Title of Invention	"A PROCESS FOR IMPROVING THE IMMUNOGENICITY OF A WEAK ANTIGEN"
Abstract	The invention discloses a process for enhancing the immunogenicity of a weak antigen by entrapping the said antigen in a biodegradable polymer matrix and immunizing alongwith alum using single point contact.

Full Text

Field of the invention: The present invention relates to the development of vaccine formulation using admixture of biodegradable polymer particles entrapping antigen and a permissible adjuvant. Immunization of polymer entrapped antigen in combination with alum resulted in considerable synergistic improvement of the immune response of a weak immunogen. Such immunization procedures can be applied to improve the immunogenicity of sub-unit and recombinant vaccine antigens, which in general are poorly irnmunogenic. Background The majorities of vaccines currently in clinical use require repeated administration and use alum as an adjuvant. In spite of their use, these adjuvants suffer from batch to batch variability, variable efficacy with different antigen and are poor stimulators of cell mediated immune response. There is a need to develop novel adjuvants and delivery system to generate long lasting immune response particularly for poorly immunogenic sub-unit and recombinant antigen vaccine candidates. Liposomes, ISCOMS, live recombinant organisms, lipids emulsion and polymeric microparticles based immunization are some of the alternatives available to improve the immunogenicity of vaccine candidates. Biodegradable polymeric microparticles using polylactide -co-glycolide (PLGA) and polylactide (PLA) polymers have attracted considerable attention in recent times as vehicles towards the development of single dose vaccine formulation. Antigen loaded polymer particles have adjuvant effects because of their size and uptake by antigen presenting cells. However the adjuvant effect is not as high as achieved with Complete Freund's Adjuvant (CFA ) or Incomplete Freund's Adjuvant (IFA ) based immunization. Alum, the most commonly use adjuvant for clinical use is a poor adjuvant for many proteins. For many sub-unit and recombinant vaccine antigens, use of clinically approved adjuvant does not result in the generation of high bio-neutralizing antibody liters. Thus, there is a need to develop new adjuvant or delivery system to improve the immunogenicity of weak antigen to make them suitable in clinical uses. Another advantages of these polymeric vaccine delivery system is that the immune response from the same particle can further be enhanced by use of additional adjuvant or by using different immunization modalities. No such studies have been conducted on very weakly immunogenic protein which elicit very poor immune response with alum as an adjuvant. Potentiation of immune response in such situation could have significant clinical implications. Such modalities of immunization can be used for generating immune responses from sub-unit and recombinant vaccine antigens, which are extensively explored nowadays for clinical use. The present invention relates to the judicious combination of the above aspects resulting in eliciting high bio-neutralizing antibody titers from a single dose vaccine formulation of a weak antigen. Description of Prior Art Alum has been traditionally used as an adjuvant to potentiate the immune response of many vaccine candidates (Ramanathan et al., 1979, Cox and Coulter, 1997). Inspite of extensive research undertaken in recent years very few novel adjuvant has been approved for clinical uses. One of them is the use of bio-degradable polymer particles entrapping immunoreactive antigens (O;Hagan et al., 1998). Even though extensive work has been carried out using polymer based particle formulation for improvement in immunogenicity of the entrapped antigen several problems exit (Hanes et al., 1997, Cleland 1999). One of the major issues of the polymeric based vaccine formulation concerns is the stability of the entrapped antigen (Wreet et al., 2000 ). Antigen stability during formulation, storage and release form the polymer matrix is of paramount importance for the successful development of polymeric base vaccine formulation ( Zhu et al., 2000 ). It is important that the antigen entrapped and release from the polymer particles retain its native conformation so that a bioneutralizing antibody response can be generated. Problems associated with the stability of polymeric vaccine delivery system have been extensively reported (Putney and Bruke 1998, Zhu et al .,2000 ). We have recently reported the organic solvent induced denaturation of tetanus toxoid (TT) during particle formulation and subsequent formulation development for the preparation of immunoreactive polymeric particles (Raghuvanshi et al, 1998, Raghuvanshi et al, 2001). Development of single dose vaccine using biodegradable polymer needs the optimization of parameters that are necessary for the generation of improved immune response. It is essential that the particles should entrap immunoreactive antigens (Raghuvanshi et al., 2001) and release of antigen should mimic the conventional vaccination schedule thus providing in vivo auto boosting to elicit desired antibody response. Polymer particle should be smaller in size ( antigens released from polymer particles are immunogenic. This is most important as polymer particles release soluble antigen and it is widely documented that soluble antigens are weak immunogen. Hence in spite of the adjuvant effect, immune response from polymer entrapped antigen are generally low in comparison to the alum adsorbed counterpart. Thus to developed single dose vaccine, it is not only imperative to release immunoreactive antigen from polymer particles but also essential to make them more immunogenic either by delivering to macrophages or immunizing along with adjuvant for better presentation to antigen presenting cells. Probably, because of the failure to meet these requirements, many reports aiming at developing single dose vaccine using polymer particle based immunization have not been successful. The earlier reports on TT were on large size particles (10-100 µm) and continuous release of TT from PLGA particles was shown for one-month time (Alonso et al., 1994; Tobio et al., 2000, Sasiak et al.,2001). There are reports that decreasing particle size (Eldridge et al., 1991) and increasing hydrophobocity of polymeric particles improve the immunogenicity of entrapped antigen (Conway et al., 1997). However very little has been reported on the combined effect of parameters such as hydrophobiocity, particle size and effect of additional adjuvant on immune response from polymer entrapped antigen (Johansen et al., 2001). Another advantages of these polymeric vaccine delivery system is that the immune response from the same particle can further be enhanced by use of additional adjuvant or by using different immunization modalities. Improved immune response from particles entrapping tetanus toxoid employing alum as additional adjuvant and different immunization protocol has been reported (Singh et al., 1997). Improved imunogenicity of polymer entrapped HBsAg in presence of additional adjuvant alum has been reported recently (Shi et al., 2002). However no such studies have been conducted on very weekly immunogenic protein. Two reports exploring the improved immunogenicity of entrapped p-hCG has been reported very recently but none of them use the adjuvant approved for human (Sefarian and Martinez, 2001, Zhu et al., 2001).Thus the need for enhancing the immunogenicity of poor immunogen is largely unmet. Objects of invention We have made an attempt to develop a single-shot polymeric formulation of a weakly immunogenic model antigen, the beta subunit of human chorionic gonadotropin hormone (p-hCG). The antigen was entrapped in poly lactide polymeric microparticles using a multiple emulsion-solvent evaporation technique and was studied for its potential to generate antibody response in rats. Further, an attempt was made to enhance the immunogenicity by the administration of a particle-alum admixture in a single dose. Immune responses were compared with p-hCG adsorbed on alum, or as emulsions in CFA or IFA. Single dose of polymer particles immunized along with alum gave rise to long lasting high antibody liters. These results are an indication that by proper choice of polymer particles and immunization protocol using a clinically approved adjuvant, immunogenicity of an weak antigen can be improved considerably. Accordingly, the inventors have developed a process for improving the immunogenicity of a weak antigen using a combination of clinically approved adjuvants. One object of invention is the development of single dose vaccine from weak immunogens such as peptide, sub-unit vaccine and recombinant antigen by entrapping them in biodegradable polymer matrix and immunizing along with alum. Another object of invention provides a process for preparing particles using hydrophobic polymers while entrapping immunoreactive antigen/vaccine inside it. Another object of the invention is the improvement of the immunogenicity of P subunit of human chorionic gonadotropin hormone (P hCG) using human permissible adjuvant One object of the invention is the generation of high bio-neutralizing antibody titer using an immunization protocol with admixture of alum and polymer particles entrapping a weakly immunoreactive antigen. Another object of invention is the generation of high affinity antibodies comparable to that achieved using CFA or IFA while immunizing with admixture of alum and polymer particles entrapping immunoreactive antigen. Another object of invention is to formulate polymer particles of suitable size for efficient uptake by immune cells and generation of improved immune response. Another object of invention is formulation of weak antigens in particulate form with the help of polymer and alum for improved immune response. Yet, another object of invention is to formulate an immunization protocol using the polymer entrapped antigens for obtaining long lasting sustained antibody responses in experimental animal with single dose immunization. Detailed description of the invention The inventors have developed an immunization process for improving the immunogenicity of an weak antigen by a synergistic combination of two clinically approved adjuvants such as a biodegradable polylactide polymer, and alum. Solvent evaporation methods was used to prepare polymer particles entrapping a weak antigen such as p-sub-unit of human chorionic gonadotropin (P-hCG). The denaturation of the antigen during the primary emulsification step of particle formulation was prevented by use of suitable stabilizers hi the internal aqueous phase. The particles were made of required size using polylactide polymer. Enhanced and sustained immune responses were generated by immunizing experimental animals with particles in combination with alum. Polylactide polymer of molecular weight of 45 KD at a concentration of 50 mg/ml was used for the preparation of the particles. The aqueous phase containing native P-hCG and stabilizers such as serum albumin at optimal concentration was used for the preparation of the primary emulsion. The resulting primary emulsion was again emulsified in presence of 2 % poly-vinyl alcohol to prepare the particles entrapping immunoreactive P-hCG. Particle size of 3-4 µM were prepared using polylactide polymers. The particles were lyophilized and stored in powdered form. Immunization with polymer particles entrapping immunoreactive P-hCG elicited very weak immune response. Alum adsorbed P-hCG also elicited very little antibody response lasting for a week. Microparticle-entrapped P-hCG elicited a much better immune response (400 ng/ml) as compared to alum-adsorbed ß-hCG (2 ng/ml) and the antibody titers peaked around 90 days. Single dose intramascular immunization of the particles in combination with alum resulted in long lasting immune responses. Use of alum along with PLA particles improved both the kinetics and the magnitude of the immune response, due to unexpected synergistic interaction between the two. Serum antibody titers from microparticles admixed with alum were around 800 ng/ml on day 60 post immunization in comparison to 150 ng/ ml achieved by particle immunization. Antibodies titres were maintained for 180 days, with peak titres observed once again on day 90 (1238 ng/ml). The antibody responses from P- hCG emulsified in either CFA or IFA was higher than that from the particles alum mixture. The affinity of the antibodies towards hCG were in the range of 1010 L/M, irrespective of the adjuvant employed. Bioneutralization capacity, as a function of immunoreactivity, equalled that observed upon CFA, IFA or particle based immunization, indicating that crucial epitopes of P-hCG were not adversely affected during its entrapment in the polymer matrix. These results demonstrate that admixtures of microparticles and alum can be employed to generate high titre, high affinity, bioneutralizing responses against weakly immunogenic antigens. The invention covers patent application 852/Del/2000 which is hereby incorporated by reference in its entirety. In an embodiment the process describes the immunization of admixture of polymer entrapped antigen and alum for generating improved immune response from an weak antigen. In an embodiment the antigen tried here are sub-unit vaccine, recombinant antigens and P sub unit of human chorionic gonadotropin hormone ( p-hCG). In another embodiment the preferred weak antigen is P-hCG. In another embodiment the preferred polymer is PDLL-lactic acid. In another embodiment the process described the formulation of hydrophobic polymer particles entrapping immunorecative antigen inside it. In another embodiment the process describe polymer particles based immunization where the entrapped antigen is released in a desired way with an initial burst release as described in patent application 852/Del/2000. In another embodiment the process describes the generation of high bioneutralizing antibody titers using admixture of polymer entrapped antigen and alum. In another embodiment, intramuscular immunization of admixture of alum and polymer particles entrapping antigen elicit improved immune response. In another embodiment the process describes the generation of high affinity antibodies comparable to that achieved using CFA or IFA while immunizing with admixture of alum and polymer particles entrapping immunorective antigen. In another embodiment, immunization with polymer entrapped antigen and alum results in early generation of immune response. In another embodiment the process describe a method of immunization with admixture of polymer entrapped antigen and alum without using multiple injection for achieving improved immune response for a longer period of time. Yet another embodiment, the immunization protocol describes here results in generation of long lasting immune response from single point immunization. In another embodiment, the process describes the generation of immune response from polymer entrapped immunogen in the presence of permissible adjuvant. In particular the applicant has described an immunization process using polymer entrapped antigen and alum for generation of effective immune response form a poor immunogen. Improvement in the immunogenicity of a weak antigen can be achieved by entrapping them in biodegradable polymers and immunizing them with alum. Use of hydrophobic polymers for particle formulation, preparation of polymer particles of required size entrapping immunoreactive antigen, and use of the right immunization protocol while using alum as an adjuvant resulted in remarkable improvement in the immunogenicity of the p-sub-unit of human chorionic gonadotropin hormone. The present invention relates to the judicious combination of the above aspects resulting in eliciting high bio-neutralizing antibody titers from a single dose vaccine formulation of a weak antigen. The invention is described in detail with the aid of following examples and the accompanying drawings and tables. Various modification that may be apparent to one in the art are intended to fall within the scope of invention. Brief Description of the Accompanying Drawing Fig. 1 is a photomicrograph, scanning electron micrograph of PLA particles entrapping immunorecative phCG. The average size of the particles are around 3-5 µm. Figure 2. Anti-hCG antibody titers (hCG binding capacity in ng/ml) in rat sear immunized with various adjuvants (saline --X-- Alum♦particles --■-- , particles with alum - ▲ - ). Rats were immunized with 25 µg of phCG intramascularly and were bled at different time point to evaluate the antibody titers, Figure 3. Comparison of Anti-hCG antibody titers (hCG binding capacity in ng/ml) in rat sear immunized with various adjuvants (particles with alum ~X --, CFA --■— , IFA--■-- , particles - ▲ -). Rats were immunized with 25 (µg of phCG intramascularly with adjuvant and were bled at different time point to evaluate the antibody titers, Figure 4. Bioneutrlization capacity of anti-hCG sera obtained by imuunization with different adjuvants (particles with alum --X --, CFA --♦--, IFA --■-- , particles - A -, CFA (SC) --*--). Table 1: In vitro characterization of mciroparticles(Table Removed) Table 2: (Table Removed) • Antibody association constant (K) in litre/mole Example : Immunization process for improving the immunogenicity ofß-hCG The beta subunit of human chorionic gonadotropin (ßhCG) was entrapped into poly (D,L-lactide) (PLA) (45KD) polymer particles using solvent evaporation method. Immunoreactivity of the antigen was protected using an optimal concentration of rat serum albumin (RSA) in the internal aqueous phase during the primary emulsiflcation stages of particle formulation. Incorporation of RSA resulted in uniform particle formulation and enhancement of serum antibody response in rats when immunized with a single-dose of 25 µg of entrapped antigen. Microparticle-entrapped ßhCG elicited a much better immune response (400 ng/ml) as compared to alum-adsorbed phCG (2 ng/ml) and the antibody liters peaked around 90 days. Use of alum along with PLA particles improved both the kinetics and the magnitude of the immune response. Serum antibody titers from microparticles admixed with alum were around 800 ng/ml on day 60 post immunization in comparison to 150 ng/ ml achieved by particle immunization. Antibodies litres were maintained for 180 days, with peak litres observed once again on day 90 (1238 ng/ml). Antibody response from ß-hCG adsorbed on to either CFA or IFA were higher than that from particles alum mixture. The affinity of the antibodies towards hCG were in the range of 1010L/M, irrespective of the adjuvant employed. Bioneutralization capacity, as a function of immunoactivity, equaled that observed upon CFA, IFA or particle based immunization, indicating that crucial epitopes of ß-hCG were not adversely affected during its entrapment in the polymer matrix. These results demonstrate that admixtures of microparticles and alum can be employed to generate high titre, high affinity, bioneutralizing responses against weakly immunogenic antigens. Methods Preparation of And Characterization Microparticles Poly (D,L-lactide) (PLA) (45 Kd) was obtained from Birmingham Polymers Inc., Birmingham, Alabama, USA. Aluminum hydroxide gel (Alhydrogel®) was from Superfos, Copenhagen, Denmark. Rat serum albumin and all other chemicals were obtained from Sigma Chemicals, St. Louis, M.O., USA. ß-hCG was purified from the native hCG molecule as described (Singh et a!.., 1989). Polymeric microparticles were prepared using a multiple emulsion (w/o/w)-solvent evaporation technique. Briefly, the organic phase constituted of 200 mg PLA dissolved in 4 ml of dichloromethane (DCM). The aqueous phase contained 5 mg of p-hCG and rat serum albumin (RSA) (2.5% w/v of the inner aqueous phase). The aqueous: organic ratio was kept at 1:10. This aqueous-organic mixture was sonicated for 2 min. at 20 cycles/min. (20W, 80% duty cycle) (Sonifier 450, Branson Ultrasonics, Danbur, CT, USA) in a ice bath to form a primary emulsion (w/o). The emulsion was homogenized in 10 ml of 1% PVA solution in an ice bath (10,000 rpm for 10 min.) (Cyclone I.Q.2, Virtis, USA) to form a w/o/w emulsion. This emulsion was stirred overnight at room temperature to evaporate DCM. Polymeric microparticles were then separated and washed by centrifugation at 15,000 rpm at 4°C for 30 min. (Sorval RC250 plus, DuPont, USA). Microparticles were lyophilized and stored in a desiccater. Microparticles were characterized for their size, shape and encapsulation efficiency. Size was determined using a laser particle size analyser (GALAI-CIS-1) and surface morphology observed on a scanning electron microscope (Jeol, Japan). The amount of antigen (P-hCG) entrapped was calculated by dissolving 30 mg of the polymer particle in 1 ml of acetronitrile, followed by centrifugation (15,000 rpm/4°C/20 min.) (Plastocrafts, USA). The resultant pellet was dissolved in 1 ml of 1% sodium dodecyl sulphate (SDS) solution. This solution was appropriately diluted and estimated for protein content using micro Bicinchoninic acid assay (mBCA) (Pierce, USA). In Vivo Animal Studies Immunization Studies The antibody responses obtained upon immunization of Wistar rates with polymer-entrapped ß-hCG were compared to those obtained upon the use of other adjuvants. Each group consisted of 6 rats. Group 1 was administered ß-hCG in saline, Group 2 alum adsorbed ß-hCG, Group 3 received polymer particles entrapping ß-hCG, Group 4 was immunized with ß-hCG containing particles admixed with alum prior to injection, while Group 5 and 6 received P-hCG emulsified in CFA and IFA respectively. All animals received a single intramuscular injection of 25µg equivalent of ß-hCG. Animals were bled from retro orbital plexus at different time intervals; the serum was separated and stored at -20°C. Characterization Of The Antibody Response Anti-hCG antibodies were estimated by direct binding radioimmunoassay (RIA) as per the method reported earlier (Pal et al, 1990). Briefly, each assay tube contained 100 µl of diluted sera, 100 µl of 125I-hCG (approximately 12,000 cpm), 100 ul of 20% horse serum and 200 ul of assay buffer (50 mM PBS with 0.1% BSA and sodium azide, pH 7.2). Tubes were incubated at 4°C for 18 h and the antibody-bound fraction was precipitated by adding 500 µl of polyethylene glycol (PEG) 8000 (25% w/v). The pellet was separated following centrifugation at 2,500 rpm for 20 min. at 8°C. The resultant pellet was counted in a multi-gamma counter (LKB-1260; Turku, Finland). Antigen binding capacity was calculated at dilutions resulting in specific binding of 5-15%, taking into account the specific activity of 125I-hCG (Singh et al, 1989, Pal et al, 1990). The avidity of antibodies for hCG was determined by the cold displacement method as described earlier (Pal et al, 1990). Briefly, diluted antisera were incubated with labeled hCG in the presence of increasing amounts of unlabeled hCG at 4°C for 24h. Bound fractions precipitated by 12.% PEG 8000 were separated by centrifugation at 2500 rpm for 20 min. and radioactivity counted in an LKB multi-y-counter. Association constants were computed by nonlinear regression analysis using a computer programme. The in vitro bioneutralization potency of the sera was determined by a method reported earlier (Singh et al, 1989, Pal et al, 1990). Briefly, testes of 2-3 month old Wistar inbred rats were decapsulated and homogenized in 50 mM Tris-HCl buffer (pH 7.4) using a Polytron homogenizer. The homogenate was filtered through a nylon mesh and centrifuged at 5000 rpm for 10 min. at 4°C. The resulting pellet was resuspended in 8 ml of Tris buffer (pH 7.4) per pair of testes; 50 µ1 of testicular homogenate was incubated with lOOµl of varying dilution of antisera and 50µ.l of 125I-hCG (approximately 20,000 cpm) for 2 h at 37°C. A common batch of radioiodinated hCG was used for all sera and 50 ng unlabelled hCG was used to assess nonspecific binding. The assay was terminated by the addition of 1 ml of cold Tris buffer (pH 7.4). Tubes were centrifuged at 2,500 rpm for 20 min. at 4°C, the resulting pellet was counted in a LKB gamma counter. Regression analysis was employed to determine the amount of sera required to achieve half-maximal binding, and neutralization capacity was expressed in ng/ml of undiluted serum, taking into account the specific activity of 125I-hCG (). The bioneutralizing potency of the antibodies generated by different adjuvants was determined by calculating the B/I ratio : B = Bioneutralizing capacity (ng/ml) I Antigen binding capacity (ng/ml) Results and Discussion In vitro characterization of microparticles The size of the microparticles as observed through a laser scattering particle size analyzer was found to be 2-5 µm. The particles were spherical and smooth and showed a homogenous distribution when observed under a scanning electron microscope (Photomicrograph-1). Entrapment efficiency, calculated with respect to the initial amount of ß-hCG, was 0.41% per mg of polymer. As reported for TT, incorporation of RSA in the internal aqueous phase during particle formulation resulted in preparation of particles containing immunoreactive P-hCG. The particles released immunoreactive P-hCG in in vitro release experiments (data not shown). Quantitation of Anti-hCG Antibody Response Figure 2 and 3 show antibody titres generated by a single dose of 25 µg of ß-hCG in various adjuvant formulations. The antibody titres produced were in order with 25 µg of P-hCG in particles with alum > 25 µg of P-hCG in particles > 25 µg of P-hCG on alum > 25 µ,g of ß-hCG on saline (Figure 2). No antibodies were detected when ß-hCG was administered in saline. Alum adsorbed P-hCG elicited very low antibody titres (2ng/ml), detectable only Day 30 and declining to background levels thereafter. Microencapsulated P-hCG generated significantly higher titres, which peaked at Day 90, and were still detectable at Day 180, when the experiment was terminated. Immunization of polymer particles along with alum changes both the kinetic and magnitude of immune response in rats. Immunization of particle and alum mixture resulted in Significantly higher ß-hCG antibody titers were observed compared with those generated by either component In addition appreciable antibody titres were observed on Day 30, indicating a reduced lag period. On day 90 of post immunization the average antibody titers in this group of animals was 1200 ng/ml in comparison to 400 ng/ml achieved by only particle immunization . As expected, animals immunized with single dose of ß-hCG emulsified with either CFA or IFA generated significantly high antibody titers (Figure 3). Antibody titres from all adjuvant groups peaked on Day 90, and were significantly higher than those obtained with microencapsulated 3-hCG. Admixture of particle and alum titers were lower than that obtained by immunization with either CFA or IFA. Significantly, affinity of the antibodies raised following immunization with microparticles or microparticles admixed with alum were comparable with those obtained with CFA and IFA. In all cases, the affinity was in the range 1010 L/M (Table 2). In vitro receptor binding inhibition assay was carried out to determine the bioneutralizing capacity of the antibodies following immunization of ß-hCG with various adjuvants. Day 90 sera was chosen for this analysis as peak titres were observed for all groups at this time. Serum samples were subjected to the in vitro receptor binding inhibition assay and the percent inhibition capacity of the sera at various dilutions was calculated (Figure 4). All immunization schedules, irrespective of adjuvant, generated a bioneutralizing antibody response. The ability to inhibit hCG-receptor interaction was a function of antibody titres, with antiserum generated using CFA being the most potent, followed by IFA, the microparticle-alum admixture, and alum alone. Bioneutralizing capacity (ng/ml) was calculated to further compare the different adjuvants (Table 2). As expected, these results confirmed the correlation between antibody titres and neutralization ability. The bioneutralization capacity of CFA generated serum was high (1716 ng/ml), followed by IFA (888 ng/ml) and particles admixed with alum (684 ng/ml), while immunization with particles alone generated antibodies of low bioneutralization capacity (215 ng/ml). B/I ratios showed that the bioneutralizing capacity per unit of immunoactivity virtually remained unchanged (≈ 0.5) irrespective of the adjuvant used (Table 2). Alum adsorbed vaccines, however, require repeated administration, since they are not capable of producing a prolonged immune response (). However in this case, polymeric microparticles entrapping P-hCG have shown improved adjuvant activity in comparison to alum. Polymeric microparticles containing P-hCG showed immune response on Day 30 (15 ng/ml) which prolonged over a period of 180 days (210 ng/ml) with a peak on day 90 (408 ng/ml). The antibody titres generated by polymeric microparticles were far superior (100 times) to those generated using alum (2 ng/ml on day 30) (Fig. 2 and 3) and saline. Immunization of ß-hCG entrapped microparticles admixed with alum resulted in significantly higher antibody titers. This not only increased the magnitude of the antibody titers but also changed the kinetics of antibody production. Immunization of particle admixed with alum resulted in very early titers in comparison to the particles alone. Animals immunized with microparticles admixed with alum showed a shortening of the lag phase, with significant titres being observed on Day 30 (60ng/ml) which peaked on Day 90 (1238 ng/ml). This may be due to much better antigen presentation by the mixture of particles and alum. Further, the antibody level was maintained over a period of 180 days. Improvement in immune response from polymer particles based immunization with help of additional adjuvant have been reported for TT and DT particles (Singh et a/., 1997). Similar enhancement in antibody titres upon the addition of other external adjuvants (squalene and Pluronic® L121) have been recently reported for P-hCG with use of chitosan (Seferian and Martinez 2001). However, the immune response generated in the present formulation is far superior in comparison to the reported value using chitosan. Antibody titer generated by single injection of admixture of ß-hCG particles with alum was higher than the reported value using chitosan formulation. The fact that B/I ratios hi the microcarrier-based immunization were of the same order as those observed with CFA- or IFA- based immunization (≈0.5) deserves comment. We believe these results indicate that crucial epitopes upon ß-hCG (i.e. those capable of eliciting a bioneutralizing antibody response) were not altered in the process of encapsulation. Based on previous results from our laboratory. Antibody titers were also long lasting following single dose immunization. Thus with proper combination of two clinical approved adjuvant, immunogenicity of ß-hCG could be improved substantially and the antibody affinity were comparable to that achieved with that of CFA or IFA. We speculate such an immunization modalities can be applied to sub-unit vaccine candidates, polysaccharides based vaccines and DNA vaccines, which in general elicit poor immune response and need repeated immunization for immunity. Such modalities can also be applied for existing multi does vaccines for improved immune response. References Alonso MJ, Cohen S, Park TG, Gupta RK, Siber GR, Langer R. Determinants of release rate of tetanus vaccine from polyester microspheres. Pharm. 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Singh 0, Rao LV, Gaur A, Sharma NC, Alam A, Talwar GP Antibody response and characteristics fo antibodies in women immunized with three contraceptive vaccines inducing antibodies against human chorionic gonadotropin. Fertil. Steril. 198952: 739-744. Shi, Li, Caufield MJ, Chern RT, Wilson RA, Sanyal G, Volkin DV,, Pharmaceutical and immunological evaluation of single shot hepatitis B vaccine formulated with PLGA microsphere. J. Pharm. Sc. 2002, 91, 1019-1035 Tobio M, Nolley J, Guo Y, Mclver J, Alonso MJ. A novel system. based on a poloxamer/PLGA blend as a tetanus toxoid delivery vehicle. Pharm. Res. 1999; 16:682-85. Weert, van de M., Hennink, W.E. and Jiskoot, W. Protein instability in poly (lactic-co-glycolic acid) microparticles. Pharm. Res. 2000: 17, 1159-1167. Zhu, KJ, Jiang. HL, Du XY, Wang. J.Xu, WX and Liu SF . Preparation and characterization of hCG loaded polylactide or polylactide-co-glycolide microsphere using a modified water -in-oil-in-water emulsion solvent evaporation techniques. J. Microencapsulation, 2001, 18, 247-260 Zhu, G., Mallery, S. R. and Schwendenman, S. P. Stabilization of proteins encapsulated in injectable poly (lactide-co-glycolide). Nature Biotechnology, 2000 :18, 52-57. We claim : 1. A process for enhancing the immunogenicity of a weak antigen by entrapping the said antigen in a biodegradable polymer matrix and immunizing along with alum using single point contact. 2. A process as claimed in claim 1 wherein the said polymer is biodegradable and the antigen is entrapped as described in Patent application 852/Del/2000. 3. A process as claimed in claim 1 where in the weak antigen is selected from |3-hCG, sub-unit vaccine or recombinant antigens. 4. A process as claimed in 1 where the polymer particles are hydrophobic in nature and have average size less than 5 µm. 5. A process of immunization as claimed in claim 1 where the antigen is uniformly entrapped inside the polymer particles. 6. A process of immunization by using polymer entrapped antigen and alum thus presenting the antigen in particulate manner to the immune cells for better antigen presentation. 7. A process by which intramuscular immunization of particle and alum combination results in improved immune response while using an weak antigen. 8. An immunization process claimed in claim 1 which produces high affinity bioneutralizing antibody titers comparable to that achieved with either using CFA or IF A based immunization, 9. A process of immunization using admixture of polymer particles entrapping immunoreactive antigen and human permissible adjuvant for improving the immunogenicity of a poor immunogen. 10. A process as claimed in claim 9 where the preferred adjuvant for immunization is alum. 11. A process of immunization claimed in 1 and 9 where single point immunization results in long lasting immune response. 12. A process as claimed in 1 and 9 where immunization of admixture of polymer entrapped antigen and alum results in generation of early immune response. 13. A process as claimed in claim 1 and 9 where long lasting immune response can be generated without use of multiple injection. 14. A process as claimed in claim 1 for generation of improved immune response from antigens which need repeated immunization for immunity. 15. An admixture of polymer entrapped antigen and alum for generation of long lasting immune response with single point immunization. 16. An admixture of polymer entrapped antigen and alum for generation of early immune response using single point immunization as described in claim 1 and 9 17. A method for use of formulation for improved immune response from weak antigen as described in claim 16.

Documents:

794-del-2001-abstract.pdf

794-del-2001-claims.pdf

794-del-2001-correspondence-others.pdf

794-del-2001-correspondence-po.pdf

794-del-2001-description (complete).pdf

794-del-2001-drawings.pdf

794-del-2001-form-1.pdf

794-del-2001-form-19.pdf

794-del-2001-form-2.pdf

794-del-2001-form-3.pdf

794-del-2001-form-4.pdf

794-del-2001-form-5.pdf

794-del-2001-gpa.pdf