United States Patent:   6,022,728

Assignee:  Board of Trustees operating Michigan State University (East Lansing, MI)

Filed:  November 10, 1997

A new method for the preparation of a whole cell bacterial vaccine for improved protection against bacterial pathogens, particularly against those pathogens which have multiple antigenic serotypes is described. The method particularly involves disruption of the whole cells using a French press of the whole cells of virulent strains of the bacteria. The preferred vaccine prevented infection of swine with Actinobacillus pleuropneumoniae (APP), which causes porcine contagious pleuropneumonia. The vaccine has good safety and few side effects even at a higher dose than commonly used for bacterins and improved protection against the homologous serotype of the pathogen; and improved cross-protection against heterologous serotypes. The whole cell vaccine, is most useful for veterinary (lower mammal) vaccines.

GENERAL DESCRIPTION OF THE INVENTION

The present invention relates to a method for producing a vaccine from cells of a pathogenic bacterium which produces a disease in a host which comprises: isolating a strain of the bacterium from the host in a virulent form by passage of the strains through the host; growing the isolated strain in a growth medium; harvesting the strain from the growth medium; disrupting the cells of the strain in an aqueous solution for a short period of time to expose capsular and subcapsular antigenic materials; and preserving the antigenic materials in the aqueous solution with a preservative agent which kills the bacterium to provide the vaccine wherein the vaccine provides immunity to the disease when injected into the host.

The present invention relates to a method for producing a vaccine from a pathogenic bacterium which produces a disease in a host which comprises: isolating a strain of the bacterium from the host in a virulent form by passage of the strains through the host; growing the isolated strain in a growth medium; harvesting the strain from the growth medium; sonicating the strain in an aqueous solution for a short period of time to expose capsular and subcapsular antigenic materials; and preserving the antigenic materials in the aqueous solution with a preservative agent which kills the bacterium, preferably without impairing the antigenic materials, to provide the vaccine, wherein the vaccine provides immunity to the disease when injected into the host. It was particularly unobvious to one skilled in the art that sonicated whole cells could provide a useful vaccine, since disrupted cells contain so many cellular components, and that purification of the outer membrane was unnecessary. It was further unobvious that cross-reactivity against various strains of APP could be achieved using this method. The invention also relates to vaccines prepared by this method, particularly vaccines containing hemolysin enhanced supernatant.

It is preferred to use a hemolysin enriched supernatant (HES) which is produced by APP. The HES contains hemolysin and cytotoxins (Hly/Cly) produced by APP, which are high molecular weight extra-cellular protein toxins. These include Hly/Cly I which is 105 Kd; Hly/Cly II which is 103 Kd and Cly III which is 120 Kd. There are most likely other toxins produced. These proteins kill host red and/or white blood cells. HES is desirable for use in the vaccines of the present invention to insure broad immunity, but is not necessary.

In the present invention, the following factors are important in producing a commercially useful vaccine:

1. The selection of bacterial strains. Through several years of experience working with bacteria, particularly Actinobacillus pleuropneumoniae (APP), it has been learned that it is very important to use carefully selected strains as freshly isolated from the host animal as possible. Virulence and thus expression of factors necessary for virulence which should be included in any vaccine as important antigens, can be increased up to 1,000-10,000 fold in this manner.

2. The media and growth conditions. Media and growth conditions are selected which optimize the production of important virulence factors of this bacterium, including capsular polysaccharide, hemolysins, and common outer membrane proteins.

3. The choice of conditions for production of sonicated cells.

4. The choice of adjuvant and vaccination schedule.

5. The choice of vaccine preservative. The most commonly used preservative, formalin, reduced the efficacy of the vaccine against heterologous serotypes of APP.

6. The maximum safe dosage. A safety trial was conducted with APP vaccine comparing different dosages for both safety and efficacy in inducing an immune response.

7. Safety and immunogenicity in the field. A field trial was conducted to test the efficacy of APP vaccine in a swine herd with a chronic problem with APP and judged to be effective.

The new method for the preparation of bacterial vaccines is designed to produce a vaccine that provides improved protection against bacterial pathogens, particularly against those pathogens which have multiple antigenic serotypes. Because the product is a modified whole cell vaccine, this procedure is most useful for veterinary rather than human vaccines wherein whole cell vaccines may be perceived as too risky.

The particular pathogen-host system that was utilized in the development of the method of the present invention is Actinobacillus pleuropneumoniae infection in swine. This is a representative gram-negative bacterial pathogen that causes pleuropneumonia. There are at least twelve (12) different antigenic serotypes of this organism. Current commercial vaccines are generally formalinized or heat killed whole cell bacterins. These bacterins provide moderate protection against infection with the homologous serotype, that is, the serotype used to produce the vaccine, but minimal protection against heterologous serotypes. In contrast, infection with the live organism generally elicits an immune response that protects against subsequent infection with any serotype. These data suggested that there are common antigens shared among the serotypes that might elicit cross-protective immunity, but that these antigens are not well exposed in whole cell bacterin vaccines. The method of the present invention enhances exposure of these antigens, which in APP are subcapsular outer membrane proteins and lipopolysaccharides and hidden capsular antigens, to produce a more effective, cross-protective vaccine.

An important component of this procedure is disruption of the bacterial cells prior to the addition of any preservative in a manner which enhances the exposure of important subcapsular antigens. The methods are well known and include sonication or the use of a French press which disrupts the cells by a sudden reduction of pressure on a fluid. Once bacterial cultures have been grown and harvested, cells are sonicated to rupture the cells and enhance exposure of outer membrane antigens. After sonication, preservatives, such as sodium azide, are added.

It is important to use either fresh clinical isolates or type strains that have been freshly passaged through a host animal to regenerate production of virulence factors. All strains used should be isolated from infected animal tissues and frozen at -70^oC. or lyophilized immediately. For production of vaccine, bacteria should be plated from the storage vial and used immediately for vaccine production, with no subsequent subculture.

The conditions for sonicating the cells are important. Preferably the sonication is between about 30 and 90 seconds at about 20 KHz output. The duty cycle is preferably 30 (percent of time of sonication) and the output is preferably 40 (percent of maximum amplification). A suitable instrument is a Branson Model 250 Sonifier (Dansbury, Conn.). This enables the exposure of the capsular and subcapsular antigenic materials without destroying them. Sonication for too long a time reduces the antigenic character of the vaccine. Usually sonication for less than two (2) minutes is sufficient.

The media for growth depends upon the bacterium being used and is selected to provide maximum virulence. Bergey's Manual of Determinative Bacteriology (8th Edition 1974) or later editions describe media for bacteria. The bacteria which are disease producing and thus suitable for vaccine production are also described.

The preservative agent which is preferred is sodium azide. This compound blocks electron transport within the membrane of the cells so that there is no generation of energy for the cells and thus the cells die. The compound is a "metabolic poison" which does not tan the proteins. Other useful preservative agent compounds are beta propiolactone, thimerosal, and binary ethyleneimine. The compounds assure that the cells are non-living. Preservatives that are metabolic inhibitors rather than fixatives are preferably used.

The preferred adjuvant for the vaccine was Emulsigen.TM. (MVP Labs, Ralston, Nebr.), which is a paraffin oil in water emulsion, since it can be used in food animals. Freunds Incomplete Adjuvant. which is 15 percent by weight mannide monooleate and 85% paraffin oil, available from Difco, Detroit, Mich. can be used in non-food (i.e. laboratory animals). The adjuvants aid in slowly releasing the vaccine into the animal and in potentiating the immune response. Any commercial oil emulsion adjuvants can be used but not aluminum hydroxide.

Claim 1 of 11 Claims

1. A method for producing a disrupted whole cell vaccine from cells of Actinobacillus pleuropneumoniae for injection into swine to produce immunity which comprises:

(a) isolating cells of a strain of the Actinobacillus pleuropneumoniae from a swine infected with the strain which are virulent as a result of passage of the strain through the first swine;

(b) growing the strain which is isolated in a growth medium;

(c) harvesting the virulent cells of the strain from the growth medium;

(d) disrupting the harvested virulent cells of the strain using a French press which disrupts the cells by a sudden reduction of pressure on the growth medium in an aqueous solution to obtain a mixture containing disrupted cells, capsular and subcapsular antigens; and

(e) preserving the mixture with a preservative which kills the Actinobacillus pleuropneumoniae to provide said vaccine.

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