Elucidating the Role of Antigen-presenting Cells in the Immunopathogenesis of the Porcine Reproductive and Respiratory Syndrome Virus
Darbellay, Joseph Andikpeme 1989-
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The porcine reproductive and respiratory syndrome virus (PRRSV) is a positive sense single-stranded RNA virus of the arteriviridae family and is one of the most economically devastating pathogens in the swine industry today. The PRRSV was discovered in the early 1990s in Europe and the United States, with strains being divided into Type 1 and Type 2 genotypes, respectively. Disease outcomes range from being asymptomatic to upwards of 100% mortality in herds, being attributable to the pathogenicity of the PRRSV strain, to co-infections with opportunistic pathogens, and to the age and breed of the pig. Animals subject to infection with PRRSV exhibit an array of clinical symptoms, including but not limited to, respiratory difficulty and pneumonia, weight loss, immune suppression leading to secondary bacterial infections, and spontaneous abortions/fetal mummification, from which the majority of the economic losses stem. Immunization with modified live vaccines are the most popular intervention to control disease, and although they are effective in improving health status of animals, the currently distributed vaccines pose a risk of reversion to virulence and afford limited cross protection amongst circulating strains of the virus. Thus, there is a high demand for a safe and effective vaccine. The goal of this study was to investigate the role of specific antigen-presenting cell (APC) subsets during the pathogenesis of PRRSV and to further understand the progression to T cell immunity in response to PRRSV. To accomplish this, we chose to investigate the susceptibility of bone marrow-derived dendritic cells (BMDCs), monocyte-derived dendritic cells (MoDCs), and monocyte-derived macrophages (MoMΦs). After successfully differentiating and characterizing BMDCs from hematopoietic stem cells isolated from the sternum of animals, we demonstrated that PRRSV infection is restricted only to APCs that express CD163. Furthermore, we showed that PRRSV replicates more quickly in MoMΦ cell cultures than in CD163+ BMDC cultures and potentially in MoDCs. We continued to investigate PRRSV infection of APCs and discovered that in non-infected MoDCs, the cellular protein gamma actin 1 associates closely with MHCII. When MoDCs were infected with PRRSV, gamma actin 1 was no longer associated with MHCII. We hypothesize that PRRSV could be manipulating the actin cytoskeleton, potentially interfering with MHCII peptide presentation. Ultimately, we were interested in the interaction of APCs with T cells. In order to study this interaction, we developed an assay utilizing a mixed leukocyte reaction (MLR). Our hypothesis was confirmed in the MLR that showed M1 MoMΦs (IFN-γ stimulated) are more potent inducers of cytotoxic lymphocyte (CTLs) and CD4α+ T cell proliferation than M2 MoMΦs (IL-4 stimulated) or M0 MoMΦs (non-stimulated). In addition, our results indicated that gamma delta (γδ) T cells did not participate in the MLR. Next we proceeded with an animal trial investigating the interaction of APCs with T cells in a PRRSV-antigen specific manner. The objective of the trial was to investigate the progression to T cell immunity during a PRRSV infection. We used a commercial swine-influenza vaccine as a positive control antigen, and as a comparative measure for the T cell immune response to PRRSV. Our results indicated that PRRSV infection of APCs does not interfere with the ability of APCs to promote T cell proliferation. We detected IFN-γ secreting cells in PBMCs two weeks post infection, and T cell proliferation was evident in all lymphoproliferative cell cultures two weeks post infection. A comparison between MoMΦ-T cell co-cultures and MoDC-T cell co-cultures indicated that MoDCs may be more potent stimulators of central memory Th cell proliferation. Lastly, PRRSV infected animals showed a higher capacity to promote the proliferation of T cells specific for swine-influenza A virus, potentially signifying that the general monocyte population from PRRSV infected animals acquired an activated state as a result of the infection. Overall, the work in this thesis allowed us to formulate a theory regarding the dysregulated immune response to PRRSV. A characteristic adaptive immune response to PRRSV includes the early appearance of non-neutralizing antibodies (within a week), a delayed induction of T cell immunity (2-3 weeks post infection), with neutralizing antibodies becoming detectable roughly 4 weeks post infection, correlating to the resolution of illness. We believe that specific subsets of DCs are responsible for the induction of T cell immunity, and the rarity of this DC population(s) causes a delay in the induction of the T cell immune response. Furthermore, the delayed induction of T cell immunity would affect the humoral immune response. In the absence of CD4+ follicular T helper cells, B cells would not be able to undergo proper somatic hypermutation and the result would be a state of hypergammaglobulinemia, similar to what is observed early during a PRRSV infection with non-neutralizing antibodies and auto-antibodies. The results herein support our theory, and contribute to the general knowledge surrounding the immunopathogenesis of PRRSV. Future work investigating the mechanisms by which PRRSV hinders the progression to adaptive immunity could prove to be fundamental in the development of a novel vaccine.
DegreeDoctor of Philosophy (Ph.D.)
CommitteeMeurens, Francois; Gordon, John; Tikoo, Suresh; Hill, Janet
Copyright DateJune 2019