Improved immunogenicity of a core-coated tetanus toxoid delivery system.

A new microparticulate delivery system composed of a stabilizing gelatin/poloxamer microcore surrounded by a PLGA coat was designed to improve the stability of tetanus toxoid (TT) encapsulated in PLGA microspheres. Microcores were prepared by a spray-congealing technique and encapsulated within PLGA using an oil-in-oil (o/o) solvent evaporation technique. SEM analysis of the cross-sections of the microcapsules revealed the adequate encapsulation of the cores, showing an intimate contact between the core and the coating. This structure was responsible for an osmotic phenomenon observed in vitro, which led to the release of the encapsulated TT in a short period of time. Nevertheless, it was observed that the release was affected by the presence of the poloxamer in the core: microspheres without poloxamer in the core exhibit a faster release (2 h) than those that incorporate the surfactant (24 h). The in vivo evaluation of this system showed that the encapsulated toxoid induced a low but continuous levels of neutralizing antibodies (Nt), whereas those obtained for the control (aluminum phosphate-adsorbed toxoid) decreased after reaching the maximum level at 14 weeks. Moreover, the administration of a mixture of encapsulated and adsorbed TT led to significant higher and more prolonged Nt levels than those measured for the adsorbed toxoid.     

An in vitro immune response model to determine tetanus toxoid antigen (vaccine) specific immunogenicity: Selection of sensitive assay criteria

Many vaccines employed in childhood vaccination programmes are produced by conventional techniques, resulting in complex biological mixtures for which batch-related quality control requires in vivo potency testing. Monitoring consistency via in vitro tests during the vaccine production has the capacity to replace certain of the in vivo methods. In this respect, determining vaccine antigen immunogenicity through functional immunological tests has high potential. Advances in immunology have made it possible to analyse this biological activity by in vitro means. The present study established such an in vitro test system for tetanus toxoid (TT). This measured vaccine immunogenicity through an antigen-specific secondary (recall) response in vitro, using a porcine model growing in value for its closeness to human immune response characteristics. Discrimination between the specific recall TT antigen and diphtheria toxoid (DT) was possible using both peripheral blood mononuclear cell cultures and monocyte-derived dendritic cells in co-culture with autologous specific lymphocytes. TT-specific activation was detected with highest discrimination capacity using proliferation assays, as well as IFN-gamma and TT-specific antibody ELISPOTS (measuring secreting T and B lymphocytes, respectively). These in vitro systems show a high potential for replacing animal experimentation to evaluate the immunogenicity of complex vaccines.     

Injectable polymer microspheres enhance immunogenicity of a contraceptive peptide vaccine

Advanced contraceptive peptide vaccines suffer from the unavailability of adjuvants capable of enhancing the antibody response with acceptable safety. We sought to overcome this limitation by employing two novel poly(lactic-co-glycolic acid) (PLGA) microsphere formulations to deliver a synthetic human chorionic gonadotropin (hCG) peptide antigen co-synthesized with a T-cell epitope from tetanus toxoid (TT), C-TT2-CTP35: surface-conjugated immunogen to induce phagocytosis; and encapsulated peptide to provide a depot effect, with MgCO(3) co-encapsulated in the polymer to neutralize acidity from the biodegrading PLGA polyester. A single immunization of encapsulated peptide in rabbits elicited a stronger antibody response with equivalent duration relative to a positive control--three injections of the peptide administered in a squalene-based water-in-oil emulsion. Surface-conjugated peptide was less effective but enhanced antibody levels at 1/5 the dose, relative to soluble antigen. Most remarkable and unexpected was the finding that co-encapsulation of base was essential to attain the powerful adjuvant effect of the PLGA-MgCO(3) system, as the MgCO(3)-free microspheres were completely ineffective. A promising contraceptive hCG peptide vaccine with acceptable side effects (i.e., local tissue reactions) was achieved by minimizing PLGA and MgCO(3) doses, without significantly affecting antibody response.     

Evaluation of two GnRH-I based vaccine formulations on the testes function of entire Suffolk cross ram lambs

A modified GnRH peptide (CHWSYGLRPG-NH(2)) was conjugated to tetanus toxoid (TT) or diphtheria toxoid (DT) and formulated with Quil A saponin or a sustained release injectible PLGA (poly(lactide-co-glycolide)/triacetin). For the Quil A formulations, two administrations of TT conjugate at 3-weekly intervals were followed by two booster injections with the DT conjugate in entire ram lambs. With the PLGA formulations, only two injections were administered; the first containing TT and the second DT at 6-weekly intervals. Evaluation was carried out by comparing the specific antibody levels produced in relationship to hormone profiles and testicular changes. The Quil A formulation was considered the most effective, as it caused significant reduction in testosterone and follicle stimulating hormone levels, resulting in marked suppression of spermatogenesis.     

A preliminary evaluation of alternative adjuvants to alum using a range of established and new generation vaccine antigens

Although alum is the most commonly used vaccine adjuvant, it has some limitations for use with the next generation recombinant antigens. We explored the use of alternative adjuvant formulations (poly lactide co-glycolide (PLG) microparticles, MF59 emulsion, CAP and l-tyrosine suspension) in comparison with five different vaccine antigens--namely, diphtheria toxoid (DT), tetanus toxoid (TT), HBsAg, Men C conjugate and MB1. The results indicated that although alum was optimal for bacterial toxoid based vaccines, it was not highly potent for MB1, Men C or HBsAg antigens. MF59 emulsion stood out as a good alternative to alum for TT, HBsAg, MB1 and Men C vaccines. On the other hand l-tyrosine suspension and CAP did not enhance immune responses over alum with most antigens. PLG microparticles were comparable or better than alum with both MB1 and Men C conjugate vaccine. The study indicates that it is possible to replace alum with other adjuvant formulations like MF59 and PLG and maintain and/or improve immune responses with some vaccine antigens.     

Strong systemic and mucosal immune responses to surface-modified PLGA microspheres containing recombinant hepatitis B antigen administered intranasally

Surface-modified DL-lactide/glycolide copolymer (PLGA) microspheres with chitosan (CS) were developed for nasal immunization using recombinant Hepatitis B (HBsAg) surface protein for the induction of humoral, cellular and mucosal immunity. Modified PLGA microspheres were characterized in vitro for their size, shape, entrapment efficiency and zeta potential. The nasal clearance rate was evaluated by gamma scintigraphy in rabbits. The antigen integrity, in vitro release and its stability at 37 degrees C were also evaluated. The designed cationic microspheres possessed 27.2 mV zeta potential and an average size less than 10 microm with antigen loading efficiency of 80+/-5%. However, zeta potential of unmodified PLGA microspheres was measured to be negative (-8.7 mV). The modified PLGA microspheres showed the lowest nasal clearance rate when compared with unmodified PLGA microspheres and lactose powder. The antigen integrity was retained intact in encapsulated form as well as on release. The immune-stimulating activity was studied by measuring anti-HBsAg titre, secretory IgA level in serum, vaginal, nasal and salivary secretions (mucosal secretions) and cytokine level (interleukin-2 (IL-2) and interferon-gamma (IFN-gamma)) in spleen homogenates following nasal administration of modified PLGA microspheres in Balb/c mice and compared with alum-HBsAg vaccine injected subcutaneously. The serum anti-HBsAg titre obtained after nasal administration of modified PLGA microspheres was comparable with titre recorded after alum-HBsAg was administered subcutaneously. Moreover, alum-HBsAg vaccine did not elicit sIgA in mucosal secretions as it was induced and measured in the case of nasal administration of modified PLGA microspheres. Similarly, there was no cellular response (cytokine level) in case of alum-HBsAg vaccine. Modified PLGA microspheres (cationic microspheres) thus produced humoral (both systemic and mucosal) and cellular immune responses upon nasal administration. These data demonstrate high potential of modified PLGA microspheres for their use as a carrier adjuvant for nasal subunit vaccines.     

Immunogenicity of single-dose diphtheria vaccines based on PLA/PLGA microspheres in guinea pigs.

Biodegradable polyester microspheres (MS) have shown potential for single-dose vaccines. This study examined the immunogenicity of diphtheria toxoid (Dtxd) microencapsulated in different types of poly(lactide) (PLA) and poly(lactide-co-glycolide) (PLGA) MS prepared by the methods of spray-drying and coacervation. We investigated the influence of polymer type (PLGA 50:50 of low M(w); PLA of high M(w); end-group stearylated PLAs of low M(w)) and co-encapsulated excipients (BSA and/or trehalose) on Dtxd content, in vitro release and immunogenicity in guinea pigs. The co-encapsulated trehalose lowered the Dtxd entrapment efficiency in the spray-dried particles from 75 to 56%, whereas albumin alone had no effect in the spray-drying, but improved the encapsulation in the coacervation process. With the hydrophobic, end-group stearylated PLAs, Dtxd could only be encapsulated in the presence of albumin. Guinea pigs immunised with Dtxd-MS made with the relatively hydrophilic PLGA 50:50 exhibited specific and sustained antibody responses over 40 weeks, comparable to the responses to alum-adjuvanted toxoid. In contrast, undetectable or very low antibody responses were determined after immunisation with MS made with hydrophobic polymers. Surprisingly, large (15-60 microm) and small (1-5 microm) MS gave comparable primary antibody responses. In conclusion, the data presented confirm the feasibility of MS vaccines to induce strong, long-lasting protective antibody responses after a single immunisation.     

The adjuvant effect of synthetic oligodeoxynucleotide containing CpG motif converts the anti-Haemophilus influenzae type b glycoconjugates into efficient anti-polysaccharide and anti-carrier polyvalent vaccines

Synthetic oligodeoxynucleotides containing CpG immunostimulatory sequences (ISS) have been shown to act as potent adjuvants of type 1 immune responses when co-administered with protein or peptide vaccines. We have recently shown that ISS can increase the anti-polysaccharide (CHO) and anti-tetanus toxoid (TT) or anti-diphtheria (CRM) toxoid antibody levels if used as adjuvant of anti-Haemophilus influenzae type b (Hib) CHO vaccine conjugated with TT or CRM. The analysis of anti-TT and anti-CRM IgG subclasses showed a significant increase in IgG2a, IgG2b and/or IgG3 in the presence of ISS. Anti-TT and anti-CRM antibodies were shown to neutralize the activity of both the tetanus and diphtheria toxin in vivo or in vitro tests respectively. These data show that ISS have the potential to increase host antibody response against both the CHO and the protein component of a conjugated vaccine, and encourage the investigation to identify strategies of vaccination with schedules aimed at the valuation of protein carriers as protective immunogens.     

Adverse event reporting rates following tetanus-diphtheria and tetanus toxoid vaccinations: data from the Vaccine Adverse Event Reporting System (VAERS), 1991-1997

Since 1966, the Advisory Committee on Immunization Practices (ACIP) has recommended tetanus-diphtheria toxoid (Td) be used instead of single antigen tetanus toxoid (TT) because, while both vaccines protect against tetanus, only Td protects against diphtheria. Despite this recommendation, approximately 2.5 million doses of TT were distributed annually from 1991 to 1997. One possible explanation for the continued use of TT is concern about the relative safety of Td. Small clinical trials found Td to be associated with a higher rate of local vaccine-associated adverse events (VAEs) than TT. To determine if the findings from the trials would hold up on a larger scale, we compared the rate of reporting to the Vaccine Adverse Event Reporting System (VAERS), a passive reporting system, after either vaccine from 1991 to 1997. There were 40 reports per million doses of Td, and 27 reports per million doses of TT, for a reporting rate ratio of 1.4. Reporting rates to VAERS are lower than the rates of VAEs identified in the clinical trials, but the magnitude of the difference in VAEs following TT versus Td is similar. While reporting rates are lower after TT than Td, rates of reported VAEs after both vaccines are low.     

Remarkably high antibody levels and protection against P. falciparum malaria in Aotus monkeys after a single immunisation of SPf66 encapsulated in PLGA microspheres

Single dose immunisation is a major goal in vaccine design. The purpose of this study was the development of a single dose delivery system for the SPf66 malaria vaccine, based on this antigen's microencapsulation in PLGA microspheres by double emulsion method.Results indicate that a single immunisation in mice and monkeys with the SPf66 malaria vaccine, encapsulated in a mixture of two formulations of PLGA microspheres, induced a remarkably high and long-lasting immune response as assessed by ELISA and Western Blott. This immune response was associated with a good protective capacity in Aotus monkeys, after experimental challenge, indicating that antigen integrity lasted following the microencapsulation process. PLGA biodegradable microspheres thus serve as an effective delivery system for the design of a single dose immunisation vaccine, such as the SPf66 synthetic malaria vaccine.     

In vivo uptake of an experimental microencapsulated diphtheria vaccine following sub-cutaneous immunisation

Previous studies demonstrated in vitro phagocytosis of poly(lactide-co-glycolide) (PLGA) microspheres (MS) by macrophages and dendritic cells and the biodistribution of fluorescent PLGA particles following oral or intranasal administration. In this study, we report the uptake and biodistribution of sub-cutaneously administered fluorescent labelled PLGA MS loaded with diphtheria toxoid (DT). The cell type and percentage of fluorescent positive cells were determined by flow cytometry and confirmed by fluorescent microscopy. Fluorescent particles were detected inside cells of the peritoneal flush as early as 10 min post-inoculation, predominantly in cells of macrophage morphology. In vivo trafficking of PLGA particles following a sub-cutaneous immunisation of mice appeared to be governed by macrophages. However, in the first week after inoculation, dendritic cells played a significant role in the uptake and digestion of the microspheres, thereby triggering the immune response against the antigen. Fluorescent PLGA MS were also observed in cells of lymphoid tissues such as mesenteric lymph nodes (MLN) and spleen (S). However, microsphere fluorescence in lymphoid tissues decreased rapidly, as they were degraded inside the cells, thereby enabling the presentation of the antigen to specific cells of the immune system. To our knowledge, this is the first time the fate of immunogenic PLGA microspheres was studied in vivo following a sub-cutaneous injection route.     

Spectroscopic characterization of antibodies adsorbed to aluminium adjuvants: correlation with antibody vaccine immunogenicity

MMA383 is an anti-idiotypic antibody designed as an immunogenic surrogate for the cancer specific Lewis Y antigen. Lewis Y is expressed in 70-90% of tumours of epithelial origin with limited expression in normal tissue. Five different MMA383 vaccines were prepared by mixing a MMA383 antibody solution with an Alhydrogel aluminium hydroxide adjuvant and tested on the biological activity in a rat model. The immunogenicity increased when: (i) the adjuvant was sterilized at 121 degrees C compared to no sterilization, (ii) the adjuvant was suspended in a phosphate buffer compared to water and (iii) the MMA383 solution was at a pH of 7.2. The immunogenicity of a ready-to-use MMA383 aluminium hydroxide suspension was the lowest. The in vivo data show that small differences in vaccine formulations before injection can generate significant changes in immunogenicity. Prior to mixing with the adjuvant, the physical and chemical characteristics of MMA383 antibodies were the same in all vaccines. Fluorescence and light scattering methods were developed to characterize antibodies in the presence of the adjuvant. Compared to the least active vaccines, the two most biologically active vaccines showed an increase in the antibody Trp fluorescence intensity, anisotropy, fluorescence lifetime, 90 degrees light-scatter, sedimentation velocity and rotational correlation time. Analysis of the 90 degrees light-scatter sedimentation kinetics indicates that stronger immune responses of vaccines can be related to the stronger binding of the antibodies to the adjuvants and the formation of more compact and condensed particles. Taken together, these results show a correlation between the in vitro fluorescence and light-scatter data and the in vivo immune response of the five MMA383 vaccines. The spectroscopic techniques described offer a new in vitro approach for the prediction of immune responses of different vaccine formulations.     

Collagen minipellet as a controlled release delivery system for tetanus and diphtheria toxoid

The use of biodegradable polymer matrices as a single-dose vaccine delivery system was investigated using tetanus toxoid (TT) and diphtheria toxoid (DT). BALB/c mice were immunized with TT or DT in different formulations including individual, in minipellet and aluminum hydroxide (alum), and the antibody responses were monitored for 48 weeks. Antigens entrapped in minipellet elicited higher antibody responses compared to those obtained with individual antigens and antigens adsorbed to alum and the antibody levels remained elevated over 48 weeks. In addition, minipellet formulations induced the same subclasses of antibodies induced by alum formulations. These results raise the possibility to obtain optimal and long-lasting immune responses by a single administration of minipellet formulations.     

Biodegradable PLGA microspheres as a delivery system for malaria synthetic peptide SPf66

SPf66 is the first chemically synthesised vaccine to elicit a partial protective immune response against malaria. The aluminium hydroxide (alum)-adsorbed SPf66 vaccine is weakly immunogenic and of poor to moderate efficacy in humans. To investigate the possibility of improving SPf66 vaccine immunogenicity, a delivery system based on poly-D,L-lactide-co-glycolide (PLGA) microspheres was developed and the immune response induced after its subcutaneous administration into mice was evaluated. Microspheres were prepared by a solvent extraction/double emulsion (w/o/w) method and characterised for morphology, size, peptide loading, release profile and peptide integrity. The in vitro and in vivo results obtained showed that there was no apparent effect of the encapsulation procedure on SPf66 integrity and immunogenicity. The subcutaneous administration of microspheres showed a significantly higher immune response (serum IgG levels) than that obtained with alum adsorbed SPf66 and it was comparable to that of SPf66 emulsified with Freund's adjuvant (FA). These observations illustrate the potential of PLGA microspheres as a delivery system for chemically synthesised antigens.     

Leptospira immunoglobulin-like protein A variable region (LigAvar) incorporated in liposomes and PLGA microspheres produces a robust immune response correlating to protective immunity

Subunit vaccines are attractive as an intervention strategy against leptospirosis, an important zoonotic disease afflicting both humans and livestock. However, the success of subunit vaccines has been hampered by weak or short-term immunity and unavailability of nontoxic, potent adjuvants. In the present study, the variable region of recombinant Leptospira immunoglobulin like protein A (LigAvar) incorporated into conventional liposomes and PLGA microspheres produced robust immune responses that induced significant protection against virulent Leptospirainterrogans serovar Pomona challenge in hamsters. Four-week-old hamsters were immunized subcutaneously with LigAvar incorporated into conventional liposomes or adsorbed on aluminum hydroxide (alum) and subsequently boosted after 3 weeks. Additionally, LigAvar incorporated into PLGA microspheres was evaluated as a single dose vaccine. All animals were challenged intraperitoneally 3 weeks after booster with a lethal dose (10 × MLD₅₀) of virulent L. interrogans serovar Pomona. Animals were bled at various time points to evaluate antibody response, then sacrificed. Splenocytes were isolated and assayed for lymphocyte proliferation and cytokine profiles in response to recall antigen. Our results indicate that both liposomes and microspheres prove to be better adjuvants compared to conventional alum as revealed by enhanced antibody titers, lymphocyte proliferation and significant enhancement in both Th1(IL-12, IFN-γ) and Th2 (IL-4, IL-10) cytokines. Moreover, LigAvar associated with liposomes and microspheres is able to provide better protection than LigAvar with alum as revealed by enhanced survival and reduced histopathological lesions in vital organs. Taken together, the data of the present study suggests that both liposomes and PLGA microspheres are promising adjuvants for use with future subunit vaccines for prevention of leptospirosis.     

Use of tetanus toxoid as a differentiating infected from vaccinated animals (DIVA) strategy for sero-surveillance of avian influenza virus vaccination in poultry

Strategies for differentiating infected from vaccinated animals (DIVA) require improvement for increased surveillance of avian influenza (AI), where vaccination is employed to control disease. We propose a novel DIVA approach for chickens using tetanus toxoid (TT) as an exogenous marker independent of serotype and relatedness of circulating and vaccine strains. Of 1779 chickens tested from Australia, Hong Kong and China, 100% were seronegative for TT-specific antibodies without vaccination. Tetanus toxoid adjuvanted to mineral oil was immunogenic in chickens. Co-delivery of both TT and inactivated LPAI (H6N2) vaccines in chickens elicited strong TT and influenza-specific antibody responses, which persisted to 53 weeks post-vaccination. Furthermore, immunization with a combined vaccine composed of TT and AI induced high levels of antibodies to both antigens. We conclude that TT is a highly suitable exogenous marker for AI vaccination in chickens allowing simple and effective monitoring of AI vaccination status.     

Co-delivery of cancer-associated antigen and Toll-like receptor 4 ligand in PLGA nanoparticles induces potent CD8+ T cell-mediated anti-tumor immunity

The purpose of this study was to evaluate the efficacy of poly(lactic-co-glycolic acid) (PLGA)-based vaccines in breaking immunotolerance to cancer-associated self-antigens. Vaccination of mice bearing melanoma B16 tumors with PLGA nanoparticles (NP) co-encapsulating the poorly immunogenic melanoma antigen, tyrosinase-related protein 2 (TRP2), along with Toll-like receptor (TLR) ligand (7-acyl lipid A) was examined. Remarkably, this vaccine was able to induce therapeutic anti-tumor effect. Activated TRP2-specific CD8 T cells were capable of interferon (IFN)-gamma secretion at lymph nodes and spleens of the vaccinated mice. More importantly, TRP2/7-acyl lipid A-NP treated group has shown immunostimulatory mileu at the tumor microenvironment, as evidenced by increased level of pro-inflammatory cytokines compared to control group. These results support the potential use of PLGA nanoparticles as competent carriers for future cancer vaccine formulations.     

Immunogenicity of IRIV- versus alum-adjuvanted diphtheria and tetanus toxoid vaccines in influenza primed mice

The immunogenicity and protectivity of two different toxoid vaccines were compared in mice. In one formulation, toxoids (diphtheria or tetanus) were adsorbed to alumoxid, whereas in the other formulation the toxoids were crosslinked to immunopotentiating reconstituted influenza virosomes (IRIVs). A preimmunization with influenza antigens is necessary for a good anti-toxoid antibody response when the IRIV formulation was administered. After two immunizations with the IRIV- or alum-based vaccines, the IRIV-based formulation induced a higher humoral immune response than toxoids adsorbed to alum. Using an in vitro test, diphtheria toxin neutralizing antibodies were tested. Di-IRIV induced a significantly (p = 0.002) higher titer of diphtheria toxin neutralizing antibodies than Di-alum. Tetanus challenge experiments showed, that the IRIV-based tetanus vaccine induced a threefold higher titer of protective antibodies than the tetanus toxoid adsorbed to alum. Therefore, the IRIV-based formulations appeared to be superior to the alum-based vaccines in terms of immunogenicity and protectivity.     

Antihepatitis B response to hepatitis B vaccine administered simultaneously with tetanus toxoid in nonresponder individuals

In this prospective study, our aim was to test the effect of simultaneous administration of preS2 and S containing recombinant hepatitis B vaccine (S2SRHB) with tetanus toxoid (TT) to the individuals who did not respond after three doses of hepatitis B vaccine previously.There were three groups (healthy individuals, pregnant women, hemodialysis patients), each was divided into two subgroups as groups A and B. Group A received S2SRHB+TT and group B received only S2SRHB.We found that in groups receiving both vaccines, both seroconversion rate and antibody titer level were significantly higher (P<0.05).In conclusion, simultaneous administration of S2SRHB+TT is more effective than administration of S2SRHB alone.     

Potential of polymeric lamellar substrate particles (PLSP) as adjuvants for vaccines.

In recent years microspheres or microparticles produced from biodegradable polymers such as poly(D,L-lactide) (PLA) and poly(D, L-lactide-co-glycolide) (PLGA) containing encapsulated vaccine antigens have been investigated for administration via parenteral, oral, and intranasal routes. These microparticles allow the controlled release of vaccines with an aim to reduce the number of doses for primary immunisation or to develop single dose vaccines. The polymer materials have been widely regarded as being of minimal toxicity. Evaluation of candidate systems in animal studies have shown antibody levels and cell responses similar to or greater than those observed with adjuvants such as alum. However, there are concerns regarding the integrity and immunogenicity of the antigen during the encapsulation process when the antigen is exposed to organic solvents, high shear stresses and the exposure of antigen to low pH which is caused by polymer degradation. An alternative approach would be to adsorb antigens to the surface of biodegradable polymer particles. Polymeric lamellar substrate particles (PLSP), produced by a simple precipitation of PLA, are suitable for this purpose. The adsorption of antigens onto these particles is a simple procedure. It avoids pH changes due to bulk polymer degradation and the use of solvents and therefore will be less damaging to the vaccine. Moreover, such systems will be much easier to scale up for a clinical study and eventual manufacture. The aim of this article is to discuss the preparation and physical characteristics of PLSP, antigen adsorption, in vivo efficacy of PLSP antigen systems and to consider the potential of PLSP as controlled release adjuvants for protein, peptide or viral vaccines.