Title of Invention	"A PROCESS FOR THE PREPARATION OF A VACCINE FOR THE DEVELOPING IMMUNOLOGICAL FROM SINGLE DOSE"
Abstract	A process for the preparation of a vaccine for the development of immunological memory from a single dose which comprises entrapping in any known manner, vaccine antigens in a polymer matrix, and optionally adding thereto , a conventional adjuvant characterized by directly entrapping the vaccine antigen and that said polymer matrix comprises biodegradable polymer matrix having average size less than 5um.

Title of Invention

"A PROCESS FOR THE PREPARATION OF A VACCINE FOR THE DEVELOPING IMMUNOLOGICAL FROM SINGLE DOSE"

Abstract

A process for the preparation of a vaccine for the development of immunological memory from a single dose which comprises entrapping in any known manner, vaccine antigens in a polymer matrix, and optionally adding thereto , a conventional adjuvant characterized by directly entrapping the vaccine antigen and that said polymer matrix comprises biodegradable polymer matrix having average size less than 5um.

Full Text	Field of the invention The present invention relates to the development of immunological memory following single point immunization with polymer-entrapped antigens. High secondary antibody response was observed upon re-exposure to the same antigen in soluble form after 6-10 months of primary immunization with polymer-entrapped antigen. Such immunization protocol can be used to develop immunological memory using single dose of polymer entrapped antigens. Background For many vaccinations, multiple immunization results in development of desired immune response. Successful vaccination for tetanus, diphtheria, hepatitis and many other infectious diseases need more than two injections to achieve protective immunity., Many times boosters are required at different time of human life cycle to develop bio-neutralizing antibody titers for protective immunity. Particularly in developing countries, logistic problems associated with mass immunization program results in low vaccination coverage. It will be ideal if the vaccines for many infectious diseases can elicit long term immunological memory from single dose administrations. Immunological memory is characterized by a rapid and robust immune response after re-exposure of the original immunizing antigen and is an ideal aim of many immunization protocols used for infectious diseases. Many newly developed or under developed recombinant vaccine candidates are poorly immunogenic thus need repeated vaccine administration achieve desired immunity. If immunological memory can be achieved using single point immunization, it will be of great help toward improving the vaccine efficacy particularly for infectious diseases. Thus there is a need to develop vaccination protocol which can give rise to effective, desired immune response from a single point immunization. Biodegradable polymer particles have been under extensive evaluation in current times to develop single dose vaccine, however none of them have tried to explore the utility of these polymer entrapped antigen for the generation of immunological memory using single point immunization. Tetanus toxoid was entrapped in Polylactide polymer particles entrapping immunoreactive tetanus toxoid of different sizes were used for single dose immunization. Single dose of admixture of polymer entrapped antigen and alum upon immunization elicited anti-TT antibody titers comparable to that achieved by two doses of alum adsorbed tetanus toxoid. Single doses of alum adsorbed tetanus toxoid elicited weak primary as well as secondary immune response . Where as animals immunized in single dose with polymer entrapped antigen alone or with an admixture of particles and alum elicited very high secondary response upon re-exposure of the antigen after 6-9 months of primary immunization. Use of polymer particle based immunization thus apart form providing immunity from single dose immunization helped in the development of immunological memory. Development of immunological memory will be required for many infectious diseases where multiple immunizations do not result in development of protective immunity for longer duration of time. Prior Art Relating to the Invention Use of poly lactide-co-glycolide (PLGA) and polylactide (PLA) polymers for entrapping vaccine/antigens provide a viable alternative to the multidose vaccine injection schedule for immunization (O.Hagan et al. 1998; Cleland 1999; Johansen et al. 2000). These polymer particles provide adjuvant activity to the antigen and thereby improve the immune response (Hanes et al.1997; Lofthouse 2002). Development of single dose vaccine using biodegradable polymer entrapping tetanus toxoid, diphtheria toxoid, Hepatitis B surface antigen and many other potential vaccine antigens have been studied extensively (Hanes et al. 1977 ; Cleland 1999). In order to develop single dose vaccine, it is not only imperative to release immunoreactive antigen from polymer particles but it is also essential to make then more immunogenic either by delivering to macrophages or immunizing along with adjuvant for better presentation to antigen presenting cells (O'Hagan et al. 2001). This is most important as polymer particles release soluble antigen and it is widely documented that soluble antigens are weakly immunogenic. It is essential that the particles should entrap and release immunoreactive antigens (Zhu et al. 2000 ; wreet et al. 2000, Raghuvanshi et al. 2001), release of the 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 ( One of the most important developments in vaccine administrations will be development of immunological memory from a single point immunization. Immunological memory is defined as the immediate immune response from the system upon re-exposure to the same antigen (Ahmed and Gray, 1996, Sprent, 1997). It will be very useful if long lasting immunological memory can be achieved from minimum number of injections. Most of the time, repeated immunization at particular interval is needed to activate the immune system and to develop immunological memory. Molecular mechanism which leads to development of immunological memory is still an unsolved problem in immunology (Ahmed and Gray, 1996 ; Gray, 2002; Zinkernagel 2002). From the available published information on memory responses from different vaccines, different mechanism have been proposed by different groups actively working to understand the molecular mechanism of immunological memory. There are reports that continuous presence of antigen is an essential requirement for the development of immunological memory (Gray 2002; Ochsenbein et al. 2000). Presence of long lived plasma cell have also been attributed to the development of B cell memory (Slifka et al. 1998, Manz and Radbruch 2002). Some researcher claims memory is due to polyclonal activation of memory B cells (Bernasconi et al. 2002). Persistance of memory B cells in the absence of immunizing antigen have also been suggested (Maruyama et al. 2000). Whatever may be the real reason for the development of immunological memory, it will be worthwhile to demonstrate the development of memory response from single point immunization. It was expected that polymer particulate based immunization may end up in the development of tolerance rather than memory, as it exposes the immune system with low doses of antigen continuously. When memory response was tested by administering low dose of original soluble antigen to the animals after a long period of time (half of life span of experimental animals) of primary immunization, the secondary response was very high. The antibodies generated after secondary exposure were not only high but also have high toxin neutralizing capacity capacity, which is an essential indicator of protective immunity. This indicated the suitability of polymer entrapped system for the development of memory response from a single dose immunization. High secondary response from polymer particles immunized animals have been reported for tetanus toxoid, while boosting the immunized animal with antigens along with adjuvants (Esparaza and Kissel 1992; Gupta et al. 1997). The real test of immunological memory is the rapid and robust immune response on re-exposure to the original antigen in soluble form, which has not been reported to date using polymer entrapped antigens. We while working extensively on polymer entrapped antigens, observed high memory response in experimental animals with different antigens. It was also observed that, particle made of a particular size are able to develop memory response more effectively than other size particles. Such memory response from single dose immunization with polymer entrapped antigen was observed for TT, HBsAg, DT, and ß hCG. It was observed that single dose of above mentioned antigens entrapped in polymer particles could give rise to very high secondary antibody response upon re-exposure to low amount of soluble antigens. The secondary response was sustained for a longer period in comparison to that observed for alum adsorbed vaccine immunization. It was also observed that memory response generated from polymer entrapped vaccine immunization is better than that observed while immunizing with multi dose alum adsorbed vaccines. Immunization with single dose of polymer entrapped TT, upon boosting with very low amount of soluble TT, elicited very high antibody titers in Wistar rats. The secondary antibody titers were higher than the primary peak anti-TT antibody titers. The affinities of the antibodies generated during secondary response were very high and the types were mostly IgG indicating the presence of memory cells upon immunization with polymer entrapped antigen. More importantly, re-exposure with soluble antigen resulted antibody response which was higher and more sustained in comparison to that elicited with alum adsorbed vaccination. Development of such memory response was demonstrated for many potential vaccine candidates. Objects of invention The main object of invention is to provide a single point immunization process which results in long term immunological memory. Vaccine antigens were entrapped in polylactide (PLA) polymer in the form of particles having different sizes. However immunization with particles less than 5 urn entrapping vaccine antigen resulted in high memory response. Experimental animals were immunized with polymer entrapped antigen and the development of immunological memory was tested by boosting the animals with very low dose of soluble antigen after 6-9 months of primary immunization. It was observed that, animal immunized with polymer entrapped antigens elicited very high secondary antibody response in comparison to that achieved with alum adsorbed immunization. Improved secondary response on immunization with polymer entrapped antigens was observed for many vaccines currently used in humans and other candidate vaccines also. Notable among them are tetanus toxoid, diphtheria toxoid and hepatitis B surface antigen. Single dose immunization of these above antigens in polymer entrapped form elicited high secondary response upon boosting with very low amount of soluble form of the respective antigen. These results indicated that long term immunity can be achieved for many infectious diseases using single dose administration of polymer entrapped antigens. Accordingly, the inventors have developed a process for improving immunological memory with single dose immunization. Another object of invention is the generation of high secondary immune response with the use of very little soluble antigen using polymer particle based immunization. One object of the invention is the generation of high bioneutralizing secondary antibodies titers using polymer particles entrapped antigens for immunization. Another object of invention provides a process for generation of immunological memory using single point immunization for infectious diseases. Yet, another object of invention is to formulate an immunization protocol using the polymer entrapped antigens for obtaining long lasting sustained primary as well as secondary antibody responses in experimental animals with single point immunization. Another object of invention is development of single point immunization protocol which generates higher secondary response in comparison to immunization with alum adsorbed vaccine. Another object of invention is the development of single point immunization protocol which elicit high and sustained secondary immune response upon boosting the animals with soluble form of antigen. Description of the invention: The inventors have developed a single step immunization process for improving immunological memory using biodegradable polymer particles. Polymer particles entrapping antigens such as tetanus toxoid (TT) and Hepatitis B surface antigen (HBsAg) of required size were prepared using polylactide polymer. Immunizing experimental animals with particles in combination with alum generated enhanced and sustained immune responses. After 6-10 months of primary immunization wistar rats were injected with low amount of soluble TT or HBsAg to check the secondary immune response. Rats immunized with polymer particle based vaccines elicited very high immune response in comparison to rats immunized with conventional alum adsorbed vaccine. Rats boosted with as low as 0.5 Lf of soluble TT after 6-10 months of primary immunization resulted in very high anti TT antibody titters in comparison to alum adsorbed immunized groups. The secondary anti TT antibody response generated by polymer particles based immunization was higher than that achieved during the primary immunization. Similar high secondary response was observed upon immunization with polymer particles entrapping antigen alone. The antibody generated during secondary response from particle based TT immunization has high toxin neutralizing capacity. Secondary antibody response was found to be dependant on dose of antigens given during primary immunization. Higher doses of polymer entrapped antigen during primary immunization resulted in very high secondary antibody response. High secondary response was observed only when the polymer particles entrapping antigens have size less than 5 urn. Larger sized particles upon immunization elicited low secondary response upon re-exposure to soluble antigen. This indicated polymer particle based immunization helps in generation of immunological memory. Similar high secondary antibody response from polymer entrapped HBsAg immunization was also observed. Antibody generated during secondary response from single dose immunization with polymer entrapped HBsAg have comparable bioneutralizing capacity to that observed from animals immunized with three doses of conventional alum adsorbed vaccine. Secondary immune response generated from single point immunization with polymer entrapped TT was always higher than that observed from immunization with alum adsorbed antigen. It is hypothesized that use of polymer entrapped antigen in particles of appropriate size, continuous release of immunoreactive antigen from the particles and elicitation of high early immune response from particles based immunization acts cooperatively to develop immunological memory upon single point immunization. Generation of very high antibody titers of high affinity upon boosting the immunized animals with soluble antigen testifies the establishment of memory cells capable of producing high antibody response upon single point immunization. Such immunization protocols can be applied to generate long lasting immunological memory where repeated immunization is essential for developing immunity. Such polymer based single point immunization can be applied to most of the infectious diseases such as Hepatitis B, Rabies, Anthrax, Diphtheria, Cholera, Tetanus, foot and mouth disease (FMD) in animals, Measles and for many other recombinant antigen vaccine candidates which elicit poor primary and secondary response. The invention covers patent application 852/Del/2000 and patent application 794/Del/2001 which is here by incorporated by reference in its entirety. In an embodiment the invention describes the process of immunization which results in development of immunological memory from single point immunization. In an embodiment, the vaccine tried here are tetanus toxoid, Hepatitis B surface antigen, vaccines for infectious disease and many recombinant vaccine antigens. In an embodiment the immunization involves the entrapment of the said vaccine antigen in biodegradable polymer particles. In an embodiment the process of immunization involves polymer entrapped particles of size less than 5 µrn with or without admixture of alum. In an embodiment, the process describes the immunization with polymer entrapped antigens where the polymers are biodegradable and hydrophobia In other embodiments, the process describes the immunization with polymer entrapped antigens as describes in patent application 852/Del/2002 and 749/Del/2001. In another embodiment the process describes the immunization process where boosting with very low amount of soluble antigen after 6-10 months of primary immunization result in very high secondary response. In an embodiment the process describes the immunization process where the affinities of antibodies generated during secondary response are very high. In an embodiment, the process of single point immunization results in high secondary response in comparison to the alum adsorbed vaccination. In an embodiment, the process describes the generation of high secondary antibody response whose affinities are comparable with that observed with multiple immunization with alum adsorbed vaccine. In another embodiment the process describes the generation of bioneutralizing memory antibody titers from a single dose of polymer entrapped antigen In another embodiment the process describes the generation of long term immunological memory from single point immunization of polymer entrapped antigens. In another embodiments these antigen could be any vaccine candidates for human and animal use where repeated immunization is required to achieved long term immunity. In another embodiment, the process describes the generation of both long lasting primary and secondary antibody response while using combination of permissible adjuvant such as biodegradable polymer and alum. In particular the applicant has described a single dose immunization procedure using polymer entrapped antigen for improving immunological memory. Vaccine antigens entrapped in biodegradable polymer particles were used for immunization. The secondary immune response generated form such immunization upon reexposure to the soluble antigen was very high in comparison that observed with alum adsorbed vaccination. High secondary immune response could be generated from single point immunization with polymer entrapped antigen. Single point immunization with polymer particle entrapped tetanus toxoid, diphtheria toxoid, hepatitis B surface antigen when boosted with corresponding soluble antigen elicited very high secondary antibody response. These results are of indication that, the described immunization protocol can be effectively used for generation of long term memory for many vaccine candidates which need repeated immunization to achieve desired immunity. The invention is described in detail with the aid of following examples and the accompanying drawings and tables. Various modifications that may be apparent to one in the art are intended to falls within the scope of the invention. Description of the accompanying drawing Figure 1. Comparison of serum anti TT antibody concentrations in male wistar rats immunized with single dose of polylactide microparticles ( 4µm average size ,-■-) and nanoparticles entrapping TT ( 0.75µm average size -●-) (15 Lf /rat in each group) and conventional two doses of alum adsorbed TT (-▲-) (5 Lf /rat /dose). 4-6 weeks old outbred wistar male rats were immunized with microencapsulated and conventional vaccines. Figure 2. Anti TT antibody concentrations in male wistar rats immunized with single dose of polylactide microparticles (-▲-), nanoparticles (-●-) entrapping TT (15 Lf /rat in each group), their combination (-T-) given with alum and conventional two doses of alum adsorbed TT (-■-) (5 Lf /rat /dose in interval of four weeks). Wistar male rats were immunized intramuscularly with microencapsulated and conventional vaccines. Each group was consisting of 6 rats. Figure 3. Primary and secondary anti TT antibody response in rats immunized with single dose of TT entrapping microparticles (-▲-), combination of microparticles and nanoparticles (-▼-) (15 Lf/rat in each group) and conventional two doses of alum adsorbed TT (-■-) (5 Lf/rat/dose). After 6 months of primary immunization animals were given 5 Lf TT. Figure 4. Primary and secondary anti TT antibody response elicited by polylactide microparticles entrapping TT of different sizes. Out bred wistar female rats were immunized with particles of sizes 140 µm (-■-), 41µm (-●-), 25µm (-▲-), 4µm(-▼-) and 0. 75µm (-♦-). 8 months after primary immunization animals were re-exposed to 0.5 Lf soluble TT to check memory response. Figure 5. Secondary antibody TT antibody response in female wistar rats immunized different doses of alum adsorbed TT and polylactide microparticle (average size 4µm ) entrapping TT in presence of alum. Two groups of 6 animals were immunized with single dose of 1.0 (-●-) and 0.1 Lf (-▲-) alum adsorbed TT and one group was immunized with two doses of 5 Lf alum adsorbed TT given at 0 and 1 month (-■-). Other groups of rats were immunized with single dose of 15Lf (-T-), 10 Lf (-+-), 5 Lf (-+-), 1 Lf (-X-), 0.5 Lf (--) and 0.1 Lf (--) microencapsulated TT along with alum as adjuvant. One group was immunized with 10 Lf soluble TT (-I-). All the groups were given 0.5 Lf soluble TT after 9 months of primary immunization to check memory response. Figure 6.Affinity of anti TT IgG in rats immunized with single dose of 10 Lf microencapsulated TT with alum before checking memory response (-●-) and during secondary response (-▲-). Equal amounts of antibody from different groups were incubated with different concentrations of antigens for two hours at 37 °C and unbound antibody was assayed using ELISA. Affinity measurements were from groups of rats used for dose response studies. Lower absorbance at different antigen concentration indicates the presence of low unbound antibody which reflects the high affinity of antibodies. Affinities of the secondary antibodies (290 days data) are higher than the antibodies present before soluble exposure ( 275 days).I Table 1. Peak and lowest serum anti TT IgG concentrations before testing memory and peak serum anti TT IgG concentrations during secondary immune responses following administration of 5 Lf soluble TT in different groups of rats. Table 2. Peak and lowest serum anti TT IgG concentrations before testing memory and peak serum anti TT IgG concentrations during secondary immune responses following administration of 0.5 Lf soluble TT in different groups of rats. Table 3. Anti-HBsAg antibody titres (AUSAB method) generated upon immunization of HBsAg vaccine. Three doses of alum adsorbed vaccine was compared with one dose of polymer entrapped vaccine. After 9 months of primary immunization, animals were immunized with 5 µg of soluble antigen to check the memory antibody response. Example 1: Immunological memory from single point immunization of polymer entrapped TT Extensive studies with the use of PLGA/PLA particles have been carried out to explore the possibilities of developing a single shot vaccine for tetanus toxoid (TT) (Kersten et al. 1996 ; Audran et al. 1998 ; Tobio et al. 2000 ; Diwan et al. 2001). Results of antibody titres obtained from groups immunized with two injections of 5 Lf TT each adsorbed on alum and equivalent doses of microencapsulated TT showed that the conventional immunization schedule gives significantly higher antibody titre than immunization with microencapsulated TT (Kersten et al. 1996 ; Johansen et al. 2001). There are reports that smaller size and hydrophobia polymeric particles improve the immunogenicity of the entrapped antigen ((Eldridge et al. 1991; Conway et al. 1997). With proper analysis of these above factor related to the development of single dose vaccine using biodegradable polymer, it is possible to generate long lasting immune response from a single point immunization. However till now no one has evaluated the potential of polymer entrapped vaccination for development of immunological memory. There are reports where people have demonstrated the amnestic response while boosting with alum adsorbed antigen. However the real test of immunological memory is the rapid and robust immune response on re-exposure to the original antigen in soluble form, which mimics the natural infections. We have demonstrated here that, immunization with polymer particles entrapping immunoreactive TT leads to development of better immunological memory. Wistar rats were immunizes with single dose of PLA entrapped TT and the memory response was evaluated after 6-9 months time while boosting with soluble TT. It was observed that polymer particles based immunization leads to very high secondary antibody response in comparison to that observed with alum adsorbed vaccine immunization. The affinity of the antibodies generated during secondary immunization was very high thus indicating the development of immunological memory from single point immunization using polymer entrapped Materials and Methods Materials Poly D,L-lactigle (PLA, 45 KD) were purchased from Birmingham Polymer Inc. USA. TT (3000 Lf/ml, protein content 8 mg/ml) was purchased from Serum Institute of India, Pune. Rat serum albumin (RSA) [A-6272] and polyvinyl alcohol, MW 30,000 (PVA) were from Sigma Chemicals USA. Micro BCA protein assay kit was from Pierce, USA. Polyclonal antibodies to TT and goat anti-TT HRPO conjugate were from Reagent Bank of National Institute of Immunology, New Delhi, India. Preparation of polymer particles Polylactide polymers particles were prepared using solvent evaporation method (Raghuvanshi et al. 2001). Primary emulsion between internal aqueous phase (TT in PBS, pH 7.4) and organic phase (polymer solution in DCM) was prepared by sonication (20 watts, 80% duty cycle, 20 cycles) (Brason, Sonifier 450, USA). Secondary emulsification was carried out either by sonication to obtain nanoparticles or by homogenization (10,000 rpm for 10 minutes) (Virtis, Cyclone I.Q., USA) for microparticles. The resulting polymeric particles were washed twice with cold PBS and lyophilized to get free flowing powder. The size of the particles was measured using GALAI-CIS-1 particle size analyzer. All particles were prepared using RSA as a stabilizer in the internal aqueous phase during particle formulation. Estimation of protein content of polymer particles To measure the protein content of particles, accurately weighed particles were dissolved in acetonitrile to solubilize the polymer while precipitating the encapsulated protein. The precipitated protein was pelleted by spinning at 5,000 rpm for ten minutes and was dissolved in 1% SDS solution. The protein content of the solution was determined by micro BCA assay. The protein loading was calculated as the per cent weight of protein per unit weight of polymer. In vivo animal studies Immunogenicity of the encapsulated TT in particles was checked in animal model using Wistar rats. Rats were injected intramuscularly with 30 µg (15 Lf) of either free, alum adsorbed or microparticle encapsulated TT. PLA polymer particles (both nanoparticles and microparticles) entrapping TT were used for animal experiments (six animal in each group). Physical mixture of polymer particle of different size were also used for immunization, the total dose of TT was 15Lf Similar groups of animals were immunized with particles along with alum. For immunization with admixture of alum, 50 µl of 2 % alum (Superfos Biosector a/s, Denmark) was mixed with PLA particles just before immunization and used. Two injections of TT (5 Lf each,) adsorbed on alum was given at four weeks (0 and 28 days) interval. Higher dose of TT in particles were taken for comparison to take care of the loss and degradation form particles. For the dose response experiments admixture of microparticles and alum was used. The total dose of TT was 15, 10, 5, 2, 1 and 0.5 Lf. Similarly for experiments related to size, particles of the following sizes were prepared and used for immunization. The dose of TT was 5 Lf and sizes are 140µm, 49 µm, 25 µm, 10 µm, 5 µm and 0.75 µm. For checking the memory response animals were immunized with single dose of polymer entrapped antigen and after 8 months of primary immunization boosted with 1 Lf of soluble TT. The antibody titers after soluble boosting were determined By ELISA and the affinities were also determined using standard protocol (Friguet et al. 1997). Animals were maintained according to the guidelines established by the Institute Animal Ethics Committee of the National Institute of Immunology, New Delhi and serum anti -TT antibody titers were determined by ELISA. Anti -TT antibody titers were defined as µg of antibody/ml of the sera employing affinity purified antibody as references in ELISA (Raghuvanshi et al. 1998). Antibody titers of individual animal (n = 6) were estimated in duplicates and their concentrations (µg/m) were determined as geometric mean. To analyze the statistical significance of the antibody titers students't-test at 95% confidence level was carried out. RESULTS Immune response from PLA particles encapsulating TT Micro-particles and nano- particles entrapping immunoreactive TT were made from PLA polymer (Raghuvanshi et al. 2001). The average size of nano particles were around 750 nm where as that of micro particles was 4 um. rats were immunized with single dose of polymer entrapped alum (15 Lf) and was compared with that of two doses of alum adsorbed TT. It was found even though polymer entrapped TT elicited immune response , the antibody titers was lower than that observed from two doses of alum adsorbed TT ( Figure 1). To improve the immunogenicity of the encapsulated TT particles, alum as an adjuvant was added along with the particles during single dose immunization. It was found that incorporation of alum in the particle suspension improved the antibody titers significantly (Figure 2). In nanoparticles the antibody response peaked early where as maicroparticles along with alum gave titers little later. Immunization with admixture of alum and particles improved the immune response significantly (Figure 2). High and early immune response from admixture of nanoparticles and alum was achieved. Antibody titers around 80µg/ml could be detected around 15 day post immunization. However the antibody titers decreased considerably after 75 days of post immunization. Early higher antibody titers were also found in group receiving admixture of nanoparticles, micrparticles and alum. Immunization with physical mixture of particles resulted in sustained antibody response for longer period of time. In groups receiving microparticles along with alum the peak response was observed at 80 days which was very high and comparable to that of TT/alum. Anti-TT antibody titers around 350 ng/ml could be detected around 80 days after immunization. More importantly high anti-TT antibody titers persisted for a longer period of time particularly in the later part of of the post immunization period. Two doses of alum adsorbed TT gave anti-TT antibody titers around 400µg/ml on day 40-45 where as admixture of microparticles and alum elicited around 350 µg/ml of anti-TT antibody titers, which peaked around day 80 (Figure 2). These results suggest that, with proper combination of polymer entrapped TT and alum; it is possible to generate antibody response using single point immunization that is comparable to that of two doses of alum adsorbed TT injection. Memory response from polymer entrapped TT immunization To check the memory response generated by particles based immunization, animals were immunized with polymer entrapped TT along with alum. Anti TT antibody titers were estimated every month and after 6-9 months of primary immunization animals were boosted with 5 Lf of soluble antigen. It was observed that groups immunized with microparticle entrapped TT gave rise to very high secondary antibody response on being re-exposed to soluble antigen (Figure 3). Memory response generated from all groups of immunization involving microparticles, nanoparticles and physical mixture of particles both given with or without alum are presented in table 1. It was observed that single dose immunization with polymer entrapped TT generated considerable memory response upon re-exposure to the same antigen. Even with re exposure of animals immunized with particulate TT with low level of soluble TT (0.5 Lf), very high secondary response was observed (Table 2). It was observed that polymer entrapped TT immunization resulted in very high secondary immune response indicating the development of immunological memory in the animals. It was also observed that polymer particles based immunization without alum even though elicited very low primary immune response, the corresponding secondary response were higher than that observed for two dose of alum adsorbed TT immunization (Table 1 and 2). Memory responses generated from particles based immunization having different doses and sizes are presented in figure 4 and figure 5. Polymer particles having size less than 5 urn was found to elicit better secondary response than larger size particles (Figure 5). It was observed that maximum memory response was observed for particles size were less than 5 urn. Nanoparticles gave substantial high secondary response upon boosting, but lower than that observed for 1-5 urn particles. It was observed that secondary response generated upon re-exposure to the same amount of soluble antigens was strictly dependent upon the doses used during primary immunization. Secondary response generated from animals immunized with microencapsulated TT was higher than that observed from animals immunized with conventional alum adsorbed TT (Figure 5). Even with single doses of microencapsulated TT lower than the combined two doses of conventional alum adsorbed vaccine gave much better secondary response upon re-exposure to the soluble antigen. The affinity of antibodies generated during memory response to the immunized antigen was high, indicating the generation of high affinity antibodies upon polymer particle based immunization (Figure 6). In all the cases, single point immunization of polymer entrapped TT thus helped in development of immunological memory. The antibody concentrations in serum during memory response were longer lasting in animals immunized with microencapsulated antigens than those generated from animals immunized with alum adsorbed TT, which declined rapidly. Antibodies generated during memory response have high toxin neutralizing capacity in comparison to that observed for alum adsorbed immunization (data not shown). Rapid response to the intra muscular injections of low doses of soluble antigen high antibody titer, high affinities of the secondary antibodies and maintenance of high antibody titers for longer period of time suggested the development of immunological memory from single point polymer particles based immunization. Example 2 Memory response from polymer entrapped HBsAg Immunization Immunogenicity of the microencapsulated HBsAg in particles was checked in animal model using Wistar rats (six animals in each group). Rats were injected intramuscularly with 10 µg of either free, alum adsorbed or particle encapsulated HBsAg. Three injections of HBsAg (3.3 µg each total of 10 µg) adsorbed on alum was given at day 0, 30, 180 days as control. Animals were bled at different time interval through retro-orbital plexus and the serum was analyzed for anti-HBs Ag antibody titers by AUSAB assay using ABBOT Lab ELISA Kit. Antibody titers were estimated in duplicates and their concentration was expressed as mlU/ml. It was observed that single immunization of polymer entrapped HBsAg elicited higher bioentralizing titers ( > 60 000 mlU/ml) than the required value of 10 mlU/ml for protection ( Table 3). After 287 days ( > 9 months) of primary immunization, the animals were boosted with soluble HBsAg (5 ug) to check the memory response. High memory response was observed from animal immunized with particle entrapped HBsAg upon re-exposure to soluble antigen (Table 3). In polymer particles based immunized animals, the bioneutralizing HBsAg antibody titers were improved from 60 000 to 403 000 mlU/ml upon re-exposure to the soluble antigen. Similar memory repose was observed from animal immunized with three doses of alum adsorbed HBsAg where the antibody titers increased from 170, 000 to 426 000 mlU/ML. The extent of secondary response in three doses of alum adsorbed vaccination increased 2.5 times the existing titer upon re-exposure to soluble HBsAg. Where as in the case of single dose polymer particles based immunization, the antibody titers increased more than 6.5 times upon re-exposure to the soluble antigen. This indicated that single dose immunization with polymer entrapped antigen gives better memory response than observed with three doses of conventional vaccine immunization. Such immunization can be applied to humans to elicit sero-protection titers from single doses of polymer entrapped HBsAg rather than using three doses of alum adsorbed vaccine. References Ahmed, R. and Gray, D., 1996. Immunological memory and protective immunity: Understanding their relation. Science, 272:54-60. 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Table 1 Peak and lowest serum Ig G concentrations before and after boosting (Table Removed) Table 2 Peak and lowest serum IgG (Table Removed) Table 3 SERUM ANTI-HBsAg NTIBODY TITRES IN RATS IMMUNIZED WITH FII(1:1O) PARTICLES IN ALUM AND ALUM ABSORBED HBsAg (AUSAB KIT) (Table Removed) Doses given at 0, 1 and 6 months WE CLAIM: 1. A process for the preparation of a vaccine for the development of immunological memory from a single dose which comprises entrapping in any known manner, vaccine antigens in a polymer matrix, and optionally adding thereto, a conventional adjuvant characterizedby directly entrapping the vaccine antigen in that said polymer matrix comprises biodegradable polymer matrix having average size less than 5µm 2. A process as claimed in claim 1 wherein the vaccine which are entrapped inside the polymer matrix used for immunization are tetanus, hepatitis B surface antigen and other infectious diseases. 3. A process as claimed in claim 1 wherein said polymer comprises biodegradable and hydrophobic polymers. 4. A process for the preparation of a vaccine for the development of immunological memory from a single dose substantially as described herein.

Full Text

Field of the invention
The present invention relates to the development of immunological memory following single point immunization with polymer-entrapped antigens. High secondary antibody response was observed upon re-exposure to the same antigen in soluble form after 6-10 months of primary immunization with polymer-entrapped antigen. Such immunization protocol can be used to develop immunological memory using single dose of polymer entrapped antigens.
Background
For many vaccinations, multiple immunization results in development of desired immune response. Successful vaccination for tetanus, diphtheria, hepatitis and many other infectious diseases need more than two injections to achieve protective immunity., Many times boosters are required at different time of human life cycle to develop bio-neutralizing antibody titers for protective immunity. Particularly in developing countries, logistic problems associated with mass immunization program results in low vaccination coverage. It will be ideal if the vaccines for many infectious diseases can elicit long term immunological memory from single dose administrations. Immunological memory is characterized by a rapid and robust immune response after re-exposure of the original immunizing antigen and is an ideal aim of many immunization protocols used for infectious diseases. Many newly developed or under developed recombinant vaccine candidates are poorly immunogenic thus need repeated vaccine administration achieve desired immunity. If immunological memory can be achieved using single point immunization, it will be of great help toward improving the vaccine efficacy particularly for infectious diseases. Thus there is a need to develop vaccination protocol which can give rise to effective, desired immune response from a single point immunization. Biodegradable polymer particles have been under extensive evaluation in current times to develop single dose vaccine, however none of them have tried to explore the utility of these polymer entrapped

antigen for the generation of immunological memory using single point immunization. Tetanus toxoid was entrapped in Polylactide polymer particles entrapping immunoreactive tetanus toxoid of different sizes were used for single dose immunization. Single dose of admixture of polymer entrapped antigen and alum upon immunization elicited anti-TT antibody titers comparable to that achieved by two doses of alum adsorbed tetanus toxoid. Single doses of alum adsorbed tetanus toxoid elicited weak primary as well as secondary immune response . Where as animals immunized in single dose with polymer entrapped antigen alone or with an admixture of particles and alum elicited very high secondary response upon re-exposure of the antigen after 6-9 months of primary immunization. Use of polymer particle based immunization thus apart form providing immunity from single dose immunization helped in the development of immunological memory. Development of immunological memory will be required for many infectious diseases where multiple immunizations do not result in development of protective immunity for longer duration of time.
Prior Art Relating to the Invention
Use of poly lactide-co-glycolide (PLGA) and polylactide (PLA) polymers for entrapping vaccine/antigens provide a viable alternative to the multidose vaccine injection schedule for immunization (O.Hagan et al. 1998; Cleland 1999; Johansen et al. 2000). These polymer particles provide adjuvant activity to the antigen and thereby improve the immune response (Hanes et al.1997; Lofthouse 2002). Development of single dose vaccine using biodegradable polymer entrapping tetanus toxoid, diphtheria toxoid, Hepatitis B surface antigen and many other potential vaccine antigens have been studied extensively (Hanes et al. 1977 ; Cleland 1999). In order to develop single dose vaccine, it is not only imperative to release immunoreactive antigen from polymer particles but it is also essential to make then more immunogenic either by delivering to macrophages or immunizing along with adjuvant for better presentation to antigen presenting cells (O'Hagan et al. 2001). This is most important as polymer particles release soluble antigen and it is widely documented that

soluble antigens are weakly immunogenic. It is essential that the particles should entrap and release immunoreactive antigens (Zhu et al. 2000 ; wreet et al. 2000, Raghuvanshi et al. 2001), release of the 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 ( One of the most important developments in vaccine administrations will be development of immunological memory from a single point immunization. Immunological memory is defined as the immediate immune response from the system upon re-exposure to the same antigen (Ahmed and Gray, 1996, Sprent, 1997). It will be very useful if long lasting immunological memory can be achieved from minimum number of injections. Most of the time, repeated immunization at particular interval is needed to activate the immune system and to develop immunological memory. Molecular mechanism which leads to development of immunological memory is still an unsolved problem in immunology (Ahmed and Gray, 1996 ; Gray, 2002; Zinkernagel 2002). From the available published information on memory responses from different vaccines, different mechanism have been proposed by different groups actively working to

understand the molecular mechanism of immunological memory. There are reports that continuous presence of antigen is an essential requirement for the development of immunological memory (Gray 2002; Ochsenbein et al. 2000). Presence of long lived plasma cell have also been attributed to the development of B cell memory (Slifka et al. 1998, Manz and Radbruch 2002). Some researcher claims memory is due to polyclonal activation of memory B cells (Bernasconi et al. 2002). Persistance of memory B cells in the absence of immunizing antigen have also been suggested (Maruyama et al. 2000). Whatever may be the real reason for the development of immunological memory, it will be worthwhile to demonstrate the development of memory response from single point immunization. It was expected that polymer particulate based immunization may end up in the development of tolerance rather than memory, as it exposes the immune system with low doses of antigen continuously. When memory response was tested by administering low dose of original soluble antigen to the animals after a long period of time (half of life span of experimental animals) of primary immunization, the secondary response was very high. The antibodies generated after secondary exposure were not only high but also have high toxin neutralizing capacity capacity, which is an essential indicator of protective immunity. This indicated the suitability of polymer entrapped system for the development of memory response from a single dose immunization. High secondary response from polymer particles immunized animals have been reported for tetanus toxoid, while boosting the immunized animal with antigens along with adjuvants (Esparaza and Kissel 1992; Gupta et al. 1997). The real test of immunological memory is the rapid and robust immune response on re-exposure to the original antigen in soluble form, which has not been reported to date using polymer entrapped antigens. We while working extensively on polymer entrapped antigens, observed high memory response in experimental animals with different antigens. It was also observed that, particle made of a particular size are able to develop memory response more effectively than other size particles. Such memory response from single dose immunization with polymer entrapped antigen was observed for TT, HBsAg, DT, and ß hCG. It was

observed that single dose of above mentioned antigens entrapped in polymer particles could give rise to very high secondary antibody response upon re-exposure to low amount of soluble antigens. The secondary response was sustained for a longer period in comparison to that observed for alum adsorbed vaccine immunization. It was also observed that memory response generated from polymer entrapped vaccine immunization is better than that observed while immunizing with multi dose alum adsorbed vaccines. Immunization with single dose of polymer entrapped TT, upon boosting with very low amount of soluble TT, elicited very high antibody titers in Wistar rats. The secondary antibody titers were higher than the primary peak anti-TT antibody titers. The affinities of the antibodies generated during secondary response were very high and the types were mostly IgG indicating the presence of memory cells upon immunization with polymer entrapped antigen. More importantly, re-exposure with soluble antigen resulted antibody response which was higher and more sustained in comparison to that elicited with alum adsorbed vaccination. Development of such memory response was demonstrated for many potential vaccine candidates.
Objects of invention
The main object of invention is to provide a single point immunization process which results in long term immunological memory. Vaccine antigens were entrapped in polylactide (PLA) polymer in the form of particles having different sizes. However immunization with particles less than 5 urn entrapping vaccine antigen resulted in high memory response. Experimental animals were immunized with polymer entrapped antigen and the development of immunological memory was tested by boosting the animals with very low dose of soluble antigen after 6-9 months of primary immunization. It was observed that, animal immunized with polymer entrapped antigens elicited very high secondary antibody response in comparison to that achieved with alum adsorbed immunization. Improved secondary response on immunization with polymer entrapped antigens was observed for many vaccines currently used in humans

and other candidate vaccines also. Notable among them are tetanus toxoid, diphtheria toxoid and hepatitis B surface antigen. Single dose immunization of these above antigens in polymer entrapped form elicited high secondary response upon boosting with very low amount of soluble form of the respective antigen. These results indicated that long term immunity can be achieved for many infectious diseases using single dose administration of polymer entrapped antigens.
Accordingly, the inventors have developed a process for improving immunological memory with single dose immunization.
Another object of invention is the generation of high secondary immune response with the use of very little soluble antigen using polymer particle based immunization.
One object of the invention is the generation of high bioneutralizing secondary antibodies titers using polymer particles entrapped antigens for immunization.
Another object of invention provides a process for generation of immunological memory using single point immunization for infectious diseases.
Yet, another object of invention is to formulate an immunization protocol using the polymer entrapped antigens for obtaining long lasting sustained primary as well as secondary antibody responses in experimental animals with single point immunization.
Another object of invention is development of single point immunization protocol which generates higher secondary response in comparison to immunization with alum adsorbed vaccine.

Another object of invention is the development of single point immunization protocol which elicit high and sustained secondary immune response upon boosting the animals with soluble form of antigen.
Description of the invention:
The inventors have developed a single step immunization process for improving immunological memory using biodegradable polymer particles. Polymer particles entrapping antigens such as tetanus toxoid (TT) and Hepatitis B surface antigen (HBsAg) of required size were prepared using polylactide polymer. Immunizing experimental animals with particles in combination with alum generated enhanced and sustained immune responses. After 6-10 months of primary immunization wistar rats were injected with low amount of soluble TT or HBsAg to check the secondary immune response. Rats immunized with polymer particle based vaccines elicited very high immune response in comparison to rats immunized with conventional alum adsorbed vaccine. Rats boosted with as low as 0.5 Lf of soluble TT after 6-10 months of primary immunization resulted in very high anti TT antibody titters in comparison to alum adsorbed immunized groups. The secondary anti TT antibody response generated by polymer particles based immunization was higher than that achieved during the primary immunization. Similar high secondary response was observed upon immunization with polymer particles entrapping antigen alone. The antibody generated during secondary response from particle based TT immunization has high toxin neutralizing capacity. Secondary antibody response was found to be dependant on dose of antigens given during primary immunization. Higher doses of polymer entrapped antigen during primary immunization resulted in very high secondary antibody response. High secondary response was observed only when the polymer particles entrapping antigens have size less than 5 urn. Larger sized particles upon immunization elicited low secondary response upon re-exposure to soluble antigen. This indicated polymer particle based immunization helps in generation of immunological memory. Similar high secondary antibody response from polymer entrapped HBsAg immunization was also observed. Antibody generated

during secondary response from single dose immunization with polymer entrapped HBsAg have comparable bioneutralizing capacity to that observed from animals immunized with three doses of conventional alum adsorbed vaccine. Secondary immune response generated from single point immunization with polymer entrapped TT was always higher than that observed from immunization with alum adsorbed antigen. It is hypothesized that use of polymer entrapped antigen in particles of appropriate size, continuous release of immunoreactive antigen from the particles and elicitation of high early immune response from particles based immunization acts cooperatively to develop immunological memory upon single point immunization. Generation of very high antibody titers of high affinity upon boosting the immunized animals with soluble antigen testifies the establishment of memory cells capable of producing high antibody response upon single point immunization. Such immunization protocols can be applied to generate long lasting immunological memory where repeated immunization is essential for developing immunity. Such polymer based single point immunization can be applied to most of the infectious diseases such as Hepatitis B, Rabies, Anthrax, Diphtheria, Cholera, Tetanus, foot and mouth disease (FMD) in animals, Measles and for many other recombinant antigen vaccine candidates which elicit poor primary and secondary response. The invention covers patent application 852/Del/2000 and patent application 794/Del/2001 which is here by incorporated by reference in its entirety.
In an embodiment the invention describes the process of immunization which results in development of immunological memory from single point immunization.
In an embodiment, the vaccine tried here are tetanus toxoid, Hepatitis B surface antigen, vaccines for infectious disease and many recombinant vaccine antigens.
In an embodiment the immunization involves the entrapment of the said vaccine antigen in biodegradable polymer particles.

In an embodiment the process of immunization involves polymer entrapped particles of size less than 5 µrn with or without admixture of alum.
In an embodiment, the process describes the immunization with polymer entrapped antigens where the polymers are biodegradable and hydrophobia
In other embodiments, the process describes the immunization with polymer entrapped antigens as describes in patent application 852/Del/2002 and 749/Del/2001.
In another embodiment the process describes the immunization process where boosting with very low amount of soluble antigen after 6-10 months of primary immunization result in very high secondary response.
In an embodiment the process describes the immunization process where the affinities of antibodies generated during secondary response are very high.
In an embodiment, the process of single point immunization results in high secondary response in comparison to the alum adsorbed vaccination.
In an embodiment, the process describes the generation of high secondary antibody response whose affinities are comparable with that observed with multiple immunization with alum adsorbed vaccine.
In another embodiment the process describes the generation of bioneutralizing memory antibody titers from a single dose of polymer entrapped antigen
In another embodiment the process describes the generation of long term immunological memory from single point immunization of polymer entrapped antigens.

In another embodiments these antigen could be any vaccine candidates for human and animal use where repeated immunization is required to achieved long term immunity.
In another embodiment, the process describes the generation of both long lasting primary and secondary antibody response while using combination of permissible adjuvant such as biodegradable polymer and alum.
In particular the applicant has described a single dose immunization procedure using polymer entrapped antigen for improving immunological memory. Vaccine antigens entrapped in biodegradable polymer particles were used for immunization. The secondary immune response generated form such immunization upon reexposure to the soluble antigen was very high in comparison that observed with alum adsorbed vaccination. High secondary immune response could be generated from single point immunization with polymer entrapped antigen. Single point immunization with polymer particle entrapped tetanus toxoid, diphtheria toxoid, hepatitis B surface antigen when boosted with corresponding soluble antigen elicited very high secondary antibody response. These results are of indication that, the described immunization protocol can be effectively used for generation of long term memory for many vaccine candidates which need repeated immunization to achieve desired immunity.
The invention is described in detail with the aid of following examples and the accompanying drawings and tables. Various modifications that may be apparent to one in the art are intended to falls within the scope of the invention.
Description of the accompanying drawing
Figure 1. Comparison of serum anti TT antibody concentrations in male wistar rats immunized with single dose of polylactide microparticles ( 4µm average size ,-■-) and nanoparticles entrapping TT ( 0.75µm average size -●-) (15 Lf /rat in each group) and conventional two doses of alum adsorbed TT (-▲-) (5 Lf /rat /dose). 4-6 weeks old outbred wistar male rats were immunized with microencapsulated and conventional vaccines.
Figure 2. Anti TT antibody concentrations in male wistar rats immunized with single dose of polylactide microparticles (-▲-), nanoparticles (-●-) entrapping TT (15 Lf /rat in each group), their combination (-T-) given with alum and conventional two doses of alum adsorbed TT (-■-) (5 Lf /rat /dose in interval of four weeks). Wistar male rats were immunized intramuscularly with microencapsulated and conventional vaccines. Each group was consisting of 6 rats.
Figure 3. Primary and secondary anti TT antibody response in rats immunized with single dose of TT entrapping microparticles (-▲-), combination of microparticles and nanoparticles (-▼-) (15 Lf/rat in each group) and conventional two doses of alum adsorbed TT (-■-) (5 Lf/rat/dose). After 6 months of primary immunization animals were given 5 Lf TT.
Figure 4. Primary and secondary anti TT antibody response elicited by polylactide microparticles entrapping TT of different sizes. Out bred wistar female rats were immunized with particles of sizes 140 µm (-■-), 41µm (-●-), 25µm (-▲-), 4µm(-▼-) and 0. 75µm (-♦-). 8 months after primary immunization animals were re-exposed to 0.5 Lf soluble TT to check memory response.
Figure 5. Secondary antibody TT antibody response in female wistar rats immunized different doses of alum adsorbed TT and polylactide microparticle (average size 4µm ) entrapping TT in presence of alum. Two groups of 6 animals were immunized with single dose of 1.0 (-●-) and 0.1 Lf (-▲-) alum adsorbed TT and one group was immunized with two doses of 5 Lf alum adsorbed TT given at 0 and 1 month (-■-). Other groups of rats were immunized with single dose of 15Lf (-T-), 10 Lf (-+-), 5 Lf (-+-), 1 Lf (-X-), 0.5 Lf (-*-) and 0.1 Lf (--) microencapsulated TT along with alum as adjuvant. One group was immunized with 10 Lf soluble TT (-I-). All the groups were given 0.5 Lf soluble TT after 9 months of primary immunization to check memory response.
Figure 6.Affinity of anti TT IgG in rats immunized with single dose of 10 Lf microencapsulated TT with alum before checking memory response (-●-) and during secondary response (-▲-). Equal amounts of antibody from different groups were incubated with different concentrations of antigens for two hours at 37 °C and unbound antibody was assayed using ELISA. Affinity measurements were from groups of rats used for dose response studies. Lower absorbance at different antigen concentration indicates the presence of low unbound antibody which reflects the high affinity of antibodies. Affinities of the secondary antibodies (290 days data) are higher than the antibodies present before soluble exposure ( 275 days).I
Table 1. Peak and lowest serum anti TT IgG concentrations before testing memory and peak serum anti TT IgG concentrations during secondary immune responses following administration of 5 Lf soluble TT in different groups of rats.
Table 2. Peak and lowest serum anti TT IgG concentrations before testing memory and peak serum anti TT IgG concentrations during secondary immune responses following administration of 0.5 Lf soluble TT in different groups of rats.
Table 3. Anti-HBsAg antibody titres (AUSAB method) generated upon immunization of HBsAg vaccine. Three doses of alum adsorbed vaccine was compared with one dose of polymer entrapped vaccine. After 9 months of primary immunization, animals were immunized with 5 µg of soluble antigen to check the memory antibody response.
Example 1: Immunological memory from single point
immunization of polymer entrapped TT
Extensive studies with the use of PLGA/PLA particles have been carried out to explore the possibilities of developing a single shot vaccine for tetanus toxoid (TT) (Kersten et al. 1996 ; Audran et al. 1998 ; Tobio et al. 2000 ; Diwan et al. 2001). Results of antibody titres obtained from groups immunized with two injections of 5 Lf TT each adsorbed on alum and equivalent doses of microencapsulated TT showed that the conventional immunization schedule gives significantly higher antibody titre than immunization with microencapsulated TT (Kersten et al. 1996 ; Johansen et al. 2001). There are reports that smaller
size and hydrophobia polymeric particles improve the immunogenicity of the entrapped antigen ((Eldridge et al. 1991; Conway et al. 1997). With proper analysis of these above factor related to the development of single dose vaccine using biodegradable polymer, it is possible to generate long lasting immune response from a single point immunization. However till now no one has evaluated the potential of polymer entrapped vaccination for development of immunological memory. There are reports where people have demonstrated the amnestic response while boosting with alum adsorbed antigen. However the real test of immunological memory is the rapid and robust immune response on re-exposure to the original antigen in soluble form, which mimics the natural infections. We have demonstrated here that, immunization with polymer particles entrapping immunoreactive TT leads to development of better immunological memory. Wistar rats were immunizes with single dose of PLA entrapped TT and the memory response was evaluated after 6-9 months time while boosting with soluble TT. It was observed that polymer particles based immunization leads to very high secondary antibody response in comparison to that observed with alum adsorbed vaccine immunization. The affinity of the antibodies generated during secondary immunization was very high thus indicating the development of immunological memory from single point immunization using polymer entrapped
Materials and Methods Materials
Poly D,L-lactigle (PLA, 45 KD) were purchased from Birmingham Polymer Inc. USA. TT (3000 Lf/ml, protein content 8 mg/ml) was purchased from Serum Institute of India, Pune. Rat serum albumin (RSA) [A-6272] and polyvinyl alcohol, MW 30,000 (PVA) were from Sigma Chemicals USA. Micro BCA protein assay kit was from Pierce, USA. Polyclonal antibodies to TT and goat anti-TT HRPO conjugate were from Reagent Bank of National Institute of Immunology, New Delhi, India.
Preparation of polymer particles
Polylactide polymers particles were prepared using solvent evaporation method (Raghuvanshi et al. 2001). Primary emulsion between internal aqueous phase (TT in PBS, pH 7.4) and organic phase (polymer solution in DCM) was prepared by sonication (20 watts, 80% duty cycle, 20 cycles) (Brason, Sonifier 450, USA). Secondary emulsification was carried out either by sonication to obtain nanoparticles or by homogenization (10,000 rpm for 10 minutes) (Virtis, Cyclone I.Q., USA) for microparticles. The resulting polymeric particles were washed twice with cold PBS and lyophilized to get free flowing powder. The size of the particles was measured using GALAI-CIS-1 particle size analyzer. All particles were prepared using RSA as a stabilizer in the internal aqueous phase during particle formulation.
Estimation of protein content of polymer particles
To measure the protein content of particles, accurately weighed particles were dissolved in acetonitrile to solubilize the polymer while precipitating the encapsulated protein. The precipitated protein was pelleted by spinning at 5,000 rpm for ten minutes and was dissolved in 1% SDS solution. The protein content of the solution was determined by micro BCA assay. The protein loading was calculated as the per cent weight of protein per unit weight of polymer. In vivo animal studies
Immunogenicity of the encapsulated TT in particles was checked in animal model using Wistar rats. Rats were injected intramuscularly with 30 µg (15 Lf) of either free, alum adsorbed or microparticle encapsulated TT. PLA polymer particles (both nanoparticles and microparticles) entrapping TT were used for animal experiments (six animal in each group). Physical mixture of polymer particle of different size were also used for immunization, the total dose of TT was 15Lf Similar groups of animals were immunized with particles along with alum. For immunization with admixture of alum, 50 µl of 2 % alum (Superfos Biosector a/s, Denmark) was mixed with PLA particles just before immunization and used. Two injections of TT (5 Lf each,) adsorbed on alum was given at four weeks (0 and 28 days) interval. Higher dose of TT in particles were taken for comparison to take
care of the loss and degradation form particles. For the dose response experiments admixture of microparticles and alum was used. The total dose of TT was 15, 10, 5, 2, 1 and 0.5 Lf. Similarly for experiments related to size, particles of the following sizes were prepared and used for immunization. The dose of TT was 5 Lf and sizes are 140µm, 49 µm, 25 µm, 10 µm, 5 µm and 0.75 µm. For checking the memory response animals were immunized with single dose of polymer entrapped antigen and after 8 months of primary immunization boosted with 1 Lf of soluble TT. The antibody titers after soluble boosting were determined By ELISA and the affinities were also determined using standard protocol (Friguet et al. 1997). Animals were maintained according to the guidelines established by the Institute Animal Ethics Committee of the National Institute of Immunology, New Delhi and serum anti -TT antibody titers were determined by ELISA. Anti -TT antibody titers were defined as µg of antibody/ml of the sera employing affinity purified antibody as references in ELISA (Raghuvanshi et al. 1998). Antibody titers of individual animal (n = 6) were estimated in duplicates and their concentrations (µg/m) were determined as geometric mean. To analyze the statistical significance of the antibody titers students't-test at 95% confidence level was carried out.
RESULTS
Immune response from PLA particles encapsulating TT
Micro-particles and nano- particles entrapping immunoreactive TT were made from PLA polymer (Raghuvanshi et al. 2001). The average size of nano particles were around 750 nm where as that of micro particles was 4 um. rats were immunized with single dose of polymer entrapped alum (15 Lf) and was compared with that of two doses of alum adsorbed TT. It was found even though polymer entrapped TT elicited immune response , the antibody titers was lower than that observed from two doses of alum adsorbed TT ( Figure 1). To improve the immunogenicity of the encapsulated TT particles, alum as an adjuvant was added along with the particles during single dose immunization. It was found that incorporation of alum in the particle suspension improved the antibody titers
significantly (Figure 2). In nanoparticles the antibody response peaked early where as maicroparticles along with alum gave titers little later. Immunization with admixture of alum and particles improved the immune response significantly (Figure 2). High and early immune response from admixture of nanoparticles and alum was achieved. Antibody titers around 80µg/ml could be detected around 15 day post immunization. However the antibody titers decreased considerably after 75 days of post immunization. Early higher antibody titers were also found in group receiving admixture of nanoparticles, micrparticles and alum. Immunization with physical mixture of particles resulted in sustained antibody response for longer period of time. In groups receiving microparticles along with alum the peak response was observed at 80 days which was very high and comparable to that of TT/alum. Anti-TT antibody titers around 350 ng/ml could be detected around 80 days after immunization. More importantly high anti-TT antibody titers persisted for a longer period of time particularly in the later part of of the post immunization period. Two doses of alum adsorbed TT gave anti-TT antibody titers around 400µg/ml on day 40-45 where as admixture of microparticles and alum elicited around 350 µg/ml of anti-TT antibody titers, which peaked around day 80 (Figure 2). These results suggest that, with proper combination of polymer entrapped TT and alum; it is possible to generate antibody response using single point immunization that is comparable to that of two doses of alum adsorbed TT injection.
Memory response from polymer entrapped TT immunization
To check the memory response generated by particles based immunization, animals were immunized with polymer entrapped TT along with alum. Anti TT antibody titers were estimated every month and after 6-9 months of primary immunization animals were boosted with 5 Lf of soluble antigen. It was observed that groups immunized with microparticle entrapped TT gave rise to very high secondary antibody response on being re-exposed to soluble antigen (Figure 3). Memory response generated from all groups of immunization involving microparticles, nanoparticles and physical mixture of particles both given with or
without alum are presented in table 1. It was observed that single dose immunization with polymer entrapped TT generated considerable memory response upon re-exposure to the same antigen. Even with re exposure of animals immunized with particulate TT with low level of soluble TT (0.5 Lf), very high secondary response was observed (Table 2). It was observed that polymer entrapped TT immunization resulted in very high secondary immune response indicating the development of immunological memory in the animals. It was also observed that polymer particles based immunization without alum even though elicited very low primary immune response, the corresponding secondary response were higher than that observed for two dose of alum adsorbed TT immunization (Table 1 and 2).
Memory responses generated from particles based immunization having different doses and sizes are presented in figure 4 and figure 5. Polymer particles having size less than 5 urn was found to elicit better secondary response than larger size particles (Figure 5). It was observed that maximum memory response was observed for particles size were less than 5 urn. Nanoparticles gave substantial high secondary response upon boosting, but lower than that observed for 1-5 urn particles.
It was observed that secondary response generated upon re-exposure to the same amount of soluble antigens was strictly dependent upon the doses used during primary immunization. Secondary response generated from animals immunized with microencapsulated TT was higher than that observed from animals immunized with conventional alum adsorbed TT (Figure 5). Even with single doses of microencapsulated TT lower than the combined two doses of conventional alum adsorbed vaccine gave much better secondary response upon re-exposure to the soluble antigen. The affinity of antibodies generated during memory response to the immunized antigen was high, indicating the generation of high affinity antibodies upon polymer particle based immunization (Figure 6). In all the cases, single point immunization of polymer entrapped TT thus helped
in development of immunological memory. The antibody concentrations in serum during memory response were longer lasting in animals immunized with microencapsulated antigens than those generated from animals immunized with alum adsorbed TT, which declined rapidly. Antibodies generated during memory response have high toxin neutralizing capacity in comparison to that observed for alum adsorbed immunization (data not shown). Rapid response to the intra muscular injections of low doses of soluble antigen high antibody titer, high affinities of the secondary antibodies and maintenance of high antibody titers for longer period of time suggested the development of immunological memory from single point polymer particles based immunization.
Example 2
Memory response from polymer entrapped HBsAg Immunization
Immunogenicity of the microencapsulated HBsAg in particles was checked in animal model using Wistar rats (six animals in each group). Rats were injected intramuscularly with 10 µg of either free, alum adsorbed or particle encapsulated HBsAg. Three injections of HBsAg (3.3 µg each total of 10 µg) adsorbed on alum was given at day 0, 30, 180 days as control. Animals were bled at different time interval through retro-orbital plexus and the serum was analyzed for anti-HBs Ag antibody titers by AUSAB assay using ABBOT Lab ELISA Kit. Antibody titers were estimated in duplicates and their concentration was expressed as mlU/ml. It was observed that single immunization of polymer entrapped HBsAg elicited higher bioentralizing titers ( > 60 000 mlU/ml) than the required value of 10 mlU/ml for protection ( Table 3). After 287 days ( > 9 months) of primary immunization, the animals were boosted with soluble HBsAg (5 ug) to check the memory response. High memory response was observed from animal immunized with particle entrapped HBsAg upon re-exposure to soluble antigen (Table 3). In polymer particles based immunized animals, the bioneutralizing HBsAg antibody
titers were improved from 60 000 to 403 000 mlU/ml upon re-exposure to the soluble antigen. Similar memory repose was observed from animal immunized with three doses of alum adsorbed HBsAg where the antibody titers increased from 170, 000 to 426 000 mlU/ML. The extent of secondary response in three doses of alum adsorbed vaccination increased 2.5 times the existing titer upon re-exposure to soluble HBsAg. Where as in the case of single dose polymer particles based immunization, the antibody titers increased more than 6.5 times upon re-exposure to the soluble antigen. This indicated that single dose immunization with polymer entrapped antigen gives better memory response than observed with three doses of conventional vaccine immunization. Such immunization can be applied to humans to elicit sero-protection titers from single doses of polymer entrapped HBsAg rather than using three doses of alum adsorbed vaccine.
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Table 1 Peak and lowest serum Ig G concentrations before
and after boosting

(Table Removed)
Table 2 Peak and lowest serum IgG
(Table Removed)
Table 3 SERUM ANTI-HBsAg NTIBODY TITRES IN RATS IMMUNIZED WITH FII(1:1O)
PARTICLES IN ALUM AND ALUM ABSORBED HBsAg (AUSAB KIT)

(Table Removed)
* Doses given at 0, 1 and 6 months

WE CLAIM:
1. A process for the preparation of a vaccine for the development of immunological memory from a single dose which comprises entrapping in any known manner,
vaccine antigens in a polymer matrix, and optionally adding thereto, a conventional

adjuvant characterizedby directly entrapping the vaccine antigen in that said polymer
matrix comprises biodegradable
polymer matrix having average size less than 5µm
2. A process as claimed in claim 1 wherein the vaccine which are entrapped inside the
polymer matrix used for immunization are tetanus, hepatitis B surface antigen and
other infectious diseases.
3. A process as claimed in claim 1 wherein said polymer comprises biodegradable and
hydrophobic polymers.
4. A process for the preparation of a vaccine for the development of immunological
memory from a single dose substantially as described herein.

Documents:

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754-del-2002-correspondence-others.pdf

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754-del-2002-form-19.pdf

754-del-2002-form-2.pdf

754-del-2002-form-26.pdf

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Patent Number

217115

Indian Patent Application Number

754/DEL/2002

PG Journal Number

13/2008

Publication Date

31-Mar-2008

Grant Date

25-Mar-2008

Date of Filing

17-Jul-2002

Name of Patentee

NATIONAL INSTITUTE OF IMMUNOLOGY

Applicant Address

1860, OR ARUNA ASAF ALI MARG, NEW DELHI-110067, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	AMULYA K. PANDA	NATIONAL INSTITUTE OF IMMUNOLOGY, ARUNA ASAF ALI MARG, NEW DELHI-110067, INDIA.
2	YOGESH K KATARE	NATIONAL INSTITUTE OF IMMUNOLOGY, ARUNA ASAF ALI MARG, NEW DELHI-110067, INDIA.

PCT International Classification Number

A61K 9/16

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA