Table 2.
Toxoplasma antigens based on DNA vaccines studies
| Antigen | Toxoplasma strain | Plasmid and the cloning organism | Processing and purification method | Route of injection and the location and dose | ||||
|---|---|---|---|---|---|---|---|---|
| SAG1 DNA |
Mice challenged with 80 tissue cysts of ME49 strain Rats with VEG strain oocysts resulted (brain cysts) highly virulent RH strain tachyzoites for mice challenge and also to derive the SAG1 cDNA clone |
pCMVInt expression vector | By double-banding on CsCl |
Intramuscular Hindquarters 100 mg of pCMVToxo or pCMVInt in 100 mL of PBS with 25% sucrose + 50 mg of pGM-CSF |
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| ROP16 DNA |
Highly virulent RH strain of T. gondii The DNA sequence of the Ag from T. gondii RH strain |
pVAX➔ pGEM | Not mentioned | Intramuscularly (i.m.) with 100 µg of plasmid DNA suspended in 100 µl sterile PBS, 100 µl in each thigh skeletal muscle, | ||||
| MIC8 | Challenge by RH strain of T. gondii genomic DNA of T. gondii RH strain | pGEM-T easy vector➔ pVAX vector | Not mentioned | Intramascular, pVAXMIC8 plasmid or empty pVAX I vector into each anterior tibial muscle (final plasmid concentration, 100 μg/100 μl) | ||||
| GRA4 | Oral challenge by 76 K T. gondii RH strain cysts | MLPIX➔ pcDNA3 expression vector | All the plasmids were purified from transformed E. coli DH5a by anion exchange chromatography | The tibialis anterior (TA) muscles of both hind legs each 50 mg pGRA4 in 50 ml PBS | ||||
| GRA14 DNA (boosted by recombinant protein) |
Highly virulent RH strain of T. gondii for challenge |
Cloning vector pTG19-T➔ eukaryotic expression vector pcDNA3 |
Plasmids were purified by EndoFree Plasmid Giga Kit | Intramuscularly with 100 μg pcGRA14 into thigh skeletal muscle the first time | ||||
| ROP54 | Intraperitoneally T. gondii RH (acute infection) and oral Prugniaud (PRU) strains tissue cists for chronic | pVAX I plasmid | Not mentioned |
Route, location, and dose 100 μg of plasmid DNA dissolved in 100 μl sterile PBS intramuscular injection into the quadriceps |
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| TgCDPK2 |
T. gondii RH strain (type I) for the DNA sequence and challenge intraperitoneally |
pMD18-T➔ vectorpVAX I vector |
Anion exchange chromatography |
Bilateral intramuscular injection into the quadriceps with 100 mL (1 mg/mL) |
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| MYR1 DNA | T. gondii RH strain for the DNA sequence and challenge | pMD19-T ➔vector pVAX1 vector | End o-free plasmid giga kit | Subcutaneous injection with 100 mL of sterile PBS containing 100 mg pVAX1-MYR1 | ||||
| PLP1 and ROP18 DNA | The RH (used to produce the PLP1 and ROP18 clones) 59 and PRU strains (used to challenge mice with tissue cysts) | PIRESneo➔ pVAX | Not mentioned | Eight groups of mice (30 mice/group) were vaccinated intramuscularly with 100 μg of 82 plasmid dissolved in 100 μl of PBS | ||||
| GRA2 and GRA5 DNA (separate groups, not in combination) | The virulent T. gondii RH strain for lethal challenge | pcDNA 3.1C | Not mentioned | Intramuscular at tibialis anterior muscle of both leg with 100 μL (50 μL in each leg) of PBS containing 100 μg of pcGRA2, or 100 μg of pcGRA5 | ||||
| GRA17 and GRA23 DNA |
Challenge infection with the highly virulent RH strain of T. gondii DNA sequence from RH strain of T. gondii |
pMD18-T➔ pVAX | Not mentioned | Intramuscular injections 100 ml (100 mg) of pVAX-TgGRA17, pVAXTgGRA23, and pVAX-TgGRA17 + pVAX-TgGRA23 | ||||
| GRA7 and ROP2 DNA (each alone or in combination) |
DNA sequence from the T. gondii RH strain Challenge by |
pTOPO➔ pcDNA3.1 plasmid Mass replication in (E. coli), strain TOP10 |
Mass replication was extracted from the bacteria using endotoxin-free plasmid extraction kit | Intramuscularly with 100 μg of plasmid DNA in 100 μl PBS, 50 μl in each thigh skeletal muscle | ||||
| tgHSP60 | Two T. gondii strains RH and PRU for acute and chronic disease challenge, respectively | pMD18-T vector➔ pVAX | Not mentioned | Thigh muscle, 100 μg pVAX-HSP60 | ||||
|
ROP21 DNA |
Challenged with tachyzoite cells of RH T. gondii and cysts of T. gondii PRU strain |
pMD19-T vector ➔ pVAX plasmid Escherichia coli DH5a |
Purification by A commercial kit (TianGen, Beijing, China) |
Intramuscular injection containing 100 mg of recombined plasmids | ||||
| GRA14 DNA | RH strain tachyzoites for challenge and DNA sequence | pTG19-T➔ pcDNA3 | End o-free plasmid mega kit | Intramuscularly (anterior tibial muscle).Concentration 100 µg/100 µl plasmid DNA | ||||
| TgHSP-40 | DNA sequence from T. gondii RH tachyzoites challenge |
pMD18-T linear vector➔ pVAX1 |
Not mentioned |
intramuscularly (i.m.) 100 µL of PBS containing 100 µg pVAX1-HSP40 |
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| SAG1 DNA | Virulent T. gondii RH for challenged DNA sequence from tachyzoites of T. gondii RH | pTZ57 R/T cloning vector➔ pVAX1 E. coli DH5α | Anion exchange chromatography (end-free plasmid mega kit, Qiagen) | Intramuscular, each thigh skeletal muscle 100 μg of plasmid DNA, and different adjuvants suspended in 100 μl sterile PBS | ||||
| SAG1 and SAG3 DNA | T. gondii, strain RH |
pcDNA3 E. coli, strain TOP10 |
Plasmid purification kit (Qiagen) |
Intramuscularly with 100 μg of plasmid DNA suspended in 100 μL sterile PBS bilateral biceps | ||||
| GRA16 DNA | Challenge by T. gondii RH (acute) and PRU (chronic) strain |
pVAX E. coli DH5α cells |
Anion exchange chromatography (EndoFree Plasmid Giga Kit, Qiagen | Intramuscularly injected with pVAXGRA16 plasmids 100 µl (1 μg/μl) | ||||
| ROM4 DNA (alone or in combination with a peptide derived from its gene) Or SAG1 DNA as control | Challenge by T. gondii RH and PRU strain |
pEASY-T1 vector pEGFP-C1 expression plasmid Escherichia coli DH5α |
Endotoxin-free mega kit following the manufacturer’s instructions (Qiagen) |
Intramuscular route, location,and dose | ||||
| ROP35 DNA | Virulent T. gondii RH strain and PRU strain for challenge |
pMD19-T vectorpVAXI vector E. coli DH5α cells |
A commercial kit (TianGen, Beijing, China) to isolate the plasmid and to eliminate endotoxin contamination |
Intramuscular injections containing 100 μg of recombined plasmids (1 μg/ μl) |
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| GRA8 DNA |
DNA sequence from RH strain tachyzoites Challenged with highly virulent T. gondii GFP-RH strain |
pGEM-T Easy vectorression plasmid pEGFP-C1HEK-293 T cell pDsRed2-N1 vector Escherichia coli DH5α |
Endotoxin-Free mega kit according to the manufacturer’s instructions (Qiagen) |
50 μg pDsRed2-GRA8 into the tibialis anterior muscles of both hind legs (100 μg/per mouse) | ||||
| ROP19 DNA |
DNA sequence from T. gondii PRU strain tachyzoites Challenge by T. gondii strain PRU cysts |
pEASY-T1 vector ➔ expression plasmid pEGFP-C1 HEK-293 T cells |
The endotoxin-free mega plasmid kit (Qiagen) | Intramuscullar, buttocks injection 100 µl | ||||
| TgGRA24, TgGRA25 and TgMIC6 DNA | DNA sequence from T. gondii RH strain challenge with the T. gondii RH and Pru strains | pMD-18 T Vector➔ pVAX I vector | Not mentioned | Intramuscular injection into the quadriceps, 100 µL (1 µg/µL) of pVAXGRA24 | ||||
| SAG5D DNA |
DNA sequence from T. gondii (RH strain) Challenge by T. gondii RH strain |
pEASY-T1 vector➔ pEGFP-C1 HEK 293-T cells |
Endotoxin-free mega kit according to the manufacturer’s instructions (Qiagen) |
Intramuscularly, pEGFP-C1-SAG5D 100 μg/ each (1 µg/µl) |
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| Booster | Adjuvants sequence or chemical | Immune response elicited by vaccine | Animal used for vaccine response evaluation | Year of publication | ||||
|---|---|---|---|---|---|---|---|---|
| Boosted with the same dose at 3 and 6 weeks after the first injection | A plasmid encoding murine GM-CSF (pGM-CSF) |
SAG1 specific AbA Th1 dominant response (in contrast to the peptide vaccine) Splenocytes produce IL-2 and IFN-g but not IL-4 following specific Ag re-stimulation Increased survival and reduced brain cyst |
C57BL/6 mice and Sprague–Dawley rat | 1999 | ||||
| With the same protocols at weeks 2 and 4 | No additional adjuvant, The DNA CpG sequence itself |
In the pVAX-ROP16 group: Significant anti-rop16 Ab production splenocytes significant proliferative response and high degree of CTL activity higher IFN-g, IL-2, IL-4, and IL-10 (cell-mediated immunity) and lower pro-inflammatory cytokines IL-6 and IL-12 Increased survival time |
Kunming mice | 2011 | ||||
| The same protocol 2 and 4 weeks after vaccination | No additional adjuvant, The DNA CpG sequence itself |
In the VAXMIC8 group high levels of specific IgG splenocytes proliferative response to MIC8 significant increase in IFN-γ, IL-2, IL-4, and IL-10 Increased survival time |
Kunming mice | 2010 | ||||
| The mice were boosted in the same way on days 14 and 28 | GM-CSF plasmid adjuvant (also pIL-12) |
In the pGRA4 or pGRA4 + pGM-CSF group: strong antibody response significant proliferative response of splenocytes Increased IFN-g and IL-10 (& low amounts of IL-2) (a modulated involvement of Th1 response) pGRA4 mixed pIL-12-, which intensifies the Th1 response, dramatically decreases the survival rate |
C57BL/6 | 2000 | ||||
| Boosted by 20 μg rGRA14 two times (pcGRA14 + rGRA14 or pcGRA14 + rGRA14 + nanoadjuvants). at … | Alum and calcium phosphate (CaPNs) for the recombinant boost |
Increased survival time in immunized mice CaPNs adjuvanted DNA prime-protein boost vaccination induce both humoral and Th1 type cellular immune responses and high levels of total IgG, IgG2a isotype and IFN-γ (a Th-1 type response) and reduced brain parasitic load Alum adjuvanted DNA prime-protein boost vaccination: predominance of IgG1 over IgG2a and increased IL-4 (a Th-2 type response) |
BALB/c mice | 2019 | ||||
| Boosted twice with the same dose at 2-week intervals | No additional adjuvant, The DNA CpG sequence itself | In the pVAX-ROP54 group: High levels of IgG antibodies higher ratio of IgG2a/ IgG1(and higher IgG2a) higher level of IFN-γ, IL-2, and IL-12 (p70) and slighter increase in IL-4 and IL-10 (collectively, mixed Th1/Th2 with Th1 predominance) elevate splenocyte proliferation prolonged survival time reduced brain cyst number | Kunming mice | 2017 | ||||
|
Twice at 2-week intervals the same dose |
No additional adjuvant, The DNA CpG sequence itself |
In the pVAX-TgCDPK2 plasmids immunized mice: significant IgG response higher levels of IgG1 and IgG2a (elevated IgG2a/ IgG1)➔ mixed Th1/Th2, with a predominant Th1 higher proliferation of splenocytes increased % of CD4 + and CD8 + T cells in the splenocytes Increased IFN-g, IL-12(p70) and IL-10 but not IL-4 in spleen cell cultures longer survival time of the mice |
Specific pathogen-free (SPF) female BALB/c | 2017 | ||||
| Twice with 2-week intervals | No additional adjuvant, The DNA CpG sequence itself |
In the group immunized with pVAX1-MYR1: Specific IgG and IgG Isotypes (high IgG2a at first) Th1 response at 2 weeks after vaccination and a mixed Th1/Th2 immune response at 6 weeks after vaccination Higher proliferation of splenocytes significantly higher levels of IFN-g, IL-12, and IL-10 but not IL-4 higher levels of CD4 + and CD8 + T cells in the splenocytes Increased Expression of p65 and T-bet in spleen lymphocytes mRNA Increase in CTL activity Increased survival time |
BALB/c mice | 2019 | ||||
| Boosted twice at 2-week intervals | pVAX/IL-18 adjuvant | Significantly increased serum IgG (and IgG2a levels), lymphocyte counts and Th1-type cytokine (IL-2, IL-12, and IFN-γ) levels longer survival times | Kunming mice | 2018 | ||||
| 2 booster injections at 3-week intervals | No additional adjuvant, The DNA CpG sequence itself |
In both the vaccinated groups: predominant Th1-like response➔ cellular-mediated immune response with significantly higher levels of interferon-gamma, interleukin-2 (IL-2), IL-4, and IL-10 Increased splenocyte Ag-specific proliferation slightly prolonged survival No elevation of IgG was detected |
BALB/c mice | 2017 | ||||
| Booster immunizations 2 and 4-week later | No additional adjuvant, the DNA CpG sequence itself | Specific humoral and cellular responses, with higher level of IgG antibody, increased levels of Th1-type cytokines IFN-g and IL-12 (p70), and CD3þCD4þCD8, and CD3þCD8þCD4 T cells, as well as prolonged survival time | BALB/c mice | 2017 | ||||
| Twice with 3-week intervals | No additional adjuvant, The DNA CpG sequence itself |
In the vaccinated groups after specific Ag re-stimulation: Increased IFN-γ levels And decreased IL-4 expression level Increased spleen lymphocyte proliferation Significant high levels of IgG in the serum Predominance of the levels of IgG2a over IgG1 increased survival time |
BALB/c mice | 2018 | ||||
| 2 times with 2-week intervals | No additional adjuvant, The DNA CpG sequence itself |
In the HSP60 DNA-immunized mice: increase of CD3+CD4+ and CD3+CD8+ T cells in spleen increased levels of IL-2, IL-4, IL-10, IL-12p70, and IFN-γ Increased proliferation of splenocytes higher levels of specific antibodies in sera Increased survival time (in the acute infection) Decreased brain cyst (in the chronic infection) |
Specific pathogen-free (SPF) grade Kunming mice |
2018 | ||||
| Twice at 2nd and 4th weeks | No additional adjuvant, The DNA CpG sequence itself |
In the pVAX-ROP21 vaccinated animals: increased levels of IgG, IgG1, and IgG2a (IgG2a predominant) IFN-g was significantly increased (while no significant changes were detected in IL-2, IL-4, and IL-10) prolonged survival time (virulent T. gondii RH strain challenge) The number and size of brain cyst decreased |
BALB/c mice | 2018 | ||||
| Boosted at days 14 and 28 with the same protocol | Calcium phosphate nanoparticles |
Increased levels of level of IgG1 and IgG2a (IgG2a predominance) Increased proliferation of splenocytes Increased IFN-g levels Increased survival time Reduced tissue parasite load |
BALB/c mice | 2017 | ||||
| Boosted twice at a 2-week intervals | No additional adjuvant, The DNA CpG sequence itself |
In immunized mice: increase in T lymphocyte subclasses (CD3+CD4+ T and CD3+CD8+ T lymphocytes) in splenic tissues reduction in the parasite cyst burden in the brain Pru strain–infected mice No difference in survival time in challenge with the virulent RH strain No difference in the level of antibodies, lymphocyte proliferation and concentration of cytokines (IFN-g, IL-2, IL-4, IL-10, and IL-12p70) |
Kunming mice | 2018 | ||||
| Boosted using the same protocol twice at 3-week intervals on days 21 and 42 and |
FliC of Salmonella typhimurium plasmid (Toll-like receptor 5 agonist) and (alum and saponin) |
The pVAX1-SAG1 + pVAX1-fliC group (compared to both traditional adjuvants and controls): Higher IgG with a predominance of IgG2a over IgG2b and IgG1 higher levels of IFN-γ, IL-12 and IL-10 cytokines and low levels of IL-4 production higher splenocyte proliferation response increased survival time |
BALB/c mice | 2019 | ||||
| Boosted twice 3-week intervals | Alum and MMT |
Cocktail DNA group: higher total IgG and the isotypes of IgG1 and IgG2a higher levels of IFN-γ (the immune response was shifted toward Th1) increase antigen-specific lymphocyte proliferation of splenocytes increased survival time and rate |
BALB/cmice | 2019 | ||||
| Boosted twice with 2-week interval (the same dose) | No additional adjuvant, The DNA CpG sequence itself |
The pVAX-GRA16 group: higher levels of specific IgG antibody high Ag-specific proliferation of spleen lymphocytes increased levels of IFN-γ, IL-2, IL-4, and IL-10 cytokines higher percentages of CD4 + and CD8 + T cells reduced numbers of tissue cysts no change in the survival time |
Specific pathogen-free (SPF) grade inbred Kunming mice |
2017 | ||||
|
Boosted three times 2-week intervals (in the pROM4/peptide group the first 2 times by plasmid and the second 2 times by peptide) |
No additional adjuvant, The DNA CpG sequence itself |
The vaccinated groups: high levels of IgG, IgG2a (predominant), and interferon (IFN)-γ, IL-12, and IL-2. (IgG, IgG2a, and IFN-γ, IL-12, and IL-2 levels were highest in the pROM4/peptide group) Prolonged survival times and reduced numbers of brain cysts (especially those in the pROM4/peptide group) |
BALB/c mice | 2017 | ||||
| Boosted twice at 2nd and 4th week | No additional adjuvant, The DNA CpG sequence itself |
In the pVAX-TgROP35 group: Higher IgG (both IgG2a and IgG1) IFN-γ, IL-2, and IL-10 levels were significantly increased, while there were no significant differences in IL-4 expression increased survival time Reduced brain cysts number and size |
BALB/c mice | 2018 | ||||
| Booster immunizations 2 and 4 weeks later | No additional adjuvant, The DNA CpG sequence itself |
Higher IgG (both IgG2a and IgG1 increased predominant IgG2a) Higher splenocyte proliferation Increased IL-10, IL-12 (p70), IFN-γ, and TNF-α but not IL-4 Increased survival time |
Specific pathogen-free (SPF) female BALB/c mice | 2018 | ||||
| Boosted with the same protocoltwice with 2-week intervals | No additional adjuvant, The DNA CpG sequence itself |
Higher levels of IgG antibodies higher levels of IFN-g reduced brain cysts |
BALB/c mice | 2016 | ||||
| Boosted twice with 2-week intervals | No additional adjuvant, The DNA CpG sequence itself |
In the immunized groups (more apparently in the multi-antigenic groups): Increased IgG titer higher IgG2a to IgG1 ratio Increased IL-2, IFN-g, IL-12 and IL-23 levels (but not IL-4 and IL-10) Increased percentages of CD3+ CD4+CD8− and CD3+CD8+CD4− T lymphocytes Increased survival time Increased spleen lymphocytes proliferation Decreased brain cyst |
Kunming mice | 2019 | ||||
|
Boosted twice at 2-week intervals pEGFP-C1-SAG5D 100 μg/ each (1 µg/µl) with α-GalCer at the 3rd time |
Alpha-Galactosylceramide (α-GalCer) (2 μg/mouse) |
In both pEGFPC1- SAG5D or α-GalCer/pEGFP-C1-SAG5D groups: increase of IgG (IgG2a over IgG1) higher level of IFN-γ higher IL-4 (only in α-GalCer-treated groups) longer survival time |
BALB/cmice | 2014 | ||||