Induction of Early and Bioeffective AntibodyResponse in Rodents With the Luteinizing

Hormone-Releasing Hormone Vaccine Given asa 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 GeneticEngineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India

BACKGROUND. Previous studies in animals and phase I/phase II clinical trials in humanshave shown the suppressive effect of immunization with the luteinizing hormone-releasinghormone (LHRH) vaccine on prostatic hypertrophy and hyperplasia. A drawback of thisvaccine was a delay of about 8 weeks in buildup of antibody titers to efficacy level and therequirement of three injections of the vaccine given at monthly interval for full primaryimmunization.METHODS. LHRH vaccine was encapsulated in poly-lactic-co-glycolic acid (PLGA) 50:50copolymer microspheres of reproducible physicochemical characteristics. Immunogenicitystudies were carried out in rodents and prostate weights were determined at various antibodytiters.RESULTS. The vaccine entrapped in biodegradable microspheres generated high antibodyresponse in rats, persisting for 5–7 months following a single immunization. One hundredmicrograms was the optimum dose, and the intramuscular route was more immunogenicthan the subcutaneous. It was further observed that coadministration of 75% of the vaccineentrapped in microspheres with 25% adsorbed on alum generated higher antibody responsein rodents, exceeding the bioeffective threshold as early as day 15 postimmunization.CONCLUSIONS. Coadministration of the LHRH vaccine in biodegradable PLGA micro-spheres with a quarter of the dose adsorbed on alum generates high antibody titers within 15days, 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; poly-lactide-co-glycolic acid copolymer

INTRODUCTION

Gonadotropin-releasing hormone or luteinizinghormone-releasing hormone (LHRH) is a hypotha-lamic hormone, regulating fertility and production ofsex steroid hormones. The decapeptide is identical inmales and females; thus, a vaccine against LHRH iseffective in both sexes. Since the molecule is essen-tially conserved in most mammalian species analyzed,the anti-LHRH vaccine has applications in both hu-mans as well as animals. An obvious application of the

vaccine is in hormone-dependent cancers, as a cheapand convenient alternative to the LHRH agonists and

Contract grant sponsor: European Community; Contract grantnumber: ECSTD III TS*-CT94-0292; Contract grant sponsor: TalwarResearch Foundation, India.*Correspondence to: G.P. Talwar, Department of ReproductiveHealth and Vaccinology, International Centre for Genetic Engineer-ing and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067,India. E-mail: talwar@icgebnd.ernet.inReceived 8 August 1997; Accepted 21 November 1997

The Prostate 35:279–284 (1998)

© 1998 Wiley-Liss, Inc.

antagonists. Immunization of rats with the LHRH vac-cine causes a dramatic atrophy of the prostate of rats[1–3]. A marked reduction in the size of the prostatewas also observed in monkeys [4]. Following toxico-logical studies and getting approvals from the DrugRegulatory and Ethical Committees of the All IndiaInstitute of Medical Sciences New Delhi; PostgraduateInstitute of Medical Education and Research, Chandi-gash; and General Hospital, Salzburg, Germany, thevaccine has undergone phase I and phase II clinicaltrials in advanced cases of carcinoma of the prostate inIndia and Austria. With the rise in anti-LHRH anti-bodies, the testosterone and the prostate-specific anti-gen (PSA) levels were reduced. Ultrasonography dem-onstrated a reduction in prostatic tissue volume andclinical alleviation of symptoms [5]. Two major limi-tations of this vaccine were 1) the necessity of givingfrequent injections, and 2) the lag period in buildup ofantibody titers at the time of primary immunization,which delayed the onset of therapeutic action. Theobjective of this work was to develop an approachwhich could shorten the latency period of antibodygeneration and achieve complete immunization by asingle contact point delivery. The polylactic-co-glycolic acid (PLGA) copolymer, whose biocompat-ibility and biodegradability are known [6], was usedfor the preparation of microspheres encapsulating theLHRH vaccine.

MATERIALS AND METHODS

Materials

Amino acids used for peptide synthesis were pur-chased from Bachem Chemicals (Torrance, CA). Diph-theria toxoid (DT, 3,000 Lf/ml) was obtained from theSerum Institute of India (Pune, India). All other chemi-cals were of analytical grade unless otherwise stated.

Preparation of LHRH Vaccine

The analog (D-Lys6) LHRH was synthesized by re-placing Gly at position 6 with D-lysine [7]. This waslinked to aminohexanoic acid as the spacer moleculeto which the carrier, diphtheria toxoid, was attachedvia its epsilon-amino group [8].

Encapsulation of LHRH Vaccine in Microspheres

Microspheres encapsulating the vaccine were pre-pared using PLGA polymers of 50:50 monomeric ra-tios (Medisorb Technologies, Cincinnati, OH). Briefly,a primary emulsion (w/o type) of the polymer solu-tion (6% w/v) in dichloromethane and antigen (Ag) inphosphate-buffered saline (PBS, 0.05 M, pH 7.4) wasprepared 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 solutionin water (10% w/v) to make a secondary emulsion(w/o/w type). The organic solvent, dichloromethane,was removed by keeping the reaction mixture stirringin an open beaker at room temperature overnight toobtain microspheres. The microspheres were washed4 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 microsphereswas measured by using a particle size analyzer (CIS-1,Galai, Migdal Haemek, Israel), based on the laser dif-fraction principle.

Antigen entrapment. The amount of antigen encap-sulated in the microspheres was estimated by employ-ing the ‘‘two-step’’ extraction technique. A knownamount of microspheres was taken in acetonitrile. Asthe polymer is soluble in acetonitrile, the microspherestructure was disrupted and the encapsulated antigenwas released in a precipitate form. It was pelleteddown by centrifugation. The supernatant-containingpolymer was discarded and the pellet was redispersedin PBS (0.05 M, pH 7.4). The supernatant was obtainedby centrifugation and the residue was further dis-solved in 0.1 M NaOH. The protein in the aqueous andalkaline extractions was determined by using the mi-croBCA protein estimation kit (Pierce Chemical Co.,Rockford, IL) and expressed as the amount of antigenentrapped per milligram of microspheres.

Animal Studies

Outbred Wistar rats 6–8 weeks old were immu-nized intramuscularly or subcutaneously with LHRH-DT conjugate, either encapsulated in microspheres oradsorbed on alum (Alhydrogel 2%, Superfos, Kvist-gaard, Denmark), supplemented with 200 mg ofSPLPS, the sodium phthalylated derivative of Salmo-nella enteritidis lipopolysaccharide (Difco Laboratories,Detroit, MI), prepared according to the method of Elinet al. [9]. In each group, 3–6 animals were employed.Blood samples were collected by retroorbital plexuspuncture at different time intervals. Serum was sepa-rated by centrifugation and stored at −20°C. The ani-mal studies were consistent with the principles oflaboratory animal care issued by the InternationalCentre for Genetic Engineering and Biotechnology,New Delhi, India.

Enzyme-Linked Immunosorbent Assay (ELISA) forAnti-LHRH Antibodies

Serum samples were assayed for antibodies againstLHRH by ELISA. Briefly, a 96-well ELISA plate

280 Diwan et al.

(Nunc-immunosorb, Maxisorb, Roskilde, Denmark)was coated with 100 ng/well of LHRH solution in 0.1M bicarbonate buffer, pH 8.3, and incubated for 2 hr at37°C. After washing, the unreacted sites of the platewells were blocked with 2% (w/v) lactogen (300 ml/well). Dilutions of sera samples were added to wells induplicate and incubated for 1 hr at 37°C. After wash-ing 4 times, goat anti-rat immunoglobulin fraction G(IgG) conjugated with horseradish peroxidase (Re-agent bank of the National Institute of Immunology,New Delhi, India) was added at 1:25,000 dilution (100ml/well), and the ELISA plate was incubated for 1 hrat 37°C, followed by 4 washings. To a 0.2% chromogensolution (12 ml) of o-phenylene diamine in citrate-phosphate buffer (0.1 M, pH 5.5), 24 ml of 30% H2O2were mixed immediately before use. One hundred mi-croliters of this solution were added to each well andthe color was allowed to develop for 15 min in thedark. The reaction was stopped by the addition of 50ml/well of 5 N H2SO4, and absorbance was measuredat 492 nm in an ELISA reader (HT2 Anthos Hill, Sal-zburg, Austria). All washings were done with 0.05 MPBS, pH 7.4, containing 0.2% of Tween-20 (PBS-T),using an ELISA plate washer (Denley, Billingshurst,UK). Antibody titers were expressed as absorbancevalues obtained at 1:100 sample dilution by the ELISAagainst a pooled serum sample from hyperimmuneanimals as an interassay reference standard. The coef-ficients of variance of inter- and intraassay were ø23.9× 10−4 and ø2.6 × 10−4.

RESULTS

Physicochemical Characteristics of LHRHMicrospheres

Three batches of microspheres were prepared bythe process described. The mean particle size of mi-crospheres varied from 0.85–0.90 mm. Figure 1 showsthe typical Gaussian particle size distribution of mi-

crospheres of a representative batch. The amounts ofantigen entrapped in microspheres in the threebatches was quantified and is given in Table I. Theamount encapsulated from batch to batch was fairlyconsistent and varied from 2.45–2.66 mg per milligramof the microspheres.

Immunogenicity Studies

Previous work by our group [10] as well as of oth-ers [11] has shown that a higher antibody response canbe elicited by coadministration of a part of the antigendose adsorbed on alum along with the rest as encap-sulated in microspheres, than is the case when theentire dose of the vaccine is given in microspheres. Todetermine the proportions of LHRH-DT, which maybe given on alum along with the rest given in micro-spheres, experiments were conducted with two differ-ent preparations. In one, only 10% of the LHRH-DTwas given adsorbed on alum and the remaining 90%was given in microspheres. In the second preparation,the proportions were 25% and 75%. The kinetics ofantibody titers generated in rats with these two com-binations are shown in Figure 2. Both formulationsinduced anti-LHRH antibodies in all animals (100%positivity of response). The rise in antibody titers wasslower in the 10% + 90% group but the titers weresustained for a longer period. On day 210 postimmu-nization, the mean antibody titers were above thethreshold required to achieve prostatic atrophy. Onthe other hand, high and early antibody response wasobtained by using the 25% + 75% combination. Thepeak antibody titers on day 60 were higher in thisgroup than those obtained with the 10% + 90% formu-lation. These were sustained above the bioefficacythreshold up to 150 days, but declined to below effi-cacy threshold level by day 210. These results indicatethe necessity of booster immunization on day 150, ifthe 25:75 formulation is employed for immunization.The 10:90 formulation, on the other hand, gave alonger-lasting antibody response, with booster immu-nization not required for at least 210 days. These in-

Fig. 1. Particle size distribution of microspheres made of poly-lactic-co-glycolic acid encapsulating the LHRH vaccine.

TABLE I. Encapsulation Efficiency and Particle Size ofThree Batches of the LHRH-DT Vaccine

Batchno.

Antigen entrapped(mg/mg of Ms)a

± SDEntrapment

efficiency (%)Mean particle

size (mm)

EC21 2.45 ± 0.18 43.2 0.88EC22 2.66 ± 0.83 60.5 0.84EC23 2.57 ± 0.25 58.2 0.90

aMs, mean values of duplicate samples.

Anti-LHRH Response With Encapsulated Vaccine 281

vestigations show the feasibility of obtaining anti-LHRH antibody response by a single-contact point im-munization.

It was imperative to determine the extent of anti-body required to cause prostatic atrophy. Prostatec-tomy was performed in rats carrying different titers ofantibodies, and the prostatic weight was determinedin each case. Figure 3 gives prostate weights as a func-tion of body weight in normal and anti-LHRH anti-body-bearing animals. Antibody titers at and above0.150 absorbance by the method followed caused anoptimum atrophy of the prostate.

Route of Immunization

The vaccine was administered by intramuscular orsubcutaneous routes. The intramuscular route wasfound more immunogenic than the subcutaneousroute (Fig. 4). The antibody titers generated by intra-muscular immunization exceeded the bioefficacythreshold by day 30 and remained higher for the entireperiod of observation, i.e., up to day 210.

Dose Response

Having determined the advantage of giving the an-tigen as 75% entrapped in microspheres and 25% ad-sorbed on alum, and having determined that the in-tramuscular route was better than the subcutaneousroute, the dose of antigen producing optimum titerswas investigated. Figure 5 gives the antibody titers ondifferent days in animals given varying doses of the

vaccine. Antibody response was detectable by day 15in all animals receiving 100 mg or 200 mg doses, andthe titers were above the therapeutic efficacy level. Onthe other hand, animals receiving the 50 mg dose of thevaccine had lower antibody titers on day 15. The peakattained was also lower than that with the 100 or 200mg dose. The anti-LHRH IgG titers increased in allgroups and reached their peak by day 60, after whichtiters started declining slowly but remained in thebioeffective range even after day 164 of single immu-nization. There was no significant difference in theimmune response generated at the 100 mg or 200 mgdose levels of the vaccine. Thus, the 100 mg dose wasselected as optimal for further experiments.

Fig. 2. Effect of differing proportions of the vaccine adminis-tered in microspheres and adsorbed on alum on antibody titers.Wistar rats were administered 100 µg LHRH-DT intramuscularlyby a single injection given either as 90% encapsulated in polylactic-co-glycolic acid microspheres and 10% adsorbed on alum, or as75% in microspheres and 25% adsorbed on alum. In both cases,SPLPS was a coadjuvant. Also shown is the geometric mean (4–6animals in each group) of antibody titers on different days postim-munization.

Fig. 3. Relationship between anti-LHRH antibody titers andprostate atrophy. Prostate weights were determined in rats bear-ing different titers of anti-LHRH antibodies.

Fig. 4. Effect of route of administration on antibody titers. Onehundred micrograms of LHRH-DT were given to rats by either theintramuscular (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 givenafter immunization. Bars bearing asterisks have statistically signifi-cant higher titers by the intramuscular route than the correspond-ing titers by the subcutaneous route.

282 Diwan et al.

Generation of Early Antibody Response

In earlier studies [1–5], the LHRH-DT vaccine wasgiven adsorbed on alum. This mode of immunizationdemanded a three-injection schedule to obtain opti-mum antibody titers. There was a perceptible delay inbuildup of antibody titers, on the order of 8 weeks inhumans [5]. An interesting feature of giving 75% of thevaccine encapsulated in biodegradable microspheresalong with 25% adsorbed on alum was the early in-duction of antibody titers, which were well above thebioefficacy threshold on day 15 in rats immunizedwith either 100 mg or 200 mg of the vaccine (Fig. 5).Table II gives the individual antibody titers in ratsimmunized with 100 mg of LHRH vaccine given onalum, or 25% adsorbed on alum and 75% entrapped inPLGA microspheres.

DISCUSSION

Previous studies have shown that immunizationagainst LHRH causes a marked atrophy of the rodentand monkey prostates [1–4]. The vaccine [7] generatedantibodies that inactivate LHRH, with consequentdownregulation of the pituitary-testis axis. Testoster-one declines to castration levels in immunized ani-mals, which may be the primary reason for prostaticatrophy. However, recent studies by Fuerst et al. [12]showed that immunization with this vaccine not onlysuppresses the growth of androgen-sensitive DunningR3327-PAP tumor cells in rodents but also had a lowbut significant effect on inhibition of the proliferationof androgen-independent Dunning R3327-AT2.1 cells.Orchiectomy (and hence testosterone deprivation) hadno such effect on the proliferation of R3327-AT2.1 cells

[5]. It is therefore likely that LHRH has a local actionon the prostatic cells over and above its effect via an-drogens. Srkalovic et al. [13] reported the presence ofLHRH receptors on an androgen dependent DunningR3327 (H) prostate adenocarcinoma. The ensemble ofthese experimental studies shows that immunizationagainst LHRH with a vaccine such as the one devisedby us [7] is effective not only in causing the atrophy ofnormal prostate but also in suppressing the growth ofDunning prostatic tumor cells in rodents. Thus thevaccine has applications both in benign prostatic hy-pertrophy (especially in cases where surgery is con-traindicated) and in prostate cancers.

Toxicology studies on this vaccine have shown itssafety and lack of side effects. It has been approved bythe Drug Regulatory Authorities and Ethical Commit-tees for clinical trials in India and Austria in advancedD2 stage carcinoma of prostate cases. These trialsshowed that in patients in whom the vaccine gener-ated adequate antibody titers, there was a drastic re-duction of testosterone and prostate-specific antigen(PSA). These changes were accompanied by a reduc-tion in the volume of the prostate and improvement ofassociated clinical symptoms. Further improvementsof the vaccine require: 1) inclusion of a safe and com-patible adjuvant which can potentiate the antibodyresponse in low responders, 2) simplification of theimmunization schedule to, if possible, a single injec-tion instead of the three previously required for pri-mary immunization, and 3) curtailment of the time forantibody-titer buildup, which with the previously-used vaccine delivery system was 4–8 weeks.

The work reported here addresses two of these is-sues. It has been demonstrated that the vaccine can beencapsulated in biodegradable microspheres wherebyprimary immunization can be completed with a singleinjection. The data further indicate the possibility of

Fig. 5. Effect of dose of LHRH vaccine on antibody response.Wistar rats (3–4 animals in each group) were immunized intra-muscularly with 50, 100, or 200 µg of the vaccine, given 75% inPLGA microspheres and 25% on alum. Geometric mean titers ofthe antibodies generated in the animals are given as a function oftime.

TABLE II. Comparative Antibody Titers Obtained inRats on Day 15 With LHRH-DT Vaccine (100 µg) GivenEither Adsorbed on Alum or as 75% Entrapped in PLGA

Microspheres and 25% Adsorbed on Alum

GroupAnimal

no.Anti-LHRH IgG titer

absorbance value (492 nm)

Microsphere-alum 5 0.44137 0.22948 0.3083

Alum 17 0.037818 0.108319 0.193320 0.1811

Anti-LHRH Response With Encapsulated Vaccine 283

getting a bioeffective, high antibody response with theLHRH-DT vaccine, giving three fourths in biodegrad-able microspheres and a quarter on alum. By follow-ing this regime, antibody titers adequate to cause at-rophy of the prostate are generated within 15 days.The antibody response is sustained at bioeffective lev-els for about 6 months. Thereafter, a booster injectioncan be given to maintain the high titers. Both the poly-lactic-co-glycolic acid copolymer as well as alum areapproved for human use by the FDA, and the mostwidely used vaccine of the world, the tetanus toxoid,is given adsorbed on alum. Thus a combined deliveryof the LHRH vaccine with a part in PLGA micro-spheres and a part adsorbed on alum should be ac-ceptable for human use. Nonetheless, a limited toxi-cology study may be required for the new formula-tion, even though the vaccine and other ingredientsindividually have no toxicity.

The early induction of antibody response by thealum-microspheres combined mode of delivery maybe attributed to the synergistic adjuvanticity of alum[14] and the small size of the microspheres [15]. It isconceived that a part of the antigen (about 15%), re-leased as a burst from the microspheres, may serve forthe priming of the immune system. Eldridge et al. [15]showed that PLGA microspheres below 10 mm in sizeare taken up by macrophages. The microspheres pre-pared with LHRH vaccine, being smaller than 2 mm,are expected to be taken up by dendritic cells andmacrophages, leading to efficient antigen presentationand stimulation of B cells for early antibody response.However, the direct uptake, presentation, and activa-tion of B cells by this mode of antigen delivery is notruled out.

REFERENCES

1. Jayashankar R, Chaudhari MK, Singh O, Alam A, Talwar GP:Semisynthetic anti-LHRH vaccine causing atrophy of the pros-tate. 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-releasinghormone on the fertility and histoarchitecture of the reproduc-tive organs of male rat. Urol Res 1992;20:323–334.

3. Giri DK, Chaudari MK, Jayashankar R, Neelaram GS, Jayara-

man S, Talwar GP: Histopathological changes in reproductiveorgans of male Wistar rats following active immunizationagainst LHRH. Exp Mol Pathol 1990;52:54–62.

4. Giri DK, Jayaraman S, Neelaram GS, Jayashankar R, Talwar GP:Prostatic hypoplasia in Bonnet monkeys following active immu-nization with semisynthetic anti-LHRH vaccine. Exp Mol Pathol1991;54:255–264.

5. Talwar GP, Diwan M, Davar H, Frick J, Sharma SK, WadhwaSN: Counter GnRH vaccine. In Rajalakshmi M, Griffin PD (eds):‘‘Male Contraception: Present and Future,’’ New Delhi: NewAge International 1998:309–318.

6. Visscher GE, Robison RL, Maulding HV, Fong JW, Pearson JE,Argentieri GJ: Biodegradation of and tissue reaction to 50:50poly(D, L-lactide-co-glycolide) microcapsules. J Biomed MaterRes 1985;20:667.

7. Talwar GP, Chaudhari MK, Jayashankar R: Antigenic derivativeof GnRH. UK Patent 2228262, 1992.

8. Chaudhari MK, Talwar GP: Synthesis of muramyl dipeptidederivatives of a decapeptide gonadotropin releasing hormone. JIndian Chem Soc 1989;66:255–257.

9. Elin RJ, Wolff SM, McAdam KPWJ, Chedid I, Audibert F, Ber-nard C, Oberling F: Properties of reference Escherichia coli endo-toxin and its phthalylated derivatives in humans. J Infect Dis1981;144:329–336.

10. Raghuvanshi RS, Misra A, Ganga S, Mehta S, Diwan M, TalwarGP: Antigen loaded microspheres—A strategy for improved im-mune response. ‘‘Proceedings of the 46th Indian PharmaceuticalCongress, 1995 December 28–30; Chandigarh (India),’’ A-32.

11. Singh M, Li X-M, McGee JP, Zamb T, Koff W, Wang CY,O’Hagan DT: Controlled release microparticles as a single dosehepatitis B vaccine: Evaluation of immunogenicity in mice. Vac-cine 1997;15:475–481.

12. Fuerst J, Fiebiger E, Jungwirth A, Mack D, Talwar GP, Frick J,Rovan E: Effect of active immunization against luteinizing hor-mone-releasing hormone on the androgen-sensitive DunningR3327-PAP and androgen-independent Dunning R3327-AT2.1prostate cancer sublines. Prostate 1997;32:77–84.

13. Srkalovic G, Bokser L, Radulovic S, Korkut E, Schally AV: Re-ceptors for luteinizing hormone-releasing hormone in DunningR3327 prostate cancers and rat anterior pituitaries after treat-ment with a sustained delivery system of LHRH antagonist SB-75. Endocrinology 1990;127:3052–3060.

14. Gupta RK, Rost BE, Siber GR: Adjuvant properties of aluminumand calcium compounds. In Powell MF, Newman MJ (eds):‘‘Vaccine Design: The Subunit and Adjuvant Approach,’’ NewYork: Plenum Press, 1995:229–248.

15. Eldridge JH, Staas JK, Meulbroek JA, Tice TR, Gilley RM: Bio-degradable and biocompatible poly(D,L-lactide-co-glycolide)microspheres as an adjuvant for staphylococcal enterotoxin Btoxoid which enhances the level of toxin-neutralizing activity.Infect Immun 1991;59:2978–2986.

284 Diwan et al.