Patent Grant
9522171
The present invention relates to methods of treating and preventing the inflammatory effects of viral infection of the upper and lower respiratory tracts, including infection by SARS coronovirus (SARS), pandemic Influenza A H5N1 (avian influenza) and influenza A H1N1 (swine 'flu).
All documents mentioned in the text and listed at the end of this description are incorporated herein by reference.
The mortality associated with SARS and pandemic influenza is linked to rapidly progressive respiratory failure causing acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). In some cases, multi-organ failure is also a feature. In the case of pandemic H5N1 influenza, the mortality due to respiratory and multi-organ failure is around 60%. The primary lung pathology of fatal H1N1 influenza has recently been described and is characterised by necrotising alveolitis and dense neutrophil infiltration [1].
Originally, it was assumed that respiratory failure associated with SARS and pandemic influenza was due to rapid viral replication leading to cytolytic destruction of target cells of the respiratory tract, such as alveolar epithelial cells, or to escape of the virus to tissues and organs remote from the respiratory system, such as the central nervous system. Recent evidence has shown, however, that the development of respiratory failure is not, in fact, associated with high viral titres. Investigators have instead found that respiratory failure is associated with significant elevation of pro-inflammatory cytokines such as TFNa and IFNβ. This has led experts to propose that the pathogenesis of these complications is inappropriate stimulation of the innate immune system triggering a so-called ‘cytokine storm’ [2, 3].
Current treatments for respiratory failure involve increasing the patient's oxygen levels using an oxygen mask, mechanical oxygenation using a ventilator or, in the most severe case, extracorporeal membrane oxygenation (ECMO) which involves circulating the patient's blood outside the body and adding oxygen to it artificially
There is a great need for agents that improve upon the currently available treatments for the respiratory failure caused by the inflammatory effects of viral infection of the respiratory tract.
Accordingly, the invention provides a method of treating or preventing the inflammatory effects of viral infection of the respiratory tract comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an agent that inhibits the classical complement pathway, the alternative complement pathway and the lectin complement pathway.
The invention also provides a therapeutically or prophylactically effective amount of an agent that inhibits the classical complement pathway, the alternative complement pathway and the lectin complement pathway for treating or preventing the inflammatory effects of viral infection of the respiratory tract.
The complement system is an essential part of the body's natural defence mechanism against foreign invasion and is also involved in the inflammatory process. More than 30 proteins in serum and at the cell surface are involved in complement system function and regulation. Recently it has become apparent that, as well as the ˜35 known components of the complement system which may be associated with both beneficial and pathological processes, the complement system itself interacts with at least 85 biological pathways with functions as diverse as angiogenesis, platelet activation, glucose metabolism and spermatogenesis
The complement system is activated by the presence of foreign antigens. Three activation pathways exist: (1) the classical pathway which is activated by IgM and IgG complexes or by recognition of carbohydrates; (2) the alternative pathway which is activated by non-self surfaces (lacking specific regulatory molecules) and by bacterial endotoxins; and (3) the lectin pathway which is activated by binding of manna-binding lectin (MBL) to mannose residues on the surface of a pathogen. The three pathways comprise parallel cascades of events that result in the production of complement activation through the formation of similar C3 and C5 convertases on cell surfaces resulting in the release of acute mediators of inflammation (C3a and C5a) and formation of the membrane attack complex (MAC). The parallel cascades involved in the classical and alternative pathways are shown in
The complement system is recognised as being an early activator of innate immune responses initiating many inflammatory cascades. However it has not previously been implicated as being a cause of the respiratory complications of viral infection of the respiratory system. Surprisingly, the data presented in the current application show for the first time that an agent that inhibits the alternative, classical and lectin complement pathways reduces the inflammatory effects of viral infection of the respiratory tract.
Reduction of the inflammatory effects of viral infection of the respiratory tract may be assessed by reduction in inflammatory cytokines and/or neutrophils in a subject suffering from such a viral infection. In one aspect of the invention, administration of the agent that inhibits the alternative, classical and lectin complement pathways may thus reduce levels of inflammatory cytokines, such as CXCL2, IL-1β, and/or IL-6, in a subject suffering from viral infection of the respiratory tract compared to an untreated subject. Administration of the agent that inhibits the alternative, classical and lectin complement pathways to a subject suffering from a viral infection of the respiratory tract may also reduce levels of neutrophils compared to an untreated subject. Cytokine levels and neutrophil levels may, for example, be assessed in bronchoalveolar lavage (BAL) fluid from the subject.
In one aspect of the invention, the agent may bind complement C5. The agent may act to prevent the cleavage of complement C5 by C5 convertase into complement C5a and complement C5b-9. The agent may act to reduce C5a levels in a subject suffering from a viral infection of the respiratory tract, for example in the BAL fluid from such a subject, compared to an untreated subject. Surprisingly, the data presented in the current application show for the first time that there is a significant increase in C5a in BAL fluid following viral infection of the respiratory tract.
The complement C5 protein, also referred to herein as C5, is cleaved by the C5 convertase enzyme, itself formed from C3a, an earlier product of the alternative pathway (
MAC is associated with other important pathological processes including rheumatoid arthritis [5;6], proliferative glomerulonephritis [7], idiopathic membranous nephropathy [8], proteinurea [9], demyelination after acute axonal injury [10] and is also responsible for acute graft rejection following xenotransplantation [11].
C5a has become a target of particular interest in the field of complement-associated disorders [12]. Although C5a has many well-recognised pathological associations, the effects of its depletion in humans appear to be limited. Monoclonal antibodies and small molecules that bind and inhibit C5a or C5a receptors have been developed to treat various autoimmune diseases. These molecules do not, however, prevent the release of MAC.
In contrast, the agent used in the current invention inhibits both the formation of C5a peptide and the MAC. Since C5 is a late product of the classical and alternative complement pathways, inhibition of C5 is less likely to be associated with risks of concomitant infection that exist when targeting earlier products in the cascade [13].
The ability of an agent to bind C5 may be determined by standard in vitro assays known in the art, for example by western blotting following incubation of the protein on the gel with labelled C5. Preferably, the agent according to the invention binds C5 with an IC50 of less than 0.2 mg/ml, preferably less than 0.1 mg/ml, preferably less than 0.05 mg/ml, preferably less than 0.04 mg/ml, preferably less than 0.03 mg/ml, preferably 0.02 mg/ml, preferably less than 1 μg/ml, preferably less than 100 ng/ml, preferably less than 10 ng/ml, more preferably still, less than 1 ng/ml.
According to one embodiment of the invention, the agent that binds C5 is not an anti-05 monoclonal antibody.
The invention also provides a method of treating or preventing the inflammatory effects of viral infection of the respiratory tract comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an agent that inhibits eicosanoid activity.
The invention also provides a therapeutically or prophylactically effective amount of an agent that inhibits eicosanoid activity for treating or preventing the inflammatory effects of viral infection of the respiratory tract.
The agent according to this aspect of the invention may inhibit leukotrine B4 (LTB4) activity. In particular, the agent according to this aspect of the invention may bind LTB4. The ability of an agent to bind LTB4 may be determined by standard in vitro assays known in the art, for example by western blotting following incubation of the protein on the gel with labelled LTB4. The agent according to the invention may bind LTB4 with an IC50 of less than 0.2 mg/ml, preferably less than 0.1 mg/ml, preferably less than 0.05 mg/ml, preferably less than 0.04 mg/ml, preferably less than 0.03 mg/ml, preferably 0.02 mg/ml, preferably less than 1 μg/ml, preferably less than 100 ng/ml, preferably less than 10 ng/ml, more preferably still, less than 1 ng/ml
In one aspect, the invention provides a method of treating or preventing the inflammatory effects of viral infection of the respiratory tract comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an agent that:
The invention also provides a therapeutically or prophylactically effective amount of an agent that inhibits:
According to one embodiment of this aspect of the invention, the agent binds both C5 and LTB4. The agent according to this embodiment may thus act to prevent the cleavage of complement C5 by C5 convertase into complement C5a and complement C5b-9 (MAC), and also to inhibit LTB4 activity.
The methods and uses of the invention described herein may be used to treat or prevent the inflammatory effects of viral infection of the upper or lower respiratory tracts. In particular, the methods and uses of the invention described herein may be used to treat or prevent respiratory failure caused by viral infection, including acute lung injury or acute respiratory distress syndrome. The methods and uses of the invention may also be used to treat or prevent the sequelae of respiratory failure caused by viral infection, including multi-organ failure.
The inflammation may be caused by any viral infection of the upper or lower respiratory tracts. In particular, the methods and uses of the invention may treat or prevent inflammatory effects caused by infection by pandemic influenza virus, such as influenza A H5N1 (avian influenza) and influenza A H1N1 (swine 'flu). The methods and uses of the invention may also be used to treat or prevent inflammatory effects caused by infection with SARS coronavirus.
Preferably, the agent of the invention is derived from a haematophagous arthropod. The term “haematophagous arthropod” includes all arthropods that take a blood meal from a suitable host, such as insects, ticks, lice, fleas and mites. Preferably, the agent is derived from a tick, preferably from the tick Ornithodoros moubata.
According to one embodiment of the invention, the agent is a protein comprising amino acids 19 to 168 of the amino acid sequence in
According to an alternative embodiment, the protein used according to this embodiment of the invention may comprise or consist of amino acids 1 to 168 of the amino acid sequence in
The protein having the amino acid sequence given in
The EV576 protein and the ability of this protein to inhibit complement activation has been disclosed in [14], where the EV576 protein was referred to as the “OmCI protein”. The EV576 protein has also been shown to be effective in the treatment of myasthenia gravis [15], respiratory disorders [16] and peripheral nerve disorders [17]. The ability of the EV576 protein to bind eicosanoids including LTB4 and its use in the treatment of diseases mediated by a leukotriene or hydroxyeicosanoid has been suggested in [18]. None of these disclosures suggest that the EV576 protein could be useful in the treatment or prevention of viral infection and in particular in the treatment or prevention of the inflammatory effects of viral infection of the respiratory tract.
It has now been found that the EV576 protein is surprisingly effective in the treatment and prevention of the inflammatory effects of viral infection of the respiratory tract. The data presented herein demonstrate that, in a murine model of human H1N1 influenza infection of the respiratory tract, mice treated with EV576 had significantly lower levels of protein and total cells, lower levels of complement C5a, lower levels of inflammatory cytokines IL-6, IL-1β, and CXCL2, and highly significantly lower neutrophils compared with vehicle-treated mice. EV576 thus represents a potential human therapy for the treatment and prevention of the inflammatory effects of viral infection of the respiratory tract. The surprising effectiveness of EV576 in the treatment of respiratory disorders may be due to the fact that it acts by binding C5, thus inhibiting the formation of C5a and MAC, or due to its LTB4 binding activity.
According to a further embodiment of the invention, the agent may be a nucleic acid molecule encoding the EV576 protein or a functional equivalent thereof. For example, gene therapy may be employed to effect the endogenous production of the EV576 protein by the relevant cells in the subject, either in vivo or ex vivo. Another approach is the administration of “naked DNA” in which the therapeutic gene is directly injected into the bloodstream or into muscle tissue.
Preferably, such a nucleic acid molecule comprises or consists of bases 55 to 507 of the nucleotide sequence in
The EV576 protein has been demonstrated to bind to C5 and prevent its cleavage by C5 convertase in rat, mouse and human serum with an IC50 of approximately 0.02 mg/ml. Preferably, functional equivalents of the EV576 protein which retain the ability to bind C5 with an IC50 of less than 0.2 mg/ml, preferably less than 0.1 mg/ml, preferably less than 0.05 mg/ml, preferably less than 0.02 mg/ml, preferably less than 1 μg/ml, preferably less than 100 ng/ml, preferably less than 10 ng/ml, more preferably still, less than 1 ng/ml.
The EV576 protein has also been demonstrated to bind LTB4. Functional equivalents of the EV576 protein may also retain the ability to bind LTB4 with a similar affinity as the EV576 protein.
In one respect, the term “functional equivalent” is used herein to describe homologues and fragments of the EV576 protein which: a) retain its ability to bind C5, and to prevent the cleavage of complement C5 by C5 convertase into complement C5a and complement C5b-9; and/or b) retain its ability to bind LTB4.
The term “functional equivalent” also refers to molecules that are structurally similar to the EV576 protein or that contain similar or identical tertiary structure, particularly in the environment of the active site or active sites of the EV576 protein that binds to C5 and/or LTB4, such as synthetic molecules. Amino acids in EV576 that are likely to be required for LTB4 binding are described in [18].
The term “homologue” is meant to include reference to paralogues and orthologues of the EV576 sequence that is explicitly identified in
Methods for the identification of homologues of the EV576 sequence given in
Typically, greater than 30% identity between two polypeptides (preferably, over a specified region such as the active site) is considered to be an indication of functional equivalence and thus an indication that two proteins are homologous. Preferably, proteins that are homologues have a degree of sequence identity with the EV576 protein sequence identified in
Homologues of the EV576 protein sequence given in
Fragments of the EV576 protein and of homologues of the EV576 protein are also embraced by the term “functional equivalents” providing that such fragments retain the ability to bind C5 and/or LTB4. Fragments may include, for example, polypeptides derived from the EV576 protein sequence which are less than 150 amino acids, less than 125 amino acids, less than 100 amino acids, less than 75 amino acids, less than 50 amino acids, or even 25 amino acids or less, provided that these fragments retain the ability to bind to complement C5.
Included as such fragments are not only fragments of the O. moubata EV576 protein that is explicitly identified herein in
A functional equivalent used according to the invention may be a fusion protein, obtained, for example, by cloning a polynucleotide encoding the EV576 protein in frame to the coding sequences for a heterologous protein sequence. The term “heterologous”, when used herein, is intended to designate any polypeptide other than the EV576 protein or its functional equivalent. Example of heterologous sequences, that can be comprised in the soluble fusion proteins either at N- or at C-terminus, are the following: extracellular domains of membrane-bound protein, immunoglobulin constant regions (Fc region), multimerization domains, domains of extracellular proteins, signal sequences, export sequences, or sequences allowing purification by affinity chromatography. Many of these heterologous sequences are commercially available in expression plasmids since these sequences are commonly included in the fusion proteins in order to provide additional properties without significantly impairing the specific biological activity of the protein fused to them [19]. Examples of such additional properties are a longer lasting half-life in body fluids, the extracellular localization, or an easier purification procedure as allowed by a tag such as a histidine or HA tag.
The EV576 protein and functional equivalents thereof, may be prepared in recombinant form by expression in a host cell. Such expression methods are well known to those of skill in the art and are described in detail by [20] and [21]. Recombinant forms of the EV576 protein and functional equivalents thereof are preferably unglycosylated.
The proteins and fragments of the present invention can also be prepared using conventional techniques of protein chemistry. For example, protein fragments may be prepared by chemical synthesis. Methods for the generation of fusion proteins are standard in the art and will be known to the skilled reader. For example, most general molecular biology, microbiology recombinant DNA technology and immunological techniques can be found in [20] or [22].
The subject to which the agent is administered in the method or use of the invention is preferably a mammal, preferably a human. The subject to which the agent is administered may also be suffering from a viral infection of the upper or lower respiratory tract, such as pandemic influenza virus, including influenza A H5N1 (avian influenza) and influenza A H1N1 (swine 'flu), or SARS coronavirus.
The agent is administered in a therapeutically or prophylactically effective amount. The term “therapeutically effective amount” refers to the amount of agent needed to treat or ameliorate the inflammation associated with the viral infection The term “prophylactically effective amount” used herein refers to the amount of agent needed to prevent inflammation associated with the viral invention.
Preferably, the dose of the agent is sufficient to bind as much available C5 as possible in the subject, more preferably, all available C5. The dose of the agent may alternatively be sufficient to bind as much available LTB4 as possible in the subject, more preferably, all available LTB4. In some aspects, the dose of the agent is sufficient to binds as much available C5 and LTB4 as possible, for example all available C5 and LTB4. The dose of the agent supplied is at least twice the molar dose needed to bind all available C5 and/or LTB4 in the subject. The dose of the agent supplied may be 2.5 times, 3 times or 4 times the molar dose needed to bind all available C5 and/or LTB4 in the subject. Preferably, the dose is from 0.0001 mg/kg (mass of drug compared to mass of patient) to 20 mg/kg, preferably 0.001 mg/kg to 10 mg/kg, more preferably 0.2 mg/kg to 2 mg/kg.
The frequency with which the dose needs to be administered will depend on the half-life of the agent involved. Where the agent is the EV576 protein or a functional equivalent thereof, the dose may be administered as a continuous infusion, in bolus doses or on a daily basis, twice daily basis, or every two, three, four days, five, six, seven, 10, 15 or 20 days or more.
The exact dosage and the frequency of doses may also be dependent on the patient's status at the time of administration. Factors that may be taken into consideration when determining dosage include the severity of the disease state in the patient, the general health of the patient, the age, weight, gender, diet, time and frequency of administration, drug combinations, reaction sensitivities and the patient's tolerance or response to therapy. The precise amount can be determined by routine experimentation, but may ultimately lie with the judgement of the clinician.
The agent will generally be administered as part of a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier”, as used herein, includes genes, polypeptides, antibodies, liposomes, polysaccharides, polylactic acids, polyglycolic acids and inactive virus particles or indeed any other agent provided that the carrier does not itself induce toxicity effects or cause the production of antibodies that are harmful to the individual receiving the pharmaceutical composition. Pharmaceutically acceptable carriers may additionally contain liquids such as water, saline, glycerol, ethanol or auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like. The pharmaceutical carrier employed will thus vary depending on the route of administration. Carriers may enable the pharmaceutical compositions to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions to aid intake by the patient. A thorough discussion of pharmaceutically acceptable carriers is available in [23].
The agent may be delivered by any known route of administration. The agent may be delivered nasally, by inhalation, for example, using a metered-dose inhaler, nebuliser, dry powder inhaler, or nasal inhaler. The agent may be delivered by a parenteral route (e.g. by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue). The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications, needles, and hyposprays.
The agent may be administered alone or as part of a treatment regimen also involving the administration of other drugs currently used in the treatment of patients with respiratory disorders. For example, the agent may be administered in combination with the infusion of additional anti-viral drugs and/or oxygen treatment.
The agent may be administered simultaneously, sequentially or separately with the other drug(s). For example, the agent may be administered before or after administration of the other drug(s).
The invention thus provides an agent that binds C5 and/or LTB4, preferably the EV576 protein or a functional equivalent thereof, for treating or preventing the inflammatory effects of viral infection of the respiratory tract in a subject, wherein said subject has been pre-treated with an anti-viral drug. Examples of anti-viral drugs that may be used in this aspect of the invention include zanamivir (Relenza) and oseltamivir (Tamiflu).
Various aspects and embodiments of the present invention will now be described in more detail by way of example. It will be appreciated that modification of detail may be made without departing from the scope of the invention.
EV576 protein having the amino acid sequence shown in
Methods
BalbC mice were infected transnasally with either a sub-lethal dose (104 PFU) or a lethal dose (106 PFU) of human H1N1 virus obtained from the Institut Pasteur or sham infected using phosphate buffered saline (PBS).
Signs of lower respiratory tract infection (weight loss and respiratory congestion) developed within 4 days in low inoculum animals and more rapidly in the high inoculum group who all succumbed to the virus by the third day.
EV576 protein having the amino acid sequence shown in
High inoculum mice received only the first two doses as they were all dead by Day 3. Bronchoalveolar lavage (BAL) was performed on Day 3 for high inoculum mice. Bronchoalveolar lavage (BAL) was performed on Day 6 for low inoculum mice, 24 hours after the last dosing and immediately prior to sacrifice. Lavage fluid was assayed for total cells, neutrophils, total protein and cytokines (IL-1β, IL-6 and CXCL2).
The results are shown in
All parameters were found to be highly significantly raised in vehicle treated mice compared to mock (sham) treated mice.
Results
Mice treated with EV576 had significantly lower levels of protein (
At 104 PFU vehicle treated and rEV131 mice had highly significantly (p<0.001) raised total cells, neutrophils and protein compared to sham (mock) treated animals. Animals treated with EV576 had significantly less elevation of total cells (p<0.05) and neutrophils (p<0.01) than vehicle treated ones (
In the low inoculum group levels of the inflammatory cytokines IL-1β, IL-6 and CXCL2 in BAL fluid were found to be significantly (p<0.05) or highly significantly (p<0.01) elevated in vehicle and rEV131 treated groups compared to sham (mock) treated animals (
The sum of all the histopathological scores of lung inflammation is show in
These data demonstrate that EV576 significantly inhibits the inflammatory effects of H1N1 infection. EV576 is known to inhibit the complement system via binding of C5 and to inhibit eicosanoid activity. These data suggest that EV576 and other agents having similar C5 binding properties may be effective in reducing the lung inflammatory effects of viral infection.
EV576 protein having the amino acid sequence shown in
Methods
BalbC mice were infected and treated as described above in Example 1.
C5a levels in BALF were measured in mock inoculated animals on day, on days 1, 4, 7, and 10 in animals exposed to a sub-lethal dose (104 PFU), and on days 1, 3 and 5 in animals exposed to a lethal dose (106 PFU) of human H1N1 virus.
On day 6, C5a levels in BALF were measured in low inoculum animals that had also been treated with vehicle, EV131 or EV576. On day 3, C5a levels in BALF were measured in high inoculum animals that had also been treated with vehicle or EV576.
Results are shown in
Results
There is a significant rise in C5a in BAL fluid for up to 10 days following infection at both the high and low inoculum levels (
This is the first time that a direct role for complement has demonstrated in inflammation caused by H1N1 influenza infection. This rise in complement can be blocked using the complement inhibitor. EV576. The ability of EV576 to block complement and associated inflammation suggests that it will be effective in the treatment of H1N1 influenza infection.
The cytokine storm and associated inflammation that follows H1N1 infection is also present following H5N1 infection. The ability of EV576 to block complement rise and associated inflammation following H1N1 infection is therefore expected to be replicated in following H5N1 infection. EV576 protein having the amino acid sequence shown in
Methods
The experimental protocol is similar to that reported in reference 24.
Pathogen-free, female, 8 week old mice with a standardised body weight of 20-23 g (e.g. FVB/J strain) are infected transnasally with human H5N1 virus or H1N1 virus obtained from the Institut Pasteur or sham infected using phosphate buffered saline (PBS).
EV576 protein having the amino acid sequence shown in
The following signs of infection and readouts of treatment effects can be measured:
Time points for collection of readouts will be day 2, day 3 and day 4 post infection after H5N1 or day 2, day 4, day 6, day 7, day 8 post infection after H1N1. Pre-infection values will also be determined.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1000318.4 | Jan 2010 | GB | national |
| 1005071.4 | Mar 2010 | GB | national |
This application is a continuation of U.S. application Ser. No. 13/521,011, filed on Jul. 6, 2012, which is a National Stage Application of PCT Application No. PCT/GB2011/000022, filed Jan. 10, 2011, which in turn, claims priority from Great Britain application Serial No. 1000318.4, filed Jan. 8, 2010, and Great Britain application Serial No. 1005071.4, filed Mar. 25, 2010. The entire disclosure of each of the above-referenced applications is incorporated herein by reference in its entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 7884066 | Ting | Feb 2011 | B2 |
| 7884069 | Schaebitz et al. | Feb 2011 | B2 |
| 20070141573 | Nunn | Jun 2007 | A1 |
| 20100105611 | Hamer | Apr 2010 | A1 |
| 20110059885 | Lea et al. | Mar 2011 | A1 |
| Number | Date | Country |
|---|---|---|
| WO-9317099 | Sep 1993 | WO |
| WO-2007028968 | Mar 2007 | WO |
| WO-2007117241 | Oct 2007 | WO |
| WO-2008029167 | Mar 2008 | WO |
| WO-2008029169 | Mar 2008 | WO |
| WO-2009098454 | Aug 2009 | WO |
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| Number | Date | Country | |
|---|---|---|---|
| 20150196619 A1 | Jul 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13521011 | US | |
| Child | 14588616 | US |
