Immunology and Infection
Published: October 30th, 2018
Here we describe a protocol for the immunization of the adult zebrafish (Danio rerio) with a DNA-based vaccine and demonstrate the validation of a successful vaccination event. This method is suitable for the preclinical screening of vaccine candidates in various infection models.
The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected antigen or a combination of antigens into a plasmid, which enables a tailor-made and safe design. The administration of DNA vaccines into host cells leads to the expression of antigens that stimulate both humoral and cell-mediated immune responses. This report describes a protocol for the cloning of antigen sequences into the pCMV-EGFP plasmid, the immunization of adult zebrafish with the vaccine candidates by intramuscular microinjection, and the subsequent electroporation to improve intake. The vaccine antigens are expressed as green fluorescent protein (GFP)-fusion proteins, which allows the confirmation of the antigen expression under UV light from live fish and the quantification of expression levels of the fusion protein with ELISA, as well as their detection with a western blot analysis. The protective effect of the vaccine candidates is tested by infecting the fish with Mycobacterium marinum five weeks postvaccination, followed by the quantification of the bacteria with qPCR four weeks later. Compared to mammalian preclinical screening models, this method provides a cost-effective method for the preliminary screening of novel DNA-based vaccine candidates against a mycobacterial infection. The method can be further applied to screening DNA-based vaccines against various bacterial and viral diseases.
The first DNA vaccine studies were performed in the 1990s1, and since then, DNA vaccines have been tested against various infectious diseases, cancer, autoimmunity, and allergy2. In mammals, a DNA vaccine against West Nile virus in horses and a therapeutic cancer vaccine for canine oral melanoma have been licensed, but these are not currently in clinical use2. In addition to the interest evoked by mammalian studies, DNA vaccination has turned out to be a convenient way to immunize farmed fish against viral diseases. A vaccine against fish infectious hematopoietic necrosis virus (IHNV) has been in commercial use since 2005, and a vaccine against infectious pancreatic necrosis virus (IPNV) was recently licensed3. In addition, several DNA vaccines against fish pathogens are being developed.
As traditional vaccines often contain inactivated or live attenuated pathogens, they pose a potential risk of transmitting the disease2. DNA vaccines, in turn, avert this risk, as they are based on the administration of plasmid encoding bacterial or viral antigens, rather than the whole pathogen itself2,4. DNA vaccines are produced with DNA recombination techniques, which allows the precise design of vaccine antigens and the flexible formulation of antigen combinations and adjuvants in a single vaccine construct5. Furthermore, the production of DNA vaccines is faster, easier and more cost-efficient than that of protein-based recombinant vaccines, which is a major advantage for vaccine candidate screening purposes, but also, for example, in the case of pandemic outbreaks2.
In fish, the most common administration routes for DNA vaccines are intraperitoneal, intramuscular, and oral3,6,7, while in mammals, subcutaneous and intradermal routes are additional options2. After an intramuscular injection, the administrated DNA plasmids enter the cells at the administration site (e.g., mostly myocytes, but also resident antigen-presenting cells [APCs]). The proportion of transfected cells can be significantly increased by electroporation2,8. After entering the cell, some plasmid DNA enters the nucleus, where the genes encoded by the plasmid are transcribed2. In this protocol, we utilize the pCMV-EGFP plasmid that has a strong ubiquitous promoter optimized for eukaryotic expression9. In this construct, the antigens are translated as a fusion protein with a GFP. The GFP enables the confirmation of a successful vaccination and the correct antigen product by the simple visualization of antigen expression with a fluorescence microscope in live fish.
In mammals, DNA vaccines have been shown to stimulate different types of immune responses depending on the transfected cell types2,5. Transfected myocytes secrete antigens into the extracellular space or release them upon cell death, and the antigens engulfed by APCs are, subsequently, presented on major histocompatibility complex II molecules2. This triggers CD4 and CD8 T cell responses, especially, in addition to B cell responses2,5,10. In fish, T and B lymphocytes, as well as dendritic cells (DCs), have been identified, yet their division of labor in antigen presentation is less well understood11. Zebrafish DCs, however, have been shown to share conserved phenotypic and functional characteristics with their mammalian counterparts12. Furthermore, DNA vaccination has been shown to elicit similar immune responses in fish and in mammals, including T and B cell responses6,13,14,15,16.
Both larvae and adult zebrafish are widely used to model different infectious diseases, such as the fish M. marinum infection model of tuberculosis used in this protocol17,18,19,20,21,22. In comparison with mammalian model organisms, the advantages of zebrafish include their small size, fast reproducibility, and low housing expenses23. These aspects make the zebrafish an ideal animal model for large-scale preclinical screening studies for novel vaccines and pharmaceutical compounds23,24,25.
In this protocol, we describe how novel vaccine antigen candidates against mycobacteriosis can be evaluated by the DNA-based vaccination of adult zebrafish. First, we describe how antigens are cloned into the pCMV-EGFP expression plasmid, followed by a detailed protocol for the intramuscular injection of vaccine plasmids and the subsequent electroporation into muscle. The expression of each antigen is confirmed by fluorescence microscopy one-week postimmunization. The efficacy of the antigen candidates is then tested by experimentally infecting vaccinated fish with M. marinum.
Experiments including adult zebrafish require a permission for animal experimentation for both the vaccination and the subsequent studies with the infection model. All methods and experiments described here are approved by the Animal Experiment Board of Finland (ESAVI) and the studies are carried out in accordance with EU directive 2010/63/EU.
1. Cloning of DNA Vaccine Antigens
2. Pulling the Microinjection Needles
3. Filling the Micropipette Needles
4. Setting the Microinjector and Electroporator for Immunization
5. Injection of the DNA Vaccine and Electroporation
6. Visualization and Imaging of Antigen Expression
7. Quantification of the Expression Level and Size of the Antigens
8. Combining the Vaccination Protocol with an M. marinum Infection Model
The steps involved in the DNA vaccination protocol of adult zebrafish are illustrated in Figure 3. At first, the selected antigen sequences are cloned into a pCMV-EGFP plasmid and plasmid DNA is produced and purified24 (Figure 3).Vaccine candidates are then injected intramuscularly with a microinjector and the injection site is electroporated to improve the intake of the plasmid into cells (Figure 3). The used vaccination dose was optimized by injecting different amounts of the pCMV-EGFP plasmid and measuring the GFP expression with ELISA (Supplementary Figure 1). Two to seven days postvaccination, the expression of the fusion protein is detected under UV light and visualized with fluorescence microscopy (Figure 3 and Figure 4). The expression levels of different antigens may vary from very intensive (antigen 1) to a faint expression (Figure 4). In addition, GFP expression can be observed across the dorsal muscle (antigen 1), or in a more limited area (antigen 2) (Figure 4). However, if no fluorescence is detected within 10 days, it is recommended to make sure that there are no mistakes in antigen cloning or primer design. To confirm that the expressed fusion protein is of the correct size, proteins can be extracted from the muscle tissue around the injection site and used for a western blot analysis.
The effect of the vaccine candidates is evaluated by challenging the fish with a low dose of M. marinum by an intraperitoneal injection (Figure 5). Four to five weeks postinfection, the bacterial counts are determined with qPCR and compared to bacterial loads in the control group (Figure 5). Furthermore, the effectiveness of the most promising vaccine candidates can be tested by monitoring the survival after a high dose M. marinum infection(Figure 5). However, in addition to giving a quantitative result on the progression of the infection, instead of merely a status of alive or dead, the qPCR-based cfu quantification requires less time and smaller group sizes and is, therefore, a more ethical approach for a primary screen. Overall, this protocol facilitates the screening of the effectiveness of novel vaccine antigens within 12 weeks (Figure 5).
Figure 1: Close-up (12X) of aluminosilicate needles used in the adult zebrafish intra muscular injections. The tip below has been cut with tweezers and is ready to be used for microinjections. This figure has been adapted from Oksanen35. Please click here to view a larger version of this figure.
Figure 2: Microinjection equipment and set-up. The main components of the equipment needed for the DNA vaccination of adult zebrafish are highlighted in bold. The critical adjustments are indicated. Please click here to view a larger version of this figure.
Figure 3: Preparing the DNA vaccine plasmids and the immunization procedure. (1) Selected antigens are cloned adjacent to the GFP tag in the pCMV-EGFP plasmid. (2) The vaccine construct is produced microbiologically, concentrated, and purified. (3) 12 µg of plasmid is injected into the dorsal muscle of an anesthetized adult zebrafish with a microinjector, and the injection site is subsequently electroporated with six 40-V, 50-ms pulses. (4) Two to seven days postvaccination, the GFP expression of the antigen-GFP fusion protein is visualized with a fluorescence microscope. (5) The fluorescent part of the dorsal muscle can be dissected and used for protein extraction. The size of the fusion protein is confirmed with a western blot analysis and the expression level with GFP-ELISA. Please click here to view a larger version of this figure.
Figure 4: Visualizing the expression of the antigen-EGFP fusion protein. Anesthetized adult zebrafish are vaccinated with 12 µg of experimental vaccine antigens (antigen 1–3) and the injection site is electroporated, subsequently, with six 40 V, 50 ms pulses. Two to seven days postvaccination, the injection site is imaged with a microscope. First, the expression of GFP is detected under a fluorescence microscope. The area is inspected using a 2X magnitude objective and imaged and saved in .tiff form. The light microscope image of the same area is merged with the fluorescence image using the ImageJ software. The quantity and position of the antigen expression may vary between antigens and individual fish. For example, the expression of antigen 1 is observed across the dorsal muscle and the expression of antigen 2 is seen as small spots, whereas antigen 3 is strongly expressed in a more limited area. Please click here to view a larger version of this figure.
Figure 5: Testing the effectiveness of vaccine candidates against a mycobacterial infection. (1) Adult zebrafish are vaccinated with experimental DNA vaccines against mycobacteriosis. (2) Five weeks postvaccination, the fish are infected with a low dose of Mycobacterium marinum (~30 cfu). (3) Four weeks later, the internal organs are dissected and used for DNA extraction. (4) The bacterial count in each fish is quantified with qPCR using M. marinum-specific primers. Immunization with antigen 1 led to a significant decrease in the bacterial counts (p < 0.01, two-way ANOVA), while antigens 2 and 3 had no effect. (5) The protective effect of the most promising vaccine candidate (antigen 1) is further evaluated in a survival experiment, where fish are infected with a high dose (~10,000 cfu) of bacteria and their survival is monitored for 12 weeks. Consistent with the decrease in the bacterial burden observed in panel 4, vaccination with antigen 1 also improved the survival of the fish upon an M. marinum infection (p <0.01), suggesting this antigen could be a promising candidate for a novel vaccine against tuberculosis. Please click here to view a larger version of this figure.
Supplementary Figure 1: Amount of plasmid DNA affects plasmid-derived EGFP expression in adult zebrafish. Groups of fish (n = 5 in each group) were injected with 0.5–20 μg of pCMV-EGFP, and electroporation (six pulses of 50 V) was used to enhance the transfection. Control fish (CTRL) were injected with 2 μg of the empty pCMV plasmid not containing the EGFP gene. GFP-ELISA was performed 3 days postinjection to define the relative EGFP expression in fish homogenates. P-values: *p <0.03, **p <0.004. The error-bars represent standard deviations. NS = not significant. This figure has been adapted from Oksanen35. Please click here to view a larger version of this figure.
The procedure of immunizing adult zebrafish with DNA-based vaccines requires some technical expertise. Even for an experienced researcher, vaccinating a single fish takes approximately 3 min, excluding preparations. Thus, a maximum of roughly 100 fish can be immunized within a day. If more than 100 fish are required for the experiment, the immunizations can be divided between up to 3 days. In addition to the quality of the experiment, sufficient training of the researcher(s) for handling the fish and performing the immunization is essential for the well-being of the fish. Make sure to follow local legal and animal welfare rules and guidelines when it comes to housing the fish, planning the experiments, and the qualifications required for the personnel carrying out the experiments.
In summary, there are several critical steps to avoid complications in the immunization protocol. For the successful immunization, ensure that 1) the fish to be immunized are healthy and sufficient in age and size (the immunization of more juvenile fish can require down-scaling the vaccine volume and the electroporation settings); 2) the fish are properly anesthetized with no stronger than 0.02% 3-aminobenzoic acid ethyl ester, and they remain anesthetized throughout the entire procedure (anesthesia should be kept as short as possible to ensure the recovery of the fish); 3) the sponge pad is properly soaked; 4) liquid is injected in each pulse from the pneumatic pump and, if not, the pulse length is adjusted (pulling the needle slightly backwards along the y-axis can help); 5) there are no air bubbles with the vaccine solution; 6) the electroporation settings and the actual pulse voltage and length are correct; 7) the electrodes do not cause skin damage on the electroporation site (during the electroporation, keep the electrodes in gentle contact with the fish, and release the fish immediately into the recovery tank after electroporation).
It is important to monitor the fish after the electroporation in the recovery tank and to euthanize any fish showing signs of discomfort. Furthermore, it is necessary to practice the procedure before starting a large-scale experiment, to ensure a fluent workflow. If possible, ask a sufficiently trained colleague for assistance with filling the needles and the electroporation.
The DNA vaccination method enables the tailor-made design of vaccine antigens. It is possible to clone the whole antigen or, preferably, select parts of the antigen based on cellular localization and immunogenicity24. In addition, the method enables combining several antigens or adjuvants into one vaccine construct or injecting several separate plasmids at the same time2. By including a stop codon after the antigen sequence or by excising the EGFP gene from the plasmid, it is possible to utilize the same plasmid vector also to express the antigen without the subsequent N-terminal GFP tag. This may be reasonable in confirming the positive screening results, as the relatively large size of GFP can affect the folding of the antigen and, thus, restrict humoral responses potentially evoked by the vaccination.
A higher antigen expression has been linked to DNA vaccine immunogenicity2. Electroporation after injection has, thus, been included in this protocol, as it has been shown to increase the expression of antigens or reporter genes from fourfold to tenfold in zebrafish32. Furthermore, electroporation as a technique causes moderate tissue injury, thus inducing local inflammation that further promotes the vaccine-induced immune responses2. On the other hand, electroporation is generally well-tolerated. With the equipment used here, practically 100% of adult zebrafish will recover well from the six pulses of 40 V used in this protocol35.
In addition to using electroporation to enhance the entry of the vaccine plasmid into the cells, we use a strong ubiquitous promoter in the vaccine plasmid and a polyA tail at the 3' end of the antigen to improve antigen expression in the transfected fish cells. In some cases, if the codon usage of the target pathogen significantly differs from the vaccinated species, codon optimization has been found useful in further increasing target gene expression2. In this zebrafish-M. marinum model, however, codon optimization had no significant effect on the expression levels of two mycobacterial model genes, ESAT-6 and CFP-10, and has, thus, been deemed unnecessary in this model35.
Target gene expression profiles have some temporal variation between the antigens, depending, for instance, on the size and the structure of the antigens in question. However, antigen expression is usually similar within a group of fish immunized with the same vaccine. Typically, the brightest EGFP expression is observed four days to one-week postvaccination, but a scale of 2–10 days is possible. It is recommended to validate the expression of each antigen-EGFP fusion protein in a small group of fish (2–3) before including the antigen in a large-scale experiment. If no GFP expression is observed at any point 2–10 days after immunization, make sure that 1) the immunization protocol was carefully followed. Always have a group of fish immunized with the empty pCMV-EGFP plasmid as a positive control and make sure that 2) the antigen design and molecular cloning was carried out correctly (adequate primer design; the antigen and the EGFP tag are both in the same reading frame and no intervening stop codons are included). In some cases, despite the correct antigen design, GFP cannot be detected. This may be due to the incorrect folding or rapid breakdown of the fusion protein. In these circumstances, it may be necessary to redesign the antigen.
In vaccines that are used to immunize farmed fish, the plasmid dose used is typically 1 µg or less7,33,34. In zebrafish, reporter gene expression can also be detected after at least a 0.5 µg plasmid injection following electroporation; however, the relative target gene expression significantly increases with a higher amount of plasmid per fish (Supplementary Figure 1). In fish injected with the pCMV-EGFP reporter plasmid, an injection with 5–20 µg of plasmid resulted in four to eight times higher EGFP levels in comparison with fish injected with 0.5 µg. Therefore, to ensure a high enough target gene expression, yet have injection volumes that are small enough (≤7 µL) to prevent any excess tissue damage or vaccine leakage, we chose to use 5 to 12 µg per fish for the preliminary screening purposes. In addition to vaccine immunogenicity, a high enough target gene expression is required to detect reporter gene expression with a fluorescence microscope and with western blot, which is necessary for screening purposes to confirm the correct in vivo translation of the target antigen. However, lower plasmid doses (0.5–1 µg) can be useful for other types of experimental uses.
In conclusion, this protocol for the immunization of adult zebrafish with a DNA plasmid can be used in the preclinical testing of novel vaccine candidates against various bacterial or viral infections. The expression of the vaccine antigen as a GFP-fusion protein allows the visualization of a successful immunization event and antigen expression. We apply this method for the preclinical screening of novel vaccine antigen candidates against tuberculosis. For this, we infect the zebrafish five weeks postvaccination and determine the bacterial counts in each fish with qPCR20,24.
The authors have nothing to disclose.
The authors are thankful to the members of the Experimental immunology research group, and especially to Leena Mäkinen and Hannaleena Piippo, for all the work they have done in developing and optimizing the vaccination protocol, and their help in actual experiments using the protocol.
This work was supported by Jane ja Aatos Erkon Säätiö (Jane and Aatos Erkko Foundation; to M.R.), Sigrid Juséliuksen Säätiö (Sigrid Juselius Foundation; to M.R.), the Competitive State Research Financing of the Expert Responsibility area of Tampere University Hospital (to M.R.), Tampereen Tuberkuloosisäätiö (Tampere Tuberculosis Foundation; to M.R., H.M., and M.N.), Suomen Kulttuurirahasto (Finnish Cultural Foundation; to H.M.), Suomen Tuberkuloosin Vastustamisyhdistyksen Säätiö (Finnish Anti-Tuberculosis Foundation; to H.M.), Väinö ja Laina Kiven säätiö (Väinö and Laina Kivi Foundation; to M.N.) and the Tampere City Science Foundation (to M.N.).
|Ampicillin sodium salt
|ECM Electro Square Porator
|BTX Harvard apparatus
|BTX ECM 830
|Flaming/brown micropipette puller
|Sutter Instrument Co.
|Pulling of needles
|GeneJet PCR Purification kit
|GFP ELISA kit
|Cell Biolabs, Inc.
|Guanidine thiocyanate (FW 118.2)
|freely available software
|LB Broth (Miller)
|Nightsea Full adapter system w/Royal Blue Color light head
|Electron Microscopy Sciences
|Phenol red sodium salt
|PV830 Phneumatic Pico Pump
|QIAGEN Plasmid Maxi kit
|Sodium citrate (FW294.1)
|Molecular Research Center, Inc.
|Tricaine (ethyl 3-aminobenzoate methanesulfonate salt)
|anestesia and euthanasia solution
|Tris (free base) (FW121.14)
|VWR Life Science
|Tweezertrodes Electrodes (7mm) Kits
|BTX Harvard apparatus
|tweezer type electrodes
|2.8 mmCeramic beads
|2ml Tough tubes with caps
|Microloader 20 µl
|Petri dishes, 16 mm
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