WO2000023106A1

WO2000023106A1 - Bacterial extract as oral adjuvant

Info

Publication number: WO2000023106A1
Application number: PCT/GB1999/003345
Authority: WO
Inventors: William Leslie Porter
Original assignee: William Leslie Porter
Priority date: 1998-10-19
Filing date: 1999-10-19
Publication date: 2000-04-27
Also published as: AU6349799A; GB2358352A; GB0108406D0; GB9822619D0

Abstract

A killed bacterial preparation for oral administration to humans or animals, especially to enhance immune stimulation, more particularly comprising a lipid of controlled particle size such that the components of the bacterial cells are made available to immune sensitive areas of the body.

Description

BACTER IAL EXTRACT AS ORAL ADJUVANT

This invention relates generally to immune response stimulation in man and animals and more especially to a bacterial preparation for enhancing immune response, effective by oral administration.

It is known that certain killed bacteria, and extracts thereof, given orally to animals, can enhance immune response to certain test antigens. Enhanced immune response caused by certain bacterial peptidoglycans, especially n-muramyl dipeptide, has also been reported.

Enhancement of growth rate in young animals and reduction of the incidence of diarrhoea, in animals orally receiving killed bacterial preparations, has also been reported, and it is also known that enhancement of growth rate is achievable in animals receiving lipid-containing extracts of such bacterial preparations adsorbed into paniculate matter, as well as in animals receiving similarly administered preparations of whole killed bacteria.

It is a feature of the above-described known art that the demonstrated effects of killed bacterial preparations and extracts thereof on antibody response to test antigens, on animal growth rate and on the incidence of diarrhoea are inconsistent, of indeterminate origin and are still generally poorly understood.

Nevertheless, control of infectious diseases in man and animals is a subject of major interest and research, since existing means of control have known deficiencies. Thus, antibiotics are continually rendered ineffective by the development of resistant strains of micro-organisms, while vaccines are highly specific. Their effectiveness is vulnerable to challenge by organisms of different or altered antigenicity. There thus exists a place for the broad spectrum enhancement of the natural immune response of man and animals to infections and to vaccines.

A principal aim of the present invention is therefore to facilitate access to the body's immune system of bacterial lipid factors from orally administered killed bacterial preparations, whereby to enhance the immune response to antigenic stimulus.

It is also known that many antigenic substances gain access from the gut to the reticulo-endothelial system via lymphoid tissue in the wall of the intestine, for instance Peyer's patches. It is also known that such materials gain access more readily via the lymphoid tissue in the intestine wall if they are of a paniculate nature, since particles of a few microns dimension may adhere, for instance, to the Peyer's patches.

However, in the production of killed bacterial preparations intended or proposed for use in oral immune enhancement or growth enhancement or diarrhoea control hitherto, no regard has been paid to particle size.

The present invention, in its broadest aspect, therefore provides for the production of killed bacterial preparations or extracts thereof for the purpose of enhancing immune response, wherein the bacteria are subjected to a degradation step to render lipid components of the bacterial cells available to receptive areas of the gut wall, as well as the digestive, reactive and absorptive processes of the stomach, by oral administration, the degradation step being controlled so that the particle size of said components, either separately or in physical association with other materials, lies within predetermined limits.

The degradation step may typically be by use of an enzyme, by autolysis, by heat treatment such as boiling, autoclaving, irradiation (e.g. gamma radiation) or microwaving, or by chemical treatment.

In accordance with the present invention, the particle size of the degraded or partially degraded bacteria or particle-associated extracts thereof is controlled within the predetermined limits by:-

a) stopping the enzymatic action or autolysis at the appropriate point, e.g. by application of heat or other enzyme-degrading means; or

b) stopping at the appropriate point heat treatments, e.g. boiling or autoclaving or irradiation procedures, or chemical treatment; or

c) attaching lipid extracts of the bacteria to particles such as liposomes or other appropriately sized particulate material.

That the particle size is within the predetermined limits may be established by known methods, e.g. microscopic examination or by sizing techniques employing beams of radiation such as laser beams, and by readily available sizing instruments such as the Malvern Mastersizer.

Particle size of degraded or partially degraded bacteria may be further controlled by standard filtration techniques, so that particles of sizes outside the predetermined limits are completely excluded. Particles of differing densities may also be separated by centrifugation, for the same purpose.

The predetermined limits on degraded or partially degraded bacteria particle size may be 0.1 microns to 50 microns and more specifically 0.2 to 25 microns. Ideally, 0.2 microns up to 10 microns or even up to only 5 microns is preferred. In this connection it is to be understood that early termination of the degradation step can result in components of particle size greater than the original bacterium. The lipid bacterial extracts or degraded bacteria may be incorporated into paniculate formulations of dry or wet powder or granular mixes, where in the latter case the liquid may be water, an edible oil, milk, a juice, alcohol or any beverage. The degraded bacteria may also be incorporated as suspensions in any of the aforementioned liquids.

The lipid bacterial extracts or finely degraded bacteria may also be mixed with oil, which may be dispersed in water or other suitable liquid with or without the aid of emulsifying agents such as Crillet 4, forming an emulsion in which the bacterial extracts or degraded bacterial product is contained within or bound to the dispersed phase. Oily bacterial extracts may be mixed directly with emulsifying agents to form emulsions.

In the case of carrier particles, these should preferably have a size range of 0.2 to 100 microns, preferably 0.3 to 50 microns and most preferably 3 to 10 or even to only 5 microns.

Preferably, after completion of the degradation step, one or more lipid components of the degraded or partially degraded bacteria are extracted. The extracted component or components, or paniculate materials to which the component or components are attached, will be of a particle size within the said predetermined limits. Lipid extraction can be achieved for example, by treating the degraded or partially degraded bacteria with a chloroform/methanol mixture, but other chemical treatments such as butanol can achieve the same result.

A preferred aspect of the invention thus provides a killed bacterial extract comprising lipid particles of dimensions within the above stated predetermined limits.

Two verification examples are now described. Example 1

Comparison is made between two tests of immune globulin production in mice, following oral administration of a test antigen, where the mice received daily doses of partially autolysed and autoclaved Bacillus subtilis.

In test A, Bacillus subtilis was grown in a clear liquid medium containing yeast extract, sucrose, magnesium and other soluble nutrients, according to well established procedures. The bacterial cells were separated from the culture medium by centrifugation and allowed to stand for 24 hours at room temperature. The cells were examined grossly and microscopically at intervals of about 6 hours. After this period, and before any visible physical breakdown of the bacteria was observed, the cells were autoclaved at 115 degrees C for 20 minutes. Microscopically, the cells all remained substantially intact after autoclaving, with approximate dimensions of 0.5 to 5 x 1 to 5 microns.

The killed bacterial slurry was diluted in water and administered by gastric lavage, daily for 7 days, to 5 mice, at a daily dose rate equivalent to 400 micrograms of bacterial dry matter.

A similar group of 5 mice serving as a control group received daily oral doses of physiological saline.

Both groups were then autogenically challenged with keyhole limpet haemocyanin with cholera toxin. Both groups were then assayed for immune globulins.

The IgA response in the group receiving the Bacillus subtilis preparation exceeded that in the control group by a factor of 9.

In test B, the above procedure was repeated, except in that the Bacillus subtilis preparation was allowed to stand at room temperature for a period of 72 hours, so that autolytic changes progressed to a stage of microscopically visible breakdown of the bacterial cells. After the autoclaving, microscopy revealed that the bacterial preparation had broken down to a fine silt, of maximum particle size 0.25 to 0.5 microns.

The antigenic challenge and subsequent assay of immune globules was then carried out as in test A.

In test B, IgA response to the bacterial preparation was found to exceed that in the control group by a much reduced factor of 2.

Example 1 thus clearly demonstrates the beneficial effect of a killed bacterial preparation given orally as particles of 0.5 to 5 microns, compared to a similarly administered preparation of sub 0.5 micron particles.

Example 2

A comparison is made between two separate tests. In test C, chickens of from 1 to 21 days of age received food supplemented with a methanol/chloroform extract of Bacillus subtilis, at the rate of the extract obtained from 100 mg of Bacillus subtilis dried biomass per kg of feed.

The methanol/chloroform extract, substantially lipid in nature, was applied to a dusty and finely granular preparation of expanded mica, before being incorporated into the feed. Growth rate of treated chickens exceeded that of controls by 14.1 per cent.

In test D, mice received daily a dose of a methanol/chloroform lipid extract of Bacillus subtilis administered by gastric lavage. The daily dose was the extract obtained from approximately 400 mg of dried biomass. Compared to controls, no weight gain enhancement, or enhancement of immune response, was detected.

Example 2 clearly demonstrates the beneficial effect of using an orally administered bacterial preparation or extract in particulate form as compared to a non-particulate form.

Example 3

Bacillus subtilis cells, cultured and subjected to partial autolysis, as in Example 1, test A, were agitated for 48 hours in a mixture of equal parts of chloroform and methanol. Following centrifugation, the supematent was concentrated by evaporation to yield a lipid extract.

The extract was combined with a liposome in a buffered saline solution to give dispersed particles of lipid/liposome of approximately 1 to 3 microns in diameter. This was administered to 5 mice by daily gastric lavage for 5 days, in a total daily dose per mouse of 0.2 ml, giving a total dose per mouse of 0.59 mg of extracted lipid. Five control mice received the same liposome preparation, but without the bacterial lipid extract, suspended in identical buffered saline and also administered at 0.2 ml per mouse per day.

On days 7 and 15, all the mice were immunised by gastric lavage with keyhole limpet haemocyanin (5 mg) in 200 ml of physiological saline containing cholera toxin (10 μg). On day 21, all the mice were bled and mesenteric lymph nodes removed.

Tests of ELISA of specific antibodies in serum showed levels of IgA in the lipid extract treated mice to exceed the control mice by a factor of 8. Cell cultures of mesenteric lymph nodes showed T cell proliferation in nodes of treated animals in response to keyhole haemocyanin to exceed that in control animals by factors of 35 and 16 following 3 and 5 days of culture.

The results therefore showed marked enhancement of immune response through oral pre-treatment with bacterial lipid extracts given in particles of approximately 1 to 3 microns.

Claims

1. A killed bacterial preparation for oral administration to enhance immune stimulation in man and animals, comprising a bacterial preparation or extract thereof wherein the bacteria are subjected to a degradation step to render lipid components of the bacterial cells available to receptive areas of the gut wall, as well as the digestive, reactive and absorptive processes of the gut, by oral administration, the degradation step being controlled so that the particle size of said components, either separately or in physical association with other materials, lies within predetermined limits.

2. A preparation according to claim 1, constituted by or incorporating a lipid, either separately or in physical association with other materials, of combined particle size controlled to lie within the predetermined limits.

3. A preparation according to claim 1 or claim 2, wherein bacteria are subjected to degradation by any one or combination of heat treatment by boiling or autoclaving, irradiation such as gamma radiation, microwaving or chemical treatment.

4. A preparation according to claim 3, wherein the panicle size is controlled within the predetermined limits by either one or both of:-

a) stopping the enzymatic process or autolysis at a predetermined point, as by application of heat or other enzyme degrading means; or

b) stopping at a predetermined point the heat treatment, irradiation procedure, or chemical treatment; or c) attaching lipid extracts of the bacteria to particles such as liposomes or other appropriately sized particulate material.

5. A preparation according to any of claims 1 to 4, wherein the particle size is at least partly additionally controlled by filtration techniques.

6. A preparation according to claim 4 or claim 5, wherein the particle size is determined to lie within the predetermined limits by a conventional sizing technique such as microscopic examination.

7. A preparation according to any of claims 1 to 6, wherein the predetermined limits are 0.1 microns to 50 microns.

8. A preparation according to claim 7, wherein the predetermined limits are 0.2 to 25 microns.

9. A preparation according to claim 8, wherein the predetermined limits are 0.2 to 10 microns.