Delayed-Type Hypersensitivity (DTH) model in the Cynomolgus monkeyJean-François Le Bigot 1, Caroline Bouchez 1, Frédéric Gervais 1, Renaud Fleurance 1, Bernard Palate 1 and Jacques Descotes 2

1 CiToxLAB in France, BP 563 - 27005 Evreux cedex, France; 2 Poison Center, 69424 Lyon cedex 03, France

www.citoxlab.com

IntroductIonAlthough T-dependent antibody response (TDAR) assays are currently first-line functional assays in nonclinical immunotoxicity evaluation, second-line assays may be helpful to obtain additional data on mechanisms and further insight on risk assessment of drug candidates. The delayed-type hypersensitivity (DTH) assay measuring cell-mediated immune response is one of these assays. Limited information, however, is available on DTH assays in monkeys. The aim of this study was to validate a DTH assay in Cynomolgus monkeys.

MaterIals and MethodsThree groups of 3 male Cynomolgus monkeys each were immunised intramuscularly with eitherone or two injections of 1 mg keyhole limpet hemocyanin (KLH) or two injections of tetanus vaccine (40 UI tetanus toxoid plus 0.6 mg aluminum hydroxide). All animals were then challenged by an intradermal injection of the same antigen on days 42, 56 and 70 (i.e. 4, 6 and 8 weeks after the last immunisation). In addition, some animals immunised with tetanus vaccine were challenged with aluminum hydroxide under the same conditions (see Figure 1). Skin reactions at the intradermal injection sites were recorded 24, 48 and 72 hours after each challenge in all animals. Thereafter, skin biopsies of challenge sites were collected, preserved and stained for histopathological examination and immunohistochemistry analysis with an immunoperoxidase technique using CD3, CD19 and CD68 markers. Figure 1: Study Design

resultsNo systemic signs of toxicity were observed in any animal during the study. Following intradermalchallenges, local reactions consisting of erythema and/or edema at the injection sites were observed whatever the selected antigen (see Table 1).

Table 1: Skin reactions (Mean score values - 9 challenge sites from 3 animals)

Erythema: no erythema = 0 / very slight erythema (barely perceptible) = 1 / well-defined erythema = 2Edema: no edema = 0 / slight edema = 1 / moderate edema = 2

The standard histopathological examination evidenced perivascular mononuclear inflammatory cell infiltrates around small veins and venules at the challenge sites (photos 1 & 2). Immunohistochemistry analysis showed this infiltrate was comprised of CD3+ (photo 3) and CD68+ (photo 4) cells corresponding to T lymphocytes and macrophages, a typical finding in DTH. No CD19+ lymphocytes were seen. Similar local reactions and histopathological findings post-challenge were obtained after one or two injections of KLH, but they were more marked following immunisation with tetanus vaccine. No clear differences in inflammatory response were seen in-between challenges. Aluminum hydroxide induced no local reactions and only a barely detectable inflammatory response at histopathologic examination with few mononuclear cells in the dermis.

Photo 1: Challenge site from a monkey immunised twice and challenged with KLH on day 42(Hematoxylin and Eosin stained, 100-fold magnification)

Photo 2: Challenge site from a monkey immunised twice and challenged with tetanus vaccine on day 42(Hematoxylin and Eosin stained, 100-fold magnification)

Photo 3: Challenge site from a monkey immunised and challenged with KLH on day 42(CD3 immunophenotyping, 200-fold magnification)

Photo 4: Challenge site from a monkey immunised and challenged with KLH on day 42(CD68 immunophenotyping, 200-fold magnification)

D 1 D 42D 14 D 56 D 70

Immunisation(s)(Intramuscular injection)

Challenges(Intradermal injection)

Clinical examinationof challenge sites

BiopsiesBiopsies Biopsies

Groups 1 2 3

Antigen KLH(2 immunisations)

KLH(1 immunisation)

Tetanus vaccine(2 immunisations)

hours after challenge 24 48 72 24 48 72 24 48 72

First challenge

Erythema 0.9 0.3 0.3 0.7 0.2 0.2 1.3 1.1 1.1

Edema 1.1 1.1 1.1 0.3 0.3 0.3 0.8 1.2 1.3

Second challenge

Erythema 0.4 0.7 0.0 1.1 1.1 0.0 1.2 1.3 1.1

Edema 0.8 0.8 0.3 0.9 0.9 0.3 1.3 1.7 1.2

Third challenge

Erythema 0.0 0.0 0.0 0.0 0.0 0.0 1.3 1.3 0.2

Edema 1.0 1.0 0.0 1.0 1.0 0.0 1.0 1.0 0.7

conclusIon This study demonstrates that a stronger DTH response can be induced in Cynomolgus monkeyswith tetanus toxoid than KLH. Thus, a DTH assay can easily be included in the immunotoxicityevaluation of pharmaceuticals in monkeys.