18, NUMBER 6
Effect of different adjuvants in equines for the
production of equine rabies immunoglobulin
SHIVALI ARORA, SOURABH SHARMA, SUNIL K. GOEL, USHA
of the recommended post-exposure prophylaxis by vaccination
and specific immunoglobulin therapy for rabies is largely hampered
by its high cost and inadequate production. Therefore, the
development and availability of an economic preparation of
rabies immunoglobulin is a high priority for India, where rabies
is a major cause of death. We studied the efficacy of four
different adjuvants in raising antibodies to rabies antigen
in older, discarded equines.
Methods. Eleven equines, 23–26 years
old, were divided into 4 groups to receive four different adjuvants in small
amounts (1–2 ml)—Freund complete adjuvant with Mycobacterium
tuberculosis, Freund complete adjuvant with M. butyricum, Freund incomplete
adjuvant and bentonite—along with purified chick embryo cell vaccine. The
immunization schedule was spread over 105 days and the antibody titres were measured
on days 56, 91 and 119.
Results. On day 119 (third sampling), Freund
complete adjuvant with M. tuberculosis provided a geometric mean titre of 654.03
IU/ml in comparison with a titre of 459.19 IU/ml with Freund complete adjuvant
with M. butyricum, 630.95 IU/ml with Freund incomplete adjuvant and
172.18 IU/ml with bentonite.
Conclusion. Purified chick embryo cell
vaccine in combination with Freund complete adjuvant containing M. tuberculosis
and Freund incomplete adjuvant were better at eliciting an immune response. The
low quantity of adjuvants used possibly helped by causing very few side-effects
but without compromising the antibody titres.
Natl Med J India 2005;18:289–92
Rabies in humans is always fatal. Therefore, effective post-exposure prophylaxis
becomes the mainstay in the prevention and control of the disease. Vaccination
is a powerful tool to prevent the disease and immunization of humans is widely
rabies immune globulin (HRIG), a homologous immunoglobulin, is safer but not
available in the quantities required. Equine rabies immune globulin (ERIG)
is thus the mainstay and should be used much more widely and produced on a
much larger scale.
ERIG for human use is produced at the Central Research Institute
(CRI), Kasauli by using tissue culture vaccines and gives good results.
increase the immunogenicity of the antigen as well as the volume of ERIG, adjuvants
used with vaccines could be useful. In India, antirabies serum (ARS) was first
produced in young healthy ponies, and bayol and falba as adjuvants were first
used in 1957 and have since then been in constant use at the CRI, Kasauli.
In 1923, Ramon showed that it was possible to artificially increase the level
of diphtheria or tetanus antitoxin by adding bread crumbs, agar, tapioca, starch
oil, lecithin or saponin to the vaccines.3 In
1955, Christensen used bentonite-adsorbed Cape cobra venom for the immunization
of horses.4 With
the use of adjuvants, a smaller quantity of antigen is required to evoke the
immune response, thus reducing production costs.
In 1930, the adjuvant properties of Mycobacterium tuberculosis were
observation led Freund in the 1940s to develop the most widely used adjuvant—Freund
complete adjuvant (FCA) containing M. tuberculosis. Freund incomplete adjuvant
(FIA) is prepared in the same manner as the complete adjuvant except that M.
tuberculosis is not added. Another variant of FCA contains M. butyricum.
A vaccine adjuvant can influence the immune response in many ways.
The ability of adjuvants to influence several para-meters of the immune response
complicates the process of finding an effective adjuvant, because our knowledge
of how an adjuvant operates at the cellular level is insufficient to support
a completely rational approach to match the vaccine antigen with a proper adjuvant.
When different adjuvants provide equally good immunogenicity, the choice may
depend upon other factors such as cost, availability, reactogenicity and induction
of the desired immune response.
We studied the addition of 4 different adjuvants to a common vaccine
antigen—the purified chick embryo cell vaccine (PCECV). We aimed to identify
the best adjuvant for the production of ERIG.
Eleven equines (10 mules and 1 mare), 23–26 years old and in good health
were included in the study. These animals were received after being discarded
from the Indian Army and the Police Department. On their arrival at our centre,
they were subjected to a quarantine of 15 days followed by an injection of
tetanus toxoid (10 ml i.m.). They were kept under observation for 1 month before
being included in the study. The routine care and management of the equines
was done as per the guidelines of the Committee for the Purpose of Control
and Supervision of Experiments on Animals (CPCSEA).6
None of the animals had been immunized against rabies before, nor
did they have any rabies-related incident during the course of the study.
PCECV (Rabipur, Batch No. 626, date of manufacture April 2000, date of expiry
March 2005) manufactured by Chiron Behring Vaccines, Ankleshwar, Gujarat was
used as the common vaccine (antigen) in combination with the four different
adjuvants. The vaccine was available in lyophilized form with 1 ml diluent
per dose and having a potency >2.5 IU/dose. The mean potency of this batch
of vaccine was 10.5 IU/dose (information provided by manufacturer).
The following adjuvants were used:
1. FCA containing M. tuberculosis (H 37 Ra) (Lot No. 139520, Difco Laboratories,
2. FCA containing M. butyricum (Lot No. 146825, Difco Labora-tories, USA)
3. FIA containing bayol and falba (prepared in-house)
4. Bentonite (2%) adjuvant (aluminium silicate hydrate) (Lot No. 48319, Loba
Eleven equines were divided into 4 groups and immunized as per the schedule
given in Table I. The equines were grouped as follows:
|Table I. Immunization schedule by the subcutaneous
||Dose of PCECV
|21, 27, 35, 42
||2 ml 2% bentonite
|63, 71, 77, 84
||Both sides of neck
|91, 98, 105
||Both sides of neck
|PCECV purified chick embryo cell vaccine *
Group I Freund complete adjuvant with M. tuberculosis, group
II Freund complete adjuvant with M. butyricum, group III
Freund incomplete adjuvant and group IV 2% bentonite
Group I: Three equines (numbers 478, 482 and 506) immunized with PCECV and
FCA containing M. tuberculosis
Group II: Four equines (numbers 484, 486, 511 and 512) immunized with PCECV
and FCA containing M. butyricum
Group III: Two equines (numbers 494 and 495) immunized with PCECV and FIA
Group IV: Two equines (numbers 496 and 498) immunized with PCECV and 2% bentonite
The schedule of immunization for all the equines was the same, except for the
change in adjuvant which was administered in a dose of 1 ml based on the group
to which the equine belonged. The FCA and FIA adjuvants were used only with
the first (priming) dose owing to their propensity for causing adverse reactions.
Two ml of 2% bentonite was used in all the groups on days 21, 27, 35 and 42.
Blood samples were drawn from the equines on days 56, 91 and 119
of the immunization schedule.
Analysis of blood samples
The samples were collected on the days mentioned, sera separated aseptically
and subjected to the mouse neutralization test (MNT)7 for
estimation of the antibody titre as recommended by the Indian Pharmacopoeia,
1996. The statistical evaluation for determination of antibody titre was done
by the Reed and Muench method.8 The National Reference Standard (Batch no.
1/95) calibrated against the International Standard and having a unitage of
85 IU/ml was received from the Central Drugs Laboratory, CRI, Kasauli
and the Challenge Virus Strain (Batch no. 2/2000, titre 108/0.03 ml/mouse dose)
from the Rabies Research Centre, CRI, Kasauli. The ND50 of each sample was calculated
along with National Reference Standard Serum, and antibody titres calculated
and expressed in international units (IU)/ml.
The sera samples collected from the 11 equines on days 56, 91 and 119 were
assayed for estimation of antibody titre against rabies by the MNT. On day
56, the highest geometric mean titres (GMT) were obtained in group I equines
(162.55 IU/ml) while the lowest were in group IV (38.68 IU/ml; Table II). This
trend in these 2 groups continued over the complete immunization schedule
|Table II. Antibody titres on days 56, 91 and
||Antibody titre (IU/ml)
||Geometric mean titre (IU/ml)
(Fig. 1). However, groups II and III, which showed a low GMT on day 56, had
a substantial rise in GMT till day 119. In fact, the GMT in group III almost
equalled that in group I, which had the highest GMT on day 119.
Mild side-effects were observed only in equines in groups I and II (Table III).
|Fig 1. Trends in geometric mean titres in the four groups
We found that all the adjuvants enhanced the immune response but FCA containing
M. tuberculosis (group I) led to the development of the highest antibody titre
as estimated by the MNT. The GMT of the antibody on day 119 was highest (654.03
IU/ml) in group I equines. In this group of equines the GMT of the antibody
on day 56 (162.55 IU/ml) was also the highest. This shows that the use of FCA
containing M. tuberculosis leads to high antibody titres in a short time. FCA
is also known to stimulate an antibody response in low responder animals, probably
through activation of T helper cells. On day 56, only equines in group IV had
a GMT <38.68 IU/ml but the equines in this group were not withdrawn from
the donor population as suggested by Luekrajang et al.9
FCA containing M. butyricum (group II) trailed behind FCA containing
M. tuberculosis and FIA (group III) in our study with regard to eliciting antibody
titres against rabies, although it potentiated the immune response (GMT 459.19
IU/ml on day 119). Similar findings have been reported with regard to the formation
of neutralizing antibodies after subcutaneous injections of inactivated rabies
vaccines with killed M. butyricum.10 However, some studies have reported that
FCA with M. butyricum is better than FCA with M. tuberculosis especially against
Salmonella typhi.11 The
main difference between the Wax D of M. tuberculosis and Wax D of other mycobacteria
is the presence of a peptide moiety in the former, which may be responsible for
the enhanced immunogenicity. As the same PCECV was used in all the four groups
of equines, the variation in potency, if any, of the different batches was not
a factor in the production of antibodies.
FIA is currently used for the production of ERIG at our centre. There
was little difference in the antibody titre of FIA compared with FCA with M.
tuberculosis (GMT on day 119 of 630.95
IU/ml v. 654.03 IU/ml). However, FIA lags behind FCA with M. tuberculosis in
providing an early rise in antibody titres (GMT on day 56 of 91.09 IU/ml v.
Bentonite (group IV) had the lowest GMT titres and on day 119 this
was 172.18 IU/ml. Though bentonite is non-ulcerogenic, it is not as efficacious
as either FCA or FIA.
The main constraint while using adjuvants in vaccine formulations
are the side-effects that accompany their use. The Freund adjuvants cause granuloma
formation but the intensity of this reaction varies with the quantity of oil
used. However, granuloma formation may also contribute to the adjuvant action,
as granulomas contain a large number of macrophages which may be important in
producing an immune response. Mycobacteria present in FCA stimulate macrophage
proliferation at the site of injection, in the lymph nodes and even in the lungs.12 Contrary
to this generally held view, we did not encounter much granuloma formation in
our equines. No abscess formation was seen immediately after inoculation of the
first dose with FCA. This may be because of the small amount (1 ml) of adjuvant
In 1 equine each in the 3 groups receiving Freund adjuvants (Table
III), a slight swelling was noticed 3–4 days after inoculation which healed
over the next 7–10 days with the local application of BIPP formulation
(boric acid, suphanilamide powder, zinc oxide and iodoform). No other side-effect
or toxic reaction was observed in the equines.
|Table III. Side-effects observed in the equines
||Freund complete adjuvant containing M. tuberculosis
||Mild abscess at the site of inoculation 3–4 days
after day 42 of immunization
||Freund complete adjuvant containing M. butyricum
||Abscess at the site of inoculation after day 42 of immunization
||Abscess on the right side of neck after day 27 of immunization
|No side-effects were seen in the equines in
groups III and IV
The antibody response of all equines to the adjuvants is not similar. While
some equines may be good responders, others may show a moderate response
and yet others may show a poor response. The response may be related to
the nutritional status, general health and immunization history of the
equine. The equines used in our study were 23–26 years of age but
this did not seem to affect their ability to mount an immune response to
PCECV. Similar findings have been reported by Chowdhuri and Thomas13 and
Goel et al.2 This
can lead to considerable cost savings by using old discarded horses from
the army, police and equestary.
The quantity and number of doses of antigen are important as lower
doses may not stimulate an appropriate immune response whereas very high doses
may cause immune tolerance. The route of inoculation also affects the immune
response. A given dose of antigen is usually more effective when injected subcutaneously
than when given intravenously. The mode of inoculation also matters as, for example,
immunization via ballistic propulsion with a gene gun results in higher primary
antibody titres than by the intradermal route.14
Therefore, FCA is better in eliciting an immune response to
rabies antigen but FIA is also comparable in raising antibody titres in equines.
If antibodies need to be developed in a short time, FCA serves the purpose but
over a longer duration FIA provides equally good results. Bentonite is clearly
a poor adjuvant. Though it did not elicit a high antibody response with a priming
dose, it is safe and non-ulcerogenic and was used till day 42 of the immunization
schedule in all the equines.
In India there is a need to produce large quantities of ERIG that is affordable
and gives minimal adverse reactions. Our results show that this can be achieved
using a small quantity of FCA with M. tuberculosis or FIA, and using
older equines donated by agencies which do not have any use for them. This
will result in substantial savings in the cost of production of ERIG.
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|Central Research Institute, Kasauli 173204, Himachal Pradesh,
SHIVALI ARORA, SOURABH SHARMA, SUNIL K. GOEL,
USHA SOREN SINGH Anti Rabies Vaccine Unit
Correspondence to SOURABH SHARMA