Screening And Immunogenicity Analysis Of Neospora Caninum Host Cell Binding Proteins

Neosporosis, a newly discovered intracellular protozoan disease caused by Neosporacaninum, can cause abortion or stillbirth in pregnant domestic animal, the neonatus’ movementdisorders or neurological diseases. Neosporosis is worldwide and harmful. Though it has led togreat economic loss, no effective vaccine is available for prevention of neosporosis. To search foreffective vaccine antigen gene of N.caninum, a T7phage display library was constructed usingT7select10-3b vector and the library was screened using Vero cells. We constructed proteinvaccine and DNA vaccine, then evaluated the protective efficacy by individual immunity orcombined immunity. It will be useful for the prevention of neosporosis.The construction and screening of the T7phage display library of tachyzoite of Neosporacaninum. A T7phage display library was constructed using T7select10-3b vector. The titer of theamplified library was5.24×1012pfu/mL and the storage capacity was8.38×1012with arecombination rate of100%. After five rounds of selection using Vero cells, nine positive cloneswere obtained, including one GRA7gene, five ribosome genes and three hypothetical proteingenes identified by sequencing and blast analysis. One of the hypothetical protein and the GRA7were chosen in the present study to evaluate their protective efficacy as vaccine. The hypotheticalprotein, which encoded a protein of78KDa and had80%homology with the acylglycerollipase-like gene of Toxoplasma gondii ME49(XP₀02370319.1, was named as NcP78in thispaper.Protein vaccines were constructed for NcP78using the prokaryotic expression vectorpGEX-4T-1and for GRA7using pET-28a. The expressions of NcP78and GRA7genes wereidentified by SDS-PAGE and Western Blot. DNA vaccines for these two genes were alsoconstructed using the eukaryotic expression vector pVAX1and the expressions were identified byWestern Blot and IFA.The protein vaccine, DNA vaccine and DNA/protein vaccines of NcP78as well as GRA7were then used to immunize BALB/c mice and the levels of IgG, IgG1, IgG2a, IL-4and IFN-γ inserum of immunized mice were measured. The results indicated these vaccines could elicit both Th1immune response with elevated levels of IgG2a and IL-4, and Th2immune response withelevated levels of IgG1and IFN-γ. Moreover, the levels of IgG were also elevatedThe inhibitory effects on invasion of the immune serum were measured in vitro and theresults indicated the inhibition rate with immune serum were40%for NcP78protein vaccine and55%for GRA7protein vaccine,40%for NcP78DNA vaccine and50%for GRA7DNA vaccine,and40%for NcP78DNA/Protein vaccine and70%for GRA7DNA/Protein vaccine, whichindicated that the immune serum could inhibit the invasion of N. Caninum, and the NcP78andGRA7were host cell binding proteins of Neospora caninum.To evaluate the protective efficacy, dams with10days pregnancy were infected with N.caninum and the survival rate and the brain parasite load were measured.（1）. NcP78and GRA7protein vaccines could also improve the survival rates of mice infected with N. caninum （60%inNcP78group and70%in GRA7group in infected pregnant dams compared to40%in the controlgroup, and68.4%in NcP78group and73.7%in GRA7group in offspring compared to42%in thecontrol group）. The vaccine could also reduce the brain parasite load as shown by the results ofReal-time PCR （the number of N.caninum per20ng brain tissue of dams were356.66±14.94inNcP78group and259.59±19.02in GRA7group compared to521.6±7.1in the control group, andthe number in offspring was64.53±4.47in NcP78group and48.83±2.76in GRA7groupcompared to106.78±7.67in the control group）. Moreover, these two proteins could also relievethe symptoms of weight lose, miscarriages and reduced litter size. The results indicated thatNcP78and GRA7had protective efficacies against neosporosis, and the protective efficacy ofGRA7was better than that of NcP78when used as the protein vaccines.（2）. The protective efficacies of NcP78and GRA7DNA vaccines indicated the survival ratesof mice infected with N. caninum were also improved （60%in both the NcP78vaccine group andGRA7vaccine group compared to40%in the control group in dams,53.8%in NcP78vaccinegroup and58.3%in GRA7vaccine group compared to42%in the control group in offspring）. Thevaccine could reduce the brain parasite load as shown by the Real-time PCR results （the numberof N.caninum per20ng brain tissue of dams was378.8±27.95in NcP78vaccine group and287.17±32.51in GRA7group compared to521.6±7.1in control group, and the number inoffspring was58.37±5.77in NcP78group and45.39±6.73in GRA7vaccine group compared to 86.78±7.67in control group）. The results indicated that the protective efficacy of GRA7was betterthan that of NcP78as the DNA vaccines.（3）. The protective efficacies of NcP78and GRA7DNA/Protein indicated these vaccinescould also improve the survival rates of mice infected with N. caninum （60%in NcP78vaccinegroup and70%in GRA7vaccine group compared to40%in control group in dams,68.4%inNcP78vaccine group and66.7%in GRA7vaccine group compared to42%in control group inoffspring）. The brain parasite loads were also reduced as detected by Real-time PCR （the numberof N.caninum per20ng brain tissue of dams was338.8.45±10.09in NcP78vaccine group and308.99±53.47in GRA7vaccine group compared to521.6±7.1in control group, and the number inoffspring was69.39±4.82in NcP78vaccine group and52.27±3.74in GRA7vaccine groupcompared to86.78±7.67in control group）. The results indicated that the DNA/Proteinimmunization with NcP78and GRA7could provide protective efficacies against neosporosis, andthe protective efficacy of GRA7was better than that of NcP78as the DNA/Protein vaccine.However, the protective efficacies of the DNA/Protein vaccines were not as good as the subunitvaccines or the DNA vaccines for both genes. Results in the present study suggest that NcP78andGRA7could serve as effective vaccine candidates against N. caninum.