WO2019043232A1

WO2019043232A1 - A fasciola hepatica antigen and vaccine

Info

Publication number: WO2019043232A1
Application number: PCT/EP2018/073640
Authority: WO
Inventors: Grace MULCAHY; Laura GARZA-CUARTERO; John Dalton
Original assignee: The Queen's University Of Belfast; University College Dublin, National University Of Ireland, Dublin
Priority date: 2017-09-01
Filing date: 2018-09-03
Publication date: 2019-03-07
Also published as: GB201714057D0

Abstract

The present invention is related to a Fasciola hepatica antigen and vaccine. Specifically, the present invention relates to a Fasciola hepatica antigen comprising a polypeptide, wherein the polypeptide comprises amino acid sequences from Fasciola hepatica, or fragments each thereof. Also disclosed are use of the antigen as a vaccine against Fasciola hepatica infection, and use of the antigen in the manufacture of a vaccine for vaccinating a subject against Fasciola hepatica infection.

Description

Title of the Invention

A Fasciola hepatica antigen and vaccine Field of the Invention

The present invention relates to a Fasciola antigen and vaccine. In an embodiment, the present invention relates to a Fasciola hepatica antigen and vaccine. The antigen finds utility in the field of use as a vaccine against Fasciola hepatica infection. Also disclosed are methods of vaccinating a subject against Fasciola hepatica infection.

Background to the Invention

The trematode parasite Fasciola hepatica is found almost on every continent of the world causing important economic losses in livestock, as well as zoonotic infection, with 180 million of people at risk. F. hepatica infection (fasciolosis) is recognised as neglected tropical disease. Fasciolosis also causes important economic losses in livestock and food industries around the world. In the Republic of Ireland, fasciolosis has 78% prevalence in cattle and, in the UK, estimated prevalence is 76%. Due to the increase of anthelmintic resistance and the inherent difficulties in developing anthelmintic agents, alternative approaches to control of fasciolosis are urgently needed.

There have been many putative protein candidates for potential vaccines against F. hepatica, such as fatty acid-binding proteins (FhFABP), and glutathione S-transferases (FhGST). Thioredoxin peroxidase (FhPrx) has also been shown to induce variable levels of vaccine protection in goats. Other vaccines, such as leucine aminopeptidase (FhLAP), have also been demonstrated to induce high levels of vaccine protection in sheep. Another group of proteases, the cathepsins, have been a major vaccine target due to their proteolytic actions and potential for immunoregulation. Members of this family are secreted by the juvenile parasite stage (FhCL3) and adult parasite (FhCL1 , FhCL2, FhCL5). FhCL1 and FhCL2, in their native state, were shown to induce 50-55%, and up to 72.4%, of fluke burden reduction when administered with a haem-containing (Hb) fraction in cattle. Specifically, FhCL1 is the major component found within the excretory and secretory products from adult F. hepatica. FhCL1 is involved in parasite nutrition and migration, as well as acting to suppress proinflammatory cytokines. FhCL1 is found as the inactive procathepsin L1 in secretory vesicles in the parasite gut that, only after secretion in the lumen, is activated by cleavage of the propeptide.

A recombinant mutant version of the full-length protease cathepsin-L 1 (rmFhCL.1 ), expressed in Saccharomyces cerevisiae or Pichia pastoris, which does not auto-digest, is potentially useful as an immunodiagnostic tool in F. hepatica infections in cattle, and is a potential vaccine capable of reducing fluke burdens in cattle. However, other trials have not shown such protection in terms of fluke burden. Inconsistency in antigen efficacy between trials hinders development of a vaccine. These differences may be due to multiple factors - for example adjuvant effects, F. hepatica strain or immunological state of the animal. Specifically, differential epitope recognition by individual animals could be a potential source of variable levels of protection both within and between trials of F. hepatica antigens.

Summary of the Invention According to a first aspect of the present invention, there is provided a Fasciola antigen comprising a polypeptide, wherein the polypeptide comprises the amino acid sequence WHQWKRM, or a fragment thereof.

Optionally, the Fasciola antigen is a F. hepatica antigen comprising a polypeptide, wherein the polypeptide comprises the amino acid sequence WHQWKRM, or a fragment thereof.

Optionally, the polypeptide comprises the amino acid sequence DLWHQWKRM, or a fragment thereof. Optionally, the polypeptide comprises the amino acid sequence WHQWKRMYNKE, or a fragment thereof.

Optionally, the polypeptide comprises the amino acid sequence DLWHQWKRMYNKE, or a fragment thereof. Optionally or additionally, the F. hepatica antigen further comprises a polypeptide, wherein the polypeptide comprises the amino acid sequence YMKNERTSISF, or a fragment thereof.

Optionally, the polypeptide comprises the amino acid sequence WHQWKRM and the amino acid sequence YMKNERTSISF, or a fragment each thereof.

Optionally, the polypeptide comprises the amino acid sequence WHQWKRMYMKNERTSISF, or a fragment thereof.

Optionally, the polypeptide comprises the amino acid sequence DLWHQWKRMYMKNERTSISF, or a fragment thereof.

Optionally or additionally, the F. hepatica antigen further comprises a polypeptide, wherein the polypeptide comprises the amino acid sequence VKNSWGLSWGE, or a fragment thereof. Optionally or additionally, the F. hepatica antigen further comprises a polypeptide, wherein the polypeptide comprises at least one amino acid sequence selected from VKNSWGLSWGE , VKNSWGSYWGE, VKNSWGTYWGE, or a fragment each thereof. Optionally, the polypeptide comprises the amino acid sequence WHQWKRM and the amino acid sequence VKNSWGLSWGE, or a fragment each thereof.

Optionally, the polypeptide comprises the amino acid sequence WHQWKRM and at least one amino acid sequence selected from VKNSWGLSWGE , VKNSWGSYWGE, VKNSWGTYWGE, or a fragment each thereof.

Optionally, the polypeptide comprises the amino acid sequence WHQWKRM, the amino acid sequence YMKNERTSISF, and the amino acid sequence VKNSWGLSWGE, or a fragment each thereof.

Optionally, the polypeptide comprises the amino acid sequence WHQWKRM, the amino acid sequence YMKNERTSISF, and at least one amino acid sequence selected from VKNSWGLSWGE , VKNSWGSYWGE, VKNSWGTYWGE, or a fragment each thereof. Optionally, the polypeptide comprises the amino acid sequence

WHQWKRMYMKNERTSISFVKNSWGLSWGE, or a fragment each thereof. Further optionally, the polypeptide comprises the amino acid sequence DLWHQWKRMYMKNERTSISFVKNSWGLSWGE, or a fragment each thereof. Optionally, the polypeptide comprises the amino acid sequence WHQWKRM, the amino acid sequence YMKNERTSISF, and at least one amino acid sequence selected from VKNSWGLSWGE , VKNSWGSYWGE, VKNSWGTYWGE, or a fragment each thereof. Further optionally, the polypeptide comprises the amino acid sequence DLWHQWKRM, the amino acid sequence

YMKNERTSISF, and at least one amino acid sequence selected from VKNSWGLSWGE ,

VKNSWGSYWGE, VKNSWGTYWGE, or a fragment each thereof.

Optionally, the polypeptide comprises at least one amino acid sequence selected from

WHQWKRMYMKNERTSISFVKNSWGLSWGE, WHQWKRMYMKNERTSISFVKNSWGSYWGE, and WHQWKRMYMKNERTSISFVKNSWGTYWGE, or a fragment each thereof. Further optionally, the polypeptide comprises at least one amino acid sequence selected from

DLWHQWKRMYMKNERTSISFVKNSWGLSWGE,

DLWHQWKRMYMKNERTSISFVKNSWGSYWGE, and

DLWHQWKRMYMKNERTSISFVKNSWGTYWGE, or a fragment each thereof. Optionally, the F. hepatica antigen comprises a polypeptide, wherein the polypeptide consists of the amino acid sequence WHQWKRM, or a fragment thereof. Optionally, the polypeptide consists of the amino acid sequence DLWHQWKRM, or a fragment thereof. Optionally, the polypeptide consists of the amino acid sequence WHQWKRMYNKE, or a fragment thereof.

Optionally, the polypeptide consists of the amino acid sequence DLWHQWKRMYNKE, or a fragment thereof.

Optionally or additionally, the F. hepatica antigen further comprises a polypeptide, wherein the polypeptide consists of the amino acid sequence YMKNERTSISF, or a fragment thereof.

Optionally, the polypeptide consists of the amino acid sequence WHQWKRM and the amino acid sequence YMKNERTSISF, or a fragment each thereof.

Optionally, the polypeptide consists of the amino acid sequence WHQWKRMYMKNERTSISF, or a fragment thereof. Optionally, the polypeptide consists of the amino acid sequence DLWHQWKRMYMKNERTSISF, or a fragment thereof.

Optionally or additionally, the F. hepatica antigen further comprises a polypeptide, wherein the polypeptide consists of the amino acid sequence VKNSWGLSWGE, or a fragment thereof.

Optionally or additionally, the F. hepatica antigen further comprises a polypeptide, wherein the polypeptide consists of at least one amino acid sequence selected from VKNSWGLSWGE , VKNSWGSYWGE, VKNSWGTYWGE, or a fragment each thereof. Optionally, the polypeptide consists of the amino acid sequence WHQWKRM and the amino acid sequence VKNSWGLSWGE, or a fragment each thereof.

Optionally, the polypeptide consists of the amino acid sequence WHQWKRM and at least one amino acid sequence selected from VKNSWGLSWGE , VKNSWGSYWGE, VKNSWGTYWGE, or a fragment each thereof.

Optionally, the polypeptide consists of the amino acid sequence WHQWKRM, the amino acid sequence YMKNERTSISF, and the amino acid sequence VKNSWGLSWGE, or a fragment each thereof. Optionally, the polypeptide consists of the amino acid sequence WHQWKRM, the amino acid sequence YMKNERTSISF, and at least one amino acid sequence selected from VKNSWGLSWGE , VKNSWGSYWGE, VKNSWGTYWGE, or a fragment each thereof. Optionally, the polypeptide consists of the amino acid sequence

WHQWKRMYMKNERTSISFVKNSWGLSWGE, or a fragment each thereof. Further optionally, the polypeptide consists of the amino acid sequence DLWHQWKRMYMKNERTSISFVKNSWGLSWGE, or a fragment each thereof. Optionally, the polypeptide consists of the amino acid sequence WHQWKRM, the amino acid sequence YMKNERTSISF, and at least one amino acid sequence selected from VKNSWGLSWGE , VKNSWGSYWGE, VKNSWGTYWGE, or a fragment each thereof. Further optionally, the polypeptide consists of the amino acid sequence DLWHQWKRM, the amino acid sequence YMKNERTSISF, and at least one amino acid sequence selected from VKNSWGLSWGE ,

VKNSWGSYWGE, VKNSWGTYWGE, or a fragment each thereof.

Optionally, the polypeptide consists of at least one amino acid sequence selected from

WHQWKRMYMKNERTSISFVKNSWGLSWGE, WHQWKRMYMKNERTSISFVKNSWGSYWGE, and WHQWKRMYMKNERTSISFVKNSWGTYWGE, or a fragment each thereof. Further optionally, the polypeptide consists of at least one amino acid sequence selected from

DLWHQWKRMYMKNERTSISFVKNSWGLSWGE,

DLWHQWKRMYMKNERTSISFVKNSWGSYWGE, and

DLWHQWKRMYMKNERTSISFVKNSWGTYWGE, or a fragment each thereof. Optionally, the F. hepatica antigen comprises at least one polypeptide. Further optionally, the F. hepatica antigen comprises at least two polypeptides. Still further optionally, the F. hepatica antigen comprises at least three polypeptides.

Optionally, the F. hepatica antigen comprises at least one of a first, second, and third polypeptide. Optionally, the first polypeptide comprises or consists of the amino acid sequence WHQWKRM or a fragment thereof. Optionally, the second polypeptide comprises or consists of the amino acid sequence YMKNERTSISF or a fragment thereof. Optionally, the third polypeptide comprises or consists of at least one amino acid sequence selected from VKNSWGLSWGE, VKNSWGSYWGE, and VKNSWGTYWGE, or a fragment each thereof.

Optionally, the F. hepatica antigen comprises a single polypeptide. Further optionally, the F. hepatica antigen comprises a single fusion polypeptide. Still further optionally, the F. hepatica antigen comprises a single fusion polypeptide comprising or consisting of at least one of the amino acid sequence WHQWKRM or a fragment thereof, the amino acid sequence YMKNERTSISF or a fragment thereof, and at least one amino acid sequence selected from VKNSWGLSWGE,

VKNSWGSYWGE, and VKNSWGTYWGE, or a fragment each thereof. Optionally, the F. hepatica antigen comprises a single polypeptide. Further optionally, the F. hepatica antigen comprises a single fusion polypeptide. Still further optionally, the F. hepatica antigen comprises a single fusion polypeptide comprising or consisting of at least one amino acid sequence selected from WHQWKRMYMKNERTSISFVKNSWGLSWGE,

WHQWKRMYMKNERTSISFVKNSWGSYWGE, and WHQWKRMYMKNERTSISFVKNSWGTYWGE, or a fragment each thereof. Further optionally, the F. hepatica antigen comprises a single polypeptide. Further optionally, the F. hepatica antigen comprises a single fusion polypeptide. Still further optionally, the F. hepatica antigen comprises a single fusion polypeptide comprising or consisting of at least one amino acid sequence selected from

DLWHQWKRMYMKNERTSISFVKNSWGLSWGE,

DLWHQWKRMYMKNERTSISFVKNSWGSYWGE, and

DLWHQWKRMYMKNERTSISFVKNSWGTYWGE, or a fragment each thereof. Optionally or additionally, the F. hepatica antigen further comprises a polypeptide, wherein the polypeptide comprises at least one amino acid sequence selected from CGSCWAFST,

YMKNERTSISF, and VDCSRPWGNNG, or a fragment each thereof. Further optionally or additionally, the F. hepatica antigen further comprises a polypeptide, wherein the polypeptide comprises at least one amino acid sequence selected from CGSCWAFST, YMKNERTSISF, VDCSRP, and WGNNG, or a fragment each thereof.

Optionally or additionally, the F. hepatica antigen further comprises a polypeptide, wherein the polypeptide comprises at least one amino acid sequence selected from CGSCWAFST,

YMKNERTSISF, and VDCSGPWGNNG, or a fragment each thereof. Further optionally or additionally, the F. hepatica antigen further comprises a polypeptide, wherein the polypeptide comprises at least one amino acid sequence selected from CGSCWAFST, YMKNERTSISF, VDCSGP, and WGNNG, or a fragment each thereof.

Optionally, the F. hepatica antigen comprises a polypeptide, wherein the polypeptide comprises the amino acid sequence WHQWKRM, the amino acid sequence DLWHQWKRMYNKE, or a fragment each thereof; and further comprises a polypeptide, wherein the polypeptide comprises at least one amino acid sequence selected from CGSCWAFST, YMKNERTSISF, and VDCSRPWGNNG, or a fragment each thereof. Optionally, the F. hepatica antigen comprises a polypeptide, wherein the polypeptide comprises the amino acid sequence WHQWKRM, the amino acid sequence DLWHQWKRMYNKE, or a fragment each thereof; and further comprises a polypeptide, wherein the polypeptide comprises at least one amino acid sequence selected from CGSCWAFST, YMKNERTSISF, and VDCSGPWGNNG, or a fragment each thereof. Optionally, the F. hepatica antigen comprises a polypeptide, wherein the polypeptide comprises the amino acid sequence WHQWKRMCGSCWAFST, or a fragment thereof.

Optionally, the F. hepatica antigen comprises a polypeptide, wherein the polypeptide comprises the amino acid sequence DLWHQWKRMYNKECGSCWAFST, or a fragment thereof.

Optionally, the F. hepatica antigen comprises a polypeptide, wherein the polypeptide comprises the amino acid sequence DLWHQWKRMYNKEYMKNERTSISF, or a fragment thereof. Optionally, the F. hepatica antigen comprises a polypeptide, wherein the polypeptide comprises the amino acid sequence WHQWKRMVDCSRP, or a fragment thereof.

Optionally, the F. hepatica antigen comprises a polypeptide, wherein the polypeptide comprises the amino acid sequence DLWHQWKRMYNKEVDCSRPWGNNG, or a fragment thereof.

Optionally or additionally, the F. hepatica antigen further comprises a polypeptide, wherein the polypeptide consists of at least one amino acid sequence selected from CGSCWAFST,

YMKNERTSISF, and VDCSRPWGNNG, or a fragment each thereof. Further optionally or additionally, the Fasciola hepatica antigen further comprises a polypeptide, wherein the polypeptide consists of at least one amino acid sequence selected from CGSCWAFST, YMKNERTSISF, VDCSRP, and WGNNG, or a fragment each thereof.

YMKNERTSISF, and VDCSGPWGNNG, or a fragment each thereof. Further optionally or additionally, the F. hepatica antigen further comprises a polypeptide, wherein the polypeptide consists of at least one amino acid sequence selected from CGSCWAFST, YMKNERTSISF, VDCSGP, and WGNNG, or a fragment each thereof. Optionally, the F. hepatica antigen comprises a polypeptide, wherein the polypeptide consists of the amino acid sequence WHQWKRM, the amino acid sequence DLWHQWKRMYNKE, or a fragment each thereof; and further comprises a polypeptide, wherein the polypeptide consists of least one amino acid sequence selected from CGSCWAFST, YMKNERTSISF, and VDCSRPWGNNG, or a fragment each thereof.

Optionally, the F. hepatica antigen comprises a polypeptide, wherein the polypeptide consists of the amino acid sequence WHQWKRM, the amino acid sequence DLWHQWKRMYNKE, or a fragment each thereof; and further consists of a polypeptide, wherein the polypeptide comprises at least one amino acid sequence selected from CGSCWAFST, YMKNERTSISF, and VDCSGPWGNNG, or a fragment each thereof. Optionally, the F. hepatica antigen comprises a polypeptide, wherein the polypeptide consists of the amino acid sequence WHQWKRMCGSCWAFST, or a fragment thereof.

Optionally, the F. hepatica antigen comprises a polypeptide, wherein the polypeptide consists of the amino acid sequence DLWHQWKRMYNKECGSCWAFST, or a fragment thereof.

Optionally, the F. hepatica antigen comprises a polypeptide, wherein the polypeptide consists of the amino acid sequence DLWHQWKRMYNKEYMKNERTSISF, or a fragment thereof. Optionally, the F. hepatica antigen comprises a polypeptide, wherein the polypeptide consists of the amino acid sequence WHQWKRMVDCSRP, or a fragment thereof.

Optionally, the F. hepatica antigen comprises a polypeptide, wherein the polypeptide consists of the amino acid sequence DLWHQWKRMYNKEVDCSRPWGNNG, or a fragment thereof.

Optionally or additionally, the F. hepatica antigen further comprises a polypeptide, wherein the polypeptide comprises at least one amino acid sequence selected from HRRNIWEKN and MVRNRGNMC, or a fragment each thereof. Optionally or additionally, the F. hepatica antigen further comprises a polypeptide, wherein the polypeptide comprises at least one amino acid sequence selected from HRRNIWEKN,

HRRNIWEEN, MVRNRGNMC, and MARNRGNMC, or a fragment each thereof.

Optionally, the F. hepatica antigen comprises a polypeptide, wherein the polypeptide comprises the amino acid sequence WHQWKRM, the amino acid sequence DLWHQWKRMYNKE, or a fragment each thereof; and further comprises a polypeptide, wherein the polypeptide comprises at least one amino acid sequence selected from HRRNIWEKN and MVRNRGNMC, or a fragment each thereof.

Optionally, the F. hepatica antigen comprises a polypeptide, wherein the polypeptide comprises the amino acid sequence WHQWKRM, the amino acid sequence DLWHQWKRMYNKE, or a fragment each thereof; and further comprises a polypeptide, wherein the polypeptide comprises at least one amino acid sequence selected from HRRNIWEKN, HRRNIWEEN, MVRNRGNMC, and

MARNRGNMC, or a fragment each thereof. Optionally, the F. hepatica antigen comprises a polypeptide, wherein the polypeptide comprises the amino acid sequence WHQWKRMHRRNIWEKN, or a fragment thereof.

Optionally, the F. hepatica antigen comprises a polypeptide, wherein the polypeptide comprises the amino acid sequence WHQWKRMHRRNIWEEN, or a fragment each thereof. Optionally, the Fasciola hepatica antigen comprises a polypeptide, wherein the polypeptide comprises the amino acid sequence DLWHQWKRMYNKEHRRNIWEKN, or a fragment each thereof.

Optionally, the Fasciola hepatica antigen comprises a polypeptide, wherein the polypeptide comprises the amino acid sequence DLWHQWKRMYNKEHRRNIWEEN, or a fragment each thereof.

Optionally, the Fasciola hepatica antigen comprises a polypeptide, wherein the polypeptide comprises the amino acid sequence WHQWKRMMVRNRGNMC, or a fragment thereof. Optionally, the Fasciola hepatica antigen comprises a polypeptide, wherein the polypeptide comprises the amino acid sequence WHQWKRMMARNRGNMC, or a fragment thereof.

Optionally, the Fasciola hepatica antigen comprises a polypeptide, wherein the polypeptide comprises the amino acid sequence DLWHQWKRMYNKEMVRNRGNMC, or a fragment thereof.

Optionally, the Fasciola hepatica antigen comprises a polypeptide, wherein the polypeptide comprises the amino acid sequence DLWHQWKRMYNKEMARNRGNMC, or a fragment thereof.

Optionally or additionally, the Fasciola hepatica antigen further comprises a polypeptide, wherein the polypeptide consists of at least one amino acid sequence selected from HRRNIWEKN and

MVRNRGNMC, or a fragment each thereof.

Optionally or additionally, the Fasciola hepatica antigen further comprises a polypeptide, wherein the polypeptide consists of at least one amino acid sequence selected from HRRNIWEKN,

HRRNIWEEN, MVRNRGNMC, and MARNRGNMC, or a fragment each thereof.

Optionally, the Fasciola hepatica antigen comprises a polypeptide, wherein the polypeptide consists of the amino acid sequence WHQWKRM, the amino acid sequence DLWHQWKRMYNKE, or a fragment each thereof; and further consists of a polypeptide, wherein the polypeptide comprises at least one amino acid sequence selected from HRRNIWEKN and MVRNRGNMC, or a fragment each thereof.

Optionally, the Fasciola hepatica antigen comprises a polypeptide, wherein the polypeptide consists of the amino acid sequence WHQWKRM, the amino acid sequence DLWHQWKRMYNKE, or a fragment each thereof; and further consists of a polypeptide, wherein the polypeptide comprises at least one amino acid sequence selected from HRRNIWEKN, HRRNIWEEN, MVRNRGNMC, and MARNRGNMC, or a fragment each thereof.

Optionally, the Fasciola hepatica antigen comprises a polypeptide, wherein the polypeptide consists of the amino acid sequence WHQWKRMHRRNIWEKN, or a fragment thereof. Optionally, the Fasciola hepatica antigen comprises a polypeptide, wherein the polypeptide consists of the amino acid sequence WHQWKRMHRRNIWEEN, or a fragment each thereof.

Optionally, the Fasciola hepatica antigen comprises a polypeptide, wherein the polypeptide consists of the amino acid sequence DLWHQWKRMYNKEHRRNIWEKN, or a fragment each thereof.

Optionally, the Fasciola hepatica antigen comprises a polypeptide, wherein the polypeptide consists of the amino acid sequence DLWHQWKRMYNKEHRRNIWEEN, or a fragment each thereof. Optionally, the Fasciola hepatica antigen comprises a polypeptide, wherein the polypeptide consists of the amino acid sequence WHQWKRMMVRNRGNMC, or a fragment thereof.

Optionally, the Fasciola hepatica antigen comprises a polypeptide, wherein the polypeptide consists of the amino acid sequence WHQWKRMMARNRGNMC, or a fragment thereof.

Optionally, the Fasciola hepatica antigen comprises a polypeptide, wherein the polypeptide consists of the amino acid sequence DLWHQWKRMYNKEMVRNRGNMC, or a fragment thereof.

Optionally, the Fasciola hepatica antigen comprises a polypeptide, wherein the polypeptide consists of the amino acid sequence DLWHQWKRMYNKEMARNRGNMC, or a fragment thereof.

Optionally or additionally, the Fasciola hepatica antigen further comprises a polypeptide, wherein the polypeptide comprises at least one amino acid sequence selected from DYWIVKNSWGLSWGERGY and SLPMVARFP, or a fragment each thereof.

Optionally or additionally, the Fasciola hepatica antigen further comprises a polypeptide, wherein the polypeptide comprises at least one amino acid sequence selected from DYWIVKNSWGLSWGERGY and SVAMVARFP, or a fragment each thereof. Optionally or additionally, the Fasciola hepatica antigen further comprises a polypeptide, wherein the polypeptide comprises at least one amino acid sequence selected from

DYWIVKNSWGSYWGERGY.DYWIVKNSWGLSWGERGY, TDYWIVKNSWGTYWGERGY, SLPMVARFP, and SVAMVARFP, or a fragment each thereof. Optionally, the Fasciola hepatica antigen comprises a polypeptide, wherein the polypeptide comprises the amino acid sequence WHQWKRM; the amino acid sequence DLWHQWKRMYNKE, or a fragment each thereof; and further comprises a polypeptide, wherein the polypeptide comprises at least one amino acid sequence selected from

DYWIVKNSWGSYWGERGY.DYWIVKNSWGLSWGERGY, TDYWIVKNSWGTYWGERGY, and SLPMVARFP, SVAMVARFP, or a fragment each thereof. Optionally, the polypeptide comprises the amino acid sequence

WHQWKRMDYWIVKNSWGLSWGERGY, or a fragment thereof.

Optionally, the polypeptide comprises the amino acid sequence WHQWKRM

DYWIVKNSWGSYWGERGY, or a fragment thereof.

Optionally, the polypeptide comprises the amino acid sequence WHQWKRM

DYWIVKNSWGTYWGERGY, or a fragment thereof. Optionally, the polypeptide comprises the amino acid sequence WHQWKRMSLPMVARFP, or a fragment thereof.

Optionally, the polypeptide comprises the amino acid sequence DLWHQWKRMYNKESLPMVARFP, or a fragment thereof.

Optionally, the polypeptide comprises the amino acid sequence DLWHQWKRMYNKESVAMVARFP, or a fragment thereof.

Optionally, the Fasciola hepatica antigen comprises a polypeptide, wherein the polypeptide comprises the amino acid sequence WHQWKRM, the amino acid sequence DLWHQWKRMYNKE, or a fragment each thereof; and further comprises a polypeptide, wherein the polypeptide comprises at least one amino acid sequence selected from DYWIVKNSWGLSWGERGY and SLPMVARFP, or a fragment each thereof. Optionally or additionally, the Fasciola hepatica antigen further comprises a polypeptide, wherein the polypeptide consists of at least one amino acid sequence selected from

DYWIVKNSWGLSWGERGY and SLPMVARFP, or a fragment each thereof.

Optionally or additionally, the Fasciola hepatica antigen further comprises a polypeptide, wherein the polypeptide consists of at least one amino acid sequence selected from

DYWIVKNSWGLSWGERGY and SVAMVARFP, or a fragment each thereof.

DYWIVKNSWGSYWGERGY.DYWIVKNSWGLSWGERGY, TDYWIVKNSWGTYWGERGY, SLPMVARFP, and SVAMVARFP, or a fragment each thereof.

Optionally, the Fasciola hepatica antigen comprises a polypeptide, wherein the polypeptide consists of the amino acid sequence WHQWKRM; the amino acid sequence DLWHQWKRMYNKE, or a fragment each thereof; and further consists of a polypeptide, wherein the polypeptide comprises at least one amino acid sequence selected from DYWIVKNSWGSYWGERGY.DYWIVKNSWGLSWGERGY, TDYWIVKNSWGTYWGERGY, and SLPMVARFP, SVAMVARFP, or a fragment each thereof.

Optionally, the polypeptide consists of the amino acid sequence

WHQWKRMDYWIVKNSWGLSWGERGY, or a fragment thereof.

Optionally, the polypeptide consists of the amino acid sequence WHQWKRM

DYWIVKNSWGSYWGERGY, or a fragment thereof. Optionally, the polypeptide consists of the amino acid sequence WHQWKRM

DYWIVKNSWGTYWGERGY, or a fragment thereof.

Optionally, the polypeptide consists of the amino acid sequence WHQWKRMSLPMVARFP, or a fragment thereof.

Optionally, the polypeptide consists of the amino acid sequence DLWHQWKRMYNKESLPMVARFP, or a fragment thereof.

Optionally, the polypeptide consists of the amino acid sequence DLWHQWKRMYNKESVAMVARFP, or a fragment thereof.

Optionally, the polypeptide comprises the amino acid sequence

MYRSGIYQSQTCSPLRVNHAVLAVGYGTQDGTDYWIVKNSWGSY, or a fragment thereof. Optionally, the polypeptide comprises the amino acid sequence

TCSPLRVNHAVLAVGYGTQDGTDYWIVKNSWGSY, or a fragment thereof.

Optionally, the polypeptide comprises the amino acid sequence

MYRSGIYQSQTCSPLRVNHAVLAVGYGTQDGTDYWIVKNSWGSY, or a fragment thereof.

Optionally, the polypeptide comprises the amino acid sequence

RNIWEKNVKHQEHNLRHDLGLVTYTLGLNQFTDMTFEEFKAKYLTEMSR ASDILSHGVPYEANNRAVPDK, or a fragment thereof. Optionally, the polypeptide comprises not more than 31 1 amino acid residues. Further optionally, the polypeptide comprises not more than 310 amino acid residues. Still further optionally, the polypeptide comprises not more than 300 amino acid residues. Still further optionally, the polypeptide comprises not more than 250 amino acid residues. Still further optionally, the polypeptide comprises not more than 200 amino acid residues. Still further optionally, the polypeptide comprises not more than 150 amino acid residues. Still further optionally, the polypeptide comprises not more than 100 amino acid residues. F. hepatica is known to elicit chronic infections in hosts through a number of mechanisms including using "decoy" antigens to divert the host immune system from protentially protective antigens.

Vaccine technology based on the inclusion of protective B- and T-cell epitopes and the elimination of such decoy epitopes within the protein sequence of FhCL1 and FhCL3 can, unlike whole protein vaccines, specifically eliminate such decoy responses. Decoy antigens may include amino-acids within the pro-peptide region of the protein, which is cleaved during activation, and/or epitopes recognised non-specifically and/or by infected animals. Optionally, there is provided a Fasciola hepatica vaccine comprising an antigen according to the first aspect of the present invention.

Optionally, the Fasciola hepatica vaccine comprises at least 200 g of the antigen according to the first aspect of the present invention.

Optionally, the Fasciola hepatica vaccine comprises an antigen according to the first aspect of the present invention and a pharmaceutically acceptable adjuvant.

Optionally, the Fasciola hepatica vaccine comprises at least 200 g of the antigen according to the first aspect of the present invention and a pharmaceutically acceptable adjuvant.

Optionally, the Fasciola hepatica vaccine comprises an antigen according to the first aspect of the present invention and at least 2ml_ of the pharmaceutically acceptable adjuvant. Optionally, the Fasciola hepatica vaccine comprises at least 200 g of the antigen according to the first aspect of the present invention and at least 2ml_ of the pharmaceutically acceptable adjuvant.

Optionally, the Fasciola hepatica vaccine comprises 100 g of the antigen according to the first aspect of the present invention for each 1 mL of the pharmaceutically acceptable adjuvant.

Optionally, the Fasciola hepatica vaccine is administered parenterally. Further optionally, the Fasciola hepatica vaccine is administered by parenteral injection. Still further optionally, the Fasciola hepatica vaccine is administered subcutaneously. Further optionally, the Fasciola hepatica vaccine is administered by subcutaneous injection.

Optionally, the Fasciola hepatica vaccine is administered orally (including sublingually, sublabially and/or buccal) and/or nasally.

Optionally or additionally, the Fasciola hepatica vaccine is administered at least once. Further optionally or additionally, the Fasciola hepatica vaccine is administered at least twice. Optionally or additionally, the Fasciola hepatica vaccine is administered parenterally at least once. Further optionally or additionally, the Fasciola hepatica vaccine is administered parenterally at least twice. Still further optionally or additionally, the Fasciola hepatica vaccine is administered subcutaneously at least once. Still further optionally or additionally, the Fasciola hepatica vaccine is administered subcutaneously at least twice.

Optionally or additionally, the Fasciola hepatica vaccine is administered at least twice, wherein each administration is at least one week apart. Further optionally or additionally, the Fasciola hepatica vaccine is administered at least twice, wherein each administration is at least two weeks apart. Still further optionally or additionally, the Fasciola hepatica vaccine is administered at least twice, wherein each administration is at least three weeks apart. Still further optionally or additionally, the Fasciola hepatica vaccine is administered at least twice, wherein each administration is at least four weeks apart. According to a second aspect of the present invention, there is provided an antigen according to the first aspect of the present invention for use as a vaccine against Fasciola infection.

Optionally, the antigen according to the first aspect of the present invention is for use as a vaccine against Fasciola hepatica infection.

Optionally or additionally, the antigen according to the first aspect of the present invention is for use as a vaccine against Fasciola gigantica infection.

Optionally, the antigen according to the first aspect of the present invention is for use as a vaccine against juvenile Fasciola hepatica infection. Further optionally, the antigen according to the first aspect of the present invention is for use as a vaccine against newly excysted juvenile Fasciola hepatica infection. Stll further optionally, the antigen according to the first aspect of the present invention is for use as a vaccine against infection by immature, migratory stage Fasciola hepatica, optionally which are found in the liver parenchyma, optionally in vivo (optionally referred to as 21 day worms).

Optionally, the antigen according to the first aspect of the present invention is for use as a vaccine against fasciolosis. Optionally, the antigen according to the first aspect of the present invention is for use in the treatment or prophylaxis of fasciolosis.

According to a third aspect of the present invention, there is provided a method of vaccinating a subject against Fasciola infection, the method comprising the step of administering to the subject an antigen according to the first aspect of the present invention. Optionally, the method is a method of vaccinating a subject against Fasciola hepatica infection.

Optionally or additionally, the method is a method of vaccinating a subject against Fasciola gigantica infection.

Optionally, the method of vaccinating the subject against Fasciola hepatica infection is a method of vaccinating a subject against Fasciolosis, the method comprising the step of administering to the subject an antigen according to the first aspect of the present invention. Optionally, the method of vaccinating the subject against Fasciola hepatica infection is a method for the treatment or prophylaxis of fasciolosis, the method comprising the step of administering to the subject an antigen according to the first aspect of the present invention.

According to a fourth aspect of the present invention, there is provided use of an antigen according to the first aspect of the present invention in the manufacture of a vaccine for vaccinating a subject against Fasciola hepatica infection.

Optionally, the use of an antigen according to the first aspect of the present invention is in the manufacture of a vaccine for vaccinating a subject against fasciolosis.

Optionally, the use of an antigen according to the first aspect of the present invention is in the manufacture of a vaccine for the treatment or prophylaxis of fasciolosis.

Also disclosed is a method of preparing a Fasciola hepatica vaccine, the method comprising the step of providing an antigen according to the first aspect of the present invention.

Optionally, the method further comprises the step of providing a pharmaceutically acceptable adjuvant. Optionally, the method further comprises the step of admixing the antigen according to the first aspect of the present invention with the pharmaceutically acceptable adjuvant.

Also disclosed is an expression vector comprising a nucleic acid sequence encoding the polypeptide of the Fasciola hepatica antigen according to the first aspect of the present invention.

Optionally, the step of providing an antigen according to the first aspect of the present invention comprises providing an expression vector comprising a nucleic acid sequence encoding the polypeptide of the Fasciola hepatica antigen according to the first aspect of the present invention; expressing the expression vector in a host cell; and isolating the polypeptide of the Fasciola hepatica antigen according to the first aspect of the present invention. Optionally, an antigen according to the first aspect of the present invention comprises a polypeptide, wherein the polypeptide comprises an amino acid sequence as defined herein and one or more amino acid substitutions or modifications. Optionally, the polypeptide comprises an amino acid sequence as defined herein and at least one amino acid substitutions or modifications. Further optionally, the polypeptide comprises an amino acid sequence as defined herein and at least two, optionally at least three, further optionally, at least four, still further optionally at least five, still further optionally at least six, still further optionally at least seven, still further optionally at least eight, still further optionally at least nine, still further optionally at least ten amino acid substitutions or modifications.

Optionally, the one or more amino acid substitutions or modifications comprise one or more amino acid insertion, one or more amino acid deletion, or one or more amino acid substitution. Optionally, the one or more amino acid substitutions or modifications comprise one or more conservative amino acid substitution.

By "conservative amino acid substitution" is meant an amino acid residue that has been replaced by another, biologically similar amino acid residue. Optionally, by "conservative amino acid substitution" is meant an amino acid residue that has been replaced by another, biologically similar amino acid residue, and which does not alter or have a deleterious effect on the ability of the antigen to elicit an immune response. A skilled person can recognize the nature of such conservative amino acid substitutions. Examples of conservative amino acid substitutions include the substitution of a hydrophobic amino acid residue such as isoleucine, valine, leucine, or methionine for another hydrophobic amino acid residue; or the substitution of a polar amino acid residue for another polar amino acid residue such as between arginine and lysine, between glutamic acid and aspartic acid, or between glutamine and asparagine; or the substitution of a positively charged amino acid for another positively charged amino acid such as between arginine and lysine; or the substitution of a negatively charged amino acid for another negatively charged amino acid such as between glutamic acid and aspartic acid. Optionally, an amino acid residue selected from any of the following groups can be replaced by another, biologically similar amino acid residue selected from the same group: Alanine (A), Serine (S), and Threonine (T); Aspartic acid (D), and Glutamic acid (E); Asparagine (N), and Glutamine (Q); Arginine (R), and Lysine (K); Isoleucine (I), Leucine (L), Methionine (M), and Valine (V); and Phenylalanine (F), Tyrosine (Y), and Tryptophan (W).

Optionally, the polypeptide comprises an amino acid sequence as defined herein, wherein at least one, optionally at least two, further optionally at least three amino acid residues have been deleted (for example proteolytically cleaved), and which does not alter or have a deleterious effect on the ability of the antigen to elicit an immune response. A skilled person can recognize the nature of such amino acid residue deletions. Optionally, the Fasciola hepatica vaccine comprises one or more antigen. Optionally, the Fasciola hepatica vaccine is a multivalent vaccine. By the term "multivalent" is meant the Fasciola hepatica vaccine comprises more than one antigen according to a first aspect of the present invention. The more than one antigen according to a first aspect of the present invention can be linked, optionally covalently linked, via a linker or tether, or can be complexed (either ionic or non-ionic), depending on the nature of the Fasciola hepatica vaccine. By the terms "linker" or "tether" is meant the chemical groups that are interposed between each more than one antigen according to a first aspect of the present invention. Suitable linkers or tethers include short stretches of amino acids, optionally neutral amino acids, such as glycine; for example, a stretch of between about 3 and about 10 amino acids, optionally, neutral amino acids.

The Fasciola hepatica vaccine may or may not include adjuvants. The Fasciola hepatica vaccine may also be used adjuvant free, with a sterile carrier.

Optionally, the Fasciola hepatica vaccine comprises an antigen according to the first aspect of the present invention and one or more pharmaceutically acceptable adjuvant selected from well-known adjuvants and adjuvant systems. Suitable adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.; Hamilton, MT); alum; aluminum hydroxide gel; aluminum phosphate; oil-in water emulsions; water-in-oil emulsions such as Freund's complete and incomplete adjuvants; Block copolymer (CytRx; Atlanta, GA); SAF-M (Chiron; Emeryville, CA); AMPHIGEN® adjuvant; killed Bordetella; saponins such as Stimulon™ QS-21 (Antigenics, Framingham, MA. described in US Patent No. 5,057,540); particles generated from saponins such as ISCOMS (immunostimulating complexes), GPI-0100 (Galenica Pharmaceuticals, Inc.; Birmingham, AL) or other saponin fractions; monophosphoryl lipid A (MPL-A); avridine; lipid-amine adjuvant; heat-labile enterotoxin from

Escherichia coli (recombinant or otherwise); cholera toxin; and muramyl dipeptide. Also useful is MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, MT), which is described in US Patent No. 4,912,094. Also suitable for use are other well known adjuvants including: synthetic lipid A analogs or aminoalkyl glucosamine phosphate (AGP) compounds, or derivatives or analogs thereof, which are available from Corixa (Hamilton, MT), and which are described in US Patnet No 6, 1 13,918,; L121/squalene; D-lactide-polylactide/glycoside; pluronic polyols; muramyl dipeptide; extracts of Mycobacterium tuberculosis; bacterial lipopolysaccharides generally; pertussis toxin (PT); and an E. coli heat-labile toxin (LT), particularly LT-K63, LT-R72, PT-K9/G129; see, e.g.,

International Patent Publication Nos. 1993/013302 and 1992/019265. Synthetic polynucleotides, such as oligonucleotides containing CpG motifs (as described in US Patent No 6,207,646), can also be used as adjuvants. CpG oligonucleotides, such as P-class immunostimulatory oligonucleotides, are useful, including E-modified P-class immunostimulatory oligonucleotides. Sterols can also be useful as adjuvants. Those suitable for use can include sitosterols, stigmasterol, ergosterol, ergocalciferol, and cholesterol.

Optionally, the Fasciola hepatica vaccine can generally further include one or more polymers such as, for example, DEAE Dextran, polyethylene glycol, polyacrylic acid, and polymethacrylic acid (e.g., CARBOPOL®). Optionally, the Fasciola hepatica vaccine can also further include one or more Th2 stimulants such as, for example, BayR1005(R) and aluminum. Optionally, the Fasciola hepatica vaccine can additionally or alternatively further include one or more immunomodulatory agents, such as quaternary ammonium compounds (e.g., DDA), interleukins, interferons, or other cytokines. A number of cytokines or lymphokines have been shown to have immune-modulating activity, and thus may be used as adjuvants. These can include, but are not limited to: the interleukins 1 -α, 1-β, 2, 4, 5, 6, 7, 8, 10, 12 (as described in US Patent No 5,723, 127), 13, 14, 15, 16, 17 and 18 (and its mutant forms); the interferons-a, β and gamma; granulocyte- macrophage colony stimulating factor (as described in US Patent No 5,078,996, and ATCC

Accession Number 39900); macrophage colony stimulating factor; granulocyte colony stimulating factor, GSF; and the tumor necrosis factors a and β. Still other useful adjuvants include chemokines, including without limitation, MCP-1 , ΜΙΡ-1α, ΜΙΡ-1 , and RANTES. Adhesion molecules, such as a selectin, e.g., L-selectin, P-selectin, and E-selectin may also be useful as adjuvants. Still other useful adjuvants include, without limitation, a mucin-like molecule, e.g., CD34, GlyCAM-1 and MadCAM-1 ; a member of the integrin family such as LFA-1 , VLA-1 , Mac-1 and p150.95; a member of the immunoglobulin superfamily such as PECAM, ICAMs (e.g., ICAM-1 , ICAM-2 and ICAM-3), CD2 and LFA-3; co-stimulatory molecules such as CD40 and CD40L; growth factors including vascular growth factor, nerve growth factor, fibroblast growth factor, epidermal growth factor, B7.2, PDGF, BL-1 , and vascular endothelial growth factor; receptor molecules including Fas, TNF receptor, Fit, Apo-1 , p55, WSL-1 , DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, and DR6. Still another adjuvant molecule includes Caspase (ICE) (as described in International Patent Publication Nos. 1998/017799 and 1999/043839). Suitable adjuvants also include, without limitation, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, MT), which is described in U.S. Patent No. 4,912,094. Also suitable for use as adjuvants are synthetic lipid A analogs or aminoalkyl glucosamine phosphate compounds (AGP), or derivatives or analogs thereof, which are available from Corixa (Hamilton, MT), and which are described in United States Patent No. 6,1 13,918. One such AGP is 2-[(R)-3-Tetradecanoyloxytetradecanoylamino] ethyl 2- Deoxy-4-0-phosphono-3-0-[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3- tetradecanoyloxytetradecanoyl-amino]-b-D-glucopyranoside, which is also known as 529 (formerly known as RC529). The RC529 adjuvant is formulated as an aqueous form or as a stable emulsion.

Additional adjuvants useful in the practice of the present invention include cholera toxins (CT) and mutants thereof, including those described in published International Patent Publication No

2000/018434 (wherein the glutamic acid at amino acid position 29 is replaced by another amino acid, other than aspartic acid, preferably a histidine). Similar CT toxins or mutants are described in published International Patent Publication No 2002/098368 (wherein the isoleucine at amino acid position 16 is replaced by another amino acid, either alone or in combination with the replacement of the serine at amino acid position 68 by another amino acid; and/or wherein the valine at amino acid position 72 is replaced by another amino acid). Other CT toxins are described in International Patent Publication No 2002/098369 (wherein the arginine at amino acid position 25 is replaced by another amino acid; and/or an amino acid is inserted at amino acid position 49; and/or two amino acids are inserted at amino acid positions 35 and 36). Said CT toxins or mutant can be included in the immunogenic compositions either as separate entities, or as fusion partners for the polypeptides of the present invention.

In a further example, adjuvant components are selected from a combination of lecithin in light mineral oil, and also an aluminum hydroxide component. Details concerning the composition and formulation this Amphigen®, Pfizer and Zoetis, (as representative lecithin/mineral oil component) are as follows. A preferred adjuvanted may be provided as a 2ML dose in a buffered solution further comprising about 5% (v/v) Rehydragel® (aluminum hydroxide gel) and "20% Amphigen" ® at about 25% final (v/v). Amphigen® is generally described in U.S Patent No 5,084,269 and provides de- oiled lecithin (preferably soy) dissolved in a light oil, which is then dispersed into an aqueous solution or suspension of the antigen as an oil-in-water emulsion. Further, the oil used in the adjuvant formulations is preferably a mineral oil. As used herein, the term "mineral oil" refers to a mixture of liquid hydrocarbons obtained from petrolatum via a distillation technique. The term is synonymous with "liquefied paraffin", "liquid petrolatum" and "white mineral oil." The term is also intended to include "light mineral oil," i.e., oil which is similarly obtained by distillation of petrolatum, but which has a slightly lower specific gravity than white mineral oil. See, e.g., Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990, at pages 788 and 1323). Mineral oil can be obtained from various commercial sources, for example, J. T. Baker (Phillipsburg, Pa.), USB Corporation (Cleveland, Ohio). Preferred mineral oil is light mineral oil commercially available under the name DRAKEOL®. Typically, the oily phase is present in an amount from 50% to 95% by volume; preferably, in an amount of greater than 50% to 85%; more preferably, in an amount from greater than 50% to 60%, and more preferably in the amount of greater than 50-52% v/v of the vaccine composition. The oily phase includes oil and emulsifiers (e.g., SPAN® 80, TWEEN® 80 etc), if any such emulsifiers are present.

Amphigen® has been improved according to the protocols of U.S. Patent No 6,814,971 (see columns 8-9 thereof) to provide a so-called "20% Amphigen"® component for use in the final adjuvanted vaccine compositions of the present invention. Thus, a stock mixture of 10% lecithin and 90% carrier oil (DRAKEOL®, Penreco, Karns City, PA) is diluted 1 : 4 with 0.63% phosphate buffered saline solution, thereby reducing the lecithin and DRAKEOL® components to 2% and 18% respectively (i.e. 20% of their original concentrations). Tween 80® and Span 80® surfactants are added to the composition, with representative and preferable final amounts being 5.6% (v/v) Tween 80® and 2.4% (v/v) Span 80®, wherein the Span® is originally provided in the stock DRAKEOL component, and the Tween is originally provided from the buffered saline component, so that mixture of the saline and DRAKEOL® components results in the finally desired surfactant concentrations. Mixture of the DRAKEOL®/lecithin and saline solutions can be accomplished using an In-Line Slim Emulsifier apparatus, model 405, Charles Ross and Son, Hauppauge, NY, USA. The vaccine composition also includes Rehydragel® LV (about 2% aluminum hydroxide content in the stock material), as additional adjuvant component (available from Reheis, NJ, USA, and ChemTrade Logistics, USA). With further dilution using 0.63% PBS, the final vaccine composition contains the following compositional amounts per 2ML dose; 5% (v/v) Rehydragel® LV; 25% (v/v) of "20% Amphigen®", i.e. it is further 4-fold diluted); and 0.01 % (w/v) of merthiolate. As is understood in the art, the order of addition of these particular components can be varied to provide the equivalent final vaccine composition. For example, an appropriate dilution of virus in buffer can be prepared. An appropriate amount of Rehydragel® LV (about 2% aluminum hydroxide content) stock solution can then be added, with blending, in order to permit the desired 5% (v/v) concentration of Rehydragel® LV in the actual final product. Once prepared, this intermediate stock material is combined with an appropriate amount of "20% Amphigen"® stock (as generally described above, and already containing necessary amounts of Tween 80® and Span 80®) to again achieve a final product having 25% (v/v) of "20% Amphigen"®. An appropriate amount of 10% merthiolate can finally be added. The vaccine compositions of this embodiment of the invention permit variation in all of the ingredients, such that the total dose of antigen may be varied preferably by a factor of 100 (up or down) compared to the antigen dose stated above, and most preferably by a factor of 10 or less (up or down),. Similarly, surfactant concentrations (whether Tween® or Span®) may be varied by up to a factor of 10, independently of each other, or they may be deleted entirely, with replacement by appropriate concentrations of similar materials, as is well understood in the art. Rehydragel® concentrations in the final product may be varied, first by the use of equivalent materials available from many other manufacturers (i.e. Alhydrogel® .Brenntag; Denmark), or by use of additional variations in the Rehydragel® line of products such as CG, HPA or HS. Using LV as an example, final useful concentrations thereof including from 0% to 20%, with 2-12% being more preferred, and

4- 8% being most preferred, Similarly, the although the final concentration of Amphigen® (expressed as % of "20% Amphigen"®) is preferably 25%, this amount may vary from 5-50%, preferably 20-30% and is most preferably about 24-26%.

Non-natural, synthetic emulsifiers suitable for use also include sorbitan-based non-ionic surfactants, e.g. fatty-acid-substituted sorbitan surfactants (commercially available under the name SPAN® or ARLACEL®), fatty acid esters of polyethoxylated sorbitol (TWEEN®), polyethylene glycol esters of fatty acids from sources such as castor oil (EMULFOR®); polyethoxylated fatty acid (e.g., stearic acid available under the name SIMULSOL® M-53), polyethoxylated isooctylphenol/formaldehyde polymer (TYLOXAPOL®), polyoxyethylene fatty alcohol ethers (BRIJ®); polyoxyethylene nonphenyl ethers (TRITON® N), polyoxyethylene isooctyl phenyl ethers (TRITON® X). Preferred synthetic surfactants are the surfactants available under the name SPAN® and TWEEN®, such as TWEEN®- 80 (Polyoxyethylene (20) sorbitan monooleate) and SPAN®-80 (sorbitan monooleate). Generally speaking, the emulsifier(s) may be present in the vaccine composition in an amount of 0.01 % to 40% by volume, preferably, 0.1 % to 15%, more preferably 2% to 10%.

In an alternative embodiment, the final vaccine composition contains SP-Oil® and Rehydragel® LV as adjuvants (or other Rehydragel® or Alhydrogel® products), with preferable amounts being about

5- 20% SP-Oil (v/v) and about 5-15% Rehydragel® LV (v/v), and with 5% and 12%, respectively, being most preferred amounts. In this regard it is understood that % Rehydragel refers to percent dilution from the stock commercial product. (SP-Oil ® is a fluidized oil emulsion with includes a polyoxyethylene-polyoxypropylene block copolymer (Pluronic® L121 , BASF Corporation, squalene, polyoxyethylene sorbitan monooleate (Tween®80, ICI Americas) and a buffered salt solution.). It should be noted that the present invention may also be successfully practiced wherein the adjuvant component is only Amphigen®.

In another embodiment, the final vaccine composition contains TXO as an adjuvant; TXO is generally described in International Patent Publication No 2015/042369. All TXO compositions disclosed therein are useful in the preparation of vaccines of the invention. In TXO, the

immunostimulatory oligonucleotide ("T"), preferably an ODN, preferably containing a palindromic sequence, and optionally with a modified backbone, is present in the amount of 0.1 to 5 ug per 50 ul of the vaccine composition (e.g., 0.5 - 3 ug per 50 ul of the composition, or more preferably 0.09- 0.1 1 ug per 50 ul of the composition). A preferred species thereof is SEQ ID NO: 8 as listed (page 17) in International Patent Publication No 2015/042369. The polycationic carrier ("X") is present in the amount of 1-20 ug per 50 ul (e.g., 3-10 ug per 50 ul, or about 5 ug per 50 ul). Light mineral oil ("O") is also a component of the TXO adjuvant. In certain embodiments, TXO adjuvants are prepared as follows: a) Sorbitan monooleate, MPL-A and cholesterol are dissolved in light mineral oil. The resulting oil solution is sterile filtered; b) The immunostimulatory oligonucleotide, Dextran DEAE and Polyoxyethylene (20) sorbitan monooleate are dissolved in aqueous phase, thus forming the aqueous solution; and c) The aqueous solution is added to the oil solution under continuous homogenization thus forming the adjuvant formulation TXO.

Particularly preferred adjuvant formulations suitable for use with all the peptide and polypeptide formulations of the invention are disclosed in U.S. Patent No. 8,580,280 which generally provides complex mixtures of adjuvant components, and subcombinations thereof. Preferred adjuvants thus contain combinations of a saponin (such as Quil A); a sterol (such as cholesterol); an

immunomodulator molecule such as dimethyl dioctadecyl ammonium bromide (:DDA") known to stimulate strong cell mediated immune response; a polymer (such as polyacrylic acid and cross linked forms thereof, i.e. Carbopol®); and an adjuvant component known to stimulate Th2 response (such as N-(2-Deoxy-2-L-leucylamino-b-D-glucopyranosyl)-N-octadecyldodecanoylamide hydroacetate, known by the tradename Bay R1005®(Bayer); further in combination with an

ORN/ODN compound such as CpG. Preferred subcombinations also include those where either an ORN/+ODN compound is used, or a compound such as DDA is used, but not both. Further related component subcombinations of the above are described in published patent application

US2013/084306, pertaining to use of cholesterol and CpG as sole adjuvant components. Published patent application US2014/0056940 discloses subcombinations of Quil A, cholesterol, and CpG, for example. Adjuvant components as described in U.S. Patent No. 7,049,302 are also highly useful in the practice of the present invention. All the adjuvant compositions of the invention can be used with any of the antigen formulations covered by the present disclosure. Optionally, the Fasciola hepatica vaccine further comprises pharmaceutically acceptable carriers, excipients and/or stabilizers (see e.g. Remington: The Science and practice of Pharmacy (2005) Lippincott Williams), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as Mercury((o-carboxyphenyl)thio)ethyl sodium salt (THIOMERSAL), octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;

benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as

polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non- ionic surfactants such as polyethylene glycol (PEG), TWEEN® or PLURONICS®.

Liposomes can also be used to provide for the sustained release of antigenic proteins Details concerning how to make and use liposomal formulations can be found in, among other places, U.S. Pat. No. 4,016, 100; U.S. Pat. No. 4,452,747; U.S. Pat. No. 4,921 ,706; U.S. Pat. No. 4,927,637; U.S. Pat. No. 4,944,948; U.S. Pat. No. 5,008,050; and U.S. Pat. No. 5,009,956.

Also disclosed is an antibody capable of recognising an antigen according to the first aspect of the present invention. Optionally, the antibody is capable of specifically recognising an antigen according to the first aspect of the present invention. Further optionally, the antibody is capable of binding an antigen according to the first aspect of the present invention. Still further optionally, the antibody is capable of specifically binding an antigen according to the first aspect of the present invention.

Also disclosed is an antibody raised against an antigen according to the first aspect of the present invention.

Also disclosed is an antigen according to the first aspect of the present invention, or an antibody capable of recognising the antigen, for use in diagnosing Fasciola infection.

Also disclosed is a method of diagnosing Fasciola infection using an antigen according to the first aspect of the present invention or an antibody capable of recognising the antigen. Optionally, the method of diagnosing Fasciola infection is an in vitro method of diagnosing Fasciola infection.

Optionally, the method of diagnosing Fasciola infection comprises:

(a) providing a sample to be tested;

(b) providing an antigen according to the first aspect of the present invention or an antibody capable of recognising the antigen; (c) contacting the sample to be tested with the antigen according to the first aspect of the present invention or an antibody capable of recognising the antigen;

(d) determining the quantitative or qualitative amount of the antigen according to the first aspect of the present invention or an antibody capable of recognising the antigen; and

(e) attributing the quantitative or qualitative amount of the antigen according to the first aspect of the present invention or an antibody capable of recognising the antigen to the quantitative or qualitative amount of Fasciola infection.

In addition, the immunodominant polypeptides disclosed as B-cell epitopes can also be used as diagnostic antigens in a variety of assay formats including ELISA, lateral flow and multiplexed assays. Peptides specifically recognised by vaccinated but not infected animals can, furthermore, be used diagnostically to distinguish between infected and vaccinated animals.

Optionally, the antigen according to the first aspect of the present invention or an antibody capable of recognising the antigen is immobilised on or at a solid support.

Optionally, the antigen according to the first aspect of the present invention or an antibody capable of recognising the antigen is immobilised on or at a solid support by:

(a) labelling the antigen according to the first aspect of the present invention or an antibody capable of recognising the antigen with, for example, dinitrophenol (DNP);

(b) contacting the solid support with the labelled antigen according to the first aspect of the present invention or antibody capable of recognising the antigen, optionally in the presence of sulfo-N-hydroxysuccinimide, optionally in the presence of 1-Ethyl-3-(3-dimethylamino- propyl)-carbodiimide (EDC); and

(c) optionally incubating for 2 hours, optionally at room temperature.

Optionally, the solid support is Thermo Scientific Nunc CovaLink NH.

Optionally, the polypeptide or the antigen has an amino acid sequence as defined in any of SEQ ID NO: 1 - SEQ ID NO:107, or any combinations each thereof.

SEQ ID NO: Amino Acid Sequence

SED ID NO: 1 WHQWKRM

SED ID NO: 2 YMKNERTSISF

SED ID NO: 3 DLWHQWKRM

SED ID NO: 4 VKNSWGLSWGE

SED ID NO: 5 VKNSWGSYWGE

SED ID NO: 6 VKNSWGTYWGE

SED ID NO: 7 WHQWKRMYMKNERTSISFVKNSWGLSWGE

SED ID NO: 8 WHQWKRMYMKNERTSISFVKNSWGSYWGE

SED ID NO: 9 WHQWKRMYMKNERTSISFVKNSWGTYWGE SED ID NO: 10 DLWHQWKRMYNKE

SED ID NO: 1 1 DLWHQWKRMYNKEYMKNERTSISFVKNSWGLSWGE

SED ID NO: 12 DLWHQWKRMYNKEYMKNERTSISFVKNSWGSYWGE

SED ID NO: 13 DLWHQWKRMYNKEYMKNERTSISFVKNSWGTYWGE

SED ID NO: 14 CGSCWAFST

SED ID NO: 15 WHQWKRMCGSCWAFST

SED ID NO: 16 DLWHQWKRMYNKECGSCWAFST

SED ID NO: 17 DLWHQWKRMYNKEYMKNERTSISF

SED ID NO: 18 VDCSRP

SED ID NO: 19 WHQWKRMVDCSRP

SED ID NO: 20 WGNNG

SED ID NO: 21 DLWHQWKRMYNKEVDCSRPWGNNG

SED ID NO: 22 HRRNIWEKN

SED ID NO: 23 HRRNIWEEN

SED ID NO: 24 WHQWKRMHRRNIWEKN

SED ID NO: 25 WHQWKRMHRRNIWEEN

SED ID NO: 26 DLWHQWKRMYNKEHRRNIWEKN

SED ID NO: 27 DLWHQWKRMYNKEHRRNIWEEN

SED ID NO: 28 MVRNRGNMC

SED ID NO: 29 MARNRGNMC

SED ID NO: 30 WHQWKRMMVRNRGNMC

SED ID NO: 31 WHQWKRMMARNRGNMC

SED ID NO: 32 DLWHQWKRMYNKEMVRNRGNMC

SED ID NO: 33 DLWHQWKRMYNKEMARNRGNMC

SED ID NO: 34 DYWIVKNSWGLSWGERGY

SED ID NO: 35 DYWIVKNSWGSYWGERGY

SED ID NO: 36 DYWIVKNSWGTYWGERGY

SED ID NO: 37 WHQWKRMDYWIVKNSWGLSWGERGY

SED ID NO: 38 WHQWKRMDYWIVKNSWGSYWGERGY

SED ID NO: 39 WHQWKRMDYWIVKNSWGTYWGERGY

SED ID NO: 40 SLPMVARFP

SED ID NO: 41 SVAMVARFP

SED ID NO: 42 WHQWKRMSLPMVARFP

SED ID NO: 43 WHQWKRMSVAMVARFP

SED ID NO: 44 DLWHQWKRMYNKESLPMVARFP

SED ID NO: 45 DLWHQWKRMYNKESVAMVARFP

SED ID NO: 46 MRLFVLAVLTVGVLGSNDDL

SED ID NO: 47 VGVLGSNDDLWHQWKRMYNK

SED ID NO: 48 WHQWKRMYNKEYNGADDQHR

SED ID NO: 49 EYNGADDQHRRNIWEKNVKH

SED ID NO: 50 RNIWEKNVKHIQEHNLRHDL SED ID NO: 51 IQEHNLRHDLGLVTYTLGLN

SED ID NO: 52 GLVTYTLGLNQFTDMTFEEF

SED ID NO: 53 QFTDMTFEEFKAKYLTEMSR

SED ID NO: 54 KAKYLTEMSRASDILSHGVP

SED ID NO: 55 ASDILSHGVPYEANNRAVPD

SED ID NO: 56 YEANNRAVPDKIDWRESGYV

SED ID NO: 57 KIDWRESGYVTEVKDQGNCG

SED ID NO: 58 TEVKDQGNCGSCWAFSTTGT

SED ID NO: 59 SC WAFSTTGTM EGQYM KN E R

SED ID NO: 60 MEGQYMKNERTSISFSEQQL

SED ID NO: 61 TSISFSEQQLVDCSRPWGNN

SED ID NO: 62 VDCSRPWGNNGCGGGLMENA

SED ID NO: 63 GCGGGLMENAYQYLKQFGLE

SED ID NO: 64 YQYLKQFGLETESSYPYTAV

SED ID NO: 65 TESSYPYTAVEGQCRYNKQL

SED ID NO: 66 EGQCRYNKQ LGVAKVTG F YT

SED ID NO: 67 GVAKVTGFYTVHSGSEVELK

SED ID NO: 68 VHSGSEVELKNLVGAEGPAA

SED ID NO: 69 NLVGAEGPAAVAVDVESDFM

SED ID NO: 70 VAVDVESDFMMYRSGIYQSQ

SED ID NO: 71 MYRSGIYQSQTCSPLRVNHA

SED ID NO: 72 TCSPLRVNHAVLAVGYGTQG

SED ID NO: 73 VLAVGYGTQGGTDYWIVKNS

SED ID NO: 74 GTDYWIVKNSWGLSWGERGY

SED ID NO: 75 WGLSWGERGYIRMVRNRGNM

SED ID NO: 76 IRMVRNRGNMCGIASLASLP

SED ID NO: 77 RGNMCGIASLASLPMVARFP

SED ID NO: 78 WHQWKRMYNKEYNGADDEHR

SED ID NO: 79 EYNGADDEHRRNIWEKNVKH

SED ID NO: 80 RNIWEKNVKHIEEHNLRHDR

SED ID NO: 81 IEEHNLRHDRGLVTYKLGLN

SED ID NO: 82 GLVTYKLGLNQFTDLTFEEF

SED ID NO: 83 QFTDLTFEEFKAKYLMEMSL

SED ID NO: 84 KAKYLMEMSLVSESLSDGIS

SED ID NO: 85 VSESLSDGISYEAEGNDVPA

SED ID NO: 86 YE AEG N DVPASVD WRE YGYV

SED ID NO: 87 SVDWREYGYVTEVKDQGQCG

SED ID NO: 88 TEVKDQGQCGSCWAFSAVGA

SED ID NO: 89 SCWAFSAVGAIEGQYLRKFQ

SED ID NO: 90 IEGQYLRKFQNQTLFSEQQL

SED ID NO: 91 NQTLFSEQQLVDCTRRFGNH SED ID NO: 92 VDCTRRFGNHGCGGGWMENA

SED ID NO: 93 GCGGGWMENAYKYLKNSGLE

SED ID NO: 94 YKYLKNSGLETASDYPYQGW

SED ID NO: 95 TASDYPYQG W E YQCQYRKE L

SED ID NO: 96 EYQCQYRKELGVAKVTGAYT

SED ID NO: 97 GVAKVTGAYTVHSGDEMKLM

SED ID NO: 98 VHSGDEMKLMQMVGREGPAA

SED ID NO: 99 QMVGREGPAAVAVDAQSDFY

SED ID NO: 100 VAVDAQSDFYMYESGIFQSQ

SED ID NO: 101 MYESGIFQSQTCTSRSVTHA

SED ID NO: 102 TCTSRSVTHAVLAVGYGTES

SED ID NO: 103 VLAVGYGTESGTDYWILKNS

SED ID NO: 104 GTDYW I LKNSWG KWWG E DGY

SED ID NO: 105 WGKWWGEDGYMRFARNRNNM

SED ID NO: 106 MRFARNRNNMCAIASVASVP

SED ID NO: 107 RNNMCAIASVASVPMVERFP

Materials and Methods

Experimental Design

Twenty male castrated Holstein Friesian cattle between 3 and 6 months of age (for Trial 1 / Kalamazoo) and between 5 and 1 1 months of age (for Trial 2 / Dublin), were purchased from areas where F. hepatica infection was not reported. Animals were housed under uniform conditions at Kalamazoo's facility Veterinary Medicine Research and Development, Zoetis Inc, 333 Portage St, Kalamazoo, Ml 49009, USA) (Trial 1 ) and at University College Dublin (UCD) Lyons Research Farm, Newcastle, County Kildare, Ireland (Trial 2). In both trials, to ensure that animals were free from F. hepatica infection before starting the trial, animals were serologically screened by ELISA using recombinant mutant F. hepatica cathepsin L1 (rmFhCLI ) and by faecal egg examination, as previously described by Golden et al. (Protection of cattle against a natural infection of Fasciola hepatica by vaccination with recombinant cathepsin L1 (rFhCLI ). Vaccine 2010;28:5551-7). Animals from each trial were then randomly divided into two groups, 10 animals in a control group, and 10 in a vaccinated group.

Vaccination and F. hepatica infection

Recombinant F. hepatica cathepsin L1 (rmFhCLI ) mutant was expressed in Pichia pastoris as previously described by Collins et al. (Cathepsin L1 , the Major Protease Involved in Liver Fluke (Fasciola hepatica) Virulence: PROPEPTIDE CLEAVAGE SITES AND AUTOACTIVATION OF THE ZYMOGEN SECRETED FROM GASTRODERMAL CELLS. J Biol Chem 2004;279:17038-46). Recombinant F. hepatica Cathepsin L3 (rmFhCL3) was a purified protein derived from (Chinese hamster ovary) CHO cells using chromatography over Sepharose Fast Flow (GE Healthcare) resin in accordance with the manufacturer's instructions. In both trials, a combination of both rmFhCL.1 and rmFhCL3 antigens was used to formulate a vaccine containing 200 g of each antigen per dose plus 2ml of an adjuvant. The adjuvant can comprise a saponin, a sterol, a quaternary ammonium compound, a polymer, and an ORN/ODN, wherein the saponin can be Quil A or a purified faction thereof, the sterol can be cholesterol, the quaternary ammonium compound can be dimethyl dioctadecyl ammonium bromide (DDA), the polymer can be polyacrylic acid, and the ORN/ODN can be a CpG (as described in United States Patent No 8580280 - the vaccine termed CL1/CL3/ZA1. For the control group, 2ml of a sterile saline solution was administered as a sham antigen. Animals in both trials were vaccinated twice subcutaneously, four weeks apart.

In Trial 1 , animals were infected with a total of 750 F. hepatica metacercariae (Baldwin Aquatics, (Oregon)) by administering 40 metacercariae orally in accordance with the manufacturer's instruction, starting 3 weeks post-2^nd vaccination, every second day and over a period of 5 weeks. Blood samples were collected by jugular venepuncture at Day 0, 3 weeks post-2^nd vaccination (pre- infection phase), at 7 weeks post-infection (7wpi) and at 13 weeks post-infection (7wpi). The number of weeks post-infection was calculated from the first day on which the trickle infection commenced.

In Trial 2, animals were infected with a total of 200 F. hepatica metacercariae (Baldwin Aquatics, (Oregon) at 2 weeks post-2^nd vaccination over two consecutive days (100 metacercariae per day). The metacercariae were dispersed in 10ml of dH₂0 and were administered by oral route via a 20ml syringe. Blood samples were collected at Day 0, 2 weeks post-2^nd vaccination (pre-infection), 2 weeks post-infection (2wpi), 6 weeks post-infection (6wpi), 10 weeks post-infection (10wpi) and 14weeks post-infection (14wpi).

Liver fluke collection

At 13 weeks (Trial 1 ) or 14 weeks (Trial 2) post-infection, the animals were killed and the livers collected. Flukes in each liver were counted as previously described in the Great Britain, Ministry of Agriculture F and F Manual of veterinary parasitological laboratory techniques (London: H.M.

Stationery Office; 1986). Briefly, flukes were collected by gently squeezing the thickened bile ducts and cutting the liver at the central portion of the liver, close to the gall bladder. Then, the larger ducts were opened longitudinally and any flukes present collected. Next, the liver was cut into slices 2-4cm thick and the bile ducts were thoroughly examined to extract smaller flukes. After this, liver slices were gently squeezed in warm water. Then, the slices were cut into 1-2cm cubes and placed inside a gauze sheet to create a bag. The closed bags were squeezed again in warm water and the liver cubes discarded. Gauzes were then rinsed to collect any potential attached flukes. Finally, the warm water containing recovered flukes from the squeezed liver portions was sieved through a series of strainers, and flukes were counted.

Measurement of lgG1 and lgG2 anti- rmFhCLI by ELISA lgG1 and lgG2 levels were measured in serum from all time points in both trials by using an in-house ELISA, wherein 96 well-plates (Corning) were coated with 5 g/ml of rmFhCL.1 in carbonate-coating buffer at pH 9.6, and incubated overnight at 37°C. Next, plates were washed 3x with Phosphate Buffered Saline plus 0.05% Tween 20 (PBS-T) and then blocked by adding "Ι ΟΟμΙ of 5% milk in PBS- T for 30min at 37°C. After washing 3X, serum samples were diluted 1 :20 in PBS-T, added into the plate (in 100μΙ volume) in duplicate and then serial dilutions (1 :3) were carried out. Once samples were incubated for 30min at 37°C and washed as before, HRP-conjugated monoclonal anti-lgG1 (Prionics) or anti-lgG2 (Bio-rad) were added at a concentration of 1 : 100 (anti-lgG1 ) in PBS-T or 1 : 1000 (anti-lgG2) in PBS without Tween. After incubation at the same conditions as previously described, and washing, 100μΙ of 3,3',5,5'-Tetramethylbenzidine (TMB) (Sigma) were added for 10min in the dark. Finally, 50μΙ of Stop solution (H₂S0₄) were added onto each well and absorbance was measured at 450_nm- Negative and positive serum controls were included in each plate. Endpoint titres were calculated for each sample and then transformed into log 10.

Linear B-Cell Epitope mapping of FhCL1

For epitope mapping studies, 5 animals from each group (5 controls and 5 vaccinated) in each trial were selected based on a representative distribution of fluke burden of each vaccinated or control group. For the purpose of comparing both trials, the following time points were selected: 3wks post- 2^nd vaccination (pre-infection), 7wpi and 13wpi from Trial 1 , and 2wks post-2^nd vaccination (pre- infection), 6wpi and 14wpi from Trial 2.

A total of 160 overlapping peptides of FhCL1 , 9 amino-acids in length, and with an overlap of 7 amino-acids between successive peptides were synthesised (Mimotopes Pty. Ltd. Australia), each with a biotin tag.

The FhCL1 reference sequence was taken from UniProtKB - Q24940, which included the whole FhCL1 protein, containing the signal peptide (positions 1-15), the pro-peptide (activation peptide) (positions 16-106), and the mature enzyme (107-126). The active site is formed by amino acids at positions 132, 269, 289.

Peptides were used as the solid-phase antigen in ELISA-based assays. Lyophilised peptides were solubilised in 50% Acetonitrile (Fisher Chemical) in H₂0 (HyClone GE Life Sciences) and then further diluted (1/20) in 0.1 % Sodium Azide (Sigma) plus 0.1 % in BSA (Sigma) in PBS. Next, 96-well plates (Nunc, high binding) were coated with 5 g/ml of Streptavidin (Sigma) overnight at 4°C and washed 4x with PBS-T. Biotinylated peptides were added at a final concentration of 50 g/ml in PBS- T. After incubation overnight at 4°C, and blocking with BSA (2% BSA in PBS-T) for 1.5h, each serum sample was diluted to 1/50 in 2% BSA PBS-T and added to the wells. The plates were then incubated for 1 .5h at room temperature with shaking, and washed as before. Next, peroxidase- conjugated secondary antibody (anti-bovine total anti-lgG (cat: A5295 Sigma)) was added at a dilution of 1 :3000 in blocking buffer and incubated for another 1.5h. After washing plates as previously, TMB (Sigma) was added and incubated for 15min in the dark. All the volumes were 10ΟμΙ . Absorbance was measured at 450_nm- Control sera, along with blank wells without peptide and with peptide but without serum, were included in each plate. Background (OD from blank wells) was subtracted from the wells containing peptides in each plate.

Statistical analysis and data processing

For fluke burden analysis, percentage of fluke burden reduction was calculated based on the geometric mean from each of the groups. Mann-Whitney U test was used to analyse the statistical significance of the fluke burden reduction applying 2-tailed test. For lgG1/lgG2 and epitope mapping studies, 2-way ANOVA and Bonferroni post-test was used to compare differences between groups and time points. Brief Description of the Drawings

The present invention will now be described with reference to the following non-limiting examples and the accompanying drawings in which: Figure 1 illustrates lgG1 and lgG2 levels specific to rmFhCL.1 induced after F. hepatica infection in CL1 CL3 ZA1 -vaccinated and controls (non-vaccinated) cattle, wherein lgG1 (a, b) and lgG2 (c, d) specific to rmFhCL.1 were measured by end-point titration in Trial 1 (a, c) and Trial 2 (b, d); Control group = dashed green line; CL1 CL3 ZA1 = purple line; n=10 for all groups except for Trial 2 CL1 CL3 ZA1 group (n=9); ^Λ indicates 1^st and 2^nd vaccinations; Red arrow indicates F. hepatica infection. ^*= p<0.05, ^**=p<0.01 ; ^***=p<0.001 ; ^*****=p<0.0001 ;

Figure 2 illustrates fluke burden in control and vaccinated animals with CL1 CL3 ZA1 ; wherein total fluke burden is shown in controls non-vaccinated (black dots) and vaccinated (CL1 CL3 ZA1 ) animals (white dots) in Trial 1 (a) and Trial 2 (b); Fluke burden reduction was calculated based on geometric means (bars). p=0.07 . n=10 for all groups, except n=9 for vaccinated group in Trial 2;

Figure 3 illustrates FhCL1 -Epitope mapping profile in sera from the control group, wherein in Trials 1 (a) and 2 (b), serum was examined at one timepoint pre-infection, and two timepoints post-infection in each case; at the early infection (7wpi and 6wpi) and late infection (13wpi and 14wpi); ^A=single peptide and * = consecutive peptides that are recognised significantly different at post-infection (p<0.05- p<0.0001 ); n=5 for each group;

Figure 4 illustrates FhCL1 -Epitope mapping profile in sera from cattle vaccinated with CL1 CL3 ZA1 and then infected with F. hepatica; wherein in Trials 1 (a) and 2 (b), serum was examined at one timepoint pre-infection (at 3wks or 2wks post-2nd vaccination with CL1 CL3 ZA1 , for Trial 1 (a) or Trial 2 (b), respectively), and at 2 timepoints post-infection (7wpi, 13wpi (Trial 1 ) and 6wpi, 14wpi (Trial 2)); ^A=single peptide and * = consecutive peptides that are recognised significantly different at post-infection (p<0.05- p<0.0001 ); n=5 for each group;

Figure 5 illustrates localization of the peptides recognised in the linear FhCL1 sequence and comparison between both trials; wherein the highlighted regions represent the peptide binding regions that are significantly recognised post-infection; 1 Con = Trial 1 Control group; 2 Con= Trial 2 Control group; 1 Vac= Trial 1 CL1 CL3 ZA1 -vaccinated group; 2 Vac= Trial 2, CL1 CL3 ZA1- vaccinated group; Grey=epitopes binding only at the early time points (7wpi for Trial 1 or 6wpi for Trial 2); Red= epitopes binding at the late time points only (13wpi for Trial 1 or 14wpi for Trial 2; Blue= epitopes binding at both time points (early and late); Dark green= non-specific binding of epitopes at pre-infection in the control group of Trial 2; Purple = active sites of the protein;

Figure 6 illustrates B-cell Epitope Mapping pattern of animals vaccinated with recombinant FhC L1 and subjected to low-dose field challenge through grazing naturally infected pasture. Yellow highlights indicate regions to which specific antibody binding is detected, indicative of B-cell epitopes; and

Figure 7 illustrates Interferon-gamma production by hepatic lymph node cells from animals infected with F. hepatica, taken at post-mortem examination 14 weeks post-infection. Yellow highlights indicate overlapping peptides that can stimulate cell proliferation and Interferon-gamma induction, thus indicating presence of T-cell epitopes; and wherein peptides 36-38 are also indicative of T-cell epitopes, and wherein these peptides represent the corresponding sequence to peptides 6-1 1 , but in FhCL3 - both sequences showing considerable homology; and Figure 8 illustrates T-cell epitopes which are aligned to the FhCL1 sequence, and which are capable of inducing lymphocyte proliferation and IFN-gamma production using cells from lymph nodes of F. hepatica infected cattle, green highlights.

Examples

Example 1

Antibody levels after vaccination and F. hepatica infection At 3wks and 2wks post-2^nd vaccination, in both Trial 1 and Trial 2, the vaccinated group had a higher anti-CL1 lgG1 response in comparison with the control group (p<0.001 ) (see Figure 1 a, b). These differences between vaccinated and control groups persisted until 7 and 6wpi in both trials. However, at the latest time point examined (Weeks 13 or 14 post-infection in Trials 1 and 2, respectively), the vaccinated group in Trial 1 had a greater lgG1 response than controls (P<0.001 ) whereas in Trial 2 (14wpi) the difference between the groups was no longer significant (see Figure 1 a, b). When comparing control groups at 2wks post-2^nd vaccination with saline, Trial 2 showed some lgG1 production (see Figure 1 b) which is not present in the control group from Trial 1 at 3wks post-2^nd vaccination (see Figure 1 a). Anti-rmFhCL1 lgG2 production was also higher at 3wks and 2wks post- 2^nd vaccination (p<0.01-p<0.05) in the vaccinated groups in comparison to the controls in both trials. However, at 13wpi, this higher lgG2 production was maintained in vaccinated animals from Trial 1 (p<0.001 ), but not in Trial 2 at 14wpi (see Figure 2 c, d).

Example 2

Fluke burden in vaccinated and control groups

The vaccinated group in Trial 1 showed a fluke burden reduction of 37.6% in comparison with the non-vaccinated group (p=0.07) (see Figure 2 a). In Trial 2, there were no differences in the numbers of flukes between groups (see Figure 2 b). Example 3

Peptides from FhCL1 recognised by F. hepatica infected and/or vaccinated-cattle

Peptides from FhCL1 recognised by cattle infected with F. hepatica but not vaccinated

Serum from cattle infected with F. hepatica (control-unvaccinated group) recognised linear epitopes from FhCL1 that were not recognised by the same animals pre-infection (see Figure 3 a, b).

Specifically, peptides 9 -13, 20-22, 90-91 , 107, 144, 148-149, and 151 -152 were recognised (p<0.05 - p<0.0001 ) by non-vaccinated animals at 7wpi or 13wpi in Trial 1 (see Figure 3 a). In Trial 2, peptides 10 to 12 were also recognised after infection. In this Trial, peptide 160 showed increased specific binding to serum at 14wpi in comparison to both pre-infection and to 6wpi (p<0.001- pO.0001 ) (see Figure 3 b).

Peptides from FhCL1 recognised by cattle infected with F. hepatica and vaccinated with CL1 CL3 ZA1

In Trial 1 , serum from vaccinated animals specifically recognised peptides 10-12, 65, 73-74, 81-82 and 89 at 7wpi and 13wpi (see Figure 4 a). Peptides 152 and 160 also showed higher serum binding at 13wpi than at 7wpi (p<0.0001 ) for Trial 1. In Trial 2, peptides 10-12 were also recognised by vaccinated animals, but in this case, a higher binding was found at 6wpi than at 14wpi (see Figure 4 b). These peptides bound to sera from all groups from both trials, and from both controls and vaccinated animals. In addition, in this Trial 2, peptides 20, 145 and 152 were specifically recognised by vaccinated animals at 6wpi (p<0.0001 ), however this reactivity declined to non-significant levels by 14wpi (see Figure 4b). When comparing peptide recognition between vaccinated and control groups in Trial 1 , peptides 145, 152 and 160 were more highly recognised by the vaccinated group than the control group at 3wk post-2^nd vaccination (for 145) and at 13wpi (for 152, 160). However, these peptides were recognised by both control and vaccinated animals in Trial 2 (see Figures 3 and 4).

Peptides from FhCL1 recognised non-specifically

Some evidence of non-specific recognition of some sequences within FhCL1 was also found, as peptides 144-145 were recognised by sera from control animals prior to infection in Trial 2 (see Figure 3b). This pattern of non-specific recognition pre-infection was not seen in Trial 1. Example 3

Localization of epitopes

Localization of epitopes recognised after F. hepatica infection

The region centred on residue numbers 21-27 (WHQWKRM), which corresponds to peptides 10-12, was consistently recognised by vaccinated and control animals in both trials at all time points postinfection. This region forms part of a larger domain (DLWHQWKRMYNKE) which contains peptides that also react at various time points of infection. This region is situated within the pro-peptide of the molecule and at the periphery of the 3D structure.

Localization of epitopes recognised by vaccinated animals

Epitopes recognised by vaccinated group with reduced fluke burden

In Trial 1 , where vaccination induced partial protection, the regions spanning positions 120-137, 145- 155, 161-171 (CGSCWAFST, YMKNERTSISF, VDCSRPWGNNG) were specifically recognised by vaccinated animals 13wpi (p<0.05-p<0.001 ), but binding was not significantly different at 7wpi from pre-vaccination. These regions were not as reactive with the control group from the same trial (Trial 1 ) nor with the vaccinated and control groups from Trial 2.

Epitopes recognised by vaccinated group with high fluke burden

In Trial 2, two regions (39-47 HRRNIWEKN and 310-31 1 MVRNRGNMC), were recognised by the vaccinated group which were not reactive in the control animals at 6wpi in this Trial. However, these regions were also reactive in both the vaccinated and control groups from Trial 1 at 13wpi in comparison to pre-infection.

Epitopes not recognised in vaccinated animals as compared with controls Recognition of some regions of the molecule was found to be switched off in vaccinated animals (Trial 1 ), such as the region 283-300 (DYWIVKNSWGLSWGERGY) that was recognised by the control group but not in the vaccinated group at 7 or 13wpi, or region SLPMVARFP which was reactive in the control group from Trial 2 but not in the vaccinated group.

Localization of epitopes recognised non-specifically

In Trial 2, the peptides 144 and 145, were found to be reactive in the control group pre-infection, which corresponds to position 287-299 VKNSWGLSWGE in the protein. However, this non-specific recognition was not seen in Trial 1 (see Figure 3 a, Figure 4 a). This latter region includes one part of the active site of FhCL1 at position 289, situated at the centre of the molecule (see Figure 10).

For many years, attempts have been ongoing to produce a vaccine capable of protecting ruminants against F. hepatica infection. In various trials, vaccination with cathepsins from F. hepatica, fluke burden reductions from 30-72% have been achieved. However, other trials have not shown this level of protection with these antigens. This inconsistency among trials is still unexplained. Therefore, the present study attempts to address this issue by comparing linear B-cell epitope recognition of FhCL1 by sera from vaccinated and control animals in two separate trials, as well as comparing the kinetics of the serological responses.

Firstly, elevated levels of anti-FhCL1 lgG1 induced after F. hepatica infection and higher levels post- vaccination indicated successful vaccination and infection in both trials. Interestingly, in Trial 2, control animals pre-infection induced some lgG1 , potentially from maternal antibodies or other exposure to similar pathogens. These cross-reactive antibodies could be present also in the immunised animals from the same trial. However, they would be masked by the effect of the antigen.

Secondly, vaccinated animals from both trials showed anti-CL1 lgG2 production after immunisation. However, in Trial 1 only, where vaccination provided partial protection, lgG2 levels were maintained until 13wpi. In contrast, in Trial 2, specific lgG2 production by vaccinated animals peaked at 2wpi and disappeared by 14wpi.

The disclosed epitope mapping analysis has shown that the sequence WHQWKRM (aa 21-27) is consistently and highly recognised within 6 or 7 weeks post-infection, and that this reactivity is maintained as the infection moves into the chronic stage. This sequence, found in the pro-peptide of FhCL1 is also found in the pro-peptide of FhCL3 (UniprotKB-Q9GRW4). This region is very exposed in the periphery of the 3D molecule, and is therefore accessible for antibody recognition. Antibodies to this region are also likely to account for the usefulness of FhCL1 as a diagnostic antigen.

Significantly, the mutant form of the molecule (rmFhCL.1 ), in which the active site has a single mutation and which therefore does not auto process, retaining the pro-peptide, unlike the native CL1 molecule, is as a consequence superior for use in diagnostic assays. In Trial 1 , the vaccinated, but not the control group, specifically recognised the regions spanning amino-acids 120-137, 145-155, 161-171 (CGSCWAFST, YMKNERTSISF, VDCSRPWGNNG) at 14wpi. These regions were not highly recognised by either group in Trial 2. To note as above, that the first of these 3 peptides (120-137) and partially the last one (161-163), are found within the sequence of FhCL3.

In Trial 2, two regions specifically reacted with serum from the vaccinated group at 6wpi but not from the control group (39-47 HRRNIWEKN and 310-31 1 MVRNRGNMC). However, these regions were also reactive in both the vaccinated and control groups from Trial 1 , at 13wpi.

Surprisingly, in Trial 2, there was a highly reactive region (position 287-299 VKNSWGLSWGE) recognised non-specifically pre-infection by the control group. Individual animals recognising these peptides pre-infection also showed some anti-FhCL1 lgG1 in standard ELISA at this time point, which, without being bound by theory, could be consequent to cross-reaction with maternal antibodies still present. A second explanation for this non-specific recognition, without being bound by theory, is the cross-reactivity with other antigens to which the animals had been exposed at that time point, such as Tritrichomonas foetus (accession number: OHT10616) with an identity of 91 %, or Paramecium tetraurelia (accession number: XP_001436024) with an identity of 100%. Higher anti-rmFhCL1 lgG2 levels were induced at the chronic infection stage by the vaccinated group that showed some degree of protection. The epitope WHQWKRM (aa 21-27) that is contained in the pro-peptide of FhCLI (and FhCL3) was highly immunogenic following F. hepatica infection in both vaccinated and control animals in two separate trials. Additionally, the vaccinated group from Trial 1 , in which a reduction of fluke burden was seen, strongly recognised the aa regions 120-137, 145-155, 161-171 (CGSCWAFST, YMKNERTSISF, VDCSRPWGNNG). However, recognition of these peptides in Trial 2, where no protection was demonstrated, was not so strong. Hence, these two regions of the protein, together with the induction of specific lgG2, could potentially be useful targets for improving vaccine antigen synthesis strategies. The present study characterises linear B-cell epitopes recognised within the FhCLI protein by sera from F. hepatica-^'mfecled and/or vaccinated cattle from two independent trials. Results showed that all F. hepatica infected animals recognised the region 19-31 of FhCLI , included as part of the propeptide. Vaccinated animals that showed fluke burden reduction recognised the regions 120-137, 145-155, 161-171 of FhCLI , which were not recognised by non-protected animals. This together with the high production of specific lgG2 in animals showing antigen efficacy suggest important targets for antigen development. The present study demonstrates that the epitope WHQWKRM from FhCLI is highly immunogenic in F. hepatica infected cattle, epitopes within the region 120-171 of FhCLI are recognised by protected calves, and production of specific lgG2 at the chronic infection is produced by protected calves.

Example 4 B-cell epitope mapping on cattle immunized with FhCL1

In addition to studying the epitopes recognized by cattle after experimental challenge, B-cell epitope mapping studies have also been conducted on cattle immunized with FhCL1 as described herein, but challenged by natural exposure to fluke infection on pasture, instead of by experimental challenge. Using two different adjuvants, 47.2 % and 49.2 % protection was found in the two groups, as described in Golden et al., 2010 (Vaccine. 2010 Aug 2;28(34):5551-7. doi:

10.1016/j. vaccine.2010.06.039). In this example, infection was acquired through animals grazing contaminated pastures over 13 weeks, beginning three weeks after a second dose of vaccine. Total mean fluke number was 16.4 +/1 5.1 1 in the non-vaccinated control animals and 8.67 +/1 2.89 and 8.22 +/- 4.43, respectively, in each of the two vaccinated groups. These numbers are more typical of the kinetics and levels of liver fluke infection acquired by cattle under normal grazing conditions, than typical burdens following experimental infection, which are of the order of 5-10 times higher. B-cell epitope recognition was measured, using the same biotinylated peptides and methods as described herein, in both control and vaccinated animals at eight weeks post-infection, and compared the results with those obtained previously in experimentally-infected animals.

As with experimentally-infected animals, it was found that both naturally-infected controls and vaccinates in this example recognized peptides within the pro-peptide region of the FhCL1 molecule. The degree of recognition was greater in the vaccinated animals, reflecting the comparatively lower antigenic stimulation of the natural/trickle infected control group. Vaccinated animals recognized the core motif of WHQWKRM represented by peptides 21-27 within the pro-peptide of the FhCL1 molecule, but also in addition regions flanking this motif including in total peptides 1-43.

Peptides 120-137 were also recognized specifically in the two vaccinated and protected groups. In addition, Peptides 102-1 15 were also strongly recognized by these groups.

Conversely, recognition of peptides 145-155, seen previously, was not so clear in the trickle-infected groups, except for the peptides 144-145. Peptide 159 was also strongly recognized.

Example 5

T-cell Epitopes

In order to generate antibody responses that are effective in protecting animals against liver fluke challenge, in terms of isotype, avidity, and memory responses, vaccines need to include specific T- cell epitopes in addition to B-cell epitopes. T-cell epitopes are contiguous sequences of amino-acids from the secondary structure of an antigenic protein which, following intra-cellular enzymatic digestion, bind to MHC molecules on the surface of antigen-presenting cells and are cross-linked to specific T-cell epitopes on T-lymphocytes. Unlike B-cell epitopes, and because of the need for intra- cellular processing, T-cell epitopes are not conformational/represented by dis-continuous stretches of amino-acids. They are thus amenable to identification through the use of synthetic peptides.

Overlapping synthetic peptides representing the entire sequence of FhCL1 , and that of FhCL3 were used to identify T-cell epitopes within these sequences. In order to do this, peptides, 20aa long, with an overlap of 10 aa, across the sequences were prepared. These peptides were then incubated with leucocytes obtained from hepatic lymph nodes from animals infected with F. hepatica, at 14 weeks post-infection. The mixtures of lymphocytes and antigen-presenting cells in these preparations would be expected to be enriched with cells having receptors specific to antigens of the parasite, being situated in the lymph node draining the site of infection. Lymphocytes bearing receptors binding to peptides containing or constituting T-cell epitopes will be stimulated to proliferate following binding. Lymphocyte proliferation can be detected thereafter by incorporating a modified constituent of DNA, 5-bromo-2-deoxyuridine (BrdU) into cultures, and measuring BrdU incorporation through a colorimetric reaction. Lymphocyte binding to specific peptides can additionally or alternatively be identified by measuring the production of specific cytokines in the culture medium of lymphocytes and APCs incubated as above.

The procedure used lymph node (LN) cells from infected animals which had been preserved in liquid nitrogen. Specifically, on day 1 , LN cells were thawed from storage in liquid nitrogen. Cold RPMI was added to vials of cells and cells were then carefully transferred to 50ml Falcon flasks and spun at 300g for 10 minutes (brake off). The cells were re-suspended and fresh medium was added to a total of 27ml. This medium was then carefully and slowly added to 18ml of Ficoll, layering the cell mixture on top of the Ficoll. This mixture was then spun at 400g for 10 minutes (brake off). The cloudy interface containing the cells was then removed with a Pasteur pipette and added to 7ml of medium. This was then spun at 300g (brake on) and the pellet re-suspended in full medium containing fetal calf serum, glutamine, pen-strep, beta mercaptoethanol and IL-2, counted and plated at 200,000 cells per well in a 96-well microtitre plate. On day 2, peptides were added (4ul/well) and on day 4 BrdU (Roche diagnostics cat no. 1 1 647 229 001 ) was added. On day 5 after 24 hours with BrdU, a colorimetric cell-proliferation assay (Sigma-Alrich) was carried out according to the manufacturer's protocol.

Using culture supernatants from the same cells, IFNy levels were measured using the bovigam kit (Bovigam™ TB Kit), Thermo-Fisher Lyd. It was found that overlapping peptides 6-1 1 from the Fh CL1 sequence, representing amino-acids 42 - 1 1 1 stimulated both cell proliferation and IFN-gamma production from cell supernatants from F. hepatica infected animals, indicating that T-cell epitopes are located within this region of the FhCL1 protein. Note that this region is immediately down-stream of an area of the protein previously shown to contain strongly-recognised B-cell epitopes. Similarly, we found that peptides 28-30, representing amino-acids 262-300 of the secondary sequence of Fh, CL1 , similarly, stimulated both cell proliferation and and IFN-gamma production. Part of this latter sequence also constitutes a linear B-cell epitope. Also disclosed is a Fasciola antigen comprising a polypeptide, wherein the polypeptide comprises an amino acid sequence selected from the following amino acid sequences:

Entry Protein Peptide Name Nterm peptide Cterm

1 FhCL1 Fh.001 H MRLFVLAVLTVGVLGSNDDL OH

2 FhCL1 Fh.002 H VGVLGSNDDLWHQWKRMYNK OH

3 FhCL1 Fh.003 H WHQWKRMYNKEYNGADDQHR OH

4 FhCL1 Fh.004 H EYNGADDQHRRNIWEKNVKH OH

5 FhCL1 Fh.005 H RNIWEKNVKHIQEHNLRHDL OH

6 FhCL1 Fh.006 H IQEHNLRHDLGLVTYTLGLN OH

7 FhCL1 Fh.007 H GLVTYTLGLNQFTDMTFEEF OH

8 FhCL1 Fh.008 H QFTDMTFEEFKAKYLTEMSR OH

9 FhCL1 Fh.009 H KAKYLTEMSRASDILSHGVP OH

10 FhCL1 Fh.010 H ASDILSHGVPYEANNRAVPD OH

1 1 FhCL1 Fh.01 1 H YEANNRAVPDKIDWRESGYV OH

12 FhCL1 Fh.012 H KIDWRESGYVTEVKDQGNCG OH

13 FhCL1 Fh.013 H TEVKDQGNCGSCWAFSTTGT OH

14 FhCL1 Fh.014 H SCW AFSTTGTM EGQYM KN E R OH

15 FhCL1 Fh.015 H MEGQYMKNERTSISFSEQQL OH

16 FhCL1 Fh.016 H TSISFSEQQLVDCSRPWGNN OH

o 17 FhCL1 Fh.017 H VDCSRPWGNNGCGGGLMENA OH

18 FhCL1 Fh.018 H GCGGGLMENAYQYLKQFGLE OH

CD 19 FhCL1 Fh.019 H YQYLKQFGLETESSYPYTAV OH

20 FhCL1 Fh.020 H TESSYPYTAVEGQCRYNKQL OH

21 FhCL1 Fh.021 H EGQCRYNKQ LGVAKVTG F YT OH

22 FhCL1 Fh.022 H GVAKVTGFYTVHSGSEVELK OH co

o 23 FhCL1 Fh.023 H VHSGSEVELKNLVGAEGPAA OH co

CD 24 FhCL1 Fh.024 H NLVGAEGPAAVAVDVESDFM OH

_C0 25 FhCL1 Fh.025 H VAVDVESDFMMYRSGIYQSQ OH

O

Ό

in 26 FhCL1 Fh.026 H MYRSGIYQSQTCSPLRVNHA OH co

27 FhCL1 Fh.027 H TCSPLRVNHAVLAVGYGTQG OH

m

CD

;g 28 FhCL1 Fh.028 H VLAVGYGTQGGTDYWIVKNS OH

CD 29 FhCL1 Fh.029 H GTDYWIVKNSWGLSWGERGY OH

30 FhCL1 Fh.030 H WGLSWGERGYIRMVRNRGNM OH

_C0 31 FhCL1 Fh.031 H IRMVRNRGNMCGIASLASLP OH

CD

> 32 FhCL1 Fh.032 H RGNMCGIASLASLPMVARFP OH

o 33 FhCL3 Fh.033 H WHQWKRMYNKEYNGADDEHR OH

34 FhCL3 Fh.034 H EYNGADDEHRRNIWEKNVKH OH

35 FhCL3 Fh.035 H RNIWEKNVKHIEEHNLRHDR OH

36 FhCL3 Fh.036 H IEEHNLRHDRGLVTYKLGLN OH

37 FhCL3 Fh.037 H GLVTYKLGLNQFTDLTFEEF OH

38 FhCL3 Fh.038 H QFTDLTFEEFKAKYLMEMSL OH

39 FhCL3 Fh.039 H KAKYLMEMSLVSESLSDGIS OH

40 FhCL3 Fh.040 H VSESLSDGISYEAEGNDVPA OH

41 FhCL3 Fh.041 H YEAEGNDVPASVDWREYGYV OH

42 FhCL3 Fh.042 H SVDWREYGYVTEVKDQGQCG OH

43 FhCL3 Fh.043 H TEVKDQGQCGSCWAFSAVGA OH

44 FhCL3 Fh.044 H SCWAFSAVGAIEGQYLRKFQ OH

45 FhCL3 Fh.045 H IEGQYLRKFQNQTLFSEQQL OH

46 FhCL3 Fh.046 H NQTLFSEQQLVDCTRRFGNH OH

O 47 FhCL3 Fh.047 H VDCTRRFGNHGCGGGWMENA OH

48 FhCL3 Fh.048 H GCGGGWMENAYKYLKNSGLE OH

49 FhCL3 Fh.049 H YKYLKNSGLETASDYPYQGW OH

50 FhCL3 Fh.050 H TASDYPYQGWEYQCQYRKEL OH

51 FhCL3 Fh.051 H EYQCQYRKELGVAKVTGAYT OH co

O 52 FhCL3 Fh.052 H GVAKVTGAYTVHSGDEMKLM OH co

o 53 FhCL3 Fh.053 H VHSGDEMKLMQMVGREGPAA OH co

54 FhCL3 Fh.054 H QMVGREGPAAVAVDAQSDFY OH

55 FhCL3 Fh.055 H VAVDAQSDFYMYESGIFQSQ OH

Ό 56 FhCL3 Fh.056 H MYESGIFQSQTCTSRSVTHA OH co

57 FhCL3 Fh.057 H TCTSRSVTHAVLAVGYGTES OH

58 FhCL3 Fh.058 H VLAVGYGTESGTDYWILKNS OH

59 FhCL3 Fh.059 H GTDYWILKNSWGKWWGEDGY OH

60 FhCL3 Fh.060 H WG KWWG EDG YMRF ARN RN N M OH

TO 61 FhCL3 Fh.061 H MRFARNRNNMCAIASVASVP OH

62 FhCL3 Fh.062 H RNNMCAIASVASVPMVERFP OH

As outlined above, in order to induce effective, protective responses of long duration, and effective memory responses, vaccines against complex pathogens such as F. hepatica can include T-cell epitopes in addition to B-cell epitopes.

An effective vaccine can be designed by covalently coupling, by solid-phase synthesis, several copies of the T-cell epitopes identified here, to known B-cell epitopes, and using these, together with appropriate adjuvants, for parenteral administration as vaccines. Epitopes which are discontiguous in the CL1/CL3 sequence can be combined and linked covalently using solid-phase synthesis and/or by coupling to expogenous proteins such as ovalbumin, or proteins from other Th1 -inducers, for example surface proteins of T. gondii. As is well known in the art, peptide function (whether to trigger T-cell or B-cell pathways or both) can be improved by various modifications to the antigen or epitope structures. See generally M.

Skwarczynski et al., "Peptide based synthetic vaccines", Chem. Sci., 2016, vol 7(2) , pp. 842-854. In regard of B-cell epitopes where maintaining natural conformation may improve immune response, such can often be achieved by incorporation of the peptide into longer polypeptide (polymer) structures, containing optionally multiple different B- or T-cell eptitopes. Alternatively, smaller

(monomeric epitope) peptides can simply be flanked with additional chemical groups. For example, amino acid residues from the GCN4 protein have been attached to heterologous peptides to facilitate and maintain alpha helical structure therein (J.Cooper et al., Mol. Immunol., 1997, vol 34, pp. 433- 440).

Various other strategies are recognized in order to maintain antigenic structure of peptide epitopes and to prevent enzymatic degradation thereof once administered as a vaccine. For example, The N- terminus of an antigenic peptide can be acetylated, the C-terminus can be amidated, or the entire peptide can be cyclized. Small peptide antigens can also be stabilized for enhanced metabolic stability or antigenic efficacy by covalent addition of D-ala, L-cyclohexylalanine, or aminocaproic acid residues, or be"stapled" to entirely unrelated foreign epitopes( for example , use of HIV-1 gp41 protein epitopes, and see generally G. Bird et al., 2014, Nat. Struct. Mol. Biol. vol. 21 , pp. 1058- 1067)

The peptide vaccines of the invention can also be adapted to include various other substances that facilitate immune response. For example, it is well known that peptide antigens (containing one or more B- cell epitopes and optionally one or more T- cell epitopes) can be assembled as lipid -peptide conjugates, optionally as nanoparticles, but in any case wherein the lipid functions as an in situ adjuvant. Use of Pam2Cys and LCP lipid components is well described, see for example, W. Zeng et. al., 2015, Vaccine, vol. 33., pp. 3626-3532; and F. Ahmad et. al., Bioorg. Med. Chem., 2015, vol. 23, pp. 1307-1312.

Accordingly, the present invention provides identification of specific linear peptide epitopes which may have specific uses as part of a vaccine to protect ruminants against liver fluke infection. The invention can also find utility as a vaccine comprising multivalent antigens against other pathogens. In each case, a single antigen regime could be used to prevent, rather than treat (liver fluke) infection.

Claims

1. A Fasciola hepatica antigen comprising a polypeptide, wherein the polypeptide comprises the amino acid sequence WHQWKRM, or a fragment thereof.

2. A Fasciola hepatica antigen according to Claim 1 , wherein the polypeptide comprises the amino acid sequence DLWHQWKRMYNKE, or a fragment thereof.

3. A Fasciola hepatica antigen according to Claim 1 or 2, wherein the Fasciola hepatica antigen further comprises a polypeptide, wherein the polypeptide comprises the amino acid sequence YMKNERTSISF, or a fragment thereof.

4. A Fasciola hepatica antigen according to any one of Claims 1-3, wherein the Fasciola

hepatica antigen further comprises a polypeptide, wherein the polypeptide comprises the amino acid sequence VKNSWGLSWGE, or a fragment thereof.

5. A Fasciola hepatica antigen according to Claim 3 or 4, wherein the Fasciola hepatica antigen comprises a single fusion polypeptide.

A Fasciola hepatica antigen according to Claim 1 or 2, wherein the Fasciola hepatica antigen further comprises a polypeptide, wherein the polypeptide comprises at least one amino acid sequence selected from CGSCWAFST, YMKNERTSISF, and VDCSRPWGNNG, or a fragment each thereof.

A Fasciola hepatica antigen according to Claim 1 or 2, wherein the Fasciola hepatica antigen further comprises a polypeptide, wherein the polypeptide comprises at least one amino acid sequence selected from HRRNIWEKN and MVRNRGNMC, or a fragment each thereof.

8. A Fasciola hepatica antigen according to Claim 1 or 2, wherein the Fasciola hepatica antigen further comprises a polypeptide, wherein the polypeptide comprises at least one amino acid sequence selected from DYWIVKNSWGLSWGERGY and SLPMVARFP, or a fragment each thereof.

9. A Fasciola hepatica vaccine comprising an antigen according to any one of Claims 1-8.

10. A Fasciola hepatica vaccine according to Claim 9, wherein the Fasciola hepatica vaccine comprises the antigen, and a pharmaceutically acceptable adjuvant.

1 1. A Fasciola hepatica vaccine according to Claim 10, wherein the Fasciola hepatica vaccine comprises 100 g of the antigen according to the first aspect of the present invention for each 1 ml_ of the pharmaceutically acceptable adjuvant.

12. A Fasciola hepatica vaccine according to any one of Claims 9-1 1 , wherein the Fasciola hepatica vaccine is administered parenterally.

13. A Fasciola hepatica vaccine according to any one of Claims 9-1 1 , wherein the Fasciola hepatica vaccine is administered at least twice, wherein each administration is at least four weeks apart.

14. An antigen according to any one of Claims 1-8 for use as a vaccine against Fasciola

hepatica infection.

15. Use of an antigen according to any one of Claims 1-8 in the manufacture of a vaccine for vaccinating a subject against Fasciola hepatica infection.