WO2011048041A1

WO2011048041A1 - Streptococcal combi-vaccine

Info

Publication number: WO2011048041A1
Application number: PCT/EP2010/065611
Authority: WO
Inventors: Laura Labrie; Brian Sheehan; Yeng Sheng Lee; Wong Fong Sian
Original assignee: Intervet International B.V.
Priority date: 2009-10-19
Filing date: 2010-10-18
Publication date: 2011-04-28
Also published as: BR112012009309A2; CN107648600A; BR112012009309B1; CN102740878A; TWI495478B; TW201119671A

Abstract

The present invention relates to a combination vaccine for the protection of fish against streptococcal infection, the use of streptococci for the manufacture of such a vaccine, to methods for the preparation of such a combination vaccine and to a kit-of-parts comprising such a vaccine.

Description

Streptococcal combi-vaccine

Over the last decades, world-wide a strong increase is seen in the consumption of fish. This is the case for both the consumption of cold water fish such as salmon, turbot, halibut and cod, and tropical fish such as Asian sea bass, tilapia, milkfish, yellowtail, amberjack, grouper and cobia. As a consequence, an increase has been seen in the number and the size of fish farms, in order to meet the increasing needs of the market.

As is known from e.g. animal husbandry, large numbers of animals living closely together are vulnerable to all kinds of diseases, even diseases hardly known or seen, or even unknown, before the days of large-scale commercial farming. This is equally true for fish farming.

Examples of notorious commercially important fish pathogens are Vibrio anguillarum,

Photobacterium damselae subspecies piscicida, Tenacibaculum maritimum, Flavobacterium sp., Flexibacter sp., Streptococcus sp., Lactococcus garviae, Edwardsiella tarda, E. ictaluri, Viral Necrosis virus, iridovirus and Koi Herpesvirus.

Several species of the bacterial genus Streptococcus are currently known to cause infections in fish, more specifically in fish that are kept in aquaculture. Examples of such streptococcal species are Streptococcus iniae, S. difficile, S. agalactiae, S. dysgalactiae and S. phocae.

There have recently been some changes in opinion about the correct nomenclature of

Streptococcus difficile and Streptococcus agalactiae. Vandamme et al (Int. J. Syst. Bacteriology 47: 81 -85 (1995)) have suggested that Streptococcus difficile is in fact a non-hemolytic

Streptococcus agalactiae.

Streptococcus iniae is frequently found in Tilapia, Rainbow trout, European sea bass and bream, Asian sea bass, Red drum, Rabbit fish, Japanese flounder, Yellowtail and hybrid striped bass. The annual impact of Streptococcus iniae infection to aquaculture exceeds 100 million US- dollars.

Streptococcus difficile is frequently found in Tilapia, snapper and even ray.

Streptococcus agalactiae, although found in many fish species, is currently mainly found to infect Tilapia.

Vaccines for combating Streptococcal infection in fish are known in the art.

Many of the Streptococcus vaccines are based upon killed whole cells. Streptococcus agalactiae vaccines are i.a. described in the US-Patent Application US 2005/0208077. Streptococcus iniae vaccines are i.a. described in US-Patent US 6,379,677. Vaccines for combating Streptococcus iniae infection are also commercially available. An example is Norvax Strep Si, a vaccine against Streptococcus iniae infection and sold by Intervet Int. B.V. Eldar et al (Vaccine 13: 867-870 (1995)) have described a vaccine against Streptococcus difficile on the basis of killed whole cells. To improve the knowledge of streptococcal disease in tropical fish, more specifically in tilapia, the inventors have performed extensive epidemiological studies in the major tilapia producing countries in Asia and Latin America. These studies have yielded almost five hundred streptococcal isolates recovered from approximately fifty sites in thirteen countries over the last eight years. The isolates were identified using standard biochemical and bacteriological identification methods and subsequently analysed by cluster analysis (unweighted pair-group average based on percent disagreement). Interestingly, out of the almost 500 streptococcal isolates recovered from tilapia, 82% were identified as Streptococcus agalactiae and 18% were identified as Streptococcus iniae. Streptococcus iniae is a significant fish pathogen causing disease and mortality in many marine and fresh- water cultured fish species in tropical and sub-tropical areas. Vaccines to combat S. iniae infection in a variety of fish species including tilapia are available and there is a large body of literature on the pathogenesis of this organism in a variety of fish species. Considerably less information is available for fish-pathogenic S. agalactiae.

S. agalactiae is a so-called Group B streptococcus (GBS). It is an important pathogen for humans and animals. Although more commonly associated with disease in human and bovine hosts, fish- pathogenic S. agalactiae have been documented from as early as 1966 when a non-hemolytic group B streptococcus was identified as the cause of two epizootics in golden shiners

(Notemigonus crysoleucas). Today, with the intensification of aquaculture, S. agalactiae is known to be a significant cause of mortality and morbidity in both marine and fresh water cultured species and in particular in tilapia. Detailed analysis of the tilapia S. agalactiae isolates as found by the inventors suggests the presence of two distinct clusters which differ in a variety of biochemical and phenotypic characteristics. These distinct clusters are referred to as Biotypes and on this basis differentiation can be made between 'classical' S. agalactiae (hereafter referred to as S. agalactiae Biotype 1) and typically ηοη-β-haemolytic S. agalactiae (hereafter referred to as S. agalactiae Biotype 2). These latter strains were previously classified as S. difflcile/S. difficilis but have subsequently been reclassified as ηοη-β-haemolytic variants of S. agalactiae. Streptococcus agalactiae infections in farmed tilapia are now known to be responsible for significant morbidity, mortality and economic loss. Infections with S. agalactiae result in septicemia and colonization of various internal organs particularly the brain leading to clinical signs. Clinical signs of S. agalactiae infection include abnormal swimming, 'C-'-shaped body posturing, and inappetance. S. agalactiae is prevalent throughout temperate and tropical regions and the inventors have recovered it from diseased tilapia in Europe, Central and Latin America, and throughout Asia.

The two S. agalactiae Biotypes cause subtly distinct disease syndromes, with Biotype 1 infecting fish throughout the production cycle from juvenile to grow-out, while Biotype 2 causes disease predominantly in larger fish.

In the inventors' epidemiological surveys to date, with almost 500 streptococcal isolates from 13 countries the majority of all streptococcal isolates of tilapia are S. agalactiae Biotype 2. The inventors have identified S. agalactiae Biotype 2 in diseased fish from most of the major tilapia- producing countries including Indonesia, China, Vietnam, Philippines, Ecuador, Honduras, Mexico and Brazil. Analysis of the Biotype 1 strains isolated from fish revealed that two serotypes exist: serotype la strains and serotype III strains (Suanyuk, N. et al., Aquaculture 284: 35-40 (2008)).

Both strains however share the same biochemical characteristics that make them Biotype 1 strains and they are both haemolytic.

Most importantly, both strains contain the gene encoding the surface-associated alpha-C protein {bed). For this protein it was extensively shown already in 1991 that it induces protective immunity against S. agalactiae (Michel, J.L. et al., Inf. & Immun. 59: 2023-2028 (1991)).

Even monoclonal antibodies were raised against a specific protective alpha-C protein epitope in group B streptococci, that was capable of killing Group B Streptococci (Madoff, L.C. et al., Inf. & Immun. 59: 204-210 (1991)).

The alpha-C-protein is commonly found on S. agalactiae serotype la strains, but is uncommon on the surface of human S. agalactiae serotype III strains. However, it however turned out to be always present on the surface of all fish-pathogenic S. agalactiae serotype III strains isolated so far.

Therefore, it could safely be concluded that the existing whole cell vaccines, based upon the serotype of e.g. the Mullet isolate la serotype are capable of inducing a protective immune response against both the known S. agalactiae serotype la strains and the more recently found S. agalactiae serotype III strains.

It was surprisingly found now, that in spite of their joined Biotype, the joined β-haemolytic characteristics and the joined surface-associated alpha-C protein, a whole cell vaccine based upon the known Mullet (la) strain does only provide partial protection against the more recently found S. agalactiae serotype III strains and vice versa.

This unexpected finding has remained unnoticed so far.

The equally unexpected consequence is that the current vaccines are not sufficiently effective to avoid or treat S. agalactiae infection in fish.

It is an objective of the present invention to provide a solution to this problem, by providing a combination vaccine for the protection of fish against S. agalactiae infection that has as a characteristic that it comprises immunogenic amounts of two S. agalactiae Biotype 1 strains: one belonging to serotype la and one belonging to serotype III. A first embodiment of the present invention thus relates to a combination vaccine for the protection of fish against streptococcal infection, wherein said vaccine comprises an

immunogenic amount of Streptococcus agalactiae Biotype 1 serotype la cells and an immunogenic amount of Streptococcus agalactiae Biotype 1 serotype III cells, and a pharmaceutically acceptable carrier.

An immunogenic amount of Streptococcus agalactiae cells is the amount of cells necessary to introduce an immune response that is at least capable of reducing the severity of the disease, compared to non-vaccinated fish.

A pharmaceutically acceptable carrier can be as simple as water or a buffer, or an emulsion such as e.g. an oil-in-water or water-in-oil emulsion. Although both Streptococcus agalactiae Biotype 1 serotype la cells and Streptococcus agalactiae Biotype 1 serotype III cells are readily available to the skilled person, an example of a

Streptococcus agalactiae serotype III strain has been deposited with the Collection Nationale de Cultures de Microorganisms (CNCM), Institut Pasteur, 25 Rue du Docteur Roux, F-75724 Paris Cedex 15, France, under accession number CNCM 1-4232, with the name and address of Intervet International B.V., Wim de Korverstraat 35, 5831 AN, Boxmeer, The Netherlands.

In a combination vaccine according to the invention, the bacteria may be present in a live attenuated form or in an inactivated form, e.g. as a bacterin. What is important is the fact that the immunogenic properties of the bacteria are still present. This can easily be assured by using whole bacterial preparations. As said above, provided that the immunogenic properties of the bacteria are still present, it is not hugely important if the bacterium in the preparation is alive, killed or even fragmented (e.g. by pressing it through a French Press).

Live attenuated bacteria are very suitable, because they by definition carry the immunogenic characteristics. And live attenuated bacteria have the advantage over bacterins, that they can easily be given without an adjuvant. Moreover they self-replicate to a certain extent until they are stopped by the immune system, as a result of which a lower number of cells can be given. On the other hand, the immunogenic characteristics are also present on bacteria when these bacteria are in the form of a bacterin. And bacterins have the advantage over live attenuated bacteria that they are very safe.

Therefore, in a preferred form of this embodiment, the invention relates to a combination vaccine wherein the Streptococcus agalactiae cells are inactivated. Preferably, the cells are in the form of a bacterin.

A bacterin is defined here as bacteria in an inactivated form. The method used for inactivation appears to be not relevant for the activity of the bacterin. Classical methods for inactivation such as heat-treatment, treatment with formalin, binary ethylene imine, thimerosal and the like, all well-known in the art, are applicable. Inactivation of bacteria by means of physical stress, using e.g. a French Press provides an equally suitable starting material for the manufacturing of a vaccine according to the invention.

Vaccines according to the invention can be prepared starting from a bacterial culture according to techniques well known to the skilled practitioner.

Review articles relating to fish vaccines and their manufacture are i.a. by Sommerset, I., Krossoy, B., Biering, E. and Frost, P. m Expert Review of Vaccines 4: 89-101 (2005), by Buchmann, K., Lindenstrom, T. and Bresciani, in J. Acta Parasitologica 46 : 71 -81 (2001 ), by Vinitnantharat, S., Gravningen, K. and Greger, E. in Advances in veterinary medicine 41 : 539-550 (1999) and by Anderson, D.P. in Developments in Biological Standardization 90: 257-265 (1997).

Moreover, the skilled practitioner will find guidance in the Examples below. Vaccines according to the invention basically comprise an effective amount of a bacteria and a pharmaceutically acceptable carrier.

The amount of cells administered will depend on the route of administration, the presence of an adjuvant and the moment of administration.

Otherwise, man skilled in the art finds sufficient guidance in the references mentioned above and in the information given below, especially in the Examples. Generally spoken, vaccines manufactured according to the invention that are based upon bacterins can be administered by injection in general in a dosage of 10³ to 10¹⁰, preferably 10⁶ to 10¹⁰, more preferably between 10⁸ and 10¹⁰ bacteria. A dose exceeding 10¹⁰ bacteria, although immunologically suitable, will be less attractive for commercial reasons.

For the amount of bacteria in a vaccine manufactured according to the invention and for oral application, the examples below will provide ample guidance.

Examples of pharmaceutically acceptable carriers that are suitable for use in a vaccine for use according to the invention are sterile water, saline, aqueous buffers such as PBS and the like. In addition a vaccine according to the invention may comprise other additives such as adjuvants, stabilizers, anti-oxidants and others, as described below.

Vaccines according to the present invention, especially the vaccines comprising a bacterin, may in a preferred presentation also contain an immunostimulatory substance, a so-called adjuvant. Adjuvants in general comprise substances that boost the immune response of the host in a nonspecific manner. A number of different adjuvants are known in the art. Examples of adjuvants frequently used in fish and shellfish farming are muramyl dipeptides, lipopolysaccharides, several glucans and glycans and Carbopol(^). An extensive overview of adjuvants suitable for fish and shellfish vaccines is given in the review paper by Jan Raa (Reviews in Fisheries Science 4(3): 229-288 (1996)).

The vaccine may also comprise a so-called "vehicle". A vehicle is a compound to which the bacterium adheres, without being covalently bound to it. Such vehicles are i.a. bio-microcapsules, micro-alginates, liposomes and macrosols, all known in the art.

A special form of such a vehicle, in which the antigen is partially embedded in the vehicle, is the so-called ISCOM (European Patents EP 109.942, EP 180.564, EP 242.380).

In addition, the vaccine may comprise one or more suitable surface-active compounds or emulsifiers, e.g. Span or Tween.

Oil adjuvants suitable for use in water-in-oil emulsions are e.g. mineral oils or metabolisable oils. Mineral oils are e.g. Bayol^®, Marcol^® and Drakeol^®.

An example of a non-mineral oil adjuvants is e.g. Montanide-ISA-763-A. Metabolisable oils are e.g. vegetable oils, such as peanut oil and soybean oil, animal oils such as the fish oils squalane and squalene, and tocopherol and its derivatives.

Suitable adjuvants are e.g. w/o emulsions, o/w emulsions and w/o/w double-emulsions

An example of a water-based nano-particle adjuvant is e.g. Montanide-IMS-2212.

Often, the vaccine is mixed with stabilizers, e.g. to protect degradation-prone proteins from being degraded, to enhance the shelf- life of the vaccine, or to improve freeze-drying efficiency. Useful stabilizers are i.a. SPGA (Bovarnik et al; J. Bacteriology 59: 509 (1950)), carbohydrates e.g. sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose, proteins such as albumin or casein or degradation products thereof, and buffers, such as alkali metal phosphates.

In addition, the vaccine may be suspended in a physiologically acceptable diluent.

It goes without saying, that other ways of adjuvating, adding vehicle compounds or diluents, emulsifying or stabilizing a protein are also embodied in the present invention. It is known that especially inactivated vaccines, such as bacterins show improved

immunogenicity when given as a water-in-oil emulsion.

Therefore, in a more preferred form of this embodiment, the invention relates to a combination vaccine wherein the vaccine is a water-in-oil emulsion. From a point of pharmaceutical acceptability, there is a growing reluctance against the use of mineral oils.

Thus, in an even more preferred form of this embodiment, the invention relates to a combination vaccine wherein the oil is a non-mineral oil. In a most preferred form of this embodiment, the invention relates to a combination vaccine wherein the oil is Montanide ISA 763 A.

Many ways of administration, all known in the art can be applied. The vaccine according to the invention is preferably administered to the fish via injection, immersion, dipping or per oral. Especially oral application and e.g. intraperitoneal application are attractive ways of administration. Generally spoken: if the vaccine can be improved by adding an adjuvant, the way of administration would preferably be the intraperitoneal route. From an immunological point of view, intraperitoneal vaccination with a bacterin is a very effective route of vaccination, especially because it allows the incorporation of adjuvants.

The administration protocol can be optimized in accordance with standard vaccination practice. The skilled artisan would know how to do this, or he would find guidance in the papers mentioned above. The age of the fish to be vaccinated is not critical, although clearly one would want to vaccinate against the fish-pathogenic bacteria in as early a stage as possible, i.e. prior to possible exposure to the pathogen.

Nevertheless, vaccination of very small fish is difficult and time-consuming. Generally spoken, fish from 5 grams and up can, if necessary or desired, be vaccinated by means of injection.

For oral administration the vaccine is preferably mixed with a suitable carrier for oral application i.e. cellulose, food or a metabolisable substance such as alpha-cellulose or different oils of vegetable or animals origin. Also an attractive way is administration of the vaccine to high concentrations of live- feed organisms, followed by feeding the live- feed organisms to the target animal, e.g. the fish. Particularly preferred food carriers for oral delivery of the vaccine according to the invention are live-feed organisms which are able to encapsulate the vaccine.

Suitable live-feed organisms include plankton-like non-selective filter feeders preferably members of Rotifera, Artemia, and the like. Highly preferred is the brine shrimp Artemia sp.

As follows from the Examples, the level of cross-protection between S. agalactiae biotype 1 and S. agalactiae biotype 2 is low, or non-existant.

Therefore, another preferred form of the combination vaccine according to the invention additionally comprises an immunogenic amount of Streptococcus agalactiae Biotype 2 cells, an immunogenic amount of an antigen of Streptococcus agalactiae Biotype 2 cells or genetic material encoding such an antigen. Also, a vaccine according to the invention would benefit from the additional presence of an immunogenic amount of Streptococcus iniae cells, an immunogenic amount of an antigen of Streptococcus iniae or genetic material encoding such an antigen.

Therefore, again another preferred form of the combination vaccine according to the invention additionally comprises an immunogenic amount of Streptococcus iniae cells, an immunogenic amount of an antigen of Streptococcus iniae or genetic material encoding such an antigen.

Clearly, a vaccine according to the invention would also benefit from the presence of another immunogenic amount of another fish-pathogenic microorganism or a fish-pathogenic virus, an antigen of such microorganism or virus or genetic material encoding such an antigen, for the manufacture of the vaccine.

Thus, another preferred form of the first embodiment relates to a combination vaccine according to the invention wherein that vaccine additionally comprises an immunogenic amount of another fish-pathogenic microorganism or a fish-pathogenic virus, an antigen of such microorganism or virus or genetic material encoding such an antigen.

Preferably, said other microorganism or virus is selected from the following group of fish pathogens: Vibrio anguillarum, Photobacterium damselae subspecies piscicida, Tenacibaculum maritimum, Flavobacterium sp., Flexibacter sp., Lactococcus garviae, Edwardsiella tarda, E. ictaluri, Streptococcus dysgalactiae, Viral Haemorrhagic Septicaemia virus, Viral Necrosis virus, iridovirus, Spring viremia of Carp and Koi Herpesvirus

Thus, in a more preferred form, the other microorganism or virus is selected from the following group of fish pathogens: Vibrio anguillarum, Photobacterium damselae subspecies piscicida, Tenacibaculum maritimum, Flavobacterium sp., Flexibacter sp., Lactococcus garviae,

Edwardsiella tarda, E. ictaluri, Streptococcus dysgalactiae, Viral Haemorrhagic Septicaemia virus, Viral Necrosis virus, iridovirus, Spring viremia of Carp and Koi Herpesvirus.

Another embodiment of the present invention relates to the use of an immunogenic amount of Streptococcus agalactiae Biotype 1 serotype la cells and an immunogenic amount of

Streptococcus agalactiae Biotype 1 serotype III cells for the manufacture of a vaccine for the protection of fish against streptococcal infection. Still another embodiment of the present invention relates to a method for the preparation of a combination vaccine according to the invention, wherein that method comprises the step of mixing an immunogenic amount of Streptococcus agalactiae Biotype 1 serotype la cells and immunogenic amount of Streptococcus agalactiae Biotype 1 serotype III cells, and a pharmaceutically acceptable carrier.

Again another embodiment of the present invention relates to a kit of parts, that has as a characteristic that it comprises at least two vials, wherein these vials together comprise an immunogenic amount of Streptococcus agalactiae Biotype 1 serotype la cells and an immunogenic amount of Streptococcus agalactiae Biotype 1 serotype III cells, and a pharmaceutically acceptable carrier. Such a kit of parts e.g. also includes a kit of parts that comprises at least two vials, wherein one vial comprises an immunogenic amount of

Streptococcus agalactiae Biotype 1 serotype la cells and an immunogenic amount of

Streptococcus agalactiae Biotype 1 serotype III cells, and another vial comprises Biotype 2 cells and a pharmaceutically acceptable carrier.

Examples:

Example 1

Vaccination

Vaccines:

Vaccine-1 : TI1428 S. agalactiae Biotype 1 , serotype III, also referred to as Sal (X) Pharmaceutical form: Water-in-oil: 30% water phase and 70%> Montanide ISA

763 oil

Antigen concentration: S. agalactiae 1 (X) at 1.36E+8 cells/ml

Vaccine-2: TI1422 S. agalactiae Biotype 1 , serotype la, also referred to as Sal (Y) Pharmaceutical form: Water-in-oil: 30% water phase and 70% Montanide ISA

763 oil

Antigen concentration: S. agalactiae 1 (Y) at 1.36E+8 cells/ml Standard Vaccine Diluent Buffer (SVDB)

Table 1 gives a description of the challenge strains used.

0

Table 1 : Challenge strains used for challenge

Agglutination* Agglutination* Serotype

Strain Biochemical

5". agalactiae strain with polyclonal with polyclonal Designation PCR code anlysis

rabbit anti TI1425 rabbit anti ΤΓ513

Til 428 S. agalactiae- + ΙΠ 5". 5".

Biotype 1 agalactiae agalactiae

TI1422 S. agalactiae- + l a S. S.

Biotype 1 agalactiae agalactiae

TI016 S. agalactiae- + lb S. difficilis S.

Biotype 2 agalactiae standardised slide agglutination, Til 425 is raised against Biotype 1 serotype ΠΙ,

TI513 is raised against Biotype 2.

Animals

Species: Tilapia (Oreochromis sp)

Av. weight on arrival: <0.5g

Av. weight at start of Exp. 23g

Husbandry

Water

Salinity: approximately 6 ppt after vaccination, freshwater after challenge

Temperature: 27°C ± 2°C after vaccination

32°C ± 2°C after challenge

Tank: 500L after vaccination

250L after challenge

Feed

After vaccination fish were fed at 2-4 % of body weight per day. Feeding rate of each treatment group was adjusted weekly. After challenge, fish were fed at l%-3% of their body weight per day after challenge if feed was accepted.

Fish were starved for at least 12 hours prior to any manipulation such as transfer to tanks, weighing, and 48 hours prior to injection.

Tanks

All tanks in the wet lab facilities have a unique numbering code and this code was used throughout the experiments and on all record forms used. After vaccination, fish were placed in a 500L tank. The tank was divided into 2 halves by means of a net placed vertically in the middle of the tank. Both tank halves were identified by the tank number and a letter (A and B). This numbering was fixed to the tank. Challenged fish were housed in 250L tanks separated by two nets placed vertically, dividing the tank in 3 compartments. Each compartment was identified by the tank number and a letter A, B or C. This numbering was fixed to the tank.

Assignment of animals to treatment groups

A total of 285 fish of a similar size were used for this experiment. The fish were grouped in 3 groups of 95 fish, two vaccinated groups and one group control group.

Vaccination

The vaccination was performed by IP injection. Fish were anaesthetised with AQUI-S until sedated and IP injected with 0.05 ml approximately at the end and between the tips of the pectoral fins. Immediately after injection, fish were transferred to their allocated tank and the recovery was followed. Control fish were injected with a similar volume of SVDB using the same procedure. Table 2 describes the groups used as well as the tank allocation post vaccination.

Table 2: Treatment schedule and allocation of tanks after vaccination

Vaccine volume SVDB volume

Treatment group Number offish Tank

(ml) (ml)

Vaccinated Sal

(X) 0.05 - 95 4F06B

(TI 1428)

Vaccinated Sal

00 0.05 - 95 4F07A

(TI 1422)

Control

- 0.05 95 4F08B Preparation of challenge inoculum

TI 1422 and TI 1428, strains were recovered from <-50°C frozen glycerol stock on TSA and subsequently incubated at 26-32°C for approximately 24 hours.

For TI 1422, growth was collected in TSB until an OD₆₆₀ of 0.134 - 0.147 were reached. Ten fold dilutions were performed until 10^"6 using 0.9% sterile NaCl. 0.4%> of this preparation was inoculated a larger volume of TSB. The broth was incubated at 32°C for 16-17h. When the OD₆₆₀ reached 0.879 - 0.904 the culture was used to prepare the challenge suspension by diluting the culture 200 fold in 0.9%> sterile NaCl.

The CFU in the resulting suspension used for challenge was determined to be 1.OE+7 CFU/ml and 1.3E+7 for week 3 and week 6 challenges respectively.

For TI 1428, growth was collected in SGM until an OD₆60nm of 0.162 - 0.170 was reached. The preparation was inoculated into 100ml of SGM at 1 % v/v and subsequently incubated at 32°C. After approximately 16h incubation, the OD₆60nm of cultures was 1.339 - 1.362. This culture was used to prepare the challenge suspension by diluting it 100 fold in 0.9%> sterile NaCl. The CFU in the resulting suspension used for challenge was determined to be 9. OE+7 CFU/ml and 7.4E+7 for week 3 and week 6 challenges respectively

For TI016 (S. agalactiae Biotype 2 (also referred to as Sa2)), a 1ml challenge seed vial was taken from the < -50°C freezer, thawed and its content inoculated into 100 ml of SGM. The culture was placed on an orbital shaker with a shaking speed set at 150 RPM at 32°C. After 21h-22h, the culture had an OD₆₆₀ of 0.181 - 0.177. This culture was used to prepare the challenge suspension by diluting it 1000 fold in 0.9% sterile NaCl. The CFU in the resulting suspension used for challenge was determined to be 9.9E+5 CFU/ml and 4.2E+5 for week 3 and week 6 challenges respectively The number of colony forming units in all challenge cultures were determined by standard spread plating of 100 μΐ aliquots of ten- fold diluted bacterial suspensions on TSA and subsequent incubation at 32°C for 24-48 hours. Challenge

At week 3 and week 6, fish were starved for at least 48 hours prior to the challenge to ensure complete emptying of the gastro-intestinal tract and thereby preventing injury to the internal organs as a result of the injection. They were anaesthetized using AQUI-S and challenge was performed through IP injection. For each challenge time point 15 fish were IP injected with 0.1ml of the above described challenge suspensions. Immediately after injection, the fish were transferred back to their allocated tank (half) and the recovery was followed (See Table 3 for distribution).

Table 3: Experimental groups, number of fish per group and allocated tanks after challenge for wk 3 and wk 6 challenges.

X: agglutination only with Sal antiserum, Y: agglutination only with Sa2 antiserum

Evaluation of results

The ability of the vaccine to protect fish against the various challenge strains was evaluated by calculating the Relative Percentage protection (RPP) values. The RPP value was calculated according to the following formula: % infected in vaccinates

RPP = i _ ( ) x ioo

% infected in controls

* Infected fish included fish collected dead during the observation period form which the challenge organism could be isolated as well as fish collected at the end of the study from which the challenge organism could be isolated.

Water parameters and mortality after vaccination

One fish was found dead form the Vaccinated Sal (Y), (TI 1422) group on day 8 post vaccination. The fish was cannibalised. No growth was observed from plated internal organs. The cause of death could not be confirmed. No abnormal behaviour was observed during the experimental period post vaccination.

Growth post vaccination

Growth in vaccinated and control groups over the 6 weeks observation period is given in Figure 1.

Mortality was similar in vaccinated and control groups.

Efficacy

Mortality after challenge

The mortality obtained after week 3 challenges with the various challenge strains are illustrated in Figures 2, 3, and 4 for Sal-X, Sal-Y and Sa2 challenges respectively. After week 3 challenges, mortality in the control groups was as expected (% cumulative mortality of 87%, 93% and 80% after Sal-X, Sal-Y and Sa2 challenges respectively). After challenge with Sa2 strains mortality was high in both vaccine groups (67% and

87%). After challenge with Sal-X, mortality levels were lower in the serologically homologous Sal-X vaccinated group (27%) when compared to the serologically heterologous Sal -Y vaccinated group (60% mortality).

After challenge with Sal-Y the same observation was made: mortality levels were lower in the serologically homologous Sal-Y vaccinated group (6.7%) when compared to the serologically heterologous Sal-X vaccinated group (40% mortality). See Table 4 for an overview of the %> cumulative confirmed mortality.

Table 4: Overview of % cumulative confirmed mortality, after challenge week 3 (n=15)

NA: non applicable

The mortality obtained after week 6 challenges with the various challenge strains are illustrated in Figures 5, 6, and 7 for Sal-X, Sal-Y and Sa2 challenges respectively. See Table 4 for an overview of the %> cumulative confirmed mortality.

After week 6 challenges, mortality in the control groups was as expected (%> cumulative mortality of 86.7%, 60% and 66.7% after Sal -X, Sal-Y and Sa2 challenges respectively).

The observations made at week 3 were confirmed after week 6 challenge: after challenge with Sa2 strains mortality was high in both vaccine groups (66.7% and 73.3%>). After challenge with Sal-X, mortality levels were lower in the serologically homologous Sal -X vaccinated group (6.7%>) when compared to the serologically heterologous Sal -Y vaccinated group (26.7%> mortality). After challenge with Sal-Y the same observation was made: mortality levels were lower in the serologically homologous Sal-Y vaccinated group (6.7%) when compared to the serologically heterologous Sal-X vaccinated group (53.3%> mortality). See Table 5 for an overview of the %> cumulative confirmed mortality.

Details on daily mortality and re isolations are given in Addendum 3.

Table 5: Overview of % cumulative confirmed mortality, after challenge week 6 (n=15)

NA: not applicable PP values

RPP values were calculated and are represented in Table 6.

Table 6: RPP values after S. agahctiae challenges at week 3 and 6 post vaccination.

Similarity between vaccine and

RPP*

challenge strain

Treatment group Challenge strain Week 3 Week 6 biochemically serologically

TI 1428 (Sal (X) 69.2% 92.3% 1 I

Vaccinated Sal (X)

Til 422 (Sal (Y) 57.1 % 1 1.1% 1 Ielerologous (TI 1428)

ΤΤΠ 1 ί l ¾9"> 1 6 7% n%

Vaccinated Sal (Y) TI 1428 (Sal (X) 30.8% 69.2% ί ft iHF-. I leteroloiious

When challenge was performed with the Sa2 challenge strain at week 3, protection was weak to non existent irrespective of the vaccines (RPP of 16.7% with Sal (X) vaccine, RPP of -8.3% with Sal (Y) vaccine). This observation was confirmed after week 6 challenge where no protection was seen with either vaccine (RPP of 0% (Sal-X vaccine) and -10% Sal (Y) vaccine).

When homologous Sal-X vaccination/Sa-1 X challenges were performed, good protection was obtained (RPP 69.2%> and 92.3%> at week 3 and 6 respectively). However, when heterologous Sal-X vaccination/Sal -Y challenges were performed protection was lower (RPP of 57.1% and 11.1% at week 3 and week 6 respectively).

The same observation was made when homologous Sal-Y vaccination/Sal -Y challenge was performed. When Sal-Y vaccinated fish were challenged with a Sal-Y challenge strain, good protection was observed (RPP 92.9%> and 88.9%> at week 3 and 6 respectively). However, when Sal-Y vaccinated fish were challenged with Sal-X strains protection was lower compared to the homologous challenges (RPP of 30.8%> and 69.2% at week 3 and week 6 respectively).

CONCLUSIONS

In conclusion it can be said that no protection is obtained between biochemically distinct S. agalactiae strains: S. agalactiae Biotype 1 vaccines do not cross protect against infections with S. agalactiae Biotype 2. However, S. agalactiae Biotype 1 vaccines provide protection against S. agalactiae Biotype 1 challenges. Nonetheless, a reduction in protection was seen when S. agalactiae Biotype 1 vaccines were challenged with serologically heterologous challenge strains (X-Y and Y-X). Excellent protection could be provided with serologically homologous challenges (X-X and Y- Y). Therefore, a combination vaccine comprising Streptococcus agalactiae Biotype 1 serotype la cells and Streptococcus agalactiae Biotype 1 serotype III cells provides excellent protection against both Streptococcus agalactiae Biotype 1 serotypes.

Abbreviations used

ΓΡ Intra peritoneal

Ppt Parts per thousand

RPP Relative percent survival

TSA Tryptone Soy agar

TSB Tryptone Soy Broth

SGM Streptococcus Growth Media

Legend to the figures:

Figure 1 : Growth post vaccination over the 6 weeks observation period post challenge (n= 15 expect at week 3: n=45 and week 6: n=50).

Figure 2: Percent (%) cumulative confirmed mortality in vaccinated (Vacc Sal (X), Vacc Sal (Y)) and control (Ctr) groups after challenge with Sal-X (TI1428) performed at week 3 post vaccination (n=15) inclusive the positive re isolation fish at the end of the observation period post challenge (pos).

Figure 3: Percent (%) cumulative confirmed mortality in vaccinated (Vacc Sal (X), Vacc Sal (Y)) and control (Ctr) after challenge with Sal-Y (TI1422) performed at week 3 post vaccination (n=15) inclusive the positive re isolation fish at the end of the observation period post challenge (pos).

Figure 4: Percent (%) cumulative confirmed mortality in vaccinated (Vacc) and control (Ctr) groups after challenge with Sa2 (Y) (TI 016) performed at week 3 post vaccination (n=15) inclusive the positive re isolation fish at the end of the observation period post challenge (pos).

Figure 5: Percent (%) cumulative confirmed mortality in vaccinated (Vacc) and control (Ctr) groups after challenge with Sal-X (TI1428) performed at week 6 post vaccination (n=15) inclusive the positive re isolation fish at the end of the observation period post challenge (pos). Figure 6: Percent (%) cumulative confirmed mortality in vaccinated (Vacc) and control (Ctr) groups after challenge with Sal-Y (TI1422) performed at week 6 post vaccination (n=15) inclusive the positive re isolation fish at the end of the observation period post challenge (pos).

Figure 7 : Percent (%) cumulative confirmed mortality in vaccinated (Vacc) and control (Ctr) groups after challenge with Sa2 (Y) (TI 016) at week 6 post vaccination (n=15) inclusive positive re isolation fish (pos).

Indications are Made All designations

FOR RECEIVING OFFICE USE ONLY -4 This form was received with the

international application: Yes

(yes or no)

-4-1 Authorized officer

Gatinet, Bruno

FOR INTERNATIONAL BUREAU USE ONLY -5 This form was received by the

international Bureau on:

-5-1 Authorized officer

Claims

1) Combination vaccine for the protection of fish against streptococcal infection,

characterized in that said vaccine comprises an immunogenic amount of Streptococcus agalactiae Biotype 1 serotype la cells and an immunogenic amount of Streptococcus agalactiae Biotype 1 serotype III cells, and a pharmaceutically acceptable carrier.

2) Combination vaccine according to claim 1 , characterised in that the Streptococcus agalactiae cells are inactivated.

3) Combination vaccine according to claim 1 or 2, characterised in that the vaccine is a water-in-oil emulsion.

4) Combination vaccine according to claim 3, characterised in that the oil is a non-mineral oil.

5) Combination vaccine according to claim 4, characterised in that the non-mineral oil is Montanide ISA 763.

6) Combination vaccine according to claim 1 -5, characterized in that said vaccine

additionally comprises an immunogenic amount of Streptococcus agalactiae Biotype 2 cells, an immunogenic amount of an antigen of Streptococcus agalactiae Biotype 2 cells or genetic material encoding such an antigen.

7) Combination vaccine according to claim 1 -5, characterized in that said vaccine

additionally comprises an immunogenic amount of Streptococcus iniae cells, an immunogenic amount of an antigen of Streptococcus iniae cells or genetic material encoding such an antigen.

8) Combination vaccine according to claim 1 -7, characterized in that said vaccine

additionally comprises an immunogenic amount of another fish-pathogenic

microorganism or a fish-pathogenic virus, an antigen of such microorganism or virus or genetic material encoding such an antigen.

9) Combination vaccine according to claim 8, characterized in that said another

microorganism or virus is selected from the following group of fish pathogens: Vibrio anguillarum, Photobacterium damselae subspecies piscicida, Tenacibaculum maritimum, Flavobacterium sp., Flexibacter sp., Lactococcus garviae, Edwardsiella tarda, E.

ictaluri, Streptococcus dysgalactiae, Viral Haemorrhagic Septicaemia virus, Viral Necrosis virus, iridovirus, Spring viremia of Carp and Koi Herpesvirus

10) Use of an immunogenic amount of Streptococcus agalactiae Biotype 1 serotype la cells and an immunogenic amount of Streptococcus agalactiae Biotype 1 serotype III cells for the manufacture of a vaccine for the protection of fish against streptococcal infection.

11) Method for the preparation of a combination vaccine according to claim 1 -9 ,

characterized in that said method comprises the steps of mixing an immunogenic amount of Streptococcus agalactiae Biotype 1 serotype la cells and an immunogenic amount of Streptococcus difficile Biotype 1 serotype III cells cells, and a pharmaceutically acceptable carrier.

12) Kit of parts, said kit being characterised in that it comprises at least two vials, said vials together comprising an immunogenic amount of Streptococcus agalactiae Biotype 1 serotype la cells and an immunogenic amount of Streptococcus agalactiae Biotype 1 serotype III cells, and a pharmaceutically acceptable carrier.