The objectives of this study are: (1) To compare the effectiveness of two dosing schedules of hepatitis B vaccine in achieving compliance within the vaccines; (2) To determine the immunization requirements in U.S. pharmacy schools both at admission and before the students begin clinical clerkships; and, (3) To design an immunization program for pharmacy students at the University of the Pacific in an attempt to enhance compliance.

The thermostability of vaccines is of crucial importance in Africa, where the logistical process to get the vaccine from the manufacturer to the animal may take months, and in many remote regions transport and storage is in the absence of a cold-chain. Vaccines with improved stability and less reliance on a cold-chain are needed and could improve the longevity of immune responses elicited in vaccinated animals. In South Africa, cattle in the vaccination zone neighbouring the Kruger National Park have to be vaccinated thrice annually because of declining antibody responses at three months postvaccination. FMDV is known to be unstable, especially for O and SAT2 serotypes in mildly acidic pH conditions or at elevated temperatures, leading to dissociation of the capsid (146S particle) and loss of immunogenicity. The link between rapidly declining antibody responses and capsid stability have been reported by Doel and Baccarini, 1981. We hypothesized that more stable viruses, especially thermostability, will not only improve the protective immune response in animals but also require less frequent booster vaccinations. / The residues at the capsid inter-pentamer interfaces, and their interactions, are important for the infectivity and stability of the virion and mutations adjacent to these interfaces have an effect on the conformational stability of FMDV. However, experimental studies on the relative importance of residues and molecular interactions in viral capsid assembly, disassembly, and/or stability are still very limited, especially for the SAT serotypes of FMDV. This study investigated the effects of potential residues at the pentameric interfaces that are responsible for increased thermostability and potentially improved stability candidates were tested in small (guinea pigs) and large (cattle) animal vaccination trials to understand the role of stabilised antigens on immune responses. The biological variation in biophysical stability in SAT2 viruses in the southern Africa region was investigated to determine if any naturally occurring viruses have greater capsid thermostability. Naturally occurring stable viruses could be used as prospective candidates in vaccine production and therefore potentially result in increased duration of immune responses. / Our first aim was to investigate the role of different amino acid changes at the interface and their effect on capsid stability using models derived by Oxford University. These changes were introduced by mutating the SAT2 ZIM7/83 infectious genome-length clone (pSAT2) to derive mutated chimeric SAT2 viruses. We quantified the stabilizing effects of these mutations by using various stability assays. We established the novel thermofluor shift assay that is able to quantify the capsid stability of viruses. The growth kinetics, antigenicity, genetic stability, pH and salt sensitivity were investigated for each of the genetically engineered viruses (Chapters 2 and 3). / The second aim was to further our understanding on the correlation between improved stability and immune responses by performing small animal (Chapter 2) and large animals trials in cattle (Chapter 4) and comparing stabilised and wild-type antigens. This study for the first time for SAT vaccines, determined differences in IgG1 and IgG2 profiles, interferon gamma (IFN-γ) responses and differences in total and neutralising antibodies of stabilised and wild-type antigens over a six month period in cattle (Chapter 4). Animals were intra-dermolingually challenged with live virus to determine levels of protection the antigens have afforded. / In addition, a third aim will be to better understand the inherent thermostability variation of SAT2 viruses in the Southern African region (Chapter 5) by establishing a protocol for screening field isolates as potential vaccine strains and correlating their stability to amino acid residues at the interface of the 146S particles. / Thesis (PhD)--University of Pretoria, 2016. / Agricultural Research Council / Veterinary Tropical Diseases / PhD / Unrestricted

Department of Microbiology / PhD (Microbiology)

Background Hepatitis B is a world health problem. The virus is a blood pathogen that transfers through blood and body fluids. Hepatitis B is common in the west pacific area and in Vietnam nurses are at higher risk to get infected by hepatitis B. Aim The aim with the study is to describe Vietnamese nursing student´s perception of caring for patients with hepatitis B. Method It is a qualitative study with semi-structured interviews. Result There is a lack of knowledge about hepatitis B in the community and self-education is needed in the care. Fear of getting infected among nurses and a mistrust for the vaccine were found. The awareness of getting infected by needles and sharp instrument were also found. Conclusion Lack of knowledge is a problem in the society, in the health care and also experienced by the nursing students. For the future the nursing students want to educate patients in how to live with the virus, but they see many challenges. Lack of knowledge leads to stigma that infected patients are exposed to by health care and the society, those actions of stigma affects the nursing students in how they see and treat the virus.

Understanding the evolutionary dynamics of influenza type B in human hosts is a public health concern as we strive to minimize the disease burden in seasonal epidemics. Vaccination is considered the best defense against contracting influenza, and everyone over the age of 6 months is advised to get vaccinated before each season. The effect that vaccine-acquired immunity has on the evolution of influenza B remains unclear. In the U.S., vaccine-uptake is irregular across the states, and the differing coverages present an opportunity to study how vaccination influences viral evolution. This thesis analyzes the evolutionary patterns of influenza B in the presence of vaccine-induced selective pressure. Using an ecological study design, estimates on statewide vaccination coverages from the Centers for Disease Control and Prevention were related to influenza B sequence data. The phylogenies and the frequencies of single nucleotide polymorphisms for high and low coverage states across three influenza seasons were compared to evaluate if there was evidence of vaccination influencing evolution. Overall, the results show that vaccination does not significantly impact the evolutionary dynamics of influenza B with both high and low coverage states showing interspersed phylogenetic trees and similar antigenic diversities.

The Gram-negative pathogen bacterium Bordetella pertussis is the infectious agent causing pertussis or whooping cough. The infection is dangerous to infants, often being deadly if untreated. Since whole-cell pertussis vaccines have been replaced by acellular pertussis vaccines, pertussis has become the most prevalent vaccine-preventable disease in developed countries. Therefore, the development of a new generation of pertussis vaccines has become a high priority. Opsonophagocytic assays are one method used to assess the efficacy of new vaccines. The main objective of the thesis is to develop opsonophagocytic killing and uptake assays for the measurement of functional antibody activity against Bordetella pertussis. Neutrophils from mice and humans were isolated by three different methods and used for the assessment of different human and mouse sera in opsonophagocytic killing and uptake assays. Different experimental conditions were tested, including multiplicity of infection and serum dilutions. The opsonophagocytic uptake assay proved to discriminate between naïve and immune sera. Serum from mice vaccinated with the whole-cell pertussis vaccine enhanced opsonophagocytic uptake of B. pertussis cells into neutrophils, while serum from mice immunized with the acellular pertussis vaccine did not....

Vaccination is one of the most successful public health interventions in history, and is estimated to save lives of 3 million children globally each year. Ongoing surveillance is warranted to identify further evolution of the epidemiology of vaccine preventable diseases, and to evaluate the effects of vaccines provided. This dissertation aims to explore the impact of vaccines on disease burden, and effectiveness of diagnostic tools for two important infectious diseases; pneumonia and tuberculosis (TB). The first study employed a large electronic health record data, Massachusetts Health Disparities Repository (MHDR), to evaluate impact of 13-valent conjugated pneumococcal vaccine (PCV13) on all-cause pneumonia among children who receive primary care at Boston Medical Center (BMC). We extracted all-cause pneumonia cases diagnosed at both inpatient and outpatient settings among children younger than 8 years of age. Using interrupted time-series regression analysis monthly rates estimated for years after (2011–2013) implementation of PCV13 were compared to expected rates calculated from pre-PCV13 era (2007–2009). The year of PCV13 introduction (2010) was excluded. We also extracted cases of urinary tract infection and evaluated as control outcome. At the end of 2013 compared to prePCV13 era, among children younger than 2 years of age there was a 35.3% (95% CI 5.4–65.3) reduction in all-cause pneumonia cases. In children with comorbidity, pneumonia declined by 38.8% (95% CI 11.1 to 65.4) in those younger than 2 years of age, and 28.7% (95% CI 2.9 to 54.5) in those 2 to 8 years of age. The results of this study contribute to the growing body of evidence supporting the benefit of indirect protection with conjugated vaccines, and emphasize the importance of high sustainable vaccine coverage rates. The second and the third studies used data from the Tuberculosis Epidemiologic Studies Consortium (TBESC) Study-1, a 10-site collaboration of academic institutions and state and local TB control programs that is funded and administered by the Division of Tuberculosis Elimination at the Centers for Disease Control and Prevention (CDC). The second study evaluated the impact of Bacille Calmette Guérin (BCG) vaccination, which continues to be the only vaccine available for prevention of TB, on tuberculin skin testing (TST) results. Using the data collected TBESC Study-1 between September 2012 and September 2014, we examined the association between BCG vaccination and TST positivity. Logistic regression models were used to calculate adjusted prevalence ratios (PR) and 95% confidence intervals (CI). Prior BCG vaccination had no impact on the TST results once adjusted for history of household contacts (adjusted PR 1.0, 95% CI 0.4–1.5). The results of this study add further evidence that BCG vaccination has little impact on TST results in children, particularly in older age groups. The third study examined the cost-effectiveness of three different screening strategies compared to no screening for latent tuberculosis infection (LTBI) in a population with high proportion of foreign-born individuals who have different risk levels for developing TB. In this study, everyone was tested with using all available tools for LTBI: TST, and interferon-gamma release assays (IGRAs) during their enrollment visit. We used decision tree analysis and Markov models to compare TST only, IGRA only, TST followed by IGRA among those who were TST positive, and no screening strategies. Regardless of the assumptions and tests used, screening provided better health outcomes such as less TB cases and less TB related mortality compared to no screening. The incremental cost-effectiveness ratio (ICER) of TST followed by IGRA compared to no screening was $75,094 per QALY gained. The results of this study suggest that prioritizing certain groups for targeted LTBI screening such as foreign-born individuals, and using TST followed by IGRA can maximize the impact of public health resources allocated to eradicate TB in the U.S. The findings from these studies will contribute to the further understanding of the impact of the vaccines and the changing epidemiology of vaccine-preventable diseases providing more insight to formulate new strategies to improve overall health of children.

Background: Worldwide efforts have been made by some countries to offer HPV vaccination since its introduction in 2006. Population effectiveness of HPV vaccines is presently an active area of research. We review available evidence on the effectiveness of HPV vaccine uptake among female adolescents to prevent HPV infection. Methods: A comprehensive search of published and grey literature was conducted in several electronic databases using a pre-defined search strategy related to HPV prevalence following vaccination. The database searches were complemented by hand-searches of reference lists of eligible studies. Data were extracted onto a purpose-designed data extraction form, pooled in a meta-analysis and stratified by continent considering vaccine type, cross protective and (high/low) risk HPV types as subgroups. Results: Our search yielded 1680 studies, of which thirteen met with our inclusion criteria (8332 vaccinated women aged 12 to 34 years from across the world). The pooled HPV (comprising types 6, 11, 16 and 18) prevalence among vaccinated female adolescents was 7% (95% Confidence Interval (CI): 5% to 9%, 13 studies, n=8,332). The 13 studies were conducted across 3 continents: HPV prevalence for North America was 5% (95% CI: 3% to 7%, 9 studies, n=5781, age range =13 to 34); Europe, 14% (95% CI: 9% to 18%, 3 studies, n=2213, age range =13 to 29) and Australia 5% (95% CI: 3% to 8%, 1 study, n=5781, age range=13 to 34). Of the studies which reported the effect of vaccination on other non-vaccine HPV type prevalence (known as cross protective types) HPV (31, 33, 45, 51 & 58), the overall pooled cross protective HPV prevalence was 9% (95% CI: 6% to 12%, 4 studies, n=3081 age range=13 to 29), by continent North America had 14% (95% CI: 12 to 17%, 1 study, n=753 age range=14 to 24), Europe 7% (95% CI: 6 to 8%, 2 studies, n=1990, age range=13 to 29) and Australia with 8% (95% CI: 5% to 11%, 1 study, n=338 age range=18 to 26). Conclusion: This study showed an HPV prevalence of 7% in women vaccinated against HPV types 6,11,16 and 18, which represents a substantial difference to the 22% HPV prevalence in non-vaccinated women. There was no statistically significant difference between HPV prevalence across the continents. There is however, still a dearth of information on vaccinated women and HPV prevalence, highlighting the need for further studies in this area. Strengths and limitation of this review • The review comprehensively searched multiple databases and bibliographies. We had no language restrictions. • We were stringent in the selection of studies as far as vaccination status was concerned. Studies considering HPV prevalence in unvaccinated women were excluded. • A variety of methods was utilised in collecting data across the studies. However, some of the study participants were not representative of the general population. Caution therefore, needs to be considered when using these results to make inferences or conclusions about prevalence of certain populations.

Bacteria are coated with repeating units of oligosaccharides that exhibit remarkable diversity. Often, glycan units of three or even two sugars are sufficient to identify a species of bacteria. Such specificity makes bacterial surface glycans attractive vaccine targets. However, efforts to create effective vaccines against carbohydrates have been hampered by poor vaccine design as well as the human immune tendency to respond to glycan antigens with non-specific, T-cell independent mechanisms. As a result, carbohydrate vaccines have historically produced only adequate memory responses in healthy individuals and poor responses in the elderly or immunocompromised. To circumvent these issues, a novel conjugate vaccine was developed that utilizes theQβ virus-like particle carrier that displays both a carbohydrate antigen as well as a Natural Killer T cell adjuvant. This unique vaccine has been reported to stimulate the production of high affinity (nanomolar) antibodies against carbohydrate antigens. To further conjugate vaccine research, the present work synthesizes two bacterial surface antigens: a trisaccharide from Streptococcus pneumoniae serotype 23F (Sp23F), and a pentasaccharide from Ruminococcus gnavus (Rg). Sp23F has been characterized as one of the more virulent and disease-causing strains of S. pneumoniae. Rg secretes highly immunostimulatory proteins and is associated with irritable bowel syndrome. The Sp23F antigen is synthesized with an alkyne at the reducing end of the sugar to facilitate coupling to Qβ. A selection reagent for Sp23F is also synthesized to enable the extraction of antibodies and B cells that bind the antigen. In conjunction with providing a conjugate vaccine antigen, the Rg pentasaccharide will be examined as a TLR4 ligand and was therefore synthesized without an alkyne. The Rg conjugate vaccine shows promise in treating irritable bowel syndrome as well as facilitating research into the role Rg plays in the human microbiome.

Malaria claims millions of lives annually. This global killer causes approximately 2.7 million annual deaths worldwide; addressing this problem has become more and more crucial. Due to pathogen evolution no efficient vaccine for treatment of malaria currently exists. As infection has developed as a field of study, it became ever more clear that infections could only be understood within the context of the host-pathogen community. This project aims to predict possible drug targets based on host-pathogen interactions rather than just protein-protein interactions within a single organism. Similar to Lee et al. (2008) pathogen-host interaction predictions are based on orthology, these interactions are then analysed to identify potential drug targets. This could potentially aid researchers in their continuous battle against malaria and the larger scale battle against pathogen evolution. To predict in vitro host-pathogen interactions DISCOVERY uses an ortholog clustering method called ORTHOMCL. ORTHOMCL is very suitable for ortholog clustering of malaria data for two reasons. Firstly, it is capable of distinguishing between recent paralogs and ancient paralogs, which enables the inclusion of recent paralogs together with orthologs. Secondly, ORTHOMCL was initially developed for the use of malaria data. Identification of in vitro interactions is followed by scoring methods to determine the possible in vivo interactions that might occur between the Plasmodium parasite and the human and mosquito hosts. Scoring measures and weights were applied to 5 different factors to calculate a final score. These final scores allow user input to define the preferred stringency when viewing possible interactions with a single protein. These different factors are sequence similarity, PEXEL/VTS motif presence, microarray expression, metabolic map sharing and sub-cellular locations boundaries. DISCOVERY’S results and results from two other (Dyer et al. and Lee et al.) in silico prediction methods were compared with Vignali et al’s experimental interactions which are based on a yeast two-hybrid approach. Similar to results shown by Doolittle and Gomez these comparisons had poor results. The next step was to compare the in silico results with each other. Dyer et al’s and Lee et al‘s results compared poorly with each other. Although DISCOVERY did not compare well with Dyer et al’s results, comparisons with Lee et al. showed more promise. Poor comparisons with Dyer et al. may be due to their unique approach to predict in vitro host-pathogen interactions. This project identified the lack of enough valid and reliable experimental data to evaluate in silico prediction methods as a definite challenge for host-pathogen interaction predictors. Although this is a major problem, DISCOVERY improved on older prediction methods with the use of a more applicable ortholog clustering technique and the use of more assessment methods during in vivo interaction predictions. DISCOVERY also used scoring methods rather than exclusion methods during the identification of in vivo interactions. This allows a user to specify a threshold of sensitivity when viewing interactions. The true potential of host-pathogen interaction predictions would only be realized when the gap between predictions and evaluation data is bridged. / Dissertation (MSc)--University of Pretoria, 2010. / Biochemistry / unrestricted