Name: immunization of adult zebrafish for the preclinical screening of dna-based vaccines
Uploaded: 2018-10-30T14:45:00.000+00:00
Duration: 5 min 39 s
Description: Watch this Scientific Journal Video about Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines at JoVE.com

The authors are thankful to the members of the Experimental immunology research group, and especially to Leena M&#228;kinen 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 S&#228;&#228;ti&#246; (Jane and Aatos Erkko Foundation; to M.R.), Sigrid Jus&#233;liuksen S&#228;&#228;ti&#246; (Sigrid Juselius Foundation; to M.R.), the Competitive State Research Financing of the Expert Responsibility area of Tampere University Hospital (to M.R.), Tampereen Tuberkuloosis&#228;&#228;ti&#246; (Tampere Tuberculosis Foundation; to M.R., H.M., and M.N.), Suomen Kulttuurirahasto (Finnish Cultural Foundation; to H.M.), Suomen Tuberkuloosin Vastustamisyhdistyksen S&#228;&#228;ti&#246; (Finnish Anti-Tuberculosis Foundation; to H.M.), V&#228;in&#246; ja Laina Kiven s&#228;&#228;ti&#246; (V&#228;in&#246; and Laina Kivi Foundation; to M.N.) and the Tampere City Science Foundation (to M.N.).

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
<ol>
	<li>Select an expression plasmid optimized for the eukaryotic expression of the antigen(s) of interest under a strong, constitutive promoter, such as the cytomegalovirus (CMV) immediate early promoter. To enable the in vivo verification of the antigen expression, select a vaccine plasmid that encodes a fluorescent tag, such as the pCMV-EGFP plasmid9 used in this protocol.</li>
	<li>Use appropriate web-based and bioinformatic tools to select a potentially immune-protective antigen sequence (or several sequences) of approximately 90&#8211;600 nt (30&#8211;200 aa)26 from the gene(s)-of-interest of the pathogen that will be used in the subsequent infection model.</li>
	<li>Use available software, or manually design primers to amplify the antigen genes from the pathogen genome and to clone the antigen sequence(s) into the multiple-cloning site of the expression plasmid. Make sure to preserve the correct reading frame when selecting the antigen.</li>
	<li>Include both a Kozak sequence (CCACC)27 and a start codon (ATG) in the 5&#8217; primer. To preserve the C-terminal EGFP tag, avoid intervening stop codons (TAG, TAA, TGA) in the antigen sequence and the 3&#8217; primer. Also, ensure that the GFP tag remains in the same reading frame with the antigen of interest.</li>
	<li>Use RNA or DNA extracted from the pathogen as a template to generate an adequate amount of PCR product with the cloning primers. Preferably, use a proofreading DNA polymerase to preserve the correct antigen sequence.</li>
	<li>Purify the PCR product and check the correct size of the product by gel electrophoresis. Digest and ligate the PCR product with the digested vaccine plasmid.</li>
	<li>Transform the ligation mix into competent bacterial cells according to a suitable protocol. Use an appropriate antibiotic selection for positive colonies and plasmid production in E. coli; the pCMV-EGFP plasmid, for example, contains an ampicillin resistance gene for this.</li>
	<li>Use Sanger sequencing to confirm the insertion of the correct antigen sequence. The following primers can be used for sequencing antigens in the pCMV-EGFP plasmid: CMV forward 5&#8217;- CGCAAATGGGCGGTAGGCGTG-3&#8217; and EGFP-N reverse 5&#8217; CGTCGCCGTCCAGCTCGACCAG-3&#8217;.</li>
	<li>Produce and purify a sufficient amount of the vaccine construct. Dissolve or elute the plasmid in sterile water. Make sure that the produced plasmid DNA is of high quality and the concentration is at least 0.72 &#181;M or 2,000 ng/&#181;L.</li>
</ol>
2. Pulling the Microinjection Needles
<ol>
	<li>Prepare microinjection needles in advance. Use 10-cm aluminosilicate glass capillaries. Note that borosilicate glass capillaries are too brittle for injecting adult fish.</li>
	<li>Pull the needles with a micropipette-needle-fabricating device. 
	NOTE: The needles should look similar to the one presented in Figure 1. With the device used in this protocol (Table of Materials), the following settings result in the desired kind of needles:
	<ol>
		<li>Set the glass capillary in the V-groove in the puller bar and tighten the clamping knob lightly.</li>
		<li>Move the holder next to the filament and gently push the capillary through the filament into the puller bar on the other side of the filament. Avoid touching the filament with the capillary.</li>
		<li>Tighten the clamping knobs, set down the safety glass, and press the pull button. 
		CAUTION: The filament is hot.</li>
	</ol>
	</li>
	<li>Place the needles on a piece of reusable adhesive inside a 15-cm Petri dish plate to protect the needle tips. Keep the dish covered to keep the needles clean.</li>
</ol>
3. Filling the Micropipette Needles
<ol>
	<li>Prepare the vaccine mix. Use 0.5&#8211;12 &#181;g of plasmid per dose. If combining several different plasmids in one vaccination mix, use a maximum total DNA concentration of 12 &#181;g per fish.</li>
	<li>Calculate the volume of the vaccine &#8220;master mix&#8221; according to the number of fish in each group (see below). Add 1 &#181;l of sterile-filtered phenol red to each injection dose to ease both the filling of the capillary needles and the observation of the injection. Add sterile 1x PBS up to a maximum total volume of 5&#8211;7 &#181;l in one injection dose8. 
	NOTE: Injection volumes higher than 7 &#181;L can result in the occasional leakage of the injection solution and should, therefore, be avoided.</li>
	<li>Place a piece of tape, glue side up, on an appropriate holder, for example, the side of an empty tip box. Gently attach the capillary needles to the tape.</li>
	<li>Pipet a maximum of 7 &#181;L of the vaccine mix onto a piece of laboratory film. Using a loading tip, transfer the vaccine from the film into the needle. Pipet slowly and carefully, avoid pipetting air bubbles into the needle.</li>
	<li>Let the needles settle for 15&#8211;30 min to remove possible remaining small air bubbles.</li>
</ol>
4. Setting the Microinjector and Electroporator for Immunization
<ol>
	<li>Set the micromanipulator and a light source into the right position. Switch the air pressure tap to the open position.</li>
	<li>Adjust the parameters for the pneumatic pump (see also Figure 2) as follows.
	<ol>
		<li>Set the vent knob on the Eject port to hold to prevent backfilling of the pipette by capillary action. Set the tubing from the eject port to the micropipette. Do not use the Vacuum port in this protocol.</li>
		<li>Adjust the pulse length: use the timed mode, where an electronic timer controls the duration of the time the pressure solenoid stays open. Check that the green lamp next to the Eject pressure gauge illuminates when the pressure solenoid is open (energized).</li>
		<li>Set the pulse range to 10 s; with this setting, the pulses may be further set from 100 ms to 10.1 s. Use the 10-turn period dial for fine-tuning the pulse length&#8212;every turn of the dial is 1.0 s. If needed, this can also be adjusted during an injection.</li>
		<li>Use the pulse initiator (&#8220;start button&#8221;) on the front panel of the pneumatic pump, or a remote foot switch (recommended). This is connected to the front panel of the pneumatic pump.</li>
	</ol>
	</li>
	<li>Set the needle onto the micropipette holder of the micromanipulator. Cut the tip of the needle with tweezers so that liquid can be pushed out, and use the microscope to view the correct position. Press the foot switch once to see that a 1-s pulse pushes a small droplet out of the needle.</li>
	<li>Use the following settings for the electroporator: voltage = 40 V; pulse length = 50 ms, number of pulses = 6. Connect the tweezers to the electroporator. See that the actual voltage and pulse length shown on the monitor do not differ significantly from the settings.</li>
</ol>
5. Injection of the DNA Vaccine and Electroporation
<ol>
	<li>For immunizations according to this protocol, use healthy, 6&#160;to 12 month-old adult zebrafish. Keep the fish in a flow-through system with a 14/10 h light/dark cycle, with a maximum of seven fish per 1 L of water.</li>
	<li>Prepare a 0.02% 3-aminobenzoic acid ethyl ester (tricaine) solution (pH 7) in tank water for anesthetizing the fish28. Use a 10 cm Petri dish or something similar.</li>
	<li>Prepare a recovery tank by filling a 5 L beaker with 3 L of clean system water.</li>
	<li>Prepare a vaccination padding to keep the fish in a fixed position during the vaccination. Take a 5 x 7 cm2 piece of a 2&#160;to 3 cm-thick sponge. Cut a groove into the sponge with a scalpel blade or sharp scissors. 
	NOTE: The same vaccination padding can be used in multiple experiments. Disinfect the sponge between the experiments by soaking it in 70% ethyl alcohol and allow to dry.</li>
	<li>Thoroughly soak the sponge in the system water and set the sponge on a Petri dish.</li>
	<li>Fast the fish 24 h before the vaccinations.</li>
	<li>Anesthetize one zebrafish by placing it on a Petri dish containing 0.02% tricaine. Wait until the fish does not respond to touch stimulation and until there is no movement of the gills. Anesthetize a single fish at a time.</li>
	<li>Using a plastic spoon, transfer the anesthetized zebrafish onto the wet sponge and set the fish&#8217;s ventral side down into the groove. In the correct position, ensure that the head and most of the body of the fish are inside the groove and the dorsal fin and the tail are protruding out from the groove.</li>
	<li>Under the microscope, carefully place the needle in an approximately 45&#176; angle close to the zebrafish&#8217;s dorsal muscle, using the x- and y-axis fine-tuning wheels on the micromanipulator.</li>
	<li>Find the small spot without scales in front of the dorsal fin, where pushing the needle does not demand force. If resistance is felt, try an adjacent spot. Avoid injuring the spine, the dorsal fin, or the scales. 
	NOTE: If the needle bends while pushing, shorten the needle slightly by cutting it.</li>
	<li>Use the foot switch to gradually inject the vaccine solution into the muscle. Observe the injection through the microscope: phenol red is visible as it enters the muscle tissue. Adjust the duration of the pulse if needed. 
	NOTE: Alternatively, use the pulse initiator button on the front panel of the pneumatic pump for the injection. However, the use of a remote foot switch allows using the other hand for adjusting the pulse length by the 10-turn dial wheel. Avoid injecting the solution too fast, since this may cause excessive tissue damage. Make sure not to inject any air.</li>
	<li>Electroporate the fish immediately after the injection. Make sure that the fish is still under anesthesia. Keep the fish on the sponge and set the fish between tweezer-type electrodes, so that the electrodes are located on each side of the injection site. Do not press the electrode tweezers too tight but keep both electrodes in contact with the fish.
	<ol>
		<li>Press the start button on the electroporator to give six 40 V, 50 ms pulses.</li>
		<li>Gently transfer the fish to the recovery tank.</li>
		<li>Clean the electrodes after each electroporation by swiping them with 70% ethanol. 
		NOTE: Carefully monitor the well-being of the fish after the vaccination. Euthanize any fish showing signs of discomfort (a slow recovery from anesthesia, aberrant swimming, gasping) in 0.04% tricaine. After recovery, transfer the fish to the flow-through unit and feed it normally.</li>
	</ol>
	</li>
</ol>
6. Visualization and Imaging of Antigen Expression
<ol>
	<li>Anesthetize the fish in 0.02 % tricaine 2&#8211;7 days after immunization, and use a UV-light to see EGFP expression near the injection site. 
	NOTE: Visual inspection under a UV light is an easy and non-invasive operation that is suitable for the routine verification of successful vaccinations, also in large-scale experiments. If no images of the fish are required, this step can also be performed in regular fish tanks without the need to anesthetize or move the fish.</li>
	<li>To capture images, use a fluorescence microscope to visualize EGFP expression at the injection site8. Use a 2X objective lens and select the correct filter to visualize fluorescence or visible light views.</li>
	<li>Keep the anesthetized fish still by pressing the ventral fin gently with tweezers toward a Petri dish bottom. Take both light-microscope and fluorescence images of the same area.</li>
	<li>Merge the light-microscope and fluorescence images using the appropriate software29. Add a scale bar.</li>
</ol>
7. Quantification of the Expression Level and Size of the Antigens
<ol>
	<li>Euthanize the fish in 0.04% tricaine. Dissect the fluorescent part of the dorsal muscle with a scalpel and tweezers under UV-light. Extract proteins from the samples8.</li>
	<li>Verify the correct size of in vivo-produced proteins with a western blot analysis, using a horseradish peroxidase (HRP)-conjugated GFP antibody (or similar)8. Include a negative control (unimmunized fish) to exclude any background signals and unspecific binding, together with a control expressing EGFP without a fused antigen. 
	NOTE: For the western blot analysis, the expected size (in kDa) of the antigen-fusion proteins can be calculated, for example, by the equation: 
	Molecular Weight (MW) of dsDNA = (number of nucleotides x 607.4) + 157.9; or by using web-based tools.</li>
	<li>Quantify the expression of each antigen using a GFP-ELISA8 (optional).</li>
</ol>
8. Combining the Vaccination Protocol with an M. marinum Infection Model
<ol>
	<li>Determine the group size required for the reliable determination of the effectiveness of a novel vaccine candidate in the infection model and the assay used (see, for example, Myllymaki et al.24 and Charan and Kantharia30). Carry out the appropriate power calculations while planning the experiments.</li>
	<li>To evaluate the effectiveness of vaccine candidates, infect the fish 5 weeks postimmunization. Use an intraperitoneal infection with approximately 30 colony-forming units (cfu) of M. marinum, which leads to a latent infection in most fish8,20,31. 
	NOTE: When using M. marinum, follow a BSL2 safety protocol. The preparation of the bacterium or virus depends on the pathogen.</li>
	<li>Quantify the number of pathogens in each fish. Euthanize the fish 4 weeks postinfection and determine the bacterial burden in each fish from the extracted DNA with qPCR using primers specific for M. marinum20,24. 
	NOTE: Be sure to include an appropriate control group and use the correct statistical methods for analyzing the results. Generally, a group of fish immunized with the empty pCMV-EGFP plasmid is a suitable negative control.</li>
	<li>Confirm positive results with antigens without the GFP tag. Clone the antigens as described in step 1 and repeat the vaccination experiment.</li>
</ol>

<ol>
	<li>Tang, D. C., DeVit, M., Johnston, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+immunization+is+a+simple+method+for+eliciting+an+immune+response.">Genetic immunization is a simple method for eliciting an immune response.</a> Nature. 356 (6365), 152-154 (1992).</li><li>Tregoning, J. S., Kinnear, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Plasmids+as+DNA+Vaccines+for+Infectious+Diseases.">Using Plasmids as DNA Vaccines for Infectious Diseases.</a> Microbiology Spectrum. 2 (6), (2014).</li><li>Evensen, O., Leong, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+vaccines+against+viral+diseases+of+farmed+fish.">DNA vaccines against viral diseases of farmed fish.</a> Fish Shellfish Immunology. 35 (6), 1751-1758 (2013).</li><li>Sommerset, I., Lorenzen, E., Lorenzen, N., Bleie, H., Nerland, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+DNA+vaccine+directed+against+a+rainbow+trout+rhabdovirus+induces+early+protection+against+a+nodavirus+challenge+in+turbot.">A DNA vaccine directed against a rainbow trout rhabdovirus induces early protection against a nodavirus challenge in turbot.</a> Vaccine. 21 (32), 4661-4667 (2003).</li><li>Li, L., Petrovsky, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+for+enhanced+DNA+vaccine+immunogenicity.">Molecular mechanisms for enhanced DNA vaccine immunogenicity.</a> Expert Review of Vaccines. 15 (3), 313-329 (2016).</li><li>Embregts, C. W. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramuscular+DNA+Vaccination+of+Juvenile+Carp+against+Spring+Viremia+of+Carp+Virus+Induces+Full+Protection+and+Establishes+a+Virus-Specific+B+and+T+Cell+Response.">Intramuscular DNA Vaccination of Juvenile Carp against Spring Viremia of Carp Virus Induces Full Protection and Establishes a Virus-Specific B and T Cell Response.</a> Frontiers in Immunology. 8, 1340 (2017).</li><li>Lorenzen, N., LaPatra, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+vaccines+for+aquacultured+fish.">DNA vaccines for aquacultured fish.</a> Revue scientifique et technique (International Office of Epizootics). 24 (1), 201-213 (2005).</li><li>Oksanen, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+adult+zebrafish+model+for+preclinical+tuberculosis+vaccine+development.">An adult zebrafish model for preclinical tuberculosis vaccine development.</a> Vaccine. 31 (45), 5202-5209 (2013).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+and+RNA+interference+in+the+rodent+retina+in+vivo+and+in+vitro.">Electroporation and RNA interference in the rodent retina in vivo and in vitro.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (1), (2004).</li><li>Cho, J. H., Youn, J. W., Sung, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-priming+as+a+predominant+mechanism+for+inducing+CD8(+)+T+cell+responses+in+gene+gun+DNA+immunization.">Cross-priming as a predominant mechanism for inducing CD8(+) T cell responses in gene gun DNA immunization.</a> The Journal of Immunology. 167 (10), 5549-5557 (2001).</li><li>Lewis, K. L., Del Cid, N., Traver, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on+antigen+presenting+cells+in+zebrafish.">Perspectives on antigen presenting cells in zebrafish.</a> Developmental &amp; Comparative Immunology. 46 (1), 63-73 (2014).</li><li>Shao, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+surface+phenotypic+molecules+of+teleost+dendritic+cells.">Characterization of surface phenotypic molecules of teleost dendritic cells.</a> Developmental &amp; Comparative Immunology. 49 (1), 38-43 (2015).</li><li>Utke, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-mediated+immune+responses+in+rainbow+trout+after+DNA+immunization+against+the+viral+hemorrhagic+septicemia+virus.">Cell-mediated immune responses in rainbow trout after DNA immunization against the viral hemorrhagic septicemia virus.</a> Developmental &amp; Comparative Immunology. 32 (3), 239-252 (2008).</li><li>Cuesta, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+active+DNA+vaccine+against+infectious+pancreatic+necrosis+virus+(IPNV)+with+a+different+mode+of+action+than+fish+rhabdovirus+DNA+vaccines.">An active DNA vaccine against infectious pancreatic necrosis virus (IPNV) with a different mode of action than fish rhabdovirus DNA vaccines.</a> Vaccine. 28 (19), 3291-3300 (2010).</li><li>Castro, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+vaccination+against+a+fish+rhabdovirus+promotes+an+early+chemokine-related+recruitment+of+B+cells+to+the+muscle.">DNA vaccination against a fish rhabdovirus promotes an early chemokine-related recruitment of B cells to the muscle.</a> Vaccine. 32 (10), (2014).</li><li>Iwanami, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+for+understanding+the+evolution+of+the+vertebrate+immune+system+and+human+primary+immunodeficiency.">Zebrafish as a model for understanding the evolution of the vertebrate immune system and human primary immunodeficiency.</a> Experimental Hematology. 42 (8), 697-706 (2014).</li><li>Patterson, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+zebrafish+model+of+bacterial+meningitis+in+Streptococcus+agalactiae+infection.">Adult zebrafish model of bacterial meningitis in Streptococcus agalactiae infection.</a> Developmental &amp; Comparative Immunology. 38 (3), 447-455 (2012).</li><li>Cronan, M. R., Tobin, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fit+for+consumption:+zebrafish+as+a+model+for+tuberculosis.">Fit for consumption: zebrafish as a model for tuberculosis.</a> Disease Model &amp; Mechanisms. 7 (7), 777-784 (2014).</li><li>Tobin, D. M., Ramakrishnan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+pathogenesis+of+Mycobacterium+marinum+and+Mycobacterium+tuberculosis.">Comparative pathogenesis of Mycobacterium marinum and Mycobacterium tuberculosis.</a> Cellular Microbiology. 10 (5), 1027-1039 (2008).</li><li>Parikka, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterium+marinum+causes+a+latent+infection+that+can+be+reactivated+by+gamma+irradiation+in+adult+zebrafish.">Mycobacterium marinum causes a latent infection that can be reactivated by gamma irradiation in adult zebrafish.</a> PLoS Pathogens. 8 (9), e1002944 (2012).</li><li>Rounioja, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defense+of+zebrafish+embryos+against+Streptococcus+pneumoniae+infection+is+dependent+on+the+phagocytic+activity+of+leukocytes.">Defense of zebrafish embryos against Streptococcus pneumoniae infection is dependent on the phagocytic activity of leukocytes.</a> Developmental &amp; Comparative Immunology. 36 (2), 342-348 (2012).</li><li>Myllymaki, H., Bauerlein, C. A., Ramet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zebrafish+Breathes+New+Life+into+the+Study+of+Tuberculosis.">The Zebrafish Breathes New Life into the Study of Tuberculosis.</a> Frontiers in Immunology. 7, 196 (2016).</li><li>Myllymaki, H., Niskanen, M., Oksanen, K. E., Ramet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+in+tuberculosis+research+-+where+is+the+beef?.">Animal models in tuberculosis research - where is the beef?.</a> Expert Opinion on Drug Discovery. 10 (8), 871-883 (2015).</li><li>Myllymaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+novel+antigen+candidates+for+a+tuberculosis+vaccine+in+the+adult+zebrafish+(Danio+rerio).">Identification of novel antigen candidates for a tuberculosis vaccine in the adult zebrafish (Danio rerio).</a> PLoS One. 12 (7), e0181942 (2017).</li><li>Myllymaki, H., Niskanen, M., Luukinen, H., Parikka, M., Ramet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+protective+postexposure+mycobacterial+vaccine+antigens+using+an+immunosuppression-based+reactivation+model+in+the+zebrafish.">Identification of protective postexposure mycobacterial vaccine antigens using an immunosuppression-based reactivation model in the zebrafish.</a> Disease Model &amp; Mechanisms. 11 (3), (2018).</li><li>Ingolotti, M., Kawalekar, O., Shedlock, D. J., Muthumani, K., Weiner, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+vaccines+for+targeting+bacterial+infections.">DNA vaccines for targeting bacterial infections.</a> Expert Review of Vaccines. 9 (7), 747-763 (2010).</li><li>Kozak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+AUG+and+alternative+initiator+codons+is+augmented+by+G+in+position++4+but+is+not+generally+affected+by+the+nucleotides+in+positions++5+and++6.">Recognition of AUG and alternative initiator codons is augmented by G in position +4 but is not generally affected by the nucleotides in positions +5 and +6.</a> The EMBO Journal. 16 (9), 2482-2492 (1997).</li><li>Matthews, M., Varga, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+and+euthanasia+in+zebrafish.">Anesthesia and euthanasia in zebrafish.</a> ILAR Journal. 53 (2), 192-204 (2012).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Charan, J., Kantharia, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+calculate+sample+size+in+animal+studies?.">How to calculate sample size in animal studies?.</a> Journal of Pharmacology and Pharmacotherapeutics. 4 (4), 303-306 (2013).</li><li>Hammaren, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adequate+Th2-type+response+associates+with+restricted+bacterial+growth+in+latent+mycobacterial+infection+of+zebrafish.">Adequate Th2-type response associates with restricted bacterial growth in latent mycobacterial infection of zebrafish.</a> PLoS Pathogens. 10 (6), e1004190 (2014).</li><li>Rao, N. M., Rambabu, K. M., Rao, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+of+adult+zebrafish.">Electroporation of adult zebrafish.</a> Methods in Molecular Biology. 423, 289-298 (2008).</li><li>McCaffrey, J., Donnelly, R. F., McCarthy, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microneedles:+an+innovative+platform+for+gene+delivery.">Microneedles: an innovative platform for gene delivery.</a> Drug Delivery and Translational Research. 5 (4), 424-437 (2015).</li><li>Lorenzen, E., Lorenzen, N., Einer-Jensen, K., Brudeseth, B., Evensen, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+study+of+in+situ+expression+of+antigens+following+DNA-vaccination+against+VHS+in+rainbow+trout+(Oncorhynchus+mykiss+Walbaum)+fry.">Time course study of in situ expression of antigens following DNA-vaccination against VHS in rainbow trout (Oncorhynchus mykiss Walbaum) fry.</a> Fish and Shellfish Immunology. 19 (1), 27-41 (2005).</li><li>Oksanen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+Zebrafish+Model+for+Studying+DNA-based+Vaccination+against+Mycobacterial+Disease.">Adult Zebrafish Model for Studying DNA-based Vaccination against Mycobacterial Disease.</a> Tampereen yliopisto. , (2011).</li></ol>

The authors have nothing to disclose.

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&#160;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&#160;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&#39; 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&#8211;10 days is possible. It is recommended to validate the expression of each antigen-EGFP fusion protein in a small group of fish (2&#8211;3) before including the antigen in a large-scale experiment. If no GFP expression is observed at any point 2&#8211;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 &#181;g or less7,33,34. In zebrafish, reporter gene expression can also be detected after at least a 0.5 &#181;g 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&#8211;20 &#181;g of plasmid resulted in four to eight times higher EGFP levels in comparison with fish injected with 0.5 &#181;g. Therefore, to ensure a high enough target gene expression, yet have injection volumes that are small enough (&#8804;7 &#181;L) to prevent any excess tissue damage or vaccine leakage, we chose to use 5 to 12 &#181;g 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&#8211;1 &#181;g) 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 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.

<table><tbody><tr><td>pCMV-GFP plasmid</td><td>Addgene</td><td>#11153</td><td></td></tr><tr><td>2-propanol</td><td>Sigma-Aldrich</td><td>278475-2L</td><td>DNA extraction</td></tr><tr><td>Ampicillin sodium salt</td><td>Sigma-Aldrich</td><td>A0166-5G</td><td></td></tr><tr><td>Chloroform</td><td>Merck</td><td>1.02445.2500</td><td>DNA extraction</td></tr><tr><td>ECM Electro Square Porator</td><td>BTX Harvard apparatus</td><td>BTX ECM 830</td><td></td></tr><tr><td>FastPrep-24 5G</td><td>MP Biomedicals</td><td>116005500</td><td>homogenizer</td></tr><tr><td>Flaming/brown micropipette puller</td><td>Sutter Instrument Co.</td><td>P-97</td><td>Pulling of needles</td></tr><tr><td>GeneJet PCR Purification kit</td><td>ThermoFischer Scientific</td><td>K0701</td><td></td></tr><tr><td>GFP ELISA kit</td><td>Cell Biolabs, Inc.</td><td>AKR-121</td><td></td></tr><tr><td>Guanidine thiocyanate (FW 118.2)</td><td>Sigma-Aldrich</td><td>G9277-500G</td><td>DNA extraction</td></tr><tr><td>ImageJ2</td><td>imagej.net/Downloads</td><td></td><td>freely available software</td></tr><tr><td>LB Agar</td><td>Sigma</td><td>L2897-1kg</td><td></td></tr><tr><td>LB Broth (Miller)</td><td>Sigma</td><td>L3522-1kg</td><td></td></tr><tr><td>Micromanipulator</td><td>Narishige</td><td>MA-153</td><td></td></tr><tr><td>Microscope</td><td>Nikon</td><td>AZ100</td><td>fluorescense microscope</td></tr><tr><td>Microscope</td><td>Olympus</td><td>ZS61</td><td></td></tr><tr><td>Nightsea Full adapter system w/Royal Blue Color light head</td><td>Electron Microscopy Sciences</td><td>SFA-RB</td><td></td></tr><tr><td>PBS tablets</td><td>VWR Chemicals</td><td>E404-200TABL.</td><td></td></tr><tr><td>Phenol red sodium salt</td><td>Sigma-Aldrich</td><td>114537-5G</td><td></td></tr><tr><td>PV830 Phneumatic Pico Pump</td><td>WPI</td><td>SYS-PV830</td><td></td></tr><tr><td>QIAGEN Plasmid Maxi kit</td><td>Qiagen</td><td>ID:12163</td><td>plasmid extraction</td></tr><tr><td>Sodium citrate (FW294.1)</td><td>VWR Chemicals</td><td>27833.294</td><td>DNA extraction</td></tr><tr><td>Tri Reagent</td><td>Molecular Research Center, Inc.</td><td>TR 118</td><td>DNA extraction</td></tr><tr><td>Tricaine (ethyl 3-aminobenzoate methanesulfonate salt)</td><td>Sigma</td><td>A5040-100g</td><td>anestesia and euthanasia solution</td></tr><tr><td>Tris (free base) (FW121.14)</td><td>VWR Life Science</td><td>0497-500G</td><td>DNA extraction</td></tr><tr><td>Tweezertrodes Electrodes (7 mm) Kits</td><td>BTX Harvard apparatus</td><td>BTX 450165</td><td>tweezer type electrodes</td></tr><tr><td>2.8 mm Ceramic beads</td><td>Omni International</td><td>19-646-3</td><td>DNA extraction</td></tr><tr><td>2 mL Tough tubes with caps</td><td>Omni International</td><td>19-649</td><td>DNA extraction</td></tr><tr><td>Aluminosilicate capillaries</td><td>Harvard apparatus</td><td>30-0108</td><td></td></tr><tr><td>Microloader 20 &amp;micro;L</td><td>eppendorf</td><td>5242956.003</td><td>loading tips</td></tr><tr><td>Petri dishes, 16 mm</td><td>Sarsted</td><td>82.1473</td><td></td></tr><tr><td>Scalpels</td><td>Swan Morton</td><td>0501</td><td></td></tr><tr><td>Parafilm</td><td>Bemis</td><td></td><td>laboratory film</td></tr><tr><td>Pins</td><td></td><td></td><td></td></tr><tr><td>Plastic spoon</td><td></td><td></td><td></td></tr><tr><td>Spatula</td><td></td><td></td><td></td></tr><tr><td>Sponge</td><td></td><td></td><td></td></tr><tr><td>Styrofoam workbench</td><td></td><td></td><td></td></tr><tr><td>Tweezers</td><td></td><td></td><td></td></tr></tbody></table>

immunization of adult zebrafish for the preclinical screening of dna-based vaccines

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 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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58453/58453fig1.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/58453/58453fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58453/58453fig2.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/58453/58453fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58453/58453fig3.jpg" /> 
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 &#181;g 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. <a href="https://www.jove.com/files/ftp_upload/58453/58453fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58453/58453fig4v2.jpg" /> 
Figure 4: Visualizing the expression of the antigen-EGFP fusion protein. Anesthetized adult zebrafish are vaccinated with 12 &#181;g of experimental vaccine antigens (antigen 1&#8211;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. <a href="https://www.jove.com/files/ftp_upload/58453/58453fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58453/58453fig5.jpg" /> 
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 &#60; 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 &#60;0.01), suggesting this antigen could be a promising candidate for a novel vaccine against tuberculosis. <a href="https://www.jove.com/files/ftp_upload/58453/58453fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure S1" src="/files/ftp_upload/58453/58453figS1.jpg" /> 
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&#8211;20 &#956;g of pCMV-EGFP, and electroporation (six pulses of 50 V) was used to enhance the transfection. Control fish (CTRL) were injected with 2 &#956;g 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 &#60;0.03, **p &#60;0.004. The error-bars represent standard deviations. NS = not significant. This figure has been adapted from Oksanen35. <a href="https://www.jove.com/files/ftp_upload/58453/58453figS1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines at JoVE.com

Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines

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 ...

immunization-adult-zebrafish-for-preclinical-screening-dna-based

Research

JoVE Journal

Immunology and Infection

11.9K Views.  University of Tampere. We gebruiken dit protocol om te zoeken naar nieuwe vaccinkandidaten tegen tuberculose, maar ik kan worden toegepast op andere infectieziekten. In vergelijking met zoogdiermodellen biedt deze methode een kosteneffectieve procedure voor de voorlopige screening van nieuwe op DNA gebaseerde vaccinkandidaten. Het aantonen van de procedure zal Hannaleena Piippo zijn, een technicus van ons laboratorium.Om te beginnen zet u de glazen capillaire in de V-groef in de poolbalk en draai de klemknop...

Video: Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

Genetics

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Cancer Research

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

Developmental Biology

Optimized Intravenous Injection in Adult Zebrafish

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in mammalian research models. However, its application in adult zebrafish for drug delivery, stem cell transplantation, and regenerative and cancer studies has been limited due to the challenges posed by their small body size and intricate blood vessels. To overcome these limitations, alternative injection techniques such as intracardiac and retro-orbital (RO) injection have been explored in the past for stem cell transplantation in adult zebrafish. However, these techniques have their drawbacks, including the need for meticulous injection techniques or increased risk of mortality.
In this study, we have developed a refined and optimized IV injection procedure specifically tailored to adult zebrafish, addressing the challenges associated with their unique anatomy. To demonstrate the effectiveness of this technique, we performed successful IV injections of whole kidney marrow cells from Tg(mpo: EGFP) fish and FITC-dextran dye into adult Casper fish. The subsequent visualization of injected cells and dyes using a fluorescence microscope confirmed their successful delivery and engraftment within the zebrafish. Furthermore, we demonstrated that compared with the intracardiac and RO injections, the IV injection resulted in improved survival rates and engraftment efficiency in treated zebrafish. This approach enables precise delivery and localization of substances and holds great potential for large-scale drug and chemical screening using adult zebrafish. Additionally, the ability to visually track the injected cells and dyes provides invaluable insights into their engraftment, migration, and interactions with host tissues, enabling a more comprehensive evaluation of therapeutic effects and biological processes in zebrafish models.

Here, we present an optimized and effective protocol for the intravenous injection of cells or pharmacological agents into adult zebrafish, resulting in enhanced cell engraftment and increased survival rates of the treated zebrafish.

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in ...

Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water environment. Developing effective methods for oral delivery of immunostimulants or vaccines, which activate the immune system against infectious diseases, is highly desirable. In devising prophylactic tools, good experimental models are needed to test their performance. Here, we show a method for oral intubation of adult zebrafish and a set of procedures to dissect and prepare the intestine for cytometry, confocal microscopy and quantitative polymerase chain reaction (qPCR) analysis. With this protocol, we can precisely administer volumes up to 50 &#181;L to fish weighing approximately 1 g simply and quickly, without harming the animals. This method allows us to explore the direct in vivo uptake of fluorescently labelled compounds by the intestinal mucosa and the immunomodulatory capacity of such biologics at the local site after intubation. By combining downstream methods such as flow cytometry, histology, qPCR and confocal microscopy of the intestinal tissue, we can understand how immunostimulants or vaccines are able to cross the intestinal mucosal barriers, pass through the lamina propria, and reach the muscle, exerting an effect on the intestinal mucosal immune system. The model could be used to test candidate oral prophylactics and delivery systems or the local effect of any orally administered bioactive compound.

The protocol describes intubating adult zebrafish with a biologic; then dissecting and preparing the intestine for cytometry, confocal microscopy and qPCR. This method allows administration of bioactive compounds to monitor intestinal uptake and the local immune stimulus evoked. It&#160;is relevant for testing the intestinal dynamics of oral prophylactics.

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

Biology

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Optimized Intraperitoneal Injection for Compound Delivery in Adult Zebrafish

An Intraperitoneal Injection Technique in Adult Zebrafish that Minimizes Body Damage and Associated Mortality

The adult zebrafish (Danio rerio), which is genetically accessible, is being employed as a valuable vertebrate model to study human disorders such as cardiomyopathy. Intraperitoneal (IP) injection is an important method that delivers compounds to the body for either testing therapeutic effects or generating disease models such as doxorubicin-induced cardiomyopathy (DIC). Currently, there are two methods of IP injection. Both&#160;methods have limitations when handling toxic compounds such as doxorubicin, which result in side effects manifesting as severe damage to the body shape and fish death. While these shortcomings could be overcome by extensive investigator training, a new IP injection method that has minimal side effects is desirable. Here, a unique IP injection method that is able to handle toxic compounds is reported. Consistently reduced cardiac function can result without incurring significant fish death. The technique can be easily mastered by researchers who have minimal experience with adult zebrafish.

Author Spotlight: Novel Intraperitoneal Injection Method for Zebrafish Cardiotoxicity Models

A new intraperitoneal (IP) injection method in adult zebrafish is described. When handling toxic compounds such as doxorubicin, this procedure is more effective than the two previously reported IP methods. The technique is designed to be easily adopted by researchers with limited experience in the zebrafish model.

The adult zebrafish (Danio rerio), which is genetically accessible, is being employed as a valuable vertebrate model to study human disorders such as ...

Medicine

Small Molecule Screening and Toxicity Testing in Early-stage Zebrafish Larvae

Zebrafish have become a prominent model organism for human translational research, toxicity testing of small molecules and environmental pollutants, therapeutic drug discovery, and other biomedical research applications. The presented protocol aims to provide a clear guide for drug screening and toxicity testing in early-stage zebrafish larvae (aged 3-7 days post fertilization, dpf). Several methodologies describing toxicity testing in zebrafish larvae have been published, albeit lacking a standardized protocol for exposure durations, rate of media changes, and morphology scoring, amongst others. We experimentally determined the ideal larvae ages for conducting the experiments, the plate sizes that work best for those ages, the rate of media changes that provide reliable results while limiting wastage&#160;of test compound, and a morphology scoring system that is easily quantifiable in a statistical manner. The manuscript describes in detail a protocol for toxicity testing in zebrafish larvae aged between&#160;3 to 7 dpf, aiming to target the gap in drug toxicity testing and taking the first step towards a standardized method similar to the Fish Embryo Acute Toxicity test.

This protocol describes a drug screening procedure in zebrafish larvae 3-7 days post fertilization followed by morphological assessment.

Zebrafish have become a prominent model organism for human translational research, toxicity testing of small molecules and environmental pollutants, ...

An Introduction to the Zebrafish: Danio rerio

Zebrafish (Danio rerio) are small freshwater fish that are used as model organisms for biomedical research. The many strengths of these fish include their high degree of genetic conservation with humans and their simple, inexpensive maintenance. Additionally, gene expression can be easily manipulated in zebrafish embryos, and their transparency allows for observation of developmental processes.
This overview video first introduces basic zebrafish biology, including their phylogeny, life cycle, and natural environment, before presenting the features that make them so useful in the lab. A brief history of zebrafish research is also provided through a review of major discoveries made in fish, ranging from the early establishment of methods for efficient genetic screening to the discovery of novel therapeutics for human diseases such as cancer. Finally, some of the many avenues of experimentation performed in zebrafish are discussed, including immunological and developmental studies.

Danio rerio Zebrafish as a Model Organism

Zebrafish (Danio rerio) are small freshwater fish that are used as model organisms for biomedical research. The many strengths of these fish include their ...

Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines

Samenvatting

Meer video's verkennen

PLAYLIST