Use of Adjuvants on Research Animals

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General Policy

This policy describes the acceptable use of adjuvants in small animal species (rodents and rabbits). Requests to use methodologies not outlined in this policy must be presented to the IACUC for review and approval. Adjuvants are vehicles employed to enhance the immune response of specific immunogens, which are rarely sufficient to induce a satisfactory antigenic response alone. They work through a number of mechanisms serving as antigen-depot-forming substances, as delivery vehicles or inert carriers, as immunostimulators or modifiers, or any combination of these. In addition, the source of the antigen preparation must be considered. Many immunogens are identified and isolated from polyacrylamide gels. Polyacrylamide is inflammatory and has adjuvant properties by itself. Ideally, the immunogen should be eluted from the gel before immunization. If this is not possible, the gel should be trimmed so that the least amount of gel is administered. Lastly, preparation of antigens for injection in aqueous solution should be performed aseptically such that contaminants, such as unwanted toxins and pyrogens, are excluded. Unintended bacteria and other pathogens need to be eliminated as well and the pH of the injection solution should be within physiological limits.

Many adjuvants can lead to moderate to severe inflammatory responses at the site of administration. The Principal Investigator should first consider whether an adjuvant is actually required to induce the desired immune response. Highly aggregated antigens are likely to induce the appropriate immune response without the aid of an adjuvant. Soluble, relatively pure small molecule antigen preparations are less likely to induce an adequate response alone thereby justifying the need for adjuvant-mediated enhancement.

Adjuvant type and administration site should be selected based on three criteria:

  • adequate antigenic response;
  • least amount of inflammatory response at the administration site;
  • best site for minimally affecting the normal posture and movement of the animal. Non-inflammatory adjuvants such as aluminum compoundsand subcutaneous implanted chambers should be considered first since they cause less inflammation.

Well-developed alternative adjuvants commonly used in immunology studies include:

  • RIBI adjuvant system (oil-in-water emulsion),
  • Titermax (copolymer water-in-oil emulsion),
  • Montanide ISA Adjuvant (oil/surfactant-based).

Water-in-oil emulsions such as:

  • Complete Freund Adjuvant (CFA)
  • Incomplete Freund Adjuvant (IFA)

will induce more severe inflammation and should be used only when alternatives do not elicit an acceptable immune response.

Consideration should be given to the amount of adjuvant and the location of the injection site so that the animal is exposed to the least pain/distress possible.
Footpad injections using CFA and/or IFA are particularly painful/distressful and specifically discouraged. Because antigens injected into the footpad are processed by the popliteal lymph node, injections made at the tail base or in the area of the popliteal node are a more humane alternative. Footpad injections with water-in-oil adjuvants will be approved only when it is documented that there are no other acceptable methods. In summary, immunization techniques should provide for high quality antibody production while limiting the adverse effects on the animal.

In addition to selection of the appropriate adjuvant, animal manipulations and monitoring of the administration site must be considered. Injection sites should be aseptically prepared prior to antigen-adjuvant administration. The area should be clipped of hair, surgically scrubbed, and allowed to dry. Compounds can be administered intravenously (preferably small particulate antigens), subcutaneously (preferred for CFA), or intradermally; intramuscular injection is discouraged. Intraperitoneal immunization is permissible only in mice and should only be administered as a single immunization; intraperitoneal administration of CFA must be scientifically justified. Aerosol, oral, and intranasal routes are also utilized when an a response is desired. When administering compounds subcutaneously or intradermally, injecting small volumes into multiple injection sites is more beneficial than injection of larger volumes in fewer sites, from both a humane and scientific perspective. Care should be taken that there is adequate separation between injection sites to avoid the coalescing of inflammatory lesions that could lead to tissue sloughing or abscess formation. Booster immunizations may be given to maintain adequate antibody levels long-term. Frequency of booster immunization should be based upon the time required for the animal to process the immunogen and should be given when the titer has peaked and started to decrease. Booster injections containing antigens such as bacteria, virions or cells may be given intravenously as long as they are not likely to cause anaphylaxis; booster injections involving CFA must be scientifically justified. Soluble antigens with a higher risk of causing anaphylaxis should be administered subcutaneously. Booster injections should be given at sites different from the primary immunizations whenever possible. Animals should not be boosted if adverse reactions were noted during a prior immunization.

Animals used in antibody production need to be monitored closely after administration of primary and booster adjuvant-antigen injections. Anaphylaxis is a common adverse effect that will determine whether further injections should be performed. Animals should be monitored immediately after injection, one hour later and then 2-3 times a day for the first two days post-injection. Post-procedural instructions and monitoring should be documented on the animals cage card and/or the individual animal record. Daily observations are necessary to ensure that the approved protocol is followed particularly as it relates to appropriate levels of pain/distress. In many cases it is appropriate to administer analgesics to animals in immunization studies. These should be given prophylatically to minimize the pain/distress. If animals exhibit pain/distress during the study (e.g., not ambulating normally, not eating, depressed activity, self-trauma), then additional analgesics should be provided or the animal should be euthanized. Consultation with veterinarians is recommended.

Blood collection for antibody harvest is a critical aspect of the immunization process. The goal of collection is to obtain a suitable volume of undamaged blood while minimizing adverse physiologic effects on the animal. The total blood volume of the animal limits the volume of blood that should be collected. Most species can tolerate losing < 15% of their total blood volume over the course of every two weeks. Collection of this volume will minimize hypovolemia and anemia to allow the animal to recover and generate sufficient erythrocytes before the next collection. Excessive bleeding frequency or volume collected may cause anemia or even hypovolemic shock. The blood collection site is also important, depending on the species and amount of blood to be collected. Animals should be acclimated to any restraint devices used for blood collection and should be restrained for the minimum amount of time necessary. Large blood volumes (up to 70% of total blood volume) are appropriate for non-survival procedures, however the animal must be anesthetized prior to phlebotomy because of the stress associated with severe acute hypovolemia. The preferred method of euthanasia associated with terminal blood collections is exsanguination. Death of the animal should be confirmed, in accordance with the current IACUC Policy on Euthanasia.

If investigators are unfamiliar with any of these techniques, they must contact the veterinarians to schedule training or to request veterinary technical services to perform these procedures.

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