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The overall aim of this thesis is to provide a better understanding of knowledge, attitudes, consent, and decision-making regarding Human papillomavirus (HPV) vaccination, seen from the perspectives of concerned parties – high school students, school nurses, and parents. Two quantitative studies were performed: one descriptive cross-sectional study and one quasi-experimental intervention study. Qualitative studies using focus group interviews and individual interviews were also performed. High school students’ knowledge about HPV and HPV prevention was low but their attitudes toward HPV vaccination were positive. An educational intervention significantly increased the students’ knowledge regarding HPV and HPV prevention. Their already positive attitudes toward condom use and HPV vaccination remained unchanged. The students wanted to receive more information about HPV from school nurses. The school nurses were also positive to HPV vaccination but identified many challenges concerning e.g. priorities, obtaining informed consent, culture, and gender. They saw an ethical dilemma in conflicting values such as the child’s right to self-determination, the parents’ right to make autonomous choices on behalf of their children, and the nurse’s obligation to promote health. They were also unsure of how, what, and to whom information about HPV should be given. Parents, who had consented to vaccination of their young daughters, reasoned as follows: A vaccine recommended by the authorities is likely to be safe and effective, and the parents were willing to do what they could to decrease the risk of a serious disease for their daughter. Fear of unknown adverse events was overweighed by the benefits of vaccination. Parents also saw the school nurse as an important source of HPV information. Conclusions: Positive attitudes toward HPV vaccination despite limited knowledge about HPV, are overarching themes in this thesis. School nurses have a crucial role to inform about HPV prevention. It is important that the concerned parties are adequately informed about HPV and its preventive methods, so that they can make an informed decision about vaccination. A short school-based intervention can increase knowledge about HPV among students. From a public health perspective, high vaccination coverage is important as it can lead to a reduced number of HPV-related disease cases.

Animal trypanosomosis is a major constraint in livestock production in Sub-Saharan Africa. With the emergence of resistance against trypanocidal drugs, the cost and environmental concerns raised by vector control, and the challenge of antigenic variation in vaccine development, alternative control measures are being sought. An anti-disease strategy, whereby the immune response or chemotherapy is aimed towards pathogenic factors rather than the parasite itself, constitutes such a novel approach. Congopain is the major cysteine protease in Trypanosoma congolense, and upon release in the bloodstream of infected cattle, acts as a pathogenic factor. It is therefore an attractive candidate for an anti-disease vaccine. It was hence deemed necessary to investigate the variability of congopain-like cysteine proteases before attempting to design drugs and vaccines based on the inhibition of congopain. Most congopain-like cysteine protease genes of T. congolense exist in a single locus of 12-14 copies organised as tandem repeats of 2 kb gene units. A gene unit library of 120 clones was constructed out of several cosmid clones selected in a previous study that contained various lengths of the congopain locus. Some 24 gene unit clones were sequenced, and it was found that congopain genes cluster in three sub-families, named CP1 (8 clones), CP2 (12 clones) and CP3 (4 clones). The latter most characteristically shows a substitution of the active site cysteine by a serine. Isoform specific primers were designed and used to verify the proportions of the three isoforms (one third CP1, half CP2 and a sixth CP3) in the remaining clones of the library. Since this first study was conducted in one isolate, IL 3000, the results were subsequently validated in a large array of isolates, of T. congolense, as well as T. vivax and T. brucei subspecies, by a PCR approach. Finally, to gain access to copies of congopain genes that are not present in the locus, but rather scattered in the genome, an attempt was made to construct a 2 kb size-restricted genomic library. Only 206 clones could be produced, of which a mere 8 coded for congopain-like proteases. The fact that 7 out of 8 of these clones belong to CP3 (thought to be inactive) suggested a cloning artefact, possibly related to the activity of the cloned proteases. Overall, all congopain genes appear very conserved in a given species, with 87-99% identity at protein level. The pre- and pro-region were the most conserved, while the catalytic domain was the most variable, especially around the active site cysteine, with frequent replacement by a serine residue, and in one instance by phenylalanine. The histidine residue of the catalytic triad was also substituted by either a serine or a tyrosine in some instances. The proenzyme cleavage site sequence was also variable, with APEA being the predominant N-terminal sequence. RT-PCR analyses indicated that CP1, CP2 and CP3 mRNA are all present in the bloodstream forms of T. congolense, showing that these variants are likely to be expressed. The conclusion of this study is that, given the high overall conservation of congopain genes in the genome, for the purpose of anti-disease vaccine, it is likely that a single immunogen will suffice to raise antibody able to inhibit all circulating congopain-like cysteine proteases. For chemotherapy however, a more in-depth enzymatic characterisation of the mutants, involving functional recombinant expression, will have to be undertaken. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

African trypanosomiasis is caused by protozoan parasites known as trypanosomes, which are transmitted by the tsetse fly, affecting both humans and animals. Trypanosoma congolense is one of the main trypanosome species affecting cattle and causes the disease known as nagana. Control of animal African trypanosomiasis currently relies on chemotherapy and vector control methods, neither of which has proven satisfactory. An effective vaccine against trypanosomiasis would be the most cost effective solution to control the disease; however, due to the phenomenon of antigenic variation, intrinsic to the parasite’s outer coat of variable surface glycoprotein, this has not yet been achieved. Recent vaccine efforts have been centred on identification of invariant parasite antigens for use as vaccine candidates. Trypanosome cytoskeleton components have in recent years been shown to be capable of providing a protective immune response against trypanosome infection. These include tubulin proteins, which form the main components of the cytoskeleton, as well as microtubule associated proteins (MAPs) and paraflagellar rod proteins. In the present study α- and β-tubulin from T. congolense were recombinantly expressed and their immuno-protective potential in mice assessed. Amplification of both α- and β-tubulin ORFs from T. congolense genomic DNA was followed by cloning of the amplicons into the T-vector pTZ57R/T, and thereafter sub-cloning into the bacterial expression vector, pET238a and the yeast expression vector pPICZαA28. Only the α-tubulin amplicon was successfully sub-cloned into pICZAαA28; however, no protein expression was achieved upon transfection of the methylotrophic yeast, Pichia pastoris, with this construct. Subcloning of both α- and β-tubulin inserts into pET28a was successful. Expression of recombinant α- and β-tubulin as fusion proteins with a histidine tag, both at a size of 55 kDa, was achieved in Escherichia coli host BL21 (DE3). Recombinant proteins were successfully purified using nickel chelate chromatography under denaturing conditions. Refolding was first attempted by dilution of purified denatured proteins in a refolding buffer followed by reconcentration, but was largely unsuccessful. A second, more successful refolding method was performed wherein denatured proteins were refolded by application of a decreasing gradient of urea, while bound to a nickel chelate column. Native tubulin from cultured T.congolense procyclics was successfully purified and renatured using a polymerisation/depolymerisation method for use as a control for immunisation. Mice were immunised separately with refolded recombinant α- and β-tubulin, native tubulin or an irrelevant protein VP4AA expressed in the same way as the tubulins. ELISA analysis confirmed the production of antibodies against each protein. Parasitaemia developed in all mice following challenge with T. congolense. Only the group immunised with β-tubulin recorded no deaths during the monitoring period despite the presence of parasitaemia, with 60% of mice immunised with α-tubulin or VP4AA and the no antigen control and no mice from the native tubulin immunised group surviving. The results showed that partial protection against trypanosomiasis caused by T. congolense infection was achieved in the group immunised with β-tubulin and suggest that β-tubulin may have vaccine potential. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

<p>Genetic modification of dendritic cells with plasmid DNA is plagued with low transfection efficiencies because DNA taken up by non-dividing dendritic cells rarely reaches the nucleus. But this difficulty can be overcome by the use of messenger RNA (mRNA), which exerts its biological function in the cytoplasm and obviates the need to enter the nucleus. Since pioneering work of Boczkwoski et al, the ex-vivo application of mRNA-transfected dendritic cells as a vaccine has been evaluated in numerous phase I trials worldwide and is still currently being actively optimized in clinical trials. </p><p> However, a major disadvantage of using mRNA-transfected DCs as a vaccine is that it requires patients to undergo at least one 4-hour leukapheresis procedure, followed by separation of the peripheral blood mononuclear cells (PBMCs), from which monocytes are isolated and cultured for a week in a defined medium with cytokines. The resulting DCs are matured after being loaded with mRNA and frozen for storage. Aliquots are subsequently thawed prior to administration to patients. This process of harvesting, culturing and loading DCs is more time- and resource-intensive than Provenge, the first FDA approved cell based tumor vaccine in 2011.Recent evidence has confirmed a lack of broad translation of Provenge due to complexity and cost of treatment. This predicates a similar fate for mRNA-transfected dendritic cell vaccine going forward. </p><p> This thesis presents alternative delivery strategies for mRNA mediated tumor vaccination. Through the application of synthetic and natural biomaterials, this thesis demonstrates two viable approaches that reduce or eliminate the need for extensive manipulation and cell culture.</p><p> The first approach is the direct in vivo delivery of mRNA encapsulated in nanoparticles for tumor vaccination. A selected number of synthetic gene carriers that have been shown to be effective for other applications are formulated with mRNA into nanoparticles and evaluated for their ability to transfect primary DCs. The best performing formulation is observed to transfect primary murine and human dendritic cells with an efficiency of 60% and 50% (based on %GFP+ cells) respectively. The in vivo transfection efficiency and expression kinetics of this formulation is subsequently evaluated and compared with naked mRNA via various routes of delivery. Following this, a proof-of-concept study is presented for a non-invasive method of mRNA tumor vaccination using intranasally administered mRNA encapsulated in nanoparticles. Results show that intranasally administered mRNA induces tumor immunity only if it is encapsulated in nanoparticles. And anti-tumor immunity is observed in mice intranasally immunized under both prophylactic as well as therapeutic models. </p><p> The second approach evaluates whole blood cells as alternative cell based mRNA carriers. A method is developed to encapsulate intact and functional mRNA in murine whole blood cells. Whole blood cells loaded with mRNA not only include erythrocytes but also T cells (CD3+), monocytes (CD11b), antigen presenting cells (MHC class II) as well as plasmacytoid DCs (CD45R-B220). Mice immunized with mRNA-loaded whole blood cells (intravenously) develop both humoral and cellular antigen-specific immune responses, and demonstrate delayed tumor onset and progression in a melanoma therapeutic immunization model (using tyrosinase related protein -2, TRP-2, as an antigen). Importantly, the therapeutic efficacy of mRNA-loaded whole blood cell vaccine formulation is found to be comparable to mRNA-transfected dendritic cell vaccine.</p><p> In conclusion, this thesis presents new methods to the delivery of mRNA tumor vaccines that reduce or eliminates the need for extensive cell manipulation and culture. Results presented in this thesis reveal viable research directions towards the development and optimization of mRNA delivery technologies that will address the problem of broad translation of mRNA tumor vaccines in the clinics.</p> / Dissertation

Untersuchungen zu Schlachtkörperqualität und Ebergeruchsstoffen bei mit einem GnRH-Analogon geimpften, chirurgisch kastrierten und intakten männlichen Mastschweinen Franziska Sauer Universität Leipzig, Medizinische Tierklinik, Leipzig, Deutschland Zielstellung Ziel der vorliegenden Studie war, Auswirkungen der Impfung gegen Ebergeruch bei männlichen Mastschweinen in konventioneller Haltung zu untersuchen und mit intakten Mastebern und Kastraten zu vergleichen. Tiere und Methode Insgesamt 348 männliche Mastschweine wurden in vier Gruppen wie folgt unterteilt: Zwei Gruppen enthielten mit Improvac® geimpfte Schweine (1. Impfung 11. Lebenswoche (LW)): Gruppe 1: n=84, 2. Impfung 18. LW, Gruppe 2: n=83, 2. Impfung 21. LW. Gruppe 3 bestand aus 90 Kastraten und Gruppe 4 aus 91 unkastrierten männlichen Mastschweinen. Die Schweine wurden im Alter von 26 bzw. 27 Wochen geschlachtet. Mast- und Schlachtleistung, das Fettsäuremuster im Rückenfett sowie Hodengewicht, Hodenhistologie und Ebergeruchsstoffe im Rückenfett wurden untersucht. Ergebnisse GnRH-geimpfte Schweine wiesen eine bessere Futterverwertung als chirurgisch kastrierte auf. Die Schlachtkörper der Geimpften hatten einen signifikant höheren Magerfleischanteil und weniger Rückenfett als die der Kastraten und erzielten dadurch einen höheren Schlachterlös. Das Fettsäuremuster der geimpften Schweine gleicht im Hinblick auf die Menge an PUFA eher den intakten als den chirurgisch kastrierten. In früherem Alter geimpfte Schweine zeigen histologisch eher eine Hodenhypoplasie, später geimpfte eher eine Hodenatrophie. Ebergeruchsstoffe im Rückenfett waren in beiden Impfgruppen und bei den Kastraten signifikant niedriger als bei intakten Mastebern. Schlussfolgerung Die Impfung männlicher Schweine mit Improvac® ist eine tierschutzgerechte, praktikable und wirtschaftliche Alternative zur Vermeidung von Ebergeruch. Literatur Sattler et al: BMTW 2014; 127:10-16 Sauer et al: WTM 2014;101:103-109 franziska.sauer@vetmed.uni-leipzig.de

Influenza is a major concern across healthcare environments. Annual vaccination of healthcare workers (HCWs) remains essential for maintaining the health and availability of HCWs, as well as influenza prevention in healthcare environments. Yet, annual vaccination coverage among HCWs continues to be below recommended standards during pandemic (pH1N1) and non-pandemic (sINFLU) influenza seasons. The primary aim of this research is to inform the design and implementation of effective HCW targeted influenza vaccination campaigns via a 1) systematic review of the existing literature on HCW pH1N1 vaccination, 2) qualitative content analysis of motivators and barriers to HCW pH1N1 and sINFLU vaccination, as well as 3) quantitative regression analysis of modifiable factors predicting pH1N1 and sINFLU vaccination. The qualitative and quantitative analysis processes were applied to data collected from a large-scale multi-professional sample of HCWs. Findings from all analysis sections were found to be consistent. Most attitudes, beliefs, motivators, and barriers influencing HCW influenza vaccination were similar for pH1N1 and sINFLU vaccinations. Yet, a number of notable differences were also identified. HCWs were likely to accept vaccination if they perceived, 1) vaccination to be safe, 2) vaccination to be protective against influenza for self, loved ones, patients or communities, and 3) influenza to be a serious and severe infection to self and others. Additionally, encouragement from supervisors and colleagues, physicians, and loves ones also enhanced vaccine uptake. Most HCWs avoided vaccination because of 1) limited knowledge or misinformation about vaccination, 2) concern for vaccine induced side-effects and 3) assuming vaccination was not a requirement for healthy adults. With respect to pH1N1 vaccination, mass media communications, perceptions of novel vaccinations, and rapid vaccine development processes especially deterred HCW pH1N1 vaccination. Future vaccination programs targeting HCWs should look towards influencing HCWs’ vaccination attitudes and promoting pro-vaccination cultures in healthcare workplaces.

Theileria parva is a complex protozoan parasite causing East Coast fever in Eastern and Central Africa. Vaccination using live parasites is an effective control measure and has been used in Zambia based on locally isolated and introduced T. parva stocks. Diversity among T. parva populations was investigated in parasites from two Zambian provinces with different disease epidemiologies and control histories. Isolates from the pre-vaccination era, local and exotic stocks used for vaccination, and one recent field isolate were cloned and passaged in vitro to study genomic stability over time. The results of the data from three genome-wide probes indicate a marked homogeneity and stability among the Zambian isolates in contrast to East African isolates. Results from Southern blot profiles and the polymorphic immunodominant molecule (PIM) sequence analysis suggest a common origin for the Zambian isolates from the pre-vaccination era, except for one isolate (Zam5) from Southern Province. This isolate showed characteristics suggesting a buffalo origin. Assays for genotype characterisation were developed using five allelic markers. Multilocus characterisation revealed identical profiles in a recent Zambian isolate from Southern Province and two components of an exotic cocktail vaccine, indicating the escape of one of the vaccine stocks in the field. Characterisation of T. parva field populations by RFLP-PCR assays after immunisation revealed the presence of dominant genotypes from those that had been used for vaccination. Circumstantial evidence for the involvement of one of the exotic vaccine parasites in epidemics in Southern Province is presented and a hypothesis formulated for the rapid spread of this genotype. Analysis of the characterisation data suggested the existence of two groups of T. parva parasites of different origin. The classic T. parva group, characterised by a dimorphism of the p150, p104 and p32 loci and the absence of a p67 insert and a buffalo-derived group which showed a polymorphism of p150, p104 and p32 and the presence of a p67 insert. There is evidence that recombination occurs, resulting in parasites that have characteristics of both groups. The relevance of these recombinant parasites in the epidemiology of the disease seems low. Characterisation of larger samples from areas of regular buffalo-cattle contact is necessary to clarify this. Sequence analysis of the most discriminative locus (PIM) was undertaken and gene conversion could be the main mechanism generating diversity. A more appropriate nomenclature for T. parva is proposed based on the growing evidence of molecular differences among isolates and stocks.

Aim: Thisstudy aimedto investigatethe knowledge and attitudes of healthcareworkers regardinginfluenza, influenza vaccination,and vaccination practicesin emergency departments in Gothenburg, Sweden. Method: This cross-sectional studyuseda self-administered questionnaire distributed tonurses, assistant nurses,and physiciansin three emergency departments atThe Sahlgrenska University Hospital in January–February2014. Results: Among214 participants, 56% were nurses, 27% assistant nurses,and 17% physicians. The response ratewas 77%. A total of 66 participants (31%)werevaccinated against influenza during the previous12 months.The highest vaccination coverage occurredin the oldestage group(56%;P&lt;0.05).Past vaccinationstrongly predicted future vaccinationbehavior (P&lt;0.001). Ourdata revealed nosignificant difference invaccination coverage betweenprofession, work experience, hospital,or gender. The mean knowledge score was higher among vaccinated vs. unvaccinated health care workers (17.9 ± 2.7vs.16.8 ± 2.6, respectively; P&lt; 0.05). Moreover, influenza risk perception was higher among participants who were vaccinated during the previous12 months compared to unvaccinated participants (P&lt; 0.001). Interestingly, more un vaccinated health care workers believed that personal behavior determines health (higher internal locus of control) compared to vaccinated workers(P&lt; 0.05). More than half of vaccinated health care workers stated that they got vaccinated to avoid influenza. Almost half of the unvaccinated workers voiced concern about vaccine side effects. Fourteen percent of all respondents mentioned patient protection as an important factor in their decision to receive influenza vaccination. Conclusion:This study demonstrates a need for improved knowledge about influenza and influenza vaccinationin health care workers. Increased risk perception of influenza can increase vaccination coverage in emergency department personnel,and may reduce the incidence of healthcare-associated influenza. / <p>ISBN 978-91-86739-77-5</p>

These two studies investigate the immune responses of lung and kidney transplant recipients to the influenza vaccine. The study involving kidney transplant recipients developed a novel flow cytometry assay to measure cell-mediated immunity in response to influenza vaccination. The activation of T-cells was assessed through the change in T-cell production of interferon gamma after vaccination. In lung transplant recipients, the study examined the formation of de novo anti-HLA antibodies following influenza vaccination. Anti-HLA antibodies were classified as donor-specific or not. The study in kidney transplant recipients found that the influenza vaccine is effective at stimulating the immune response and producing long-lived memory in these patients, as evidenced by high baseline T-cell activity. The study of lung transplant recipients found that receiving the influenza vaccine did not result in the production of anti-HLA antibodies. Both studies found vaccine to be safe for use in these populations. / Experimental Medicine