Antiviral function of LL-37 on respiratory syncytial virus

Abstract

Recurrent infection with human respiratory syncytial virus (RSV) is one of the most common causes for lower respiratory tract illness (LRI) in infants, the elderly, and immunocompromised individuals. Due to lack of vaccines and therapeutic interventions, medical care of acute RSV bronchiolitis is mostly limited to supportive measures. Thus, novel treatment options to control RSV infection are desperately required. The cationic host defence peptide human cathelicidin LL-37 possesses both microbicidal and immunomodulatory properties. This essential effector of the innate immune system holds potent antiviral activity against a variety of viruses, including influenza virus, and has been proposed as a promising candidate for antiviral drug development. Previous studies revealed that lower cathelicidin levels put RSV infected infants at risk for more severe RSV disease, while infection of lung epithelial cells induced cathelicidin up-regulation. These findings suggest that LL-37 might possess antiviral activity against RSV. However, its potential antiviral function on RSV remains to be elucidated. This thesis therefore aimed to evaluate the antiviral activity of cathelicidins against RSV, by assessing its relevance in vitro and in vivo and elucidating the underlying antiviral mechanism. Firstly, the antiviral effects of human cathelicidin LL-37 against RSV were addressed in vitro. Presence of LL-37 during infection potently reduced viral titres and protected cells against virus-associated cytopathic effects. Experiments revealed that only the core region of LL-37 holds antiviral activity against RSV. Antiviral effects were also observed for the murine LL-37 orthologue mCRAMP. Administration of LL-37 at different stages in the infection cycle provided evidence that LL-37 can be used preventatively, protecting against RSV infection by directly acting on both cells and viral particles. When given therapeutically, once an infection was established, LL-37 also limited viral spread. Next, the molecular mechanism mediating the peptide’s antiviral activity was investigated. It was demonstrated that LL-37 does not affect the interferon-mediated cellular antiviral immune response to RSV. Experiments established that LL-37 does not contribute to viral clearance by inducing epithelial cell death. Further mechanistic studies revealed that the peptide directly binds to RSV particles, destabilises the integrity of the viral envelope, and prevents adsorption of RSV to epithelial cells during the entry stage of infection. Finally, the in vivo relevance of LL-37 treatment and endogenous cathelicidin expression was examined, employing both murine and human model systems. It was established that LL-37 has protective antiviral effects against RSV in vivo. In contrast to the cell culture model, only co-administration of LL-37 and RSV, but not treatment prior or post infection, protects mice from clinical signs of infection. Levels of the murine LL-37 orthologue mCRAMP were increased in RSV infected lungs, pointing towards its importance in antiviral defence. In keeping with this, mCRAMP-deficient mice were more susceptible to RSV induced disease. Equally, individuals with low nasal LL-37 baseline levels that were experimentally challenged with RSV, were more susceptible to infection. This highlights the importance of endogenous cathelicidin expression to fight and control RSV infection. Overall, these results identify LL-37 as an important antiviral agent against RSV in vitro and in vivo, and emphasise the role of endogenous cathelicidins in the defence against this pathogen. Moreover, unravelling the underlying antiviral mechanism of LL-37 against RSV adds to our understanding of how CHDP act on enveloped viruses, thus supporting the development of new antiviral treatment options.