PARASITIC NEMATODE VACCINE

Abstract

There is discussed nematode antigens capable of causing an immune response in a host such that a protective effect is provided to the host in relation to the nematode. Antigens, compositions for the treatment of parasitic nematode infections, methods of prophylaxis and treatment of parasitic nematode infections, and vaccines to reduce and/or control parasitic nematode infections are provided.

Claims

1. An exWAGO protein or fragment thereof from a parasitic nematode or a nucleic acid sequence capable of expressing exWAGO or a fragment thereof for use in raising a protective immune response in an animal.

2. An exWAGO protein for use in raising a protective immune response in an animal as claimed in claim 1 wherein the exWAGO protein comprises a sequence of SEQ ID NO: 1 or has a sequence identity of at least 60% to a portion of SEQ ID NO: 1.

3. An exWAGO protein for use in raising a protective immune response in an animal as claimed in claim 1 wherein the exWAGO protein comprises a sequence of SEQ ID NO: 1 or has a sequence identity of at least 60% to SEQ ID NO: 1.

4. A composition comprising an exWAGO protein or a fragment thereof or a nucleic acid sequence capable of expressing exWAGO comprising a sequence of SEQ ID NO: 1 or with a sequence identity of at least 60% to SEQ ID NO: 1 and optionally an adjuvant for use in the treatment or prophylaxis of a parasitic nematode.

5. A composition as claimed in claim 4 further comprising at least a second parasitic antigen to which an immune response may be raised.

6. An exWAGO protein for use in raising an immune response in an animal as claimed in claim 1 wherein the parasitic nematode is selected from the group comprising: parasitic nematodes of Clade V and Clade III, optionally wherein Clade V includes: Strongylida Rhabditida Diplogasterida and Clade III includes: Oxyurida Spirurida Rhigonematida Ascaridida.

7. An exWAGO protein for use in raising an immune response in an animal as claimed in claim 1 wherein the parasitic nematode is selected from the group comprising Nippostrongylus brasiliensis (rodents) Ancylostoma caninum Ancylostoma ceylanicum Ancylostoma duodenale Oesophagostomum dentatum Angiostrongylus costaricensis Dictyocaulus viviparous Symphacia muris (rodents) Enterobius vermicularis Anisakis simplex Ascaris suum Ascaris lumbricoides Thelazia callipaeda Brugia malayi Wuchereria bancrofti Onchocerca flexuosa Onchocerca ochengi, and Acanthocheilonema viteae (rodents).

8. An exWAGO protein for use in raising an immune response in an animal as claimed in claim 1 wherein the parasitic nematode is selected from the group comprising Heligmosomoides polygyrus, Heligmosomoides bakeri, Teladorsagia circumcincta, Ancylostoma caninum, Ancylostoma ceylonicum, Nippostrongylus brasiliensis, Necator americanus, Oesophagostomum dentatum, Haemonchus contortus, Ancylostoma duodenale, Angiostrongylus cantonensis, Angiostrongylus costaricensis, Nematodirus, Ostertagia ostertagi, and Dictyocaulus viviparous.

9. An exWAGO protein for use in raising an immune response in an animal as claimed in claim 1 wherein the parasitic nematode is selected from the group comprising Ascaris lumbricoides, Ascaris suum, Thelazia callipaeda, Brugia malayi, Litomosoides sigmodontis, Acanthocheilonema viteae, Dirofilaria immitis, Loa loa, Onchocerca flexuosa, Onchocerca ochengi, Enterobius vermicularis, Onchocerca volvulus, Wuchereria bancrofti, and Symphacia muris.

10. An exWAGO protein for use in raising an immune response in an animal as claimed in claim 1 wherein an immune response is against a parasitic nematode of a different clade of parasitic nematode to that of Heligmosomoides polygyrus.

11. An exWAGO protein for use in raising an immune response in an animal as claimed in claim 1 wherein an immune response is against a parasitic nematode of the same clade of parasitic nematode to that of Heligmosomoides polygyrus.

12. An exWAGO protein for use in raising an immune response in an animal as claimed in claim 1 wherein the animal is selected from human, cat, dog, camelid, equid, equine, or ovine or bovine.

13. A method of raising an immune response against a parasitic nematode in an animal said method comprising the step of administering to the animal exWAGO protein or fragment thereof or a nucleic acid sequence capable of expressing exWAGO or a fragment thereof sufficient to induce an immune response against the parasitic nematode.

14. A method of raising an immune response against a parasitic nematode in an animal as claimed in claim 13 said method comprising the step of administering to the animal a composition comprising an exWAGO protein or a fragment thereof or a nucleic acid sequence capable of expressing exWAGO comprising a sequence of SEQ ID NO: 1 or with a sequence identity of at least 60% to SEQ ID NO: 1.

15. A method to determine an epitope of exWAGO to which antibodies or aptamers can be raised wherein the antibodies or aptamers provide a protective immune response to a host subsequently infected with a parasitic nematode the method comprising the steps of determining an antibody or antibodies that associate with exWAGO under native in vivo conditions, determining with the antibody or antibodies that block the ability of exWAGO to interact with host or microbial genes in vivo.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0098] Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying figures in which:

[0099] FIG. 1: The exWAGO protein sequence from H. bakeri.

[0100] FIG. 2: Closest sequence identity to exWAGO protein sequence from H. bakeri in human genome—alignment to piwi domain of human Argonaute 1 protein.

[0101] FIG. 3: Comparative protein sequence alignment of H. bakeri exWAGO protein with the orthologue of N. americanus (SEQ ID NO: 2) showing a high level of identity.

[0102] FIG. 4: Comparative sequence alignment of H. bakeri exWAGO protein with the orthologue of T. circumcinta (SEQ ID NO: 3) showing a high level of identity.

[0103] FIG. 5: Western blot from an immunoprecipitation of exWAGO from excretory-secretory material of the H. bakeri parasite showing very strong signal in EV-depleted HES (A) (meaning a large portion is not in classically purified extracellular vesicles and is accessible to capture/binding by antibodies) and a lower signal in EVs (B) (10× more protein used for EV-depleted than HES IP). The exWAGO protein is immunopurified from the HES using a polyclonal antibody that only isolates this (and not other) Argonaute proteins, which is confirmed by mass spectrometry analysis of the IP. The antibody is conjugated to Protein L magnetic beads and the complex eluted in SDS/loading buffer prior to being run on a SDS PAGE gel and probed for presence of exWAGO using another antibody that recognizes the denatured form.

[0104] FIG. 6: Vaccination strategy using recombinant H. bakeri exWAGO protein in mice and subsequent challenge with H. bakeri, in comparison to HES which is all H. bakeri Excretory-Secretory products. Mice are primed with recombinant exWAGO protein or HES or PBS with Alum at day 0 and then boosted 28 and 35 days later prior to infection on day 42. The mice are sacrificed at 14 and 28 days post infection to assess larval and worm burden, faecal egg count and antibody and cellular response.

[0105] FIG. 7: Vaccination results following the strategy of FIG. 4; worm counts per gut section were counted following inoculation and subsequent challenge with H. bakeri at day 14. Total worm counts in the intestine are shown on the left. The normal host niche for H. bakeri is the top section of the intestine (duodenum) and worms in the process of expulsion may be more localized in the bottom section therefore all were counted. The results reveal a significant reduction in adult worm burden at day 28 post infection.

[0106] FIG. 8: Vaccination results following the strategy of FIG. 4; egg counts from stool samples following inoculation and subsequent challenge with H. bakeri. Total egg counts in the intestine are shown on the left. The results reveal a significant reduction in faecal egg counts at day 28 post infection.

[0107] FIG. 9: Western blot to demonstrate the recognition of post-vaccination, post-worm challenge mouse sera of exWAGO protein which demonstrates that the antibodies against this protein are present in the sera.

[0108] FIG. 10: Measurement of IgG1 antibody response to exWAGO at 28 days post infection, n=5 mice per group by ELISA. The recombinant exWAGO protein is used to coat plates so we can measure IgG1 antibodies specific to exWAGO, and then sera from each mouse per group is screened at different dilutions as noted. A secondary IgG antibody is used for detection.

[0109] FIG. 11: Comparative expression of various Argonaute proteins in different nematode parasites. Relative expression levels of Argonautes from RNAseq data of the adult free-living parasitic nematode worms. Data are based on the sum of tpm reads of RNAseq data for each orthogroup, normalized to tpm for OG1273 orthogroup (ALG-1/2). The total number of distinct transcripts in each orthogroup in each species is noted below each column. The known Caenorhabditis elegans Argonaute names are used where applicable, or exWAGO. This shows that exWAGOs are the most abundantly expressed of all Argonautes (Supplementary Table S3) in the sheep parasites H. contortus and Teladorsagia circumcincta and the lungworm Angiostrongylus cantonensis.

[0110] FIG. 12: Illustrates that exWAGO exists in T. circumcinta in an immune system accessible form (can be captured from the Excretory-secretory products by antibody).

[0111] FIG. 13: Provides details of exWAGO orthologues and their sequence identity to SEQ ID NO:1.

[0112] FIG. 14: Provides illustrative sequences of exWAGO proteins of noted in FIG. 13.

[0113] FIG. 15: indicates protection in H. bakeri model observed at day 28 post infection when using adjuvant Alum.

[0114] FIG. 16 indicated exWAGO-specific IgG detected upon infection of sheep with T. circumcinta.

[0115] FIG. 17: Provides sequence comparison of H. Corntorus sequence (SEQ ID NO: 8).

[0116] Definitions

[0117] Throughout the specification, unless the context demands otherwise, the terms ‘comprise’ or ‘include’, or variations such as ‘comprises’ or ‘comprising’, ‘includes’ or ‘including’ will be understood to imply the includes of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0118] As used herein, the articles “a” and “an” refer to one or to more than one (for example to at least one) of the grammatical object of the article.

[0119] “About” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements.

[0120] As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a condition or disease or disorder or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a condition or disease or disorder and/or adverse symptom or effect attributable to the condition or disease or disorder. For either prophylaxis (prevention) or to cure or reduce the extent of or likelihood of occurrence of the infirmity or malady or condition or event in the instance where the patient is afflicted.

[0121] “Treatment” for example, covers any treatment of a condition or disease in a mammal, particularly in a human, and includes: (a) preventing the condition or disease, disorder or symptom thereof from occurring in a subject which may be predisposed to the condition or disease or disorder but has not yet been diagnosed as having it; (b) inhibiting the condition or disease, disorder or symptom thereof, such as, arresting its development; and (c) relieving, alleviating or ameliorating the condition or disease or disorder or symptom thereof, such as, for example, causing regression of the condition or disease or disorder or symptom thereof. As used herein, the term “effective amount” means that amount of an agent, for example protein that will elicit the biological or medical response of a tissue, system, animal or human that is being sought, for instance, by a researcher, clinician or veterinarian.

[0122] As used herein, “pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” mean a pharmaceutically acceptable material, composition or vehicle involved in giving form or consistency to the composition for example the protein and adjuvant. Each excipient must be compatible with the other ingredients of the pharmaceutical composition when commingled such that interactions which would substantially reduce the efficacy of the compound of the invention when administered to a patient and interactions which would result in pharmaceutical compositions that are not pharmaceutically acceptable are avoided. In addition, each excipient must of course be pharmaceutically-acceptable e.g. of sufficiently high purity.

[0123] The term “combination” as used herein refers to either a fixed combination in one dosage unit form, or non-fixed combination. The term “fixed combination” means that the active ingredients, e.g. a protein and a combination partner, for example an antihelmintic or another antigen are both administered to a patient simultaneously in the form of a single entity or dosage.

[0124] The term “non-fixed combination” means that the active ingredients, e.g. an exWAGO protein (1) and a combination partner, (e.g. another drug or antigen as explained below, also referred to as “therapeutic agent” or “co-agent”) are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no, specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient.

[0125] Herein, “Heligmosomoides polygyrus” and “Heligmosomoides bakeri” are used interchangeably.

[0126] As used herein, a peptide for example a fragment of exWAGO protein can include any polymer consisting of at least two amino acids which are covalently linked to each other, preferably via a peptide bond. Preferably a peptide consists of two to ten amino acids. Suitably, the peptide can be an oligopeptide which comprises from 10 to 100 amino acids, suitably covalently linked to each other, preferably via a peptide bond. Suitably, the peptide can be a protein comprising a plurality of amino acids which are covalently linked to each other and which comprise at least 100 amino acids. Suitably a protein may also be two or more peptides each comprising of at least two amino acids, non-covalently linked to each other.

DETAILED DESCRIPTION OF THE INVENTION

[0127] Embodiments of the present invention will now be described by way of example only with reference to the accompanying figures.

EXAMPLE 1

Screening H. bakeri Excretory-Secretory Material (HES) for exWAGO in the Presence and Absence of EVs

[0128] ExWAGO protein was captured from excretory-secretory products using immunopurification with exWAGO antibodies attached to beads. The ES products that are screened were either vesicles (where the ES is first ultracentrifuged and the pellet is used) or non-vesicular material that is the supernatant of the ultracentrifugation. The Immunoprecipitate products are then eluted from the beads and run on a gel.

EXAMPLE 2

Protective Role of Inoculation by exWAGO Against Future Parasitic Infection

[0129] A vaccination protocol was proposed (as shown in FIG. 6), using a vehicle only control of PBS and alum, a positive control of H. bakeri HES and alum, and recombinant H. bakeri exWAGO and alum. On day 0, 10 female C57BL/6 mice aged 6-8 weeks per condition were inoculated via intraperitoneal (IP) injection with 10 μg of each condition. At days 28 and 35, booster IP inoculations of 2 μg were provided to the mice.

[0130] On day 42 (day 0 of post-challenge, p.c.) the mice were each provided with 200 H. bakeri larval infective (L3) nematode worms by gavage.

[0131] Samples were collected and measurements taken of: [0132] 1) Worm counts (d56 [d14 p.c.] (n=5), d70 [d28 p.c.] (n=4)). [0133] 2) Egg counts (d63 [d21 p.c.] (n=5)) [0134] 3) Serum [0135] 4) Extra stools [0136] 5) Tissue and gut sections (d56 [d14 p.c.] (n=5), d70 [d28 p.c.] (n=4)).

[0137] Vaccination with Recombinant exWAGO Results in Protection against Subsequent Infection at 28 Days Post Infection

[0138] Following the above inoculation protocol, gut sections of mice were sampled at day d56 [d14 p.c.] (n=5) and at day d70 [d28 p.c.] (n=4), and screened for the number of adult H. bakeri worms was counted.

[0139] As shown in FIG. 7, vaccination with recombinant H. bakeri exWAGO results in protection from subsequent infection at 28 days post infection. This shows a 58% reduction in adult worm counts. Those found only in the bottom section are presumably in the process of exiting the gut. FIG. 7A: 14 days post-infection,

[0140] FIG. 7B: 28 days post-infection.

[0141] As shown in FIG. 8, stool samples were taken at days 14, 21 and 28 and the number of H. bakeri eggs were then counted. This clearly demonstrates that inoculation with recombinant H. bakeri exWAGO results in protection from subsequent infection with a 92% (average) reduction in egg counts in the exWAGO experiment. FIG. 8A: Change in egg count at 14, 21 and 29 days, FIG. 8B: collated egg count/day data.

[0142] As shown in FIG. 9, antibodies generated during vaccination recognise exWAGO: Western blot of 1 μg worm lysate, 1 μg total excretory-secretory material (HES) or the noted concentrations of the recombinant exWAGO protein, incubated with the sera of mice vaccinated with exWAGO and infected.

[0143] As shown in FIG. 10, mice which had been vaccinated with recombinant exWAGO demonstrated a significantly higher IgG1 antibody response to exWAGO (by ELISA, coated with recombinant exWAGO) at 28 days post infection, n=5 mice per group.

[0144] As shown in FIG. 15, using Alum as an adjuvant, exWAGO provides protection in the model.

EXAMPLE 3

Comparative Expression of Various Argonaute Proteins in Different Nematode Parasites

[0145] As shown in FIG. 11, relative expression levels of different classes of Argonautes from adult free-living parasitic nematode worms were compared, using RNAseq data. Data was based on the sum of tpm reads of RNAseq data for each orthogroup, normalized to tpm for 0G1273 orthogroup (ALG-1/2). The total number of distinct transcripts in each orthogroup in each species is noted below each column. The known Caenorhabditis elegans Argonaute names are used where applicable, or exWAGO. This shows that exWAGOs are the most abundantly expressed of all Argonautes in the sheep parasites H. contortus and Teladorsagia circumcincta and the lungworm Angiostrongylus cantonensis.

EXAMPLE 4

exWAGO Specific IgG Detected upon Infection of Sheep with T. circumcinta

[0146] As shown in FIG. 16, using a sheep model instead of a mouse model with exWAGO from the parasite T. circumcinta, it was considered that IgG levels were modulated in the host following treatment. Thus, it is considered that the protective effect of exWAGO as illustrated in the H. bakeri model will be observed in other animals.

[0147] Preferred compositions, features and embodiments of each aspect of the invention are as for each of the other aspects mutatis mutandis unless context demands otherwise.

[0148] Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in the text is not repeated in this text is merely for reasons of conciseness.

[0149] Reference to cited material or information contained in the text should not be understood as a concession that the material or information was part of the common general knowledge or was known in any country.

[0150] Although the invention has been particularly shown and described with reference to particular examples, it will be understood by those skilled in the art that various changes in the form and details may be made therein without departing from the scope of the present invention.