A method is described for preparing an antigenic product which incorporates exposing an aerosol of a microbial suspension to temperatures at which substantially only the heat stable components of the microbial suspension which retain their immunogenic properties remain. More specifically, the aerosol is exposed to an elevated temperature which denatures all labile components and removes the liquid portion of the aerosol by evaporation.

FIELD OF THE INVENTION 
This invention relates to vaccines or antigenic compositions and methods of 
preparation thereof. These methods are particularly useful for preparing 
animal vaccines, more specifically fish vaccines. 
BACKGROUND OF THE INVENTION 
Vaccines and/or bacterins (formalin inactivated whole cell suspensions) are 
widely used to prevent diseases in humans and other animals. They usually 
consist of the organism and/or its metabolites responsible for a 
particular disease problem. The organisms may be in either their natural 
form, dead whole cells or live or attenuated or in non-native forms 
comprised of structural subunits of the pathogen or crucial metabolites. 
Almost all gram negative bacteria have a heat stable cell wall structural 
component referred to as lipopolysaccharide (LPS), that may function as a 
protective antigen. That an immune response directed against these 
structural components is sufficient to protect the animal against 
subsequent challenge by the pathogen. As with other animals, fish reared 
for food also suffer from systemic diseases that require prevention and/or 
treatment. 
These diseases are due to variety of pathogens, including viruses, 
bacteria, fungi, protozoans and metazoans. The types of diseases depend 
upon geography (where the fish are being reared), genetics (the species 
and genetic makeup), the environment (i.e., fresh versus brackish versus 
salt water) and the management/husbandry philosophies of those individuals 
rearing the fish. Disease prevention is always preferable to reactive 
disease treatment. Many of the primary bacterial pathogens that affect 
fish can readily be prevented by immunization. 
Fish vaccines generally consist of formalin inactivated suspensions of 
whole cells. These preparations are diluted and administered to fish by 
injection or immersion. Alternative methods include bathing the fish in 
highly dilute suspensions or spraying fish directly with the diluted 
preparation, which is then absorbed through the gill membranes. 
Virtually all commercially available fish vaccines are in liquid form and 
are prepared by either continuous culture or batch fermentation and are 
subsequently inactivated with formaldehyde. They are whole cell-based 
products that depend on a stable cell wall component (LPS) for their 
protective properties. Due to their liquid nature, these products have 
strict refrigeration requirements. Accordingly, a relatively short 
shelf-life can be expected and the products may become contaminated with 
extraneous organisms or even occasionally with living organisms contained 
in the vaccine, particularly if the container of the liquid product is 
opened and resealed improperly. The liquid products are also bulky and 
inconvenient to transport in large quantities. Furthermore, the use of 
liquid based bacterins in oral vaccine preparations may be hampered by 
rancidity problems, standardization of antigen levels and stability 
problems. 
Exemplary of the currently available methods for the preparation of 
vaccines are the following: 
U.S. Pat. No. 3,862,313 to Fryer et al. describes a wet whole cell vaccine 
of Vibrio anguillarum which may be administered orally or by injection. 
The actual preparation of the vaccine comprises growing the bacterial 
cells in a suitable medium, killing the cells by formaldehyde addition, 
harvesting the cells by centrifugation and finally freezing the resultant 
wet cells. For use, the frozen vaccine is thawed into a paste. 
U.S. Pat. No. 3,755,557 to Jacobs teaches the use of lecithin as a 
stabilizer to disperse dry antigens in a liquid propellant for spray 
vaccines. The antigens must be worked up into the vaccine in a dry 
condition. The techniques of freeze-drying and vacuum drying are 
described. 
U.S. Pat. No. 3,608,066 to Illartein describes a pharmaceutical preparation 
of finely ground, killed, lyophilized microorganisims and a suitable 
carrier. It is designed to be used in the upper and lower respiratory 
tracts. The preparation of the antigenic component includes heat killing 
and centrifuging the chosen bacteria, lysing the cells and then 
lyophilizing the resultant suspension by conventional means. The powdered 
antigen is then prepared into an aerosol formulation with a lubricant and 
a propellant. 
U.S. Pat. No. 4,287,179 to Amend provides a liquid vaccine in which whole 
fish are immersed which comprises killed Yersinia ruckeri. 
It is an object of the present invention to obviate or mitigate the above 
disadvantages. 
SUMMARY OF THE INVENTION 
The present invention provides a method of preparing an antigenic product 
which comprises exposing an aerosol of a microbial suspension to 
temperatures at which substantially only the heat stable components of the 
pathogen culture which retain their immunogenic properties remain. More 
specifically, the aerosol is initially exposed to a temperature at which 
substantially only the heat stable components of the microbial suspension 
remain immunogenic and subsequently the components are cooled to a second, 
lower temperature prior to harvesting the product so formed. 
This method is suitable for preparing vaccines which are lipopolysaccharide 
(LPS) based, that is, those in which LPS comprises the primary protective 
antigen. The method is also suitable for preparing vaccines based upon any 
heat stable antigenic determinants. This method is particularly useful in 
preparing animal including, but not limited to, fish vaccines. 
The method of preparation described herein will be referred to as the 
"spray-drying" method. Also included within the scope of the present 
invention are the antigenic products formed by the methods described 
herein. 
The antigenic products of the present invention are particules and retain 
their biological activity after prolonged storage even in non-refrigerated 
conditions. Therefore, unlike the previous liquid based products, they do 
not appear to have a date limitation. This stability makes the product 
invaluable in environments where refrigeration is unavailable and 
significantly reduces the cost associated with transporting the product. 
With respect to the immunization of fish, the heat stability of the 
antigenic products of the present invention makes them attractive 
candidates for milling into feeds where significant heat can be generated 
during the milling process. Furthermore, due to the very low moisture 
content, the antigenic products are resistent to contamination with 
bacteria and/or viable fungi. 
Pathogenic cells contained in conventional freeze-dried vaccine preprations 
retain their structural integrity. Were it not for treatment with 
formaldehyde, these cells would remain viable. In contrast, cells under 
going the spray-drying process of the present invention are rendered 
structurally different from the starting material. This structural 
alteration is due to the heating step within the process which denatures 
the majority of proteins found in the cells. In doing so, the cells are 
rendered non-viable.

EXAMPLES 
Example 1: Preparation of a vaccine for spray drying 
A pure culture of the bacterial fish pathogen, Vibrio anguillarum, was used 
to generate a large volume suspension of V. anguillarum by batch 
fermentation (continuous culture fermentation may also be used). The exact 
fermentation parameters depend on the organisms and strains employed. 
After the fermentation reached the desired endpoint, usually determined by 
biomass, the bacterial culture was inactivated using standard methods in 
many, but not all, cases, by the addition of 0.3 to 0.6% formaldehyde. The 
fermentor vessel was then used to agitate the mixture. After enough time 
had passed to allow for inactivation of the bacterial culture (12-36 
hours), the cell mass was harvested by centrifugation. The use of 
concentrated materials is not in itself critical to the spray drying 
process itself but relates only to the time involved in processing the 
materials. 
The bacterin was then pumped through a commercial spray drier--Niro 
Atomizer, serial #8410 which was fitted with a size 4 nozzle (0.99 um 
diameter). The vaccine was pumped through this nozzle. The water in the 
bacterin was evaporated as the vaccine was deposited on the walls of the 
spray drying vessel. This spray dried material was then harvested as 
discussed herein. The bacterin was exposed to a temperature of no less 
than 70.degree. C. and no higher than 160.degree. C. during the drying 
process. The powdered product was packaged and tested. 
______________________________________ 
Example 2: Oral application of the spray dried bacterin 
Results 
Organ- 
Method of (mortality).sup.4 
ism.sup.1 
Vaccination.sup.2 
Dosage.sup.3 
Control Vaccinates 
______________________________________ 
VA Oral 2 mg/gram diet 
73% 17% 
10 feedings 
2%/day 
top dress 
Oral 5 mg/gram feed 
100% 11% 
10 feedings 
2%/day 
milled into feed 
______________________________________ 
.sup.1 VA Vibrio anguillarum. 
.sup.2 and .sup.3 Fish were fed the vaccine either mixed in oil and mixe 
manually onto feed referred to as top dressing or milled into the feed 
when the feed was manufactured. 
.sup.4 N = 30; approximately 14 days post vaccination, the fish were 
exposed to a virulent suspension of Vibrio anguillarum by being bathed in 
5 .times. 10.sup.6 CFU/ml for 20 min. A group of fish that were fed a die 
that had been handled exactly the way that the experimental diets were 
i.e. top dresscoated with oil containing no bacterin, milledsame diet 
except with no vaccine in it were the control fish. These fish were 
challenged using the same culture and under the same conditions as the 
vaccinates were. The values expressed here are based on thirty fish 
samples and reflect the numbers of fish dying up to 14 day post challenge 
______________________________________ 
Example 3: Immersion vaccination with a spray dried bacterin 
Results 
Organ- 
Method of (mortality).sup.4 
ism.sup.1 
Vaccination.sup.2 
Dosage.sup.3 
Control Vaccinates 
______________________________________ 
VA Immersion 5 mg/ml 20 sec. 
68% 0% 
Immersion 5 mg/ml 20 sec. 
100% 17% 
VO Immersion 5 mg/ml 20 sec. 
68% 0% 
______________________________________ 
.sup.1 VA Vibrio anguillarum; VOVibrio ordalii. 
.sup.2 and .sup.3 The fish were immunized by being immersed in a 
suspension of the material at a concentration of 5 mg of bacterin/ml of 
water for 20 seconds duration. 
.sup.4 Approximately 14 day post vaccination the fish were challenged by 
being exposed to virulent pathogens. Refer to Example 2 for procedure use 
with Vibrio anguillarum. For Vibrio ordalii, the fish were injected 
intraperitoneally with approximately 10.sup.6 bacteria and observed for 1 
days. 
______________________________________ 
Example 4: Stability testing of a spray dried vaccine. 
Results 
Organ- 
Method of (mortality).sup.4 
ism.sup.1 
Vaccination.sup.2 
Dosage.sup.3 
Control Vaccinates 
______________________________________ 
VA Immersion 5 mg/ml 20 sec. 
73% 0% 
(2 yr 
stability) 
______________________________________ 
.sup.1 VA Vibrio anguillarum. 
.sup.2 and .sup.3 The bacterin had been on the shelf for two years (room 
temperature mean approximately 20.degree. C.) and was tested by immersio 
using the same protocol as described in Example 3). 
.sup.4 See previous examples for explanation. 
______________________________________ 
Example 5: Evidence of duration of immunity 
Results 
Organ- 
Method of (mortality).sup.4 
ism.sup.1 
Vaccination.sup.2 
Dosage.sup.3 
Control Vaccinates 
______________________________________ 
VA Immersion 5 mg/ml 20 sec. 
71% 20% 
(156 days post 
vaccination) 
VO Immersion 5 mg/ml 20 sec. 
90.6% 38% 
(180 days post 
vaccination) 
Immersion 5 mg/ml 20 sec. 
100% 40% 
(114 days post 
vaccination) 
______________________________________ 
.sup.1 VA Vibrio anguillarum; VOVibrio ordalii. 
.sup.2 and .sup.3 The fish were immunized by being immersed in a 
suspension of the material at a concentration of 5 mg of bacterin/ml of 
water for 20 seconds duration. 
.sup.4 At the number of day indicated post vaccination the fish were 
challenged by being exposed to virulent pathogens. Consult previous 
examples for procedure used with both organisms.