*2.8. Human Papilloma Virus*

Most α-defensins possess some level of anti-human papilloma virus (HPV) activity, with HD-6 being a notable exception [38]. Due to their low toxicity and high efficacy, HNP-1 and HD-5 have been most frequently tested as anti-HPV candidates. Recent studies have focused solely on HD5 as it is secreted by epithelial cells in the genitourinary tract. The antiviral activity of HNP-1 and HD5 (5 μg/mL) against HPV is time-independent, with robust inhibition even when peptides are introduced to cells six hours post infection in vitro [38]. Employing immunofluorescent confocal microscopy, the authors demonstrated that the peptides do not inhibit viral entry but rather prevent virion escape from cytoplasmic vesicles [38]. During the course of HPV entry, cleavage of HPV L2 capsid protein by furin, a cellular protease, is required for successful infection [39]. HD5 directly disrupts this proteolytic processing step, thereby preventing HPV genome escape from endosomes [39]. Interestingly, the use of a furin-cleaved HPV does not result in abrogation of HD5 activity. HD5 can still block HPV infection by preventing viral capsid dissociation from the genome, and by reducing viral trafficking within the host cells [40]. These results indicate that α-defensins, particularly HD5, demonstrate robust anti-HPV activity by targeting multiple steps during viral life cycle.

#### **3. Cathelicidin, LL-37**

#### *3.1. Expression*

Cathelicidins are peptides with a conserved 100-amino-acid cathelin domain, a protein sequence first identified in porcine leukocytes that is capable of inhibiting the protease cathepsin-L [9]. Cathelicidins are typically linear peptides that fold into amphipathic α-helical structures which are frequently cleaved from the highly variable C-terminal antimicrobial domain [12]. In humans, cathelicidin is produced by a vitamin D-dependent antimicrobial pathway [41]. Although there are multiple cathelicidins found in nature, humans only express a single cathelicidin, known as human cationic antimicrobial peptide 18 (hCAMP-18), hCAP18, or LL-37. LL-37 was identified and isolated in 1995 from neutrophils [1,42]. Like α-defensins, cathelicidins are synthesized as pre-propeptides; following proteolytic removal of the signal peptide, the inactive propeptide is tagged for storage in neutrophil granules. The active cationic molecule is generated by cleavage of the C-terminus end of the hCAP18 precursor protein yielding a linear 37-amino-acid-long peptide [1]. The name hCAP18 alludes to the molecular weight of the polypeptide (18 kDa) and the cationic character of the structure, whereas LL-37 refers to the 37 amino acid length of the peptide along with a Leu-Leu motif located at the N-terminus [1]. The peptide can also be produced in epithelial cells and may play an important role in the initial immune response to various pathogens [10,11]. In epithelial skin cells, LL-37 is further cleaved into shorter segments exhibiting potent antimicrobial activity [43]. LL-37 is also produced by monocytes, NK cells, mast cells, B cells, colon enterocytes, and keratinocytes [1], and has been detected in numerous tissues and biological fluids such as sweat, breast milk, wound fluid, vernix, tracheal aspirates of newborns, and seminal plasma [9]. The concentration of LL-37 and its precursor in tissues and body fluids can range between 2 and 5 μg/mL (0.4–1 μM); however, concentrations can increase up to 20 μg/mL (2.2 μM) during infections in bronchoalveolar fluid [44]. In nasal secretions LL-37 concentrations can vary from 1.2–80 μg/mL [44]. The expression of LL-37 is regulated by a number of endogenous factors including pro-inflammatory cytokines and growth factors such as the active form of vitamin D [1]. LL-37 functions as a chemoattractant for neutrophils, monocytes, dendritic and T-cells and is rapidly released by epithelial cells and leukocytes following infection [11,45]. LL-37 can stimulate IL-6 production in human dendritic cells and may act as both an anti and pro-inflammatory factor during the immune response to various infections [45]. Individuals with cathelicidin deficient neutrophils display an increased susceptibility to infection [11].
