Local immune responses to influenza antigen are synergistically enhanced by the adjuvant ISCOMATRIX®
Introduction
The local (draining) lymph node is the first lymphoid organ encountered by antigen following subcutaneous injection, and is the site of primary immune induction [1], [2]. Central to this process is the co-ordinated interaction of antigen presenting cells (APCs) with lymphocytes, mediated through the regulation of lymphocytes trafficking from blood into the node [3]. In a resting node, 10% of lymphocytes enter from tissues via the afferent lymphatics, and 90% enter through a process of selective recruitment from blood [4]. Following administration of antigen with certain adjuvants, there is a consistent pattern of lymphocyte migration from the node into efferent lymph [3], [4], [5]. This involves an immediate initial period of reduced cell output, termed “node shutdown”, followed 24–48 h later by a period in which cell outputs increase markedly above resting levels. This increased output is indicative of the recruitment of lymphocytes into the node. Lymphocyte recruitment, therefore, provides a mechanism to maximise the number of low frequency antigen-specific cells entering the lymph node and interacting with antigen and APCs. Activated cells then differentiate into effector (blast) cells within the node. A second cell output peak coincides with an increased proportion of lymphoblasts and plasma cells in efferent lymph [3]. Shutdown and recruitment can be influenced by the type and quantity of antigen [6], adjuvant [7] (Windon et al., unpublished data), or, as shown in the results reported here, the combination of antigen and adjuvant. Orchestration of the local immune responses is thought to be mediated through cytokines [3].
Procedures involving cannulation of the efferent lymphatic ducts from peripheral nodes in sheep have been used to investigate local immune responses to antigens and adjuvants [7], [8]. The creation of a chronic fistula, by surgically inserting a cannula into the efferent duct of the draining peripheral node, allows output from the node to be collected and the dynamics of immune induction to be characterised [3], [4].
Immune stimulating complexes (ISCOMs®) are open cage-like structures, typically about 40 nm in diameter, formed from cholesterol, phospholipid, Quillaja saponins and immunogen [9]. ISCOMATRIX® (IMX) is identical to ISCOMs® but does not include the immunogen [10]. IMX has adjuvant properties when admixed with antigen and has been tested in pre-clinical and phase I trials [9]. In an earlier study [7], we demonstrated that IMX was capable of inducing lymph node activation in the absence of antigen. Injection of IMX provoked cytokine production and influenced lymphocyte trafficking through the node by causing shutdown and subsequent increased lymphocyte recruitment. It was hypothesised that this may be one of the mechanisms by which ISCOM®-based adjuvants achieve their immunomodulatory properties. In the current experiments, lymphatic duct cannulation procedures in sheep were used to extend these earlier findings and assess the wider applicability of the cannulation model by investigating local immune responses to human influenza virus antigen (Flu ag), a complex native antigen [11], adjuvanted with IMX. Our results demonstrate that, compared to Flu ag or IMX alone, Flu ag admixed with IMX (Flu ag+IMX) caused synergistic responses for a number of local immunological parameters.
Section snippets
Sheep
Mature medium-wool Merino ewes, aged 1–2 years, were brought from pasture into pens to acclimatise prior to the commencement of experimental procedures. Following surgery, the animals were maintained in metabolism cages, fed a mixture of lucerne chaff and commercial pellets, and had access to water ad libitum. At the conclusion of each experiment, the animals were sacrificed by lethal injection of sodium pentobarbitone (Lethabarb, Boehringer Ingelheim). All experimental procedures in these
Cell outputs
Total cell output response patterns in two sheep following administration of Flu ag alone (Sheep 1), Flu ag+IMX (Sheep 4), and IMX alone (Sheep 4), are shown in Fig. 1. After injection, total cell outputs displayed an initial shutdown for 24–48 h followed by a period of increased cell output. Shutdown after injection of Flu ag (66±11% of pre-injection cell outputs, n=3) was not as severe and of shorter duration than Flu ag+IMX (32±9%, n=6; P=0.037) or IMX alone (20±5%, n=3; P=0.018). The
Discussion
The studies described here have used lymphatic duct cannulation procedures in sheep to demonstrate that Flu ag+IMX, when compared to Flu ag or IMX alone, induced synergistically enhanced, rather than additive, local immune responses. Furthermore, this work has shown the potential of the sheep lymphatic duct cannulation model to evaluate antigen/adjuvant combinations for vaccine development.
Following injection of Flu ag alone, the immediate decrease (node shutdown) and the subsequent increase in
Acknowledgements
The authors wish to thank Bob Geyer for the care of experimental animals used in this study. This work was supported by the Cooperative Research Centre for Vaccine Technology, Australia.
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Cited by (0)
- 1
Present address: Bavarian Nordic Research Institute, GmbH, Fraunhoferstrasse 18b, D-82152 Martinsried, Germany.
- 2
Present address: The Centre for Animal Biotechnology, The University of Melbourne, Parkville 3010, Vic., Australia.
- 3
Present address: Veterinary Division, CSL Limited, 45 Poplar Road, Parkville 3052, Vic., Australia.