Development status of vector-mediated Eg95 vaccine of Echinococcus granulosus / 中华地方病学杂志

Cystic echinococcosis (CE), caused by the larva of Echinococcus granulosus (Eg), is one of the key parasitic diseases to prevent and control in China. The Eg95 protein is a recognized vaccine candidate molecule, and the development of vector-mediated Eg95 vaccine is one of the current research hotspots in CE. This article summarizes the development status of Eg95 vaccine such as vaccinia virus, orf virus, goatpox virus, rabies virus, Salmonella typhimurium, Bacille Calmette-Guerin, Agrobacterium tumefaciens and Bifidobacteria bifidum, in order to provide valuable information for the immune prevention of CE.

Variability of the EG95 antigen-coding gene of Echinococcus granulosus in animal and human origin: implications for vaccine development

In the present study, the genetic variability of the EG95 protein-coding gene in several animal and human isolates of Echinococcus granulosus was investigated. A total of 24 isolates collected from cattle, buffalo, sheep, goat, dog and man were amplified by Eg95-coding gene-specific primers. From the generated sequence information, a conceptual amino acid sequence was deduced. Phylogenetically, the Eg95 coding gene belongs to the Eg95-1/Eg95-2/Eg95-3/Eg95-4 cluster. Further confirmation on the maximum composite likelihood analysis revealed that the overall transition/transversion bias was 2.913. This finding indicated thatthere is bias towards transitional and transversional substitution. Using artificial neural networks, a B-cell epitope was predicted on primary sequence information. Stretches of amino acid residues varied between animal and human isolates when hydrophobicity was considered. Flexibility also varied between larval and adult stages of the organism. This observation is important to develop vaccines. However, cytotoxic T-lymphocyte epitopes on primary sequence data remained constant in all isolates. In this study, agretope identification started with hydrophobic amino acids. Amino acids with the same physico-chemical properties were present in the middle. The conformational propensity of the Eg95-coding gene of 156 amino acid residues had α-turns and β-turns, and α-amphipathic regions up to 129, 138–156 and 151–155 residues, respectively. The results indicated potential T-cell antigenic sites. The overall Tajima’s D value was negative (−2.404165), indicative of negative selection pressure.

Genetic diversity and phylogenetic analysis of EG95 sequences of Echinococcus granulosus: Implications for EG95 vaccine application

OBJECTIVE@#To analyse the genetic variability of EG95 sequences and provide guidance for EG95 vaccine application against Echinococcus granulosus (E. granulosus).@*METHODS@#We analysed EG95 polymorphism by collecting total 97 different E. granulosus isolates from 12 different host species that originated from 10 different countries. Multiple sequence alignments and the homology were performed by Lasergene 1 (DNASTAR Inc., Madison, WI), and the phylogenetic analysis was performed by using MEGA5.1 (CEMI, Tempe, AZ, USA). In addition, linear and conformational epitopes were analysed, including secondary structure, NXT/S glycosylation, fibronectin type III (FnIII) domain and glycosylphosphatidylinositol anchor signal (GPI-anchor). The secondary structure was predicted by PSIPRED method.@*RESULTS@#Our results indicated that most isolates overall shared 72.6-100% identity in EG95 gene sequence with the published standard EG95 sequence, X90928. However, EG95 gene indeed has polymorphism in different isolates. Phylogenetic analysis showed that different isolates could be divided into three subgroups. Subgroup 1 contained 87 isolates while Subgroup 2 and Subgroup 3 consisted of 3 and 7 isolates, respectively. Four sequences cloned from oncosphere shared a high identity with the parental sequence of the current vaccine, X90928, and they belonged to Subgroup 1. However, in comparison to X90928, several amino acid mutations occurred in most isolates besides oncosphere, which potentially altered the immunodominant linear epitopes, glycosylation sites and secondary structures in EG95 genes. All these variations might change their previous antigenicity and thereby affecting the efficacy of current EG95 vaccine.@*CONCLUSIONS@#This study reveals the genetic variability of EG95 sequences in different E. granulosus isolates, and proposed that more vaccination trials would be needed to test the effectiveness of current EG95 vaccine against distinct isolates in different countries.

Genetic diversity and phylogenetic analysis of EG95 sequences of Echinococcus granulosus: Implications for EG95 vaccine application

Objective To analyse the genetic variability of EG95 sequences and provide guidance for EG95 vaccine application against Echinococcus granulosus (E. granulosus). Methods We analysed EG95 polymorphism by collecting total 97 different E. granulosus isolates from 12 different host species that originated from 10 different countries. Multiple sequence alignments and the homology were performed by Lasergene 1 (DNASTAR Inc., Madison, WI), and the phylogenetic analysis was performed by using MEGA5.1 (CEMI, Tempe, AZ, USA). In addition, linear and conformational epitopes were analysed, including secondary structure, NXT/S glycosylation, fibronectin type III (FnIII) domain and glycosylphosphatidylinositol anchor signal (GPI-anchor). The secondary structure was predicted by PSIPRED method. Results Our results indicated that most isolates overall shared 72.6–100% identity in EG95 gene sequence with the published standard EG95 sequence, X90928. However, EG95 gene indeed has polymorphism in different isolates. Phylogenetic analysis showed that different isolates could be divided into three subgroups. Subgroup 1 contained 87 isolates while Subgroup 2 and Subgroup 3 consisted of 3 and 7 isolates, respectively. Four sequences cloned from oncosphere shared a high identity with the parental sequence of the current vaccine, X90928, and they belonged to Subgroup 1. However, in comparison to X90928, several amino acid mutations occurred in most isolates besides oncosphere, which potentially altered the immunodominant linear epitopes, glycosylation sites and secondary structures in EG95 genes. All these variations might change their previous antigenicity and thereby affecting the efficacy of current EG95 vaccine. Conclusions This study reveals the genetic variability of EG95 sequences in different E. granulosus isolates, and proposed that more vaccination trials would be needed to test the effectiveness of current EG95 vaccine against distinct isolates in different countries.

Immunogenicity of tandem expression recombinant EG95s protein of Echinococcus g ranulosus / 中国人兽共患病学报

In order to analyze the immunogenicity of the recombinant EG95s protein ,the recombinant plasmids of pET-1EG95s ,pET-2EG95s and pET-3EG95s which containing respectively 1 ,2 ,and 3 copies EG95s were induced to express HIS-1EG95s ,HIS-2EG95s and HIS-3EG95s ,and then the proteins were purified and identified by western-blotting .The same im-mune process was used ,and 8 weeks-old BALB/c mice were immunized ,then its immunogenicity was analyzed by detecting an-tibody levels in mice by indirect ELISA method .Results showed that for recombinant EG95s proteins after transformation , HIS-1EG95s ,HIS-2EG95s ,and HIS-3EG95s also retained immunogenicity and could induce specific antibodies in mice .One week's late after the first immunization with HIS-1EG95s ,the antibody level of was significantly higher than HIS-2EG95s and HIS-3EG95s .But began from 2 weeks after immunization ,the antibody level of HIS-3EG95s was always higher than that of HIS-1EG95s group during the period of the immune .Both the final antibody titers after immunization of HIS-1EG95s and HIS-2EG95s groups was 1∶819 200 ,while HIS-3EG95s group was 1∶163 840 0 .HIS-1EG95s ,HIS-2EG95s and HIS-3EG95s all induced IFN-γin immune mice ,but the difference was not significant .The HIS-1EG95s showed lower response to Echinococ-cus granulosus positive serum than HIS-2EG95s and HIS-3EG95s .It’s indicated that the HIS-1EG95s and HIS-3EG95s also had good immunogenicity .HIS-3EG95s make recombinant protein immunic effects more lasting ,and benefit to generate more long-lasting protective immunity .This study provides the scientific basis for the immunization of echinococcosis (hydatidosis) .

Recombinant protein of Echinococcus g ranulosus EG95 / 中国人兽共患病学报

Echinococcosis (hydatid disease) caused by the metacestodes of Echinococcus granulosus tapeworm is a seri-ous zoonotic infection ,which leads to a large amount of economic loss in human and animals .It needs to be prevented urgently . The EG95 protein is highly conserved and crucial for survival and development of E .granulosus in the host ,which means that it is one of the potential candidate antigens for vaccines characterized so far .Great effort has been made to construct and ex-press the recombinant EG95 (rEG95) gene in various kinds of expression systems in order to obtain an efficient defined antigen . This review will summarize research progress on expression of rEG95 in different vector systems .

Induction of protective T-helper 1 immune responses against Echinococcus granulosus in mice by a multi-T-cell epitope antigen based on five proteins

In this study, we designed an experiment to predict a potential immunodominant T-cell epitope and evaluate the protectivity of this antigen in immunised mice. The T-cell epitopes of the candidate proteins (EgGST, EgA31, Eg95, EgTrp and P14-3-3) were detected using available web-based databases. The synthesised DNA was subcloned into the pET41a+ vector and expressed in Escherichia coli as a fusion to glutathione-S-transferase protein (GST). The resulting chimeric protein was then purified by affinity chromatography. Twenty female C57BL/6 mice were immunised with the antigen emulsified in Freund's adjuvant. Mouse splenocytes were then cultured in Dulbecco's Modified Eagle's Medium in the presence of the antigen. The production of interferon-γ was significantly higher in the immunised mice than in the control mice (> 1,300 pg/mL), but interleukin (IL)-10 and IL-4 production was not statistically different between the two groups. In a challenge study in which mice were infected with 500 live protoscolices, a high protectivity level (99.6%) was demonstrated in immunised BALB/C mice compared to the findings in the control groups [GST and adjuvant (Adj) ]. These results demonstrate the successful application of the predicted T-cell epitope in designing a vaccine against Echinococcus granulosus in a mouse model.

Changes of splenocyte subsets in mice immunized with recombinant Bb-Eg95-EgA31 vaccine against Echinococcus granulosus / 中国人兽共患病学报

To investigate the weight reduction of hydatid cyst and level of splenocyte subsets in mice immunized with recombinant Bifidobacteria bifidum (Bb) Bb-Eg95-EgA31 vaccine of Echinococcus granulosus (Eg) and challenged with Eg protoscoleces,BALB/c mice were immunized with the vaccine subcutaneously,intramuscularly,intranasally or orally.Then the mice were challenged with 50 Eg protoscoleces on 8~(th) week after vaccination and killed on 25~(th) week after infection.The weight of hydatid cyst was measured and the reduction of the weight was calculated.The spleens were collected for splenocytes preparation.CD_4~+ and CD_8~+ T cells were determined by flow cytometry(FCM),and the blank vector,Bb or MRS were set as control.The hydatid cyst weight was reduced by 45.33%,41.33%,70.67% and 62.67% respectively for the 4 immunization groups.Number of CD_4~+ and CD_8~+ subsets in the immunization groups was more than that in the control group.CD_4~+ and the ratio of CD_4~+/CD_8~+ in the intranasal or oral immunization group was much higher than that in the subcutaneous or intramuscular immunization group.It's concluded that CD_4~+ and CD_8~+T cells may play an important role in the protection induced by recombinant Eg95-EgA31 vaccine of Eg.In addition,intranasal and oral vaccinations could be the better ways for immunization.

Construction and expression efficiency of the recombinant plasmid pBI-Eg95 of Echinococcus granulosus in the agrobacterium tumefaciens LBA4404 strain / 重庆医科大学学报

Objective:To construct the recombinant plasmid pBI-Eg95 of E.granulosus,and to study its expression efficiency in the Agrobacterium tumefaciens(At) LBA4404 strain.Methods:Total RNA was extracted from hydatid cyst protoscoleces shattered by supersound.The Eg95 coding gene was amplified by RT-PCR,and then was cloned into the plant expression vector pBI121 to construct the recombinant plasmid pBI-Eg95.The recombinant plasmid was electroporated into At(LBA4404) strain.The recombinant At(rAt) was induced by IPTG for 0,1,3,5,7,9,11or13 hour,respectively.The expression product was identified by SDS-PAGE and Western blot assay.Results:The 471bp Eg95 coding gene was successfully amplified,and the recombinant pBI-Eg95 plasmid was successfully constructed,which was confirmed by restriction endonuclease digestion and PCR.The recombinant plasmid pBI-Eg95 was expressed successfully in the rAt,and the expression product with an approximately Mr of 16.5 kD could be detected by sera from mice infected by E.granulosus,and the expression efficiency was about 20 percent of the total bacterial protein.Conclusion:The recombinant plasmid could provide the basis for further research of the transgenic plant vaccine of E.granulosus.

The reduced apoptosis of spleen cells in infected mice by immunization with recombinant BCG-Eg95 vaccine against Echinococcus granulosus / 中国免疫学杂志

Objective:To investigate apoptosis of spleen cells in infected mice by immunization with recombinant BCG-Eg95 vaccine of Echinococcus granulosus(Eg) and against challenge with Eg protoscoleces.Methods:BALB/c mice were vaccinated with the vaccine subcutaneously,intranasally,orally and intramuscularly respectively.The mice were then challenged with Eg protoscolexes at 8w of vaccination and sacrificed in 18w of infection to get spleen.Spleen cells were separated to measure apoptotic rate by FACsort with BCG and PBS served as control.Results:Apoptotic rate in the immunization group was lower than that in the control.Apoptotic rates in the oral or intramuscular group were significantly lower than that in the subcutaneous or intranasal group.Conclusion:Apoptosis of spleen cells in mice may be induced by infection with hydatid cyst,but is inhibited by immunization with rBCG-Eg95 vaccine.Oral or intramuscular vaccination may be the good regimen.

Construction of the Recombinant Secretion Type BCG-Eg95 Vaccine of Echinococcus granulosus / 中国寄生虫学与寄生虫病杂志

Objective To construct the recombinant secretion type BCG-Eg95 vaccine of Echinococcus granulosus (rsBCG-Eg95). Methods BCG-Ag85B signal sequence with 117 bp and Eg95 gene with 471 bp were amplified from the genome of BCG and pGEX-4T-Eg95 by PCR,respectively. BCG-Ag85B signal coding gene and Eg95 gene were cloned into E. coli-BCG shuttle-vector pMV261 to get the recombinant plasmid pSMEg95,which was confirmed by restriction endonuclease digestion,PCR amplification and gene sequencing. These recombinant plasmids were introduced into BCG by electroporation for the construction of rsBCG-Eg95 vaccine. The rsBCG-Eg95 positive clones were screened by Kan+ and identified by PCR amplification. Results BCG-Ag85B signal sequence coding gene and Eg95 coding gene were successfully cloned into pMV261,which was confirmed by restriction endonuclease digestion,PCR amplification and sequencing of the plasmid pSMEg95. The plasmids were introduced into BCG and confirmed as the recombinant secreting BCG-Eg95 vaccine of E. granulosus (rsBCG-Eg95). Conclusion The recombinant secretion type BCG-Eg95 vaccine (rsBCG-Eg95) of E. granulosus with BCG-Ag85B signal sequence and Eg95 gene has been constructed.

Change of Splenocyte Lymphokines in Mice Induced by Recombinant BCG-Eg95 Vaccine against Echinococcus granulosus / 中国寄生虫学与寄生虫病杂志

Objective To investigate the reduction of hydatid cyst weight and change of splenocyte lymphokines in mice immunized with recombinant BCG-Eg95 vaccine of Echinococcus granulosus(Eg). Methods BALB/C mice were subcutaneously, intranasally, orally and intramuscularly vaccinated respectively, with BCG and PBS served as controls. The mice were challenged with Eg protoscolices 8 weeks after vaccination and sacrificed in 18 weeks after infection. The weight of hydatid cyst was measured and reduction of the weight was obtained, spleens were used to separate splenocytes which were cultured under stimulation with EgAg or ConA. The supernatant was collected to measure the level of IL-2、IFN-?、TNF- and IL-4 by ELISA. Results The hydatid cyst weight reduced by 45.77%, 18.20%, 88.05% and 92.46% respectively in the 4 immunization groups. In comparison with PBS control, the level of IL-2、IFN-?and TNF-?was (30.0?0) pg/ml, (65.0?0) pg/ml and (425.0?10.7) pg/ml respectively in the intramuscular group with a significant increase, but that of IL-4 decreased, with a value of (10.0?0) pg/ml. Conclusion The recombinant BCG-Eg95 vaccination induces Th1 response in mice against challenge infection.

Construction and identification of the recombinant pCD-Eg95 plasmid of Echinococcus granulosus / 重庆医科大学学报

Objective: To construct and identify the recombinant pCD-Eg95 plasmid of Echinococcus granulosus. Methods: Total RNA was extracted from hydatid cyst protoscoleces shattered by supersound. The Eg95 coding gene was amplified by RT-PCR, and then was cloned into the eukaryotic expression vector pCDNA3.1 to construct the recombinant pCD-Eg95 plasmid. The recombinant plasmid was electroporated into E. coliBL21(DE3) strain. Results: The 471 bp Eg95 coding gene was successfully amplified, and the recombinant pCD-Eg95 plasmid was successfully constructed, which was confirmed by restriction endonuclease digestion and PCR. Conclusion: The results could provide the basis for the further research of the nucleic acid vaccine of Echinococcus granulosus.

Dynamic observation on IgG,its subclass and IgE in sera of mice immunized by recombinant BCG-Eg95 vaccine of Echinococcus granulosus / 重庆医科大学学报

Objective:To dynamically observe changes of IgG,its subclass and IgE in sera of mice immunized by recombinant BCG-Eg95 vaccine of Echinococcus granulosus(Eg).Methods:BALB/C mice were intranasally or orally vaccinated and killed on 0、2、4、6、8、10、12、14、16 and 18w of immunization,with 4 in each killing.Sera were gathered from the eyeball to measure IgG, its subclass and IgE by routine ELISA,with PBS as control.Results:In the intranasal group,levels of IgG、IgG2a and IgG2b increased obviously on 2~18w,and reached the highest level on 10、4 and 6w respectively;while levels of IgG1、IgG3 and IgE decreased remarkably on 2~18w,and came to the lowest level on 8、12 and 10~12w respectively.In the oral group,levels of IgG、IgG2a and IgG2b rose remakably on 2~18w,and came to the peak value on 14、8 and 8w respectively;while levels of IgG1、IgG3 and IgE decreased on 2~18w,and reached the lowest level on 8、6 and 16~18w respectively.Conclusion:TH1 response could be induced in mice immunized by recombinant BCG-Eg95 vaccine of Echinococcus granulosus in early immunization.

Dynamic observation on Th1/Th2 type immune response in mice immunized with recombinant Bb-Eg95-EgA31 vaccine of Echinococcus granulosus / 重庆医科大学学报

Objective:To dynamically observe changes of Th1/Th2 type immune responses in mice immunized with recombinant Bifidobacteria bifidum(Bb)-Eg95-EgA31 vaccine of Echinococcus granulosus(Eg).Methods:BALB/c mice were vaccinated by 5?108 colony forming unit(CFU) orally and 5?105CFU intranasally respectively.Mice were killed on week 2,4,6,8,10,12,14,16,18 and 20 after immunization respectively,sera were collected to measure the levels of IgG,its subclasses and IgE by enzyme linked immunosorbent assay(ELISA);Spleens were removed to determine the levels of IFN-?,IL-12,TNF-?and IL-10 of splenocyte culture supernatant by ELISA.Results:Oral immunization mice showed higher levels of IgG,IgG2a,IgG2b,IgG1,IgG3 and IgE on the 8～10th week,2～20th week,2～20th week,4～8th week,6～12th week and 10th week respectively,while IFN-?,IL-12,TNF-?and IL-10 on the 2～16th week,2～12th week,2～6th week and 4～12th week post vaccination respectively;Intranasal immunization mice showed higher levels of IgG,IgG2a,IgG2b,IgG1,IgG3 and IgE during the 4～10th week,4～20th week,2～20th week,2～12th week,4～12th week and 10～12th week respectively,while IFN-?,IL-12,TNF-?and IL-10 on the 2～8th week,2～12th week,2～8th week and 6～16th week post vaccination respectively.Conclusion:Mixed Th1/Th2 type immune responses may be induced in mice immunized with the recombinant Bb-Eg95-EgA31 vaccine of Echinococcus granulosus.Oral and intranasal inoculation are two better immune routes,and the former is better than the latter.