Controlled release microparticles for vaccine development

D.T. O ' H a g a n * , H. Jeffery, M.J .J . Rober t s , J .P. M c G e e and S.S. Davis

The primary and secondary sera IgG antibody responses to ovalbumin ( 0 VA) entrapped in biodegradable poly( lactide-co-ctlycolide) ( PLG A ) microparticles were compared with the responses obtained with soluble 0 VA. In addition, 0 VA in PLGA microparticles was also a~hninistered aJ?er dispersion in an immunostimulatory vehicle, Freund's incomplete adjuvant (FIA). The primary I,qG re,wonses to OVA in microparticles/FIA were signi.l~cantly greater than the responses m soluble OVA fi 'om day 14 to day 42, when booster immuni=ations were administered. From day 49 to the end q/" the study at day 84, the responses to OVA, both in microparlicles alone and in microparticles/FIA, were significantly greater than the responses to soluble OVA. Nevertheless, the responses ohtained /br 0 VA in microparticles or microparticles/FIA were, in ,qeneral, not as high as those obtained with 0 VA in Freuml's complete adjuvant.

Keywords: Biodcgradable microparticles: vaccine: ovalbumin

I N T R O D U C T I O N

Worldwide~ there is currently considerable interest in the development of biodegradable microparticles as controlled release vaccines, since the major disadvantage of several currently available vaccines is the need for repeated administration 1. Controlled release vaccines could obviate the need for booster immunizations and help to overcome this problem. Clearly, controlled release vaccines would be particularly advantageous in the Third World where repeated contact between the health-care worker and the vaccine recipient is often difficult to achieve 1

Over the years, there have been several reports describing the adjuvant effect achieved by the association of antigens with microparticles. Kreuter and Speiser 2 described the use of polymethylmethacrylate nano- particles as adjuvants for incorporated inactivated influenza virus. Subsequently, the adjuvant effect of the insoluble particles was related to their physicochemical characteristics 3'~. In addition, the adjuvant effect of microparticles has also been demonstrated by Artursson et al. ~ using polyacryl starch microparticles, by Schroder and Stahl ~ using crystallized dextran nanospheres and Martin et al. ~ using polymerized serum albumin beads. Alternatively, Preis and Langer 8 described the use of an ethylene vinyl acetate (EVA) copoiymer implant as a controlled release antigen delivery system. However, the EVA implant was non-degradable and required surgical removal after completion of the immunization process.

Department of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK. *To whom correspondence should be addressed. (Received 5 April 1991; revised 15 May 1991; accepted 21 May 1991)

0264--410X/91/100768-04 ( 1991 Butterworth-Heinemann Ltd

768 Vaccine, Vol. 9, October 1991

Consequently, biodegradable poly(CTTH-iminocarbon- ate) polymeric implants were developed which degraded in vivo to release tyrosine derivatives 9, since tyrosine posseses innate adjuvanticity 1°.

However, although these studies have highlighted the tremendous potential of controlled release technology in vaccine development, acceptability by regulatory authorities remains a significant obstacle for any 'novel' antigen delivery system. Aluminium hydroxide remains the only adjuvant approach for use in humans 11 Therefore, we have adopted an alternative approach to the development of controlled release antigen delivery systems and have entrapped antigens in microparticles prepared from poly(lactide-co-glycolide) (PLGA) polymers. PLGA polymers are biodegradable and biocompatible polyesters which are non-toxic and have been used as resorbable sutures for many years 12. Furthermore, the excellent tissue compatibility of PLGA microparticles has been demonstrated by Visscher et al.13 PLGA polymers have been extensively investigated as controlled release drug delivery systems for many years 14 and several PLGA based systems are currently licensed for administration to humans.

In this preliminary study, we have investigated the immunogenicity of a model protein antigen, ovalbumin (OVA), entrapped in PLGA microparticles, following subcutaneous administration to rats. Since a previous brief report indicated that PLGA microparticles alone did not serve as an effective primary vaccine, but functioned well as a booster vaccine 15, PEGA micro- particles were also administered after emulsification in an immunostimulatory vehicle, Freund's incomplete adjuvant (FIA). In addition, OVA was administered in Freund's complete adjuvant (FCA) for comparison.

MATERIALS AND M E T H O D S

Animals

Male Wistar rats weighing about 200 g were used and maintained on a normal rat diet throughout the study.

Microparticle preparation

Microparticles with entrapped OVA (Grade V, Sigma Chemical Company, Poole, Dorset) were prepared using a PLGA polymer which was obtained as a gift from Boehringer Ingelheim KG (Resomer RG503, Ingelheim, Germany) and the microparticles were prepared using a water-in-oil-in-water (WOW) solvent evaporation tech- nique. Briefly, a 6% w/v solution of the polymer in dichloromethane (HPLC grade, May and Baker, Dagen- ham) was emulsified together with a 6% w/v solution of OVA in double distilled water using a Silverson homogenizer (Silverson Machines Ltd, Chesham, Bucks) to produce a water-in-oil emulsion. This emulsion was then added to a much larger volume of an aqueous solution of 5% w/v polyvinyl alcohol (PVA) (88% hydrolysed, Aldrich Chemical Company, Poole) and homogenized to produce a stable WOW emulsion. The double emulsion was then stirred overnight at ambient temperature and pressure to allow solvent evaporation to proceed, with resultant microparticle formation. Full details of the effect of formulation variables on OVA incorporation efficiency and microparticle size will be published elsewhere (Jeffery, Davis and O'Hagan, unpublished results).

Following preparation, the microparticles were col- lected by centrifugation, washed three times to remove non-entrapped OVA and freeze-dried. The protein content of the microparticles was determined in a bicinchoninic acid protein assay (Sigma), after dissolution of an aliquot of the microparticles in dichloromethane. The microparticles contained an average of 1% w/v OVA and the volume mean diameter of the microparticles, as measured by laser diffractometry, was 5.34 #m (Malvern laser sizer 2600D).

Immunization protocols

Immediately before administration, the required dose of freeze-dried microparticles (~ 10 mg PLGA micro- particles per rat, containing 100 #g OVA) was weighed and resuspended in physiological saline (SAL).

Four groups of eight rats were each immunized with 100#g OVA (1) dissolved in SAL, (2) entrapped in PLGA microparticles, (3) entrapped in PLGA microparticles and dispersed in FIA, or (4) emulsified in FCA. Booster immunizations of OVA in identical vehicles were administered to each study group 6 weeks (day 42) after the primary immunization, except for group 4, which received a booster dose of OVA emulsified in FIA.

Blood samples were collected from the tail vein of the rats weekly for 12 weeks following primary immunization. The blood samples were centrifuged and sera were collected and stored frozen at -20°C until assayed by ELISA.

Measurement of lgG by ELISA

The specific anti-OVA IgG antibody content of each serum sample was determined in an ELISA and standard-

Biodegradable microparticles as vaccines: D.T. O'Hagan et al.

ized against a positive control antiserum obtained by hyperimmunizing a male Wistar rat with OVA in FCA/FIA. The hyperimmunization schedule involved three intraperitoneal injections at days 0, 14 and 28 of OVA (100 #g in FCA for primary injection and 100/tg in FIA for booster injections) and a blood sample was collected by cardiac puncture 7 days after the final injection; serum was obtained as described above.

The ELISA was performed as follows; microtitre plates (Dynatech M 129B) were coated overnight with 190 #1 per well of OVA 10 #g ml- 1 in carbonate buffer, pH 9.8; they were washed three times in phosphate-buffered saline, pH 7.4 (PBS) containing 0.05% Tween 20 (T20) and blocked for 2 h at room temperature with 0.3% T20 in PBS (215 #1 per well). Serum samples (120/21) from the study animals were added to the wells at four dilutions (1:500, 1:1000, 1:2000 and 1:4000) in T20/PBS and incubated overnight at 4°C. The hyperimmunization serum samples were incubated under the same conditions at eight different dilutions on each plate ranging from 1:250 to 1:32000. The plates were washed three times in PBS/T20 and 110#1 sheep anti-rat IgG horseradish peroxidase conjugate (Serotec, Kidlington, Oxford) diluted 1:6400 in T20/PBS was added to the wells and incubated at 37°C for 2h (the conjugate was pre- adsorbed with OVA 0.01% w/v prior to use). The plates were washed three times in PBS/T20 and 100#1 of o-phenylene diamine 0.4mgm1-1 (Sigma) in citrate/ phosphate buffer (pH 5) containing 0.4 #1 ml - 1 hydrogen peroxide was added to each well. The reaction was stopped within 40 min by the addition of 50/11 4M H2SO 4 per well and the plates were read at 492 nm in a Titertek Multiscan plus ELISA reader.

The results are expressed as antibody units calculated from the standard curve obtained from the hyperimmune mouse serum diluted between 1:250 and 1:32000, the value for each serum sample dilution falling in the standard curve and the value for the sample taken as the mean of the four separate dilutions of that sample.

Statistical analysis

The results are expressed as mean + s.e. for eight rats. An unpaired Student's t-test was used to compare the means for each study group at the different sample times and to assess statistical significance. Results were considered statistically significant if p <0.05.

RESULTS

Microparticles alone

The primary IgG antibody responses to OVA entrapped in PLGA microparticles were significantly greater than the responses to soluble OVA 14 and 21 days after immunization. There were no significant differences in the responses at days 28 through to 42, but the responses to the microencapsulated antigen remained higher. Following secondary immunization, the IgG responses to OVA in microparticles remained significantly greater than the responses to soluble OVA from day 49 to day 84 when the study was terminated (Figure I).

Microparticles in Freund's incomplete adjuvant

Both the primary and the secondary IgG responses to OVA in PLGA microparticles dispersed in FIA remained

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Biodegradable microparticles as vaccines: D.T. O'Hagan et al.

3000

200C

a c

~ooc

14 21 28 35 42 49 56 63 70 77 84 Study day

Figure 1 The sera IgG antibody responses to primary and secondary subcutaneous injections of 100 fig ovalbumin administered to four groups of rats: (1) m, dissolved in physiological saline (soluble OVA), (2) ~ , entrapped in poty (O,L-lactide-co-glycolide) microparticles (PLGA particles), (3) I~, entrapped in PLGA microparticles and dispersed in Freund's incomplete adjuvant (PLGA/FIA) or (4) [~, emulsified in Freund's complete adjuvant (FCA). All four groups were boosted after 6 weeks with 100 Hg ovalbumin in the same vehicles as the primary injection, except group 4, which was boosted with OVA in FlA. Each column represents the mean responses _+s.e. of eight animals

significantly greater than the responses to soluble OVA from day 14 to the completion of the study at day 84.

The primary lgG antibody responses to OVA in microparticles/FIA were significantly greater than the responses to OVA in micropartieles alone from day 14 through to day 42, while the secondary IgG antibody responses to OVA in micropartides/ 'FIA were significantly greater than the responses to OVA in microparticles alone from day 56 through to day 70 and at day 84 when the study was terminated (Figure I).

Freund's complete adjuvant

The responses obtained by OVA in FCA were generally greater than those obtained by OVA in microparticles and microparticles/FIA. Only at day 56 was the response to OVA in microparticles/FIA greater than the response to OVA in FCA and the difference was not significant (Figure I ).

D I S C U S S I O N

The results obtained in this study are in general agreement with the earlier findings of Altman and Dixon ~5, who investigated the immunogenicity in mice of a peptide entrapped in PLGA microparticles. Altman and Dixon 1~ reported that microencapsulated peptide elicited weak primary antibody responses, unless they were dispersed in immunost imulatory vehicles such as FlA. However, the microencapsulated peptide induced efficient secondary immune responses in the absence of additional vehicles. In our study also, potent primary antibody responses were obtained only when the PLGA microparticles were administered after dispersion in FIA. Nevertheless, PLGA microparticles alone were able to induce potent secondary immune responses when ad- ministered in the absence of F1A (Fixture 1). The

dispersion of PLGA microparticles in FIA resulted m significantly greater secondary immune responses than those obtained with microparticles alone. Hence, the findings of this study indicate that microencapsulated antigen alone may not function as an efffcctive primary vaccine, but may serve well as a vaccine for booster injections. However, effective primary immtinization may be achieved by the dispersion of microparticles in immunostimulatory vehicles such as FlA.

The responses obtained with microencapsulated OVA alone were generally much less than thosc obtained with OVA in FCA (Figure 1 ). This was a predictable finding. Nevertheless, a previous study in mice using the same batch of microparticles had indicated that micro- encapsulated OVA was capable of inducing antibody responses that were greater than, or at least comparable to, those induced by FCA TM. The reasons why such differences in response were obtained in these similar studies in two closely related species are as yet unknown. The immune responses to microparticles dispersed in FIA were generally comparable to the responses obtained by FCA, which was an encouraging finding. Interestingly, the responses to microparticles in FIA appeared to be still climbing at the termination of the study, while the responses to FCA had peaked earlier (Figure 11. This may reflect controlled release from the microparticles dispersed in FlA.

Although PLGA microparticles alone may not function as an effective primary vaccine, the results obtained here indicated that PLGA microparticles dispersed in FIA may be more effective. Many papers reviewing the use of FIA in humans have been published and trials involving more than one million vaccinees have demon- strated the safety and efficacy of FIA ~ ~. However, reports of the appearance of tumours in mice administered mineral oil adjuwlnts resulted in the curtailing of the use of FIA in humans ~ ~'. Nevertheless, two follow-up studies of 10 and 18 years duration in 18000 recipients of FIA influenza vaccines have shown no evidence of increased tumour formation ~ and interest in the use of FIA in humans as an adjuwint for an inactivated HIV vaccine has recently been revived ~s

It should be noted that the PLGA polymer selected for microparticle preparation for this preliminary study (molecular weight ~ 9000, 50:50 ratio of lactide:glycolide) was deliberately chosen to degrade relatively rapidly and to release the entrapped antigen over several wecks. Rapid antigen release was appropriate, since this study was simply designed to assess the immttnogenicity of a protein antigen entrapped in PLGA microparticles. No attempt was made to develop a delivery system suitable for the induction of long-lasting immunity. This will be the objective of later studies, in which alternative PLGA polymers with slower degradation kinetics will be used for microparticle preparation. PLGA microparticles release entrapped drugs by one or more mecfianislns as a function of time, but the release of macromolecules is mainly controlled by bulk degradation of the polymer ~ e. Consequently, depending on copolymer composition and molecular weight, PLGA microparticles can be prepared that will release antigens over a period of weeks to morc than 1 year. Thercforc, mixed populations of PLGA microparticles can bc prepared for simultaneous injection, with different polymeric compositions and wlriable molecular weights, which can be designed to release antigens at predetermined intervals to mimic booster

770 Vaccine, Vol. 9, October 1991

immunizations. The exciting potential of these controlled release antigen delivery systems to induce potent long-term immune responses to clinically relevant anti- gens remains to be further assessed, as does the possible requirement for immunostimulatory vehicles in which the microparticles may need to be dispersed.

In conclusion, PLGA microparticles are extremely flexible delivery systems capable of encapsulating a wide range of antigens, advances in pharmaceutical technology may allow the development of safe, single-dose vaccines against a number of infectious diseases. Finally, since particulates are taken up across the intestine through Peyer's patches after oral administration 19, controlled release microparticles can also be used for oral immuniz- ation z°'21. We are currently assessing the ability of PLGA microparticles to act as antigen delivery systems following oral administration, and initial results are very encouraging.

A C K N O W L E D G E M E N T S

Vaccine development research at Nottingham is currently funded by the Medical Research Council AIDS Directed Programme and the World Health Organisation. Miss Jeffery is the recipient of a research studentship from the Science and Engineering Research Council. The authors are grateful to Dr Daniel Rafferty for his valuable assistance with the ELISA.

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