Nematode vaccine

Abstract

The present invention relates to a vaccine comprising a recombinant antigen derived from a parasitic nematode, Stronchus annulata, which will produce an immune response in cultured and wild ruminants susceptible to or prone to being infected with one or more nematode helminth species.

Description

Technical Field

[0001] This invention relates to a vaccine comprising an antigen that stimulates or enhances acquired immunity against parasitic nematode infection, particularly in domesticated or wild ruminants such as sheep, cattle, goats, deer, buffalo, bison, camels, llamas, etc. Background Technology

[0002] Parasitic nematode infections are one of the biggest health problems in farmed ruminants worldwide. There are many reasons why parasitic nematode infections are harmful to their host animals. For example, they steal the host's food, damage internal tissues and organs, leading to anemia, weight loss, diarrhea, dehydration, and loss of appetite. Such parasitic infections cause costly production losses; if left untreated, animals will die, resulting in further economic losses for farmers.

[0003] Currently, farmers rely on anthelmintics (such as benzimidazoles, levamisole, methamidophos, mononitrate, oxifenesole, or ivermectin) to control parasitic nematodes; however, resistance to one or more of these agents is now widespread. In fact, a recent industry-funded survey in New Zealand found that 64% of sheep farms and 94% of beef cattle farms now have parasites resistant to at least one of these anthelmintics.

[0004] It is estimated that this resistance costs New Zealand's livestock industry approximately $700 million annually in terms of control and productivity losses.

[0005] Alternative approaches to controlling the impact of parasite infections in farms have been proposed, including changes in grazing management, the use of fungi to trap nematodes, dietary supplements, breeding animals with host resistance, and vaccination.

[0006] Attempts to develop recombinant vaccines against parasitic nematodes have achieved limited success, and to date, there are no commercially available recombinant vaccines that can be used against any nematode parasite. However, the development of such a vaccine is seen by the industry as a solution to the problem of drug resistance.

[0007] One target of protective vaccines is essential worm metabolic enzymes. Parasitic nematode larvae grow rapidly, and adult worms lay a large number of eggs; both require highly active nitrogen and energy metabolism. Essential worm enzymes involved in these pathways and absent in the host are therefore potential targets for parasite control. Essential enzymes involved in hemodigestion and other pathways crucial to the worm's life cycle can also be targeted, either individually or as multiple targets.

[0008] Vaccination with antigens containing such metabolic enzymes can theoretically generate circulating host antibodies that bind to and disrupt the function of essential parasite metabolic enzymes, potentially leading to a significant reduction in worm load and fecal egg count (FEC).

[0009] A vaccine (Barbervax) based on an extract of *H. contortus* has recently been marketed in Australia. While the vaccine is effective in protecting sheep against infection, its use presents several real and potential challenges. First, the vaccine does not provide any long-term protection against infection, thus requiring multiple administrations during the risk period. Second, there is a significant risk of degradation if the natural antigen is exposed to high temperatures in the wild. Third, because the antigen is extracted from worms derived from donor sheep, there is a potential risk of cross-contamination with infectious agents such as viruses.

[0010] Recombinant antigens can overcome these problems. A group of researchers successfully tested a *T. circumcincta* vaccine comprising eight recombinant proteins (APY-1; MEP-1; ES20; CF-1; TGH-2; ASP-1; MIF-1; and SAA-1). These recombinant proteins were carefully selected using a three-step method. First, antigens excreted / secreted at key stages of worm growth were selected; then, SAA-1, an immunogenic homolog of a protective antigen from *Hookworm*, was selected; finally, a potential immunosuppressive molecule antigen (WO2013 / 117912) was selected. While the antigen containing all eight recombinant proteins showed reduced worm load and FEC, similar to the effects of vaccination with a detergent extract of *T. circumcincta*, a later publication indicated that the antibody responses induced by these recombinant antigens did not reach optimal levels due to structural and / or post-translational modifications between the natural antigen and its recombinant form (Longhi-Browne, 2014).

[0011] Attempts to prepare commercial vaccines from recombinant antigens have so far failed due to such problems in recombinant protein technology. Therefore, there is a need in the art to provide such recombinant vaccines.

[0012] The purpose of this invention is to overcome this need in some way and / or to provide the public with useful options. Summary of the Invention

[0013] This invention relates to a vaccine comprising a recombinant antigen derived from the parasitic nematode *Teladorsagia circumcincta*, which will induce an immune response in farmed and wild ruminants susceptible to or prone to infection by one or more nematode species. The recombinant antigen used in this invention is conserved among nematode species, therefore the vaccine will provide protection against multiple types of nematodes.

[0014] In a first embodiment, the present invention provides a composition or vaccine composition comprising recombinant *Nematoda circinatesii* antigen:

[0015] (i) Enolase (EN);

[0016] (ii) Arginine kinase (AK);

[0017] (iii) Ornithine decarboxylase (ODC);

[0018] (iv) Seryl tRNA synthetase (SRS-2);

[0019] (v) Macrophage migration inhibitory factor 2 (MIF-2);

[0020] (vi) aldolases; and

[0021] (vii) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH),

[0022] Or its antigen fragments, and veterinary-acceptable carriers or diluents.

[0023] The composition or vaccine composition may further include one or more recombinant *Nematoda* antigens selected from the group consisting of:

[0024] (viii) Isocitrate lyase (ICL);

[0025] (ix) malate synthase;

[0026] (x) chitinase (Cht); and

[0027] (xi) Glutathione S-transferase (GST)

[0028] Preferably, the composition or vaccine composition includes at least one, at least two, at least three, or at least four of the antigens (iv) to (xi) described above.

[0029] In a second embodiment, the present invention provides a composition or vaccine composition comprising recombinant antigen of *Nematoda circinate*:

[0030] (i) Enolase (EN);

[0031] (ii) Arginine kinase (AK);

[0032] (iii) Ornithine decarboxylase (ODC);

[0033] (iv) Seryl tRNA synthetase (SRS-2);

[0034] (v) Macrophage migration inhibitory factor 2 (MIF-2);

[0035] (vi) Aldolase;

[0036] (vii) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH);

[0037] (viii) Isocitrate lyase (ICL);

[0038] (ix) malate synthase;

[0039] (x) chitinase (Cht); and

[0040] (xi) Glutathione S-transferase (GST)

[0041] Or its antigen fragments, and veterinary-acceptable carriers or diluents.

[0042] The composition or vaccine composition may further include adjuvants such as: alum, Quil A, Freund's complete adjuvant, Freund's incomplete adjuvant, lipopolysaccharide, monophospholipase A, montanide, lipovant, bacterial flagella protein, adjuvant 65, γ-inulin, algammulin, imiquimod, gademod, murimyl dipeptide, etc.

[0043] The composition or vaccine composition may further include a carrier, such as:

[0044] Chitosan-based sustained-release compounds (sol-gels), hollow mesoporous silica nanoparticles (HMSN), poly(d,l-lactide-co-glycolic acid) (PGC) nanoparticles, poly(d,l-l-lactic acid-co-glycolic acid) (PGCA) nanoparticles, liposomes, virions, and cochleate delivery vehicles, etc.

[0045] In a third embodiment, the present invention provides a method for reducing parasitic nematode load in farmed or wild ruminants, the method comprising administering an effective amount of the composition or vaccine composition of the present invention to the ruminant at one or more times, thereby measuring the reduction in parasitic nematode load by a decrease in fecal egg count (FEC) and / or an increase in the excretion of larvae and / or adult nematodes.

[0046] In a fourth embodiment, the present invention provides a method for inducing an immune response in farmed or wild ruminants to treat or protect the animals from parasitic nematode infection, the method comprising administering an effective amount of the composition or vaccine composition of the present invention to the animals at one or more times, wherein the induction of the immune response is measured by the presence of protective antibodies against one or more specific antigens present in the composition or vaccine composition.

[0047] In a fifth embodiment, the present invention provides a method for stimulating or enhancing acquired immunity in farmed or wild ruminants to treat or protect the animals from parasitic nematode infection, the method comprising administering an effective amount of the composition or vaccine composition of the present invention to the animal at one or more times, wherein the stimulation or enhancement of acquired immunity is measured by one or more of the following: the presence of protective antibodies against one or more specific antigens present in the composition or vaccine composition; elevated cytokine levels; reduced FEC; and / or the shedding of larvae and / or adult nematodes.

[0048] In a sixth embodiment, the present invention provides a method for treating or preventing nematode infection in farmed or wild ruminants, comprising administering an effective amount of the composition or vaccine composition to the animal.

[0049] In a seventh embodiment, the present invention provides the use of the following recombinant *C. annularis* antigens in the preparation of compositions or vaccine compositions for reducing nematode-parasitic worm load in farmed or wild ruminants: (i) enolase (EN), (ii) arginine kinase (AK), (iii) ornithine decarboxylase (ODC), (iv) serine tRNA synthetase (SRS-2), (v) macrophage migration inhibitory factor 2 (MIF-2), (vi) aldolase, and (vii) glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or antigenic fragments thereof.

[0050] In an eighth embodiment, the present invention provides the use of the following recombinant *C. annularis* antigens in the preparation of compositions or vaccine compositions for stimulating or enhancing acquired immunity in farmed or wild ruminants to treat or protect said animals from parasitic nematode infection: (i) enolase (EN), (ii) arginine kinase (AK), (iii) ornithine decarboxylase (ODC), (iv) serine tRNA synthetase (SRS-2), (v) macrophage migration inhibitory factor 2 (MIF-2), (vi) aldolase, and (vii) glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or antigenic fragments thereof.

[0051] In a ninth embodiment, the present invention provides the use of the following recombinant Haemaphysema contortus antigens in the preparation of compositions or vaccine compositions for the treatment or prevention of nematode infection in farmed or wild ruminants: (i) enolase (EN), (ii) arginine kinase (AK), (iii) ornithine decarboxylase (ODC), (iv) serine tRNA synthetase (SRS-2), (v) macrophage migration inhibitory factor 2 (MIF-2), (vi) aldolase, and (vii) glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or antigenic fragments thereof.

[0052] In a tenth embodiment, the present invention provides the use of the following recombinant *C. annularis* antigens in the preparation of compositions or vaccine compositions for inducing an immune response in farmed or wild ruminants to treat or protect said animals from parasitic nematode infection: (i) enolase (EN), (ii) arginine kinase (AK), (iii) ornithine decarboxylase (ODC), (iv) serine tRNA synthetase (SRS-2), (v) macrophage migration inhibitory factor 2 (MIF-2), (vi) aldolase, and (vii) glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or antigenic fragments thereof.

[0053] The compositions or vaccine compositions used in these embodiments of the invention may further include one or more of the antigens (viii) to (xi) described above.

[0054] Domesticated or wild ruminants are selected from the group consisting of: sheep, cattle, goats, deer, buffalo, bison, camels, llamas, etc. Domesticated or wild ruminants are preferably young animals under one year old, i.e., lambs, calves, goats, etc. On the one hand, domesticated or wild ruminants are less than 6 months old. On the other hand, domesticated or wild ruminants are at least 3 months old.

[0055] Parasitic nematodes that can be treated by the method of this invention include *Trichostrongylus colubriformis*, *Haemonchus contortus*, *Haemonchus placei*, *Ostertagia (Teladorsagia) circumcincta*, *Cooperia curticei*, *Nematodirus spathiger*, *Trichostrongylus axi*, *Trichostrongylus vitrini*, *Ostertagia ostertagia*, *Cooperia oncophera*, *Nematodirus brasiliensis*, *Dictyocaulus eckerti*, *Strongylus vulgaris*, and *Toxascaris*. vitolorum, Nematodirus filicollis, Ashworthius sidemi, Mecistocirrus digitatus, Bunostomum trigonocephalum, Trichuris discolor, Toxacara vitulorum, etc. Attached Figure Description

[0056] The invention will now be described in more detail with reference to the accompanying drawings, in which:

[0057] Figure 1a -c: indicates the metabolic enzyme AK ( Figure 1a ), EN ( Figure 1b ) and ODC ( Figure 1c The homology of transnexacarp species is as follows:

[0058] Figure 1aComparisons of the predicted arginine kinase amino acid sequences are shown, from members of the order Strongylida: *Haemaphysalis contortus* (Genebank accession number AFT82971), *Teladorsagia circumcincta* (AFT82970), *Necator americanus* (ETN81593), and *Ancylostoma ceylanicum* (EYC23758; EYC23757; EYB91576), and from members of the family Rhabditidae: *Caenorhabditis elegans* (CAB00062; NP509217; NP507054; CCD73398; CCD79398), and *Caenorhabditis brigandi*. briggsae)(CAP24981; CAP24932), Caenorhabditis brenneri)(EGT52941; EGT41918 and Caenorhabditis remanei(EFP12066; EFO86450; EFO82749);

[0059] Figure 1b Comparison of the predicted enolase amino acid sequences is shown, from members of the order Strongyloides: *Haemaphysalis contortus* (Genebank accession number AGC24386; ADK47524; CDJ96217), *Nematoda annularis* (derived from the *Nematoda annularis* genome sequence), *Lysimachia digitata* (BAN67669), *Ahedrus srilochetes* (EYB81234), *Angiostrongylus cantonensis* (AGO81688), *Ahedrus gracilis* (ETN80540), and members of the family Nematodae: *C. elegans* (NP495900; NP871916; NP001022349), *C. brygius* (EGT35078), *C. brygius* (CAP23453), and *C. roe* (EFO85696);

[0060] Figure 1cComparison of the predicted amino acid sequences of ornithine decarboxylases is shown, with sequences from members of the order Strongyloides: *Haemaphysalis contortus* (Genebank accession number AAC27893), *Nematoda annularis* (AGH70348), and *Ahedrosoma srilosum* (EYC11973; EYC11971; EYC11970), and members of the family Nematodae: *C. elegans* (P41931), *C. brigandri* (CAP36352), *C. brigandri* (EGT47038), and *C. roe* (EFP05480).

[0061] Figure 1a The alignment in -c was performed using the Muscle alignment option in Geneious 5.6.5 (Biomatters Ltd), and the Blosum 62 similarity matrix was used to identify 100% similar residues (shaded). The shared sequences shown are the most common residues with the least ambiguity;

[0062] Figure 2a -g: This shows the serum antibody response in sheep against seven recombinant antigens (7AgV) including AK, EN, MIF-2, SRS-2, GAPDH, ODC, and aldolase when Quil A is used as an adjuvant (G1 / G2) or when Montanide is used as an adjuvant (G3 / G4). Groups G2 and G4 are adjuvant-only groups, i.e., control groups.

[0063] Figure 3 The following is a comparison of salivary IgA responses in sheep treated with 7AgV, compared to the control, in two adjuvants (QuilA vs Montanide).

[0064] Figure 4 The total (male and female) worm load in sheep treated with 7AgV antigen is shown in two adjuvants (QuilA vs Montanide) compared to the control.

[0065] Figure 5 The figure shows the predicted mean fecal egg count (FEC) of sheep treated with 7AgV compared to the control group in two adjuvants (Quil A vs Montanide).

[0066] Figure 6 The following figures show the worm load in sheep treated with 7AgV, 8aAgV, 8bAgV, or 11AgV of Montanide adjuvant compared to the control group in the second sheep trial.

[0067] Figure 7aThe figure shows the mean FEC predictions of sheep treated with 7AgV, 8aAgV, 8bAgV, and 11AgV in Montanide adjuvant compared to the control group.

[0068] Figure 7b The results show the serum IgG levels in the mixed serum samples from the 11AgV group and the control group throughout the entire experiment.

[0069] Figure 8 This shows the IgG levels in serum samples from sheep treated with 11AgV after being challenged with susceptible and drug-resistant worms at a lower dose of antigen, compared to the control group in the third sheep trial.

[0070] Figure 9 The predicted mean fecal egg counts for the vaccine group and the control group are shown.

[0071] Figure 10 The total worm counts for the control and treatment groups are shown.

[0072] Figure 11 The figure shows the IgG levels in serum samples from calves treated with 11AgV, compared to the control group, in the first calf trial.

[0073] Figure 12 The average fecal egg counts for the vaccine group and the control group are shown.

[0074] Figure 13 The table shows the male, female, and total worm counts for the vaccine and control groups.

[0075] Figure 14 This shows the IgG levels in serum samples from deer treated with 11AgV, compared to the control group, in the first deer experiment.

[0076] Figure 15 The image shows the IgA levels in saliva samples from deer treated with 11AgV compared to the control group.

[0077] Figure 16 The figure shows the average fecal egg count for the vaccine group and the control group.

[0078] Figure 17 The total worm counts for the vaccine group and the control group are shown.

[0079] Figure 18 The following figure shows the anti-recombinant AK IgG levels in serum samples of deer treated with two adjuvants (Montadide vs QuilA / Sol gel) at 3AgV, 7AgV, and 11AgV, compared with the control group, in the second calf trial.

[0080] Figure 19 The table shows the levels of anti-recombinant GAPDH IgG in serum samples of deer treated with 3AgV, 7AgV, and 11AgV of the two adjuvants compared to the control group.

[0081] Figure 20 The figure shows the average fecal egg count for the vaccine group and the control group.

[0082] Figure 21 The figure shows the average body weight of calves in the vaccine group and the control group;

[0083] Figure 22 The total worm counts for the control and vaccine groups are shown.

[0084] Figure 23 The total stalled L4 counts are shown for the control and vaccine groups.

[0085] Figure 24 The figure shows the predicted mean fecal egg count (FEC) of calves treated with 7AgV and 11AgV compared with the control group in the third calf trial.

[0086] Figure 25 The total worm counts are shown for the vaccine group, control group, and treatment group.

[0087] Figure 26 The total stalled L4 counts for the control and vaccine groups are shown; and

[0088] Figure 27 The figure shows the IgG levels in serum samples tested for all 11 recombinant antigens compared to the control group. Detailed Implementation

[0089] Nematode infections in farmed and wild ruminants are a major global problem, and *Teladorsagia circumcincta* is a leading cause of parasitic gastroenteritis in small ruminants in cool temperate regions.

[0090] There are currently no commercially available protein vaccines against *Nematodea annularis*.

[0091] This invention provides for the first time an effective vaccine against nematode infection in farmed and wild ruminants, comprising a mixture of recombinant antigens.

[0092] The recombinant antigen corresponds to a metabolic enzyme in the annular nematode, which is essential for the survival of the worm.

[0093] The term “antigen” as used in this article refers to a molecule that triggers an immune response involving antibody production.

[0094] Without being bound by theory, it is assumed that antibodies generated by immunization using the vaccine composition of the present invention function in two main ways. First, for nematodes that cause intestinal wall damage, such as *Nematoda circinate*, the worms are continuously immersed in inflammatory exudate, some of which they ingest. Therefore, it is assumed that the worms will ingest antibodies. The ingested antibodies will bind to target antigens, in this case, essential metabolic enzymes present in the nematode's intestinal wall or secreted into the intestinal lumen, thereby inhibiting their activity, causing the worms to weaken, and then clearing these worms from the host animal's intestine by peristalsis. Other blood-sucking nematodes, such as *Haemonchus*, will also ingest antibodies from the host's bloodstream during feeding. Additionally, the antibodies generated by the vaccine composition of the present invention include antibodies against antigens found in the worm's somatic tissues and / or secretions / excretions that affect the worm's ability to survive in the host's intestine. The worms become weakened and are expelled.

[0095] The efficacy of the vaccine composition of the present invention can be measured by an increase in the excretion of larvae and / or adult nematodes, and / or a decrease in fecal egg count (FEC), as well as the presence of one or more protective antibodies targeted by the antigens present in the vaccine composition.

[0096] The antigens present in the compositions or vaccine compositions of the present invention include (i) recombinant *C. annularis* enolase (EN), (ii) recombinant *C. annularis* arginine kinase (AK), (iii) recombinant *C. annularis* ornithine decarboxylase (ODC), (iv) recombinant *C. annularis* aldolase; (v) recombinant *C. annularis* serine tRNA synthetase (SRS-2), (vi) recombinant *C. annularis* macrophage migration inhibitory factor 2 (MIF-2), and (vii) recombinant *C. annularis* glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or antigen fragments thereof.

[0097] Enolases are enzymes involved in the glycolytic pathway and are secretory enzymes that form part of the excretory / secretive (ES) complex. Enolases play a crucial role in the metabolism of nematodes (Han et al., 2012). Arginine kinases are believed to be present in the intestinal lining cells of parasites and play a vital role in maintaining ATP levels. Ornithine decarboxylase catalyzes the conversion of ornithine to putrescine and is the rate-limiting enzyme in polyamine biosynthesis. Inhibition of ODC leads to loss of cell proliferation. Aldolases and GAPDH are essential enzymes involved in energy metabolism. Seryl-tRNA synthetase is involved in translation, and macrophage migration inhibitory factor 2 is involved in innate and acquired nematode immunity. Nematode chitinases are induced during helper responses and contribute to nematode immunity. Isocitrate lyase and malate synthase are key glycolytic enzymes. Glutathione transferase protects parasites from oxidative stress and the toxic and carcinogenic effects of endogenous substances. The inhibitory effect of antibodies generated in response to vaccination with the vaccine of the present invention on these enzymes has for the first time shown a significant reduction in fecal egg count (FEC), worm load, and other symptoms of annular dorsal nematode infection in sheep.

[0098] Enolase and arginine kinase are highly conserved enzymes among transnephrite worm species; therefore, the vaccine of this invention is expected to be effective against a large number of nematodes infecting farmed and wild ruminants, including *Bunostomum*, *Strongylus*, *Trichostrongylus*, *Haemonchus*, *Teladorsagia (Ostertagia)*, *Toxascaris*, *Nematodirus*, *Trichuris*, *Dictyocaulus*, *Toxocara*, *Strongyloides*, *Cooperia*, *Ashworthius*, and *Mecistrocirrus*.

[0099] The degree of homology among EN, AK, ODC, SRS-2, MIF-2, aldolase, GAPDH, ICL, malate synthase, Cht, and GST transnexacarp worm species is shown in Figures 1a-1c The results are shown in Table 1 below.

[0100] As understood by those skilled in the art, it is anticipated that, due to the high conservation of EN and AK among species, vaccines containing these annular dorsal nematode antigens will produce antibodies that recognize the EN and AK of other nematode species and block enzyme activity in the same manner, thereby adversely affecting the survival of the worms.

[0101] Table 1 lists the common amino acid residues found in *T. circumcincta* arginine kinase (T. circumcincta AK; GenBank accession number JX422017), *T. circumcincta* enolase (GenBank accession number KX452941), and *T. circumcincta* ornithine decarboxylase (GenBank accession number KC484698).

[0102] % homology of *C. annularis*

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109] Examples of specific nematode species for which the vaccine of the present invention can be used include: *Trichoderma serpentina*, *Haemaphysalis contortus*, *Haemaphysalis burgdorferi*, *Osteria annularis*, *Coprea coccinea*, *Trichoderma ehrlich*, *Trichoderma hyaline*, *Osteria ostreatus*, *Cooperia oncophera*, *Coperia brasiliensis*, *Dictyocaulus viviparus*, *Dictyocaulus deer*, *Strongyloides scabra*, *Toxascaris vitolorum*, *Strongyloides scabra*, *Ashworthius sidemi*, *Longyloides digitata*, *Nematoda evodia*, *Nematoda evodia*, *Trichoderma variegata*, *Toxascaris vitolorum*, *Strongyloides scabra*, *Ashworthius sidemi*, *Nematoda evodia*, *Nematoda evodia*, *Nematoda evodia*, *Toxocara caerulea*, etc.

[0110] Preferably, the parasitic nematode is *Nematoda circinata*.

[0111] In addition to (i) EN, (ii) AK, (iii) ODC, (iv) SRS-2, (v) MIF-2, (vi) aldolase, and (vii) GAPDH, the compositions or vaccine compositions of the present invention may further comprise one or more recombinant *Nematoda* antigens selected from the group consisting of:

[0112] (viii) Isocitrate lyase (ICL);

[0113] (ix) malate synthase;

[0114] (x) chitinase (CHT); and

[0115] (xi) Glutathione S-transferase (GST)

[0116] Or its antigen fragments.

[0117] Preferably, the composition or vaccine composition of the present invention further comprises at least one, at least two, at least three, or all at least four of the above-mentioned antigens (viii) to (xi).

[0118] % sequence identity of EN, EK, and ODC transnexacarp worm species is shown in Figures 1a-1c The homology of the remaining antigens (iv) to (xi) is shown in Table 1 above.

[0119] The compositions or vaccine compositions of the present invention having additional antigens other than (i) EN, (ii) AK, (iii) ODC, (iv) SRS-2, (v) MIF-2, (vi) aldolase and (vii) GAPDH are expected to result in a stronger immunogenic response and improved reduction in FEC and worm load due to the additive effect of at least each individual antigen.

[0120] However, both the 7-antigen vaccine and the 11-antigen vaccine generally resulted in similar reductions in FEC in sheep and deer (72% reduction in sheep treated with 7-AgV and 50% in sheep treated with 11-AgV, see Figure 7); and a 70% reduction in sheep treated with 11-AgV (see Figure 7). Figure 9 ); while FEC in deer decreased by 49% (see Figure 16 However, a 50-70% reduction in FEC is still considered a significant reduction, demonstrating the efficacy of the 7AgV and 11AgV vaccine compositions of the present invention.

[0121] The worm load in animals vaccinated with the vaccine compositions of the present invention was also reduced. Compared with the control group, the total adult worms, as well as the total male and female worms, were significantly reduced after vaccination with seven and eleven antigens in sheep, deer, and calves. Similarly, the reduction in worm load was similar for each vaccine (7AgV or 11AgV), with a 65% reduction in total worm load observed in animals vaccinated with 7AgV in the first sheep trial (see...). Figure 4 In the second sheep trial, the use of 7AgV reduced the incidence by 54% in sheep, while the use of 11AgV reduced it by 46% (see [link to relevant documentation]). Figure 6 In further sheep trials using 11AgV, a 69% reduction in sheep worm load was observed (see...). Figure 10 A 41% reduction in worm load was observed in deer inoculated with 11AgV (see [link to article]). Figure 17 ); and a 56% reduction was observed in calves vaccinated with 11AgV (the reduction increased to 71% after removing one outlier animal) (see Figure 22The reduction in adult worm counts using the vaccine composition of the present invention is significant and sufficient to demonstrate the efficacy of the vaccine composition comprising seven to eleven antigens.

[0122] It is also foreseeable that, as those skilled in the art will understand, the vaccine compositions of the present invention using antigen fragments of *Nematoda circinata* EN, AK, ODC, SRS-2, MIF-2, aldolase, and GAPDH will also be effective.

[0123] The antigen fragments of the optional recombinant antigens (viii) to (xi) described above may also be used in the vaccine compositions of the present invention.

[0124] An antigen fragment is understood to refer to any one or more fragments of antigens (i) to (xi) that will possess effective antigenic properties, i.e., will result in the generation of antibodies that recognize and bind to the corresponding worm protein. Those skilled in the art can readily test fragments of antigens (i) to (xi) to determine antigenicity, i.e., antibody response, using a standard procedure to perform enzyme-linked immunosorbent assay (ELISA) on immunized or non-immunized sheep saliva and serum.

[0125] Optionally, as those skilled in the art will understand, species-specific recombinant homologs of *C. annularis* antigen or fragments of recombinant homologs of *C. annularis* antigen having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity can be used. Such recombinant homologs can be identified and produced using known techniques.

[0126] In addition, as those skilled in the art will understand, taking into account the issues related to natural antigens as discussed in the background section, the corresponding natural antigen may be used in place of or in conjunction with the recombinant antigen disclosed herein.

[0127] In a further embodiment, the composition or vaccine composition of the present invention comprises recombinant annular nematode antigen: (i) EN of SEQ ID NO:1, (ii) AK of SEQ ID NO:2 and (iii) ODC of SEQ ID NO:3; (iv) SRS-2 of SEQ ID NO:4; (v) MIF-2 of SEQ ID NO:5; (vi) aldolase of SEQ ID NO:6; (vii) GADPH of SEQ ID NO:7, or an antigen fragment thereof, and a veterinary acceptable carrier or diluent.

[0128] The composition or vaccine composition may further include one or more recombinant *Nematoda* antigens selected from the group consisting of:

[0129] (viii) ICL of SEQ ID NO:8;

[0130] (ix)MS of SEQ ID NO:9;

[0131] (x)CHT of SEQ ID NO:10; and

[0132] (xi)GST of SEQ ID NO:11;

[0133] See Table 2 below, or its antigen fragments.

[0134] In one embodiment, the composition or vaccine composition of the present invention may include an antigen having at least 70% sequence identity with SEQ ID NOS:1-11.

[0135] Table 2. Antigen protein sequences of *Nematodea annularis*

[0136] Enolase (EN) protein sequence of *C. annularis* (SEQ ID NO:1)

[0137] MPITKIHARQIYDSRGNPTVEVDLYTDKGVFRAAVPSGASTGVHEALELRDKDKKVHHGKGVLKAVANINEKIAPALIAKNFCVTQQRDIDQFMLALDGTENKSNLGANAILGV SLAVAKAGAVHKGMPLYKYLAELAGVSKVILPVPAFNVINGGSHAGNKLAMQEFMILPVGASSFHEAMRMGSEVYHHLKAEIKKRYGLDATAVGDEGGFAPNIQDNKEGLDLLKT AIDLAGYTGKISIGMDVAASEFYKEGKYDLDFKNPKSDPSKWLTGDQLAALYQTFIKEYPVVSIEDAFDQDDWDNWGKLKAATNIQLVGDDLTVTNPKRIRQAIDKKSCNCLLL KVNQIGSVTESIEAAKLSRSNGWGVMVSHRSGETEDTFIADLVVGLATGQIKTGAPCRSERLAKYNQLLRIEEELGKDAVYAGQNFRNPVAAAGAPVPYPDPLEPRAAAHHHHHH

[0138] Arginine kinase (AK) protein sequence of *C. annularis* (SEQ ID NO:2)

[0139] MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDHPFTMSVPPEIIKKIEDGYQTLQNAKDCHSLLKKYLTKEVVDQLKDKKTKLGATLWDVIQSGVANLDSGVGVYAPDAEAYTLFKPLFDPLIQDYHNGFSPSQKQPATDLGEGKTAQLVDLDPEGKYINSTRVRCGRSLQGYPFNPCLTEANYLEMEAKVKKIFENISDPELQGTYYPLDGMTKEVQNQLIKDHFLFKEGDRFLQAANACRYWPKGRGIFHNKNKTFLVWANEEDHLRIISMQNGGNVGQVLERLIKGVKIIQAQAPFSRDDRLGWLTFCPSNLGTTVRASVHIRLPKISAKPDFKKICDDLKLQIRGIHGEHSDSEGGVYDISNKARLGLTEFEAVKQMYDGVKHLIELEKKA

[0140] Ornithine decarboxylase (ODC) protein sequence of *Strongyloides cricitidis* (SEQ ID NO: 3)

[0141] MTMITQMELIGDSKVAIADGEVDAISMCQEIAHSYDQDNIDDAFMLVDLDVIFERFLLWKREMPMIEPFYAVKCNTDRVLVRTLAALGAGFDCASREEIDIVMDMGVRAEKIIYANPCKTRSFITHAKEKNVSMMTFDSVEELAKIAHLHPDAKMILRIAVSDPTARCPLNLKFGVDPVTKAPHLLVHAKELGVNVIGISFHVGSGCNDPTAFREALTHARHLTELGRGLGFDMNLVDLGGGYPGTLQQTSFEDIAAVIRSAVDEFLPPEFGVRLIAEPGRFFAAAPFTLVCNIIHATEVSAEKITKRPEDVDHRGFMYYVNDGVYGSFNCILFDHVDPVGAPLFDEIVEEYPSTIWGPTCDSLDKIEDQKMMRMMSVGEWIVYQNMGAYTCSASTTFNGFQRPNAVYVISRKNWARISTSPNV

[0142] Serine tRNA synthetase (SRS-2) protein sequence of *Strongyloides cricitidis* (SEQ ID NO: 4)

[0143] MVLDMDLFREEKGGNPELIRSSQRQRYSDPSIVDKVIELDQAWRKERFLLDVLNRQKNVLSKAIGEKMKKKEAQGTDENVDGSIISQLESLKMEDLSALTVTQIKKLRVLLDEKMNDTKVSMEQLEEDRHQSLIQIGNIVHHSVPVSDDEANNRVERTHGDITSRKKYSHVDLVVMIDGFDGERGTTVAGGRGYFLKGPLVFLEQAIIQLALQKLGEKGFTPLYTPFFMRKEVMQEVAQLNQFDEELYKVCGKGSEMLGDSSVDEKYLIATSEQPIAAFHRNEWIKESDLPIKYAGISTCFRQEVGSHGRDTRGIFRVHQFEKVEQFVICSPLNNESWKMFDEMIFNAEECCQLLGIPYQIMCIVSGELNNAASKKLDLEAWFPGSGAFRELVSCSNCTDYQARRLKVRYGMTKKMDGEVPFVHMLNATMCATTRVLCALLENYQTEDGITVPEVLHPFMPEKYRTFIPFVKPAPIDEEVKKKGGKAAAGAPVPYPDPLEPRAAAHHHHHH

[0144] Macrophage migration inhibitory factor 2 (MIF-2) protein sequence of Caenorhabditis catenaria (SEQ ID NO:5)

[0145] MPMVRVATNIPDKDVPPNFEERLTDILAESMNKPRTRIAVEVYAGQRIMHGGVRNPVVIIKIESIGALEPDKNIRHTERVTQLCQDVLHVPKDKVVISYFDLAPTNVGFGGTTVAAATVAAAGAPVPYPDPLEPRAAAHHHHHH

[0146] Aldolase protein sequence of Caenorhabditis catenaria (SEQ ID NO:6)

[0147] MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDHPFTMASYSQPPPKEKEDELRGIANAIVAPGKGILAADESTGSMDKKMKGIGTENTEEQRRKYRQLLFTASPEMSKHISGVIMFHETFYQKCDDGTRFVDALKKQGIIPGIKVDKGVVPMAGTVGEGTTQGMDDLNARCAQYKKDGAQFAKWRCVHKISATTPSHMAL VEIAEVLARYASICQQNGLVPIVEPEILPDGEHDIDRCRKITETVLSYCYRALNDHHVYLEGTLLKPNMVTAGQAFKGKKPSHDEIALATITALQRSVPAAVPGVVFLSGGQSEEDATLNLNAMNKLDTKKPWALTFSYGRALQASCMSKWGGKDENVKDAQAVFMQRAQANSLAALGKYSGDPNADKAASQSLFVANHAY

[0148] Protein sequence of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from *C. annularis* (SEQ ID NO:7)

[0149] MGKAKVGINGFGRIGRLVLRAAVEKDTVEVVSVNDPFINIDYMVYLFKYDSTHGRFKGSVSHEGDHLVVSKEGKSTHRIKVHNSKDPAEIPWGADGAEYIVESTGVFTTVDKASAHLKGGAKKVVISAPSADAPMFVMGVNNETYNASNNHIISNASCTTNCLAPLAKVI HDNFGIIEGLMTTVHATTATQKTVDGPSGKLWRDGRGAAQNIIPAATGAAKAVGKVIPALNGKLTGMAFRVPTPDVSVVDLTCRLEKPASMDDIKKVIKSASEGAMKGILGYTEDQVVSTDFLSDTHSSIFDAGACISLNPHFVKLISWYDNEYGYSHRVVDLLTYIASKA

[0150] The protein sequence of isocitrate lyase (ICL) from *Nematoda annularis* (SEQ ID NO:8).

[0151] MAQAAKNWYQVVKAAPKGRFQGIKRDYQVEDVLKLRGSVEIEYTLATRGANKLWQLLHTEPFVPALGAQTGNQAVQMVRAGLKAIYLSGWQVAADANTAGDMYPDQSLYPANSGPELCRRINRSLRRADQIEAVEAEDYLAQRDWYAPIVADAEAGFGGALNCFELMKSYIEAGAAGVHFEDRLGSEKKCGHMGGKVLIPTAQHIRHLNAARLAADVCGAPTIVVARTDAESSRLLTSDVDERDHPYIDRQAGRTVEGFHRLKDSTALQYCIDRAINYAPYCDLIWMETSHPTIADAREFAEGVRKVYPDKMFAYNCSPSFNWKQHLSPTQLEKFQKELGALGFKYQFITLAGFHANSYSMFDLARNYKEKGMLAYSSLQQQEFAAEQHGYSAVKHQREVGTGYFDHISNAVTGGQSSTTALAGSTEEAQFHTATASSEDEEILAAAGAPVPYPDPLEPRAAAHHHHHH

[0152] Malate synthase (MS) protein sequence of Criconemella annulata (SEQ ID NO:9)

[0153] MTLTAPMAAGDEKILTPDALRFIKDLNKKFDGKRRELLKKRQQVQIEINDGVYFPDFSSETAHLREDMGWKGSEIPQDLQDRRVEITGPTDRKMVINALNSGANVFMADFEDSNTPSWRNQLEGQVNLYDAVRDNISYMHPTTKKEYTLNQKVAVLNVRPRGWHLPEKHVLIHNKPTSGSLFDFGLFVYHNAKALKDKGSGPYFYLPKLQNAEEAKLWAEVFAYSEDRLDLPRGTIKCTVLIEHLLATFQMNEIIYALKDHIVGLNCGRRDYIFSYIKTFQNHRKFLLPDRFQIGMTSPFMRAYSLLCIKTCHQRGIHAMGGMAAQIPIKNDDVANSKALALVHQDKEREATDGHDGTWVAHPGLVPIARKVFDDCMPSANQIEKQLQSFFVTNQELTAIPEGTRTDHGFRHNISVTLGYLDSWLRGVGCVPLYNLMEDAATAEISRSQLWQWLRHDARLEDGRTIDAQLVKQTIAAETERRLIRAGSVVSRLPEAAELLEKFALEEHMSDFLTLDAYDKLVSEGHAAAGAPVPYPDPLEPRAAAHHHHHH

[0154] Chitinase (CHT) protein sequence of Criconemella annulata (SEQ ID NO:10)

[0155] MVAETPVRRKVFINSAIAFVRQWDFDGIDIDWEYPSGPADVRNYASFISELRQACEAEATSSQKPRLLVTAAVSAGESTIDAGYDVPAIADHLDFILLMNYDFHGAWSTETGFNSPLYAREDMRESEKVWNIDWSANHWHQKGMAKEKIIIGIPTYGRGWTLKDKSNITVGAEGSPAKITPYTQEAGVASFYEFCEMLATGATRYWSSEQQVPYLVQGDQWWSYDDEESIANKMAWIKRNKYGGAFVWTLDFDDFNAKCSNSDGQLYPLISIIAKELGGVTIPKVSTCSTWGNMDDVVDKGLSIAAAGAPVPYPDPLEPRAAAHHHHHH

[0156] Glutathione S-transferase (GST) protein sequence of *C. annularis* (SEQ ID NO:11)

[0157] MVHYRLLYFDGRGRAEVARQLFALANQEYVDVRITHEEWPKHKPEMPFGQLPVLDVDGKLLGQSHAINRYLARQFGFAGKSPFEEALVDAFADQYRDFYTEAQPYLYAVWGFVKG DVNALENEKFAPARDKFFNLMTKHLKASKSGFLVGDSVTWADLQLAELATFTEKYATLYVGFPEVKAHSEKVRSIPEIKKRIETRKNTPFAAAGAPVPYPDPLEPRAAAHHHHHH

[0158] The compositions or vaccine compositions of the present invention optionally include adjuvants.

[0159] As used in this article, the term "adjuvant" refers to a reagent used to enhance the immune response of an immunized host to an immunizing composition.

[0160] Suitable adjuvants for vaccinating farmed or wild ruminants include, but are not limited to: oil emulsions, such as Freund's complete adjuvant, Freund's incomplete adjuvant, squalane, or squalene; mineral gels, such as aluminum hydroxide, aluminum phosphate, calcium phosphate, calcium phosphate, and alum; surfactants, such as hexadecylamine, lysophosphatidylcholine, and methoxyhexadecylglycerol; polyanionic agents, such as dextran sulfate and carbomer; peptides, such as muramyl dipeptide and dimethylglycine; or other adjuvants, including QuilA, lipopolysaccharide, montanide, lipovant, bacterial flagellar proteins, adjuvant 65, imiquimod, gamma inulin, gademod, etc.

[0161] Preferred adjuvants are Montanide or Quil A.

[0162] The compositions or vaccine compositions of the present invention optionally include a carrier.

[0163] The compositions or vaccine compositions of the present invention may include carriers selected from, but not limited to, the following: sol-gel (chitin-based sustained-release compounds), hollow mesoporous silica nanoparticles (HMSN), poly(d,l-lactide-co-glycolic acid) (PGC) nanoparticles, poly(d,l-l-lactic acid-co-glycolic acid) (PGCA) nanoparticles, liposomes, virions, lipid-carrying delivery carriers, etc.

[0164] The compositions of the present invention or vaccine compositions can be given to animals for preventative purposes before any infection is detected, or they can be given to infected animals as treatment.

[0165] The compositions or vaccine compositions of the present invention are preferably administered to animals via subcutaneous or intramuscular injection.

[0166] The compositions or vaccine compositions of the present invention will be formulated as parenteral acceptable aqueous solutions for subcutaneous or intramuscular administration, and will be pyrogen-free and have suitable pH, isotonicity and stability.

[0167] As understood by those skilled in the art, the composition or vaccine composition may contain salts, buffers, adjuvants or other substances required to improve the efficacy of the composition.

[0168] The compositions or vaccine compositions of the present invention will be administered to animals in a therapeutically effective amount, i.e., an amount that elicits an immune response such as the production of desired antibodies. As can be seen from the examples, sustained antibody responses to vaccines comprising seven and eleven antigens have been demonstrated in sheep, deer, and calves (see...). Figures 2a-2g (8, 11, 13-15, 18, and 19). These antibody responses were associated with reduced worm load in sheep, deer, and calves, demonstrating the efficacy of the vaccine. Based on these results, the vaccine is expected to be effective in other ruminants.

[0169] Typically, the amount of antigen administered to animals is about 30 μg to 250 μg per antigen, preferably about 50 μg to 200 μg, and more preferably about 75 μg to 150 μg per antigen.

[0170] The compositions or vaccine compositions of the present invention can be administered as a single or multiple doses in a therapeutically effective amount. Preferably, the compositions or vaccine compositions are administered twice, wherein an initial immunization is performed, followed by a booster immunization 2-8 weeks later, preferably 3 weeks later. In some ruminants, as understood by those skilled in the art, a second booster immunization may be required approximately 4-8 weeks after the first booster immunization, depending on antibody levels.

[0171] As will be understood by those skilled in the art, additional doses may be administered as needed to treat or prevent infection.

[0172] The compositions or vaccine compositions of the present invention can be administered together with anthelmintics such as levamisole, methamidophos, oxifenedazole, mononitrate and / or ivermectin to increase the reduction rate of total FEC and worm load in treated animals at the time of vaccination.

[0173] The compositions or vaccine compositions of the present invention may also be administered in conjunction with other vaccine treatments commonly used in ruminants, such as clostridial diseases (including myeloid nephropathy, tetanus, malignant edema, blackleg, and blackleg); bovine viral diarrhea (BVD); foot rot; leptospirosis; salmonellosis; stomatitis, etc.

[0174] The compositions or vaccine compositions of the present invention are formulated for the treatment or prevention of nematode infections in farmed or wild ruminants, particularly sheep, cattle, goats, deer, buffalo, bison, camels, and llamas. Preferably, the compositions or vaccine compositions of the present invention are formulated for the treatment or prevention of nematode infections in farmed animals, including cattle, sheep, goats, and deer, particularly young animals under one year of age. On one hand, the animals may be less than 6 months old. On the other hand, the animals are at least 3 months old.

[0175] As used in this specification and claims, the term "comprising" means "consisting of at least a portion of...". When interpreting statements in this specification and claims containing the term "comprising", it should be understood that in each statement or claim, additional features may be present besides those introduced by the term. Related terms such as "comprise" and "comprised" are interpreted in a similar manner.

[0176] References to the numerical ranges disclosed herein (e.g., 1 to 10) also include references to all rational numbers within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) and any ranges of rational numbers within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7), and thus all subranges of all ranges explicitly disclosed herein are explicitly disclosed. These are merely examples of specific intentions, and all possible combinations of numerical values ​​between the listed minimum and maximum values ​​are to be considered as expressly stated in a similar manner in this application.

[0177] The invention can also be used for portions, elements and features that are individually or jointly mentioned or indicated in the specification of this application, and any or all combinations of any two or more of said portions, elements or features, and wherein a particular integer mentioned herein has an equivalent in the field to which the invention relates, such known equivalents being considered as separately stated herein.

[0178] The invention will now be described by way of the following representative methods and embodiments, which are provided to further illustrate the subject matter of the invention. The use of any and all embodiments or exemplary language (e.g., “such as” or “including”) provided herein is intended only for the purpose of better describing the invention. The presence of embodiments and the use of exemplary language do not limit the scope of the invention disclosed herein, unless otherwise specifically stated. Unless explicitly stated otherwise, the language used throughout the disclosure of this application should not be construed as indicating that any particular element or feature as disclosed herein is necessary for the practice of the invention. For example, those skilled in the art will be able to modify the reagent amounts and processing times in the exemplified methods and processes, as known in the art, based on the inventive concepts disclosed herein. Such modifications are considered to be within the scope of the invention.

[0179] Example

[0180] Experiment 1

[0181] Sheep were immunized with a vaccine composition comprising enolase (EN) from the annular nematode, arginine kinase (AK), ornithine decarboxylase (ODC), serine tRNA synthetase (SRS-2), macrophage migration inhibitory factor 2 (MIF-2), aldolase, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0182] Materials and methods

[0183] Preparation of antigens

[0184] Recombinant AK, EN, ODC, SRS-2, MIF-2, aldolase, and GAPDH were purified as described (Han et al., 2012; Umair et al., 2013a,b). The proteins were individually identified on coomassie blue-stained gels to confirm their size and solubility.

[0185] Recognizing vaccine antigens in immunized lambs

[0186] Prior to the start of the experiment, mucosal and systemic antibody responses against recombinant EN, MIF-2, aldolase, GST, CHT, ICL, MS, and GAPDH were assessed by enzyme-linked immunosorbent assay (ELISA) of saliva and serum from immunized or non-immunized sheep using a standard procedure (Umair et al., 2021a; 2021b; 2020a; 2020b; 2017; 2016; 2023 pending publication).

[0187] animal

[0188] The use of laboratory animals was approved by the AgResearch Animal Ethics Committee. Thirty-six male Romney crossbred lambs (approximately 3 months old) were purchased from Ballantrae Farm, AgResearch Ltd, and transported to the Grasslands Large Animal facility for deworming, weighing, and tagging. The animals were randomly assigned to four groups of nine animals each. Prior to the experiment, all groups had the same mean weight. The animals were housed and fed indoors. The animals were divided into the following groups:

[0189] Group 1: Vaccine in QuilA and sol-gel (VacQuil)

[0190] Group 2: QuilA control group (QuilA in sol-gel) (CtQuil)

[0191] Group 3: Vaccine in Montanide ISA 71VG (1:2 ratio) (VacMon)

[0192] Group 4: Montanide ISA 71VG control group (CtMon)

[0193] Vaccine trial

[0194] Each animal in the vaccinated groups received 50 μg of each antigen via SC injection. Animals in groups 3 and 4 received three doses of vaccine or adjuvant at 3-week intervals, while animals in groups 1 and 2 received the vaccine or adjuvant at weeks 0 and 6. This trial was initially conducted in vitro, but due to some animals' failure to adapt to the indoor management system, they were transferred to AgResearch's Aorangi farm shortly after the third vaccination. They were grazed in a parasite-free pasture and challenged with 12,000 *L. annularis* nematodes over 3 days, 4 weeks after the third vaccination. Animals in groups 3 and 4 were euthanized 5 weeks post-infection, but since there was no difference in egg production between groups 1 and 2, these groups were challenged again with 20,000 *L. annularis* nematodes over 3 weeks, and euthanized 3 weeks after the second parasitic challenge. Abomasums were collected from the euthanized animals for parasitological studies (counting of adult males and female worms).

[0195] Immunoassay and Parasitology

[0196] Antibody levels in serum and saliva were measured by ELISA. Eggs per gram of feces (EPG) were counted using a modified McMaster method, where each counted egg represented 50 eggs per gram of feces (Lyndal-Murphy, 1993). Adult worms were recovered from the abomasum in a 7-liter volume, with 10% used for worm count, male-to-female ratio, and worm length. Worm length was calculated using ImageJ software.

[0197] Functional activity of recombinase

[0198] Purified recombinant AK, EN, ODC, SRS-2, MIF-2, aldolase, and GAPDH were incubated in the immune serum of animals in the Quil A vaccine group at 25°C for one hour, followed by enzyme assays to determine whether the antibodies in the serum could inhibit the function of these enzymes. The recombinant enzymes were also incubated in non-immunized serum as a control. AK, EN, ODC, SRS-2, MIF-2, aldolase, and GAPDH were measured according to the described protocols (Umair et al., 2013a; Han et al., 2013; Umair et al., 2013b).

[0199] Results and discussion

[0200] Antibodies in the saliva and serum of naturally immunized sheep reacted strongly with seven recombinant proteins, indicating that the immune host was able to recognize these antigens.

[0201] antibody response

[0202] Serum IgG antibody responses against recombinant AK, EN, ODC, SRS-2, MIF-2, aldolase, and GAPDH were measured by ELISA. Compared with the corresponding control groups, the enzyme-specific IgG levels in the QuilA and MontanideISA 71VG vaccine groups were significantly higher in pooled serum at dilutions of 1:6400 or 1:1600. Figure 2a -g). Compared with the corresponding control, saliva antibodies (IgA) also increased in the Quil A and Montanide ISA 71VG vaccine groups after vaccination. Figure 3 ).

[0203] Parasitology and Adult Worm Counting

[0204] The results of this experiment were remarkable; compared with the control group 4, the number of adult worms (males and females) in group 3 was significantly reduced (P < 0.05). Figure 4 The difference in fecal egg counts was nearly significant (P = 0.06). Figure 5Compared with the control group (Group 4), the VacMon group (Group 3) had a greater weight gain (~1.5 kg, although this was not statistically significant - results not shown).

[0205] discuss

[0206] This study demonstrated that immunization of 3-4 month old sheep with a mixture of seven recombinant proteins induced protective levels against *Strombus annularis*. The recombinant 7-antigen vaccine (VacMon) in the Montanide ISA 71VG adjuvant resulted in a significant reduction in adult worm counts (both male and female). VacMon also resulted in a reduction in fecal excretion compared to the control (P = 0.06). When the same recombinant protein mixture was used with another adjuvant, Quil A, VacQuil and CtQuil showed no difference in fecal egg excretion and adult worm counts. However, two animals in the CtQuil group had to be excluded from the study due to a lack of weight gain, which may have contributed to the lack of a significant reduction in the VacQuil group.

[0207] in conclusion

[0208] The results of this trial are very promising, showing for the first time that recombinant antigens that elicit a significant immune response when injected into sheep can be prepared.

[0209] Further experiments on a larger scale will help to ultimately determine whether these antigens are protective and suitable vaccine targets.

[0210] Experiment 2

[0211] Sheep were immunized with a vaccine composition containing 7, 8, or 11 recombinant antigens of the annular nematode.

[0212] Materials and methods

[0213] Animals and Experimental Design

[0214] The use of laboratory animals was approved by the AgResearch Animal Ethics Committee. Ninety newly weaned female Romney crossbred lambs (approximately 3 months old) were purchased from a private farm and transported to the Aorangi Farm of AgResearch Ltd., where they were dewormed, weighed, and tagged. The animals were randomly assigned to five groups (InfCt, 7AgV, 8aAgV, 8bAgV, and 11AgV), with 15 animals in each group. An additional 15 animals served as a control group (-Ct). All groups had the same mean weight before the experiment began. The animals were housed on pastures with no sheep grazing history for at least the past three years. Cattle grazed on these pastures before and during the experiment.

[0215] This experiment was designed to test whether administering recombinant Teladorsagia antigens in different combinations could improve vaccine efficacy compared to previous experiments. Animals were divided into the following groups:

[0216] Group 1: Uninfected control group (-Ct)

[0217] Group 2: Infection Control Group (PostCt)

[0218] Group 3: 7-antigen vaccine (7AgV) in Montanide ISA 71VG

[0219] Group 4: 8-antigen vaccine (8aAgV) in Montanide ISA 71VG

[0220] Group 5: 8-antigen vaccine (8bAgV) in Montanide ISA71VG

[0221] Group 6: 11-antigen vaccine (11AgV) in Montanide ISA 71VG

[0222] Vaccine antigens and preparations

[0223] Seven-antigen vaccine (7AgV)

[0224] (i) Arginine kinase (AK)

[0225] (ii) Enolase (EN)

[0226] (iii) Ornithine decarboxylase (ODC);

[0227] (iv) Seryl tRNA synthetase (SRS-2); (v) Macrophage migration inhibitory factor 2 (MIF-2); (vi) Aldolase;

[0228] (vii) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) octaantigen vaccine (8aAgV)

[0229] (ix) malate synthase;

[0230] (viii)ICL;

[0231] (iii) ODC;

[0232] (iv)SRS-2;

[0233] (v)MIF-2;

[0234] (vi) Aldolase;

[0235] (vii)GADPH;

[0236] And (x)Cht.

[0237] Eight-antigen vaccine (8bAgV)

[0238] (i)AK;

[0239] (ii)EN;

[0240] (iii) ODC;

[0241] (iv)SRS-2;

[0242] (viii)ICL;

[0243] (x)Cht;

[0244] (ix) malate synthase;

[0245] And (xi)GST.

[0246] 11AgV vaccine

[0247] (i)AK

[0248] (ii)EN

[0249] (iii) ODC

[0250] (iv)SRS-2

[0251] (v)MIF-2

[0252] (vi) Aldolase

[0253] (vii)GADPH; and

[0254] (viii) Isocitrate lyase (ICL);

[0255] (ix) malate synthase;

[0256] (x) chitinase (Cht); and

[0257] (xi) Glutathione S-transferase (GST)

[0258] The recombinant antigen was purified as previously described (Han et al., 2012; Umair et al., 2013a,b). The proteins were identified separately on coomassie blue-stained gels, and their size and solubility were confirmed.

[0259] The recombinant proteins were expressed in *E. coli* (see Appendix 2). Each antigen was prepared with an equal volume of adjuvant (Montanide ISA71 VG). Each animal received a 3 ml dose per vaccination, for a total of three vaccinations at 3-week intervals. Each dose consisted of 50 μg of each antigen.

[0260] Vaccine trial

[0261] Animals in the 7AgV, 8aAgV, 8bAgV, and 11AgV groups received 50 μg of each antigen in a subcutaneous dose (3 ml per animal) prepared with Montanide ISA71 VG. Vaccinations were administered three times weekly for three weeks (days 0, 7, and 21). The control group received neither adjuvant nor vaccine.

[0262] Two weeks after the last vaccination, all animals except the -Ct group were infected with 2,000 L3 annular dorsal nematode larvae orally three times a week for four weeks. The -Ct animals served as uninfected controls to identify potential pasture contamination.

[0263] All animals were euthanized 6 weeks after infection, and the abomasum was collected for parasitology. The total number of adult male and female *Zygodium annularis* was counted, as was the number of eggs in the adult female worms.

[0264] result

[0265] The results of this experiment were at the highest point of our expectations, where treatment with 7AgV and 11AgV significantly reduced the number of adult worms (males and females) compared to the PosCt group. Figure 6 (P < 0.05). Furthermore, the difference in fecal egg count between the 7AgV group and the PosCt group was also significant (P < 0.05). Figure 7a ), and each antigen elicits a significant antibody response ( Figure 7b Compared with the InfCt group, the 7AgV group had a higher weight gain (~1.5 kg, although this was not statistically significant - results not shown).

[0266] discuss

[0267] This trial demonstrated the efficacy of the 7- and 11-recombinant vaccines against the mucosal-feeding nematode *Nematoda* annularis in young animals. Vaccination with a combination of recombinant parasite antigens induced a significant increase in antibody levels and a significant reduction in egg count and worm load. Importantly, vaccine efficacy was achieved in young lambs highly susceptible to parasite infection under field conditions. Here, immunization of young lambs (approximately 3 months old) with a mixture of recombinant proteins induced protective levels against *Nematoda* annularis.

[0268] in conclusion

[0269] The vaccine of this invention (7AgV), comprising seven core antigens, produced a prototype vaccine with or without the addition of multiple recombinant antigens, which demonstrated efficacy in young lambs under stringent field conditions exposed to various environmental stresses. Interestingly, two eight-antigen vaccines (8aAgV and 8bAgV) excluding the seven core antigens were ineffective, demonstrating that the 7AgV core antigen is essential for the efficacy of the vaccine of this invention.

[0270] Experiment 3

[0271] Sheep were immunized with a vaccine composition containing recombinant antigens of 11 annular nematodes.

[0272] Materials and methods

[0273] Animals and Experimental Design

[0274] The use of laboratory animals was approved by the AgResearch Animal Ethics Committee. Eighty newly weaned lambs (approximately 3 months old, female) were grazed outdoors in the Grasslands for one week, introduced with pelleted feed, alfalfa pellets, and alfalfa hay. The lambs were then moved indoors for one week, provided with cut hay, pelleted feed, alfalfa pellets, and alfalfa hay to help them acclimatize. To complete acclimatization, the animals were fed only pelleted feed, alfalfa pellets, and alfalfa hay. Sixty animals that acclimatized to the indoor environment were selected for further testing.

[0275] This study was designed to determine the efficacy of a *Nematoda* vaccine against monodrug-resistant or tripledrug-resistant wild-type strains of *Nematoda*; to determine the efficacy of a *Nematoda* vaccine when administered intramuscularly; and to determine the efficacy of a *Nematoda* vaccine when administered at a low dose (20 μg / antigen / animal / vaccine, instead of the usual dose of 50 μg / antigen / animal / vaccine).

[0276] The animals were divided into the following groups:

[0277]

[0278] Vaccine antigens and preparations

[0279] 11AgV vaccine

[0280] In this group, the vaccine includes eleven antigens, namely:

[0281] (i)AK

[0282] (ii)EN

[0283] (iii) ODC

[0284] (iv)SRS-2

[0285] (v)MIF-2

[0286] (vi) Aldolase

[0287] (vii)GADPH; and

[0288] (viii) Isocitrate lyase (ICL);

[0289] (ix) malate synthase;

[0290] (x) chitinase (Cht); and

[0291] (xi) Glutathione S-transferase (GST)

[0292] The recombinant antigen was purified as previously described (Han et al., 2012; Umair et al., 2013a,b). The proteins were identified separately on coomassie blue-stained gels, and their size and solubility were confirmed.

[0293] The recombinant proteins were expressed in *E. coli* (see Appendix 2). Each antigen was prepared with an equal volume of adjuvant (Montanide ISA 71VG). Each animal received a 3 ml dose per vaccination, for a total of three vaccinations at 3-week intervals. Each dose consisted of 50 μg of each antigen.

[0294] Vaccine trial

[0295] Animals in groups 2, 3, 5, and 6 received 50 μg of each antigen in a subcutaneous dose (3 ml per animal) prepared with Montanide ISA 71VG. Animals in group 4 received a low dose of only 20 μg of each antigen. Vaccinations were administered three times a week for three weeks (days 0, 7, and 21). Animals in control group 1 did not receive any adjuvant or vaccine.

[0296] Two weeks after the third vaccination, all lambs were orally administered 3,000 L3 annular dorsal nematode larvae daily (as shown in the table above) for five days. All animals were slaughtered 8–10 weeks after the attack, and the abomas were collected for parasitology.

[0297] result

[0298] antibody response

[0299] Compared with the control group, vaccination induced a significantly higher antibody response in the vaccine group. Figure 8Serum samples from LG2 / VS, LG3 / V-IM, and LG6 / V-3R were tested at weeks 0, 10, 14, and 18 and compared with the control (LG1 / Ct). All three vaccinations induced significantly higher antibody levels compared with the control; however, there was no difference in antibody titers between the vaccination groups. Antibody titers increased after each vaccination, peaking after the last vaccination.

[0300] Fecal worm egg count

[0301] Vaccination resulted in a significant reduction in fecal egg counts in LG6 / V-3R, decreasing by 70% compared to the control group. Figure 9 Although vaccination did result in a 26% and 29% reduction in FEC for LG2 / VS and LG3 / VIM-S, respectively, this was not statistically significant. No other vaccine groups showed significantly lower fecal egg counts compared to the control group. Saliva samples from this trial were not analyzed, but saliva samples from animal study 1 showed a significant increase in IgA antibodies in the vaccine group compared to the control group.

[0302] Adult worm count

[0303] When adult parasites (male and female) were counted from the abomasum collected from slaughtered animals, LG3 / VIM and LG6 / V-3R had significantly fewer adult worms compared to LG1 / Ct. Figure 10 Vaccination had no adverse effects on the animals' weight, and there was no significant difference in the weight of lambs between the vaccinated group and the control group.

[0304] discuss

[0305] Vaccination significantly reduced fecal egg counts in the treatment group infected with triple-drug-resistant *Nematoda* strains. Vaccination resulted in a significant reduction in adult worm counts in LG3 / VIM-S and LG6 / V-3R. Intramuscular delivery of the vaccine provided better protection compared to the SC route. Interestingly, lower antigen concentrations offered no protection.

[0306] in conclusion

[0307] The vaccine of this invention (11AgV), which includes eleven recombinant antigens, produced a prototype vaccine that demonstrated efficacy against multidrug-resistant worms in young sheep under strict field conditions in which the animals were exposed to a variety of environmental stresses.

[0308] Experiment 4

[0309] Calves were immunized with a vaccine composition containing recombinant antigens of 11 annular nematodes.

[0310] Materials and methods

[0311] Animals and Experimental Design

[0312] The use of laboratory animals was approved by the AgResearch Animal Ethics Committee. Thirty newly weaned male Friesian calves (approximately 3 months old) were grazed on a parasite-free pasture at the AgResearch Aorangi farm. The calves remained outdoors throughout the experiment. The calves were divided into two groups of 15 animals each.

[0313] This experiment was designed to demonstrate the concept that recombinant Nematode genus vaccines can protect cattle from Ostertagia infection.

[0314] The animals were divided into the following groups:

[0315] Group Quantity (n) deal with CG1 / Ct 15 Control group, attack only CG2 / V 15 Inoculate with 11 antigens and attack.

[0316] Vaccine antigens and preparations

[0317] 11AgV vaccine

[0318] In this group, the vaccine includes eleven antigens, namely:

[0319] (i)AK

[0320] (ii)EN

[0321] (iii) ODC

[0322] (iv)SRS-2

[0323] (v)MIF-2

[0324] (vi) Aldolase

[0325] (vii)GADPH; and

[0326] (viii) Isocitrate lyase (ICL);

[0327] (ix) malate synthase;

[0328] (x) chitinase (Cht); and

[0329] (xi) Glutathione S-transferase (GST)

[0330] The recombinant antigen was purified as previously described (Han et al., 2012; Umair et al., 2013a,b). The proteins were identified separately on coomassie blue-stained gels, and their size and solubility were confirmed.

[0331] The recombinant proteins were expressed in *E. coli* (see Appendix 2). Antigens were prepared using an equal volume of adjuvant (Montanide ISA71VG). Each animal received a 4 ml dose per vaccination, for a total of three vaccinations at 3-week intervals. Each dose consisted of 150 μg of each antigen.

[0332] Vaccine trial

[0333] The control group (CG1 / Ct) and the vaccinated group (CG2 / V) calves received 150 μg of each antigen in a subcutaneous dose (4 ml per animal) prepared with Montanide ISA71VG, three times at three-week intervals. Two weeks after the third vaccination, all animals were attacked with a total of 15,000 L3 Osterella ostreatus larvae, administered for more than 4 days. Adult worms from the abomasum were recovered and counted after slaughter.

[0334] result

[0335] Although vaccination does lead to significantly higher antibody levels ( Figure 11 However, the fecal egg count between the two groups ( Figure 12 ) or adult worm load ( Figure 13 There was no significant reduction in weight gain, and there was no difference in weekly weight gain between the two groups. This appears to suggest that antibodies produced against the antigen of *Streptococcus osternii* are ineffective against *Oestera osternii* infection, and that *Streptococcus osternii* vaccines may be species-specific and may not be effective against other parasites of the same family or genus.

[0336] discuss

[0337] Surprisingly, despite the striking similarity between the two parasites, the vaccine failed to even partially protect against *Osteria osternii* infection in cattle. Currently, no publicly available *Osteria osternii* gene sequences are available in databases, making it difficult to compare homology between *C. annularis* and *Osteria osternii* gene sequences. A possible reason for its ineffectiveness is that, although most gene sequences may be closely related, the epitopes of the recombinant proteins differ between the two parasite species. Another possible reason for the vaccine's ineffectiveness is the choice of adjuvant. Our previous experience with parasite vaccines suggests that vaccine efficacy is highly dependent on the choice of adjuvant. The Montanide ISA 71VG adjuvant, which works in sheep, may not work in cattle. Another cattle trial with a different adjuvant is planned (see Trial 6 below).

[0338] Experiment 5

[0339] Deer were immunized with a vaccine composition containing recombinant antigens of 11 annular dorsal nematodes.

[0340] Materials and methods

[0341] Animals and Experimental Design

[0342] The use of laboratory animals was approved by the AgResearch Animal Ethics Committee. Throughout the experiment, 30 male red deer, aged 3-4 months, were grazed on a standard pasture at the AgResearch farm in Inverme. The deer were divided into two groups of 15 each.

[0343] This experiment was designed to demonstrate the concept that recombinant Nematodea vaccines can protect deer from Osteria cauda infection.

[0344] The animals were divided into the following groups:

[0345] Group Quantity (n) deal with DG1 / Ct 15 Control group, natural attacks only DG2 / V 15 Inoculate with 11 antigens and allow for natural attack.

[0346] Vaccine antigens and preparations

[0347] 11AgV vaccine

[0348] In this group, the vaccine includes eleven antigens, namely:

[0349] (i)AK;

[0350] (ii)EN;

[0351] (iii) ODC;

[0352] (iv)SRS-2;

[0353] (v)MIF-2;

[0354] (vi) Aldolase;

[0355] (vii)GADPH;

[0356] (viii) Isocitrate lyase (ICL);

[0357] (ix) malate synthase;

[0358] (x) chitinase (Cht); and

[0359] (xi) Glutathione S-transferase (GST).

[0360] The recombinant antigen was purified as previously described (Han et al., 2012; Umair et al., 2013a,b). The proteins were identified separately on coomassie blue-stained gels, and their size and solubility were confirmed.

[0361] The recombinant protein was expressed in Escherichia coli (see Appendix 2).

[0362] The control group (DG1 / Ct) was not vaccinated, while the vaccinated group (DG2 / V) received 150 μg of each antigen in a subcutaneous dose (3 ml per animal) prepared with Montanide ISA71VG, three times at three-week intervals.

[0363] Vaccine trial

[0364] The control group was not vaccinated (DG1 / Ct), while the treatment group received three doses of each antigen prepared with Montanide ISA71VG at three-week intervals.

[0365] Compared to the artificial parasite attack used in the lamb and calf trials described above, these animals were naturally attacked by grazing on a standard deer pasture, where deer parasite contamination was expected to be relatively high. All animals were weighed, and blood, saliva, and fecal samples were taken monthly. The trial was terminated 8–10 weeks after the last vaccination, and samples were collected for analysis.

[0366] result

[0367] antibody response

[0368] Compared with the control group, vaccination resulted in serum samples from all animals in the vaccinated group ( Figure 15 ) and saliva samples ( Figure 16 The antibody titers were significantly higher in the group receiving the third vaccination. Antibody titers peaked after the third vaccination but began to decline three weeks after the last vaccination.

[0369] Fecal egg and adult worm count

[0370] Compared with the control group, vaccination resulted in a decrease in the amount of fecal eggs excreted in the vaccine group. Figure 16 ), and it appears that the vaccine resulted in a reduction of ~49%. Vaccination resulted in a 41% reduction in adult worms in DG2 / V (excluding 1 outlier). Figure 17 ).

[0371] discuss

[0372] The results of the first deer trial were very encouraging, and it appears that the recombinant *Nematoda* vaccine partially protects weaned red deer from *Oestera* type infection. Parasites are a major problem in farmed deer, and current anthelmintics are ineffective. *Oestera* type worms are the most important parasites in farmed deer in New Zealand. Further experiments are needed to improve vaccine efficacy and optimize vaccine concentration and delivery routes.

[0373] in conclusion

[0374] The vaccine of this invention (11AgV), which includes eleven recombinant antigens, produced a prototype vaccine that demonstrated efficacy in young fawns under strict field conditions in which the animals were exposed to a variety of environmental stresses.

[0375] Experiment 6

[0376] Cows were immunized with vaccine compositions containing 7 or 11 recombinant antigens of *Nematoda* annularis* in two different adjuvants (Montanide ISA 61VG and QuilA / Sol gel).

[0377] Materials and methods

[0378] Animals and Experimental Design

[0379] The use of laboratory animals was approved by the AgResearch Animal Ethics Committee. Sixty-nine male calves, approximately 5-6 months old, were purchased and brought to the AgResearch Aorangi farm two weeks prior to the outdoor trials. All animals, except for the negative control, were marked with pink and randomly assigned to one of seven groups (see below).

[0380] This experiment was designed to determine the efficacy of a *C. dorsi* vaccine against *O. ostella* infection in cattle using multiple antigen combinations; and to determine the efficacy of a *C. dorsi* vaccine against *O. ostella* infection in cattle using antigen combinations formulated with two different adjuvants, including the novel Montanide adjuvant (Montanide ISA 61VG), because Montanide ISA 71VG had previously been ineffective in cattle (see Experiment 4 above).

[0381] Divide the calves into the following groups:

[0382]

[0383]

[0384] Vaccine antigens and preparations

[0385] Seven-antigen vaccine (7AgV)

[0386] (i) Arginine kinase (AK)

[0387] (ii) Enolase (EN)

[0388] (iii) Ornithine decarboxylase (ODC); (iv) Seryl tRNA synthetase (SRS-2); (v) Macrophage migration inhibitory factor 2 (MIF-2); (vi) Aldolase; and

[0389] (vii) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0390] 11AgV (i)AK vaccine;

[0391] (ii)EN;

[0392] (iii) ODC;

[0393] (iv)SRS-2;

[0394] (v)MIF-2;

[0395] (vi) Aldolase;

[0396] (vii)GADPH;

[0397] (viii) Isocitrate lyase (ICL);

[0398] (ix) malate synthase;

[0399] (x) chitinase (Cht); and

[0400] (xi) Glutathione S-transferase (GST).

[0401] The recombinant antigen was purified as previously described (Han et al., 2012; Umair et al., 2013a,b). The proteins were identified separately on coomassie blue-stained gels, and their size and solubility were confirmed.

[0402] The recombinant proteins were expressed in *E. coli* (see Appendix 2). Antigens were prepared with equal volumes of adjuvants (such as Montanide or QuilA as shown in the table above). Each animal received a 4 ml dose per vaccination, for a total of three vaccinations at 3-week intervals. Each dose consisted of 150 μg of each antigen.

[0403] Vaccine trial

[0404] Candidate vaccines (150 μg of each purified antigen) were administered via subcutaneous injection (SC) in the neck. For the Montanide vaccine group (Vac1 and Vac3), 2–3 ml was administered in three divided doses at 3-week intervals using an 18-gauge needle. For the Solgel vaccine group (Vac2 and Vac4), two doses were administered at 6-week intervals. The injection site was monitored and examined twice in the same week following vaccination, and weekly thereafter. Blood was collected and weighed weekly from all animals, and saliva samples were collected every two weeks. Fecal samples were collected monthly prior to parasite infection and twice weekly starting from day 18 post-infection to count eggs per gram of feces.

[0405] Animals were grazed on new, parasite-free pastures. Once the animals began excreting eggs in their feces after the attack, they were moved to different pastures weekly, with isolation fences erected at both ends to prevent reinfection. All animals except -Ct were attacked with 15,000 Osterella ostreatus larvae. All animals were slaughtered on day 35 post-infection, and their abomasums were collected for the recovery of adult worms and L4.

[0406] result

[0407] antibody response

[0408] Vaccination resulted in antigen-specific antibodies in all vaccine groups. The Montanide adjuvant group received three injections of each vaccine at 3-week intervals, while the QuilA / Sol gel adjuvant group received two injections. The QuilA / Sol gel animals had antibody titers equal to or higher than those of the Montanide group, despite receiving only two injections. Antibody responses were generally high in all vaccinated animals. Figure 18 and 19 Two embodiments are shown in the figure. Figure 19 The antibody responses in four vaccine groups when vaccinated with recombinant GAPDH and other antigens (as described in the methods above) are shown. Animals vaccinated with 11AgV showed the highest antibody response at week 12 compared to the control animals (mean values ​​are shown in...). Figure 19 middle).

[0409] Antibody levels were measured by ELISA on serum diluted 1:4000 at an optical density of 450 nm.

[0410] Fecal worm egg count

[0411] Fecal samples were collected from all animals twice weekly for two weeks. The vaccine did not result in a significant reduction in fecal ova. There were no differences in fecal ova excretion between the different vaccine groups. Similarly, there were no differences in fecal ova excretion between the Montanide or QuilA / Solgel groups. Figure 20 ).

[0412] weight

[0413] The average body weight of all vaccine groups and control groups is shown in the figure. Figure 21 There were no significant differences in weight gain among the groups, and the average weight gain was similar across all groups, although the average weight gain of the positive control group was 3-4 kg more than any of the vaccine groups, but this was not statistically significant.

[0414] Adult worm count

[0415] The adult worm count was the most interesting aspect of this trial, and vaccination resulted in a significant reduction in adult worm counts in the 11AgV (Quila / Sol gel) group. Compared to the control group, this group saw a 56% reduction in adult worm counts. One outlier animal in this group was excluded from the analysis; if that animal had been excluded, the reduction would have increased to 71%. No other vaccine groups showed significant differences compared to the control group. Figure 22 ).

[0416] Stagnant larvae recovery and counting

[0417] Detained L4 was recovered from the abomas of animals in all vaccine groups by incubating each abomas in 1 L of water containing 30 ml hydrochloric acid and 10 g pepsin at 37°C. Vaccination resulted in a significant reduction in L4 in the abomas of animals vaccinated with 7 AgV (in Montanide adjuvant) and 11 AgV (in Quila / Sol gel adjuvant), with reductions of 59% and 54%, respectively. When one outlier was removed from each of these groups, the percentage reduction in L4 was approximately 70% in both groups compared to the control group. Figure 23 ).

[0418] discuss

[0419] This trial is the first to demonstrate the efficacy of vaccines containing recombinant *O. ostella* antigens against *O. ostella* infection in calves. Although the vaccine failed to reduce fecal egg excretion in any vaccine group, the number of adult worms (male and female *O. ostella*) was significantly reduced by approximately 70%. *O. ostella* larvae arrest in development within the mucosa of the abomasum and develop into adult worms and complete their life cycle several months later (when external host conditions are favorable). Vaccination with this invention significantly reduced the number of arrested L4 larvae by approximately 70%, meaning fewer larvae will develop into adult worms in the spring. This aspect of the *O. ostella* vaccine has significant implications for livestock farming. In New Zealand, due to the extreme cold and frost of winter, most larvae on pastures die, leaving arrested larvae in the host as a reserve source that develops into adult worms in the spring and begins to infect pastures. This vaccine can potentially eliminate most of the arrested L4 population, meaning that in the following season, there will be no or very few eggs excreted on pastures.

[0420] Compared to the control group, both the QuilA / Solgel and Montanide ISA 61VG vaccine groups produced high antibody titers. The QuilA / Solgel groups (7AgV and 11AgV) performed better than the three vaccine groups in the Montanide adjuvant group because they had similar antibody levels, but these animals received only two injections compared to the three injections in the Montanide group. Furthermore, animals in the Montanide group had severe and adverse vaccine site reactions compared to the QuilA group. Animals vaccinated with QuilA / Solgel had significantly fewer and smaller masses compared to Montanide. The data suggest that the greater the amount of antigen (regardless of adjuvant), the larger the mass. Histological examination of samples taken from the vaccine site showed calcification and eosinophilic infiltration.

[0421] in conclusion

[0422] The vaccines of this invention, comprising seven or eleven recombinant antigens (7AgV, 11AgV), yielded prototype vaccines that demonstrated efficacy in young calves under stringent field conditions, where the animals were exposed to a variety of environmental stresses. Both adjuvants (Montanide ISA 61VG and QilA / Solgel) were useful. We also showed that the novel Montanide adjuvant (ISA 61VG) was successfully used in calves, while the original Montanide adjuvant (ISA 71VG) was successfully used in sheep and deer. This is important information for vaccine formulations targeting these animals.

[0423] Experiment 7

[0424] Calves are immunized with a vaccine composition containing recombinant antigens of 7 or 11 annular nematodes.

[0425] Materials and methods

[0426] Animals and Experimental Design

[0427] The use of laboratory animals was approved by the AgResearch Animal Ethics Committee. Thirty-five Jersey calves, aged 4 months, were brought to the Aorangi farm two weeks prior to the experiment. The animals were dewormed to remove existing worm loads, weighed, and had their saliva collected. Throughout the experiment, the animals were housed in a parasite-free pasture. The animals were randomly assigned to four treatment groups.

[0428] This experiment was designed to determine the efficacy of *C. dorsiflora* vaccines against *O. ostella* infection in cattle using multiple antigen combinations; and to determine the efficacy of *C. dorsiflora* vaccines against *O. ostella* infection in cattle using antigen combinations formulated in QuilA and chitosan-based extended-release formulations.

[0429] Divide the calves into the following groups:

[0430] Group Quantity (n) deal with NegCt 2 No treatment, no infection control PosCt 11 Infection control group Vac1(7AgQ) 11 A vaccine containing 7 antigens, in QuilA and sol-gel. Vac2(11AgQ) 11 Solgel 1 contains 7 antigens (chitosan + QuilA).

[0431] Seven weeks after the second vaccination, eight animals from Group 1 (NegCt) and eight from each of the remaining groups were culled for adult worm recovery and tissue collection. The remaining three animals from each group were then grazed for another six months. Serum samples were collected every two weeks to obtain antibody levels and determine the duration of protection.

[0432] result

[0433] Fecal worm egg count

[0434] From day 21 to day 31 post-infection, the amount of fecal eggs excreted by animals in the vaccinated group was significantly lower than that in any of the positive control groups. Figure 24 The 11AgV group showed significantly lower egg excretion at all sampling time points, while the 7AgV group showed significantly reduced egg excretion at both sampling time points. Animals in the negative control group ( Figure 24 (Not shown in the image) was not attacked by parasites, therefore the amount of fecal worm eggs excreted was zero.

[0435] Adult worm count

[0436] Compared with the control group, the total number of adult worms and the total number of male and female worms were significantly reduced in the 11AgV animals. Figure 25 Compared to PosCt, the 11AgV group showed an overall reduction of 67% in adult worms.

[0437] Stagnant larvae recovery and counting

[0438] Detained L4 was recovered from the abomas of calves in all vaccine groups by incubating each abomas in 1 L of water containing 30 ml hydrochloric acid and 10 g pepsin at 37°C. Vaccination significantly reduced L4 in the abomas of the 7 AgV and 11 AgV groups. Figure 26 ).

[0439] antibody response

[0440] Serum samples collected weekly throughout the trial were tested against all 11 antigens, and serum antibody responses to vaccination were measured in individual samples. Serum samples were diluted 1:1000, 1:2000, and 1:8000, with significantly higher antibody levels detected in all treated animals at all three dilutions. Saliva samples were collected from individual animals prior to the start of the trial, and anti-CarLA antibodies were measured to assess pre-existing parasite exposure levels. Animals were assigned to control and treatment groups based on body weight and CarLA levels. CarLA levels were very low in most animals. Vaccination induced significantly higher serum antibody responses than PosCt in both treatment groups. Serum samples were collected every two weeks from two calves in each group for seven months following the second vaccination to assess antibody levels. Antibody responses at the 1:2000 dilution are shown in [Figure / image / image]. Figure 27 middle.

[0441] discuss

[0442] In this calf trial, vaccination resulted in a significant reduction in adult worm counts in the 7Ag and 11Ag groups. Compared to the control group, vaccination resulted in a 67% reduction in adult worm counts in the 11AgV group. Vaccination had a positive effect on vaccinated calves, with fewer parasite eggs excreted in their feces. Furthermore, vaccination resulted in a significant increase in serum antibodies in all vaccinated animals, and antibody titers remained stable for over eight months after the second vaccination, indicating that the protective effect against *Oestera ostreatus* infection can last for several months after vaccination. The calves grazed on high-quality pastures, and the total daily weight gain per calf was 1.2–1.5 kg / day (no statistically significant difference was observed between the vaccinated and control groups due to the small sample size). These results are encouraging and will contribute to illustrating the patent claim that a *Nematoda* vaccine protects young calves from *Oestera ostreatus* infection under field conditions.

[0443] in conclusion

[0444] The vaccines of this invention, comprising seven or eleven recombinant antigens (7AgV, 11AgV), have produced prototype vaccines that have demonstrated efficacy in young calves under stringent field conditions, where the animals are exposed to a variety of environmental stresses. This is important information for vaccine formulations targeting these animals.

[0445] General Conclusion

[0446] *Typhae annularis*, also known as the brown stomach worm, is one of the most prominent nematode parasites affecting livestock worldwide. As described above, the recombinant *Typhae annularis* vaccine of this invention has shown truly promising results in lambs. In particular, the vaccine has consistently shown a significant reduction in fecal egg excretion and adult worm counts in young, vaccinated lambs. Preliminary trials of this invention have also shown promising results against *Oesterus* parasites in deer and cattle. To date, there are no *Typhae annularis* vaccines on the market. This vaccine has great potential, particularly against drug-resistant *Typhae annularis*, in which the vaccine has already shown a reduction in drug-resistant worm counts in young, vaccinated lambs, and also provides broader protection against the corresponding *Oesterus* genus worms in deer and calves.

[0447] Appendix 1.

[0448] Discovery of vaccine antigens for nematode parasites (Nematode annularis)

[0449] Overview of Antigen Screening

[0450] The basic principle of this research is the discovery that proteins (genes) essential for the survival of the parasitic nematode *Nematoda* can be used as antigens for vaccine development. This discovery was made from two directions.

[0451] 1. Select homologs of three antigens that correspond to the antigens used in our early Haemaphysalis vaccines, namely EN, AK, and ODC (see NZ 780917).

[0452] 2. Select the remaining antigens based on available literature.

[0453] Appendix 2

[0454] Summary of the expression and purification of recombinant proteins from *C. annularis*.

[0455]

[0456]

[0457]

[0458] References

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[0460] Umair,S.,Knight,J.S.,Bland,R.J.,Simpson,H.V.,2013a.Molecular andbiochemical characterisation of arginine kinases in Haemonchus contortus andTeladorsagia circumcincta.Experimental Parasitology 134,362-367.

[0461] Umair,S.,Knight,J.S.,Simpson,H.V.,2013b.Molecular and biochemicalcharacterisation of ornithine decarboxylases in the sheep abomasal nematodeparasites Teladorsagia circumcincta and Haemonchus contortus.ComparativeBiochemistry and Physiology B 165,119-124.

[0462] Umair,S.,Ria,C.,Knight,J.S.,Bland,R.J.,Simpson,H.V.,2013c.Sarcosinemetabolism in Haemonchus contortus and Teladorsagia circumcincta.ExperimentalParasitology 134,1-6.

[0463] Han,K.,L.Xu,et al.(2012)."Vaccination of goats with glyceraldehyde-3-phosphate dehydrogenase DNA vaccine induced partial protection againstHaemonchus contortus."Veterinary Immunology and Immunopathology 149(3-4):177-185.

[0464] Longhi-Browne,C.W.(2014)using Caenorhabditis elegans as a novelexpression system for the generation of recombinant Teladorsagia circumcinctavaccine candidates.Abstract PhD thesis.

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[0467] Weygand-Durasevic I.,Ban,N.,Jahn,D.and Soll,D.(1993).Yeast seryl-tRNAsynthetase expressed in Escherichia coli recognises bacterial serine-specifictRNAs in vivo.Eur.J.Biochem.214:869-877

[0468] Umair,S.,Bouchet,C.L.G.,Baten,A.2021.Characterisation and recognitionby immune hosts of a sheep nematode parasite Teladorsagia circumcinctachitinase.Journal of Veterinary Science and Medicine 9,1-5.

[0469] Umair,S.,Bouchet,C.L.G.,Palevich,N.,Simpson,H.V.2021b.Teladorsagiacircumcincta 1.6-Biphosphate aldolase:Molecular and biochemicalcharacterisation,structure analysis and recognition by immunehosts.Parasitologia 1,1-9.

[0470] Umair,S.,Bouchet,C.L.G.,Palevich,N.,Simpson,H.V.2020a.Characterisation and structural analysis of glyoxylate cycle enzymeof Teladorsagia circumcincta.Molecular and Biochemical Parasitology 240,111335.

[0471] Umair,S.,Bouchet,C.L.G.,Deng,Q.,Palevich,N.,Simpson,H.V.,2020b.Characterisation of a Teladorsagia circumcincta glutathionetransferase.Molecular and Biochemical Parasitology,239,111316.

[0472] Umair,S.,Bouchet,C.B.G.,Knight,J.S.,Pernthaner,A.,Simpson,H.V.,2017.Molecular and biochemical characterisation and immune recognition ofTeladorsagia circumcincta glyceraldehyde 3-phosphate dehydrogenase(GAPDH).Experimental Parasitology 181,40-46.

[0473] Umair,S.,Bouchet,C.B.G.,Knight,J.S.,Pernthaner,A.,Simpson,H.V.,2017a.Molecular and biochemical characterisation of Teladorsagia circumcinctaenolase and its recognition by the immune host.Experimental Parasitology 172,30-38.

[0474] Umair,S.,Bouchet,C.B.G.,Knight,J.S.,Pernthaner,A.,Simpson,H.V.,2017b.Molecular and biochemical characterisation and immune recognition ofTeladorsagia circumcincta glyceraldehyde 3-phosphate dehydrogenase(GAPDH).Experimental Parasitology 181,40-46.

[0475] Umair,S.,Knight,JS,Simpson,HV,2013a.Molecular and biochemical characterization of ornithine decarboxylases in the sheep abomasal nematodeparasites Teladorsagia circumcincta and Haemonchus contortus.ComparativeBiochemistry and Physiology B 165,119-124.

[0476] Umair, S., Knight, JS, Bouchet, CLG, Palevich, N., Cleland, S., Grant, W., Simpson, HV, 2022. Characterization of macrophage inhibitory factor-2 (MIF-2) in Haemonchus contortus and Teladorsagia circumcincta. Parasitologia, 2, 338-349.

[0477] In this specification, which has already referenced patent specifications, other external documents, or other sources of information, this is generally to provide context for discussing the features of the invention. Unless otherwise specifically stated, references to such external documents should not be construed as an admission that such documents or such sources are prior art or part of common general knowledge in any jurisdiction.

Claims

1. A composition or vaccine composition comprising recombinant Haemonchus contortus antigens: (i) enolase (EN); (ii) arginine kinase (AK); (iii) ornithine decarboxylase (ODC); (iv) seryl tRNA synthetase (SRS-2); (v) macrophage migration inhibitory factor 2 (MIF-2); (vi) aldolase; and (vii) glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or antigenic fragments thereof, and a veterinarily acceptable carrier or diluent.

2. The composition or vaccine composition of claim 1, further comprising one or more recombinant Haemonchus contortus antigens selected from the group consisting of: (viii) isocitrate lyase (ICL); (ix) malate synthase (MS); (x) chitinase (CHT); and (xi) glutathione S-transferase (GST) or antigenic fragments thereof.

3. The composition or vaccine composition of claim 2, comprising at least one, at least two, at least three, or at least four of the antigens (viii) isocitrate lyase (ICL), (ix) malate synthase, (x) chitinase (CHT), and (xi) glutathione S-transferase (GST).

4. A composition or vaccine composition comprising Haemonchus contortus recombinant antigens: (i) enolase (EN); (ii) arginine kinase (AK); (iii) ornithine decarboxylase (ODC); (iv) seryl tRNA synthetase (SRS-2); (v) macrophage migration inhibitory factor 2 (MIF-2); (vi) aldolase; (vii) glyceraldehyde-3-phosphate dehydrogenase (GAPDH); (viii) isocitrate lyase (ICL); (ix) malate synthase (MS); (x) chitinase (CHT); and (xi) glutathione S-transferase (GST) or antigenic fragments thereof, and a veterinarily acceptable carrier or diluent.

5. The composition or vaccine composition of any one of claims 1 to 4, further comprising an adjuvant.

6. The composition of claim 5, wherein the adjuvant is selected from one or more of the group consisting of: alum, Quil A, Freund's complete adjuvant, Freund's incomplete adjuvant, lipopolysaccharide, monophosphoryl lipid A, montanide, lipovant, bacterial flagellin protein, adjuvant 65, gamma inulin, algammulin, imiquimod, gardiquimod, and murimyl dipeptide.

7. The composition or vaccine composition of any one of claims 1 to 6, further comprising a carrier. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 8. The composition of claim 7, wherein the carrier is selected from one or more of the group consisting of: a chitin-based slow release compound (sol-gel), hollow mesoporous silica nanoparticles (HMSN), poly(d,l-lactide-co-glycolide) (PGC) nanoparticles, poly(d,l-lactic acid-co-glycolic acid) (PGCA) nanoparticles, liposomes, virosomes, and cochleates delivery vehicles.

9. A method of reducing the parasitic nematode worm burden in a farmed or wild ruminant, the method comprising administering to the ruminant at one or more occasions an effective amount of the composition or vaccine composition of any one of claims 1 to 8, whereby the reduction in parasitic worm burden is measured by a reduction in fecal egg count (FEC) and / or an increase in larval and / or adult nematode worm expulsion.

10. A method of inducing an immune response in a farmed or wild ruminant to treat or protect the animal from infection by a parasitic nematode, the method comprising administering to the animal at one or more occasions an effective amount of the composition or vaccine composition of any one of claims 1 to 8, wherein the induction of an immune response is measured by the presence of protective antibodies against one or more of the specific antigens present in the composition or vaccine composition.

11. A method of stimulating or enhancing acquired immunity in a farmed or wild ruminant to treat or protect the animal from infection by a parasitic nematode, the method comprising administering to the animal at one or more occasions an effective amount of the composition or vaccine composition of any one of claims 1 to 8, wherein the stimulation or enhancement of acquired immunity is measured by one or more of: the presence of protective antibodies against one or more of the specific antigens present in the composition or vaccine composition; elevated cytokine levels; a reduced FEC; and / or expulsion of larval and / or adult nematodes.

12. A method of treating or preventing nematode infection in a farmed or wild ruminant, comprising administering to the animal an effective amount of the composition or vaccine composition of any one of claims 1 to 8.

13. Use of the following recombinant Haemonchus contortus antigens: (i) enolase (EN), (ii) arginine kinase (AK), (iii) ornithine decarboxylase (ODC), (iv) seryl tRNA synthetase (SRS-2), (v) macrophage migration inhibitory factor 2 (MIF-2), (vi) aldolase; and (vii) glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or antigenic fragments thereof, in the manufacture of a composition or vaccine composition for reducing the nematode parasitic worm burden in a farmed or wild ruminant.

14. Use of the following recombinant Haemonchus contortus antigens: (i) enolase (EN), (ii) arginine kinase (AK), (iii) ornithine decarboxylase (ODC), (iv) seryl tRNA synthetase (SRS-2), (v) macrophage migration inhibitory factor 2 (MIF-2), (vi) aldolase; and (vii) glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or antigenic fragments thereof, in the preparation of a composition or vaccine composition for stimulating or enhancing acquired immunity in a farmed or wild ruminant to treat or protect said animal from infection by a parasitic nematode.

15. Use of the following recombinant Haemonchus contortus antigens: (i) enolase (EN), (ii) arginine kinase (AK), (iii) ornithine decarboxylase (ODC), (iv) seryl tRNA synthetase (SRS-2), (v) macrophage migration inhibitory factor 2 (MIF-2), (vi) aldolase; and (vii) glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or antigenic fragments thereof, in the preparation of a composition or vaccine composition for treating or preventing nematode infection in a farmed or wild ruminant.

16. Use of the following recombinant Haemonchus contortus antigens: (i) enolase (EN), (ii) arginine kinase (AK), (iii) ornithine decarboxylase (ODC), (iv) seryl tRNA synthetase (SRS-2), (v) macrophage migration inhibitory factor 2 (MIF-2), (vi) aldolase; and (vii) glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or antigenic fragments thereof, in the preparation of a composition or vaccine composition for inducing an immune response in a farmed or wild ruminant to treat or protect said animal from infection by a parasitic nematode.

17. The use according to any one of claims 13 to 16, wherein the composition or vaccine composition further comprises one or more of the antigens (viii) isocitrate lyase (ICL), (ix) malate synthase, (x) chitinase (Cht), and (xi) glutathione S-transferase (GST) as claimed in claim 2.

18. The method according to any one of claims 9 to 12, or the use according to any one of claims 13 to 16, wherein the farmed or wild ruminant is selected from the group consisting of sheep, cattle, goats, deer, water buffalo, bison, camels and llamas.

19. The method or use according to claim 18, wherein the farmed or wild ruminant is a young animal less than one year of age.

20. The method or use according to claim 19, wherein the farmed or wild ruminant is less than 6 months of age.

21. The method according to any one of claims 9 to 12 and 18 to 20, or the use according to any one of claims 13 to 17, wherein the parasitic nematode worm is selected from one or more of the group consisting of: Haemonchus contortus, Haemonchus placei, Haemonchus nematorum, Ostertagia (Periostomum) circumcincta, Cooperia curticei, Nematodirus helvolus, Trichostrongylus axei, Trichostrongylus capricola, Trichostrongylus colubriformis, Cooperia onchophora, Nematodirus brasiliensis, Dictyocaulus eckerti, Strongyloides papillosus, Toxascaris vitolorum, Nematodirus spathiger, Ashworthius sidemi, Setaria cervi, Oxyurovastrongylus quadriradiatus, Trichuris trichiura, and Bunostomum phlebotomum.

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