Vaccine 28 (2010) 1463–1467
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Evaluation of the immunogenicity of a recombinant glycoprotein-based
Chandipura vaccine in combination with commercially available DPT vaccine
C.H. Venkateswarlu, V.A. Arankalle∗
Hepatitis Division, National Institute of Virology, Microbiological Containment Complex, Sus Road, Pashan, Pune 411021, India
a r t i c l e i n f o
Received 12 May 2009
Received in revised form
19 November 2009
Accepted 24 November 2009
Available online 14 December 2009
Baculovirus expression system
Recombinant G protein-based vaccine
Commercially available DPT vaccine
CHP–DPT combination vaccine
a b s t r a c t
Chandipura virus (CHPV) belongs to family Rhabdovoridae and has emerged as an encephalitis causing
pathogen with high mortality among pediatric population from three Indian states. The recombinant
glycoprotein (rGp) was shown to be an excellent vaccine candidate as evaluated in a murine model. As
the disease is predominantly rural, to ensure maximum coverage for Chandipura vaccine, an attempt
was made to evaluate combination of rGp and a commercially available DPT vaccine (CHP–DPT). When
CHP–DPT was used for immunization of mice, 90% seroconversion against rGp with high antibody titers
(1:1200 by ELISA and 1:320 by neutralization test) was observed and did not differ from mice immunized
with rGp alone (P>0.05). Similarly seroconversions and antibody titers against DPT were comparable in
mice immunized with DPT alone or in combination with rGp. Seroconversions and antibody titers ranged
resulted in 90% survival in rGp alone and CHP–DPT groups. Lymphocyte proliferative responses were also
to the other component. Substantial decrease of CHPV RNA and absence of histopathological changes in
the brains of surviving immunized mice after challenge than the unimmunized controls further confirm
efficacy of the vaccine even after intracerebral challenge. In conclusion, a combination vaccine seems
feasible for use in a restricted area where the disease is endemic and should be subjected to additional
studies required for future use in humans.
© 2009 Elsevier Ltd. All rights reserved.
Chandipura virus (CHPV) was isolated in 1965 from the sera of
two patients during an outbreak of febrile illness in a locality called
“Chandipura”, Maharashtra state, India  and subsequently from
an encephalopathy patient in Raipur, Madhya Pradesh state, India
sents an emerging pathogen causing epidemics and sporadic cases
of encephalitis with high mortality (55–75%) in pediatric popula-
tion in three states from India ([4–6] and our unpublished data).
of symptoms, most dying within 24h. Treatment is mainly symp-
Considering the rapid mortality of the disease in children and
restricted distribution, vaccination was considered as an impor-
tant control measure. We successfully prepared a recombinant G
protein-based candidate vaccine showing ∼90% efficacy in mice
challenged intracerebrally with the virus . In order to avoid
increased needle pricks to young children and to ensure maximum
∗Corresponding author. Tel.: +91 20 26006330; fax: +91 20 25871895.
E-mail address: email@example.com (V.A. Arankalle).
coverage, we tried to develop a combination vaccine containing
DPT [component of Extended Program of Immunization (EPI)] and
recombinant G protein of Chandipura virus. This study reports the
evaluation of such a combination vaccine (CHP–DPT).
2. Materials and methods
2.1. Vaccine components and immunization
The animal ethical committee of the institute approved this
study. Expression and purification of rGp was carried out as
described earlier . Briefly, the G-gene of Chandipura virus was
cloned and expressed using baculovirus expression system. The
recombinant protein was purified employing HPLC HiPrep 16/60
Gel filtration column and characterized by ELISA and immunoblot.
The DPT vaccine manufactured by the Serum Institute of India,
Pune, India was purchased from the market and dose/mouse was
calculated based on the average weight of the mice used for the
experiments (25?l/mouse). Aluminium phosphate (AlPO4) was
used as adjuvant. 1?g of rGp was adsorbed on 3.25?g of AlPO4
(Sigma Chemicals, St. Louis, MO, USA) by vortexing at a low speed
for 1h at RT followed by centrifugation at 5000rpm for 5min at RT.
The pellet was re-suspended in 0.01M PBS, pH 7.2 and diluted to
0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.
C.H. Venkateswarlu, V.A. Arankalle / Vaccine 28 (2010) 1463–1467
the required rGp dose at the time of immunization. This rGp (1 or
2?g/dose) adsorbed on AlPO4mixed with commercially available
DPT vaccine (25?l/dose).
The rGp (1 and 2?g) with or without DPT vaccine was used for
immunization (3 doses, intramuscular, 4 weeks apart) of mice (10
mice/group, Swiss albino, Female, 6–8 weeks old). DPT alone and
adjuvant alone were administered to two groups of mice.
2.2. Evaluation of immune response
Serum samples were collected periodically by retro-orbital
plexus bleeding and subjected to ELISA for detection/quantitation
of IgG-anti-CHP antibodies as well as tissue culture based in vitro
virus neutralization test (NT) according to the methods described
earlier . Detection/quantitation of antibodies against each com-
ponent of DPT vaccine was carried out by Serion ELISA classic test
(Institut Virion\Serion GmbH, Germany) according to the manu-
facturer’s instructions except that horseradish peroxidase (HRP)
conjugated goat anti-mouse IgG (Sigma Chemicals, St. Louis, MO,
USA) replaced the anti-human-IgG-HRP conjugate. Antibody titers
were determined for individual mouse from each group and the
reciprocal of the highest dilution was taken as the titer against the
respective component of the vaccine. The titers are presented as
geometric mean titers (GMTs) and log-transformed antibody titers
with standard error.
For determining IgG isotypes, rGp was used as coating anti-
gen. The reaction with test serum (37◦C, 30min) in different wells
was followed by incubation at 37◦C for half an hour with goat
anti-mouse IgG isotype antibodies (IgG1, IgG2a, and IgG2b) (Sigma
Chemicals, St. Louis, MO). HRP-conjugated rabbit anti-goat IgG
CHPV-specific titers for IgG isotypes namely IgG1, IgG2a and IgG2b
were determined for individual mouse from each group using 2-
fold serum dilutions. The reciprocal of the highest dilution that had
an absorbance greater than or equal to the ELISA cut-off was taken
as the CHPV-specific antibody titer. All the analyses were carried
out on the log-transformed antibody titers with standard errors.
Lymphocyte proliferative response against rGp was determined
index (SI) was determined as ratio of counts per minutes (CPM) in
the presence of rGp/CPM in absence of rGp. Mice with SI value ≥3
were considered to be the responders.
Mice lethal dose 50 (LD50) of homologous CHPV strain
(CIN034627) was determined in 16–18 weeks female Swiss albino
mice . All immunized/control mice were challenged with
100LD50of CHPV at 2 weeks after the last immunization.
2.3. CHPV RNA quantitation
CHPV RNA quantitation in the brains of mice harvested on 5th
day after challenge was carried out employing real time PCR in the
following groups: (1) unimmunized, challenged mice (just prior
to death n=6), (2) immunized, challenged, surviving mice (n=6,
day 5 and n=6, day 14) (3) one immunized mouse succumbing
to challenge. RNA was isolated by using Ribopure RNA isolation kit
of total RNA was used per reaction. Real time one-step RT-PCR was
performed in a 96 well format using 7300 real time PCR system
(Applied Biosystems International, Foster City, CA) and SDS soft-
ware version 1.3.1., according to the protocol described by Kumar
et al. .
Mice (n=3/group) were euthanized, brains removed, fixed in
10% neutral buffered formalin and embedded in paraffin. 6-?m
vertical sections were stained with hematoxylin and eosin. The
groups included (1) unimmunized mice (n=3) and (2) mice immu-
nized with 1?g of rGp alone (n=3) or 1?g rGp+DPT (n=3) and
challenged with 100LD50of CHPV.
2.5. Statistical analysis
ELISA and NT titers were compared using t-test employing SPSS
9.0 software. Non-responders in each group were included in the
3.1. Antibody responses to different vaccine components
Table 1 documents that percent seroconversions in mice immu-
nized with different immunogens were comparable with respect
to Chandipura as well as Diphtheria, Pertussis and Tetanus. Thus,
addition of rGp to DPT did not alter seroconversion rates produced
by both components of the vaccine. Administration of 1 and 2?g
rGp resulted in similar seroconversion rates (90%).
Both ELISA and NT detected anti-rGp antibodies as early as 2
weeks after the first dose in mice receiving single or combina-
tion vaccine. Fig. 1 depicts that anti-CHP titers were comparable
irrespective of combination with DPT, maximum titers in NT
and ELISA being 1:320 and 1:1200 respectively. The NT titers
were consistently lower than the ELISA titers. Seroconversion
rates to Diphtheria, Pertussis and Tetanus components were 100%,
90–100% and 90–100% respectively when administered along with
rGp while 100% (D), 90% (P) and 90% (T) seroconversions were
observed when immunized with DPT alone (Table 1). The maxi-
mum antibody titers against Diphtheria and Tetanus were 1:1200
whereas against Pertussis a titer of 1:2400 was recorded (Fig. 1).
We further studied the effect of combination on the persistence
of antibodies to individual components over a period of 6 months.
As evident from Fig. 2, addition of rGp to DPT had no significant
effect on the antibody titers to all the 4 components during the
observation period of 6 months.
3.1.1. Isotype analysis
In mice immunized with rGp (n=6), IgG1 isotype was sig-
nificantly higher than IgG2a (P=0.0035). Similarly, IgG1 was the
predominant isotype in mice surviving challenge after immu-
nization with DPT along with rGp (n=6) as compared to IgG2a
(P=0.0153) (Fig. 3). However the only mouse succumbing to the
Fig. 1. The geometric mean of reciprocal anti-CHPV, Diphtheria, Pertussis and
dose of rGp. Bar represents standard error. All antibody positives were subjected to
titration for respective antibodies.
C.H. Venkateswarlu, V.A. Arankalle / Vaccine 28 (2010) 1463–1467
Percent seroconversion in mice immunized with rGp and/or DPT.
IgG positives (%)
1?g of rGp+25?l of DPT
2?g of rGp+25?l of DPT
1?g of rGp alone
25?l of DPT
Fig. 2. The geometric mean of reciprocal anti-CHPV IgG titers (ELISA), anti-Diphtheria, anti-Pertussis, anti-Tetanus antibody titters (log10) and CHPV neutralizing antibody
titers in mice (n=10 for each group) immunized with 1?g of rGp+25?l of DPT and monitored for 6 months after the last dose. Bar represents standard error.
infection despite immunization with DPT along with rGp, IgG1 was
1.24-fold less when compared to surviving mice. The IgG2b titers
specific IgG and all its isotypes. IgG1 predominance is suggestive of
Th2 type of immune response in mice immunized with rGp alone
or in combination with DPT.
3.2. Lymphocyte proliferative responses
rGp or 1?g rGp alone (60%) (Fig. 4). The control mice remained
3.3. Challenge studies
immunized with rGp and DPT or rGp alone resulted in 90% survival
in all groups. All survived mice were healthy during the observa-
Fig. 3. Serum CHPV-specific anti-rGp IgG1 and IgG2a antibody mean log(10) titers
detected in ELISA at 2–3 weeks post-last dose (third dose) in mice (n=6).
tion period of 21 days post-challenge. There was 100% mortality
in control-unimmunized mice with 7.5 days of cumulative survival
(2–7 days). The immunized mice succumbing to challenge died at
times similar to the controls (Fig. 5).
Brain samples of immunized (1?g of rGp+DPT), challenged
and surviving mice (n=4) succumbing to challenge despite immu-
nization (n=1) and control non-immunized mice infected with
CHPV (n=4) were subjected to real time one-step RT-PCR. Of
the four surviving mice, two exhibited low titres (4.46×103
and 1.41×103copies/500ng RNA) while two were negative.
The immunized mouse succumbing to the challenge exhibited
1.6×106copies/500ng RNA while CHPV RNA in the control
mice varied from 1.15×107to 1.44×106copies/500ng RNA
(P=0.007). Thus, clear reduction in the replication was noted
in immunized and challenged mice when compared to the
As evident from Fig. 6, control mice (unimmunized, CHPV
infected) showed distinct pathological changes such as congested
Fig. 4. Stimulation indices measured in spleenocytes from mice immunized with
different concentrations of rGp and/or DPT and stimulated with rGp antigen. SI
values>3 indicate responders. Each point represents a single mouse.
C.H. Venkateswarlu, V.A. Arankalle / Vaccine 28 (2010) 1463–1467
Fig. 5. Kaplan–Meier survival curve showing survival/mortality pattern of mice
immunized with rGp singly or in combination with DPT and challenged intracere-
brally with 100LD50homologous strain of CHPV as compared to unimmunized mice
infected with the same dose of the virus.
blood vessels, foci of gliosis, paranchymatous lymphocyte infil-
Overall, 1 and 2?g purified recombinant G protein expressed
employing baculovirus expression system showed excellent effi-
cacy following immunization along with commercially available
DPT vaccine. Specific antibody titers were comparable (P>0.05)
when the immunogens were administered singly, or in combina-
This study provides important data to suggest that combination
of recombinant G protein of Chandipura virus and commercially
individual components. Earlier, we showed that rGp represents an
excellent vaccine candidate emphasizing utility of this vaccine in
controlling fatal encephalitis caused by this virus . Neither rGp
nor DPT had any significant effect on the antibody titers of anti-rGp
or all the three components of DPT vaccine.
Subtype analysis showed the predominance of IgG1, i.e., Th2
type of immune response in mice immunized with rGp alone and
in combination with DPT. Importance of antibodies against each
component of DPT vaccine has been well documented [10–13].
In the present study, immunized mice not mounting detectable
lymphocyte proliferative response but positive for anti-CHP anti-
bodies did survive vial challenge suggesting the important role of
antibody-mediated protection in this infection. We did not check
for cell-mediated immunity against DPT antigens. The immune
response generated by the vaccine could reduce the viral load in
brain by ∼4logs or to undetectable levels confirming efficacy of
the vaccine following even intracerebral challenge.
Being a pediatric disease, immunization of children represents
an ideal way of control. The affected children mainly come from
rural areas with poor nutritional status. For ensuring most efficient
coverage of Chandipura vaccine to this at-risk population, admin-
istration with vaccine constituents of EPI was thought to be an
appropriate approach. We chose DPT as it is given intramuscularly
at the age of 3, 4 and 5 months and if CHP vaccine is given at this
time, we will be able to protect children at-risk without requiring
an additional injection with resultant high compliance. Both for-
mats of the vaccine, i.e., Chandipura alone and as a combination
with DPT must undergo pre-clinical and clinical trials on priority
Fig. 6. Hematoxylin- and eosin-stained vertical brain sections from unimmunized (A) and immunized (B) mice infected with intracerebrally with CHPV. The samples were
collected on 5th day post-infection for both groups at indicated magnifications. Histopathological changes observed in unimmunized control mice were absent in immunized
mice surviving challenge.
C.H. Venkateswarlu, V.A. Arankalle / Vaccine 28 (2010) 1463–1467 Download full-text
and used in the affected areas to save lives of a large number of
The authors thank Dr. A.C. Mishra, Director, National Institute of
Virology for all the encouragement for constant support. Financial
support for this work was provided by the Indian Council of Med-
ical Research (ICMR). Venkateswarlu C.H. acknowledges ICMR for
providing Senior Research Fellowship.
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