Mechanism Of Thymus Lesions Induced By Highly Pathogenic PRRSV Infection And Regulation Of Its Anti-idiotypic Antibodies

Porcine reproductive and respiratory syndrome virus(PRRSV) is the causative agent of porcine reproductive and respiratory syndrome(PRRS) that can result in reproductive failure in pregnant sows andrespiratory complications in piglets. PRRS was first described in North America in 1987 and confirmed in China in 1996.PRRSV is a single-stranded, positive RNA virus of the Arteriviridae family, which can cause severe host immune response disorders. From 2006, HP-PRRS attacked the swine industry, which causedhigh temperature and high morbidity in pigs of all ages, leading to huge economic loss to the swine industryin our country. Studies have shown that HP-PRRSV has a much stronger tropism to the central and peripheral immune organs than classical PRRSV, besides the severe thymic atrophy, it can also result in serious damage and cell apoptosis to the peripheral immune organs. PRRS continues to be a threat to the swine industry worldwide and is still the most economically important infectious swine disease in our country.PRRSV/HP- PRRSV infection can induce strong humoral immune responses, however, the initial antibody cannot against the virus infection, it maybe mediate the antibody dependent enhancement effect and cause severe lesions the infected pigs. In this study, we will investigate the cell autophagy and apoptosis induces by HP-PRRSV in the thymus of infected-piglets, research the lesions mechanism of thymus of infected piglets; study the immune responses and protection ofclassical and highly pathogenic PRRSV attenuated live vaccine to highly pathogenic PRRSV; investigate the role of the anti-idiotypic antibody specific for antibody against GP5 in regulating the immune responses and the immunological properties of sc Fv generated from the Mab2-5G2. The results generated from this study will be useful for clarify the lesion mechanism of immune organs and immune regulation mechanism to HP-PRRSV infection. 1、Highly pathogenic PRRSV infection induced apoptosis and autophagy in thymi of infected pigletsApoptosis is a programmed cell death(PCD), which is regulated by genes. Apoptosis is an initiative cell death for maintaining the balance of body and homeostasis of itself. Autophagy, a process initially described to represent another type of programmed cell death, is a series of biochemical events that involve the degradation of unnecessary or dysfunctional cellular components through the actions of lysosomes. Autophagy and apoptosis each have distinct mechanisms and pathways. In addition to cellular homeostasis, autophagy is also important for physiological and pathological processes. As an important primary lymphoid organ, the thymus contains thymocytes at various stages of T cell differentiation and maturation, which is related to the immune organs.HP-PRRSV Hu N4 strain causes severe thymic atrophy in infected piglets after birth, which is a unique feature of HP-PRRSV infection. Previously, we demonstrated that HP-PRRSV induced severe thymic atrophy in infected piglets. In this study, we investigated apoptosis and autophagy in the thymus of piglets infected with the HP-PRRSV Hu N4 strain, and found that both apoptosis and autophagy occurred in the thymus of piglets infected with HP-PRRSV. In addition to a few virus-infected cells, CD14+ cells, the main autophagic cells in the thymus were thymic epithelial cells. These findings demonstrated that HP-PRRSV induces apoptosis in bystander cells, and induces autophagy in both infected and bystander cells in the thymus of infected piglets. Herein, we first present new data on the thymic lesions induced by HP-PRRSV, and show that apoptosis and autophagy are key mechanisms involved in cell survival and determinants of the severity of thymic atrophy in infected piglets. Finally, future studies of the mechanism underlying immune responses are proposed based on our current understanding of PRRSV-host interactions. 2 、 Immune responses and protection ofclassical and highly pathogenic PRRSV attenuated live vaccine to highly pathogenic PRRSVSince HP- PRRS outbreak in China in 2006, the PRRSV vaccines widely used in the pig farms. However, due to the immune mechanism of PRRSV is not yet clear, the controversy of the choice and use of PRRSV vaccines is still exist. At present, HP-PRRSV is the major epidemic strain in the pig farm, however, because of the safety and cross protection to HP-PRRSV, classical PRRSV attenuated vaccine is still used in the pig farms. In this study, to clarify immune responses and protection ofclassical and highly pathogenic PRRSV attenuated live vaccine to highly pathogenic PRRSV, PRRSV negative piglets were vaccinated with the attenuated PRRSV vaccine CH-1R and the attenuated HP-PRRSV vaccine Hu N4 F112, respectively, and then challenged with the highly pathogenic PRRSV Hu N4 strain. The animals were evaluated for clinical signs, pathological changes of the thymus and lungs, viremia, levels of serum antibodies and cytokines. The results showed that both CH-1R and Hu N4 F112 vaccine have the good efficacy against highly pathogenic PRRSV challenge, these piglets show mild symptom and pathological change in lung and thymus compared to the piglets infected with Hu N4. However, piglets vaccinated with Hu N4 F112 generated the higher level antibodies against PRRSV during immunization and have the lower virus load in serum after Hu N4 infection than that of CH-1R group. Whether the virus load in serum related to the level of neutralizing antibody is unclear and need to the further investigation. 3、Anti-idiotypic antibodies reduce efficacy of the attenuated vaccine against highly pathogenic PRRSV challengeThe reason why the inability of current vaccines to provide effective protection against PRRSVinfection is not fully understood. One of the reasons might be the presence of anti-idiotypic antibodies(Ab2s) to the envelop glycoprotein GP5 induced by PRRSV infection since our previous studied demonstrated the presence of auto-Ab2s(a Ab2s) in pigs infected with PRRSV. To test this hypothesis, PRRSV negative piglets were injected with a monoclonal Ab2(Mab2-5G2) and a Ab2 s that are specific for anti-GP5 antibody, vaccinated with the attenuated PRRSV vaccine CH-1R and then challenged with the highly pathogenic PRRSV Hu N4 strain. The animals were evaluated for clinical signs, pathological changes of the thymus and lungs, viremia, levels of serum antibodies and cytokines.The piglets injected with Mab2-5G2 or a Ab2, and who received the attenuated PRRSV vaccine CH-1R before challenge, produced high levels of anti-N antibodies, IL-2 and IL-4, but low levels of neutralizing antibodies. After PRRSV Hu N4 challenge, the animals showed obvious clinical signs, including lung lesions, severe thymus atrophy and decreased production of IL-4 and higher level of viremia. When anti-GP5 Ab2 s are present, the use of attenuated PRRSV vaccine CH-1R against HP-PRRSV infection is not recommended. It can result in the poor health status with pneumonia and thymus atrophy. 4、Single-chain anti-idiotypic antibody retains it specificity to porcine reproductive and respiratory syndrome virus GP5Monoclonal anti-idiotypic antibody(Mab2-5G2) raised against idiotypic antibodies to membrane glycoprotein GP5 of PRRSV. MAb2-5G2, as “internal image” of PRRSV GP5, can imitate the antigen GP5 epitope play to the role of immune responses and has potential application value. Compared to the full Mab2-5G2 molecule, the sc Fv protein has a simple structure and binding constants that are similar to the antibody binding constants. The simple structure of sc Fv can be easily produced by E. coli cells and generated from the high affinity parental monoclonal antibody. Our previous study showed that the renatured sc Fv-His protein of Mab2-5G2 retained the same characteristics of specific recognition of Mab2-5G2 and binding to a specific protein on Marc-145 cells. Furthermore, renatured sc Fv-His along with Mab2-5G2 were used to immunize rabbits to produce anti-anti-idiotypic antibodies(Ab3) that neutralized PRRSV infection of Marc-145 cells. These results demonstrated that the expressed sc Fv-His protein possessed the same characteristics of Mab2-5G2 and will be suitable for future investigations of Mab2-5G2 antibody structure and its ability to interact with potential PRRSV cellular receptor as well as immunological properties against PRRSV infection. To further investigated immunological characteristics of Mab2-5G2 sc Fv, PRRSV negative piglets were injected with Mab2-5G2 or sc Fv, respectively, vaccinated with the attenuated PRRSV vaccine CH-1R and then challenged with the highly pathogenic PRRSV Hu N4 strain. The animals were evaluated for clinical signs, pathological changes of the thymus and lungs, viremia and levels of serum antibodies.The results showed that sc Fv of Mab2-5G2 retains the same immunogenicity as Mab2-5G2 mimicking of PRRSV GP5, it can induce bodies produced high levels of anti-GP2/GP5 antibodies after HP-PRRSV infection, and improve pig’s survival after HP-PRRSV infection.