Antiviral Activities of Human Host Defense Peptides

Abstract

Peptides with broad-spectrum antimicrobial activity are found widely expressed throughout nature. As they participate in a number of different aspects of innate immunity in mammals, they have been termed Host Defense Peptides (HDPs). Due to their common structural features, including an amphipathic structure and cationic charge, they have been widely shown to interact with and disrupt microbial membranes. Thus, it is not surprising that human HDPs have activity against enveloped viruses as well as bacteria and fungi. However, these peptides also exhibit activity against a wide range of non-enveloped viruses as well, acting at a number of different steps in viral infection. This review focuses on the activity of human host defense peptides, including alpha- and beta-defensins and the sole human cathelicidin, LL-37, against both enveloped and non-enveloped viruses. The broad spectrum of antiviral activity of these peptides, both in vitro and in vivo suggest that they play an important role in the innate antiviral defense against viral infections. Furthermore, the literature suggests that they may be developed into antiviral therapeutic agents.

1. INTRODUCTION

Much of the world’s population is currently infected with at least one virus, with essentially every person having a viral infection at one point in life. While not all viral infections lead to disease, a new pathogen introduced to a person can lead to disease, and the history of past pandemics leads everyone at risk for new infections. Even well-characterized diseases caused by viral infections either still do not have cures or treatments, or do not have effective vaccines or treatments. There is evidence that current antiviral treatments are losing efficacy: an alarming parallel to the rise of many antibiotic-resistant bacterial strains [1]. The need for new ways to treat or potentially prevent viral infections, or prepare for future emerging ones, is evident.

One such study for the prevention and treatment of viral infections is that of host defense peptides or HDPs. HDPs, also known as antimicrobial peptides, or AMPs, represent an early form of innate immunity, with all known forms of life, from bacteria to humans, expressing these peptides [2]. HDPs are small, mostly cationic and amphipathic peptides with a broad spectrum in vitro antimicrobial activities [3]. In humans, these include alpha- and beta-defensins, along with the lone human cathelicidin, LL-37. While these HDPs exhibit potent activity against pathogenic bacteria [4], as well as multi-drug resistant fungi [5], they are also known to directly inhibit viral infections. While other host defense peptides possess antiviral activities, such as mucins, lactoferrin, and secretory leukocyte protease inhibitor (SLPI), the bulk of this review will focus on the main classes of human HDPs: defensins and LL-37.

These peptides were initially characterized by their broad-spectrum antibacterial and antifungal activities, which have been extensively described [6]. In contrast, their antiviral activities are not as well understood, especially with respect to their mechanism of action. For inhibiting bacterial or fungal infections, HDPs seem to focus on perturbing the membrane of the microorganism [7]. However, HDPs seem to have a greater repertoire of known activities against viruses; not only do HDPs disrupt viral envelopes, but also directly affect host cells and specific steps of viral life cycles. These more specialized roles of HDPs for viral infections showcase not only the evolutionary maturity of these peptides to battle viruses but also the potential to use them to fight infections for which we do not currently have proper methods to prevent or treat. Human HDPs have been shown to be effective not only in vitro, but also in vivo to inhibit viral infections. While a number of studies have investigated the potential to use these HDPs exogenously, induce their expression endogenously, or even use small molecule mimetics of the peptides, the capacity of HDPs to inhibit viral infections in a number of ways warrants further examination in these areas in the context of activities against viruses.

2. ANTIVIRAL ACTIVITIES OF DEFENSINS

Defensins are cationic, cysteine-rich peptides, characterized by specific cysteine motifs and the presence of arginines and leucines (reviewed extensively previously [8, 9]). Based on structural and evolutionary characteristics, defensins are divided into alpha-defensins (α-defensins), beta-defensins (β-defensins) and θ-defensins (θ-defensins) (which are only found in certain non-human primates [10]). α-defensins are found in high concentrations in human neutrophils, as well as in human small-intestinal Paneth cells. In mice, α-defensins are found highly expressed in the Paneth cells (although not in the neutrophils). While α-defensins have only been found in mammals, β-defensins are much more broadly expressed, present in epithelial and myeloid cells from a number of fish, bird, and mammal species [11]. Human β-defensins (HBD)1, 2, 3, and 4 are expressed in a variety of mucosal epithelial cells, and as such, are proposed to participate in the mucosal innate immune defense against microbial colonization.

Human neutrophil α-defensins are generally referred to as human neutrophil peptide (HNP)1, 2, 3 and 4, with HNP1, 2 and 3 being the most abundant. As with many host defense peptides, they are produced as inactive prepropeptides. The peptides undergo proteolytic maturation during neutrophil differentiation, and the mature, active peptides are found only in mature neutrophils, where they exist at high concentrations in the azurophilic granules. Enteric α-defensins, human defensins (HD)5 and HD6, are expressed in the Paneth cell as inactive precursors, which are secreted into the lumen of the intestine, where they are proteolytically activated by trypsin into the mature form.

In contrast, human β-defensin genes are transcriptionally regulated, producing pre-peptides, which are predicted to be cleaved to the active form by signal peptidase [12]. Expression of human β-defensin genes can be regulated as part of innate immune response. They are induced by the TLR-mediated response to bacteria, viruses, and other microbial patterns. While defensins are more well-known for their activity against bacteria and fungi, antiviral properties of defensin have been well-studied [13–19], including human defensins specifically [20–22]. These antiviral activities of defensins go beyond the main mechanism of inhibiting bacteria defenses by disrupting the microorganism outer membrane and instead act on a wide variety of both viral and host cell components.

2.1. Human Defensin Activity Against DNA Viruses

2.1.1. Human Defensin Activity Against Enveloped DNA Viruses

While vaccinia virus infection of the African green monkey kidney cell line BS-C-1 was shown to increase HBD3 expression and the subsequent production of the peptide inhibited viral gene expression and overall infection [23], all other antiviral studies of human defensins against enveloped DNA viruses have focused on herpesviruses (Table 1). Herpesviruses represent some of the most ubiquitous infections known to humankind, with around 98% of all people infected with at least one virus of the Herpesviridae family [24–26]. The initial discovery of human α-defensins, and first ever use of the term “defensin,” in 1985 involved the ability of the newly characterized peptides, HNP1-3, to inhibit herpes simplex virus type 1 (HSV-1), among other microbes, infection of the monkey kidney epithelial cell line Vero [27]. One year later, the same group further characterized the antiviral activities of HNP1 by demonstrating the ability of the peptide to inhibit other herpesviruses: HSV-2 and cytomegalovirus (CMV) [28]. This study also was the only examination of anti-CMV activity by HDPs besides a later paper demonstrating the decrease in neutrophil inactivation of the virus upon addition of anti-HNP1-3 and anti-LL-37 antibodies [29]. Almost twenty years passed, with only a few more studies of anti-herpesvirus activities of HNP1 and HNP2 [30, 31], before a potential mechanism for the anti-HSV activity of HNP1-3 was hypothesized: the peptides were found to decrease the expression of the viral transcription factors VP16 and ICP4 during HSV-2 infection of the human metastatic cervical cell line ME-180 [32].

Table 1.

Known antiviral activities of human defensins against enveloped DNA viruses [21].

Virus\Defensin	HNP1	HNP2	HNP3	HNP4	HD5	HD6	HBD1	HBD2	HBD3	HBD4
Herpesviruses	-	-	-	-	-	-	-	-	-	-
CMV	U	U	U	*	*	*	*	*	*	*
HSV-1	U	U	U	*	U	*	U	*	U	*
HSV-2	Binds gB to prevent attachment, inhibits VP16 transport and viral gene expression post-infection	Binds gB to prevent attachment, inhibits VP16 transport and viral gene expression post-infection	Binds gB to prevent attachment, inhibits VP16 transport and viral gene expression post-infection	Binds to heparan sulfate to prevent attachment	Binds gB to prevent attachment, inhibits viral gene expression post-infection	Binds to heparan sulfate to prevent attachment	NE	NE	Binds to gB and heparan sulfate to prevent attachment	*
VZV	*	*	*	*	*	*	*	U	*	*
Vaccinia	NE	*	*	*	*	*	NE	NE	U	*

NE: Tested HDP had no effect, U: Antiviral mechanism is undetermined,

^*:Yet to be tested.

Table shows most common result of HDP antiviral test.

The ability of the other human α-defensins (HNP4, HD5, and HD6) to prevent HSV infection was then demonstrated for the first time using another human metastatic cervical cell line CaSki [33]. This same study also provided the first evidence of human β-defensin anti-HSV activity where HBD3, but not HBD1 or HBD2, inhibited HSV-2 infection. Interestingly, despite the overall homology between HSV-1 and HSV-2, all the peptides tested inhibited the two viruses in different manners: defensin activity against HSV-1 was effective post-entry, while anti-HSV-2 activity was observed both before and after internalization of the virus into the host cell. HNP1-3 and HD5 all bound to the gB envelope protein of HSV-2 to prevent attachment of the virus to the cell in addition to specifically inhibiting immediate-early and late viral protein expression, with HNP2 and HD5 exhibiting the ability to bind directly to viral DNA. HNP4 and HD6 also prevented viral attachment, but as opposed to the other α-defensins, these peptides bound to the heparan sulfate on the surface of the host cell. HBD3 directly interacted with both gB and heparan sulfate to prevent viral binding.

Despite the earlier study in which only HBD3 was able to inhibit HSV-2 infection [33], later evidence suggested that HBD1 also possessed anti-HSV-2 activity, albeit at a much lower efficacy than HBD3 [34]. This investigation also determined the regions of the peptides responsible for the observed antiviral activity, with HBD1 retaining anti-HSV-2 capabilities with the internal region of the peptide and HBD3 with the N-terminal region. Using only these regions also did not diminish the chemotactic ability of the peptides, suggesting a link between the direct antiviral and chemotactic regions of β-defensins. A later study demonstrating the ability of HSV-1 infection of human peripheral blood mononuclear cells (PBMCs) and plasmacytoid dendritic cells (PDCs) to increase HBD1 expression further implicates HBD1, and not only HBD3 and α-defensins, in the antiviral response against herpesviruses [35]. While HBD2 has not of yet been found to inhibit HSV infections, the peptide was able to diminish replication of varicella-zoster virus (VZV), another herpesvirus, in HaCaT human skin cell line [36]. With pre-incubation of the virus and peptide not enhancing the antiviral activity of HBD2, the peptide most likely exerts its activity either via the host cell or after the virus enters, much like the other herpesviruses and defensins, suggesting a common evolutionary trait between the peptides in terms of innate antiviral defenses against herpesviruses.

The only in vivo data for human defensin activity against herpesviruses came from a study examining the anti-HSV-2 capabilities of HD5 [37]. In addition to HD5 preventing HSV-2-induced CaSki cell death in vitro, both intravaginal prophylactic and therapeutic treatment of mice infected with HSV-2 decreased both local tissue pathology and overall lethality of the virus. In terms of endogenous human defensins, HNP1-3 levels also correlated with the ability of cervicovaginal lavages (CVLs) of human patients to inhibit HSV-2 infection of CaSki cells [38], while both HNP1-3 and HBD1-3 levels were associated with decreased infection of Vero cells [39]. These in vivo data hold promise for future studies of human defensins in the potential treatment and prevention of herpesvirus infections.

2.1.2. Human Defensin Activity Against Nonenveloped DNA Viruses

2.1.2.1. Human Defensin Activity Against Human Adenoviruses

Previous work in defensin research, especially their antiviral activities, was of particular interest to those studying adenoviral gene therapy vectors since these peptides might affect the efficacy of their potential viral treatments. The first study of any HDP against adenoviruses found that transfection of plasmids containing genes encoding HBD1 and HD5 into the human carcinoma cell line HT-29 inhibited the ability of human adenovirus type 1 (HAdV-1) to deliver the CFTR gene into the cells [40]. This investigation was the only evidence for anti-HAdV activity of human β-defensins, as other studies have shown the inability of HBD1 and HBD2 to inhibit HAdV types 3, 5, 8, or 19 [41–43] and HBD2 to inhibit HAdV-35 [43] (Table 2).

Table 2.

Known antiviral activities of human defensins against nonenveloped DNA viruses [21].

Virus\Defensin	HNP1	HNP2	HNP3	HNP4	HD5	HD6	HBD1	HBD2	HBD3	HBD4
HAdV	-	-	-	-	-	-	-	-	-	-
A	U	*	*	*	Binds to virus, inhibits endosomal lysis and viral uncoating	*	*	*	*	*
B	U	*	*	*	Binds to virus, inhibits endosomal lysis and viral uncoating	*	NE	NE	*	*
C	Inhibits endosomal lysis	*	*	*	Binds to virus, inhibits endosomal lysis and viral uncoating	*	U	NE	*	*
D	NE	*	*	*	NE	*	NE	NE	*	*
E	U	*	*	*	Binds to virus, inhibits endosomal lysis and viral uncoating	*	*	*	*	*
F	NE	*	*	*	NE	*	*	*	*	*
HPV	Binds to virus, inhibits endosomal lysis and viral uncoating	U	U	U	Binds to virus, inhibits endosomal lysis and viral uncoating	NE	NE	NE	*	*
Polyomavirus	-	-	-	-	-	-	-	-	-	-
BKV	U	*	*	*	Binds to virus, inhibits attachment	*	NE	U	*	*
JCV	U	*	NE	*	U	*	U	U	U	NE
SV40	NE	*	*	*	U	*	NE	NE	*	*

NE: Tested HDP had no effect, U: Antiviral mechanism is undetermined,

^*:Yet to be tested.

Table shows most common result of HDP antiviral test.

The most well-characterized interactions of human defensins and HAdVs have been between HNP1/HD5 and HAdV-5/HAdV-35 (reviewed previously [44]). HNP1 was found to inhibit HAdV-5 during in vitro experiments using the human embryonic kidney cell line HEK293 [41] and later the human cancerous lung cell line A549 [43]. This study of HNP1 against HAdV-5 in A549 cells found that the peptide prevents endosomal lysis by the virus [43], an important step of the viral life cycle once internalized. Further investigation into the role of HNP1 activity against HAdV-5 demonstrated a more efficacious ability to inhibit viral infection when the virus was already bound to the host cell as opposed to pre-incubating the virus and peptide together before infection [45], a phenomenon dictated by the total number of peptide bound to the virion as opposed to affinity of the peptide to the virus.

HD5 activity against HAdVs, especially in terms of mechanism of action, has been the most well defined of all human HDPs. Compared to HNP1, HD5 also exhibited greater efficacy in inhibiting HAdV-5 and HAdV-35 infections of A549 cells [43]. HD5 also decreased viral protein production in human enteroid cells [46]. The peptide was found to bind directly to HAdV-5 virions, leading to inhibition of entry into A549 cells and virus-mediated endosome penetration and subsequent disassembly of the viral particle necessary for later stages of the viral life cycle [43, 47]. The mechanism behind this particular inhibition was further elucidated with the discovery that HD5 binds to the HAdV-5 capsid [48], specifically at the penton base [45], decreasing infection but not via aggregation of the virus. This binding was mediated by the avidity of the hydrophobic face of the peptide as opposed to the overall affinity of HD5 to the virion [45]. The specific interactions of HD5 at the fiber and penton base viral capsid proteins prevented the vertex removal and release of viral protein VI used in endosomalysis [49]. Further resolution of HD5 binding to the HAdV-5 capsid [50], along with similar interaction of the peptide with the capsid of HAdV-35 and downstream endosomal lysis have also been studied [51].

In addition to HAdV-5 and HAdV-35, HNP1 and HD5 were also able to inhibit HAdV species A, B1, B2, C, and E, but not D or F [49] and HD5 failed to inhibit HAdV41p [46]. HNP1 itself was shown to prevent HAdV-3 infection [42]. Interestingly, HAdV-3-produced free penton-dodecahedra capsid proteins, and prevented the antiviral activity of HD5 [52]. HNP1 and HNP2 have also exhibited the only known HDP antiviral activity against another common viral gene therapy vector, Adeno-Associated Virus (AAV), where a combination of the peptides inhibited the ability of AAV to deliver GFP to the cystic fibrosis cell line IB3-1 [53]. These examples of host-pathogen interaction, along with the other antiviral activities of human defensins against HAdV and AAV, underscore the importance of this peptide, and defensins and HDPs in general, in innate defenses against this group of viruses, especially in the context of potential viral vector-based therapies.

2.1.2.2. Human Defensin Activity Against Human Papillomaviruses

While most human papillomavirus (HPV) infections are cleared without symptoms, some go on to lead to cancers most notably of the cervix, mouth, and throat [54]. The majority of these cancers are caused by the HPV16 and HPV18 types of the virus, with HPV6 and HPV11 also associated with genital and pharyngeal non-cancerous lesions [54]. Understandably more of the focus of defensin antiviral activity has been placed on the cancer-causing types, with only one study to date examining HPV6 and HPV11, where the addition of a plasmid containing the gene encoding HNP1 to condylomaacuminatum tissue infected with HPV6 and HPV11 led to a specific decrease in viral gene expression and apoptosis of infected cells [55].

HNP1-4 and HD5 were found to inhibit HPV16 infection of the cervical cancer cell line HeLa, while HD6, HBD1, and HBD2 peptides had no effect [56]. The idea that these human defensins could act as an innate antiviral defense against HPV was supported by a study later that year that demonstrated the upregulation of HDPs in HPV-infected lesions [57], and a subsequent study in amniotic epithelial cells [58]. However, the defensins that were upregulated in this study were HBD1-3, which were previously shown to have no direct effect against HPV16 [56]. Another study found that, despite not having a direct antiviral effect, HBD2, as well as HNP2, acted as chemotactic agents for dendritic cells to infiltrate HPV-infected keratinocyte tissue to potentially expedite the adaptive immune response to the virus [59]. Similarly, polymorphisms in the gene encoding for HBD1 have been associated with susceptibility to HPV infection [60]. Levels of CVL HNP1-3 correlated with HPV infection of the human immunodeficiency virus (HIV)+ individuals compared to non-HPV infection, further suggesting defensins as an endogenous innate antiviral response against HPV.

More direct effects by human defensins were found with HNP1 and HD5 (Table 2), with similarities to those against another class of nonenveloped DNA viruses, HAdV. Even though HNP1 and HD5 did not prevent viral binding to HeLa cells, the peptides blocked HPV16 virion escape endocytic vesicles [56]. In addition, the avidity of the hydrophobic face of HD5 allowed it to bind to the HPV16 capsid, leading to antiviral activity, which was determined by the total number of peptides bound as opposed to affinity to the capsid [45]. In addition to the HAdV-5-related stabilization of the HPV16 capsid and redirection of the virion to the lysosome by HD5 [61], the peptide also inhibited HPV16 infection of HeLa cells by preventing cell surface furin from cleaving the viral capsid protein L2, a major step in the internalization process of the virus [62]. Overall, Arg28 seemed to be the most important amino acid for HD5 inactivation of both HAdV-5 and HPV16, while changing Arg9 was only detrimental in defending against HAdV-5 [48]. Understanding the potential of human defensins to prevent HPV infections could lead to a decrease in the number of people diagnosed with cancers associated with this viral infection.

2.1.2.3. Human Defensin Activity Against Polyomaviruses

Some of the more well-known members of the Polyomaviridae family include two viruses named after the initials of patients from which the viruses were isolated: B.K. [63] and J.C. [64, 65] (BKV and JCV or BKPyV and JCPyV). Most people infected with these viruses are asymptomatic, but immunosuppression can lead to mild symptoms in the case of BKV [66] and Progressive Multifocal Encephalopathy (PML), a fatal inflammation of multiple sites of white matter in the brain, during JCV infection [67]. The other well-known virus in the family is simian vacuolating virus 40 (SV40), a virus originally discovered as a contaminant in poliovirus vaccines, which used already infected rhesus monkey kidney cells during vaccine production [68]. The connection between this virus and a number of human cancers is still being explored [69]. Human defensins have been the only human HDP studied for antiviral activity against these viruses. HD5 seemed to have the best activity against the polyomaviruses as it was the only defensin tested that was able to inhibit BKV, JCV, and SV40 (Table 2) [70, 71]. The peptide was also the most studied in terms of antiviral mechanisms against polyomaviruses, where HD5 was found to bind to the BKV virion and inhibit attachment to the host Vero cell [70]. HD5 also bound to and stabilized the JCV capsid after entry into the human astrocyte cell line SVG-A as well as decreased traffic of the virus to the endoplasmic reticulum where the virus normally uncoats [71]. HNP1 was also able to inhibit BKV infection of Vero cells at early points of infection, but was either not as effective against JCV as HD5 or not effective at all based on the cell type used to infect and had no activity against SV40 [70, 71]. This same phenomenon was also observed with HBD2 [70, 71]. While neither HNP2, HNP4, nor HD6 was tested against any polyomavirus, HNP3 did not possess any activity against JCV [71]. For the remaining human β-defensins, HBD1 had no effect on either BKV or SV40 [70] and was only able to inhibit JCV with less efficacy than HD5 for infection of Vero cells [70], but had no antiviral activity during JCV infection of SVG-A cells [71]. HBD3 and HBD4 were only examined during JCV infection with HBD3 showing antiviral activity while HBD4 had no activity [71]. Overall, HD5 displayed the greatest ability to inhibit not only these polyomaviruses, but all the nonenveloped DNA viruses studied to date, suggesting a potential broad-spectrum therapy based on this peptide to prevent or treat a number of infections associated with these nonenveloped DNA viruses.

2.2. Human Defensin Activity Against RNA Viruses

2.2.1. Human Defensin Activity Against Group IV Viruses

One of the first ever studies of human defensins measured the ability of HNP1 to inhibit a number of viruses, including a number of enveloped viruses [28]. This particular paper also demonstrated the inability of HNP1 to act against the nonenveloped reovirus (Group III) and echovirus (Group IV) and was the only insight into potential human HDP activity against any Group III virus or echovirus [28]. Similarly, HBD2 was found to be ineffective against another Group IV virus, human rhinovirus (HRV) [72]. The only evidence of a direct inactivation of a Group IV virus by human defensins for many years, until a recent paper demonstrated the ability of HBD3 to inactivate a number of picornaviruses [73], came from a study examining hepatitis C virus (HCV) [74] a member of the Flaviviridae family responsible for its namesake hepatitis C along with downstream liver cancers and lymphomas [75]. This paper demonstrated the ability of combinations of either HNP1-4 or HBD1-4 to diminish viral gene expression and cytotoxicity associated with infection in PBMCs and the HuH-7.5 liver cell line [74]. While both sets of defensins acted directly on the virion to prevent viral entry into the host cell (Table 3), the HNP1-4 set had a higher efficacy, suggesting an inherent ability of α-defensins in general to defend HCV infections more effectively than β-defensins. Concentrations of both α- and β-defensins have been demonstrated to be elevated in the sera of HCV-infected individuals compared to healthy controls [76], despite α-defensins not displaying as much of an inhibitory effect. While other Group IV viruses have not been examined in the context of direct inactivation studies using defensins, a number of correlative studies have found connections between defensins and immune responses against dengue virus [77, 78] (DENV), hepatitis B virus [79] (HBV), HRV [80, 81], and Middle East respiratory syndrome-related coronavirus [82] (MERS-CoV). More direct experiments will determine the efficacy of defensins against these viruses.

Table 3.

Known antiviral activities of human defensins against group IV viruses [21].

Virus\Defensin	HNP1	HNP2	HNP3	HNP4	HD5	HD6	HBD1	HBD2	HBD3	HBD4
Enveloped	-	-	-	-	-	-	-	-	-	-
HCV	Prevent viral entry	Prevent viral entry	Prevent viral entry	Prevent viral entry	*	*	Prevent viral entry	Prevent viral entry	Prevent viral entry	Prevent viral entry
Nonenveloped	-	-	-	-	-	-	-	-	-	-
Picornavirus	-	-	-	-	-	-	-	-	-	-
Coxsackievirus	*	*	*	*	*	*	*	*	U	*
Echovirus	U	*	*	*	*	*	*	*	*	*
Enterovirus	*	*	*	*	*	*	*	*	Prevent viral entry	*
Poliovirus	*	*	*	*	*	*	*	*	U	*
HRV	*	*	*	*	*	*	*	NE	*	*

NE: Tested HDP had no effect, U: Antiviral mechanism is undetermined,

^*:Yet to be tested.

Table shows most common result of HDP antiviral test.

2.2.2. Human Defensin Activity Against Group V Viruses

Influenza A virus is one the most well-studied viruses in the antiviral defensin field (reviewed previously [83]), beginning with one of the first human defensin experiments [28], which demonstrated the ability of HNP1 to inhibit influenza A virus infection of Vero cells. HNP1, as well as HNP2, remains as the most studied human defensin in anti-influenza research. In addition to several correlative studies between human defensins and anti-influenza immune responses [35, 80], HNP1 and HNP2 specifically were able to suppress viral protein production in the canine kidney cell line MDCK [84], in addition to inhibiting overall viral infection of MDCK and human bronchial/tracheal epithelial (HBTE) cells [85]. HNP1 also inhibited influenza A virus infection of human small airway epithelial (SAE) cells [86]. These inhibitions were determined to be due to the ability of HNP1 and HNP2 to aggregate the influenza virions [85] (Table 4). This aggregation of influenza virus by HNP1 and HNP2, along with HD5, was also shown to lead to increased viral uptake by neutrophils to clear the virus, preventing further infection [87]. Despite this study where HNP3, as well as HBD1 and HBD2, was unable to lead to increased influenza uptake by neutrophils, a later paper demonstrated HNP3 capable of doing so [88]. This same study also exhibited the only evidence of anti-influenza activity of HNP4, HD6, HBD1, and HBD2, where these peptides inhibited virus infection of A549 cells [88]. Although these antiviral mechanisms were not defined, others have been hypothesized beyond virion aggregation and increased uptake by neutrophils: inhibition of cellular protein kinase C (PKC) activation [89] and inhibition of viral membrane fusion to cellular endosomes by HNP1 [90].

Table 4.

Known antiviral activities of human defensins against group V viruses [21].

Virus\Defensin	HNP1	HNP2	HNP3	HNP4	HD5	HD6	HBD1	HBD2	HBD3	HBD4
Influenza A virus	Aggregates and increases neutrophil uptake of virus, inhibits PKC activation and viral envelope fusion with endosome	Aggregates and increases neutrophil uptake of virus	Aggregates and increases neutrophil uptake of virus	U	Increases neutrophil uptake of virus	U	U	U	Inhibits HA-mediated fusion and membrane protein mobility	*
HPIV	*	*	*	*	*	*	*	U	*	*
RSV	*	*	*	*	*	*	NE	Disrupt envelope	*	*
VHSV	Binds to viral envelope G protein and induce TLR3 and ISG expression	*	*	*	*	*	*	*	*	*
VSV	Prevent viral entry	*	*	*	*	*	*	*	*	*

NE: Tested HDP had no effect, U: Antiviral mechanism is undetermined,

^*:Yet to be tested.

Table shows most common result of HDP antiviral test.

In addition to influenza A virus, other Group V viruses have also been shown to be inhibited by human defensins. Shortly after its discovery, HNP1 was found to prevent vesicular stomatitis Indiana virus (VSIV or VSV) infection of Vero cells [28], later determined to act via inhibiting viral entry into host cells [90]. HNP1 also decreased viral hemorrhagic septicemia virus (VHSV) infection of in vitro fish cells by not only binding to viral envelope G protein to inhibit viral attachment, but also activating TLR3, leading to the production of downstream antiviral interferon-stimulated genes (ISGs) [91]. Antiviral activity of HBD2 was also tested against the respiratory syncytial virus (RSV) and human parainfluenza virus (HPIV) [92]. While both pre-treating the virus or cell with HBD2 led to the diminished infection of A549 cells, RSV infection seemed to be even more sensitive to peptide pre-incubation with the virus due to envelope disruption and subsequent prevention of viral entry. HPIV infection, however, was equally affected by pre-treatment of either the virus or cells with HBD2, suggesting some aspect of the HPIV envelope makeup or morphology preventing HBD2 disruption of the viral lipid bilayer. The idea of HBD2 as an innate defender against Group V viruses was further bolstered by a later study in which serum HBD2 levels of patients with Crimean-Congo Hemorrhagic Fever Virus (CCHFV) were found to be elevated compared to healthy controls [93]. Overall, human defensins possess seemingly novel methods of antiviral defense against this particular group of viruses that could eventually be used in conjunction with standard vaccination plans against influenza and treatments of other related diseases.

2.3. Human Defensin Activity Against HIV

HIV, the etiological agent of Acquired Immune Deficiency Syndrome (AIDS), currently infects around 40 million people around the world, with about two million new cases every year [94]. While Highly Active Antiretroviral Therapies (HAART) have helped to decrease the number of new cases by preventing already infected individuals from spreading the virus, there is still no approved vaccine to protect uninfected individuals from becoming infected [95]. After the initial discovery of the virus leading to AIDS [96, 97], human defensins were tested in vitro as a potential way to protect people from infection [98]. Overall, HIV (more specifically HIV-1) has become the most studied virus in the context of antiviral activities of HDPs, including human defensins (reviewed extensively [99–106]). Early papers examined the ability of both α- and β-defensins in breast milk to prevent transmission of HIV-1 from mother to child [107–110]. Polymorphisms in the gene encoding HBD1 were also found to correlate with risk of HIV-1 infection in both Italian children [111] and Mexican women [112], while levels of defensins in the genital tracts of HIV-negative women from the United States were significantly greater than HIV-negative women from Africa [113], potentially contributing to the overall greater rate of HIV infection in Africa. The idea that defensins could act as a natural defense against HIV was further supported by a number of studies linking HIV-1 infection to increased defensin production [114–117].

Early in vitro studies of antiviral human defensins against HIV-1 led to confusion as to the source of HNP1-3 that was found to have anti-HIV activity. HNP1-3 was initially thought to be secreted from CD8⁺ T cells as part of their overall antiviral adaptive immune response [118], but this was ultimately due to neutrophil contamination of CD8⁺T cell isolations [119, 120]. Regardless of the source of the peptides, HNP1 was determined to act through the host cell, as pre-treatment of HeLa cells was sufficient for peptide inhibition of the virus [121]. However, when using human CD4⁺ T cells as the target for HIV-1 infection, HNP1-3 seemed to act directly on the virion [120]. HBD2 and HBD3, but not HBD1, also directly affected the virion, in addition to downregulating the expression of CXCR4 (another CD4⁺ T cell surface protein used by HIV-1 for initial binding) in GHOST X4/R5 cells [122, 123], while HBD3 both bound to, and initiated internalization of CXCR4 in human T cell line CEM X4/R5 [124]. This downregulation was also observed with treatment of GHOST X4/R5 cells with HNP1 [123]. HD5 inhibition of HIV-1 infection of the modified Jurkat cell line JLTRG [37] was later hypothesized to be due to downregulation CXCR4 in addition to binding to CD4 [125]. CD4 downregulation was observed with HNP1-2 treatment of the mouse embryo fibroblast cell line NIH-3T3 transfected to express CD4 [126].

Much of human defensin anti-HIV activity revolves around binding to viral and/or cellular surface proteins to prevent initial virus attachment and internalization (Table 5). HNP1-4 were found to inhibit HIV-1 infection of human PBMCs [127, 128], with HNP4 having the greatest inhibitory effect most likely due to its relative lack of binding to serum [127]. HNP1-3 bound to HIV gp120, while HNP1 and HNP2 bound to both T cell CD4 along with gp120, two important surface receptors in the infection process [127, 129]. The presence of amino acid W26 in HNP1 [130], in addition to oligomerization of the peptide [131, 132] was later determined to be the most important for HNP1 binding to HIV gp120. At sub-inhibitory concentrations, HNP1 also bound to HIV envelope protein gp41 to stabilize the viral protein, leading to enhanced inhibition by anti-gp41 antibodies [133]. HBD2 and HBD3 were shown to compete with gp120 for heparan sulfate proteoglycans (HSPGs), which are a site of viral binding to host cells [134]. A more intricate method defensins use against HIV-1 binding was outlined when it was discovered that HNP1 and HNP2 increased the release of CCL3 and CCL4 by human monocytes, which bind to CCR5 on the surface of CD4⁺ T cells and prevent HIV-1 attachment [135].

Table 5.

Known antiviral activities of human defensins against HIV [21].

Virus\Defensin	HNP1	HNP2	HNP3	HNP4	HD5	HD6	HBD1	HBD2	HBD3	HBD4
HIV-I	Binds to CD4 and gp120, downregulates CD4 and CXCR4 expression, induces expression of CCR5 ligands, inhibits PKC activation, prevents viral entry	Binds to CD4 and gp120, downregulates CD4 expression, induces expression of CCR5 ligands	Binds to gp120	Binds to CD4 and gp120	E	E	U	Binds to virus, competes with virus for HSPGs, downregulates CXCR4, induces APOBEC3G expression	Binds to virus, competes with virus for HSPGs, downregulates CXCR4	*

E: Tested HDP enhanced viral infection, NE: Tested HDP had no effect, U: Antiviral mechanism is undetermined,

^*:Yet to be tested.

Table shows most common result of HDP antiviral test.

Human defensins have also demonstrated anti-HIV activities beyond those mediated by preventing viral attachment to host cells. HNP1 activity seems to be affected by serum: without serum, the peptide acts directly on the virus, but in the presence of serum, HNP1 exerts its anti-HIV-1 activity through the host cell both before and after viral entry [90, 136]. HNP1 binding to CD4 and HIV-1 is independent of serum availability while its anti-endocytic activity is diminished by serum. HNP1 may act as serum-sensitive oligomers (as described above) for certain aspects of anti-HIV-1 activity and not for others, which may lead to the discrepancy between observations of virus-targeting or cell-targeting aspects of the peptide from previous experiments (also described above). While HBD1-3 have been shown to decrease HIV-1 viral replication in human PBMCs [137, 138], only the mechanism of HBD2 anti-HIV-1 activity was further examined. HBD2 possessed longer-lasting anti-HIV-1 properties compared to HBD1 and inhibited not only early, non-virus-cell-fusion, aspects of infection [137], but also led to the expression of ISG protein product apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G (APOBEC3G), which interferes with multiple steps of HIV-1 replication [138].

Fairly unique in the antiviral HDP field of research, HIV infection has, on multiple occasions, been positively associated with certain defensins. HNP1 [139], along with HD5 and HD6 [140–143] have been shown to increase HIV-1 infection in vitro. The route to determine HD5 and HD6 as potentially pro-HIV-1 was especially circuitous. After the initial finding that the two peptides increased HIV-1 entry [140], specifically attachment [141], another group suggested the non-physiologically relevant bovine serum used in the experiments altered the potential normal antiviral abilities of the peptides [125]. The first group responded to this criticism by demonstrating that serum blocked the cytotoxicity of HD5 [142], which was mistaken for antiviral activity in the contrasting paper. The idea of HD5 and HD6 normally acting to enhance HIV-1 infection was supported by a later study outlining the specific regions of the peptides responsible for their pro-HIV-1 activities [143]. The report also outlined which amino acids of HD5 and HD6 would need to be changed in order to lead to a decrease in HIV-1 infection. Overall, use of these new peptides along with other endogenous or engineered human defensins, based on their robust activities on both the virus and host cell, could potentially be used as a method of preventing HIV-1 infections in people not already in contact with the virus and could also be used in conjunction with current treatments of HIV-infected individuals to further suppress viral loads and spread of the virus.

3. ANTIVIRAL ACTIVITIES OF LL-37

Cathelicidins are a group of cationic host defense peptides mostly found in the myeloid and epithelial cells of mammals, but this class of peptide has also been observed in fish, reptiles, birds, and amphibians [144]. The lone known human cathelicidin is human cathelicidin antimicrobial peptide, 18 kDa (hCAP-18) [145], which is encoded by the CAMP gene. Humans constitutively express CAMP in epithelial cells, with elevated levels of the mature peptide observed during inflammatory responses, as part of the first line of defense against pathogens [146]. Phagocytic cells store the precursor peptide hCAP-18 in granules and release the preproprotein upon stimulation with TLR agonists or certain cytokines [146].

LL-37 has mostly been studied in the context of inhibiting bacterial infections, with the main antibacterial mechanism being disruption of the bacterial membrane after electrostatic interactions between the cationic peptide and the negatively charged membrane and subsequent pore formation due to LL-37’s amphipathicity [147]. However, the field of cathelicidin research has grown to incorporate studies of the class of peptides in the context of antiviral activities [15, 148]. Interestingly, while the main mechanism of viral inhibition of LL-37 seems to be disruption of the viral envelope (much like its perturbation of bacterial and fungal membranes), LL-37 plays a role in inhibiting specific aspects of the viral life cycle in addition to enhancing the innate immune response to viruses (Table 6).

Table 6.

Known antiviral activities of LL-37.

Virus	CMV	HSV-1	HSV-2	KSHV	VZV	Vaccinia	HAdV A	B	C	D	E	F	HPV
LL-37	U	Increase IFNB expression, inhibit virus binding	U	Disrupt envelope, prevent viral entrance	U	Disrupt envelope	*	NE	NE	U	NE	*	U

Virus (con.)	DENV	HCV	VEEV	Aichivirus A	Echovirus	HRV	Influenza A virus	HPIV	RSV	VHSV	VSV	HIV-I
LL-37	Binding to envelope protein	Disrupt enve lope	U	U	*	U	Disrupt envelope, increase neutrophil NET	*	Disrupt envelope, prevent viral exit	*	*	Inhibits reverse transcriptase

NE: Tested HDP had no effect, U: Antiviral mechanism is undetermined,

^*:Yet to be tested.

Table shows most common result of HDP antiviral test.

3.1. LL-37 Activity Against DNA Viruses

3.1.1. LL-37 Activity Against Enveloped DNA Viruses

3.1.1.1. LL-37 Activity Against Herpesviruses

As is the case with defensins, LL-37 has been shown to inhibit infections of a number of herpesviruses. The first evidence of any antiviral activity of LL-37 came in 2000 [30], when the peptide was found to inhibit HSV-2-mediated cytotoxicity of human cervical carcinoma cells during a screen of a number of human and other animal host defense peptides, including defensins. This study, though, used supraphysiological-concentrations of LL-37 (200 μg/ml) and concluded the peptide did not inhibit HSV-2 as well as the other HDPs tested. However, this initial investigation into the antiviral activities of LL-37 led to a host of other studies to further uncover the potential of the peptide to inhibit viral infections, including other herpesviruses.

In addition to LL-37 inhibiting HSV-2 [30, 149], the peptide has also been shown to have activity against HSV-1. HSV-1 overall plaque formation was decreased by LL-37 in both A549 [150] and Vero cells [151]. Viral genome replication of HSV-1 was decreased by LL-37 in the human lung fibroblast cell line MRC-5, regardless of whether the peptide was added to the cells before, during, or after infection [152]. The efficacy of the inhibition was decreased during the post-infection condition, suggesting earlier stages of viral infection as the main target of LL-37 for HSV-1 infections, a hypothesis bolstered by a later study measuring LL-37 inhibition of HSV-1 replication in primary human skin keratinocytes [153]. However, a study investigating the delivery of LL-37 to cells using liposomes found the peptide to be able to protect human immortalized keratinocytes from HSV-1-mediated tissue damage when introduced to cells seven days after infection [154]. Investigations into the antiviral mechanisms of LL-37 against HSV-1 found the peptide can act even before infection, where LL-37 increased the delivery of dsRNA mimetic polyinosinic:polycytidylic acid, poly(I:C), leading to enhanced interferon beta production in normal human epidermal keratinocytes [155]. An early infection step, viral binding, was tested and LL-37 in hydrogel protected human corneal epithelial cells from HSV-1 infection by inhibiting viral attachment to cells [156].

LL-37 is known to inhibit other herpesviruses, although the majority of antiviral studies of the peptide revolve around HSV-1. In addition to inhibiting CMV [29] and VZV [36] through mechanisms yet to be defined, LL-37 was also shown to decrease Kaposi’s sarcoma-associated herpesvirus (KSHV) internalization into, and subsequent infection of, the human oral epithelial cell line OKF6/Tert-1, most likely due to the peptide’s ability to disrupt the viral envelope [157]. This inhibition of KSHV corroborates earlier studies that demonstrate LL-37 upregulation in Kaposi’s sarcoma skin lesions [158], a phenomenon the authors suggested may be evidence for the antiviral potential for those peptides against KSHV, as was previously shown for HSV-1 and HSV-2 as described in this section.

3.1.1.2. LL-37 Activity Against Vaccinia Virus

The first ever tests of any HDP against vaccinia virus found that LL-37 inhibited viral gene expression and overall infection of BS-C-1 cells, with enhanced antiviral capabilities compared to HNP1, HBD1, and HBD2 [159]. This study also is the first example of LL-37 inhibiting infections through disrupting the viral envelope, a phenomenon seen again in later tests with vaccinia [160, 161]. The KS-30 region of LL-37 was found to have the greatest inhibitory effect against vaccinia virus infection of BS-C-1 cells, along with antimicrobial activities against Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, and Candida albicans [162]. The ability of the peptide, especially a specific region of the peptide, to broadly inhibit a number of microorganisms provides further evidence for membrane perturbation as the main mechanism of LL-37 inhibition of vaccinia virus. However, LL-37 was also shown to inhibit vaccinia gene expression in human keratinocytes, even when the peptide was added after viral infection [163], suggesting an antiviral role of LL-37 against vaccinia virus beyond membrane disruption.

3.1.2. LL-37 Activity Against Nonenveloped DNA Viruses

While the majority of LL-37 antiviral activities seem to stem from disrupting viral envelopes, the peptide has been shown to also act on nonenveloped viruses. Earlier work with human defensin activities against adenoviruses prompted studies to see if LL-37 had similar activities. The first activity of LL-37 against a nonenveloped virus was discovered during a screen of human HDPs against viruses that cause disease at the ocular surface, including HSV and HAdVs. LL-37 inhibited HAdV-19 in a time-dependent manner of pre-incubation of the virus and peptide before infection of A549 cells [150], suggesting the virus—despite its lack of envelope was the target of the peptide as part of its antiviral activity. In addition to further evidence of inhibitory effects against HAdV-19, another study also found LL-37 restricts genome replication of HAdV-3 and HAdV-8 in A549 cells [164]. These inhibitions, however, occur at supraphysiological concentrations of the peptide: >100 μg/ml. LL-37 activity against another nonenveloped DNA virus, HPV16, though, occurs within normal peptide levels in humans [56]. Structural differences between the capsids of these viruses may be the cause of the vastly disparate efficacies of LL-37 inhibition.

3.2. LL-37 Activity Against RNA Viruses

3.2.1. LL-37 Activity Against Group IV Viruses

Group IV viruses (+ssRNA genome) are unique in LL-37 antiviral research in that just as much, if not more, is known about the peptide’s activity against non-enveloped viruses than those with envelopes. HRV, a member of the picornavirus family and predominant etiological agent of the common cold, has been shown to increase LL-37 expression in a three-dimensional model of the human airway [80]. The peptide suppresses overall viral infection in the human lung fibroblast cell line WI-38 [165] and specifically viral RNA genome replication in primary bronchial epithelial cells of patients with cystic fibrosis [166]. Addition of LL-37 either to HRV or WI-38 cells before infection led to similar levels of inhibition by the peptide, suggesting LL-37 has some antiviral effect via interaction with host cells for HRV infections [167]. Another nonenveloped group IV virus of the picornavirus family, Aichivirus A, is also inhibited by LL-37. The peptide exerts its antiviral effect after the virus enters the host cell [151].

The enveloped group IV viruses HCV and DENV, in addition to Venezuelan equine encephalitis virus (VEEV) [168], are also known to be inhibited by LL-37. After initial studies connecting serum LL-37 levels and HCV infections [169], the peptide was foundto disrupt the viral envelope and prevent binding to HuH-7 human liver cell line [170]. LL-37 also displayed a direct effect on DENV, as pre- or post-infection treatment of Vero E6 cells did not exhibit the same antiviral phenotype as when the virus and peptide were added at the same time. Computer modeling showed a potential binding site for LL-37 in the DENV envelope protein dimer [171], which could be the mechanism by which the peptide binds to, and subsequently inhibits, the virus. The hypothesis that LL-37 could act as an early step in DENV innate antiviral immunity gained further traction when it was discovered that human keratinocytes [172] and monocytes [78] upregulated the expression of LL-37 upon infection with DENV.

3.2.2. LL-37 Activity Against Group V Viruses

Two enveloped members of the group V viruses (-ssRNA genome)-influenza A virus and RSV-have been shown to be inhibited by LL-37. Currently, influenza A virus is the most studied virus in the context of LL-37 antiviral investigation and is overall well characterized in the field of antiviral HDP research in general [173]. The first inhibitory effect of LL-37 against influenza A virus was discovered during a series of experiments studying the anti-influenza characteristics of leukotriene B4, LTB4, stimulation of neutrophils. Addition of antibodies against certain HDPs, including LL-37, inhibited the antiviral capabilities of LTB4 [174], suggesting the peptide was carrying out the antimicrobial activity previously seen with LTB4 treatment of neutrophils [175]. Further testing of LL-37’s ability to enhance neutrophil-mediated anti-influenza activity led to the discovery that the peptide binds to both the virion and the formyl peptide receptor, leading to increased neutrophil intracellular calcium concentration along with H₂O₂ and neutrophil extracellular trap (NET) production [176], with the GI-20 region of the peptide retaining these activities [177]. H₂O₂ and NET production appear to be the main mechanism by which LL-37 increases neutrophil anti-influenza activities, in contrast to HNP1 [178] and HNP2 [176] increasing neutrophil uptake of the virus. A direct effect on the virion by LL-37 was also observed, where the peptide disrupted the viral envelope. Interestingly, although this disruption did not prevent viral uptake by the MDCK or HBTE cell lines, the peptide did inhibit the influenza life cycle specifically before RNA synthesis [85], leading to an overall decrease in influenza infection of these cells [86]. LL-37 was also found to inhibit infection of a number of influenza A strains in human monocytes [178]. In one of the only in vivo studies of LL-37 antiviral activity, the peptide was introduced to mice via nebulization along with intranasal infection of influenza A virus. Mice treated with LL-37 were protected from infection-associated lung inflammation [179]. Although whether LL-37 was acting on local neutrophils in the mouse lung, the virions, or both were not tested, this experiment provides strong evidence for the potential use of LL-37 as a treatment for certain viral infections. As in the case of HRV, influenza A virus also has been shown to increase LL-37 production in a three-dimensional model of the human airway [80], further suggesting the potential of LL-37 to be used for antiviral defenses against influenza.

The initial finding that RSV infections of respiratory epithelial cells increased LL-37 expression [180] led to further studies of the potential antiviral activity of the peptide. LL-37 was found to be most effective as a prophylactic measure against RSV infection of an air-liquid interface Calu-3 human lung carcinoma cell line system [181], suggesting the peptide acted on the virion. Not only did LL-37 disrupt the RSV envelope structure [182], as seen with influenza A virus, but the peptide also displayed antiviral activities at later stages of the virus life cycle by preventing the release of the virus from infected Hep-2 cells [183]. This investigation also found the antiviral activity of LL-37 against RSV is conserved in the KI-22 central region of the peptide [183]. As with studies of influenza virus, RSV in vivo infections of mice were challenged with LL-37. Treatment of mice with LL-37 during RSV infection led to an increase in body weight recovery along with a decrease in viral gene and IFNB expressions in the lungs [182]. Along with the in vivo data for influenza virus, this data further demonstrates the potential for LL-37 to be the target for antiviral treatments.

3.3. LL-37 Activity Against HIV

With the need for new types of antivirals against HIV, a number of studies have looked to associate antimicrobial peptide expression with HIV infection. LL-37, found in both seminal plasma [184] and Cervicovaginal Secretions (CVS) [185], seemed to be a candidate for the study of preventing sexually acquired HIV. However, in vivo data from these correlative experiments were inconclusive: while HIV load in sexual partners was associated with LL-37 levels in CVS of HIV-negative patients and recombinant LL-37 enhanced in vitro neutralization HIV in cationic peptide fractions of CVS, the ability of the CVS to inhibit HIV in vitro did not correlate with LL-37 levels within the secretion [128]. Similarly, the connection between LL-37 presence in CVS and HIV acquisition [185] has also been questioned as the production of the peptide might be in response to other sexually transmitted infections. Overall, these in vivo data provide a starting point for research in LL-37 as a potential method of preventing or treating HIV infections but require a more thorough investigation into synergistic effects with other HDPs.

A more conclusive anti-HIV picture of LL-37 was formulated through in vitro studies. LL-37 was found to inhibit HIV infection not only in HEK293 cells [186], but also primary CD4⁺ T cells [187]. This inhibition was more potent than HNP1-3 activity against HIV [128] with the GI-20 region of the peptide retaining the antiviral capability [188], similar to studies with influenza A virus. The main hypothesized mechanism by which LL-37 inhibits HIV infection is via binding of the viral protease and subsequent decrease in reverse transcriptase activity [189]. This targeting of reverse transcriptase, especially due to its specificity toward HIV, could lead to further developments of HDP-based strategies for HIV therapies.

CONCLUSION

HDPs act as the most innate form of immunity against pathogens, with all forms of life producing this class of molecule. Similarly, viruses can infect all types of organisms, allowing this host-pathogen relationship to grow over millions, if not billions, of years. Studying the endogenous HDPs in humans allows us to read glimpses of the story told by our co-evolution with pathogens, including viruses, and learn how we naturally defend against infections. This knowledge can then allow us to effectively elicit their response and potentially improve on their activities in the form of therapies or treatments to use in the battle against viruses and the diseases they cause.

Defensins and LL-37 are the main HDPs found in humans and have broad-spectrum activity against not only bacteria and fungi, but also viruses. One of the noteworthy trends from the overview of antiviral activities of human HDPs (Tables 7, 8) includes the consistent ability of HD5 to inhibit nonenveloped DNA viruses, except for certain types of HAdV, with the highest efficacy compared to other HDPs. HD5, with such a conserved antiviral activity throughout that particular subset of viruses lends itself toward both a potential target for strategies to inhibit pathogenic viruses and a potential obstacle for HAdV-based gene therapy vectors. Similarly, more anti-AAV HDP studies, beyond the single one to date, would be helpful with the influx of AAV-based gene therapy vectors to engineer viruses that evade HDP activities, much like the research done with endogenous antibody escape mutants of AAV [190].

Table 7.

Direct comparisons of human HDP activity against viruses.

Virus	Direct comparison of HDP activity
HSV-1	HNP1, HNP2, and HNP3 were about equal in effectiveness
HSV-2	HNP1 and HD5 were most effective among defensins tested. HBD3 was more effective than HBD1
VZV	HBD2 and LL-37 had similar activities, but unlike with HBD2, LL-37 pre-incubation of virus and peptide before infection increased antiviral activity compared to after infection
Vaccinia	HBD2, but not HBD1 or HNP1/2 had effect. This effect was more potent than LL-37
HAdV	For HNP1 and HD5, viral species A, B1, B2, C, and E susceptible, but D and F are not. HD5 was more effective than HNP1 against HAdV5 and HAdV35. HBD1/2 and HD6 mostly ineffective. Defensins and LL-37 inhibit different species.
HPV	HD5 had highest efficacy. HBD1/2 and HD6 had no effect. HNP1-4 and LL-37 had similar efficacies
BKV	HD5 had highest efficacy. HNP1 and HBD2 had similar effect, while HBD1 had no effect
JCV	HD5 had highest efficacy. Discrepancy for activity of HNP1 and HBD1/2. No effect seen in HNP3 or HBD4
SV40	HD5 inhibited, while HNP1 and HBD1/2 did not
HCV	Mixture of HNP1-4 (about as effective as LL-37) was more effective than mixture of HBD1-5+116
Influenza A virus	HNP3, HBD1, and HBD2 did not increase IAV uptake by neutrophils, unlike HNP1/2 and HD5. HNP1/2 (with slight edge to HNP2) more effective than HBDs or LL-37. HNP1/2 work synergistically with LL-37
RSV	HBD2 had most activity while HBD1 was ineffective. LL-37 was less effective than HBD2
HIV-I	LL-37 and HNP4 are more effective than HNP1-3, most likely due to HNP4 less binding to serum. Combination of HBD2 and HBD3 was more effective than either HBD alone. Conflicting data about HBD1 activity, with HBD2/3 having more activity. HBD2 inhibits single stage infection and provides long term antiviral activity, unlike HBD1.

Comparisons are only between HDPs tested in the same experiment.

Table 8.

Direct comparisons of viral sensitivity to human HDPs.

HDP	Direct Comparison of Viral Sensitivity
HNP1	HIV-1 and VSV inhibition happens before uptake, while influenza inhibition happens after. HAdV species A, B1, B2, C, and E susceptible, but D and F are not
HD5	HAdV species A, B1, B2, C, and E susceptible, but D and F are not
HBD2	Similar activities against RSV and HPIV, but no activity against vaccinia
LL-37	More effective against HSV-2 than HSV-1

Comparisons are only between viruses tested in the same experiment.

While the vast majority of the studies outlined in this paper have focused on direct antiviral activities of human HDPs, there are other manners by which HDPs can enhance the overall antiviral defenses. HDPs and chemokines overlap in structure, function, and therefore specificity for immune cell receptors, as evident in the main mechanism of defensin inhibiting HIV-1 infection, competing with the virus for CD4⁺ T cell receptors to ultimately prevent viral binding. HDPs possess a wide range of immunomodulatory activities such as stimulating TLR and downstream interferon-stimulated genes and recruiting neutrophils to the site of infection [191]. Vaccines containing HDPs as adjuvants have also been shown to increase immune memory against a number of viruses [82, 192, 193].

Manufacturing HDPs for use in vaccines, or alone as treatments for infection, can be expensive and the peptides are subject to endogenous proteolytic cleavage. In addition to continuing to study ways of increasing natural HDP production in humans-such as with vitamin D₃ [194, 195]-engineering peptide mimetics represents a promising field of research, with much of the work stemming from basic science studies of structures and domains of HDPs that retain antiviral activity [196–201]. Both of these approaches will hopefully lead to an increase in in vivo studies and subsequent novel treatments or prophylaxes for viral infections.

Despite human defensins and LL-37 garnering the majority of attention in the HDP field, there are a number of other human proteins listed in the antimicrobial peptide database [202] (APD) that have known antiviral activities including hepcidin, alpha-melanocyte-stimulating hormone (alpha-MSH), histatin 5, elafin, amyloid beta 40 and 42, secretory leukocyte protease inhibitor (SLPI), RNase 2 and 3, and high mobility group nucleosomal binding domain 2 (HMGN2). These proteins-along with others not on the list, but which also have antiviral activity-could potentially serve as a starting point, just as human defensins and LL-37 have, for research into defending against viral infections. Overall, human HDPs represent not only one of the first lines of natural defense against viruses, but also one of the first steps in our quest in creating potential treatments and preventions of diseases associated with these pathogens.

LIST OF ABBREVIATIONS

AMP

Antimicrobial Peptide

APD

Antimicrobial Peptide Database

CAMP

Cathelicidin Antimicrobial Peptide

CCHFV

Crimean-Congo Herorrhagic Fever Virus

CMV

Cytomegalovirus

CVL

Cervicovaginal Lavage

DENV

Dengue Fever Virus

HAART

Highly Active Antiretroviral Therapy

HAdV

Human Adenovirus

HBD

Human Beta-defensin

HBTE

Human Bronchial/Tracheal Epithelial

HBV

Hepatitis B Virus

HCV

Hepatitis C Virus

HD

Human Defensin

HDP

Host Defense Peptide

HIV

Human Immunodeficiency Virus

HMGN2

High Mobility Group Nucleosomal Binding Domain 2

HPIV

Human Parainfluenza Virus

HPV

Human Papillomavirus

HRV

Human Rhinovirus

HSV

Herpesvirus

ISG

Interferon Stimulated Gene

MSH

Melanocyte-stimulating Hormone

NET

Neutrophil Extracellular Trap

PBMC

Peripheral Blood Mononuclear Cell

RSV

Respiratory Syncytial Virus

SLPI

Secretory Leukoprotease Inhibitor

SV40

Simian Virus 40

TLR

Toll-like Receptor

VHSV

Viral Hemorrhagic Septicemia Virus

VSV

Vesticular Stomatitis Indiana Virus

VZV

Varicella Zoster Virus