Rotavirus diarrhea causes a disproportionate amount of the world's childhood mortality. Approximately 611,000 children die each year due to complications of rotavirus infections. In this study we evaluate rotavirus vaccination using four different methods. We look at the epidemiological history of the disease and vaccination against the disease, then we evaluate the effectiveness of vaccination first using a cost-benefit analysis, then using an ordinary differential equations based model, and last through computer simulations in Matlab. We do a traditional cost-benefit analysis as suggested by the Public Health Service of the United States to evaluate the costs and benefits of implementing a rotavirus vaccination program in Egypt with the RotaRix vaccine. Our results show that given the current standards of care in Egypt, it would be more cost-beneficial for Egypt not to use the rotavirus vaccine. We formulate a model of the spread of rotavirus diarrhea based on a continuous time ordinary differential equations model of two viral strains of influenza. We expand this influenza model to include the case of co-infection. We further expand the original model to explore the effects of vaccination. We used computer simulations to further analyze the effect of vaccination as a control method. These simulations show that the spread of the disease is highly sensitive to the levels of cross-immunity between the strains, and the level of vaccination in the population. We found that the dynamics observed in the new model are similar to the dynamics observed in the original model. We found the minimum levels of vaccination necessary in this model to eradicate severe rotavirus disease and minimum levels of cross-immunity between the strains

Although it is known that cell-mediated immunity (CMI) plays a key role in protection against brucellosis, the exact immune mechanisms leading to protection are still not fully understood. Better understanding of the mechanisms would help in the development of a human Brucella vaccine and help in improving animal vaccines. In this research, B. abortus strain RB51 and a closely-related, nonpathogenic Ochrobactrum anthropi (strain 49237) bacterium were used to study the immune response against brucellosis in mice. Both O. anthropi strain 49237 and recombinant strain 49237 expressing Brucella protective antigen copper-zinc superoxide dismutase (Cu/Zn SOD) induced a mix of Th1 and Th2 type immune responses but failed to provide protection against virulent Brucella challenge. After changing the immune response to a predominantly Th1 type of response using CpG oligonucleotides as an adjuvant, both strains provided protection with the recombinant strain inducing significantly higher protection. It was also demonstrated that vaccination with strain RB51 induced Th1 immune responses characterized by high interferon-gamma (IFN-g) production with no interleukin-4 (IL-4) secretion as well as high IgG2a and minimal IgG1 production. A colorimetric cytotoxic T lymphocytes (CTL) assay was developed to demonstrate that strain RB51 induced an antigen-specific CTL reaction that probably plays an important role in protection. The results suggest that optimal protection against brucellosis requires IFN-g-secreting T cells and antigen-specific CTLs. Recombinant strain RB51 overexpressing Brucella Cu/Zn SOD and simultaneously expressing mycobacterial 85A antigen induced higher IFN-g production and CTL activity than the parent RB51 strain. The combined results suggest that the recombinant O. anthropi strain could be used as a human vaccine against brucellosis and that the recombinant RB51 strain could be used as an effective vaccine against both brucellosis and tuberculosis in animals. / Ph. D.

Malaria is a severe public health concern in certain regions, causing 445,000 deaths and over 200 million cases in 2016 (Ashley et al., 2018). The vast majority of these cases and deaths are located in warm climates where the Anopheles mosquito is present, especially in sub-Saharan Africa and southeast Asia. Current efforts aim to prevent the disease through vaccination, which has proven to be challenging. Plasmodium falciparum, the pathogen responsible for malaria, is transmitted between humans via the female vector Anopheles mosquito. This parasite has a complex life cycle that is not fully understood, making it difficult to treat the infection and even more difficult to inoculate a population through vaccination. P. falciparum is also capable of polymorphism, changing structure once a host antibody has identified a pathogenic antigen. For this reason, it is very technically challenging to develop a vaccine that is able to confer a high enough immune response through sufficient host antibody production. This thesis will begin with a review on the prevalence and severity of malaria and an overview of the principles of immunology and vaccine development. We will then discuss the parasitic life cycle and how it results in the pathogenesis of the disease. Current antimalarial treatments and resistance to those treatments will be analyzed. This thesis will conclude with an in-depth analysis of the current prophylactic vaccines against P. falciparum, focusing on their mechanism, efficacy, and probability of success.

African horse sickness (AHS) is a life-threatening disease of equids caused by African horse sickness virus (AHSV), a member of the genus Orbivirus in the family Reoviridae. The virus is transmitted by midges (Culicoides spp.) and the disease is most prevalent during the time of year, and in areas where vector Culicoides spp. are most abundant, namely in late summer in the summer rainfall areas of endemic regions. The disease is of importance to health and international trade in horses worldwide. Effective surveillance is critical in order to establish transparent criteria for animal trade from a country or region where AHS occurs. / The 2011 outbreak of African horse sickness in the African horse sickness controlled area in South Africa: An outbreak of AHS caused by AHSV type one (AHSV1) occurred in the surveillance zone of the AHS controlled area of the Western Cape during the summer of 2011. The epicentre of the outbreak was the town of Mamre in the magisterial district of Malmesbury, and the outbreak was confined to a defined containment zone within this area through movement control of all equids and a blanket vaccination campaign. A total of 73 confirmed cases of AHS were reported during this outbreak, which included four subclinical cases confirmed by virus isolation (VI). The estimated morbidity rate for the outbreak was 16% with an estimated mortality rate of 14% and a case fatality rate of 88% based on the figures above. Outbreak disease surveillance relied on agent identification using AHSV group specific reverse transcriptase quantitative polymerase chain reaction (GS RT-qPCR) based assays, which was novel for an AHS outbreak in South Africa. The source of this outbreak was not confirmed at the time, but was believed to be associated with an illegal 2 movement of an infected animal into the Mamre area. A detailed description of the outbreak is given in Chapter 2, and the outbreak provided an opportunity to assess decision making in future AHS outbreaks in the AHS controlled area of South Africa and in countries where AHS is an exotic or emerging disease. This outbreak further highlighted deficiencies and complications of available AHSV diagnostic testing and surveillance methods, and the need for further refinement of these assays and strategies. / Development of three triplex real-time reverse transcription PCR assays for the qualitative molecular typing of the nine types of African horse sickness virus: The typing of the specific AHSV involved in the Mamre outbreak was initially done by partial, direct sequencing of the S10 gene (encoding the non-structural protein NS3) and the L2 gene (encoding the type-specific outer capsid protein VP2) which confirmed the virus to be AHSV1. This process is time consuming and it became evident that a faster alternative was needed. This led to the development of type specific RT-qPCR (TS RT-qPCR) assays to supplement the GS RT-qPCR assay that had already been developed, characterized and validated. Blood samples collected during routine diagnostic investigations from South African horses with clinical signs suggestive of AHS were subjected to analysis with the GS RT-qPCR assay and VI with subsequent serotyping by plaque inhibition (PI) assays using AHSV type-specific antisera. Blood samples that tested positive by AHSV GS RT-qPCR were then selected for analysis using AHSV TS RT-qPCR assays. The TS RT-qPCR assays were evaluated using both historic stocks of the South African reference strains of each of the 9 AHSV types, as well as recently derived stocks of these same viruses. Of the 503 horse blood samples tested, 156 were positive by both AHSV GS RT-qPCR and VI assays, whereas 135 samples that were VI negative were positive by AHSV GS RT-qPCR assay. The virus isolates made from the various blood samples included all 9 AHSV types, and there was 100% agreement between the results of conventional serotyping of individual virus isolates by PI assay and AHSV TS RT-qPCR typing results. Results of this study confirmed that the AHSV TS RT-qPCR assays for the identification of individual AHSV types are applicable and practicable and therefore are potentially highly useful and appropriate for virus typing in AHS outbreak situations in endemic or sporadic incursion areas, which can be crucial in determining appropriate and timely vaccination and control strategies. / Evaluation of the use of foals for active surveillance in an AHS containment zone during the season following an AHS outbreak: In order to further evaluate the AHS status of horses in the Mamre area after the outbreak of 2011, a targeted surveillance strategy was developed. Serial serum and whole blood samples were collected on a monthly basis from January to June, 2012 from foals (identified by microchip) that were born in the Mamre 3 district after the end of the outbreak. Sera were evaluated using traditional serological methods and the results were compared to the results obtained using the newly developed molecular assays for virus detection and identification. This study confirmed that AHSV was eradicated in the Mamre area after the outbreak and, therefore, that the control measures implemented in the area by the State Veterinary Authorities were effective. / Characterization of the dynamics of African horse sickness virus in horses by assessing the RNAaemia and serological responses following immunisation with a commercial polyvalent live attenuated vaccine: As was shown in the 2011 Mamre outbreak, detection of AHSV during outbreaks has become more rapid and efficient with the recent development of quantitative GS RT-qPCR assays to detect AHSV nucleic acid. Use of this assay together with the TS RT-qPCR assays described in Chapter 3, will not only expedite diagnosis of AHS but also facilitate further evaluation of the dynamics of AHSV infection in the equine host. A potential limitation to the application of these assays is that they detect viral nucleic acid originating from any AHSV live attenuated vaccine (AHSVLAV), which is the vaccine type routinely administered to horses in South Africa. A study was, therefore, designed to characterize the dynamics and duration of the RNAaemia as compared to the serological responses of horses following vaccination with a commercial AHSV-LAV, using GS and TS RT-qPCR assays and serum neutralisation tests. This study provided baseline data on the GS and TS nucleic acid dynamics in weanling foals vaccinated for the first time, yearlings vaccinated for a second time and adult mares following a booster to multiple previous vaccinations. These data are fundamental to interpreting results of AHSV GS RT-qPCR testing of vaccinated horses within an area where virological surveillance is being applied. / African horse sickness caused by genome reassortment and reversion to virulence of live, attenuated vaccine viruses, South Africa, 2004 - 2014: In 2014 a further outbreak of AHS caused by AHSV1 occurred in the Porterville area of the AHS protection zone (PZ), spreading into the Wellington area in the AHS surveillance zone (SZ). Further involvement of the Robertson area (AHS PZ) subsequently also occurred. The case fatality rate was much lower than that of the Mamre outbreak. The clinical signs in infected horses were also generally milder in the 2014 outbreak, as compared to the 2011 outbreak. Whole genome sequencing of samples from the Porterville outbreak confirmed that causative virus was a recombination (reassortant) of AHSV types 1 and 4, with genes derived from the relevant vaccine strains contained in OBP comb1 of the commercial polyvalent AHSV-LAV used in South Africa. This led to further analysis of 39 AHSV strains from field cases of AHS that originated from outbreaks within the controlled area, which confirmed reversion to virulence 4 of AHSV type 1 vaccine in two outbreaks (2004 and 2011) and multiple reassortment events in two outbreaks (2004 and 2014) with genes derived from all three AHSV vaccine strains (types 1, 3 and 4). This study provided a molecular and epidemiological comparison of the five unique AHSV type 1 outbreaks in the AHS controlled area. It was shown that all the outbreaks in the AHS controlled area attributed to AHSV type 1 since the inception of the area in 1997, have been due either to reversion to virulence of the AHSV type 1 vaccine strain, or recombination of AHSV type 1 vaccine strain with one or both of the other vaccine strains in OBP comb1 of the commercial AHSV-LAV. / Thesis (PhD)--University of Pretoria, 2016. / ERC / Racing South Africa (Pty) Ltd / Equine Health Fund / Mary Slack and Daughters Foundation / THRIP / National Research Foundation / Veterinary Tropical Diseases / PhD / Unrestricted

La leishmaniose est une maladie tropicale négligée à transmission vectorielle qui est endémique dans 98 pays dont les plus pauvres. Vingt espèces de Leishmania sont capables d’établir une infection intracellulaire au sein des macrophages humains, provoquant différentes manifestations cliniques. Le développement d'un vaccin contre les leishmanioses est étayé par des preuves d'immunité naturelle contre l'infection, induite par une réponse à médiation cellulaire de type Th1 dominante associée à la production d'IFN-γ, d'IL-2 et de TNF-α par des cellules T polyfonctionnelles TCD4+ et TCD8+, conduisant à l'activation classique des macrophages entrainant la destruction des parasites. Induire une protection robuste et durable et déterminer les épitopes immunodominants responsables de la protection naturelle représente un véritable défi.Les protéines sécrétées sont des facteurs de virulence jouant un rôle important dans le cycle de vie des leishmanies et sont capables d’induire une protection durable chez le chien, un bon modèle pour l’infection humaine. Notre objectif est de développer, à partir du sécrétome de Leishmania, un vaccin de seconde génération reproductible et facile à produire à bas prix dans les zones d’endémie, avec des rendements de production rendant possible son utilisation à grande échelle.Les sécrétomes des six espèces les plus pathogènes de leishmanie (plus L. tarentolae) ont été analysés et comparées par spectrométrie de masse. Les antigènes candidats ont été recherchés dans l'ensemble des données disponibles (analyses protéomiques, littérature…). 52 antigènes candidats vaccin ont ainsi été sélectionnés, dont 28 avaient déjà été décrits et 24 sont nouveaux et découverts grâce à une approche de vaccinologie réverse.Une analyse de la prédiction de liaison des épitopes in silico HLA-I et –II a été réalisée sur tous les antigènes candidats vaccin, prenant ainsi en compte le polymorphisme HLA de la population mondiale. Pour sélectionner les meilleurs épitopes parmi des milliers d’épitopes potentiels, un script R automatisé a été développé en interne, selon des critères rationnels stricts. Ainsi, 50 épitopes de classe I et 24 épitopes de classe II ont été sélectionnés et synthétisés sous forme de peptides individuels. Des essais de toxicité in vitro ont montré l’absence de toxicité cellulaire de ces peptides.Les individus guéris par chimiothérapie généralement développent des réponses immunitaires protectrices à Leishmania. Des tests de stimulation des PBMC ont donc été réalisés avec des échantillons biologiques provenant de donneurs guéris de Tunisie et la production d'IFN-γ a été évaluée par ELISpot. De plus, il était important d'inclure dans l'étape de validation expérimentale des peptides des échantillons provenant d’individus naïfs, population cible à vacciner avec un vaccin prophylactique. Les résultats montrent que des peptides spécifiques de Leishmania induisent avec succès la production d'IFN-γ par les PBMC totaux provenant de donneurs guéris et par les lymphocytes T spécifiques amplifiés à partir du répertoire naïf.Globalement, la validation expérimentale des peptides réalisée exclusivement sur des échantillons humains nous fournira une base préclinique très solide pour développer un vaccin efficace capable de protéger les populations touchées par ces maladies. Elle constituera un moyen sûr et rentable de mieux sélectionner les candidats retenus pour le vaccin et d'éliminer ceux qui présentent un risque d'échec élevé au tout début du processus de développement du vaccin.Grâce à la combinaison de l'analyse protéomique et d'outils in silico, des candidats peptidiques prometteurs ont été rapidement identifiés pour le développement d'un vaccin. Le « pipeline » de développement préclinique du vaccin proposé fournit une sélection rapide de peptides immunogènes, offrant une approche puissante pour accélérer le déploiement d'un vaccin pan-spécifique efficace contre les leishmanioses. / Leishmaniasis is a vector-borne neglected tropical disease endemic to 98 countries worldwide. Twenty Leishmania species are capable of establishing intracellular infection within human macrophages, causing different clinical presentations. Vaccine development against leishmaniases is supported by evidence of natural immunity against infection, mediated by a dominant cellular Th1 response and production of IFN-γ, IL-2 and TNF-α by polyfunctional TCD4+ and TCD8+ cells, ultimately leading to macrophage activation and parasite killing.Excreted-secreted proteins are important virulence factors present throughout Leishmania life stages and are able to induce durable protection in dogs, a good model for human infection. We aim to develop a second generation vaccine from the Leishmania secretome, with the potential for large scale dissemination in a cost-effective, reproducible approach.The secretome of six main pathogenic species (plus L. tarentolae) was analysed by Mass-Spectrometry and conserved candidate antigens were searched in the complete dataset. A total of 52 vaccine antigen candidates were selected, including 28 previously described vaccine candidates, and an additional 24 new candidates discovered through a reverse vaccinology approach.In silico HLA-I and –II epitope binding prediction analysis was performed on all selected vaccine antigens, with world coverage regarding HLA restriction. To select the best epitopes, an automated R script was developed in-house, according to strict rational criteria. From thousands of potential epitopes, the automated script, in combination with optimal IC50, homology to host and solubility properties, allowed us to select 50 class I and 24 class II epitopes, synthesized as individual peptides. In vitro toxicity assays showed these selected peptides are non-toxic to cells.The peptides’ immunogenicity was evaluated using immunoscreening assays with immune cells from human donors, allowing for the validation of in silico epitope predictions and selection, and the assessment of the peptide’s immunogenicity and prophylactic potential. Healed individuals, which had active infection and received treatment, possess Leishmania-specific memory responses and are resistant to reinfection, being considered the gold standard of protective immunity. On the other hand, the naive population is extremely important to include in the experimental validation step since it is the target population to vaccinate with a prophylactic vaccine. Importantly, a minimum specific T-cell precursor frequency is needed to induce long-lasting memory protective responses. Furthermore, there is also a positive correlation between immunodominant epitopes and a high frequency of specific T-cell precursors. Peptides able to induce Th1 and/or cytotoxic immune responses in both background are promising candidates for a vaccine formulation. Altogether,experimental validation exclusively in human samples will provide us a very strong base for a vaccine formulation and allow to accelerate translation to the field.Results show Leishmania-specific peptides successfully induce IFN-γ production by total PBMC from healed donors, and by specific T cells amplified from the naïve repertoire. Preliminary evidence exists for peptides which are immunogenic in both immune backgrounds (eight HLA-class I 9-mer peptides and five class II 15-mer peptides) which are, for now, the most promising candidates to advance for the multi-epitope peptide design.Through the combination of proteomic analysis and in silico tools, promising peptide candidates were swiftly identified and the secretome was further established as an optimal starting point for vaccine development. The proposed vaccine preclinical development pipeline delivered a rapid selection of immunogenic peptides, providing a powerful approach to fast-track the deployment of an effective pan-specific vaccine against Leishmaniases.

Respiratory disease complex is a major cause of mortality and economic losses in the commercial broiler industry. In 1991 a previously unidentified bacterium associated with respiratory disease and cranial cellulitis was isolated from broilers in the then Transvaal Province (van Beek, van Empel, van den Bosch, Storm, Bongers, du Preez, 1994. ). In 1994 the organism was named Ornithobacterium rhinotracheale (Vandamme, Segers, Vancanneyt, van Hove, Mutters, Hommez, Dewhirst, Paster, Kersters, Falsen, Devriese, Bisgaard, Hinz, Mannheim, 1994.). Since then Ornithobacterium rhinotracheale has been isolated worldwide from chickens and turkeys showing respiratory signs and has become well established as contributing to the respiratory disease complex in both species (van Empel, Hafez, 1999). In South Africa respiratory disease and Ornithobacterium rhinotracheale in particular is routinely controlled by the inclusion of antibiotics such as Oxtetracycline into the feed of broilers during rearing. Concerns about antibiotic residues in poultry meat for human consumption as well as evidence that suggests that Ornithobacterium rhinotracheale readily develops resistance to antibiotics (Devriese, Hommez, Vandamme, Kersters, Haesebrouck, 1995), make this strategy unsustainable. It was with a view to reducing producers’ dependence on long term prophylactic antibiotic therapy that this study to determine the safety and efficacy of an OR bacterin vaccine was carried out. Injection of the bacterin into broilers was deemed impractical on a commercial scale, so it was applied to broiler breeder parent stock in order that they could protect their progeny through vertically transmitted immunity developed as a result of vaccination. Breeder flocks were vaccinated intramuscularly at nine and 18 weeks with a monovalent bacterin based on OR serotype A with oil adjuvant. Vaccine safety was evaluated by palpation of vaccination sites and clinical observation of breeders for two weeks after vaccination. The serological response of breeders to vaccination was monitored using an ELISA test for Ornithobacterium rhinotracheale optimised for use under South African conditions. Vaccine efficacy was determined by monitoring of broiler progeny of vaccinated breeders raised under commercial conditions as well as through controlled challenge studies with Ornithobacterium rhinotracheale under laboratory conditions. In order to determine the financial consequences of using the test vaccine, a partial farm budget was drawn up from available broiler data and possible outcomes were modelled using a stochastic model. The vaccine proved to be safe for use in commercial broiler breeders and vaccinated birds developed a good humoral response to vaccination. As a result of cross-contamination of isolators with Ornithobacterium rhinotracheale the results of the challenge studies were inconclusive. No evidence of protection of broiler progeny of vaccinated breeder flocks could be detected through the challenge trials. In the absence of in-feed medication, broilers hatched from vaccinated breeders did, however, performed better under commercial conditions than those hatched from unvaccinated breeder flocks. The partial farm budget showed that broilers raised from OR vaccinated breeder flocks were more profitable than the negative control flocks. The quantitative risk analysis showed that the probability of making a relative profit from broilers as a result of OR vaccination of parent stock was 74%, from the use of in-feed medication in broilers from unvaccinated parents was 70% and from a combination of the interventions was 99%. It can be concluded that the last of these options was most profitable. / Dissertation (MSc (Veterinary Sciences))--University of Pretoria, 2003. / Veterinary Tropical Diseases / unrestricted

Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the causative agents of botulism and represents a family of seven structurally similar but antigenically different serotypes (A to G). The BoNTs are expressed in C. botulinum as a single polypeptide chain and then posttranslationally nicked, forming a di-chain polypeptide chain consisting of a 100-kDa heavy chain and a 50-kDa light chain held together by a disulfide bond. Topologically, the neurotoxins are composed of three domains, a binding domain (HC), a translocation domain (HN) and a catalytic domain. The BoNTs act preferentially on cholinergic nerve endings in both humans and animals and thus produce a flaccid paralysis that may result in death. In southern Africa, BoNT types C and D have been associated with botulism in cattle. To combat the disease, a bivalent vaccine consisting of formalin-inactivated type C and D holotoxins is currently available, and although it is efficacious, several concerns regarding its production has been raised, most notably its cost. The development of efficacious recombinant subunit vaccines may provide a means whereby many of the production problems may be eliminated or minimized. Consequently, the aim of this investigation was to produce a recombinant botulinum neurotoxin serotype D binding domain [BoNT/D(HC)] vaccine candidate for preventing BoNT/D intoxication. Towards this end, the gene fragment for the heavy chain (HC) of the BoNT produced by the C. botulinum type D vaccine strain D-50 was amplified, cloned in Escherichia coli and characterized by nucleotide sequence analyses. An alignment of the deduced amino acid sequence with that of characterized clostridial type C and D neurotoxins demonstrated that the heavy chains are composed of highly conserved domains interceded with tracts of amino acids exhibiting little overall relatedness, although considerable identity between the components ofa specific pair is apparent in certain of the regions. The deduced amino acid sequence exhibited 99, 66 and 73% identity with the reported amino acid sequences of BoNT/D-SA, BoNT/D and BoNT/C1, respectively. Attempts at expressing the native gene sequence for the HC from BoNT/D-50 in Brevibacillus brevis 47-5Q were unsuccessful. This may have been due to differences in codon bias between the heterologous gene and B. brevis. Consequently, a completely synthetic codonoptimized gene encoding the HC of BoNT/D-SA was constructed and expressed using a B. brevis 47-5Q mutant as expression host, obtained on mutagenesis with N-methyl-N’-nitro-Nnitrosoguanidine (NTG). Extracellular expression of the 48-kDa recombinant protein was verified by Western blot analyses with anti-BoNT/D antibodies. The recombinant BoNT/DSA(HC) protein was purified from the culture supernatant and used to vaccinate mice, after which their survival against challenge with active toxin was evaluated. Mice given two subcutaneous vaccinations were protected against intraperitoneal administration of 4 X 102 mouse lethal dosages (MLDs) of 16S BoNT/D-50 toxin. Antibody levels in mice surviving challenge were determined by enzyme-linked immunosorbent assays and confirmed that BoNT/D-SA(HC) was successful in evoking a protective immune response, whilst Western blot analyses indicated the presence of anti-16S BoNT/D-50 toxin antibodies in the serum. From these results it could be concluded that the recombinant BoNT/D-SA(HC) protein is an effective immunogen, able to protect against a high challenge dose of BoNT/D-50 neurotoxin. / Dissertation (MSc)--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

A shotgun genome sequencing project was undertaken in the expectation that access to the entire protein coding potential of E. Ruminantium (Welgevonden) will facilitate the identification of vaccine candidate genes against heartwater. The 1,516,355 bp sequence is predicted to encode 888 proteins and 41 stable RNA species. The most prominent feature is the large number of tandemly repeated and duplicated sequences, some of continuously variable copy number. These repeats have mediated numerous translocation and inversion events and seem to be responsible for the generation of both new full and partial protein coding sequences. There are 32 predicted pseudogenes, most of which are truncated fragments of genes associated with repeats. Of the 13 members of the order Rickettsiales compared in this study, E. Ruminantium has the lowest coding capacity (62%), lowest GC content (27.5%), but the highest proportion of repetitive sequences, which comprise 8.5% of the genome. Metabolic reconstruction of E. Ruminantium revealed the metabolic and biosynthetic capabilities typical of an obligate intracellular organism. We identified a number of genes unique to E. Ruminantium, most of which are not functionally characterised in any organism, and those shared with 12 other members of the Rickettsiales. Bioinformatic tools were used to identify possible vaccine candidates from the annotated genome sequence. The protective properties of seven open reading frames (ORFs), which induced cellular immune responses in vitro, were tested in vivo Only 20% survival was obtained in sheep immunised with a DNA formulation consisting of three ORFs. We found that the levels of peripheral blood mononuclear cell proliferation and interferon-gamma (IFN-γ) production did not correlate with each other, nor with the levels of protection, suggesting that the current assays are just not reliable and that IFN-γ expression alone is not an indicator of protection. Therefore more cytokines and different assays will have to be investigated to define in detail what constitutes a protective immune response against E. Ruminantium infection. However, the data generated from the genome sequence will continue to facilitate novel approaches to study the organism and to develop an efficacious vaccine against heartwater. / Thesis (PhD)--University of Pretoria, 2010. / Veterinary Tropical Diseases / unrestricted

Seasonal influenza outbreaks are associated with morbidity and mortality in the United States. Though children are the most susceptible to influenza infection and are most likely to transmit the illness to others, many children are not vaccinated. The purpose of this study was to examine the relationship between seasonal influenza vaccination Advisory Committee on Immunization Practices (ACIP) recommendations, demographic characteristics, socioeconomic factors, and vaccine type among children over 3 consecutive flu seasons. This quantitative cross-sectional study was guided by the social ecology of health model. Secondary data from 3 consecutive flu seasons (2014-2015, 2015-2016, and 2016-2017) provided by the National Health Interview Survey was used for this study. Binary logistic regression and chi-square were used to analyze the data. A relationship between socioeconomic status, demographics (age, race, and family income) and vaccine type (live-attenuated influenza vaccine [LAIV]/inactivated influenza vaccine) was established among U.S. children; those who received LAIV were most likely to be White elementary school age children with a higher family income. Demographic and socioeconomic status was not considered influential in LAIV uptake for race, health insurance status, or family income. ACIP recommendations by age and year had the greatest impact on flu vaccine choice for this sample population. The results of this study can lead to social change by providing information for policy that can increase vaccine uptake, which can result in lower health cost and reduced illness and death rates associated with the flu, especially for those most at risk.

The development of a safe, effective prophylactic HIV vaccine remains a major global health priority. Stabilized, soluble trimers that mimic the native functional HIV trimer have been developed that elicit strain-specific neutralizing HIV antibodies in animal models, and are currently being evaluated in several human clinical trials. Identifying whether multiple immunogens could be administered to facilitate the broadening of responses represents a pivotal challenge. In this thesis, we characterized the antibody response in individuals infected with multiple HIV strains to inform the development of polyvalent and sequential HIV vaccine regimens. We found that conventional approaches to detect HIV co- and superinfection are confounded by recombination. Therefore, we developed an automated, Bayesian approach to detect superinfection explicitly accounting for recombination. Using simulated and real sequence data, we demonstrated that this approach is sensitive, highly specific, and robust to recombination. Furthermore, analyzing previously published sequence datasets, we identified cases of superinfection that previously went undetected, indicating that superinfection occurs more frequently than previously estimated. We characterized the development of antibodies in five superinfected individuals identified in the CAPRISA 002 acute infection cohort. Specifically, we evaluated whether superinfection re-engaged cross-reactive memory B cells, promoting the development of cross-neutralizing antibodies. By comparing the breadth of the neutralizing antibody response in superinfected individuals to those that typically develop in singly infected individuals, we showed that HIV superinfection was not sufficient to broaden responses. By characterizing the kinetics and specificity of autologous neutralizing antibody responses, we show that responses to the superinfecting viruses failed to efficiently recruit neutralizing memory B cells. Instead, the secondary infection elicited strain-specific, de novo responses. This occurred even though the superinfecting viruses were relatively closely related (from the same subtype). To determine whether the co-exposure to diverse Env antigens favours the development of cross-neutralizing antibodies better than sequential exposure, we characterized the development of neutralizing antibodies in HIV co-infected individuals where several divergent viruses were transmitted prior to seroconversion. We identified three cases of co-infection that encompassed immunological exposure to: (i) two diverse, unlinked Envs, (ii) two related Envs with diversity uniformly distributed over the trimer, and (iii) two diverse but recombined Envs such that clusters of high homology were preserved in the presence of high diversity elsewhere. We found that, like superinfection, co-infection was not sufficient to broaden neutralizing antibody responses. Co-exposure to two HIV Env antigens did not necessarily produce additive or cross-neutralizing antibody responses, and in some cases was subject to immunological interference. This was most evident in the case of co-infection with two related Envs where diversity was uniformly distributed across the Env trimer; in this case neutralizing antibody responses to one variant arose to the near exclusion of responses to the other. However, in the case of co-exposure to diverse Envs but where the trimer apex was conserved in both variants through recombination, potent neutralization of both variants was evident. This was the co-infected participant who developed the broadest neutralizing antibody response, and we show that cross-neutralization was mediated, in part, by trimer apextargeting neutralizing antibodies. In conclusion, we find that HIV superinfection fails to efficiently recruit neutralizing memory B cells and, at best, results in additive nAb responses rather than a synergistic effect leading to cross-neutralization; a distinction that is highly relevant for vaccine design. While sequential immunizations with heterologous Env immunogens may be able to improve the potency of elicited responses, alone, they are unlikely to promote the development of bnAbs. Our observations from cases of co-infection suggests that cocktails of divergent stabilized Env trimers are unlikely to drive the development of cross-neutralizing antibodies, and may be subject to interference. However, the rational design of more similar immunogen cocktails where conserved epitopes are preserved across immunogens may be able to facilitate neutralizing antibodies to these targets, as seen in one individual. Thus, the use of related, stabilized Env trimers with diversity introduced in key regions together with strategies to reduce the immunogenicity of immunodominant, strain-specific epitopes may represent one path to a cross-neutralizing antibody response to multiple Envs within a cocktail.