Induction of Allergy to Nonvaccine or Food Proteins

This is a particularly important test when examining the suitability of an adjuvant for inclusion in an oral vaccine, as there could be a reaction to food proteins (23). With the interest in the oral route as a means of stimulating mucosal immunity, there is a possibility that an adjuvant could induce an allergic response to dietary proteins. In this study, both lactalbumin and gluten failed to elicit an IgE response in the presence of the original Freund's Complete or Incomplete Adjuvants (FCA or FIA) in HAM1/CR mice or Dunkin Hartley guinea pigs. On the other hand, the guinea pigs showed increased IgE production after oral administration of ovalbumin or soy bean protein, both unusual proteins in their normal pellet diet. Such tests are valid only if all of the previous toxicity tests are negative.

1. Groups of mice or guinea pigs are fed freely with moistened ovalbumin or lactal-bumin as the main food supply for 24 h. This does not affect their normal weight gain or health. Subsequently, the mice are dosed orally with 0.2 mL of the

Table 2

Style of Report for the Toxicity Tests

Mouse groups

Weight (g) at the start of the test Weight (g) at the end of the test Difference in weight (g) Deaths

Autopsy report Histology report Assessment. P = Pass; F = Fail adjuvanted formulation or with physiological saline, as the control, containing 2.0 mg of the test protein. The dose in guinea pigs is 0.3 mL of adjuvanted formulation, administered in a gelatin capsule, whereas the saline is delivered from a syringe without a needle at the back of the oral cavity.

2. All animals are fed on the protein diet for a further 24 h after adjuvant dosing and then returned to their normal pellet diet and water. The guinea pigs are also given ascorbic acid to prevent vitamin C deficiency. All animals were bled out on day 21.

3. Passive cutaneous anaphylaxis (PCA) reactions are measured in hairless mice, hrhr, injected intradermally with 0.05 mL of serum diluted 1 in 2 at four sites on the dorsal surface. For IgG PCA tests, the sera are heated for 2 h at 56°C to inactivate IgE. After 2 h for IgG and 48 h for IgE, 1.0 mg of the respective protein in 0.2 mL saline containing 0.5% (w/v) Evans Blue dye is injected into the caudal vein, and after 30 min the areas of blueing on the skin are measured. The hair is clipped from the dorsal surface of the guinea pig, injected with 0.1 mL of the serum and after 4 h for IgG and 12 d for IgE injected with 0.1 mL saline containing 1.0% dye and 1.0 mg of the respective protein. The zones of blueing are measured after 2 h, intensively staining zones of >0.5 cm2 are indicative of a positive reaction, although the positive zones appear more diffuse with the IgE response in the guinea pig.

4. The sera may also be examined for the presence of antibodies by a standard ELISA.

3.5. Standard Adjuvants and Antigens, Routes, and Volumes of Injection Mixtures for Use with New Adjuvant Formulations in Tests to Measure the Stimulation of Humoral and Cell-Mediated Responses

3.5.1. Standard Adjuvants

Those recommended were Alhydrogel and the FCA produced by the Statens Serum Institute, Copenhagen (1). A suitable alternative for the latter is a "Non-

Ulcerative Freund's Complete Adjuvant (NUFCA)" which contains BCG vaccine BP, BNF intradermal (see Note 3). The BCG vaccine for sc injection should not be used as this will cause local ulceration. The id BCG vaccine is reconstituted according to the manufacturer's instructions and 0.1 mL is added to 0.9 mL of the aqueous phase-containing antigen. Note that this is a major difference between FCA and NUFCA as the Mycobacterium tuberculosis in FCA was suspended in the oil phase. The aqueous phase is emulsified with the oil phase before use. The manufacturers indicate that this NUFCA can be administered by id, im, or sc routes and agree with the WHO (22) that the im route produces fewer adverse reactions and creates a longer-lived slow-release depot which tends to provide a better immune response.

3.5.2. Standard Antigens

For the comparative biological testing of immunomodulators, the antigens chosen were ovalbumin (Ovalbumin,grade V crystallized and lyophilized, Sigma), and influenza H3N2 type A hemagglutinin (1), however, it was pointed out that the latter antigen is an unsuitable standard for guinea pigs (2) (see Note 5).

3.5.3. Animals for Standard Antibody Production Tests

The guinea pig was the animal of choice for biological tests. In regard to mice, the influence of the animal's genetic background and MHC haplotype must also be considered. For this reason, animals with either similar genetic background and variable H-2 haplotype (e.g., C3H H-2k and C3H.B10 H-2b) or variable genetic background and similar H-2 haplotype (e.g., Balb/c H-2d and DBA/c H-2d) should be included in comparative tests.

3.5.4. Route of Injection

In most instances, researchers have their own preferences in regard to the site of injection of an adjuvant-formulated, experimental vaccine, however, consideration should be given to whether the vaccine is for human or veterinary use. It is doubtful whether patients would be willing to accept ip or iv injections as a routine vaccination procedure. Consequently, it is advisable to give the injections either subcutaneously or intramuscularly. Similarly, in no circumtances should an oil or alum-adjuvanted veterinary vaccine be injected intravenously nor booster injections administered iv or ip as there is a danger of inducing anaphylactic shock in the animals. Intraperitoneal injection of adjuvanted mixtures into some animals may result in decreased weight gain over 7 d. This inflammation may resolve itself after 7 d, but later postmortem

Table 3

Some Recommended Routes and Volumes for Injection Doses

Injection Sites

Maximum volume per

Species injection site Primary Secondary

Mice or hamsters Guinea pigs or rats

Rabbit Large animal Chicken

300 |L 250 | L (if in multiple sites <25 |L/site*) 500 | L (if in multiple sites <250 |L/site*) 250 | L

sc; im sc; im. into one hindlimb sc; im. into one thigh muscle; id* sc; im into one hindlimb; id* sc; im sc; im oral sc; im into one hindlimb oral sc; im into one thigh muscle; id* sc; im into one hindlimb; id* sc; im sc: subcutaneous; im: intramuscular.

* If the intradermal (id) multiple injection site schedule is used.

examination may reveal macroscopic evidence of such a response. There may be specific tests where there is a requirement for another route, for example, id injections.

3.5.5. Volume of Injection Mixture

For animals the maximum volumes per site of injection are shown in Table 3. These dosages are based on the use of an adjuvanted vaccine which has already been shown to be nontoxic and nonpyrogenic.

3.5.6. Dose of Adjuvant

The upper limit of adjuvant per dose may be dictated by the results obtained in the toxicity and pyrogenicity tests, although it may be preferable for economic reasons to determine a lower dose at which an adjuvant response is obtained. In general however, the weight in the injection mixture should not exceed 25 ^g for a mouse and 200 ^g for guinea pigs, rats, or rabbits. If the dose is to be spread among multiple injection sites in larger animals, the volume should be not more than 250 ^L per site and preferably as little as 25 ^L. If a new adjuvant is being developed for veterinary use, it is important that, if possible, the animal species ear-marked for the vaccine is tested during the development phase.

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