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ssessment of different formulations of oral Mycobacterium bovis Bacillealmette-Guérin (BCG) vaccine in rodent models for immunogenicity androtection against aerosol challenge with M. bovis
imon Clarka, Martin L. Crossb, Alan Smithc, Pinar Courtd, Julia Viponda, Allan Nadiand,. Glyn Hewinsond, Hannah K. Batchelorc, Yvonne Perriec, Ann Williamsa,rank E. Aldwellb, Mark A. Chambersd,∗
Health Protection Agency, Porton Down, Salisbury SP4 0JG, United KingdomCentre for Innovation, University of Otago, PO Box 56, Dunedin, New ZealandSchool of Life and Heath Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, United KingdomTB Research Group, Department of Statutory and Exotic Bacterial Diseases, Veterinary Laboratories Agency Weybridge, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom
r t i c l e i n f o
rticle history:eceived 14 July 2008eceived in revised form 19 August 2008ccepted 19 August 2008
eywords:CGuberculosisaccinationralipid
a b s t r a c t
Bovine tuberculosis (bTB) caused by infection with Mycobacterium bovis is causing considerable economicloss to farmers and Government in the United Kingdom as its incidence is increasing. Efforts to controlbTB in the UK are hampered by the infection in Eurasian badgers (Meles meles) that represent a wildlifereservoir and source of recurrent M. bovis exposure to cattle. Vaccination of badgers with the human TBvaccine, M. bovis Bacille Calmette-Guérin (BCG), in oral bait represents a possible disease control tool andholds the best prospect for reaching badger populations over a wide geographical area. Using mouse andguinea pig models, we evaluated the immunogenicity and protective efficacy, respectively, of candidatebadger oral vaccines based on formulation of BCG in lipid matrix, alginate beads, or a novel microcapsularhybrid of both lipid and alginate. Two different oral doses of BCG were evaluated in each formulation fortheir protective efficacy in guinea pigs, while a single dose was evaluated in mice. In mice, significantimmune responses (based on lymphocyte proliferation and expression of IFN-�) were only seen with thelipid matrix and the lipid in alginate microcapsular formulation, corresponding to the isolation of viableBCG from alimentary tract lymph nodes. In guinea pigs, only BCG formulated in lipid matrix conferredprotection to the spleen and lungs following aerosol route challenge with M. bovis. Protection was seen
with delivery doses in the range 106–107 CFU, although this was more consistent in the spleen at the higherdose. No protection in terms of organ CFU was seen with BCG administered in alginate beads or in lipid inalginate microcapsules, although 107 in the latter formulation conferred protection in terms of increasingbody weight after challenge and a smaller lung to body weight ratio at necropsy. These results highlightthe potential for lipid, rather than alginate, -based vaccine formulations as suitable delivery vehicles for
gers.
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. Introduction
Bovine tuberculosis (bTB) caused by infection with Mycobac-erium bovis adversely affects animal health and welfare. Between996 and 2006, there was an estimated 225% increase in the
umber of bTB incidents in Great Britain [1], which is a cause ofonsiderable economic loss to farmers and Government. Althoughransmission of M. bovis between cattle is an important factor inpread of the disease [2], efforts to control bTB in the UK are
ampered by the infection in Eurasian badgers (Meles meles) thatepresent an additional source of recurrent M. bovis infection toattle in both the UK and Ireland [3–5]. Strategies to reduce theumber of infected badgers through culling is controversial [6], ands a means to reducing bTB, has been demonstrated to have bothositive and negative effects on the incidence of bTB [5,7].
As an alternative to reducing the number of badgers, their vac-ination against TB is an attractive option as a possible means to
educe and control bTB. In the short to medium term, the licenseduman TB vaccine, M. bovis Bacille Calmette-Guérin (BCG), repre-ents the best candidate for vaccination of wildlife [8,9]. Delivery ofhe vaccine in oral bait holds the best prospect for reaching badgeropulations over a wide geographical area [8] and has proven par-
icularly effective for the mass vaccination of other wildlife speciesgainst rabies [10]. However, BCG has little to no efficacy if deliv-red in a non-viable state [11,12], which is exacerbated in the casef oral delivery by inactivation in the low pH environment of thetomach [12–14]. The success of an oral BCG vaccine for wildlife willepend in large part on the ability to formulate BCG so it maintainsiability on transit through the stomach and is delivered efficientlyo immune induction sites in the gastrointestinal tract [12].
Recently, we demonstrated that BCG delivered to guinea pigsrally in a lipid matrix [15–21] was immunogenic and that liveacilli were recovered from the mesenteric and cervical lymphodes. Protection against aerosol challenge with virulent M. bovisas demonstrated for BCG only when protected by the lipid [22].
odium alginate is a naturally occurring polysaccharide which willross-link into a solid matrix following the addition of divalentations such as calcium [23]. Calcium alginate gels are biocom-atible with living cells and stable at typical gastric pH and haveherefore been used to formulate live bacterial cells for oral deliv-ry in therapeutic settings (reviewed in [24]). Recently, live BCG wasncapsulated in microspherical alginate beads and used to success-ully immunise BALB/c mice via the oral route [25]. BCG formulatedn this way was protected from inactivation in simulated gastricuid [26].
The titre of BCG needed orally to protect animals against chal-enge with virulent M. bovis is unknown, although studies in micend possums using BCG in a lipid matrix clearly indicate that suf-cient BCG must be delivered to establish replicating populationsf mycobacteria in the lymphatics in order to generate protectivemmune responses [12,27]. Another unknown for the oral deliveryf BCG vaccine is the precise role played by the oral formulationtself in delivery of live BCG to the appropriate gut-associated lym-hoid tissue (GALT). In this study, we compared BCG in a lipid matrixith BCG in alginate beads, and a novel hybrid approach wherebyCG in a lipid matrix was coated with alginate to form microcap-ules of approximately 1.2–1.7 mm diameter. The lipid formulationill be exposed to the action of gastric lipase and acid, whereas BCG
n both formulations that utilise alginate will be protected fromtomach acid. In the case of the hybrid microcapsular formulation,he alginate will also protect the lipid matrix from the action of gas-ric lipase thereby potentially delivering a relatively larger cargof lipid-encapsulated BCG to the small intestine compared withhe use of lipid matrix alone. The effect of these differences in theral BCG formulations was assessed by their protective efficacy inguinea pig aerosol M. bovis challenge model.
The nature of the immune response in BALB/c mice followingral immunisation with BCG-lipid is now well described [18,28], soe evaluated all formulations for the nature and magnitude of the
mmune response after oral delivery and looked to correlate thisith the isolation of viable BCG from the cervical and mesenteric
ymph nodes (MLNs). In a previous guinea pig experiment, BCGas used at a relatively high dose (107 CFU) in a lipid matrix [22].
o ascertain differences in the efficiency of each formulation foronferring vaccine protection in the present study, a 10-fold lowerose of BCG was also tested.
. Materials and methods
.1. Bacteria and media
Lyophilised BCG Danish strain (1331) was obtained from thetatens Serum Institute, Copenhagen, Denmark and subsequentlyultured in Middlebrook 7H9 medium + albumin-dextrose-catalaseADC) + 0.05% (v/v) Tween 80 to an optical density at 600 nmf approximately 0.2 (mid-log-phase growth). Virulent M. bovis
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(2008) 5791–5797
solate AF2122/97 [29] was propagated in Middlebrook 7H9edium and stored as frozen aliquots prior to aerosol infection
f guinea pigs. For routine bacterial enumeration, BCG was platednto modified Middlebrook 7H11 + oleic acid-albumin-dextrose-atalase (OADC) + glycerol, and M. bovis was plated onto modifiediddlebrook 7H11 + OADC + pyruvate.
.2. Formulation of M. bovis BCG
BCG was formulated in Lipid PK, a pharmaceutical grade, non-ydrogenated, vegetable-derived lipid containing a mixture ofriglycerides of fatty acids as previously described [16,22] at aarget concentration of 106 and 107 CFU/ml. Portions of the BCG-ipid matrix were further formulated to produce lipid in alginate
icrocapsules as follows. Lipid-cored alginate microcapsules wereroduced using an Inotech IE-50R encapsulator (Inotech Labor AG,witzerland) equipped with 300/600 �m concentric nozzle. BCG-ipid PK formulations were used as core-forming phase and sodiumlginate (1.5% (w/v), Keltone LVCR, ISP) as shell-forming phase withflow rate of 2.25 and 19.5 ml/min, respectively. The amplitude
nd frequency were set at 4 and 170 Hz, respectively. The cap-ules were hardened in 100 mM CaCl2 for 30 min. BCG was alsoormulated in alginate beads by external gelation using the syringeroplet technique. BCG suspension was mixed with 2% (w/v) HighAlginate solution (protanal SF200, FMC) in a 1:1 ratio. This mix-
ure was then cross-linked by drop-wise addition into a solution of00 mM CaCl2 using a syringe with a 30 gauge needle producingead sizes ∼1.6 mm in diameter. The beads were removed from theaCl2 solution after 15 min, washed with distilled H2O and stored in00 mM CaCl2. To facilitate the oral delivery of the alginate formu-ation in mice a smaller particle size was required. To achieve this,CG loaded alginate beads (600 �m diameter) were prepared fromhe alginate BCG mixture (by external gelation in 500 mM CaCl2)sing the Inotech encapsulator (300 �m nozzle). The loaded algi-ate beads were recovered, washed with distilled H2O and stored
n 100 mM CaCl2.
.3. Extraction of BCG from the formulations
The delivered dose of BCG was determined by plating an aliquotf the vaccine preparation. BCG was extracted from the lipidatrix by dispersal of the aqueous phase in non-ionic detergent
as described previously; [18]), prior to agar plating and enumer-tion. BCG was extracted from alginate beads by incubation in 2%w/v) sodium citrate for 45 min. BCG was extracted from the lipidn alginate microcapsules by a combination of both methods. The
icrocapsules were washed with chilled distilled water to removexcess CaCl2 and to harden the lipid core. The washed microcap-ules were incubated in 3% (w/v) ice cold sodium citrate for 60 mino dissolve the alginate coating and the sodium citrate solutionemoved by aspiration. The BCG from the solid lipid cores was thenxtracted as described [18].
.4. Post-vaccination BCG quantitative bacteriology and immunessessment
To study the delivery of the different vaccine formulations tohe upper and lower alimentary tract lymphatics and the result-ng immunogenicity, 8–10-week-old female BALB/c mice weremmunised individually with the three oral vaccine formulations
n volumes of 100–500 �l. The oral vaccines were flavoured byhe addition of 10% peanut butter (to encourage swallowing), anddministered to the rear of the buccal cavity via a dropper pipetten an attempt to avoid excessive mastication. Oral immunisations
ere administered to mice on a single day (Time 0), in two separate
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oses (morning and evening); injected BCG was administered as aingle 100 �l inoculum, delivered to mice via subcutaneous injec-ion at the nape of the neck (3.1 × 106 CFU, actual dose). All fouraccine groups comprised six mice per group. A group of nine miceerved as negative controls (i.e. no BCG delivered), of which threeeceived Lipid PK alone, three received alginate beads alone, andhree received lipid in alginate microcapsules alone.
Eight weeks post-vaccination, mice were euthanized via CO2sphyxia; their spleens were removed and prepared as singleell suspensions in Dulbeccos’s Modified Eagles Medium (DMEM;ibco-BRL, Auckland, New Zealand) for immunological assays,hile cervical-region lymph nodes (CLNs) and mesenteric lymphodes were excised and homogenised in Middlebrook 7H9 broth
or quantitative bacteriology.Murine splenocyte suspensions were enumerated and resus-
ended to a concentration of 106 lymphocytes per ml inMEM supplemented with 10% foetal calf serum, 20 mM HEPES,00 U/ml penicillin/100 �g/ml streptomycin, and 5.5 × 10−5 M-mercaptoethanol. For lymphocyte stimulation and secretoryytokine assays, cells were stimulated for a total period of 4 days,n the presence or absence of 60 �g/ml bovine Purified Proteinerivative (PPD-B, Prionics Inc., Switzerland) at 37 ◦C/5% CO2. Lym-hocyte proliferation was determined via thymidine incorporationver the final 18 h of assay, and data were expressed as a stimu-ation index (SI) as described previously [15]. For assessment ofecretory cytokine levels, cell-free supernatants were harvestedrom 96 h-cultured cells and assessed via capture ELISAs, usingeagents as described previously for the measurement of IL-2, IL-2 and IFN-� [27,30]; data were expressed as secreted pg or ngf cytokine per ml of cell culture supernatant. For ELISPOT assay,plenocytes were co-cultured in the presence or absence of PPD-
for 18 h, before IFN-positive cell spots were identified usingnti-murine IFN-� reagents, as described previously [31]; dataere expressed as the frequency of IFN-�-secreting cells per 106
plenocytes.
.5. Vaccination of guinea pigs for evaluation of vaccine efficacy
Eight separate groups of vaccinated guinea pigs were used tovaluate vaccine efficacy. Out-bred female Dunkin Hartley guineaigs (weighing between 250 and 300 g) free from inter-current
nfection were obtained from Harlan, UK. There were eight animalsn each group. Oral vaccines were pre-loaded into small volume1–2 ml) syringes and delivered at the back of the mouth slowlyo ensure the full dose was swallowed. Animals in a negative con-rol group received Lipid PK in the same way but without BCG. Thefficacy of each oral vaccine was evaluated against lyophilised BCGaccine as the positive control. A single vial of lyophilised BCG vac-ine (2–8 × 106 CFU) was reconstituted with 1 ml Sauton diluentupplied by the Statens Serum Institute with the vaccine and deliv-red subcutaneously in a volume of 0.1 ml in the nape of the necks.c. BCG).
.6. Aerosol challenge with M. bovis
Twelve weeks after vaccination all guinea pigs were challengedia the aerosol route with M. bovis strain AF2122/97 using a fullyontained nose-only exposure Henderson apparatus as previouslyescribed [32,33]. A fine particle aerosol with a mean diameterange of 2 �m (diameter range, 0.5–7 �m) [34] was generated with
saline suspension containing 1 × 106 CFU/ml in order to obtain
n estimated retained inhaled dose of approximately 10–20 CFUelivered to the lungs of each animal. The Henderson apparatusllows a controlled delivery of aerosols to the animals and theeproducibility of the system and relationship between numbers of
titdo
ig. 1. Light micrograph of the BCG-lipid in alginate microcapsule formulation. Thearker lipid core containing BCG (L) is surrounded by an outer shell of alginate (A).ize bar represents 500 �M. Examples are shown of microcapsules containing single,ouble and triple lipid droplets encased in alginate.
esions and concentration of bacilli in the Collison nebuliser haveeen described previously [32,35].
.7. Monitoring of animals post-challenge and necropsy
Following aerosol challenge, the guinea pigs were housed atCDP containment level 3 and weighed regularly as a marker ofisease progression. The animals were euthanized by peritonealverdoses of sodium pentobarbitone 5 weeks after challenge orhen an individual weighed 20% less than its maximal bodyeight (humane endpoint). Whenever a guinea pig was eutha-ized, the spleen and lungs were aseptically removed and the lungseighed after removal of the trachea and pulmonary associated
ymphoid tissues. Spleen and lungs were placed into separate ster-le tubes for storage at −20 ◦C until processed for bacteriologicalnalysis. Frozen tissues were thawed and homogenised in sterileeionised water using a rotating blade macerator system. Viableounts were performed by preparing decimal dilutions in sterileeionised water and plating 100 �l aliquots onto Middlebrook 7H11gar + OADC + pyruvate. Plates were incubated at 37 ◦C for 5 weeksefore counting the number of M. bovis colonies (CFU).
.8. Statistical analyses
For comparing response variables between groups, data setsere first assessed for distribution. Those data with either a nor-al distribution, or with a distribution that could be normalised by
og10 transformation, were assigned to parametric testing of meanalues via one-way ANOVA (with Dunnett post-hoc testing to iden-ify significance against the negative control). Those data with aon-normal distribution were analysed by the Kruskal–Wallis testnon-parametric ANOVA) followed by Dunn’s Multiple Comparisonost-hoc test. For assessing correlative relationships between inde-endent variables, Spearman’s rank correlation tests were applied.
. Results
.1. Appearance of BCG-lipid in alginate microcapsules
Fig. 1 shows the appearance of the novel microcapsular formula-
ion. Light dense lipid cores containing BCG are visibly encapsulatedn a light transmissible outer layer of alginate. The majority ofhe microcapsules were roughly spherical, containing a single lipidroplet encased in alginate, but in some cases, doublets or tripletsf lipid were found. It was estimated that the microcapsules con-
5794 S. Clark et al. / Vaccine 26 (2008) 5791–5797
Table 1CMI responses in BALB/c mice, 4 weeks following vaccination with BCG via parenteral or oral routes
Control groups Test oral BCG vaccine groups (N = 6 per group)
No BCG (N = 9) BCG s.c. (N = 6) Lipid matrix Lipid in alginate microcapsules Alginate beads
Lymphocyte proliferation meanstimulation index (S.E.M.)
FN-� median secreting cellser 106 splenocytes (range)
1 (0–20) 372 (260–672)**
sterisks refer to P values indicating significant differences between vaccine groups
aining a single lipid droplet were 1.2 mm in diameter, rising topproximately 1.7 mm where lipid doublets or triplets had formed.n all cases, the thickness of the alginate shell was estimated to be0 �M.
.2. BCG formulation delivery and immunogenicity in mice
The actual doses of viable BCG delivered per mouse, based onetrospective counts of BCG extracted from the various matrices,ere as follows: (a) in Lipid PK, 3.1 × 107 CFU; (b) in lipid in alginateicrocapsules, 4.6 × 107 CFU; (c) in alginate beads, 6.8 × 106 CFU;
esponses were recorded in mice which had received BCG in lipidatrix or microcapsular formulation (Table 1). Among the gen-
ral lymphocyte responses, mice which had received BCG in lipidatrix or microcapsules exhibited a significantly-elevated prolif-
rative response to PPD-B compared to control (non-vaccinated)ice (Table 1). Mice that had received BCG in alginate beads did
ot produce any significant specific immune responses (i.e. IFN-�r IL-2 production, lymphocyte proliferation). Levels of IL-12 werelevated in all vaccinated mice groups with the exception of BCGn alginate beads compared to controls (Table 1), however, nonef the differences were statistically significant (indicating that theverwhelming majority of IL-12 secretion due to PPD-B occurred
ndependently of specific antigen recognition).
Fig. 2 shows the isolation of the different BCG formulationsrom the CLN and MLN of mice 8 weeks post-vaccination. BCGas isolated from the CLN of all mice immunised with BCG s.c.
nd orally in lipid matrix and microcapsules. In contrast, BCG
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ig. 2. Recovery of viable BCG from the cervical (CLN) and mesenteric (MLN) lymph nodeas cultured (limit of detection = 5 CFU). Bars represent group mean values. Data refer to
ontrol mice (non-vaccinated). BCG CFU were assessed 4 weeks after homogenising and p
262 (100–496)* 442 (32–944)** 0 (0–12)
ontrols: *** P < 0.001; ** P < 0.01; * P < 0.05.
as isolated occasionally from the MLN of mice that received.c. BCG and BCG in lipid–alginate microcapsules but from all theice immunised with BCG in lipid alone. BCG was not isolated
rom either the CLN or MLN of any mice dosed with BCG-alginateeads.
Correlation analyses were applied to immunological and bacte-iological data from orally-vaccinated mice, in an attempt to discernny association between BCG load (in the alimentary tract lymphat-cs) and any of the ensuing CMI responses. The presence of BCGn the alimentary tract lymphatics of mice was positively and sig-ificantly correlated with the magnitude of IFN-� responses (bothecretory and cell frequency), as well as with lymphocyte prolifer-tive responses (P values < 0.001, 0.002 and 0.010, respectively). Inontrast, BCG presence was not correlated with the magnitude ofL-2 responses (P = 0.755).
.3. Protection conferred by different BCG oral formulations touinea pigs against aerosol challenge with M. bovis
The target doses for each oral vaccine formulation were 107
nd 106 CFU. However, this was technically difficult to achievecross all formulations and the actual doses of viable BCG deliv-red per guinea pig were as follows: (a) in Lipid PK, 1.7 × 107 or.4 × 106 CFU; (b) in lipid in alginate microcapsules, 1.0 × 107 or.5 × 106 CFU; (c) in alginate beads, 6.4 × 107 or 9.6 × 106 CFU; (d)
ubcutaneously, 3.4 × 105 CFU.
Some guinea pigs from the following groups were euthanizedn the 5th week after challenge as they had reached their humanendpoint: control group (N = 1); BCG in alginate beads (both doses)N = 2 per group); and BCG in microcapsules (N = 1 low dose, N = 2
s of mice vaccinated as shown. Open symbols represent individuals where no BCGN = 6 mice per group for vaccination groups or N = 9 mice per group for negative
lating lymphatic tissues onto 7H11 agar.
S. Clark et al. / Vaccine 26 (2008) 5791–5797 5795
Fig. 3. Influence of vaccination expressed as the percentage change in body weightfrom the pre-challenge weight. The mean value plus S.E.M. for each group at eachtf
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Fig. 5. Influence of vaccination on bacterial load of M. bovis in the spleens of guineapigs infected by the aerosol route. The data are presented as group median andrange with the first to third inter-quartile range as a box, as they were found to benon-normally distributed. Significant differences between group medians and thatof the Lipid PK (Control) group were demonstrated using Dunn’s Multiple Compar-ibmo
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mhigher than in the spleen, consistent with the route of challenge.Only the BCG-Lipid PK formulations were protective, and the great-est reduction in mean lung bacterial load of all groups (2.10 log10)was observed with the higher dose of BCG-Lipid PK.
ime point is shown. LD = lower dose of BCG and HD = higher dose of BCG, in theormulation used for vaccination.
igh dose). No animals were lost from the BCG s.c. group nor fromither of the BCG-lipid groups.
There were clear differences in disease progression betweenroups. Groups of animals that received BCG in lipid matrix showedn increase in body weight over the period of study (Fig. 3). Thereas no difference between the higher dose group and the s.c. BCG
ontrol. Animals gained weight less rapidly in groups immunisedith the lower dose of BCG-Lipid PK and with the higher dose ofCG in the microcapsular hybrid formulation. The animals in theemaining groups had all lost between 5 and 10% of their startingody weight by the end of the experiment. In this respect thereas no difference between the negative control group and those
eceiving BCG in alginate beads (at either dose), nor BCG in theicrocapsular formulation at the lower dose.The ratio of lung weight to body weight at necropsy was calcu-
ated as another general way to express the clinical advancementf TB in individual animals [13,36–38] (Fig. 4). Consistent withhe data in Fig. 3, the best protection (lowest lung/body weightatio) was seen with BCG in lipid matrix. Both doses were indistin-uishable from the BCG s.c. control in giving significant protectionompared with the negative control group (P < 0.01). Of the twoipid in alginate formulations, only that at the higher dose of BCGave protection (P < 0.05). The two groups vaccinated with BCG
lginate beads were indistinguishable from the negative control,espite the higher dose group representing the highest titre of BCGelivered across all groups (6.4 × 107 CFU).
ig. 4. Influence of vaccination on the ratio of lung weight (in grams) to bodyeight (in kilograms) at necropsy. The mean + S.E.M. is shown. Significant differ-
nces between group means and that of the Lipid PK alone (Control) group wereemonstrated using Dunnett Multiple Comparisons test following ANOVA and areepresented by asterisks (*P < 0.05, **P < 0.01). LD = lower dose of BCG and HD = higherose of BCG, in the formulation used for vaccination.
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sons test following non-parametric ANOVA (Kruskal–Wallis) and are representedy asterisks (*P < 0.05, **P < 0.01). NS = not significant. The log10 difference in groupedians compared with the Control group are shown numerically. LD = lower dose
f BCG and HD = higher dose of BCG, in the formulation used for vaccination.
Fig. 5 shows the concentration of M. bovis in the spleens fromach group at the termination of the experiment. BCG formulatedt either dose in lipid matrix conferred significant protection com-ared to the negative control group. The significance was greaterP < 0.01) with the higher dose than with the lower (P < 0.05). Inerms of the median count, both BCG-Lipid PK formulations wereower than even that of the positive control (s.c. BCG), although thisas not statistically significant. No other formulation gave protec-
ion to the spleen, although the lipid in alginate formulation at theigher dose of BCG reduced the median spleen count by 1.38 log10ompared with the negative control.
Almost identical results were found in the lungs (Fig. 6). Theean bacterial loads in the lung were approximately one log10
ig. 6. Influence of vaccination on bacterial load of M. bovis in the lungs of guineaigs infected by the aerosol route. The mean ± S.E.M. are shown for each group. Sig-ificant differences compared with the Lipid PK (Control) group were demonstratedsing the Dunnett Multiple Comparisons test following ANOVA and are representedy asterisks (*P < 0.05, **P < 0.01). NS = not significant. The log10 difference in groupeans compared with the Control group are shown numerically. LD = lower dose of
CG and HD = higher dose of BCG, in the formulation used for vaccination.
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. Discussion
In an effort to identify a strategy suitable for the formulation ofive BCG for oral delivery to badgers, we used a mouse and a guineaig pulmonary challenge model to determine the immunogenicitynd protective efficacy, respectively, of BCG in lipid matrix, alginateeads, and a novel microcapsule formulation based on the encap-ulation of BCG-lipid in alginate. The microcapsules were madesing a commercial laboratory-scale encapsulator employing therinciple that a laminar liquid jet can be broken into equally sizedroplets by a superimposed vibration. Through the use of concen-ric nozzles, molten BCG-lipid solution was passed through theentre of a stream of sodium alginate, disrupted into droplets byibration, which fell into a bath of CaCl2 solution, causing the algi-ate to cross-link and form stable lipid in alginate microcapsules.his approach was used recently to produce alginate microcapsulesith a drug-containing self-microemulsifying core [39], although
o our knowledge, ours is the first application of the method toicroencapsulate live bacterial cells within a lipid core. In this case,
he encapsulation of the lipid matrix with alginate actually reducedhe efficacy of the lipid-BCG, although the immunogenicity of the
icrocapsules was fully retained in mice.The best levels of protection were achieved with oral BCG simply
dministered in the Lipid PK matrix, supporting previous observa-ions of the efficacy of this formulation in guinea pigs [22]. LipidK is a mixture of triglycerides of fatty acids. Most of the digestionf triglycerides is brought about by pancreatic lipase in the upperart of the intestinal lumen, although the action of both lingual andastric lipases together with the emulsifying action of the stomachre important prerequisites for efficient hydrolysis by pancreaticipase [40]. How these influence the survival and uptake of BCG byhe GALT is unclear, but the lower efficacy seen with the lipid inlginate microcapsules points to exposure of the Lipid PK matrix toipase as a contributory factor in the efficacy of the BCG-lipid for-
ulation, although at this stage this cannot be separated from anyeneficial effect caused by additional uptake of the BCG throughhe oro-pharyngeal region. In fact, the isolation of BCG in relativelyarge numbers from both the MLN and CLN of mice (this study)nd guinea pigs [22] given BCG in the lipid matrix suggests thatoth routes of uptake may be contributing to the overall efficacy ofCG-lipid.
No small animal model is an ideal representation of the naturalost. In this case for efficacy testing we used a guinea pig aerogenichallenge model [35] since the respiratory route is considered therincipal route of exposure to natural M. bovis infection in the bad-er [41]. However, the guinea pig has relatively less gastric lipasectivity compared to other species [42], so empirical evaluation inhe badger is still necessary.
BCG in alginate beads failed to protect the guinea pigs against. bovis challenge by any measure used, despite the fact that this
ormulation actually contained the highest titres of BCG. Sincehese experiments were conducted, Ajdary et al. reported the oralmmunisation of BALB/c mice with live BCG encapsulated in micro-pherical alginate beads, resulting in immune responses (notably,ymphocyte proliferation and IFN-� production) and protectiongainst systemic homologous challenge with BCG [25]. Their studyiffered from ours in that they produced alginate beads withiameters approximately 6 to 150 times less than ours [26] anddministered 10-fold more BCG per mouse by intragastric intuba-ion. Any of these things may have accounted for the difference
n results; for example the size of microparticles, such as alginateeads, are important determinants in their uptake by cells [43],
ncluding those of the gut [44]. With regard to size, work with algi-ate encapsulated Bifidobacterium longum actually demonstrated
mproved protection from artificial gastric fluid and bile salts with
(2008) 5791–5797
ncreasing size of the alginate beads [45], although it is possible thathe volume of the mouse and guinea pig small intestine was toomall to permit adequate dissolution of the larger alginate beadssed in the present study and the release of the BCG for uptakey the GALT. By whichever mechanism, the failure of the alginateeads to deliver BCG to the GALT of the mice was consistent withheir lack of protective efficacy in guinea pigs.
The immunogenicity of the different vaccine formulations inice was associated with the isolation of viable BCG from theLN and CLN; especially the latter. This is consistent with previ-
us observations [18], although in that study, colonisation of theLN with BCG following oral dosing was less frequently observed.
n the present study IFN-� responses were strongly correlated withCG load, although the data may have been biased by the failure ofhe alginate beads to deliver BCG to the intestinal lymph nodes andpso facto to induce any CMI. In previous studies in mice we haveailed to demonstrate a statistically valid correlation between intra-ymphatic BCG load and CMI responses. Furthermore, in the presenttudy, the BCG-lipid in alginate microcapsules generated an almostquivalent magnitude of immune response to BCG in lipid matrixlone despite the fact that considerably fewer BCG were isolatedrom the alimentary tract lymph nodes with the former; especiallyn the case of the MLN. These data suggest that the alginate coatingf the lipid matrix may have limited the efficient release of BCGnd/or its uptake by the GALT, although surprisingly this did notmpact on the stimulation of splenic IFN-� responses. Clearly theumerical relationship between BCG load in the alimentary tract
ymph nodes following oral delivery and the magnitude of induc-ion of type 1 immune responses is complicated [46], however,esults here confirm earlier reports that delivery and establish-ent of live BCG in the lymphatic system is necessary to invoke
MI [27].In conclusion, the oral delivery of live BCG in Lipid PK matrix
o mice resulted in colonisation of alimentary tract lymph nodesnd type 1 systemic immune responses equivalent to subcu-aneous BCG. In guinea pigs, BCG in Lipid PK gave significantrotection against aerogenic challenge with M. bovis that was indis-inguishable from subcutaneous BCG. Alternative approaches tohe formulation of BCG for oral delivery were less effective. This,ogether with the fact that a relatively low dose of BCG (i.e. onlyne log10 more than the BCG s.c. dose) was still protective whendministered orally in lipid matrix, give further support to the fea-ibility of this simple formulation as an oral vaccine for badgers andther wildlife. Studies in the target species are clearly warrantednd planned for the future.
cknowledgements
This work was funded by the Department for Environment, Foodnd Rural Affairs (Defra), Great Britain. All animal procedures werearried out under licences issued according to the U.K. Animals (Sci-ntific Procedures) Act 1986 following local ethical review, or theew Zealand University of Otago Animal Ethics Committee.
The Authors gratefully acknowledge the technical animal andaboratory skills of Matt Lambeth and Yvonne Coughlan (Universityf Otago) in this study.
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