|
Studies
of immune response by antigen loaded biodegradable polymer particles |
| Principal Investigator :
Amulya K Panda Research Associates PhD 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. To evaluate immune responses
from polymer particles entrapping TT, HBsAg or plasmid DNA expressing HBsAg
protein. The specific objectives of the project are (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 strategies, and (iii)
induction of immune responses by polymer entrapped plasmid DNA expressing
protein. A.
Immune response with polymer entrapped TT particles To
further improve the immune response from PLA entrapped TT particles,
formulation variables were optimized. It was observed that incorporation of
stabilizer and basic salt helped in better entrapment of TT in polymer
particles during formulation using solvent evaporation method. With
incorporation of these excipients during formulation, entrapment efficiency of
TT was increased from 35% to around 75%. These particles showed improved
immune response upon immunization and the in vitro release profiles of
antigen correlated well with the in vivo response. Such improvement in
formulation parameter while protecting the immunogenicity of the entrapped
antigen were also achieved for other antigen like DT, b-hCG,
HBsAg and for lysozyme. It
was also repeatedly observed that use of alum along with particles improved
the antibody titers in Wistar rats. Use of alum along with antigen entrapped
polymer particles not only improved the initial antibody titers very rapidly
but also helped in maintaining high antibody titer throughout the post
immunization period. 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 (1 lf TT),
particles based immunization with alum generated almost 5 time higher antibody
titers than the alum based immunization. Similar high antibody titers were
also observed with different doses of TT entrapped in PLA particles. Improved
anti-TT titers from particles based immunization were reproducible and similar
trend in antibody response were observed for three set of animal experiments
each involving 48 rats. These results showed that with proper particle
formulation and immunization protocol, single dose polymer particles
entrapping TT can elicit antibody response comparable to the two dose schedule
of alum adsorbed TT vaccination. Such improvement in immune response with
combined immunization of particles along with alum was observed for DT and
HBsAg. The exact role of alum in improving the immune response from polymer
particles based immunization is not known. Possibilities of different
mechanism of alum on improving immune response from polymer entrapped antigen
is under evaluation. Rats
immunized with polymer entrapping TT particles were boosted with 5lf 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 the groups of animal immunized with particles in comparison to alum
adsorbed TT group. Higher secondary response was generated from particles base
immunization while boosting with 0.5 lf of soluble antigen after 8 months of
primary immunization. Among different particles formulations, the ones which
gave high initial primary response gave very high secondary antibody response
upon boosting with soluble antigen. These results indicated that particles
based immunization results in improved immunological memory in comparison to
alum based immunization. The exact nature of memory is not known. 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 based immunization. B.
Improved immune response from weak antigen using polymer particles The
immuno-stimulatory potential of polymer particles based immunization was
investigated using a poor immunogen; b-hCG without
conjugation to an immunogenic carrier. On immunizing rats with b-hCG
with alum as adjuvant no detectable anti-hCG antibody titers were detected.
Single dose immunization with PLA particles entrapping b-hCG
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 during TT particles immunization,
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 was 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,
immunogenicity of a weak immunogen could 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. Particles based immunization,
co-encapsulating both b-HCA
and TT generated better antibody response than the single antigen. This
indicated that co-encapsulation of antigen and conjugate in polymer particles
can be a substitute for chemical conjugation for improved immune response. C.
Development of single dose vaccine for HBsAg Polymer
particles entrapping HBsAg were used 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. As the HBsAg is
particulate in nature, different formulation variables were optimized to check
its in vitro release characteristic from polymer particles. Rats were
immunized with different formulation and the antibody titers were found to be
lower than that achieved with three doses of HBsAg injection on alum. The
seroprotective nature of HBsAg antibody generated by polymer-based
immunization were evaluated using AUSAB Kit (Abbot Lab, USA). It was observed
that for more than 4 months particles based immunization resulted in >10
000 mIU/ml of HBsAg antibody titers after which it declined. Antibody titers
over a period of six months were higher than the desired units required for
sero-protection (10 mIU/ml). Improved formulation and dose response studies
using HBsAg are currently under progress to evaluate the feasibility of single
dose vaccine. D.
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. Results from DNA
immunization have not been reproducible and we are currently optimizing
formulation parameter for improving the immune response. Publications Original
peer-reviewed articles 1.
*Raghuvanshi RS, Om Singh and Panda AK (2001) Formulation and
characterization of immunoreactive tetanus toxoid biodegradable polymer
particles. Drug Delivery 8:99-106 (*in
press last year, since published). 2.
Upadhyay PK, Patra AK, Mukhopadhyay R and Panda AK (2001) Real time
detection and quantification of inclusion bodies expressed in E. coli
by impedance measurements. Biotechnol Lett 23:839-843. Review/Proceedings 1.
Katare YK and Panda AK (2001) Effect of alum on in vitro release
profile and immunogenicity of microencapsulated tetanus toxoid. Proc Int
Sym Control Rel Bioact Mater 28:1059-1060. Patents 1.
Panda AK. A process for the formulation of single dose vaccine.
Indian patent application No. 852/Del/2000 filed in Sep 2001. 2.
Panda AK, Pal R, Venkatasen N and Om Singh O. A process for improving
the immunogenicity of a weak antigen. Indian patent application No.
794/Del/2001 filed in Jul 2001. |