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D Sesardic

Publications and source records attributed to D Sesardic.

At least 19 recordsLinked to original sources
PubMed2000

Ambiguities in the preclinical quality assessment of microparticulate vaccines.

Vaccination techniques do not always stimulate immunity because of the inappropriate mobilization of immune responses, and the frequency of vaccinations required is impractical in many developing countries. Such limitations have spurred the development of new vaccine-delivery approaches. Microparticles made of poly(lactide-co-glycolide) can induce adaptive immunity after a single administration of a vaccine. However, the preclinical assessment of such vaccines is not standardized, making it difficult to compare pharmaceutical with immunological data. The relevance of and the ambiguity in the assessment of microparticulate vaccines with respect to the current knowledge on immunity are discussed, in addition to the application of this knowledge to rational vaccine design.

Antigens↗
PubMed1999

Non-pertussis components of combination vaccines: problems with potency testing.

Vaccines comprising combinations of diphtheria, tetanus and pertussis (DTP) with Haemophilus influenzae type b polysaccharide-protein conjugate (Hib), inactivated poliomyelitis virus (IPV) and hepatitis B virus (HBV) are already available, and new combinations using acellular pertussis components in a triple vaccine (DTaP) are under development. Evidence to date has shown that control of the efficacy, safety and stability of combination vaccines cannot be based on information already available on the individual components or existing licensed formulations. Several examples of immunological interference between components of a combination vaccine have been observed both in clinical trials and in laboratory tests. Examples of these for D, T and Hib components in DTP and DTaP combinations have been investigated.

Animals↗
PubMed1999

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.

Animals↗
PubMed1999

Validation of in vitro assays for botulinum toxin: a case study.

Ensuring the reliability and precision of assay results requires careful attention to assay design. In this case study we describe validation studies of an in vitro assay for botulinum neurotoxin type A based on its endopeptidase activity towards immobilised synthetic substrate. This assay, in common with many in vitro assays, is sensitive to changes in reagents and assay conditions and is time dependent. In addition, the toxin is not stable in solution. Differences in estimates of potency, resulting from positional factors, which are not significant in individual assays, are shown to be consistent and statistically significant over a longer series of assays. This study emphasizes that assay validation should not be viewed as a single step in assay development but must be considered as a continuing process if assay results are to be reliable and reproducible.

Analysis of Variance↗
PubMed1999

Serological assays as alternatives to the Ph Eur challenge test for batch release of tetanus vaccines for human use.

According to the European Pharmacopoeia (Ph Eur) monograph on Tetanus Vaccine (adsorbed) (1997: 0452), assessment of potency is based on a challenge test in guinea pigs or mice. The end-point is taken as paralysis or death. The test requires a large number of animals and causes severe distress. The aim of the present study was to refine the test, and reduce the number of animals needed, for batch release purposes. Serological assays having the potential of being internationally accepted, have been compared with Ph Eur assays. The study included five tetanus vaccines of various combinations and produced by different manufacturers. The results indicated an excellent correlation between enzyme-linked immunosorbent assay (ELISA) and the challenge test (about 93% predictive value), as well as between the toxin binding inhibition (ToBI) test and the challenge test (about 95% predictive value) and between ELISA and the ToBI test (r = 0.92). Antitoxin concentrations determined by ELISA and ToBI were generally in the same range. An overall good correlation was also seen for serum pools of the guinea pigs injected with equal vaccine doses, between toxin neutralisation test in mice (TNT) and ELISA (r = 0.93) as well as between TNT and ToBI (r = 0.97). The ultimate goal of this project was to determine whether serological assays can be used for testing combined vaccines, particularly for tetanus and diphtheria components, using sera from the same animals.

Animal Testing Alternatives↗
PubMed1999

Alternatives to the use of animals for bacterial toxins and antitoxins.

The potency of several novel botulinum toxin-derived biological therapeutic products now in routine medical use is determined exclusively by in vivo methods. In addition, large numbers of animals continue to be used for the potency and safety testing of therapeutic antitoxins and toxoid vaccines. There is thus an increasing need to develop acceptable alternative assays for toxicity testing of clostridia neurotoxins which could be applied to different toxin derived therapeutic agents, including vaccines. Scientific advances in the understanding of the mode of action of clostridial neurotoxins have now provided the basis for improving conventional testing procedures.

Animal Testing Alternatives↗
PubMed1998

Comparison of phenotypic and genotypic methods for detection of diphtheria toxin among isolates of pathogenic corynebacteria.

We have compared molecular, immunochemical, and cytotoxic assays for the detection of diphtheria toxin from 55 isolates of Corynebacterium diphtheriae and Corynebacterium ulcerans originally isolated in five different countries. The suitabilities and accuracies of these assays for the laboratory diagnosis of diphtheria were compared and evaluated against the "gold standard" in vivo methods. The in vivo and Vero cell cytotoxicity assays were accurate in their abilities to detect diphtheria toxin but were time-consuming; however, the cytotoxicity assay is a suitable in vitro alternative to the in vivo virulence test. There was complete concordance between all the phenotypic methods. Genotypic tests based upon PCR were rapid; however, PCR must be used with caution because some isolates of C. diphtheriae possessed toxin genes but failed to express a biologically active toxin. Therefore, phenotypic confirmation of toxigenicity for the microbiological diagnosis of diphtheria is recommended.

Animals↗
PubMed1998

Modulation of the immune response to tetanus toxoid by polylactide-polyglycolide microspheres.

The cellular and antibody responses in mice to tetanus toxoid (TT), alone or adsorbed to aluminium hydroxide TT were compared to those obtained with TT incorporated in polylactide-polyglycolide (PLGA) microspheres, a candidate single dose vaccine. After subcutaneous injection, the serum IgM antibody to TT followed similar overall patterns for all preparations although the highest levels were achieved with the alum-adsorbed material. A similar pattern was observed with the overall IgG antibody responses. The isotype distribution of antibodies was broadly similar for all types of preparation although considerable qualitative differences were observed. IgG1 antibodies predominated although IgG2a and IgG2b made a substantial contribution, especially at three months in the case of the two adjuvanted preparations. The IgG3 responses to each type of vaccine were very low. Both alum and microsphere vaccines induced proliferative responses to TT in splenic lymphocytes at three months after vaccination. Each type of vaccine was much less effective in inducing proliferative responses in lymph node cells. There was evidence of induction of IL2, IL4 and interferon-gamma genes by microsphere vaccines in splenic but not lymph node cells. There were indications that PLGA microspheres alone exerted a modulating effect on cellular responses after immunization. These results suggest that TT encapsulated in microspheres induces a pattern of cellular and antibody responses qualitatively similar to those induced by conventional TT vaccines.

Animals↗
PubMed1997

Two-dimensional structure of the Opc invasin from Neisseria meningitidis.

A two-dimensional structural model was devised for the Opc outer membrane protein invasin which contains 10 transmembrane strands and five surface-exposed loops. One continuous epitope recognized by three monoclonal antibodies was localized to the tip of loop 2 by synthetic peptides and site-directed mutagenesis while a second, discontinuous epitope recognized by a fourth antibody was localized to loops 4 and 5 by insertion mutagenesis. These monoclonal antibodies are bactericidal and inhibit adhesion and invasion. Most of the T-cell epitopes defined by Wiertz et al. (1996) were localized to the transmembrane strands. Oligonucleotides encoding a foreign epitope (nabla) from Semliki Forest virus were inserted into BglII restriction sites created by site-directed mutagenesis. The nabla epitopes inserted in all five predicted loops were recognized on the cell surface of live Escherichia coli bacteria by a monoclonal antibody and are exposed while nabla epitopes in the N-terminus or three predicted turns were not. The results thus confirm important predictions of the model and define five permissive sites within surface-exposed loops which can be used to insert foreign epitopes.

Adhesins, Bacterial↗
PubMed1996

Estimation of antigenic tetanus toxoid extracted from biodegradable microspheres.

Microspheres made from poly (lactic/glycolic acid) polymers have been considered as a new delivery system for single-dose tetanus toxoid (TT) vaccines. One of the most critical properties of the proposed vaccines is the loading and distribution of TT as this will have a profound effect on immunogenicity. As the concentration of TT in microspheres is very low sensitive assay methods are required. An assay incorporating monoclonal antibody (MAb) recognizing a neutralizing epitope and cross-reacting with TT was developed (MAp capture ELISA) which provided a sensitivity of 0.001 Lf/ml. An extraction procedure was devised which did not destroy the antigenicity and gave a recovery of 90.6 +/- 3.39% when applied to different preparations. The extracted TT was then quantified by MAb capture ELISA which was estimated to be 250-fold more sensitive than single-site ELISA for toxoid. The loading of 20 microspheres preparations (12 filled and 8 placebo) was determined by both protein micro-BCA assay and the developed assay for TT. The TT content obtained for the 12 filled microspheres preparations from different sources varied up to 400-fold (range 0.01-4.0 Lf/mg microspheres). The utility of the MAb capture ELISA for detection of total antigenic content in microspheres was confirmed by the observation that the determine TT loading correlated with the theoretical loading predicted from the protein content for the best preparations. Preparations with high loading gave the greatest peak response. There was no relationship between dose and the in vivo immunogenic response, suggesting that encapsulated vaccines with differential loading, release properties and presence of excipients will have different response curves in vivo. Hence, the present assay, when combined with information on toxoid release rate and presence and effect of excipients may be of value in predicting in vivo response.

Animals↗
PubMed1996

Therapeutic botulinum type A toxin: factors affecting potency.

The type A neurotoxin produced by Clostridium botulinum is a potent neuromuscular blocking agent which causes paralysis by preventing the release of neurotransmitter from motor neurons. This property has resulted in the use of the toxin in the treatment of a number of neuromuscular diseases involving muscle spasms. At present, the only recognised assay to estimate accurately the potency of botulinum toxin in clinical preparations is bioassay, in which lethality is used as the endpoint. Such bioassay is inherently variable and large interlaboratory variability has been reported, highlighting problems for standardisation of activity in the absence of any commonly used reference preparation. In the present study, we have confirmed that many different assay conditions can affect potency estimates of clinical formulations of type A botulinum toxin. Further, our studies indicate that different preparations, because of their unique formulation and stability, are differentially affected by some of these assay conditions and that these differences might well contribute to the differences observed in their clinical use.

Analysis of Variance↗
PubMed1996

Physicochemical and immunological studies on the stability of free and microsphere-encapsulated tetanus toxoid in vitro.

The stability of tetanus toxoid released from experimental, slow-release microsphere vaccines was compared with that of free toxoid under different conditions over a 3 month period. The amount of antigenicity remaining, as measured by ELISA, correlated well with loss of toxoid structure, as determined by circular dichroism and fluorescence spectroscopy. At 37 degrees C and low pH, pH 2.5 for free toxoid, or under the acidic conditions generated by the hydrolysis of fast-release microspheres, a gradual unfolding of the polypeptide chain was observed within the first few weeks with more rapid denaturation beyond 30 days.

Drug Stability↗
PubMed1996

Refinement and validation of an alternative bioassay for potency testing of therapeutic botulinum type A toxin.

The type A neurotoxin produced by Clostridium botulinum is a potent neuromuscular blocking agent which causes paralysis by preventing the release of neurotransmitter from motor neurones. This property has led to the use of the toxin in the treatment of a number of neuromuscular diseases involving muscle spasms. At present, the only recognised assay with the specificity and sensitivity to estimate accurately the potency of botulinum toxin in clinical preparations is bioassay, in which lethality is used as the end point. Refinement of this assay, with respect to the end point, was explored on the basis of the development of flaccid paralysis of muscles following subcutaneous injection of the toxin at the inguinocrural region. Potency estimates, relative to in house reference preparations, for different therapeutic preparations obtained using flaccid paralysis as a scored response gave excellent agreement with estimates obtained in independent assay using the currently required control method. This study demonstrates that an alternative, more humane bioassay for potency testing of clostridia neurotoxins gives valid estimates equivalent to those currently in use.

Animals↗
PubMed1996

Requirements for valid alternative assays for testing of biological therapeutic agents.

Several novel toxin-derived biological therapeutic products are now available or under development. There is thus an increasing need to develop alternative assays for their efficacy testing, which currently still depends largely on in vivo methods. Scientific advances in the understanding of the mode of action of these substances has not only fuelled the pace of their development as therapeutic agents but has also provided the basis for improving upon conventional testing procedures.

Animal Testing Alternatives↗