction enzymes. (Mavi et al., 2019)
Vaccination Protocol:
84 BALB/c mice were divided randomly into seven groups of 12 mice. Negative control groups, including Groups I to III, received 100 µL of PBS, 100 µg of empty pVitro2-neo-mcs vector in 100 µL of PBS and 30 µg of CpG-ODN in 100 µL of PBS, respectively. Experimental groups of IV to VII were injected with 100 µg of pVitro-SAG1 plasmid in 100 µL of PBS, 100 µg of pVitro-GRA7 plasmid in 100 µL of PBS, 100 µg of pVitro-SAG1-GRA7 plasmid in 100 µL of PBS and 100 µg of pVitro-SAG1-GRA7 plasmid with 30 µg of CpG-ODN in 100 µL of PBS, respectively. Mice were immunized intramuscularly (IM) thrice on days 1, 21 and 42. (Mavi et al., 2019)
Immune Response:
Humoral: higher IgG levels in pVitro-SAG1-GRA7 and pVitro-SAG1-GRA7 with CpG-ODN groups, higher IgG levels in pVitro-SAG1 group than in pVitro-GRA7 group. IgG2a levels significantly increased in all experimental groups. Cellular: higher lymphocyte proliferation and IFN-γ levels in pVitro-SAG1-GRA7 and pVitro-SAG1-GRA7 with CpG-ODN groups. Difference of SI was statistically significant in mice immunized with pVitro-SAG1-GRA7 with CpG-ODN, compared to that in mice received pVitro-SAG1-GRA7 alone (P<0.001). (Mavi et al., 2019)
Challenge Protocol:
Four weeks after the last immunization, nine immunized BALB/c mice from each group were randomly selected and involved in infection experiments. Each mouse was inoculated intraperitoneally (IP) with a single dose of 1×10^3 tachyzoites of the virulent T. gondii, RH strain. Mice were monitored twice daily and the time of death was recorded until all mice were dead. (Mavi et al., 2019)
Efficacy:
All mice in control groups died within 5–8 days after confronting lethal challenge. The survival time in experimental groups was significantly (P<0.05) longer than that in control groups. Mice immunized with pVitro-SAG1–GRA7 vaccine alone and pVitro-SAG1-GRA7 with CpG-ODN vaccine, respectively, survived 9–15 days (13.25±1.85) and 11–18 days (15.0±2.29) after lethal challenges which were significantly longer than that mice immunized with pVitro-SAG1 or pVitro-GRA7 groups did, including 8–14 (10.87±2.02) and 8–12 (10.0±1.41) days after lethal challenges, respectively. Furthermore, in pVitro-SAG1-GRA7 with CpG-ODN group, a longer survival time was observed, compared to that in pVitro-SAG1-GRA7 group. (Mavi et al., 2019)
Efficacy:
All control pVR1020-vaccinated mice succumbed to infection after challenge, while 75% of the mice vaccinated with pVR1020-GRA1 were protected against infection with strain IPB-G. In the second experiment, all mice vaccinated with GRA1 DNA survived, while 75% of the mice vaccinated with control DNA succumbed to infection (P < 0.05) (Scorza et al., 2003).
Immune Response:
splenocytes from mice immunized with ROP8-pVAX-1 secreted significantly high levels of IFN-γ (816 ± 26.3 pg/mL) compared with spleen cells cultured from mice immunized with pVAX-1 (48 ± 10.8) or PBS (45 ± 6.6). Only a low level of IL-4 was detected in the culture supernatant of splenocytes from mice immunized with ROP8-pVAX-1 (148 ± 18.3) but was significantly higher compared with mice immunized with pVAX-1 (50 ± 13.6) or PBS (47 ± 6.1) (Parthasarathy et al., 2013).
Efficacy:
Mice immunized with ROP8-pVAX-1 DNA had a 100% survival rate until Day 9, whereas all the control mice died. On top of that, mice immunized with ROP8-pVAX-1 DNA showed a significant increase in the survival time (29 days, P < 0.05) (Parthasarathy et al., 2013).
Efficacy:
The T.g.HSP70 gene vaccination induced protective immunity against T.g.HSP70-induced anaphylactic reaction not only at the acute phase but also at acute exacerbation of the chronic phase of infection, although the protective effect was partial (Kikumura et al., 2010).
Vaccination Protocol:
Mice were immunized with 5 × 10^4 ΔGRA17 tachyzoites or mock-immunized in a volume of 200 µl PBS intraperitoneally (i.p.) (Wang et al., 2017)
Immune Response:
Humoral: Higher IgG and IgG2a levels at day 28 postimmunization. Increased IgG1 level at 70 days postimmunization. (Wang et al., 2017) Cellular: Significantly higher levels of Th1-type cytokines (IFN-γ, IL-2, and IL-12) and significant increase in the level of Th2-type cytokine (IL-10) (Wang et al., 2017)
Challenge Protocol:
The efficacy of ΔGRA17 was tested against acute, latent, and congenital infections in mice 70 days after vaccination. Acute: mice were challenged with 1 × 10^3 RH tachyzoites of T. gondii RH, ToxoDB#9 PYS, or ToxoDB#9 TgC7 strain. Chronic mice werechallenged with 20 Pru cysts. Congenital: female mice were mated to males on a 2:1 ratio 70 days post vaccination. Compared between five groups: (A) ΔGRA17-immunized and orally gavaged with 10 tissue cysts of Pru on day 12 of gestation, (B) ΔGRA17-immunized and i.p. inoculated with 200 RH tachyzoites on day 18 of gestation, (C) non-immunized, and orally gavaged with 10 tissue cysts of Pru on day 12 of gestation, (D) non-immunized and i.p. inoculated with 200 RH tachyzoites on day 18 of gestation, and (E) non-immunized and uninfected. (Wang et al., 2017)
Efficacy:
Acute: All immunized mice survived the challenge, whereas all challenged naïve mice died within 10 dpi. Significantly higher levels of IFN-γ and IL-12 in the sera and peritoneal washes in non-immunized and infected mice than in the immunized and infected, and non-immunized and uninfected mice. Chronic: All ΔGRA17-immunized mice survived compared to 40% survival of non-immunized and infected mice. Immunized mice had significantly less cyst burden (53 ± 15 cysts/brain compared to 4,296 ± 687 cysts/brain) (P < 0.05). Brain cysts were not found in 4 out of 10 (40%) immunized mice. Congenital: The mean sizes of viable pups born to immunized and infected mice were similar to pups born to non-immunized and uninfected mice and in both groups, and no abortions were observed. The mean litter sizes of viable neonates born to non-immunized and infected mice were significantly reduced, and abortions were observed. Five days after birth, the survival rate and mean BW of pups born to non-immunized and uninfected, and immunized and infected mice were significantly higher than that from non-immunized and infected mice. Non-immunized and infected dams showed clinical signs of toxoplasmosis, whereas immunized and infected, and non-immunized and uninfected dams did not show any clinical signs. T. gondii DNA was found in all examined non-immunized and infected dams, but not in any of the 10 examined pups. However, parasites DNA was detected in some aborted fetuses. The pups from the immunized and infected mice had equivalent mean BW and survival rate to that from non-immunized and uninfected dams on day 35 of age. (Wang et al., 2017)
44. T. gondii irradiated sterilised tachyzoites
a. Type:
Live, attenuated vaccine
b. Status:
Research
c. Host Species for Licensed Use:
Human
d. Immunization Route
Intraperitoneal injection (i.p.) or oral gavage (v.o.)
e. Description
Viable irradiated tachyzoites of T. gondii, maintains morphology and physiology but abolishes reproduction (Zorgi et al., 2016)
Vaccination Protocol:
Groups of five BALB/c mice received three doses of 10^7 irradiated T. gondii tachyzoites (ME-49 or VEG strain) biweekly; the parasites were administered either by i.p. injection or via oral gavage (v.o.) of 10^7 irradiated tachyzoites suspended 1:1 (v/v) in 6 % aluminum hydroxide. The unimmunized mice served as negative controls, and mice chronically infected by ME-49 or VEG strain served as positive control. (Zorgi et al., 2016)
Immune Response:
Humoral: Significant increase in the total IgG and IgM levels in i.p. immunized groups. Higher IgA levels in oral immunized groups. (Zorgi et al., 2016) Cellular: Higher levels of IL-10, TNF-α, IFN-ɤ, IL-4, IL-2, and IL-6 in i.p. immunized groups (Zorgi et al., 2016)
Challenge Protocol:
The immunized or control mice were challenged with ten cysts of the respective strain (ME49 or VEG) administered by oral gavage 90 days after the last immunizing dose; the mice were observed for daily survival determination. After 30 days, the surviving animals were killed and the brains were macerated in sterile saline solution. The solution containing the brain was subjected to DNA extraction to analyze the absolute number of parasites. (Zorgi et al., 2016)
Efficacy:
The challenge with both the ME-49 strain and the VEG strain in the intraperitoneally and orally immunized animals showed a significant decrease in the number of parasites in the brain tissue compared with the infected mice without immunization. (Zorgi et al., 2016)
45. T. gondii LDH1 and LDH2 mutant vaccine
a. Type:
Live, attenuated vaccine
b. Status:
Research
c. Host Species for Licensed Use:
Human
d. Immunization Route
Intraperitoneal injection (i.p.)
e. Description
T. gondii attenuated vaccine: deleted LDH1 and LDH2 gene in T. gondii PruΔKu80::hxgprt strain (Abdelbaset et al., 2017)
Vaccination Protocol:
Mice were vaccinated with a low-dose with 10^3 tachyzoites of the parental PruΔKu80::hxgprt strain or LDH knockout strains (ΔLDH1, ΔLDH2, or ΔLDH1ΔLDH2). A group of non-vaccinated mice was kept as control. (Abdelbaset et al., 2017)
Side Effects:
Infection study: Infection with 10^6 tachyzoites: parental or Δldh2 strain infected mice demonstrated a 10 and 0% survival rate, respectively. On the other hand, mice challenged with Δldh1 and Δldh1Δldh2 strains demonstrated a 60 and 30% survival rate, respectively. Average number of cysts in each group was 680 (parental, n = 1), 177 ± 62 (Δldh1, n = 6), no data (Δldh2, n=0) and 60 ± 40 (Δldh1Δldh2, n = 3). Infection with 10^4 tachyzoites: parental and Δldh2 strain infected mice demonstrated a 60 and 40% survival rate, respectively. On the other hand, Δldh1 and Δldh1Δldh2 strain infected mice showed 100% survival. Average number of cysts in each group was 231 ± 116 (parental, n = 6), 27 ± 7 (Δldh1, n = 10), 52 ± 11 (Δldh2, n = 4), and 1 ± 1 (Δldh1Δldh2, n = 10) Infection with 10^3 tachyzoites: parental, Δldh1 and Δldh1Δldh2 strain infected mice showed 100% survival. Δldh2 infected mice showed low (10%) mortality. Average number of cysts in each group was 640 ± 72 (parental, n = 4), 56 ± 5 (Δldh1, n = 5), 330 ± 34 (Δldh2, n = 5), and 0 (Δldh1Δldh2, n = 5) (Abdelbaset et al., 2017)
Challenge Protocol:
30 days after vaccination, mice were challenged with 100 highly virulent (LD100) RH strain parasites. (Abdelbaset et al., 2017)
Efficacy:
All mice in the naïve group died within 11 days after challenge, one mouse died in parental strain infected group, and all mice survived in the other vaccinated groups. (Abdelbaset et al., 2017)
Efficacy:
A mic2 mutant induces protection in mice from challenge with wild type T. gondii (Huynh and Carruthers, 2006).
Host Gene Response of
Ccl2
Gene Response:
Levels of serum MCP-1 (Ccl2) were higher in mice infected with MIC2 deletion mutants than in uninfected mice on days 4, 6 and 8 post infection (Huynh and Carruthers, 2006).
Gene Response:
Levels of serum IFN-gamma were higher in mice infected with MIC2 deletion mutants than in uninfected mice on days 4, 6 and 8 post-infection (Huynh and Carruthers, 2006).
Gene Response:
Levels of serum IL-6 were higher in mice infected with MIC2 deletion mutants than in uninfected mice on days 4, 6 and 8 post infection (Huynh and Carruthers, 2006).
Gene Response:
Levels of serum IL-12p40 (IL-12b) were higher than those of uninfected mice on days 4, 6 and 8 post-infection (Huynh and Carruthers, 2006).
Gene Response:
Levels of serum TNF-alpha were higher in mice infected with MIC2 deletion mutants than in uninfected mice on days 4, 6 and 8 post infection (Huynh and Carruthers, 2006).
Vaccination Protocol:
Mice were either vaccinated once with 500 freshly harvested Pru:Δcdpk2 tachyzoites or mock-vaccinated in a total of 200 µL phosphate-buffered saline (PBS) ip. (Wang et al., 2018)
Immune Response:
Humoral: Higher levels of IgG and IgG2a antibodies 28 dpv, increased level of IgG1 at 70 dpv (Wang et al., 2018) Cellular: Significantly higher levels of Th1-type cytokines (IFN-γ, IL-2, and IL-12) and Th2-type cytokine (IL-10) at 70 dpv. (Wang et al., 2018)
Challenge Protocol:
At 70 dpv, both vaccinated and age-matched naive mice were challenged. Acute: mice were challenged ip with 200 μL PBS containing 1000 tachyzoites of RH, or ToxoDB#9 (PYS or TgC7) strain Chronic: mice were inoculated orally with 20 Pru cysts. Congenital: female mice were mated to males on a 2:1 ratio 70 days post vaccination. Compared between three groups: (A) Δcdpk2-immunized and orally gavaged with 10 Pru cysts on day 12 of gestation, (B) non-immunized, and orally gavaged with 10 Pru cysts on day 12 of gestation, and (C) non-immunized and uninfected. (Wang et al., 2018)
Efficacy:
Acute: All nonvaccinated and infected mice died within 10 dpi, whereas all mice vaccinated survived. Significantly elevated levels of IL-12 and IFN-γ were found in the nonvaccinated + RH-infected mice, whereas only modestly elevated levels of the same cytokines was observed in Pru:Δcdpk2 vaccinated + RH-infected mice. Chronic: All vaccinated mice survived, whereas only 40% of nonvaccinated infected mice survived. Pru:Δcdpk2-vaccinated mice had significantly less brain cyst burden(78 ± 48 cysts/brain compared to 4296 ± 687 cysts/brain) (P < .001). Congenital: Body weight of neonates born to Pru:Δcdpk2-vaccinated dams was similar to neonates born to nonvaccinated uninfected dams. In contrast, the litter size and body weight of pups of nonvaccinated + infected mice were significantly lower. The average brain cyst number in all neonates (n = 19) born to nonvaccinated + infected dams was 919 ± 339. In contrast, 41.4% (24/58) of neonates born to vaccinated dams had an average brain cyst number of (60 ± 33). The brain of 58.6% (34/58) of neonates born to vaccinated + infected dams revealed no cysts. The average brain cyst number was significantly higher in nonvaccinated + infected dams (3287 ± 569 cysts/brain) than in that of vaccinated dams (77 ± 58 cysts/ brain). (Wang et al., 2018)
50. T. gondii recombinant vaccine vector rBCGpMV361-TgCyP
Immune Response:
Greater proliferation of CD4+ and CD8+ T cells; levels of Th1-type IFN-γ, IL-2 and IL-12 were significantly increased (Yu et al., 2013)
Efficacy:
rBCGpMV361-TgCyP i.v. inoculation protected the BALB/c mice, as shown by the 17% increased survival rate compared to those mice not inoculated (Yu et al., 2013)
51. T. gondii Subunit Rop2-Hsp83 Fusion Protein Vaccine
Vaccination Protocol:
The immunization doses and boosters of each antigen were the following: Rop2 (44 kDa, 2 μg), LiHsp83 (86 kDa, 6 μg), Rop2-LiHsp83 (not, vert, similar120 kDa, 9 μg) and the mixture of Rop2 and LiHsp83 (2 + 6 μg, respectively). Balb/c, C57BL/6 and C3H mice were immunized by footpad injections on days 0, 21, 35 and 45 with PBS (control) or recombinant proteins (Echeverria et al., 2006).
Challenge Protocol:
To analyze protection against virulent T. gondii strain (lethal challenge) immunized mice were challenged intraperitoneally with 10^5 T. gondii RH tachyzoites 2 weeks after the last immunization. To analyze protection against non-lethal challenge, immunized mice were orally infected with 20 cysts of the ME49 T. gondii strain 2 weeks after the last booster. The brain parasite load was evaluated 1 month after infection (Echeverria et al., 2006).
Efficacy:
Vaccination with fusion protein conferred a remarkable resistance against oral infection with ME49 cysts in C57BL/6 and C3H mice, in comparison to mice immunized with Rop2 alone or the protein mixture. Following lethal challenge, a significant survival rate was observed in Rop2-83 immunized Balb/c and C57BL/6 mice in comparison to control groups (Echeverria et al., 2006).
Description:
MIC1 was PCR amplified and was cloned into pDONR201 vector and yielded pENTR-TgMIC1. Inserts from pENTR-TgMIC1 were transferred into pDEST17 vectors and yielded pEXP17-TgMIC1. The proteins were expressed by E. coli BL21 (DE3) (Novagen) cells transformed with pDEST17-MIC1. (Pinzan et al., 2015)
Vaccination Protocol:
Mice were subcutaneously (s.c.) injected with TgMIC1 (10 μg), TgMIC4 (10μg), TgMIC6 (10μg), TgMIC1-4 (5 μg of each protein), TgMIC1-4-6 (3.3μg of each protein), or lactose-binding proteins (Lac+, 10 μg) emulsified in Freund’s complete adjuvant. Animals were boosted at the same dose and regimen on day 15 and 30 after first injection, now emulsified in Freund’s incomplete adjuvant. A control group was injected at the same regimen with PBS emulsified in Freund’s adjuvant (vehicle). (Pinzan et al., 2015)
Challenge Protocol:
One month after the last immunization procedure, the mice were orally infected with 80 cysts of the ME49 strain and were monitored and recorded for 30 days to compute the survival rates. The brain of the mice infected with 40 cysts was removed 1 month after the challenge and the mean number of cysts per brain was determined. (Pinzan et al., 2015)
Efficacy:
Mice immunized with TgMIC1, compared with non-immunized mice, showed reductions in brain cysts by 52%. Non-immunized control mice started dying 6 days after T. gondii infection, and they were all dead by day 11 post infection. Immunization with TgMIC1 was associated with a survival rate of about 50%, indicating that these vaccines confer partial protection against T. gondii infection. (Pinzan et al., 2015)
Description:
MIC1 was PCR amplified and was cloned into pDONR201 vector and yielded pENTR-TgMIC1. Inserts from pENTR-TgMIC1 were transferred into pDEST17 vectors and yielded pEXP17-TgMIC1. The proteins were expressed by E. coli BL21 (DE3) (Novagen) cells transformed with pDEST17-MIC1. (Pinzan et al., 2015)
Description:
MIC4 was PCR amplified and was cloned into pDONR201 vector and yielded pENTR-TgMIC4. Inserts from pENTR-TgMIC4 were transferred into pDEST17 vectors and yielded pEXP17-TgMIC4. The proteins were expressed by E. coli BL21 (DE3) (Novagen) cells transformed with pDEST17-MIC4. (Pinzan et al., 2015)
Vaccination Protocol:
Mice were subcutaneously (s.c.) injected with TgMIC1 (10 μg), TgMIC4 (10μg), TgMIC6 (10μg), TgMIC1-4 (5 μg of each protein), TgMIC1-4-6 (3.3μg of each protein), or lactose-binding proteins (Lac+, 10 μg) emulsified in Freund’s complete adjuvant. Animals were boosted at the same dose and regimen on day 15 and 30 after first injection, now emulsified in Freund’s incomplete adjuvant. A control group was injected at the same regimen with PBS emulsified in Freund’s adjuvant (vehicle). (Pinzan et al., 2015)
Challenge Protocol:
One month after the last immunization procedure, the mice were orally infected with 80 cysts of the ME49 strain and were monitored and recorded for 30 days to compute the survival rates. The brain of the mice infected with 40 cysts was removed 1 month after the challenge and the mean number of cysts per brain was determined. (Pinzan et al., 2015)
Efficacy:
Mice immunized with TgMIC1-4, compared with non-immunized mice, showed reductions in brain cysts by 59%. Non-immunized control mice started dying 6 days after T. gondii infection, and they were all dead by day 11 post infection. Effective and highly significant protection was demonstrated in mice immunized with TgMIC1-4, since 70% of the mice survived to the acute phase of infection. (Pinzan et al., 2015)
Description:
MIC1 was PCR amplified and was cloned into pDONR201 vector and yielded pENTR-TgMIC1. Inserts from pENTR-TgMIC1 were transferred into pDEST17 vectors and yielded pEXP17-TgMIC1. The proteins were expressed by E. coli BL21 (DE3) (Novagen) cells transformed with pDEST17-MIC1. (Pinzan et al., 2015)
Description:
MIC4 was PCR amplified and was cloned into pDONR201 vector and yielded pENTR-TgMIC4. Inserts from pENTR-TgMIC4 were transferred into pDEST17 vectors and yielded pEXP17-TgMIC4. The proteins were expressed by E. coli BL21 (DE3) (Novagen) cells transformed with pDEST17-MIC4. (Pinzan et al., 2015)
Vaccination Protocol:
Mice were subcutaneously (s.c.) injected with TgMIC1 (10 μg), TgMIC4 (10μg), TgMIC6 (10μg), TgMIC1-4 (5 μg of each protein), TgMIC1-4-6 (3.3μg of each protein), or lactose-binding proteins (Lac+, 10 μg) emulsified in Freund’s complete adjuvant. Animals were boosted at the same dose and regimen on day 15 and 30 after first injection, now emulsified in Freund’s incomplete adjuvant. A control group was injected at the same regimen with PBS emulsified in Freund’s adjuvant (vehicle). (Pinzan et al., 2015)
Challenge Protocol:
One month after the last immunization procedure, the mice were orally infected with 80 cysts of the ME49 strain and were monitored and recorded for 30 days to compute the survival rates. The brain of the mice infected with 40 cysts was removed 1 month after the challenge and the mean number of cysts per brain was determined. (Pinzan et al., 2015)
Efficacy:
Mice immunized with TgMIC1-4-6, compared with non-immunized mice, showed reductions in brain cysts by 67.8%. Non-immunized control mice started dying 6 days after T. gondii infection, and they were all dead by day 11 post infection. Effective and highly significant protection was demonstrated in mice immunized with TgMIC1-4-6, since 80% of the mice survived to the acute phase of infection. (Pinzan et al., 2015)
Description:
MIC4 was PCR amplified and was cloned into pDONR201 vector and yielded pENTR-TgMIC4. Inserts from pENTR-TgMIC4 were transferred into pDEST17 vectors and yielded pEXP17-TgMIC4. The proteins were expressed by E. coli BL21 (DE3) (Novagen) cells transformed with pDEST17-MIC4. (Pinzan et al., 2015)
Vaccination Protocol:
Mice were subcutaneously (s.c.) injected with TgMIC1 (10 μg), TgMIC4 (10μg), TgMIC6 (10μg), TgMIC1-4 (5 μg of each protein), TgMIC1-4-6 (3.3μg of each protein), or lactose-binding proteins (Lac+, 10 μg) emulsified in Freund’s complete adjuvant. Animals were boosted at the same dose and regimen on day 15 and 30 after first injection, now emulsified in Freund’s incomplete adjuvant. A control group was injected at the same regimen with PBS emulsified in Freund’s adjuvant (vehicle). (Pinzan et al., 2015)
Challenge Protocol:
One month after the last immunization procedure, the mice were orally infected with 80 cysts of the ME49 strain and were monitored and recorded for 30 days to compute the survival rates. The brain of the mice infected with 40 cysts was removed 1 month after the challenge and the mean number of cysts per brain was determined. (Pinzan et al., 2015)
Efficacy:
Mice immunized with TgMIC4, compared with non-immunized mice, showed reductions in brain cysts by 46.9%. Non-immunized control mice started dying 6 days after T. gondii infection, and they were all dead by day 11 post infection. Immunization with TgMIC4 was associated with a survival rate of about 50%, indicating that these vaccines confer partial protection against T. gondii infection. (Pinzan et al., 2015)
Vaccination Protocol:
Mice were subcutaneously (s.c.) injected with TgMIC1 (10 μg), TgMIC4 (10μg), TgMIC6 (10μg), TgMIC1-4 (5 μg of each protein), TgMIC1-4-6 (3.3μg of each protein), or lactose-binding proteins (Lac+, 10 μg) emulsified in Freund’s complete adjuvant. Animals were boosted at the same dose and regimen on day 15 and 30 after first injection, now emulsified in Freund’s incomplete adjuvant. A control group was injected at the same regimen with PBS emulsified in Freund’s adjuvant (vehicle). (Pinzan et al., 2015)
Challenge Protocol:
One month after the last immunization procedure, the mice were orally infected with 80 cysts of the ME49 strain and were monitored and recorded for 30 days to compute the survival rates. The brain of the mice infected with 40 cysts was removed 1 month after the challenge and the mean number of cysts per brain was determined. (Pinzan et al., 2015)
Efficacy:
Mice immunized with TgMIC6, compared with non-immunized mice, showed reductions in brain cysts by 27.2%. Non-immunized control mice started dying 6 days after T. gondii infection, and they were all dead by day 11 post infection. Immunization with TgMIC6 was associated with a survival rate of about 40%, indicating that these vaccines confer partial protection against T. gondii infection. (Pinzan et al., 2015)
Description:
TgPF gene was amplified by PCR using a forward primer introducing EcoRI site and a reverse primer introducing XhoI site. The amplified TgPF was digested and purified and was inserted into the pGEX-4T1 vector. Recombinant TgPF was expressed as a glutathione-S-transferase (GST) fusion protein in the E. coli DH5α strain. Endotoxins were removed from the purified protein fraction. TgPF was encapsulated in OMLs. (Tanaka et al., 2014)
Vaccination Protocol:
Mice were inoculated subcutaneously with 40 pmol of TgPF encapsulated in OMLs (TgPF-OML), OMLs, 40 pmol of TgPF in PBS (TgPF), or PBS alone (each 100 μl). Booster immunizations were administered 14 and 28 days after the first immunization. (Tanaka et al., 2014)
Immune Response:
Significantly higher levels of total IgG and IgG2c antibodies, but not IgG1 antibodies, were detected in the sera of mice immunized with TgPF-OML. (Tanaka et al., 2014)
Challenge Protocol:
Fourteen days after the third immunization, the mice were challenged with the 1 × 10^3 tachyzoites of PLK strain. DNA was isolated from the brain samples 30 days after infection, and parasite numbers were analyzed with PCR. (Tanaka et al., 2014)
Efficacy:
The survival rate of the mice immunized with TgPF-OML (66.7%) was significantly higher than that of mice treated with PBS (25.0%), OML (25.0%), or TgPF (16.7%). The parasite burden in the brains of the surviving mice in the TgPF-OML-immunized group (386 ± 336 parasites/50 ng of DNA) was significantly lower than that in the group injected with TgPF (2707 ± 929 parasites/50 ng of DNA) or PBS (2521 ± 1540 parasites/50 ng of DNA). (Tanaka et al., 2014)
58. UV-attenuated Toxoplasma gondii with DSCG adjuvant
a. Type:
Live, attenuated vaccine
b. Status:
Research
c. Host Species for Licensed Use:
Human
d. Immunization Route
Intraperitoneal injection (i.p.)
e. Description
UV-attenuated RH strain T. gondii tachyzoites. DSCG was used as the adjuvant (Li et al., 2018)
Vaccination Protocol:
Mice were randomly divided into 7 groups (10 mice per group) and received immunization of 10^5 UV-irradiated T. gondii tachyzoites alone or 10^5 UV-irradiated T. gondii tachyzoites co-administrated with DSCG (Sigma-Aldrich, 25 mg/kg). Each mouse received three times of immunization at 10-day interval. 1) naive mice without vaccination or infection, 2) mice injected with DSCG without vaccination or infection, and 3) mice immunized with 10^5 UV-Tg without challenge. 4) mice immunized with UV-Tg without DSCG and then challenged, 5) mice immunized with UV-Tg with DSCG and then challenged, 6) naive mice challenged (without DSCG), and 7) mice injected with DSCG without vaccination and then challenged. (Li et al., 2018)
Immune Response:
Higher level of TNF-α, IFN-ɤ, IL-2, IL-4, IL-10, IL-17 (Li et al., 2018)
Challenge Protocol:
Group (4) - (7) were i.p. challenged with 10^2 RH tachyzoites after the final immunization (Li et al., 2018)
Efficacy:
All mice infected alone (Tg) died at 5–7 days post-infection (p.i.). Tg + DSCG group had survival times ranged from 7 to 16 days and UV-Tg + Tg group had survival times ranged from 6 to 17 days, significantly longer (P < 0.01) compared to Tg group but no significant difference between these two groups (P > 0.05). UV-Tg + DSCG + Tg group had survival times ranged from 7 to 27 days, significantly longer compared to Tg group (P < 0.0001), Tg + DSCG group (P < 0.01), and UV-Tg + Tg group (P < 0.01). Compared to that of Tg group 5-7 days p.i., parasite burdens were significantly decreased in the livers of Tg + DSCG group (P < 0.01), and UV-Tg + Tg group (P < 0.01) and UV-Tg + DSCG + Tg group (P < 0.001) at 7–9 days p.i.. Compared to UV-Tg + Tg group, the parasite burden in UV-Tg + DSCG + Tg group was significantly decreased (P < 0.05) at 7–9 days post-challenge. (Li et al., 2018)
V. References
1. Bhopale, 2003: Bhopale GM. Pathogenesis of toxoplasmosis. Comparative immunology, microbiology and infectious diseases. 2003; 26(4); 213-222. [PubMed: 12676122].
2. Buxton and Innes, 1995: Buxton D, Innes EA. A commercial vaccine for ovine toxoplasmosis. Parasitology. 1995; 110 Suppl; S11-16. [PubMed: 7784124].
3. Ching et al., 2017: Ching XT, Fong MY, Lau YL. Evaluation of the Protective Effect of Deoxyribonucleic Acid Vaccines Encoding Granule Antigen 2 and 5 Against Acute Toxoplasmosis in BALB/c Mice. The American journal of tropical medicine and hygiene. 2017; 96(6); 1441-1447. [PubMed: 28719288].
4. Cong et al., 2008: Cong H, Gu QM, Yin HE, Wang JW, Zhao QL, Zhou HY, Li Y, Zhang JQ. Multi-epitope DNA vaccine linked to the A2/B subunit of cholera toxin protect mice against Toxoplasma gondii. Vaccine. 2008; 26(31); 3913-3921. [PubMed: 18555564].
5. Cui et al., 2008: Cui YL, He SY, Xue MF, Zhang J, Wang HX, Yao Y. Protective effect of a multiantigenic DNA vaccine against Toxoplasma gondii with co-delivery of IL-12 in mice. Parasite immunology. 2008; 30(5); 309-313. [PubMed: 18331395].
6. Czarnewski et al., 2017: Czarnewski P, Araújo ECB, Oliveira MC, Mineo TWP, Silva NM. Recombinant <i>Tg</i>HSP70 Immunization Protects against <i>Toxoplasma gondii</i> Brain Cyst Formation by Enhancing Inducible Nitric Oxide Expression. Frontiers in cellular and infection microbiology. 2017; 7; 142. [PubMed: 28487847].
7. Echeverria et al., 2006: Echeverria PC, de Miguel N, Costas M, Angel SO. Potent antigen-specific immunity to Toxoplasma gondii in adjuvant-free vaccination system using Rop2-Leishmania infantum Hsp83 fusion protein. Vaccine. 2006; 24(19); 4102-4110. [PubMed: 16545504].
8. Fox and Bzik, 2010: Fox BA, Bzik DJ. Avirulent uracil auxotrophs based on disruption of orotidine-5'-monophosphate decarboxylase elicit protective immunity to Toxoplasma gondii. Infection and immunity. 2010; 78(9); 3744-3752. [PubMed: 20605980].
9. Huynh and Carruthers, 2006: Huynh MH, Carruthers VB. Toxoplasma MIC2 is a major determinant of invasion and virulence. PLoS pathogens. 2006; 2(8); e84. [PubMed: 16933991].
11. Kikumura et al., 2010: Kikumura A, Fang H, Mun HS, Uemura N, Makino M, Sayama Y, Norose K, Aosai F. Protective immunity against lethal anaphylactic reaction in Toxoplasma gondii-infected mice by DNA vaccination with T. gondii-derived heat shock protein 70 gene. Parasitology international. 2010; 59(2); 105-111. [PubMed: 20346412].
12. Liu et al., 2010: Liu MM, Yuan ZG, Peng GH, Zhou DH, He XH, Yan C, Yin CC, He Y, Lin RQ, Song HQ, Zhu XQ. Toxoplasma gondii microneme protein 8 (MIC8) is a potential vaccine candidate against toxoplasmosis. Parasitology research. 2010; 106(5); 1079-1084. [PubMed: 20177910].
13. Liu et al., 2017: Liu Y, Cao A, Li Y, Li X, Cong H, He S, Zhou H. Immunization with a DNA vaccine encoding Toxoplasma gondii Superoxide dismutase (TgSOD) induces partial immune protection against acute toxoplasmosis in BALB/c mice. BMC infectious diseases. 2017; 17(1); 403. [PubMed: 28592247].
14. Parthasarathy et al., 2013: Parthasarathy S, Fong MY, Ramaswamy K, Lau YL. Protective Immune Response in BALB/c Mice Induced by DNA Vaccine of the ROP8 gene of Toxoplasma gondii. The American journal of tropical medicine and hygiene. 2013; 88(5); 883-887. [PubMed: 23509124].
15. Picchio et al., 2018: Picchio MS, Sánchez VR, Arcon N, Soto AS, Perrone Sibilia M, Aldirico MLA, Urrutia M, Moretta R, Fenoy IM, Goldman A, Martin V. Vaccine potential of antigen cocktails composed of recombinant Toxoplasma gondii TgPI-1, ROP2 and GRA4 proteins against chronic toxoplasmosis in C3H mice. Experimental parasitology. 2018; 185; 62-70. [PubMed: 29309783].
16. Qu et al., 2008: Qu D, Wang S, Cai W, Du A. Protective effect of a DNA vaccine delivered in attenuated Salmonella typhimurium against Toxoplasma gondii infection in mice. Vaccine. 2008; 26(35); 4541-4548. [PubMed: 18590785].
17. Scorza et al., 2003: Scorza T, D'Souza S, Laloup M, Dewit J, De Braekeleer J, Verschueren H, Vercammen M, Huygen K, Jongert E. A GRA1 DNA vaccine primes cytolytic CD8(+) T cells to control acute Toxoplasma gondii infection. Infection and immunity. 2003; 71(1); 309-316. [PubMed: 12496180].
19. Xiang et al., 2009: Xiang W, Qiong Z, Li-peng L, Kui T, Jian-wu G, Heng-ping S. The location of invasion-related protein MIC3 of Toxoplasma gondii and protective effect of its DNA vaccine in mice. Veterinary parasitology. 2009; 166(1-2); 1-7. [PubMed: 19800170].
20. Xue et al., 2008: Xue M, He S, Zhang J, Cui Y, Yao Y, Wang H. Comparison of cholera toxin A2/B and murine interleukin-12 as adjuvants of Toxoplasma multi-antigenic SAG1-ROP2 DNA vaccine. Experimental parasitology. 2008; 119(3); 352-357. [PubMed: 18442818].
21. Yu et al., 2013: Yu Q, Huang X, Gong P, Zhang Q, Li J, Zhang G, Yang J, Li H, Wang N, Zhang X. Protective immunity induced by a recombinant BCG vaccine encoding the cyclophilin gene of Toxoplasma gondii. Vaccine. 2013; ; . [PubMed: 24176493].
22. Yuan et al., 2013: Yuan ZG, Ren D, Zhou DH, Zhang XX, Petersen E, Li XZ, Zhou Y, Yang GL, Zhu XQ. Evaluation of protective effect of pVAX-TgMIC13 plasmid against acute and chronic Toxoplasma gondii infection in a murine model. Vaccine. 2013; ; . [PubMed: 23707448].
