Zabeen Lateef

The chemokine-binding protein encoded by the poxvirus orf virus inhibits recruitment of dendritic cells to sites of skin inflammation and migration to peripheral lymph nodes

Orf virus (ORFV) is a zoonotic parapoxvirus that induces acute pustular skin lesions in sheep and humans. ORFV can reinfect its host and the discovery of several secreted immune modulatory factors that include a chemokine‐binding protein (CBP) may explain this phenomenon. Dendritic cells (DC) are professional antigen presenting cells that induce adaptive immunity and their recruitment to sites of infection in skin and migration to peripheral lymph nodes is critically dependent on inflammatory and constitutive chemokine gradients respectively. Here we examined whether ORFV‐CBP could disable these gradients using mouse models. Previously we established that ORFV‐CBP bound murine inflammatory chemokines with high affinity and here we show that this binding spectrum extends to constitutive chemokines CCL19 and CCL21. Using cell‐based chemotaxis assays, ORFV‐CBP inhibited the movement of both immature and mature DC in response to these inflammatory and constitutive chemokines respectively. Moreover in C57BL/6 mice, intradermally injected CBP potently inhibited the recruitment of blood‐derived DC to lipopolysaccharide‐induced sites of skin inflammation and inhibited the migration of ex vivo CpG‐activated DC to inguinal lymph nodes. Finally we showed that ORFV‐CBP completely inhibited T responsiveness in the inguinal lymph nodes using intradermally injected DC pulsed with ovalbumin peptide and transfused transgenic T cells.

Orf virus-encoded chemokine-binding protein is a potent inhibitor of inflammatory monocyte recruitment in a mouse skin model.

The parapoxvirus orf virus causes pustular dermatitis in sheep and is transmissible to humans. The virus encodes a secreted chemokine-binding protein (CBP). We examined the ability of this protein to inhibit migration of murine monocytes in response to CC inflammatory chemokines, using chemotaxis assays, and its effects on monocyte recruitment into the skin, using a mouse model in which inflammation was induced with bacterial lipopolysaccharide. CBP was shown to bind murine chemokines CCL2, CCL3 and CCL5 with high affinity by surface plasmon resonance and it completely inhibited chemokine-induced migration of monocytes at a CBP:chemokine molar ratio of 4:1. In the mouse, low levels of CBP potently inhibited the recruitment of Gr-1+/CD11b+ monocytes to the site of inflammation in the skin but had little effect on neutrophil recruitment, suggesting that this factor plays a role in disrupting chemokine-induced recruitment of specific immune cell types to infection sites.

Orf virus IL-10 reduces monocyte, dendritic cell and mast cell recruitment to inflamed skin.

Orf virus (ORFV) is a zoonotic parapoxvirus that causes pustular dermatitis of sheep, and occasionally humans. Despite causing sustained infections, ORFV induces only a transient increase in pro-inflammatory signalling and the trafficking of innate immune cells within the skin seems to be impaired. An explanation for this tempered response to ORFV infection may lie in its expression of a homolog of the anti-inflammatory cytokine, interleukin (IL)-10. Using a murine model in which inflammation was induced by bacterial lipopolysaccharide, we examined the effects of the ORFV-IL-10 protein on immune cell trafficking to and from the skin. ORFV-IL-10 limited the recruitment of blood-derived Gr-1(int)/CD11b(int) monocytes, CD11c(+ve)/MHC-II(+ve) dendritic cells and c-kit(+ve)/FcεR1(+ve) mature mast cells into inflamed skin. ORFV-IL-10 also suppressed the activation of CD11c(+ve)/MHC-II(+ve) dendritic cells within the skin, reducing their trafficking to the draining lymph node. These findings suggest that expression of IL-10 by ORFV may contribute to the impaired trafficking of innate immune cells within infected skin.

Deletion of the Chemokine Binding Protein Gene from the Parapoxvirus Orf Virus Reduces Virulence and Pathogenesis in Sheep

Orf virus (ORFV) is the type species of the Parapoxvirus genus of the family Poxviridae and infects sheep and goats, often around the mouth, resulting in acute pustular skin lesions. ORFV encodes several secreted immunomodulators including a broad-spectrum chemokine binding protein (CBP). Chemokines are a large family of secreted chemotactic proteins that activate and regulate inflammation induced leukocyte recruitment to sites of infection. In this study we investigated the role of CBP in vivo in the context of ORFV infection of sheep. The CBP gene was deleted from ORFV strain NZ7 and infections of sheep used to investigate the effect of CBP on pathogenesis. Animals were either infected with the wild type (wt) virus, CBP-knockout virus or revertant strains. Sheep were infected by scarification on the wool-less area of the hind legs at various doses of virus. The deletion of the CBP gene severely attenuated the virus, as only few papules formed when animals were infected with the CBP-knock-out virus at the highest dose (107 p.f.u). In contrast, large pustular lesions formed on almost all animals infected with the wt and revertant strains at 107 p.f.u. The lesions for the CBP-knock-out virus resolved approximately 5–6 days p.i, at a dose of 107 pfu whereas in animals infected with the wt and revertants at this dose, lesions began to resolve at approximately 10 days p.i. Few pustules developed at the lowest dose of 103 p.f.u. for all viruses. Immunohistochemistry of biopsy skin-tissue from pustules showed that the CBP-knockout virus replicated in all animals at the highest dose and was localized to the skin epithelium while haematoxylin and eosin staining showed histological features of the CBP-knockout virus typical of the parent virus with acanthosis, elongated rete ridges and orthokeratotic hyperkeratosis. MHC-II immunohistochemistry analysis for monocytes and dendritic cells showed greater staining within the papillary dermis of the CBP-knock-out virus compared with the revertant viruses, however this was not the case with the wt where staining was similar. Our results show that the CBP gene encodes a secreted immunodulator that has a critical role in virulence and pathogenesis.

Transcriptomic analysis of human norovirus NS1-2 protein highlights a multifunctional role in murine monocytes.

BackgroundThe GII.4 Sydney 2012 strain of human norovirus (HuNoV) is a pandemic strain that is responsible for the majority of norovirus outbreaks in healthcare settings. The function of the non-structural (NS)1-2 protein from HuNoV is unknown.ResultsIn silico analysis of human norovirus NS1-2 protein showed that it shares features with the murine NS1-2 protein, including a disordered region, a transmembrane domain and H-box and NC sequence motifs. The proteins also contain caspase cleavage and phosphorylation sites, indicating that processing and phosphorylation may be a conserved feature of norovirus NS1-2 proteins. In this study, RNA transcripts of human and murine norovirus full-length and the disordered region of NS1-2 were transfected into monocytes, and next generation sequencing was used to analyse the transcriptomic profile of cells expressing virus proteins. The profiles were then compared to the transcriptomic profile of MNV-infected cells.ConclusionsRNAseq analysis showed that NS1-2 proteins from human and murine noroviruses affect multiple immune systems (chemokine, cytokine, and Toll-like receptor signaling) and intracellular pathways (NFκB, MAPK, PI3K-Akt signaling) in murine monocytes. Comparison to the transcriptomic profile of MNV-infected cells indicated the pathways that NS1-2 may affect during norovirus infection.

A Broad-Spectrum Chemokine-Binding Protein of Bovine Papular Stomatitis Virus Inhibits Neutrophil and Monocyte Infiltration in Inflammatory and Wound Models of Mouse Skin.

Bovine papular stomatitis virus (BPSV) is a Parapoxvirus that induces acute pustular skin lesions in cattle and is transmissible to humans. Previous studies have shown that BPSV encodes a distinctive chemokine-binding protein (CBP). Chemokines are critically involved in the trafficking of immune cells to sites of inflammation and infected tissue, suggesting that the CBP plays a role in immune evasion by preventing immune cells reaching sites of infection. We hypothesised that the BPSV-CBP binds a wide range of inflammatory chemokines particularly those involved in BPSV skin infection, and inhibits the recruitment of immune cells from the blood into inflamed skin. Molecular analysis of the purified protein revealed that the BPSV-CBP is a homodimeric polypeptide with a MW of 82.4 kDa whilst a comprehensive screen of inflammatory chemokines by surface plasmon resonance showed high-affinity binding to a range of chemokines within the CXC, CC and XC subfamilies. Structural analysis of BPSV-CBP, based on the crystal structure of orf virus CBP, provided a probable explanation for these chemokine specificities at a molecular level. Functional analysis of the BPSV-CBP using transwell migration assays demonstrated that it potently inhibited chemotaxis of murine neutrophils and monocytes in response to CXCL1, CXCL2 as well as CCL2, CCL3 and CCL5 chemokines. In order to examine the effects of CBP in vivo, we used murine skin models to determine its impact on inflammatory cell recruitment such as that observed during BPSV infection. Intradermal injection of BPSV-CBP blocked the influx of neutrophils and monocytes in murine skin in which inflammation was induced with lipopolysaccharide. Furthermore, intradermal injection of BPSV-CBP into injured skin, which more closely mimics BPSV lesions, delayed the influx of neutrophils and reduced the recruitment of MHC-II+ immune cells to the wound bed. Our findings suggest that the CBP could be important in pathogenesis of BPSV infections.

Treatment of limb wounds of horses with orf virus IL-10 and VEGF-E accelerates resolution of exuberant granulation tissue, but does not prevent its development

Bandaging of limb wounds in horses leads to formation of exuberant granulation tissue (EGT) that retards healing due to protracted inflammation, aberrant vascularisation and delayed epithelialisation. EGT is not observed if wounds are left undressed or when wounds are on the body. A previous study showed that short-term administration of proteins derived from orf virus dampened inflammation and promoted epithelialisation of open wounds in horses. Here, we investigated the impact of orf virus interleukin-10 and vascular endothelial growth factor-E on the development and resolution of EGT. Excisional wounds were created on the forelimb of four horses, and bandages were maintained until full healing to induce EGT formation. Matching body wounds were created to ensure EGT was limited to the limb, and to differentiate the effects of the viral proteins on normal healing and on EGT formation. Viral proteins or the hydrogel vehicle control were administered topically to site-matched wounds at day 1, with repeat administration at day 8. Wound healing and EGT formation were monitored macroscopically. Wound margin samples were harvested at 2, 7 and 14 days, and at full healing, with histology used to observe epithelialisation, immunofluorescence used to detect inflammatory cells, angiogenesis and cell death, and qPCR to measure expression of genes regulating inflammation and angiogenesis. Limb wounds developed EGT, and exhibited slower healing than body wounds. Viral protein treatment did not accelerate healing at either location nor limit EGT formation in limb wounds. Treatment of limb wounds did however increase epithelialisation and angiogenesis, without dampening inflammatory cell infiltration or gene expression. The healed wounds also had less occlusion and death of blood vessels and fewer epidermal rete ridges following viral protein treatment. These findings indicate that the viral protein treatment does not suppress wound inflammation or EGT formation, but does promote vascular and epidermal repair and EGT resolution.

Virus-like particle vaccines: immunology and formulation for clinical translation

ABSTRACT Introduction: Virus-like particle (VLP) vaccines face significant challenges in their translation from laboratory models, to routine clinical administration. While some VLP vaccines thrive and are readily adopted into the vaccination schedule, others are restrained by regulatory obstacles, proprietary limitations, or finding their niche amongst the crowded vaccine market. Often the necessity to supplant an existing vaccination regimen possesses an immediate obstacle for the development of a VLP vaccine, despite any preclinical advantages identified over the competition. Novelty, adaptability and formulation compatibility may prove invaluable in helping place VLP vaccines at the forefront of vaccination technology. Areas covered: The purpose of this review is to outline the diversity of VLP vaccines, VLP-specific immune responses, and to explore how modern formulation and delivery techniques can enhance the clinical relevance and overall success of VLP vaccines. Expert commentary: The role of formation science, with an emphasis on the diversity of immune responses induced by VLP, is underrepresented amongst clinical trials for VLP vaccines. Harnessing such diversity, particularly through the use of combinations of select excipients and adjuvants, will be paramount in the development of VLP vaccines.