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Studies of immune response by antigen loaded biodegradable polymer particles |
| Principal Investigator : Amulya K Panda
Project Associates
/ Assistants Ph D Students Collaborators The
theme of the project is to evaluate the immunostimulatory activities
associated with polymer entrapped antigens such as Tetanus Toxoid (TT),
Hepatitis B surface antigen (HBsAg) and plasmid DNA expressing antigens for
development of single shot vaccines. The main objective is to study the immune
response from biodegradable polymer particles entrapping antigens and plasmid
DNA. Immune responses from polymer particles entrapping TT, HBsAg or plasmid
DNA expressing HBsAg protein will be evaluated to (i) develop single shot
vaccination for TT and extension of such immunization approach for Hepatitis B
surface antigen (HBsAg), (ii) analyse the immune response from antigen loaded
particles by employing different polymer composition, particle size and usage
of various immunization protocols; evaluate long term memory response using
polymer particles base immunization; generate cell mediated immune response
using antigen entrapped polymer particles, (iii) induce immune responses by
polymer entrapped plasmid DNA expressing protein. A. Immune response with polymer entrapped TT particles Parameters
which influence the immunogenicity of polymer entrapped TT particles were
investigated in detail. Apart from protection of immunoreactivity of antigen
during particles formulation, the other parameter which influences the
immuniogenicity are, size of the particles, loading of antigen and doses of
antigen. These above three parameter were extensively evaluated using polymer
entrapped TT particles in single doses. It was repeatedly observed that use of
alum along with particles improved the antibody titers in Wistar rats. Thus
all immunization studies were carried out with admixture of polymer particles
and alum for development of single dose vaccine. It was observed that higher
size particles (> 100 µm) elicited very low level of immune response. High
anti-TT antibody titers were achieved as the size of the particles reduced
from 40 to 4 µm. Nanoparticles (<1 µm size) helped in eliciting very
early antibody response but the titers dropped within two months time.
Particles size of 2-5 µm not only gave very high anti –TT antibody titers
but also for a longer period of time from single point immunization. Immune
response from particles having different loading of antigen do not gave rise
to different titers upon single point immunization. Dose response studies with
very low dose of TT in particles were carried to evaluate the immuno-stimulatory
potential of particle based immunization. It was observed that at lower doses
(5 to 1 lf TT), particles based immunization with alum generated higher
antibody titers than the alum base immunization. Use of alum along with
antigen entrapped polymer particles not only improve the initial antibody
titers very rapidly but also helped in maintaining high antibody titer
throughout the post immunization period. Adsorption studies of TT on to alum
in presence of dummy polymer particles, its in vitro release and in vivo
evaluation are under progress to understand mechanism of alum on improving
immune response from polymer entrapped antigen. Rats
immunized with polymer entrapping TT particles were boosted with 0.5 lf of
saline TT after a period of eight months to evaluate the secondary responses.
It was observed that secondary immune responses generated after boosting was
higher in groups of animal immunized with particles in comparison to alum
adsorbed TT group. Secondary immune response from particles based immunization
having different doses, loading and size were evaluated. Among different
particles formulation, the ones which gave high initial primary response gave
very high secondary antibody response upon boosting with soluble antigen. Even
very low amount of entrapped TT (1 lf) from single point immunization gave
rise to very high secondary immune response in comparison to the alum based
immunization. Secondary immune response from polymer entrapped antigen was
five times higher than that observed for alum adsorbed TT. These results
indicated that particles base immunization results in improved immunological
memory in comparison to alum based immunization. Experiments involving
cytokines profile of primed T cells, affinity of antibody generated after
boosting are under investigation to have better understanding of the high
secondary response generated from particles base immunization. B. Development of single dose vaccine for HBsAg Polymer
particles entrapping HBsAg in PLA particles were evaluated for generation of
antibody response. The aim was to develop single dose HBsAg vaccine using
polymer particles, which can be an alternative to the three-dose vaccination
schedule. Rats were immunized with different polymer formulation and doses and
antibody titers were compared with three doses of HBsAg injection on alum. The
seroprotective nature of HBsAg antibody generated from polymer particle base
immunization were evaluated using AUSAB Kit (Abbot Lab, USA). For more than 9
months particles based immunization resulted in > 50 000 mIU/ml of HBsAg
antibody titers. Antibody titer obtained from three injection of alum adsorbed
HBsAg was however higher (170,000 mIU/ml) than single dose particle base
immunization. However the polymer particles base immunization resulted in
generation of bio-neutralizing antibody titer higher than the desired units
required for sero-protection (10 mIU/ml). Immunization studies with different
doses of polymer entrapped antigens (preferably lower doses) and pulsed
release formulation to mimic the conventional immunization schedule are under
progress. Secondary immune response generated from immunization with polymer
entrapped antigen was compared with three injection schedule of alum adsorbed
HBsAg. Animals were boosted with 2 mg of soluble HBsAg after 9 months of post
immunization and the antibody titers were evaluated. Very high anti-HBsAg
titers (400,000 mIU/ml) were achieved within 15-20 days of post booster
injection both for polymer entrapped as well alum adsorbed immunization. This
indicated that single dose of polymer entrapped antigen is equally suitable to
the three dose alum adsorbed HBsAg immunization for generation of high
secondary immune response. Polymer entrapped HBsAg probably helps in
generation of immunological memory as reported for tetanus immunization. C. Immunization of Plasmid DNA vaccine using polymer particles Formulation
and entrapment of HBsAg plasmid DNA in polymer matrix have been attempted to
develop single dose DNA vaccine. Currently we are developing large-scale
plasmid DNA preparation methods for immunization. We have been able to develop
an improved process for the purification of pharmaceutical grade plasmid DNA
for immunization process. The new method does not use ultracentrifugation,
RNAse addition or column chromatography steps for the purification of plasmid
DNA. From shaker flask culture, 2-3 mg of pure plasmid free from RNA and
having negligible amount of endotoxin could be achieved. Pure plasmid yields
are comparable to that of plasmid preparation using commercially available
purification column. Process optimization studies combining high cell density
fermentation and purification are under investigation to produce 10-20 mg pure
plasmid from one litre of bacterial culture. Publications Original
peer-reviewed articles 1. Raghuvanshi RS, Singh O and Panda AK (2002) Correlation between in vitro release and in vivo immune response from biodegradable polymer particles entrapping tetanus toxoid. Drug Delivery 9:113-120. 2.
Raghuvanshi RS, Katare Y, Lalwani K, Ali MM, Singh O and Panda AK (2002)
Improved immune response from biodegradable polymer particles entrapping
tetanus toxoid by use of different immunization protocols and adjuvants. Int J
Phram 245: 109-121. 3.
Gupta V, Eshwari ANS, Panda AK and Aggarwal GP (2002) Optimization of IMAC for
single step purification of recombinant ovine growth hormone from inclusion
bodies of E.coli. J Chromatogr (in press). Reviews/Proceedings 1.
Panda AK (2002) Biochemical Engineering. In: CBSE book on Biotechnology. (Ed.
Kannan K) CBSE Publication, New Delhi. 2.
Panda AK (2003) Bioprocessing of therapeutic protein from the inclusion bodies
of E.coli In: Advances in biochemical engineering/biotechnology (Ed. Ghose TK)
Springer Verlag,Germany (in press). Patents 1.
Panda AK and Katare YK (2002) A process for developing immunological memory
from single point immunization Indian patent application no. 754/DEL/2002. 2.
Panda AK, Sheikh GB, Eshwari ANS and Garg LC (2002) A process for
solubilization and refolding of inclusion body. Indian patent application no.
921/DEL/2002 filed in Sep 2002. |