WO1982002491A1

WO1982002491A1 - Fish vaccines

Info

Publication number: WO1982002491A1
Application number: PCT/US1982/000039
Authority: WO
Inventors: Regents Univ Washington Board; Jorge Homero Crosa
Original assignee: Univ Washington
Priority date: 1981-01-16
Filing date: 1982-01-15
Publication date: 1982-08-05
Also published as: ES508767A0; IT1154265B; JPS58500026A; IT8247575A0; DK414082A; ES8304801A1; FR2509177A1; SE8205247D0; SE8205247L; NO823124L

Abstract

Method for making a vaccine against Vibrio anguillarum and other closely related vibrios. Cells containing a virulence plasmid are grown under iron limitation to allow expression of the 86, 000 dalton protein OM2 present in the outer membrane of Vibrio anguillarum when the cells are grown under iron limitation. These cells, or isolated OM2, are then used as a vaccine.

Description

FISH VACCINES This invention relates generally to vaccines and, more particularly, to a method for making a vaccine against Vibrio anguillarum and other closely related vibrios. This invention was made under a Federal contract NOAA Sea Grant No. NA79AA-D-00054.

A major obstacle to the success of commercial fish farming or aquaculture is disease. One particularly high mortality rate disease which affects salmon (salmonids) and many other anadromous and catadro ous species of fish is vibriosis (1) . This disease is caused by infection with Vibrio anguillarum and other closely related vibrios. The disease is characterized by a he orrhagic septicemia, and morbidity is accompanied by a massive destruction of a variety of cells and tissue types (2).

Although numerous attempts have been conducted to develop a successful vaccine against this disease, results have been disappointing. More particularly, attempts to protect against the more virulent forms of Vibrio anguillarum have been either failures or of very limited success (3) (4) .

It is an object of the present invention to provide an improved vaccine against Vibrio anguillarum and other closely related vibrios.

Another object of the invention is to provide an improved method for making a vaccine against Vibrio anguillarum and other closely related vibrios.

A further object of the invention is to provide a vaccine against Vibrio anguillarum and other closely related vibrios which is highly effective against many of the virulent strains.

Other objects of the invention will become apparent to those skilled in the art from the following description, taken in connection with the illustrations wherein:

FIGURE 1 is a graph illustrating the kinetics of radioactive iron uptake by nongrowing cells of Vibrio anguillarum under conditions of iron limitation; and FIGURE 2 is a photographic reproduction illustrating the range of iron chloride concentrations at which production of specific surface proteins of Vibrio anguillarum are induced.

Very generally, one form of the method of the invention comprises growing cells expressing the 86,000 dalton protein 0M2 which is present in the outer membrane of Vibrio anguillarum grown under iron limitation. Such cells may be either Vibrio anguillarum, or cells which are a nonpathogenic host having replicatable DNA coding for at least a substantial portion of the OM2 protein. The cells are subsequently either attenuated or killed and used as a vaccine, or the 0M2 protein is isolated from the cells and used as a vaccine. it has been shown that virulence in Vibrio anguillarum is associated with the presence of a particular plasmid in the pathogen (5) . This specific plas id class is absent from low virulence strains (6). The association of this specific plasmid class and the virulence of Vibrio anguillarum has been demonstrated (7) . It has further been reported that there is evidence showing that Vibrio anguillarum virulence plasmid specifies a very efficient iron sequestering system (7) . This enables the bacteria to survive in conditions of limited iron availability. The significance of this fact is that the bacteria are therefore enabled to survive and grow even though the host defense mechanisms cause binding of iron to the proteins transferrin and lactoferrin. It has been reported that many of the virulent strains of Vibrio anguillarum biotype I isolated in the Pacific Northwest of the United States harbor a specific plasmid class pJMl, which is absent from low virulence strains (5). The experiments suggested that virulence was associated with this particular plasmid class. The virulence mechanism appears to be the ability, in the virulent strains containing the plasmid, to take up iron even though the infected host fish's defense mechanism produces transferrin and lactoferrin to bind with the iron (6). Because the Vibrio anguillarum containing this plasmid therefore has the ability to grow under conditions of iron limitation, by scavenging the bound iron from the iron binding protein, the invading bacteria are able to proliferate in body fluids and tissues.

It has been discovered that those strains of Vibrio anguillarum which are characterized by the plasmid mediated uptake system have certain specific outer membrane proteins at least one of which is only produced when the virulence plasmid is present. These outer membrane proteins appear to be associated with the iron uptake function. It is believed that these outer membrane proteins form a receptor for ferric siderophore (iron binding compounds produced by the bacteria) necessary for its transport into the cell.

Analysis of outer membrane proteins present in Vibrio anguillarum containing the virulence plasmids demonstrates that at least two outer membrane proteins are induced under growth conditions in which iron is limited to certain levels. One of these proteins, designated OM2, is 86,000 dalton molecular weight and is associated with the presence of the pJMl plasmid. Although this protein is present only in strains containing the plasmid, it is as yet uncertain as to whether this protein is actually coded for by th plasmid and induced in response to a decrease in the iron concentration, or is a chromosonal product that is regulated by a plasmid specific substance.

The second protein detected in the outer membrane has been designated OM3 and is 79,000 dalton molecular weight. The association of this protein with the plasmid pJMl is as yet unclear.

In accordance with the invention, the vaccine is prepared from cultures of cells which are grown in such a way that the OM2 protein is expressed. Preferably, these cells are Vibrio anguillarum cells grown under iron limitation sufficient to induce expression of the OM2 protein. However, the protein may also be produced by producing a host cell having a replicatable ONA coding for at least a substantial portion of the OM2 protein. Recombinant DNA techniques are well known to those skilled in the art and may be followed to produce such host cells. In either case, the cells are then attenuated or killed and used as the vaccine, or the OH2 protein is isolated from the cells and itself used as the vaccine. By way of example, strains of Vibrio anguillarum, some of which contain the virulence plasmid and some of which were cured derivatives of plasmid carrying strains were studied as follows:

Correlation between iron-uptake, presence of plasmid and virulence of strains of Vibrio anguillarum. Vibrio anguillarum Presence of^a Uptake of LD50^C

H775-8 - 450 2.5 x 10⁶

E775-100 - 600 2 x 10⁶

Plasmid ONA was determined by an agarose electrophoretic method as described in (6) . Values were obtained from kinetic experiments similar to that in FIGURE 1 by determinining the ratio of 55Fe cpm/time at the 20 minute time point. ^c LD50 values were determined as described below.

As set out in the table, the particular strains were studied for the ability to take up iron and for the mean lethal dose. Measurement of iron uptake was carried out as follows:

Bacterial strains were grown for several generations at 22°C in a low-iron minimal medium (14) (iron content 2μM) supplemented with 0.5%' (wt/vol) glucose and the required amino acids, aspartic acid and histidine, at 20 μg/ml. Exponentially growing cultures were centrifuged, cells were washed and resuspended to a

D density of 0.4 x 10 cells in similar medium except that the iron content was now less than 0.5μM iron.

After further incubation for 2 hours at 22^βC to deplete iron intracellular pools, cultures were centrifuged, cells were washed and resuspended to a density of 4 x g

10. cells/ml in similar medium lacking essential amino acids but containing lOOμM sodium nitrilotriacetate. In some experiments 2mM KCN {a respiratory inhibitor) was included in this stage. Carrier-free 55FeCl, (ly

Ci/ml) was added to shaking cell suspensions, 1 ml samples were removed at intervals and filtered through Millipore membrane filters (0.45μM pore size). Filters were washed with 100 mM sodium nitrate, dried and counted in a Packard-TriCarb liquid scintillation counter using a toluene-based scintillation cocktail containing Omnifluor (New England Nuclear) at 4 grams/liter.

In order to determine whether the ability of plasmid-carrying high-virulence Vibrio anguillarum strains to grow in iron-limited conditions is due to an efficient iron-uptake system, the uptake of radioactive iron by non-growing cells was directly measured. The plasmid-carrying Vibrio anguillarum 775 (pJMl) and the plasmidless derivative H775-3 were grown in a minimal medium containing about 2 μM FeCl₃ (minimal iron concentration at which the H775-3 strain can grow) for several generations. Exponentially growing cultures were centrifuged, cells were washed with low-iron medium (iron content less than 0.5.μM) and resuspended to 0.4 x 10 cells/ml in similar medium containing less than 0.5 μM iron. After incubation in this medium to deplete intracellular iron pools, cultures were centrifuged, cells were washed and resuspended to a density of 4 x

Q

10 cells in similar medium lacking essential amino acids but containing nitrilotriacetate and then exposed

55 to Fe as described in Materials and Methods. FIGURE

1 and the preceding table show that under the same uptake conditions non-growing cells of the plasmid-carrying high-virulence Vibrio anguillarum 775

(pJMl) , 133S (pJMl) or LS174 (pJMl) take up radioactive iron more rapidly than the isogenic plasmidless low-virulence derivative H775-3. Similar low uptake was obtained with other plasmidless low-virulence derivatives shown in the table.

Additional information regarding the events leading to iron accumulation was obtained by utilizing the respiratory inhibitor KCN. An energy-dependent process like transport of iron inside the cell is inhibited by KCN whereas binding of iron to the bacterial membrane which is energy-independent is not. FIGURE 1 shows that iron accumulation by the plasmid- carrying strain of Vibrio anguillarum 775 (pJMl) is greatly inhibited by 2mMKCN suggesting taht the process must be uptake rather than simply binding to the bacterial membranes, although the presence of the small amount of energy-independent accumulation of iron (in the presence of KCN) could be due to some kind of association step which also appears to be plasmid-mediated.

These results demonstrate that presence of the virulence plasmid in Vibrio anguillarum indeed involves a more rapid and efficient iron-uptake. This observation explains the ability of plasmid-carrying Vibrio anguillarum to grow in the presence of iron-chelators. In order to further investigate the presence of a plasmid-mediated iron uptake system in Vibrio anguillarum, an investigation was made as to whether any specific cell envelope protein was induced under conditions of iron-limitation. Such is the case for iron-uptake systems in certain enteric bacteria. To this end, Vibrio anguillarum 775 (pJMl) and its plasmidless low-virulence derivative H775-3 were grown in minimal medium to which iron as FeCl, was added to various concentrations. In some cases 3μM transferrin was added to the growth medium. Total cell envelopes as well as outer membranes were prepared from cells grown under these various conditions and were analyzed by SDS-polyacrylamide gel electrophoresis. FIGURE 2 lanes A-H show total cell envelopes for both strains at different FeCl. concentrations; while lanes I and J are outer-membrane proteins obtained from cells grown at 2μM FeCl₃. There are five major outer-membrane proteins (0M1-5) present in the plasmid-carrying strains grown at 2;-M FeCl₃. one of these OM2 is missing from the plasmidless derivative.

^■ Analysis of total cell envelopes of the plasmid- containing strain shows clearly that there is a threshold of FeCl₃ concentration (below 4 M) at which both OM2 and OM3 are inducible while these proteins are not detectable at higher iron concentrations in plasmid- carrying strains (lanes A-F) . Lane A shows that both OM2 and OM3, as expected, are present in the plasmid-carrying strain grown in the presence of 3-.μM transferrin. In the case of the plasmidless strain, the OM3 protein is the only protein induced at 2μ M FeCl₃ (lowest iron concentration to allow growth of this strain) (compare Lanes G, J and H) . Thus iron limitation conditions which allow a very rapid uptake of radioactive iron by plasmid-carrying Vibrio anguillarum strains also induce the synthesis of two specific outer-membrane proteins OM2 and OM3 of 86,000 and 79,000 daltons respectively (as measured with protein molecular weight standards (not shown)) . The OM2 protein is associated with the presence of the plasmid pJMl. An elevated synthesis of the OM2 protein under iron limitation conditions could be a reflection of an increased number of copies of the virulence plasmid under these same conditions. However, this is not the case. Plasmid copy numbers determined in the presence of transferrin and at various iron concentrations

(0.05-12 μM) indicated that there are no significant changes of copy number, while at this same range of concentrations there are dramatic changes in the induction of the OM2 protein (FIGURE 2) . The foregoing results demonstrate that the surface protein OM2 and possibly the surface protein OM3 are significant factors in the virulence quality of the pathogen of interest. Their presence, therefore, is significant in producing an antibody response to these proteins in an infected host fish species. Prior art vaccines do not take this fact into account. Accordingly, vaccines may be prepared from bacterial strains expressing this protein, by attenuating or killing the strains by conventional techniques if necessary. As an alternative, vaccines may be prepared by purifying the surface protein itself as the vaccinating agent.

It may be seen, therefore, that vaccines prepared in accordance with the invention confer more precise immunity to the highly virulent strains of

Vibrio anguillarum and other closely related vibrios.

Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying illustrations. For instance, products described herein may be utilized with products of other vibrio serotypes to provide broader immunity. Such modifications are intended to fall within the scope of the appended claims. REFERENCES

1. Fryer et al., "Vibriosis in Fish", Prog. Fish Food Sci., 1:129-131 (1972).

2. Harbell et al., "Studies on the Pathology of Vibriosis in Coho Salmon (Oncorhynchus kisutch)", J. Fish. Dis. .2:527-535 (1979).

3. Gunnels et al., "Failure of Vaccines to Protect Salmon from Vibriosis Enzootic in Puget Sound, Washington", Am. J. Vet. Res., 32:737-740 (1976).

4. Gould et al., "Immersion Vaccination of Sockeye Salmon (Oncorhynchus. nerka) with Two Pathogenic

Strains of Vibrio anguillarum", J. Fish Res. Board Can., 21:222-225 (1979)

5. Crosa et al., "Evidence for Plasmid Contribution to the Virulence of the Fish Pathogen Vibrio anguillarum". Infect. Immun. 18:509-513 (1977).

Iff^* 6. Crosa et al., "Curing of a Plasmid is Correlated with an Attenuation of Virulence in the Marine Fish Pathogen Vibrio anguillarum". Infect.

Immun._27:897-902 (1980).

7. Crosa et al., "A Plasmid Associated with Virulence in the Marine Fish Pathogen Vibrio anguillarum Specifies an Iron Sequestering System", Nature, 284:566-567 (1980).

\

Claims

CLAIMS :

1. A method for making a vaccine against Vibrio anguillarum and other closely related vibrios, comprising, growing Vibrio anguillarum cells under iron limitation sufficient to induce the expression of the 86,000 dalton outer membrane protein OM2, and attenuating or killing such cells.

2. A method according to Claim 1 wherein the iron content of the growth medium in which the cells are grown is less than about 4 μM.

3. A method according to Claim 1 wherein the

79,000 dalton outer membrane protein OM3 is also expressed.

4. A method for making a vaccine against Vibrio anguillarum and other closely related vibrios, comprising, growing cells expressing the 86,000 dalton protein OM2 which is present in the outer memorane of Vibrio anguillarum grown under iron limitation.

5. A method according to Claim 4 wherein said cells are Vibrio anguillarum.

6. A method according to Claim 4 wherein said cells are a nonpathogenic host cell having replicatable DNA coding for at least a substantial portion of the OM2 protein.

7. A method according to Claim 4 wherein said cells are attenuated.

8. A method according to Claim 4 wherein said cells are killed.

9. A method according to Claim 4 wherein the OM2 protein is isolated from the cells.

10. A vaccine produced in accordance with

Claim 1.

11. A vaccine produced in accordance with Claim 4,