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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 achieve the objectives of (i)
development of single shot vaccination for TT and extension of such
immunization approach for Hepatitis B surface antigen (HBsAg), (ii) analysis
of immune response from antigen loaded particles by employing different
polymer composition, particle size and usage of various immunization
protocols, evaluation of long term memory response using polymer particles
base immunization, generation of cell mediated immune response using
antigenentrapped polymer particles., (iii) induction of immune responses by
polymer entrapped plasmid DNA expressing protein and (iv) structure function
analysis of inclusion body proteins from E.coli for improved recovery
of bioactive proteins. A.
Immune response with polymer entrapped TT particles Formulation parameters which influences the
immunogenicity of polymer entrapped antigens are, size of the particles,
loading and doses of antigen and mode of immunization. These parameters were
extensively evaluated using polylactic acid (PLA) entrapped TT particles in
single doses. It was observed that TT entrapped in PLA particles immunized
with admixture of alum generates high and long lasting immune response.
Nanoparticles (<1 µm size) helped in eliciting very early antibody
response but the titers dropped within two months time. Very large particles
(> 40 µm) do not elicit sustained antibody response. Particles in the size
ranges of 4-10 µm generated long lasting immune response from single point
immunization. Optimization of these above formulation parameters resulted in
developing single dose of PLA entrapped tetanus toxoid formulation which
elicit comparable antibody response with two doses of alum adsorbed TT. Extensive studies were carried out to evaluate the
secondary response generated from single point immunization with polymer
entrapped TT. Rats immunized with polymer entrapping TT particles were boosted
with 0.5 lf of saline TT after a period of nine months to evaluate the
secondary responses. Secondary immune response from particles 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 (1lf) 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. Secondary antibody
response generated upon soluble boosting sustained at high level for
considerable period of time in comparison to alum based immunization. Antibody
generated during secondary response have high affinity and were predominantly
of IgG types. As observed with primary response, use of higher size particles
(> 40 µm) did not elicit higher secondary response upon soluble boosting.
Immunization of dummy polymer particle adsorbing equivalent dose of TT failed
to elicit high antibody response indicating that entrapment of antigen inside
the polymer particles is necessary for generation of high secondary response.
Particles entrapping antigen inside the polymer matrix only and not in its
surface elicited high secondary response upon soluble boosting. These results
indicated that particles base immunization results in improved immunological
memory in comparison to alum base immunization. Detail mechanism of particle
uptake and immune response using polymer entrapped TT are under investigation. B.
Development of single dose vaccine for HBsAg Polymer particles entrapping HBsAg in 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). Rats were immunized with lower doses of HBsAg and the antibody
response were monitored for one year. It was observed that at lower doses,
polymer entrapped HBsAg immunized along with alum elicited comparable HBsAg
antibody titers obtained from three doses of alum adsorbed HBsAg. Immunization
with very low amount of polymer entrapped HBsAg (1mg) gave rise to >500 mIU/ml of antibody titers.
Secondary immune response generated from single dose 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 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. Further improvement in single
dose HBsAg vaccine formulation is currently under evaluation in collaboration
with Shantha Biotech, Hyderabad. C.
Immunization of plasmid DNA vaccine using polymer
particles 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. From shaker flask culture, 2-3 mg of pure plasmid
(encoding HBsAg gene) 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. Different
plasmids have been purified using this above procedure and their ability to
transfect mammalian cells has been characterized. Currently we are optimizing
process for large scale preparation of plasmid DNA for vaccination. D.
Structure function analysis of inclusion body
protein from E.coli We are also investing the refolding of inclusion
bodies protein from E.coli. Previously, we have developed a novel
solubilization and refolding protocol for the high through put recovery of
bioactive protein from the inclusion bodies of E.coli. To improve
further the purification efficiency we are anlysing the pure, intact inclusion
bodies of different protein expressed in E.coli. These inclusion bodies
are being analysed in terms of size, density, charge distribution and dominant
forces causing protein aggregation. It is expected that a detail analysis of
the above parameters will not only help in recovering proteins from inclusion
bodies but also provide information about the nature of protein aggregation.
Different inclusion body solubilization process without using very high
concentration of urea or guanidine hydrochloride are under progress for
efficient recovery of bioactive protein from the inclusion bodies of E.coli.
Publications Original peer-reviewed articles 1. Katare YK, Lalwani K, Haque IU, Ali MM and Panda AK (2003) Potentiation of immune response from polymer entrapped TT particles : towards the development of single dose vaccine. Drug Delivery 10:231-238. 2. *Gupta V, Eshwari ANS, Panda AK and Aggarwal GP (2003) Optimization of IMAC for single step purification of recombinant ovine growth hormone from inclusion bodies of E.coli. J Chromatogr A 998:93-101 (*in press last year, since published). 3. Chatopadhyay S, Srivastava AK, Panda AK and Bisaria VS (2004) Cytotoxicity of in vitro produced Podophylotoxin from P.hexandrum cell culture on human cancer cell line. Nat Product Res 18:51-57. 4.
Dhiman HK, Ray AK and Panda AK (2004) Characterization and evaluation
of chitosan matrix for in vitro growth of MCF-7 breast cancer cell
lines. Biomaterials (in press).
Reviews/Proceedings 1. Panda AK (2003) Microbial culture and its applications. In: CBSE book on biotechnology for Class XII (Ed: Kannan K), CBSE Publication, New Delhi. 2. *Panda AK (2003) Bioprocessing of therapeutic protein from the inclusion bodies of E.coli. Adv Biochem Eng Biotechnol 85:43-93 (*in press last year, since published). 3. Panda AK (2004) High throughput recovery of proteins from the inclusion bodies of E.coli. In: Methods in molecular biology: Therapeutic proteins. The Humana Press Inc, USA (in press).
Patents 1.
Panda AK (2003) A process for preparation of pharmaceutical grade
plasmid DNA for therapeutic application. Indian patent application #
847/DEL/2003. |