EffectofCo-expressingChickenIL-2,IL-18,IFN-γ Or Built CpG DNA In The Plasmid Backbone As Adjuvants On DNA Vaccines Against H5 Subtype Avian Influenza Delivered By Attenuated Salmonella And Production Of Anti-chicken CD4 And CD8 Monoclonal Antibodies

H5 subtype of highly pathogenic avian influenza (HPAI), is a disease of poultry with high morbidity and mortality caused by specific influenza virus type A, and also threats to human health. HPAI is classified as one of the list A animal disease by OIE and China Ministry of Agriculture. Vaccination is frequently used to prevent and control the disease in some countries when the disease spreads widely. Current inactivated whole viral vaccines against HAPI in poultry have been proved to provide adequate protection against the disease. However, there are several disadvantages for these vaccines. Most of all, the antibodies induced by the vaccination interfere with the serological surveillance. A number of studies have shown that DNA vaccines can effectively induce cellular, humoral and mucosal immune responses against their encoded antigens. An HA DNA vaccine against HPAI only induces specific antibodies to HA, so the vaccine will not interfere with the serological surveillance. It can also overcome the interference of maternal antibodies. Although significant progress has been achieved in delivering HA DNA vaccines, immunogenicity of these DNA vaccines remains relatively low in chickens compared to inactivated whole viral vaccines. Several approaches have been used to influence not only the magnitude but also the type of immune response induced by DNA vaccines. Among these, the use of CpG DNA, cytokines, chemokines, costimulatory molecules, HSP, and c3d genes as adjuvants to DNA vaccines have been found to be important determinants of immunogenicity. One of main purposes of this paper was to evaluate the protective efficacy of HA DNA vaccines delivered by attenuated Salmonella typhimurium strain SL7207 strain with the co-expression of chicken IL-2, IL-18, IFN- γ, or built-in CpG DNA as adjuvant.In many studies on DNA vaccines against HPAI where no antibody was found, the vaccinated chickens were still partially to fully protected from challenge, indicating that an adaptive immune response other than humoral (presumably cellular immunity) was induced by the vaccination. There are no studies to date directly addressing the role of CD4+ or CD8+ T lymphocytes in the protection. To understand which subtypes of T cells were involved, It is necessary to develop mouse anti-chicken CD4 and CD8 mAbs.Chicken CD3, CD4 and CD8 are transmembrane glycoproteins on T lymphocytes, and they are also important cell surface markers of these cells. During antigen recognition, CD3 complex are involved in signal transduction, which ultimately leads to T-lymphocyte activation after the initial recognition steps. CD4 and CD8 molecules act as co-receptors for TCR in recognition of antigens. T cells that express CD4 recognize antigens in context with MHC class II, whereas T cells that express CD 8 recognize antigens in the context with MHC class I. Whether differences of the CD molecules at extracellular domain in chicken has any role in extending the antigen recognition capacity of TCR or in evading viral infections remain to be seen. There are rich resources of local breeds in China, and they are useful materials to study the polymorphism of chicken CD3, CD4 and CD8. In this study, chicken CD3, CD4 and CD8 a cDNA of Leghorn, ISA, and Chinese local breeds would be cloned and sequenced, and the polymorphism of these sequences would also be compared. These materials are useful to study the structures and functions of these molecules and to guild the applications of monoclonal antibodies against chicken CD3, CD4 or CD8 molecules.Anti-chicken CD3, CD4 or CD8 mAbs are useful tools to study the functions of T lymphocytes expressing CD3, CD4 or CD8 molecules and cellular immunity in chicken. It is difficult to obtain these mAbs using whole lymphocytes as immunogen. In this study, we attempt to prepare monoclonal antibodies against chicken CD4 and CD8 molecules based on DNA immunization strategy.1. Construction of expression vectors which co-expressing AIV HA with chicken IL-2, IL-18 or IFN- γ, or incorporating CpG DNA and HA into pVAXlChicken IL-2, IL-...