CSL324, a granulocyte colony-stimulating factor receptor antagonist, blocks neutrophil migration markers that are upregulated in hidradenitis suppurativa.

BACKGROUND: Neutrophils have been shown to contribute to the pathophysiology of hidradenitis suppurativa (HS), a chronic, painful and debilitating inflammatory skin disease, yet their exact role remains to be fully defined. Granulocyte colony-stimulating factor (G-CSF), a major regulator of neutrophil development and survival, can be blocked by the novel, fully human anti-G-CSF receptor (G-CSFR) monoclonal antibody CSL324. OBJECTIVES: We investigated the activation and migration of neutrophils in HS and the impact of blocking G-CSFR with CSL324. METHODS: Biopsy and peripheral blood samples were taken from participants of two studies: 2018.206, a noninterventional research study of systemic and dermal neutrophils and inflammatory markers in patients with neutrophilic skin diseases, and CSL324_1001 (ACTRN12616000846426), a single-dose ascending and repeated dose, randomized, double-blind, placebo-controlled study to assess the safety, pharmacokinetics and pharmacodynamics of CSL324 in healthy adult subjects. Ex vivo experiments were performed, including neutrophil enumeration and immunophenotyping, migration, receptor occupancy and transcriptome analysis. RESULTS: The number of cells positive for the neutrophil markers myeloperoxidase (MPO) and neutrophil elastase (NE) was significantly higher in HS lesions compared with biopsies from healthy donors (HDs) (P < 0.0001 and P = 0.0223, respectively). In peripheral blood samples, mean neutrophil counts were significantly higher in patients with HS than in HDs (2.98 vs. 1.60 × 109 L-1, respectively; P = 8.8 × 10-4). Neutrophil migration pathways in peripheral blood were increased in patients with HS and their neutrophils demonstrated an increased migration phenotype, with higher mean CXCR1 on the surface of neutrophils in patients with HS (24453.20 vs. 20798.47 for HD; P = 0.03). G-CSF was a key driver of the transcriptomic changes in the peripheral blood of patients with HS and was elevated in serum from patients with HS compared with HDs (mean 6.61 vs. 3.84 pg mL-1, respectively; P = 0.013). Administration of CSL324 inhibited G-CSF-induced transcriptional changes in HDs, similar to those observed in the HS cohort, as highlighted by expression changes in genes related to neutrophil migratory capacity. CONCLUSIONS: Data suggest that neutrophils contribute to HS pathophysiology and that neutrophils are increased in lesions due to an increase in G-CSF-driven migration. CSL324 counteracted G-CSF-induced transcriptomic changes and blocked neutrophil migration by reducing cell-surface levels of chemokine receptors.

Evaluation of the bioactivity of influenza vaccine strains in vitro suggests that the introduction of new strains in the 2010 Southern Hemisphere trivalent influenza vaccine is associated with adverse events.

In Australia, during the 2010 Southern Hemisphere (SH) influenza season, there was an unexpected increase in post-marketing adverse event reports of febrile seizures (FS) in children under 5 years of age shortly after vaccination with the CSL trivalent influenza vaccine (CSL 2010 SH TIV) compared to previous CSL TIVs and other licensed 2010 SH TIVs. The present study describes the outcomes of a series of in vitro experiments directed at elucidating the root cause. The scientific investigations found that a subset of paediatric donors displayed elevated cytokine/chemokine responses to the CSL 2010 SH TIV but not to previous CSL TIVs nor other 2010 SH TIVs. The induction of elevated cytokines/chemokines in paediatric whole blood correlated with elevated NF-κB activation in a HEK293 cell reporter assay. The data indicate that the introduction of the B/Brisbane/60/2008 strain within the CSL manufacturing process (such as occurred in the preceding 2009/10 NH season) appears to have raised the pyrogenic potential of the CSL 2009/10 NH TIV but that this was insufficient to elicit FS in children <5 years. The 2010 SH season coincided with the first introduction of the H1N1 A/California/07/2009 in combination with the B/Brisbane/60/2008 strain. Our data demonstrates that the introduction of the H1N1 A/California/07/2009 (and to a much lesser degree, H3N2 A/Wisconsin/15/2009) in combination with B/Brisbane/60/2008 (as expressed through the CSL method of manufacture) combined and likely compounded the bioactivity of the CSL 2010 SH TIV. This was associated with stronger immune responses, which in a proportion of children <5 years were associated with FS. The assays and systems developed during these investigations should greatly assist in determining the bioactivity of new influenza strains, and thus aid with the manufacture of CSL TIVs indicated for use in the paediatric population.

Role of viral RNA and lipid in the adverse events associated with the 2010 Southern Hemisphere trivalent influenza vaccine.

In Australia, during the 2010 Southern Hemisphere (SH) influenza season, there was an unexpected increase in post-marketing adverse event reports of febrile seizures (FS) in children under 5 years of age shortly after vaccination with the CSL 2010 SH trivalent influenza vaccine (CSL 2010 SH TIV) compared to previous CSL TIVs and other licensed 2010 SH TIVs. In an accompanying study, we described the contribution to these adverse events of the 2010 SH influenza strains as expressed in the CSL 2010 SH TIV using in vitro cytokine/chemokine secretion from whole blood cells and induction of NF-κB activation in HEK293 reporter cells. The aim of the present study was to identify the root cause components that elicited the elevated cytokine/chemokine and NF-κB signature. Our studies demonstrated that the pyrogenic signal was associated with a heat-labile, viral-derived component(s) in the CSL 2010 SH TIV. Further, it was found that viral lipid-mediated delivery of short, fragmented viral RNA was the key trigger for the increased cytokine/chemokine secretion and NF-κB activation. It is likely that the FS reported in children <5 years were due to a combination of the new influenza strains included in the 2010 SH TIV and the CSL standard method of manufacture preserving strain-specific viral components of the new influenza strains (particularly B/Brisbane/60/2008 and to a lesser extent H1N1 A/California/07/2009). These combined to heighten immune activation of innate immune cells, which in a small proportion of children <5 years of age is associated with the occurrence of FS. The data also demonstrates that CSL TIVs formulated with increased levels of splitting agent (TDOC) for the B/Brisbane/60/2008 strain can attenuate the pro-inflammatory signals in vitro, identifying a potential path forward for generating a CSL TIV indicated for use in children <5 years.

Scientific investigations into febrile reactions observed in the paediatric population following vaccination with a 2010 Southern Hemisphere Trivalent Influenza Vaccine.

During the 2010 Southern Hemisphere (SH) influenza season, there was an unexpected increase in the number of febrile reactions reported in the paediatric population in Australia shortly after vaccination with the CSL 2010 trivalent influenza vaccine (TIV) compared to previous seasons. A series of scientific investigations were initiated to identify the root cause of these adverse events, including in vitro cytokine/chemokine assays following stimulation of adult and paediatric whole blood, as well as mammalian cell lines and primary cells, profiling of molecular signatures using microarrays, and in vivo studies in rabbits, ferrets, new born rats and rhesus non-human primates (NHPs). Various TIVs (approved commercial vaccines as well as re-engineered TIVs) and their individual monovalent pool harvest (MPH) components were examined in these assays and in animal models. Although the scientific investigations are ongoing, the current working hypothesis is that the increase in febrile adverse events reported in Australia after vaccination with the CSL 2010 SH TIV may be due to a combination of both the introduction of three entirely new strains in the CSL 2010 SH TIV, and differences in the manufacturing processes used to manufacture CSL TIVs compared to other licensed TIVs on the market. Identification of the causal component(s) may result in the identification of surrogate assays that can assist in the formulation of TIVs to minimise the future incidence of febrile reactions in the paediatric population.