CN114569711B - ME49 delta cdpk3 attenuated live vaccine for preventing toxoplasmosis as well as preparation method and application thereof - Google Patents
ME49 delta cdpk3 attenuated live vaccine for preventing toxoplasmosis as well as preparation method and application thereof Download PDFInfo
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- CN114569711B CN114569711B CN202210298777.5A CN202210298777A CN114569711B CN 114569711 B CN114569711 B CN 114569711B CN 202210298777 A CN202210298777 A CN 202210298777A CN 114569711 B CN114569711 B CN 114569711B
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- toxoplasmosis
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Classifications
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Abstract
The application relates to the technical field of toxoplasmosis vaccines, in particular to an ME49 delta CDPK3 attenuated live vaccine for preventing toxoplasmosis, which is an ME49 delta CDPK3 insect strain constructed by knocking out CDPK3 on ME49 through a CRISPR/Cas9 mediated gene editing technology, and the preparation steps of the attenuated live vaccine comprise (1) constructing a targeted Cas9 plasmid, (2) constructing homologous donor DNA, (3) constructing a mixed clone insect strain of ME49 delta CDPK3 and (4) screening to obtain an ME49 delta CDPK3 monoclonal insect strain. In the present application, the use of the tachyzoites of ME49 delta cdpk3 to immunize BALB/c mice, the protective immunization of the ME49 delta cdpk3 attenuated strain as a vaccine was evaluated by a mouse model, and studies have found that the immune response resulting from ME49 delta cdpk immunization can protect mice from acute and latent toxoplasma infection, indicating that the tachyzoites of ME49 delta cdpk3 can provide effective protection against different parasite strains, and that ME49 delta cdpk3 can therefore be used as a potential candidate vaccine against toxoplasma.
Description
Technical Field
The invention relates to the technical field of toxoplasmosis vaccines, in particular to an ME49 delta cdpk3 attenuated live vaccine for preventing toxoplasmosis, a preparation method and application thereof.
Background
Toxoplasma gondii (t.gondii) is a common zoonotic intracellular parasite capable of infecting almost all warm-blooded animals including humans. About 30% of the world's population is infected with toxoplasma gondii, most of which are asymptomatic in healthy individuals, but in immunocompromised or defective populations can lead to the appearance of toxoplasmosis encephalitis, retinochoroiditis, and maternal gestational infections can cause congenital toxoplasmosis. In addition, primary infections of many important domestic animals such as goats can lead to abortion and stillbirth, which can present significant economic losses and serious challenges to the animal industry. In animals or humans, a fixed combination of pyrimethamine and sulfadiazine for tachyzoites is currently the drug of choice for the treatment of acute stage toxoplasmosis, but this drug has proven ineffective against bradykinin or latent infection.
Toxoplasma has complex life cycle and multiple transmission paths. Domestic cats and other wild cats are the ultimate host of toxoplasma gondii, and intake of oocysts excreted by cats is the primary source of infection in intermediate hosts (humans and animals). In addition, toxoplasma can also be transmitted between intermediate hosts by asexual reproduction and predation. Toxoplasma strains have a complex population structure. There are three main types of toxoplasma strains in north america and europe (I, II and III). The strain I is mainly RH and GT1, the strain II is PRU and ME49, the strain III is mainly CEP, and the toxicity difference is large. The toxoplasma strain in south america is more genetically diverse than the toxoplasma strain in north america and europe. However, genotype Chinese 1 has been identified as the major strain in east asia, particularly in china. Jensen et al found that strains from different genotypes could repeatedly infect the same host. The above several reasons present a great challenge for the control of toxoplasmosis. Therefore, the development of an effective toxoplasmosis vaccine is critical to limiting infection of various toxoplasmosis strains.
In recent years, related researchers have conducted various studies for developing safe and effective toxoplasma vaccines. Nucleic acid vaccines, recombinant protein vaccines and cocktail antigen vaccines have been developed to prevent toxoplasma infection. However, it is difficult for these vaccines to provide adequate protection. The most promising strategy to obtain an effective toxoplasma vaccine to date is the use of attenuated live vaccines which induce a higher and longer-term protective cellular and humoral immune response to prevent toxoplasma infection. At present, the current time of the process,The live attenuated S48 toxoplasma strain is the only commercial vaccine for preventing congenital toxoplasma infection of sheep and goats, and more toxoplasma attenuated vaccines are researched to expand the application range of the toxoplasma attenuated vaccine, so that the live attenuated S48 toxoplasma strain has important value and significance for both human health and development of livestock breeding.
Toxoplasma gondii has 14 genes encoding Ca 2+ -dependent protein kinase (CDPK) which are involved in a variety of functions such as movement, invasion, proliferation and escape of toxoplasma gondii. TgCDPK3 is critical for rapid induction of parasite discharge and establishment of chronic infections in mice, which is critical for endoparasite virulence. The protective immune effect of the ME49 delta cdpk3 attenuated strain as a vaccine was evaluated by a mouse model in the present application using the tachyzoites of ME49 delta cdpk to immunize BALB/c mice. Studies have found that ME49 Δ cdpk3 immunization elicits a strong immune response and protects mice from acute and latent toxoplasma infection, suggesting that tachyzoites of ME49 Δ cdpk3 can provide effective protection against different parasite strains, and that ME49 Δ cdpk3 can therefore be used as a potential candidate vaccine against toxoplasma.
Disclosure of Invention
The technical problem to be solved by the invention is how to prepare the attenuated live vaccine of ME49 delta cdpk3 for preventing toxoplasmosis, and the prepared attenuated live vaccine can effectively resist acute infection and chronic infection of toxoplasmosis.
The invention solves the technical problems by the following technical means:
The first aspect of the invention provides an ME49 delta CDPK3 attenuated live vaccine for preventing toxoplasmosis, wherein the attenuated live vaccine is used for constructing an ME49 delta CDPK insect strain by knocking out CDPK3 from the ME49 insect strain through a CRISPR/Cas9 mediated gene editing technology.
The second aspect of the invention provides a method for preparing an attenuated live vaccine of ME49 delta cdpk3 for preventing toxoplasmosis, comprising the following specific steps:
(1) Construction of a Targeted Cas9 plasmid
Plasmid pSAG for knocking out toxoplasma CRISPR/Cas 9:: cas 9-U6::: sgUPRT, target design using E-CRISPR software, useThe site-directed mutagenesis kit replaces the sgRNA in the plasmid pSAG <1 >: cas9-U6 < sgUPRT > with the sgRNA targeting CDPK3, namely a plasmid pSAG <1 >: cas9-U6 < sgCDPK >;
(2) Construction of homologous donor DNA
CDPK 35 '-and 3' -homology arm fragments will be amplified from genomic DNA of type II ME49 strain, and DHFR-TS sequence will be amplified from pUPRT-DHFR plasmid; cloning the three fragments between pUC19 linearization vectors through homologous recombination by using a multi-fragment one-step rapid cloning kit, and constructing a three-fragment donor DNA plasmid;
(3) Construction of an insect strain of ME49 delta cdpk3
Electroporation transfection of Cas9-U6: sgCDPK3 and the donor DNA fragment constructed in step (2) into purified ME49 tachyzoites, and screening with pyrimethamine to prepare a mixed clone of ME49 delta cdpk3 tachyzoites;
(4) Screening of the ME49 delta cdpk3 monoclonal insect strain
Single toxoplasma was inoculated by fold dilution into 96-well plates plated with human foreskin fibroblasts (HFF cells); positive clones were detected by PCR and immunoblotting 7-10 days later, and single cloned insect strains were obtained.
Preferably, the primers of the plasmid pSAG 1:Cas9-U6: sgUPRT in the step (1) are:
CDPK3-gRNA-F：TGTACCGAGGGTTTTAGAGCTAGAAATAGC；
CDPK3-gRNA-R：CCTCCATGACAACTTGACATCCCCATTTAC。
Preferably, the primers of DHFR-TS in step (2) are:
DHFR-TS-F：TGTCATTCGATTTTCACCCCC；
DHFR-TS-R：AGTGTGATGACTCCGCAACT
GGATCGATCCCCCCGGGCTGC。
preferably, the diagnostic PCR in step (4) includes PCR1, PCR2 and PCR3.
The primer of the PCR1 is as follows:
PCR1-F：GCCTAACAAGGATTCGATCAGTAGC；
PCR1-R：TGTCGTGGATTTACCAGTCATGGAC；
the primer of the PCR2 is as follows:
PCR2-F：TGACTCTTCATGTGGCATTTCACAC；
PCR2-R：TACTGTGTTAGGTAGCAAATGTGG；
the primer of the PCR3 is as follows:
PCR3-F：TCGTGCGTCTTCAGGCATGTACATC；
PCR3-R：GAGGTCCTTTCGCTTCCTGAGACTC。
Preferably, the immunodose of ME49 Δ cdpk3 tachyzoites is 10 3～106.
Preferably, the immune dose of the ME49 Δ cdpk3 tachyzoites is 10 3.
In a third aspect the invention provides the use of a live attenuated ME49 delta cdpk3 vaccine for the prophylaxis of toxoplasmosis in the prophylaxis of parasitic infections.
In a fourth aspect, the invention provides the use of an attenuated live vaccine of ME49 delta cdpk3 for the prevention of toxoplasmosis in the prevention of chronic infection by toxoplasmosis.
In a fifth aspect, the invention provides the use of an attenuated live vaccine of ME49 delta cdpk3 for the prevention of toxoplasmosis in the prevention of a variety of acute infections of toxoplasmosis.
The invention has the advantages that:
1. The mice vaccinated with the ME49 Delta cdpk3 tachyzoites of the application are effective against a variety of wild type strains including type I RH, type II ME49, chinese1 WH3 and WH6 infections, indicating that the ME49 Delta cdpk3 vaccination elicits a strong immune response against toxoplasma infection when challenged with different strains, and that the parasite load in the peritoneal fluid is significantly reduced when the ME49 Delta cdpk3 immunizes mice against re-infection with toxoplasma.
2. Clinical scores of 10 3 mice infected with ME49Δ cdpk3 tachyzoites according to the application showed that the status of the mice was relatively good, and that the 10 3 mice infected with ME49Δ cdpk3 tachyzoites induced a similarly high level of toxoplasma specific IgG in the serum, indicating that 10 3 mice vaccinated with ME49Δ cdpk3 tachyzoites were a safe and effective immunizing dose.
3. Toxoplasma IgG levels in the serum of mice of the application remained high and stable throughout the vaccination period; for the IgG subclass levels, igG1 and IgG2a levels were significantly elevated in immunized mice compared to non-immunized mice, and significantly higher IgG2a levels than IgG1 indicated that the ME 49.DELTA. cdpk3 vaccination triggered a Th1 and Th2 mixed immune response that was predominantly a Th1 response, indicating the potential protective immunity of the ME 49.DELTA. cdpk3 vaccination.
4. IFN-gamma, TNF-alpha, IL-12p70 and IL-10 levels were significantly elevated in the spleen cell supernatant of 75 day vaccinated mice compared to non-vaccinated mice, indicating that ME49 delta cdpk3 vaccination effectively elicits a cell-mediated immune response.
5. The present application also reduces the development of chronic toxoplasmosis in mice with ME49 delta cdpk. Survival rates were 100% after 50 ME49 encapsulates infection in vaccinated mice compared to non-vaccinated mice, with significantly reduced in-brain encapsulation in surviving mice 35 days after encapsulation.
6. The serum of mice vaccinated with the ME49 delta cdpk vaccine can reduce the proliferation of parasites, which shows that the serum of mice vaccinated with the ME49 delta cdpk vaccine can effectively resist toxoplasma infection.
In summary, the present ME49 Δ cdpk3 vaccination can elicit cellular and humoral immunity and protect mice from toxoplasma infection, suggesting that the ME49 Δ cdpk3 strain may be a viable attenuated live vaccine candidate against acute and latent toxoplasmosis.
Drawings
FIG. 1 shows the results of PCR and immunoblotting assays in example 2 of the present application.
FIG. 2 shows the results of detection of clinical symptoms of mice during parasitic infection and the levels of toxoplasma specific IgG in the serum of mice in example 9 of the present application.
FIG. 3 shows the case of the present application in example 10 where the ME 49.DELTA. cdpk3 vaccine immunizes mice against infection with Toxoplasma gondii tachyzoite.
Figure 4 is a figure showing the protection of mice from cyst infection by the administration of ME49 delta cdpk3 vaccine according to example 11 of the present application.
FIG. 5 shows cytokine levels in serum from mice 30 days, 75 days and 125 days after the immunization with ME 49.DELTA. cdpk3 in example 12 of the present application.
FIG. 6 shows toxoplasma specific total IgG and IgG subclasses (IgG 1 and IgG2 a) in serum from mice 30 days, 75 days, and 125 days after the administration of ME 49.DELTA. cdpk3 in example 12 of the present application.
FIG. 7 shows the results of the cellular immune response induced by the ME 49.DELTA. cdpk3 vaccination after toxoplasma antigen stimulation in example 13 of the present application.
FIG. 8 shows the results of passive immunization with serum from mice vaccinated with ME 49.DELTA. cdpk3 in example 14 of the present application.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1
The embodiment discloses an ME49 delta CDPK3 attenuated live vaccine for preventing toxoplasmosis, wherein the attenuated live vaccine is an ME49 delta CDPK insect strain constructed by knocking out CDPK3 in the ME49 insect strain through a CRISPR/Cas9 mediated gene editing technology.
Example 2
The embodiment discloses a preparation method of an ME49 delta cdpk3 attenuated live vaccine for preventing toxoplasmosis, which comprises the following specific steps:
(1) Construction of a Targeted Cas9 plasmid
Plasmid pSAG to Toxoplasma CRISPR/Cas 9:: cas9-U6:: sgUPRT (purchased from Addgene, U.S. A. # 54467). Target design Using E-CRISPR software and useThe site-directed mutagenesis kit (purchased from NEW ENGLAND Biolabs (NEB)) replaced the sgRNA in plasmid pSAG:: cas9-U6:: sgUPRT with the sgRNA targeting CDPK3, i.e., plasmid pSAG:: cas9-U6:: sgCDPK3 was constructed.
Wherein, the primer of the plasmid pSAG 1:Cas9-U6: sgUPRT is:
CDPK3-gRNA-F：TGTACCGAGGGTTTTAGAGCTAGAAATAGC；
CDPK3-gRNA-R：CCTCCATGACAACTTGACATCCCCATTTAC。
(2) Construction of homologous donor DNA
CDPK 35 '-and 3' -homology arm fragments will be amplified from genomic DNA of type II ME49 strain, and DHFR-TS sequences will be amplified from pUPRT-DHFR. Three fragments, CDPK 35 '-homology arm fragment, CDPK3 3' -homology arm fragment and DHFR-TS sequence, were cloned between pUC19 linearization vectors by homologous recombination using a multi-fragment one-step rapid cloning kit (ClonExpress MultiS One Step Cloning Kit, available from Vazyme Biotech company, nanjing, china) to construct three-fragment donor DNA plasmids.
Wherein, the primers of CDPK3 5' homology arm fragment UpCDPK-F are:
AAAACGACGGCCAGTGAATTC
AGCCAACATGCATTGGAGCT；
the UpCDPK-R primer is:
GGGGGTGAAAATCGAATGACA
ACGCAGCGACTGGGAGAATC。
The primers of DHFR-TS in step (2) are:
DHFR-TS-F：TGTCATTCGATTTTCACCCCC；
DHFR-TS-R：AGTGTGATGACTCCGCAACT
GGATCGATCCCCCCGGGCTGC。
Primers for CDPK3 3' homology arm fragment DnCDPK-F are:
GCAGCCCGGGGGGATCGATCC
AGTTGCGGAGTCATCACACT；
the DnCDPK-R primer is:
GACCATGATTACGCCAAGCTT
TCGTTGTGGGCTATACAGCT。
(3) Construction of an insect strain of ME49 delta cdpk3
The plasmid pSAG constructed in step (1): cas9-U6: sgCDPK and the donor DNA fragment constructed in step (2) were electroporated into purified ME49 tachyzoites and screened with 1. Mu.M pyrimethamine to prepare a mixed clone of ME 49. Delta. Cdpk3 tachyzoites.
(4) Screening to obtain ME49 delta cdpk3 monoclonal insect strain
Single toxoplasma was inoculated by fold dilution into 96-well plates plated with human foreskin fibroblasts (HFF cells); and detecting positive clones by PCR and immunoblotting after 7-10 days to obtain a single cloned insect strain, namely the ME49 delta cdpk insect strain.
Among them, diagnostic PCR includes PCR1, PCR2 and PCR3.
The primers for PCR1 were:
PCR1-F：GCCTAACAAGGATTCGATCAGTAGC；
PCR1-R：TGTCGTGGATTTACCAGTCATGGAC；
the primers for PCR2 were:
PCR2-F：TGACTCTTCATGTGGCATTTCACAC；
PCR2-R：TACTGTGTTAGGTAGCAAATGTGG；
The primers for PCR3 were:
PCR3-F：TCGTGCGTCTTCAGGCATGTACATC；
PCR3-R：GAGGTCCTTTCGCTTCCTGAGACTC。
the PCR identification results of the ME49 Delta CDPK3 strain are shown in FIG. 1A, and the ME49 Wild Type (WT) strain is used as a control, PCR1 and PCR2 verify the integration of the 5 'and 3' homology arms respectively, and PCR3 verifies the successful knockout of CDPK3 gene, so that the successful knockout of CDPK3 gene can be seen, and a monoclonal insect strain with ME49 knockout of CDPK3 is obtained.
The Western blot results of ME49 WT and ME 49.DELTA. CDPK3 strains were as shown in FIG. 1B, with toxoplasma actin (actin) antibodies as an internal control, and the blot results showed that the ME 49.DELTA. CDPK3 strain did not express CDPK3 protein.
Example 3
This example uses the ME49 Δ CDPK3 tachyzoites prepared in example 2 to perform a parasite infection assay on mice to assess the effect of CDPK3 inactivation on parasite virulence.
To test the difference in immune response between ME49 and ME49 Δ cdpk3 tachyzoite infected mice, mice were infected with ME49 and ME49 Δ cdpk3 parasites, respectively, at doses of 10 3、104、105 and 10 6, and observed for clinical symptoms over 35 days.
Clinical scores ranged from 0 (no sign) to 10 (all signs). The clinical symptoms evaluated included humpback, standing hair, behavior, insect finding, ptosis, eye depression, ataxia, slow motion, poor evacuation and tactile reflex and groveling. In addition, serum from the above infected mice was collected on day 35 post-infection and assayed for toxoplasma specific IgG levels by a 10 μg/ml soluble tachyzoite antigen coated ELISA plate.
Example 4
This example uses the ME 49.DELTA. cdpk3 tachyzoites prepared in example 2 to conduct acute and chronic toxoplasma infection tests on mice.
BALB/c mice were immunized by intraperitoneal injection of 10 3 ME49Δ cdpk3 tachyzoites. On day 75 post immunization, different mice were injected with 10 3 Chinese1 WH3 (10 mice per group), 10 5 Chinese1 WH6 (10 mice per group) or 50 ME49 capsules (10 mice per group), respectively. On day 125 post immunization, mice were injected with 10 3 RH and 10 5 ME49 tachyzoites (10 mice per group). Unvaccinated mice infected with the same dose and route served as controls. The re-infected mice were then monitored daily for clinical symptoms and survival over 35 days. On day 7 after toxoplasma infection, the peritoneal fluid parasite burden of the mice was determined. For chronic infection, the number of cysts in the brains of surviving mice was detected on day 35 post challenge.
Example 5
This example uses the ME 49.DELTA. cdpk3 tachyzoites prepared in example 2 to immunize mice and then detects cytokine and toxoplasma specific IgG levels in the serum of immunized mice.
BALB/c mice were immunized by intraperitoneal injection of 10 3 ME49Δ cdpk3 tachyzoites and mock-vaccinated with 300 μl PBS, with mouse serum samples taken 30, 75 and 125 days after vaccination, respectively. And the levels of total toxoplasma serum specific IgG and IgG subtypes (IgG 1 and IgG2 a) were detected by enzyme-linked immunosorbent assay (ELISA).
The specific operation is as follows: 10. Mu.g/ml soluble toxoplasma antigen diluted in coating buffer (50 mM carbonate buffer, pH 9.6) was used to coat 96-well ELISA plates, which were then incubated overnight at 4℃and then washed 5 times with phosphate buffered saline containing 0.05% Tween-20 (PBST, pH 7.4); nonspecific binding was blocked with 3% BSA for 1 hour at 37℃and then washed five times with PBST.
Then, after diluting the collected serum at 1:50, incubating for 1 hour at 37 ℃; after washing, HRP-sequestered goat anti-mouse IgG and IgG subtypes (IgG 1 or IgG2 a) were diluted with PBST (1:1000) and then added to 96-well plates, 100 μl per well, and incubated for one hour at 37 ℃; each well was then washed 5 times with PBST.
Finally, the reaction was performed using TMB (100. Mu.l/well) as a substrate, and 2M H 2SO4 was added to stop the reaction. Optical Density (OD) was measured at 450nm in triplicate using an ELISA reader, and all serum samples were analyzed. Meanwhile, the production levels of cytokines IFN-. Gamma.IL-12 p70, TNF-. Alpha.and IL-10 were measured using ELISA kits according to the recommendations of the kit instructions.
Example 6
This example evaluates the level of cytokine production in spleen cell supernatants isolated from vaccinated and unvaccinated mice 75 days after immunization in example 5.
Immunized and non-immunized mice were anesthetized and sacrificed, and spleens were harvested to assess the level of spleen cytokine production. The specific operation is as follows: spleen cells were isolated by 70 μm wire mesh screen and lysed in lysis buffer for 5 min to obtain a single spleen cell suspension; spleen cells were cultured in 24-well plates and stimulated with 10 μg/mL toxoplasma soluble tachyzoite antigen (STAg) of ME49 strain; cell-free supernatants were then collected, and levels of TNF- α and interleukin 10 (IL-10) at 72 hours post-culture, and levels of interleukin 12p70 (IL-12 p 70) and interferon γ (IFN- γ) at 96 hours post-culture were measured by ELISA, with non-immunized mouse spleen cells as negative controls.
Example 7
This example uses mice vaccinated with ME49 delta cdpk tachyzoites from example 5 for passive immunization.
BALB/c mice were intraperitoneally injected with 10 6 type II ME49 tachyzoites, and serum from 125 days of normal mice or immunized mice was injected into infected mice (100 μl/mouse) by tail vein on days 0 and 3 after challenge, and serum from normal mice was used as negative control. Parasite burden in peritoneal fluid was detected by fluorescent quantitative PCR 7 days after challenge to measure parasite proliferation under passive immunization and daily record of mortality for 35 days in each remaining group of mice.
Example 8
The parasite burden in infected mice was measured in this example.
Genomic DNA was extracted from the peritoneal fluid of parasite infected mice using Steady Pure Genomic DNA Kit and the parasite burden was determined by qPCR amplification of Tg-529 gene (forward primer 5'-CGTCCAGGGAGGAAGACGAAAGTTG-3', reverse primer 5'-CGCTGCAGACAGAGTGCATCTGGATT-3') using corresponding genomic DNA samples. The mouse actin gene in each sample was used as an internal reference (forward primer 5'-AGCTTCTTTGCAGCTCCTTCGT-3', reverse primer 5'-TACACGCTAGGCGTAAAGTTGG-3'). According to the specification, useThe Premix Ex Taq TM II kit was subjected to real-time qPCR and all reactions were run on the Roche LC480II system.
Standard curves for quantification of parasites were obtained: genomic DNA from 0, 100, 10 1、102、103、104、105、106 and 10 7 parasites was extracted, then CT values of each sample were obtained as ordinate, lg (tachyzoites number) as abscissa using qPCR based on Tg529, and standard curves were generated from the CT values and Lg, and parasite loads were calculated from the standards.
Example 9
Clinical symptoms were monitored 35 days after mice in example 3 were infected with ME49 and ME49 delta cdpk3, and uninfected mice were used as controls, with the monitoring results shown in fig. 2A. The results show a significant attenuation of the virulence of the ME49 Δ cdpk tachyzoites compared to the Wild Type (WT) strain ME49, with a survival rate of 100% even at an infectious dose of 1×10 6 tachyzoites.
The results of clinical evaluation 35 days after infection of 10 3、104、105 and 10 6 parent strains ME49 or ME 49.DELTA. cdpk3 tachyzoite-infected BALB/c mice in example 3 are shown in FIG. 2B. Preliminary experimental results showed that lower clinical scores were observed in 10 3、104、105 and 10 6 mice infected with ME49 a cdpk tachyzoites, while 10 3、104、105 and 10 6 mice infected with ME49 exhibited more severe clinical symptoms.
IgG was detected 35d after infection of 10 3、104、105 and 10 6 parental strains ME49 or ME49 a cdpk3 tachyzoite infected BALB/C mice in example 3 and the results were shown in fig. 2C (statistical differences p <0.0001, 6 mice per group) with uninfected serum as a control. The results indicate that at 35 days post-infection, toxoplasma specific IgG was induced to have similarly high levels in serum of 10 3、104、105 and 10 6 mice infected with ME49 a cdpk3 tachyzoites or 10 3、104、105 ME 49. These represent similar immunogenicity, combined with clinical scores, suggesting that 10 3 ME49 Δ cdpk3 tachyzoite vaccinations are a safe and effective immunizing dose.
Example 10
Mortality of vaccinated mice infected with 10 3 Chinese 1WH 3 or 10 5 Chinese 1WH6 at 75 days post-inoculation in example 4 was assessed for the protective efficacy of the ME49 a cdpk3 immunization against tachyzoite infection and the non-immunized mice were considered negative controls, the results are shown in fig. 3A-B. It can be seen that the survival rate of mice infected with Chinese 1WH6 was 100%. However, when challenged with highly toxic Chinese 1WH 3, only 30% of vaccinated mice survived, although the survival time of mice had been significantly prolonged.
Vaccinated mice were infected with 10 3 type I (RH) or 10 5 type II (ME 49) at 125 days post-vaccination in example 4 for mortality, examined whether the ME49 a cdpk3 vaccination provided long-term protective immunity, and the non-immunized mice were considered negative controls, and the results are shown in fig. 3D-E. It can be seen that no mortality was observed in immunized mice challenged with ME49 tachyzoites, whereas the survival rate of vaccinated mice re-challenged with RH tachyzoites was about 30%, indicating that the ME49 delta cdpk3 vaccination has a longer lasting and stronger protective effect against the less virulent parasite strains, but a weaker protective effect against the more virulent strains.
Mice were immunized with ME49 delta cdpk3 collected 7 days after infection in example 4 for secondary infection of peritoneal fluid to assess the peritoneal fluid parasite burden in mice, and the parasite burden in mice was determined using the quantitative PCR technique of example 8 as a control, as shown in fig. 3C, 3F (statistical differences were calculated as p <0.001, p <0.01, p <0.05, ns: no difference). It can be seen that in non-immunized mice, RH, ME49 and Chinese1WH3 and Chinese1WH 6 infections resulted in rapid parasite proliferation, whereas very few parasites were detected in RH, ME49 and Chinese1WH3 and Chinese1WH 6 challenged me49Δ cdpk3 vaccinated mice, indicating that the me49Δ cdpk3 vaccination promoted rapid elimination of infected parasites, further demonstrating that the ME49 Δ cdpk3 vaccination could produce an effective and safe protective immunity to clear challenging parasites, thereby allowing the host to survive.
Example 11
75 Days after immunization of 10 3 ME49Δ cdpk3 tachyzoite-vaccinated BALB/c mice in example 4, mice were orally infected with 50 ME49 encapsulates, and survival of the mice was recorded to evaluate the protective efficacy of the ME49 Δ cdpk3 immunization against chronic infection, and the results were shown in FIG. 4A with unvaccinated mice as a control. It can be seen that mice vaccinated with the ME49 delta cdpk vaccine survived 100% whereas unvaccinated mice survived only 50%.
The parasite encapsulation load in the brains of surviving mice, including immunized and non-immunized mice, was assessed 35 days after infection encapsulation, as shown in fig. 4B (statistical differences P <0.001, 5 mice per group). It can be seen that the number of cysts per mouse brain not vaccinated was 1640± 638.7, whereas the number of cysts per mouse brain vaccinated with ME49 Δ cdpk3 was 94.00±21.04 (p < 0.001), indicating that the ME49 Δ cdpk3 strain also induced a protective immune response against bradyzoite infection.
Example 12
The immune response mechanisms provided by the ME49 a cdpk3 vaccination were elucidated by serum assessment of IL-12p70, IL-10, IFN- γ and TNF- α levels 30 days, 75 days and 125 days after the BALB/c mice were vaccinated with ME49 a cdpk tachyzoites in example 5, and the results were shown in fig. 5 with serum samples of non-immunized mice as negative controls (statistical differences were calculated as p <0.001, < p <0.01, < p <0.05, ns: no difference, 6 mice per group). Wherein, A corresponds to IFN-gamma, B corresponds to IL-12p70, C corresponds to TNF-alpha, and D corresponds to IL-10. It can be seen that the levels of the pro-inflammatory cytokines IL-12p70, IFN-gamma and TNF-alpha were significantly elevated at 30, 75 and 125 days post-immunization compared to non-immunized mice. However, the cytokine levels at day 75 were lower than those at day 30, probably due to activation of the anti-inflammatory response, as evidenced by elevated IL-10 levels in vaccinated mice 30 days post-infection. At day 125, the cytokine levels of vaccinated mice were comparable to those of unvaccinated mice.
The serum samples of mice from example 5 were tested for toxoplasma specific IgG and IgG subclass levels and serum samples from non-immunized mice were used as negative controls and the results were shown in fig. 6 (statistical differences were defined by p <0.001, <0.01, <0.05, < ns: no difference, 6 mice per group). Toxoplasma specific IgG levels were assessed and an increase in mouse IgG levels was observed at day 30, day 75 and day 125 after ME49 delta cdpk3 immunization. The type of immune response triggered by ME49 delta cdpk was determined by detecting the levels of IgG subclasses (IgG 1 and IgG2 a). The results indicate that mice vaccinated with ME49 Δ cdpk3 induced high levels of IgG1 and IgG2a antibodies at day 30, day 75, and day 125 post-infection, and IgG2a titers were significantly higher than IgG1, indicating that ME49 Δ cdpk3 induced a Th1 biased immune response. The above results indicate that ME49 delta cdpk immunity can also activate cellular and humoral immune responses to control toxoplasma infection.
Example 13
Spleen cells from vaccinated and unvaccinated mice were isolated 75 days after mice were vaccinated with ME49 delta cdpk3 in example 6 to evaluate cell-mediated immune responses in ME49 delta cdpk3 vaccinated animals. Spleen cells were cultured in vitro and stimulated with toxoplasma gondii tachyzoite soluble antigen, the supernatant was collected and cytokine levels estimated by ELISA, as shown in figure 7 (6 mice per group). It can be seen that the toxoplasma soluble antigen induced high levels of IFN-gamma, TNF-alpha and IL-12p70 and IL-10 compared to spleen cells obtained from unvaccinated mice, and these results indicate that ME 49.DELTA. cdpk3 immunization can activate an effective cellular immune response.
Example 14
Example 12 mice immunized with ME49 delta cdpk have been shown to have high levels of toxoplasma specific IgG, and to determine the role of this antibody in further limiting parasitic infections, protective immunization of immune mice serum against toxoplasma infection was determined by example 7 as shown in figure 8 (statistical differences p < 0.05). Wherein A is parasite burden and B is survival rate of mice. As can be seen, the mice vaccinated with ME 49.DELTA. cdpk3 immunized mice serum had significantly reduced parasite burden on the peritoneal fluid compared to the non-immunized mice vaccinated with normal serum, and the immunized mice had significantly higher survival rates than the non-immunized mice by 60%. Taken together, these results indicate that serum from mice vaccinated with ME49 delta cdpk reduced parasite proliferation to some extent.
The use principle and the advantages are that: mice were infected with 10 3、104、105 and 10 6 parental strains, ME49 and ME49 a cdpk3 toxoplasma tachyzoites and monitored for survival, which indicated that mice infected with ME49 a cdpk3 toxoplasma remain 100% over 35 days at an infectious dose of 1 x 10 6 tachyzoites/mouse (fig. 2A). 10 3 mice infected with ME49 Δ cdpk3 showed lighter clinical symptoms, and induced toxoplasma specific IgG in the serum of 10 3 mice infected with ME49 Δ cdpk3 tachyzoites had similarly high levels as the serum of 10 3、104、105 mice infected with ME49 and 10 4、105、106 mice infected with ME49 Δ cdpk3 (fig. 2B-C).
In order to obtain the appropriate immunogenicity required for the attenuated ME49 Δ cdpk3 strain without generating excessive immune responses in immunized mice, the present application administered 10 3 ME49 Δ cdpk3 tachyzoites per mouse. The results indicate that mice vaccinated with ME49 Δ cdpk3 tachyzoites are effective against a variety of wild type strains including type I RH, type II ME49, chinese1 WH3 and WH6 infections. The ME49 delta cdpk vaccination was shown to elicit an immune response against toxoplasma infection when challenged with different strains. Notably, protection of the more virulent type I RH and Chinese1 WH3 strains was significantly reduced (30% viability) compared to the less virulent type II ME49 and Chinese1 WH6 strains (100% viability) (FIGS. 3A, B, D, E). Furthermore, the parasite burden was significantly reduced in the peritoneal fluid of the ME 49.DELTA. cdpk3 vaccinated mice (p < 0.001) (FIG. 3C, F).
The present application also reduces the development of chronic toxoplasmosis in mice with ME49 delta cdpk. Survival was 100% after 50 ME49 cysts in vaccinated mice compared to non-vaccinated mice, and the brain parasite cysts were significantly reduced (figure 4).
To evaluate the potential protective immunity of the ME49 delta cdpk vaccination, the present application examined the levels of cytokines and antibodies in mouse serum. Toxoplasma IgG levels remained high and stable throughout the vaccination period (fig. 6). Upon testing the IgG subclass, it was found that the levels of IgG1 and IgG2a (based on IgG2 a) were significantly increased in immunized mice compared to non-immunized mice (FIG. 6). These indicate that the ME49 delta cdpk3 vaccination triggered a Th1 and Th2 mixed immune response that was predominantly a Th1 response.
Previous studies have shown that (e.g., romagnani S et al (Annu Rev Immunol (1994) 12:227-57.Epub 1994/01. Doi:10.1146/Annurev. Iy. 12.040194.001303), liu et al (Front Microbiol (2019) 10:813.Epub 2019/05/21.Doi: 10.3389/fmicb.2019.00813)), dupont CD et al (Semin Immunopathol(2012)34(6):793-813.Epub 2012/09/08.doi:10.1007/s00281-012-0339-3)),IFN-γ,IL12,TNF-α are associated with Th1 immune responses, whereas IL-10 is associated with Th2 responses, th 1-biased immune responses are effective in preventing the occurrence of toxoplasma infection. Pro-inflammatory cytokines IFN-. Gamma., IL-12 and TNF-. Alpha.are critical for activating cell-mediated immune responses against toxoplasma infection. IFN-. Gamma.can regulate a variety of intracellular mechanisms to kill parasites and inhibit their replication, are the primary mediators of anti-toxoplasma, IL-12 can stimulate CD4 + and CD8 + T cells and natural killer cells (NK-cell release) and additional IFN-. Gamma.10-gamma.that are required to provide synergistic signals for the killing of the parasite-IFN-. Gamma.10.
30 Days post-vaccination, IFN-gamma, TNF-alpha, IL-12p70 and IL-10 levels were significantly elevated in serum of vaccinated mice compared to unvaccinated mice and returned to normal at 125 days post-vaccination (FIG. 5), indicating that the relative equilibrium levels of cytokines were maintained by the ME49 delta cdpk vaccination. This balance is also demonstrated by high levels of the pro-inflammatory cytokines IFN-gamma, IL-12p70 and TNF-alpha and the anti-inflammatory cytokine IL-10 in the splenocyte stimulation assay (FIG. 7).
The sera of the present application 125 days after mice were vaccinated with the ME49 delta cdpk vaccine were used for active resistance to toxoplasma infection by tail vein injection. Experiments have shown that serum from mice vaccinated with ME49 delta cdpk vaccine can reduce parasite proliferation (figure 8).
In summary, the present ME49 Δ cdpk3 vaccination can elicit cellular and humoral immunity protecting mice from toxoplasmosis, suggesting that the ME49 Δ cdpk3 strain may be a viable attenuated live vaccine candidate against acute and latent toxoplasmosis.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. An attenuated live vaccine of ME49 delta cdpk3 for the prevention of toxoplasmosis, characterized in that: the attenuated live vaccine is an ME49 delta CDPK insect strain constructed by knocking out CDPK3 in the ME49 insect strain through a CRISPR/Cas9 mediated genome editing technology.
2. A method for preparing a live attenuated ME49 delta cdpk3 vaccine for the prevention of toxoplasmosis according to claim 1, wherein: the method comprises the following specific steps:
(1) Construction of a Targeted Cas9 plasmid
The method comprises the steps of knocking out a plasmid pSAG of toxoplasma CRISPR/Cas9, namely, cas9-U6, namely, sgUPRT, designing a target point by using E-CRISPR software, and replacing sgRNA in a plasmid pSAG, namely, cas9-U6, namely, sgUPRT by sgRNA in a Q5 site-directed mutagenesis kit, namely, constructing a plasmid pSAG1, namely, cas9-U6, namely, sgCDPK3; the primer of the plasmid pSAG 1:Cas9-U6: sgUPRT is as follows:
CDPK3-gRNA-F：TGTACCGAGGGTTTTAGAGCTAGAAATAGC；
CDPK3-gRNA-R：CCTCCATGACAACTTGACATCCCCATTTAC；
(2) Construction of homologous donor DNA
CDPK 35 '-and 3' -homology arm fragments will be amplified from genomic DNA of type II ME49 strain, and DHFR-TS sequences will be amplified from pUPRT-DHFR; cloning the three fragments between pUC19 linearization vectors through homologous recombination by using a multi-fragment one-step rapid cloning kit, and constructing a three-fragment donor DNA plasmid; the primers for DHFR-TS are:
DHFR-TS-F：TGTCATTCGATTTTCACCCCC；
DHFR-TS-R：AGTGTGATGACTCCGCAACTGGATCGATCCCCCCGGGCTGC；
(3) Construction of an insect strain of ME49 delta cdpk3
Electroporation transfection of the plasmid pSAG 1:Cas9-U6: sgCDPK3 constructed in the step (1) and the donor DNA fragment constructed in the step (2) into purified ME49 tachyzoites, and screening with pyrimethamine to prepare a mixed clone of ME49 delta cdpk 3;
(4) Screening to obtain ME49 delta cdpk3 monoclonal insect strain
Inoculating individual toxoplasma gondii by fold ratio dilution into 96-well plates plated with human foreskin fibroblasts; detecting positive clones by PCR and immunoblotting after 7-10 days to obtain single cloned insect strains; PCR includes PCR1, PCR2 and PCR3;
the primer of the PCR1 is as follows:
PCR1-F：GCCTAACAAGGATTCGATCAGTAGC；
PCR1-R：TGTCGTGGATTTACCAGTCATGGAC；
the primer of the PCR2 is as follows:
PCR2-F：TGACTCTTCATGTGGCATTTCACAC；
PCR2-R：TACTGTGTTAGGTAGCAAATGTGG；
the primer of the PCR3 is as follows:
PCR3-F：TCGTGCGTCTTCAGGCATGTACATC；
PCR3-R：GAGGTCCTTTCGCTTCCTGAGACTC。
3. An attenuated live vaccine of ME49 delta cdpk3 for use in the prevention of toxoplasmosis according to claim 1, wherein: the immunodose of the ME49 delta cdpk3 tachyzoites was 10 3~106.
4. A live attenuated ME49 delta cdpk vaccine for use in the prevention of toxoplasmosis according to claim 3, characterised in that: the immune dose of the ME49 delta cdpk tachyzoites is 10 3.
5. A live attenuated ME49 delta cdpk vaccine for use in the prevention of toxoplasmosis according to claim 3, characterised in that: the immune dose of the ME49 delta cdpk tachyzoites is 10 5.
6. Use of an attenuated live vaccine of ME49 delta cdpk3 for the prevention of toxoplasmosis according to claim 1 in the preparation of a vaccine for the prevention of toxoplasmosis just like infection.
7. Use of an attenuated live vaccine of ME49 delta cdpk3 for the prevention of toxoplasmosis according to claim 1 in the preparation of a vaccine for the prevention of chronic infection by toxoplasmosis.
8. Use of a live attenuated ME49 delta cdpk3 vaccine for the prevention of toxoplasmosis according to claim 1 in the preparation of a vaccine for the prevention of acute toxoplasmosis infection.
9. Use of an attenuated live vaccine of ME49 delta cdpk3 for the prevention of toxoplasmosis according to claim 1 in the preparation of a vaccine for the prevention of toxoplasmosis by eliciting an immune response in a host cell.
10. Use of an attenuated live vaccine of ME49 delta cdpk3 for the prevention of toxoplasmosis according to claim 1 in the preparation of a vaccine for the prevention of toxoplasmosis infection by eliciting a humoral immune response in a host.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997017082A1 (en) * | 1995-11-09 | 1997-05-15 | Trustees Of Dartmouth College | A vaccine against toxoplasma gondii |
| CA2767664A1 (en) * | 2009-07-09 | 2011-01-13 | Irm Llc | Compounds and compositions for the treatment of parasitic diseases |
| CN104830850A (en) * | 2015-05-08 | 2015-08-12 | 中国农业科学院兰州兽医研究所 | Nucleotide sequence for preventing infection of toxoplasmas and application of nucleotide sequence |
| CN106754380A (en) * | 2016-11-24 | 2017-05-31 | 中国农业科学院兰州兽医研究所 | Weak poisonous insect mutant strain of Infection of Toxoplasma Gondii of one plant of CDPK2 gene delection and preparation method thereof |
| WO2017178755A1 (en) * | 2016-04-12 | 2017-10-19 | Universite Francois Rabelais | Novel peptide structures and use thereof in the treatment of toxoplasmosis |
| CN109999188A (en) * | 2019-03-13 | 2019-07-12 | 中国农业科学院兰州兽医研究所 | For preventing the RH of arch insect infection: Δ NPT1 attenuated live vaccine and its preparation method and application |
| CN110384797A (en) * | 2019-09-02 | 2019-10-29 | 中国农业科学院兰州兽医研究所 | A kind of attenuated live vaccine and its application for preventing arch insect infection |
| CN111939247A (en) * | 2020-08-19 | 2020-11-17 | 安徽医科大学 | Protein mixed vaccine for preventing toxoplasmosis of human and animal |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8282942B2 (en) * | 2007-10-29 | 2012-10-09 | Trustees Of Dartmouth College | Toxoplasma gondii vaccines and uses thereof |
| US9408899B2 (en) * | 2010-11-04 | 2016-08-09 | The University Of Chicago | Vaccines against Toxoplasma gondii |
-
2022
- 2022-03-24 CN CN202210298777.5A patent/CN114569711B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997017082A1 (en) * | 1995-11-09 | 1997-05-15 | Trustees Of Dartmouth College | A vaccine against toxoplasma gondii |
| CA2767664A1 (en) * | 2009-07-09 | 2011-01-13 | Irm Llc | Compounds and compositions for the treatment of parasitic diseases |
| CN104830850A (en) * | 2015-05-08 | 2015-08-12 | 中国农业科学院兰州兽医研究所 | Nucleotide sequence for preventing infection of toxoplasmas and application of nucleotide sequence |
| WO2017178755A1 (en) * | 2016-04-12 | 2017-10-19 | Universite Francois Rabelais | Novel peptide structures and use thereof in the treatment of toxoplasmosis |
| CN106754380A (en) * | 2016-11-24 | 2017-05-31 | 中国农业科学院兰州兽医研究所 | Weak poisonous insect mutant strain of Infection of Toxoplasma Gondii of one plant of CDPK2 gene delection and preparation method thereof |
| CN109999188A (en) * | 2019-03-13 | 2019-07-12 | 中国农业科学院兰州兽医研究所 | For preventing the RH of arch insect infection: Δ NPT1 attenuated live vaccine and its preparation method and application |
| CN110384797A (en) * | 2019-09-02 | 2019-10-29 | 中国农业科学院兰州兽医研究所 | A kind of attenuated live vaccine and its application for preventing arch insect infection |
| CN111939247A (en) * | 2020-08-19 | 2020-11-17 | 安徽医科大学 | Protein mixed vaccine for preventing toxoplasmosis of human and animal |
Non-Patent Citations (11)
| Title |
|---|
| Differences among the three major strains of Toxoplasma gondii and their specific interactions with the infected host;Jeroen P.J. Saeij等;TRENDS in Parasitology;第21卷;476-481 * |
| James M. McCoy等.TgCDPK3 Regulates Calcium-Dependent Egress of Toxoplasma gondii from Host Cells.PLOS PATHOGENS.2012,第8卷e1003066. * |
| Live-attenuated ME49Δcdpk3 strain of Toxoplasma gondii protects against acute and chronic toxoplasmosis;Minmin Wu等;npj Vaccines;第7卷;98 * |
| Minmin Wu等.Live-attenuated ME49Δcdpk3 strain of Toxoplasma gondii protects against acute and chronic toxoplasmosis.npj Vaccines.2022,第7卷98. * |
| Minmin Wu等.Toxoplasma gondii CDPK3 Controls the Intracellular Proliferation of Parasites in Macrophages.Frontiers in Immunology.2022,第13卷905142. * |
| TgCDPK3 Regulates Calcium-Dependent Egress of Toxoplasma gondii from Host Cells;James M. McCoy等;PLOS PATHOGENS;第8卷;e1003066 * |
| Toxoplasma gondii CDPK3 Controls the Intracellular Proliferation of Parasites in Macrophages;Minmin Wu等;Frontiers in Immunology;第13卷;905142 * |
| 动物弓形虫病疫苗研究进展;李双星等;中国兽医科学;20201231(第05期);610-616 * |
| 弓形虫CDPK3蛋白疫苗对实验性小鼠保护性免疫的研究;吴敏敏;中国优秀硕士学位论文全文数据库 医药卫生科技辑;E059-258 * |
| 弓形虫CDPK5基因多克隆抗血清的制备及功能鉴定;钟亮尹等;重庆医学;20160630;第45卷(第16期);2182-2185 * |
| 弓形虫抗原主要功能和免疫特性;王艳华;;中国人兽共患病学报;20201231(第04期);330-339 * |
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