Single dose, polymeric, microparticle-based vaccines: the influence of formulation conditions on the magnitude and duration of the immune response to a protein antigen

Elsevier PII: S026440X(96)00077-1

ELSEVIER

Vaccine, Vol. 14, No. 15, pp. 1429-1436, 1996 Copyright 0 1996 Elsevier Science Ltd. All rights resewed Printed in Great Britain 0264-41 OX/96 $15+0.00

Single dose, polymeric, microparticle-based vaccines: the influence of formulation conditions on the magnitude and duration of the immune response to a protein antigen A.G.A

Coombes*$,

E.C. Lavelle”,

P.G Jenkins?

and S.S. Davis*

Ovalbumin-loaded pol_y(D, L-luctide co-gl_ycolide) [OVA-loaded PLG] microparticles, produced bl>emulsionlsolvent evuporation stimuluted the production of high serum IgG antibodqj levels ufter a single subcutaneous (XC.) administration in mice and the duration of the immune response paralleled the degradution rate of the carrier. Formulations based on slow resorbing PLG maintained relatively constant peak antibody levels for 26 weeks and high titres jbr over I year at a level approximating the peuk response to the faster resorbing, 0 VA-loaded particles which was of lower duration. Vuccine formulations prepared by simple mixing of blank PLG microparticles and 0 VA exhibited low primaq immune responses which were only elevated blj boosting. 0 VA-loaded PLG microparticles exhibited a substantial surface protein component amounting to ca 40% and 60% of the total protein loading for slog) resorbing and fast resorbing PLG, respectively. These jindings suggest that sustained presentation of surface protein to the immune system was a mcq’or factor in the induction and long-term maintenance of high antibody titres following u single s. c. administration of 0 VA-loaded microparticles Copyright 0 1996 Elsevier Science Ltd The poor immunogenicity of many purified or inactivated antigens such as tetanus toxoid (TT) and experimental sub-unit vaccines necessitates the use of adjuvants such as alum and/or repeated (booster) administrations to induce adequate immunity. A major goal of researchers in vaccine design and formulation is, therefore, the development of sustained release or pulse release delivery systems, capable of eliminating the requirement for a cold chain and for a multiple dosing schedule which attends the administration of conventional vaccines’. This would reduce the number of contacts with the vaccinee and lessen the problem of patient non-compliance and population coverage. Vaccine formulations based on emulsions, liposomes, ISCOMS and microparticles have all been investigated in this context’. In the latter category the potential of resorbable, poly(lactide co-glycolide)-based microparticulate systems for providing improved and novel parenteral vaccines has been demonstrated for model antigens such as ovalbumin3,4 and for more clinically *Department of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK. fPresent address: Proteus Molecular Design, Lyme Green Business Park, Macclesfield, UK. $To whom correspondence should be addressed. (Received 27 July 1995; revised 3 March 1996; accepted 1 April 1996)

relevant antigens such as Mycobacterium tuberculosis 38 kDa protein5, TT&s, diptheria toxoid’ and Staphylococcal enterotoxin B (SEB)“. The resorbable, synthetic polyesters such as poly(L-lactide) (L.PLA) and co-polymers such as poly(D,L-lactide co-glycolide) (PLG) have been widely used for medical implants”-l3 and for drug delivery14,15. Their broad range of physico-chemical properties, degradation rates and biocompatibility gives wide scope for controlling implant performance. The encapsulation of proteins and peptides in PLG matrices for vaccine formulation has generally been achieved using double emulsion/solvent evaporation techniques, originally developed by Vranken and Claeys16 and modified by Ogawa et aI.” These involve the formation of a primary emulsion consisting of droplets of polymer solution containing the antigen, which is subsequently mixed with a continuous aqueous phase containing a particle stabilizer/surfactant. This approach counteracts the partition of protein into the aqueous phase and subsequently results in efficient and reproducible encapsulation. One of the main attractions of using PLG microparticles to deliver antigens is the ability to vary the degradation rate from several days to over a year by selecting polymers with a particular lactide-glycolide ratio, molecular weight and crystallinity profile. The

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Single dose microparticle-based

vaccines: A.G.A. Coombes et al.

potential exists therefore for producing pulse release of antigens by mixing two or more populations of microparticles in a single formulation which degrade at different rates and hence release entrapped antigen at predetermined times. This approach is of interest for replicating the conventional multiple dosing regimes which are generally required for many inactivated and sub-unit vaccines. Long-term, potent immune responses have already been measured after subcutaneous (s.c.) administration of antigen-loaded PLG microparticles3s which are generally considered to result from degradation of the carrier in vivo permiting gradual release of entrapped antigens to stimulate immuno-competent cells. The primary aim of the present study was to measure the capacity of fast and slow resorbing, OVA-loaded PLG microparticles for eliciting and sustaining high antibody levels after a single S.C. administration, The immune responses to blank PLG microparticles mixed with OVA solutions were also investigated since conflicting reports exist in the literature concerning the ability of adsorbed systems to stimulate immune responses. The levels of antibody elicited by immunization with OVA adsorbed to alum were assessed for comparison. The nature of protein association with each substrate and the short-term protein release profiles in vitro were determined to aid in the interpretation of the immune responses and to establish design guidelines for improving the performance of microparticulate vaccines.

MATERIALS

AND

METHODS

Two types of resorbable PLG copolymer were obtained from Boehringer Ingelheim, Germany. A SO:50 PLG copolymer containing 50% D,L-lactide and 50% glycolide (Resomer 503, A4, 34000) was selected as an example of a fast resorbing copolymer. The 75:25 PLG copolymer (Resomer 755, I& 63000) degrades more slowly than the 50:50 PLG copolymer due to the increased lactide component. [Weight average molecular weights (M,) as supplied by the manufacturer]. Alum (“Alhydrogel”, Superfos, Denmark) was provided by Medeva, Leatherhead, Surrey. Ovalbumin (Grade V), Lauryl sulfate (SDS), Freund’s complete adjuvant (FCA) and Freund’s incomplete adjuvant (FIA) were obtained from Sigma. Bicinchoninic acid reagents and Trypsin (Type 1, Bovine pancreas) were obtained from Sigma. Poly(vinyl alcohol) (PVA), M, 13-23000 was supplied by Aldrich Chemicals, Gillingham, UK. Dichloromethane (HPLC grade) was obtained from Fisons Scientific Equipment, Loughborough, UK. Vaccine formulation OVA-loaded PLG 0 VA-loaded PLG microparticles. microparticles were produced using a water-in oil-in water (w/o/w), emulsification/solvent evaporation technique with PVA as a stabilizer. An aqueous solution of ovalbumin (2.0 ml of a 30 mg ml-’ solution) were emulsified with 10 ml of a 6O/o w/v solution of the respective polymer in dichloromethane (12400 revs min-’ for 2 min) using a Silverson homogenizer (Silverson Machines, Chesham, Bucks, UK). A 10% w/v aqueous solution of PVA (40 ml) was added to the

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Vaccine 1996 Volume 14 Number 15

primary water-in-oil (w/o) emulsion and stirring was continued for a further 4 min. The resulting water-in oil-in water (w/o/w) suspension was stirred magnetically for 16 h to evaporate the solvent. The microspheres were harvested and cleaned by centrifugation at 12096g for 15 min (Beckman Model 52-21 Centrifuge) and resuspension in distilled water a total of three times. The cleaned microspheres were freeze dried and stored in sealed vials at 5°C. Blank microspheres (OVA-free) were produced by emulsifying the respective polymer solution with a PVA surfactant solution and proceeding as described above. PLG microparticlelOVA mixtures. Vaccine formulations were prepared by simple mixing of blank PLG microparticles and OVA solutions in which the amounts of each component were equivalent to those constituting OVA-loaded PLG microparticle formulations prepared by solvent evaporation (7 mg microparticles/ pug OVA). Blank PLG microparticles (84 mg) were suspended in a 0.06% w/v solution of OVA in PBS to yield a 14mgmll’ particle suspension and incubated at room temperature for 24 h. Sample tubes were rotated endover-end with a Voss mixer (Voss Instruments, Maldon, Essex, UK). An aliquot of the microparticle suspension was separated and the microparticles were isolated from the suspension medium by centrifugation. Samples of the supernate were analysed in duplicate by a bicinchoninic (BCA) protein assay (Sigma) to provide an estimate of the unbound OVA fraction and the amount of OVA adsorbed to the microparticles, respectively.

OVA adsorbed to alum OVA (4.4 mg) was added to 5 ml of an alum suspension (8.4 mg ml-‘) and incubated at room temperature for 24 h with end-over end rotation. The alum samples were isolated from the incubation medium by centrifuging (2200g) and washed once by resuspension in 5 ml of water. Duplicate samples of supernate and washing were analysed for protein content by a BCA assay and the amount of OVA adsorbed to the alum substrate was determined by subtraction. Particle size measurement Microparticle size was measured by a laser light scattering technique which is based on the theory that the observed time dependency of the fluctuations in intensity of scattered light from a colloidal dispersion, is a function of the rate of diffusion or Brownian motion of the scattering particles, and hence of their size in suspension18. The instrument used was based on a Malvern spectrometer (Malvern Instruments, Malvern, UK) incorporating a Helium-Neon Laser (Siemens, Germany). Particle size measurements were performed on microparticle suspensions at 25 *O.O5”C and each sample was analysed a total of ten times to give an average value calculated for the particle diameter. Determination of the protein content of microparticles The OVA content of microparticles was determined according to the methods described by O’Hagan et a1.19 Approximately 3.0 mg of freeze dried microparticles were accurately weighed and dispersed in 1 ml of 0.1 M

Single dose microparticle-based vaccines: A. G.A. Coombes et al. NaOH containing 5% (w/v) agitated for 24 h on a IKA centrifuged and the supernate BCA protein assay”. Each duplicate. Analysis

SDS. The sample was VIBRAX-VXR shaker, analysed for OVA by a sample was assayed in

of surface OVA

The amount of OVA associated with the surface of OVA-loaded microparticles produced by emulsification/ solvent evaporation was estimated using three approaches. Treatment with 2% SDS. Microparticles (3-3.5 mg) were accurately weighed, redispersed in 1 ml of 2% (w/v) SDS solution and agitated for 4 h using a IKA Vibrax shaker. The samples were centrifuged and the supernate was analysed for OVA by a BCA assay. At least three samples of microparticles were assayed for each formulation. Treatment with trypsin. Microparticles (1.5-l .8 mg) were accurately weighed and redispersed in 0.5 ml PBS containing 30 ,ug of trypsin. Samples were retained in a water bath at 37°C for 1 h and shaken intermittently to retain the microparticles in suspension. Following centrifugation, the supernate was analysed for OVA content by a BCA assay. Calibration curves were constructed from a series of dilutions of OVA in PBS (with each dilution containing 30 ,ug of trypsin) which had been subjected to the above treatment. At least three samples of microparticles were assayed for each formulation. Treatment u>ith BCA reagent. Microparticles (1.51.8 mg) were suspended in 0.5 ml PBS and mixed with BCA reagent [2.0 ml BCA solution, 40 ~1 Copper sulfate] by shaking for 2 min at room temperature. The microparticles were sedimented by centrifugation at 2200g and the supernate was analysed for OVA content by the BCA assay. At least three samples of microparticles were tested for each formulation. Scanning electron microscospy

(SEM)

Measurements of microparticie size and information on shape and surface morphology were obtained by SEM (JEOL 6400, Japan). Cleaned microparticle suspensions were dropped onto aluminium stubs and allowed to air dry. Specimens were sputter coated with gold prior to examination in the SEM. In vitro release rates of OVA The short-term, in vitro protein release rates from: (1) OVA-loaded PLG (2) microparticle/OVA (3) OVA adsorbed to background data in vivo studies.

microparticles; mixtures; and alum were measured to provide for analysis of the results of

OVA-loaded microparticles were incubated in release medium (PBS, pH 7.4, 37°C) containing 0.02% sodium azide as a bacteriostatic agent. Sample tubes contained ca 20 mg of freeze-dried microparticles, accurately weighed and dispersed in 2 ml of release medium. Samples were retained in a water-bath and shaken

intermittently to retain the microparticles in suspension. After 4 h, 24 h and at intervals of 3 days up to 1 month, the release medium was separated from the microparticles by centrifugation (2200g). Fresh medium was added to the microparticles and the release study was continued. Samples of release medium were stored at -20°C prior to testing in duplicate by a BCA assay. /ul vitro release studies were also performed with a mixture of blank PLG microparticles in OVA solution (21 mg microparticles, 0.9 mg OVA, 2.0 ml PBS) and a suspension of OVA-adsorbed alum (42 mg alum, 4.4 mg OVA in 5 ml PBS release medium). Protein release profiles were generated for: (1) OVA-loaded 50:50 PLG and 75:25 PLG microparticles; (2) OVA adsorbed to alum; and (3) 50:50 PLG and 75:25 PLG microparticle mixtures in OVA solution. Protein release profiles were generated for each microparticle system in terms of cumulative protein release (% w/w) vs time. Immunization

protocols

Mice were injected with OVA in 0.25 ml physiological saline S.C. at two sites. Five groups of five mice were immunized with 300 lug OVA in the following formulations: (1) adsorbed on 4.2 mg alum; (2) OVA-loaded 5050 PLG microparticles prepared by emulsification/solvent evaporation; (3) OVA-loaded 75:25 PLG microparticles; (4) 50:50 PLG microparticles (7 mg)/OVA mixture; or (5) 75:25 PLG microparticles (7 mg)/OVA mixture. Identical booster immunizations were administered to groups (4) and (5) 6 weeks after the primary immunization but animals in groups l-3 were not boosted. For groups 2 and 3 the required dose of freeze-dried microparticles was weighed and resuspended in 0.5 ml physiological saline immediately before administration. The characteristics of the OVA-loaded microparticles used for immunization are presented in Table 1 as 50:50 PLG 1 and 75:25 PLG 1, respectively. The PLG microparticle/OVA mixtures and the alum-adsorbed OVA preparation were incubated at room temperature for 24 h before administration. Blood samples were collected at periodic intervals up to 58 weeks for groups 1-3 and for up to 27 weeks for groups 4 and 5 by bleeding from the tail vein. Samples were centrifuged and the collected serum was stored frozen at -20°C until analysed by ELISA. The protocol for the preparation of the hyperimmune antiserum involved primary S.C.immunization with 100,~g OVA in FCA at two sites. This, was repeated 14 and 28 days later with OVA in Freunds incomplete adjuvant (FIA) and mice were bled by cardiac puncture 7 days after the final immunization. ELISA for measurement

of IgG

Levels of specific IgG anti-OVA antibody in test serum samples were assessed by an established ELlSA”. Results were standardized against a positive control

Vaccine 1996 Volume 14 Number 15 1431

Single dose microparticle-based Table 1 The characteristics

vaccines: A. G.A. Coombes et al.

of OVA-loaded

and blank PLG microparticles Sgace

OVA loaded 50:50 PLG

75:25 PLG

Blank 50:50 PLG 75:25 PLG

Total OVA (% w/w)

1 2 3

720.8e58.1 512.5rt32.8 575.Ok53.8

4.2 3.8 3.9

35.0*2.1 48.8k6.2 48.752.7

55.Ok6.6 58.8k7.2 64.1i3.0

50.8*9.9 61.9&l 59.224.6

1 :

893.Ok60.0 697.1 k62.5 764.8+46.7

4.2 4.3 4.1

35.1 i3.3 32.Ok2.4 31.9*3.2

43.4i1.6 37.8i4.0 39.6k7.0

46.Oe5.6 47.823.5 43.9&O

BCA

467.1k12.8 770.4k32.6

hyperimmune mouse antiserum. The ELISA was carried out as follows: microtitre plates (Nunc, InterMed Life Technologies, Glasgow, UK) were coated overnight with 100 ~1 per well of 5 ,ug ml-’ OVA in carbonatebicarbonate buffer, pH 9.6, washed three times in 200 ~1 of phosphate-buffered saline +O.OS% Tween-20 (PBST) and blocked for 2 h at 37°C with 100 ~1 of 1% bovine serum albumin (BSA). Serum samples (100 ~1) at four separate doubling dilutions from 1 in 400 and hyperimmune serum at 16 dilutions from 1 in 400 in PBST were added to wells and incubated overnight at 4°C. Plates were washed three times with PBST and 100 ~1 of peroxidase-conjugated sheep anti-mouse IgG (Sera-lab, Crawley Down, Sussex, UK), diluted 1:2000 in PBST, was added to wells and incubated at 37°C for 2 h. Plates were washed three times in PBST and 100 ~1 of chromagen (20 mg o-phenylenediamine and 20 ~1 hydrogen peroxide in 50 ml citrate-phosphate buffer, pH 5.0) was added to each well. The reaction was stopped after 5 min by the addition of 50~1 of 9M H,SO, and the absorbance values for each sample were read at 490 nm by a Titertek Multiscan ELISA reader. The results are expressed as antibody units calculated from the standard curve obtained from the hyperimmune mouse serum. The value for experimental serum samples at each of four dilutions falling in the linear portion of the standard curve was calculated as follows: OD of experimental sample at dilution a x 1000=Antibody units OD of hyperimmune serum at dilution a

Statistical analysis Results are expressed as the mean of the antibody units (measured for each sample at four dilutions) f S.E. for five mice. Means were compared using an unpaired Student’s t-test to assess statistical significance. Results were considered statistically significant if WO.05.

RESULTS Characteristics of microparticles The microparticle size and measurements of total protein loading and surface protein for OVA-loaded PLG microparticles are shown in Table 1. The particle size was sub-micron. The particle size measured for separate formulations of OVA-loaded 50:50 and 75:25 PLG microparticles (Table 1) gives an indication of the

1432

OVA (% total) Trypsin

Size (nm)

System

Vaccine 1996 Volume 14 Number 15

Figure 1 SEM of OVA-loaded 50:50 PLG microparticles

batch variation associated with the emulsification/ solvent evaporation technique used in these investigations. The scanning electron micrograph shown in Figure 1 reveals the good spherical form and smooth surfaces which are characteristic of both protein-loaded and blank (OVA-free) microparticles. The mean diameter of the blank microparticles (Table I) differed from that of the protein-loaded microparticles, reflecting the influence of protein on microparticle formulation. Protein loading and location The double emulsion technique used to prepare OVAloaded microparticles resulted in similar protein loading figures for the fast resorbing 50:50 PLG microparticles and the slow resorbing 75:25 PLG particles, lying in a fairly narrow range between 3.8 and 4.3% (w/w) (Table 1). Analysis of surface protein, however, revealed that a substantial part of the OVA content was associated with the surface of the microparticles. These levels are usually higher for the 50:50 PLG system (in the region of 60% as determined by trypsin treatment) compared with ca 40%) for the 75:25 PLG system. In contrast, analysis of the supernate after separation of microparticles from the PBS release medium at 24 h, resulted in detection of only 20% and 13% OVA for 50:50 PLG and 75:25 PLG, respectively (see Figure 2). Thus, only loosely bound surface protein is removed into the PBS release medium and a large component remains strongly bound to the microparticle surface over a 4 week incubation period.

Single dose microparticle-based

vaccines: A. G.A. Coombes et al.

75125 PLG

Opi

0

’

0 0

’

5

I

’

IO

’

’

1.5

’

’

20

I

’ / ’

25

30

I

’

35

I

IO

’

Figure 2 Cumulative protein release from OVA-loaded PLG microparticles prepared by emulsification/solvent evaporation (0, 0, batch 1; 0, W, batch 2)

The degradation of lactide polymers is generally considered to be unaffected by protease enzymes14 and as such, trypsin treatment presents a useful means of removing surface adsorbed protein. Russell-Jones and Jeffery” have reported, for example, the efficiency of simulated intestinal media in removing surface protein from PLG microparticles. In the present study, trypsin was found to be more effective than SDS in removing OVA from PLG microparticles (Table Z), which may indicate a degree of interpenetration of OVA molecules with the particle surface. Exposure of microparticle suspensions to the BCA reagent provides a powerful method for simultaneously removing and quantifying the levels of surface protein on microparticles. However, although the time of exposure in the present study was extremely short (2 min at room temperature) the possibility exists of hydrolytic surface degradation of PLG microparticles due to the highly alkaline conditions prevailing during the test. As a result, exposure of encapsulated protein to the reagents may occur, yielding an overestimate of the level of surface protein. The technique does, however, have potential for analysing protein distribution by controlled erosion of microparticles. Zfzvitro protein release 0 VA-loaded PLG microparticles. In vitro protein release profiles over 4 weeks are similar for OVA-loaded 50:50 PLG and 7525 PLG microparticles (Figure 2). A characteristic “burst release” of OVA occurs over the first 24 h which is more prominent for the 50:50 PLG microparticles and amounts, in this case, to a loss of ca 20% of the OVA initially associated with the particles. This initial phase has been well-documented and is generally accepted as arising from release of surface protein. Following the burst phase, loss of protein stabilizes after 1 week at a level around 25% and 17%, respectively, for 50:50 PLG and 75:25 PLG particles and is minimal from 14 weeks. MicroparticlelOVA mixtures Analysis of the amount of OVA adsorbed to blank PLG microparticles after mixing with OVA solutions at

30

40

so

60

71)

80

Days

40

Days

20

Figure

3

Cumulative

release of OVA from alum

room temperature revealed OVA levels of 1.2% and 0.8% (w/w) on 50:50 PLG and 75:25 PLG particles, respectively. These figures are consistent with the higher total developed (surface) area of the 50:50 PLG microparticles23. The cumulative protein release data obtained by incubating vaccine formulations prepared by simple mixing of PLG microparticles with OVA solutions (7 mg particles/300 pg OVA) revealed that only 40% and 30% of the initial protein component of the formulation remained associated with 50:50 PLG and 75:25 PLG microparticles, respectively, after 7 h in PBS at 37°C. No further loss of OVA was recorded over 6 weeks indicating retention of strongly bound protein at the microparticle surface. This behaviour indicates that only ca 120 pg and 90 rug, respectively, of an administered dose of 300 ,ug OVA can be expected to persist in contact with the microparticles and a sizeable fraction of the dose would be dispersed soon after injection. OVA adsorbed to alum The amount of OVA adsorbed on the alum substrate after incubation with OVA solution was significantly higher than that obtained using blank PLG microparticles. Complete adsorption of the available OVA had occurred giving rise to an association figure of 10.5% w/w. The cumulative protein release curve presented in Figure 3 gives an indication of the strength of association of OVA with the alum substrate. Gradual, almost linear release of adsorbed protein occured over 30 days in vitro. The release rate of OVA then tended to level off, resulting in just over 50% retention of the original protein load after 68 days. The presentation of strongly bound OVA (i.e. that remaining after burst release) at the surface of microparticles can be expected to influence the primary immune response. After 24 h, 120 pugand 80 ,ug of OVA, respectively. are estimated to lie at the surface of OVA-loaded 50:50 PLG and 75:25 PLG microparticles produced by solvent evaporation, which is similar to the levels of adsorbed OVA in PLG microparticle/OVA mixtures (Table 2). Immune response to OVA-loaded PLG microparticles and OVA-adsorbed onto alum The levels of specific IgG anti-OVA antibody in serum after administration of OVA-loaded 75:25 PLG micro-

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Table 2 Estimated surface OVA remaining in vaccine formulations after 24 h in vitro

OVA content (% w/w)

Surface OVA (% total)

Estimated surface OVA @g) remaining after 24 ha

50:50 PLG 75:25 PLG

4.0 4.2

60 40

120 80

50:50 PLG/OVA mixture 75:25 PLG/OVA mixture

1.2 0.8

100 100

120 90

OVA adsorbed to alum

10.5

100

290

System OVA-loaded OVA-loaded

DISCUSSION

alnitial OVA content: 300 pg

particles was significantly greater than that elicited by administration of OVA-loaded 50:50 PLG particles or OVA adsorbed on alum at all time points from weeks 2-34 (Figure 4). The levels of specific IgG following delivery of OVA-loaded 7525 PLG microparticles reached a maximal level 6 weeks after injection and remained relatively constant until 26 weeks after administration. Levels of antibody then declined gradually until week 47 and remained relatively constant from week 47 until week 58 when the study was terminated. After administration of OVA-loaded 50:50 PLG particles the levels of antibody induced reached a maximal level by 4/6 weeks after delivery. Thereafter a progressive decline was noted until week 58. At 4 weeks after administration the response to OVA-loaded 50:50 PLG microparticles was greater than that elicited by alumadsorbed OVA but at later time points, from week 6-34 after delivery, the differences between these groups were not significant. At weeks 47 and 58 after antigen administration the levels of antibody to OVA elicited by immunization with the OVA-loaded 50:50 PLG microparticles were less than those to alum-adsorbed OVA. Immune response to PLG microparticle/OVA

mixtures

Following primary immunization with OVA mixed with either 50:50 or 75:25 blank PLG microparticles very low levels of specific IgG were detected in serum. The antibody levels were significantly lower than those elicited by administration of OVA-loaded PLG microparticles prepared with the respective polymers (Figure 5). Boosting with the same formulations at week 6 significantly increased the responses in both groups. In contrast with the results obtained after immunization with OVA-loaded microparticles the levels of antibody elicited upon boosting with either 50:50 PLG or 75:25 PLG microparticle/OVA mixtures were not significantly different. A notable finding was the high degree of variability in the responses to microparticle/OVA mixtures which was considerably greater than was the case for OVA-loaded microparticles. The peak levels of antibody recorded after boosting with 50:50 PLG microparticle/OVA mixtures were not significantly different from the primary response to OVA-loaded 50:50 PLG microparticles prepared by emulsification/solvent evaporation (Figure 5). In contrast, the levels of IgG induced by 75:25 PLG microparticles/OVA mixtures even after boosting were considerably lower than the primary response to OVA-loaded 75:25 PLG microparticles. Furthermore unlike the response to OVA-

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Vaccine 1996 Volume 14 Number 15

loaded 75:25 PLG microparticles the response to both 50:50 and 75:25 PLG microparticles/OVA mixtures declined progressively from week 2-13 after boosting. However, the levels of serum antibody remained constant in the case of both of the latter systems from week 13-2 1 after boosting.

Adjuvants may act in a number of ways to improve the immune response for example by protecting antigens, stimulating phagocytosis, activating lymphoid cells and by retaining the antigen at the site of ‘depositionZ4. Antigen retention appears vital for repeated stimulation of the memory B-cell population and for maintaining antibody titres over long periods2’. The adjuvant effect of water-in-oil emulsions (FCA/FIA), for example, is considered to arise from the creation of a short-term “depot effect”. In contrast the considerable research effort on microparticle-based vaccines has generated a number of strategies based on optimizing antigen release rates to produce single dose delivery systems. For example, pulse release of antigen from biodegradable microparticles is considered advantageous for simulating the conventional, multi-dose vaccine delivery regime. However, most microparticulate delivery systems are considered to function on the principles of efficient phagocytosis and transport to the lymph nodes and sustained antigen release over extended time periods which may present a continuous trickle of antigen to the immune system. An initial pulse release of antigen (BSA) followed by continuous delivery from non-degradable systems has been found capable of sustaining high antibody responses for over 25 weeks following a single dose26. Raghuvanshi et 01.’ developed a single injection formulation for TT based on biodegradable PLG microparticles which resulted in immune responses over 5 months in rats comparable with the conventional twodose schedule of TT adsorbed on alum. The incremental rise in antibody titres was considered to result from sustained release of TT from microparticles presenting a small amount of antigen continuously to the immune system to stimulate immune cells and induce antibody production. The lower primary response observed when TT was adsorbed to alum was considered to be due to rapid antigen depletion and consequently reduced stimulation of immune cells. Alonso et al6 have added a further and necessary qualification, stressing the need for the release of sufficient concentrations of antigenitally intact TT to maintain high antibody levels. The potential for modulating the immune response to a protein antigen by altering formulation conditions has been confirmed by the present study. In particular a further illustration has been provided of the necessity for adequate dosing and correct antigen presentation for induction and maintenance of high antibody levels after a single s.c immunization. Administration of a single dose of OVA loaded-PLG microparticles containing 300 klg of OVA induced a considerable immune response (Figure 4). Moreover, the duration of the response corresponded closely with the resorption rate of the carrier. The peak antibody levels were maintained longer by the use of slow resorbing 75:25 PLG microparticles which result in sustained presentation of antigen to immuno-competent cells. O’Hagan et al.” measured a

Single dose microparticle-based

vaccines: A. G.A. Coombes et al w so:50 PLG 0 75:25 PLG 0 Alum

Week number Figure 4

Serum IgG levels following a single S.C. administration

80

0

50:50 PLG

q 7525 PLG

II

”

2

4

8

IO

I4

Boost Week number Figure 5 Serum IgG levels following S.C. administration of PLG microparticle/OVA mixtures. Booster doses were administered at 6 weeks

50% weight loss for 50:50 PLG microparticles in 25 days exhibited in vitro, while 75:25 PLG microparticles extended degradation times due to the increased lactide content of the copolymers with only 26% weight loss over 21 weeks. Based on the above findings, microparticles having resorption times in excess of 1 year would appear to offer opportunities for presentation of antigen over more extended time periods with, possibly, a corresponding increase in the duration of the immune

of: (1) OVA-loaded

microparticles;

and (2) OVA adsorbed to alum

response. The effectiveness of 75:25 PLG microparticles as an antigen carrier is underlined by the potent and extended immune response over 26 weeks which persists at a level equivalent to the peak response of 50:50 PLG microparticles for a further 20 weeks. In addition, while the peak response to OVA adsorbed on alum was comparable with that of OVA-loaded 50:50 PLG microparticles, it was considerably less than OVA-loaded 75:25 PLG particles. Gradual depletion of antigen as microparticle resorption occurs, could be one of the factors accounting for the eventual fall-off in antibody levels in the formulations tested. Gradual loss of immunogenicity as a result of antigen exposure to acid degradation products generated by the PLG carrier” is another possibility. Sustained antibody responses in mice have previously been measured by O’Hagan et ul.” following S.C. administration of a single dose of OVA-loaded microparticles. A total dose of 300 pug of OVA was injected (as in the present study) but using three populations of equally loaded microparticles, each type having a different degradation rate. The decrease in antibody levels to a lower but stable level at week 20 in that study may now be explained by rapid resorption of the 50:50 PLG component of the mixture and the reduced 75:25 PLG (slower resorbing) content which would be associated with at most 100 pg of OVA compared with 300 ,ug in the present study. In addition, the primary immune response started to fall away at week 6 for 50:50 PLG microparticles in the present study (Figure 4) compared with week 20 in the study of O’Hagan rf (11.The higher molecular weight of the 50:50 PLG microparticles produced by O’Hagan et al. may have resulted in a slower microparticle resorption rate. Further information on the threshold levels of OVA required to elicit high primary responses after s.c

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Single dose micropatticle-based

vaccines: A. G.A. Coombes et al.

administration is provided by the work of Uchida et a1.4 and O’Hagan et al3 who prepared OVA-loaded 50:50 PLG microparticles using a technique similar to that described here. For example, injection of 100 pg of OVA entrapped in PLG microparticles in mice resulted in extremely low primary immune responses although these were si nificantly higher than the response to soluble OVA B. Boosting doses were required at 6 weeks to elevate the serum antibody levels. The primary immune responses measured by Uchida et aL4 after administration of lower amounts of microencapsulated OVA (50 pug) resulted in antibody levels which were only two to four times higher than soluble OVA. However, in this case, boosting at 3 weeks was ineffective in raising the response. It appears from the above investigations that there is a clear dose response to OVA but in both previous studies the levels of retained surface protein on the microparticles and the influence on the immune response was not considered. Measurements of the amount of OVA associated with the surface of OVA-loaded PLG microparticles produced by solvent evaporation revealed high levels of surface protein amounting to ca 60% and 40% for the 50:50 PLG and 75:25 PLG systems, respectively (Table I). Thus, while an initial release of loosely bound surface protein occurs in vitro (Figure 2) a surface layer of strongly bound protein remains at the microparticle surface for at least 4 weeks. Verecchia el ~1.‘~ reported similar phenomena for L.PLA nanoparticles (150 nm) prepared using an emulsion/microfluidization/solvent evaporation technique with human serum albumin as a stabilizer. Adsorption/desorption studies suggested a multilayer model of protein association and the presence of an irreversibly bound albumin fraction accounting for 3545% of the originally associated protein. Verecchia et al.” considered that during nanoparticle production, the association of albumin with the particle surface probably occurred through an intermediate state involv ing accumulation then denaturation at the solvent/water interface. Production of protein-loaded microparticles by the emulsification/solvent evaporation technique investigated here will also result in protein exposure to droplets of solvent-swollen polymer. In the primary emulsion stage, PVA surfactant is absent and hydrophobic protein domains can be expected to orient preferentially at the surface of the droplets enhancing the prospect of hydrophobic interactions. Interpenetration of albumin molecules with the microparticle surface is also feasible. The presence of strongly bound albumin at the surface of microparticles invites a reassessment of the current models of protein release from resorbable PLG microparticles and has major implications for vaccine design, formulation and performance. In the present case a change of concept is clearly called for from release of entrapped antigens alone to one which encompasses loss of surface antigen with greater emphasis placed on the role of substrate stability and degradation in controlling the presentation and depletion of surface antigen and consequently the duration of the immune response. While exposure and release of encapsulated antigen from PLG microparticles could contribute to long term immunity, the possibility of the loss of immunogenicity, due to the generation of acid products as the PLG

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matrix degrades*‘, does render the general applicability of this mechanism for producing long-term immune responses highly questionable. In the case of vaccine formulations prepared by simple mixing of blank, pre-formed microparticles and OVA solutions, protein interaction with the substrate would be expected to differ from that existing in the solvent evaporation process. Protein adsorption and desorption from particulate substrates is governed by a complex interaction involving polar forces e.g. hydrogen bonding, hydrophobic interactions via the hydrophobic regions of protein molecules and weaker van der Waals forces and is influenced by the ionic strength and composition of the incubation medium28. Maximum albumin adsorption is generally observed on hydrophobic surfaces. The binding of multiple charged proteins to terminal carboxylic acid end groups of PLG is considered to impede release of encapsulated protein from PLG microparticles” and is also applicable to the case of protein desorption from micropartitle surfaces. Of particular relevance to the study reported here is the finding that serum albumin adsorbs in a multilayer mode with the layer adjacent to the surface being more strongly bound than the outermost layer which is consequently more easily removed by washing’-‘. The ability of retained surfactant molecules to change the surface hydrophobicity of PLG microparticles and consequently to influence protein adsorption has been considered by Alpar and Almeida”’ in relation to TT adsorption on PLA microparticles. In the present system of preformed PLG microparticle/OVA mixtures, the presence of PVA surfactant molecules at the microparticle surface will modulate and further complicate protein interaction with the microparticles. The simplest model would be one of steric hindrance and reduction of protein binding sites. However, protein interaction with PVA surfactant molecules may also play a significant role in protein retention at the surface. analogous to the protein binding effects of polyethylene oxidepolypropylene oxide block copolymers in emulsionbased vaccines3’. It has frequently been suggested that the higher immune response obtained when using OVA-loaded PLG microparticles could be attributed to an adjuvant (antigen retention/ presentation) effect rather than to slow release of encapsulated protein since antigens adsorbed onto microparticles have been shown to generate potent immune responses after s.c.~.~’and nasal administration3’. Conflicting reports do exist, however. In particular, Eldridge et a1.33 found that SEB mixed with blank PLG microparticles did not induce greater antibody responses than immunization with SEB alone. More recently, Uchida et a1.4 reported the absence of an immune response on administration of a mixture of OVA and blank PLG microparticles, although no details of dose or dosing regimen were provided. In contrast, O’Hagan et aL3 measured similar serum IgG antibody responses over 6 weeks following boosting, when 100 pg of OVA was either encapsulated in PLG microparticles or adsorbed to blank PLG microparticles. A finding that PLG microparticles can confer greater immunogenicity on adsorbed labile antigens after a single dose would certainly be welcomed since harsh encapsulation procedures involving high shear rates and exposure to organic solvents (factors which may

Single dose microparticle-based

denature some antigens) would be avoided. Concern also exists over the acid degradation products generated by PLG microparticles which could denature some encapsulated proteins. Antigen adsorption would also provide a means of avoiding loss of antigenicity due to irradiation of vaccine formulations for the purpose of sterilization’. Uncertainties exist, however, over protection and desorption of adsorbed antigens in viva. In agreement with the results of O’Hagan er ul.“, the present study has confirmed that a single dose of 90-120 pug of OVA adsorbed onto pre-formed, PLG microparticles is inadequate to induce a high primary response but serum antibody levels are raised significantly on boosting (Figure 5). The presentation of strongly bound OVA (i.e. that remaining after burst release) at the surface of OVA-loaded microparticles can be expected to exert a major influence on the primary immune response. In the present study, 120 ,ug and 80 ,ug of OVA, respectively, are estimated to lie at the surface of OVA-loaded 5030 PLG and 75:2.5 PLG microparticles produced by solvent evaporation, which is similar to the levels of adsorbed OVA in PLG microparticle/OVA mixtures (Table 2). However, the considerable primary immune response obtained for OVA-loaded PLG microparticles and the low primary response in animals immunized with OVA adsorbed on PLG microparticles suggests that the total OVA content (core and surface) influences the immune response in the former case. Alternatively, the presentation of surface protein in OVA-loaded particles may differ from that in the adsorbed system, for example as a result of exposure to solvent-swollen polymer during microparticle production, and subsequently influence the immune response. The ability of OVA-loaded PLG microparticles < 10 pm in size, to function as potent antigen delivery systems after S.C. administration is considered to arise from two mechanisms:

(1) efficient phagocytosis resulting in transport to the lymph nodes where antigen processing and presentation to T-helper cells occurs; and (2) controlled release of antigen from the microparticle?.

on microparticles may be beneficial in increasing particle hydrophilicity’4 and thus prolonging the process of uptake by macrophages. Consequently, this condition may result in persistence of microparticles in s.c. sites where they provide a depot for antigen. Malarial antigen, for example, has been detected at the injection site 80 days after administration to rats when formulated with liposomes and encapsulated in alginate poly( L-lysine) microparticles” providing further support for antigen-loaded microparticles functioning as “depot-type” systems for sustained retention and presentation of antigens to the immune system.

ACKNOWLEDGEMENTS This work was supported by the Medical Research Council, UK, the Welkome Trust and the World Health Organization.

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Microparticles < 10 pm in diameter with entrapped antigen (SEB) have been reported to induce more potent antibody responses than microparticles 10 pm due to the ability of the smaller microparticles to be efficiently phagocytosed and transported to the draining lymph nodes33. However, high immune responses have also been induced when using large (72 pm) protein-loaded microparticle? demonstrating that phagocytosis and transport to lymph nodes is not absolutely necessary for achieving high serum antibody titres, although it was recognized that antigen-containing fragments removed from large microparticles could be phagocytosed. The sub-micron particles used in the present study would be expected to be rapidly phagocytosed, stimulating a primary immune response. However, the close correlation between the duration of the immune response measured and the in vitro degradation rate of PLG microparticles suggests that substrate resorption by hydrolysis is largely controlling the immune response through its effect on antigen retention and presentation. The surface presence of both OVA and PVA molecules

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