Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
  • C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
  • C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/0003—Invertebrate antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
  • A61K2039/552—Veterinary vaccine
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to vaccines for the prevention and/or treatment of filarial nematode infections, and to methods of prevention and/or treatment using such vaccines.
• the invention also relates to novel proteins, suitable for use in the prevention and/or treatment of filarial nematode infections.
• the invention further relates to pharmaceutical compositions comprising proteins of the invention, or nucleic acids encoding such proteins.
• the various aspects of the present invention are applicable to the prevention and/or treatment of filarial nematode infections in canine subjects, and also in human subjects.
• Nematodes are frequent infectious agents of both human and veterinary animal subjects.
• Filarial nematodes (belonging to the superfamily Filarioidea) are responsible for a global health burden of approximately 6.3 million disability-adjusted life-years, which represents the greatest single component of morbidity attributable to helminths affecting humans.
• the rodent filaria Litomosoides sigmodontis has become a popular experimental model over the past two decades, as BALB/c mice are fully permissive for its development and reproduction.
• Lymphatic filariasis or "elephantiasis", which is distributed across Africa, South Asia, the Pacific, Latin America and the Caribbean, accounts for 92% of this toll; while the remainder is caused by onchocerciasis or "river blindness", primarily in sub-Saharan Africa.
• the major human filarial pathogens are Wuchereria bancrofti (which is responsible for 90% of LF cases), Brugia malayi and Brugia timori (geographically restricted causes of LF), and Onchocerca volvulus (the sole agent of human onchocerciasis).
• Loa loa affects ⁇ 13 million people in West and Central Africa, generally causing a relatively mild disease, although infection has been associated with severe and sometimes fatal adverse events following chemotherapy.
• Filarial parasites are primarily drivers of chronic morbidity, which manifests as disabling swelling of the legs, genitals and breasts in LF; or visual impairment and severe dermatitis in onchocerciasis. Furthermore, filarial parasites are also a major problem in small animal veterinary medicine, with ⁇ 0.5 million dogs in the USA alone infected with Dirofilaria immitis, the cause of potentially fatal heartworm disease.
• the invention provides a polypeptide comprising a ShK domain of a filarial nematode protein, or a variant thereof, for use as a vaccine for the prevention and/or treatment of a filarial nematode infection.
• the invention provides an artificial polypeptide comprising a plurality of ShK domains of a filarial nematode protein, or variants of such domains, and an artificial spacer separating the ShK domains or variants.
• the invention provides a nucleic acid encoding a polypeptide according to the second aspect of the invention.
• the invention provides a nucleic acid encoding a polypeptide comprising a ShK domain of a filarial nematode protein, or a variant thereof, for use as a vaccine for the prevention and/or treatment of a filarial nematode infection.
• the invention provides a pharmaceutical composition comprising a polypeptide that comprises a ShK domain of a filarial nematode protein, or a variant thereof.
• the invention provides pharmaceutical composition comprising a nucleic acid encoding a polypeptide that comprises a ShK domain of a filarial nematode protein, or a variant thereof.
• the invention provides a method of preventing and/or treating a filarial nematode infection, the method comprising providing to a subject in need of such prevention and/or treatment a therapeutically effective amount of a polypeptide comprising a ShK domain of a filarial nematode protein, or a variant thereof.
• the various aspects of the invention have utility in the prevention and/or treatment of filarial nematode infections in human subjects, or in veterinary subjects such as dogs.
• Filarial nematodes are those most commonly responsible for diseases in humans, and to a lesser extent, other animal hosts. A good deal of information is available regarding the proteome of filarial nematodes.
• ShK domains which are so called due to their similarity to the Stichodactyla toxin produced by the sea anemone Stichodactyla helianthus, contain six cysteine residues with a characteristic spacing.
• ShK domains present in an amino acid sequence are readily identified using a bioinformatics approach. For example, they are defined in the Pfam database by the identifier "PF01549" and in the InterPro database by the identifier "IPR003582".
• the inventors have found that proteins from filarial nematode species that vary quite significantly in terms of their sequence across the protein as a whole share notably higher levels of similarity in their ShK domains. This opens the possibility of using polypeptides comprising ShK domains (or variants thereof) derived from a first filarial nematode pathogen in the prevention and/or treatment of diseases caused by infection with a second, different, filarial nematode pathogen.
• ShK domains in filarial nematode proteins are illustrated in the sequence information and comparison section of this specification. Here the characteristic arrangement of six cysteines within the ShK domains can be seen, as can the increased degree of sequence identity within ShK domains of different nematodes (as compared to sequence identity shared by the proteins as a whole).
• the L. sigmodontis ShK domain protein nl_s_04059 represents an example of a filarial nematode protein; a ShK domain of which may be employed in the various aspects of the invention.
• ShK domains from this protein or orthologues of this protein, or variants thereof, may be employed in the various aspects of the invention.
• nl_s_04059 orthologues including suitable ShK domains, or variants thereof, are shown in Figure 12. These include proteins derived from filarial nematodes such as: L. sigmodontis (for example isoform nl_S.2.1.2.t04059 (Gene ID nLs.2.
• B. malayi for example isoforms Bm8157b (Gene ID WBGene00228418, Species Brugia malayi (PRJNA 10729), Location Bmal_v3_scaffold110:1 12156-1 16304), Bm8157d (Gene ID WBGene00228418 Species Brugia malayi (PRJNA10729) Location Bmal_v3_scaffold1 10: 1 12156-1 16304), Bm8157c (Gene ID WBGene00228418, Species Brugia malayi (PRJNA10729) Location Bmal_v3_scaffold1 10:1 12156-1 16304) and Bm12896a), A.
• viteae for example isoform nAV.1.0.1.t09742, (Gene ID nAv.1.0.1.g09742, Species Acanthocheilonema viteae (PRJEB4306) Location nAv.1.0.scaf00135:58565-62790), D.
• nDi.2.2.2.t03402 for example isoforms nDi.2.2.2.t03402 (Gene ID nDi.2.2.2.g03402 Species Dirofilaria immitis (PRJEB1797) Location nDi.2.2.scaf00051 :142071-145588), and nDi.2.2.2.t04314 (Gene ID nDi.2.2.2.g04314, Species Dirofilaria immitis (PRJEB1797) Location nDi.2.2.scaf00083:48251-52806), L.
• ochengi for example isoforms nOo.2.0.1.t12220 (Gene ID nOo.2.0.1.g12220 Species Onchocerca ochengi (PRJEB1809) Location nOo.2.0.Scaf09993:244-1668), nOo.2.0.1.t06172 (Gene ID nOo.2.0.1.g06172, Species Onchocerca ochengi (PRJEB1809) Location nOo.2.0.Scaf01844: 10830-1 1939), and nOo.2.0.1.t06343 (Gene ID nOo.2.0.1.g06343, Species Onchocerca ochengi (PRJEB1809) Location nOo.2.0.Scaf01943:3177-7069).
• O. volvulus for example isoforms OVOC 0000232701.1 , and OVOC 000102301.1
• W. bancrofti for example isoforms WUBG
• a variant of a ShK domain of a filarial nematode protein should be considered to encompass sequences sharing at least 70% identity with a ShK domain of a filarial nematode protein.
• variants may share at least 75% identity, at least 80% identity, at least 85% identity, or at least 90% identity with a ShK protein.
• variants may share at least 91 % identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity with a Shk domain of a filarial nematode protein.
• a polypeptide of the invention should exhibit the ability to induce a protective immune response.
• a variant may retain at least 70% of the immunogenic capacity of the ShK domain from which it is derived.
• a variant may retain at least 80%, at least 90%, or even at least 95% of the immunogenic capacity of the ShK domain from which it is derived.
• a variant may have a greater immunogenic capacity than the ShK domain from which it is derived.
• prevention and/or treatment The medical uses, methods of treatment, and pharmaceutical compositions of the invention may be used to establish protective immunity that prevents the establishment of a filarial nematode infection in a subject.
• This prophylactic use exemplifies the "prevention" of a filarial nematode infection as this term is used in the present disclosure.
• references to "a polypeptide of the invention” or to “polypeptides of the invention” should be taken as encompassing not only the artificial polypeptides of the second aspect of the invention, but also the polypeptides for medical use (as vaccines for the prevention and/or treatment of a filarial nematode infection) defined by the first aspect of the invention.
• the medical uses of the first aspect of the invention may employ naturally occurring polypeptides, or may make use of artificial polypeptides such as those of the second aspect of the invention.
• polypeptides of the invention are suitable for use as vaccines, where the vaccine is for the prevention and/or treatment of a filarial nematode infection.
• a polypeptide of the invention may comprise an ShK domain from L. sigmodontis.
• the inventors have believe that polypeptides comprising ShK domains from L. sigmodontis (and specifically nucleic acids encoding such polypeptides) are surprisingly able to act as vaccines conferring protective immunity in respect of infections by filarial nematodes other than L. sigmodontis.
• a polypeptide in accordance with the invention comprises an ShK domain from a filarial nematode infection which is to be prevented and/or treated (or a variant of such an ShK domain).
• a polypeptide for of the invention may be for use in the prevention and/or treatment of canine heartworm.
• the polypeptide may comprise a ShK domain from D. immitis, or a variant thereof.
• a polypeptide of the invention may be for use in the prevention and/or treatment of a disease in a human subject, the disease being selected from the group consisting of: lymphatic filariasis (also referred to as “elephantiasis”); onchocerciasis (also referred to as “river blindness”); and loiasis.
• lymphatic filariasis also referred to as “elephantiasis”
• onchocerciasis also referred to as “river blindness”
• loiasis loiasis.
• a polypeptide of the invention for use in the prevention and/or treatment of lymphatic filariasis will comprise a ShK domain from a filarial nematode selected from the group consisting of: Wuchereria bancrofti; Brugia malayi; and Brugia timori, or a variant thereof.
• W. bancrofti is responsible for approximately 90% of lymphatic filariasis cases, and so polypeptides comprising an ShK domain from W. bancrofti may be preferred for use in the prevention and/or treatment of lymphatic filariasis.
• a polypeptide of the invention in an embodiment in which a polypeptide of the invention is for use in the prevention and/or treatment of onchocerciasis, it may comprise a ShK domain from Onchocerca volvulus, or a variant thereof.
• a suitable polypeptide for use in the prevention and/or treatment of loiasis may comprise a ShK domain from Loa loa, or a variant thereof.
• a polypeptide of the invention comprises a plurality of ShK domains, or variants thereof.
• a polypeptide of the invention may comprise at least two, at least three, at least four, at least five, or at least six ShK domains or variants thereof.
• a polypeptide of the invention may comprise two, three, four, five or six ShK domains or variants thereof.
• a polypeptide according to the invention comprises a plurality of ShK domains (or variants thereof), it may comprise a plurality of the same ShK domain (or variants of the same ShK domain). In an embodiment utilising variants of the same ShK domain these variant may be the same variant, or may comprise a plurality of different variants.
• a polypeptide of the invention comprising a plurality of ShK domains (or variants thereof), it may comprise a plurality of the different ShK domains (or variants of these different domains).
• each one of the plurality of ShK domains may be different, or alternatively the polypeptide may comprise more than one copy of a single ShK domain among a plurality of different domains.
• the naturally occurring ShK domain protein contains six ShK domains, each of which has its own characteristic sequence.
• a polypeptide of the invention may comprise each of these six ShK domains.
• a polypeptide of the invention may comprise six ShK domains made up of six copies of the same ShK sequence, such as the sixth of the sequences found in the native protein.
• the sixth ShK sequence found in the native ShK domain protein of D. immitis may represent a preferred ShK domain to be included (either directly, or in variant form) in a polypeptide of the invention.
• a polypeptide of the invention may comprise one or more ShK domains (or variants thereof) selected from ShK domains one to five of the native protein, in addition to the sixth ShK domain (or a variant thereof).
• the single ShK domain may be the sixth ShK domain from the ShK domain protein of D. immitis (or a variant based upon this domain).
• a polypeptide of the invention may be a branched protein.
• each branch of the protein may carry an antigenic sequence.
• Some, and potentially all, of these antigenic sequences may comprise ShK domains, or their variants.
• a polypeptide of the invention may further comprise an additional antigen that is able to confer protective immunity on a subject to whom the additional antigen is provided.
• a polypeptide may comprise an additional antigen that does not comprise an ShK domain.
• the additional antigen incorporated in such a polypeptide may be a further nematode antigen.
• the additional antigen capable of conferring protective immunity is derived from the same filarial nematode as the ShK domain incorporated in the polypeptide.
• a polypeptide to be employed in accordance with the various aspects or embodiments of the invention may be a naturally occurring polypeptide.
• the second aspect of the invention provides an artificial polypeptide comprising a plurality of ShK domains of a filarial nematode protein, or variants thereof, and an artificial spacer separating the ShK domains or variants.
• a suitable artificial spacer serves to expose the ShK domains to cells of the immune system, thereby allowing the development of protective immunity.
• the spacer itself need not contribute to the development of the protective immunity and may itself be immunologically inert.
• the artificial spacer may be any spacer, other than naturally occurring sequence found between ShK domains in a natural protein, that serves to separate the ShK domains, or variants, within the artificial protein.
• an artificial spacer suitable for use in the artificial polypeptides of the invention may comprise a sequence of amino acid residues that separates the ShK domains or variant.
• the spacer may comprise poly-L-lysine.
• Artificial polypeptides of the invention may comprise a plurality of artificial spacers, as necessitated by the number of ShK domains (or variants thereof) incorporated in the artificial polypeptide.
• a polypeptide to be employed in accordance with the various aspects or embodiments of the invention may be an artificial polypeptide, such as an artificial polypeptide of the 2 nd aspect of the invention.
• an artificial polypeptide of the invention may be a chimeric polypeptide.
• Artificial polypeptides of the invention may comprise a ShK domain, or variant thereof, and an additional antigen that is not found in the polypeptide from which the ShK domain is derived.
• an artificial protein of the invention may comprise an ShK domain (or variant thereof) and an additional antigen from a nematode that the ShK domain is derived from, or an additional antigen from a nematode other than that which the ShK is derived from, or an additional antigen that is derived from a source other than a nematode.
• Chimeric polypeptides of the invention comprising an ShK domain or variant thereof, and an additional antigen from a source other than the filarial nematode from which the ShK domain was derived are able to induce protective immunity against more than one pathogen.
• Artificial polypeptides of the invention may comprise a plurality of the same ShK domain, or variants thereof. Alternatively, artificial polypeptides of the invention may comprise a plurality of different ShK domains, or variants thereof.
• an artificial polypeptide of the invention further comprises an additional vaccine antigen.
• the additional vaccine antigen may be derived from an antigen that does not comprise a ShK domain.
• An artificial polypeptide of the invention may comprise an additional vaccine antigen derived from the same filarial nematode (or filarial nematodes) as the ShK domains incorporated in the polypeptide.
• Suitable additional vaccine antigens that may be incorporated in artificial polypeptides of the invention include cysteine proteinase inhibitor (CPI) and/or abundant larval transcript (ALT). As discussed elsewhere in the specification, these proteins represent secreted immunomodulators secreted by female filarial nematodes, and targeting of these immunomodulators by vaccination leads to greatly reduced microfilaremia!. Accordingly, introduction of these additional vaccine antigens into artificial polypeptides of the invention will be expected to confer therapeutic advantages that go beyond the surprising benefits provided by the polypeptides of the invention.
• CPI cysteine proteinase inhibitor
• ALT abundant larval transcript
• Therapeutic vaccination of Onchocerca vo/vulus -infected hosts with vaccines comprising CPI and/or ALT in combination with the ShK domain-containing polypeptides of the invention may provide further suppression of microfilaria! production, prevent the progression of disease, reduce morbidity and block transmission, even if adult worm burden remains unaffected.
• a polypeptide, medical use, or method of treatment of the invention utilising as a vaccine a polypeptide comprising a ShK domain of a filarial nematode protein, or a variant thereof, may be used in conjunction with a vaccine comprising CPI and/or ALT.
• a vaccine comprising CPI and/or ALT.
• the polypeptide of the invention may be provided in the same vaccine as the CPI and/or ALT.
• the polypeptide of the invention and the CPI and/or ALT may be provided in separate vaccines.
• the invention provides a nucleic acid encoding a polypeptide of the invention.
• the nucleic acid may encode an artificial polypeptide in accordance with the second aspect of the invention.
• the invention also provides a vector comprising a nucleic acid of the invention, and such a vector may be adapted for expression in bacteria, such as E. coli.
• compositions of the invention may comprise a polypeptide of the invention and/or a nucleic acid of the invention.
• the nucleic acid may be provided in the form of a vector.
• Suitable pharmaceutical compositions of the invention may be formulated for use as a vaccine, and may be formulated for any appropriate route of administration, including (but not limited to) injection.
• suitable routes of administration include, but are not limited to, oral (e.g, by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eyedrops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuti
• compositions in accordance with the invention may be formulated such that the polypeptide or nucleic acid of the invention is delivered in alum adjuvant, or in virus-like particles.
• the seventh aspect of the invention provides a method of preventing and/or treating a filarial nematode infection, the method comprising providing to a subject in need of such prevention and/or treatment a therapeutically effective amount of a polypeptide comprising a ShK domain of a filarial nematode protein, or a variant thereof.
• methods of treatment may be referred to in the present disclosure as "methods of treatment", but, unless the context requires otherwise, it should be considered that such methods of treatment also encompass prophylactic use to prevent filarial nematode infections.
• polypeptides of the invention described herein represent suitable polypeptide to be used in such methods of treatment, and the various considerations set out in connection with the nature of such polypeptides will also be applicable to polypeptides for use in such methods of treatment.
• the methods of treatment of the invention are applicable to veterinary subjects.
• the subject is a dog
• the filarial nematode infection to be prevented and/or treated is heartworm.
• the polypeptide provided in the method suitably comprises a ShK domain from D. immitis, or a variant thereof.
• the methods of treatment of the invention are also applicable to human subjects.
• the filarial nematode infection to be prevented and/or treated is one that causes a disease selected from the group consisting of: lymphatic filariasis; onchocerciasis; and loiasis.
• the polypeptide suitably comprises a ShK domain from Wuchereria bancrofti; Brugia malayi; and Brugia timori, or a variant thereof.
• W. bancrofti is responsible for approximately 90% of lymphatic filariasis cases, and so it may be preferred that methods for the prevention and/or treatment of lymphatic filariasis make use of polypeptides comprising an ShK domain from W. bancrofti.
• a method of the invention in which it is wished to prevent and/or treat onchocerciasis may make use of a polypeptide comprising a ShK domain from Onchocerca volvulus, or a variant thereof.
• Methods of the invention in which it is desired to prevent and/or treat loiasis may make use of a polypeptide that comprises a ShK domain from Loa loa, or a variant of such a domain.
• the therapeutically effective amount of the polypeptide is provided by administration of the polypeptide.
• the therapeutically effective amount may be provided through single or multiple incidences of administration, as required.
• Such embodiments of the invention may utilise pharmaceutical compositions of the invention for the provision of the required amount of the polypeptide.
• the invention also encompasses methods of treatment in which the therapeutically effective amount of the polypeptide is provided by administration of a nucleic acid encoding the polypeptide, for example by provision of the nucleic acid in a suitable vector.
• expression of the nucleic acid by the cells of the recipient subject leads to the production of the therapeutically effective amount of the protein, thus leading to the development of protective immunity.
• These embodiments of the methods of treatment may utilise pharmaceutical compositions comprising nucleic acids, which are also aspects of the present invention.
• Factors that may be considered in the determination of a therapeutically effective amount of a polypeptide, variant, or nucleic acid of the invention may include: the nature of the agent in question (i.e. whether the agent in question is a polypeptide, a variant thereof, or a nucleic acid); the activity of the agent in question; the severity of the infection to be prevented and/or treated; the size of the subject requiring prevention and/or treatment; and the route by which the agent is to be administered.
• a therapeutically effective amount of a polypeptide comprising a ShK domain, or a variant thereof, or a nucleic acid encoding such a polypeptide or variant may be between 1.5 g and 1 ⁇ g.
• a suitable therapeutically effective amount may be between 1500 mg and 1 mg; for example between 1000 mg and 50 mg; such as between 500 mg and 100 mg.
• a suitable therapeutically effective amount may be between 100 mg and 1 mg; for example between 50 mg and 5 mg; such as between 25 mg and 10 mg.
• a suitable therapeutically effective amount may be between 500 ⁇ g and 1 ⁇ g; for example between 400 ⁇ g and 5 ⁇ g; such as between 250 ⁇ g and 10 ⁇ g.
• a suitable therapeutically effective amount may be between 200 ⁇ g and 15 ⁇ g, such as between 150 ⁇ g and 20 ⁇ g, between 100 ⁇ g and 25 ⁇ g, or between 50 ⁇ g and 30 ⁇ g.
• a therapeutically effective amount may be approximately 40 ⁇ g.
• a polypeptide comprising a ShK domain, or a variant thereof, or a nucleic acid encoding such a polypeptide or variant may be provided in one or more administrations. Incidences of administration may be provided once per 24 hours, once a week, once, a month, or as otherwise required.
• FIG. 1 a illustrates the difference in parasite burden between vaccinated and non-vaccinated mice in Study 1 below;
• FIG. 1 b Distribution of ESP proteins between life stages of L. sigmodontis. Venn diagram of the shared and stage-specific ESP proteins in each of the life stages examined.
• FIG. 2 Pfam enrichment analysis of ESP proteins against the complete theoretical proteome of L. sigmodontis. The fold-enrichment is displayed for each lifecycle stage; DUF290 represents the transthyretin-like protein family.
• FIG. 3 Relative abundance of L. sigmodontis ESP proteins compared to corresponding somatic extracts.
• ESP proteins ⁇ 2 peptides detected at p ⁇ 0.05 and ⁇ 1 % FDR, present in ⁇ 2 biological replicates
• iBAQ ion intensity
• x-axis Individual abundance values were normalised by dividing by the summed total abundance of that individual sample (life stage). The normalised abundance ratio was used as a guide to evaluate the enrichment of the protein (ESP/WBE). Note that as the data are normalised within each life stage dataset, comparing protein abundance directly between life stages is not valid.
• FIG. 4 Comparison of ESP protein abundance (iBAQ) in adult stages of L. sigmodontis.
• the top 35 most abundant proteins in each ES preparation (A, AM; B, GAF; C, PAF) are ranked by normalised iBAQ abundance (grey bars); the corresponding abundance in WBE is displayed for comparison (black bars) in a stacked format.
• Individual protein abundance values were normalised by the summed total abundance per sample.
• An asterisk indicates proteins with a predicted signal peptide, while predicted secretion through the non-classical pathway is indicated by a plus sign.
• FIG. 5 Comparison of ESP protein abundance (iBAQ) in larval stages of L. sigmodontis. Proteins in each ESP preparation (A, vL3; B, iMf) are ranked by normalised iBAQ abundance (grey bars); the corresponding abundance in WBE is displayed for comparison (black bars) in a stacked format. Individual protein abundance values were normalised by the summed total abundance per sample. An asterisk indicates proteins with a predicted signal peptide, while predicted secretion through the non-classical pathway is indicated by a plus sign.
• FIG. 6 The ShK domains from L sigmodontis protein nl_s_04059 and its orthologues in other filarial species have a distinct sequence signature.
• FIG. 7 Number of adult ES proteins detected in published studies of B. malayi adults and comparison with orthologues present in the L. sigmodontis adult secretome. The study-specific and shared proteins represent combined data from both adult sexes. Note that protein identifications are those quoted by each individual study and statistical cut-offs have not been standardised.
• Brugia malayi orthologues of L. sigmodontis proteins were identified by reciprocal BLAST of the respective theoretical proteomes (bit score >50).
• the distribution of the orthologues in adult nematode ESP across three previously published studies (B. malayi) and the current study (L sigmodontis) is displayed in (A), while the distribution of species-specific (non-orthologous) proteins is summarised in (B).
• FIG. 8 Heat-map of protein profiles for excretory-secretory preparations and whole body extracts of Litomosoides sigmodontis. Dendrograms shown in this Figure were generated by hierarchical clustering based on pair-wise distance.
• ESP excretory-secretory products
• WBE whole body extracts
• GAF gravid adult females
• PAF pre-gravid adult females
• AM adult males
• iMF immature microfilariae
• Figure 9 Domain organisation of protein nl_s_04059 from Litomosoides sigmodontis. Linear representation of the amino-acid sequence highlighting the signal peptide (italicised), six ShK toxin-like domains (open rectangles) containing six cysteine residues each (highlighted), and a predicted propeptide cleavage site (underlined). Domain six at the C-terminus is unique in containing two lysyltyrosine dyads (bold).
• Figure 10 Amino-acid sequence alignment of L. sigmodontis protein nl_s_03577 and its orthologues in other filarial nematodes. Homologues of nl_s_03577 were identified by BLASTp search of protein databases from sequenced nematode genomes and a transcriptome assembly for Setaria labiatopapillosa (G. Koutsovoulos, B. Makepeace, M. Blaxter; unpublished). No homologues were found outside the filarial nematodes. The protein sequences were aligned with ClustalOmega, and identity is indicated by a coloured scale (green, high; yellow, moderate; red, low).
• Figure 1 1 Rooted phylogenetic tree of L. sigmodontis protein nl_s_03577 and its orthologues in other filarial nematodes. Homologues of nl_s_03577 were identified by BLASTP search of protein databases from sequenced nematode genomes and a transcriptome assembly for Setaria labiatopapillosa (G. Koutsovoulos, B. Makepeace, M. Blaxter; unpublished). No homologues were found outside the filarial nematodes. The protein sequences were aligned with ClustalOmega and the alignment subjected to phylogenetic analysis using MrBayes version 3.2.
• Figure 13 Unrooted phylogenetic tree of ShK domains among predicted proteins in filarial nematodes and Ascaris suum
• Figure 14 Distribution of biotin in labelled and unlabelled specimens of adult Litomosoides sigmodontis. Fixed worm sections were incubated with streptavidin-FITC. A, Biotin-labelled works. B, An unlabelled control specimen. Scale bars represents 20 ⁇ . Experimental Results
• polypeptides comprising ShK domains of filarial nematode proteins to as vaccines conferring protective immunity in respect of filarial nematode infection, and the suitability of nucleic acids encoding such polypeptides to serve as vaccines, was demonstrated by the following study.
• the L. sigmodontis ShK domain containing protein used as an exemplary vaccine was designated LsShK for the purposes of this study.
• Immunisations and infections were performed with female BALB/c mice, starting at ages of 6-7 weeks, with five animals per experimental group. Mice were housed in individually ventilated cages and infected subcutaneously with 30 or 40 L. sigmodontis infective larvae (il_3). Naive, uninfected animals were maintained and sampled in parallel as controls for the immunological readouts.
• LsShK gene ID nl_s.2.1.2.t04059-RA
• DEC single- chain anti-DEC205 antibody
• PCR products of genes of interest were digested with Nott and Xba ⁇ (Neb laboratory, UK), then ligated into an Nott and X6al-digested anti-mouse dec-205 single chain antibody - ovalbumin construct (DEC-OVA) or antibody control Ig-OVA to replace the fragment of OVA gene, respectively. All plasmids were sequenced to confirm identity.
• Plasmids were injected in the tibialis anterior muscle of the left leg with a 27G needle, immediately followed by electroporation with an ECM 830 generator+Tweezertrodes (BTX Harvard Apparatus) using as settings 8 pulses, 200 V/cm, 40 ms duration, 460 ms interval.
• ECM 830 generator+Tweezertrodes BTX Harvard Apparatus
• Each mouse was immunised twice separated by 2 weeks interval with 40 ⁇ g of DNA total made up by equal quantities of each plasmid species, delivered in 50 ⁇ PBS.
• the quantity of each individual plasmid was reduced as the number of different plasmids incorporated into the inoculums increased. However, the quantity of each one remained in excess of the minimal efficient dose.
• Parasite survival was determined at experiment endpoint.
• Adult filariae were isolated from the pleural cavity lavage fluid in 10 ml cold PBS, fixed in hot 70% ethanol and counted. Protection was
• Microfilariae were counted in 30 ⁇ of blood after fixation in 570 ⁇ of BD FACS lysing solution (BD Biosciences) under an inverted microscope.
• the invention may further be understood by the skilled person on consideration of the following details of a study undertaking quantitative secretome analysis of a model filarial nematode (Litomosoides sigmodontis) across the parasite life cycle.
• Filarial nematodes are responsible for an annual global health burden of approximately 6.3 million disability-adjusted life-years, which represents the greatest single component of morbidity attributable to helminths affecting humans.
• the rodent filaria Litomosoides sigmodontis has become a popular experimental model, as BALB/c mice are fully permissive for its development and reproduction.
• excretory-secretory products from all parasite stages contained several uncharacterised members of the transthyretin-like protein family.
• biotin labelling revealed that redox proteins and enzymes involved in purinergic signalling were enriched on the adult nematode cuticle.
• Comparison of the L. sigmodontis adult secretome with that of the human-infective filarial nematode Brugia malayi identified differences that suggest a considerable underlying diversity of potential immunomodulators.
• the molecules identified in L. sigmodontis excretory-secretory products show promise not only for vaccination against filarial infections, but for the amelioration of allergy and autoimmune diseases.
• Filarial nematodes are the most important helminth parasites of humans in terms of overall impact on public health, with an annual global burden of ⁇ 6.3 million disability-adjusted life- years (1). Lymphatic filariasis (LF) or "elephantiasis”, which affects populations across Africa, South Asia, the Pacific, Latin America and the Caribbean, accounts for 92% of this toll. The remainder is caused by onchocerciasis or "river blindness", primarily in sub-Saharan Africa.
• LF Lymphatic filariasis
• the major human filarial pathogens are Wuchereria bancrofti (responsible for 90% of LF cases), Brugia malayi and Brugia timori (geographically restricted causes of LF), and Onchocerca volvulus (the sole agent of human onchocerciasis).
• Loa loa affects ⁇ 13 million people in West and Central Africa. This parasite usually induces a relatively mild disease, but has been associated with severe and sometimes fatal adverse events following anthelmintic chemotherapy (2).
• Filarial parasites are primarily drivers of chronic morbidity, which manifests as disabling swelling of the legs, genitals and breasts in LF; or visual impairment and severe dermatitis in onchocerciasis.
• filariae are also a major problem in small animal veterinary medicine, with ⁇ 0.5 million dogs in the USA alone infected with Dirofilaria immitis (3), the cause of potentially fatal heartworm disease.
• filarial infections are generally quite benign (4).
• the filarial lifecycle involves uptake of the first-stage larvae (microfilariae, Mf) by a haematophagous arthropod, two moults in this vector, followed by transmission of third-stage larvae (L3) to a new vertebrate host. Two further moults occur in the definitive host before the nematodes mature as dioecious adults in a species-specific, parenteral predilection site.
• Mf first-stage larvae
• L3 third-stage larvae
• L3 third-stage larvae
• Two further moults occur in the definitive host before the nematodes mature as dioecious adults in a species-specific, parenteral predilection site.
• the complete lifecycle of the New World filaria Litomosoides sigmodontis can be maintained in laboratory rodents, including inbred mice (18).
• transthyretin-like family (TTL) proteins As has been observed in other parasitic nematodes, we find transthyretin-like family (TTL) proteins to be particularly dominant in the ESP. Leakage of uterine fluid may account for the remarkable diversity of proteins that we detect in GAF ESP, and we highlight several novel proteins that warrant evaluation in vaccine trials and as anti-inflammatory mediators.
• TTL transthyretin-like family
• L. sigmodontis The life cycle of L. sigmodontis was maintained in jirds (Meriones unguiculatus) infected with vL3 harvested from the mite vector Ornithonyssus bacoti. After 70 - 90 days, GAF and AM were recovered from the pleural cavity by lavage with serum-free RPMI 1640 medium (Life Technologies), whereas PAF were recovered 32 days post-challenge. To harvest iMf liberated in vitro, GAF culture medium was removed after 24 h and centrifuged at 1 ,900 g for 20 minutes (4°C).
• Blood-derived microfilariae were obtained by overlay of blood (from cardiac puncture of jirds >75 days post-infection) onto a 25% Percoll suspension, centrifugation at 1 ,900 g for 20 minutes (4°C), and passage of the bMf fraction through a PD-10 desalting column (GE Healthcare) prior to culture.
• the vL3 larvae were dissected directly from the mite vector and washed three times in RPMI 1640 before transfer to culture vessels.
• ESP and whole body extracts were extracted and analysed separately. All parasite stages were incubated in serum-free RPMI 1640 supplemented with 100 U/ml penicillin, 100 ⁇ g ml streptomycin and 1 % glucose at 37°C (5% C0 2 ) in ultra-low attachment flasks (Corning), and were confirmed to be viable during incubation by microscopic examination.
• the medium was replaced every 24 h, and spent media recovered at 24 h and 48 h were centrifuged at 1 ,900 g for 20 minutes (4°C) in low protein-binding Oak Ridge tubes (polypropylene copolymer; Thermo Scientific Nalgene) to remove debris.
• Low protein-binding Oak Ridge tubes polypropylene copolymer; Thermo Scientific Nalgene
• hydroxylated silica slurry (StrataClean Resin, Agilent Technologies) was added at 30 ⁇ /ml and vortex-mixed at high speed for 2 min. Resin used for each 24 h incubation sample was reused for the respective 48 h sample to concentrate ESP prior to storage at -80°C.
• Soluble WBE was prepared by homogenisation in 25 mM ammonium bicarbonate, 1 % RapiGest SF surfactant (Waters) and complete Protease Inhibitor Cocktail (Roche) using a mini-pestle in a microcentrifuge tube. This was followed by 10 cycles of sonication on ice using a Vibra-Cell VCX130PB sonicator (Sonics & Materials, Inc.) with microprobe (10 sec sonication alternating with 30 sec incubation on ice). Homogenised samples were centrifuged at 13,000 g for 20 minutes (4°C) and the supernatant retained.
• the WBE preparations were obtained from single pools of parasites for all stages except GAF and AM, where two biological replicates were available. Protein concentrations were determined using the Pierce Coomassie Plus (Bradford) Protein Assay (Thermo Scientific). Surface biotinylation of live worms
• the supernatant was removed and the DTT diluted tenfold before digestion with 0.2 ⁇ g proteomic-grade trypsin (Sigma) overnight at 37°C.
• the resultant peptides were concentrated using C 18 reverse-phase spin filters (Thermo Scientific) according to the manufacturer's instructions prior to MS analysis.
• StrataClean Resin containing bound ESP was washed twice with 25 mM ammonium bicarbonate before suspension in 0.1 % RapiGest SF, 25 mM ammonium bicarbonate.
• the resin samples were heated at 80°C for 10 min, reduced with 3 mM DTT at 60°C for 10 min, cooled, then alkylated with 9 mM iodoacetamide (Sigma) for 30 min (room temperature) protected from light. All steps were performed with intermittent vortex-mixing.
• the samples were then digested using 0.2 ⁇ g proteomic-grade trypsin at 37°C overnight with rotation, centrifuged at 13,000 g for 5 min, and the supernatant removed.
• the resin was washed twice with 0.1 % RapiGest SF, 25 mM ammonium bicarbonate and the supernatants pooled.
• the samples were precipitated using TFA (final concentration, 1 %) at 37°C for 2 h and centrifuged at 12,000 g for 1 hr (4°C).
• the peptide supernatant was concentrated using C 18 reverse-phase spin filters according to the manufacturer's instructions.
• the WBE samples were reduced and alkylated as above, digested with trypsin at a protein:trypsin ratio of 50: 1 at 37°C overnight, and precipitated to remove RapiGest SF as for the ESP preparations.
• sigmodontis genome and its Wolbachia symbiont wLs [obtained from hitp:/ nematodes.org/genomes/iitomosoides sigmodontis, release nLs 2.1.2, 10,246 protein sequences (M. Blaxter, S. Kumar, G.
• Mascot search results were imported into Progenesis LC-MS as XML files and analysed according to the following criteria: at least two unique peptides were required for reporting protein identifications, and an individual protein had to be present in ⁇ 2 biological replicates to be included in the ESP dataset. Protein abundance was calculated by the iBAQ method; i.e. , the sum of all peak intensities from the Progenesis output was divided by the number of theoretically observable tryptic peptides (28). For ESP and WBE, protein abundance was normalised by dividing the protein iBAQ value by the summed iBAQ values for the corresponding sample, and the reported abundance is the mean of the biological replicates.
• ShK domains were identified in the complete predicted proteomes of the filariae B. malayi (9), D. immitis (10), L. sigmodontis, Onchocerca ochengi, Acanthocheilonema viteae (draft unpublished genomes available at http://www.nematodes.org/genomes/; Blaxter et al., unpublished), W. bancrofti and L. loa (1 1), plus the ascaridid nematode Ascaris suum (40) (which is an outgroup for the filarial species), using the Pfam hidden Markov model for the domain and hmmer (version3.1 b.1).
• Each domain was excised and a total of 531 distinct domains identified, which were aligned using ClustalOmega (41). Inspection of the alignment revealed that a subset of domains were misaligned (and therefore did not have the six cysteine residues in register with the others); these were corrected manually.
• the alignment was analysed for phylogenetic signal using MrBayes (version 3.2) (42) and two runs of four chains each were run for two million generations. The first million generations were discarded as burn- in after inspection in Tracer (version 1.5; A. Rambaut, unpublished: http://tree.bio.ed.ac.uk/software/tracer/) and a consensus tree was inferred from the remaining 10,000 samples taken every 100 generations.
• the GAF ESP displayed the most complex composition, and the majority of the abundant proteins secreted by this stage were uncharacterised or contained conserved domains associated with very limited functional information (Fig. 4b).
• the dominant GAF ESP protein (nl_s_03577) was unique to filarial nematodes and exhibited only very weak similarity to a bacterial P-type ATPase (Tables 4and 5). Twelve distinct TTL family proteins were identified in GAF ESP (Fig. 4b), although only two were unique to this lifecycle stage.
• nl_s_08836 Another abundant GAF ESP protein (nl_s_08836), also well-represented in PAF and iMf ESP, contained von Willebrand factor type-d (VWD) and cysteine-rich (C8) domains in its carboxy-terminal portion.
• VWD von Willebrand factor type-d
• C8 cysteine-rich domains in its carboxy-terminal portion.
• the best match identified for nl_s_08836 was an apolipophorin from A. suum, but nl_s_08836 lacks the expected amino-terminal lipoprotein domain, and the carboxy-terminal portion displayed weak similarity to predicted zonadhesin-like or SCO-spondin proteins (Tables 4 and 5).
• nl_s_04059 A protein that contained six metridin-like ShK toxin domains, nl_s_04059, was moderately abundant in GAF ESP and was also observed in PAF, AM and iMf secretomes ( Figure 9, 1 1 , 12). While the ShK domain has a wide phylogenetic distribution, the particular pattern apparent in nl_s_04059 is limited to filariae (Tables 4 and 5; see below for detailed analyses of this protein). Additional proteins present in GAF ESP were homologues of previously described ESP antigens from other filarial species. However, RAL-2 (44), SXP-1 (45), S3 (46) and CCG-1 (47) remain functionally obscure.
• Functionally defined components of the ESP included a small cysteine protease inhibitor [CPI (48)], the omega-class glutathione S-transferases [GST (49)], the MSPs (50), and the microfilaria! sheath protein (51) (Fig. 4b). Additionally, L. sigmodontis homologues of Av33 and ES-62, proteins known to be abundant in the ESP from adult females of other filarial species, were identified. Av33 is similar to an aspartate protease inhibitor from A. suum (52), whereas ES-62 is a secreted leucyl aminopeptidase (53).
• a secreted acid phosphatase which may be involved riboflavin metabolism and have a role in the hydrolysis of prosthetic groups such as flavin mononucleotide and/or pyridoxal 5-phosphate (54, 55), was prominent in PAF ESP.
• Three of the GAF ESP proteins had putative lipid-binding regions: ML-domain proteins have been reported to interact with cholesterol and lipid A (56, 57), the conserved filarial antigen Ov16 has a putative phosphatidylethanolamine-binding domain (58), and a novel and highly abundant vitellogenin (nl_s_07321) contained an amino-terminal lipid transport domain.
• AM ESP Abundant components of AM ESP included three isoforms of MFP2 (59) and proteins known to be highly expressed in sperm or seminal fluid, such as an extracellular superoxide dismutase (60) and a serine protease inhibitor (61) (Fig. 4a). However, AM ESP also contained several previously described but uncharacterised proteins, such as RAL-2 (44), nematode secreted protein 22U (62) and immunogenic protein 3 (63). A novel KH (RNA-binding) domain protein had homologues in other filarial species, but also weak homology to the Vasa DEAD-box helicase GLH-2 from Caenorhabditis elegans (Table 4), which is associated with spermatogenic chromatin (64).
• iMf ESP proteins of nematode origin to 36
• Fig. 4b the dominant proteins in iMf ESP closely mirrored the profile of GAF ESP
• nl_s_03443 the two most abundant parasite ESP proteins observed in bMf, a TTL protein and a nematode-specific uncharacterised protein (nl_s_03443), were not present in iMf ESP (Table 6).
• Non-unique but proportionally enriched proteins in iMf included two galectins ( ⁇ -galactoside-binding proteins 1 and 2), a fatty acid and retinoid-binding protein (FAR-1), and a nucleoside diphosphate kinase (Fig.
• vL3 ESP was composed of previously characterised filarial proteins that are known to be uniquely expressed or enriched in this stage [such as ASP-1 (71), ALT-1 (72), and cathepsin-L-like protease (73)], and other antigens that were well represented in ESP from other stages (RAL-2, CPI-2, Ov16 and ⁇ -galactoside-binding proteins) (Fig. 5a).
• the nematode secreted protein 22U was moderately abundant in the L. sigmodontis vL3 ESP preparations (Fig.
• nl_s_03577 The most abundant protein in GAF ESP, nl_s_03577, is an enigmatic, uncharacterised molecule with a predicted MW of 28.5 kDa and a lack of conserved domains, with the exception of a classical N-terminal signal peptide. Downstream of the signal peptide, moderate to high levels of sequence conservation were apparent across the Filarioidea in the N-terminal portion ( Figure 10). However, the C-terminal segment displayed low complexity and was highly variable between filariae, with two isoforms in B. malayi diverging in this region only ( Figure 10).
• the L sigmodontis protein was predicted to contain six potential /V-linked and 1 1 O-linked glycosylation sites, as well as propeptide cleavage sites at positions 31 and 147.
• the former cleavage site was absolutely conserved within the Filarioidea, despite some variation in the motif, whereas the latter (at position 154 of the consensus) was unique to L. sigmodontis.
• These observations suggest that several processed isoforms of nl_s_03577 might be secreted by L. sigmodontis.
• Phylogenetic analysis of nl_s_03577 orthologues confirmed that this protein is restricted to the Filarioidea, with no representatives in A. suum or other non-filarial nematodes.
• the ShK domain protein nl_s_04059 was a particularly distinctive molecule identified in all ESP preparations except vL3.
• the ShK domain (or metridin- like toxin domain, also known as the SXC or six-cysteine domain) was first identified in cnidarian venoms, but is particularly abundant in nematode proteomes (74), where it is associated with secreted proteins.
• the prototypic ShK peptide (from the cnidarian Stichodactyla helianthus) is a type 1 toxin that blocks voltage-gated potassium channels, and synthetic analogues are currently under development as a therapy for autoimmune diseases, in which Kv1.3 channels expressed by effector memory T-lymphocytes are specifically targeted (75).
• nl_s_04059 was not especially abundant in any ESP preparation, its presence in the secretomes of all mammalian-derived stages and its unusual domain structure (Figure 9) suggest a potentially immunomodulatory role.
• the nl_s_04059 protein has the largest number of ShK domains (six) of any protein in L. sigmodontis. We identified orthologous genes in all the other filarial nematode genomes, each containing six ShK domains ( Figure 12). The nl_s_04059-like ShK domains form a distinct subset of all filarial and A. suum ShK domains (I Figures 12 and 13), with a striking pattern of conservation particularly around the last three universally-conserved cysteine residues (Fig. 6).
• a proline residue (at position 32 of the alignment, but residue 17 of the nl_s_04095 domains) was also strikingly conserved in the nl_s_04059 domains (Fig. 6), but not common in the full set of 531 domains.
• the six ShK repeats are separated by five low-complexity spacers (27 - 104 amino acids) ( Figure9 ). Some spacer domains were conserved, but others showed variation in the pattern and length of low complexity, serine-rich regions.
• the spacer domains have no clear similarity to other proteins, but by analogy to the ShK mucins of the ascaridid Toxocara canis (76), they could be recipients of O-linked glycan decorations. There are 65 potential O-glycosylation sites on nl_s_04059. However, by geLC-MS we found that nl_s_04059 migrated exclusively at the expected molecular weight of the unmodified mature protein ( ⁇ 52 kDa), ruling out a mucin-like structure (data not shown). This protein also contained two lysyltyrosine dyads located within the C-terminal ShK domain ( Figure 9).
• a lysyltyrosine dyad is essential for binding of type-1 cnidarian peptide toxins to potassium channels (77), this could be related to Kv1 channel-blocking activity.
• one lysyltyrosine dyad in ShK domain 6 is conserved in many (although not all) orthologues in other species (Fig. 6).
• the nematode cuticle is the critical interface between the parasite and the immune system of its host (78). Surface-associated proteins may simply mirror ESP, perhaps by passive adsorption of released material, or comprise a distinct component of the exoproteome.
• Live AM and GAF nematodes were surface-labelled incubated with Sulfo-NHS-SS-Biotin and fractionated. Immunofluorescent imaging of fixed nematode sections confirmed that biotin labelling was largely confined to the cuticular layers ( Figure 14). Low levels of endogenous biotin were present within internal structures as expected. We identified five proteins that were present in biotin-labelled AM extracts but not unlabelled controls and 1 1 proteins in biotin-labelled GAF (Table 2).
• a striking feature of the surface-associated proteins was the presence of two ectoenzymes involved in purinergic signalling. These were an adenylate kinase predominant in AM extracts and a purine nucleoside phosphorylase found exclusively found in GAF extracts (79) (Table 2, Table 7).
• a homologue of complement component 1 , q subcomponent-binding protein was identified in GAF surface-labelled extracts.
• the L. sigmodontis protein contained an N-terminal mitochondrial import signal sequence, although the former is expressed in a number of extramitochondrial locations, including on the surface of lymphocytes, endothelial cells, dendritic cells and platelets (80). These proteins may play a role in immunomodulation, as purinergic signalling is known to regulate lymphocyte trafficking (79), while the complement component 1 q receptor is involved in vasodilation via the generation of bradykinin (80).
• GAF surface extracts from AM contained a homologue of the actin-binding protein, calponin, which has been localised to both striated muscle and the cuticle in adult O. volvulus (81).
• the GAF surface extracts contained two proteins, protein disulphide isomerase and a leucine-rich repeat family protein, both of which have previously been associated with cuticle synthesis in filariae and C. elegans (82, 83).
• Stress response-related proteins were also well represented on GAF (including thioredoxin peroxidase (84), aldehyde dehydrogenase, a thioredoxin-like protein and heat-shock proteins), as were several enzymes of pyruvate metabolism (Table 2 and Table 7).
• the endosymbiont-derived Wolbachia surface protein was found to be accessible to surface biotinylation in GAF.
• sigmodontis ESP (Fig. 7a). This common core included leucyl aminopeptidase, enolase, triosephosphate isomerase, ⁇ - galactoside-binding protein 1 , acetylcholine receptor protein, cyclophilin-5 and macrophage migration inhibitory factor-1.
• the 22 L. sigmodontis adult ESP proteins that lacked B. malayi orthologues included two of the most highly abundant GAF ESP molecules (the vitellogenin nLs_07321 and the VWD protein nLs_08836), together with secretory protein Ls1 10 and two superoxide dismutase isoforms. Although the B.
• malayi secretome studies identified a total of 90 proteins that did not have orthologues in L. sigmodontis (Fig. 7b), only one (cuticular glutathione peroxidase) was observed in all three studies. Since standardised quantification methods were not used for our L. sigmodontis and the published B. malayi studies, it is difficult to determine whether adult B. malayi and L. sigmodontis differ in their levels of secretion for individual ESP proteins. However, in terms of rank abundance, triosephosphate isomerase, macrophage migration inhibitory factor-1 , and ⁇ -glutamyl transpeptidase were reported to be grossly overrepresented in adult B. malayi; whereas adult L.
• sigmodontis ESP was enriched for uncharacterised protein nl_s_03577 (orthologous to Bm1_38495), TTL protein nl_s_09750 (orthologous to Bm1_43635), and homologues of Av33 and S3 (Table 3). Proteins that were apparently equally abundant in relative terms between each species included leucyl aminopeptidase and homologues of CPI-2 and Ov16.
• Filarial nematodes exact a significant burden of morbidity in human populations and are important pathogens of companion animals. While efficacious anti-filarial drugs exist, the spectre of the evolution of genetic resistance to these is ever-present (5, 6), and alternative routes to treatment are required. It would be preferable to be able to prevent infection as well as treat patent disease, and thus an anti-filarial vaccine would be an extremely valuable addition to medical and veterinary treatment options (85).
• the ESP released by parasites into their hosts have been the target of vaccine development for decades, but the understanding of these molecules in filarial nematodes is limited.
• GAF ESP The diversity of GAF ESP is consistent with the material containing not only somatic adult ESP, but also proteins released from the reproductive tract that derive from the processes of oogenesis, fertilisation and embryonic development in utero (all filarial pathogens are ovoviviparous).
• Nematode sperm are acutely sensitive to aerobic damage (86).
• the AM ESP contained proteins suggestive of roles in protection of sperm against oxidants and other stressors, including superoxide dismutase, a serine protease inhibitor and a glutaredoxin-like protein.
• Glutaredoxins are thiol-containing antioxidant proteins, and C. elegans GLRX-21 plays a key role in mitigating selenium toxicity (87). Mammalian seminal fluid accumulates selenium, which if in excess, can impede sperm motility (88).
• a homologue of the serine protease inhibitor is secreted by A.
• the mature microfilarial secretome is dominated by host proteins
• microfilariae are enclosed in a proteinaceous sheath comprising an inner layer that originates from the eggshell and an outer layer that is produced by secretions in the distal portion of the uterus.
• Five major structural proteins have been identified in the L. sigmodontis sheath, some of which are synthesised in the developing embryo and others in the uterine epithelium (51), but none of these were found in iMf ESP, indicating that they are stable components.
• Many host serum proteins were released from bMf in culture. These are likely to derive from specific interactions with the parasite surface, perhaps reflecting a tension between the nematode exploiting the host and the host immune system recognising the parasite.
• Ls1 10 a protein localised in the uterine lumen and variably present on iMf, but absent from bMf (67)] and two possible proteoglycan core proteins. Accordingly, large glycoproteins ( ⁇ 200 kDa) have been described from B. malayi ESP (100).
• sigmodontis iMf-derived CPG-like protein is predicted to have chitin-binding domains and may function in eggshell and sheath development.
• CPGs form an inner layer that binds to the central chitinous layer of the eggshell, maintaining the perivitelline space around the embryo (101) forming a barrier to prevent polyspermy (102).
• chitin has been detected in the oocytes and zygotes, although it is absent from the iMf sheath (103). The degradation of chitin during Mf sheath development in utero may release the underlying CPG, which is highly soluble (101), into the surrounding milieu.
• C. elegans of the PAN domain protein The closest homologue in C. elegans of the PAN domain protein is SRAP-1 , which is expressed in the hypodermis, central nervous system and vulva of developing larvae and is secreted onto the cuticle surface during moulting (104).
• peroxidasin PXN-2 is located in the extracellular matrix and is required for late embryonic elongation, muscle attachment, and motoneuron axon guidance choice (105).
• nl_s_03577 which displayed a significant match to a P-type ATPase (but lacked an ATPase domain)
• nl_s_08836 which showed some similarity to zonadhesin, a VWD protein located in the head of mammalian sperm (106).
• nl_s_08836 is not an orthologue of the C. elegans zonadhesin-domain protein, DEX-1 (107).
• the third novel protein, nl_s_07321 is a vitellogenin.
• vitellogenins are expressed exclusively in the intestine, where they bind cholesterol and transport it via the body cavity to the gonad (108). Subsequently, oocytes internalise the protein and its lipid cargo by receptor-mediated endocytosis and store it in yolk granules (108).
• vitellogenins have also been identified in ESP derived from adults of the oviparous gastrointestinal nematode, Heligmosomoides polygyrus (109).
• Uterine fluid as a source of nematode and endosymbiont products
• Proteins excreted or secreted from filarial nematodes could be derived from a number of routes.
• nematodes also secrete material from the anterior sensory glands (amphids) (1 14) and the secretory pore, and may also void material from the genital openings during copulation and release of Mf. Proteins can also be released from the hypodermis through transcuticular secretion (1 15), especially during moulting, and exosome release may also be important (1 16).
• Wolbachia may be present in uterine fluid (1 18), inside degenerating embryos (1 19), or exit via the secretory pore (120). Additionally, they may secrete proteins into structures that lack bacterial cells, such as the cuticle (121). lVo/6ac/7/ ' a-derived proteins were present in very low amounts in B. malayi secreted products (17).
• GroEL is the most abundant protein in Wolbachia (13, 15), and its detection in ESP may be through release of whole bacterial cells, for example in the female uterus from degenerating oocytes or embryos, or through secretion. GroEL, as a chaperonin, would be expected to be confined to the cytosol, although GroEL homologues have been reported to "moonlight" on the surface of some bacterial species (122).
• This protein is a putative ligand of Toll-like receptors 2 and 4 (1 19), and these findings support the hypothesis that Wolbachia modifies and perhaps misdirects the immune response to filariae (123). Whether Wolbachia GroEL also stimulates proinflammatory Toll-like receptors has not been evaluated, but a precedent exists in other bacteria (124), and antibodies against this protein are associated with pathology in LF (125).
• Mf Mf
• Vaccination with a combination of ALT-1 and CPI-2 delivered as a DNA vaccine reduced circulating Mf levels by up to 90% in L. sigmodontis. Importantly, this protection was only achieved if immunomodulatory domains of the antigens were ablated (by mutation or deletion of the coding sequence) and was maintained even when the adult nematode burden was not significantly reduced.
• L. sigmodontis especially the GAF stage, releases a remarkable diversity of proteins into the external milieu and the majority of these molecules are uncharacterised. Although many of these proteins may be involved in fundamental aspects of embryogenesis, a subset are likely to be active immunomodulatory agents that protect the nematodes (and especially the circulating Mf) from the host immune response.
• the abundant ESP protein, CPI may represent an archetype for this dual functionality, as it plays fundamental roles in oogenesis and fertilisation not only in parasitic nematodes but also in C. elegans (132). This suggests that its immunomodulatory properties are an example of secondary adaptation to a radically different environment.
• the pharmacopeia released by GAF may provide the ideal set of molecule(s) to target for immunoprophylaxis and chemotherapy of filariases; moreover, it could provide new compounds to tackle proinflammatory and autoimmune diseases (22)
• nLs _03968 Nematode cuticle collagen N-terminal domain containing protein
• nLs _06052 Translationally controlled tumor protein nLs_ 00526 Glutathione reductase
• ESP excretory-secretory products
• PAF pre-gravid adult female
• AM adult male
• vL3 vector- derived third-stage larvae
• iMf immature microfilariae.
• ESP excretory-secretory products
• AM adult male
• OG octyl ⁇ -D-glucopyranoside
• GAF gravid adult female
• FMN flavin mononucleotide
• Onchocerciasis the role of Wolbachia bacterial endosymbionts in parasite biology, disease pathogenesis, and treatment. Clin. Microbiol. Rev. 24, 459-468
• NPC2 Niemann-Pick C2
• Onchocerca volvulus a major hydrogen peroxide detoxifying enzyme in filarial parasites. Mol. Biochem. Parasitol. 91, 221-235
• Nematode sperm maturation triggered by protease involves sperm- secreted serine protease inhibitor (Serpin). Proc. Natl. Acad. Sci. U. S. A 109, 1542- 1547
• Litomosoides carinii extraction of the microfilaria! sheath components and antigenicity of the sheath fractions. Parasitol. Res. 78, 501-508
• ROS1 is required for epithelial development in C. elegans. Genesis. 51, 545-561
• CPI-2a cystatin-like inhibitor has an essential regulatory role during oogenesis and fertilization. 7. Biol. Chem. 281, 28415-28429
• nl_s.2.1.2.t04059-RA from Litomosoides sigmodontis
• nDi.2.2.2.t03402-RA from Dirofilaria immitis

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