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A sheep cannulation model for evaluation of nasal vaccine delivery
Methods 38 (2006) 117–123 www.elsevier.com/locate/ymeth
A sheep cannulation model for evaluation of nasal vaccine delivery Hung-Hsun Yen, Jean-Pierre Y. Scheerlinck, Susie Gekas, Phil Sutton ¤ Centre for Animal Biotechnology, University of Melbourne, Parkville, Vic. 3010, Australia Accepted 28 September 2005
Abstract We have developed and validated a novel model to investigate the eYcacy of nasal vaccine delivery. Based on lymphatic cannulation of the tracheal lymph trunk of sheep, the model allows collection of lymph draining from the Nasal Associated Lymphoid Tissue. The model is suitable for determining both the amount of material that is absorbed into the lymphatic system, following intra-nasal delivery and the immune response that occurs following vaccination into the nasal area. The cell populations that track in this duct were phenotyped and found to be similar to those previously reported to be present in eVerent lymph draining from peripheral lymph nodes. Following intra-nasal spray, we demonstrated that the amount of material recovered in draining lymph is only a very small fraction of the total delivered. Nevertheless, intra-nasal spraying of a vaccine could activate local immune cells. The method described will be invaluable for optimising intra-nasal delivery systems by allowing a separate optimisation of vaccine uptake and immune responses induction. 2005 Elsevier Inc. All rights reserved. Keywords: Sheep; Lymphatic cannulation; Nasal delivery
1. Introduction From early times to modern day, the most common mode for vaccine delivery has been via injection. This, however, has some limitations including occupational risk associated with transmission of infections through needle-stick injuries or via the repeated use of needles as sometime occurs in poorer countries. There is also a perception that some individuals may avoid immunisations due to a fear of needles, which could potentially impact on vaccine eYcacy at both the individual and cohort level. However, perhaps the key argument for exploring alternative routes of vaccine delivery is our increasing realisation of the compartmentalisation of the immune system. Immune responses at mucosal sites are best induced by exposure of antigen at mucosal sites. In fact, it has been demonstrated that mucosal immune cells induced at one site can selectively home into distinct and distant mucosal sites [1,2]. A key implication of this is that while injected vaccines are typically very *
Corresponding author. Fax: +61 3 9347 4083. E-mail address: [email protected] (P. Sutton).
1046-2023/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2005.09.011
eYcient at inducing systemic immunity, they are less capable at producing an eVective immune response at mucosal surfaces. Thus, mucosal delivery may be important when aiming to induce a response at a particular mucosal surface. Pathogens generally infect a host via one of two routes, either via a break in the skin or via a mucosal surface. The reality is that the majority of pathogens of signiWcance infect via a mucosal surface, be that intestinal (i.e., cholera), respiratory (tuberculosis, inXuenza) or reproductive (HIV). Development of techniques to improve immunity at mucosal surfaces would therefore potentially be highly valuable in inducing protection against such pathogens. While it is now generally accepted that induction of eVective immunity at mucosal surfaces is more eYciently induced by mucosal vaccines than by injected immunisations, eVective mucosally delivered vaccines are extremely rare. The immune systems of mucosal surfaces are essentially designed to be generally immunologically non-responsive to antigenic stimulation. In the intestine, the bulk of antigens present are from ingested food or commensal and useful bacteria against which reaction would be detrimental to the host. Large quantities of antigens, bacteria, fungal spores etc., are
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also constantly inhaled into the respiratory system, where, similarly, development of a strong inXammatory response may be harmful. In the vagina, reaction to foreign proteins such as found on sperm may interfere with reproduction. The immune regulation mechanisms that have evolved to prevent these events mean that it is not simple to induce an eVective, protective immune response via the mucosal route. The best example of a mucosal vaccine is the Sabin polio vaccine, which is a live-attenuated virus, delivered via its natural route of infection. Thus, in this case, the vaccine is eVective because it takes advantage of mechanisms developed by the pathogen itself to overcome the tolerance present in the gut. If eVective mucosal immunisations could be achieved, there would be signiWcant potential for improving protection against mucosal pathogens. Such approaches need to address two key points: (i) how to overcome the natural immune regulation processes present at mucosal sites and (ii) to ensure that potentially harmful severe adverse reactions are not induced. The delivery of vaccines via the nasal cavity is one approach which has been investigated for many decades, but more recently has attracted increasing attention. One such example is the recently released Flumist vaccine produced by MedImmune, which comprises a cold-adapted live-attenuated virus sprayed intra-nasally as a mist [3]. As a rule, eVective mucosal vaccines have tended to be live attenuated organisms, where a specialised infectious organism is used to circumvent the mucosal defences and induce an immune response. Much less success has been achieved with subunit or killed vaccines. Indeed, the nasal mucosa has evolved many mechanisms to exclude substances entering via the nostril from traversing its surface. The nasal mucosal surface is covered with mucus to limit access to the epithelial surface. In addition, the nasal epithelial cells are covered with cilia and joined by tight junctions, which prevent passage between them. Mucociliary action (beating of the cilia, which causes the mucus to Xow) directs the vast bulk of inhaled substances down the oesophagus into the intestine. The immune systems of various mucosal surfaces appear to be linked, such that immune cells induced at one mucosal site drain via the lymphatics into the blood circulation and migrate to distant mucosal regions. It has been shown previously that nasal immunisations may be of use for inducing immune responses at other mucosal sites [4–6]. The spread of these mucosal immune cells between mucosal sites has been clearly demonstrated in murine models, which have been extremely useful at demonstrating the presence of the common mucosal immune system. However, murine models only provide a snapshot within a single animal and do not readily allow for real-time evaluations of the kinetics of these processes, nor do they allow to directly measure material transfer across mucosal barriers. Another limitation of murine models for nasal vaccine delivery is that the majority of studies use a delivery volume that is far from representative of that which would translate to humans. It has been calculated,
based on comparisons of nasal cavity volumes alone, that substances delivered intra-nasally in mice should be delivered in a Wnal volume of 3 L, to be comparative with humans. However, most mouse vaccination studies frequently use 30 L or more [7]. Due to the small size of mice, intra-nasal delivery of these larger volumes typically results in exposure of the delivered substances at multiple sites including the intestinal tract and the lungs [8].This latter situation makes true interpretation of nasally delivered vaccines diYcult as it is unclear at which site any observed eVect is induced. Anaesthesia can also signiWcantly impact on the eYcacy of nasal vaccinations in mice [9]. The Nasal Associated Lymphoid Tissue (NALT) of sheep, unlike rodents, has many similarities to that found in humans. Sheep have at least three diVerent lymphoid nodules in the nasal-pharynx region, speciWcally pharyngeal tonsils, lymphoid tissue in the mid-nasopharynx, and also at the opening of the auditory tube [10]. These nodules are highly structured and organised with distinct T- and B-cell follicles possessing follicle associated epithelium with characteristic M-cells [11]. These NALTs appear to be positioned predominantly for sampling antigens present in small, inhaled particles, which traverse the turbinate structures and reach the nasopharynx region. Most liquids sprayed nasally will tend to encounter the turbinates, where they are exposed to mucociliary action and channelled away from the NALT. There are thus many challenges to overcome for eVective nasal vaccination, but there is clear evidence that with the correct technology and approach this is possible. For example, it has been shown that the addition of chitosan, an excipient which opens tight junctions and inhibits mucociliary action can assist the induction of eVective immunity, following nasal vaccination [12]. We have developed a novel large animal surgical procedure, which is intended to assist with the development of nasally delivered vaccines. This procedure uses sheep, recognised as a good animal model for a range of human diseases [13], providing several advantages. In addition to the similar NALT structures to humans mentioned above, their size allows surgical cannulation of lymphatic ducts to be performed, which permits draining immune responses to be measured continuously from a single animal. We have previously used sheep cannulation models of the prefemoral lymphatic duct to examine the induction of immune responses following subcutaneous vaccine delivery [14,15] and have now extended this approach to the nasal region. This model has applications for the evaluation of new technologies, delivery systems or adjuvants, in particular for kinetic studies involving vaccine uptake, induction of local immunity and the dissemination of this immunity to distant mucosal sites. 2. Description and validation of method 2.1. Animals The sheep used in these studies were Merino ewes, which were initially housed in pens within the School of
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Veterinary Science animal facility, The University of Melbourne, Parkville. Following surgery, the animals were maintained in metabolism cages to prevent cannula removal. Sheep were fed lucerne chaV mixed with commercial pellets and allowed access to water ad libitum. All experimental procedures were approved by the University of Melbourne Animal Experimentation Ethics Committee. 2.2. Lymphatic duct cannulation: surgical procedure To prevent regurgitation during anaesthesia, sheep were fasted 24 h prior to surgery. Sheep were initially anaesthetised by an intra-venous injection of thiopentone (10 mg/ kg) into the cephalic vein in the foreleg, thus avoiding possible haematoma formation in the neck area often seen when injecting into the jugular vein. The animals were then intubated through the mouth with a balloon catheter, which was attached to an open circuit anaesthetic apparatus. Anaesthesia was maintained with 1.5–2% halothane in oxygen and monitored regularly by checking for the absence of an eye reXex. Sheep were positioned on their backs and a 5 cm incision made on the median plane of the ventral neck to expose the trachea and the sternothyreohoideus (with/without sternohyoid) muscles. The incision was made 8–10 cm posterior and ventral to the thyroid cartilage. Using blunt dissection, the common carotid artery distal to the heart after the bicarotid trunk was identiWed through the interval between the sternocephalicus and sternothyreohoideus muscles. The tracheal lymph trunks (including lateral and medial ducts) running along each of the left and right common carotid arteries were identiWed and localised (Fig. 1A). In initial experiments, 200 L of 1% (w/v) patent blue dye in phosphate buVered saline (PBS) was injected into the outer rim of each of the nostrils to facilitate the localisation of the ducts (Fig. 1A). The migration of the dye was very fast with colouration appearing in the draining lymphatic ducts within 5 min. We have found that the number of draining ducts vary between sheep, but in most cases consist of two ducts identiWable on each side of the neck. When more than one lymphatic duct was identiWed alongside the common carotid arteries, only the largest lymphatic was cannulated and the other lymphatic was tied oV using suture. Heparinised polyvinyl tubing with an inner diameter of 0.4 mm and an outer diameter of 0.8 mm (CBAS-coated, Carmeda AB, Stockholm, Sweden) was used as cannula tubing. While heparinisation of the cannula is expensive, it substantially prevents coagulation of lymph in the cannula and therefore increases the success rate of the procedure, plus extends the period during which lymph can be collected. Cannulation was performed at an exposed site along the lymphatic duct, at a location before the ducts joined the aVerent (caudal deep cervical) lymph nodes. Using nylon suture (1 metric, 5/0 USP) all identiWed ducts were ligated at sites as proximal as possible to the aVerent lymph nodes (Fig. 1B). Ligation resulted in swelling of the ducts (Fig. 1B) at the upstream end. To facilitate this ligation, we used a
Fig. 1. Illustration of surgical procedure to cannulate the tracheal lymph trunks. (A) The lymphatic duct located along the common carotid artery is visible as a 2–3 mm translucent line. (B) The swollen duct is exposed (following downstream constriction using suture ). The long ends of the suture are attached to the drape to provide tension allowing insertion of the cannula. A home-made ligature instrument composes of a microbiological loop-holder holding the sharp end of a curved surgical needle . Two loose overhand knots are placed around the duct and will serve to secure the inserted cannula. (C) Fine ocular scissors are positioned to allow incising of the exposed duct , to a depth of half its diameter. (D) An incision in the duct allows the insertion of the cannula into the lymphatic duct. Two loose overhand knots are tightened to hold the cannula in place.
home-made ligature instrument (Fig. 1B) composed of a curved surgical needle inserted into the end of a microbiological loop-holder. It is suggested that after the ligation, suYcient length of suture be reserved temporarily at both ends, to create a tension on the duct thereby facilitating the insertion of the cannula into the lymphatic duct (Fig. 1B). Later these same ends are also used for the formation of another square knot securing the inserted cannula. The largest and/or most exposed duct was chosen for cannulation. Connective and adipose tissue along a 1.5 cm length of this duct was carefully removed using blunt forceps and cotton tips. This is a delicate and critical procedure as the duct collapses immediately, following incision and a very clean Weld is required to be able to insert the cannula. Two sutures (Fig. 1B) were placed around the duct at the distal end to the aVerent lymph node and about 1 cm from the Wrst ligation, each made with a loose overhand knot. A slight tension was applied on the duct, by attaching the ends of the Wrst ligation to the drape, in line with the orientation of the duct. This facilitates insertion of the cannula into the duct. Using delicate scissors (Aesculap, FD100R), a transverse incision was made on the duct to a depth of approaching half the diameter of the duct (Fig. 1C). This incision was
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located between the Wrst ligation and the two loose ligatures around the duct (Fig. 1C). Using a syringe Wtted with a 22 gauge needle, the cannula was then Wlled with saline containing 50 U/mL of heparin. This facilitates the Xow of lymph following insertion of the cannula into the duct. The cannula was then cut at a 45 ° angle and inserted into the small incision made previously in the lymphatic duct. Following insertion, the loose overhand knots were tightened and the needle/syringe removed from the cannula (Fig. 1D). Typically, immediately following removal, lymph commenced to Xow, particularly when the external end of the cannula was lowered below the point of surgery. After the insertion of the cannula into the duct, the cannula was secured to the duct by tightening these two loose ligatures and Wxing them Wrmly with a second overhand knot. Using the reserved ends of the Wrst ligation, the cannula was secured one more time. Using an autopsy needle, the cannula was threaded through the skin to the outer side of the initial incision and secured with suture at the point where it exits the skin and again with simple interrupted sutures about 5 cm from the exit point. Synthetic, rapidly setting glue was used to secure the cannula to the suture threaded through the skin, which prevented it from being pulled out through movement of the sheep. The end of the cannula was wiped with 70% ethanol and placed into sterile plastic bottles containing 2000 IU heparin (Pharmacia, Bentley, Australia), attached to the neck using an elastic bandage (SurgiWx size 6, Beiersdforf Australia, North Ride, Australia). 2.3. Post-surgical period As the main application of the above cannulation method is to evaluate nasally delivered vaccines, it was essential to demonstrate that the surgical procedure itself did not exert any eVect on immune cells draining through the cannulated duct. Any surgical procedure can result in a short period of inXammation, plus there was the possibility of blood contamination, either of which could impact upon the interpretation of samples collected in the lymph draining. To address this, we characterised cells present in the draining lymph collected daily following surgery. In order to test for blood contamination, overnight lymph samples were collected daily from over 20 sheep following surgery, centrifuged (650 g for 5 min) and then examined under light microscopy for the presence of red blood cells. The majority of cannulated samples were free of blood contamination at all times post-surgery. On the rare occasion that blood was present in the lymph, this always disappeared by around day 4 or 5 post-cannulation. However, following subcutaneous injection of the vaccine into the outer rim of the nostril, a transient (1–2 days) presence of red blood cells was often observed, consistent with previous observations that red blood cells can occur in draining lymph following delivery of a vaccine or adjuvant [14,15].
Analysis of these parameters, and in particular the occasional observation of red blood cell contamination of the lymph up to day 5, led us to adopt the principle that all sheep should be left for a minimum of 6 days after surgery before any immunisations or measurement of immune parameters. During this period following surgery, the daily body temperature was measured to assess the general health status of the animals. The body temperature was found to be well within the normal range for sheep during this period conWrming that the animals were not adversely impacted by the surgical procedure (data not shown). Waiting this period reduces the possibility that the surgical procedure would interfere with immunological readouts. In a number of cannulations, the lymph Xow gradually slowed and eventually stopped. This appeared to be caused by the formation of clots in the cannulae leading to blockage. 2.4. Lymph cell phenotyping We next used Xow cytometry to deWne the phenotypes of cells normally found circulating through this eVerent lymphatic system. Six sheep were cannulated and, commencing 6 days after surgery, overnight cells were collected daily for 6 days. These cells were phenotyped by Xow cytometry using a panel of monoclonal antibodies. The monoclonal antibodies used were speciWc for the T-cell markers CD4 (clone 44–97), CD8 (38–65) and the T-cell receptor (86D), the B-cell marker CD45Rp220 (20.96) and for MHC class II (49.1). These antibodies are all of mouse origin and of either the IgG1 or IgG2a subclass. All originated at the Centre for Animal Biotechnology [16–18] except for 86D [19]. For lymph cell phenotyping, cells collected overnight in draining lymph were counted and resuspended at 4 £ 106/ mL in PBS/FCS (PBS plus 2% v/v foetal calf serum [JRH Biosciences, Melbourne. Australia] and 0.02% w/v sodium azide). Twenty-Wve microlitres/well were placed into round bottomed 96-well plates (Greiner) with either 25 L of hybridoma supernatant, or culture medium as a negative control. Following incubation at 4 °C for 15 min, plates were washed three times with PBS/FCS before addition of 50 L/well of the appropriate secondary antibody (either 1/1000 phycoerythrin-conjugated Goat anti-mouse IgG1, or 1/500 FITC-conjugated goat antimouse IgG2a diluted in PBS/FCS). After a further 15 min at 4 °C, plates were washed three times and cells resuspended in 150 L PBS/FCS for acquisition on a Becton– Dickinson FACSCalibur Xow cytometer. Two colour analysis was used to determine the cellular co-expression of CD45Rp220 and MHC-II. The proportion of cells present in lymph draining from the cannulated duct is shown in Table 1. The predominant cell type in the draining lymph in this model was T-cells which comprised 72% of the total cells, including 41% CD4+, 13% CD8+, and 9% T-cells. The T-cell population included over 22% naïve T-cells, which expressed the CD45Rp220 marker but lacked MHC-II. B-cells comprised
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Table 1 Phenotypes of cells in lymph draining the nasal region Cell population
Phenotypic markers
Percentage positive (Mean § SD)
T-cells T-helper cells T-cytotoxic cells T-cells
CD5+ CD4+ CD8+ T-cell receptor+ CD45Rp220+ MHC-II¡ CD45Rp220+ MHC-II+ CD11b+
71.7 § 3.6 40.8 § 3.2 12.9 § 3.8 9.3 § 1.4
Naïve T-cellsa B-cellsa B1 cells, monocytes, macrophages and granulocytesb
22.6 § 3.7 21.3 § 1.9 0.4 § 0.2
Data shown represent the averages of cell phenotypes from six cannulations, analysed daily for 6 days. Cells were stained with monoclonal antibodies, and the percentages of cells labelled with Xuorescent conjugates determined by FACS analysis. a CD45Rp220 is expressed in sheep only on B-cells and naïve T-cells [23]. B-cells, but not naïve T-cells also express MHC-II [17]. b Sheep CD11b is expressed by B1 cells, monocytes, macrophages and granulocytes [24].
21% of the total cells, while levels of CD11b expressing cells were similar to background, indicating an absence or extremely low frequency of monocytes, macrophages, and granulocytes. This further demonstrated the absence of blood contamination and localised inXammation. These Wgures are highly comparable with other studies analysing cell populations in diVerent lymphatic systems (reviewed in [20,21]). Thus, the data generated by our analysis of cells in the lymph eVerent to the retropharyngeal lymph node is typical for that reported for other sheep lymphatics. 2.5. Nasal delivery drains the cannulated lymphatics To directly assess whether nasally delivered preparations drain to the cannulated lymphatics we delivered 500 mg of patent blue violet dye in 1 ml PBS, into the nasal cavity. This was performed using a syringe-mounted 10 cm dog catheter (3.3 mm outer diameter, Arnolds, UK) blocked at the end with glue, and pierced laterally close to the blocked tip (eight holes). This devise was used to compensate for the considerable length of the sheep nose. The 10 cm catheter allows delivery of preparations deep into the nasal cavity, into a nasal region similar to that to which preparations would be delivered nasally into humans. Despite this approach, only a very small proportion of the patent blue violet ended up in the lymph. Using optical density at a wavelength of 620 nm to estimate dye concentration, we calculated that approximately 1 part per 10 million parts delivered, is recovered in the lymph. Blue colouring of the lymph started about 10 min following intra-nasal delivery (Fig. 2) and stopped within 2 h. This is in sharp contrast to previous unpublished observations in which patent blue violet (1% w/v) was injected subcutaneously into an area draining a cannulated prefemoral lymph node [14,15] and observed in collected lymph from about 5 min post-
Fig. 2. Time course of the amount of patent blue in the cannulated lymph following intra-nasal spray. A solution containing 500 mg of patent blue violet in 1 ml of PBS was sprayed into the nasal cavity of a cannulated sheep. Lymph was collected at regular time intervals and the volume was measured. The concentration of patent blue violet in lymph was assessed by measuring the optical density of the stained lymph (620 nm) and comparison with a standard curve. The total amount as shown on the graph was obtained for each time-point by multiplying the patent blue violet concentration by the collected volume.
injection but for a period of 24–48 h. Following injection, the dye leaks out of the site of injection over an extended period, while following intra-nasal spray there appeared to be no reservoir eVect and most of the delivered dye was cleared quite rapidly from the mucosal surface. Using a Wbre-optics bronchoscope we directly observed that the vast majority of the sprayed dye is channelled by mucus Xow into narrow streams which Xowed along the top, bottom and sides of the nasal turbinate and then neatly Xowed around the NALT completely avoiding them. These streams of material are directed into the oesophagus and are therefore swallowed by the animal (data not shown), explaining the extremely ineYcient uptake of material from the nasal cavity into the lymphatic system. 3. Application 3.1. Immune responses in eVerent lymph following intra-nasal vaccine delivery Following establishment of the cannulation model, we tested whether this procedure could be used to examine immune responses induced by nasal immunisations. As discussed above, nasally delivered vaccines have generally been found to be poorly eYcacious. Hence, for our proof of concept study using a model antigen, we primed two animals with two subcutaneous immunisations of 100 g recombinant protein adjuvanted in 1 mg of Quil A (1 ml volume), 3 weeks apart. These primed animals were boosted by intra-nasal delivery of 1 ml of PBS containing 1 mg of protein adjuvanted with 1 mg of Quil A using the same devise as for the delivery of patent blue violet (see above). The rationale for this was that boosting an immune response would require less immune stimulation than
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initiating an immune response and hence we would maximise our chances of observing an eVect following intranasal vaccine delivery, despite the very low uptake eYciency observed with patent blue violet. The immune response in eVerent lymph was measured daily through ex vivo proliferation. Lymph cells collected overnight were resuspended at 4 £ 106/mL in complete medium (Dulbecco’s ModiWed Eagles Medium plus 10% foetal calf serum (JRH Biosciences), supplemented with 100 U/mL penicillin, 100 g/mL streptomycin and 292 g/ mL glutamine (GibcoBRL, Grand Is., NY, USA) and 50 M -mercaptoethanol). Aliquots of 200 L were placed into six replicate wells of Falcon 96-well Xat bottom tissue culture plates (Becton–Dickinson Labware, Franklin Lakes) and immediately pulsed with 1 Ci/well of Methyl[3H]Thymidine ([3H]T, Pharmacia; TRK120) in 20 L of complete medium. After 24 h, the 96-well plates were stored frozen until samples were harvested onto EasyTab-C SelfAligning Filters (Packard, Groningen, Netherlands), using an Inotech Cell Harvester. After drying, Wlters were placed in a cartridge and 25 L of Microscint scintillation liquid (Packard) added to each well. The incorporation of [3H]T into DNA of dividing cells was measured by reading the glass Wbre Wlters on a Packard Topcount Microplate Scintillation Counter. Following surgery, but before vaccine delivery, there was a constant (background) level of proliferation (Fig. 3A). Five to six days following intra-nasal spray of the vaccine, we observed a small increase in the proliferative response of cells from the collected lymph. This weak response was also reXected in the lack of humoral response in lymph from the cannulated animals following intra-nasal delivery (Fig. 3B). The humoral response was measured using standard ELISA techniques to calculate the antigen-speciWc antibody titre in lymph at an ELISA OD of 0.5 as previously described [22]. The data showed that the antibody titre declined over the period lymph was collected. This may have been due to a natural decline in antibody levels occurring after immunisation or, perhaps more likely, was an artefact introduced by the cannulation. The constant collection of lymph containing antibodies in the absence of further antigenic stimulation is likely over time to deplete circulating antibodies. Thus, our immunisation results indicate that intra-nasal spray was ineVective at inducing signiWcant immunity to a model antigen using one particular adjuvant and delivery system. However, our dye experiments presented above had shown that intra-nasal spray delivery did result in delivered substances entering the draining lymphatic system. Since the induced immune response was only marginal following intra-nasal spray we wanted to conWrm that the animals were able to induce high immune responses in the collected lymph provided suYcient vaccine could reach the cannulated nodes. Using a 29 gauge needle, we injected the vaccine subcutaneously into the outer rim of the nostril, an area we knew (from observing colouring of ducts during surgery following injection into that area) drained to the cannu-
Fig. 3. Immune responses in lymph from the tracheal lymph trunk following nasal vaccine delivery. Following intra-nasal spray of the vaccine, exvivo proliferative (A) and antigen speciWc humoral (B) responses were measured in lymph. The ex-vivo proliferative (C) and antigen speciWc humoral (D) responses to injection into the outer rim of the nostril were measured. The data of two diVerent sheep (䊊 and 䊉) are presented.
lated duct. The injected vaccine was composed of 100 g of protein adjuvanted with 1 mg of Quil A in 200 l of PBS. Following this vaccination, we did observe an increased proliferative response (Fig. 3C) as well as a dramatic increase in the antibody titre (Fig. 3D) in the collected lymph. Thus, provided the vaccine reaches the node in suYcient quantities, substantial local immune responses can be mounted in eVerent lymph draining the site of vaccine delivery. 4. Concluding remarks An interesting feature of this lymphatic cannulation model is that collected cells and lymph are removed from the animal, hence mainly local immune responses are measured (i.e., immune responses originating in one or a limited number of local lymph nodes). This characteristic of our model results in very sharp changes in immune responses following vaccination compared to measuring immune responses in serum for example. Indeed, when measuring immune responses in blood or spleen, an accumulated response was measured over a period, as immune cells and mediators gradually accumulate and re-circulate in the body. In contrast, since all lymph collected is removed from the animal mainly de novo generated responses are measured in the collected lymph in real-time, as they occur.
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This advantage allows precise kinetics of the immune response to be assessed, as is illustrated in our experiment in which following injection of the vaccine, proliferative responses appear 1 day before the antibody response is observed (Figs. 3C and D). This is expected, as cells become activated and start proliferating within the local lymph node to suYcient numbers before they produce large amounts of antibodies within the local lymph node. Some of these proliferating cells would be transported to the rest of the body through eVerent lymph and these are the cells we collect and assay in our ex vivo proliferation assay. In conclusion, we have developed a methodology to investigate in real time the eVect of nasal immunisation on the local immune response. Using our model vaccine system, the uptake of material into the lymphatic system via the nasal mucosa was ineYcient, as very poor immune responses were generated following nasal spray in comparison with vaccine injected in the same proximity. This cannulation model therefore provides an ideal experimental model for the evaluation of strategies aimed at increasing either the uptake of substances delivered via the nasal mucosa or the immune responsiveness induced by such vaccines. Examples of such approaches include excipients aimed at increasing uptake, such as chitosan, mechanical devises that deliver droplets of sizes most suitable for reaching the NALT and novel adjuvants, which may induce improved immune responses. Thus, both the adjuvanticity of additives as well as their eVects on uptake through the NALT can be separately assessed. Acknowledgment This work was supported by an Australian Research Council Linkage grant. References [1] P. Brandtzaeg, I.N. Farstad, G. Haraldsen, Immunol. Today 20 (1999) 267–277.
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[2] P. Brandtzaeg, E.S. Baekkevold, I.N. Farstad, F.L. Jahnsen, F.E. Johansen, E.M. Nilsen, T. Yamanaka, Immunol. Today 20 (1999) 141–151. [3] R.B. Belshe, K.L. Nichol, S.B. Black, H. ShineWeld, J. Cordova, R. Walker, C. Hessel, I. Cho, P.M. Mendelman, Clin. Infect. Dis. 39 (2004) 920–927. [4] Z.Q. Xiang, S. Pasquini, H.C. Ertl, J. Immunol. 162 (1999) 6716–6723. [5] H.Y. Wu, M.W. Russell, Vaccine 16 (1998) 286–292. [6] E.L. Johansson, L. Wassen, J. Holmgren, M. Jertborn, A. Rudin, Infect. Immun. 69 (2001) 7481–7486. [7] J.E. Eyles, I.D. Spiers, E.D. Williamson, H.O. Alpar, Vaccine 16 (1998) 2000–2009. [8] A. Visweswaraiah, L.A. Novotny, E.J. Hjemdahl-Monsen, L.O. Bakaletz, Y. Thanavala, Vaccine 20 (2002) 3209–3220. [9] L. Janakova, H. Bakke, I.L. Haugen, A.K. Berstad, E.A. Hoiby, I.S. Aaberge, B. Haneberg, Infect. Immun. 70 (2002) 5479–5484. [10] W. Chen, M.R. Alley, B.W. Manktelow, J. Comp. Pathol. 101 (1989) 327–340. [11] A.C. Stanley, J.F. Huntley, M. JeVrey, D. Buxton, J. Comp. Pathol. 125 (2001) 262–270. [12] L. Illum, I. Jabbal-Gill, M. HinchcliVe, A.N. Fisher, S.S. Davis, Adv. Drug Deliv. Rev. 51 (2001) 81–96. [13] W.R. Hein, P.J. Griebel, Nat. Rev. Immunol. 3 (2003) 79–84. [14] R.G. Windon, P.J. Chaplin, L. Beezum, A. Coulter, R. Cahill, W. Kimpton, D. Drane, M. Pearse, A. Sjolander, J.M. Tennent, J.Y. Scheerlinck, Vaccine 19 (2000) 572–578. [15] R.G. Windon, P.J. Chaplin, P. McWaters, M. Tavarnesi, M. Tzatzaris, W.G. Kimpton, R.N. Cahill, L. Beezum, A. Coulter, D. Drane, A. Sjolander, M. Pearse, J.P. Scheerlinck, J.M. Tennent, Vaccine 20 (2001) 490–497. [16] J.F. Maddox, C.R. Mackay, M.R. Brandon, Immunology 55 (1985) 739–748. [17] N.K. Puri, C.R. Mackay, M.R. Brandon, Immunology 56 (1985) 725–733. [18] C.R. Mackay, J.F. Maddox, M.R. Brandon, Cell Immunol. 110 (1987) 46–55. [19] C.R. Mackay, M.F. Beya, P. Matzinger, Eur. J. Immunol. 19 (1989) 1477–1483. [20] D.M. Haig, J. Hopkins, H.R. Miller, Immunology 96 (1999) 155–163. [21] A.J. Young, Semin. Immunol. 11 (1999) 73–83. [22] J.P. Scheerlinck, J. Karlis, T.E. Tjelle, P.J. Presidente, I. Mathiesen, S.E. Newton, Vaccine 22 (2004) 1820–1825. [23] C.R. Mackay, W.L. Marston, L. Dudler, J. Exp. Med. 171 (1990) 801–817. [24] V.K. Gupta, I. McConnell, M. Pepin, W.C. Davis, R.G. Dalziel, J. Hopkins, J. Comp. Pathol. 112 (1995) 339–349.
A sheep cannulation model for evaluation of nasal vaccine delivery Hung-Hsun Yen, Jean-Pierre Y. Scheerlinck, Susie Gekas, Phil Sutton ¤ Centre for Animal Biotechnology, University of Melbourne, Parkville, Vic. 3010, Australia Accepted 28 September 2005
Abstract We have developed and validated a novel model to investigate the eYcacy of nasal vaccine delivery. Based on lymphatic cannulation of the tracheal lymph trunk of sheep, the model allows collection of lymph draining from the Nasal Associated Lymphoid Tissue. The model is suitable for determining both the amount of material that is absorbed into the lymphatic system, following intra-nasal delivery and the immune response that occurs following vaccination into the nasal area. The cell populations that track in this duct were phenotyped and found to be similar to those previously reported to be present in eVerent lymph draining from peripheral lymph nodes. Following intra-nasal spray, we demonstrated that the amount of material recovered in draining lymph is only a very small fraction of the total delivered. Nevertheless, intra-nasal spraying of a vaccine could activate local immune cells. The method described will be invaluable for optimising intra-nasal delivery systems by allowing a separate optimisation of vaccine uptake and immune responses induction. 2005 Elsevier Inc. All rights reserved. Keywords: Sheep; Lymphatic cannulation; Nasal delivery
1. Introduction From early times to modern day, the most common mode for vaccine delivery has been via injection. This, however, has some limitations including occupational risk associated with transmission of infections through needle-stick injuries or via the repeated use of needles as sometime occurs in poorer countries. There is also a perception that some individuals may avoid immunisations due to a fear of needles, which could potentially impact on vaccine eYcacy at both the individual and cohort level. However, perhaps the key argument for exploring alternative routes of vaccine delivery is our increasing realisation of the compartmentalisation of the immune system. Immune responses at mucosal sites are best induced by exposure of antigen at mucosal sites. In fact, it has been demonstrated that mucosal immune cells induced at one site can selectively home into distinct and distant mucosal sites [1,2]. A key implication of this is that while injected vaccines are typically very *
Corresponding author. Fax: +61 3 9347 4083. E-mail address: [email protected] (P. Sutton).
1046-2023/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2005.09.011
eYcient at inducing systemic immunity, they are less capable at producing an eVective immune response at mucosal surfaces. Thus, mucosal delivery may be important when aiming to induce a response at a particular mucosal surface. Pathogens generally infect a host via one of two routes, either via a break in the skin or via a mucosal surface. The reality is that the majority of pathogens of signiWcance infect via a mucosal surface, be that intestinal (i.e., cholera), respiratory (tuberculosis, inXuenza) or reproductive (HIV). Development of techniques to improve immunity at mucosal surfaces would therefore potentially be highly valuable in inducing protection against such pathogens. While it is now generally accepted that induction of eVective immunity at mucosal surfaces is more eYciently induced by mucosal vaccines than by injected immunisations, eVective mucosally delivered vaccines are extremely rare. The immune systems of mucosal surfaces are essentially designed to be generally immunologically non-responsive to antigenic stimulation. In the intestine, the bulk of antigens present are from ingested food or commensal and useful bacteria against which reaction would be detrimental to the host. Large quantities of antigens, bacteria, fungal spores etc., are
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also constantly inhaled into the respiratory system, where, similarly, development of a strong inXammatory response may be harmful. In the vagina, reaction to foreign proteins such as found on sperm may interfere with reproduction. The immune regulation mechanisms that have evolved to prevent these events mean that it is not simple to induce an eVective, protective immune response via the mucosal route. The best example of a mucosal vaccine is the Sabin polio vaccine, which is a live-attenuated virus, delivered via its natural route of infection. Thus, in this case, the vaccine is eVective because it takes advantage of mechanisms developed by the pathogen itself to overcome the tolerance present in the gut. If eVective mucosal immunisations could be achieved, there would be signiWcant potential for improving protection against mucosal pathogens. Such approaches need to address two key points: (i) how to overcome the natural immune regulation processes present at mucosal sites and (ii) to ensure that potentially harmful severe adverse reactions are not induced. The delivery of vaccines via the nasal cavity is one approach which has been investigated for many decades, but more recently has attracted increasing attention. One such example is the recently released Flumist vaccine produced by MedImmune, which comprises a cold-adapted live-attenuated virus sprayed intra-nasally as a mist [3]. As a rule, eVective mucosal vaccines have tended to be live attenuated organisms, where a specialised infectious organism is used to circumvent the mucosal defences and induce an immune response. Much less success has been achieved with subunit or killed vaccines. Indeed, the nasal mucosa has evolved many mechanisms to exclude substances entering via the nostril from traversing its surface. The nasal mucosal surface is covered with mucus to limit access to the epithelial surface. In addition, the nasal epithelial cells are covered with cilia and joined by tight junctions, which prevent passage between them. Mucociliary action (beating of the cilia, which causes the mucus to Xow) directs the vast bulk of inhaled substances down the oesophagus into the intestine. The immune systems of various mucosal surfaces appear to be linked, such that immune cells induced at one mucosal site drain via the lymphatics into the blood circulation and migrate to distant mucosal regions. It has been shown previously that nasal immunisations may be of use for inducing immune responses at other mucosal sites [4–6]. The spread of these mucosal immune cells between mucosal sites has been clearly demonstrated in murine models, which have been extremely useful at demonstrating the presence of the common mucosal immune system. However, murine models only provide a snapshot within a single animal and do not readily allow for real-time evaluations of the kinetics of these processes, nor do they allow to directly measure material transfer across mucosal barriers. Another limitation of murine models for nasal vaccine delivery is that the majority of studies use a delivery volume that is far from representative of that which would translate to humans. It has been calculated,
based on comparisons of nasal cavity volumes alone, that substances delivered intra-nasally in mice should be delivered in a Wnal volume of 3 L, to be comparative with humans. However, most mouse vaccination studies frequently use 30 L or more [7]. Due to the small size of mice, intra-nasal delivery of these larger volumes typically results in exposure of the delivered substances at multiple sites including the intestinal tract and the lungs [8].This latter situation makes true interpretation of nasally delivered vaccines diYcult as it is unclear at which site any observed eVect is induced. Anaesthesia can also signiWcantly impact on the eYcacy of nasal vaccinations in mice [9]. The Nasal Associated Lymphoid Tissue (NALT) of sheep, unlike rodents, has many similarities to that found in humans. Sheep have at least three diVerent lymphoid nodules in the nasal-pharynx region, speciWcally pharyngeal tonsils, lymphoid tissue in the mid-nasopharynx, and also at the opening of the auditory tube [10]. These nodules are highly structured and organised with distinct T- and B-cell follicles possessing follicle associated epithelium with characteristic M-cells [11]. These NALTs appear to be positioned predominantly for sampling antigens present in small, inhaled particles, which traverse the turbinate structures and reach the nasopharynx region. Most liquids sprayed nasally will tend to encounter the turbinates, where they are exposed to mucociliary action and channelled away from the NALT. There are thus many challenges to overcome for eVective nasal vaccination, but there is clear evidence that with the correct technology and approach this is possible. For example, it has been shown that the addition of chitosan, an excipient which opens tight junctions and inhibits mucociliary action can assist the induction of eVective immunity, following nasal vaccination [12]. We have developed a novel large animal surgical procedure, which is intended to assist with the development of nasally delivered vaccines. This procedure uses sheep, recognised as a good animal model for a range of human diseases [13], providing several advantages. In addition to the similar NALT structures to humans mentioned above, their size allows surgical cannulation of lymphatic ducts to be performed, which permits draining immune responses to be measured continuously from a single animal. We have previously used sheep cannulation models of the prefemoral lymphatic duct to examine the induction of immune responses following subcutaneous vaccine delivery [14,15] and have now extended this approach to the nasal region. This model has applications for the evaluation of new technologies, delivery systems or adjuvants, in particular for kinetic studies involving vaccine uptake, induction of local immunity and the dissemination of this immunity to distant mucosal sites. 2. Description and validation of method 2.1. Animals The sheep used in these studies were Merino ewes, which were initially housed in pens within the School of
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Veterinary Science animal facility, The University of Melbourne, Parkville. Following surgery, the animals were maintained in metabolism cages to prevent cannula removal. Sheep were fed lucerne chaV mixed with commercial pellets and allowed access to water ad libitum. All experimental procedures were approved by the University of Melbourne Animal Experimentation Ethics Committee. 2.2. Lymphatic duct cannulation: surgical procedure To prevent regurgitation during anaesthesia, sheep were fasted 24 h prior to surgery. Sheep were initially anaesthetised by an intra-venous injection of thiopentone (10 mg/ kg) into the cephalic vein in the foreleg, thus avoiding possible haematoma formation in the neck area often seen when injecting into the jugular vein. The animals were then intubated through the mouth with a balloon catheter, which was attached to an open circuit anaesthetic apparatus. Anaesthesia was maintained with 1.5–2% halothane in oxygen and monitored regularly by checking for the absence of an eye reXex. Sheep were positioned on their backs and a 5 cm incision made on the median plane of the ventral neck to expose the trachea and the sternothyreohoideus (with/without sternohyoid) muscles. The incision was made 8–10 cm posterior and ventral to the thyroid cartilage. Using blunt dissection, the common carotid artery distal to the heart after the bicarotid trunk was identiWed through the interval between the sternocephalicus and sternothyreohoideus muscles. The tracheal lymph trunks (including lateral and medial ducts) running along each of the left and right common carotid arteries were identiWed and localised (Fig. 1A). In initial experiments, 200 L of 1% (w/v) patent blue dye in phosphate buVered saline (PBS) was injected into the outer rim of each of the nostrils to facilitate the localisation of the ducts (Fig. 1A). The migration of the dye was very fast with colouration appearing in the draining lymphatic ducts within 5 min. We have found that the number of draining ducts vary between sheep, but in most cases consist of two ducts identiWable on each side of the neck. When more than one lymphatic duct was identiWed alongside the common carotid arteries, only the largest lymphatic was cannulated and the other lymphatic was tied oV using suture. Heparinised polyvinyl tubing with an inner diameter of 0.4 mm and an outer diameter of 0.8 mm (CBAS-coated, Carmeda AB, Stockholm, Sweden) was used as cannula tubing. While heparinisation of the cannula is expensive, it substantially prevents coagulation of lymph in the cannula and therefore increases the success rate of the procedure, plus extends the period during which lymph can be collected. Cannulation was performed at an exposed site along the lymphatic duct, at a location before the ducts joined the aVerent (caudal deep cervical) lymph nodes. Using nylon suture (1 metric, 5/0 USP) all identiWed ducts were ligated at sites as proximal as possible to the aVerent lymph nodes (Fig. 1B). Ligation resulted in swelling of the ducts (Fig. 1B) at the upstream end. To facilitate this ligation, we used a
Fig. 1. Illustration of surgical procedure to cannulate the tracheal lymph trunks. (A) The lymphatic duct located along the common carotid artery is visible as a 2–3 mm translucent line. (B) The swollen duct is exposed (following downstream constriction using suture ). The long ends of the suture are attached to the drape to provide tension allowing insertion of the cannula. A home-made ligature instrument composes of a microbiological loop-holder holding the sharp end of a curved surgical needle . Two loose overhand knots are placed around the duct and will serve to secure the inserted cannula. (C) Fine ocular scissors are positioned to allow incising of the exposed duct , to a depth of half its diameter. (D) An incision in the duct allows the insertion of the cannula into the lymphatic duct. Two loose overhand knots are tightened to hold the cannula in place.
home-made ligature instrument (Fig. 1B) composed of a curved surgical needle inserted into the end of a microbiological loop-holder. It is suggested that after the ligation, suYcient length of suture be reserved temporarily at both ends, to create a tension on the duct thereby facilitating the insertion of the cannula into the lymphatic duct (Fig. 1B). Later these same ends are also used for the formation of another square knot securing the inserted cannula. The largest and/or most exposed duct was chosen for cannulation. Connective and adipose tissue along a 1.5 cm length of this duct was carefully removed using blunt forceps and cotton tips. This is a delicate and critical procedure as the duct collapses immediately, following incision and a very clean Weld is required to be able to insert the cannula. Two sutures (Fig. 1B) were placed around the duct at the distal end to the aVerent lymph node and about 1 cm from the Wrst ligation, each made with a loose overhand knot. A slight tension was applied on the duct, by attaching the ends of the Wrst ligation to the drape, in line with the orientation of the duct. This facilitates insertion of the cannula into the duct. Using delicate scissors (Aesculap, FD100R), a transverse incision was made on the duct to a depth of approaching half the diameter of the duct (Fig. 1C). This incision was
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located between the Wrst ligation and the two loose ligatures around the duct (Fig. 1C). Using a syringe Wtted with a 22 gauge needle, the cannula was then Wlled with saline containing 50 U/mL of heparin. This facilitates the Xow of lymph following insertion of the cannula into the duct. The cannula was then cut at a 45 ° angle and inserted into the small incision made previously in the lymphatic duct. Following insertion, the loose overhand knots were tightened and the needle/syringe removed from the cannula (Fig. 1D). Typically, immediately following removal, lymph commenced to Xow, particularly when the external end of the cannula was lowered below the point of surgery. After the insertion of the cannula into the duct, the cannula was secured to the duct by tightening these two loose ligatures and Wxing them Wrmly with a second overhand knot. Using the reserved ends of the Wrst ligation, the cannula was secured one more time. Using an autopsy needle, the cannula was threaded through the skin to the outer side of the initial incision and secured with suture at the point where it exits the skin and again with simple interrupted sutures about 5 cm from the exit point. Synthetic, rapidly setting glue was used to secure the cannula to the suture threaded through the skin, which prevented it from being pulled out through movement of the sheep. The end of the cannula was wiped with 70% ethanol and placed into sterile plastic bottles containing 2000 IU heparin (Pharmacia, Bentley, Australia), attached to the neck using an elastic bandage (SurgiWx size 6, Beiersdforf Australia, North Ride, Australia). 2.3. Post-surgical period As the main application of the above cannulation method is to evaluate nasally delivered vaccines, it was essential to demonstrate that the surgical procedure itself did not exert any eVect on immune cells draining through the cannulated duct. Any surgical procedure can result in a short period of inXammation, plus there was the possibility of blood contamination, either of which could impact upon the interpretation of samples collected in the lymph draining. To address this, we characterised cells present in the draining lymph collected daily following surgery. In order to test for blood contamination, overnight lymph samples were collected daily from over 20 sheep following surgery, centrifuged (650 g for 5 min) and then examined under light microscopy for the presence of red blood cells. The majority of cannulated samples were free of blood contamination at all times post-surgery. On the rare occasion that blood was present in the lymph, this always disappeared by around day 4 or 5 post-cannulation. However, following subcutaneous injection of the vaccine into the outer rim of the nostril, a transient (1–2 days) presence of red blood cells was often observed, consistent with previous observations that red blood cells can occur in draining lymph following delivery of a vaccine or adjuvant [14,15].
Analysis of these parameters, and in particular the occasional observation of red blood cell contamination of the lymph up to day 5, led us to adopt the principle that all sheep should be left for a minimum of 6 days after surgery before any immunisations or measurement of immune parameters. During this period following surgery, the daily body temperature was measured to assess the general health status of the animals. The body temperature was found to be well within the normal range for sheep during this period conWrming that the animals were not adversely impacted by the surgical procedure (data not shown). Waiting this period reduces the possibility that the surgical procedure would interfere with immunological readouts. In a number of cannulations, the lymph Xow gradually slowed and eventually stopped. This appeared to be caused by the formation of clots in the cannulae leading to blockage. 2.4. Lymph cell phenotyping We next used Xow cytometry to deWne the phenotypes of cells normally found circulating through this eVerent lymphatic system. Six sheep were cannulated and, commencing 6 days after surgery, overnight cells were collected daily for 6 days. These cells were phenotyped by Xow cytometry using a panel of monoclonal antibodies. The monoclonal antibodies used were speciWc for the T-cell markers CD4 (clone 44–97), CD8 (38–65) and the T-cell receptor (86D), the B-cell marker CD45Rp220 (20.96) and for MHC class II (49.1). These antibodies are all of mouse origin and of either the IgG1 or IgG2a subclass. All originated at the Centre for Animal Biotechnology [16–18] except for 86D [19]. For lymph cell phenotyping, cells collected overnight in draining lymph were counted and resuspended at 4 £ 106/ mL in PBS/FCS (PBS plus 2% v/v foetal calf serum [JRH Biosciences, Melbourne. Australia] and 0.02% w/v sodium azide). Twenty-Wve microlitres/well were placed into round bottomed 96-well plates (Greiner) with either 25 L of hybridoma supernatant, or culture medium as a negative control. Following incubation at 4 °C for 15 min, plates were washed three times with PBS/FCS before addition of 50 L/well of the appropriate secondary antibody (either 1/1000 phycoerythrin-conjugated Goat anti-mouse IgG1, or 1/500 FITC-conjugated goat antimouse IgG2a diluted in PBS/FCS). After a further 15 min at 4 °C, plates were washed three times and cells resuspended in 150 L PBS/FCS for acquisition on a Becton– Dickinson FACSCalibur Xow cytometer. Two colour analysis was used to determine the cellular co-expression of CD45Rp220 and MHC-II. The proportion of cells present in lymph draining from the cannulated duct is shown in Table 1. The predominant cell type in the draining lymph in this model was T-cells which comprised 72% of the total cells, including 41% CD4+, 13% CD8+, and 9% T-cells. The T-cell population included over 22% naïve T-cells, which expressed the CD45Rp220 marker but lacked MHC-II. B-cells comprised
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Table 1 Phenotypes of cells in lymph draining the nasal region Cell population
Phenotypic markers
Percentage positive (Mean § SD)
T-cells T-helper cells T-cytotoxic cells T-cells
CD5+ CD4+ CD8+ T-cell receptor+ CD45Rp220+ MHC-II¡ CD45Rp220+ MHC-II+ CD11b+
71.7 § 3.6 40.8 § 3.2 12.9 § 3.8 9.3 § 1.4
Naïve T-cellsa B-cellsa B1 cells, monocytes, macrophages and granulocytesb
22.6 § 3.7 21.3 § 1.9 0.4 § 0.2
Data shown represent the averages of cell phenotypes from six cannulations, analysed daily for 6 days. Cells were stained with monoclonal antibodies, and the percentages of cells labelled with Xuorescent conjugates determined by FACS analysis. a CD45Rp220 is expressed in sheep only on B-cells and naïve T-cells [23]. B-cells, but not naïve T-cells also express MHC-II [17]. b Sheep CD11b is expressed by B1 cells, monocytes, macrophages and granulocytes [24].
21% of the total cells, while levels of CD11b expressing cells were similar to background, indicating an absence or extremely low frequency of monocytes, macrophages, and granulocytes. This further demonstrated the absence of blood contamination and localised inXammation. These Wgures are highly comparable with other studies analysing cell populations in diVerent lymphatic systems (reviewed in [20,21]). Thus, the data generated by our analysis of cells in the lymph eVerent to the retropharyngeal lymph node is typical for that reported for other sheep lymphatics. 2.5. Nasal delivery drains the cannulated lymphatics To directly assess whether nasally delivered preparations drain to the cannulated lymphatics we delivered 500 mg of patent blue violet dye in 1 ml PBS, into the nasal cavity. This was performed using a syringe-mounted 10 cm dog catheter (3.3 mm outer diameter, Arnolds, UK) blocked at the end with glue, and pierced laterally close to the blocked tip (eight holes). This devise was used to compensate for the considerable length of the sheep nose. The 10 cm catheter allows delivery of preparations deep into the nasal cavity, into a nasal region similar to that to which preparations would be delivered nasally into humans. Despite this approach, only a very small proportion of the patent blue violet ended up in the lymph. Using optical density at a wavelength of 620 nm to estimate dye concentration, we calculated that approximately 1 part per 10 million parts delivered, is recovered in the lymph. Blue colouring of the lymph started about 10 min following intra-nasal delivery (Fig. 2) and stopped within 2 h. This is in sharp contrast to previous unpublished observations in which patent blue violet (1% w/v) was injected subcutaneously into an area draining a cannulated prefemoral lymph node [14,15] and observed in collected lymph from about 5 min post-
Fig. 2. Time course of the amount of patent blue in the cannulated lymph following intra-nasal spray. A solution containing 500 mg of patent blue violet in 1 ml of PBS was sprayed into the nasal cavity of a cannulated sheep. Lymph was collected at regular time intervals and the volume was measured. The concentration of patent blue violet in lymph was assessed by measuring the optical density of the stained lymph (620 nm) and comparison with a standard curve. The total amount as shown on the graph was obtained for each time-point by multiplying the patent blue violet concentration by the collected volume.
injection but for a period of 24–48 h. Following injection, the dye leaks out of the site of injection over an extended period, while following intra-nasal spray there appeared to be no reservoir eVect and most of the delivered dye was cleared quite rapidly from the mucosal surface. Using a Wbre-optics bronchoscope we directly observed that the vast majority of the sprayed dye is channelled by mucus Xow into narrow streams which Xowed along the top, bottom and sides of the nasal turbinate and then neatly Xowed around the NALT completely avoiding them. These streams of material are directed into the oesophagus and are therefore swallowed by the animal (data not shown), explaining the extremely ineYcient uptake of material from the nasal cavity into the lymphatic system. 3. Application 3.1. Immune responses in eVerent lymph following intra-nasal vaccine delivery Following establishment of the cannulation model, we tested whether this procedure could be used to examine immune responses induced by nasal immunisations. As discussed above, nasally delivered vaccines have generally been found to be poorly eYcacious. Hence, for our proof of concept study using a model antigen, we primed two animals with two subcutaneous immunisations of 100 g recombinant protein adjuvanted in 1 mg of Quil A (1 ml volume), 3 weeks apart. These primed animals were boosted by intra-nasal delivery of 1 ml of PBS containing 1 mg of protein adjuvanted with 1 mg of Quil A using the same devise as for the delivery of patent blue violet (see above). The rationale for this was that boosting an immune response would require less immune stimulation than
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initiating an immune response and hence we would maximise our chances of observing an eVect following intranasal vaccine delivery, despite the very low uptake eYciency observed with patent blue violet. The immune response in eVerent lymph was measured daily through ex vivo proliferation. Lymph cells collected overnight were resuspended at 4 £ 106/mL in complete medium (Dulbecco’s ModiWed Eagles Medium plus 10% foetal calf serum (JRH Biosciences), supplemented with 100 U/mL penicillin, 100 g/mL streptomycin and 292 g/ mL glutamine (GibcoBRL, Grand Is., NY, USA) and 50 M -mercaptoethanol). Aliquots of 200 L were placed into six replicate wells of Falcon 96-well Xat bottom tissue culture plates (Becton–Dickinson Labware, Franklin Lakes) and immediately pulsed with 1 Ci/well of Methyl[3H]Thymidine ([3H]T, Pharmacia; TRK120) in 20 L of complete medium. After 24 h, the 96-well plates were stored frozen until samples were harvested onto EasyTab-C SelfAligning Filters (Packard, Groningen, Netherlands), using an Inotech Cell Harvester. After drying, Wlters were placed in a cartridge and 25 L of Microscint scintillation liquid (Packard) added to each well. The incorporation of [3H]T into DNA of dividing cells was measured by reading the glass Wbre Wlters on a Packard Topcount Microplate Scintillation Counter. Following surgery, but before vaccine delivery, there was a constant (background) level of proliferation (Fig. 3A). Five to six days following intra-nasal spray of the vaccine, we observed a small increase in the proliferative response of cells from the collected lymph. This weak response was also reXected in the lack of humoral response in lymph from the cannulated animals following intra-nasal delivery (Fig. 3B). The humoral response was measured using standard ELISA techniques to calculate the antigen-speciWc antibody titre in lymph at an ELISA OD of 0.5 as previously described [22]. The data showed that the antibody titre declined over the period lymph was collected. This may have been due to a natural decline in antibody levels occurring after immunisation or, perhaps more likely, was an artefact introduced by the cannulation. The constant collection of lymph containing antibodies in the absence of further antigenic stimulation is likely over time to deplete circulating antibodies. Thus, our immunisation results indicate that intra-nasal spray was ineVective at inducing signiWcant immunity to a model antigen using one particular adjuvant and delivery system. However, our dye experiments presented above had shown that intra-nasal spray delivery did result in delivered substances entering the draining lymphatic system. Since the induced immune response was only marginal following intra-nasal spray we wanted to conWrm that the animals were able to induce high immune responses in the collected lymph provided suYcient vaccine could reach the cannulated nodes. Using a 29 gauge needle, we injected the vaccine subcutaneously into the outer rim of the nostril, an area we knew (from observing colouring of ducts during surgery following injection into that area) drained to the cannu-
Fig. 3. Immune responses in lymph from the tracheal lymph trunk following nasal vaccine delivery. Following intra-nasal spray of the vaccine, exvivo proliferative (A) and antigen speciWc humoral (B) responses were measured in lymph. The ex-vivo proliferative (C) and antigen speciWc humoral (D) responses to injection into the outer rim of the nostril were measured. The data of two diVerent sheep (䊊 and 䊉) are presented.
lated duct. The injected vaccine was composed of 100 g of protein adjuvanted with 1 mg of Quil A in 200 l of PBS. Following this vaccination, we did observe an increased proliferative response (Fig. 3C) as well as a dramatic increase in the antibody titre (Fig. 3D) in the collected lymph. Thus, provided the vaccine reaches the node in suYcient quantities, substantial local immune responses can be mounted in eVerent lymph draining the site of vaccine delivery. 4. Concluding remarks An interesting feature of this lymphatic cannulation model is that collected cells and lymph are removed from the animal, hence mainly local immune responses are measured (i.e., immune responses originating in one or a limited number of local lymph nodes). This characteristic of our model results in very sharp changes in immune responses following vaccination compared to measuring immune responses in serum for example. Indeed, when measuring immune responses in blood or spleen, an accumulated response was measured over a period, as immune cells and mediators gradually accumulate and re-circulate in the body. In contrast, since all lymph collected is removed from the animal mainly de novo generated responses are measured in the collected lymph in real-time, as they occur.
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This advantage allows precise kinetics of the immune response to be assessed, as is illustrated in our experiment in which following injection of the vaccine, proliferative responses appear 1 day before the antibody response is observed (Figs. 3C and D). This is expected, as cells become activated and start proliferating within the local lymph node to suYcient numbers before they produce large amounts of antibodies within the local lymph node. Some of these proliferating cells would be transported to the rest of the body through eVerent lymph and these are the cells we collect and assay in our ex vivo proliferation assay. In conclusion, we have developed a methodology to investigate in real time the eVect of nasal immunisation on the local immune response. Using our model vaccine system, the uptake of material into the lymphatic system via the nasal mucosa was ineYcient, as very poor immune responses were generated following nasal spray in comparison with vaccine injected in the same proximity. This cannulation model therefore provides an ideal experimental model for the evaluation of strategies aimed at increasing either the uptake of substances delivered via the nasal mucosa or the immune responsiveness induced by such vaccines. Examples of such approaches include excipients aimed at increasing uptake, such as chitosan, mechanical devises that deliver droplets of sizes most suitable for reaching the NALT and novel adjuvants, which may induce improved immune responses. Thus, both the adjuvanticity of additives as well as their eVects on uptake through the NALT can be separately assessed. Acknowledgment This work was supported by an Australian Research Council Linkage grant. References [1] P. Brandtzaeg, I.N. Farstad, G. Haraldsen, Immunol. Today 20 (1999) 267–277.
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