No mês de março de 2013 foram reportados pela Organização Mundial da Saúde (OMS) os primeiros casos de infecções humana pelo vírus Influenza aviário A(H7N9). Em maio de 2017 já haviam sido registrados 1439 casos. As infecções em humanos causadas por este vírus são associadas a consideráveis níveis de mortalidade e morbidade. A vacinação é atualmente o meio mais efetivo para prevenir as infecções e as severas complicações na saúde humana causada pelo vírus da Gripe. Desenvolver e produzir vacinas contra a Gripe pandêmica é a estratégia principal para uma resposta contra a pandemia (WORLD HEALTH ORGANIZATION, 2005). Este projeto produziu os bancos de vírus H7N9, um lote piloto e três lotes industriais em condições (BPF) dos monovalentes utilizados para a produção da vacina contra a gripe pandêmica, A(H7N9). Estes lotes foram caracterizados, avaliados em relação aos padrões de qualidade e testados quanto a imunogenicidade em ensaios com camundongos. / In March 2013 it was reported by the World Health Organization (WHO) the first cases of human infections with avian influenza virus A (H7N9). In May 2017 they had been registered 1439 cases. The infections in humans caused by this virus are associated with considerable morbidity and mortality levels. Vaccination is currently the most effective way to prevent infections and severe complications on human health caused by the flu virus. Developing and producing vaccines against pandemic influenza is the main strategy for a response to the pandemic (WORLD HEALTH ORGANIZATION, 2005). This project produce the seeds virus A(H7N9), a pilot batch and three industrial lots (GMP) of the monovalents used to produce the pandemic influenza vaccine against A(H7N9). These batches were characterized, evaluated for quality standards and tested for immunogenicity in mouse assay.

Introdução: As ações de imunização merecem destaque mundial pelo grande impacto do uso de vacinas na prevenção das doenças imunopreveníveis. A necessidade de um diagnóstico da prevalência de perdas vacinais constitui uma etapa fundamental para o gerenciamento de vacinas no município, permitindo organizar adequadamente o sistema, evitando assim desperdícios dos recursos públicos. Objetivo: Diagnosticar e caracterizar as perdas vacinais das Unidades Básicas de Saúde da Região Oeste do município de São Paulo. Método: Trata-se de um estudo descritivo, retrospectivo, quanti-qualitativo, no qual foi utilizado como abordagem metodológica o estudo de caso. A partir dos registros de movimentação de imunobiológicos e relatórios de doses aplicadas do ano de 2015, foi calculada a taxa total das perdas, prevalência das perdas técnicas, prevalência de perdas físicas e os motivos das perdas físicas, prevalência das perdas não categorizadas e a razão das doses aplicadas por doses utilizadas. Foram realizadas entrevistas com os funcionários das salas de vacina, das UBSs da Região Oeste do município de São Paulo. Resultados: A taxa total de perdas foi de 71,3 %, a prevalência de perda técnica 18,6% e a prevalência de perda física 28,4 %. Dentre os motivos das perdas físicas, obteve-se destaque para a falta de energia elétrica, representando 18,4 % das mesmas; as perdas não categorizadas totalizaram 24,2 %. Quanto à razão de doses aplicadas, por doses utilizadas, a vacina que apresentou o maior percentual de perdas foi a vacina BCG, visto que para cada dose aplicada foram perdidas 4,86 doses. Nas entrevistas realizadas com os profissionais que trabalham nas salas de vacina, observou-se que 60% deles acreditavam que a maior causa de perda vacinal em sua unidade se devia à perda técnica. Já 36,67 % referiram que a maior causa de perda se deveu às perdas físicas, causada por falta de energia elétrica. Os profissionais apontaram sugestões para minimizar as perdas vacinais; 50 % dos entrevistados relataram que a presença de vacinas unidoses amenizaria as perdas, 46,67 % dos profissionais sugeriram a existência de gerador elétrico e 16,67 % sugeriram o agendamento de vacinas com maior percentual de perda técnica. Conclusão: Os resultados demonstraram que a taxa total de perdas vacinais na Região Oeste do município de São Paulo foi de 71,3 % e na avaliação da prevalência de perdas, obteve-se uma maior prevalência de perdas físicas. Já na opinião dos profissionais entrevistados, a maior causa de perdas deveu-se às perdas técnicas. O presente trabalho propiciou a realização de uma cartilha educativa, que propõe ações que visem diminuir as perdas vacinais nas UBSs da Região Oeste do município de São Paulo. Essa cartilha será apresentada aos órgãos da Secretaria Municipal da Saúde de São Paulo, a saber: Coordenadoria de Saúde da Região Oeste (SUVIS Oeste) e Gerência de Imunização (COVISA). / Introduction: Immunization actions deserve worldwide focus due to the great impact of the use of vaccines in the foresight of immune preventable diseases. The need of prevalence of vaccine losses diagnosis constitutes a fundamental step for the vaccines management in the city, allowing the system adequate organization, therefore avoiding public resources waste. Objective: To diagnose and feature the vaccine losses at the basic health units of Sao Paulo\'s Western Area. Method: This is a descriptive, retrospective, quantitative-qualitative study, where the case study was used as a methodological approach. The total loss rate, the technical losses prevalence, the physical losses prevalence and the physical losses reasons proportion, the unclassified losses prevalence and the dose ratio applied by the doses used were calculated from the immune-biological records movement and dose reports for the year 2015. Interviews were carried out with the employees of the basic health units\' vaccine rooms of Sao Paulo\'s Western Area. Results: The total losses prevalence was of 71.3%, the technical loss prevalence was of 18.6%, the physical loss prevalence was of 28.4%. Of the reasons for physical loss, the lack of electricity represents 18.4%. Non-categorized losses totaled 24.2%. Regarding the dose ratio applied by doses used, the vaccine that presented the highest percentage was the BCG vaccine, for each applied dose there is a loss of 4.86 doses; regarding Yellow Fever vaccine, for each applied dose, 1.63 doses are lost; as for the Triple Viral vaccine, for each applied dose, 1.31 doses are lost. From the interviews with vaccine rooms employees, it was observed that 60% of them believe that vaccine loss greatest reason in their unit is due to technical loss, and 36.67% reported that the greatest cause of loss, it is due to physical loss caused by lack of electricity. The employees gave suggestions to minimize vaccine losses; 50% of the interviewees reported that the presence of unit dose vaccines would reduce losses, 46.67% of the employees suggested the need of an electric generator, and 16.67% suggested scheduling vaccines which have a higher percentage of technical loss. Conclusion: The results showed that the vaccine losses total rate in São Paulo\'s Western Area was of 71.3%, and that in the losses prevalence evaluation, a higher prevalence of physical losses was obtained, as for the opinion of employees interviewed, the greatest cause of loss is due to technical losses. Vaccine losses monitoring is important in order to find new alternatives for the production and distribution of immune-biological agents in order to reduce losses without missing the opportunity to vaccinate. The present work led to the realization of an educational booklet, which will propose actions aimed at reducing vaccine losse at the Health Basic Units of Sao Paulo\'s West Region. This booklet will be presented to Sao Paulo\'s City Health Department agencies, namely: Western Region Health Coordination (SUVIS Oeste) and Immunization Management (COVISA).

A leptospirose é considerada a zoonose mais difundida do mundo, assim como uma doença reemergente. Esta enfermidade, causada por bactérias patogênicas do gênero Leptospira, possui altas taxas de infecção em países em desenvolvimento, ocasionando graves prejuízos econômicos e de saúde pública. Até o momento, não existem vacinas humanas licenciadas contra leptospirose. Após o seqüenciamento do genoma de três espécies de Leptospiras vários genes foram apontados como candidatos vacinais promissores. Uma categoria importante de genes candidatos são aqueles com possível atividade hemolítica. Neste trabalho, clonamos e expressamos diversas proteínas com possível atuação hemolítica. As proteínas recombinantes obtidas, no entanto, não exibiram atividade hemolítica. Uma destas proteínas, TlyC, foi investigada quanto à sua capacidade de interagir com os componentes da matriz extracelular (MEC). Os resultados obtidos indicam que TlyC liga-se com alta afinidade a diversos componentes da MEC, e que esta proteína é capaz de inibir competitivamente a adesão de Leptospiras à um material biológico que se assemelha à MEC. A transcrição e expressão destas proteínas foi detectada em cultura de Leptospira. Algumas das proteínas recombinantes foram utilizadas em um desafio animal contra leptospirose, mas nenhum delas foi protetora. Concluímos que estas proteínas não parecem ser bons candidatos vacinais e que TlyC é uma proteína que interage com componentes da MEC. / Leptospirosis is considered the most disseminated zoonosis of the world, and also a reemerging disease. This disease, caused by pathogenic bacteria of the genus Leptospira, has high rates of infection in developing countries, leading to severe economic and medical costs. There is not a licensed vaccine against leptospirosis for human use. After the genome sequencing of three species of leptospires, several genes were pointed to be promising vaccinal candidates. An important category of these candidates are those with putative hemolytic activity. In this work, we cloned and expressed some proteins with putative hemolytic activity. The recombinant proteins obtained, however, did not show hemolytic activity. One of these proteins, TlyC, was investigated with regard to its possible ability to interact to extracellular matrix (ECM) components. The results obtained indicate that TlyC binds with high affinity to several ECM components and that this protein can inhibit the Leptospira bind to a biological material that ressambles the ECM. The transcription and expression of these proteins were detected in leptospires cultures. Some of the recombinant proteins were used in an animal challenge against leptospirosis, but none of them were protective. We conclude that these proteins do not seem to be good vaccine candidates and that TlyC is a protein that interacts with the ECM and its components.

O câncer cervical representa a terceira causa de morte por câncer em mulheres e está associado a infecções persistentes pelo Vírus do Papiloma Humano (HPV) em mais de 99% dos casos. Evidências sólidas mostram que a expansão de Células Mielóides Supressoras (MDSC) e fatores solúveis como a interleucina IL-10 são importantes mediadores da evasão tumoral à resposta imunológica contra tumores, que limita a eficácia de imunoterapias. Dada a importância epidemiológica dos tumores induzidos por HPV e a necessidade eminente do desenvolvimento de imunoterapias ativas contra essas lesões, desenvolvemos durante os últimos 12 anos, uma estratégia vacinal terapêutica baseada em DNA. Esta vacina codifica a proteína E7 do HPV-16 fusionada à glicoproteína D (gD) do Vírus Herpes Simplex do tipo 1 (HSV-1), denominada pgDE7h. O presente trabalho propôs a investigação de estratégias que restringem a imunossupressão, baseadas em um plasmídeo que codifica o receptor solúvel da citocina IL-10 (pIL10R) e da quimioterapia com gencitabina, na potencialização da vacina pgDE7h para controlar o crescimento de tumores que expressam as proteínas E6 e E7 do HPV-16 em modelo murino (células TC-1). Foi possível observar um atraso no crescimento do tumoral a partir da combinação da vacina com o pIL-10R. Adicionalmente, a utilização da eletroporação como método de entrega dos plasmídeos aumentou a proteção terapêutica para 90% e 60% quando os animais foram imunizados 5 ou 14 dias após o desafio, respectivamente. A combinação dos plasmídeos foi ainda capaz de elevar o número de linfócitos T CD8+ E7-específicos ativados, encontrados sistemicamente e no sítio tumoral. No consócio da imunização com a pgDE7h à gencitabina, foi possível observar um efeito sinérgico dessa associação, que aumentou a proteção antitumoral de 20% para 100% de animais livres de tumor, concomitante ao controle da expansão de populações imunossupressoras no baço. Além disso, a quimioimunoterapia administrada até 14 dias após o desafio com as células tumorais mostrou-se protetora em modelo de recidivas, simuladas por meio de transplantes subsequentes de células tumorais. Na busca de um protocolo vacinal com maior viabilidade clínica, combinamos a coadministração dos plasmídeos pgDE7h e pIL- 10R com o tratamento com gencitabina. Esta combinação terapêutica mostrou-se mais eficiente, onde se observou um aumento robusto da ativação de linfócitos T CD8+ E7-espefíficos, com regressão completa de tumores pré-estabelecidos. Em suma, esses resultados evidenciam que a associação de abordagens terapêuticas pode superar barreiras imunológicas presentes no ambiente tumoral e aumentar as chances de sucesso clínico do tratamento proposto. / Cervical cancer represents the third cause of cancer death among women, which is associated with persistent Human Papilloma Virus (HPV) infection in more than 99% of cases. Solid evidences show that the expansion of Myeloid-derived Suppressor Cells (MDSC) and soluble factors, such as IL-10, are important mediators of immune evasion mechanisms expressed by tumor cells, that limits the efficacy of immunotherapeutic approaches. Given the epidemiological importance of HPVinduced tumors and the crucial importance of developing active immunotherapies against neoplasias, during the past 12 years, we have been developing a DNA-based therapeutic vaccine strategy encoding the HPV-16 E7 protein fused to the Herpes Simplex Virus type 1 (HSV-1) glycoprotein D (gD), named pgDE7h. This work aimed to investigate the potential role of immunossupression restriction using a plasmid encoding the soluble IL-10 receptor (pIL10R) and the chemotherapeutic drug gemcitabine to enhance in enhancing the pgDE7h vaccine potency in the control of tumors expressing HPV-16 E6 and E7 proteins in a murine model (TC-1 cells). It was possible to observe a tumor growth delay after combining the vaccine with pIL-10R. Additionally, the use of electroporation as a plasmid delivery method provided a therapeutic protection of 90% and 60% when the animals were immunized 5 or 14 days after challenge, respectively. The combination of plasmids was also capable to increase substantially the numbers of activatedE7-specific CD8+ T lymphocytes both systemically and at the tumor site. When pgDE7h immunization was combined to chemotherapy, we observed a synergistic effect, which increased the antitumor protection from 20% to 100% and promoted the control of immunosuppressive cell populations in the spleen. It was possible to maintain the high antitumour protective effects of the chemo-immunotherapy when the animals were immunized 14 days after tumor cell challenge and tumor relapses after subsequent challenges with tumor cells. To search a vaccine protocol with greater applicability in a clinical setting, we combined co-administration of pgDE7h and pIL-10R and gemcitabine treatment. Our data showed that the combined treatment induced a robust increase in the activation of CD8+ T lymphocytes and a complete regression of pre-established tumors. All together, these results show that the combination of therapeutic approaches can overcome immunological barriers present in the tumor environment and increase the chances of clinical success of the propose therapeutic treatment.

As formulações vacinais contra o Vírus Respiratório Sincicial Humano, HRSV, estão associadas à indução de eosinofilia pulmonar mediada por uma resposta de células TCD4+ Th2, após exposição ao HRSV selvagem. Foi identificado um peptídeo da proteína viral G, que modificado perde a capacidade de predispor à eosinofilia, tornando-o um imunógeno atraente. Células T CD8+ específicas para HRSV reduzem a resposta Th2, mediam resistência a desafio com o vírus, e estão relacionadas à redução dos sintomas. Assim, neste trabalho buscamos e identificamos epítopos de células TCD8+ na polimerase viral, utilizando programas de predição, imunização com peptídeos e avaliação da resposta celular. Também construímos vacinas de DNA contendo a seqüência nucleotídica do peptídeo da proteína G modificado. A caracterização da resposta imune estimulada por essas vacinas e por peptídeos purificados revelou que o plasmídio pTGMCTB, bem como os peptídeos GM e GMCTB, foram capazes de induzir anticorpos que, porém, não se mostraram neutralizantes de HRSV e protetores frente a desafio. / Vaccines against human respiratory syncytial virus (HRSV) are associated with pulmonary eosinophilia induction mediated by a TCD4+ Th2 response, after exposition to wild HRSV. A peptide from the viral protein G was identified to predispose to eosinophilia and loses this ability when mutated, making it an interesting immunogen. CD8+ T cells specific to HRSV reduce the Th2 response, mediate resistance to virus challenge, and are related to symptom-reduction. Thus, in the present work, we searched for and identified CD8+ T cell epitopes in the viral polymerase; using prediction programs, peptide immunization and evaluation of the cellular response. We also constructed DNA vaccines containing the nucleotide sequence of the mutated peptide from G protein mentioned above. The characterization of the immune response elicited by these vaccines and purified peptides showed that the pTGMCTB plasmid, as well as GM and GMCTB peptides were able to induce antibody response; however they are not neutralizing and protective against HRSV challenge.

A gripe é causada pelo vírus Influenza e é um problema de saúde pública mundial, que pode levar a problemas sérios em idosos e crianças. O Brasil implantou a vacinação anual contra influenza a partir de 1999, como ação preventiva contra a doença. A vacina é produzida pelo Instituto Butantan e contém três cepas diferentes do vírus Influenza fragmentado para induzir resposta imune adaptativa, com produção de anticorpos específicos e neutralizantes. A literatura tem mostrado que a exposição à xenobióticos com potencial imunossupressor pode comprometer a eficácia de imunizações ativas, como a imunização contra a gripe. Nosso grupo de pesquisa tem mostrado que a exposição à hidroquinona (HQ), um composto tóxico presente em altas concentrações na fumaça do cigarro, prejudica a resposta imune inata e adquirida. Assim, este trabalho avaliou o efeito da exposição à HQ sobre a resposta imune à vacinação contra influenza. Camundongos machos da linhagem C57BL/6 foram diariamente expostos à HQ (2500 ppm) ou PBS, por 1 hora, por nebulização, por um período de 8 semanas. Durante este período, foram imunizados nas semanas 6 e 8 do início das exposições, pela injeção i.m. de 100µL da vacina. Os parâmetros tóxicos e imunológicos foram avaliados 7, 35 e 70 dias após a segunda dose da vacina. A exposição à HQ não alterou o peso corpóreo dos animais e nem causou alterações morfológicas no pulmão, fígado e rins (histologia por H&E); reduziu a frequência de hemácias (11%), hematócrito (14%), hemoglobina (14%) e volume celular (4%); causou estresse oxidativo no baço (citometria de fluxo); aumentou a área dos folículos de células B no baço e linfonodomegalia (histologia por H&E). Em conjunto, os dados aqui obtidos mostram que a exposição à HQ afetou mecanismos envolvidos na gênese da imunidade ativa contra influenza. Assim, os dados deste trabalho mostram mecanismos tóxicos ainda não descritos para a HQ, e ressalta a HQ como um poluente ambiental que deve ser considerado nas avaliações de risco. / The flu is a health problem worldwide which is caused by the Influenza virus and may result in severe illness in infants and the elderly. The annually vaccination against influenza was implemented in Brazil in 1999 as a preventive measure. The vaccine is produced by Butantan Institute and contains three different strains of the inactivated Influenza virus which induce the adaptive immune response along with production of specific and neutralizing antibodies. The literature has shown that exposure to immunosuppressive xenobiotics may compromise the efficacy of active immunizations, such as influenza. Our research group has shown that exposure to hydroquinone (HQ), a toxic constituent of cigarette smoke, impairs both innate and adaptive immune response. Thus, the aim of this work was to evaluate the effects of HQ on the immune response induced by the influenza vaccine. Male C57BL/6 mice were daily exposed to HQ (2500 ppm) or PBS by nebulization, for 1 hour, for 8 weeks. During the exposure period, the animals were vaccinated on weeks 6 and 8 with 100µL of the vaccine. Toxicologic and immunological parameters were assessed 7, 35 and 70 days after boost administration. HQ exposure did not alter body weight and did not cause morphological alterations in the lungs, liver and kidneys (H&E staining); reduced the frequency of erythrocytes (11%), hematocrit (14%), hemoglobin (14%) and cellular volume (4%) and caused oxidative stress on the spleen (Flow Cytometry); increased the area of B cell follicles in the spleen and increased the size of draining lymph nodes (H&E staining). Altogether, these data show that HQ exposure affected mechanisms involved in the genesis of the adaptive immune response. Thus, the data presented in this work show toxic mechanisms of HQ that have not yet been described, and it also points out HQ as an environmental pollutant which should be considered on risk assessments.

Rotavirus is the most common cause of severe gastroenteritis among children <5 years of age worldwide and is responsible for 453,000 deaths among children in this age group. More than half of these deaths occur in sub-Saharan Africa. Because of the tremendous global burden of rotavirus, vaccine development and introduction has been a high priority for several international agencies, including the World Health Organization (WHO) and GAVI. Two live, attenuated, orally administered rotavirus vaccines, a pentavalent bovine-human reassortant vaccine (RV5; RotaTeq® (Merck and Co, Inc, Pennsylvania)) and a monovalent vaccine (RV1; Rotarix™ (GSK Biologicals, Rixensart, Belgium)) based on a human rotavirus strain, are licensed and available for use in many countries worldwide. Pre-licensure clinical trials of each of these vaccines in high and middle-income countries demonstrated high efficacy (85-98%) against severe rotavirus disease. Further studies conducted in low-income countries of Asia and Africa found modest efficacy (50%-70%) of these vaccines against severe rotavirus disease. However, the public health impact of vaccination (in terms of burden of severe rotavirus disease prevented by vaccinating a given number of children) is greater in developing countries because of the substantially higher baseline rotavirus disease burden in these settings. In 2009, the World Health Organization recommended the inclusion of rotavirus vaccine in the national immunization programs of all countries globally and particularly in those countries with high child mortality due to diarrhea. Of the 16 countries recently approved by GAVI for rotavirus vaccine introduction, 12 countries are located in Africa. As rotavirus vaccines are introduced into national immunization programs, monitoring their impact is a high priority for several reasons. There is a need to assess the effectiveness of these vaccines in routine use to ensure it parallels that of pre-licensure trials, particularly when used in developing countries. Assessing the impact of vaccination on disease burden in countries such as Rwanda will be vital to understanding the full public health benefit of the vaccine. The primary purpose of this program evaluation is to determine the impact of pentavalent rotavirus vaccine on rotavirus and all-cause diarrhea morbidity following introduction into the national immunization program in Rwanda in May 2012. Additionally, this evaluation will document changes in circulating strains over time pre- and post-vaccine introduction. It will also strengthen support for economic evaluation of treating diarrhea versus introduction of new vaccine in routine immunization. Methodology Various studies have been implemented since 2011 in the health sector in Rwanda to reach the goal of this thesis. First, we analyzed data for all-cause, non-bloody diarrheal disease among children <5 years of age from the routine health management information system (HMIS) in Rwanda from January 2008 through December 2011, The objective of this analysis was to determine whether routinely collected health information on national diarrhea hospitalizations, in-hospital deaths, and outpatient visits can be used to monitor the impact of rotavirus vaccine. We used data from the health management information system (HMIS) in Rwanda to describe trends in all-cause, non-bloody diarrhea hospitalizations and outpatient visits among children <5 years of age from 2008 to 2011 prior to vaccine introduction. Second, we evaluated the economic burden attributable to hospitalization for diarrhea among children aged less than 5 years in Rwanda. This was a prospective costing study where medical records and hospital bills for children admitted with diarrhea at 3 hospitals were collected to estimate costs. Interviews with the child’s caregivers provided medical costs incurred before and after hospitalization and the household costs. Third, we analyzed and tried to understand the introduction and delivery cost per dose or per child of the three new vaccines in Rwanda including the rotavirus vaccine for domestic and external financial resource mobilization. Fourth, we determined the rotavirus prevalence rates and circulating genotypes directly pre- and post-introduction of the RotaTeq rotavirus vaccine in May 2012. Stool samples were collected from 1,847 children <5 admitted to 8 surveillance sites for acute gastroenteritis (AGE) and tested for rotavirus antigens by enzyme immunoassay. Fifth, to monitor the effect of rotavirus vaccine in Rwanda, we studied trends in the number of hospital admissions for diarrhea and rotavirus before and after the introduction of the rotavirus vaccine. We conducted a time-series analysis to examine trends in admissions to hospital for non-bloody diarrhea in children younger than 5 years in Rwanda between Jan 1, 2009, and Dec 31, 2014, using monthly discharge data from the HMIS.Result All-cause, non bloody diarrheal hospitalizations and outpatient visits among children <5 years of age in Rwanda from 2008 to 2011 peaked during the June to August dry season, coinciding with the rotavirus season. The bulk of the diarrheal disease burden occurred in children <1 year of age. Average medical costs for each child for the hospitalization were $44.22 ± $23.74 and the total economic burden per hospitalization was $101, of which 65% was borne by the household. The unit cost of introducing rotavirus vaccines 2012 was 22.69 US. Among the 397 stool samples that were genotyped, 5 G types (G1, G4, G8, G9, and G12) and 3 P types (P[4], P[6], and P[8]) were identified. G8 (30.3%), G9 (28.0%), and G1 (19.7%) were the most prevalent G types, while P[8] (52.0%) and P[4] (32.6%) were the most prevalent P types. There was a significant amount of mixed G genotypes (12.1%), while mixed P types were less common (5.1%). G8P[4], G9P[8], and G1P[8] were the most prevalent strains, accounting for 27.8%, 24.3%, and 15.3% of all specimens, respectively.Compared with the 2009–11 pre vaccine baseline, hospital admissions for non-bloody diarrhea captured by the HMIS fell by 17–29% from a pre-vaccine median of 4051 to 2881 in 2013 and 3371 in 2014, admissions for AGE captured in pediatric ward registries decreased by 48–49%, and admissions specific to rotavirus captured by active surveillance fell by 61–70%. The greatest effect was recorded in children age-eligible to be vaccinated, but we noted a decrease in the proportion of children with diarrhea testing positive for rotavirus in almost every age group.ConclusionGiven the stable and consistent trends and the prominent seasonality consistent with that of rotavirus, HMIS data should provide a useful baseline to monitor rotavirus vaccine impact on the overall diarrheal disease burden in Rwanda. Active, sentinel surveillance for rotavirus diarrhea will help interpret changes in diarrheal disease trends following vaccine introduction. Other countries planning rotavirus vaccine introduction should explore the availability and quality of their HMIS data.Households often bear the largest share of the economic burden attributable to diarrhea hospitalization and the burden can be substantial, especially for households in the lowest income quintile.The cost of introduction of new vaccines (rotavirus) is less than the cost of treating the diarrhea diseases. The number of admissions to hospital for diarrhea and rotavirus in Rwanda fell substantially after rotavirus vaccine implementation, including among older children age-ineligible for vaccination, suggesting indirect protection through reduced transmission of rotavirus. These data highlight the benefits of routine vaccination against rotavirus in low-income settings. / Doctorat en Santé Publique / info:eu-repo/semantics/nonPublished

Les entérobactéries Salmonella sont divisées en plusieurs sérovars dont les quatre principaux Typhimurium, Enteritidis, Typhi et Paratyphi sont responsables soit de gastroentérites soit de fièvres typhoïdes, à raison de plus de 90 millions de cas et 400 000 décès par an. L’apparition de souches multi-résistantes nécessite la mise en place d’une vaccination prophylactique muqueuse. L’environnement intestinal est caractérisé par une balance entre tolérance immunitaire et réaction inflammatoire régulée par les immunoglobulines (Ig) A sécrétoires. Les IgA des sécrétions muqueuses sont dimériques, les IgA sériques sont monomérique et deux isotypes ont été décrits chez l’Homme: IgA1 et IgA2. Nous avons tout d’abord exploré les fonctions des différents isotypes et isoformes des IgA humaines. Nous avons pu noter un rôle anti-inflammatoire des IgA1 à l’inverse d’un rôle pro-inflammatoire des IgA2 et nous avons souligné un processus de régulation de l’expression des récepteurs aux IgA par les IgA elles-mêmes ainsi qu’un axe IgA/lymphocytes T CD8 cytotoxiques. Nous avons ensuite mis en place un vaccin multivalent composé des antigènes SseB et OmpC de Salmonella liés à des Ig sécrétoires. Cette étude a mis en évidence une solide réponse immunitaire humorale et cellulaire spécifique aux antigènes couplés à des IgA ou IgM après vaccination intra-nasale au niveau systémique et muqueux. Par ailleurs, de plus fortes réponses humorales et systémiques spécifiques ont été observées en couplant à la fois OmpC et SseB sur l’IgA. Ce travail de thèse ouvre de nouvelles perspectives pour la mise en place de vaccins muqueux multivalents et pourrait apporter des réponses quant au rôle des IgA. / The enterobacteria Salmonella species are divided into several serovars such as Typhimurium, Enteritidis, Typhi and Paratyphi which are the major causative agents of either gastroenteritis or typhoid fever. They are responsible for more than 90 million cases and 400 000 deaths each year. The increase in multi-drug resistant strains requires the implementation of prophylactic mucosal vaccines. Besides, the intestinal environment is characterized by a balance between immune tolerance and inflammatory response tightly regulated by secretory immunoglobulins (Ig) A. Mucosal IgA are mainly dimeric, serum IgA monomeric and two IgA isotypes have been described in humans: IgA1 and IgA2. We firstly explored the functions of the different isotypes and isoforms of human IgA. We pointed out a pro-inflammatory role of IgA2 whereas IgA1 rather oriented the immunity towards an anti-inflammatory response. We have also highlighted both the regulation of IgA receptors expression by IgA and an IgA/CD8 cytotoxic T cells axis. We also designed a multivalent vaccine against Salmonella by coupling two antigens – SseB and OmpC – to secretory Ig. We pointed out solid specific humoral and cellular responses against both these antigens coupled to either IgA or IgM after intra-nasal immunization in mucosal but also systemic compartments. We have also demonstrated the possibility to preserve and increase the antigen immunogenicity with a multivalent vaccine. This thesis thus paves the way for new secretory Ig-vectorized mucosal vaccines. In addition, the immune response could be modulated through the chosen isotype or isoform and the differences in immune activation generated by structural changes in IgA could shed some light on their role in mucosal homeostasis.

Schistosomiasis is a water-borne parasitic disease of great public health importance mainly in sub-Saharan African countries. The majority of current control programmes use the antihelminthic drug praziquantel to reduce disease burden in endemic areas. Praziquantel treatment has been reported to accelerate the development of protective immunity against re-infection that otherwise takes years to develop. To date, there is no licensed vaccine for schistosomiasis in humans but an attenuated schistosome parasite vaccine has been tested in animal models. Employing systematic review and meta-analysis approaches, my PhD research has four main objectives relating to attenuated schistosome vaccine and praziquantel treatment: 1) to identify predictors that determine protection levels after treatment with attenuated Schistosoma mansoni vaccines in the mouse model, 2) to quantify the influence of host and schistosome parasite species on attenuated parasite vaccine efficacy, 3) to explore the direction of change (increase/decrease) in schistosome parasite-specific antibody isotypes after praziquantel treatment in humans, 4) to identify predictors of praziquantel efficacy in humans. My analyses revealed three factors that have an influence on the protection levels provided by attenuated schistosome parasite vaccines: increasing numbers of immunizing parasites had a positive effect on the levels of protection whereas increasing the radiation dose and the time to challenge infection had negative effects. Analyses showed that the attenuated schistosome vaccine has the potential to achieve protection levels as high as 79% after a single dose in mice. Alongside this, baboon studies consistently reported protective effects of attenuated schistosome vaccines against re-infection. These results show there is a high potential for an attenuated schistosome parasite vaccine to be effective in humans. A meta-analysis of the influence of praziquantel treatment on the direction of change in schistosome-specific antibody isotypes was conducted. The analysis revealed considerable variability in the antibodies’ direction of change among populations. The results also demonstrated an increase of anti-worm IgA and IgE in the majority of studies. These antibodies have been reported to have a protective effect against re-infection. The combination of age and infection intensity, and the number of days after treatment were identified as influential predictors for some antibody isotypes, but there was no single predictor that consistently affected all antibody isotypes in the same way. Praziquantel efficacy levels in humans were investigated and the analyses revealed that cure rates for schistosomiasis increase with praziquantel dose, and were affected by the identity of the schistosome parasite species (S. mansoni vs. S. haematobium) and the age of the participants (children: 0-19 years old vs. adults: ≥ 20 years old). There has been no clear efficacy level reduction over the treatment years (1979-2013) suggesting that praziquantel is still effective in the treatment of schistosomiasis despite concerns about possible resistance. The development of a schistosome vaccine will benefit from a closer investigation into the mechanisms through which protection is acquired in attenuated schistosome parasite vaccine studies showing high potential efficacy in animal models. Nevertheless, it will take time to develop a schistosome vaccine for human use. The uptake of the vaccine will be made even more challenging by the lack of adequate infrastructure in schistosomiasis endemic areas. In the meantime, close monitoring of praziquantel efficacy levels is necessary to confirm the effectiveness of schistosomiasis control in endemic areas.

Uma vacina liofilizada, em comparação a uma líquida, possui inúmeras vantagens, tais como: a melhora na estabilidade do produto às variações de temperatura aumentando sua vida de prateleira, possibilitando uma melhor logística do produto aos locais onde o acesso à rede refrigerada é difícil. O presente trabalho visou investigar as estratégias do processo para a conservação da potência da vacina rábica de uso veterinário por liofilização. O material testado foi a vacina rábica inativada (VRI). Ao total foram testados 13 excipientes com três concentrações diferentes. O processo de triagem utilizado para a escolha das melhores formulações (VRI + excipiente) foi liofilização em microplacas próprias para cultivo celular adaptadas ao liofilizador, sendo posteriormente verificada a preservação da antigenicidade da vacina por meio do teste de ELISA. A análise estatística foi realizada no programa SPSS® v.17, aplicando análise de regressão para dados categóricos. A arginina 0,5%, o PEG 3350 0,5%, a sacarose 2%, a maltose 1% e a trealose 1% foram os que deram melhores resultados, um alto \"score\" de antigenicidade em comparação à vacina liofilizada sem adição de excipientes. A temperatura de colapso da VRI e de suas diversas formulações foi determinada por meio de um microscópio acoplado a um módulo de liofilização. A análise térmica foi realizada em Calorimetria Exploratória Diferencial (Differential Scanning Calorimetry - DSC). Realizou-se os seguintes testes para avaliação da VRI liofilizada em frasco ampola: microscopia eletrônica de varredura (MEV) para análise da estrutura da pastilha e o antibody-binding test (ABT) - Teste de ligação do anticorpo modificado para determinação da potência da VRI e suas formulações. A umidade residual da VRI com PEG3350 0,5% liofilizada foi determinada em temperatura constante de 100ºC resultou em 1,60%. A pastilha da vacina rábica + PEG 3350 0,5% liofilizada em frasco ampola apresentou a elegância farmacêutica esperada de um produto liofilizado. / A lyophilized vaccine, compared to a liquid form, has many advantages, such as the improvement in product stability to changes in temperature by increasing its shelf life, allowing better logistics for product to locations where access to chill is difficult to achieve. This work has investigated the strategies of the process for the conservation of the potency of rabies vaccine for veterinary use by lyophilization. The material tested is an inactivated rabies vaccine (VRI). Altogether 13 excipients were tested with three different concentrations. The screening process used to choose best formulation (s) (VRI + excipient) was lyophilization in microplates suitable for cell culture adapted to the lyophilizer, and subsequently verified the preservation of the antigenicity of the vaccine through the ELISA test. Statistical analysis was performed using SPSS® v.17, applying regression analysis for categorical data. Arginine 0.5%, PEG 3350 0.5%, Sucrose 2%, Maltose 1% and Trehalose 1% were those that gave better results, a high score of antigenicity compared to the lyophilized vaccine with no added excipients. The temperature of collapse of VRI and its various formulations was determined using a microscope coupled to a module of lyophilization. Thermal analysis was performed in Differential Scanning Calorimetry (DSC). The following tests were used for evaluation of freeze-dried VRI: Scanning electron microscopy (SEM), Antibody-binding test (ABT). The residual moisture of lyophilized samples were determined in the moisture analyzer at constant temperature of 100 °C and resulted in 1.60%. The cake of lyophilized rabies vaccine + 0.5% PEG 3350 in vial showed the pharmaceutical elegance expected of a lyophilized product.