Patent Application
20130216589
The present invention relates to an oral vaccine for aquatic animals, particularly an oral vaccine with an enhanced immune response.
Oral vaccine containing a viral antigen is the most desirable immunization method to prevent fish disease at early larvae and fingerling stage, as it is easy to use, safe and stress-free for fish [Campbell R et al., Uptake of Vibrio anguillarum vaccine by Artemia salina as a potential oral delivery system to fish fry. Fish Shellfish Immunol. 3:451-459, 1993; and Vervarcke S et al., Oral vaccination of African catfish with Vibrio anguillarum O2: effect on antigen uptake and immune response by absorption enhancers in lag time coated pellets. Fish Shellfish Immunol. 16:407-414, 2004]. For example, fish larvae are developed in water environment that contains pathogens, also encounters diseases, and thus oral vaccine encapsulated in rotifer or Artemia was developed as the natural starting feed to enable the early and easy immunization. However, fish oral vaccines were known to have low efficacy and thus the potency needed to be bettered [Shao Z J; Aquaculture pharmaceuticals and biologicals: current perspectives and future possibilities. Adv Drug Deliv Rev. 50:229-243, 2001; and Lin C-C et al.; An oral nervous necrosis virus vaccine that induces protective immunity in larvae of grouper (Epinephelus coioides). Aquaculture 268:265-273, 2007].
Conventionally, adjuvant, either a chemical composition or a physical structure, that can enhance efficacy of a particular vaccine rare used in injective vaccine. Oral vaccine often cannot use chemical based adjuvant, as chemical based adjuvant will cause side effect, especially, deformity in fish larva that is still at the embryonic stage. A biological based adjuvant that can be digested and with no side effect would be preferred for a oral vaccine.
To develop aquaculture industry, a method to enhance the efficacy of the oral vaccine is desirable.
The present invention relates to a new approach to enhance the protective efficacy of an oral vaccine for aquatic animals by expressing a specific antigen in a bacterial expression host that has activity to enhance innate immunity and confers adjuvant function, and used to construct an oral vaccine [Lin C-C et al.; An oral nervous necrosis virus vaccine that induces protective immunity in larvae of grouper (Epinephelus coioides). Aquaculture 268:265-273, 2007]. It is found in the invention that the prophylactic and therapeutic efficacy of the antigen contained in an oral vaccine if Listonella sp., such as L. anguillarum, is used as an expression host for the antigen.
In one aspect, the invention provides an oral vaccine with an enhanced protective efficacy for aquatic animals comprising an antigen, which is expressed in Listonella sp., such as L. anguillarum, as an expression host transformed with an expression vector comprising a specific nucleic acid sequence encoding the antigen, such as a viral subunit.
In another aspect, the invention provides an oral vaccine with an enhanced protective efficacy for aquatic animals comprising an antigen, which is expressed in L. anguillarum as an expression host transformed with an expression vector comprising the antigen, and then is encapsulated in rotifer sp., such as Brachionus plicatilis.
In another aspect, the invention provides an oral vaccine with an enhanced protective efficacy for aquatic animals comprising an antigen, which is expressed in L. anguillarum as an expression host transformed with an expression vector comprising the antigen, and then is encapsulated in Artemia sp., such as Artemia nauplii.
In the other aspect, the invention provides a method for preparing the oral vaccine preparation to have concurrent adjuvant activity, the invention.
The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments, which are presently preferred. It should be understood, however, that the invention is not limited to the embodiments shown.
Selecting a bacterium that when inactivated can stimulate innate immunity and function as adjuvant for vaccine preparation
Selection of shuttle promoter that can express in the bacterium
Construct a recombinant plasmid by fusing gene or part of gene of a specific target antigen onto the promoter, as exampled in the drawings:
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs.
As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a sample” includes a plurality of such samples and equivalents thereof known to those skilled in the art.
As used herein, the term “vaccine” refers to a biological preparation that induces immunity to a particular disease in a subject, which typically contains an agent that resembles a disease-causing microorganism, and is often made from weakened or killed forms of the microbe, its toxins or one of its surface proteins, and can stimulate the immune system to recognize the agent as foreign, destroy it and remember it so that the immune system can more easily recognize and destroy any of these microorganisms that infect the subject later. In the invention, the vaccine is prophylactic and/or therapeutic.
As used herein, the term “aquatic animal” refers to either vertebrate or invertebrate living in water for most or all of its life; particularly fishes or shrimps.
As used herein, the term “natural starting feed” refers to multiple cell animals living in water and usually are food for fish or shrimp larvae or fingerlings; particularly Artemia or Rotifers.
As used herein, the term “promoter” refers to a regulatory region of DNA located upstream of a gene, providing a control point for regulated gene transcription in a host. In the invention, the promoter includes any regulatory region for expression of an antigen in a Listonella host.
According to the invention, an oral vaccine with an enhanced protective efficacy for aquatic animals is provided. The oral vaccine comprises an antigen, which is expressed in Listonella sp., such as L. anguillarum, as an expression host transformed with an expression vector comprising the nuclei acid encoding the antigen as illustrated in
The present invention relates to an oral vaccine of the invention is either for prophylactic vaccination or for therapeutic vaccination. In the invention, the antigen used in the oral vaccine may be any agent resembles a disease-causing microorganism, and is often made from weakened or killed forms of the antigen. One example of the antigens in the invention is a foreign polypeptide of interest. In one example, the antigen is a viral subunit. The viral subunit includes non-recombinant or recombinant viral subunit. Recombinant viral subunits are usually used for preparing oral vaccine for aquatic animals. In one example of the invention, the antigen is a subunit of Nervous Necrosis Virus (NNV).
According to the invention, the invention is characterized by using Listonella sp. (formerly Vibrio sp.) as an expression host, providing an unexpectedly prophylactic and therapeutic efficacy against infection. In one example of the invention, Listonella anguillarum was used as an expression host to prepare an oral vaccine for aquatic animals, providing an improved protective efficacy of the oral vaccine.
The oral vaccine may include a multiple-cell organism that has fed on, and therefore contains, a single-cell organism. The single-cell organism fed to the multiple-cell organism has been transformed to express a recombinant antigen that can induce an immune response in the aquatic animal. The just-described multiple-cell organism, fed to the aquatic animal, serves as an oral vaccine to the animal. In other words, an antigen, being expressed in a single-cell, multiple-cell organism or in a aquatic animal, is delivered to the aquatic animal via two steps of feeding, i.e., the above-described single-cell organism fed to the multiple-cell organism, and the multiple-cell organism from previous step fed to the aquatic animal. This has been invented by Yang et a.l and were patented in U.S. Pat. No. 6,872,386 B2, ORAL VACCINES. Mar. 29, 2005 and U.S. Pat. No. 7,807,144 B2 Oct. 5, 2010
In the invention, the nucleic acid encoding the antigen that triggers the immune response, is cloned into a recombinant vector. The nucleic acid encoding the antigen is operatively linked to one or more regulatory sequences. The regulatory sequences can be those that direct constitutive expression of the antigen, as well as inducible sequences. The recombinant vector can be designed based on such factors for expression in Listonella sp. It may contain more than one nucleic acid encoding different antigens. For example, a recombinant vector contains nucleic acids encoding two antigens, which can induce immune responses against the same or different pathogens. Alternatively, the recombinant vector may contain a nucleic acid encoding a polypeptide (e.g., a helper epitope) that is not antigenic, but itself or its encoded peptide serves to enhance an immune response against a targeted pathogen.
According to the invention, a nucleic acid encoding a viral subunit is constructed in an expression vector, which is operatively linked to a promoter for Listonella sp., particularly, L. anguillarum HP-60 promoter.
Furthermore, the oral vaccine of the invention may be encapsulated in Artemia sp., such as Artemia nauplii. The process for the encapsulation was disclosed in U.S. Pat. Nos. 6,872,386 and 7,807,144 invented by one of the inventors, which are incorporated by reference herein in its entirety. According to the invention, the oral vaccine of the invention is encapsulated in Artemia sp., such as Artemia nauplii, which provides an effective way to provide a solution to the problem due to difficult antigen degradation in the digestive tract of the aquatic animal.
The following examples are offered to illustrate this invention and are not to be construed in any way as limiting the scope of the present invention.
1.1 Bacteriaum and Fish
L. anguillarum strain TW0302 was isolated from seawater in fish farm at Tainan. All experimental fish were produced from the indoor NNV-free hatchery and were NNV free as confirmed by RT-PCR. All challenge trails were carried out in our indoor NNV-free fish experimental facility.
1.2 Construction of pgroEp-NNVcp Fusion Plasmid
A p groEp-NNVcp (NNV coat protein) fusion plasmid based on the pGL3 plasmid (as shown in
1.3 Construction of Shuttle pgroEp-NNVcp Plasmid and Transformation to L. anguillarum
The expression hosts used to produce recombinant NNVcp were L. anguillarum strain TW0302 isolated in our laboratory or E. coli strain BL21 (DE3). Recombinant pgroEp-NNVcp plasmid was transformed to L. anguillarum by electroporation [Cutrin J M et al., Genetic transformation of Vibrio anguillarum and Pasteurella piscicida by electroporation. FEMS Microbiol Lett 128:75-80, 1995], then selected for the ampicillin resistant trait using a TSB agar plate supplemented with 0.1 mg ml−1 ampicillin sodium. The bacterial colonies grown on the plate were further analyzed by PCR using temperature profiles described in section 2.1, with the set of primers listed in Table 1, to verify the presence and ensure the percentage of the NNVcp containing plasmid.
1.4 Analysis of the Optimal Temperature Induction Condition of pgroEp-NNVcp in L. anguillarum
The transformed L. anguillarum was inoculated into a 2 L flask containing 600 ml TSB broth supplemented with 0.1 mg ml−1 sodium ampicillin, cultivated by shaking (200 rpm) at 25° C. until the OD600 reached 1.0, then incubated for inducing NNVcp.
1.5 Measurement of Antigen Dosage in Oral Vaccines
The oral vaccines were prepared by encapsulating the inactivated bacteria and the mock versions in Artemia nauplii according to Lin's reference (Aquaculture 268:265-27312007).
In order to determine how number of bacteriim and the quantity of antigen encapsulated in one Artemia, we used a real time PCR method to quantify the number of bacterium, and SDS-PAGE/west blot method to measure the amount of antigen in these two bacterial hosts. A method based on real time PCR technology was designed to determine the amount of L. anguillarum or E. coli encapsulated in each Artemia . Briefly, duplicate samples of 105 to 109 CFU of L. anguillarum or E. coli, and Artemia individuals encapsulated with E. coli or L. anguillarum were collected, washed thoroughly with deionized water, treated according to Prol M J et al. (Real-time PCR detection and quantification of fish probiotic Phaeobacter strain 27-4 and fish pathogenic Vibrio in microalgae, rotifer, Artemia and first feeding turbot (Psetta maxima) larvae. J Appl Microbiol 106:1292-1303, 2009) to obtain purified DNA, and analyzed by real time PCR using the temperature profiles described in Rojo I et al. (Innate immune gene expression in individual zebrafish after Listonella anguillarum inoculation. Fish Shellfish Immunol 23:1285-1293, 2007) with an adequate set of primers designed for E. coli or L. anguillarum (Table 1).
Standard curves relating Ct values to bacterial number (CFU) were obtained by analyzing the PCR measurements with Microsoft Excel software, and quantification of bacteria in each Artemia was conducted by running the same real-time PCR analysis with an adequate set of primers and standard curves.
The SDS-PAGE/western blotting was performed according to Chen Y M et al. (Grouper Mx confers resistance to nodavirus and interacts with coat protein. Dev Comp Immunol. 32:825-836, 2008). The protein extracted from 4×107 CFU recombinant E. coli or L. anguillarum was loaded onto the same gel, and the relative amount of antigen was determined according to the analysis of western blot staining image by using Image J software (NIH, Bethesda, Md., USA).
1.6 Preparation of NNVcp Oral Vaccine Using E. Coli and L. Anguillarum Expression Host
To prepare L. anguillarum based oral vaccine, the cells with maximal NNVcp content were washed by centrifugation (2,500×g, 15 min), rinsed with 600 ml sterile seawater, re-suspended in sterile seawater at 1×1010 CFU ml−1, deactivated at 65° C. in a water bath for 10 min, and cooled on ice for 10 min to obtain the subunit vaccine product. A L. anguillarum mock subunit vaccine was also produced by using non-transformed L. anguillarum following the same procedures.
To prepare an E. coli-based NNV subunit vaccine, the same p groEp-NNVcp fusion plasmid was transformed into E. coli strain BL21 (DE3), and the transformed E. coli cells were harvested and deactivated by the same procedures for the preparation of the L. anguillarum-based subunit vaccine, with the exception of the use of sterile PBS buffer (137 mM NaCl; 2.7 mM KCl; 10 mM Na2HPO4; 2 mM KH2PO4; adjusted to pH 7.4 with 6 N HCl) instead of sterile seawater for rinsing and suspending cells. A mock subunit vaccine was prepared by using non-transformed E. coli following the same protocol used for preparing E. coli-based subunit vaccine.
1.7 Immunization of Larvae
Thirty days post-hatch (dph) larvae of E. coioides, which were confirmed free of NNV by RT-PCR, were used in the study. Five hundred larvae were randomly divided into five groups (n=100) and immunized. One group was a placebo group fed Artemia only, the other four groups were fed with Artemia encapsulated mock) of E. coli based oral vaccines, and two parallel groups fed with mock (transformed with pgroEp,) and NNVcp subunit vaccine produced in L. anguillarum (transformed with pgroEp-NNVcp). Fish were kept in five separate 20-L tanks with aeration with temperature maintained at 26±1° C. and larvae were acclimated for 3 days before immunization. A single dose of four individual oral vaccines or placebo was given 4 times at 33.0, 33.5, 34.0, and 34.5 dph. After immunization all groupers were fed with untreated Artemia twice a day until the end of the trial.
1.8 Assay the Expression of Innate Immunity Related Genes by RT-PCR
To evaluate the immune-stimulant effects of the subunit vaccine expressed in L. anguillarum, 3 grouper larvae were randomly selected from experimental groups fed with mock E. coli or L. anguillarum from 0 to 96 h after immunization (from 34.5 to 38.5 dph), killed with an overdose of anesthetic, and dissected to remove the brain and viscera.
RNAs were isolated and subjected to real time PCR to measure their expression levels of the three innate-immunity related genes i.e. IL-1β, TNF-α, or Mx isolated and sequenced from E. coioides in our laboratory. The total RNA was extracted from these samples, cDNA was synthesized, and real-time PCR was performed, wherein the set of primers used are given in Table 1. The significant of difference of expression of immune genes were statistically analyzed by one-way ANOVA and Duncan's new multiple range test.
1.9 Measurement of NNVcp Antibody
At 40 dph, 1 week after the beginning of oral vaccination, three fish were sampled randomly from each group, killed, homogenized, and centrifuged to obtain humoral extracts, and analyzed by ELISA to determine anti-NNVcp antibody titer. The ELISA analysis was performed, and the statistical analysis of antibody titer was proceeded by using one-way ANOVA and Dunnett's tests.
1.10 Challenge
NNV was used for challenging trail. At 40 dph, 40 fry were taken randomly from each group and injected intraperitoneally with 10 μl of virus solution with TCID50 as 1.0×105.5, pre-determinded as a lethal dosage. The mortality of grouper fry in each group was recorded daily for 3 weeks post-challenge, and the final cumulative mortality rates were used to calculate relative percent survival (RPS).
2.1 Construction of pgroEp-NNVcp Fusion Plasmid
The NNVcp gene was cloned under the control of pgroEp promoter isolated from L. anguillarum and put on a shuttle vector pGL 3, The genetic construct, the promoter region of recognition sites between two PCR primers—RV4 and Co-RV4 and the resulted pgroEp-NNVcp plasmid were illustrated in
2.2 The Optimal Temperature Induction Condition of pgroEp-NNVcp in L. anguillarum
It was found that the best induction temperature for NNVcp production was 37° C. The recombinant L. anguillarum was cultivated at 25° C. then shift to 37° C. to induce the synthesis of NNVcp. The heat induction resulted in significant NNVcp production in recombinant L. anguillarum as shown in
2.3 Measurement of the Number of Bacterium in Each Artemia
As bacterium once eaten bt Artemia, it could be rapidly digested, so that the conventional bacterial colony counting method to measure the number of bacteraium encapsulated into Artemia could be not accurate. The number of bacterium in each Artemia was then measured by assaying the copy of bacterial gene in Artemia using RT-PCR. The relationship between Ct value and bacteria number can be fit well with equation obtained from non-linear regression (
2.4 The Determination of NNVcp Antigen in Each Dosage of Oral Vaccines
The SDS-PAGE/western blot analysis confirmed the production of NNVcp in the E. coli and L. anguillarum expression hosts (indicated by solid arrow in
2.5 Innate-Immunity Stimulation Effects Related to Expression Hosts
The intake of both mock E. coli and L. anguillarum significantly upregulated TNF-α and IL-1β genes in viscera of grouper fry. The transcription levels of the two genes peaked at 24 h post-vaccination and then declined immediately (
As known in fish oral vaccine, no considerable amount of NNVcp serum specific antibody was found after the group immunized with E. coli based oral vaccine. However, it was found that L. anguillarum-based oral vaccine significantly elicited antigen-specific antibody level at 7 days post-vaccination (P<0.05) (
2.7 Challenge Test
Fish challenged with NNV died from 1-18 days post-challenge, with maximal mortality occurring during the first week in all groups (
In conclusion, the protective efficacy for aquatic animals could be improved by using L. anguillarum as an expression host with an improved immunogenicity.
It is believed that a person of ordinary knowledge in the art where the present invention belongs can utilize the present invention to its broadest scope based on the descriptions herein with no need of further illustration. Therefore, the descriptions and claims as provided should be understood as of demonstrative purpose instead of limitative in any way to the scope of the present invention.
