Protection of mice from Bordetella pertussis respiratory infection using microencapsulated pertussis fimbriae

Vaccine, Vol. 13, No. 7, pp. 675481, Copyright Printed

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Protection of mice from Bordetella pertussis respiratory infection using microencapsulated pertussis fimbriae D.H. Jones*, B.W. McBride*, and G.H. Farrar*

H. Jeffery+, D.T. O’Hagan?‘,

A. Robinson*

Conditions have been established which allow the efficient entrapment of Bordetella pertussisJimbriae in poly( lactide-co-glycolide) microspheres. Fimbriae released from the matrix were found to have retained some degree of conformational structure, as determined by assessing the capacity ofjmbrial protein to bind to untibodies mapping to either conformational or denatured structures on the jimbriae. Following a single intraperitoneal injection, equivalent amounts of fimbriue, either encapsulated in microspheres with a mean diameter of 24 ,um and an estimated in vitro protein release rate of approximately 42 days, or conventionally adjuvanted with alhydrogel, elicited vigorous immune responses in mice. The encapsulated$mbriae appear to elicit marginally lower serum antibody levels than those induced by equivalent amounts of alhydrogeladjuvanted fimbriae. Mic*e immunised with both preparations were, however, protected against intranasal infection with live B. pertussis as evidenced by the signljicant reduction in levels of bacterial colonisation observed in the lungs and tracheas of immunised unimuls when compared to the immunologically naive controls. Keywords: Microencapsulation;

acellular

pertussis

vaccine;

protection

The microencapsulation of proteins in biodegradable polymers is now well established’. However, the application of this technology to vaccines has only recently been investigated2-5. The most widely used formulation is the co-polymer poly(D,L-lactide-coglycolide) (PLG) which is tissue compatible, degrading by non-enzymic hydrolysis to the metabolites lactic and glycolic acids6,‘. The encapsulation of antigens in PLG microspheres may have several advantages for vaccines. First, encapsulation Iof the immunogen into microspheres can stabilise protein; second, the slow hydrolysis of the polymer Ipermits the sustained release of immunogen; third, microspheres can be prepared with diameters which facilitate their uptake into macrophages and other antigen presenting cells; and finally, protected from degradation within the polymers, microencapsulated immunagens can be delivered orally, offering the possibility of inducing specific mucosal immunity. The recent observations that immunogens can be encapsulated without loss of epitopes responsible for the induction of protection*-‘” reinforces the *Experimental Vaccines Section, Microbial Antigens Research Division, Centre for Applied Department, Microbiology and Research, Porton Down, Salisbury SP4 OJG, UK. ‘Department of Pharmaceutical Science, University of Nottingham, University Park, Nottingham, NG7 2RD, UK. %urrent address: United Biomedical Inc., 25 Davids Drive, Hauppage, NY 11788, USA. (Received 30 June 1994; revised 9 September 1994; accepted 9 September 1994)

potential of the system for the development of novel, convenient delivery systems for vaccination. We now report the microencapsulation and characterisation of Bordetellupertussis fimbrial proteins in PLG microspheres, data comparing and contrasting the immune responses in mice following the intraperitoneal injection of equivalent amounts of microencapsulated and alhydrogel-adjuvanted fimbriae, and the protection of mice against intranasal challenge with live B. pertussis after the single injection of microencapsulated fimbriae. The implication of these results for the development of mucosal vaccines is also discussed.

METHODS Reagents

Poly(D,L-lactide-co-glycolide) (Resomer RG506, 50:50 ratio of lactide:glycolide, mol. wt 22 000 kDa) was obtained from Boehringer Ingelheim KG (Ingelheim, Germany). Dichloromethane (HPLC grade) was obtained from Fisons plc (Loughborough, UK) and polyvinyl alcohol (PVA: 88% hydrolysed, 13 00623 000 mol. wt) was obtained from Aldrich Chemical Company (Poole, UK). Alhydrogel was obtained from Superfos Biosector a/s (Denmark). Mouse IgM monoclonal antibodies specific for and Agg3a, Agg2a conformational B. pertussis fimbrial epitopes’ ’ were obtained from Dr L.E.A. Ashworth, (Centre for Applied Microbiology and Research, CAMR); rabbit polyclonal anti-peptide IgG (R326) specific for a highly conserved linear amino acid sequence present in the

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fimbriae” was obtained from Dr R.N. Seabrook, CAMR; and mouse IgG monoclonal MM4, generated by immunisation of mice with fimbriae denatured at 70°C in the presence of 2% (w/v) SDS, was obtained from M.A. Matheson, CAMR. Peroxidase-conjugated anti-rabbit IgG, anti-mouse from Jackson IgM and IgG were obtained Labs (Pennsylvania, USA); ImmunoResearch peroxidase-conjugated anti-mouse IgA was obtained from Sigma Chemicals (Poole, UK). TMBlue soluble substrate was obtained from Universal Biologics (London, UK). Charcoal agar plates containing 10% (v/ v) defibrinated horse blood and 40 mg/l cephalexin were obtained from Becton Dickinson (Oxford, UK).

in 0.05 M NaOH and 1% (w/v) SDS at 100°C’“. Encapsulation of protein was expressed as a percentage of total particle weight. The rate of protein release from microspheres was determined as follows: microspheres (25 mg) were incubated in sterile PBS containing 0.05% (w/v) sodium azide (500 pl), at 37°C on a rotating wheel. At various times throughout a seven week period, aliquots of supernatant buffer were removed, clarified by centrifugation at 1OOOgfor 15 min and assayed for protein by the BCA method. The rate at which protein was released, expressed as a percentage of the known loading of the microspheres, and the time taken to release half of the total protein load were determined.

B.pertussis culture

Characterisation

Bacteria (Wellcome 28 strain of B. pertussis (serotype 1, F2, 3)), stored lyophilised, were reconstituted in distilled water and grown at 35°C for 48 h on charcoal agar plates containing 10% (v/v) defibrinated horse blood and 40 mg/l cephalexin. Bacteria harvested from these plates were used to generate cultures for both the preparation of fimbriae and the establishment of challenge stocks. Bacteria used for the preparation of fimbriae were cultured in CL medium containing 1 g/l (2,6-O-dimethyl)/3-cyclodextrin’3 and incubated at 35°C for 48 h”. Bacteria were harvested by centrifugation at 50000g for 30 min and homogenised to release fimbriae which were purified by repeated ammonium sulphate precipitation as previously described’“. Fimbriae prepared in this way showed no obvious contamination when examined by SDS-PAGE, and were composed of Fim2 and Fim3 in the approximate ratio of 3:214. The challenge stock of live B. pertussis was prepared by inoculation of bacteria onto charcoal agar plates as above and incubated at 35°C for 24 h. Bacteria were scraped from plates, dispersed in 1% (w/v) casamino acids, adjusted to a concentration of 2x10’ organisms/ ml (estimated by opacity and subsequently confirmed by viable counting) and used immediately for intranasal challenge.

Fimbriae were released from microspheres by incubation at 37°C for 3 weeks in PBS containing 0.05% (w/v) sodium azide. Parallel samples containing lyophilised fimbriae dissolved in PBS containing 0.05% (w/v) sodium azide, were also incubated at 37°C for 3 weeks. Control samples contained either native fimbriae or firnbriae denatured by treatment with 0.125 M ethanolamine buffer pH 10.5 containing 5 M guanidinium hydrochloride’7. Protein in all samples was estimated as described above and the concentration adjusted to 1 ,&ml before assessment of denaturation. ELISA plates (Nunc Maxisorb) were coated with aliquots (50 ~1) of solutions of fimbrial protein derived from particles or controls at 37°C for 16 h. Plates were then washed with PBS containing 0.05% (v/v) Tween 20 (PBST) and blocked with PBST containing 4% (v/v) New Born Calf Serum and 1% (w/v) BSA (Blocking buffer) at 20°C for 1 h. After washing with PBST, plates were incubated with optimal dilutions of anti-fimbrial antibodies (Agg2a, Agg3a, R326 and MM4) diluted in blocking buffer (50 pi/well) at 37°C for 2 h. Plates were again washed with PBST and bound antibody detected by incubation with the appropriate peroxidase conjugated antibody (50 pi/well of a 1:5000 dilution) at 37°C for 30 min. Finally, plates were washed with PBST before the addition of TMBlue soluble substrate (50 ~11 well). Colour development was stopped after 15 min by addition of 1 M H,SO, (50 pi/well) and the absorbance measured at 450 nm.

Microencapsulation

of fimbriae

Fimbriae were microencapsulated by the (water-inoil)-in-water solvent evaporation technique as previously described15. Briefly, 4.15 ml of a 6% (w/v) solution of the polymer in dichloromethane was emulsified with 0.25 ml of a 6 mg/ml aqueous solution of fimbriae using a Silverson homogeniser fitted with an emulsor screen (Silverson Machines Ltd, Chesham, UK). This emulsion was immediately added to an aqueous solution of 10% (w/v) PVA (50 ml) and emulsified as above. The resulting double emulsion was stirred at 20°C overnight to remove dichloromethane and to allow the formation of the microspheres. Microspheres were collected by centrifugation, washed three times in distilled water and lyophilised. Characterisation

of microspheres

Freeze-dried microspheres were dispersed in distilled water and their size determined by laser diffractometry using a Malvern Laser Sizer 2600D (Malvern, UK). The efficiency of fimbrial encapsulation was determined using the BCA protein assay (Pierce), following dissolution of a known weight of microspheres for 3 min

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of protein released from microspheres

Immunisation

Immediately before injection of mice, lyophilised microspheres were resuspended in PBS to give a final protein concentration of 20 pg fimbriae/ml. Four groups, each of five NIH-Porton mice (68 weeks), were immunised with single intraperitoneal (i.p.) injections of either 0.5 ml of microsphere suspension, 10 g of fimbriae in 0.5 ml of a 25% (v/v) suspension of Alhydrogel in PBS or, as controls, 0.5 ml of PBS alone. Prior to immunisation and at 3, 6, 10, 14, 18, 21 and 24 weeks post immunisation, mice were anaesthetised by ether inhalation and 30 ~1 of blood collected in capillary tubes by retro-orbital puncture. Blood was allowed to clot at 2O”C, centrifuged for 15 min in a Haematocrit Centrifuge (Hawksley and Sons, Lansing, UK) and the serum transferred to Eppendorf tubes. Samples were stored at -70°C until the completion of the studies when samples were assayed en bloc.

D.H. Jones et al.

Protection of mice from B. pertussis:

the rate of release of protein from a known weight of microspheres. The rate of release, expressed as a percentage of total encapsulated protein, was plotted against time (Figure I) and found to approximate to a straight line with 50% of the encapsulated protein being released within 6 weeks. The size distribution of an aqueous suspension of microspheres, determined by laser diffractometry, was found to have a volume mean diameter of 24 pm within the range 1140 pm.

Intranasal challenge

Groups of mice were challenged by intranasal instillation at 6, 10, 14 or 24 weeks post immunisation. Mice were lightly anaesthetised with ether and 50 ~1 of bacterial suspension (approx. lo6 bacteria) was pipetted onto the nostrils and allowed to be inhaled”. Mice were killed 7 days later by cervical dislocation and their lungs and tracheas removed. Tissues were homogenised in 10 ml of 1% (w/v) casam:ino acids using a Silverson Homogeniser, and the viable organisms in homogenates assessed by plating out serial dilutions onto charcoal agar plates supplemented with defibrinated horse blood and cephalexin as above. Plates were read after incubation at 35°C for 6 days. Determination

of specific anti-fimbrial

Assessment of fimbrial denaturation during encapsulation

To assess whether the process of encapsulation had any denaturing effect on fimbriae, four well characterised anti-fimbrial antibodies were used to detect changes to their corresponding protein epitopes. Using ELISAs, the binding of these antibodies to fimbriae released from microspheres by hydrolysis was compared and contrasted with the binding to native and chemically denatured fimbriae. The results, presented in Figure 2, show that both Agg2a and Agg3a, which recognise conformational epitopes, bound strongly to native fimbriae and fimbriae released from particles but did not bind to denatured fimbriae (Figure 2). In

responses

Specific anti-fimbrial responses were assayed by ELBA using plates coated with native or chemically denatured fimbriae as described above. After blocking, two-fold serial dilutions of sera (from 1:25 for determination of IgA, 1:500 for IgG and 1:50 for IgM) were made across the plates (50 @l/well) and the plates incubated at 37°C for 2 h. Plates were further developed as above except that peroxidase conjugated anti-mouse IgA (1: IOOO), IgG (1:5000’) or IgM (1:5000) were used to detect antigen-specific serum immunoglobulins. End point titres were determined for each serum titration using each animals corresponding non-immune serum to determine the cut off point.

RESULTS Characterisation

of microspheres

The microencapsulation of fimbriae was monitored by several criteria and the conditions described above were found to maximise both the efficiency of encapsulation and the overall recovery of fimbriae with minimal denaturation. Following solubilisation of a weighed sample of microspheres in SDS, under alkaline conditions, the protein content of the particles was found to be 0.4% (expressed as a percentage per unit weight of microspheres), giving an efficiency of protein encapsulation of 60%. The half-life of fimbrial protein release from microspheres was determined by following

OS/

I

0

10

I

I

20

30

Incubation

time

I

I

40

50

(days1

Figure 1 The release of B. pett~~.ssis fimbriae from microparticles with time in aqueous suspension at 37°C. Fimbriae released into the supernatant, was determined by protein assay and the rate of release expressed as a percentage of the total encapsulated protein

I

n 0

AgO

AMa

MM4

Native Microencapsulated Denatured

(released)

R326

Antibody Figure 2 Binding of antibodies Agg2a and Agg3a (both reactive with native fimbriae), MM4 and R326 (both reactive with denatured fimbriae), to native, denatured and microencapsulated fimbriae released by hydrolysis. The binding of each antibody, at a predetermined optimal working dilution, to all fimbriae samples was determined by ELISA

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Protection of mice from B. pertussis:

D.H. Jones et al. alhydrogel-adjuvanted fimbriae showed a significant reduction in the numbers (greater than 97%) of organisms recovered from lungs and tracheas compared to control mice (Figure 4).

contrast both MM4 and R326, antibodies which recognise linear epitopes on denatured fimbriae, bound strongly to denatured fimbriae but only weakly to both the native fimbriae and the fimbriae recovered from microspheres.

DISCUSSION

Immune responses elicited by microencapsulated fimbriae

This study was initiated with the distinct aims of optimising the conditions for the microencapsulation of Bordetella pertussis fimbriae and comparing the immune responses elicited by this formulation directly with conventionally (alhydrogel) adjuvanted fimbriae. As this particular fimbriae preparation has previously been developed, characterised, and assessed in a mouse animal model, as a prelude to its promotion as a component of acellular pertussis vaccine12. 14, this afforded an excellent opportunity to investigate the potential problems and advantages of this approach to antigen formulation. The initial stages in the microencapsulation of proteins involves emulsification of aqueous solutions of proteins with polymers dissolved in an organic solvent. Characteristics of the protein such as its concentration, hydrophobicity, charge, molecular weight, and degree of glycosylation are all understood to contribute to the partitioning of the molecules during this stage. External factors such as the ratio of volumes of the phases, molecular weight of the polymer, the type of organic solvent used, the concentration of salts present in the aqueous phases and the mechanical conditions of homogenisation, may also influence the efficiency of encapsulation. The conditions described here for the encapsulation of fimbriae resulted in an efficiency of protein encapsulation, i.e. the percentage of the initial protein concentration incorporated into the polymer, of 60%, with a total protein load of approximately 1% w/w of the microspheres. These figures are comparable to those obtained by other workers using different protein molecules2. 6. 15.19.21.

A intraperitoneal injection of single microencapsulated fimbriae elicited a substantial systemic immune response in mice. The data presented in Figure 3 are average values from groups of five animals. Titres of specific anti-fimbrial serum IgM had reached a maximum by 3 weeks post immunisation and fell steadily for the remainder of the study consistent with a primary immune response. End point titres of specific anti-fimbrial IgG reached a maximum at 6 weeks post immunisation, falling only slightly during the next 12 weeks, and remained at a constant level, significantly above pre-immune titres, from 18 weeks post immunisation. Titres of antigen-specific serum IgA were relatively low compared to those of IgM and IgG over the 24-week period of study. A single i.p. injection of alhydrogel-adsorbed fimbriae elicited a stronger immune response. Throughout the 24 weeks of study, end point titres of specific serum antifimbrial antibodies were consistently higher than those elicited by microencapsulated fimbriae (Figure 3). None the less, end point titres of IgA, IgG and IgM elicited by alhydrogel-adsorbed fimbriae followed the trend resulting from immunisation with microencapsulated fimbriae. Protection against challenge with

B.pevtussis

Immunisation with microencapsulated fimbriae and alhydrogel-adsorbed fimbriae protected mice against infection when challenged by intranasal instillation of live B. pertussis for a period of up to 24 weeks following immunisation. Control animals were highly susceptible compared to the immunised groups. All animals immunised with microencapsulated fimbriae or with Anti - fimbrial

IgA

Anti - fimbrial

A very important aspect of this technique, which has largely been ignored in other studies, is the potential denaturation of the protein by the process of IgG

Anti -

fimbrial IgM

5.

HAlurn qEncap

5

5

3 6 10 14 18 21 24

’

3 6 IO 14 18 21 24 Weeks post immunisation

L

6 10 14 18 21 24

Figure 3 Specific anti-fimbrial antibody responses in serum elicited by a single intraperitoneal injection of microencapsulated or alhydrogeladjuvanted fimbriae were determined by ELISA. Log,, reciprocal end point titres (using pre-immune sera to determine cut-off values) were measured for individual animals and the mean for each group of animals plotted against time (weeks) post immunisation

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Protection of mice from B. pertussis: TRACHEAS

LUNGS 120

120 r

H Control c] PLG/fim q Alum&m

100 i? 2 g 0

8o

1 100

2 8o 2 & 60 L

60

g

40 20

\o

40 20

0I

D.H. Jones et al.

IfIt 6

L

IO

14

24

Weeks post imnwnisation

0

;I

10 14 24 6 Weeks post immunisation

Figure 4 Protection of mice against intranasal challenge with live bacteria was assessed by recovery of viable organisms from lungs and tracheas of mice immunised with microencapsulated or alhydrogel-adsorbed fimbriae, and expressed as a percentage of the recovery from tissues of non-immunised control mice. Means for each group of animals are presented

encapsulation, in particular the effects of organic solvent and the physical damage resulting from emulsification. Although it is believed that the protein solution is uniformly dispersed throughout the organic phase and consequently that the protein is not actually in contact with the organic solvent, the possibility of denaturation at the interface surrounding the aqueous phase cannot be discounted. As this present study was intended to compare microencapsulated fimbrial protein directly with a non-denaturing formulation (alhydrogel), it was important to address as far as possible changes in the properties of the protein. We have examined changes to discrete regions of the fimbrial subunit by monitoring the binding of antibodies that recognise epitopes which are only present on native or denatured fimbriae, respectively. The data suggest that the conditions leading to described here maintain the encapsulation conformational structure recognised by antibodies Agg2a and Agg3a (monoclonal antibodies generated using alhydrogel-formulated fimbriae) and do not unfold the tertiary structure to the extent of exposing the denatured epitopes recognised by antibodies MM4 and R326. This suggests that in the regions analysed, the fimbriae possess same microencapsulated the their alhydrogel-formulated conformation as counterparts which are known to elicit antibody which recognises these structures. A single i.p. injection of microencapsulated fimbriae elicited strong humoral immune responses which persisted throughout the 24-week experimental period. However, the specific antibody titres following injection with microencapsulated fimbriae did decline over the experimental period. This mirrors the trends seen in the with alhydrogel formulated animals immunised fimbriae, suggesting that 1:he expected sustained release did not continuously stirnulate the immune response. Similar results have been reported following a single injection of PLG microencapsulated systemic the proteins”. “‘, while other studies have observed durability of responses following priming and boosting with microencapsulated antigens*‘. Further studies have reported that PLG encaps#ulated antigens elicited better antigens”, than alhydrogel-formulated responses after boosting”. The variability in particularly

responses to different proteins formulated in alhydrogel is not surprising since the range of antigens for which alhydrogel is an effective adjuvant is restricted*‘. The high antibody titres attained in this study confirm this; however, when the kinetics of release of fimbriae from the PLG microspheres, determined in vitro, are taken into consideration (5 clg in 42 days), the corresponding antibody response to microencapsulated fimbriae is encouragingly vigorous, perhaps indicating that the predicted sustained release rate may have potentiated the immune response. It must, however, be stressed that the exact fate of microspheres in vivo is largely unknown and studies on their uptake and retention times are urgently required. The protection against challenge with live bacteria afforded by a single systemic injection of PLG microencapsulated fimbriae was comparable to that of a single injection of alhydrogel-adsorbed fimbriae throughout the duration of the study. When compared to control groups of mice, both formulations of fimbriae induced a significant reduction in viable bacteria recovered from the normally susceptible tissues of immunised mice. There have been few attempts to demonstrate protection against an infectious agent following administration of PLG microencapsulated antigens. One study ‘O, in which hamsters immunised i.p. with microencapsulated human parainfluenza virus and boosted one week later demonstrated protection. The microencapsulated vaccine elicited almost complete protection but no attempt to assess the durability of the protective effect was reported. Another study’ administered microencapsulated whole influenza virus to mice, systemically or orally, and elicited protection against challenge with live virus, but a primary and booster dose of the vaccine was required to elicit protection and again the durability of the response was not investigated. A further study” demonstrated that regimes comprising administration of microencapsulated whole simian immunodeficiency virus (SIV) (three intramuscular doses followed by four doses orally or intratracheally), protected animals against intravenous challenge with live SIV. It is thus highly significant that fimbriae are not only relatively stable to microencapsulation, but also when administered as a

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single i.p. injection, microencapsulated fimbriae elicit strong and durable immune responses, comparable to an accepted and established adjuvant. The microencapsulation of antigens is likely to find application in the delivery of vaccines in the future. However, the precise role to be played still has to be established. Although adjuvanting properties of PLG have been reportedz2, the most apparent immune potentiation stems from the depot effect and the capacity to sustain release of antigen over a period of time. As such, the system may not have many advantages over alhydrogel type adjuvants which have for years elicited extremely potent immune responses. A possible advantage of microencapsulated antigen systems is in the combining of particles which degrade at different times and with variable rate so that, at least in theory, induction of primary and secondary immunity can be programmed into a single administration of vaccine’*. In reality this is proving very difficult to achieve and often immunity can only be attained by multiple administration of encapsulated protein or combination strategies involving encapsulated and conventionally adjuvanted protein. Even if precise delivery could be built in at the formulation stage there may be some very real problems to overcome regarding the deposition of often very bulky, albeit biodegradable, material intravascularly or subcutaneously. However, the potential protein stabilising properties of microencapsulation may have a very important impact on the supply of crucial health care products, many of which, by virtue of their present production methods, arrive at the recipient spoiled. The most promising application for microencapsulation of antigens is in the oral delivery of vaccines and the stimulation of mucosal immunity. The majority of pathogens enter the host via a mucosal surface and the potentiation of mucosal immunity may offer an effective first line of defence. Antigens formulated in PLG pass safely through the stomach and, as the size of the particles can be accurately determined at the time of manufacture, they can be made optimal for uptake by the immune inductive tissue (Peyer’s patches) of the gut. Orally administered microencapsulated antigen cannot only induce vigorous systemic immunity but also elicits a disseminated mucosal response23-28. With the recent report that orally administered SIV can elicit protection against intravaginal challenge” and that PLG delivery systems are capable of producing cellular immune responsesZ7, further experiments are required to test the general appropriateness of this approach so that the delivery of human vaccines and vaccine responses by this route can be assessed. There are increasing numbers of immunogens becoming available for childhood and possibly future adolescent and adult vaccination regimes. Even if the combined formulation of these antigens is possible without loss of individual component performance, the cost and inconvenience implications of extended vaccination schedules are likely to be great. Consequently it is an important time to begin to assess the feasibility of alternative formulations and methods of delivery of which microencapsulation in PLG is a very strong contender.

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ACKNOWLEDGEMENTS This project was supported by the Public Health Laboratory Service. The authors also wish to thank Mary Matheson and Simon Funnel1 for their helpful discussions and advice.

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Challacombe, S.J., Rahman, D., Jeffery, H., Davis, S.S. and O’Hagan, D.T. Enhanced secretory IgA and systemic IgG antibody responses after oral immunisation with biodegradable microparticles containing antigen. /mmuno/cgy 1992, 78, 164-168 27 O’Hagan, D.T., McGee, J.P., Holmgren, J., Mowat, A.Mcl., Donachie, A.M., Mills, K.H.G. et al. Biodegradable microparticles for oral immunisation. Vaccine 1993, 11, 149-154 28 Maloy, K.J., Donachie, A.M., O’Hagan, D.T. and Mowat, A. MCI. Induction of mucosal and systemic immune responses by immunisation with ovalbumin entrapped in poly(lactide-coglycolide) microparticles. immunology 1994, 81, 661-667 29 Miller, C.J.,, Kang, D.W., Marthas, M., Moldoveanu, Z., Kiyono, H., Marx, P. eta/. Genital secretory immune response to chronic simian immunodeficiency virus (SIV) infection: a comparison between intravenously and genitally inoculated rhesus macaques. C/in. Exp. Immunol. 1992, 88, 520-526

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