Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
  • A61K39/092—Streptococcus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to the use of bacteria of the genus Streptococcus for the manufacture of a vaccine for combating Rickettsia-like organism infection.
• Piscirickettsia salmonis infection of white sea bass has recently been shown by Arkush, K.D. et al., in Diseases of Aquatic Organisms 64: 107-119 (2005).
• Rickettsia-likc organisms also referred to further as RLOs have now been described i.a. in Tilapia (Chern, R.S. and Chao, C.B., Fish Pathology 29: 61-71 (1994), Chen, S.C. et al., J. Fish Diseases 17: 591-599 (1994), Mauel et al., Diseases of aquatic organisms, 53: 249-255 (2003) , Fryer, JX and Mauel, MJ., Emerging Infectious Diseases 3: 137-144 (1997)), grouper, (Chen, S.C. et al., Journ. Fish Dis. 23: 415-418 (2000)), blue-eyed plecostomus (Khoo, L. et al., J. Fish Diseases 18:35-48 (1995)) and Sea bass (Comps, M. et al., Bull.
• Prophylactic treatment i.a. includes i.p. injection of brood stock before spawning, and incorporation of antibiotics in the water during hardening of the eggs.
• bacteria of the genus Streptococcus i.e. non-RLO Gram- positive bacteria, are capable of providing a high level of long-lasting protection against RLO infection. This protection is conferred by Streptococcal bacteria when given as a bacterin and/or when given in a live attenuated form.
• the invention relates to the use of a bacterium of the genus Streptococcus for the manufacture of a vaccine for combating RLO infection.
• the status of the bacterium; live or inactivated is not really important. What is important is the fact that the stimulator of cross-specific immunity in fish against RLO is still present. This can be assured by using whole bacterial preparations. As said above, it is not important if the bacterium in the preparation is alive, killed or even fragmented (e.g. by pressing it through a French Press). What is important, is that the components making up the bacterium are still present in the vaccine.
• Live attenuated bacteria are very suitable, because they by definition carry the factor stimulating the cross-specific immunity against RLO. 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.
• the invention relates to the use of live attenuated Streptococcal bacteria for the manufacture of a vaccine for combating Rickettsia-like organism infection according to the invention.
• the factor stimulating the cross-specific immunity against RLO is also present on bacteria when these bacteria are in the form of a bacterin.
• Bacterins have the advantage over live attenuated bacteria that they are very safe. Therefore, in another preferred form, the invention relates to the use of a Streptococcal bacterin for the manufacture of a vaccine for combating Rickettsia-like organism infection according to the invention.
• the genus Streptococcus comprises i.a. Streptococcus iniae, Streptococcus difficile and Streptococcus agalactiae.
• Vaccines for use according to the invention can be prepared starting from a bacterial culture according to techniques well known to the skilled practitioner.
• a live attenuated bacterium is a bacterium that is less pathogenic than its wild-type counterpart, while nevertheless inducing an efficacious immune response.
• Attenuated strains can be obtained along classical routes, long known in the art such as chemical mutagenesis, UV -radiation and the like, or by site-directed mutagenesis.
• 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 equally 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 basically comprise an effective amount of a bacterium for use according to the invention and a pharmaceutically acceptable carrier.
• the term "effective” as used herein is defined as the amount sufficient to induce an immune response in the target fish.
• the amount of cells administered will depend on the Streptococcus species used, the presence of an adjuvant, the route of administration, the moment of administration, the age of the fish to be vaccinated, general health, water temperature and diet. When starting from commercially available vaccines, the manufacturer will provide this information.
• vaccines for use according to the invention that are based upon bacterins can be given in general in a dosage of 10 3 to 10 10 , preferably 10 6 to 10 9 , more preferably between 10 8 and 10 9 bacteria.
• a dose exceeding 10 10 bacteria, although immunologically suitable, will be less attractive for commercial reasons.
• Vaccines according to the invention that are based upon live attenuated bacteria can be given in a lower dose, due to the fact that the bacteria will continue replicating for a certain time after administration.
• a vaccine for use according to the invention examples include sterile water, saline, aqueous buffers such as PBS and the like.
• a vaccine according to the invention may comprise other additives such as adjuvants, stabilisers, anti-oxidants and others, as described below.
• Vaccines for use according to the present invention 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 non-specific manner.
• a number of different adjuvants are known in the art. Examples of adjuvants frequently used in fish and shellfish farming are muramyldipeptides, lipopolysaccharides, several glucans and glycans and CarbopolW.
• 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).
• 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 ® .
• non-mineral oil adjuvants 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
• Very suitable w/o emulsions are e.g. obtained starting from 5-50% w/w water phase and 95-
• a water-based nano-particle adjuvant is e.g. Montanide-IMS-2212.
• the amount of adjuvant added depends on the nature of the adjuvant itself, and information with respect to such amounts will be provided by the manufacturer.
• the vaccine is mixed with stabilisers, 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 stabilisers 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.
• 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.
• the vaccines according to the invention are preferably administered to the fish via injection, immersion, dipping or per oral. It should be kept in mind however that the route of administration highly depends on the way the Streptococcal vaccine for use according to the invention is usually given.
• the Streptococcus bacterium now used for the manufacture of a vaccine for combating RLO infection is e.g. a live attenuated bacterium
• the vaccine could i.a. be administered by immersion or bath vaccination, due to the ease of administration.
• Such vaccines are often applied by immersion vaccination.
• Streptococcus bacterium now used for the manufacture of a vaccine for combating RLO infection is in the form of a bacterin, oral application and e.g. intraperitoneal application are attractive ways of administration.
• the way of administration would preferably be the intraperitoneal route.
• 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 RLO in as early a stage as possible, i.e. prior to possible exposure to the pathogen.
• Immersion vaccination would be the vaccination of choice especially when fish are still small, e.g. between 2 and 5 grams. Fish from 5 grams and up can also be vaccinated by means of injection.
• the vaccine is preferably mixed with a suitable carrier for oral administration i.e. cellulose, food or a metabolisable substance such as alpha-cellulose or different oils of vegetable or animals origin.
• a suitable carrier for oral administration i.e. cellulose, food or a metabolisable substance such as alpha-cellulose or different oils of vegetable or animals origin.
• an attractive method is administration of the vaccine to high concentrations of live-feed organisms, followed by feeding the live-feed organisms to 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.
• the Streptococcal bacterium for use according to the invention is of the species Streptococcus iniae, agalactiae or difficile.
• the bacterium for use according to the invention is of the species Streptococcus difficile.
• the vaccines for use according to the invention will preferably be vaccines that protect against RLO and additionally against more than one non-RLO pathogenic microorganism. It would be beneficial to use, next to Streptococcal bacteria for the manufacture of the vaccine, also at least one other fish-pathogenic microorganism or virus, an antigen of such microorganism or virus or genetic material encoding such an antigen, in a combination- vaccine.
• the advantage of such a vaccine is that it not only provides protection against RLO and against Streptococcal infection, but also against other diseases.
• a preferred form of this embodiment relates to a vaccine wherein that vaccine comprises at least one other microorganism or virus that is pathogenic to fish, or one other antigen or genetic material encoding said other antigen, wherein said other antigen or genetic material is derived from a virus or microorganism pathogenic to fish.
• Examples of commercially important fish pathogens in tropical and/or Mediterranean fish are Vibrio anguillarum, Photobacterium damselae subspecies piscicidae, Tenacibaculum maritimum, Flavobacterium sp., Flexibacter sp., Lactococcus garvieae, Edwardsiella tarda, E. ictaluri, Viral Necrosis virus, iridovirus and Koi Herpesvirus.
• Examples of commercially important cold water fish pathogens are Vibrio anguillarum, Aeromonas salmonicidae, Vibrio salmonicidae, Moritella viscosa, Vibrio ordalii, Flavobacterium sp., Flexibacter sp., Streptococcus sp., Lactococcus garvieae, Edwardsiella tarda, E. ictaluri, Piscirickettsia salmonis, Salmon Pancreatic Disease virus, Sleeping Disease virus, Viral Nervous Necrosis virus, Infectious Pancreatic Necrosis virus and iridoviruses. Parasites infecting Salmonids are i.a.
• a parasite infecting freshwater fish is e.g. Ichthyophthirius multifiliis.
• Tilapia parasites are e.g. Dactylogyrus spp. and Trichodina spp. Marine fish may suffer i.a from the parasite Benedenia seriolae
• the other microorganism or virus is selected from the following group of fish pathogens: Vibrio anguillarum, Photobacterium damselae subspecies piscicidae, Tenacibaculum maritimum, Flavobacterium sp., Flexibacter sp., Lactococcus garvieae, Edwardsiella tarda, E.
• Viral Necrosis virus Viral Necrosis virus, iridovirus and Koi Herpesvirus, Aeromonas salmonicidae, Vibrio salmonicidae, Moritella viscosa, Vibrio ordalii, Piscirickettsia salmonis, Salmon Pancreatic Disease virus, Sleeping Disease virus, Viral Nervous Necrosis virus, Infectious Pancreatic Necrosis virus, iridoviruses, Lepeophtherius salmonis, Caligus elongatus, Cryptobia salmositica, Myxobolus cerebralis, Kudoa thyrsites, Ichthyophthirius multifiliis, Dactylogyrus spp. , Trichodina spp. and Benedenia seriolae.
• Example 1 In this Example, a comparison is given between the efficacy of RLO-based vaccines, Streptococcus -based vaccines and a non-Streptococcal Gram-negative vaccine
• RLO-based vaccines A RLO-strain originally isolated from diseased tilapia, cultured in Indonesia showing typical RLO-disease signs was used both as vaccine strain and as challenge strain. This strain was sub-cultured on Chocolate agar and subsequently inoculated and grown in a liquid broth on an orbital shaker at 26°C - 150 RPM. After approximately 24 h incubation the culture reached an OD 66 o nm of 0.674 and was inactivated by the addition of 0.5% formalin and transferred to a new vessel. The total amount of bacterial cells in the inactivated culture was 3.7 x 10 9 cells/ml.
• V. anguillarum vaccines were prepared starting from a V. anguillarum serotype 02 A strain. Standard V. anguillarum growth methods were applied. Vaccines comprising 2.4 x 10 9 and 1.0 xlO 10 cells were prepared.
• the S. iniae culture and the S. difficile culture were mixed with standard buffer and brought to a total count of 5 x 10 9 cells per ml each.
• the RLO used for challenge was grown as described under preparation of challenge material.
• Vaccination was done by IP-injection of tilapia with 0.1 ml of the respective vaccines (See table 1). Fish were maintained for 3 weeks after vaccination and subsequently challenged with a viable RLO culture. Challenged fish were kept separate and mortality was followed over a 1 week period post-challenge. The efficacy was evaluated by expression the relative percent survival of vaccinated fish as compared to the controls.
• Table 1 Vaccine composition with regard to number of cells and presence of adjuvant.
• a vial of RLO seed glycerol stock was thawed and inoculated onto Chocolate agar and incubated at 26°C for 24 hours. Growth from the plates was collected and inoculated as solid growth into 100 ml of liquid broth and incubated on an orbital shaker (150 RPM) at 26°C. After approximately 24 hours of growth the purity of the culture was checked and the OD 660 was measured.
• Challenge Groups of approximately 15 fish were taken from the tanks and injected with the challenge suspension as described above. Fish were IP-challenged by injection with 0.1 ml of the suspension. The same procedure was followed for all groups. After challenge fish were transferred to different tanks and kept for observation and the occurrence of mortality.
• the vaccines tested were found to be safe.
• a challenge of vaccinated fish was performed 3 weeks after the IP-vaccination with RLO 's grown in artificial media.
• the disease as well as the typical disease signs could be reproduced and the challenge organism could be re-isolated from dead fish.
• the objective of this experiment was to evaluate whether protection against RLO infections is obtained when tilapia are vaccinated with oil-based bivalent vaccines containing Streptococcus iniae and S. difficile. Also, the relation between the level of protection and the number of bacteria present in the Streptococcus -based vaccine was determined in this Example.
• Two different vaccines, containing both Streptococcus iniae and S. difficile at different concentrations in a non-mineral water-in-oil emulsion were IP-injected in juvenile tilapia.
• the antigen levels in the vaccines were 6.8 x 10 8 and 1.7 x 10 8 cells of each antigen per ml of vaccine.
• fish from each vaccine group and non- vaccinated control groups were challenged with a suspension of Rickettsia-Hkc bacteria.
• Vaccines were prepared starting from a S. iniae fermentor culture and a S. difficile fermentor culture. All vaccine formulations were prepared in a non-mineral water-in-oil adjuvant. The vaccines used in the study are presented in Table 2.
• the antigen concentrations specified in Table 2 represent the final vaccine concentration, i.e. after mixing with the PBS/oil emulsion.
• the fish were starved for 48 h prior to vaccination to assure complete emptying of the intestinal tract and thereby reducing the possibility of damaging the internal organs when injecting.
• the vaccination was performed by IP injection.
• the experiment included 3 experimental groups (2 vaccine groups and 1 control group), each consisting of 25 fish. Each fish from the vaccine groups was injected with 0.1 ml of vaccine according to Table 2. Control fish were not injected.
• Vaccines were prepared as described in Examples 1 and 2. The following challenges with RLO were performed:
• Figure 4 shows, that at 6 weeks post-vaccination with a monovalent Streptococcus difficile vaccine followed by injection challenge, cumulative mortality in control fish reached 90%, whereas of the vaccinated fish only 20% died. This results in a RPS of 78%.
• Figure 1 RPS values per vaccine group as calculated compared to the PBS- mock vaccinated control group
• Figure 2 Cumulative percentage mortality over time, in relation to the vaccine dose given. The effect of the number of cells in the vaccine on the relative percentage survival after RLO -challenge is given.
• Figure 3 Final cumulative percentage survival (compare figure 2), in relation to the vaccine dose given.
• Streptococcus difficile vaccine comprising 6.8 x 10 7 Sd cells in a non-mineral water-in- oil emulsion.

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