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Oral immunization of mice with ricin toxoid vaccine encapsulated in polymeric microspheres against aerosol challenge
Vaccine 20 (2002) 1681–1691
Oral immunization of mice with ricin toxoid vaccine encapsulated in polymeric microspheres against aerosol challenge Meir Kende∗ , Changhong Yan, John Hewetson, Matthew A. Frick, Wayne L. Rill, Ralph Tammariello United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, MD 21702-5011, USA Received 21 November 2000; received in revised form 1 August 2001; accepted 10 October 2001
Abstract Mucosal (oral) immunization of mice with carrier-delivered ricin toxoid (RT) vaccine was accomplished by one long (7 weeks) or two short (4 weeks) immunization schedules. For the long and short immunization schedule two lots of vaccine were administered prepared with the same procedure but at different occasions. The long schedule consisted of a total of seven doses of 50 g of vaccine in microencapsulated (lot #108) or aqueous form administered on days 1, 2, 3, 28, 29, 30 and 49. With the short schedule a total of seven or six doses of 25 g (lot #111) were administered on days 1, 2, 3, 14, 15, 16 and 30, or on 1, 2, 14, 15, 30, 31 and 32, respectively. Mice immunized orally with the long schedule, 50 g of RT vaccine incorporated into poly-dl-lactide-co-glycolyde (dl-PLG) microspheres (MS) produced serum IgG, IgG2a and IgA ELISA antibodies. All mice immunized with RT in dl-PLG MS (RT-MS) were protected against a lethal ricin aerosol challenge. In contrast, with the same schedule and with the same dose, the aqueous vaccine (RT) failed to stimulate IgG, IgG2a and IgA antibodies, and these mice were not protected against an aerosol ricin toxin challenge. With the shorter immunization scheme, seven doses of 25 g RT-MS stimulated a significant, though reduced, protection with the microencapsulated, but not with the aqueous vaccine. When the first and second 3-day cycles of the short immunization schedule was reduced to two doses, and the 3-day cycle was administered at the end of the schedule, neither RT-MS nor RT stimulated protection against the challenge. These results indicated that successful oral immunization with RT-MS depended on both the dose and the schedule, consisting of three consecutive days of administration in two cycles, 4 weeks apart. Altering this schedule and the dose, resulted in a reduced protection or no protection at all. Furthermore, under the conditions of this study, the advantage of the microencapsulated RT vaccine over the aqueous vaccine for effective oral immunization was well demonstrated. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Oral immunization; Ricin toxoid; Polymeric microspheres; Humoral immune response; Protection
1. Introduction Antigen presentation via the intestinal tract possesses all the prerequisites for simple, safe and effective oral immunization. Although, the mucosal surface of the gastrointestinal tract represents a large area, only the ileum with its neutral pH has the proper environment for effective presentation of antigens via orally delivered vaccines. The dominant immunoglobulin at the mucosal surface of the gastrointestinal tract is secretory IgA (sIgA) [1,2] produced in the lamina propria regions underlying the mucous membranes [3,4]. Secretory immunity can be achieved with some, but not with every aqueous antigen by administering the immunogen to the gut-associated lymphoid tissue, of which the largest mass is represented by the Peyer’s patches of the gastrointestinal tract. The Peyer’s patches ∗
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are covered by unique epithelial cells, the M cells, which possess high pinocytotic activity that allows the transport of the antigen into the antigen processing cells of the underlying dome region, with subsequent stimulation of B and T lymphocytes. Numerous studies have demonstrated that for oral stimulation of high immunoglobulin levels frequent administration of high doses of the aqueous vaccine is required [5–7], which may render oral immunization with the aqueous vaccine impractical. Studies with orally administered biodegradable polymeric microspheres (MS) have demonstrated the usefulness of this approach for effective stimulation of the immune response with lower doses of antigen [8–13]. BALB/c mice immunized perorally with three doses of 100 g of formalinized staphylococcal enterotoxin B (SEB) incorporated into dl-PLG MS stimulated circulating IgG, IgA and IgM anti-SEB antibodies in the serum, and a concurrent mucosal IgA response in lung lavage, gut-washing and in the saliva [8]. Parallel oral immunization with doses of aqueous unencapsulated
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SEB toxoid ranging from 1 to 100 g was ineffective at inducing a detectable immune response in any of the above test fluids. A single priming dose of 0.1 mg ovalbumin encapsulated in MS made of derivatized alpha-amino acids, induced ovalbumin-specific IgG antibodies in the sera, sIgA in the intestinal secretions, and a consequent antigen-dependent proliferation of splenic CD4+ T cells [9]. In the same study, enhancement of the protective efficacy of the vaccine by co-encapsulated cholera toxin was demonstrated against infectious bursal virus infection in chickens. Multiple oral immunization of mice with ovalbumin in dl-PLG MS resulted in ovalbumin-specific cytotoxic lymphocyte activity and an ovalbumin-specific intestinal IgA response [10]. For primary immunization the mice received by gastric intubation ovalbumin entrapped in dl-PLG MS or aqueous albumin on three consecutive days, and the 3-day cycle was repeated as a secondary boost 4 weeks later [10]. Although, the cytotoxic lymphocyte subset induced by ovalbumin in MS was not characterized, it is presumed to be the conventional class I MHC-restricted cytotoxic T lymphocytes. In this study, the target cells of ovalbumin-sensitized T lymphocytes do not express class II MHC antigens. The responses stimulated by microencapsulated ovalbumin appear very similar to those found after immunization with ovalbumin in ISCOM adjuvant, which are known to be due to CD4+ T cells [11,12]. The demonstration that ovalbumin in dl-PLG MS can stimulate cytotoxic T lymphocyte activity and prime a variety of systemic and mucosal responses has important implications in viral vaccine development. The drawback of those studies in which ovalbumin was employed is the lack of in vivo challenge; therefore, no conclusion can be made regarding the protective efficacy of the elicited immune response. Recently, we reported on the stimulation of mucosal immunity via intranasal administration of ricin toxoid (RT) vaccine encapsulated in dl-PLG MS [13]. Mice immunized intranasally were completely protected for at least 1 year against lethal ricin challenge delivered by aerosol. In the present study, we explored the oral route of immunization to evoke anti-ricin immunity by microencapsulated vaccine (RT-MS). The efficacy of dl-PLG-encapsulated toxoid was compared to the efficacy of the aqueous vaccine.
2. Materials and methods 2.1. Polymers Polymers dl-PLG of 50:50 lactide to glycolyde ratio with a molecular mass of 72 kDa was purchased from Medisorb Technologies International L.P., Cincinnati, OH. Ricin toxin was supplied by Vector Laboratories, Burlingame, CA. The Bio-Rad protein assay kit was purchased from Bio-Rad Laboratories, Richmond, CA. Polyvinyl alcohol (PVA) (31–50 kDa, 88% hydrolyzed) was purchased from Aldrich, Milwaukee, WI.
2.2. Formalin-inactivated ricin toxoid vaccine Ricin was diluted to 1 mg ml−1 in phosphate-buffered saline (PBS) and dialyzed against 4.2% formaldehyde at 47 ◦ C for 18 h and at 42 ◦ C for 30 h. The dialysis was continued against distilled water at 4 ◦ C, with 12 changes over 4 days. After centrifugation at 600 × g for 20 min, the precipitate was resuspended in an equal volume of distilled water and centrifuged. The supernatant was lyophilized and stored at −40 ◦ C until its use. 2.3. Microencapsulaton of RT vaccine PLG MS containing RT vaccine were prepared by a water-in-oil-in-water (w/o/w) solvent extraction procedure as reported previously [14]. Briefly, 100 l of an aqueous RT solution (protein concentration 50 mg ml−1 ) was added to 200 mg of PLG in 1 ml of methylene chloride. The mixture was emulsified with a Vortex mixer to form the water-in-oil (w/o) emulsion. This emulsion was then added to 10 ml of 2% aqueous PVA and was emulsified by a Bronson sonicator probe at output 4 (50 W) for 30 s to form the w/o/w emulsion. The organic solvent was extracted by adding 100 ml of 5% isopropanol, and the mixture was stirred at a moderate speed at ambient temperature for 30 min. The RT-loaded MS were centrifuged at 5000 × g for 5 min and washed three times with Milli-Q-grade water (Millipore, Bedford, MA). The MS were then lyophilized and stored at 4 ◦ C. Core loading of RT in MS was determined by digesting the MS and assaying their protein content. Briefly, a known amount of MS (∼20 mg) was suspended in 2 ml of 0.5 N sodium hydroxide, and the suspension was stirred at ambient temperature for about 20 min. When the MS were completely hydrolyzed, the transparent hydrolysate solution was immediately neutralized to pH 7.4 with HCl. The protein content of the solution was determined by the Bio-Rad protein assay. The diameter of the MS was determined with a Coulter Multisizer II (Coulter ELectronics Ltd., Luton, Beds, UK). Two lots of MS (#108 and #111) were prepared for the study. The mean volume size of two MS lots and the percent size distribution are plotted in Fig. 3. The encapsulation efficiency and the core loading of lot #108 was 35 and 1.17%, respectively. In study 2, lot #111 of the microencapsulated RT vaccine was used for oral and subcutaneously (s.c.) immunization. The encapsulation efficiency and the core loading of lot #111 was 62 and 2.51%, respectively. 2.4. Immunization and challenge studies Female, 8-week-old, NIH Swiss or CD1 mice were obtained from the National Cancer Institute, Frederick Research and Development Center, Frederick, MD and Charles River Inc., Wilmington, MA, respectively. Doses of 50 or 25 g of RT, either in MS resuspended in Mili-Q-grade water or in water, were administered into the stomach of the
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mice (8–10 per group) by a blunt-tipped gavage needle on days specified below. For about 6 h prior to oral immunization no food was given to the mice to reduce the vaccine reflux and the bowel movement. In schedule 1 of study 1, 50 g of lot #108 microencapsulated or aqueous RT was given orally in 0.2 ml volume on days 1, 2, 3, 28, 29, 30 and 49. Polymer mass of 4.22 mg was needed to deliver 50 g RT. A single dose of 15 g of RT in MS or in water was administered s.c. in 0.1 ml volume to separate group of mice on day 1 to assess the potency of the lot. In study 2, a lower dose and two shorter immunization schedules were used. The mice were immunized orally with 25 g of RT in MS or in water in a volume of 0.2 ml on days 1, 2, 3, 14, 15, 16 and 30; or on days 1, 2, 14, 15, 30, 31 and 32. Polymer mass of 0.965 mg was needed to deliver 25 g RT. A single dose of 15 g of RT in MS or in water was administered s.c. in 0.1 ml to a separate group of mice on day 1. Immunized and control mice were challenged on the specified day with an estimated inhaled dose of 60 g/kg of ricin toxin (∼15 LD50 ) [15]. The mice were exposed to ricin in a dynamic, whole-body exposure chamber with a total system airflow rate of 19.5 l/min. The aerosol was sampled during the entire exposure period of 10 min in sterile saline, and the protein concentration of the sample was determined by protein analysis (Pierce Micro BCA, Pierce Labs, Rockford, IL). Exposed mice were observed daily for 3–4 weeks postchallenge. Efficacy was established with survival as endpoint. Statistical significance was calculated by Fisher’s two-tailed exact test. 2.5. Determination of antibodies by ELISA Blood samples were collected from the tail veins of the mice at the times indicated in the appropriate experiment. The levels of anti-ricin IgG, IgG2a and IgA in the serum of each mouse were measured by direct ELISA according to
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previously published methods by using 0.25 g per well of ricin as the capture antigen [13,14]. Briefly, to the two-fold serial dilutions or to a single 1:2500 dilution of 50 l of the test serum, 50 l of peroxidase-labeled anti-mouse IgG (Kirkegard and Perry Laboratories, Gaithersburg, MD) or IgA or IgG2a (ICN Biomedicals Inc., Irvine, CA) was added. After addition of 50 l of o-phenylenediamine (OPD) substrate (Sigma, St. Louis, MO), the plates were incubated for 20 min at room temperature, 10 l stop buffer (0.1 N NaOH) was added, and the absorbency of each well was read at 405 nm with ELISA plate reader (Model MR 600, Dynatech Co., Chantilly, VA). Antibody levels were expressed as the reciprocal of the geometric mean titer (GMT) based on the last serum dilution with an o.d. twice that of the control (normal) serum on the same dilution. In that case, the P value is 0.01, provided that the o.d. of the normal serum is ∼0.1–0.2. In titrations of immune and normal serum, the initial serum dilution was 1:16, and if that dilution was negative, the serum’s base-line titer was taken as 1:8. The P values of the GMS were calculated with the Wilcoxon nonparametric procedure.
3. Results 3.1. Study 1: oral immunization with higher dose of RT administered on extended schedule 3.1.1. Stimulation of IgG antibodies The anti-ricin total IgG isotype antibodies in the serum was assayed with a single dilution (1:2500) level (Fig. 1). Based on previous studies, the mice were protected against ricin challenge with an anti-ricin IgG titer of 1:2500; therefore, as a quick reference for the immune status of the mice, only a single serum dilution of 1:2500 was used. Only s.c. administration of the microencapsulated RT
Fig. 1. Mean o.d. of anti-ricin serum IgG (1:2500 dilution) in mice immunized orally with 50 g of RT in MS (RT-MS) or in aqueous solution on days 1, 2, 3, 28, 29, 30 and 49; or s.c. on day 1 with 15 g of RT-MS or aqueous vaccine (n = 6).
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Fig. 2. Geometric mean anti-ricin serum IgG2a titers in mice immunized orally with 50 g of RT in MS (RT-MS) or in aqueous solution on days 1, 2, 3, 28, 29, 30 and 49; or s.c. on day 1 with 15 g of RT-MS or aqueous vaccine (n = 5–6).
stimulated IgG antibodies at 4 weeks (o.d. = 0.706±0.014), which remained unchanged at 7 (o.d. = 0.785 ± 0.041) and 10 weeks (o.d. = 0.793 ± 0.032). Orally administered RT in MS induced IgG antibodies at 7 weeks, (o.d. = 0.475 ± 0.052) which were increased by 10 weeks (o.d. = 0.612 ± 0.055). In contrast, aqueous RT did not stimulate ricin-specific IgG, either by single s.c. or by multiple oral administrations (o.d. = 0.149 ± 0.012, and 0.167 ± 0.030, respectively). 3.1.2. Stimulation of IgG2a antibodies Anti-ricin serum IgG2a immunoglobulin titer was determined by terminal dilution at 4, 7 and 10 weeks after administration of the first oral dose (Fig. 2). By the 4, 7 and the 10 weeks bleedings, three, six and seven oral doses were administered, respectively. At 4 weeks, a single dose of parentally administered RT-MS stimulated IgG2a antibodies, yielding a GMT of 1:1783 ± 205, which remained at that level for 10 weeks (1:1351 ± 324). A single s.c. administration of RT-solution did not stimulate this subisotype at all during 10 weeks of observation. With oral administration, RT stimulated IgG2a immunoglobulins when it was incorporated into the MS: 1:128 ± 43 and 1:446 ± 202 GMT at 7 and 10 weeks, respectively. At 10 weeks, the GMT of 1:446 ± 202 stimulated by oral administration of RT-MS was not significantly different (P = 0.08) than the four-fold higher GMT stimulated by RT-MS injected parentally (1:1783 ± 205). Orally administered RT-solution did not stimulate anti-ricin IgG2a immunoglobulins, except for a small marginal GMT of 1:21 ± 13 at the 10th week. The size distribution of lot #108 used in study 1 was quite favorable; close to 75% of the MS had a size distribution less than 11.4 m, and 50% of the MS sizes were below 5.1 m (Fig. 3).
3.1.3. Stimulation of serum IgA antibodies The anti-ricin IgA isotype in the serum was assayed with a single dilution (1:80) level (Fig. 4). A significant amount of serum IgA (twice the mean o.d. of the normal serum) was stimulated only with orally administered RT-MS: 0.342 ± 0.059 and 0.342 ± 0.033 at 7 and 10 weeks, while the control serum o.d. were: 0.049 ± 0.011 and 0.048 ± 0.009, respectively. By definition, at 7 and 10 weeks, twice, or almost twice, the levels of IgA were stimulated by oral administration of aqueous RT (0.147 ± 0.014 and 0.109 ± 0.011), and by s.c. administration of microencapsulated (0.142 ± 0.035 and 0.095 ± 0.021) or by the aqueous (0.085 ± 0.016 and 0.112 ± 0.027) RT. However, at 7 and 10 weeks, the o.d. of the serum control from naive mice was at least twice lower than the o.d. of the control serum at 4 weeks (0.129). The lower o.d. value accounts for defining the GMT as positive in those mice that were treated orally or s.c. with the aqueous RT, or s.c. with RT-MS. Furthermore, with the exception of the orally administered RT-MS, the o.d. values in all other immunization groups did not increase at all by 7 and 10 weeks relative to the o.d. at 4 weeks. 3.1.4. Resistance of immunized mice to lethal aerosol challenge In study 1, the mice were challenged 10 weeks after the first oral dose (day 1), which was 3 weeks after the seventh (last) oral administration of the microencapsulated or the aqueous vaccine (Fig. 5). All nonimmunized control mice (n = 9) died within 3 days. All mice (n = 9) immunized orally with seven doses of 50 g of RT-MS survived the lethal aerosol ricin challenge. In contrast, none of the mice (n = 10) that were immunized orally with seven doses of 50 g aqueous RT survived. Immunization by s.c. with a single dose of aqueous vaccine failed to protect any mice
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Fig. 3. Mean volume size (m) and size distribution of lots #108 and 111 dl-PLG MS containing RT vaccine.
Fig. 4. Mean o.d. of anti-ricin serum IgA (1:80 dilution) in mice orally immunized with 50 g of RT in MS (RT-MS) or in aqueous solution on days 1, 2, 3, 28, 29, 30 and 49; or s.c. on day 1 with 15 g of RT-MS or aqueous vaccine (n = 6).
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Fig. 5. Resistance of mice to aerosol ricin challenge 10 weeks postimmunization elicited orally with 50 g of RT in MS (RT-MS) or in aqueous solution on days 1, 2, 3, 28, 29, 30 and 49; or s.c. on day 1 with 15 g of RT-MS or aqueous vaccine (n = 9–10).
(n = 10), however a single s.c. dose of 15 g RT-MS protected all mice (n = 10). 3.2. Study 2: Immunization with reduced dose and shorter schedule 3.2.1. Stimulation of IgG antibodies The mice were immunized orally with seven or six doses of 25 g of RT in MS, or in an aqueous solution using two schedules on days 1–3, 14–16, and 30 (schedule A), or on
days 1, 2, 14, 15, and 30–32 (schedule B), respectively. The two schedules differed somewhat in the length of administration, but mainly at the timing and the number of the 3-day cycle (Fig. 6). The total IgG antibody response assayed with a single dilution (1:2500) exhibited the pattern observed for IgG2a (see Fig. 7) with the exception of the IgG response stimulated by the aqueous RT (Fig. 6). Compared to the o.d. (0.269 ± 0.049) of the serum of the nonimmunized mice, aqueous RT administered orally by schedule A also stimulated statistically significant IgG responses
Fig. 6. Mean o.d. of anti-ricin IgG (1:2500 dilution) of mice 6 weeks after multiple oral immunization with 25 g of RT vaccine in MS (RT-MS) or in aqueous solution; or s.c. with a single dose of 15 g of RT-MS or aqueous vaccine (n = 6). The respective schedules are indicated in the figure.
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Fig. 7. Anti-ricin IgG2a titers in the sera of mice 6 weeks after multiple oral immunization with 25 g of RT vaccine in MS (RT-MS) or in aqueous solution; or s.c. with a single dose of 15 g of RT-MS or aqueous vaccine (n = 6). The respective schedules are indicated in the figure.
Fig. 8. Resistance of mice to aerosol ricin challenge 6 weeks postimmunization stimulated orally with multiple doses of 25 g of RT vaccine in MS (RT-MS) or in aqueous solution; or s.c. with a single dose of 15 g of RT-MS or aqueous vaccine (n = 8–10). The respective schedules are indicated in the figure.
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(o.d. = 0.684 ± 0.199) as did the RT-MS with schedule A (o.d. = 0.710 ± 0.261) but not with schedule B (o.d. = 0.459 ± 0.137). 3.2.2. Stimulation of IgG2a antibodies Oral administration of 25 g of RT-MS under schedule A stimulated seven-fold higher IgG2a antibodies at 42 days than the RT-MS stimulated by schedule B (Fig. 7). With schedules A and B the GMTs stimulated by RT-MS were: 1:114 ± 330 and 1:16 ± 12, respectively (P = 0.03). The aqueous RT administered orally by schedules A and B stimulated no (1:8) or very low GMT (1:13 ± 5) of IgG2a antibodies, respectively. Thus, RT-MS administered with schedule A stimulated significantly higher IgG2a antibodies than the aqueous RT did with the same schedule (P = 0.0026). However, there was no difference in the IgG2a antibodies stimulated by RT-MS or the aqueous antigen when the 3-day cycle was administered under schedule B. In comparison with the aqueous RT, a single s.c. dose of 15 g of RT-MS stimulated 21-fold higher IgG2a antibodies, a GMT of 1:1625 ± 256 and 1:72 ± 159, respectively (P = 0.0057). In comparison with RT-MS administered orally with schedule A, s.c. administration of the same lot of RT-MS by s.c. route stimulated 14-fold higher GMT of IgG2a antibodies. Lot #111 in study 2 had the best size-distribution among the two lots; 90% of the MS had diameters less than 6.9 m (Fig. 3), and 75% of the MS had sizes less than 4.4 m. 3.2.3. Stimulation of serum IgA antibodies The IgA responses were not meaningful (results not shown), most likely due to the dilution of the serum (1:80) assayed, or due to the suboptimal dose and schedule. 3.2.4. Resistance of immunized mice to aerosol challenge As shown in Fig. 8, a significant number (50%) of the mice (n = 10) were protected against the challenge (P = 0.039) when they were immunized orally with 25 g of RT-MS according to schedule A (n = 10), but only 10% survived (n = 10) when RT-MS was administered schedule B (P = 0.4). Only marginal, non-significant protection was stimulated by orally administered 25 g aqueous vaccine with either schedule 25% (n = 8) and 13% (n = 8), P = 0.46 and 1.0, respectively. Subcutaneous immunization with RT-MS protected all the mice (n = 9), whereas 50% (P = 0.076) of the mice (n = 8) were protected following s.c. immunization with 15 g aqueous RT. 4. Discussion The results of this study shows that, with an extended 7 weeks optimal oral immunization schedule, RT vaccine in dl-PLG microspheric carrier was a much more effective mucosal immunogen than the aqueous vaccine was by oral
administration. The extended oral immunization schedule, with slight modification, was similar to the schedule reported previously for ovalbumin entrapped in dl-PLG MS, which employed a daily dose for three consecutive days, and it was repeated 4 weeks later [10,16]. Presumably, administering a daily dose for three consecutive days, at least one, but perhaps all three doses taken up by the antigen-processing cells in the Peyer’s patches. Such schedule assured the availability of sufficient immunogen in spite of reduced resident time due to clearance by the bowel movement. The dose of 50 g RT was chosen arbitrarily, but, it was prompted by the good antibody response and protection stimulated by that dose upon intranasal administration of microencapsulated RT [13]. In contrast to the ineffective aqueous RT, oral administration of 50 g of RT-MS by the extended schedule (the last oral dose was administered on day 49) stimulated high IgG, IgG2a and IgA responses and induced complete protection against a lethal aerosol ricin challenge (study 1). The size of the MS in our recent study was similar to those used in previous publications [8,16,17]. Among the two lots we used, lot #111 (used in study 2) had a better size distribution of ∼1–10 m than lot #108 (used in study 1) had (Fig. 3). These sizes are considered to be optimal for uptake by antigen-processing cells, while MS with size >10 m are too large for uptake. The core loading and encapsulation efficiency of lot #108 was twice lower than that of lot #111, consequently two times more of polymeric mass was needed to deliver the same dose. The difference in size distribution and core loading were not decisive factors, since only lot #108 stimulated full protection by oral administration, although the two lots were administered with two different doses and schedules in two separate tests. However, by s.c. administration using the same dose regimen, both lots were equally effective in the microencapsulated form indicating equal potency. As was observed for intranasal immunization with RT-MS [13], oral immunization provided a very good correlation between the stimulation and the presence of anti-ricin serum IgG2a subisotype and subsequent protection against aerosol challenge with ricin toxin. In study 1, at 10 weeks, just 2 days before the challenge, the GMT of IgG2a elicited by the microencapsulated RT was 21-fold higher than that elicited by the aqueous vaccine. IgG2a immunoglobulin subisotype is believed to be the principal protective antibody against tumor cells and tumors [18–23], trypanosomes [24], hepatitis B [25] and influenza [26] virus infections and candida albicans [27]. F protein of the respiratory syncytial virus (RSV) with QS-21 adjuvant but not with aluminum hydroxide (AlOH) stimulated significantly higher titer of IgG2a immunoglobulins and virus neutralizing antibodies similarly to those stimulated by natural RSV infection [28]. Enhancement of IgG2a was well demonstrated by the results obtained with intranasal immunization using RT in MS [13], and by the results in Figs. 2 and 5 of the present study. These observations equates PLG MS with known mucosal adjuvant (QS-21), and surpasses the ability of AlOH to stimulate
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IgG2a antibodies. IgG2a immunoglobulin is the humoral marker of TH1-type of immune response, thus, presentation of RT by PLG-MS appears to stimulate both B and T cell responses. A similar TH1 type pattern of immune response occurred following vaccination with PLG-entrapped M. tuberculosis protein, characterized by higher levels of IgG2a antibodies and by upto 10 times higher levels of interferon-␥ secretion by T cells [29]. Strong enhancement of IgG2a and the protection occurred by vaccination with DNA encoding the glycoprotein E2 of bovine viral diarrhoea virus [30]. In contrast to IgG2a, the total IgG response to RT did not always correlate with the protection against aerosol challenge, as was the case in study 2. IgG was assayed with a single dilution (1:2500) for a quick determination of the kinetics of the IgG response (or lack of it). In study 2, the mean o.d. of IgG antibodies stimulated by RT-MS or the aqueous antigen were very much the same, indicating, that endpoint titration would provide similar titers. The increase in IgG level could have been caused by a subisotype other than IgG2a, which is about 8–10% of the total IgG. IgG1 constitutes upto 80% of the total IgG. In our study, IgG1 was not determined; however, IgG1 determinations performed after immunization with aqueous solution of chain A subunit of ricin showed no correlation between its presence and protection (M. Kende, unpublished observations). Therefore, while an increased IgG1 response might elevate the total IgG response it may not provide an accurate predictive value regarding the resistance to ricin. In the present study, we did not assess other IgG subclasses, because in rats immunized with RT-MS or RT with alum adjuvant, IgG2b and IgG2c were below level of detection, although the GMT of IgG, IgG2a and IgG1 were >1:10,000 [31]. Anti-ricin IgM determinations did not reveal a relationship between its presence stimulated by RT with or without dl-PLG and between the protection against ricin (M. Kende, unpublished observations). Since intranasal immunization by RT-MS or aqueous RT showed a good agreement between the number of IgA-positive samples from the lung (the target organ of the aerosol-delivered ricin) and the number of IgA-positive serum samples [13], in the present study, we measured IgA only in the serum with a single (1:80) dilution. Only 50 g RT-MS administered orally with the long optimal schedule stimulated serum IgA, while 25 g RT-MS did not, although both doses stimulated IgG and IgG2a. The orally administered aqueous vaccine, s.c. administered microencapsulated or aqueous RT failed to stimulate IgA. Although at the present time, the relationship between serum IgA and sIgA in the lung is not established, serum IgA and perhaps IgG2a and other IgG subisotypes might be transported to the lung through the circulation. The transported immunoglobulins are disseminated with the capillary vessels very close to the mucosal surfaces, where they could neutralize the ricin toxin delivered by aerosol challenge. Thus, increased presence of IgA in the serum could possibly indicate an enhanced protection due to oral–mucosal-immunization, while s.c.
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immunization stimulated systemic immunity only. At this time, the anti-ricin neutralizing activity of IgA is not known, but it’s role is suggested in the protection against microbial infections. Intranasal treatment with mouse monoclonal IgA antibody directed against respiratory syncytial viral glycoprotein reduced the upper and lower respiratory tract infection in mice and monkeys [32,33]. Oral administration of monoclonal IgA directed against the lipopolysaccharide component of vibrio, protected neonatal mice against oral challenge with Vibrio cholerae, as measured by reduced intestinal colonization [34]. Oral immunization with recombinant Helicobacter pylori urease induced sIgA, and the sIgA level correlated with the protection against H. pylori challenge [35]. It appears that 3-day cycle of primary immunization and most likely the secondary immunization cycle as well was a prerequisite for successful immunization. Marginal GMT at 28 days clearly indicated the need for a second immunization cycle. After administration of the seventh oral dose at day 49 the antibody response was not enhanced further, therefore, presumably six immunization doses (or less) are sufficient to stimulate full protection by 50 g RT-MS. Attempts were made in the present study to reduce the dose and shorten the immunization time. In study 2, a high GMT of IgG2a was stimulated with a condensed, shorter immunization schedule, which stimulated significant, but reduced protection (50% survivors, P = 0.037). Presumably, the condensed and shorter immunization schedule together with lower dose of 25 g RT-MS was suboptimal, therefore, it was not sufficient to stimulate the development of full protection, which was not better than the 50% protection stimulated with a single s.c. dose of aqueous ricin (P = 0.076, above the significance level). Efficacy of RT-MS was further reduced when primary and secondary stimulation with two cycles of 2 days (2 weeks apart) was followed 2 weeks later by a tertiary boost of one cycle of 3 days (schedule B). Further studies are needed for conclusive determination if 50 and 25 g doses administered with the short or long schedules, respectively, would be equally effective or not. Our results indicated that PLG MS is an effective mucosal carrier/adjuvant for a vaccine of plant toxin origin by oral administration, and extended previous studies in which the immune response to dl-PLG-incorporated model antigens [10,17], viral [36,37], bacterial [38–41] and other protein vaccines [42] were enhanced.
Acknowledgements The research was conducted in compliance with the Animal Welfare Act and other Federal statues and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996. The facility where the research was conducted is fully accredited by the Association for Assessment and Accredita-
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tion of Laboratory Animal Care International. The views of the authors do not purport to reflect the position of the Department of the Army or the Department of the Defense. This work was performed while Dr. C. Yan held a National Research Council—US Army Medical Research and Material Command research associateship. References [1] Tomasi TB, Zigelbaum S. The selective occurrence of gamma 1A globulin in certain body fluids. J Clin Invest 1963;42:1552–60. [2] Tomasi TB, Tam E, Salomon A, Prendergast RA. Characteristics of an immune system common to certain external secretions. J Exp Med 1965;121:101–24. [3] Brandtzaeg P, Baklien K. Immunohistochemical studies of the formation and epithelial transport of immunoglobulins in normal and diseases human intestinal mucosa. Scand J Gastroenterol 1976;11(Suppl 36):1–45. [4] Brandtzaeg P. Immune functions of human nasal mucosa and tonsils in health and disease. In: Bienenstock J, editor. Immunology of the lung and upper respiratory tract. New York: McGraw-Hill, 1984. p. 28–39. [5] Mestecky J, McGhee JR, Arnold RR, Michalek SM, Prince SJ, Babb JL. Selective induction of immune response in human external secretion by ingestion of bacterial antigen. J Clin Invest 1978;61:731– 7. [6] Bienenstock J, Befus AD. Mucosal immunology. Immunology 1980;41:249–70. [7] Mestecky J, McGhee JR, Crago SS, Jackson S, Kilian M, Kiyono H, et al. Molecular–cellular interactions in the secretory IgA response. J Reticuloendothel Soc 1980;28:455s–60s. [8] Eldridge JH, Staas JK, Meulbroek JA, McGhee JR, Tice TR, Gilley RM. Biodegradable microspheres as vaccine delivery system. Mol Immunol 1991;28:287–94. [9] Haas S, Miura-Fraboni J, Zavala F, Murata K, Leone-Bay A, Santiago N. Oral immunization with a model protein entrapped in microspheres prepared from derivatized alpha-amino acids. Vaccine 1996;14:785– 91. [10] Maloy KJ, Donachie AM, O’Hagan DT, Mowat AMcI. Induction of mucosal and systemic immune responses by immunization with ovalbumin entrapped in poly(lactide-co-glycolide) microparticles. Immunnology 1994;81:661–7. [11] Mowat AMcI, Donachie AM, Reid G, Jarrett O. Immune-stimulating complexes containing Quil A and protein antigen prime class I MHC-restricted T lymphocytes in vivo and immunogenic by the oral route. Immunology 1991;72:317–22. [12] Heeg K, Kuon W, Wagner H. Vaccination of class I major histocompatibility complex (MHC)-restricted murine CD8+ cytolytic T lymphocytes toward soluble antigens: immunostimulating ovalbumin complexes enter class I MHC-restricted antigen pathway and allow sensitization against the immunodominant peptide. Eur J Immunol 1992;21:1523–7. [13] Yan C, Rill WL, Malli R, Hewetson J, Naseem H, Tammariello R, et al. Intranasal stimulation of long-lasting immunity against aerosol ricin challenge with ricin toxoid vaccine encapsulated in polymeric microspheres. Vaccine 1996;14:1031–8. [14] Yan C, Resau JH, Hewetson J, West M, Rill WL, Kende M. Characterization and morphological analysis of protein-loaded poly(lactide-co-glycolide) microparticles prepared in water-in-oil emulsion technique. J Contr Rel 1994;32:231–41. [15] Guyton AC. Measurement of the respiratory volumes of laboratory animals. Am J Phys 1947;150:70–7. [16] Challacombe SJ, Rahman D, Jeffery S, Davis S, O’Hagan DT. Enhanced secretory IgA and systemic IgG antibody respones after oral immunization with biodegradable microparticles containing antigen. Immunology 1992;76:164–8.
[17] Eldridge JH, Hammond CJ, Meulbroek JA, Staas JK, Gilley RM, Tice TR. Controlled vaccine release in the gut-associated lymphoid tissues. I. Orally administered biodegradable microspheres target the Peyer’s patches. J Contr Rel 1990;11:205–14. [18] Bernstein ID, Nowinski RC, Tam MR, McMaster B, Houston LL, Clark EA. In: Kenneth RH, Kearn TJ, Bechtol KB, editors. Monoclonal antibodies. New York: Plenum Press, 1980. p. 275–91. [19] Herlyn D, Kaprowski H. IgG2A monoclonal antibodies inhibit human tumor growth through interaction with effector cells. Proc Natl Acad Sci USA 1982;76:4761–4. [20] Seto M, Takahashi T, Nakamura S, Matsudaira Y, Nishizuka Y. In vivo antitumor effects of monoclonal antibodies with different immunoglobulin classes. Cancer Res 1983;43:4768–73. [21] Samuel J, Budzynsky WA, Reddish MA, Ding L, Zimmermann GL, Krantz MJ, Koganty RR, Longenecker BM. Immunogenicity and antitumor activity of a liposomal MUC1 peptide-based vaccine. J Inter du Cancer 1998;5(2):295–302. [22] Weiner GH, Liu HM, Wooldridge JE, Dahle CE, Krieg AM. Immunostimulatory oligodeoxynucleotides containing the CpG motif are effective as immunoadjuvants in tumor antigen immunization. Proc Natl Acad Sci USA 1997;94(20):10833–7. [23] Rovero S, Boggio K, DiCarlo E, Amici A, Qugliano E, Porcedda P, Musiani P, Forni G. Insertion of the DNA for the 163–171 peptide of IL-Ibeta enables a DNA vaccine encoding p185neu to inhibit mammary carcinogenesis in Her-2/neu transgenic BALB/C mice. Genether 2001;8(6):447–52. [24] Wechsler DS, Kongshavn PAL. Heat-labile IgG2a antibodies affect cure of Trypanosoma musculi infection in C57BL/6 mice. J Immunol 1986;137:2968–72. [25] Byars NE, Nakano G, Welch M, Lehman D, Allison AC. Improvement of hepatitis B vaccine by the use of a new adjuvant. Vaccine 1991;9:309. [26] Arulandam BP, Mittler JN, Lee WT, O’Toole M, Metzger DW. Neonatal administration of IL-12 enhances the protective efficacy of antiviral vaccines. J Immunol 2000;164(7):3698–704. [27] Cardenas-Freytag L, Cheng E, Mayeaux P, Domer JE, Clements JD. Effectiveness of a vaccine composed of heat-killed Candida albicans and a novel mucosal adjuvant. Infect Immunol 1999;67(2):826–33. [28] Hanckok GE, Speelman DJ, Heers K, Bortell E, Smith J, Cosco C. Generation of atypical pulmonary responses in BALB/c mice after immunization with a native attachmaent (G) glycoprotein of respiratory syncytial virus. J Virol 1996;70(11):7783–91. [29] Vordermeier HM, Coombes AGA, Jenkins P, McGee JP, O’Hagan DT, Davis SS, Singh M. Synthetic delivery system for tuberculosis vaccine: immunological evaluation of the M. tuberculosis 38 kDa protein entrapped in biodegradable PLG microparticles. Vaccine 1995;13:1576–82. [30] Nobiron I, Thompson I, Brownlie J, Collins ME. Co-administration of IL-2 enhances antigen-specific immune responses following vaccination with DNA encoding the lipoprotein E2 of bovine viral diarrhoea virus. Vet Microbiol 2000;76(2):129–42. [31] Kende M, Yan C, Hewetson J, Loving J, Frick M, Tamariello R, Kockes J, Assaad A, editors. Enhancement of ricin toxoid (RT) efficacy in rats by polylactide-co-glycolide (PLG) microspheres. In: Proceedings of the 26th International Symposium on Controlled Release of Bioactive Materials, Boston, MA, 1999. p. 37–8. [32] Weltzin R, Traina-Dorge V, Soike K, Zhang JY, Mack P, Soman G, Drabik G, Monath TP. Intranasal monoclonal IgA antibody to respiratory syncytial virus protects rhesus monkeys against upper and lower respiratory tract infection. J Infect Dis 1996;174:256–61. [33] Hall CB, Douglas RG. Modes of transmission of respiratory syncytial virus. J Pediatr 1981;99:100–3. [34] Lee CK, Weltzin R, Soman G, Georgakopoulos KM, Houle DM, Monath TP. Oral administration of polymeric immunoglobulin A prevents colonization with Vibrio cholerae in neonatal mice. Infect Immunol 1994;62:887–91.
M. Kende et al. / Vaccine 20 (2002) 1681–1691 [35] Lee CK, Weltzin R, Thomas Jr WD, Kleanthous H, Ermak TH, Soman G, Hill JE, Ackerman SK, Monath TP. Oral immunization with recombinant Helicobacter pylori urease induces secretory IgA antibodies and protects mice from challenge with Helicobacter Felis. J Infect Dis 1995;172:161–72. [36] Moldoveanu Z, Novak M, Huang WQ, Gilley RM, Staas JK, Schafer D, Compans RW, Mestecky J. Oral immunization with influenza virus in biodegradable microspheres. J Infect Dis 1993;167:84–90. [37] Ray R, Novak M, Duncan JD, Matsuoka Y, Compans RW. Microencapsulated human parainfluenza virus induces a protective immune response. J Infect Dis 1993;167:752755. [38] McQueen CE, Boedeker EC, Reid R, Jarboe D, Wolf M, Le M, Brown WR. Pili in microspheres protect rabbits from diarrhoea induced by E. coli strain RDEC-1. Vaccine 1993;11:201–6.
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Oral immunization of mice with ricin toxoid vaccine encapsulated in polymeric microspheres against aerosol challenge Meir Kende∗ , Changhong Yan, John Hewetson, Matthew A. Frick, Wayne L. Rill, Ralph Tammariello United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, MD 21702-5011, USA Received 21 November 2000; received in revised form 1 August 2001; accepted 10 October 2001
Abstract Mucosal (oral) immunization of mice with carrier-delivered ricin toxoid (RT) vaccine was accomplished by one long (7 weeks) or two short (4 weeks) immunization schedules. For the long and short immunization schedule two lots of vaccine were administered prepared with the same procedure but at different occasions. The long schedule consisted of a total of seven doses of 50 g of vaccine in microencapsulated (lot #108) or aqueous form administered on days 1, 2, 3, 28, 29, 30 and 49. With the short schedule a total of seven or six doses of 25 g (lot #111) were administered on days 1, 2, 3, 14, 15, 16 and 30, or on 1, 2, 14, 15, 30, 31 and 32, respectively. Mice immunized orally with the long schedule, 50 g of RT vaccine incorporated into poly-dl-lactide-co-glycolyde (dl-PLG) microspheres (MS) produced serum IgG, IgG2a and IgA ELISA antibodies. All mice immunized with RT in dl-PLG MS (RT-MS) were protected against a lethal ricin aerosol challenge. In contrast, with the same schedule and with the same dose, the aqueous vaccine (RT) failed to stimulate IgG, IgG2a and IgA antibodies, and these mice were not protected against an aerosol ricin toxin challenge. With the shorter immunization scheme, seven doses of 25 g RT-MS stimulated a significant, though reduced, protection with the microencapsulated, but not with the aqueous vaccine. When the first and second 3-day cycles of the short immunization schedule was reduced to two doses, and the 3-day cycle was administered at the end of the schedule, neither RT-MS nor RT stimulated protection against the challenge. These results indicated that successful oral immunization with RT-MS depended on both the dose and the schedule, consisting of three consecutive days of administration in two cycles, 4 weeks apart. Altering this schedule and the dose, resulted in a reduced protection or no protection at all. Furthermore, under the conditions of this study, the advantage of the microencapsulated RT vaccine over the aqueous vaccine for effective oral immunization was well demonstrated. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Oral immunization; Ricin toxoid; Polymeric microspheres; Humoral immune response; Protection
1. Introduction Antigen presentation via the intestinal tract possesses all the prerequisites for simple, safe and effective oral immunization. Although, the mucosal surface of the gastrointestinal tract represents a large area, only the ileum with its neutral pH has the proper environment for effective presentation of antigens via orally delivered vaccines. The dominant immunoglobulin at the mucosal surface of the gastrointestinal tract is secretory IgA (sIgA) [1,2] produced in the lamina propria regions underlying the mucous membranes [3,4]. Secretory immunity can be achieved with some, but not with every aqueous antigen by administering the immunogen to the gut-associated lymphoid tissue, of which the largest mass is represented by the Peyer’s patches of the gastrointestinal tract. The Peyer’s patches ∗
Corresponding author. Tel.: +1-301-619-7494; fax: +1-301-619-2348. E-mail address: [email protected] (M. Kende).
are covered by unique epithelial cells, the M cells, which possess high pinocytotic activity that allows the transport of the antigen into the antigen processing cells of the underlying dome region, with subsequent stimulation of B and T lymphocytes. Numerous studies have demonstrated that for oral stimulation of high immunoglobulin levels frequent administration of high doses of the aqueous vaccine is required [5–7], which may render oral immunization with the aqueous vaccine impractical. Studies with orally administered biodegradable polymeric microspheres (MS) have demonstrated the usefulness of this approach for effective stimulation of the immune response with lower doses of antigen [8–13]. BALB/c mice immunized perorally with three doses of 100 g of formalinized staphylococcal enterotoxin B (SEB) incorporated into dl-PLG MS stimulated circulating IgG, IgA and IgM anti-SEB antibodies in the serum, and a concurrent mucosal IgA response in lung lavage, gut-washing and in the saliva [8]. Parallel oral immunization with doses of aqueous unencapsulated
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SEB toxoid ranging from 1 to 100 g was ineffective at inducing a detectable immune response in any of the above test fluids. A single priming dose of 0.1 mg ovalbumin encapsulated in MS made of derivatized alpha-amino acids, induced ovalbumin-specific IgG antibodies in the sera, sIgA in the intestinal secretions, and a consequent antigen-dependent proliferation of splenic CD4+ T cells [9]. In the same study, enhancement of the protective efficacy of the vaccine by co-encapsulated cholera toxin was demonstrated against infectious bursal virus infection in chickens. Multiple oral immunization of mice with ovalbumin in dl-PLG MS resulted in ovalbumin-specific cytotoxic lymphocyte activity and an ovalbumin-specific intestinal IgA response [10]. For primary immunization the mice received by gastric intubation ovalbumin entrapped in dl-PLG MS or aqueous albumin on three consecutive days, and the 3-day cycle was repeated as a secondary boost 4 weeks later [10]. Although, the cytotoxic lymphocyte subset induced by ovalbumin in MS was not characterized, it is presumed to be the conventional class I MHC-restricted cytotoxic T lymphocytes. In this study, the target cells of ovalbumin-sensitized T lymphocytes do not express class II MHC antigens. The responses stimulated by microencapsulated ovalbumin appear very similar to those found after immunization with ovalbumin in ISCOM adjuvant, which are known to be due to CD4+ T cells [11,12]. The demonstration that ovalbumin in dl-PLG MS can stimulate cytotoxic T lymphocyte activity and prime a variety of systemic and mucosal responses has important implications in viral vaccine development. The drawback of those studies in which ovalbumin was employed is the lack of in vivo challenge; therefore, no conclusion can be made regarding the protective efficacy of the elicited immune response. Recently, we reported on the stimulation of mucosal immunity via intranasal administration of ricin toxoid (RT) vaccine encapsulated in dl-PLG MS [13]. Mice immunized intranasally were completely protected for at least 1 year against lethal ricin challenge delivered by aerosol. In the present study, we explored the oral route of immunization to evoke anti-ricin immunity by microencapsulated vaccine (RT-MS). The efficacy of dl-PLG-encapsulated toxoid was compared to the efficacy of the aqueous vaccine.
2. Materials and methods 2.1. Polymers Polymers dl-PLG of 50:50 lactide to glycolyde ratio with a molecular mass of 72 kDa was purchased from Medisorb Technologies International L.P., Cincinnati, OH. Ricin toxin was supplied by Vector Laboratories, Burlingame, CA. The Bio-Rad protein assay kit was purchased from Bio-Rad Laboratories, Richmond, CA. Polyvinyl alcohol (PVA) (31–50 kDa, 88% hydrolyzed) was purchased from Aldrich, Milwaukee, WI.
2.2. Formalin-inactivated ricin toxoid vaccine Ricin was diluted to 1 mg ml−1 in phosphate-buffered saline (PBS) and dialyzed against 4.2% formaldehyde at 47 ◦ C for 18 h and at 42 ◦ C for 30 h. The dialysis was continued against distilled water at 4 ◦ C, with 12 changes over 4 days. After centrifugation at 600 × g for 20 min, the precipitate was resuspended in an equal volume of distilled water and centrifuged. The supernatant was lyophilized and stored at −40 ◦ C until its use. 2.3. Microencapsulaton of RT vaccine PLG MS containing RT vaccine were prepared by a water-in-oil-in-water (w/o/w) solvent extraction procedure as reported previously [14]. Briefly, 100 l of an aqueous RT solution (protein concentration 50 mg ml−1 ) was added to 200 mg of PLG in 1 ml of methylene chloride. The mixture was emulsified with a Vortex mixer to form the water-in-oil (w/o) emulsion. This emulsion was then added to 10 ml of 2% aqueous PVA and was emulsified by a Bronson sonicator probe at output 4 (50 W) for 30 s to form the w/o/w emulsion. The organic solvent was extracted by adding 100 ml of 5% isopropanol, and the mixture was stirred at a moderate speed at ambient temperature for 30 min. The RT-loaded MS were centrifuged at 5000 × g for 5 min and washed three times with Milli-Q-grade water (Millipore, Bedford, MA). The MS were then lyophilized and stored at 4 ◦ C. Core loading of RT in MS was determined by digesting the MS and assaying their protein content. Briefly, a known amount of MS (∼20 mg) was suspended in 2 ml of 0.5 N sodium hydroxide, and the suspension was stirred at ambient temperature for about 20 min. When the MS were completely hydrolyzed, the transparent hydrolysate solution was immediately neutralized to pH 7.4 with HCl. The protein content of the solution was determined by the Bio-Rad protein assay. The diameter of the MS was determined with a Coulter Multisizer II (Coulter ELectronics Ltd., Luton, Beds, UK). Two lots of MS (#108 and #111) were prepared for the study. The mean volume size of two MS lots and the percent size distribution are plotted in Fig. 3. The encapsulation efficiency and the core loading of lot #108 was 35 and 1.17%, respectively. In study 2, lot #111 of the microencapsulated RT vaccine was used for oral and subcutaneously (s.c.) immunization. The encapsulation efficiency and the core loading of lot #111 was 62 and 2.51%, respectively. 2.4. Immunization and challenge studies Female, 8-week-old, NIH Swiss or CD1 mice were obtained from the National Cancer Institute, Frederick Research and Development Center, Frederick, MD and Charles River Inc., Wilmington, MA, respectively. Doses of 50 or 25 g of RT, either in MS resuspended in Mili-Q-grade water or in water, were administered into the stomach of the
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mice (8–10 per group) by a blunt-tipped gavage needle on days specified below. For about 6 h prior to oral immunization no food was given to the mice to reduce the vaccine reflux and the bowel movement. In schedule 1 of study 1, 50 g of lot #108 microencapsulated or aqueous RT was given orally in 0.2 ml volume on days 1, 2, 3, 28, 29, 30 and 49. Polymer mass of 4.22 mg was needed to deliver 50 g RT. A single dose of 15 g of RT in MS or in water was administered s.c. in 0.1 ml volume to separate group of mice on day 1 to assess the potency of the lot. In study 2, a lower dose and two shorter immunization schedules were used. The mice were immunized orally with 25 g of RT in MS or in water in a volume of 0.2 ml on days 1, 2, 3, 14, 15, 16 and 30; or on days 1, 2, 14, 15, 30, 31 and 32. Polymer mass of 0.965 mg was needed to deliver 25 g RT. A single dose of 15 g of RT in MS or in water was administered s.c. in 0.1 ml to a separate group of mice on day 1. Immunized and control mice were challenged on the specified day with an estimated inhaled dose of 60 g/kg of ricin toxin (∼15 LD50 ) [15]. The mice were exposed to ricin in a dynamic, whole-body exposure chamber with a total system airflow rate of 19.5 l/min. The aerosol was sampled during the entire exposure period of 10 min in sterile saline, and the protein concentration of the sample was determined by protein analysis (Pierce Micro BCA, Pierce Labs, Rockford, IL). Exposed mice were observed daily for 3–4 weeks postchallenge. Efficacy was established with survival as endpoint. Statistical significance was calculated by Fisher’s two-tailed exact test. 2.5. Determination of antibodies by ELISA Blood samples were collected from the tail veins of the mice at the times indicated in the appropriate experiment. The levels of anti-ricin IgG, IgG2a and IgA in the serum of each mouse were measured by direct ELISA according to
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previously published methods by using 0.25 g per well of ricin as the capture antigen [13,14]. Briefly, to the two-fold serial dilutions or to a single 1:2500 dilution of 50 l of the test serum, 50 l of peroxidase-labeled anti-mouse IgG (Kirkegard and Perry Laboratories, Gaithersburg, MD) or IgA or IgG2a (ICN Biomedicals Inc., Irvine, CA) was added. After addition of 50 l of o-phenylenediamine (OPD) substrate (Sigma, St. Louis, MO), the plates were incubated for 20 min at room temperature, 10 l stop buffer (0.1 N NaOH) was added, and the absorbency of each well was read at 405 nm with ELISA plate reader (Model MR 600, Dynatech Co., Chantilly, VA). Antibody levels were expressed as the reciprocal of the geometric mean titer (GMT) based on the last serum dilution with an o.d. twice that of the control (normal) serum on the same dilution. In that case, the P value is 0.01, provided that the o.d. of the normal serum is ∼0.1–0.2. In titrations of immune and normal serum, the initial serum dilution was 1:16, and if that dilution was negative, the serum’s base-line titer was taken as 1:8. The P values of the GMS were calculated with the Wilcoxon nonparametric procedure.
3. Results 3.1. Study 1: oral immunization with higher dose of RT administered on extended schedule 3.1.1. Stimulation of IgG antibodies The anti-ricin total IgG isotype antibodies in the serum was assayed with a single dilution (1:2500) level (Fig. 1). Based on previous studies, the mice were protected against ricin challenge with an anti-ricin IgG titer of 1:2500; therefore, as a quick reference for the immune status of the mice, only a single serum dilution of 1:2500 was used. Only s.c. administration of the microencapsulated RT
Fig. 1. Mean o.d. of anti-ricin serum IgG (1:2500 dilution) in mice immunized orally with 50 g of RT in MS (RT-MS) or in aqueous solution on days 1, 2, 3, 28, 29, 30 and 49; or s.c. on day 1 with 15 g of RT-MS or aqueous vaccine (n = 6).
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Fig. 2. Geometric mean anti-ricin serum IgG2a titers in mice immunized orally with 50 g of RT in MS (RT-MS) or in aqueous solution on days 1, 2, 3, 28, 29, 30 and 49; or s.c. on day 1 with 15 g of RT-MS or aqueous vaccine (n = 5–6).
stimulated IgG antibodies at 4 weeks (o.d. = 0.706±0.014), which remained unchanged at 7 (o.d. = 0.785 ± 0.041) and 10 weeks (o.d. = 0.793 ± 0.032). Orally administered RT in MS induced IgG antibodies at 7 weeks, (o.d. = 0.475 ± 0.052) which were increased by 10 weeks (o.d. = 0.612 ± 0.055). In contrast, aqueous RT did not stimulate ricin-specific IgG, either by single s.c. or by multiple oral administrations (o.d. = 0.149 ± 0.012, and 0.167 ± 0.030, respectively). 3.1.2. Stimulation of IgG2a antibodies Anti-ricin serum IgG2a immunoglobulin titer was determined by terminal dilution at 4, 7 and 10 weeks after administration of the first oral dose (Fig. 2). By the 4, 7 and the 10 weeks bleedings, three, six and seven oral doses were administered, respectively. At 4 weeks, a single dose of parentally administered RT-MS stimulated IgG2a antibodies, yielding a GMT of 1:1783 ± 205, which remained at that level for 10 weeks (1:1351 ± 324). A single s.c. administration of RT-solution did not stimulate this subisotype at all during 10 weeks of observation. With oral administration, RT stimulated IgG2a immunoglobulins when it was incorporated into the MS: 1:128 ± 43 and 1:446 ± 202 GMT at 7 and 10 weeks, respectively. At 10 weeks, the GMT of 1:446 ± 202 stimulated by oral administration of RT-MS was not significantly different (P = 0.08) than the four-fold higher GMT stimulated by RT-MS injected parentally (1:1783 ± 205). Orally administered RT-solution did not stimulate anti-ricin IgG2a immunoglobulins, except for a small marginal GMT of 1:21 ± 13 at the 10th week. The size distribution of lot #108 used in study 1 was quite favorable; close to 75% of the MS had a size distribution less than 11.4 m, and 50% of the MS sizes were below 5.1 m (Fig. 3).
3.1.3. Stimulation of serum IgA antibodies The anti-ricin IgA isotype in the serum was assayed with a single dilution (1:80) level (Fig. 4). A significant amount of serum IgA (twice the mean o.d. of the normal serum) was stimulated only with orally administered RT-MS: 0.342 ± 0.059 and 0.342 ± 0.033 at 7 and 10 weeks, while the control serum o.d. were: 0.049 ± 0.011 and 0.048 ± 0.009, respectively. By definition, at 7 and 10 weeks, twice, or almost twice, the levels of IgA were stimulated by oral administration of aqueous RT (0.147 ± 0.014 and 0.109 ± 0.011), and by s.c. administration of microencapsulated (0.142 ± 0.035 and 0.095 ± 0.021) or by the aqueous (0.085 ± 0.016 and 0.112 ± 0.027) RT. However, at 7 and 10 weeks, the o.d. of the serum control from naive mice was at least twice lower than the o.d. of the control serum at 4 weeks (0.129). The lower o.d. value accounts for defining the GMT as positive in those mice that were treated orally or s.c. with the aqueous RT, or s.c. with RT-MS. Furthermore, with the exception of the orally administered RT-MS, the o.d. values in all other immunization groups did not increase at all by 7 and 10 weeks relative to the o.d. at 4 weeks. 3.1.4. Resistance of immunized mice to lethal aerosol challenge In study 1, the mice were challenged 10 weeks after the first oral dose (day 1), which was 3 weeks after the seventh (last) oral administration of the microencapsulated or the aqueous vaccine (Fig. 5). All nonimmunized control mice (n = 9) died within 3 days. All mice (n = 9) immunized orally with seven doses of 50 g of RT-MS survived the lethal aerosol ricin challenge. In contrast, none of the mice (n = 10) that were immunized orally with seven doses of 50 g aqueous RT survived. Immunization by s.c. with a single dose of aqueous vaccine failed to protect any mice
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Fig. 3. Mean volume size (m) and size distribution of lots #108 and 111 dl-PLG MS containing RT vaccine.
Fig. 4. Mean o.d. of anti-ricin serum IgA (1:80 dilution) in mice orally immunized with 50 g of RT in MS (RT-MS) or in aqueous solution on days 1, 2, 3, 28, 29, 30 and 49; or s.c. on day 1 with 15 g of RT-MS or aqueous vaccine (n = 6).
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Fig. 5. Resistance of mice to aerosol ricin challenge 10 weeks postimmunization elicited orally with 50 g of RT in MS (RT-MS) or in aqueous solution on days 1, 2, 3, 28, 29, 30 and 49; or s.c. on day 1 with 15 g of RT-MS or aqueous vaccine (n = 9–10).
(n = 10), however a single s.c. dose of 15 g RT-MS protected all mice (n = 10). 3.2. Study 2: Immunization with reduced dose and shorter schedule 3.2.1. Stimulation of IgG antibodies The mice were immunized orally with seven or six doses of 25 g of RT in MS, or in an aqueous solution using two schedules on days 1–3, 14–16, and 30 (schedule A), or on
days 1, 2, 14, 15, and 30–32 (schedule B), respectively. The two schedules differed somewhat in the length of administration, but mainly at the timing and the number of the 3-day cycle (Fig. 6). The total IgG antibody response assayed with a single dilution (1:2500) exhibited the pattern observed for IgG2a (see Fig. 7) with the exception of the IgG response stimulated by the aqueous RT (Fig. 6). Compared to the o.d. (0.269 ± 0.049) of the serum of the nonimmunized mice, aqueous RT administered orally by schedule A also stimulated statistically significant IgG responses
Fig. 6. Mean o.d. of anti-ricin IgG (1:2500 dilution) of mice 6 weeks after multiple oral immunization with 25 g of RT vaccine in MS (RT-MS) or in aqueous solution; or s.c. with a single dose of 15 g of RT-MS or aqueous vaccine (n = 6). The respective schedules are indicated in the figure.
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Fig. 7. Anti-ricin IgG2a titers in the sera of mice 6 weeks after multiple oral immunization with 25 g of RT vaccine in MS (RT-MS) or in aqueous solution; or s.c. with a single dose of 15 g of RT-MS or aqueous vaccine (n = 6). The respective schedules are indicated in the figure.
Fig. 8. Resistance of mice to aerosol ricin challenge 6 weeks postimmunization stimulated orally with multiple doses of 25 g of RT vaccine in MS (RT-MS) or in aqueous solution; or s.c. with a single dose of 15 g of RT-MS or aqueous vaccine (n = 8–10). The respective schedules are indicated in the figure.
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(o.d. = 0.684 ± 0.199) as did the RT-MS with schedule A (o.d. = 0.710 ± 0.261) but not with schedule B (o.d. = 0.459 ± 0.137). 3.2.2. Stimulation of IgG2a antibodies Oral administration of 25 g of RT-MS under schedule A stimulated seven-fold higher IgG2a antibodies at 42 days than the RT-MS stimulated by schedule B (Fig. 7). With schedules A and B the GMTs stimulated by RT-MS were: 1:114 ± 330 and 1:16 ± 12, respectively (P = 0.03). The aqueous RT administered orally by schedules A and B stimulated no (1:8) or very low GMT (1:13 ± 5) of IgG2a antibodies, respectively. Thus, RT-MS administered with schedule A stimulated significantly higher IgG2a antibodies than the aqueous RT did with the same schedule (P = 0.0026). However, there was no difference in the IgG2a antibodies stimulated by RT-MS or the aqueous antigen when the 3-day cycle was administered under schedule B. In comparison with the aqueous RT, a single s.c. dose of 15 g of RT-MS stimulated 21-fold higher IgG2a antibodies, a GMT of 1:1625 ± 256 and 1:72 ± 159, respectively (P = 0.0057). In comparison with RT-MS administered orally with schedule A, s.c. administration of the same lot of RT-MS by s.c. route stimulated 14-fold higher GMT of IgG2a antibodies. Lot #111 in study 2 had the best size-distribution among the two lots; 90% of the MS had diameters less than 6.9 m (Fig. 3), and 75% of the MS had sizes less than 4.4 m. 3.2.3. Stimulation of serum IgA antibodies The IgA responses were not meaningful (results not shown), most likely due to the dilution of the serum (1:80) assayed, or due to the suboptimal dose and schedule. 3.2.4. Resistance of immunized mice to aerosol challenge As shown in Fig. 8, a significant number (50%) of the mice (n = 10) were protected against the challenge (P = 0.039) when they were immunized orally with 25 g of RT-MS according to schedule A (n = 10), but only 10% survived (n = 10) when RT-MS was administered schedule B (P = 0.4). Only marginal, non-significant protection was stimulated by orally administered 25 g aqueous vaccine with either schedule 25% (n = 8) and 13% (n = 8), P = 0.46 and 1.0, respectively. Subcutaneous immunization with RT-MS protected all the mice (n = 9), whereas 50% (P = 0.076) of the mice (n = 8) were protected following s.c. immunization with 15 g aqueous RT. 4. Discussion The results of this study shows that, with an extended 7 weeks optimal oral immunization schedule, RT vaccine in dl-PLG microspheric carrier was a much more effective mucosal immunogen than the aqueous vaccine was by oral
administration. The extended oral immunization schedule, with slight modification, was similar to the schedule reported previously for ovalbumin entrapped in dl-PLG MS, which employed a daily dose for three consecutive days, and it was repeated 4 weeks later [10,16]. Presumably, administering a daily dose for three consecutive days, at least one, but perhaps all three doses taken up by the antigen-processing cells in the Peyer’s patches. Such schedule assured the availability of sufficient immunogen in spite of reduced resident time due to clearance by the bowel movement. The dose of 50 g RT was chosen arbitrarily, but, it was prompted by the good antibody response and protection stimulated by that dose upon intranasal administration of microencapsulated RT [13]. In contrast to the ineffective aqueous RT, oral administration of 50 g of RT-MS by the extended schedule (the last oral dose was administered on day 49) stimulated high IgG, IgG2a and IgA responses and induced complete protection against a lethal aerosol ricin challenge (study 1). The size of the MS in our recent study was similar to those used in previous publications [8,16,17]. Among the two lots we used, lot #111 (used in study 2) had a better size distribution of ∼1–10 m than lot #108 (used in study 1) had (Fig. 3). These sizes are considered to be optimal for uptake by antigen-processing cells, while MS with size >10 m are too large for uptake. The core loading and encapsulation efficiency of lot #108 was twice lower than that of lot #111, consequently two times more of polymeric mass was needed to deliver the same dose. The difference in size distribution and core loading were not decisive factors, since only lot #108 stimulated full protection by oral administration, although the two lots were administered with two different doses and schedules in two separate tests. However, by s.c. administration using the same dose regimen, both lots were equally effective in the microencapsulated form indicating equal potency. As was observed for intranasal immunization with RT-MS [13], oral immunization provided a very good correlation between the stimulation and the presence of anti-ricin serum IgG2a subisotype and subsequent protection against aerosol challenge with ricin toxin. In study 1, at 10 weeks, just 2 days before the challenge, the GMT of IgG2a elicited by the microencapsulated RT was 21-fold higher than that elicited by the aqueous vaccine. IgG2a immunoglobulin subisotype is believed to be the principal protective antibody against tumor cells and tumors [18–23], trypanosomes [24], hepatitis B [25] and influenza [26] virus infections and candida albicans [27]. F protein of the respiratory syncytial virus (RSV) with QS-21 adjuvant but not with aluminum hydroxide (AlOH) stimulated significantly higher titer of IgG2a immunoglobulins and virus neutralizing antibodies similarly to those stimulated by natural RSV infection [28]. Enhancement of IgG2a was well demonstrated by the results obtained with intranasal immunization using RT in MS [13], and by the results in Figs. 2 and 5 of the present study. These observations equates PLG MS with known mucosal adjuvant (QS-21), and surpasses the ability of AlOH to stimulate
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IgG2a antibodies. IgG2a immunoglobulin is the humoral marker of TH1-type of immune response, thus, presentation of RT by PLG-MS appears to stimulate both B and T cell responses. A similar TH1 type pattern of immune response occurred following vaccination with PLG-entrapped M. tuberculosis protein, characterized by higher levels of IgG2a antibodies and by upto 10 times higher levels of interferon-␥ secretion by T cells [29]. Strong enhancement of IgG2a and the protection occurred by vaccination with DNA encoding the glycoprotein E2 of bovine viral diarrhoea virus [30]. In contrast to IgG2a, the total IgG response to RT did not always correlate with the protection against aerosol challenge, as was the case in study 2. IgG was assayed with a single dilution (1:2500) for a quick determination of the kinetics of the IgG response (or lack of it). In study 2, the mean o.d. of IgG antibodies stimulated by RT-MS or the aqueous antigen were very much the same, indicating, that endpoint titration would provide similar titers. The increase in IgG level could have been caused by a subisotype other than IgG2a, which is about 8–10% of the total IgG. IgG1 constitutes upto 80% of the total IgG. In our study, IgG1 was not determined; however, IgG1 determinations performed after immunization with aqueous solution of chain A subunit of ricin showed no correlation between its presence and protection (M. Kende, unpublished observations). Therefore, while an increased IgG1 response might elevate the total IgG response it may not provide an accurate predictive value regarding the resistance to ricin. In the present study, we did not assess other IgG subclasses, because in rats immunized with RT-MS or RT with alum adjuvant, IgG2b and IgG2c were below level of detection, although the GMT of IgG, IgG2a and IgG1 were >1:10,000 [31]. Anti-ricin IgM determinations did not reveal a relationship between its presence stimulated by RT with or without dl-PLG and between the protection against ricin (M. Kende, unpublished observations). Since intranasal immunization by RT-MS or aqueous RT showed a good agreement between the number of IgA-positive samples from the lung (the target organ of the aerosol-delivered ricin) and the number of IgA-positive serum samples [13], in the present study, we measured IgA only in the serum with a single (1:80) dilution. Only 50 g RT-MS administered orally with the long optimal schedule stimulated serum IgA, while 25 g RT-MS did not, although both doses stimulated IgG and IgG2a. The orally administered aqueous vaccine, s.c. administered microencapsulated or aqueous RT failed to stimulate IgA. Although at the present time, the relationship between serum IgA and sIgA in the lung is not established, serum IgA and perhaps IgG2a and other IgG subisotypes might be transported to the lung through the circulation. The transported immunoglobulins are disseminated with the capillary vessels very close to the mucosal surfaces, where they could neutralize the ricin toxin delivered by aerosol challenge. Thus, increased presence of IgA in the serum could possibly indicate an enhanced protection due to oral–mucosal-immunization, while s.c.
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immunization stimulated systemic immunity only. At this time, the anti-ricin neutralizing activity of IgA is not known, but it’s role is suggested in the protection against microbial infections. Intranasal treatment with mouse monoclonal IgA antibody directed against respiratory syncytial viral glycoprotein reduced the upper and lower respiratory tract infection in mice and monkeys [32,33]. Oral administration of monoclonal IgA directed against the lipopolysaccharide component of vibrio, protected neonatal mice against oral challenge with Vibrio cholerae, as measured by reduced intestinal colonization [34]. Oral immunization with recombinant Helicobacter pylori urease induced sIgA, and the sIgA level correlated with the protection against H. pylori challenge [35]. It appears that 3-day cycle of primary immunization and most likely the secondary immunization cycle as well was a prerequisite for successful immunization. Marginal GMT at 28 days clearly indicated the need for a second immunization cycle. After administration of the seventh oral dose at day 49 the antibody response was not enhanced further, therefore, presumably six immunization doses (or less) are sufficient to stimulate full protection by 50 g RT-MS. Attempts were made in the present study to reduce the dose and shorten the immunization time. In study 2, a high GMT of IgG2a was stimulated with a condensed, shorter immunization schedule, which stimulated significant, but reduced protection (50% survivors, P = 0.037). Presumably, the condensed and shorter immunization schedule together with lower dose of 25 g RT-MS was suboptimal, therefore, it was not sufficient to stimulate the development of full protection, which was not better than the 50% protection stimulated with a single s.c. dose of aqueous ricin (P = 0.076, above the significance level). Efficacy of RT-MS was further reduced when primary and secondary stimulation with two cycles of 2 days (2 weeks apart) was followed 2 weeks later by a tertiary boost of one cycle of 3 days (schedule B). Further studies are needed for conclusive determination if 50 and 25 g doses administered with the short or long schedules, respectively, would be equally effective or not. Our results indicated that PLG MS is an effective mucosal carrier/adjuvant for a vaccine of plant toxin origin by oral administration, and extended previous studies in which the immune response to dl-PLG-incorporated model antigens [10,17], viral [36,37], bacterial [38–41] and other protein vaccines [42] were enhanced.
Acknowledgements The research was conducted in compliance with the Animal Welfare Act and other Federal statues and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996. The facility where the research was conducted is fully accredited by the Association for Assessment and Accredita-
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