GB1580539A

GB1580539A - Orally administrable vaccine

Info

Publication number: GB1580539A
Application number: GB2602477A
Authority: GB
Original assignee: Institut National de la Sante et de la Recherche Medicale INSERM
Current assignee: Institut National de la Sante et de la Recherche Medicale INSERM
Priority date: 1976-06-25
Filing date: 1977-06-22
Publication date: 1980-12-03
Also published as: BE855776A; FR2355507B1; DE2728806A1; NL7706871A; FR2355507A1

Description

(54) ORALLY-ADMINISTRABLE VACCINE (71) We, INSTITUT NATIONAL DE LA SANTE ET DE LA RE CHERCHE MEDICALE (I.N.S.E.R.M.) of 13-17, rue Camille Guerin, 59000 Lille, France, a National Institution organised and existing according to the laws of the French Republic, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- The present invention relates to an orally-applicable antiparasite vaccine, to a process for preparing such a vaccine as well as its method of application to mammals.

Trichostrongylides and strongylides, at the digestive entry gate in mammals, and Trichinella spiralis even now represent a serious problem in human and veterinary pathology.

Parasitic illnesses at the digestive entry gate affect man and numerous wild and domestic animals. Among the nematodae, the digestive and respiratory stronglylatae in ruminants, which are endemic in all the emerging countries, have a considerable effect on the economy of these countries by the losses which they cause, either directly or indirectly. They cause mortality, ante-natal mortality, miscarriages, loss of meat and/or milk by lowering performance and by causing seizures.

In France, the losses solely attributable to stronglylatae in sheep and cows was evaluated, in 1974, at 811 million francs.

The Trichinella spiralis parasite belongs to another family of nematodae and infests its host, like the strongyldatae, through the digestive tract. The infesting larvae attack the muscles of the animals attacked with trichinosis. In France, the repercussions of trichinosis are quite negligible. However, Trichinella spiralis, is an extremely good experimental model for studying immunity and immunoprophylaxis because its life cycle can be easily maintained in the laboratory.

Trichinella spiralis, which is an ubiquitous parasite in many mammals, causes two .problems to man. Firstly, infection of pork necessitates forced freezing of the meat, which affects the value of the meat. The other problem is medical, in that infection of man causes a disease which is sometimes fatal and for which there is no specific remedy.

This parasitic disease does not exist, so to speak, in France. However, there have been two recent epidemics, in summer 1975 and in February 1976, each of which affected about one hundred people. The disease is still a major problem in the Middle East, the Far East, Inter-tropical Africa, Spain, Portugal and the United States of America. The actual prophylaxis of trichinosis is solely based on the track ing down of infected pigs, this being done by veterinary control (trichinoscopy) and the isolation from breeding stock and from abattoirs of the sources of the virus (wild rodents). In practice, this Is somewhat difficult.

For stronglyloses in mammals, particularly ruminants, the prophylaxis is, in practice, based on sterilisation of the grazing grounds and prophylactic medical treat ment. Very rarely, it is possible to administer a vaccine made from infesting larvae irradiated with X-Rays.

The present invention seeks to provide a method of immunoprophylaxis, which can be extended to the treatment of all animal parasites at the digestive entry gate.

According to the present invention, there is provided a process for the preparation of anti-parasitic oral vaccines comprising the steps of collecting infesting larvae of the parasite, culturing the infesting larvae of the parasite in a synthetic culture medium free of proteins and of antigenic substances for a period sufficient to obtain an optimum yield of secretion and excretion "metabolic" antigens of the parasite, separating the "metabolic" antigens from the culture medium and formulating the metabolic antigens into an orally acceptable form to give optimum protection against the parasites.

The stages of the process will now be described in greater detail.

The collection is effected from animal material infected with parasites as a function of the biological cycle of the parasite. For example, in the case of Trichinella spiralis, it is collected from the muscles of laboratory animals experimentally infected.

In the case of Haemoncus contortus, it is collected from infested larvae of the type obtained from a culture. The culturing of the previously collected parasitic material may be effected at 37 C. The culture is continued for a time sufficient to obtain, in the medium, the secretion and excretion metabolic antigens of the parasite, which antigens are subsequently used to form the active constituent of the vaccine.

The separation of the active constituents from the culture medium may be effected as follows:- a- Separating the parasites from the culture medium by centrifuging at 4" C.

b- Collecting the supernatent liquid from the centrifuging operation.

c- Removing of the elements of the original medium by dialysis with distilled water or other liquids at 40 C.

d- Collecting the dialysed medium containing the components of the vaccine.

The vaccine may be made up into unitary dosage form or into bottles or sachets.

There is thus obtained an orally-assimilatable vaccine which can be mixed with animal feeds, and can thus be easily administered.

The invention will be further described, by way of example, with reference to a specific process for preparing a vaccine in accordance with the present invention which is active against Trichinella spiralis. Thereafter, the manner in which pigs and mice can be vaccinated with this vaccine, and the results obtained thereby will be described.

Vaccination of Pigs and Mice Against Trichinella spiralis.

1) Preparation of Antigens of Trichinella spiralis.

All the antigens used are prepared from types of infesting larvae of Trichinella spirals. These are obtained from rats experimentally infected in a laboratory. The rats are slaughtered one month after infection, entirely dismembered, the whole carcass is then passed through a mincer and the meat thus obtained in put into a very acid chlorhydropepsic solution at 37 C and subjected to continuous agitation tor a period of 16 hours. After 16 hours, the meat is totally digested and is filtered through gauze to remove moulds formed during such digestion. The media containing the larvae is recovered and decanted into ampules. The larvae are recovered after half an hour to an hour by sedimentation. These larvae are then washed three times in a physiological medium, (9% sodium chloride) for the preparation of the antigens.

Different types of antigens have been used, for example, somatic antigens prepared by the extraction from the larvae. This preparation, which has been used for notwholly-satisfactory vaccination trials also for the carrying-out of surveillance tests for retarded hypersensitivity, will not be described in detail save that it is effected by crushing the larvae in a 1% solution of sodium chloride in a Potter-type apparatus followed by six successive freezings and thawings in 1% sodium chloride. The extraction product is subsequently centrifuged and the supernatent dialysed for 24 to 36 hours with distilled water at 4" C. The product is lyophilized and there is obtained a powder known as soluble somatic antigen which is titrated for nitrogen in accordance with the Dumas method. This first antigen has a nitrogen content of 10 to 12%. A second type of somatic antigen known as insoluble somatic antigen is obtained from this process. This is the insoluble product recovered after the centrifuging stage mentioned hereinbefore.

Metabolic antigen is obtained in a similar manner from the larvae infesting the muscles. This antigen is obtained by the following method. The larvae are collected, washed and then cultured, at 37 C, in a 199 synthetic media deprived of proteins and all antigenic substances. A "199" synthetic culture medium is the mediumascribed that number in the Standard International Nomenclature of culture media.

The culturing is effected for 24 to 36 hours, the larvae thereafter being separated by centrifuging the culture medium. The culture medium is then dialysed with distilled water at 4" C for 48 hours. The dialysate is then lyophilised and a white powder which is metabolic antigen is obtained. This is titrated for nitrogen in accordance with the Dumas method, and has a total nitrogen content of from 1 to 2%. Adjuvants have been used in certain related experiments, usually Freund's complete adjuvant, sold under the Registered Trade Mark Difco or Corinebacterium parvum adjuvant containing 2 mg of bacteria for 0.1 ml of a 9% saline physiological serum.

2) Infestation of Animals.

All the animals were infected with muscle-attacking larvae and by gastric cannulation in accordance with the technique described by Larsh and Kent in 1949. The implantation of millipore chambers has been made in certain experimental tests in accordance with the technique described by Despommier and Wostmann in 1968.

Moreover, certain animals were infected by intravenous injection with new-born larvae in accordance with the technique described by Dennis and collaborators in 1970.

3) Parasitological Control and Study of the Protection obtained.

Mice were slaughtered one month after being infected and the carcasses digested individually in accordance with the precedingly described Larsh and Kent method.

250 g of muscle from each piglet was used, the muscle being taken from 25 different areas, always the same areas being utilised. The percentage protection is calculated in accordance with the following formula: % of protection=

number of larvae recovered from test animals X X 100 number of larvae recovered from control animals 4) Inhibition Test and Display of Macrophages.

This test was conducted in accordance with the method described by Vernes and collaborators in 1975. The display index of the macrophages is calculated in accordance with the following formula: % of Macrophages Displayed in the Presence of Antigen X 100 % of Macrophages Displayed in the Absence of Antigen All of the results were submitted to a statistical study using variance analysis and test " t " of Student and Fisher for the comparison of the series.

lI-RESULTS.

1. Retarded Hypersensitivity.

The results obtained in the different experiments are set forth in Table 1.

TABLE 1.

Kinetics of the retarded hypersensitivity of the CBA mouse as a function of the method of immunisation. The index of the display of macrophages (I.E.M.) of the non-infected control animals is 98.7.

I.E.M. at day Experimental conditions 4 7 14 28 60 120 180 Infection 96.1 47.2 82.4 68.3 69.2 108.1 Diffusion chamber - 50 69.7 59.7 67.1 - - New born larvae - 86.6 59.6 56.9 67.2 - - Ag. sol. som 2mgS/C - 102.9 100.3 97.2 - - - Ag. Ins. -som 2mgS/C - 49.4 50.4 42.9 65.1 - - Ag. Metab.

0.5 mgS/C i! 68.8 64.4 48.7 63.7 - - Ag. metab. 63.155 2 mg S /C 83.3 47.1 59.7 46.4 Ag. metab. -51.965.7 2mgOral 72.8 72.8 53.6 43.6 The immunisation of mice with diffusion chambers containing the same number of muscle-attacking larvae as that given for the infections produced an identically retarded hypersensitive state.

The infection of mice with the newly born larvae gives comparable results with a delay of one week. The immunisation with antigenic extracts of muscle-attacking larvae produces from the first week following a single injection, a significantly retarded hypersensitive state except when soluble somatic antigen is used. It is to be noted that the metabolic antigens are very efficacious in the production of retarded hypersensitivity, especially when immunisation is effected orally. It is finally also to be noted that mice immunised orally with metabolic antigens, a very significantly lowered macrophage display index is observed six months after immunisation, whilst in infected mice, one notes a normal index from the fourth month following infection.

2. Attempt to Protect Mice.

A/ Immunisation with antigens alone. The results are collated in Table II: TABLE II.

Immunity against oral infection by Trichinella spiralis in CBA mice immunised by a primo-infection or by different antigens. The percentage protection is calculated from the test animals on the 30th day following the infection. The testing of the immunised mice is taken at variable times after immunisation.

% of protection after infestation of test at moment X (days) after immunisation Experimental conditions 4 7 14 21 28 60 120 Primo infestation 59 59.6 - 72.5 - - Ag. sol. som. 2mg S/C 23.7 26.7 10.7 55.2 67.5 50.6 26.6 Ag. Inn. som. 2mg S/C 25.8 43.5 34.5 51 64.6 55.2 21.3 Ag. Metab. 2mg S/C 54.9 63 61.8 88.4 93.1 63.4 33.8 Ag. Metab. 3 x2mg S/C - - 64.1 79.4 92.3 Ag. Metab. Ag. 2 mg Oral 90.5 98 98 98.9 97.4 - 100 The immunisations with the somatic extracts do not succeed in producing an immunity any greater than that induced by a primo-infestation. On the other hand, the metabolic antigens produce a very significant protection. Subcutaneous im munisation produces protection which attains its maximum level at the end of the first month following immunisation. Oral immunisation gives good protection from the first day following the immunisation, this immunity persists during throughout the experimentation period and attaining 100% protection at the end of 120 days.

B/ Effect of Adjuvants.

The adjuvants of Freund and Cornebacterium parvum have been tested to ascertain their ability to augment the protection achieved utilising antigenic extracts.

Tables III and IV summarise the results obtained from these tests.

TABLE III.

Effect of adjuvants on immunity against an oral infestation by Trichinella sptralis in CBA mice immunised with somatic antigen larvae with and without Corynebacterium parvum. The percentage protection is calculated from tests carried out on the 30th day following infestation.

% of protection after infestation of test at moment X (days) after immunisation l Experimental conditions 4 7 14 21 28 60 120 AG. Sol. som. S/C 23.7 26.7 10.7 55.2 67.5 50.6 26.6 Ag. Sol. som. Ag. +Cp S/C 18.5 23.4 8.1 53.7 60.7 45.4 20.2 Ag. Ins. som 25.8 43.5 34.5 51 64.6 55.2 21.3 Ag.Ins.som+Cp S/C 21.9 38.2 31.9 48.7 61.5 54.7 17.9 TABLE IV.

The effect of adjuvants on the immunity against oral infestation by Trichinella spiralis in CBA mice immunised with metabolic larvae antigens with or without Corynebacterium parvum or a complete adjuvant of Freund. The percentage protection is calculated from tests carried out on the 40th day following infestation.

% of protection after infestation of test at moment X (days) after immunisation Experimental conditions 14 21 28 60 120 Metab. Ag. 2mg S/C 61.8 88.4 93.1 63.4 33.8 Metab. Ag. 2mg + Cp S/C 59.9 84.4 92.6 59.3 3.1 Metab. Ag.3 x2mgS/C 64.1 79.4 92.3 65.9 Metab. Ag. 3 X2mg +FCA S/C 63.5 84.5 93 72 The adjuvants utilised do not show a significant increase in degree of protection obtained compared with the antigen used alone. It can, however, be noted that Corynebacterium parvum lowers the degree of protection as predicted by Ruitenberg and Steerenberg's publication in 1973.

3. Attempt to Protect Piglets.

The results observed in test piglets immunised one mouth before infestation are summarised in Table V.

TABLE V.

Immunity against an oral infestation by Trichinella spiralis in the piglets immunised orally with metabolic antigens one month before infestation.

Experimental conditions No. of animals No. of Larvae/g. % protection Animals infested by 5000 larvae 3 943 Metab. Ag. 50 mg Oral 5 64.6 * 93.2 Metab. Ag. 3 x 50 mg Oral 3 33 * 96.5 * Only 15% of the larvae found in the immunised animals are infestants for mice.

The results are substantially identical to those obtained in CBA mice. It is to be noted that only 15% of the muscle-attacking larvae found in the immunised animals are infestants for CBA mice. This means that the percentage protection observed is 99% and 99.5% in the animals immunised with a dose of 50 mg and with three doses of 50 mg. respectively.

III CONCLUSIONS.

The results obtained by Despamier and Wostmann in 1968 suggest that the immunity is produced by the metabolic products of the muscle-attacking larvae.

The results show that, under the same conditions, retarded hypersensitivity becomes apparent from the first week following immunisation. On the other hand, Denham has demonstrated by the intermediary of infestations cut short by methyridine that these induced a fairly solid immunity. The results that the inventor has obtained with metabolic antigens are in accordance with the results obtained in these preceding works.

It is to be noted that the percentage protection varies generally with retarded hypersensivity. One could think that the immunity observed by these artificial immunisations is essentially mediated by cellular phenomena, that it is principally localised at the level of the intestine and that it is directed against infesting larvae.

To be efficacious, the immunisation must stop infection before the infesting larvae are transformed into adult worms. Immunisation orally with metabolic antigenic extracts of infesting larvae appears to attain this object. The desired protection should approach 100% and the duration thereof should be 6 months. The obtaining of an efficacious oral vaccination in the piglet against Trichinella spiralis, utilising metabolic antigenic extracts of infesting larvae, can be extended to other parasites at the digestive entry gate, such as the strongyloses of ruminants.

Anti-parasitic substances such as L-tetramisole and the Levamisole administered simultaneously with the vaccines can be utilised as adjuvants.

WHAT WE CLAIM IS: 1. Process for the preparation of anti-parasitic oral vaccines comprising the steps of collecting infesting larvae of the parasite, culturing the infesting larvae of the parasite in a synthetic culture medium free of proteins and of antigenie substances for a period sufficient to obtain an optimum yield of secretion and excretion metabolic antigens of the parasite, separating the " metabolic " antigens from the culture medium and formulating the metabolic antigens into an orally acceptable form to give optimum protection against the parasites.

2. A process as claimed in claim 1 wherein one or more adjuvants are added to the vaccine.

3. A process as claimed in claim 1, wherein the collection is effected from a parasitic animal material as a function of the biological cycle of the parasite, 4. A process as claimed in claim 1, wherein the culturing is effected at 37 C, the culture medium being a synthetic survival medium free of proteins or of antigenic substances, the culturing being of forms of the parasite previously collected the said culture heated for a time sufficient to obtain metabolic secretion and excretion products of the parasite in the media.

5. A process as claimed in claim 1, wherein the separation of the active products from the culture medium comprises the steps of separating the parasites from the culture media by centrifuging at 40 C, collecting the supematent thus obtained, removing the culture media by dialysis with distilled water or other liquids at 40 C, and collecting the dialysate containing the active constituents of the vaccine.

6. A process as claimed in Claim 1, wherein the vaccine is put into unitary dosage form, bottles or sachets.

7. An anti-parasitic orally-administrable vaccine prepared by a process as claimed in any one of claims 1 to 6.

8. A process for immunising mammals other than human beings, comprising orally administring a vaccine as claimed in claim 7.

9. A process as claimed in claim 8 wherein an anti-parasitic adjuvant is administered simultaneously with the vaccine.

10. A process as claimed in claim 9 wherein the adjuvant is L-tetramisole or Levamisole.

11. A process for preparing a vaccine as claimed in claim 1 substantially as hereinbefore described.

12. A vaccine when prepared by the process claimed in claim 11.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. The results show that, under the same conditions, retarded hypersensitivity becomes apparent from the first week following immunisation. On the other hand, Denham has demonstrated by the intermediary of infestations cut short by methyridine that these induced a fairly solid immunity. The results that the inventor has obtained with metabolic antigens are in accordance with the results obtained in these preceding works. It is to be noted that the percentage protection varies generally with retarded hypersensivity. One could think that the immunity observed by these artificial immunisations is essentially mediated by cellular phenomena, that it is principally localised at the level of the intestine and that it is directed against infesting larvae. To be efficacious, the immunisation must stop infection before the infesting larvae are transformed into adult worms. Immunisation orally with metabolic antigenic extracts of infesting larvae appears to attain this object. The desired protection should approach 100% and the duration thereof should be 6 months. The obtaining of an efficacious oral vaccination in the piglet against Trichinella spiralis, utilising metabolic antigenic extracts of infesting larvae, can be extended to other parasites at the digestive entry gate, such as the strongyloses of ruminants. Anti-parasitic substances such as L-tetramisole and the Levamisole administered simultaneously with the vaccines can be utilised as adjuvants. WHAT WE CLAIM IS:

1. Process for the preparation of anti-parasitic oral vaccines comprising the steps of collecting infesting larvae of the parasite, culturing the infesting larvae of the parasite in a synthetic culture medium free of proteins and of antigenie substances for a period sufficient to obtain an optimum yield of secretion and excretion metabolic antigens of the parasite, separating the " metabolic " antigens from the culture medium and formulating the metabolic antigens into an orally acceptable form to give optimum protection against the parasites.

3. A process as claimed in claim 1, wherein the collection is effected from a parasitic animal material as a function of the biological cycle of the parasite,

4. A process as claimed in claim 1, wherein the culturing is effected at 37 C, the culture medium being a synthetic survival medium free of proteins or of antigenic substances, the culturing being of forms of the parasite previously collected the said culture heated for a time sufficient to obtain metabolic secretion and excretion products of the parasite in the media.

12. A vaccine when prepared by the process claimed in claim 11.