REVIEW ARTICLE

The Role of Serpin and Cystatin Antiproteases in Mucosal Innate Immunity and their Defense against HIV Lindsay Aboud1, Terry Blake Ball1,2,3, Annelie Tjernlund4, Adam Burgener1,3 1

Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada; Department of Immunology, University of Manitoba, Winnipeg, Manitoba, Canada; 3 National HIV and Retrovirology laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada; 4 Department Medicine, Karolinska Institute, Solna, Sweden 2

Keywords Cystatins, female genital tract, HIV, innate mucosal immunity, serpins Correspondence Adam Burgener, Department of Medical Microbiology, University of Manitoba, 507-745 Bannatyne Ave. Basic Medical Sciences Building, Winnipeg, Manitoba R3E 0W3, Canada. E-mail: [email protected] Submission July 22, 2013; accepted September 16, 2013. Citation Aboud L, Ball TB, Tjernlund A, Burgener A. The role of serpin and cystatin antiproteases in mucosal innate immunity and the defense against HIV. Am J Reprod Immunol 2014; 71: 12–23

Antiproteases play diverse roles in nature, from regulating protease activity to innate defense against microorganisms. Recently, antiproteases have been shown to play important roles in HIV pathogenesis including, inhibiting HIV binding and replication and reducing activation and inflammation of susceptible cells. They have also been implicated as one of the initial host responders, in plasma, to control replication of HIV. More recently, antiproteases expressed at the mucosal surface have been linked to reduced susceptibility to HIV infection in HIV-exposed seronegative individuals. These factors are expressed in the epithelial layer of the female genital tract, thus at the frontline of defense against mucosal infection. This review focuses on the specific antimicrobial roles of antiproteases, focusing on serpins and cystatins, with an emphasis on their known and potential roles in HIV infection. Their potential as therapeutic interventions to combat HIV is also discussed.

doi:10.1111/aji.12166

Introduction Mucosal Protection Mechanisms in the Female Genital Tract from HIV The mechanism of HIV transmission, through vaginal mucosa, is not entirely understood. HIV is thought to cross the intact epithelial barrier through either direct infection of CD4+ T cells following migration of these cells to the epithelial layer of the mucosa or through infection of intermediary cells (Dendritic cells (DCs) or monocytes) present within the mucosal layer, before infection of CD4+ T cells. HIV may also directly infect epithelial cells followed by transcytosis through the epithelial layer or HIV 1 can directly disrupt the integrity of the mucosal epithelial barrier, resulting in translocation of additional virus particles 12

through the mucosal layer.2 Once the virus has infected its target cells, it undergoes local amplification, followed by dissemination to draining lymph nodes and subsequent systemic infection.3 One important component of protection offered by the FGT is conferred by the soluble components in mucosal secretions. The FGT produces a hydrophilic layer of glycoproteins and glycolipids, referred to as the glycocalyx, as well as a layer of thick hydrophobic glycoprotein mucus, both of which act as barriers to invading potential pathogens.4 Within these secretions, there is a plethora of antimicrobial agents, the majority of which are broad-spectrum microbicides. Many of these factors, including antileukoproteinase (SLPI), specific serpins and cystatins, defensins, and heat-shock proteins, have shown in vitro inhibitory activity against HIV.5–8 In spite of American Journal of Reproductive Immunology 71 (2014) 12–23 ª 2013 John Wiley & Sons Ltd

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these mechanisms of protection, HIV is still able to bypass these barriers to establish an infection in 1:100–1:1000 coital acts.2 As with any infectious disease not all individuals exposed become infected, in the case of HIV, this has been a well-described phenomenon. Many groups of individuals around the world, including intravenous drug users, sero-discordant couples and commercial sex workers, have been identified as being HIV-exposed sero-negative (HESN).9 Arguably, the most well described are those from the Pumwani sex worker cohort in Kenya, where approximately 150 individuals have been identified as HESN. These women are considered high-risk for exposure to HIV and have been exposed for more than 7 years yet remain uninfected. Recent evidence has shown that, within the FGT of these women, specific mucosal antiproteases are elevated, including specific serpins and cystatins.10–12 Many of these factors have shown antiviral activity against HIV,5,13 they have also been shown to be key players in the regulation of inflammatory responses.14–18 This article will focus primarily on the biological function of specific serpins and cystatins in the context of HIV pathogenesis and inflammation, and propose a functional role for these innate factors, at mucosal surfaces. Serpins Role in HIV Infection and Inflammation Serpins (Serine protease inhibitors) play important roles in regulating inflammation, apoptosis, and tissue development.15,16,19 Serpins primary biological function is to inhibit serine proteases (cathepsins, elastase, granzyme B) and to a lesser degree cysteine proteases.20 Serine proteases, secreted by immune cells such as cytotoxic T-lymphocytes and neutrophils, function to either directly kill invading pathogens,21,22 or to trigger inflammatory immune functions through induction of complement activation or through secretion of inflammatory mediators.23 Serpins regulate the activity of these proteases, which, if not controlled, can lead to severe inflammation, tissue damage and, potentially, a progressed disease state.24–26 It has been shown in recent studies that serpins play important roles in host defense against HIV infection, either through the inhibition of host cell protease activity or through directly inhibiting the infection of host cells through interference with viral protein binding (Fig. 1). Host cell proteases have been shown to enhance both the infectivity of HIV American Journal of Reproductive Immunology 71 (2014) 12–23 ª 2013 John Wiley & Sons Ltd

and activation of inflammatory processes. For example, the serine proteases cathepsin G and elastase enhance the susceptibility of macrophages to HIV infection and promote pro-inflammatory cytokine expression, leading to recruitment of target cells for HIV, including T cells,27 an effect that serpin A3 abrogates.28–30 Thus, the ability of this serpin to inhibit the function of cathepsin G and elastase may lead to reduction in activated target cells for HIV at the site of infection. Serpins demonstrate primarily indirect mechanisms of antiviral activity, through interference with host proteases; however, there is currently one serpin that exhibits direct antiviral activity against HIV. Serpin A1/alpha-1-antitrysin (AAT) acts as a direct binding inhibitor of HIV. The interaction of AAT, with its target protease results in the cleavage of a C-terminal fragment, designated C-36. M€ unch and colleagues identified and characterized this fragment as a CD4 competitive binding inhibitor for gp120.31,32 This fragment has also, recently been identified as a gp41 fusion protein inhibitor and is currently being used in an antiretroviral (ARV) therapy that is in Phase I/II clinical trial.33 AAT has also been shown to interfere with viral protein late processing, specifically through interference with gp120 formation and processing of p55(gal) to p24.6 Serpins reduce HIV-induced inflammation through indirect mechanisms. Serpin C1 and AAT both interfere with NF-kb signal transduction.35 The NF-kb pathway is an initiator of pro-inflammatory responses, including IL-1 and TNF-a. The ability to interfere with NF-kb activation, a pathway that is heavily relied upon by HIV for efficient virus replication, is thought to be the primary mechanism by which AAT inhibits HIV-1 replication in vitro.34,36,37 Serpin C1 (antithrombin) is also capable of interfering with the NF-kb pathway through activation of prostaglandin synthetase-2 (PTGS2).35 PTGS2 has a known ability to downregulate NFjb-mediated transcription, and thus, it is reasonable to hypothesize that this pathway may provide a route for inhibition of HIV by serpin C1.35 Lastly, AAT has been shown to inhibit directional neutrophil migration to the site of infection, via inhibition of calpain and it has also been shown to inhibit LPS-mediated stimulation of monocytes via regulation of CD14 expression.39 Anti-inflammatory functions of AAT and serpin C1 may be critical at mucosal surfaces, as demonstrated by an observed, decreased risk of infection by SIV when IL-8 levels are reduced.40 Inflammation in the 13

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Fig. 1 The intracellular and extracellular role of serpins against inflammation and HIV infection. 1. Serpin A1 inhibits elastase. Elastase has been shown to increase the infectivity of HIV-1 as well as to impair wound repair. By inhibiting this protease Serpin A1 slows the rate of infection while also aiding in the maintenance of an intact epithelial barrier at the mucosal level.26 2. Serpin A1 inhibits proinflammatory cytokines and inflammatory mediators, which limits the number of target cells brought to the site of infection.34,38 3. C-36 is also capable of direct inhibition of HIV-1 infection in certain cell cultures through competitive binding to gp120.31,32 4. Serpin A3 inhibits the neutrophil and macrophage chemoattractant and T-cell activating abilities of Cathepsin G, thus reducing the number of potential target cells for HIV-1 at the site of infection 27–30 5. Serpin A1 has been shown to inhibit directional neutrophil migration towards the site of infection.64 6. Serpin A1 inhibits the dissociation of NF-jb from Ijb, preventing migration of NF-jb to the nucleus and subsequent initiation of transcription of the HIV genes, thereby slowing the reproductive ability of HIV-1 as well as numerous other microbial species.37 7. Serpin C1 up-regulates prostacyclins through up-regulation of PTGS2, which is a known inhibitor of NF-jb-mediated transcription.35 8. The C-terminal residue C-36 becomes internalized by the cell. leading to migration to the nucleus and inhibition of HIV-1-Long Terminal Repeat (LTR)-driven transcription.34 9. Serpin A1 interferes with formation of gp120 and processing of gp 55(gal) to p24 by HIV protease, thus reducing the number of glycoproteins/receptors on the surface of the newly produced HIV-1 virus particle.6 10. Serpin B1 is primarily intracellular, within the nucleus and cytoplasm. It inhibits elastase-like and chymotrypsin-like proteases (including Cathepsin G), protecting the cell from proteases released into the cytoplasm during stress or phagocytosis 42.

female genital tract is a key determinant for risk of HIV acquisition, thus the reduction in IL-8 as well as other pro-inflammatory factors may play a role in protection against HIV-1 infection.41 The group of serpin B antiproteases is one of the other primary clades of serpins that has been defined as important regulators of the human immune response.15 Specific members of this clade were identified as being upregulated in HIV-highly exposed sero-negative (HESN) women, within the Pumwani sex worker cohort in Nairobi, Kenya.10 14

These serpin B antiproteases were upregulated at similar levels to factors such as Elafin and AAT compared with uninfected, susceptible individuals, suggesting a role for them in the protective, mucosal immune response of these HESN women.12 Serpin B1 (MNEI) has been shown to be important at mucosal surfaces. It is primarily intracellular; however, it has also been found in extracellular fluids. It has been shown to inhibit both elastase-like and chymotrypsin-like proteases, including cathepsin G and is thought to protect cells from proteases American Journal of Reproductive Immunology 71 (2014) 12–23 ª 2013 John Wiley & Sons Ltd

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released into the cytoplasm during stress or phagocytosis. Also, in the absence of serpin B1, severe inflammatory damage of the lungs was observed by uncontrolled protease activity induced during bacterial infections.42 Serpin B9 is found primarily in cells that produce granzyme B (GrB), namely cytotoxic T lymphocytes (CTLs) and natural killer cells.43 GrB cleaves cytosolic pro-apoptotic substrates, serpin B9 assists in protecting effector cells from unwanted apoptosis resulting from mislocalized GrB within the cell.15 Also, it has been demonstrated that when B9 is over-expressed in primary CTLs, there is an enhancement in these cell’s ability to kill target cells.44 Although there is no published data examining clade B serpins in HIV pathogenesis, there is a large body of support for their role in regulating inflammatory processes at mucosal surfaces, which has been implicated as a risk factor for HIV acquisition. Various serpins have been evaluated for anti-HIV activity. However, they have not been tested at physiological ranges found in mucosal fluids. Our group has tested these proteins at concentration ranges that reflect physiological concentrations in cervical mucosa as measured by Burgener et al.12 to match those performed previously by M€ unch et al.32 37 and Zhou et al. Infections performed by our laboratory utilizing serpin C1 have confirmed modest antiviral activity against HIV at these levels (Fig. 2). This suggests that specific serpins are capable of inhibiting HIV-1 at concentrations found within the natural mucosal fluids of women, providing further evidence of the potentially critical role that specific antiproteases naturally play in protection against HIV-1. SLPI (Secretory Leukocyte Protease Inhibitor) SLPI is a primarily serine protease inhibitor that is well documented in its ability to inhibit viral infection, specifically that of HIV. This serine inhibitor is functionally similar to serpins, and because it is also present at mucosal surfaces, it is also considered to be at the frontline of innate immunity. SLPI is produced by salivary glands where it has been identified as a major HIV-inhibiting component45; however, it is also present in other mucosal secretions 46 and in serum. SLPI has been shown to exhibit inhibitory activity against neutrophil serine proteases including cathepsin G and neutrophil elastase 47 while also having the ability to inhibit chymotrypsin and trypAmerican Journal of Reproductive Immunology 71 (2014) 12–23 ª 2013 John Wiley & Sons Ltd

Fig. 2 Serpin C1 exhibits inhibitory effects on HIV-1 infection in a TZM-bl cell assay Briefly, Serpin C1 was added at the same time as an R5-tropic (Bal) HIV-1 virus to TZM-bl reporter cells. Serum-free media was used for the duration of the 3 hr infection. Following removal of virus and antiprotease, fresh Dulbecco’s Eagle Modified Media (DMEM) containing 10% fetal bovine serum (FBS) was added to the cells. Plates were then incubated for 3 days and then the b-galactosidase assay was performed. Cytotoxicity assays were performed in parallel to the infection assays and no significant cytotoxic effects were observed. Results indicate that Serpin C1 inhibits HIV-1 infection at physiologically relevant concentrations (0.370–30 lg/mL). A 40% reduction in infection was observed for Serpin C1 at 10 and 30 lg/ mL. (Results represent two separate experiments performed in triplicate P < 0.05).

sin.45 It is present within numerous cell types, including epithelial cells within the female genital tract where they are secreted in response to proinflammatory stimuli.48 Various mechanisms are employed by this antiprotease to inhibit HIV. SLPI downregulates expression of pro-inflammatory factors through inhibition of NF-jb via alteration of Ijb, a pathway that, when functioning normally, is heavily relied upon by HIV for replication.49,50 Also, SLPI has been shown to block HIV-1 infection of monocytes and T cells by preventing internalization of the virus prior to reverse transcription; however, this action is independent of the antiprotease activity of this protein and is instead directly related to interactions of the protein with the target cells.45,50–52 Cystatins Role in HIV Infection and Inflammation Cystatins regulate the activity of papain-like cysteine proteases and are found in many cell types, 15

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including epithelial cells, granulocytes, monocytes, macrophages, and DCs.53 The primary function of type 1 cystatins (cystatin A and B) is thought to be regulation of cathepsins B, L, and S.53 The regulation of these proteases’ activity has proven importance, as seen in the rapid progression of disorders such as

atherosclerosis and cancer in the absence of cystatins.14 Cystatins are thought to play important roles in countering inflammation and providing innate defense against microorganisms, including HIV (Fig. 3). Cystatin expression is increased following

Fig. 3 The intracellular and extracellular role of cystatins in inflammation and HIV infection. 1. Cystatins a & b inhibit cathepsins B, L and S which are proteases released by macrophages during the inflammatory response.53 These proteases are all lysosomal proteases which are thought to assist in the degradation of proteins for antigen presentation, thus control of these proteases will reduce the level of antigen presentation and subsequently the level of immune activation at the site of infection. 2. Cystatin A & B inhibit migration of T cells and monocytes to the site of infection,58 thereby decreasing the number of potential target cells for HIV to infect. 3. Cystatin A & B increase production of reactive oxygen species (ROS), including nitric oxide (NO), in macrophages.60,61 Nitric oxide is a known antimicrobial product released by macrophages upon infection, Although there has not been direct evidence for NO having any antiretroviral activity, it is possible that the control of secondary infections by NO AIDS in the reduction of inflammation and thus target cells during initial infection with HIV-1. 4. Cystatin B is known to upregulate production of NO and has also been linked to the STAT-1 pathway, thus, a hypothesis developed by Montalyo et al. is that low levels of Cystatin B allow for the activation of a tyrosine kinase, promoting phosphorylation of STAT-1, leading to subsequent induction of IRF-1 production and a decrease in HIV-1 replication 62 (4a) Induction of IRF-1 also leads to the activation of iNOS, resulting in further production of NO 62 (4b). This hypothesis has not yet been tested, although it does state a plausible scenario for the involvement of Cystatin B in control of HIV infection 5. Cystatin A is anti-apoptotic in certain viral infections and is capable of reducing programmed cell death.65 Since apoptosis plays a role in the reproductive efficiency of HIV-1 virus, the ability of Cystatin A to reduce cell death may assist in reduction of overall viral load within an infected host.

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various immune responses, including: activation of TLRs by TNF-a, IL-1b, and IFN-b,54 stimulation of monocytes/macrophages by LPS,55 and following activation of the transcription factor IRF-8 in macrophages, CD8+ T cells and DCs.56,57 Once cystatins are present at the site of infection they function to regulate protease activity in macrophages, which will reduce the amount of damage to bystander cells and tissues while also reducing the migration of monocytes and T cells to the site of infection, thereby reducing the overall inflammation at any given site.58,59 Cystatins also assist macrophages in the controlled production of reactive oxygen species, thereby assisting in the killing of pathogens.60–62 Thus, like serpins, cystatins function primarily to control the level of inflammation, subsequently reducing the level of damage to the human host’s surrounding cells and tissues and possibly reducing the number of potential target cells for HIV. Considering that excessive inflammation is known to be a key factor in the rapid dissemination of HIV, it is not surprising that both cystatins A and B have been shown to have direct antiviral activity against HIV (Fig. 4). Cystatin B has been reported to exhibit 20% inhibitory activity against HIV-1 in vitro at 1 lg/mL and 90% at 25–35 lg/ mL45,46; however, when similar experiments were performed by our research group, we observed more modest levels of inhibition, with significant values being obtained at a concentration of 13 lg/ mL (Fig. 4b). Using human saliva samples, Cole’s research team discovered a 30–90% inhibition in antiviral activity when physiological levels of cystatin A were present in solution.46 Again similar assays by our group yielded more modest inhibitory activity. However, we too observed an antiviral effect, reaching a maximum of 50% inhibition at concentrations as high as 40 lg/mL (Fig. 4a). These differences in overall level of inhibition may be due to the differences in protein purification techniques used by the various laboratories, resulting in differing levels of purity for the exogenous protein. Varying levels of purity create margins of uncertainty when considering the exact unknown peptides/proteins present within the known (cystatin) protein solution. These unknown peptides may affect the inhibitory activity of the antiprotease to varying degrees, depending on the preparation methods used, and they may contribute to the level of non-specific antiprotease activity exhibited by the protein during an infection. American Journal of Reproductive Immunology 71 (2014) 12–23 ª 2013 John Wiley & Sons Ltd

Serpins and Cystatins are Primarily Expressed in the Epithelial Layer of the Ectocervix In order for serpins and cystatins to be considered legitimate natural factors responsible for the inhibition of efficient HIV infection, there must be a steady source of these antiproteases at the sites of infection, including the FGT. Although the specific expression levels have been well described in mucosal fluids, their abundance patterns in the FGT tissue has not been well described. Here, we show immunohistochemistry staining images of AAT and cystatin B from ectocervical tissue of the human female genital tract (Fig. 5a,b, respectively). In this tissue subset, AAT (5A) was expressed in both the epithelium and also in cells located in the lamina propria/ submucosa. In contrast, cystatin B (5B) was more localized to the epithelial layer, showing dense expression in multiple cell layers, which did not vary considerably along the length of the tissue. For both, the expression appeared to be more membrane-associated than cytoplasmic. However, this is in contrast to previous in vitro cell line studies showing that AAT enters cells relatively quickly (18 hrs), specifically when incubated with LPS and IFN-c,63 indicating that this protein is also present intracellularly. It is unknown what is the discrepancy for these results and may reflect the nature of the different assays to assess the protein’s location, although mucosal tissue may have unique sources or mechanisms of secretion for AAT. Hence, these antiproteases are positioned at the frontline of the epithelium where HIV makes first contact in male-to-female sexual transmission, thus providing these antiproteases with the perfect opportunity to become key players in the fight against efficient HIV transmission. Predictive Model for the Role of Antiproteases at Mucosal Surfaces against HIV Infection and Inflammation Due to their known biological properties in inflammation and HIV replication, their inherent antiviral activities, as well as their expression at the surface of mucosa in FGT tissues, serpins and cystatins are likely players in the host defense against HIV. A model of their function at mucosal surfaces in the context of HIV infection is illustrated in Fig. 6. Serpins and cystatins are capable of limiting virus entry into target cells through either direct interference with binding of the virus to host cell receptors 17

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(a)

(b)

Fig. 4 Cystatin A and Cystatin B demonstrate inhibitory effects on HIV-1 in a TZM-bl cell assay. Briefly, antiproteases were added at the same time as an R5-tropic (Bal) HIV-1 virus to TZM-bl reporter cells. Serum-free media was used for the duration of the three hour infection. Following removal of virus and antiprotease, fresh Dulbecco’s Eagle Modified Media (DMEM) containing 10% fetal bovine serum (FBS) was added to the cells. Plates were then incubated for three days following which a B-galactosidase assay was performed. Cytotoxicity assays were performed in parallel to the infection assays and no significant cytotoxic effects were observed. A. At varying concentrations (0.494 g/mL-40 lg/mL), Cystatin A inhibits HIV-1 infection. B. Similar results were observed with Cystatin B however a higher concentration of protein was required to observe significant results (13.33 lg/mL-40 lg/mL). A maximum of 50% reduction in infection was observed for both Cystatin A and B. (Results represent a triplicate experiment, p < 0.05).

(a)

(b)

(AAT) 31 or prevention of target cell activation (AAT & serpin C1).27 Serpin A3, AAT, cystatins A and B are all responsible for reducing the level of efficient chemotactic ability of various target cells through control of pro-inflammatory proteases at the site of infection,27,64 which in turn reduces the amount of available HIV target cells. For those cells that do 18

Fig. 5 In situ staining for antiproteases in ectocervical tissue. The bright-field images show representative human ectocervical tissue sections collected from healthy Caucasian women undergoing a hysterectomy for benign, non-inflammatory disorders. The sections are stained (in brown) for (a) Serpin A1 and (b) Cystatin B. The right column is a magnified view of the region indicated in the left column and the bottom figure in B is a negative control which was treated with only the secondary antibody (biotintylated polyclonal rabbit anti-mouse Ig Ab). All sections were stained with Hematoxylin (blue) for visualization of cell nuclei. Images were collected with a 109/ 0.30 objective. Scale bar = 200 lm.

become infected by HIV, they are offered increased protection from apoptosis by cystatin A 65 and reduction in viral replication via interference of AAT C-terminal fragment with the NF-kb pathway.6 Thus, if a virus is able to bypass the protective measure set in place at the mucosal surfaces, antiproteases assist in the control of the overall American Journal of Reproductive Immunology 71 (2014) 12–23 ª 2013 John Wiley & Sons Ltd

ROLE OF SERPINS AND CYSTATINS AGAINST HIV

(a)

(b)

(c)

(d)

(e)

Fig. 6 Hypothesis of protective mechanisms offered by antiproteases against inflammation and HIV infection at mucosal surfaces. (a) Virus entry is limited in susceptible cells; Serpin A1 C-36 fragment prevents HIV binding and uptake. Serpins inhibit the activating effect of specific proteases upon HIV-infection. Serpins primarily inhibit serine proteases such as elastase and cathepsins 23 which are all pro-inflammatory to a certain extent. (b) Higher antiprotease levels reduce protease-mediated activation of monocytes, T-cells, and DC’s. Serpins and Cystatins primarily inhibit serine proteases such as elastase and cathepsins 23 which are all proinflammatory and thus inhibit immune activation. (c) Inhibition of efficient chemotactic ability of target cells. Serpin A1 inhibits neutrophil chemotaxis through inactivation of calpain-I (cysteine protease), leading to subsequent random migration of neutrophils.63 Various Serpins and Cystatins inhibit monocyte, macrophage, neutrophils and T-cell migration, all of which either lead to the influx of more HIV target cells to the site of infection (neutrophils and macrophages) or are target cells themselves (T cells). (d) Infected cells are given increased protection, and virus replication is slowed in presence of higher antiproteases. Cystatin A has been shown to reduce the level of apoptosis,64 which could lead to reduction in the amount of viral particles being released into the human host following infection and of a single. (e) Antiproteases reduce the production of pro-inflammatory cytokines reducing migration of activated T cells, DC’s, and NK cells.

immune response, making efficient viral replication and dissemination from the initial founder population much more challenging. Potential of Utilizing Antiproteases as a Therapeutic Intervention or Preventative Treatment Current ARV therapy regimens have proven to be successful at treating HIV-infected individuals and American Journal of Reproductive Immunology 71 (2014) 12–23 ª 2013 John Wiley & Sons Ltd

have shown some promise as preventative treatments.66,67 However, with increasing duration of using these drugs, the problem of drug resistance is becoming a very common issue. Therefore, this necessitates parallel research into developing and identifying novel factors that are capable of inhibiting HIV. One such inhibitor that is currently in Phase I/II clinical trials is called VIRus-Inhibitory Peptide (VIRIP), which is a derivative of AAT. This 19

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20-peptide fragment of AAT functions to prevent gp41 fusion to the cell membrane following gp120 docking protein binding, thereby preventing release of the viral genome into the host cell. Frossmann and his colleagues cite the advantage of utilizing this antiprotease-derived peptide in ARV therapy due to the highly conserved nature of gp41 and its inability to undergo changes without complete loss of fusion function.33 Another benefit is the broad, cross-clade inhibitory activity that it exhibits against HIV. The downfalls for this therapy are the high cost associated with it and the need for it to be given intravenously,33 limiting the feasibility for administration in developing nations. This study further strengthens the argument that specific serpins have the ability to be used as preventative and treatment options for HIV. Second-generation microbicides are also beginning to employ smaller, more HIV-specific and host-cellspecific agents, compared with first generations. These microbicides are proving to be more potent in preventing HIV transmission and are also more suitable for use in non-gel-based microbicides, such as vaginal rings, making their use independent of coitus and thus more practical.68 Various natural innate factors with capabilities of interfering directly with either HIV binding or reverse transcription are currently in various stages of development for future use in microbicides. One such factor is PSC-RANTES. This antimicrobial peptide is a derivative of the natural immune factor RANTES and is a CCR5 chemokine inhibitor, it is currently in preclinical stages of development for use in a microbicide. This molecule binds to CCR5, thereby inhibiting binding of this coreceptor to the gp120 docking glycoprotein and subsequent virus entry into the host cell.68 This, as well as other trials, offers hope for further use of small natural proteins as the primary constituent of new novel microbicides. Serpins and cystatins may offer another attractive option for natural factors in microbicides. Their multifactorial effect at inhibiting HIV replication, while reducing inflammation, may provide a potential avenue of exploration as preventative technologies. Serpins, specifically, are part of the early innate immune response to viral infections and have been shown to have broad range antiviral activity against HIV, HCV, HSV, and influenza.35,69 Also, as both serpins and cystatins function by inhibiting natural, host-derived factors, there is relatively no risk of viral resistance to these antiproteases. Current ARV 20

treatment options primarily function to inhibit various viral proteins, reverse transcriptase, etc., allowing the virus the opportunity to mutate these proteins, thus becoming resistant to the treatment. However, when the primary target of the treatment is a host-derived protein, even if the virus mutates, there will be no subsequent effect on the ability of the antiprotease to inhibit their targets. Both AAT and serpin C1 are currently being used as replacement therapy options for patients deficient in these factors. These deficiencies result in an increased severity in conditions including chronic obstructive pulmonary disease, hepatic complications, asthma, and septic shock.70,71 Both of these treatments have resulted in marked improvement in the patient’s health and have been relatively well tolerated with minimal side-effects. Taken together, this data suggest that these specific serpins and potentially others are likely safe options for potential clinical use in HIV preventative/treatment options in the future. One study that is currently examining the effectiveness of a serpin as a therapeutic option for HIV involves serpin C1 (antithrombin III). Heparin-activated antithrombin III has been shown to significantly inhibit lentivirus replication and reduce levels of plasma viraemia, in a non-human primate model.72 Numerous other small, naturally occurring, molecules/peptides are currently being studied for potential use in microbicides. However, the current belief is that use of a combination of microbicides, with two or more different mechanisms of action being used together will be the most effective method for prevention of HIV-1 transmission.73 Conclusion Serpins and cystatins have demonstrated the ability to interfere with efficient transmission and dissemination of HIV through mechanisms associated with their antiprotease activity, resulting in an overall reduction in inflammation, target cell migration, and immune activation as well as through mechanisms that directly interfere with HIV binding and replication. Elevated levels of various serpins and cystatins in the cells and secretions of HESN women’s genital tracts may suggest that these antiproteases, not only are capable of interfering with HIV survival and replication at the systemic level but also at the mucosal level. The fact that these proteins are natural constitAmerican Journal of Reproductive Immunology 71 (2014) 12–23 ª 2013 John Wiley & Sons Ltd

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uents of the female genital tract makes them an ideal candidate for future microbicides. The use of natural anti-inflammatory factors in preventative strategies for HIV infection is an ideal strategy, in that it allows for protection against initial infection, prevents initiation of a strong immune responses, while reducing the risk that the virus will develop resistance to the treatment, all necessary components to an effective HIV therapy. Further studies must be performed in an effort to better understand exactly how specific serpins and cystatins affect the mucosal surfaces of the female genital tract. It is imperative to understand the specific roles of these antiproteases in controlling immune activation and inflammation within mucosal tissues. It has yet to be determined, if serpins or cystatins are capable of interfering directly with virus infection and replication within mucosally derived cells, which is an important next step to understand their suitability as microbicide candidates. An acceptable protective microbicide, which can be controlled by the receptive partner, independent of coitus remains the primary goal in the fight to reduce HIV transmission worldwide.68 This goal is achievable with the continued effort to screen and identify potential natural, innate factors capable of interfering with efficient infection and reproduction of HIV. Acknowledgments We would like to thank the study participants; Anette Hofmann for technical help; and Agnes Lagerstedt, Harry Flam, and colleagues at the St. G€ oran Hospital, Stockholm, Sweden, for the collection of clinical samples. We would also like to thank Sue Ramdahin at the University of Manitoba for her technical support. This work was funded by the Canadian Institute of Health Research (A.B., B.B) and the Swedish Society for Medical Research (A.T.). L.A. and A.B. equally contributed to writing of the manuscript. L.A. performed the neutralization assays. A.T. performed immunohistochemical analysis and assisted in editing the manuscript. B.B. assisted in writing and editing the manuscript. A.B. conceived the study and provided overall scientific guidance. Ethics statement All human participants provided informed consent and the study protocol approved by the Karolinska Institute’s ethics board. American Journal of Reproductive Immunology 71 (2014) 12–23 ª 2013 John Wiley & Sons Ltd

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