|dc.description.abstract||Poxviruses are large DNA viruses that replicate in the cytoplasm, and encode numerous host immune modulating factors. The parapoxvirus, orf virus (ORFV) is a zoonotic pathogen that causes pustular dermatitis in sheep, goats and humans. The fact that ORFV can repeatedly infect its host may be explained by the discovery of several secreted immunomodulators. One of the secreted viral proteins is a chemokine binding protein (CBP) that shares low homology with the type II poxvirus CBPs. These viral CBPs have no known mammalian homologue and are unique in their ability to specifically bind CC chemokines with high affinity. The inflammatory CC chemokines play a role in recruiting monocytes and dendritic cells to the site of infection during innate immunity, and constitutive CC chemokines are required for the migration of dendritic cells to the lymph nodes to initiate the adaptive immune response. This study examined the ability of ORFV CBP to block chemokine gradients formed during innate and adaptive immune responses.
In this study, the ability of ORFV CBP to bind murine inflammatory and constitutive CC chemokines was assessed using murine models. The immune responses in mice are well characterised and murine reagents are readily available allowing detailed analysis of both in vitro and in vivo studies. CBP bound murine chemokines CCL2, CCL3, CCL5, CCL19, and CCL21 with high affinity by surface plasmon resonance studies. The inflammatory chemokines CCL2, CCL3 and CCL5 are produced during damage and inflammation in epithelial tissues, and are involved in recruiting antigen-presenting cells to the site of acute inflammation. Transwell migration assays were used to determine optimal amounts of these chemokines required to induce migration of monocytes and immature dendritic cells. Monocytes and dendritic cells migrated in maximal numbers in response to between 12.5 – 50 ng/ml of inflammatory chemokines. In the presence of increasing amounts of CBP, the chemokine-induced migration of monocytes and dendritic cells was inhibited in a dose dependent manner. A molar ratio of chemokine to CBP of 1:4 reduced the CCL2-, CCL3-, and CCL5-induced migration of monocytes and immature dendritic cells to background levels. The chemotactic ability of CCL19 and CCL21 for mature dendritic cells was also assessed using transwell migration assays. CCL19 and CCL21 were used at 12.5 ng/ml to induce maximal migration of mature dendritic cells. A molar ratio of chemokine to CBP of 1:4 completely inhibited CCL19-induced migration of mature dendritic cells. CBP bound murine CCL21 with comparatively lower binding affinity and hence a higher chemokine to CBP ratio of 1:32 was required to reduce chemotaxis to background levels. Further functional assays utilized murine models to study the effects of CBP on cellular trafficking in vivo.
The administration of lipopolysaccharide (LPS) in skin results in a highly localised inflammation with increased expression of the inflammatory chemokines CCL2, CCL3, and CCL5. The ability of CBP to bind these inflammatory chemokines in vivo and hence block the recruitment of monocytes and dendritic cells was assessed using a skin inflammation model. CBP inhibited the recruitment of Gr-1+/CD11b+ monocytes and CD11c+/MHC-II+ dendritic cells to the skin, but had no effect on Gr-1+/CD11b- neutrophil recruitment at 24 h post administration of LPS. This suggests that CBP can inhibit the recruitment of specific immune cells by targeting inflammatory CC chemokines. The ability of CBP to bind constitutive chemokines CCL19 and CCL21 was assessed by the intradermal administration of ex vivo CpG-pulsed CCR7+ dendritic cells in the skin. The migration of antigen-pulsed dendritic cells from the skin to the draining lymph node was inhibited in the presence of CBP, with complete inhibition of migration observed when 1 μg of CBP was used. To assess the consequences of reduced numbers of draining dendritic cells, the activation of T cells at the lymph node was measured. The presence of CBP in the skin inhibited the proliferation of adoptively transferred OT-II CD4+ and OT-I CD8+ T cells in the draining inguinal lymph nodes in response to ovalbumin-pulsed dendritic cells. The results suggest that CBP can bind the constitutive chemokines in vivo to inhibit the migration of mature dendritic cells from the skin to the draining lymph nodes and hinders the initiation of the adaptive immune response.
The observations using mouse models strongly suggest that ORFV CBP will bind the inflammatory and constitutive CC chemokines during viral infection and prevent the formation of a chemokine gradient, thus delaying the recruitment of immune cells to the site of infection, and the migration of antigen presenting cells to the draining lymph nodes. This will delay both the innate and adaptive immune response of the host allowing the successful replication and survival of ORFV.
The results of the study highlight the importance of chemokines in the induction of host immunity and complement the growing body of data regarding viral manipulation of the host chemokine network. The functional study of ORFV CBP adds to the increasing repertoire of host immune modulators encoded by poxviruses.||