INTRODUÇÃO: Influenza é uma doença potencialmente grave após o transplante de células tronco hematopoiéticas (TCTH). A vacinação é a principal estratégia profilática, mas a resposta imune é menor do que em indivíduos saudáveis. Em geral os pacientes não respondem a vacinação nos primeiros seis meses após transplante, o que torna o período de maior vulnerabilidade. OBJETIVOS: Neste estudo avaliaram-se diferentes esquemas de vacinação contra influenza em TCTH alogênico relacionado, com imunização do doador e/ou do receptor no pré transplante. Determinou-se a resposta a vacina comparando-se as taxas de soroconversão entre os grupos de intervenção. Foram avaliados também os níveis de anticorpos considerados soroprotetores alcançados. MÉTODOS: Realizou-se ensaio clinico randomizado não cego, em população de candidatos ao TCTH e seus doadores do Hospital das Clínicas da Faculdade de Medicina da Universidade Federal de Minas Gerais/BH-MG e Hospital Amaral Carvalho/Jaú-SP. Quatro grupos de pares receptor-doador receberam diferentes esquemas de imunização de influenza no pré transplante: 1- sem vacinação, 2 - vacinação do doador; 3 - vacinação do receptor e 4 - vacinação de doador e receptor. Todos os pacientes receberam vacina a partir do 6º mês após transplante. Acompanhamento sorológico do par foi realizado no pré e no dia do transplante, e nos dias 30, 60, 100, 180 e após vacina apenas para os receptores. Títulos dos anticorpos sorotipo - específicos foram determinados pela reação de inibição de hemaglutinação. Níveis 1:40 foram considerados protetores e < 1:10 negativos. Soroconversão foi definida como aumento de quatro vezes ou mais dos títulos ou aumento de <1:10 para 1:40. RESULTADOS: De 08/2007 a 02/2010 131 pares receptor-doador foram incluídos e randomizados: 38 no grupo 1, 44 no grupo 2, 40 no grupo 3 e 9 no grupo 4. Não houve diferença estatística entre os grupos com relação às características clínicas. As taxas de soroproteção basal dos receptores foram de 18%, 32,8% e 63,3% para influenza A/H1N1, A/H3N2 e B, respectivamente. A condição sorológica pré transplante dos doadores foi semelhante. As taxas de soroconversão dos doadores foram de 30,1%, 41,5% e 30,9%, respectivamente para os A/H1N1, A/H3N2 e B. Nos receptores soroconversão até o dia 30 após transplante ocorreu em 16,3% dos pacientes para o A/H1N, 14,7% para A/H3N2 e 28,7%. As médias geométricas dos títulos de anticorpos neutralizantes foram calculadas para todos os subtipos virais ao longo do tempo. Para o A/H1N1 não houve diferença dos títulos até o D180 para nenhum dos grupos. Para o A/H3N2 houve diferença nos títulos no momento do transplante (p=0,019), com maiores títulos nos grupos 2 e 3 em relação ao grupo 1 e no dia 30 (p=0,018) com menores títulos para o grupo 4 que os demais. Para o subtipo B, as diferenças entre os grupos ocorreram no dia do transplante (p=0,020) e nos dias 30 (p= 0,018) e 60 (p=0,026) com menores títulos do grupo 4 em relação aos demais. As taxas de soroconversão após a vacina do dia 180 foram de 19,7% para o A/H1N1, 18% para o A/H3N2 e 8,2% para o B. Não houve diferença estatisticamente significante entre os grupos. CONCLUSÃO: A imunogenicidade da vacina de influenza em receptores de TCTH foi baixa. A estratégia de vacinação do doador e do receptor no pré transplante aumentou a média geométrica dos títulos protetores apenas para o subtipo A/H3N2 até o 30º pós transplante, em relação ao grupo sem vacinação / INTRODUCTION: Influenza is a potentially severe illness after hematopoietic stem cell transplantation (HSCT). Vaccination is the main prophylactic strategy, but the immune response is limited in the compromised host. Existing data support the recommendation of influenza vaccination after the 6th month of HSCT. OBJECTIVES: The study evaluated different schedules of influenza vaccination in HSCT. Recipient and/or their donors were randomized to receive influenza vaccine before transplant. The primary outcome was the comparison of serotype response between groups at baseline, 30 days and 6 months after transplantation. METHODS: A randomized, non-blind trial was conducted in patients undergoing HSCT and respective donors at Hospital das Clínicas, Universidade Federal de Minas Gerais /BH-MG and at Hospital Amaral Carvalho/Jaú-SP. Four groups of donor and recipient pairs received different influenza immunization at least 7 days before HSCT: group 1 no vaccination, group 2 donor received a single dose of influenza vaccine; group 3 recipients received a single dose of influenza vaccine and group 4 recipients and donor received influenza vaccine. Following transplantation, all study patients were immunized with influenza vaccine at 6 months. Donor serum samples were collected at baseline (pre transplantation) and in the day of transplantation. Recipients serum samples were taken at baseline, 30, 60, 100, 180 days after transplantation, and at least 2 weeks after 6-month vaccination. The hemagglutination inhibition assay (HIA) was performed to evaluate immune response. HI antibodies titers 1:40 were considered protective. Titers < 1:10 were considered negative. Antibody response (seroresponse) was defined as the appearance or 4-fold rise in HI antibody titers after vaccination. RESULTS: From August 2007 to February 2010 131 donor and recipient pairs were included in the study: 38 pairs in group 1, 44 in the group 2 , 40 in 3 group and 9 in group 4.There were no statistically difference between the 4 groups regarding to the clinical characteristics. At baseline 18% of recipients had protective antibody levels to A/H1N1, 32.8% to A/H3N2 and 63.3% to B. Donor serological condition was similar to recipients. Seroresponse occurred in 30,1% of donors to A/H1N1, 41,5% to A/H3N2 and 30,9% to B. Seroresponse until day 30 after transplant were detected in only 16,3% of the patients for the A/H1N, 14.7% for A/H3N2 and 28.7% for B. Comparisons of geometric mean antibody concentrations was performed at baseline and day of transplantation, and also at 30, 60, 100, 180 days after HSCT e after 6 months influenza vaccine. For the A/H1N1 there was no statistically difference between groups until 180 days after HSCT. For the A/H3N2 a significant increase in geometric mean titers in the day of transplantation (p=0,019) was observed in groups 2 and 3 in relation to group 1 and lower geometric mean titers (p=0,018) at day 30 for patients in group 4. For subtype B, the differences between groups occurred in the day of the transplantation (p=0,020) and at 30 (p= 0,018) and 60 (p=0,026) day. The geometric mean titers were lower in group 4 in relation to the others. Seroresponse after 6-month vaccination occurred in 19,7% to A/H1N1, 18% to A/H3N2 and 8.2% to B. There was no significant difference between the groups. CONCLUSION: Immunogenicity to influenza vaccine was poor in HSCT recipients. Donor or recipient vaccination strategy prior to transplantation increased the geometric mean titers only for subtype A/H3N2 at day 30 after transplant. No impact was observed in seroresponse rates after 6-month vaccination

Memoria para optar al Título Profesional de Médico Veterinario / La Influencia Aviar (I.A.) es una de las enfermedades más importantes en la avicultura, especialmente para economías emergentes y exportadoras de productos avícolas como es la chilena. Durante el año 2002 un brote de IA ocurrió por primera vez en el país, y el propósito de este estudio, fue mostrar el impacto económico de este brote. La información fue recopilada a partir del Servicio Agrícola Ganadero (SAG), la Asociación de Productores Avícolas (APA), la Oficina de Estudios y Políticas Agrarias (ODEPA) y consulta a expertos. El impacto económico se estimó a través de una análisis en niveles, definidos desde un punto de vista geográfico: Foco, área de restricción y resto del país. Al mismo tiempo se realizó una aproximación temporal, describiendo cuatro fases de análisis: Fase 1 desde el 6 al 23 de mayo (antes de la denuncia oficial del brote); Fase 2 desde el 24 de mayo al 20 de junio (desde la notificación hasta que las ultimas aves fueron sacrificadas); Fase 3 desde el 21 de junio al 19 de Diciembre (desde la eliminación de las ultimas aves hasta la declaración de país libre de I.A.); Fase 4 desde el 20 de diciembre hasta el 6 de mayo del 2003 (post-erradicación). De esta forma el país pasó a ser una matriz, y los costos fueron determinados y cuantificados a través del desarrollo y aplicación de una planilla de costos tipo en la que se determinaron 6 ítemes: I) Tratamiento II) Perdidas productivas III) Limpieza y repoblación IV) Bioseguridad y vigilancia V) Relaciones públicas, asesorias y capacitación VI) Perdidas de mercados externos y mantención del mercado interno. Las perdidas productivas fueron calculadas a través de la modelación y simulación de los planteles afectados. El costo total para el brote de I.A. fue de aproximadamente US $31.782.475,83, divididos de la siguiente forma: Perdidas del sector publico: US $390.586,43; y sector privado: US $31.391.889,4.

Class of 2016 Abstracts / Objectives: To compare the minimum vaccination age to receive an influenza vaccine of rural and urban pharmacies. Rural pharmacies are defined in all Arizona pharmacies in counties other than Pima or Maricopa, urban pharmacies are defined as all pharmacies within the Tucson city limits. Methods: Pharmacies were called for a phone interview asking what the minimum age someone needs to be to receive an influenza vaccination from their store is. Pharmacies were called during their operating hours during a 4-week period at the end of January and early February. Results: 269 pharmacies were included in the study. Pharmacies consisted of 153 rural pharmacies and 116 urban pharmacies. The median minimum vaccination age for both rural and urban pharmacies was 8. Overall, there was found to be no significant difference between the minimum vaccination age between rural and urban pharmacies (p = 0.242). Conclusions: The minimum age to receive an influenza appears to be similar between rural and urban pharmacies.

Class of 2016 Abstract / Objectives: To determine the prevalence and incidence of community pharmacies not vaccinating children down to six years of age and to identify the reasons for non-compliance with Arizona State Board administrative rule R4-23-411. Subjects: 103 community retail pharmacies from six corporations in the Tucson metropolitan area established by December 2014. Methods: Funnel questionnaire administered via phone call to collect pharmacist response if he or she would vaccinate a six-year-old child; data for reasons why he or she would or would not vaccinate and gender of pharmacist was also collected. Results: Responses were collected from 103 pharmacists (male n = 55, female n = 48). 87% (n =90) of pharmacies stated they would not vaccinate a six-year-old, while 13% (n =13) would vaccinate. The rationale for not vaccinating varied from corporate policy (45%) to state law (37%). Conclusions: The majority of six retail pharmacy chains in Tucson, Arizona are not vaccinating down to six-years old, although Arizona administrative amendments allow pharmacists to do so. More pharmacies that would choose not to vaccinate may be related to a better understanding of corporate policies versus state administrative rule change.

Magister Public Health - MPH / Avian Influenza (AI) is a contagious viral zoonotic disease with great public health implications and negative socioeconomic impact (WHO, 2006a). The highly pathogenic avian influenza (HPAI) infection is transmitted from birds to man mostly through contact with contaminated poultry and objects (INFOSAN, 2005), hence people who come in contact with birds such as live bird sellers (LBS) are the more vulnerable population (WHO, 2006a). Inadequate knowledge of AI health risks and poor practice of AI preventive measures amongst LBS increases the risk of spread of the infection in both humans and animals.The aim of this study was to describe and quantify the knowledge and practice of LBS with regards to avian influenza health risks and preventive activities in Agege, an urban area in Lagos State, Nigeria. / South Africa

Avian influenza H5N1 causes occasional but serious infections in humans and efforts to produce vaccines against this strain continue. Current influenza vaccines are prophylactic and utilize the two major antigens, hemagglutinin and neuraminidase. NP is an attractive alternative antigen because it is highly conserved across all influenza strains, has been shown to increase the rate of viral clearance, and potential therapeutic vaccines would elicit cytotoxic T lymophcyte responses in an infected person. The NP antigen from H5N1 was characterized using a variety of physiochemical methods to gain insights into both the biological and physical properties of the antigen which are important from a regulatory viewpoint when considering therapeutic vaccines. Results obtained to date show that NP is relatively unstable and indicate that the conformation of the H5N1 NP antigen is highly dependent upon purification procedure, buffer conditions, pH and the presence or absence of RNA. These factors will need to be clearly defined and taken into consideration when manufacturing and regulating NP vaccine preparations.

Vaccination is one of the major strategies available for combating viral infections in humans. However, currently available vaccines are not without pitfalls; they are laborious to produce, could potentially be unsafe, and in the case of the highly variable influenza virus need to be reformulated each season. The use of synthetic peptides thus represents an exciting alternative to traditional vaccines. However, these synthetic peptides are not highly immunogenic without the use of potent adjuvants. The lack of immunogenicity might be addressed by conjugation between T or B cell epitopes with universal or immunodominant T-helper epitopes. The construction of branched peptides, lipidated peptides, or designs combining both of these elements might also enhance the immunogenicity, as they might target Toll-like receptors and/or mimic the 3-dimensional structure of epitopes within the native protein. In this study, a recognized T-B peptide based on the hemagglutinin protein of the A/Puerto Rico/8/34 influenza virus was chosen as a backbone and modified to evaluate if the construction of branched peptides, lipidation, the addition of cysteine residues, or mutations could indeed alter reactivity. Screening the different designs with various antibody binding and cellular assays revealed that combining a branched design with the addition of lipid moieties leads to a greatly enhanced activity as compared to other similar T-B diepitope constructs.

The genetic requirements for Influenza virus to infect and adapt to new species is largely unknown. To understand the evolutionary steps required by a virus to become virulent, a human virus (A/HK/1/68) (HK), avirulent in mice, was subjected to 20 and 21 serial lung-to-lung passages in mouse. Sequence analysis revealed the emergence of eleven mutations within the NS1 gene of the new virulent strains, many of which occurred in binding sites for transcriptional and translational cellular factors. In the present study we have rescued viruses containing each of the NS1 mouse adapted mutations onto A/PR/8/34 (PR8) backbone. We found 9 of 16 NS1 mutants were adaptive by inducing mortality, body weight loss in BALB/c mice and enhanced virus replication in MDCK cells with properties of host cell interferon transcription inhibition. Sequence comparisons with the highly pathogenic A/Hong Kong/156/1997 (H5N1) and the most severe pandemic A/Brevig Mission/1/1918 (H1N1) NS1 genes showed convergent evolution with some of the mouse adapted viruses for F103L plus M106I and V226I plus R227K mutations respectively. The F103L and M106I mutations in the HK NS1 gene were shown to be adaptive by assessment with respect to replication, early viral protein synthesis, interferon-β antagonism and tropism in the mouse lung. We extended the study and proved increased virulence associated with F103L+M106I mutations in their respective H5N1 NS1 gene on the PR8 and HK backbones, as well as the PR8 NS1 gene and the H9N2 (A/Ck/Bj/1/95) gene in the PR8 and A/WSN/33 backbones respectively. However the V226I and R227K mutations in their respective HK and 1918 NS1 genes slightly enhanced virulence and viral growth at later stages of infection. This study demonstrates that NS1 is a virulence factor; involved in multiple viral processes including interferon antagonism and viral protein synthesis. Furthermore, NS1 mutations acquired during mouse adaptation are proven to be adaptive in human, mouse and avian NS1 genes.

The overarching objective of this thesis was to investigate the phenomenon of vaccine hesitancy in Canada and examine relationships among vaccine beliefs, socio-demographic characteristics, and seasonal influenza immunization. Quantitative findings were derived from a national health risk perception survey administered to adults across Canada (N = 1,125). Respondents were asked to provide their level of agreement (1 = do not agree at all to 5 = agree completely) with 2 vaccine-related behaviour statements and 21 vaccine-related beliefs statements. A principal components analysis was performed to reduce the number of belief statements into meaningful components. Two components were retained and reflected negative beliefs about ‘vaccine safety’ and positive beliefs about ‘vaccine regulation and benefits’. Descriptive results presented in the first study indicated a heightened uncertainty about the long-term side effects of vaccination, particularly with respect to the purported link between the Measles-Mumps-Rubella vaccine and the development of autism, among survey respondents. Multivariate analyses identified differences in the endorsement of numerous vaccine beliefs according to age and educational attainment. Findings revealed that older respondents and respondents without a university education demonstrated more negative attitudes towards vaccination, whereas younger respondents and respondents with a university education demonstrated more positive vaccine attitudes, respectively. Finally, both components of vaccine beliefs were significant predictors of vaccine-related behaviours, including discussing information about vaccines with others and reported receipt of the seasonal influenza vaccine. The second study investigated interrelationships among components of vaccine beliefs, socio-demographic characteristics, and reported receipt of the seasonal influenza vaccine. A mediation analysis revealed that higher levels of agreement with the statement ‘I usually get the seasonal flu vaccine’ among older adults was associated with lower levels of agreement with negative beliefs about vaccine safety in conjunction with higher levels of agreement with positive beliefs about the regulation and benefits of vaccines, whereas the opposite was true for younger adults. Also, a significant moderation analysis revealed that among respondents with greater concern about vaccine safety, those with higher educational attainment reported lower levels of agreement with the statement ‘I usually get the seasonal flu vaccine’ compared to those with lower educational attainment. Recognizing the limitations of quantitative findings, a qualitative investigation was undertaken to provide more in-depth insight on the factors driving influenza immunization among healthy adults. A thematic analysis was performed on transcripts from 6 semi-structured focus group discussions with a total of 18 participants residing in Ottawa, Ontario. Findings identified 7 themes and 8 sub-themes related to contextual, vaccine specific, and individual determinants of vaccine hesitancy. Participants predominantly discussed themes related to individual determinants of vaccine hesitancy (perceived severity, susceptibility, and likelihood of contracting the influenza virus; personal interests; interactions with healthcare professionals). The perceived novelty, severity and effectiveness of the influenza vaccine, as well as a lack of information and discontent with communication by government health authorities and the media were also discussed. Overall, findings identified salient themes informing vaccine decision-making and behaviours among a sample of educated adults, which can inform subsequent studies investigating influenza immunization in a more representative sample of Canadian adults.

The antigenic variability of the Influenza virus hinders our ability to develop new therapeutic and vaccine strategies which provide a broad protection against all influenza strains. It has been previously suggested that a means to approach this challenge is to identify conserved sequences within viral proteins and use these for future therapeutic targets. Although such conserved sequences are plentiful amongst the internal viral proteins, their lack of exposure to the host immune system makes mounting an immune response against these regions difficult. Alternatively, the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) have been shown to provide host protection against a limited number of influenza strains when used as vaccine targets; however conserved regions within these proteins which are also antibody accessible are extremely rare. My Ph.D. thesis project is focused on investigating the functional role of a conserved region within the NA protein and to further determine the protection afforded by a monoclonal antibody to this region. In a comprehensive bioinformatics analysis, the only universally conserved sequence amongst all influenza A and B viral NA has been previously identified as being located between amino acids (a.a.) 222-230 (dubbed the HCA-2 region). However, the potential role of this region remains largely unknown. Through an array of experimental approaches including mutagenesis, reverse genetics and growth kinetics, I have found that substitutions in this sequence significantly affect viral replication by impairing the catalytic activity, substrate-binding and thermostability of NA. These findings prompted me to further investigate if antibody to this region may provide protection against influenza infection. Indeed, universal monoclonal antibody (HCA-2 MAb) against this peptide provided broad inhibition against all nine subtypes of NA in vitro and heterosubtypic protection in mice challenged with lethal doses of mouse-adapted viruses. I further demonstrated that residues within this peptide that are exposed on the surface of NA and located in close proximity to the active site, I222 and E227, are indispensable for antibody-mediated inhibition. These data are the first to demonstrate a monoclonal antibody against the NA protein which provides heterosubtypic protection. Since I observed that the HCA-2 antibody provided a broad inhibition against all nine subtypes of influenza A NA, I decided to investigate whether this inhibitory effect could be extended against Influenza B. Here, I have further reported that HCA-2 MAb provides a broad inhibition against various strains of influenza B viruses of both Victoria and Yamagata genetic lineage. I also demonstrate that the growth and NA enzymatic activity of two drug resistant influenza B strains are also inhibited by the HCA-2 antibody. The findings of my Ph.D. thesis project have thus demonstrated that the HCA-2 region is paramount to optimal viral function. Additionally, my data show that antibodies generated against this region provide heterosubtypic protection both in vitro and in vivo and against drug resistant strains. These results indicate that this universally conserved epitope should be further explored as a potential target for future antiviral intervention and vaccine-induced immune responses.