No description available.

The poultry red mite Dermanyssus gallinae (De Geer) is a blood feeding ectoparasite that infests many bird species. Economically it is the most important parasite affecting egg-laying hens. The aim of this study was to identify vaccine candidate proteins from D. gallinae using a number of approaches. An immunisation trial was conducted using four protein fractions derived from D. gallinae. The fractions were injected into hens and immunoglobulin Y was purified from the yolk of eggs laid by the hens. An in vitro feeding assay in which mites were fed these antibodies showed a significant increase (p = 0.013) in mortality of mites fed with antibodies against a PBS-soluble fraction of D. gallinae. The PBS-soluble proteins were then fractionated by anion exchange chromatography into three fractions, which were then used to immunise hens. One fraction produced antibodies that caused the greatest percentage mortality of mites, based on the results from a single feeding assay. To identify further potential vaccine antigens, three expressed sequence tag (EST) databases were produced. A complementary DNA (cDNA) library was prepared from a mixed gender and life stage population and 46 contigs were identified with significant homology to proteins from other organisms. A second database was generated using a suppression subtractive hybridisation approach and 133 “fed mite enriched” contigs identified with significant homology to proteins in either the NCBI non-redundant (nr) protein, KEGG databases or had a domain predicted by InterProScan. The most abundant proteins in this database were vitellogenin and GP80, a precursor molecule of vitellogenin. Roche 454 sequencing technology was used to generate an EST database of 13,363 contigs, of which 26 % had significant homology to a protein in the NCBI nr protein database. The majority of contigs (45 %) were classified as Cellular Processes and Signalling proteins. Illumina Solexa sequencing technology was also used to analyse the expression levels of genes in fed and starved mites. A total of 66 contigs were obtained with a significant and greater than three-fold change in expression level between the two groups. The contig with the largest fold change was homologous to vitellogenin (fold change 110). Paramyosin and tropomyosin have been used successfully to immunise hosts against other parasitic species. Tropomyosin had previously been characterised in D. gallinae. In this study paramyosin was characterised and recombinant versions of both proteins were used in an immunisation trial. Anti-tropomyosin (p < 0.001) and anti-paramyosin (p = 0.004) antibodies fed to mites in the in vitro feeding assay caused a significant increase in the mortality of the mites when compared to controls. An in vivo challenge was performed injecting three groups of hens with the PBS-soluble native protein fraction, a recombinant protein cocktail, consisting of paramyosin, tropomyosin, cathepsin L, cathepsin D and histamine release factor, and an adjuvant only control, which were subjected to a challenge of D. gallinae. Each group consisted of three replicates based on weight. Western blot analysis of hen serum showed a significant increase (p < 0.05) in the titre of antibody from the hens immunised with the recombinant cocktail compared with controls. One of the replicates immunised with the PBS-soluble protein fraction showed a strong response but this was lacking in the other two replicates and no significant difference in IgY titre was found. No significant differences were found between the number of mites collected from the test or control groups following a large mite challenge. In conclusion the PBS-soluble protein fraction, paramyosin and tropomyosin have been shown to have potential as vaccine candidates based on in vitro studies. The bioinformatic analysis of D. gallinae has provided a large EST database and a list of 66 proteins that had a significant difference in expression levels in fed and starved mites, which can be further mined for potential vaccine candidates.

Transmission-blocking vaccines (TBVs) target Plasmodium parasite sexual stages, aiming to block further development of the parasite within the mosquito host. Plasmodium falciparum zygote/ookinete surface protein Pfs25 is one of the leading TBV candidate antigens and antibodies against Pfs25 have been shown to exhibit complete transmission-blocking activity in pre-clinical studies. Phase 1 human clinical trials have revealed that Pfs25 was a poor immunogen in humans in the formulations tested and high titers of anti-Pfs25 antibodies are required to achieve good transmission-blocking activity in the ex vivo standard membrane feeding assay which measures the functional activity of the antibodies induced. Work in this thesis describes the production of recombinant monomeric Pfs25 protein, Pfs25 based particulate vaccines (Pfs25-IMX313 nanoparticle, Pfs25-HBsAg VLP and Pfs25-Q&beta; VLP) and a Pfs25-Pfs28 multivalent protein vaccine in the Pichia pastoris protein expression system. These proteins were tested in mice using protein-in-adjuvant formulations and their immunogenicity was assessed. Pfs25-IMX313 nanoparticle induced significantly higher anti-Pfs25 antibodies than monomeric Pfs25 and the antibodies had higher avidity and transmission-blocking activity. All of the candidate vaccines generated, except for Pfs25-HBsAg VLP, were immunogenic. The Pfs25-IMX313 nanoparticle induced the highest antibody response in mice followed by the Pfs25-Pfs28 multivalent protein and Pfs25-Q&beta; VLP.

Human immunodeficiency virus (HIV) causes acquired immunodeficiency syndrome by targeting and destroying CD4+ T cells via its Envelope protein (Env), while Ebola virus (EBOV) causes a lethal hemorrhagic fever and targets antigen presenting cells (APCs) via its glycoprotein (GP). There are no licensed vaccines for either virus, posing a problem particularly in Africa, where succumbing to EBOV or HIV is a grim reality. We hypothesized that a replication-competent HIV expressing GP as a replacement for Env will redirect the virus from CD4+ T cells toward antigen presenting cells and act as a live, bivalent vaccine to induce cellular and humoral immune responses against both pathogens, and confer protection against a lethal EBOV challenge in mice. Recombinant HIV-1 molecular clones containing different truncations of the GP gene to replace HIV gp120 were generated and used to rescue three GP-expressing vaccines, HIV-EBOV, HIV-EBOVΔ1, and HIV-EBOVΔ2. These demonstrated tropism for the monocyte cell line, THP-1, and decreased tropism for the CD4+ T cell line, SupT1. While all vaccines induced HIV p24- and GP-specific IFN-γ-secreting T cell responses, HIV-EBOVΔ1 and HIV-EBOVΔ2 induced the most robust responses at 21 days post-vaccination (dpv), respectively. While all vaccines induced total anti-p24 and anti-GP IgG responses, HIV-EBOVΔ1 induced the most robust responses at 42 dpv. HIV-EBOVΔ1 demonstrated the highest protective efficacy against lethal EBOV challenge, followed by HIV-EBOVΔ2 and HIV-EBOV, providing 83%, 67%, and 50% survival in mice, respectively. HIV-EBOVΔ1 shows promise as a protective vaccine against EBOV, but may require further optimization and characterization regarding its mechanism of action and ability to protect against HIV. / October 2016

No description available.

Congenital cytomegalovirus infection is the leading cause of sensorineural hearing loss in the U.S. CMV vaccines developed to date do not protect the majority of women of childbearing age from primary CMV infection. Insufficient vaccine-induced epithelial entry neutralizing activity may be the reason for poor performance of these vaccines. CMV entry into endothelial and epithelial but not fibroblast cells requires the virion envelope complex gH/gL/UL128-131. Since current vaccines do not target this complex, epithelial entry mediators UL128-131 are attractive subunit CMV vaccine candidates, since they should target mucosal immunity. The mucosal immune response, specifically salivary epithelial entry neutralizing activity, has not been previously described. This report demonstrates that salivas from CMV seropositive children under two, adolescents, and Towne vaccine recipients do not have epithelial or fibroblast neutralizing activity. Epithelial but not fibroblast neutralizing activity was identified in half of the salivas from CMV seropositive adults tested. This activity correlated with the level of serum neutralizing activity, suggesting that salivary neutralizing activity results from passively transferred serum IgG. Furthermore, this report describes three highly immune individuals with serum and saliva neutralizing titers two- to four-fold above average. These individuals also have UL130 antibodies detectable in western blot assays. This is the first report of antibodies by western blot in CMV seropositive sera to UL128, UL130, or UL131. To determine the feasibility of UL128-131 as vaccine candidates both peptide and DNA vaccines were tested in animal models. Rabbit anti-peptide sera from UL130 and UL131 vaccinated animals induced epithelial entry neutralizing activity similar to that found following natural infection. Mixing anti-peptide UL130 and UL131 sera neutralized CMV infection of epithelial cells at titers higher than natural infection. DNA vaccination with these proteins was not as successful but based on DNA vaccination of mice UL130 is the most immunogenic of the three proteins. These data support further development of UL130 as a CMV vaccine. Future vaccines, including the vaccine candidates described in this report, should strive to induce levels of immunity seen in the three highly immune individuals, specifically serum epithelial neutralizing titers >1:7,000 and saliva epithelial neutralizing titers >1:20.

FACULTY OF HUMANITIES School of Literature and Language Studies 9511112w mokope@webmail.co.za / The scourge of HIV/AIDS continues to worsen in the country in spite of efforts made by government and other stakeholders to combat this disease. This is reflected by the everincreasing statistics of new cases of HIV infection that are reported every minute. This rate of infection is believed to be influenced by factors such as cultural constructions that inhibit efforts to educate the populace about the disease. The research focuses on the extent to which cultural ideologies, as reflected in figurative expressions, render the task of educating people about sexually related diseases difficult. Translators seem to prefer figurative instead of literal language when they translate STDs and AIDS-related education materials. The preference of the former renders the message inaccessible to the average target audience. This study neither strives to conscientise and sensitise the doubting Thomases about the danger of HIV/AIDS and STDs nor does it seek a cure or treatment but a new way of communicating about these diseases. Ratzan maintains that “until a vaccine or cure for HIV infection is discovered, communication is all that we have” (1990: 257). This study deals with communication about HIV/AIDS. It is believed that the research’s findings can be used to help reduce the rate of transmission of this life-threatening infectious disease.

Widespread use of childhood vaccination has significantly reduced the burden of childhood disease, however a subset of parents are choosing to delay or refuse available vaccines. This study analyzed data collected from a large surveillance study to examine the parents’ attitudes about vaccines and the prevalence of vaccine hesitancy (vaccine delay and refusal) in an urban clinic population. The parents of 961 children attending the Boston Medical Center Pediatrics department participated in the study. Parental responses to four vaccine questions were used to assess vaccine attitudes. Log-Binomial Regression models were used to evaluate the relationship between comorbidity status and birth order with vaccine delay or refusal. In this study population, parents reported generally positive attitudes toward vaccination. The majority (87%) believed that vaccines were necessary to protect their child. Approximately 16% of parents reported that had delayed or refused a vaccine and 24% of parents indicated that they did not believe or were unsure if other parents vaccinating their children. When asked the reason for their choice, parents who only delayed frequently cited logistic concerns like a missed appointment, whereas parents who refused more often cited personal beliefs, such as concerns that their child could become ill from vaccination. Finally, parents of children with a comorbidity were more likely to refuse a vaccine than parents of children without comorbidity (Adjusted RR=1.8, 95% CI: 1.1, 2.9). While parents were generally positive toward vaccines, for the small portion of parents refusing vaccines, further work could help to better explain their motivations.

The pathogenic actinomycete Rhodococcus equi is a facultative intracellular parasite that replicates within macrophages. This ability is dependent on the pVAP virulence plasmid, and more specifically, on the laterally acquired vap pathogenicity island (vap PAI) carried by it. R. equi has two contrasting lifestyles as a soil-dwelling microbe and as an inhabitant of the intracellular macrophage compartment. In the first part of this thesis work we analysed the nature of the signals recognised by R. equi to adapt the expression of the virulence genes of the plasmid during the transition from soil saprotroph to intracellular parasite. The expression profile of virulence plasmid genes in response to temperature and pH in vitro and to the macrophage environment was investigated by microarray analysis. A shift to 37ºC was the main stimulus involved in vap PAI gene activation and macrophage-derived signals did not further modulate the expression of the PAI genes contrary to previous suggestions. In a second part of the thesis we investigated the role of a horizontally acquired island encoding exopolysaccharide biosynthesis in the R. equi saprotroph-intracellular parasite dual lifestyle. Mutational analysis of this locus showed that it is responsible for the typical mucoid colony morphology of R. equi and the ability to produce a polysaccharide capsule. Mutations in the capsule locus favoured macrophage uptake but had no effect on intracellular proliferation and in vivo survival in mice. However, the capsule mutants showed significantly increased susceptibility to desiccation, ultraviolet radiation and heat and were outcompeted by capsulated wild-type R. equi in dry soil. Thus, while having a minor role in virulence, the R. equi capsule appears to be primarily required for survival in soil and to act as a transmission factor. The third part of this work followed the identification of a horizontally acquired locus that encodes pili appendages that promote association with macrophages and colonization of the mouse lung. The ability of a component of this structure, the RplB pilin subunit, to act as a vaccine antigen was investigated in mice and horses. Vaccinated mice produced high levels of anti-RplB IgG and showed significant protection against pulmonary challenge with virulent R. equi. The experimental RplB subunit vaccine proved also to be immunogenic in horses, eliciting a strong IgG response in pregnant mares and foals. We also demonstrated passive transfer of high levels of maternal anti-RplB antibodies from the mares to the foals via colostrum. Our results indicate that the RplB pilin subunit is a promising novel candidate R. equi vaccine antigen.

Doctor of Philosophy / Department of Chemical Engineering / John R. Schlup / Jishu N. Shi / Vaccination is one of the most essential steps in controlling and preventing economically important infectious diseases in livestock. Vaccines need to be effective at producing a high level of immune responses that protect the animal from future encounters with infectious agents. Additional requirements for veterinary vaccines include safety, inexpensive components, and feasibility for large-scale production. These factors make emulsions attractive vaccine adjuvants. The use of emulsions as adjuvants (substances that help to amplify the immune responses to the antigen) has been explored for decades. However, emulsions are commonly produced with expensive and energy-demanding devices which impact the price of the adjuvant, therefore, affecting the price of the vaccines. This study examined low-energy emulsification methods to meet the requirements for a simple and low-cost vaccine manufacture that avoided utilizing complicated equipment. Spontaneous emulsification (SE) and phase inversion composition (PIC) was explored to formulate stable emulsions with nanometer droplet sizes. The study on the impact of oil composition on the formation of emulsions produced by SE revealed that addition of medium-chain triglycerides into the oil phase is beneficial for droplet size reduction and stability of emulsions. Box-Behnken design (BBD) was used to develop mathematical relationships between formulation variables and droplet size, polydispersity, zeta potential, and stability of emulsions formulated via SE. The BBD allowed the study of a simultaneous effect of multiple variables and formulate emulsions with certain physical characteristics, an effect that suggested that there was a more effective approach in designing complex systems like emulsions. New adjuvants containing mixtures of oils and surfactants were developed to produce emulsions with nanoscale droplet diameters and multiple water-in-oil-in-water structures via the PIC approach. The strong antibody responses and the absence of injection site side effects were observed in animals that received emulsion vaccines with experimental adjuvants. Additionally, inexpensive food-grade saponin extract was examined for stabilizing and increasing immunostimulatory activity of oil-in-water emulsion-based adjuvants. The adjuvants demonstrated high immune responses in pigs after co-administration with a subunit protein antigen.