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Exploiting Old Pathogens to Create New Therapeutics
Cell Host & Microbe
In Translation Exploiting Old Pathogens to Create New Therapeutics Tiffany Bouchery1 and Nicola L. Harris1,* 1Global Health Institute, School of Life Sciences, E ´ cole Polytechnique Fe´de´rale de Lausanne (EPFL), 1015 Lausanne, Vaud, Switzerland *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chom.2016.11.007
Intestinal worms are well known for their potent immuno-modulatory capacity. In a recent study, Navarro et al. (2016) identify a secreted hookworm protein that can suppress allergic responses in both mice and humans. This represents an exciting strategy for treating chronic inflammatory disorders such as allergy. Nector americanus, more commonly referred to as the hookworm, evolved together with humans with evidence of these parasites found in many old world archaeological sites as well as in human fossils and mummies (Araujo et al., 2008). Today they can still be found at endemic levels among poor communities living without access to adequate sanitation. Recent estimates indicate that approximately 700 million people suffer from infection with Nector americanus or its close relative Ancylostoma duodenale (Hotez, 2008). A likely explanation for the spectacular success of these parasites is their ability to modulate the body’s immune system to avoid detection and to protect the host from ill health caused by long-term inflammation. In this way the parasite manages to establish a longterm relationship with its host (individual worms typically live for 1–5 years in our intestine) allowing time for the parasite to mature, mate, and produce eggs that are spread into the environment through feces. It is perhaps not surprising therefore that the absence of these parasites in industrialized regions has been linked to a rising incidence of disorders resulting from excessive or inappropriate immune responses, such as allergy and autoimmunity (Cooper, 2004). These observations have raised interest in the use of parasitic nematodes for the treatment of inflammatory diseases. Yet the therapeutic use of live parasites is hampered by problems of safety and scalability. It was on this basis that Navarro and colleagues (Navarro et al., 2016) embarked on their current work to identify and characterize the immune-modulatory factors secreted by Necator americanus. Importantly, the authors not only investigated the
disease-modulating activity of proteins isolated directly from the parasite, but also produced and tested a recombinant form of the most abundant protein, which they named anti-inflammatory protein-2 (AIP-2). This step is crucial for the translational potential of the work, as recombinant proteins allow for the large-scale production and superior purity necessary for human delivery. AIP-2 treatment could suppress the development of allergic airway inflammation in a mouse model of asthma. This effect required uptake of AIP-2 by specialized immune cells called dendritic cells (DCs) that migrated from the site of inoculation to the draining mesenteric lymph nodes and expanded a specialized subset of immune cells called regulatory T cells (Treg) (Figure 1). The DCs, which acquired AIP-2 expressed markers known to be associated with the stimulation of regulatory T cells (Tregs) (Coombes et al., 2007), and Tregs have been widely reported to play a key role in the prevention of inappropriate or excessive immune responses including allergy, autoimmunity, and inflammatory bowel disease (Lan et al., 2007). AIP-2 could also suppress the expansion of allergen reactive immune cells from human patients cultured in vitro. Whether the mechanisms allowing suppression of allergic responses are similar for mice and humans remains to be determined. Interestingly, the authors showed that the mesenteric lymph node of mice treated with AIP-2 retained a form of ‘‘imprinting’’ that allowed the long-term maintenance of regulatory immune responses. Although the exact nature of this imprinting was not determined, it is tempting to speculate that it involved an interaction between immune and stromal cells. Stromal cells provide structural
support to lymphoid organs and have recently been shown to interact with and provide a stimulatory or suppressive microenvironment for immune cells (Cording et al., 2014). Future work investigating the exact nature of the observed ‘‘imprinting’’ and it’s relevance to other lymphoid organs will be of great interest to immunologists and clinicians interested in promoting regulatory T cell responses. Other nematode products with potential to decrease inflammatory diseases have been identified, including ES-62, a glycoprotein from a filarial nematode, AV-17, a filarial cystatin, and AS-MIF, a macrophage inhibitory factor (MIF) homolog from Anisakis. All three of these parasite products are able to alleviate airway inflammation in mouse models of asthma. However, one of the advantages of using a protein from Necator americanus is that this parasite has been shown in epidemiological studies to be associated with protection against asthma in humans (Scrivener et al., 2001). By contrast, most of the other immunodulatory products identified to date have been originally isolated from parasites that naturally infect rodents or that are associated with a heightened risk of allergy in humans. A noteworthy difference between AIP-2 and ES-62 (a lead parasite-derived candidate for use as an anti-inflammatory therapeutic) is the lack of glycans or phosphorylcholine motifs on the protein. This is important, as the presence of unique motifs decorating many parasite-derived proteins has been suggested to underlie the difficulty in reproducing the therapeutic activity of proteins isolated from parasite secretions following production of the same protein in a recombinant form.
Cell Host & Microbe 20, December 14, 2016 ª 2016 Elsevier Inc. 705
Cell Host & Microbe
In Translation
Figure 1. Modulation of Allergic Airway Inflammation by the Parasitic Protein AIP-2 Intestinal-dwelling hookworms secrete an array of proteins, including AIP-2, which is taken up by migratory CD103+ dendritic cells (DCs). CD103+ DCs then migrate to the draining mesenteric lymph nodes where they promote the activation and expansion of regulatory T cells over allergy-promoting effector T cells. This process can also be mimicked by peritoneal inoculation of total parasite secreted products or recombinant AIP-2 into mice. The presence of an increased regulatory T cell pool in turn suppresses the development of allergic immune responses in the lung.
An important hurdle in the generation and use of novel protein therapeutics is the possible induction of a humoral immune response against the protein by the patient. This can lead to decreased efficacy, or worse may even cause a harmful reaction in the patient. By contrast, protein therapeutics that exhibit low immunogenicity, such as insulin, has been highly successful. Preliminary experiments in mice indicate that recombinant AIP-2 has low immunogenicity, strengthening its potential for therapeutic use. The next step will be to undertake detailed studies of solubility, distribution, and stability following AIP-2 administration to mice, and eventually in patients. Ideally, the route of administration would be optimized to avoid the use of needles. Although most of the work reported by Navarro et al. (2016) investigated delivery of AIP-2 by peritoneal injection, preliminary findings in mice indicated similar
efficacy following intranasal application, raising the possibility of its delivery to patients using an inhaler or nasal spray. Possibly the greatest challenge to developing protein therapeutics for chronic diseases is cost. Indeed, the cost of patient treatment with recombinant proteins can be greater than $100,000 per patient per year (Leader et al., 2008). This alone would render the use of AIP-2 as a therapeutic for asthma unlikely, unless improvements in recombinant protein expression, such as the use of cell-free expression systems, meet the requirement for lower cost production. Given that AIP-2 stimulates Tregs, future work could instead focus on the possible use of this protein as a prophylactic treatment that promotes allergen tolerance. Allergen tolerance defines an immunological state in which the patient ‘‘ignores’’ or ‘‘tolerates’’ an allergen rather than mounting the inflam-
706 Cell Host & Microbe 20, December 14, 2016
matory response that causes asthma. Induction of allergen tolerance through allergen desensitization currently represents one of the most efficient methods for preventing ongoing disease (Akdis, 2009). However, the practice of allergen desensitization has long been hampered by the need for the patient to undergo time-consuming and costly injections of regular small doses of allergen in the presence of a medically trained professional. Indeed, typical procedures entail regular visits to a medical practitioner over a period of 3 to 5 years. Delivery of the allergen together with a protein like AIP2 to promote the development of allergen reactive regulatory T cells could represent an exciting avenue for improving allergen tolerance. Should this strategy prove successful, the fusion of AIP-2 to other protein antigens (proteins able to activate immune cells) could prove useful in other disease states where immune tolerance
Cell Host & Microbe
In Translation is desirable, including autoimmunity, organ transplantation, and the delivery of important but highly immunogenic protein therapeutics such as L-Asparaginase, used in the treatment of acute lymphocytic leukemia.
REFERENCES Akdis, M. (2009). Curr. Opin. Immunol. 21, 700–707.
Araujo, A., Reinhard, K.J., Ferreira, L.F., and Gardner, S.L. (2008). Trends Parasitol. 24, 112–115.
Lan, R.Y., Mackay, I.R., and Gershwin, M.E. (2007). J. Autoimmun. 29, 272–280.
Coombes, J.L., Siddiqui, K.R., Arancibia-Ca´rcamo, C.V., Hall, J., Sun, C.M., Belkaid, Y., and Powrie, F. (2007). J. Exp. Med. 204, 1757–1764.
Leader, B., Baca, Q.J., and Golan, D.E. (2008). Nat. Rev. Drug Discov. 7, 21–39.
Cooper, P.J. (2004). Parasite Immunol. 26, 455–467.
Navarro, S., Pickering, D.A., Ferreira, I.B., Jones, L., Ryan, S., Troy, S., Leech, A., Hotez, P.J., Zhan, B., Laha, T., et al. (2016). Sci. Transl. Med. 8, 362ra143.
Cording, S., Wahl, B., Kulkarni, D., Chopra, H., Pezoldt, J., Buettner, M., Dummer, A., Hadis, U., Heimesaat, M., Bereswill, S., et al. (2014). Mucosal Immunol. 7, 359–368. Hotez, P.J. (2008). PLoS Negl. Trop. Dis. 2, e329.
Scrivener, S., Yemaneberhan, H., Zebenigus, M., Tilahun, D., Girma, S., Ali, S., McElroy, P., Custovic, A., Woodcock, A., Pritchard, D., et al. (2001). Lancet 358, 1493–1499.
Cell Host & Microbe 20, December 14, 2016 707
In Translation Exploiting Old Pathogens to Create New Therapeutics Tiffany Bouchery1 and Nicola L. Harris1,* 1Global Health Institute, School of Life Sciences, E ´ cole Polytechnique Fe´de´rale de Lausanne (EPFL), 1015 Lausanne, Vaud, Switzerland *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chom.2016.11.007
Intestinal worms are well known for their potent immuno-modulatory capacity. In a recent study, Navarro et al. (2016) identify a secreted hookworm protein that can suppress allergic responses in both mice and humans. This represents an exciting strategy for treating chronic inflammatory disorders such as allergy. Nector americanus, more commonly referred to as the hookworm, evolved together with humans with evidence of these parasites found in many old world archaeological sites as well as in human fossils and mummies (Araujo et al., 2008). Today they can still be found at endemic levels among poor communities living without access to adequate sanitation. Recent estimates indicate that approximately 700 million people suffer from infection with Nector americanus or its close relative Ancylostoma duodenale (Hotez, 2008). A likely explanation for the spectacular success of these parasites is their ability to modulate the body’s immune system to avoid detection and to protect the host from ill health caused by long-term inflammation. In this way the parasite manages to establish a longterm relationship with its host (individual worms typically live for 1–5 years in our intestine) allowing time for the parasite to mature, mate, and produce eggs that are spread into the environment through feces. It is perhaps not surprising therefore that the absence of these parasites in industrialized regions has been linked to a rising incidence of disorders resulting from excessive or inappropriate immune responses, such as allergy and autoimmunity (Cooper, 2004). These observations have raised interest in the use of parasitic nematodes for the treatment of inflammatory diseases. Yet the therapeutic use of live parasites is hampered by problems of safety and scalability. It was on this basis that Navarro and colleagues (Navarro et al., 2016) embarked on their current work to identify and characterize the immune-modulatory factors secreted by Necator americanus. Importantly, the authors not only investigated the
disease-modulating activity of proteins isolated directly from the parasite, but also produced and tested a recombinant form of the most abundant protein, which they named anti-inflammatory protein-2 (AIP-2). This step is crucial for the translational potential of the work, as recombinant proteins allow for the large-scale production and superior purity necessary for human delivery. AIP-2 treatment could suppress the development of allergic airway inflammation in a mouse model of asthma. This effect required uptake of AIP-2 by specialized immune cells called dendritic cells (DCs) that migrated from the site of inoculation to the draining mesenteric lymph nodes and expanded a specialized subset of immune cells called regulatory T cells (Treg) (Figure 1). The DCs, which acquired AIP-2 expressed markers known to be associated with the stimulation of regulatory T cells (Tregs) (Coombes et al., 2007), and Tregs have been widely reported to play a key role in the prevention of inappropriate or excessive immune responses including allergy, autoimmunity, and inflammatory bowel disease (Lan et al., 2007). AIP-2 could also suppress the expansion of allergen reactive immune cells from human patients cultured in vitro. Whether the mechanisms allowing suppression of allergic responses are similar for mice and humans remains to be determined. Interestingly, the authors showed that the mesenteric lymph node of mice treated with AIP-2 retained a form of ‘‘imprinting’’ that allowed the long-term maintenance of regulatory immune responses. Although the exact nature of this imprinting was not determined, it is tempting to speculate that it involved an interaction between immune and stromal cells. Stromal cells provide structural
support to lymphoid organs and have recently been shown to interact with and provide a stimulatory or suppressive microenvironment for immune cells (Cording et al., 2014). Future work investigating the exact nature of the observed ‘‘imprinting’’ and it’s relevance to other lymphoid organs will be of great interest to immunologists and clinicians interested in promoting regulatory T cell responses. Other nematode products with potential to decrease inflammatory diseases have been identified, including ES-62, a glycoprotein from a filarial nematode, AV-17, a filarial cystatin, and AS-MIF, a macrophage inhibitory factor (MIF) homolog from Anisakis. All three of these parasite products are able to alleviate airway inflammation in mouse models of asthma. However, one of the advantages of using a protein from Necator americanus is that this parasite has been shown in epidemiological studies to be associated with protection against asthma in humans (Scrivener et al., 2001). By contrast, most of the other immunodulatory products identified to date have been originally isolated from parasites that naturally infect rodents or that are associated with a heightened risk of allergy in humans. A noteworthy difference between AIP-2 and ES-62 (a lead parasite-derived candidate for use as an anti-inflammatory therapeutic) is the lack of glycans or phosphorylcholine motifs on the protein. This is important, as the presence of unique motifs decorating many parasite-derived proteins has been suggested to underlie the difficulty in reproducing the therapeutic activity of proteins isolated from parasite secretions following production of the same protein in a recombinant form.
Cell Host & Microbe 20, December 14, 2016 ª 2016 Elsevier Inc. 705
Cell Host & Microbe
In Translation
Figure 1. Modulation of Allergic Airway Inflammation by the Parasitic Protein AIP-2 Intestinal-dwelling hookworms secrete an array of proteins, including AIP-2, which is taken up by migratory CD103+ dendritic cells (DCs). CD103+ DCs then migrate to the draining mesenteric lymph nodes where they promote the activation and expansion of regulatory T cells over allergy-promoting effector T cells. This process can also be mimicked by peritoneal inoculation of total parasite secreted products or recombinant AIP-2 into mice. The presence of an increased regulatory T cell pool in turn suppresses the development of allergic immune responses in the lung.
An important hurdle in the generation and use of novel protein therapeutics is the possible induction of a humoral immune response against the protein by the patient. This can lead to decreased efficacy, or worse may even cause a harmful reaction in the patient. By contrast, protein therapeutics that exhibit low immunogenicity, such as insulin, has been highly successful. Preliminary experiments in mice indicate that recombinant AIP-2 has low immunogenicity, strengthening its potential for therapeutic use. The next step will be to undertake detailed studies of solubility, distribution, and stability following AIP-2 administration to mice, and eventually in patients. Ideally, the route of administration would be optimized to avoid the use of needles. Although most of the work reported by Navarro et al. (2016) investigated delivery of AIP-2 by peritoneal injection, preliminary findings in mice indicated similar
efficacy following intranasal application, raising the possibility of its delivery to patients using an inhaler or nasal spray. Possibly the greatest challenge to developing protein therapeutics for chronic diseases is cost. Indeed, the cost of patient treatment with recombinant proteins can be greater than $100,000 per patient per year (Leader et al., 2008). This alone would render the use of AIP-2 as a therapeutic for asthma unlikely, unless improvements in recombinant protein expression, such as the use of cell-free expression systems, meet the requirement for lower cost production. Given that AIP-2 stimulates Tregs, future work could instead focus on the possible use of this protein as a prophylactic treatment that promotes allergen tolerance. Allergen tolerance defines an immunological state in which the patient ‘‘ignores’’ or ‘‘tolerates’’ an allergen rather than mounting the inflam-
706 Cell Host & Microbe 20, December 14, 2016
matory response that causes asthma. Induction of allergen tolerance through allergen desensitization currently represents one of the most efficient methods for preventing ongoing disease (Akdis, 2009). However, the practice of allergen desensitization has long been hampered by the need for the patient to undergo time-consuming and costly injections of regular small doses of allergen in the presence of a medically trained professional. Indeed, typical procedures entail regular visits to a medical practitioner over a period of 3 to 5 years. Delivery of the allergen together with a protein like AIP2 to promote the development of allergen reactive regulatory T cells could represent an exciting avenue for improving allergen tolerance. Should this strategy prove successful, the fusion of AIP-2 to other protein antigens (proteins able to activate immune cells) could prove useful in other disease states where immune tolerance
Cell Host & Microbe
In Translation is desirable, including autoimmunity, organ transplantation, and the delivery of important but highly immunogenic protein therapeutics such as L-Asparaginase, used in the treatment of acute lymphocytic leukemia.
REFERENCES Akdis, M. (2009). Curr. Opin. Immunol. 21, 700–707.
Araujo, A., Reinhard, K.J., Ferreira, L.F., and Gardner, S.L. (2008). Trends Parasitol. 24, 112–115.
Lan, R.Y., Mackay, I.R., and Gershwin, M.E. (2007). J. Autoimmun. 29, 272–280.
Coombes, J.L., Siddiqui, K.R., Arancibia-Ca´rcamo, C.V., Hall, J., Sun, C.M., Belkaid, Y., and Powrie, F. (2007). J. Exp. Med. 204, 1757–1764.
Leader, B., Baca, Q.J., and Golan, D.E. (2008). Nat. Rev. Drug Discov. 7, 21–39.
Cooper, P.J. (2004). Parasite Immunol. 26, 455–467.
Navarro, S., Pickering, D.A., Ferreira, I.B., Jones, L., Ryan, S., Troy, S., Leech, A., Hotez, P.J., Zhan, B., Laha, T., et al. (2016). Sci. Transl. Med. 8, 362ra143.
Cording, S., Wahl, B., Kulkarni, D., Chopra, H., Pezoldt, J., Buettner, M., Dummer, A., Hadis, U., Heimesaat, M., Bereswill, S., et al. (2014). Mucosal Immunol. 7, 359–368. Hotez, P.J. (2008). PLoS Negl. Trop. Dis. 2, e329.
Scrivener, S., Yemaneberhan, H., Zebenigus, M., Tilahun, D., Girma, S., Ali, S., McElroy, P., Custovic, A., Woodcock, A., Pritchard, D., et al. (2001). Lancet 358, 1493–1499.
Cell Host & Microbe 20, December 14, 2016 707