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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
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
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.
controlled release delivery; LHRH-DT vaccine; prostatic atrophy; polylactide-co-glycolic acid copolymer
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:
Received 8 August 1997; Accepted 21 November 1997
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 [4]. 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 [5]. 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 [6], was used
for the preparation of microspheres encapsulating the
LHRH vaccine.
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 [7]. This was
linked to aminohexanoic acid as the spacer molecule
to which the carrier, diphtheria toxoid, was attached
via its epsilon-amino group [8].
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. [9]. 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
TABLE I. Encapsulation Efficiency and Particle Size of
Three Batches of the LHRH-DT Vaccine
Antigen entrapped
(mg/mg of Ms)a
± SD
efficiency (%)
Mean particle
size (mm)
2.45 ± 0.18
2.66 ± 0.83
2.57 ± 0.25
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.
Physicochemical Characteristics of LHRH
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 [10] as well as of others [11] 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-
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
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
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
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 [5]. 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.
Previous studies have shown that immunization
against LHRH causes a marked atrophy of the rodent
and monkey prostates [1–4]. The vaccine [7] 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. [12]
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
Anti-LHRH IgG titer
absorbance value (492 nm)
[5]. It is therefore likely that LHRH has a local action
on the prostatic cells over and above its effect via androgens. Srkalovic et al. [13] 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 [7] 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
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
[14] and the small size of the microspheres [15]. 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. [15]
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.
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