Controlled release microparticles for vaccine development D . T . O ' H a g a n * , H. Jeffery, M . J . J . R o b e r t s , J.P. M c G e e a n d S.S. D a v i s
The primary and secondary sera IgG antibody responses to ovalbumin ( 0 VA) entrapped in biodegradable poly( lactide-co-ctlycolide) ( P L G A ) microparticles were compared with the responses obtained with soluble 0 VA. In addition, 0 VA in P L G A microparticles was also a~hninistered aJ?er dispersion in an immunostimulatory vehicle, Freund's incomplete adjuvant (FIA). The primary I,qG re,wonses to O V A in microparticles/FIA were signi.l~cantly greater than the responses m soluble O V A 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 O V A , both in microparlicles alone and in microparticles/FIA, were significantly greater than the responses to soluble O V A . 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
INTRODUCTION 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 nanoparticles 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-iminocarbonate) polymeric implants were developed which degraded in vivo to release tyrosine derivatives9, 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 microparticles 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.
Biodegradable microparticles as vaccines: D.T. O'Hagan et al.
MATERIALS AND METHODS
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 technique. Briefly, a 6% w/v solution of the polymer in dichloromethane (HPLC grade, May and Baker, Dagenham) 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 collected 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
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 preadsorbed 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
The primary and secondary sera IgG antibody responses to ovalbumin ( 0 VA) entrapped in biodegradable poly( lactide-co-ctlycolide) ( P L G A ) microparticles were compared with the responses obtained with soluble 0 VA. In addition, 0 VA in P L G A microparticles was also a~hninistered aJ?er dispersion in an immunostimulatory vehicle, Freund's incomplete adjuvant (FIA). The primary I,qG re,wonses to O V A in microparticles/FIA were signi.l~cantly greater than the responses m soluble O V A 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 O V A , both in microparlicles alone and in microparticles/FIA, were significantly greater than the responses to soluble O V A . 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
INTRODUCTION 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 nanoparticles 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-iminocarbonate) polymeric implants were developed which degraded in vivo to release tyrosine derivatives9, 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 microparticles 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.
Biodegradable microparticles as vaccines: D.T. O'Hagan et al.
MATERIALS AND METHODS
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 technique. Briefly, a 6% w/v solution of the polymer in dichloromethane (HPLC grade, May and Baker, Dagenham) 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 collected 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
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 preadsorbed 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
