Alum-adsorbed Tetanus Toxoid Research Articles

A cell-based in vitro assay for testing of immunological integrity of Tetanus toxoid vaccine antigen

Vaccines containing inactivated toxins confer protection by eliciting a neutralizing antibody response against bacterial toxins such as tetanus and diphtheria. At present, release of tetanus toxoid (TT) and diphtheria toxoid (DT)-containing vaccines relies on in vivo experiments showing the protective vaccine response. The aim of this study was to develop a reliable in vitro assay for TT vaccine antigen characterization with the potential of replacing in vivo potency experiments. To this end, we exploited that TT elicits a recall response in vaccinated donors: human peripheral blood mononuclear cells (PBMC) were stimulated with alum-adsorbed TT bulk antigen and low concentrations of TLR9 ligand; induction of TT-specific IgG was quantified via ELISpot after 5 days. Proof-of-concept was obtained using paired samples from donors before and after vaccination; anti-TT IgG was only detected in PBMC collected after booster vaccination; specificity was demonstrated with DT stimulation as control. Notably, when using PBMC from buffy coats, the specific response to TT was reproducible in 30% of cells; responsiveness correlated with higher numbers of switched memory B cells. Consecutive results showed that TT-specific IgG was also detectable when PBMC were stimulated with DTaP final vaccine product. Thus, the assay provides a viable means to test B-cell differentiation and induction of TT-specific IgG secretion using bulk antigen and final vaccine. However, prequalification of PBMC is required for reliable performance. Along with physicochemical and immunochemical methods, the functional assay could represent a complementary tool to replace in vivo potency assays in batch release of TT-containing vaccines.

Chitosan–HPMC-blended microspheres as a vaccine carrier for the delivery of tetanus toxoid

The purpose of this research was to develop a suitable and alternate adjuvant for the tetanus toxoid (TT) vaccine that induces long term immunity after a single-dose immunization. In our study, the preformulation studies were carried out by using different ratios (7/3, 8/2, and 9/1) of chitosan–hydroxypropyl methylcellulose (HPMC)-blended empty microspheres. Moreover, TT was stabilized with heparin (at heparin concentrations of 1%, 2%, 3%, and 4% w/v) and encapsulated in ideal chitosan – HPMC (CHBMS) microspheres, by the water-in-oil-in-water (W/O/W) multiple emulsion method. The vaccine entrapment and the in vitro release efficiency of the CHBMS was evaluated for a period of 90 days. The release of antigens from the microspheres was determined by ELISA. Antigen integrity was investigated by SDS-PAGE. From the optimization studies, it was found that a chitosan/HPMC ratio of 8/2 produced a good yield, with microspheres that were spherical, regular and uniformly-sized. In the CHBMS, a heparin concentration of 3% w/v resulted in well-sustained antigen delivery for a period of 90 days. It was found that the characteristics of initial release could be observed in 2 days, followed by a constant release, and an almost 100% complete release in 90 days. From the in vitro release characteristics, the ideal batch of CHBMS (3% w/v heparin) was evaluated for in vivo studies by the antibody induction method. The antibody levels were measured for different combinations for the period of 9 months, and finally, with a second booster dose after 1 year. In conclusion, it was observed that CHBMS (combination-1) resulted in the antibody level of 4.5 IU/mL of guinea pig serum, and the level was 3.5 IU/mL for the Central Research Institute's alum-adsorbed tetanus toxoid (CRITT) (combination 2), after 1 year, with a second booster dose. This novel approach of using CHBMS may have potential advantages for single-step immunization with vaccines.

Evaluation of lactose stabilized tetanus toxoid encapsulated into alginate, HPMC composite microspheres

The utility of both in vitro and in vivo methods to assess the release pattern of alginate, HPMC (TTAL) composite microspheres containing stabilized tetanus toxoid (TT) vaccine were evaluated. TT was stabilized with different concentrations of stabilizer, lactose (1–5%, w/v) and encapsulated into alginate, HPMC composite microspheres by W/O/W multiple emulsion method. The morphology of the prepared microspheres was smooth and spherical in shape with a diameter of around 10μm. The in vitro release efficiency of microspheres was evaluated for the period of 70 days. In TTAL microspheres, 4% (w/v) lactose gave good sustained antigen delivery for the period of 70 days. Antigen releases from microspheres were determined by ELISA. Based on the results of in vitro release, the ideal batch of TTAL was used to carry out the in vivo studies by antibody induction method using alum adsorbed tetanus toxoid (CRIT) as standard. The antibody level was measured for about of 9 months and finally with one booster dose after 12 months. In this case, TTAL antibody level was rose up to 3IU/ml of guinea pig serum and 2.5IU/ml of guinea pig serum was observed in CRIT after 1 year with second booster dose. This novel approach would be helpful to replace the existing adjuvant alum in future.

Preparation and evaluation of sucrose stabilized tetanus toxoid encapsulated into chitosan microspheres

Immunization is the most cost effective weapon for disease prevention in developing countries, and advanced molecular and genetic technologies are making new types of vaccines feasible. Here, the utility of both in vitro and in vivo methods to assess the release pattern of chitosan microspheres containing tetanus toxoid (TT) vaccine were evaluated. TT was stabilized and encapsulated in chitosan (TTCH) with a water-in-oil-in-water (W/O/W) multiple emulsion method using sucrose as a protein stabilizer. The TTCH prepared was smooth and spherical in shape with a diameter of around 10μm. The in vitro release efficiency of TTCH was evaluated by differing stabilizer (sucrose) concentration (5%, 7%, 10% and 12%w/v) for a period of 70 days. The antigen release rates from the microspheres were determined by enzyme-linked immunosorbent assay. In these TTCH microspheres, a 10%w/v sucrose concentration gave good sustained antigen delivery for the period of 70 days. Based on the results of in vitro release, the in vivo studies were carried out using alum-adsorbed TT (from the Central Research Institute) as the standard. The antibody level was measured after 6 months, 9 months and finally, with one booster dose, after 12 months. In these in vivo studies, the TTCH antibody level rose up to 3.5IU/mL of guinea pig serum; this compared with 2IU/mL of guinea pig serum using the alum-adsorbed TT after 12 months with a second booster dose. The TTCH approach would be helpful to replace the existing adjuvant alum in the future.

Tetanus toxoid-loaded transfersomes for topical immunization

Topical immunization is a novel immunization strategy by which antigens and adjuvants are applied topically to intact skin to induce potent antibody and cell-mediated responses. Among various approaches for topical immunization, the vesicular approach is gaining wide attention. Proteineous antigen alone or in combination with conventional bioactive carriers could not penetrate through the intact skin. Hence, specially designed, deformable lipid vesicles called transfersomes were used in this study for the non-invasive delivery of tetanus toxoid (TT). Transfersomes were prepared and characterized for shape, size, entrapment efficiency and deformability index. Fluorescence microscopy was used to investigate the mechanism of vesicle penetration through the skin. The immune stimulating activity of these vesicles was studied by measuring the serum anti-tetanus toxoid IgG titre following topical immunization. The immune response was compared with the same dose of alum adsorbed tetanus toxoid (AATT) given intramuscularly, topically administered plain tetanus toxoid solution, and a physical mixture of tetanus toxoid and transfersomes again given topically. The results indicated that the optimal transfersomal formulation had a soya phosphatidylcholine and sodium deoxycholate ratio of 85:15%, w/w. This formulation showed maximum entrapment efficiency (87.34 +/- 3.81%) and deformability index (121.5 +/- 4.21). An in-vivo study revealed that topically administered tetanus toxoid-loaded transfersomes, after secondary immunization, elicited an immune response (anti-TT-IgG) comparable with that produced by intramuscular AATT. Fluorescence microscopy revealed the penetration of transfersomes through the skin to deliver the antigen to the immunocompetent Langerhans cells.

Role of alum in improving the immunogenicity of biodegradable polymer particle entrapped antigens

This study was aimed at understanding the role of alum in improving the immunogenicity of biodegradable polymer particle entrapped antigens. Presence of alum formed a fine network around PLA particles holding them together and promoted attachment of higher number of particles on macrophage surface for a considerable period of time. Use of alum lowered the burst release of the entrapped antigen from particles and thereafter also reduced the cumulative release of antigen from particles. Apart from this, PLA microparticles alone induced macrophages to release TNF-α similar to that induced by alum. However admixture of PLA particles and alum enhanced the secretion of TNF-α from 876 pg/ml at 6 h to 3500 pg/ml at 24 h which was higher than that induced by alum adsorbed TT. Immunization with admixture of antigen loaded polylactide (PLA) microparticles (2–8 μm) and alum improved the antibody titers almost twice than that achieved from particle alone in experimental animals. Single point immunization with particle entrapped antigens and alum also elicited antibody titers comparable to two doses of alum adsorbed tetanus toxoid (TT) or diphtheria toxoid (DT). Our results suggest that presence of alum acts in multiple ways to improve the antibody titers of polymer particles entrapped antigens. Such co-operative adjuvant action of alum and polymer particles can be exploited to improve the immunogenicity of other antigens.

Induction of high antitoxin titers against tetanus toxoid in rabbits by intranasal immunization with dextran microspheres

Poor absorption of protein antigens through the mucosal membranes necessitates the use of mucoadhesive delivery systems. Regarding the advantages of mucosal immunization and also the penetration enhancement potential of dextran microspheres, in this study the adjuvant potential of these microspheres was compared with CpG-ODN. Cross-linked dextran microspheres (CDMs) were loaded with tetanus toxoid (TT). In vitro release studies were performed in a model, simulating the nasal cavity. The immunoreactivity of encapsulated TT was assayed by ELISA. Membrane toxicity and local irritating potential of CDM was examined by erythrocyte hemolysis and nasal administration to human nose, respectively. The various formulations were nasally administered to rabbits ( n = 4). Alum-adsorbed TT (AATT) was injected as the positive control. The serum IgG and nasal lavage sIgA titers were determined by ELISA method. Serum antitoxin titers were determined by toxin neutralization (TN) bioassay method. Mean diameter of CDM was 128.1 ± 25.8 μm. Mean encapsulation efficiency was 20.3 ± 3.2% ( n = 3). Antigenicity of encapsulated TT was 90.5 ± 1.8% ( n = 3) that of original TT. Hemolysis studies showed no membrane disruption by CDM and none of the human subjects reported nasal irritation. Among the nasally immunized animals, the highest antitoxin titers was seen in the group immunized with CDM + TT ( P < 0.0001). The serum IgG titers of the CDM + TT group was higher than the TT solution group ( P < 0.05). The adjuvant potentials of CDM and CpG-ODN in inducing IgG titers was not significantly different ( P > 0.05). The lowest sIgA titers in the bronchial lavage were seen in the group of animals received AATT parenterally. Considering the proper release characteristics, desirable preservation of the antigen activity of TT, good mucoadhesion properties and also safety of CDM + TT, these microspheres could be regarded as an efficient mucosal adjuvant and antigen delivery system. These microspheres could induce very high antitoxin titers following nasal administration, while the CpG-ODN could not induce such titers. The antitoxin titers induced by CDM + TT was 175 times higher than the protective levels.

Development of novel fusogenic vesosomes for transcutaneous immunization

Transcutaneous immunization (TCI) is a novel vaccination strategy based on the application of antigen together with an adjuvant onto hydrated bare skin. This simple and non-invasive immunization procedure elicits systemic and cell mediated immune responses and therefore, it provides a viable and cost-effective strategy for disease prevention. In the present study, novel fusogenic vesicular carrier constructs, i.e. vesosomes were developed and evaluated for topical delivery of vaccines using tetanus toxoid (TTx) as a model antigen. Prepared vesosomes were characterized for size, shape, entrapment efficiency and zeta potential. The prepared novel systems were examined for in process antigen stability and long-term storage stability studies. In vitro skin permeation and fluorescence microscopy study were also preformed for prepared novel vesicular systems for the evaluation of skin penetration efficiency. The immune stimulating activity of these vesicles was studied by measuring the serum anti-tetanus toxoid IgG titer and isotype ratio IgG2a/IgG1 following topical immunization in three different protocols and results were compared with the alum adsorbed tetanus toxoid given intramuscularly and topically administered plain tetanus toxoid solution, plain liposomes and cationic fusogenic liposomes. Serum IgG titers after three consecutive topical administrations were significantly better ( * P < 0.05) than single administration of TTx antigen with vesosomal systems, suggesting an effective stimulation of serum immune response. Furthermore, notable serum anti-TTx antibody titers also occurred in animals primed with alum adsorbed TTx and subsequently boosted with topical administration of novel vesosomal systems. In each immunization studies, the vesosomal systems could elicit combined Th1 and Th2 immune responses following topical administration. These results suggest that the investigated vesosomal systems can be effective as topical delivery of vaccines.

Mannosylated Niosomes as Adjuvant-Carrier System for Oral Mucosal Immunization

The aim of the present study was to develop mannosylated niosomes as oral vaccine delivery carrier and adjuvant for the induction of humoral, cellular, and mucosal immunity. Tetanus toxoid (TT) loaded niosomes composed of sorbiton monostearate (Span 60), cholesterol, and stearylamine were prepared by the reverse-phase evaporation method. They were coated with a modified polysaccharide o-palmitoyl mannan (OPM) to protect them from bile salts caused dissolution and enzymatic degradation in the gastrointestinal tract and to enhance their affinity toward the antigen presenting cells of Peyer's patches. Prepared niosomes were characterized in vitro for their size, shape, entrapment efficiency, ligand binding specificity, and stability in simulated gastric fluid and simulated intestinal fluid. OPM-coated niosomes were found to more stable in simulated gastrointestinal conditions. The immune stimulating activity was studied by measuring serum IgG titer, IgG2a/IgG1 ratio in serum, and sIgA levels in intestinal and salivary secretions following oral administration of niosomal formulations in albino rats. The results were compared with alum-adsorbed TT following oral and intramuscular administration, and it was observed that OPM-coated niosomes produced better IgG levels as compared to plain uncoated niosomes and alum-adsorbed TT upon oral administration. Oral niosomes also elicited a significant mucosal immune response (sIgA levels in mucosal secretions). The developed formulations also elicited a combined serum IgG2a/IgG1 response, suggesting that they were capable of eliciting both humoral and cellular response. The study signifies the potential of OPM-coated niosomes as an oral vaccine delivery carrier and adjuvant. The proposed system is simple, stable, and cost-effective and may be clinically acceptable.

Non-invasive vaccine delivery in transfersomes, niosomes and liposomes: a comparative study

Non-invasive vaccine delivery is a top priority for public health agencies because conventional immunization practices are unsafe and associated with numerous limitations. Recently, the skin has emerged as a potential alternative route for non-invasive delivery of vaccine. Topical immunization (TI), introduction of antigen through topical application onto the intact skin, has many practical merits compared to injectable routes of administration. One of the possibilities for increasing the penetration of bioactives through the skin is the use of vesicular systems. Specially designed lipid vesicles are attracting intense attention and can be used for non-invasive antigen delivery. In the present study, elastic vesicle transfersomes, non-ionic surfactant vesicles (niosomes) and liposomes were used to study their relative potential in non-invasive delivery of tetanus toxoid (TT). Transfersomes, niosomes and liposomes were prepared and characterized for shape, size and entrapment efficiency. These vesicles were extruded through polycarbonate filter (50-nm pore size) to assess the elasticity of the vesicles. The immune stimulating activity of transfersomes, niosomes and liposomes were studied by measuring the serum anti-TT IgG titre following topical immunization. The immune response elicited by topical immunization was compared with that elicited by same dose of alum-adsorbed tetanus toxoid (AATT) given intramuscularly. The results indicate that optimal formulations of transfersomes, niosomes and liposomes could entrap 72.7 ± 3.4, 42.5 ± 2.4 and 41.3 ± 2.2% of antigen and their elasticity values were 124.4 ± 4.2, 29.3 ± 2.4 and 21.7 ± 1.9, respectively. In vivo study revealed that topically given TT containing transfersomes, after secondary immunization, could elicit immune response (anti-TT-IgG) that was equivalent to one that produced following intramuscularly alum-adsorbed TT-based immunization. In comparison to transfersomes, niosomes and liposomes elicited weaker immune response. Thus transfersomes hold promise for effective non-invasive topical delivery of antigen(s).

Potentiation of Immune Response from Polymer-Entrapped Antigen: Toward Development of Single Dose Tetanus Toxoid Vaccine

Poly(lactide) (PLA) polymer particles entrapping immunoreactive tetanus toxoid (TT) were used for generation of immune response using single point immunization. Immunization with different sizes of polymer particles encapsulating immunoreactive TT elicited anti-TT antibody titers that persisted for more than 5 months. However, antibody response generated by single point immunization of either nanoparticles or microparticles were lower than the conventional two doses of alum adsorbed TT. To overcome this limitation, alum was used with particles that improved anti-TT antibody response. 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 postimmunization. However anti-TT antibody titers declined rapidly with time. Immunization with admixture of microparticles and alum elicited higher antibody titers than the particles alone and the antibody titers were high particularly during the later part of the postimmunization period. Single point immunization with admixture of PLA microparticles and alum resulted in an antibody response very close to that achieved by two injection of alum-adsorbed TT. Physical mixture of both a nano- and microparticles along with alum resulted in sustained anti-TT antibody response from very early days of postimmunization until 150 days. The antibody titers were maintained around 50 μg/ml for more than 5 months. These results indicated that immune response from polymer particles can be further improved by use of additional adjuvant. Furthermore, using various size particles or physical mixture of different size particles along with alum, it is possible to modulate the kinetics of immune response using polymer particles based immunization.

Unresponsiveness to lymphoid-mediated signals at the neonatal follicular dendritic cell precursor level contributes to delayed germinal center induction and limitations of neonatal antibody responses to T-dependent antigens.

The factors limiting neonatal and infant IgG Ab responses to T-dependent Ags are only partly known. In this study, we assess how these B cell responses are influenced by the postnatal development of the spleen and lymph node microarchitecture. When BALB/c mice were immunized with alum-adsorbed tetanus toxoid at various stages of their immune development, a major functional maturation step for induction of serum IgG, Ab-secreting cells, and germinal center (GC) responses was identified between the second and the third week of life. This correlated with the development of the follicular dendritic cell (FDC) network, as mature FDC clusters only appeared at 2 wk of age. Adoptive transfer of neonatal splenocytes into adult SCID mice rapidly induced B cell follicles and FDC precursor differentiation into mature FDC, indicating effective recruitment and signaling capacity of neonatal B cells. In contrast, adoptive transfer of adult splenocytes into neonatal SCID mice induced primary B cell follicles without any differentiation of mature FDC and failed to correct limitations of tetanus toxoid-induced GC. Thus, unresponsiveness to lymphoid-mediated signals at the level of neonatal FDC precursors delays FDC maturation and GC induction, thus limiting primary Ab-secreting cell responses to T-dependent Ags in early postnatal life.

Potentiation of Immune Response from Polymer-Entrapped Antigen: Toward Development of Single Dose Tetanus Toxoid Vaccine

Poly(lactide) (PLA) polymer particles entrapping immunoreactive tetanus toxoid (TT) were used for generation of immune response using single point immunization. Immunization with different sizes of polymer particles encapsulating immunoreactive TT elicited anti-TT antibody titers that persisted for more than 5 months. However, antibody response generated by single point immunization of either nanoparticles or microparticles were lower than the conventional two doses of alum adsorbed TT. To overcome this limitation, alum was used with particles that improved anti-TT antibody response. 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 postimmunization. However anti-TT antibody titers declined rapidly with time. Immunization with admixture of microparticles and alum elicited higher antibody titers than the particles alone and the antibody titers were high particularly during the later part of the postimmunization period. Single point immunization with admixture of PLA microparticles and alum resulted in an antibody response very close to that achieved by two injection of alum-adsorbed TT. Physical mixture of both a nano- and microparticles along with alum resulted in sustained anti-TT antibody response from very early days of postimmunization until 150 days. The antibody titers were maintained around 50 μg/ml for more than 5 months. These results indicated that immune response from polymer particles can be further improved by use of additional adjuvant. Furthermore, using various size particles or physical mixture of different size particles along with alum, it is possible to modulate the kinetics of immune response using polymer particles based immunization.

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 μg/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.

Formulation strategies for the stabilization of tetanus toxoid in poly(lactide-co-glycolide) microspheres

The development of a single-dose tetanus vaccine based on Poly(Lactic acid) (PLA) or Poly(Lactide-co-Glycolide) (PLGA) microspheres has been complicated due to the instability of tetanus toxoid (TT) inside these systems. Herein we report an attempt to re-design PLGA microspheres by co-encapsulating TT in the dry solid state together with potential protein stabilizers, such as trehalose, bovine serum albumin, alginate, heparin, dextran or poloxamer 188 and by using an appropriate microencapsulation technique. These newly developed PLGA microspheres were able to release in vitro antigenically active TT for at least 5 weeks, the amount released being highly dependent on the stabilizing excipient used. More specifically, results showed that dextran and heparin provided a particularly stabilizing environment for TT inside the microspheres during the polymer degradation process. The efficacy of this strategy was demonstrated by the high, long lasting titers of neutralizing antibodies achieved after in vivo administration of dextran-containing microspheres with a small amount of alum-adsorbed TT, as compared to the commercial adsorbable tetanus vaccine. These findings suggest that future developments in the area of vaccinology depend on ability to combine a detailed knowledge of the microencapsulation technology with a rational choice of stabilizing excipient or combination of excipients.