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Vaccine 27 (2009) 2138–2143
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/vaccine
Poly(lactide-co-glycolide) microspheres: A potent oral delivery system to elicit
systemic immune response against inactivated rabies virus
R. Ramyaa,∗, P.C. Vermaa, V.K. Chaturvedia, P.K. Guptab, K.D. Pandeya, M. Madhanmohanc,
T.R. Kannakia, R. Sridevia, B. Anukumara
aBiological Products Division, Indian Veterinary Research Institute, Izatnagar 243122, UP, India
bAnimal Biotechnology Division, Indian Veterinary Research Institute, Izatnagar 243122, UP, India
cResearch and Development centre, Indian Immunologicals Ltd., Gachibowli, Hyderabad 500032, India
a r t i c l ei n f o
Received 1 October 2008
Received in revised form 23 January 2009
Accepted 29 January 2009
Available online 6 February 2009
a b s t r a c t
Rabies is an endemic, fatal zoonotic disease in the developing countries. Oral vaccination strategies are
suitable for rabies control in developing countries. Studies were performed to investigate the suitability
of poly(lactide-co-glycolide) (PLG) microspheres as an oral delivery system for ?-propiolactone inac-
tivated concentrated rabies virus (CRV). Immune responses induced by encapsulated (PLG+CRV) and
un-encapsulated inactivated rabies virus after oral and intraperitoneal route administrations were com-
neutralization test (MNT) and IgG2a and IgG1 titers of mice group immunized orally with PLG+CRV
showed significantly (p<0.001) higher response than the group immunized orally with un-encapsulated
CRV. There was no significant difference (p>0.05) between groups inoculated by intraperitoneal route.
The stimulation index (SI) obtained by lymphoproliferation assay of PLG+CRV oral group also showed
significantly (p<0.001) higher response than the group immunized orally with un-encapsulated CRV,
suggesting that oral immunization activates Th1-mediated cellular immunity. Immunized mice of all
experimental groups were challenged intracerebrally with a lethal dose of virulent rabies virus Challenge
Virus Standard (CVS). The survival rates of mice immunized orally with PLG+CRV and CRV alone were
75% and 50%, respectively, whereas intraperitoneally immunized groups showed 100% protection. The
overall results of humoral, cellular immune response and survival rates of mice immunized orally with
© 2009 Elsevier Ltd. All rights reserved.
Rabies, a fatal neuroencephalomyelitis is transmitted through
the bite of rabid animals. Human mortality from endemic canine
of stray dogs have been effective in rabies control in developed
countries, similar approaches have been difficult to implement in
humans exposed at risk of rabies is expensive. In addition wild ani-
mals which constitute reservoirs for the propagation of the virus
are not readily accessible for vaccination and hence an alternative
approach to limit the spread of the disease is warranted . Oral
immunization which has been successful in control of rabies in
∗Corresponding author. Tel.: +91 40 23000211; fax: +91 40 23005958.
E-mail address: email@example.com (R. Ramya).
wild animals uses live attenuated vectors or attenuated vaccines
[3,4]. These technologies may not be readily available for vaccine
manufactures in developing countries. However inactivated virus
vaccines are of wide use in developing countries. Hence vaccine
native (oral/mucosal) immunization strategies. To be viable, oral
vaccination may be improved by using adjuvants or gastrointesti-
system that minimizes the antigen dose, boosters and offers pro-
tection from the stomach acids is needed to control rabies using
oral vaccination. Among a variety of inert and biodegradable poly-
(PLG) particles made from lactic and glycolic acids holds promise
for delivery of variety of antigens to the immune system . PLG
microspheres have been used in the delivery of a variety of anti-
gens because of their stability, non-toxicity and ability to induce
strong immune response in various animal models [8–12].
This study was under taken to investigate the protective effi-
cacy of orally delivered ?-propiolactone (BPL) inactivated rabies
0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.
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R. Ramya et al. / Vaccine 27 (2009) 2138–2143
virus (RV) encapsulated in PLG microspheres in mice. The immune
responses induced by oral vaccination were compared with
intraperitoneal route of immunization.
2. Materials and methods
2.1. Cells and viruses
BHK-21 cell line (baby hamster kidney cell line, ATCC, USA)
was cultured at 37◦C and 5% CO2in a humidified incubator using
Dulbecco’s modified Minimum Essential Medium (DMEM, GIBCO,
USA) supplemented with 10% (v/v) heat inactivated fetal bovine
serum (FBS, GIBCO, USA) and antibiotics (100IU of penicillin/ml
and 100?g of streptomycin/ml).
Rabies virus (PV-11 strain, Institut Pasteur, France) was propa-
gated using BHK-21 C13 cells grown in 150cm2tissue culture flasks
. Challenge Virus Standard (CVS) mouse brain strain of rabies
virus (Institut Pasteur, France) was used for mouse protection test
to determine the vaccine potency and mouse neutralization test
(MNT) to determine the virus neutralizing antibody (VNA) titers.
2.2. Preparation of concentrated BPL-inactivated rabies virus
The rabies virus harvests obtained from infected BHK-21 cells
the degree of inactivation was determined by mouse inoculating
test . The concentrated, inactivated rabies virus was prepared
as described elsewhere [15,16]. The pellet was resuspended in
small volume of STE buffer (0.15M NaCl, 0.01M Tris and 0.001M
EDTA, pH 7.5) and protein was estimated using bicinchoninic acid
(BCA) protein assay kit (Bangalore Genei, India). The purified rabies
viral proteins were visualized on 10% SDS-PAGE after staining with
Coomassie brilliant blue stain. For the detection of rabies viral pro-
and transferred onto PVDF membrane (Millipore, USA). The blot
was probed with rabbit anti-rabies hyperimmune serum (1:500).
IgG-HRPO conjugate (1:5000, Bangalore Genei, India).
2.3. Preparation and characterization of PLG microspheres
PLG microsphere (Sigma, USA) containing purified CRV was pre-
pared as described by Rosas et al. . Briefly 6% (W/V) of PLG
(50:50) was dissolved in 5ml of dichloromethane (Merck, India)
and emulsified with 5mg of purified rabies virus antigen by high-
speed homogenization for 2–4min. The primary emulsion (W/O)
was mixed with 25ml of 1mM HEPES buffer, pH 7.5 containing 8%
by high-speed homogenization for 5min in order to form double
emulsion (W/O/W). Finally, 50ml of 2% isopropanol solution was
added and stirred for 1h. The microspheres were collected by cen-
water and stored at −20◦C after freeze drying. PLG microspheres
visualized by scanning electron microscopy (SEM). Briefly, a small
pinch of the lyophilized microspheres were coated on the metal-
lic slub by ion sputtering or gold coating and then observed under
different magnifications in a scanning electron microscope (JEOL
JSM 840X, Japan) and the images captured. Viral antigen load in the
microspheres was determined by measuring the protein content
using BCA method after known quantity (20mg) of microspheres
were hydrolyzed by using 3ml of 5% (W/V) SDS in 0.1M NaOH solu-
tion and stirring overnight at room temperature. The mixture was
Experimental groups of mice used in this study. The mice were inoculated with
appropriate vaccine as indicated.
Sl. no.Groups No. of miceRoute Antigen dose/mice (?g)
IP: intraperitoneal; PLG: poly(lactide-co-glycolide); ?g: microgram.
aControl groups inoculated with equivalent quantity of empty microspheres.
centrifuged and the protein content of supernatant (hydrolysate)
was determined. Antigenicity of the CRV released from micro-
spheres was determined by Dot-ELISA using rabies virus specific
rabbit polyclonal serum and compared with results obtained using
pre-encapsulated CRV .
2.4. Immunization and sample collection
Swiss albino mice (3–4 weeks old; n=105) were used in this
study. The animals were maintained and used according to the
guidelines of Council for the Purpose of Control and Supervision
of Experiments on Animals (CPCSEA). The experiment groups are
quantity of microspheres suspended in PBS. Blood samples were
obtained on day 0, 7, 14, 21, 28, 35 and 42 post-immunization.
Splenocytes from vaccinated and control Swiss albino mice were
isolated after lysis of erythrocytes using RBC lysis buffer (Sigma,
blue dye exclusion method.
2.5. ELISA and determination of rabies virus specific Ig titers
Serum IgG, IgG1 and IgG2a antibody responses were deter-
mined by ELISA. Ninety-six-well Maxisorp immunoplates (Nunc,
Denmark) were coated with appropriately diluted CRV (100?l) in
carbonate/bicarbonate buffer (pH 9.6) and incubated overnight at
4◦C. After removal of unbound antigens, the un-reacted sites were
blocked using 200?l of 3% BSA by incubation at 37◦C for 1h. The
test sera (100?l) and control sera were subjected to serial two-fold
dilutions with phosphate-buffered saline–Tween 20 (PBS contain-
ing 0.05% Tween 20, V/V; PBST) as a diluent and incubated at 37◦C
diluted HRP-conjugated IgG raised against mouse IgG, IgG1and
IgG2a (Bangalore Genei, India) were added to the plates and incu-
bated at 37◦C for 1h. After removal of unbound conjugate, positive
binding was detected by adding substrate (OPD/H2O2) and incu-
bation for 15min at 37◦C in dark. The enzyme–substrate reaction
was stopped by adding 100?l/well of 1.25M sulphuric acid and
absorbance read at 492nm. Rabies virus specific antibody titers
were expressed as the reciprocal of the highest serum dilution that
showed an OD492value above the cut-off value, which was defined
as the average OD492value of seven non-immunized sera±3 stan-
2.6. Mouse neutralization test
MNT was carried out essentially as described . Briefly, the
heat inactivated sera samples were serially diluted and incubated
with 50 LD50 of CVS at 37◦C for 90min. The presence of un-
neutralized virus in the mixture was checked by intracerebral
inoculation of mice (n=10) for each serum dilution. Control groups
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R. Ramya et al. / Vaccine 27 (2009) 2138–2143
to the major viral protein G, N, P and M are marked. (B) Western blot analysis of CRV showing immunoreactivity with hyperimmune serum raised against rabies. Lane 1:
molecular weight marker. Lane 2: CRV.
were inoculated with 100 LD50, 50 LD50or 10 LD50of CVS. The
inoculated mice were observed daily for 21 days and presence of
any symptom typical of rabies such as hind limb paralysis were
noted. The VNA titer of test sera samples was calculated based
on the percentage of survivors in each group. A standard rabies
immunoglobulin (SRIG) was included as a positive control for com-
2.7. Challenge study
The protective efficacy of the different groups of immunized
mice were assessed by challenging eight mice randomly selected
from each group with 0.03ml of 20 LD50of mouse adapted rabies
virus CVS strain. After inoculation by intracerebral route the mice
were observed for 14 days for symptoms of rabies. Protection
index was calculated based on the percentage of survivors in each
2.8. Lymphoproliferation assay
Stimulation index was measured by lymphoproliferation assay
using MTT [3-[4,5-dimethylthiazole-2-yl]2,5-diphenyl tetrazolium
bromide] dye . Briefly CRV immunized mouse splenocytes
(1×106splenocytes/ml) were seeded in 96-well flat-bottom tissue
culture plate (NunclonTM, Denmark). The splenocytes were stimu-
lated with CRV (10?g/ml) or Concanavalin A (Con A; 20?g/ml) and
incubated at 37◦C for 72h in a CO2incubator. Twenty microliters
of MTT (5mg/ml in PBS) was added to all the wells and the plates
were incubated for 4h at 37◦C in a CO2incubator. One hundred
and 50?l of dimethyl sulfoxide (DMSO) was added to dissolve the
formazon crystals formed by metabolization of MTT and the plates
were incubated for 15min at 37◦C. The optical density (OD) was
measured at 520nm with reference background color reduction at
650nm in a microplate reader.
2.9. Statistic analysis
Unpaired Student’s t-test was used to analyze differences
of means between groups. Statistical analyses were performed
using OriginPro 7.5 SR4 analysis software (Origin Lab Corporation,
3.1. Characterization of CRV
of major structural proteins such as G protein (65kDa), N protein
(57kDa), P protein (38kDa) and M protein (25kDa) (Fig. 1A). West-
ern blot analysis of the CRV produced expected polypeptide profile
3.2. Characterization of PLG microspheres
SEM observation of the PLG prepared by double emulsion
method exhibited varying size (range of 0.5–2.5?m) of micro-
spheres and majority of them were less than 1?m in diameter
(Fig. 2). All the microspheres had spherical, intact and smooth sur-
face regardless of their size. There was no difference in surface
morphology of empty and antigen loaded microspheres. The load-
ing efficiency of the rabies antigen in microspheres varied from
50 to 55% as determined by BCA assay. To check for in vitro toxic-
ity, ten mice were injected subcutaneously with a 10× vaccination
dose. No abnormal clinical signs or death were observed in these
animals during the observation period of 14 days (data not shown).
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R. Ramya et al. / Vaccine 27 (2009) 2138–2143
Fig. 3. Detection of rabies virus specific IgG antibody titer by indirect ELISA. Rabies virus specific IgG antibody titer of each immunized groups was determined by indirect
ELISA. The serum samples from each group (n=10) were collected at 0, 7, 14, 21, 28, 35 and 42 days post-immunization. The ELISA antibody titers were calculated as the
log10of the reciprocal antibody dilution that showed an OD492value above the cut-off value, which was defined as the average OD492value of seven non-immunized sera±3
standard deviation. Each symbol represents mean level of serum antibody, expressed as geometric mean±standard deviation (GMT±S.D.). Each curve represents kinetics of
antibody production induced by various immunized groups. ***p<0.001.
In Dot-ELISA the hydrolysate of the CRV loaded microspheres
showed immunoreactivity with RV specific hyperimmune sera and
positive dots were formed similar to that of the pre-encapsulated
CRV, indicating the retention of antigenicity after microencapsula-
tion (data not shown).
3.3. In vivo antibody response in mice
The ability of PLG microspheres for oral and intraperitoneal (I/P)
delivery of CRV to induce specific immune responses was evalu-
and their sera tested in ELISA to determine anti-RV antibody titer.
The anti-RV antibody titers were determined for all experimental
groups up to 42 days post-immunization (dpi). The highest mean
ELISA titer of 2.893±0.018 was observed in PLG+CRV oral group
which was significantly (p<0.001) higher than CRV (1.991±0.024)
at 42 dpi. In the case of intraperitoneal immunized group there
were no significant differences between PLG+CRV (2.870±0.033)
and CRV alone (2.893±0.011) at 42 dpi (Fig. 3).
3.4. Mouse neutralization test
MNT was done to determine VNA titers in the immune mice
sera (n=10). A VNA titer of 4IU/ml was observed in PLG+CRV oral
group which was significantly (p<0.001) higher than the group
immunized orally with CRV alone (2IU/ml) on 35 dpi. The highest
mean VNA titer of 8IU/ml was observed in the following intraperi-
toneal immunized groups CRV, PLG+CRV. There was no significant
difference (p>0.05) between encapsulated and un-encapsulated
results suggest that the PLG encapsulated CRV can induce a virus
neutralizing antibody response by oral route. However, intraperi-
toneal administration may induce a higher antibody response in
3.5. Protection against challenge
In order to compare the protective efficacy, different groups of
immunized mice were challenged intracerebrally with 20 LD50of
CVS strain on 42 dpi. In the group immunized orally with PLG+CRV
75% protection was observed whereas mice immunized with CRV
orally exhibited 50% protection. The mice groups immunized with
(100%) were protected. The unvaccinated control mice failed to
Fig. 4. Detection of rabies neutralizing antibody titer by MNT. VNA titer of 35 dpi
sera was determined by mouse neutralization test. Briefly the heat inactivated sera
samples were serially diluted and incubated with 50 LD50of CVS at 37◦C for 90min.
The presence of un-neutralized virus in the mixture was checked by inoculating
intracerebrally in mice (n=10) for each serum dilution. Virus neutralizing antibody
in each group. The VNA titer was expressed as IU/ml. ***p<0.001.
resist the challenge and died between 7 and 10 days post-challenge
3.6. Cell-mediated immune response
The induction of cellular immunity in immunized groups was
assessed in vitro by lymphoproliferation assay. As shown in Fig. 5,
the level of proliferative responses in the mice immunized orally
with PLG+CRV were significantly higher (1.855±0.03, p<0.001)
Protection levels of different groups of mice challenged with 20 LD50 ofrabies virus
CVS strain (mouse adapted).
Sl. no. Groups No. of mice challengedNo. of mice diedProtection (%)
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R. Ramya et al. / Vaccine 27 (2009) 2138–2143
ulated with Concanavalin A and BPL inactivated rabies virus (CRV). The end point
was determined by colorimetric MTT dye reduction test. The stimulation index was
calculated by OD of stimulated–OD of un-stimulated/OD of unstimulated. Data are
expressed as the mean±S.D. (n=6 mice per group). ***p<0.001.
than in the group immunized with CRV alone (1.057±0.03). Stim-
ulation index of intraperitoneal group also showed significant
difference (p<0.001) between encapsulated (2.017±0.02) and un-
encapsulated CRV (1.256±0.01) suggesting that PLG can help to
induce strong cell-mediated immune responses.
3.7. Th1 (IgG2a) and Th2 (IgG1) response
Anti-RV IgG1 and IgG2a titers were measured in mice sera
from different immunized groups by ELISA. There was significant
(p<0.001) difference of IgG2a/IgG1 response between encapsu-
lated and un-encapsulated inactivated rabies virus administered
by oral route. In the case of intraperitoneal route there was no sig-
nificant difference (p>0.05) in the IgG2a/IgG1 response (Fig. 6).
Interestingly, a group of mice immunized with CRV alone (oral)
showed low levels of IgG1 and IgG2a responses.
Taken together, these observations indicate that mice immu-
nized orally with PLG+CRV are able to respond to the inactivated
rabies virus and produce strong Th1 response.
Rabies is endemic in Asia and Africa and rabid dogs are con-
sidered responsible for more than 90% of human rabies deaths.
Various types of human and animal vaccines are used worldwide
for the prevention of rabies. This includes inactivated cell culture,
Fig. 6. Detection of rabies virus specific IgG1 and IgG2a antibody titer by indirect
ELISA. Rabies virus specific IgG1 and IgG2a antibody titer of each immunized groups
titers were calculated as the log10of the reciprocal antibody dilution that showed an
seven non-immunized sera±3 standard deviations. Each bar represents mean level
of serum antibody, expressed as geometric mean±standard deviation (GMT±S.D.).
modified live, recombinant and subunit vaccines. Among these
inactivated cell culture vaccine is the most commonly used. Vac-
cination of stray dogs has been a major obstacle for the control
of rabies in developing countries. Oral vaccination of dogs against
rabies is a promising supplementary method for dog rabies control
. There are reports describing a bait delivery system for deliv-
ering oral rabies vaccine to stray dogs in India . Baer et al. 
demonstrated that oral vaccination of foxes by inactivated vaccine
resulted in protection against live virus challenge and induction of
serum-neutralizing antibodies. Rupprecht et al.  suggested that
adjuvants or gastrointestinal protectants are necessary to deliver
inactivated rabies vaccines orally and induce a protective immune
response. Among the several antigen delivery systems available,
PLG has received considerable attention due to its potential for
protecting the antigens against adverse microenvironment in the
mucosal milieu and for controlled release of antigens . Unlike
soluble antigens, mucosally administered antigens entrapped in
PLG microspheres can induce good local and systemic antibody
response as well as cell-mediated immunity . Oral delivery of
many bacterial and viral antigens based on PLG microspheres have
been reported [8–12].
mucosal route can induce local and systemic antibody response 
as well as cell-mediated immunity .
In the present study oral vaccination of mice using PLG micro-
spheres encapsulated CRV was attempted and immune responses
were compared with intraperitoneal administered CRV. Encapsu-
lation of CRV by PLG microspheres did not result in degradation
of the CRV. Dot-ELISA and Western blot of CRV hydrolyzed from
the PLG microspheres indicated that the antigenic polypeptides
were present in the vaccine formulations. The PLG encapsulated
CRV was able to induce a humoral immune response in mice as
shown by the indirect ELISA using CRV as antigen (Fig. 3). The
antibody responses induced by oral inoculation of PLG encapsu-
lated CRV were significantly higher than responses induced by
CRV alone. Protection of antigen in the gastric environment and
presentation of antigen to lymphoid cells in the gut by PLG micro-
Degradation of the un-encapsulated CRV may have resulted in a
weaker antibody response. The glycoprotein of rabies is respon-
sible for inducing strong humoral immune response including
induction of neutralizing antibody response. It is possible that
PLG encapsulation helps in maintaining the antigen (glycoprotein)
Further, the MNT results indicate that PLG encapsulated CRV
is better than CRV alone in inducing virus neutralizing antibod-
ies and also antiviral resistance mechanisms resulting in protection
of mice. In this respect, the intraperitoneal administered CRV and
PLG encapsulated CRV were superior. Highest neutralization titers
were observed in these groups. Intraperitoneal administrations
may present CRV to a larger surface area for efficient uptake by
Cell-mediated immunity plays an important role in recovery
from attenuated rabies virus infection and in the post-exposure
prophylaxis of rabies [26–28]. Dietzschold et al.  demon-
strated that T-cell activation is a major protective component in
the immune response against RV infection, considering that anti-
body levels are low in the brain because of the blood brain barrier
, and lymphocytes infiltrate relatively easily into the brain after
virus infection. Lymphoproliferative responses as indicated by the
MTT assay were higher in the PLG encapsulated CRV immunized by
the oral route. PLG microspheres have been reported to induce cell-
mediated response against M. tuberculosis 38kDa protein  and
recombinant HIV protein . The lymphoproliferative responses
may involve induction of antigen specific T cells after engulfment
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R. Ramya et al. / Vaccine 27 (2009) 2138–2143
, these responses may involve expression of cytokines [31,32].
In this study, the T helper cell immune responses were demon-
strated to be the Th1 type as shown by the higher IgG2a responses
in mice orally immunized with PLG encapsulated CRV. This type of
[31,32]. The Th1 type response will help in protection against live
rabies virus challenge [26–28].
Earlier studies  showed that PLG encapsulated antigens
are efficiently phagocytosed by macrophages (APC) and present-
ing the antigen by cytotoxic MHC class I pathway resulting in
CMI response. Compared with un-encapsulated CRV immunized
groups, SI values of T-cell proliferation assay were much higher in
mice immunized with PLG encapsulated antigens given by oral and
intraperitoneal route (Fig. 6) and all the PLG encapsulated groups
showed significantly higher or comparable immune response with
un-encapsulated CRV group immunized by oral and I/P route,
and Th2 pathways.
Survival from lethal challenge could be predicted based on the
VNA titers obtained by MNT. Since the VNA titers in this study
ies would have been able to inhibit the binding of the virus to the
rabies virus specific receptors or the fusion process of the virus
as described by Dietzschold et al. . Hence, it is reasonable to
assume that VNA inhibits virus growth in the brain and VNA titers
predictive of challenge study results. The 50% protection observed
in CRV (oral) without PLG may largely be attributed due to the
whole viral protein remaining after degradation in the stomach.
In summary, the anti-rabies virus IgG antibody response, VNA
response obtained by MNT and IgG2a and IgG1 antibody response
of mice group immunized orally with PLG+CRV showed signif-
icantly higher response than the group immunized orally with
Results presented here show that PLG encapsulated rabies vac-
by oral route. Significantly higher immune response of the PLG
encapsulated groups to that of the CRV alone oral group suggest-
ing, that the PLG microspheres were efficiently phagocytosed by
macrophages and presented to the immune system without alter-
ing the immunogenicity of the antigen. PLG microspheres can be
useful in the mass oral immunization programme to control rabies
in stray dogs and wild animals. Future studies will determine the
effect of the booster immunization; dose–response and potency
CRV for rabies control in developing countries.
Institute, Izatnagar, India, for providing necessary facilities to carry
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