The zebrafish as a potential model for vaccine and adjuvant development

Figures & data

Figure 1. Reprinted from “zebrafish developmental timeline,” by BioRender.com (2024). Retrieved from https://app.biorender.com/biorender-templates. From Stephanie Lepage (Creator) and Ann Sanderson.

Table 1. Comparison of TLRs (and NLRs) between humans and zebrafishes.

TLR	Present in Humans	Present in Zebrafishes	Natural Ligand in Humans	Corresponding Adjuvant Ligand
TLR3	Yes	Yes	dsRNA	Poly (I:C)
TLR4	Yes	Yes, but does not bind LPS	LPS	Monophosphoryl Lipid A (MPLA) or Glucopyranosyl Lipid A (GPLA)
TLR5	Yes	Yes	Flagellin	Flagellin derivatives
TLR7/8	Yes	Yes but unknown if these bind to -ssRNAs	ssRNA	imidazoquinolines, 3M–052
TLR9	Yes	Yes	CPGs	CPGs
TLR11	Yes	Yes	Multiple ligands	N/A
Inflammasomes NLRs	Yes	Yes	Bacterial cell wall components	Alum MF59 QS21, Matrix-M

Table 2. Gap areas for evaluating acquired immunity to COVID-19 antigens in the adult zebrafish.

Activity	Rationale	Expected/Potential Outcomes
Reagents	To generate reagents (secondary antibodies) vs. all zebrafish antibody isotypes	Labeled antibodies against zebrafish IgM, IgD, IgZ/T
ELISA antibody	To develop ELISA kits for measuring zebrafish isotypes	Ability to quantitate zebrafish antibody isotypes in response to immunization
Immunization route	To optimize the immunization route for antibody measurement	Assessing oral (mucosal), immersion, intraperitoneal, intramuscular routes
Antibody kinetics	To measure the time kinetics of zebrafish antibody responses	Assessing adaptive immunity and more rapid responses upon restimulation
Functional antibody	To measure zebrafish virus neutralizing antibodies vs pseudoviruses	Assessing protective immune responses and surrogate correlates of protection
Epitope broadening	To measure zebrafish cross-neutralizing antibodies vs pseudoviruses	Assessing the effects of antigen mixing and combining antigens
Challenge studies	To measure protection vs live sarbecoviruses or beta-coronaviruses	Assessing protective immunity
Mechanistic studies	To generate transgenic zebrafish and markers	Assessing underlying mechanisms and NextGen correlates of protection

Table 3. Summary table of advantages and disadvantages of embryos versus adults for evaluating vaccine safety as well as innate and adaptive immunity.

Life History Stage	Vaccine Safety	Innate Immunity	Adaptive Immunity	Other Considerations
Embryos	Advantages: Optical transparency Observation through cell and organ live imaging Effects on all organs except for the gonad can be studied in embryos. Embryos can be used to assess the acute toxicity of any small molecule, protein, or mixture that is water soluble or can be delivered via embryo injection. This “Fish Embryo Toxicity Test” is sanctioned by the OECD. Epigenetic and other OMICs studies Potential for substituting for rodents in toxicology or vaccine screening for identifying molecules or mixtures that are toxic and should not be developed further for use in humans Availability of genetic mutants, ease of generating new mutants, to study interactions between genes and vaccine components Addresses the 3Rs for the more ethical use of animals in scientific research Disadvantages: For safety/toxicology, not approved by the FDA currently to substitute for mammalian vertebrate species. Molecules that are non-water soluble must be administered via embryo injection, which is lower throughput than adding chemicals to embryo water	Advantages: Zebrafish embryos can be infected with some pathogenic organisms resulting in innate immune responses that resemble those of humans. Optical transparency: Vaccines and adjuvants monitored through fluorescent-tagged biomarkers attached to macromolecules or transgenes expressed in restricted cell types to study mechanisms. Screen new or existing adjuvants for targeting a specific innate immune response. Rational adjuvant design includes the new field of “immuno-engineering” for developing new natural or synthetic materials. Epigenetic and other OMICs studies Availability of genetic mutants Disadvantages: Not yet validated for innate immune responses to vaccines or adjuvants Zebrafish TLR4 does not bind LPS; other TLRs may or may not bind other commonly used adjuvants.	An adaptive immune system is not developed or present in embryos.	NIH OLAW policies apply to egg-laying vertebrates such as zebrafish “only after hatching” at three days post-fertilization. Potential microinjection sites (1–3 days post-fertilization): Hindbrain ventricle Duct of Cuvier Caudal vein Tail muscle Notochord Otic vesicle
Larvae/Adults	Advantages: Evaluate general animal and organ safety and are potentially suitable for toxicology analyses, particularly for identifying molecules or mixtures that are toxic and should not be developed further for use in humans Hepatotoxicity, nephrotoxicity, neurotoxicity, and cardiac effects. Epigenetic and Other OMICs Studies DART Studies: Embryogenic & teratogenic effects Disadvantages: No optical transparency Lower throughput than embryos. For safety/toxicology, not approved by the FDA currently to substitute for mammalian vertebrate species.	Advantages: Zebrafishes can be infected with some pathogenic organisms resulting in innate immune responses that resemble those of humans. Study the mechanisms of new or existing adjuvants. Screen new or existing adjuvants for targeting a specific innate immune response, Rational adjuvant design includes the new field of “immuno-engineering” for developing new natural or synthetic materials. Epigenetic and other OMICs studies Availability of genetic mutants Disadvantages: The Zebrafish model has not yet been validated for innate immune responses to vaccines or adjuvants. Lack of optical transparency Zebrafish TLR4 does not bind LPS; other TLRs may or may not bind other commonly used adjuvants. No lymph nodes	Advantages: Evidence for immunological memory, including enhanced protection and pathogen clearance correlating with a secondary immune response upon reinfection, IgM and IgD immunoglobulin isotypes 2 recently discovered IgZ-like isotypes – IgZ-1 and IgZ-2 Both IgM and IgZ-2 are also found on zebrafish B cell surfaces Accelerated antibody production, the expression of igm and igz2 heavy chain immunoglobulin genes, and an il13 Th2 signature cytokine gene. MHC1 and MHCII genes Evidence for mucosal immunity and MALT Disadvantages: No zebrafish IgG or IgA. No class switching. Adaptive immune responses are slower in teleosts compared to mammals. Survival blood collection is technically challenging. Minimal availability of ELISA Kits and other reagents for measuring antibody	Zebrafishes can be housed in densities (up to 50 adult fish per tank) higher than mice (4–6 mice per cage) or other common laboratory mammals. Zebrafish possess a hindbrain ventricle, a cavity filled with cerebrospinal fluid into which immune cells can be recruited. This is a convenient injection site to study host immunity; other routes are as above.