Immunology 1992 76 164-168
Enhanced secretory IgA and systemic IgG antibody responses after oral immunization with biodegradable microparticles containing antigen S. J. CHALLACOMBE, D. RAHMAN, H. JEFFERY, S. S. DAVIS & D. T. O'HAGAN Departments of Oral Medicine and Pathology, UMDS, Guy's Hospital, London and Department of Pharmaceutical Sciences, University of Nottingham
Acceptedfor publication 8 January 1992
SUMMARY Intragastric immunization may lead to the induction of antibodies in the secretory immune system including saliva. The antibody response is usually short-lived. The objectives of this study were to see whether oral immunization with biodegradable microparticles containing antigen might lead to enhanced mucosal responses. Ovalbumin (OVA) was entrapped in a novel antigen delivery system comprising poly (D,L-lactide-co-glycolide) (PLGA) microparticles. Salivary IgA and serum IgG responses after three daily oral immunizations in BALB/c mice were assayed by ELISA at weekly intervals and compared with those to soluble antigen. Low levels of salivary IgA antibodies were detected at Weeks 2 and 3 in both groups and no significant differences were found. After a secondary series of intragastric immunizations at Week 4, marked differences were apparent between the groups. The mean salivary IgA titre at Week 6 was 959 + 494 U compared with 30 + 5 in the soluble OVA group (P < 000001). Significant differences were still apparent at Weeks 7-8 though the value was falling. Serum IgG antibodies were detectable and were significantly greater in the particle group (at Weeks 4 and 8) than in controls (P < 0.001). These results suggest that microparticles are taken up by antigen-presenting cells in Peyer's patches, then slowly degrade in vivo and release entrapped antigens, and thus can function as potent antigen delivery systems giving rise to both mucosal and systemic responses. Microparticles have considerable potential as a controlled released antigen delivery system for the induction of longer-term immune responses at mucosal surfaces.
INTRODUCTION Diseases of the mucosa are amongst the most common affecting man. It is probable that most bacterial and viral diseases gain access to the host via mucosal tissues and these include human immunodeficiency virus (HIV) and cholera. The mucosal system is known to be quite different from the systemic immune system both in terms of its main immunoglobulin, its method of stimulation and its function. The most common method of immunization of the secretory immune system in both animals and humans has been administration of the antigen in capsules or by gastric intubation in order to initiate immune responses via the gut-associated lymphoid tissues (GALT). This method has been shown to result in the induction of secretory antibody at various mucosal sites in the absence of stimulation of serum Abbreviations: BSA, bovine serum albumin; FCA, Freund's complete adjuvant; FIA, Freund's incomplete adjuvant; HPLC, high performance liquid chromatography; i.p., intraperitoneal; OVA, ovalbumin; PBS, phosphate-buffered saline; PLGA, poly (D,L-lactideco-glycolide); s.c., subcutaneous; T20, Tween 20. Correspondence: Professor S. J. Challacombe, Dept. of Oral Medicine and Pathology, UMDS, Guy's Hospital, London SE1 9RT, U.K.
antibody. These sites include: salivary glands,' the gut,4 the eye,25 the lungs,6 the vagina78 and mammary glands.9"0 The
potential of this route of immunization is therefore enormous. There seems no doubt that secretory IgA antibodies may function at mucosal surfaces in a manner which is independent of complement fixation or opsonization. " In secretions, IgA can prevent micro-organisms from adherence to or penetrating mucosal surfaces.'2-'4 Secretory IgA has also been shown to be able to neutralize toxins'5 and viruses'6 and modulate enzyme activity'7 or IgG-modulated antibody-dependent cellular cytotoxicity (ADCC).'8 However studies both in humans and animals have shown that the secretory immune responses are generally of low titre and are short lived.2'4"9 In both humans2 and in rhesus monkeys'9 even secondary series of immunizations led to responses only slightly greater than primary. It is envisaged however that long-term protection of mucosal surfaces would require longer-lasting immune responses. Due to the limited absorption of desired antigens from the gut lumen of orally immunized individuals repeated large doses of antigens seem to be required for an effective IgA response. Thus there is a need for antigen delivery systems which might result in such long-lived resoonses and which may not only make efficient use of antigen
164
Oral immunization with biodegradable microparticles but deliver it to the gut-associated lymphoid tissue over long time periods. One approach to overcome the problems of inducing longlasting secretory immune responses is the use of controlled release antigen delivery systems.20 In the studies described here we have entrapped a model antigen ovalbumin (OVA) in microparticles prepared from a biodegradable polymer, poly (D,L-lactide-co-glycolide) (PLGA). The biocompatibility of such PLGA microparticles has been demonstrated;2' these polymers undergo biodegradation by random enzymatic scission to form the endogenous metabolites lactic and glycolic acids. Such polymers have been used for many years as surgical sutures22 and because of this biodegradability and excellent tissue compatibility PLGA microparticles have been used as drug delivery systems.23 PLGA microparticles can be prepared that release antigens over a period of days to more than 1 year and thus the antigen delivery could be modified to allow the development of a variety of safe single-dose vaccines against a number of infectious diseases affecting mucosal surfaces. There is now much evidence of particle uptake into Peyer's patch (PP).24 Particles appear to be taken up by M cells, and can be found in the Peyer's patch for up to several weeks in the mouse.25 Since Peyer's patches are the source of IgA precursor B cells26 it could be hypothesized that a longer presence of antigen in PP might lead to increased stimulation and release of antigen-specific B cells. Previous studies in the rat using OVA in microparticles were encouraging.27 The objective of this work was to determine whether PLGA microparticles could potentiate the induction of secretory IgA antibody responses to a model poor immunogen after oral immunization using a small animal model.
MATERIALS AND METHODS Animals Male BALB/c mice (Olac Ltd, Cirencester, Oxon, U.K.) aged up to 6-8 weeks and weighing about 25 g were used and maintained on a normal mouse diet throughout the study.
Microparticle preparation Microparticles with entrapped OVA (Grade V; Sigma, Poole, Dorset, U.K.) were prepared using PLGA 50:50 ratio lactideco-glycolide, 22,000 MW obtained from Boehringer Ingelheim KG (Resomer RG506, Ingelheim, Germany). The microparticles were prepared by solvent evaporation from a water in oil emulsion as reported in ref. 23 and adapted for entrapment of macromolecules as previously reported.24 A 6% w/v solution of antigen was emulsified with a 6% solution of polymer in dichloromethane (DCM) using a Silverson homogenizer at high speed (Silverson Machines Limited, Chesham, Bucks, U.K.). The resulting water in oil emulsion was then emulsified with an aqueous continuous phase containing 10% w/v Polyvinyl alcohol (PVA) (88% hydrolysed, Aldrich Chemical Company, Poole, Dorset, U.K.). Following overnight evaporation of the volatile solvent the microparticles were collected by centrifugation, washed three times in water to remove non-entrapped OVA and freeze dried. The protein content of the microparticles was determined in a Bicinchoninic acid protein assay (BCA; Sigma), after dissolution of an aliquot of the microparticles in dichloromethane (HPLC Grade, May & Baker, Dagenham, U.K.). A known weight of microparticles
165
was dissolved in DCM, approximately 3 ml distilled water was added to the sample and shaken for 30 min to extract the OVA. The aqueous layer was retrieved following centrifugation and placed in a 10-ml volumetric flask. The process was repeated twice and the supernatants combined to give 10 ml and the total protein concentration determined using BCA. The microparticles contained an average of 4-6% w/w OVA. The volume mean diameter of the microparticles as measured by laser diffractometry was 3 Mm (Malvern laser sizer 2600D). Immunization protocols Immediately before administration, the required dose of freezedried microparticles (- 20 mg PLGA microparticles per mouse containing 1 mg OVA) was weighed and resuspended in the appropriate volume of physiological saline (SAL) (0 5 ml). Primary immunization. Two groups of 10 mice were each intragastrically immunized on 3 consecutive days28 with 1 mg OVA either entrapped in PLGA microparticles or dissolved in saline. Secondary immunization. Four weeks after the primary immunizations the two groups were reimmunized on 3 consecutive days with microparticles, or antigen in saline.
Samples Blood samples were collected from the tail veins of the mice at the fourth and eighth weeks following primary immunization. Saliva samples were collected at weekly intervals to 8 weeks following an intraperitoneal injection of 50 p1 of pilocarpine (0 5% w/v).28 Assay of salivary IgA antibodies The specific anti-OVA IgA antibody content of each individual saliva sample was determined in an established ELISA assay and standardized against a positive control antiserum obtained by hyperimmunization of mice with OVA in Freund's complete adjuvant (FCA).29 The ELISA was performed as follows: microtitre plates (Dynatech M 129B, Billingshurst, Kent, U.K.) were coated overnight with 100 ,l per well of OVA 2 pg/ml (shown previously to be an optimal concentration for assay of saliva samples), they were washed three times at 370 with 0-5% bovine serum albumin (BSA) and 0 05% Tween 20 (T20) in phosphate-buffered saline (PBS) 200 pl per well. Saliva samples (100 pl) at four separate dilutions from 1:10 in BSA/T20/PBS were added to the wells and incubated overnight at 4°. The plates were washed three times in PBS and 100 1l sheep antimouse IgA (Evai-Bios, Petworth, West Sussex, U.K.) diluted 1:250 in BSA/T20/PBS was added to the wells and incubated at 370 for 2 hr. Serum samples (100 ,l) at four separate dilutions from 1:200 in BSA/T20/PBS were added to the wells and incubated overnight at 4°. The plates were washed three times in PBS and 100 p1 sheep anti-mouse IgG (Evai-Bios) and diluted 1:2000 in BSA/T20/PBS were added to wells and incubated overnight at 4°. The plates were washed three times in PBS and 100 p1 anti-sheep IgG alkaline phosphatase conjugate (Sigma) was added to the wells at 1/300 in BSA/T20/PBS and incubated at 370 for 1 hr. The plates were washed with PBS and 100 pl ofpnitrophenyl phosphate [one tablet in 5 ml of diethanolamine buffer, (Sigma)] was added to each well. The reaction was stopped after 20 min by the addition of 50 pl 3 M NaOH per well and the plates were read at 405 nm in an ELISA reader (Biorad, Hemel Hempstead, Herts, U.K.).
S. J. Challacombe et al.
166
Mean Ab U to OVA (2y)
Mean Ab U to OVA (1y) 1000
800 600 400
200 0
Figure 1. Salivary IgA antibodies after oral immunization with microparticles. Groups of 10 BALB/c mice were orally immunized 3 consecutive days with OVA in PLGA microparticles at Week 0 and after Week 4. The specific anti-OVA IgA antibody content of each individual saliva was determined by ELISA (see Materials and Methods). ly, primary immunization; 2y, secondary immunization. Microparticles (-); soluble Ag (0). on
The results were expressed as antibody units calculated from the standard curve obtained from the hyperimmune mouse serum given an arbitrary antibody value of 2 x 106 IgG antibody U/ml and 100,000 IgA antibody U/ml. This serum was double diluted from 1/2000 to 1/64,000 to give an IgG standard curve and from 1/100 to 1/3200 for an IgA standard curve. The value for each dilution of saliva falling in the standard curve was taken and the value for the sample calculated as the mean of the four separate dilutions. Statistical analysis
The results are expressed as mean + SE for 10 mice. 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
Primary immunization In animals immunized with antigen in saline the mean salivary IgA antibodies to ovalbumin were below 10 antibody U/ml for Weeks 1 and 2 and rose to a mean of 20 U at 3 weeks and 30 U at 4 weeks (Fig. 1). This was significantly greater than the preimmune values (P < 0-02). Similar results were found with the PLGA particles and the mean salivary IgA values at Weeks 3 and 4 were 30 and 40 U respectively (Fig. 1). These values were not statistically different from the soluble antigen group. The percentage of animals responding (giving an OD above mean background+2 SD) was also similar between the two groups with eight of the 10 animals in each group responding at Week 3 and nine of 10 animals in each group responding at Week 4. Repeat immunization The antibody levels detected after secondary immunizations were dramatically different. At week 5 (since the beginning of the experiment and I week after the secondary immunizations), the values were similar to those seen at Week 4. However at Week 6 (which was 2 weeks after the secondary immunizations)
Soluble OVA at 4 weeks Soluble OVA at 8 weeks OVA in particles at 4 weeks OVA in particles at 8 weeks 0
50
o1000 1500 2000 2500 Mean IgG Ab U (x 100)
Figure 2. Serum IgG antibodies to ovalbumin after oral immunization with microparticles. BALB/c mice were orally immunized with OVA on 3 consecutive days with PLGA microparticles at Week 0 and after Week 4. Serum samples were collected at Weeks 4 and 8. The specific antiOVA IgG antibody content of each individual serum was determined by ELISA (see Materials and Methods).
value was found in the microparticle group of 950 + 494 U/ml compared with less than 30 in the soluble antigen group (P
Enhanced secretory IgA and systemic IgG antibody responses after oral immunization with biodegradable microparticles containing antigen S. J. CHALLACOMBE, D. RAHMAN, H. JEFFERY, S. S. DAVIS & D. T. O'HAGAN Departments of Oral Medicine and Pathology, UMDS, Guy's Hospital, London and Department of Pharmaceutical Sciences, University of Nottingham
Acceptedfor publication 8 January 1992
SUMMARY Intragastric immunization may lead to the induction of antibodies in the secretory immune system including saliva. The antibody response is usually short-lived. The objectives of this study were to see whether oral immunization with biodegradable microparticles containing antigen might lead to enhanced mucosal responses. Ovalbumin (OVA) was entrapped in a novel antigen delivery system comprising poly (D,L-lactide-co-glycolide) (PLGA) microparticles. Salivary IgA and serum IgG responses after three daily oral immunizations in BALB/c mice were assayed by ELISA at weekly intervals and compared with those to soluble antigen. Low levels of salivary IgA antibodies were detected at Weeks 2 and 3 in both groups and no significant differences were found. After a secondary series of intragastric immunizations at Week 4, marked differences were apparent between the groups. The mean salivary IgA titre at Week 6 was 959 + 494 U compared with 30 + 5 in the soluble OVA group (P < 000001). Significant differences were still apparent at Weeks 7-8 though the value was falling. Serum IgG antibodies were detectable and were significantly greater in the particle group (at Weeks 4 and 8) than in controls (P < 0.001). These results suggest that microparticles are taken up by antigen-presenting cells in Peyer's patches, then slowly degrade in vivo and release entrapped antigens, and thus can function as potent antigen delivery systems giving rise to both mucosal and systemic responses. Microparticles have considerable potential as a controlled released antigen delivery system for the induction of longer-term immune responses at mucosal surfaces.
INTRODUCTION Diseases of the mucosa are amongst the most common affecting man. It is probable that most bacterial and viral diseases gain access to the host via mucosal tissues and these include human immunodeficiency virus (HIV) and cholera. The mucosal system is known to be quite different from the systemic immune system both in terms of its main immunoglobulin, its method of stimulation and its function. The most common method of immunization of the secretory immune system in both animals and humans has been administration of the antigen in capsules or by gastric intubation in order to initiate immune responses via the gut-associated lymphoid tissues (GALT). This method has been shown to result in the induction of secretory antibody at various mucosal sites in the absence of stimulation of serum Abbreviations: BSA, bovine serum albumin; FCA, Freund's complete adjuvant; FIA, Freund's incomplete adjuvant; HPLC, high performance liquid chromatography; i.p., intraperitoneal; OVA, ovalbumin; PBS, phosphate-buffered saline; PLGA, poly (D,L-lactideco-glycolide); s.c., subcutaneous; T20, Tween 20. Correspondence: Professor S. J. Challacombe, Dept. of Oral Medicine and Pathology, UMDS, Guy's Hospital, London SE1 9RT, U.K.
antibody. These sites include: salivary glands,' the gut,4 the eye,25 the lungs,6 the vagina78 and mammary glands.9"0 The
potential of this route of immunization is therefore enormous. There seems no doubt that secretory IgA antibodies may function at mucosal surfaces in a manner which is independent of complement fixation or opsonization. " In secretions, IgA can prevent micro-organisms from adherence to or penetrating mucosal surfaces.'2-'4 Secretory IgA has also been shown to be able to neutralize toxins'5 and viruses'6 and modulate enzyme activity'7 or IgG-modulated antibody-dependent cellular cytotoxicity (ADCC).'8 However studies both in humans and animals have shown that the secretory immune responses are generally of low titre and are short lived.2'4"9 In both humans2 and in rhesus monkeys'9 even secondary series of immunizations led to responses only slightly greater than primary. It is envisaged however that long-term protection of mucosal surfaces would require longer-lasting immune responses. Due to the limited absorption of desired antigens from the gut lumen of orally immunized individuals repeated large doses of antigens seem to be required for an effective IgA response. Thus there is a need for antigen delivery systems which might result in such long-lived resoonses and which may not only make efficient use of antigen
164
Oral immunization with biodegradable microparticles but deliver it to the gut-associated lymphoid tissue over long time periods. One approach to overcome the problems of inducing longlasting secretory immune responses is the use of controlled release antigen delivery systems.20 In the studies described here we have entrapped a model antigen ovalbumin (OVA) in microparticles prepared from a biodegradable polymer, poly (D,L-lactide-co-glycolide) (PLGA). The biocompatibility of such PLGA microparticles has been demonstrated;2' these polymers undergo biodegradation by random enzymatic scission to form the endogenous metabolites lactic and glycolic acids. Such polymers have been used for many years as surgical sutures22 and because of this biodegradability and excellent tissue compatibility PLGA microparticles have been used as drug delivery systems.23 PLGA microparticles can be prepared that release antigens over a period of days to more than 1 year and thus the antigen delivery could be modified to allow the development of a variety of safe single-dose vaccines against a number of infectious diseases affecting mucosal surfaces. There is now much evidence of particle uptake into Peyer's patch (PP).24 Particles appear to be taken up by M cells, and can be found in the Peyer's patch for up to several weeks in the mouse.25 Since Peyer's patches are the source of IgA precursor B cells26 it could be hypothesized that a longer presence of antigen in PP might lead to increased stimulation and release of antigen-specific B cells. Previous studies in the rat using OVA in microparticles were encouraging.27 The objective of this work was to determine whether PLGA microparticles could potentiate the induction of secretory IgA antibody responses to a model poor immunogen after oral immunization using a small animal model.
MATERIALS AND METHODS Animals Male BALB/c mice (Olac Ltd, Cirencester, Oxon, U.K.) aged up to 6-8 weeks and weighing about 25 g were used and maintained on a normal mouse diet throughout the study.
Microparticle preparation Microparticles with entrapped OVA (Grade V; Sigma, Poole, Dorset, U.K.) were prepared using PLGA 50:50 ratio lactideco-glycolide, 22,000 MW obtained from Boehringer Ingelheim KG (Resomer RG506, Ingelheim, Germany). The microparticles were prepared by solvent evaporation from a water in oil emulsion as reported in ref. 23 and adapted for entrapment of macromolecules as previously reported.24 A 6% w/v solution of antigen was emulsified with a 6% solution of polymer in dichloromethane (DCM) using a Silverson homogenizer at high speed (Silverson Machines Limited, Chesham, Bucks, U.K.). The resulting water in oil emulsion was then emulsified with an aqueous continuous phase containing 10% w/v Polyvinyl alcohol (PVA) (88% hydrolysed, Aldrich Chemical Company, Poole, Dorset, U.K.). Following overnight evaporation of the volatile solvent the microparticles were collected by centrifugation, washed three times in water to remove non-entrapped OVA and freeze dried. The protein content of the microparticles was determined in a Bicinchoninic acid protein assay (BCA; Sigma), after dissolution of an aliquot of the microparticles in dichloromethane (HPLC Grade, May & Baker, Dagenham, U.K.). A known weight of microparticles
165
was dissolved in DCM, approximately 3 ml distilled water was added to the sample and shaken for 30 min to extract the OVA. The aqueous layer was retrieved following centrifugation and placed in a 10-ml volumetric flask. The process was repeated twice and the supernatants combined to give 10 ml and the total protein concentration determined using BCA. The microparticles contained an average of 4-6% w/w OVA. The volume mean diameter of the microparticles as measured by laser diffractometry was 3 Mm (Malvern laser sizer 2600D). Immunization protocols Immediately before administration, the required dose of freezedried microparticles (- 20 mg PLGA microparticles per mouse containing 1 mg OVA) was weighed and resuspended in the appropriate volume of physiological saline (SAL) (0 5 ml). Primary immunization. Two groups of 10 mice were each intragastrically immunized on 3 consecutive days28 with 1 mg OVA either entrapped in PLGA microparticles or dissolved in saline. Secondary immunization. Four weeks after the primary immunizations the two groups were reimmunized on 3 consecutive days with microparticles, or antigen in saline.
Samples Blood samples were collected from the tail veins of the mice at the fourth and eighth weeks following primary immunization. Saliva samples were collected at weekly intervals to 8 weeks following an intraperitoneal injection of 50 p1 of pilocarpine (0 5% w/v).28 Assay of salivary IgA antibodies The specific anti-OVA IgA antibody content of each individual saliva sample was determined in an established ELISA assay and standardized against a positive control antiserum obtained by hyperimmunization of mice with OVA in Freund's complete adjuvant (FCA).29 The ELISA was performed as follows: microtitre plates (Dynatech M 129B, Billingshurst, Kent, U.K.) were coated overnight with 100 ,l per well of OVA 2 pg/ml (shown previously to be an optimal concentration for assay of saliva samples), they were washed three times at 370 with 0-5% bovine serum albumin (BSA) and 0 05% Tween 20 (T20) in phosphate-buffered saline (PBS) 200 pl per well. Saliva samples (100 pl) at four separate dilutions from 1:10 in BSA/T20/PBS were added to the wells and incubated overnight at 4°. The plates were washed three times in PBS and 100 1l sheep antimouse IgA (Evai-Bios, Petworth, West Sussex, U.K.) diluted 1:250 in BSA/T20/PBS was added to the wells and incubated at 370 for 2 hr. Serum samples (100 ,l) at four separate dilutions from 1:200 in BSA/T20/PBS were added to the wells and incubated overnight at 4°. The plates were washed three times in PBS and 100 p1 sheep anti-mouse IgG (Evai-Bios) and diluted 1:2000 in BSA/T20/PBS were added to wells and incubated overnight at 4°. The plates were washed three times in PBS and 100 p1 anti-sheep IgG alkaline phosphatase conjugate (Sigma) was added to the wells at 1/300 in BSA/T20/PBS and incubated at 370 for 1 hr. The plates were washed with PBS and 100 pl ofpnitrophenyl phosphate [one tablet in 5 ml of diethanolamine buffer, (Sigma)] was added to each well. The reaction was stopped after 20 min by the addition of 50 pl 3 M NaOH per well and the plates were read at 405 nm in an ELISA reader (Biorad, Hemel Hempstead, Herts, U.K.).
S. J. Challacombe et al.
166
Mean Ab U to OVA (2y)
Mean Ab U to OVA (1y) 1000
800 600 400
200 0
Figure 1. Salivary IgA antibodies after oral immunization with microparticles. Groups of 10 BALB/c mice were orally immunized 3 consecutive days with OVA in PLGA microparticles at Week 0 and after Week 4. The specific anti-OVA IgA antibody content of each individual saliva was determined by ELISA (see Materials and Methods). ly, primary immunization; 2y, secondary immunization. Microparticles (-); soluble Ag (0). on
The results were expressed as antibody units calculated from the standard curve obtained from the hyperimmune mouse serum given an arbitrary antibody value of 2 x 106 IgG antibody U/ml and 100,000 IgA antibody U/ml. This serum was double diluted from 1/2000 to 1/64,000 to give an IgG standard curve and from 1/100 to 1/3200 for an IgA standard curve. The value for each dilution of saliva falling in the standard curve was taken and the value for the sample calculated as the mean of the four separate dilutions. Statistical analysis
The results are expressed as mean + SE for 10 mice. 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
Primary immunization In animals immunized with antigen in saline the mean salivary IgA antibodies to ovalbumin were below 10 antibody U/ml for Weeks 1 and 2 and rose to a mean of 20 U at 3 weeks and 30 U at 4 weeks (Fig. 1). This was significantly greater than the preimmune values (P < 0-02). Similar results were found with the PLGA particles and the mean salivary IgA values at Weeks 3 and 4 were 30 and 40 U respectively (Fig. 1). These values were not statistically different from the soluble antigen group. The percentage of animals responding (giving an OD above mean background+2 SD) was also similar between the two groups with eight of the 10 animals in each group responding at Week 3 and nine of 10 animals in each group responding at Week 4. Repeat immunization The antibody levels detected after secondary immunizations were dramatically different. At week 5 (since the beginning of the experiment and I week after the secondary immunizations), the values were similar to those seen at Week 4. However at Week 6 (which was 2 weeks after the secondary immunizations)
Soluble OVA at 4 weeks Soluble OVA at 8 weeks OVA in particles at 4 weeks OVA in particles at 8 weeks 0
50
o1000 1500 2000 2500 Mean IgG Ab U (x 100)
Figure 2. Serum IgG antibodies to ovalbumin after oral immunization with microparticles. BALB/c mice were orally immunized with OVA on 3 consecutive days with PLGA microparticles at Week 0 and after Week 4. Serum samples were collected at Weeks 4 and 8. The specific antiOVA IgG antibody content of each individual serum was determined by ELISA (see Materials and Methods).
value was found in the microparticle group of 950 + 494 U/ml compared with less than 30 in the soluble antigen group (P
