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This thesis work aims to improve the thermal stability of vesicular stomatitis virus (VSV) through spray drying and investigates differences in thermal stability in a matrix between enveloped and non-enveloped viral vectors. The spray drying process was used to dry and encapsulate the VSV vector within the glassy amorphous phase of a matrix of carbohydrate excipients, which imparted increased thermal stability. Viral activity was maintained when the powders were stored for 30 days at 37 °C, in contrast to the liquid control that lost all activity after 15 days. The best excipients for enhancing thermal stability of VSV were trehalose and a 3:1 blend of trehalose and dextran. Immunogenic response from the spray dried trehalose-VSV particles was detected through an in vivo study with Female BALB/c mice after storing the vaccine for 15 days at 37 °C. Two enveloped viral vectors, VSV and influenza, and a non-enveloped viral vector, human type 5 adenoviral vector (AdHu5) were spray dried with the same formulations to observe how excipients enhance thermal stability and encapsulate the different groups of viruses. The thermal stability of both enveloped viral vectors was enhanced the most when spray dried with trehalose or a 3:1 trehalose/dextran blend, and exhibited the greatest activity loss when spray dried with a mannitol/dextran blend, determined by in vitro TCID50 assays measuring GFP expression. Conversely, the best performing excipient formulation for the non-enveloped viral vector was a mannitol/dextran blend. This led to the hypothesis that the encapsulation mechanism differs between the two groups of viruses. The glass transition temperature (Tg) of the spray dried formulations (without virus) stored at 37 °C for 10 days was measured to infer the potential molecular mobility of the viral vector within the primarily amorphous matrix. Formulations containing dextran exhibited the smallest depression in Tg after storage, indicating minimal increase in molecular mobility over time. RNA leakage from aged spray dried powders containing VSV was quantified to investigate the encapsulation mechanism of enveloped viral vectors and followed a similar trend to the in vitro activity tests. VSV with poor performing excipients yielded the least detectable RNA/pfu after three days of storage at 45°C, suggesting that the lipid envelopes ruptured and released viral RNA which denatured during storage. This work demonstrates that the VSV vector can be thermally stabilized through spray drying but highlights that different carbohydrates interact differently with enveloped versus non-enveloped viral vectors, providing a guideline for future work with the advent of new vaccines. / Thesis / Master of Applied Science (MASc) / Most vaccines lose their activity when stored at room temperature and therefore are required to be stored at temperatures between 4 °C and -80°C to maintain vaccine potency. The refrigeration required to meet these temperatures is costly and limits the distribution of vaccines to resource poor areas in the world where refrigeration technology is uncommon. Spray drying, a process that rapidly dries a solution to form a dry powder, was used to trap vaccines within a sugar matrix to protect the vaccine from heat and structural changes that would otherwise deactivate it. The spray dried powders demonstrated higher thermal stability than liquid samples, which decreases the need for refrigeration during transportation and storage. Thermal stability trends for spray dried powders produced with various sugars and categories of viruses are presented.

Francisella tularensis is a highly virulent zoonotic pathogen that is the causative agent of tularemia in humans. Two subspecies of F. tularensis are the most virulent in humans: tularensis (type A) and holarctica (type B), with less than 10 organisms via aerosol of a type A strain having the ability to cause fatal infection. Over the last decade much research has been done on the pathogenesis of this unique intracellular bacterium and many different virulence factors have been identified. The goal of this dissertation has been to identify and characterize the capsule-like complex (CLC) surface antigen of F. tularensis, and to determine its role in virulence and immunoprotection in a mouse model. In addition, I have investigated the role of CLC in biofilm formation. The CLC appears as a negatively staining material surrounding F. tularensis cells during transmission electron microscopy (TEM). I found that the CLC in the type B live vaccine strain (LVS) could be significantly diminished by deleting two glycosyl transferase genes (LVSΔ1423-22) in the putative polysaccharide locus, FTL_1432-FTL_1421. In addition, I determined that the CLC was not a typical polysaccharide capsule, but was in fact composed of over 50 proteins and glycoproteins including known virulence determinants, such as GroEL, DnaK, and ClpB. Upon further evaluation of the CLC, I determined that it was composed of an increase in production of outer membrane vesicles and tubules (OMV/T). These OMV/T appeared to be self-aggregating into what I visualized through TEM as the CLC. LVSΔ1423-22 was attenuated in the mouse model, and BALB/c mice immunized with CLC and adjuvant were protected against challenge with LVS. In addition to virulence, the CLC appears to play a role in biofilm formation and development. F. tularensis type B strains lacking the surface antigens CLC or CLC and O-antigen, develop a 2-7-fold more robust biofilm than the parent strains. The biofilm matrix contains a glucan-like EPS, proteins, and extracellular DNA, and further characterization may lead to determining if the biofilm acts as an environmental survival mechanism for F. tularensis. In summary, the CLC appears to be a novel surface antigen composed of upregulated OMV/T that is present in type A and B F. tularensis. Deficiency in CLC contributes to increased biofilm formation that could contribute to the survival of F. tularensis in a wide range of environmental niches. Furthermore, the CLC contributes to virulence of type B strains and elicits a protective immune response to type B challenge. A CLC-deficient type A strain could be a candidate for a new live vaccine strain, and therefore further investigation of such a mutant is warranted. / Ph. D.

Recombinant and DNA vaccines utilizing Mycobacterium sp. antigen 85A (Ag85A) were assessed for immunostimulatory and protective effects against M. marinum. Because of their known susceptibility to piscine mycobacteriosis, Morone sp. were utilized as the models for these studies. The first study evaluated a recombinant vaccine with a Brucella abortus strain RB51 vector expressing the Mycobacterium bovis Ag85A. Striped bass (M. saxatilis) were inoculated at doses equivalent to 106, 107, 108, 109, and 1010 colony-forming units/fish. Vaccinated fish demonstrated significant specific humoral and cell-mediated immune responses towards the Ag85A in a dose-dependant manner. However, vaccinated fish failed to demonstrate cross-protective responses after live Mycobacterium marinum challenge 70 days post-vaccination. A DNA vaccine was constructed utilizing the Mycobacterium marinum Ag85A gene and a commercially-available eukaryotic expression vector. Hybrid striped bass (M. saxatilis x M. chrysops) were immunized by intramuscular (i.m.) and intraperitoneal (i.p.) injection at doses of 5 μg, 25 μg, or 50 μg plasmid. These fish produced significant Ag85A-specific antibody and lymphoproliferative responses over those of control fish injected with saline or empty plasmid. Non-specific macrophage phagocytic and respiratory burst functions failed to exhibit significant upregulation after vaccination. Fish receiving the DNA vaccine developed protective responses to high-dose M. marinum challenge 90 days post-vaccination, as demonstrated by increased relative percent survival and by reduced splenic bacterial counts over control fish. Furthermore specific immunostimulatory and protective effects were significantly increased using higher vaccine doses and using the i.m. injection route. Given these promising findings, the protective responses induced by the DNA vaccine were further investigated. Hybrid striped bass were injected with 25 μg or 50 μg plasmid i.m. and developed specific protective responses to high-dose M. marinum challenge 120 days post-vaccination. The 25 μg and 50 μg groups both developed more rapidly and significantly increased immune responses post-challenge over those of the control groups. The vaccination groups also demonstrated increased survival, reduced splenic bacterial counts, and reduced granuloma formation compared to the control groups. However, though the vaccination groups did not demonstrate the same acute effects post-challenge as the control groups, the vaccination groups ultimately developed increased splenic bacterial counts and granuloma formation, and eventually experienced 100% mortalities. Because piscine mycobacteriosis can affect virtually any species of fish, a vaccine against this disease could be widely beneficial to the aquaculture and ornamental fish industries. The vaccines in these studies exhibited significant immunostimulatory capabilities in Morone sp., but only the DNA vaccine showed promise for conferring protection against M. marinum challenge. Though the DNA vaccine only provided limited protection against high challenge doses, future studies may likely find enhanced protective effects against lower, more natural exposure doses. / Master of Science

Hemorrhagic enteritis virus (HEV) causes an acute viral disease in turkeys characterized by bloody diarrhea and death. Current live HEV virus vaccines are immunosuppressive and predispose turkeys to secondary bacterial infections. Data indicates that the capsid proteins (fiber, penton base, hexon) of HEV are capable of stimulating protective antibodies against an HEV challenge. Using tobacco as a model, we sought to determine if a plant could be used to synthesize the HEV fiber protein and produce sufficient antigen to stimulate protective antibodies. To introduce the fiber gene into plants, the coding region of the HEV fiber gene was fused to either a constitutive plant promoter (35S) or a wound inducible promoter (hmg2) on plasmids adapted for Agrobacterium-mediated transformation. Approximately sixty transgenic plants of each construct were generated and determined to contain the HEV fiber gene based on amplification of specific HEV DNA sequences by the polymerase chain reaction. Plants were screened by Northern dot blot to identify lines expressing high levels of fiber mRNA. Expression of fiber protein was observed in selected lines of transgenic tobacco by Western blot analysis using turkey anti - HEV serum. The accumulation of fiber protein in leaves of tobacco transformants was quantified by Sandwich ELISA. Fiber protein from these plants has undergoing large - scale purification and concentration for a turkey immunization trials to determine if plant expressed fiber antigen is capable of inducing protective antibodies against HEV in turkeys. / Master of Science

Yes / This qualitative study was designed to understand the impact of social distancing measures on people with dementia and carers living in the community in England and Wales during a period of social restrictions before the COVID-19 vaccination roll-out. We conducted 12 semi-structured interviews with people with dementia aged 50-88 years, living alone or with a partner, and 10 carers aged 61-78 years, all living with the person with dementia. Three of the interviews were with dyads. Participants were recruited during November and December 2020. We used framework analysis to identify themes and elicit suggestions for potential solutions. We identified three interrelated themes. People with dementia experienced a fear of decline in capabilities or mood and attempted to mitigate this. Carers noticed changes in the person with dementia and increased caring responsibilities, and for some, a change in the relationship. Subsequently, reduced confidence in capabilities to navigate a new and hostile environment created a cyclical dilemma of re-engaging where an inability to access usual activities made things worse. People with dementia and carers experienced neglect and being alone in their struggle, alongside feeling socially excluded during the pandemic, and there was little optimism associated with the upcoming vaccine programme. People found their own solutions to reduce the effects of isolation by keeping busy and being socially active, and practising skills deemed to help reduce the progression of dementia. This and some limited local public initiatives for the general public facilitated feelings of social inclusion. This study adds understanding to existing evidence about the longer-term experience of social isolation several months into the pandemic. It highlights the importance of health and community groups and suggests how services can find ways to support, include, and interact with people with dementia and carers during and after social restrictions. / Economic and Social Research Council. Grant Numbers: ES/V004964/1, L001853, V004964. National Institute for Health and Care Research. Grant Number: ES/L001853/2. Department of Health. UK Research and Innovation. Health and Care Research Wales

Today's development of novel vaccines stresses the need for edible vaccines that are inexpensive, easily administered and capable of being stored and transported without refrigeration. Without these characteristics, developing countries find it difficult to adopt vaccination as the central strategy for preventing their most devastating diseases. A promising approach is the production of vaccines in plants we commonly consume. Two major obstacles have been encountered in developing vaccines in plants. First, the expression level of foreign antigens tends to be low and second, co-expression of an adjuvant may be required to facilitate an appropriate immune response. Ricin, a plant toxin that survives the human digestive process, has been proven to stimulate an immune response and could therefore serve as a suitable adjuvant. The long-term goal is to produce a vaccine that protects against the disease entamoebic dysentery. The specific goal of this research was to produce ricin in tobacco as adjuvant for the vaccine. Vectors were constructed that fused the ricin coding sequence to different plant promoters and transgenic tobacco plants were generated by transformation with Agrobacterium tumefaciens. The levels of expression in these transgenic plants were tested using immunoblot assays. Southern blot analysis was performed for the highest expressors of each construct. The enzymatic activity of the tobacco-synthesized ricin was shown using a protein translation inhibition assay. Expression of ricin was also confirmed using transient transformation of hairy root cultures. Future experiments will address the practical use of the tobacco-synthesized ricin as adjuvant, as well as the expression of the ricin B subunit fused to a protective antigen of Entamoeba histolytica in tobacco as edible vaccine. / Master of Science

A eficácia da associação quádrupla (DPT + BCG) foi estudada através da proteção e comparação da soro conversão das vacinas, usando como adjuvante o hidróxido de alumínio e ou BCG. A potência da vacina pertussis foi avaliada pelo teste de proteção em camundongos e o BCG pelo teste de consumo de oxigênio e de vitalidade, sendo considerada satisfatória. Sendo os toxóides diftérico e tetânico considerados proteínas inertes e estáveis, suas potências não foram determinadas depois de associadas. Os níveis de anticorpos foram determinados para difteria e tétano pela reação imunoenzimática, para vacina pertussis pela reação de imunofluorescência e para o BCG pela conversão tuberculínica. Os níveis de conversão foram satisfatórios, revelando ser possível a associação sem prejuízo para nenhum dos antígenos. A associação DPT + BCG não causou reação local ou geral significante, possibilitando uma simplificação operacional. / The efficacy of the quadruple association (DPT + BCG) was studied though protection and comparison ot the conversion serum in vaccines using aluminium hydroxide or BCG as adjuvant. Both the protection power of the Pertussis vaccine evaluated by the protection test in mice and BCG by the oxygen uptake and counts of viable particles were considered satisfactory. Being difteria and tetanus toxoids considered inert and stable proteins, their protection wasn\'t determined after their combination. The antibody levels produced by the antigens were determined by the enzyme-linked immunosorbent assay to the difteria and tetanus toxoids, by the fluorescent antibody technique to the Pertussis vaccine and by the tuberculin conversion to the BCG. The conversion levels were satisfatory and there was no damage in their association. There was no meaningful local or general reaction in this association making an operational simplification possible.

Submitted by Maria Beatriz Vieira (mbeatriz.vieira@gmail.com) on 2017-08-29T11:52:09Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_thais_larre_oliveira.pdf: 616408 bytes, checksum: 404ac55ee19b90246334aac10658a361 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-08-29T19:49:36Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_thais_larre_oliveira.pdf: 616408 bytes, checksum: 404ac55ee19b90246334aac10658a361 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-08-29T19:49:43Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_thais_larre_oliveira.pdf: 616408 bytes, checksum: 404ac55ee19b90246334aac10658a361 (MD5) / Made available in DSpace on 2017-08-29T19:49:54Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_thais_larre_oliveira.pdf: 616408 bytes, checksum: 404ac55ee19b90246334aac10658a361 (MD5) Previous issue date: 2014-08-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / A leptospirose é uma doença infecciosa emergente causada por espiroquetas patogênicas do gênero Leptospira, com complicações humana e veterinária. Essa doença é transmitida através do contato direto com um animal reservatório ou com o ambiente contaminado com a urina destes animais. O desenvolvimento de vacinas seguras e multivalentes contra leptospirose, que substituam as bacterinas existentes, permanece um desafio. Bacterinas são reatogênicas e conferem imunidade sorovar-específica e de curta duração. Esforços para o desenvolvimento de vacinas recombinantes tem focado em proteínas de membrana externa (OMPs). A proteína de membrana externa OmpL37 é conservada entre diferentes sorovares de Leptospira e atua na adesão aos tecidos do hospedeiro. Neste estudo, nós relatamos pela primeira vez, a avaliação do potencial imunoprotetor de OmpL37 como antígeno vacinal em hamsters, sob diferentes estratégias: vacina de subunidade, vacina de DNA e prime-boost. A caracterização da resposta imune através de ELISA indireto e qRT-PCR demonstrou que os maiores níveis de IgG foram estimulados pela vacina de subunidade, a qual também induziu resposta inflamatória. A vacina de DNA falhou em induzir imunidade humoral, porém também estimulou TNF-α. Apesar da resposta induzida, nenhuma formulação protegeu significativamente os animais contra a doença. / Leptospirosis is an emerging infectious disease caused by pathogenic spirochetes of Leptospira genus, of human and veterinary concern. This infectious disease is transmitted through direct contact with an animal reservoir or an environmental contaminated with their urine. The development of safe and multivalent vaccines against leptospirosis, which replace existing bacterins, remains a challenge. Bacterins are reactogenic and afford serovar specific and short-term immunity. Efforts to develop recombinant vaccines against leptospirosis have focused on outer membrane proteins (OMPs). The outer membrane protein OmpL37 is conserved among different Leptospira serovars and plays a role in adherence to host tissues. In this study we report for the first time, the evaluation of OmpL37 immunoprotective potential as a vaccine antigen in hamsters, under different strategies: subunit vaccine, DNA vaccine and prime-boost. The characterization of the immune response by indirect ELISA and qRT-PCR showed that higher levels of IgG were stimulated by the vaccine subunit, which also induced an inflammatory response. The DNA vaccine failed to induce humoral immunity, but also stimulated TNF-α. Despite the induced response, no formulation significantly protected the animals against the disease.

A eficácia da associação quádrupla (DPT + BCG) foi estudada através da proteção e comparação da soro conversão das vacinas, usando como adjuvante o hidróxido de alumínio e ou BCG. A potência da vacina pertussis foi avaliada pelo teste de proteção em camundongos e o BCG pelo teste de consumo de oxigênio e de vitalidade, sendo considerada satisfatória. Sendo os toxóides diftérico e tetânico considerados proteínas inertes e estáveis, suas potências não foram determinadas depois de associadas. Os níveis de anticorpos foram determinados para difteria e tétano pela reação imunoenzimática, para vacina pertussis pela reação de imunofluorescência e para o BCG pela conversão tuberculínica. Os níveis de conversão foram satisfatórios, revelando ser possível a associação sem prejuízo para nenhum dos antígenos. A associação DPT + BCG não causou reação local ou geral significante, possibilitando uma simplificação operacional. / The efficacy of the quadruple association (DPT + BCG) was studied though protection and comparison ot the conversion serum in vaccines using aluminium hydroxide or BCG as adjuvant. Both the protection power of the Pertussis vaccine evaluated by the protection test in mice and BCG by the oxygen uptake and counts of viable particles were considered satisfactory. Being difteria and tetanus toxoids considered inert and stable proteins, their protection wasn\'t determined after their combination. The antibody levels produced by the antigens were determined by the enzyme-linked immunosorbent assay to the difteria and tetanus toxoids, by the fluorescent antibody technique to the Pertussis vaccine and by the tuberculin conversion to the BCG. The conversion levels were satisfatory and there was no damage in their association. There was no meaningful local or general reaction in this association making an operational simplification possible.