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Studies of immune response by antigen loaded
biodegradable-polymer particles |
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Principal Investigator : Amulya K Panda Research Associates Ph D Student 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. PLA particles entrapping immunoreactive TT were used
along with alum to potentiate immune response in wistar rats. Both nano
particles (< 1 mm) and microparticles (2-5 mm) were evaluated along with
alum for immune responses. It was observed that the nanoparticles gives high
early antibody responses whereas, in case of microparticles immune response
peaked around 90 days of post immunization. Physical mixture of both nano and
microparticles given along with alum generated a sustained immune response
over the period of eight months. These results indicate that by judicious use
of physical mixture of different size of particles, long lasting sustained
immune response can be generated with single step immunization. Preliminary
studies are being carried out to formulate particles having different in vitro
antigen release characteristics so that a desired type of immune response can
be generated. Rats immunized with particles entrapping TT were
boosted with 5lf of saline TT after a period of eight months to evaluate the
secondary responses. It was observed that the secondary immune responses
generated after boosting was higher in groups of animal immunized with
particles in comparison to the alum adsorbed TT group. It was also observed
that high secondary responses were generated from those particle groups where
the primary immune response was very high. Animals immunized with particles
when boosted after 8 months with low doses of TT in saline also gave high
secondary immune responses. The immunostimulatory potential of polymer particles
base immunization was also investigated using a poor immunogen b-hCG without
conjugation to an immunogenic carrier. With alum as adjuvant, no detectable
anti-hCG antibody titers were detected by immunizating rats with b-hCG. PLA
particles were formulated entrapping b-hCG and a single immunization schedule
elicited antibody response. A combination of alum and microparticles
immunization resulted in high antibody titers comparable to those achieved
when CFA or IFA were employed. As observed for TT particles, the antibody
titers from b-hCG particles gave a peak value on day 90, which was the
characteristic of the polymer formulation. The anti-hCG antibody titers
generated were bio-neutralizing as evident from a receptor binding inhibition
assay. The affinity of the antibodies towards hCG was in the range of 1010 L/M
and was of the same order as antibodies generated using CFA or IFA. With a
single dose immunization of particles along with alum, significant anti-hCG
antibody titers were measurable for six months of post immunization. These
results showed that with the judicious use of particle and adjuvant,
immunogenecity of a weak immunogen can be improved. These results also suggest
that for antigens where alum does not provide adjuvant effect, combination of
particles with alum may be a suitable alternative for generating a high
antibody response. Formulations of polymer particles entrapping HBsAg are
being evaluated for in vitro release and in vivo responses. Recombinant HBsAg
denaturation during polymer particles formulation and lyophilization are being
studied in detail to prepare required size polymer particles entrapping
immunoreactive HBsAg. Different formulation resulting in pulse release of
HBsAg is also under investigation to mimic the HBsAg immunization schedule so
that a single dose vaccine can be developed. Formulation and entrapment of
HBsAg plasmid DNA in polymer matrix have been attempted to develop single dose
DNA vaccine. Publications Original peer-reviewed articles 1.
Panda AK, Khan RH, Mishra S, Appa Rao KBC and Totey SM (2000)
Influences of yeast extract on the specific cellular yield of ovine growth
hormone in E.coli. Bioprocess Engineering 22:379-383. 2.
Patra AK, Gahlay GK, Reddy BVV, Gupta SK and Panda AK (2000)
Refolding, structural transition and spermatozoa-binding of recombinant bonnet
monkey (Macaca radiata) zona pellucida glycoprotein-C expressed in Escherichia
coli. Eur J Biochem 267:7075-7081 Reviews/Proceedings 1.
Panda AK (2000) High cell density fermentation and improved
refolding of inclusion body proteins from E.coli. In: Proceeding of AIBA
Symposium, National Conferences on Emerging Trends in Biopharmaceutical
Proteins and Role of Preparative Chromatography, New Delhi, 18-20 Dec 2000,
32-39. 2.
Panda AK (2001) Recombinant protein production from E.coli. In:
Proceeding of Biohorizons - 2001, New Delhi, 23-24 Feb 2001, 3-4. |