Om Singh

Improved immune response from biodegradable polymer particles entrapping tetanus toxoid by use of different immunization protocol and adjuvants

Poly lactide-co-glycolide (PLGA) and polylactide (PLA) particles entrapping immunoreactive tetanus toxoid (TT) were prepared using the solvent evaporation method. The effect of different formulation parameters such as polymer hydrophobicity, particle size and use of additional adjuvants on the generation of immune responses in experimental animals was evaluated. Immune responses from hydrophobic polymer particles were better than those from hydrophilic polymer. Immunization with physical mixtures of different size particles resulted in further improvement in anti-TT antibody titers in Wistar rats. Physical mixture of nano and microparticles resulted in early as well as high antibody titers in experimental animals. Immunization with polymer particles encapsulating stabilized TT elicited anti-TT antibody titers, which persisted for more than 5 months and were higher than those obtained with saline TT. However, antibody responses generated by single point immunization of either particles or physical mixture of particles were lower than the conventional two doses of alum-adsorbed TT. Immunization with nanoparticles along with alum resulted in very high and early immune response: high anti-TT antibody titers were detected as early as 15 days post-immunization. Use of a squalene emulsion along with the particles during immunization enhanced the level of anti-TT antibody titers considerably. Single point immunization with admixtures of PLA microparticles and alum resulted in antibody response very close to that achieved by two injections of alum-adsorbed TT; the antibody titers were more than 50 microg/ml over a period of 6 months. These results indicated that the judicious choice of polymer and particles size, protecting the immunoreactivity of the entrapped antigen and the appropriate design of immunization protocol along with suitable adjuvant can lead to the generation of long lasting immune response from single dose vaccine formulation using polymer particles.

Phase I clinical trials with three formulations of anti-human chorionic gonadotropin vaccine

Comparative phase I clinical trials were carried out in 5 centres with three formulations of beta-hCG-based vaccines inducing antibodies against human chorionic gonadotropin. The objectives of these trials were to determine their relative immunogenicity, duration, reversibility and safety. A total of 116 tubal ligated women volunteers were enrolled in the study and 101 subjects were followed-up for one year or more until the antibody titres declined to near zero levels. Every woman receiving the vaccine produced anti-hCG and anti-tetanus antibodies. Clinical examination carried out at intervals of 4-6 weeks revealed no abnormality. No serious side effects or adverse reactions were reported with any of the formulations during primary immunization with three monthly injections of the vaccine. Eleven women, however, demonstrated hypersensitivity to test dose at the time of the booster injection. The reaction was to tetanus toxoid; gonadotropin subunits conjugated to another carrier did not evoke any such reaction. Progesterone in bleeds taken at midluteal phase, as well as complete progesterone and estradiol done in two immunized women, indicated normal ovulatory cycles. Immunization with these formulations had no significant effect on haematological, clinical chemistry and other metabolic parameters. In summary, the results indicate that none of the three beta-hCG-based contraceptive vaccines had any adverse effects clinically, on endocrine status and metabolic parameters. Formulations A and B induced comparatively higher anti-hCG titres than M. Thus, further work can be undertaken to study the efficacy of these vaccines in humans for preventing pregnancy.

Antibody response and characteristics of antibodies in women immunized with three contraceptive vaccines inducing antibodies against human chorionic gonadotropin

Data are presented on antibody titers generated in 88 women immunized with three formulations of antihuman chorionic gonadotropin (hCG) vaccine, namely, beta-hCG (formulation B); beta-hCG associated with alpha-subunit of ovine luteinizing hormone (LH) (formulation A) and beta-hCG + beta-ovine LH (formulation M), each linked to tetanus toxoid and cholera toxin chain B as carriers. Each formulation was tested at two dose levels (100 and 500 micrograms). All women without exception developed anti-hCG antibodies having hCG-binding capacity above 20 ng mL-1 (0.5 nM), a level considered to be the threshold for prevention of pregnancy. Formulations A and B gave relatively better immunogenic response in human subjects than M. In each case, the antibody response was reversible. The mean duration of response above 20 ng was 35 to 37 weeks for formulation A, 34 weeks for B, and 17 to 20 weeks for M. Antibodies induced by three formulations of the vaccine had high-affinity (Ka 10(9)-10(10)M-1) for binding with hCG. They were devoid of cross-reaction with human follicle-stimulating hormone and thyroid-stimulating hormone but, as expected, cross-reacted with human LH. Antibodies were competent to block the hCG induced ovarian hyperemia.

Stabilization of Dichloromethane-Induced Protein Denaturation During Microencapsulation

This paper describes the denaturation of protein drugs by dichloromethane (DCM) during the primary emulsification step of the microencapsulation process using biodegradable polymer matrix for controlled-release application. It was found that interaction of proteins such as tetanus toxoid (TT), diphtheria toxoid (DT), ovine growth hormone (oGH), and human chorionic gonadotropin-based antifertility vaccine (beta-hCG-TT) with DCM during primary emulsification stages of particle formulation led to the precipitation of the proteins at the aqueous organic interface with concomitant reduction in their immunoreactivity. On the other hand, the B subunit of E. coli enterotoxin (LTB) was found to be comparatively stable toward the denaturing action of DCM. Attempts were made to overcome the DCM-induced denaturation by incorporation of stabilizers during the primary emulsification step of the particle formulation. Of the many additives tested to overcome the DCM-induced denaturation of proteins, serum albumins and polyvinyl alcohol (PVA) showed promising results in terms of retention of the immunoreactivity of the protein. TT stabilized by the incorporation of serum albumin during the primary emulsification step not only showed immunoreactivity in vitro, but also invoked antibody titers in rats comparable to those obtained for the native protein molecules. Incorporation of 2.5% of serum albumins in the internal aqueous phase not only protected the protein from the degradative action of DCM but also led to stabilized primary emulsion, which is necessary for uniform entrapment of protein drugs in the polymer matrix.

Biodegradable delivery system for a birth control vaccine : immunogenicity studies in rats and monkeys

AbstractPurpose. The purpose of this study was to develop a single administration delivery system for a model birth control vaccine, in order to reduce the need for multiple injections and enhance immunogenicity. Methods. The immunogen-loaded microspheres were prepared by solvent evaporation method and characterized for loading levels, size distribution and in vitro release kinetics. The microspheres were immunized intramuscularly in wistar rats and bonnet monkeys, and the antibody response was compared to that obtained with the same total dose of the immunogen on alum given at a monthly interval. Results. Results indicated that a single injection of the immunogen entrapped in the microspheres generated a response comparable to that obtained by the same immunogen on alum injected at a monthly interval. The antibodies generated by the microspheres in the monkeys also had a good bioneutralization capacity indicating immunogen integrity during the microencapsulation process. Conclusions. Biodegradable microspheres served as an effective delivery system for a model immunogen used in this study to reduce the need for frequent immunizations and enhance immunogenicity.

Regain of Fertility and Normality of Progeny Born During Below Protective Threshold Antibody Titers in Women Immunized With the HSD-hCG Vaccine

PROBLEM: Phase II clinical trials with the heterospecies dimer of βhCG and α‐subunit of ovine luteinizing hormone (HSD)‐human chorionic gonadotropin (hCG) vaccine showed that pregnancy was prevented at and above 50 ng/ml titers, whereas conceptions occurred below 35 ng/ml of hCG bioneutralization capacity. The effect of below‐protective threshold anti‐hCG antibodies on the progression of pregnancy and the normality of progeny was studied.

An improved immunogen for anti-human chorionic gonadotropin vaccine eliciting antibodies reactive with a conformation native to the hormone without cross-reaction with human follicle stimulating hormone and human thyroid stimulating hormone

Beta-subunit of human chorionic gonadotropin (hCG) was associated with alpha-subunit of ovine luteinizing hormone (OLH) to create a heterospecies dimer (HSD) which has a higher steroidogenic potency than the homologous dimer of alpha hCG and beta hCG in the mouse Leydig cell bioassay. The properties and merits of the antibodies induced by this HSD and beta hCG linked to carrier(s) were investigated in rodents and in a subhuman primate species. The antisera had, in both cases, high affinity for binding with hCG (K alpha = 10(9)-10(10) M-1). The mean (+/- S.E.M.) bioneutralization capacity as a percentage of immunoreactivity (determined by radioimmunoassay (RIA)) of the antibodies generated by the HSD-carrier in rats and bonnet monkeys was higher in comparison with those induced by beta hCG linked to carrier (80 +/- 2.3% vs. 63 +/- 1.5% in rats, and 65 +/- 1.9% for HSD vs. 44 +/- 3.7% in monkeys). None of the sera gave any evidence of cross-reactivity with human follicle stimulating hormone (hFSH) and human thyroid stimulating hormone (hTSH).

Analysis of menstrual records of women immunized with anti-hcg vaccines inducing antibodies partially cross-reactive with hLH

Menstrual data of 13 control subjects and 88 subjects immunized with three beta-hCG-based vaccine formulations were analysed. Immunization did not change the menstrual regularity; bleeding days were normal (3-7 days) and 89% of the menstrual cycles were within the normal range of 22-35 days. Irregular (short or long) cycles were observed in both immunized and control groups. These were, however, unrelated to prevailing anti-hCG antibody titres or to cross-reactivity of antibodies with hLH.

Immunogenicity studies on diphtheria toxoid loaded biodegradable microspheres

Diphtheria toxoid loaded polylactide microspheres were prepared using the solvent evaporation method. The vaccine loaded microspheres were subjected to in vitro antigen release studies and in vivo immune response tests. The immunogenicity of the vaccine after microencapsulation was compared to that against diphtheria toxoid given in the conventional three injection schedule on alum as an adjuvant. The antibody response seen in the two cases is compared. The duration of the immune response from the two schedules is exhibited.

Formulation and Characterization of Immunoreactive Tetanus Toxoid Biodegradable Polymer Particles

Poly lactide-co-glycolide and polylactide polymer particles entrapping immunoreactive tetanus toxoid (TT) were prepared with a view to developing a single shot controlled release vaccine formulation. Denaturation of TT by dichloromethane (DCM) during primary emulsification stage of particle formulation was minimized by incorporation of an optimal amount of rat serum albumin (RSA) in the internal aqueous phase. Incorporation of RSA as a stabilizer during the primary emulsification stage of polymer particle formulation protected the immunoreactivity of TT, enhanced its encapsulation efficiency and also led to uniform polymer particle formation. Use of sonication, both during primary and secondary emulsification processes, resulted in formation of nanoparticles whereas microparticles were formed when the secondary emulsion was carried out by homogenization. Immunoreactive TT particles made from different polymers incorporating stabilizers released antigen continuously for more than four months in vitro. Single injection of both type of particles encapsulating stabilized TT elicited anti-TT antibody titers in rats for more than five months, which was higher than that obtained with TT injected in saline. Anti-TT antibody titers in vivo were in accordance with the in vitro release characteristics of immunoreactive TT from the particles. Immune responses with hydrophobic polymer particles were better than those made using hydrophilic polymers. These results indicate the importance of protecting the immunoreactivity of TT during formation of polymer particles for sustained and improved antibody response.Poly lactide-co-glycolide and polylactide polymer particles entrapping immunoreactive tetanus toxoid (TT) were prepared with a view to developing a single shot controlled release vaccine formulation. Denaturation of TT by dichloromethane (DCM) during primary emulsification stage of particle formulation was minimized by incorporation of an optimal amount of rat serum albumin (RSA) in the internal aqueous phase. Incorporation of RSA as a stabilizer during the primary emulsification stage of polymer particle formulation protected the immunoreactivity of TT, enhanced its encapsulation efficiency and also led to uniform polymer particle formation. Use of sonication, both during primary and secondary emulsification processes, resulted in formation of nanoparticles whereas microparticles were formed when the secondary emulsion was carried out by homogenization. Immunoreactive TT particles made from different polymers incorporating stabilizers released antigen continuously for more than four months in vitro. Single injection of both type of particles encapsulating stabilized TT elicited anti-TT antibody titers in rats for more than five months, which was higher than that obtained with TT injected in saline. Anti-TT antibody titers in vivo were in accordance with the in vitro release characteristics of immunoreactive TT from the particles. Immune responses with hydrophobic polymer particles were better than those made using hydrophilic polymers. These results indicate the importance of protecting the immunoreactivity of TT during formation of polymer particles for sustained and improved antibody response.