*8.2. HIV*

In HIV-infected monocytes, α-MSH exhibited inhibitory properties against HIV-1. Previously, α-MSH inhibited pro-inflammatory cytokine production such as TNFα and IL-1β in blood from HIV-infected individuals [145]. Barcellini et al. [55] aimed to determine if α-MSH exhibits anti-HIV properties by utilizing chronically infected monocytic U1 cells and acutely infected monocyte-derived macrophages. Not only did U1 cells consistently produce α-MSH, but α-MSH and the tripeptide (KPV) both (10 μM) inhibited HIV expression at low concentrations comparable to physiological concentrations of α-MSH, with inhibition more pronounced at higher concentrations [146]. In addition, both peptides inhibited nuclear factor-kB (NF-kB) activation which is known to enhance HIV viral protein expression.

#### **9. Therapeutic Potential and Challenges of AMPs in Clinical Applications**

In recent years the antiviral properties of AMPs, both naturally occurring and synthetic, and their therapeutic potential have generated increasing interest within the academic and pharmaceutical communities. Many of the AMPs originally thought to demonstrate only antibacterial activity have been found to exhibit antiviral and immune modulatory capabilities. While to date few AMP-based therapies have been clinically approved, so far, the suitability and efficacy of only a few AMPs as therapeutic agents has been studied in clinical trials as treatments for infections as well as agents for immune modulations [147]. Clinical studies utilizing AMPs include defensin-mimetic compounds (CTIX 1278) for the treatment of drug-resistant *Klebsiella pneumoniae*, an alpha-helical AMP (PL-5) for gram negative and positive bacterial skin infections, and a cationic peptide (DPK 060) for skin dermatitis [148]. AMPs are promising therapeutics due to their relatively low toxicity and tolerance in vivo. AMPs can be delivered in a variety of ways including intravenous injections, oral administration, as topical ointments, and via inhalation. In respiratory tract infections, supplementation and treatment with AMPs such as defensins and LL-37 may provide protection in the lungs. For example, nebulizing mice with LL-37 prior to infection with IAV can reduce disease severity and result in increased survival rates [50]. The same concept could be applied against other respiratory pathogens such RSV and rhinovirus with the inclusion of AMPs such as lactoferrin and EDN, which have both previously demonstrated efficacy against RSV [79,121].

AMPs can also be applied as ointments, gels, or creams. For instance, a gel for the treatment of vaginal candidiasis currently in clinical trials is derived from human α-MSH [147]. As α-MSH has previously demonstrated antiviral activity against HIV [146], this form of treatment could be developed into a cheap and effective strategy to reduce HIV transmission. The treatment can be expanded to other sexually transmitted diseases, such as HSV, and include many of the other classes of AMPs demonstrating antiviral activity against those particular viruses. Equally, oral formulations or intravenous injections are more feasible for viral infections affecting internal organs. Lactoferrin has been used as an oral supplement to antiviral therapy against HCV infections and greatly enhances the effectiveness of treatment [98]. While most studies utilizing oral LF against other pathogens alone have not provided promising results, different AMPs may prove more effective against different pathogens. An intravenous treatment of human LF is currently in clinical trials for the treatment of bacteremia and fungal infections [147]. Interestingly, the protease inhibitor Telaprevir, which is marketed for use against HCV suggests that eosinophil peptides that have dual functionality as protease inhibitors and AMPs may possess anti-HCV properties. In addition, AMPs that directly target viruses can serve as alternatives to antivirals for which resistance has become an issue as it is a challenge for viruses to change their envelope lipid compositions. Nonetheless, more research and investigation are required to assess the full potential of AMPs as novel therapeutics.

However, despite their appeal and several successful in vitro outcomes following the use of AMPs as antiviral therapeutics, their greater application as therapeutics faces many hurdles. Little clinical data on the use, toxicity, efficacy, methods of delivery, or host uptake and the in vivo response to AMPs is currently available to guide the development of AMPs into therapeutic products [147,149]. It is unclear whether and to what extent laboratory findings will translate into antiviral activity in vivo. Additionally, the relationship between peptide concentrations used in vitro experiments and physiologically effective concentrations required for in vivo activity is unknown for many AMPs. As an example, the anti-bacterial activities of AMPs appear to heavily depend on the environmental conditions used during experiments [147]. Several AMPs have been reported to control group A Streptococcus at physiological concentrations which are ineffective in vitro [150], suggested to be partly due to the physiological ionic environment not being recapitulated fully in laboratory experiments [151]. Moreover, AMPs minimally active in in vitro assays have been shown to efficiently control infections upon in vivo administration [152,153], highlighting a lack of clear correlation between efficacy and dose-dependence of AMPs derived from laboratory experiments and animal models of infection. Further research into the correlation between antiviral activity of AMPs in vitro and the translation of this activity into in vivo efficacy will be needed for further development of AMPs as a new class of therapeutics.

Another important challenge is isolation and large scale manufacturing of AMPs. The high cost of producing AMPs impedes the applicability for clinical use as a commercial-scale manufacturing platform does not yet exists. The complex secondary structures of AMPs also serve as an obstacle for large-scale production as their activity is structure-based. The use of expression systems such as bacterial systems can generate "correctly folded peptides" in large quantities; however, the peptides are susceptible to proteolytic degradation in these systems [17]. To turn AMPs into viable therapeutics for clinical use, these production disadvantages must be overcome. Meanwhile, as an alternative, non-human AMPs are being used. For example, numerous studies are examining the activity of LF utilize bovine instead of human LF due to ease of producing bovine LF in bulk. While bovine LF demonstrates equivalent and at times enhanced efficacy, as compared to the human form, other AMPs may not function similarly or may contain sequence variations that limit or abolish their properties. Synthetic peptides may prove to be a practical alternative; however, they are expensive to generate, and future use in therapies will require development of novel synthesis methods [154,155]. Several systems have demonstrated promise in mass-production of AMPs including mammalian cells and transgenic plants. While mammalian cell culture systems are preferred to produce human AMPs, they are not very cost-effective. Plant bioreactors; however, are a cost-effective method for commercial-scale AMPs production that have demonstrated applicability with other compounds [17]. Another interesting approach that could possibly counteract these challenges that has recently gained attention is the employment of agents to induce or increase expression of AMPs. For example, vitamin D induces LL-37 production that can be used to boost the natural response to infection and has been suggested for use prior to potential DENV outbreaks [43].

Furthermore, the metabolic stability of AMPs may limit their future clinical application [156–158]. There is a lack of data assessing the pharmacokinetics of AMPs to address issues such as the physiological half-life of peptides, the necessary dosing regiments, and the aggregation of peptides in vivo. For

example, AMPs in oral and topical formulations will be subjected to enzymatic degradation, changes in pH and ionic concentrations, and face hurdles upon delivery, such as penetration of and uptake from the skin, intestinal, or nasal mucosa [159]. Intravenous administered AMPs will also face proteolytic degradation from enzymes in plasma and tissues, although the relatively shorter half-life of AMPs when compared to small-molecule drugs may be considered beneficial in some contexts due to lack of accumulation in tissues [159]. In addition, some AMPs are sensitive to salt or serum, a factor that substantially reduces their activity further limiting their applicability in clinical use.

The delivery of sufficient amounts of AMPs to an infection site may also present challenges and require careful calibration of dosing. For example, topical application of AMPs to the skin may require large-scale application of ointments, raising issues about sensitization and development of allergies against applied AMPs. Similarly, AMP activity in the context of natural innate immunity occurs in the presence numerous feedback pathways that collectively calibrate the anti-pathogen response to an appropriate level. It remains to be determined how the introduction of potentially high doses of AMPs during infections will interact with these pathways and modulate the activity of AMP therapeutics, especially as synthetic AMPs appear to elicit weaker immune responses upon administration when compared to the response produced by naturally secreted peptides during infection [160].

While these challenges may considerably impede clinical development of AMPs, the incorporation of innovative formulations can speed the process of bringing AMP products to clinical phases. The use of nanoparticles (NPs) as delivery vehicles may greatly improve the metabolic stability of AMPs. Nanoparticles can provide unique advantages, such as protection of AMPs from proteolytic degradation and control over the rate of peptide release. Several nanoparticles have been evaluated for delivery of AMPs. For example, self-assembling hyaluronic acid (HA) nanogels have been used to successfully deliver LLLKKK18, an analog of LL-37 via intra-tracheal administration in tuberculosis-infected mice [161]. Additionally, the HA nanocarrier was able to both stabilize the peptide and decrease peptide cytotoxicity in macrophages in vitro [161]. Poly lactic-co-glycolic acid (PGLA) NPs were also successfully utilized to deliver cationic AMPS [162]. Although these formulations have only been investigated in experimental animal models and in vitro applications, the outcomes for developing future AMP-based therapeutics appear promising.

#### **10. Conclusions**

Progress has been made in the last decade to elucidate the mechanisms of action of various AMPs. The primary mechanism of AMP-mediated antiviral activity has been attributed to direct interference with, and destabilization of, viral envelopes. However, AMPs have also demonstrated selective immune modulation. Antiviral activity against both enveloped and non-enveloped viruses has been reported with the latter hinting at the presence of undiscovered activities of AMPs, in addition to the known direct interaction with viral envelopes. Indeed, antiviral activity has also been reported at post entry steps affecting later stages in the viral life cycle, such as genome replication and viral protein trafficking. Additionally, studies have demonstrated that AMP treatment prior to viral infection results in peptide retention and internalization by cells which may reflect a more robust response to viruses compared to other potential therapeutics in development. Additionally, post-infection treatments have been reported to exhibit antiviral activity; however, to a lesser extent to that of treatments prior to infection. Nonetheless, these treatments play a role in altering viral replication and assembly as well as a role in accelerating immune activation or suppression. Hence, AMPs can directly impact viral infections or can modulate host processes that ultimately impact viral replication negatively. AMPs have been reported to drive interferon β (IFNβ) signaling, contributing to the induction of an antiviral state in susceptible cells. This dual functionality of AMPs is advantageous as they can be used as a prophylactic and/or as part of post-exposure antiviral measures. In vulnerable individuals, prophylactic expression of AMPs has the potential to become a preventative strategy against viral infections, especially during emerging pandemics. In addition, the simplicity of AMPs makes the development of synthetic peptide analogues a cost-effective measure to treat established viral infections. AMPs and their synthetic derivatives are a promising avenue to yield new strategies to control and treat a wide range of viral diseases but their application is still at the preliminary stages. Therefore, further research is warranted to understand AMP antiviral activity both in vivo and in vitro and to determine underlying mechanisms involved in AMP-mediated immune modulation for clinical applications.


**Table 1.** Mechanisms of actions of antiviral AMPs.

**Author Contributions:** Conceptualization, A.A., G.S.-T. and A.N.; manuscript drafting, A.A., G.S-T. and N.B.; literature search, A.A., G.S.-T., N.B. and G.H.; Editing, N.B. and A.A.

**Funding:** This work was supported by the Defense Threat Reduction Agency (HDTRA11810041).

**Conflicts of Interest:** The authors declare no conflicts of interest.
