The Prostate 35:279–284 (1998) Induction of Early and Bioeffective Antibody Response in Rodents With the Luteinizing Hormone-Releasing Hormone Vaccine Given as a Single Dose in Biodegradable Microspheres Along With Alum Manish Diwan, Hema Dawar, and G.P. Talwar* Department of Reproductive Health and Vaccinology, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India BACKGROUND. Previous studies in animals and phase I/phase II clinical trials in humans have shown the suppressive effect of immunization with the luteinizing hormone-releasing hormone (LHRH) vaccine on prostatic hypertrophy and hyperplasia. A drawback of this vaccine was a delay of about 8 weeks in buildup of antibody titers to efficacy level and the requirement of three injections of the vaccine given at monthly interval for full primary immunization. METHODS. LHRH vaccine was encapsulated in poly-lactic-co-glycolic acid (PLGA) 50:50 copolymer microspheres of reproducible physicochemical characteristics. Immunogenicity studies were carried out in rodents and prostate weights were determined at various antibody titers. RESULTS. The vaccine entrapped in biodegradable microspheres generated high antibody response in rats, persisting for 5–7 months following a single immunization. One hundred micrograms was the optimum dose, and the intramuscular route was more immunogenic than the subcutaneous. It was further observed that coadministration of 75% of the vaccine entrapped in microspheres with 25% adsorbed on alum generated higher antibody response in rodents, exceeding the bioeffective threshold as early as day 15 postimmunization. CONCLUSIONS. Coadministration of the LHRH vaccine in biodegradable PLGA microspheres with a quarter of the dose adsorbed on alum generates high antibody titers within 15 days, which are effective in causing atrophy of the prostate. Prostate 35:279–284, 1998. © 1998 Wiley-Liss, Inc. KEY WORDS: controlled release delivery; LHRH-DT vaccine; prostatic atrophy; polylactide-co-glycolic acid copolymer INTRODUCTION Gonadotropin-releasing hormone or luteinizing hormone-releasing hormone (LHRH) is a hypothalamic hormone, regulating fertility and production of sex steroid hormones. The decapeptide is identical in males and females; thus, a vaccine against LHRH is effective in both sexes. Since the molecule is essentially conserved in most mammalian species analyzed, the anti-LHRH vaccine has applications in both humans as well as animals. An obvious application of the © 1998 Wiley-Liss, Inc. vaccine is in hormone-dependent cancers, as a cheap and convenient alternative to the LHRH agonists and Contract grant sponsor: European Community; Contract grant number: ECSTD III TS*-CT94-0292; Contract grant sponsor: Talwar Research Foundation, India. *Correspondence to: G.P. Talwar, Department of Reproductive Health and Vaccinology, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India. E-mail: firstname.lastname@example.org Received 8 August 1997; Accepted 21 November 1997 280 Diwan et al. antagonists. Immunization of rats with the LHRH vaccine causes a dramatic atrophy of the prostate of rats [1–3]. A marked reduction in the size of the prostate was also observed in monkeys . Following toxicological studies and getting approvals from the Drug Regulatory and Ethical Committees of the All India Institute of Medical Sciences New Delhi; Postgraduate Institute of Medical Education and Research, Chandigash; and General Hospital, Salzburg, Germany, the vaccine has undergone phase I and phase II clinical trials in advanced cases of carcinoma of the prostate in India and Austria. With the rise in anti-LHRH antibodies, the testosterone and the prostate-specific antigen (PSA) levels were reduced. Ultrasonography demonstrated a reduction in prostatic tissue volume and clinical alleviation of symptoms . Two major limitations of this vaccine were 1) the necessity of giving frequent injections, and 2) the lag period in buildup of antibody titers at the time of primary immunization, which delayed the onset of therapeutic action. The objective of this work was to develop an approach which could shorten the latency period of antibody generation and achieve complete immunization by a single contact point delivery. The polylactic-coglycolic acid (PLGA) copolymer, whose biocompatibility and biodegradability are known , was used for the preparation of microspheres encapsulating the LHRH vaccine. MATERIALS AND METHODS Materials Amino acids used for peptide synthesis were purchased from Bachem Chemicals (Torrance, CA). Diphtheria toxoid (DT, 3,000 Lf/ml) was obtained from the Serum Institute of India (Pune, India). All other chemicals were of analytical grade unless otherwise stated. Preparation of LHRH Vaccine The analog (D-Lys6) LHRH was synthesized by replacing Gly at position 6 with D-lysine . This was linked to aminohexanoic acid as the spacer molecule to which the carrier, diphtheria toxoid, was attached via its epsilon-amino group . Encapsulation of LHRH Vaccine in Microspheres Microspheres encapsulating the vaccine were prepared using PLGA polymers of 50:50 monomeric ratios (Medisorb Technologies, Cincinnati, OH). Briefly, a primary emulsion (w/o type) of the polymer solution (6% w/v) in dichloromethane and antigen (Ag) in phosphate-buffered saline (PBS, 0.05 M, pH 7.4) was prepared by homogenization (Virtis Homogenizer, Gardiner, NY) at 10,000 rpm for 1 min. The primary emulsion was stabilized by further homogenization (18,000 rpm, 5 min) with polyvinyl alcohol solution in water (10% w/v) to make a secondary emulsion (w/o/w type). The organic solvent, dichloromethane, was removed by keeping the reaction mixture stirring in an open beaker at room temperature overnight to obtain microspheres. The microspheres were washed 4 times with distilled water before freeze-drying. These were stored at 4°C in a desiccator until used. Characterization of Prepared Microspheres Particle size analysis. The size of the microspheres was measured by using a particle size analyzer (CIS-1, Galai, Migdal Haemek, Israel), based on the laser diffraction principle. Antigen entrapment. The amount of antigen encapsulated in the microspheres was estimated by employing the ‘‘two-step’’ extraction technique. A known amount of microspheres was taken in acetonitrile. As the polymer is soluble in acetonitrile, the microsphere structure was disrupted and the encapsulated antigen was released in a precipitate form. It was pelleted down by centrifugation. The supernatant-containing polymer was discarded and the pellet was redispersed in PBS (0.05 M, pH 7.4). The supernatant was obtained by centrifugation and the residue was further dissolved in 0.1 M NaOH. The protein in the aqueous and alkaline extractions was determined by using the microBCA protein estimation kit (Pierce Chemical Co., Rockford, IL) and expressed as the amount of antigen entrapped per milligram of microspheres. Animal Studies Outbred Wistar rats 6–8 weeks old were immunized intramuscularly or subcutaneously with LHRHDT conjugate, either encapsulated in microspheres or adsorbed on alum (Alhydrogel 2%, Superfos, Kvistgaard, Denmark), supplemented with 200 mg of SPLPS, the sodium phthalylated derivative of Salmonella enteritidis lipopolysaccharide (Difco Laboratories, Detroit, MI), prepared according to the method of Elin et al. . In each group, 3–6 animals were employed. Blood samples were collected by retroorbital plexus puncture at different time intervals. Serum was separated by centrifugation and stored at −20°C. The animal studies were consistent with the principles of laboratory animal care issued by the International Centre for Genetic Engineering and Biotechnology, New Delhi, India. Enzyme-Linked Immunosorbent Assay (ELISA) for Anti-LHRH Antibodies Serum samples were assayed for antibodies against LHRH by ELISA. Briefly, a 96-well ELISA plate Anti-LHRH Response With Encapsulated Vaccine 281 TABLE I. Encapsulation Efficiency and Particle Size of Three Batches of the LHRH-DT Vaccine Batch no. Antigen entrapped (mg/mg of Ms)a ± SD Entrapment efficiency (%) Mean particle size (mm) EC21 EC22 EC23 2.45 ± 0.18 2.66 ± 0.83 2.57 ± 0.25 43.2 60.5 58.2 0.88 0.84 0.90 a Ms, mean values of duplicate samples. Fig. 1. Particle size distribution of microspheres made of polylactic-co-glycolic acid encapsulating the LHRH vaccine. (Nunc-immunosorb, Maxisorb, Roskilde, Denmark) was coated with 100 ng/well of LHRH solution in 0.1 M bicarbonate buffer, pH 8.3, and incubated for 2 hr at 37°C. After washing, the unreacted sites of the plate wells were blocked with 2% (w/v) lactogen (300 ml/ well). Dilutions of sera samples were added to wells in duplicate and incubated for 1 hr at 37°C. After washing 4 times, goat anti-rat immunoglobulin fraction G (IgG) conjugated with horseradish peroxidase (Reagent bank of the National Institute of Immunology, New Delhi, India) was added at 1:25,000 dilution (100 ml/well), and the ELISA plate was incubated for 1 hr at 37°C, followed by 4 washings. To a 0.2% chromogen solution (12 ml) of o-phenylene diamine in citratephosphate buffer (0.1 M, pH 5.5), 24 ml of 30% H2O2 were mixed immediately before use. One hundred microliters of this solution were added to each well and the color was allowed to develop for 15 min in the dark. The reaction was stopped by the addition of 50 ml/well of 5 N H2SO4, and absorbance was measured at 492 nm in an ELISA reader (HT2 Anthos Hill, Salzburg, Austria). All washings were done with 0.05 M PBS, pH 7.4, containing 0.2% of Tween-20 (PBS-T), using an ELISA plate washer (Denley, Billingshurst, UK). Antibody titers were expressed as absorbance values obtained at 1:100 sample dilution by the ELISA against a pooled serum sample from hyperimmune animals as an interassay reference standard. The coefficients of variance of inter- and intraassay were ø23.9 × 10−4 and ø2.6 × 10−4. RESULTS Physicochemical Characteristics of LHRH Microspheres Three batches of microspheres were prepared by the process described. The mean particle size of microspheres varied from 0.85–0.90 mm. Figure 1 shows the typical Gaussian particle size distribution of mi- crospheres of a representative batch. The amounts of antigen entrapped in microspheres in the three batches was quantified and is given in Table I. The amount encapsulated from batch to batch was fairly consistent and varied from 2.45–2.66 mg per milligram of the microspheres. Immunogenicity Studies Previous work by our group  as well as of others  has shown that a higher antibody response can be elicited by coadministration of a part of the antigen dose adsorbed on alum along with the rest as encapsulated in microspheres, than is the case when the entire dose of the vaccine is given in microspheres. To determine the proportions of LHRH-DT, which may be given on alum along with the rest given in microspheres, experiments were conducted with two different preparations. In one, only 10% of the LHRH-DT was given adsorbed on alum and the remaining 90% was given in microspheres. In the second preparation, the proportions were 25% and 75%. The kinetics of antibody titers generated in rats with these two combinations are shown in Figure 2. Both formulations induced anti-LHRH antibodies in all animals (100% positivity of response). The rise in antibody titers was slower in the 10% + 90% group but the titers were sustained for a longer period. On day 210 postimmunization, the mean antibody titers were above the threshold required to achieve prostatic atrophy. On the other hand, high and early antibody response was obtained by using the 25% + 75% combination. The peak antibody titers on day 60 were higher in this group than those obtained with the 10% + 90% formulation. These were sustained above the bioefficacy threshold up to 150 days, but declined to below efficacy threshold level by day 210. These results indicate the necessity of booster immunization on day 150, if the 25:75 formulation is employed for immunization. The 10:90 formulation, on the other hand, gave a longer-lasting antibody response, with booster immunization not required for at least 210 days. These in- 282 Diwan et al. Fig. 2. Effect of differing proportions of the vaccine administered in microspheres and adsorbed on alum on antibody titers. Wistar rats were administered 100 µg LHRH-DT intramuscularly by a single injection given either as 90% encapsulated in polylacticco-glycolic acid microspheres and 10% adsorbed on alum, or as 75% in microspheres and 25% adsorbed on alum. In both cases, SPLPS was a coadjuvant. Also shown is the geometric mean (4–6 animals in each group) of antibody titers on different days postimmunization. vestigations show the feasibility of obtaining antiLHRH antibody response by a single-contact point immunization. It was imperative to determine the extent of antibody required to cause prostatic atrophy. Prostatectomy was performed in rats carrying different titers of antibodies, and the prostatic weight was determined in each case. Figure 3 gives prostate weights as a function of body weight in normal and anti-LHRH antibody-bearing animals. Antibody titers at and above 0.150 absorbance by the method followed caused an optimum atrophy of the prostate. Route of Immunization The vaccine was administered by intramuscular or subcutaneous routes. The intramuscular route was found more immunogenic than the subcutaneous route (Fig. 4). The antibody titers generated by intramuscular immunization exceeded the bioefficacy threshold by day 30 and remained higher for the entire period of observation, i.e., up to day 210. Dose Response Having determined the advantage of giving the antigen as 75% entrapped in microspheres and 25% adsorbed on alum, and having determined that the intramuscular route was better than the subcutaneous route, the dose of antigen producing optimum titers was investigated. Figure 5 gives the antibody titers on different days in animals given varying doses of the Fig. 3. Relationship between anti-LHRH antibody titers and prostate atrophy. Prostate weights were determined in rats bearing different titers of anti-LHRH antibodies. Fig. 4. Effect of route of administration on antibody titers. One hundred micrograms of LHRH-DT were given to rats by either the intramuscular (i.m.) or subcutaneous (s.c.) route. In both cases, 90% of the dose was encapsulated in PLGA microspheres and 10% adsorbed on alum. The geometric mean antibody titers are given after immunization. Bars bearing asterisks have statistically significant higher titers by the intramuscular route than the corresponding titers by the subcutaneous route. vaccine. Antibody response was detectable by day 15 in all animals receiving 100 mg or 200 mg doses, and the titers were above the therapeutic efficacy level. On the other hand, animals receiving the 50 mg dose of the vaccine had lower antibody titers on day 15. The peak attained was also lower than that with the 100 or 200 mg dose. The anti-LHRH IgG titers increased in all groups and reached their peak by day 60, after which titers started declining slowly but remained in the bioeffective range even after day 164 of single immunization. There was no significant difference in the immune response generated at the 100 mg or 200 mg dose levels of the vaccine. Thus, the 100 mg dose was selected as optimal for further experiments. Anti-LHRH Response With Encapsulated Vaccine 283 TABLE II. Comparative Antibody Titers Obtained in Rats on Day 15 With LHRH-DT Vaccine (100 µg) Given Either Adsorbed on Alum or as 75% Entrapped in PLGA Microspheres and 25% Adsorbed on Alum Group Microsphere-alum Alum Fig. 5. Effect of dose of LHRH vaccine on antibody response. Wistar rats (3–4 animals in each group) were immunized intramuscularly with 50, 100, or 200 µg of the vaccine, given 75% in PLGA microspheres and 25% on alum. Geometric mean titers of the antibodies generated in the animals are given as a function of time. Generation of Early Antibody Response In earlier studies [1–5], the LHRH-DT vaccine was given adsorbed on alum. This mode of immunization demanded a three-injection schedule to obtain optimum antibody titers. There was a perceptible delay in buildup of antibody titers, on the order of 8 weeks in humans . An interesting feature of giving 75% of the vaccine encapsulated in biodegradable microspheres along with 25% adsorbed on alum was the early induction of antibody titers, which were well above the bioefficacy threshold on day 15 in rats immunized with either 100 mg or 200 mg of the vaccine (Fig. 5). Table II gives the individual antibody titers in rats immunized with 100 mg of LHRH vaccine given on alum, or 25% adsorbed on alum and 75% entrapped in PLGA microspheres. DISCUSSION Previous studies have shown that immunization against LHRH causes a marked atrophy of the rodent and monkey prostates [1–4]. The vaccine  generated antibodies that inactivate LHRH, with consequent downregulation of the pituitary-testis axis. Testosterone declines to castration levels in immunized animals, which may be the primary reason for prostatic atrophy. However, recent studies by Fuerst et al.  showed that immunization with this vaccine not only suppresses the growth of androgen-sensitive Dunning R3327-PAP tumor cells in rodents but also had a low but significant effect on inhibition of the proliferation of androgen-independent Dunning R3327-AT2.1 cells. Orchiectomy (and hence testosterone deprivation) had no such effect on the proliferation of R3327-AT2.1 cells Animal no. Anti-LHRH IgG titer absorbance value (492 nm) 5 7 8 17 18 19 20 0.4413 0.2294 0.3083 0.0378 0.1083 0.1933 0.1811 . It is therefore likely that LHRH has a local action on the prostatic cells over and above its effect via androgens. Srkalovic et al.  reported the presence of LHRH receptors on an androgen dependent Dunning R3327 (H) prostate adenocarcinoma. The ensemble of these experimental studies shows that immunization against LHRH with a vaccine such as the one devised by us  is effective not only in causing the atrophy of normal prostate but also in suppressing the growth of Dunning prostatic tumor cells in rodents. Thus the vaccine has applications both in benign prostatic hypertrophy (especially in cases where surgery is contraindicated) and in prostate cancers. Toxicology studies on this vaccine have shown its safety and lack of side effects. It has been approved by the Drug Regulatory Authorities and Ethical Committees for clinical trials in India and Austria in advanced D2 stage carcinoma of prostate cases. These trials showed that in patients in whom the vaccine generated adequate antibody titers, there was a drastic reduction of testosterone and prostate-specific antigen (PSA). These changes were accompanied by a reduction in the volume of the prostate and improvement of associated clinical symptoms. Further improvements of the vaccine require: 1) inclusion of a safe and compatible adjuvant which can potentiate the antibody response in low responders, 2) simplification of the immunization schedule to, if possible, a single injection instead of the three previously required for primary immunization, and 3) curtailment of the time for antibody-titer buildup, which with the previouslyused vaccine delivery system was 4–8 weeks. The work reported here addresses two of these issues. It has been demonstrated that the vaccine can be encapsulated in biodegradable microspheres whereby primary immunization can be completed with a single injection. The data further indicate the possibility of 284 Diwan et al. getting a bioeffective, high antibody response with the LHRH-DT vaccine, giving three fourths in biodegradable microspheres and a quarter on alum. By following this regime, antibody titers adequate to cause atrophy of the prostate are generated within 15 days. The antibody response is sustained at bioeffective levels for about 6 months. Thereafter, a booster injection can be given to maintain the high titers. Both the polylactic-co-glycolic acid copolymer as well as alum are approved for human use by the FDA, and the most widely used vaccine of the world, the tetanus toxoid, is given adsorbed on alum. Thus a combined delivery of the LHRH vaccine with a part in PLGA microspheres and a part adsorbed on alum should be acceptable for human use. Nonetheless, a limited toxicology study may be required for the new formulation, even though the vaccine and other ingredients individually have no toxicity. The early induction of antibody response by the alum-microspheres combined mode of delivery may be attributed to the synergistic adjuvanticity of alum  and the small size of the microspheres . It is conceived that a part of the antigen (about 15%), released as a burst from the microspheres, may serve for the priming of the immune system. Eldridge et al.  showed that PLGA microspheres below 10 mm in size are taken up by macrophages. The microspheres prepared with LHRH vaccine, being smaller than 2 mm, are expected to be taken up by dendritic cells and macrophages, leading to efficient antigen presentation and stimulation of B cells for early antibody response. However, the direct uptake, presentation, and activation of B cells by this mode of antigen delivery is not ruled out. REFERENCES 1. Jayashankar R, Chaudhari MK, Singh O, Alam A, Talwar GP: Semisynthetic anti-LHRH vaccine causing atrophy of the prostate. Prostate 1989;14:3–11. 2. Rovan E, Fiebiger E, Kalla NR, Talwar GP, Aulitzky W, Frick J: Effect of active immunization to luteinizing-hormone-releasing hormone on the fertility and histoarchitecture of the reproductive organs of male rat. Urol Res 1992;20:323–334. 3. Giri DK, Chaudari MK, Jayashankar R, Neelaram GS, Jayara- 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. man S, Talwar GP: Histopathological changes in reproductive organs of male Wistar rats following active immunization against LHRH. Exp Mol Pathol 1990;52:54–62. Giri DK, Jayaraman S, Neelaram GS, Jayashankar R, Talwar GP: Prostatic hypoplasia in Bonnet monkeys following active immunization with semisynthetic anti-LHRH vaccine. Exp Mol Pathol 1991;54:255–264. Talwar GP, Diwan M, Davar H, Frick J, Sharma SK, Wadhwa SN: Counter GnRH vaccine. In Rajalakshmi M, Griffin PD (eds): ‘‘Male Contraception: Present and Future,’’ New Delhi: New Age International 1998:309–318. Visscher GE, Robison RL, Maulding HV, Fong JW, Pearson JE, Argentieri GJ: Biodegradation of and tissue reaction to 50:50 poly(D, L-lactide-co-glycolide) microcapsules. J Biomed Mater Res 1985;20:667. Talwar GP, Chaudhari MK, Jayashankar R: Antigenic derivative of GnRH. UK Patent 2228262, 1992. Chaudhari MK, Talwar GP: Synthesis of muramyl dipeptide derivatives of a decapeptide gonadotropin releasing hormone. J Indian Chem Soc 1989;66:255–257. Elin RJ, Wolff SM, McAdam KPWJ, Chedid I, Audibert F, Bernard C, Oberling F: Properties of reference Escherichia coli endotoxin and its phthalylated derivatives in humans. J Infect Dis 1981;144:329–336. Raghuvanshi RS, Misra A, Ganga S, Mehta S, Diwan M, Talwar GP: Antigen loaded microspheres—A strategy for improved immune response. ‘‘Proceedings of the 46th Indian Pharmaceutical Congress, 1995 December 28–30; Chandigarh (India),’’ A-32. Singh M, Li X-M, McGee JP, Zamb T, Koff W, Wang CY, O’Hagan DT: Controlled release microparticles as a single dose hepatitis B vaccine: Evaluation of immunogenicity in mice. Vaccine 1997;15:475–481. Fuerst J, Fiebiger E, Jungwirth A, Mack D, Talwar GP, Frick J, Rovan E: Effect of active immunization against luteinizing hormone-releasing hormone on the androgen-sensitive Dunning R3327-PAP and androgen-independent Dunning R3327-AT2.1 prostate cancer sublines. Prostate 1997;32:77–84. Srkalovic G, Bokser L, Radulovic S, Korkut E, Schally AV: Receptors for luteinizing hormone-releasing hormone in Dunning R3327 prostate cancers and rat anterior pituitaries after treatment with a sustained delivery system of LHRH antagonist SB75. Endocrinology 1990;127:3052–3060. Gupta RK, Rost BE, Siber GR: Adjuvant properties of aluminum and calcium compounds. In Powell MF, Newman MJ (eds): ‘‘Vaccine Design: The Subunit and Adjuvant Approach,’’ New York: Plenum Press, 1995:229–248. Eldridge JH, Staas JK, Meulbroek JA, Tice TR, Gilley RM: Biodegradable and biocompatible poly(D,L-lactide-co-glycolide) microspheres as an adjuvant for staphylococcal enterotoxin B toxoid which enhances the level of toxin-neutralizing activity. Infect Immun 1991;59:2978–2986.