*5.3. HIV*

In earlier studies, EDN did not demonstrate direct activity against HIV; However, it indirectly induces the production of HIV-inhibiting molecules. Soluble factors produced by hosts such as chemokines, interferons, RNases, and alloantigen-stimulated factors (ASF) have been reported to inhibit HIV-1 replication or binding [123]. A test to determine the soluble HIV-1 inhibitory activity utilized supernatants from mixed lymphocytes of healthy individuals that exhibit anti-HIV-1 activity. While the supernatant exhibited anti-HIV activity, this activity was blocked with antibodies specific for EDN by 58% [123]. Antibodies to RNase A had no effect on the anti-HIV-1 activity, thus ruling out its involvement. In addition, an RNase inhibitor significantly blocked the inhibitory activity indicating that EDN or a possible closely-related RNase is responsible for the HIV-1 inhibitory activity [123]. Later studies reported EDN and a recombinant EDN, containing a four amino acid extension of the N-terminus of END, both exhibit anti-HIV-1 activity independent of time of addition, before, during, or 2 h post infection [124].

#### **6. AMPs from Immune Cells**

#### *6.1. Expression*

Immune cells have the ability to produce AMPs such as the protease inhibitors elafin and secretory leukocyte protease inhibitor (SLPI) [3]. Studies investigating AMPs produced by gamma-delta T cells (γδ T cells), recorded production of elafin [125]. Elafin is expressed in tissues such as skin, placenta, genital and gastrointestinal tracts, as well as by cells including neutrophils, epithelial cells, macrophages, and keratinocytes and thus can be found at many surfaces and in secretions [126]. Elafin is a 57-amino-acid single-polypeptide chain produced by the proteolytic cleavage of a precursor molecule called trappin-2 [127,128]. Similarly, SLPI is expressed by neutrophils, macrophages, epithelial cells of renal tubules and respiratory/alimentary tracts [129]. This peptide can be found in saliva, cervical mucus, breast milk, cerebral spinal fluid, tears, and secretions from nose and bronchi [129]. SLPI is a 107-amino-acid, cysteine-rich polypeptide chain that is highly basic [129]. The C-terminals of elafin and SLPI exhibit both high homology and similar protease activity [127] and have demonstrated antimicrobial as well as anti-inflammatory activities (Table 1) [129,130].

#### *6.2. Herpes Simplex Virus*

Elafin and its precursor trappin-2 (Tr) both exhibit anti-HSV activity. Drannik and colleagues (2013) investigated the efficacy of elafin and Tr on HSV-2 infection using a Tr-expressing, replication-deficient adenovirus vector as well as recombinant TR/elafin proteins. Cells were either infected with the adenovirus vector or treated with the recombinant proteins prior to HSV-2 infection. When endometrial and endocervical epithelial cells were pre-treated with these molecules (TR IC50~0.07 μg/mL, E IC50~0.01 μg/mL), viral titers following HSV-2 infection were significantly reduced, with the activity of Tr being more potent at inhibiting HSV-2 than elafin [126]. A decrease in viral titers correlated with a decrease in production of pro-inflammatory cytokines, whereas the antiviral IFN-β response was increased. Furthermore, the recombinant molecules decreased viral attachment to cells [126]. In vivo, Tr-transgenic mice, mice generated to express the full elafin/Tr human gene, demonstrated reduced central nervous system viral load and TNF-α expression upon intravaginal infection [126]. The data provides evidence for elafin and its precursor's potential as anti-HSV therapeutics.
