Contribution of Chinese Pekin duck complement component C3d-P29 repeats to enhancement of Th2-biased immune responses against NDV F gene induced by DNA immunization.

BACKGROUND AND AIM: C3d, a split product of C3, interacts with its receptor (CR2 or CD21) on B cells and follicular dendritic cells (FDCs) and is crucial for induction and maintenance of a normal humoral immune response. This fragment of complement protein C3 (C3d) has also been shown to enhance B cell responses when complexed with antigen and C3d fusion increased Th2-biased immune response by inducing IL-4 production. MATERIALS AND METHODS: The gene fragment coding for Chinese Pekin duck (Anas platyrhynchos) C3d gene (duC3d) was cloned and expressed as a component of fusion proteins destined for use in in vitro experiments. Two, four and six copies of CR2-binding domain duC3d-P29 were fused, respectively, to truncated Newcastle disease virus (NDV) F gene encoding soluble glycoprotein F in pcDNA3.1.All recombinant proteins were analyzed by SDS-PAGE and Western immunoblot. BALB/c mice were, respectively, immunized with recombinant plasmids, blank vector, and inactivated vaccine. RESULTS: The result of immunogenicity detections of The IL-4 level for F-C3d-P29.6 DNA immunization approached that for the inactivated vaccine. Compared to C3d-P29.6, C3d-P29.4 enhanced F DNA immunogenicity to a lesser extent. Furthermore, C3d-P29.n fusion increased Th2-biased immune response by inducing IL-4 production. CONCLUSION: We demonstrated that C3d-P29 could enhance immunogenicity by directing Th1-biased to a balanced and more effective Th1/Th2 response. The expression of the duck C3d fusion proteins in this study which was the first reported, and the detections of the cytokine level for F-C3d-P29.n in DNA immunization using the BALB/c mice as the model animal, will provide the basis for immunization trials in chicken or other poultry, studies of receptor binding and cell activation of animal lymphocytes, and investigations of new types of vaccine, including genetic recombinant and DNA vaccines for the future against relevant pathogens.

The structure, genetic polymorphisms, expression and biological functions of complement receptor type 1 (CR1/CD35).

The complement system is comprised of soluble and cell surface associated proteins that recognize exogenous, altered, or potentially harmful endogenous ligands. In recent years, the complement system--particularly component C3 and its receptors--have been demonstrated to be a key link between innate and adaptive immunity. Complement receptor type 1 (CR1), the receptor for C3b/C4b complement peptides, has emerged as a molecule of immense interest in gaining insight to the susceptibility, pathophysiology, diagnosis, prognosis and therapy of such diseases. In this review, we wish to briefly bring forth the structure, genetic polymorphisms, expression and biological functions of CR1.

Fusion of Ross chicken complement component C3d-P29 repeats with a chicken viral Newcastle disease virus F gene enhances immune responses following DNA immunization.

C3d, a split product of C3, interacts with its receptor (CR2 or CD21) on B cells and follicular dendritic cells (FDCs) and is crucial for induction and maintenance of a normal humoral immune response. The gene fragment coding for Ross chicken C3d gene (chC3d) was cloned and expressed as a component of fusion proteins destined for its application in the vaccine study of chicken, and for in vitro experiments. Three potential vaccine construct units were engineered to contain two, four, and six copies of chC3d-P29 coding gene, which was linked to the F gene of Newcastle disease virus (NDV), an economically important pathogen of chicken that is classified as a list A contagious disease of poultry by the Office International des Epizooties (OIE). The cloned chC3d protein and different repeats of C3d-P29 proteins that contained the F gene of NDV (C3d-P29.n-F) was generated separately in Escherichia coli and CEF cells with the help of expression vectors. All recombinant proteins were analyzed by SDS-PAGE and Western blot. The result of immunogenicity detection of different numbers of repeats of C3d-P29.n-F revealed that C3d-P29 could enhance immunogenicity. Six or more repeats of C3d-P29 may be necessary for efficient enhancement of antigen-specific immune responses.

Cloning of a gene fragment encoding chicken complement component C3d with expression and immunogenicity of Newcastle disease virus F gene-C3d fusion protein.

The gene fragment coding for the complement protein C3 (chC3d) from Arbor Acres (AA) chickens was cloned and expressed as a fusion protein for its application in the vaccine study of chicken, and for in vitro experiments. The chC3d fragment strengthened B-cell responses when complexed with antigen. Three potential vaccine construct units were engineered to contain two, four and six copies of the chC3d-P29 coding gene linked to the F gene of Newcastle disease virus, an economically important pathogen of chicken that is classified as a list A contagious disease of poultry by the Office International des Epizooties. Recombinant chC3d protein and C3d-P29 proteins that contained the F gene of Newcastle disease virus (C3d-F-P29.n) were generated separately in Escherichia coli and chick embryo fibroblast cells with the help of expression vectors. All recombinant proteins were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blotting. Analysis of the immunogenicity of different repeats of C3d-F-P29.n revealed that C3d-P29 had an enhancing effect on the immunogenicity of antigens, and that six or more repeats of C3d-P29 may be necessary for efficient enhancement of antigen-specific immune responses. To date, published research into the adjuvant activities of C3d has been limited to experiments in mice, rabbits and cattle. The adjuvant properties of C3d have not been assessed in poultry using homologous C3d in association with antigens relevant to the target species. The chicken C3d fusion proteins detailed in this study are the first reported and they provide a basis for immunization trials in chicken, studies of receptor binding and cell activation of chicken lymphocytes, and investigations of new types of vaccine, including genetic recombinant and DNA vaccines against various pathogens.

Immunogenicity of a chicken viral Newcastle disease virus F gene-C3d fusion protein containing a chicken homologue of C3d.

The gene fragment coding for Arbor Acres (AA) chicken C3d gene (chC3d) was cloned and expressed as a component of fusion proteins for its potential use as a vaccine for chickens and for in vitro experiments. This fragment of complement protein C3 (C3d) has been shown to strengthen B-cell responses when complexed with antigen. Three potential vaccine constructs were engineered to contain two, four, and six copies of chC3d-P29 coding gene, which was linked to the F gene of Newcastle disease virus (NDV), an economically important pathogen of chicken that is classified as a class A contagious disease of poultry by the Office international des épizooties (OIE). The cloned chC3d protein and different repeats of C3d-P29 proteins contained in the F gene of NDV (C3d-F-P29.n) were generated separately in Escherichia coli and CEF cells with the help of expression vectors. All recombinant proteins were analyzed by SDS-PAGE and Western blotting. The results with different repeats of C3d-F-P29.n revealed that C3d-P29 could enhance immunogenicity. Six or more repeats of C3d-P29 may be necessary for efficient enhancement of antigen-specific immune responses. To date, published research into the adjuvant activities of C3d has been limited to experiments in mice, rabbits, and cattle, and adjuvant properties of C3d have not been assessed in poultry using homologous C3d in association with antigens relevant to the target species. The chicken C3d fusion proteins detailed in this study are the first reports and they provide a basis for immunization trials in chicken, studies of receptor binding and cell activation of chicken lymphocytes, and investigations of new types of vaccines, including genetic recombinant and DNA vaccines for future use against chicken pathogens.

[The immune enhancement of the C3 to the Escherichia coli vaccine].

C3b was separated and purified from the SPF chicken serum. It was linked with E. coli antigen by the glutaral. 11 days aged SPF chicken were immunized by the complex antigen and the chickens of control group were immunised by the FCA- E. coli antigen . They were boosted at the age of 18 day. The immune response was monitored by an enzyme-linked immunosorbent assay(ELISA) for anti-E. coli anitibody. The ELISA results indicated that during the early several weeks, IgG titers elicited by FCA (FCA-E. coli) were higher than those elicited by C3 (C3b-E. coli), but decreased rapidly after a peak around the end of 4th week from being immunized. Chickens immunized with C3b always gave increased response, and the IgG titers were equal to that of FCA at the end of 5th week from being immunized and then higher and higher than that of FCA. Thus the adjuvant effect of C3b is different from that of FCA, it could induce production of memory cell and make the antigens stimulate immune cells consistently and stably.