WO1997019107A1 - Identification of antigenes from post-infection nematodes for the purpose of developing new anthelmintics and vaccines - Google Patents

Abstract

The invention concerns antigenes isolated from nematodes in essentially pure form and which show an immunological reaction with a protective anti-serum used against nematode larvae at the skin-shedding stage from L3 to L4, the antigenes being obtained by the in vitro cultivation of nematode larvae in the L4 stage and extraction from the surplus culture. The invention also concerns a method of obtaining such antigenes and their use in the development of new anthelmintics and vaccines.

Description

Identification of antigens from post-infectious nematodes for the development of new anthelmintics and vaccines

The invention relates to the isolation and characterization of antigens from nematodes in a substantially pure form, which react immunologically with protective antisera, and a method for obtaining these antigens. Furthermore, the present invention relates to the use of these antigens for the development of new anthelmintics and vaccines

Parasitic nematodes, which can affect human and animal lymphatic vessels and tissues, cause chronic diseases.Human pathogenic nematode species occur in tropical regions, where the WHO estimates that there are more than 100 million infected people.Nematode diseases in farm animals and pets occur throughout World and cause high economic damage and costs

Commercial anthelmintics are able to cure infested humans and animals from their parasitic nematodes, provided that the nematodes have not yet developed resistance to these agents. The development of resistance has already been observed with prophylactic administration. There is therefore no great need for the development of new ones Anthelmintics that are not resistance-breaking

A passive protective immunity by administration of antibodies against surface structures of nematodes cannot be easily achieved

There are several publications in which failures in the use of monoclonal antibodies or polyclonal antisera for passive immunization against filenic infections are described (Abraham et al (1988) J Parasitol 74 275-282, Vickery et al (1983) J Parasitol 69 478 - 485 and Tanner and Weiss (1979) Tropical Medicine and Parasitology, 30 371 - 375). Passive immunization with monoclonal antibodies can even lead to an increase in the parasite load (Janecharut et al (1991) Parasitology Research, 77 668 - 674) Eisenbeiss et al (J. Immunol. 152 (1994), 735-742) describe that the repeated administration of a small number of larvae of the parasitic filaria A. viteae in gerbils (M. unguiculatus) causes a significant reduction in the total worm burden. This partial protective immunity appears to be linked to antigens derived from the post-infectious larvae in the

L3 / L4 molting stage. No such effect was found in inactivated larvae at the L3 stage or live larvae at the L4 stage.

A protective antiserum can be isolated from immunized host animals which is directed against antigenic substances which are secreted between the L3 and L4 stages during molting. However, this secretion takes place only for a very short period and in very small amounts, so that isolation of the antigens in a pure form is not possible.

Surprisingly, however, it was found that the molting antigens which are secreted during the transition from the L3 to the L4 stage are present in a substantially larger amount of nematode larvae cultivated in vitro in the L4-

Stage are produced. The amount of antigens produced in this way is so large that isolation using conventional techniques is readily possible. Furthermore, it was surprisingly found that the antigens isolated in this way are not species-specific in their immunological properties, but that a cross-species cross-reaction between different

Antisera and various nematode species is found.

The present invention thus relates to isolated antigens from nematodes in a substantially pure form, which react immunologically with a protective antiserum which is directed against nematode larvae at the molting stage from L3 to L4, the antigens being converted by in vitro

Cultivation of nematode larvae in the L4 stage and extraction from the culture supernatant are available.

The term "protective antiserum" in the sense of the present application means that the serum is obtained from animals whose immune system can completely eliminate a new infection due to a corresponding pre-immunization. Surprisingly, it was found that antigens which react immunologically with protective antisera can be isolated from a large number of nematode species, in particular from filaria species, for example from free-living nematode species, such as Caenorhabditis elegans, or parasitic nematodes, such as, for example, Acanthocheilonema viteae, Monanemaia martini and Dirofϊlar immitis,

Wuchereria bancrofti, Brugia malayi, Loa loa, Onchocerca volvulus, Onchocerca ochengi, Dracunculus medinensis, Litomosoides sigmodontis, Haemonchus contortus, Trichinella spiralis or Trichuris muris

Protective antisera are available from host animals which are exposed to a primary infection with live nematode larvae before the L3 / L4 skin stage, and which are at least partially inhibited in their development by a second exposure infection with nematode larvae of the same species or a different species. Protective antisera from the Gerbils gerbil M unguiculatus, the African striped mouse Lemniscomys striatus or other natural hosts from nematodes are preferably used for primary infection. Repeated administration of a sufficient number of nematode larvae is preferably used, the interval between the administration of the larvae and the number of larvae can vary depending on the nematode species Immunization of M unguiculatus can, for example, live infectious L3 larvae of the nematode species A viteae 3 times at 3-day intervals and in one

Number of 5-10 larvae are administered to achieve good immunization

Furthermore, protective antisera can also be obtained by immunization with living L4 A vitaea larvae, with living in vitro cultivated L3 / L4 or L4 A vitaea larvae, with living in vitro cultivated "exsheathed" L3 H contortus

Larvae are obtained with living in vitro cultivated L3 (80% Lethargus) C elegans larvae

With the protective antiserum obtained in this way, not only antigens of the nematode species used for immunization, but also antigens of other nematode species can be detected and isolated

For example, a protective antiserum obtained from the natural host of the Filaπe A viteae, the Meriones unguiculatus, shows an immunological cross-reactivity against antigens from the nematode species M martini, L. sigmodontis, D. immitis, H. contortus and even the free-living nematode species C. elegans.

The antigens according to the invention are preferably polypeptides, which can optionally be glycosylated. Different antigens are secreted, which can be separated according to their molecular weight and obtained in isolated form. The amount of antigens obtained is easily sufficient for a partial determination of the amino acid sequence. Based on this information, recombinant DNA techniques can then also be used to isolate DNA molecules that code for the respective antigens. After isolation of the coding DNA sequences, the antigens according to the invention can also be obtained by recombinant

Methods in suitable host cells, e.g. Bacteria, yeast or insect cell culture lines, or with transgenic C. elegans.

Preferred antigens are antigens which are obtainable from A. viteae and have molecular weights of approximately 4, 14, 18, 25, 29-30, 40, 44, 52, 56, 60, 66 or 68 kD after sieve gel chromatography, or are thus immunologically cross-reactive Antigens. Also preferred are antigens which are obtainable from A viteae and have molecular weights of about 17, 40, 52, 56, 60, 63, 67, 90, 94, 1 16, 180, 212, 400 or 800 kD according to SDS-PAGE, or thus immunologically cross-reacting antigens. Immunologically cross-reacting antigens, for example, have already been found in D. immitis, M. martini, L. sigmodontis,

H. contortus or C. elegans can be detected

Also preferred are antigens which are obtainable from C elegans and have molecular weights of about 14, 16, 26, 28, 30, 46, 49, 58 and 66 kD according to sieve gel chromatography, or antigens which are obtainable from C. elegans and molecular weights of 46, 60, 97, 109, 212 or 400 kD according to SDS

PAGE exhibit, or thus immunologically cross-reactive antigens. Immunologically cross-reacting antigens have already been detected in A. vitaea, for example.

Antigens which have a molecular weight of approximately 60 kD and are particularly preferred

(a) an N-terminal amino acid sequence

Xaa-Thr- (Val, Leu) -Lys-Glu- (Glu, Leu) -Ile-Ala- (Phe, Val) -Leu- (Phe, Leu) -Xaa-Leu-Leu-Gly- where Xaa means any amino acid, or

(b) have an N-terminal amino acid sequence which is at least 80% and in particular at least 90% homologous to the sequence from (a).

These antigens can have serine / threonine protein kinase activity.

In connection with the above-mentioned molecular weights with the antigens according to the invention, it should be noted that the numerical values are only approximate values and are therefore to be understood as a numerical range of + 10% and preferably + 5% of the respectively stated value.

Another object of the present invention is a method for obtaining antigens from nematodes which react immunologically with a protective antiserum which is directed against nematode larvae at the molting stage from L3 to L4, larvae of nematodes at the L4 stage cultured in vitro, the culture supernatant separated and the antigens isolated from the culture supernatant. The isolation preferably comprises a sieve gel chromatography step. Furthermore, the use of A. viteae larvae at the stage 13 days after infection is preferred. In other organisms, suitable larval stages in which the production of the antigens according to the invention takes place can be identified in a simple manner by determining the immunological activity

Determine supernatants from larvae cultivated in vitro.

Yet another object of the present invention is a pharmaceutical composition which comprises one or more of the aforementioned antigens as an active ingredient and, optionally, pharmaceutically customary additives, carriers and auxiliaries. The antigens or a pharmaceutical containing them

Composition can be used for diagnosis, prevention and therapy of nematode infections. The present invention thus also relates to a method for immunizing against a nematode infection, in which one or more of the aforementioned antigens are administered in pharmaceutically acceptable form. The administration is preferably in a parenteral or oral formulation in a dose between 0.5 and 250 mg / kg, particularly preferably between 10 and 100 mg / kg on 1 to 4 consecutive days. Single administration is particularly preferred.

Furthermore, it was surprisingly found that administration of attenuated larvae of parasitic nematodes in the L4 stage, preferably of at least 2 days in vitro cultivated nematode larvae, to a sterile

Immunity leads. The attenuated larvae can no longer develop in the body after in vitro cultivation, but do lead to effective protection against subsequent infections with nematodes. In this way, sterile immunity to filaria larvae is achieved for the first time. The invention thus also relates to a pharmaceutical composition which contains attenuated larvae of parasitic nematodes in the L4 stage as an active ingredient and, if appropriate, pharmaceutically customary additives, auxiliaries and carriers.

The attenuated larvae and a pharmaceutical composition containing them can be used for the prevention and therapy of nematode infections, optionally in combination with one or more of the aforementioned antigens.

Furthermore, the application is to be explained by the following examples.

example 1

Obtaining protective antisera from Meriones unguiculatus

Gerbils (M unguiculatus) were subjected to a primary infection by subcutaneous administration of 10 - 20 live infectious larvae of A viteae at intervals of three days. Serum was taken 25 days after the initial infection

Protective antibodies were formed in the infected animals, giving protection against subsequent secondary infections with A viteae larvae. This procedure is described in detail by Eisenbeiss et al (J Immunol 152 (1994),, 735-742). Protective antisera could also be used in an analogous manner by

Use of living L4 larvae as well as in vitro cultivated L3 / L4 and L4 larvae of A viteae can be obtained

Also by 3 subcutaneous infections with 1000 living "exsheathed" H contortus L3 + 6 + 7 larvae ("exsheathed" L3 are cultured for 6 days in vitro, the medium is discarded and incubated in fresh medium for a further 7 days) at intervals of each 3 days and removal of the serum 25 days after the initial infection, protective antisera were obtained from M unguiculatus

Furthermore, 3 subcutaneous infections with 1000 living C elegans L3 led to 80% Lethargus + 3 hours (L3 larval populations, which were 80% in the

Lethargus phase prior to molting to L4 are cultured in vitro for 3 h) at intervals of 14 days and serum withdrawal 46 days after the initial infection, protective antisera are obtained from M unguiculatus

The antisera obtained in this way recognize nematode antigens which are secreted from L3 to L4 during the skin stage Example 2

In vitro cultivation of nematode larvae

A viteae larvae were isolated from M unguiculatus as described in Eisenbeiss, J Parasitol 77 (1991) 580-586. For this purpose, the bodies of infected and then killed gerbils in

RPMI1640 medium added and chopped. The larvae were isolated from the tissue fractions and stored in RPMI

Larvae at different stages of maturity were isolated, namely larvae in the L3 stage (0-6 days p i), larvae in the skin stage from L3 to L4 (6-7 days p i) and larvae in the L4 stage (more than 8 days p I)

The larvae were cultivated in vitro at a concentration of 500-1000 larvae / ml culture medium RPMI 1640 at 37 ° C. and 5% CO ₂

Example 3

Obtaining antigens from the supernatant of culture media

Surprisingly, antigens could be detected in the supernatant of the culture medium from 13-day-old larvae (L4 stage) in a high yield, namely with a cultivation period of 0-2, 2-4, 4-6 and 6-8 days

11 and 17 day old larvae in the L4 stage, however, showed no secretion of antigens. 14 day old L4 larvae and 6-7 day old mL3 larvae showed weak secretion of antigens in amounts that were too low for isolation

The antigens were detected by an ELISA as follows. 96-well microtiter plates were incubated with 100 μl of crude culture medium from 1000 larvae / ml overnight at 4 ° C. Three different dilutions of the crude culture medium, namely 1 10, 1 100 and 1 1000, were used in

Elution buffer (0.1 M K-phosphate buffer pH 7.0), used All wells were blocked with 150 μl 3% bovine serum albumin (RSA), 10% milk powder and 0.05% Tween 20 in elution buffer. Protective anti- mL3 antiserum from M. unguiculatus, the recovery of which is described in Example 1, diluted 1: 400, used in elution buffer with 1% RSA and 0.05% Tween 20. Rabbit anti-M was used as the second antibody. unguiculatus-immunoglobulin IgG, diluted 1: 1500, and as a third antibody a conjugate of peroxidase and anti-rabbit IgG from goat, diluted 1: 2000. Between

Antibody additions were washed three times with 100 μl elution buffer + 0.05% Tween 20. Finally, o-phenylenediamine (0.4 mg / ml in 200 mM phosphate buffer pH 5.0) and 0.05% H ₂ O _{2 was} added as chromogen and the color reaction was stopped after one minute with 10 μl 11 MH ₂ S0 ₄ .

Under these test conditions, immunoreactive antigens could already be detected with a dilution of the raw culture medium of 1: 100.

Example 4

Obtaining the secreted antigens

A total of 12 fractions could be separated from the collected culture supernatants from larvae cultured in vitro using an FPLC gel filtration method. Of these, seven fractions were of particular interest because they either appeared to be dominant and / or were recognized as antigens by antibodies from the serum of 100% immune-protected animals. The separation was carried out using a Superdex 75 HR 10/30 gel filtration column in 0.5 ml fractions. Elution was carried out with a 0.1 M potassium phosphate buffer pH 7.0. The immunologically relevant fractions were determined by means of ELISA with sera from protected animals.

Immunologically reactive components with 68 kD, 66 kD, 60 kD, 56 kD, 52 kD, 44 kD, 40 kD, 29-30 kD, 25 kD, 18 kD, 14 kD and <4 kD were the strongest Peaks for the components with molecular weights

<4 kD and 60 kD. A culture medium isolated as a control for the FPLC column was free of protein components. Example 5

Preparation of protein samples for amino acid analysis

Proteins in the FPLC fractions obtained in Example 4 were concentrated in bands via SDS-PAGE and then transferred electrophoretically to a support membrane

Preliminary experiments with samples from crude (unfractionated) culture supernatants from 13-day-old L4 larvae gave 14 protein bands with a molecular weight distribution in the range from 800, 400, 212, 180, 1 16, 94, 90, 67, 63, 60, 56, 52, 40 and 17 kD

The resulting protein bands were applied to a nitrocellulose membrane

0.1 μm pore size electrophoretically transferred. A subsequent "Western analysis" with protective M unguiculatus antiserum, rabbit anti-M unguiculatus immunoglobulin IgG and peroxidase-labeled anti-rabbit IgG as well as a suitable chromophore provided the immunologically relevant protein bands

Protein-containing FPLC fractions were then selected for the protein sequence analysis. The individual fractions contained approx. 7 pmol protein in 400 μl elution buffer

The selected fractions were transferred in a concentrated form to PVDF membranes using a suction device with which large application volumes can be filtered through small membrane areas. The filters were washed in water and stored in an argon atmosphere until protein sequencing

Sequencing of the antigen resulted in the following n-terminal amino acid partial sequence

Xaa-Thr- (Val, Leu) -Lys-Glu- (Glu, Leu) -Ile-Ala- (Phe, Val) -Leu- (Phe, Leu) -Xaa-

Leu-Leu-Gly- where Xaa means any amino acid The identified sequence region does not start with a methionine, although the native protein has been sequenced N-terminally. As a result, the protein probably originally had a leader sequence.

Example 6

Immunization experiments

Supernatants from the culture medium of 13-day-old L4 larvae (800 μl supernatant from 48 larvae as doses of immunization) showed effective protection of M. unguiculatus against subsequent stress infections.

Immunization doses of 36-48 larvae cultured in vitro per inoculation provided protection against a subsequent stress infection of 80-100%. Interestingly, none of the L4 larvae cultured in vitro for at least two days survived in the host. This is a "sterile immunity" that was first observed against filariasis.

Example 7

Identification of immunoreactive components in other nematodes

7.1. Gerbils (unguiculatus) were immunized with 15-20 mL3 larvae of the nematode species Monanema martini and Litomosoides sigmodontis. This immunization resulted in significant protection against subsequent stress infections with A. viteae.

Similarly, immunization of African striped mice resulted

(Lemniscomys striatus) with mL3 larvae of A. viteae provide significant protection against subsequent stress infections with M. martini.

The larvae administered to the pre-immunized animals during the stress infection were completely inactivated or their development was severely hampered. The response of the host animals to heterologous species was even stronger than to homologous species. 7.2 In a similar way, protection against a subsequent infection with A viteae could be achieved in a primary infection of M unguiculatus with the nematode species Haemonchus contortus and Dirofilaria immitis

7 3 Even with free-living nematodes, such as Caenorhabditis elegans, one can

Cross immunization against A viteae can be achieved in the gerbil M unguiculatus. For this purpose, C elegans larvae in the L3 / L4 stage (L3 / S0% lethargus larvae), which had preferably been incubated in vitro beforehand for 1.5 to 3 hours, were used to immunize M. unguiculatus used against a subsequent infection of A viteae

Improvement in immune protection was achieved by extending the time between the immunization doses, eg administration on days 0.30 and 73

Example 8

Immunological detection of antigens from other nematode species

8.1 Sera from M unguiculatus, which had been immunized with mL3 larvae of the nematode species M martini, L sigmodontis and H contortus and showed low recovery rates of A viteae after a stress infection, showed high speciifi c in the ELISA (Example 3) e Cross reactivities against antigens from A viteae Comparable to that as

Both the anti-L sigmodontis serum and the anti-C elegans serum reacted with positive control using homologous antiserum. The anti-M martini and anti-H contortus sera also recognized antigens in the culture medium of 13-day-old L4-A viteae larvae

8.2 From skin-shedding L3 larvae of the nematode species C elegans (L3 80%

Lethargus + 1.5 or 3 hours in vitro cultivation) secreted antigens were recognized by protective antisera, which were obtained from M unguiculatus, which had previously been immunized with A viteae, M martini and L sigmodontis

Western blot analysis of the C elegans antigens revealed bands of approx

46 kD, 60 kD, 97 kD, 109 kD, 212 kD and 400 kD (SDS-PAGE). The 60 kD antigen is expressed by adult C. elegans worms and molting C. elegans L3 larvae. The other antigens are also expressed by adult C. elegans worms and by mL3 larvae and are immunologically cross-reactive with corresponding antigens which are expressed by 13-day-old L4-A. viteae larvae. Furthermore, antigens with molecular weights of approx. 14, 16, 26, 28, 30, 46, 49, 58 and 66 kD could be identified by sieve gel chromatographic analysis of the culture supernatants.

Claims

claims 1. Isolated antigens from nematodes in a substantially pure form which react immunologically with a protective antiserum which is directed against nematode larvae at the stage of molting from L3 to L4, the antigens being cultivated in vitro by nematode larvae L4 stage and recovery from the culture supernatant are available. 2. Antigens according to claim 1, characterized in that they come from free living nematodes. 3. Antigens according to claim 2, characterized in that they originate from Caenorhabditis elegans 4. Antigens according to claim 1, characterized in that they come from parasitic nematodes. 5. Antigens according to claim 4, characterized in that they consist of Acanthocheilonema viteae, Monanema martini, Dirofilaria immitis, Wuchereria bancrofti, Brugia malayi, Loa loa, Onchocerca volvulus, Onchocerca ochengi, Dracunculus medinensis, Litomosoides sigmodontis, Haemonchus contortus, Trichinella spiralis or Trichuris muris. 6. Antigens according to one of claims 1-5, characterized in that they have molecular weights of about 4, 14, 18, 25, 29-30, 40, 44, 52, 56, 60, 66 or 68 kD after sieve gel chromatography and from A. viteae are available, or thus immunologically cross-reactive antigens. 7. Antigens according to any one of claims 1-5, characterized in that they have molecular weights of about 800, 400, 212, 180, 1 16, 94, 90, 67, 63, 60, 56, 52, 40 or 17 according to SDS Have PAGE and are available from A. viteae, or thus immunologically cross-reactive antigens. 8. Antigens according to claim 6 or 7, characterized in that they originate from D. immitis, M. martini, L. sigmodontis, H. contortus or C. elegans. 9. Antigens according to any one of claims 1-5, characterized in that they have molecular weights of about 14, 16, 26, 28, 30, 46, 49, 58 and 66 kD according to sieve gel chromatography and are obtainable from C. elegans, or so immunologically cross-reactive antigens. 10. Antigens according to any one of claims 1-5, characterized in that Have molecular weights of about 46, 60, 97, 109, 212 or 400 kD according to SDS-PAGE and are obtainable from C. elegans or thus immunologically cross-reactive antigens. 11. Antigens according to claim 6, characterized in that they have a molecular weight of about 60 kD and (a) an N-terminal amino acid sequence Xaa-Thr- (Val, Leu) -Lys-Glu- (Glu, Leu) -Ile-Ala- (Phe, Val) -Leu- (Phe, Leu) -Xaa-Leu-Leu-Gly- where Xaa means any amino acid, or (b) have an N-terminal amino acid sequence which is at least 80% homologous to the sequence from (a). 12. Antigens according to one of claims 5-1 1, characterized in that they have a serine / threonine protein kinase activity. 13. A method for obtaining antigens according to any one of claims 1-12, characterized in that the larvae of nematodes in the L4 stage are cultivated in vitro, the culture supernatant is separated off and the antigens are isolated from the culture supernatant. 14. The method according to claim 13, characterized in that the isolation comprises a Siebgelchromatographieschnitt. 15. The method according to claim 13 or 14, characterized in that larvae of A. vitaea are used in the stage 13 days post infectionem. 16 Pharmaceutical composition, characterized in that it comprises one or more antigens according to one of claims 1-12 as an active ingredient and optionally pharmaceutically customary additives, carriers and auxiliaries 17 Use of antigens according to any one of claims 1-12 Diagnostics, prevention and therapy of nematode infections 18 A method of immunization against a nematode infection, characterized in that one or more antigens according to one of claims 1-12 are administered in a pharmaceutically contractual form 19 Pharmaceutical composition, characterized in that it contains attenuated larvae of parasitic nematodes in the L4 stage as an active ingredient and, if appropriate, pharmaceutically customary additives, auxiliaries and carriers 20 Composition according to claim 19, characterized in that the attenuated larvae have been subjected to in vitro cultivation for at least 2 days 21 Use of attenuated larvae of parasitic nematodes in the L4 stage for the prevention and therapy of nematode infections 22 Use according to claim 21, characterized in that the attenuated larvae have been subjected to in vitro cultivation for at least 2 days 23 Use according to claim 21 or 22 in combination with one or more antigens according to one of claims 1-12 24 Method for immunization against a nematode infection, characterized in that attenuated larvae of parasitic nematodes are administered in a pharmaceutically contractual form A method according to claim 24, characterized in that the attenuated larvae have been subjected to in vitro cultivation for at least 2 days A method according to claim 24 or 25, characterized in that the administration is carried out in combination with one or more antigens according to one of claims 1-12