A Multiple Dose Immunisation Protocol Suitable For Edible Vaccines

摘要

Frequent administration of oral immunisation has proven to be more successful than single administration. The frequency of feeding edible vaccines, however, is limited by the maximal oral intake, the lack of nutritional value and the possible presence of toxic ingredients. Therefore, we designed a protocol in which the animals received multiple immunisations on three alternating days ('triple dose') and the protocol was compared to single immunisations. Mice were immunised via intragastric (IG) gavage with ovalbumin (OVA) mixed with cholera toxin (CT) and the effects on systemical and local immune responses were determined. Serum IgG1 and IgA titres against OVA after oral boost immunisation given three weeks after primary immunisation were significantly higher after 'triple dose' than after 'single dose' immunisation. Faecal IgA was detected only after 'triple dose' boost immunisation. A second boost did not further increase serum IgG1 and faecal IgA. Antibody responses against CT were also elicited and again, boost immunisations did not further increase this response. We concluded that oral immunisation with multiple doses was more effective than 'single dose' immunisation and it seems practical and efficient for edible vaccines. Introduction Several factors affect the immune response upon oral administration of antigen and a few can be manipulated [1].One of the most General complicating factors are degradation of the antigen in the gastro-intestinal tract and the induction of a state of oral tolerance [2,3]. Furthermore, the nature of the antigen strongly determines the outcome of oral immunisation. Oral administration of live pathogens revealed in many cases significant mucosal and systemic immune responses [4]. Oral immunisation with non-living pathogens, subunits or peptides, however, is often inefficient and requires multiple administrations with large amounts of antigen and adjuvant [5]. Another important factor is the immunisation schedule. Chalacombe [6] found that a weekly immunisation did not result in significant responses. Serial immunisations on consecutive days, however, induced sIgA. These and other data suggested that frequency and timing of immunisation are important. Detailed study on differences between single dose immunisation and multiple dose immunisation on the development of IgA and IgG1 antibodies and their course in time after priming and booster immunisation are not described yet. The goal of the present study was to establish an effective oral immunisation protocol applicable for edible vaccines for which the frequency of feeding is limited by the maximal possible oral intake. Materials and Methods Mice Swiss female mice (6 to 8 weeks old) were obtained from Charles River (Sulzfield, Germany) and housed per groups under conventional conditions. All mice were raised and kept on an OVA free diet. All animal experiments were held under auspices of the ID-Lelystad B.V. Animal Experimentation Committee according to the Dutch Law on Animal Experimentation. Antigen preparation and immunisation The antigen preparation tested consisted of 10 mg of ovalbumin (OVA; Grade V, A-5503, Sigma) mixed with 5 μg cholera toxin (CT; C-8052, Sigma) dissolved in 0.4 ml saline. Mice fasted overnight (water was provided ad libitum) and were immunised orally by intragastric intubation with OVA plus CT on Day 0, 21, and 42 (‘single dose') or on Day 0, 2, 4, 21, 23, 25, 42, 44, and 46. Collection of faeces and serum samples Pre-immune tail blood serum and faeces samples were collected before the first immunisation and on Day 14, 35, and 49. Fresh faeces pellets were immediately frozen at –20 oC. Before testing, faeces pellets were treated as described elsewhere to prevent degradation [7]. Detection of anti-OVA and anti-CT antibodies High binding ELISA plates (Greiner, Nürtingen, Germany) were coated overnight at 4oC with 100 μg ml-1 OVA or 2 μg ml-1 CT dissolved in PBS. ELISA was performed as described earlier [7]. Antibody