The development of anti-drug antibodies (ADA) against therapeutic proteins can impact upon drug safety and efficacy. This is a major challenge in the development of biotherapeutics. Various factors have the potential to contribute to protein immunogenicity and the production of ADA. Protein aggregation is one of these factors, though the mechanisms underlying aggregate immunogenicity are poorly understood. In this thesis the effect of protein aggregation on immunogenicity has been investigated. The thermal and/or mechanical stresses required in order to achieve subvisible aggregates of three test proteins were determined. Stressed preparations of proteins were characterised using a suite of biophysical techniques, including dynamic light scattering and circular dichroism. The immunogenic potential of subvisible aggregates of a humanised single chain variable fragment (scFv) and ovalbumin (OVA) was studied following intraperitoneal exposure in BALB/c strain mice. Monomeric proteins induced a T helper (Th) 2 dominant immune response, but when aggregated, the responses gained a Th1 phenotype, with a significant increase in the antigen-specific IgG2a antibody response. Cytokine profiles in supernatants taken from splenocyte-dendritic cell co-cultures were also consistent with aggregated preparations of OVA inducing a Th1-type response. Host cell protein (HCP) impurities can also contribute to immunogenicity. Mass spectrometry analysis of an scFv preparation identified the presence of the Escherichia coli (E.coli) heat shock protein DnaK, amongst other HCP, as an impurity. Protein preparations free from DnaK were spiked with recombinant E.coli DnaK to mimic the HCP impurity. The effect of DnaK on the immunogenicity of aggregated and monomeric scFv preparations was then investigated. BALB/c mice were immunised with monomeric and aggregated preparations, with and without E.coli DnaK at 0.1% by mass. Aggregation alone resulted in an enhanced IgG2a antibody response, and the presence of DnaK increased this further. Comparable investigations were also conducted using mouse albumin; here an increase in immunogenicity was observed with protein aggregation, and the presence of DnaK was found to increase the IgG2a response. Collectively, the evidence presented in this thesis shows that aggregation can impact upon the magnitude and character of induced immune responses, and that subvisible aggregation promotes a Th1 immune skewing. Additionally, E.coli HCP DnaK enhances protein aggregate immunogenicity, which indicates that heat shock proteins, as a class of HCP, could have an adjuvant-like effect on biotherapeutic aggregates.

Anthrax is a zoonotic disease affecting most warm-blooded mammals. Primarily recognized as a disease of herbivores, it is caused by a spore-forming, rod-shaped bacterium, Bacillus anthracis. The disease has a worldwide distribution though only of a sporadic nature in developed countries due to effective vaccination and control measures. The current anthrax live spore veterinary vaccine was developed by Max Sterne and its introduction in the 1940s made the control of the disease possible. The principal virulence factors of the B. anthracis are located on two plasmids, pXO1 and pXO2. The pXO1 encodes the toxic factors; protective antigen (PA), lethal and oedema factors (LF and EF) respectively while pXO2 contains the encapsulation genes. Attenuated strains lack either of the two virulence plasmids and consequently have reduced virulence. The Sterne 34F2 strain (tox+, cap-) therefore produces the PA, LF and EF components of the anthrax toxin but lacks the plasmid pXO2 encoding capsule formation and is therefore relatively safe, albeit with some residual virulence. During the development of the avirulent Sterne vaccine, few studies were conducted on the immunogenicity of the vaccine in the target animals. In vivo immunity tests (pathogenicity test) using guinea pigs were mainly used due to few and less sensitive serological diagnostic tools being available at that time. The need for serology in relation to anthrax only became apparent during the development of a human vaccine much after the introduction of the veterinary vaccine. Consequently, though anthrax is recognized as a primary disease of herbivores, there are no records of an in depth study on the immunogenicity and protectivity of the widely accepted strain 34F2 Sterne spore vaccine in any ruminant species. This study was undertaken to fill the present knowledge gap relating to the immunogenicity and duration of protectivity induced by vaccination with the Sterne live spore vaccine in a goat model, which are highly susceptible to the disease. Twenty-one age-matched Boer goats were procured and split into 3 groups of five animals each and vaccinated using different immunization regimens. Six goats served as negative controls utilized in determining the minimum infective dose (MID). Serum samples were collected at intervals for analysis in the laboratory. Bacillus anthracis virulent spore challenge was done not earlier than 3 weeks after the last vaccination and survival was monitored for 14 days. Our findings revealed the MID of virulent B. anthracis spores in naïve goats under experimental conditions to be below 36 spores using the subcutaneous route. Production of anti-anthrax immunoglobulins in the first month following Sterne vaccination was 400-fold higher than pre-vaccination levels with subsequent decline over time to a 50- fold difference, 14 months post vaccination. A similar trend was reflected in the toxin neutralizing antibody titres. There was a correlation between the toxin neutralizing antibody titres and protection against challenge with virulent anthrax spores (P = 0.01). Goats challenged 6 and 62 weeks after vaccination showed a survival rate of 60% and 80% respectively. Those revaccinated one year after the first vaccination were fully protected from virulent anthrax spore challenge. Early pre-validation data of a quantitative indirect ELISA for the detection of anti-PA antibodies was promising and should be further investigated as a tool for anthrax vaccine studies in goats. / Dissertation (MSc)--University of Pretoria, 2012. / Veterinary Tropical Diseases / MSc / Unrestricted

Heterologous enzymes have been investigated for a variety of therapeutic applications, including the treatment of a number of cancers that are sensitive to the systemic depletion of specific amino acids. One such example is acute lymphoblastic leukemia (ALL) for which enzyme-mediated L-Asparagine (L-Asn) depletion by the Escherichia coli L-Asparaginase II (EcAII) has been proven critical for treatment. However, the repeated or prolonged therapeutic administration of such enzymes is restricted by their immunogenicity, which frequently results in the generation of anti-enzyme antibodies that may in turn mediate a variety of adverse hypersensitivity reactions and neutralization of the enzymes themselves. Thus, while the therapeutic efficacy of asparaginase is well established, a significant number of patients still develop adverse immune responses to the enzyme. Here, we have developed and explored novel strategies towards engineering an asparaginase with reduced immunogenicity for ALL therapy. First, we identified and investigated human enzymes that putatively shared functional similarity to asparaginase with the long-term aim of engineering such enzymes to acquire biochemical and pharmacological properties requisite for eventual therapeutic application. In one study, we described the bacterial expression and characterization of the human asparaginase-like protein 1 (hASRGL1). We presented evidence that hASRGL1 exhibited an activity profile consistent with enzymes previously designated as [Beta]-aspartyl peptidases, which had only been previously identified in plants and bacteria. Similar to non-mammalian [Beta]-aspartyl peptidases, hASRGL1 was revealed to be an N-terminal nucleophile (Ntn) hydrolase whereby Thr168 serves as the essential Ntn for both intramolecular processing and catalysis. In a second study, we described the optimized bacterial expression and biochemical characterization of the human N-terminal asparagine amidohydrolase 1 (hNTAN1). We demonstrated that hNTAN1 catalysis is dependent upon direct involvement of a thiol group, and subsequently identified Cys75 as an essential residue that may act as the catalytic nucleophile. Further, we presented the first description of hNTAN1 kinetics, secondary structure composition, and thermal stability. Second, we devised and validated a novel therapeutic deimmunization approach by combinatorial T-cell epitope removal using neutral drift. We showed that combinatorial saturation mutagenesis coupled with a robust neutral drift screen enabled the isolation of engineered EcAII variants that contained multiple amino acid substitutions yet exhibited catalytic efficiencies nearly indistinguishable to that of the parent enzyme. Three regions of EcAII were computationally identified as putative T-cell epitopes and then subjected to saturation mutagenesis at 4 positions (per region) believed to be critical for MHC-II binding. The resulting libraries were then sequentially subjected to a neutral drift FACS screen in order to isolate EcAII mutants that retained wild-type function. Pools of neutral drift variants were then computationally evaluated for MHC-II binding and those that displayed scores indicative of compromised binding were purified and biochemically characterized. Finally, T-cell activation assays and antibody titers in HLA-transgenic mice were used to evaluate T-cell epitope removal and immunogenicity, respectively. Ultimately, we revealed that mice immunized with an EcAII neutral variant containing 8 amino acid substitutions -- 3 of which were non-phylogenetically conserved -- within computationally predicted T-cell epitopes, displayed a significant 10-fold reduction in serum anti-EcAII IgG titer relative to mice similarly immunized with the parent enzyme. / text

Despite the use of potent immunosuppressive agents, injury secondary to the alloimmune response principally directed against mismatched HLA antigens, remains a significant cause of renal transplant dysfunction and loss. One of the principal ways to limit the detrimental effect of immune alloreactivity towards the graft is through minimisation of donor-recipient HLA mismatches, but this has not changed conceptually since the early period of clinical transplantation. The central hypothesis of the research described in this thesis is that assessment of histocompatibility based on evaluation of structural differences between donor and recipient HLA molecules has the potential to improve graft outcomes after kidney transplantation. Amino acid sequence analyses have been central to gaining an understanding of the relative immunogenicity of a mismatched donor HLA antigen in the context of a recipient's HLA repertoire. While these methods offer advantages over basic enumeration of HLA mismatches, regardless of how sophisticated they may be, they are unable to fully describe the intricate nature of the B cell epitope--BCR interaction that initiates a humoral response. This interaction requires, but is not limited to, structural and electrostatic potential complementarity. The aim of this thesis is to describe a fully structural description of HLA immunogenicity through analysis of the three-dimensional physicochemical environment at the surface of the HLA molecule. First, I describe the bioinformatic techniques that integrate HLA sequence and X-ray crystallographic data to produce accurate models of HLA molecular structure and, subsequently, calculate the electrostatic potential in the three-dimensional area surrounding the molecule. Through quantitative comparison of the three-dimensional electrostatic potential on the surface of each HLA molecule, Electrostatic Mismatch Score 3D (EMS-3D) is derived. EMS-3D represents the electrostatic disparity between a group of HLA molecules. Subsequently, I demonstrate, using examples of HLA mutagenesis studies described in the literature, that patterns of antigenicity are better explained through analysis of surface electrostatic potential than by analysis of sequence. Of greater clinical interest, the ability of EMS-3D to predict the development of \textit{de novo} alloantibody responses was assessed in a cohort of patients who received a standardised immunisation and had their antibody profile characterised using Luminex single-antigen beads. This analysis validated EMS-3D as a valuable predictor of the development and magnitude of an alloantibody response, and therefore as a potentially useful clinical tool in the allocation of donor organs. The clinical utility of EMS-3D was assessed in a national cohort of kidney transplants, which found EMS-3D to better predict transplant outcome than conventional, currently-employed, measures of histocompatibility. HLA polymorphisms are implicated in many disease processes, particularly in autoimmunity. In the last chapter, the methodology developed in this thesis is employed to analyse the association between particular HLA polymorphisms and Inflammatory Bowel Disease (IBD). Analyses of the electrostatic potential within the HLA peptide binding groove found that particular patterns of electrostatic potential within the HLA peptide binding groove were associated with disease phenotype. Future work will examine how the examination of multiple discrete regions, ostensibly epitopes, can further improve the prediction of the HLA alloimmune response.

No description available.

No description available.

BACKGROUND Yellow fever (YF) is a non-contagious, mosquito borne haemorrhagic fever caused by a single-strand RNA flaviviruses. YF is endemic in the tropics primarily in South America and Africa although the vectors are present in Asia, Europe, Pacific and Middle East. Human beings serve as viraemic hosts for mosquito infection. YF carries a high burden of disease, particularly in developing countries with up to 200 000 cases reported annually and a case fatality rate of 20-50%.The pathogenesis is poorly understood and little research has been conducted .There is no known cure or specific treatment for YF and prevention remains the mainstay the public health approach in terms of effectiveness and cost. The World Health Organisation (WHO) conventions have made vaccination mandatory for travel to endemic countries to prevent outbreaks and transmission to susceptible individuals. YF vaccine is one of the oldest vaccines known and in use and is derived from an attenuated virus strain 17D originally produced in the 1930s. The vaccine has historically been considered effective and safe. However, severe life-threatening side effects to the vaccine have been reported in the past 20 years. Acute vaccinerelated viscerotropic (AVD) and neurotropic (AND) side effects have been reported globally particularly in the elderly. The adverse reactions typically present as YF- like illness resulting in multi-organ failure with death as a possible outcome. OBJECTIVES To estimate the immunogenicity and toxicity of 17D and 17DD YF vaccines by summarizing the available data from randomised controlled trials. STUDY DESIGN A summary of randomized controlled trials (RCT) of YF vaccine immunogenicity and safety and tolerability was obtained using standard meta-analysis methodologies. METHODS A comprehensive literature search was conducted in order to identify trial that met with predetermined inclusion and exclusion criteria. Features of each study were noted taking into account the type of vaccine used, the duration of follow up, assignment to intervention, blinding and randomization methods. Three studies were eventually pooled and effect size estimates reported in each study were noted and analysed using meta-analysis software, MIX. Reports on the side effects post vaccination were summarized and analysed. RESULTS The difference in outcomes between the standard 17DD YF vaccines intervention, traded as Arilvax ® and the 17D YF vaccines traded as YF-Vax ® and Stamaril ® was negligible in terms of effect size. Effect sizes that considered the means between the treatment and control groups demonstrated a difference that favoured the control group viz. Arilvax ®. The pooled results also showed significant publication bias most likely attributable to the small number of studies considered. The pooled and annotated forest plot supported the available literature in confirming the effectiveness of YF vaccines in conferring immunity. A summary of tolerability events CONCLUSIONS This study has confirmed the effectiveness of YF vaccines in terms of immunogenicity and also demonstrated that YF vaccines are well tolerated and safe The small number of study units considered in this study presented challenges for analysis and for interpretation but highlighted the need for more research to be conducted in this area. The results are in keeping with the existing body of evidence supporting the robustness of the immunological response to YF vaccination. The safety and tolerability of the vaccine established in this study was also consistent with known literature. There are important implications for further research and implementation that became evident such as the need for further studies to be conducted in African populations where the burden of disease is highest. / Dissertation (MSc)--University of Pretoria, 2010. / Clinical Epidemiology / unrestricted

No description available.

CARACTERISATION DE VACCINS A BASE DE GLUCIDESVariables influençant l'immunogénicité et propriétés physico-chimiques des vaccins glycoconjuguésDe nombreux aspects peuvent influer sur l'immunogénicité des vaccins conjugués et les principales variables étudiées jusqu'ici sont la taille du fragment saccharide et la nature des glycosides: taux de protéine dans le conjugué purifié, la stratégie de conjugaison, nature de l’espaceur et la protéine porteuse.La taille de la partie saccharidique et le ratio de cette partie / protéine a été étudiée dans différents travaux de Seppälä et Mäkelä,.Dans l'une des premières études sur l'effet de la taille sur l'immunogénicité de la protéine conjuguées à des dextrans, il a été montré que des dextrans de faible poids moléculaire conjugué à l’albumine sérique de poluet induit des réponses anti-dextran fortes chez la souris. L’augmentation de la taille du dextran, a abouti à une réduction de l’immunogénicité. Peeters et al. a montré qu’un tétramère synthétique de Hib unité capsulaire polysaccharide, conjuguée à un support protéique, induit chez des souris adultes, des niveaux d'anticorps primates non humains comparables à un conjugué Hib commercial. Ces niveaux sont plus élevées que ceux induits par un trimère répété, ce qui indique que pour le Hib un minimum de huit sucres est nécessaire pour une bonne réponse immunologique. Laferrière et al. a trouvé peu d'influence de la longueur de la chaîne glucidique sur l'immunogénicité des vaccins antipneumococciques conjugués chez la souris. Pozsgay et al. a étudié chez la souris, l'immunogénicité de l’oligosaccharides du LPS de Shigella dysenteriae de type 1 conjugué à l'albumine sérique humaine (HSA). Les auteurs ont constaté que les octa-, dodéca-, et des fragments de hexadécasaccharides induit des niveaux élevés d'anticorps IgG après trois injections. Ces niveaux sont supérieurs à ceux obtenus avec un conjugué tétrasaccharidique. L'influence du ratio glucides / protéine est différente pour les trois conjugués. Le conjugué octasaccharide-HSA avec la plus forte densité provoque une bonne réponse immunitaire, tandis que dans le cas des conjugués dodéca- et hexadécasaccharides, la densité médiane est optimal. Ces études suggèrent que la longueur de la chaîne d'oligosaccharides et le chargement de l’haptène peuvent être liés entre eux pour déterminer l'immunogénicité des vaccins glycoconjugués.L'espaceur est une molécule linéaire courte qui est généralement liée à la chaîne polysaccharidique et la protéine ou de fragments. Il ya des évidences dans la littérature qui suggèrent que les espaceurs rigides, contraints comme le maléimide cyclohexyle, provoquent une importante quantité d'anticorps indésirables, avec le risque de conduire à une réponse immunitaire éloignée de l'épitope ciblé sur la haptène. L'utilisation d'un alkyle souple type maleimido a été rapporté comme un moyen de surmonter l'immunogénicité observée précédemment. Un certain nombre de transporteurs protéiques ont été utilisés jusqu'ici dans l'évaluation préclinique et clinique de vaccins conjugués. Des protéines telles que les anatoxines diphtériques et tétaniques, qui dérivent des toxines respectives, après la décontamination chimique avec le formaldéhyde, ont été initialement choisies comme transporteur en raison de inocuité (tétanos et la vaccination contre la diphtérie). CRM 197, un mutant non toxique de la toxine 61 de la diphtérie a été largement utilisé comme support pour Hib. Un complexe protéique de la membrane externe de méningocoque du sérogroupe B a été utilisé par Merck comme support pour leur vaccin conjugué Hib. GSK dans leur vaccin antipneumococcique, conjugué multivalent, introduit la protéine D Hib liée à la plupart des polysaccharides inclus dans le vaccin. L'équipe de John Robbins fait un large usage de la forme recombinante non toxique de l’exo-toxine de Pseudomonas aeruginosa comme support contre Staphylococcus aureus de type 5 et 8 ainsi que pour Salmonella. / CHARACTERIZATION OF CARBOHYDRATE BASED VACCINES Variables influencing the immunogenicity and physicochemical properties of glycoconjugate vaccinesMany aspects can influence the immunogenicity of conjugate vaccines and the main variables investigated so far are the size of the saccharide moiety, the saccharide:protein ratio in the purified conjugate, the conjugation strategy, the nature of the spacer and the protein carrier. The size of the saccharide moiety and saccharide/protein ratio were investigated in different works such as Seppälä and Mäkelä in one of the first studies on the effect of size and chemistry on the immunogenicity of dextrans-protein conjugates found that dextrans of low molecular weight conjugated to chicken serum albumin, induced strong anti-dextran responses in mice, while increasing the dextrans' size resulted in reduced immunogenicity.47 Peeters et al. showed that a synthetic tetramer of Hib capsular polysaccharide repeating unit, conjugated to a protein carrier, induced in adult mice and non-human primates antibody levels comparable to a commercial Hib conjugate and higher than those induced by a trimer, indicating that for Hib a minimum of eight sugars is needed for a proper immunological response.48 Laferriere et al. found little influence of the carbohydrate chain length on the immunogenicity of pneumococcal conjugate vaccines in mice.49 Pozsgay et al. studied the immunogenicity in mice of synthetic Shigella dysenteriae type 1 LPS oligosaccharides conjugated to human serum albumin (HSA). The authors found that octa-, dodeca-, and hexadecasaccharide fragments induced high levels of lipopolysaccharide binding IgG antibodies in mice after three injections and were superior to a tetrasaccharide conjugate. The influence of the carbohydrate/protein ratio was different for the three conjugates. The octasaccharide-HSA conjugate with the highest density evoked a good immune response, while in the case of dodeca- and hexadecasaccharide conjugates, the median density was optimal.50 These studies suggest that oligosaccharide chain length and hapten loading might be interconnected in determining the immunogenicity of glycoconjugate vaccines. The spacer is a short linear molecule that is generally linked to the polysaccharide chain or to the protein or to both moieties, depending on the chemistry, used to facilitate the coupling between the protein and sugar. There are evidences in the literature which suggest that rigid, constrained spacers like cyclohexyl maleimide, elicit a significant amount of undesirable antibodies, with the risk of driving the immune response away from the targeted epitope on the hapten.51 52 The use of a flexible alkyl type maleimido spacer has been reported as a way to overcome the previous observed immunogenicity of cyclic maleimide linkers.53 A number of protein carriers have been used so far in preclinical and clinical evaluation of conjugate vaccines. 54 55 56 57 58 59 60Proteins such as diphtheria and tetanus toxoids, which derive from the respective toxins after chemical detoxification with formaldehyde, were initially selected as carrier because of the safety track record accumulated with tetanus and diphtheria vaccination. CRM197, a non-toxic mutant of diphtheria toxin61 which instead does not need chemical detoxification, has been extensively used as carrier for licensed Hib, pneumococcal, meningococcal conjugate vaccines and for other vaccines being developed. An outer membrane protein complex of serogroup B meningococcus has been used by Merck as carrier for their Hib conjugate vaccine.62 GSK in their multivalent pneumococcal conjugate vaccine introduced the use of the Hib-related protein D as carrier for most of the polysaccharides included into the vaccine.63 64 The team of John Robbins made extensive use of the recombinant non toxic form of Pseudomonas aeruginosa exo-toxin as carrier for Staphylococcus aureus type 5 and 8 as well as for Salmonella

Biologic therapies have revolutionized the treatment of rheumatoid arthritis, a common chronic inflammatory disease, mainly characterized by the chronic inflammation of the joints. The activity and progression of the disease are highly variable, both between subjects and between the successive assessments for the same subject. Standardized assessments of clinical variables have been developed to reflect the disease activity and evaluate new therapies. Pharmacokinetics-pharmacodynamic (PKPD) models and methods for analyzing the generated time-course data are needed to improve the interpretation of the clinical trials’ outcomes, and to describe the variability between subjects, including patients characteristics, disease factors and the use of concomitant treatments that may affect the response to treatment. In addition, good simulation properties are also desirable for predicting clinical responses for various populations or for different dosing schedules. The aim of this thesis was to develop methods and models for analyzing pharmacokinetic and pharmacokinetic-pharmacodynamic (PKPD) data from rheumatoid arthritis patients, illustrated by treatment with a new anti-TNFα biologic drug under clinical development, certolizumab pegol. Two models were developed that characterized the relationship between the exposure to the drug and the efficacy ACR variables that represent improvement of the disease; a logistic-type Markov model for 20% improvement (ACR20) and a continuous-type Markov model for simultaneous analysis of 20% (ACR20), 50% (ACR50) and 70% (ACR70) improvement. Both models accounted for the within-subjects correlation in the successive clinical assessments and were able to capture the observed ACR responses over time. Simulations from these models of the ACR20 response rate supported dosing regimens of 400 mg at weeks 0, 2 and 4 to achieve a rapid onset of response to the treatment, followed by 200 mg every 2 weeks, or alternative maintenance regimen of 400 mg every 4 weeks. The immunogenicity induced by the biologic drug was characterized by a time to event model describing the time to appearance of antibodies directed against the drug. The immunogenicity was predicted to appear mainly during the first 3 months following the start of the treatment and to be reduced at higher trough concentrations of CZP, as well as with concomitant administration of MTX. The full time-course of sequential events, such as dose-exposure-efficacy relations, is most accurately described by a simultaneous analysis of all data. However, due to the complexity and runtime limitations of such an analysis, alternatives are often used. In this thesis, a method, IPPSE, was developed and compared to the reference simultaneous method and to existing alternative methods. The IPPSE method was shown to provide accuracy and precision of estimates similar to the simultaneous method, but with easier implementation and shorter run times. In conclusion, two PKPD models and one immunogenicity model were developed for evaluation of the response of a biologic drug against rheumatoid arthritis that allowed accurate analysis and simulation of clinical trial data, as well as serving as examples for how a model-informed basis for decisions about biological drugs can be created.