TWI495478B - Streptococcal combi-vaccine - Google Patents

Description

Streptococcal combination vaccine

The present invention relates to a combination vaccine for protecting fish against streptococcal infection, the use of Streptococcus in the preparation of the vaccine, a method for preparing the combination vaccine, and a kit of parts comprising the vaccine.

Fish consumption worldwide has grown strongly over the past few decades. The consumption of cold-water fish (such as salmon, turbot, halibut and squid) and tropical fish (such as Asian catfish, tilapia, milkfish, yellow tail, amber fish, grouper and cobia) is the same. As a result, the number and size of fish farms can be seen to increase to meet growing market needs.

It is known, for example, from animal husbandry that a large number of animals living together intensively are susceptible to various diseases, even those that are barely known or rare before large-scale commercial farming, or even unidentified diseases. This is also true in fish farming.

Examples of notorious commercially important fish pathogens are Vibrio anguillarum, Photobacterium damselae subspecies piscicida, Tenacibaculum maritimum, Flavobacterium sp. .), Flexibacter sp., Streptococcus, Lactococcus garviae, Edwardsiella tarda, E. ictaluri, viral necrosis, iridescent Virus (iridovirus) and Koi Herpesvirus.

Several varieties of the genus Streptococcus are currently known to cause infection by fish, more specifically those that remain in aquaculture farms. Examples of such Streptococcus species are Streptococcus iniae, S. difficile, S. agalactiae , S. dysgalactiae, and Streptococcus faecalis. (S. phocae).

Recently, the concept of the correct naming of Streptococcus mutans and Streptococcus agalactiae has changed somewhat. Vandamme et al. (Int. J. Syst. Bacteriology 47:81-85 (1995)) have suggested that Streptococcus mutans is actually a non-hemolytic S. agalactiae.

Streptococcus mutans is commonly found in tilapia, rainbow trout, European squid and scorpion, Asian sea bream, red drum, rabbit fish, Japanese flounder, squid and hybrid striped scorpion.

Streptococcus mutans infection affects more than $100 million in aquaculture years.

Streptococcus streptans are common in tilapia, turtles, and even fins.

Although it can be found in many fish species, it has been found that S. agalactiae mainly infects tilapia.

Vaccines for controlling Streptococcus mutans infection are known in the art.

Many streptococcal vaccines are based on whole cells of death. S. agalactiae vaccines are described, for example, in U.S. Patent Application No. US 2005/0208077. Streptococcus mutans vaccines are described, for example, in U.S. Patent No. 6,379,677. Vaccines for the control of Streptococcus mutans infection are also commercially available. Norvax Strep Si, sold by Intervet International, is an example of a Streptococcus mutans infection. B.V. Eldar et al. (Vaccine 13: 867-870 (1995)) describe a vaccine against Streptococcus mutans based on whole cells of death.

In order to improve knowledge of streptococcal disease in tropical fish, more specifically tilapia, the inventors conducted extensive epidemiological studies in major tilapia producing countries in Asia and Latin America. Over the past eight years, these studies have produced approximately 500 Streptococcus isolates in 50 locations in approximately 13 countries. The isolates were identified using standard biochemical and bacteriological identification methods and analyzed by cluster analysis (unweighted pairwise group averaging based on divergence percentage). Interestingly, 82% of the nearly 500 Streptococcus isolates obtained from tilapia were identified as Streptococcus agalactiae and 18% were identified as Streptococcus mutans.

Streptococcus mutans is an important fish pathogen that causes disease and death in marine and freshwater farmed fish in many tropical and subtropical regions. Vaccines against Streptococcus mutans infection in a variety of fish species, including tilapia, are currently available and have a large body of literature on the pathogenic mechanisms of such organisms in various fish species.

There is very little information about fish pathogenic Streptococcus agalactiae. Streptococcus agalactiae is called Group B Streptococcus (GBS). This is an important pathogen for humans and animals. Although more commonly associated with diseases in human and bovine hosts, fish-borne S. agalactiae was documented as early as 1966, when the non-hemolytic streptococcus group B was identified as causing the golden American carp (Notemigonus crysoleucas). The cause of the two epidemics.

Today, with the increase in aquaculture, it is known that S. agalactiae is an important cause of mortality and morbidity in seawater and freshwater aquaculture species, particularly tilapia. A detailed analysis of the tilapia S. agalactiae isolates discovered by the present inventors suggests the existence of two different cohorts with different biochemical and phenotypic characteristics of the two different cohorts. These different groups are called biotypes, on the basis of which it is possible to distinguish between "classical" Streptococcus agalactiae (hereinafter referred to as Streptococcus agalactiae biotype 1) and typical non-beta-hemolytic Streptococcus agalactiae (below) Known as Streptococcus agalactiae biotype 2). The latter strain was previously classified as S. difficile/S. difficilis, but was later reclassified as a non-beta-hemolytic variant of Streptococcus agalactiae.

S. agalactiae infection in cultured tilapia is currently known to cause significant morbidity, mortality and economic loss. S. agalactiae infection causes sepsis and colonizes various internal organs (especially the brain) leading to clinical symptoms. Clinical signs of Streptococcus agalactiae infection include abnormal swimming, 'C'-shaped body posture, and loss of appetite. Streptococcus agalactiae is ubiquitous throughout the temperate and tropical regions and has been obtained by the inventors from diseased tilapia in Europe, Central America and Latin America, and throughout Asia.

These two S. agalactiae biotypes cause subtle differences in disease syndrome, biotype 1 can infect the entire fish production cycle from childhood to adulthood, while biotype 2 mainly causes disease in larger fish.

To date, in the epidemiological investigation of nearly 500 species of Streptococcus isolates from 13 countries, all of the tilapia Streptococcus isolates were mostly S. agalactiae biotypes 2 . The inventors have identified S. agalactiae biotypes from diseased fish in most major tilapia producing countries including Indonesia, China, Vietnam, the Philippines, Ecuador, Honduras, Mexico and Brazil.

Analysis of the biotype 1 strain isolated from fish revealed the presence of two serotypes: serotype Ia strain and serotype III strain (Suanyuk, N. et al., Aquaculture 284: 35-40 (2008)).

However, the two strains share the same biochemical characteristics, making them both biotype 1 strains and both are hemolyzed.

Most importantly, both strains contain a gene encoding the surface-associated α-C protein (bca). This protein has been widely demonstrated in 1991 to induce protective immunity against Streptococcus agalactiae (Michel, J. L. et al., Inf. & Immun. 59: 2023-2028 (1991)). It can even produce monoclonal antibodies that kill Group B streptococci against specific protective alpha-C protein epitopes in Group B Streptococcus (Madoff, LC et al., Inf. & Immun. 59:204-210 (1991)).

The α-C-protein is commonly found in the S. agalactiae serotype Ia strain, but is rare on the surface of the human S. agalactiae serotype III strain. However, to date, it has been shown to be frequently present on the surface of all fish pathogenic S. agalactiae serotype III isolates.

Therefore, it can be safely concluded that the existing whole-cell vaccine based on serotypes (eg, salmon serotype Ia isolates) can induce resistance against the known S. agalactiae serotype Ia strain and the recently discovered milkless chain. Protective immune response of cocciform serotype III strain.

Surprisingly, it has now been found that regardless of the linked biotype, the linked beta-hemolytic character and the linked surface-bound alpha-C protein, the whole cell vaccine based on the known salmon (Ia) strain is indeed only The recently discovered S. agalactiae serotype III strain provides partial protection and vice versa.

This unexpected discovery has been ignored so far.

An equally unfortunate consequence is that current vaccines are not sufficient to effectively avoid or treat S. agalactiae infection in fish.

One of the objects of the present invention is to provide a combination vaccine for protecting fish against Streptococcus agalactiae infection to solve this problem, characterized by comprising two immunogenic amounts of Streptococcus agalactiae biotype 1 strain: one belonging to serum Type Ia, another type belongs to serotype III.

Accordingly, the first system of the present invention relates to a combination vaccine for protecting fish against streptococcal infection, wherein the vaccine comprises an immunogenic amount of Streptococcus agalactiae biotype 1 serotype Ia cells and an immunogenic amount of milk-free chain Coccus biotype 1 serotype III cells and a pharmaceutically acceptable carrier.

The immunogenic amount of S. agalactiae cells is the amount of cells necessary to introduce an immune response that at least reduces the severity of the disease (compared to unvaccinated fish).

The pharmaceutically acceptable carrier can be as simple as water or a buffer, or an emulsion such as, for example, an oil-in-water or water-in-oil emulsion.

Although it is easy for those skilled in the art to obtain S. agalactiae biotype 1 serotype Ia cells and S. agalactiae biotype 1 serotype III cells, an example of S. agalactiae serotype III strain has been deposited in the French National Microorganisms. Culture Collection (CNCM) Pasteur Institute (25 Rue du Docteur Roux, F-75724 Paris Cedex 15, France), accession number CNCM I-4232, name and address: Intervet International BV, Wim de K Rverstraat 35, 5831 AN, Boxmeer, The Netherlands.

According to the combination vaccine of the present invention, the bacterium can be present in a live attenuated form or in an inactivated form (e.g., in the form of a bacterin). Importantly, the immunological properties of the bacterium still exist. This can be easily confirmed by using whole bacterial products. As described above, it is not very important that the bacteria in the article survive, die or even become fragments (e.g., squeezing bacteria through the French Press) as long as the immunogenic properties of the bacteria are still present.

Live attenuated bacteria are very suitable because they are clearly immunogenic. Live attenuated bacteria have the advantage over vaccines because they can be easily administered without the need for adjuvants. Furthermore, it replicates to some extent until it is blocked by the immune system, so fewer cells can be administered.

On the other hand, when these bacteria are in the form of a vaccine, the immunogenic properties are also present on the bacteria. The vaccine has the advantage of surpassing live attenuated bacteria because it is very safe.

Accordingly, in a preferred embodiment of the invention, the invention pertains to a combination vaccine wherein the S. agalactiae cells are inactivated. Preferably, the cell is in the form of a vaccine.

The bacterin defined herein is an inactivated form of the bacterium. The method used for inactivation seems to be independent of the activity of the vaccine. Conventional methods of inactivation, such as heat treatment, treatment with fumarin, diethyleneimine, thimerosal, etc., are well known to those skilled in the art. Inactivation of the bacteria by physical pressure (e.g., using French Press) can provide starting materials that are also suitable for use in making vaccines in accordance with the present invention.

Vaccines according to the present invention can be prepared starting from bacterial culture according to techniques well known to those skilled in the art.

Review articles on fish vaccines and their preparation methods are, for example, Sommerset, I., Kross Φ y, B., Biering, E. and Frost, P. in Expert Review of Vaccines 4: 89-101 (2005), by Buchmann , K., Lindenstr Φ m, 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, DP in Developments in Biological Standardization 90: 257-265 (1997). In addition, familiar operators will find guidance from the following examples.

A vaccine according to the invention essentially comprises an effective amount of a bacterium and a pharmaceutically acceptable carrier.

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

Alternatively, those skilled in the art can find sufficient guidance in the above references and in the following materials (especially in the examples).

In general, a vaccine prepared according to the present invention based on the vaccine can be administered by injection, and the dose is generally from 10 ³ to 10 ¹⁰ , preferably from 10 ⁶ to 10 ¹⁰ , more preferably from 10 ⁸ to 10 ¹⁰ bacterial. Dose of more than 10 ¹⁰ bacteria, although immunologically suitable, is less attractive for commercial reasons.

The following examples will provide ample guidance on the amount of bacteria and oral administration in vaccines made in accordance with the present invention.

Examples of pharmaceutically acceptable carriers suitable for use in vaccines for use in accordance with the present invention are sterile water, physiological saline, aqueous buffers such as PBS, and the like. Further, the vaccine according to the present invention may contain other additives such as adjuvants, stabilizers, antioxidants, and the like as described below.

The vaccine according to the invention, in particular the vaccine comprising the vaccine, may also comprise an immunostimulating substance (so-called adjuvant) in a preferred presentation. In general, adjuvants include substances that enhance the immune response of the host in a non-specific manner. A variety of different adjuvants are known in the art. Examples of adjuvants commonly used in fish and shellfish culture are the cell wall dipeptide, lipopolysaccharide, several glycans and dextran, and carbomer ^(R) . There are a number of reviews in Jan Raa's review article (Reviews in Fisheries Science 4(3): 229-288 (1996)) that are suitable for use in fish and shellfish vaccines.

The vaccine may also contain so-called "carriers". The carrier is a compound that is attached to the bacteria (not covalently bound to the bacteria). Such carriers are, for example, biological microcapsules, microalginates, liposomes, and macrosols, all of which are known in the art.

A particular form of such a carrier, in which the antigen is partially embedded in the carrier, is the so-called ISCOM (European Patent No. EP 109.942, EP 180.564, EP 242.380).

Furthermore, the vaccine may comprise one or more suitable surface active compounds or emulsifiers, such as Span or Tween.

Oil adjuvants suitable for use in water-in-oil emulsions are, for example, mineral oils or metabolisable oils. Mineral oil is, for example , and .

An example of a non-mineral oil adjuvant is, for example, Montanide-ISA-763-A.

Metabolisable oils are, for example, vegetable oils such as peanut oil and soybean oil, animal oils such as fish oil squalane and squalene, and vitamin E and its derivatives.

Suitable adjuvants are, for example, w/o type emulsions, o/w type emulsions, and w/o/w double emulsions. An example of a water-based nanoparticle adjuvant is, for example, Montanide-IMS-2212.

Typically, the vaccine is mixed with a stabilizer to, for example, protect the protein with degradation tendency from degradation, increase the shelf life of the vaccine, or improve freeze-drying efficiency. Useful stabilizers are, for example, SPGA (Bovarnik et al; J. Bacteriology 59: 509 (1950)), carbohydrates (eg sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose), proteins (eg white) Protein or casein or their degradation products) and buffers such as alkali metal phosphates. Additionally, the vaccine can be suspended in a physiologically acceptable diluent.

It goes without saying that the addition of an adjuvant, the addition of a carrier compound or diluent, the emulsification or stabilization of the protein in other ways is also within the system of the invention.

It is known that when a particularly inactivated vaccine (such as a vaccine) is administered in the form of a water-in-oil emulsion, it shows an improvement in immunogenicity.

Thus, in a more preferred form of this system, the invention pertains to a combination vaccine wherein the vaccine is a water-in-oil emulsion.

From the standpoint of pharmaceutically acceptable, people are increasingly reluctant to use mineral oil.

Thus, in a more preferred form of the system, the invention pertains to a combination vaccine wherein the oil is a non-mineral oil.

In a preferred form of the system, the invention pertains to a combination vaccine wherein the oil is Montanide ISA 763A.

There are a variety of methods of application that can be applied (all known in the art). The vaccine according to the invention is preferably administered by injection, immersion, soaking or via oral administration of fish. In particular, oral administration and, for example, intraperitoneal administration are attractive methods of administration.

Broadly speaking, if the vaccine can be improved by the addition of an adjuvant, the route of administration is preferably the intraperitoneal route. From an immunological point of view, inoculation of the vaccine via the intraperitoneal route is a very effective vaccination route, especially since it allows for the inclusion of adjuvants.

This dosage regimen can be idealized according to standard vaccination procedures. Those skilled in the art will know how to do this, or can find it in the guidance in the above paper.

The age at which fish are vaccinated is not critical, but it is clear that individuals will be willing to vaccinate against fish-borne pathogens as soon as possible (ie before they may be exposed to the pathogen).

However, vaccinating very small fish is difficult and time consuming. In general, 5 grams or more of fish can be vaccinated via injection if necessary or needed.

In oral administration, the vaccine is preferably admixed with a suitable vehicle for oral administration (i.e., cellulose, food or metabolizable material such as alpha-cellulose or a different vegetable or animal oil). Another attractive approach is to administer the vaccine to a high concentration of live feed organisms and then feed the live feed organisms to target animals (eg, fish). A particularly preferred food carrier for oral delivery of a vaccine according to the invention is a live feed organism that can encapsulate the vaccine. Suitable live feed organisms include non-selective filter-feeding animals like plankton, preferably members of rotifers, Artemia, and the like. Saltwater shrimp Artemia is very suitable.

It can be seen from the following examples that the degree of cross protection between S. agalactiae biotype 1 and S. agalactiae biotype 2 is low or absent.

Therefore, another preferred form of the combination vaccine according to the present invention further comprises an immunogenic amount of S. agalactia biotype 2 cells, an immunogenic amount of an antigen of S. agalactiae biotype 2 cells or a genetic material encoding the antigen .

In addition, the vaccine according to the invention will benefit from the additional immunogenic amount of Streptococcus mutans cells, the immunogenic amount of S. cerevisiae antigen or the genetic material encoding the antigen.

Thus, another preferred form of the combination vaccine according to the invention additionally comprises an immunogenic amount of Streptococcus faecium cells, an immunogenic amount of an antigen of Streptococcus faecium or a genetic material encoding the antigen.

Obviously, the vaccine according to the invention will also benefit from another fish pathogenic microorganism or fish pathogenic virus used to make the immunogenic amount of the vaccine, the antigen of the microorganism or virus or the inheritance encoding the antigen The existence of matter.

Therefore, another preferred form of the first system relates to another fish pathogenic microorganism or fish pathogenic virus in which the vaccine additionally comprises an immunogenic amount, an antigen of the microorganism or virus or a genetic material encoding the antigen A combination vaccine according to the invention.

Preferably, the other microorganism or virus is selected from the group consisting of the following fish pathogens: Vibrio anguillarum, sea bream bacterium, subspecies, marine flexor, Flavobacterium, genus, genus Lactococcus, L. edulis Spring viremia of the bacterium, E. sinensis, Streptococcus dysgalactia, viral hemorrhagic septicemia virus, viral necrosis virus, iridescent virus, squid and koi herpes virus.

Therefore, in a more preferred form, the other microorganism or virus is selected from the following fish pathogens: scorpion bacterium, sea bream bacterium, subspecies, marine flexor, flavobacterium, genus, genus Lactococcus Spring viremia of Edwards, Salmonella serrata, Streptococcus dysgalactiae, viral hemorrhagic septicemia virus, viral necrosis virus, iridescent virus, squid and koi herpes virus.

Another system of the present invention relates to an immunogenic amount of Streptococcus agalactiae biotype 1 serotype Ia cells and an immunogenic amount of S. agalactiae biotype 1 serotype III cells for producing a vaccine for protecting fish against streptococcal infection the use of.

Still another system of the present invention relates to a method for the preparation of a combination vaccine according to the present invention, wherein the method comprises immunizing an amount of S. agalactiae biotype 1 serotype Ia cells and an immunogenic amount of Streptococcus agalactiae The step of mixing the biotype 1 serotype III cells with a pharmaceutically acceptable carrier.

Another system of the present invention relates to a kit of parts characterized in that it comprises at least two vials, wherein the vials together comprise an immunogenic amount of Streptococcus agalactiae biotype 1 serotype Ia cells and an immunogenic amount S. agalactiae biotype 1 serotype III cells and a pharmaceutically acceptable carrier. The kit of parts also includes, for example, a kit of parts comprising at least two vials, one of which contains an immunogenic amount of Streptococcus agalactiae biotype 1 serotype Ia cells and an immunogenic amount of Streptococcus agalactiae biotype 1 serum Type III cells, another vial containing biotype 2 cells and a pharmaceutically acceptable carrier.

Example 1 vaccination Vaccine: Vaccine-1: TI1428 Streptococcus agalactiae biotype 1 serotype III, also known as Sa1(X)

Pharmaceutical dosage form: water-in-oil: 30% aqueous phase and 70% Montanide ISA 763 oil

Antigen concentration: 1.36E+8 cells/ml of Streptococcus agalactiae 1 (X)

Vaccine-2: TI1422 Streptococcus agalactiae biotype 1 serotype Ia, also known as Sa1 (Y)

Pharmaceutical dosage form: water-in-oil: 30% aqueous phase and 70% Montanide ISA 763 oil

Antigen concentration: 1.36E+8 cells/ml of Streptococcus agalactiae 1 (Y)

Standard Vaccine Dilution Buffer (SVDB)

Table 1 describes the challenge strains used.

* Normalized slide agglutination, TI1425 is produced for biotype 1 serotype III, and TI513 is produced for biotype 2.

animal

Variety: Tilapia (Wu Guoyu)

Average weight on arrival: <0.5g

Average weight at the start of the experiment: 23 grams

Livestock water

‧ Salinity: about 6ppt after vaccination, fresh water after challenge

‧ Temperature: 27 ° C ± 2 ° C after vaccination

32 °C ± 2 ° C after the challenge

‧ Fish tank: 500 liters after vaccination

250 liters after the challenge

feed

Feed 2-4% of the body weight of the fish daily after vaccination. The feeding rate of each treatment group was adjusted weekly. After the challenge, if the feeding is accepted, the daily weight of the fish is 1% to 3% after the challenge.

The fish are starved for at least 12 hours before any operation (such as transfer to a fish tank, weighing) and starved for at least 48 hours prior to injection.

Fish tank

All fish tanks in wet laboratory equipment have a unique numbering code that is used throughout the experiment and in all record forms. The fish was placed in a 500 liter aquarium after vaccination. Place a net vertically in the middle of the aquarium to divide the aquarium into two halves. The fish tanks of the two halves are confirmed by the number and letter (A and B) of the fish tank. This number is fixed on the fish tank. The challenged fish is housed in a 250 litre aquarium that divides the aquarium into three compartments by two vertically placed nets. Each compartment is identified by the number of the aquarium and the letters A, B or C. This number is fixed on the fish tank.

Treatment group animal distribution

A total of 285 fish of similar size were used in this experiment. The fish were divided into 3 groups, 95 in each group, the second group was the vaccination group, and the other group was the control group.

Vaccination

Vaccination was performed via IP injection. Fish were anesthetized with AQUI-S until sedated and 0.05 ml was injected via IP at the end of the tail and between the tips of the pectoral fins. Immediately after the injection, the fish is transferred to its assigned aquarium for recovery. A similar volume of SVDB was injected into the control fish using the same procedure. Table 2 describes the population used and the aquarium dispensed after vaccination.

Method for preparing challenge inoculum

The TI 1422 and TI 1428 strains were recovered from the frozen glycerol stock solution at <-50 °C TSA, which was then incubated at 26-32 °C for about 24 hours.

In terms of TI 1422, collected in the growth of the strain in TSB until the OD ₆₆₀ reached 0.134-0.147. A 10-fold dilution with 0.9% sterile sodium chloride was reached until 10 ^{-6 was} reached. A larger volume of TSB was inoculated in 0.4% of this product. The broth was incubated at 32 ° C for 16-17 hours. When the OD ₆₆₀ reached 0.879-0.904, the culture was used, and the culture was diluted 200-fold with 0.9% sterile sodium chloride to prepare a challenge suspension. The CFU in the resulting suspension for challenge was determined to be 1.0E+7 CFU/ml and 1.3E+7 CFU/ml at weeks 3 and 6, respectively.

In the case of TI 1428, the growth strains in SGM were collected until the OD _{660 nm} reached 0.162-0.170. The preparation was inoculated into 100 ml of 1% v/v SGM, followed by incubation at 32 °C. After about 16 hours of incubation, the culture had an OD _{660 nm} of 1.339-1.362. The culture was diluted 100-fold with 0.9% sterile sodium chloride to prepare a challenge suspension. The CFU in the resulting suspension for challenge was determined to be 9.0E+7 CFU/ml and 7.4E+7 CFU/ml at weeks 3 and 6, respectively.

In TI016 (S. agalactiae biotype 2 (also known as Sa2)), 1 ml of challenge seed vial was removed from the ≦-50 °C freezer, thawed and the contents were inoculated into 100 ml of SGM. The culture was placed in an orbital shaker, and the shaking speed was set at 32 ° C, 150 RPM. After 21 hours to 22 hours, the OD ₆₆₀ of the culture was 0.181-0.177. The culture was diluted 1000-fold with 0.9% sterile sodium chloride to prepare a challenge suspension. The CFU in the resulting suspension for challenge was determined to be 9.9E + 5 CFU/ml and 4.2E + 5 CFU/ml at weeks 3 and 6, respectively.

A 100 microliter 10-fold diluted bacterial suspension was inoculated on TSA by standard coated plates followed by incubation at 32 °C for 24-48 hours to determine colony forming units in all challenge cultures.

challenge

At 3 and 6 weeks, the fish were starved for at least 48 hours before the challenge to ensure complete venting of the gastrointestinal tract, thereby avoiding injury to the internal organs due to injection. Fish were anesthetized with AQUI-S and challenged by IP injection. At each challenge point, 15 fish were injected via IP into 0.1 ml of the above challenge suspension. Immediately after the injection, the fish are returned to their assigned aquarium (half tank) and allowed to recover (see Table 3 for distribution).

X: agglutination only with Sa1 antiserum, Y: evaluation of only Sa2 antiserum agglutination results

The ability of the vaccine to protect fish against various challenge strains was assessed by calculating the relative protection percentage (RPP) value. Calculate the RPP value according to the following formula:

* Infected fish include dead fish collected during the observation period from which the challenge organism can be isolated and fish collected from the end of the study from which the challenge organism can be isolated.

Water parameters and mortality after vaccination

One dead fish was found from the vaccinated Sa1(Y), (TI 1422) group on the 8th day after vaccination. This fish is disfigured by the same kind. No growth of the strain was observed in the internal organs inoculated on the plate. The cause of death cannot be confirmed. No abnormal behavior was observed during the experiment after vaccination.

Growth after vaccination

The growth of the vaccinated group and the control group during the observation period of 6 weeks is shown in Fig. 1.

The mortality rates of the vaccinated group and the control group were similar.

Effective Post-challenge mortality

In the challenges of Sa1-X, Sa1-Y and Sa2, the third week was chosen with different challenges. Mortality rates obtained after the war are illustrated in Figures 2, 3 and 4, respectively. After the third week of challenge, the mortality rate in the control group was as expected (% cumulative mortality after challenge with Sa1-X, Sa1-Y, and Sa2, respectively, 87%, 93%, and 80%). After challenge with the Sa2 strain, the mortality rates of the two groups were high (67% and 87%). After Sa1-X challenge, serologically homologous Sa1-X vaccinated group had lower mortality (27%) compared with serologically heterologous Sa1-Y vaccinated group (60% mortality) .

The same observations were made with the challenge of Sa1-Y: the serologically homologous Sa1-Y vaccinated group had a lower mortality rate compared to the serologically heterologous Sa1-X vaccinated group (40% mortality). 6.7%). See Table 4 for a review of % cumulative confirmed mortality.

After challenge with various challenge strains in Week 6, Figures 5, 6 and 7 illustrate the mortality rates obtained with the Sa1-X, Sa1-Y and Sa2 challenges, respectively. See Table 4 for a review of % cumulative confirmed mortality.

After the 6th week of challenge, the mortality rate in the control group was as expected (the cumulative mortality rates for the Sa1-X, Sa1-Y, and Sa2 challenges were 86.7%, 60%, and 66.7, respectively).

Observations at week 3 were confirmed after challenge at week 6: after challenge with the Sa2 strain, the mortality rates of the two groups were high (66.7% and 73.3%). After Sa1-X challenge, the serologically homologous Sa1-X vaccination group had a lower mortality rate (6.7%) compared with the serologically heterologous Sa1-Y vaccination group (mortality 26.7%). .

The same observations were made with the Sa1-Y challenge: mortality in the serologically homologous Sa1-Y vaccinated group (6.7%) compared to the serologically heterologous Sa1-X vaccinated group (mortality 53.3%) ) lower. See Table 5 for a review of % cumulative confirmed mortality. Appendix 3 provides detailed information on daily mortality and re-separation.

NA: Not applicable

RPP value

The RPP values were calculated and are shown in Table 6.

*Includes a positive group that re-isolated the strain at the end of the observation period

When challenged with the Sa2 challenge strain in the third week, the protection was weak to almost no matter regardless of the vaccine (RPP of 16.7% using the Sa1(X) vaccine and -8.3% of the R1 using the Sa1(Y) vaccine). This observation confirmed after the challenge of Week 6 that no vaccine was used in this challenge regardless of which vaccine was used (0% for the Sa1(X) vaccine and -10% for the Sa1(Y) vaccine).

Good protection was obtained when homologous Sa1-X vaccination/Sa-1 X challenge was performed (RPP at 3 and 6 weeks was 69.2% and 92.3%, respectively). However, when the heterologous Sa1-X vaccination/Sa1-Y challenge was performed, the protection was low (RPP at 3 and 6 weeks was 57.1% and 11.1%, respectively).

The same observations were performed when the homologous Sa1-Y vaccination/Sa1-Y challenge was performed. Good protection was observed when the Sa1-Y challenged fish were challenged with the Sa1-Y challenge strain (92.9% and 88.9% for the 3rd and 6th week, respectively). However, compared to the homologous challenge, the degree of protection observed was lower when the Sa1-Y vaccine-inoculated fish were challenged with the Sa1-X strain (30.8% and 69.2% for the 3rd and 6th week, respectively). .

in conclusion

In summary, it can be said that there is no protection between biochemically different strains of S. agalactiae: S. agalactiae biotype 1 vaccine has no cross-protective effect against S. agalactiae biotype 2 infection. However, the S. agalactiae biotype 1 vaccine provides protection against the challenge of S. agalactiae biotype 1. However, the protection of the S. agalactiae biotype 1 vaccine was reduced when challenged with serologically heterologous challenge strains (X-Y and Y-X). Excellent protection (X-X and Y-Y) is provided in the challenge of serological homology.

Thus, a combination vaccine comprising S. agalactiae biotype 1 serotype Ia cells and S. agalactiae biotype 1 serotype III cells provides excellent protection against the serotypes of the two S. agalactiae biotypes 1.

Abbreviation used

IP intraperitoneal

Ppt thousand rate

RPP relative survival percentage

TSA protein soy agar

TSB protein soy broth

SGM Streptococcus culture medium

Figure 1: Growth after challenge during the 6-week observation period following vaccination (n=15, expected at week 3: n=45 and at week 6: n=50).

Figure 2: Cumulative confirmed mortality percentage in vaccinated groups (Vacc Sa1 (X), Vacc Sa1 (Y)) and control group (Ctr) after Sa1-X (TI 1428) challenge in the third week after vaccination (%) (n=15) (including the positive group (pos) of the challenge strain re-separated at the end of the observation period after the challenge).

Figure 3: Cumulative confirmed mortality percentage in vaccinated groups (Vacc Sa1 (X), Vacc Sa1 (Y)) and control group (Ctr) after Sa1-Y (TI1422) challenge at week 3 after vaccination (%) (n=15) (including the positive group (pos) of the challenge strain re-separated at the end of the observation period after the challenge).

Figure 4: Cumulative confirmed mortality percentage (%) in the vaccinated group (Vacc) and control group (Ctr) after Sa2 (Y) (TI 016) challenge in the third week after vaccination (n=15) (Include the positive group (pos) of the challenge strain at the end of the observation period after the challenge).

Figure 5: Cumulative confirmed mortality percentage (%) in the vaccinated group (Vacc) and control group (Ctr) after Sa1-X (TI1428) challenge at week 6 after vaccination (n=15) After the challenge, the challenge group positive (pos) was re-isolated at the end of the observation period.

Figure 6: Cumulative confirmed mortality percentage (%) in the vaccinated group (Vacc) and control group (Ctr) after Sa1-Y (TI1422) challenge at week 6 after vaccination (n=15) After the challenge, the challenge group positive (pos) was re-isolated at the end of the observation period.

Figure 7: Cumulative confirmed mortality percentage (%) in the vaccinated group (Vacc) and control group (Ctr) after challenge with Sa2(Y) (TI 016) at week 6 (n=15) (Include the positive group (pos) of the challenge strain at the end of the observation period after the challenge).

Claims (8)

A combination vaccine for protecting fish against streptococcal infection, characterized in that the vaccine comprises 10 ³ to 10 ¹⁰ Streptococcus aga1 actiae biotype 1 serotype Ia cells and 10 ³ to 10 ¹⁰ lactase -free chains Coccus biotype 1 serotype III cells and a pharmaceutically acceptable carrier. The combination vaccine of claim 1, wherein the S. agalactiae cell line is deactivated. The combination vaccine of claim 1, wherein the vaccine is a water-in-oil emulsion. A combination vaccine according to claim 3, wherein the oil is a non-mineral oil. The combination vaccine of claim 4, wherein the non-mineral oil is Montanide ISA 763. A use of a Streptococcus agalactiae biotype 1 serotype Ia cell and a S. agalactiae biotype 1 serotype III cell for the manufacture of a combination vaccine for protecting fish against streptococcal infection. A method for the preparation of a combination vaccine according to any one of claims 1 to 5, characterized in that the method comprises 10 ³ to 10 ¹⁰ Streptococcus agalactiae biotype 1 serotype Ia cells and 10 ³ A step of mixing 10 ¹⁰ S. agalactiae biotype 1 serotype III cells with a pharmaceutically acceptable carrier. A kit comprising at least two vials comprising 10 ³ to 10 ¹⁰ Streptococcus agalactiae biotype 1 serotype Ia cells and 10 ³ to 10 ¹⁰ Streptococcus agalactiae biotype 1 serum Type III cells and a pharmaceutically acceptable carrier.