Tesi sul tema "Prophylactic vaccine"

Thesis (M.Sc.) (Honours) -- University of Western Sydney, Hawkesbury, 2001.
A thesis submitted for the degree of Master of Science (Honours), Centre for Farming Systems Research, University of Western Sydney, Hawkesbury, 2001. Bibliography : leaves 142-162.

Papilomavírus humanos (HPVs) são vírus de DNA que infectam células epiteliais, podendo ser responsáveis pelo aparecimento de lesões benignas e malignas. Dentre os mais de 120 tipos identificados, o HPV -16 constitui o principal agente etiológico do câncer cervical, que é uma das maiores causas de morte por câncer em mulheres no mundo. Sendo assim, infecções associadas ao HPV devem ser prevenidas por vacinas indutoras de resposta imune vírus-específicas. A proteína L1 do capsídio viral é capaz de arranjar-se em partículas morfologicamente e antigenicamente semelhantes ao vírus, denominadas \"virus-like particles\" (VLPs), que induzem altos títulos de anticorpos neutralizantes. Neste trabalho, foram clonados os genes L1 selvagem e otimizado de HPV -16 em vetores de expressão de leveduras metilotróficas como Hansenula polymorpha e Pichia pastoris. Foi observada uma expressão consistente da proteína recombinante apenas em P. pastoris, com o gene L1 otimizado. Foram realizadas diversas tentativas de purificação da proteína heteróloga, empregando técnicas de cromatografia e ultracentrifugação em gradiente descontínuo de sacarose. A correta montagem das VLPs foi confirmada por microscopia eletrônica. Problemas de agregação, heterogeneidade e adsorção a superfícies apresentados pela proteína L1 foram resolvidos após utilização de surfactante não-iônico e de um procedimento de desmontagem e remontagem das partículas, gerando preparações mais homogêneas. Ensaios de hemaglutinação e inibição da hemaglutinação comprovaram a apresentação de epítopos conformacionais na superfície das VLPs. Este trabalho demonstrou pela primeira vez a expressão da proteína L1 de HPV -16 em P. pastoris, visando ao desenvolvimento de uma vacina profilática de baixo custo para o sistema público de saúde.
Human papillomaviruses (HPVs) are DNA viruses that infect epithelial cells and can cause both benign and malignant lesions. From over 120 types catalogued so far, HPV-16 is the main etiologic agent of cervical cancer, which is the one of the most common causes of cancer deaths among women worldwide. Thus, HPV -associated infections might be prevented by vaccine inducing virus-specific immune responses. The L1 major capsid protein can self assemble into virus-like particles (VLPs), which are morphologically and antigenically indistinguishable from native viruses and induce high titers of neutralizing antibodies. In this work, we have cloned wild-type and codon-optimized L1 genes from HPV-16 in expression vectors of the methylotrophic yeasts Hansenula polymorpha and Pichia pastoris. Consistent L1 expression was only observed in P. pastoris transformed with the construction containing the codon-optimized gene. Many attempts to purify the heterologous protein were made, including chromatography and ultracentrifugation in sucrose density gradients. The correct assembly of VLPs was confirmed by electron microscopy. Some problems presented by recombinant L1 like aggregation, surface adsorption and heterogeneity were solved by using non-ionic surfactants and a procedure of disassembly and reassembly of the particles. Hemagglutination and hemagglutination inhibition assays corroborated the display of surface conformational epitopes by VLPs. This work showed for the first time the expression of the HPV-16 L1 protein in P. pastoris, aiming the development of a prophylactic vaccine free of charge for the public health system in Brazil.

abstract: Cancer is one of the most serious global diseases. We have focused on cancer immunoprevention. My thesis projects include developing a prophylactic primary and metastatic cancer vaccines, early cancer detection and investigation of genes involved in tumor development. These studies were focused on frame-shift (FS) antigens. The FS antigens are generated by genomic mutations or abnormal RNA processing, which cause a portion of a normal protein to be translated out of frame. The concept of the prophylactic cancer vaccine is to develop a general cancer vaccine that could prevent healthy people from developing different types of cancer. We have discovered a set of cancer specific FS antigens. One of the FS candidates, structural maintenance of chromosomes protein 1A (SMC1A) FS, could start to accumulate at early stages of tumor and be specifically exposed to the immune system by tumor cells. Prophylactic immunization with SMC1A-FS could significantly inhibit primary tumor development in different murine tumor models and also has the potential to inhibit tumor metastasis. The SMC1A-FS transcript was detected in the plasma of the 4T1/BALB/c mouse tumor model. The tumor size was correlated with the transcript ratio of the SMC1A-FS verses the WT in plasma, which could be measured by regular RT-PCR. This unique cancer biomarker has a practical potential for a large population cancer screen, as well as clinical tumor monitoring. With a set of mimotope peptides, antibodies against SMC1A-FS peptide were detected in different cancer patients, including breast cancer, pancreas cancer and lung cancer with a 53.8%, 56.5% and 12.5% positive rate respectively. This suggested that the FS antibody could be a biomarker for early cancer detection. The characterization of SMC1A suggested that: First, the deficiency of the SMC1A is common in different tumors and able to promote tumor initiation and development; second, the FS truncated protein may have nucleolus function in normal cells. Mis-control of this protein may promote tumor development. In summary, we developed a systematic general cancer prevention strategy through the variety immunological and molecular methods. The results gathered suggest the SMC1A-FS may be useful for the detection and prevention of cancer.
Dissertation/Thesis
Ph.D. Molecular and Cellular Biology 2012

No vaccines are available for hepatitis C virus (HCV) which infects over 71 million people worldwide. Current therapeutic options are very expensive and as a consequence, only around 1% of those diagnosed with Hepatitis C receive treatment each year. Induction of neutralizing antibody (NAb) by vaccination will be important for the successful prevention of HCV infection. HCV envelope glycoproteins E1 and E2 are required for virus entry into host cells making these proteins attractive targets to prevent virus infection. DNA-based vaccines are appealing candidates for novel vaccine development because they are not infectious and are cost-effective to manufacture on a global scale. However, despite being licensed for veterinary use, DNA vaccine have not been highly immunogenic in large animals. Virus-like particles (VLP), on the other hand, which resemble native viruses but are non-infectious because they lack viral genetic materials, have provided highly encouraging results in clinical trials. In this thesis the immunogenicity of VLPs and the ease of production of plasmid DNA were combined by designing DNA vaccines encoding VLPs consisting of HCV-core, E1 and E2 proteins (which can self-assemble into VLPs following expression). This vaccine also encoded a cytolytic gene perforin (PRF) to cause cell death and ensure the release of the VLPs from vaccine-targeted cells along with damage associated molecular patterns (DAMPs), which act as natural adjuvants. Bicistronic DNA vaccine constructs encoding HCV structural proteins and PRF were successfully generated and validated. Vaccination with the DNA construct encoding CoreE1E2-PRF generated higher adaptive immune responses in mice than vaccination with a construct unable to induce cell death, therefore confirming an adjuvant effect by PRF. However, these responses were weak compared to those reported in the literature. Antigen oligomerisation has been shown to improve vaccine immunogenicity. To improve the immunogenicity of a DNA vaccine encoding HCV-E1E2, a novel strategy that incorporates E1 and E2 into heptamers by fusion with the oligomerisation domain of a chimeric C4 binding protein (namely IMX3133 or IMX313P) was used. As the adjuvanticity of IMX313 or IMX313P requires efficient secretion of the oligomerised protein, the leader sequence of the tissue plasminogen activator (tPA) was introduced upstream of the E1 or E2 proteins (tE1 or tE2) from which the transmembrane domains were removed. The use of tE1 and tE2 proteins as separate immunogens or as a single tE1tE2 polyprotein when fused to IMX313 or IMX313P was assessed in vaccination studies in Balb/c mice using prime-boost intra-dermal DNA immunisations. Vaccination with the DNA construct encoding tE1/tE2 fused to IMX313 or IMX313P resulted in increased antibody and cell mediated immune (CMI) responses compared to the same dose of DNA without IMX313 or IMX313P, while fusion of tE1/tE2 to IMX313P resulted in the highest immune responses. DNA prime/E1E2 protein boost or DNA prime/HCV-VLP boost approaches were then used to further improve the immunogenicity of tE1/tE2-IMX313P DNA constructs. Boosting with E1E2 proteins improved overall antibody responses, compared to boosting with HCV-VLPs or plasmid DNA. However, experiments to examine the neutralisation of binding of labelled VLPs showed that the E1E2 or VLP boost did not improve the neutralising potency of these antibodies or the CMI responses. This thesis demonstrated that expression of heptamerised and secreted tE1/tE2 from DNA vaccine constructs significantly improved the antibody and CMI responses to HCV E1 and E2 proteins. Most importantly, the antibody induced by these constructs possess neutralising properties.
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2018

Tese de Doutoramento em Ciências Farmacêuticas, no ramo de Tecnologia Farmacêutica, apresentada à Faculdade de Farmácia da Universidade de Coimbra
The hepatitis B virus (HBV) killed 887 000 people in 2015. The World Health Organization (WHO) set the goal to eliminate HBV as a public health threat by 2030. The major hurdles include the high prevalence in developing countries due to limited vaccination coverage and mother-to-child transmission and, the ineffective HBV clearance from hepatocytes with currently available antivirals. Hence, therapeutic vaccines may stimulate both the neonate immature or the chronic hepatitis B exhausted immune systems, to either avoid HBV persistence or promote HBV clearance. Additionally, new vaccines can be designed to provide the antigen with increased stability to temperature variations, benefiting HBV vaccine coverage in developing countries, where the cold chain to vaccine transportation is not readily available. Thus, this thesis aimed to develop new powerful adjuvants to include in new vaccines to meet hepatitis B current needs. β-glucan particles were elected to mimic pathogen three-dimensional structure and chemical composition. The capacity to induce HBsAg-specific Th1 antiviral protection through several vaccination strategies was the main goal. Indeed, this was the first time that nonmodified natural β-glucans were used as adjuvants for HBsAg. The new formulations of HBsAg vaccines were tested through subcutaneous (SC) and oral routes, while plasmid DNA (pDNA) vaccines only through the SC route. Different β-glucan adjuvants were developed and included β-glucan particles (GPs), prepared from alkaline/acid treatment of Saccharomyces cerevisiae, β-glucan/chitosan particles (GenGluChiPs), prepared by a precipitation technique followed by genipin crosslink and, polyplexes prepared by pDNA complexation with cationic PβAE and PDMAEMA polymers (Pol) in the presence of β-glucan (GluPol) or combined with GPs (GPsPol). Notably, GenGluChiPs were produced by a new precipitation technique to combine two polymers that do not interact with each other. GPs were tested in all vaccination schedules while GenGluChiPs only for HBsAg SC vaccination and GluPol only for pDNA SC vaccination. Additionally, chitosan particles were developed for comparison purposes either by precipitation/coacervation (ChiPs) or precipitation followed by genipin crosslink (GenChiPs). Both were positively charged and had a mean diameter near 900 nm.GenGluChiPs, also positively charged, measured approximately 1.3 μm. On the other side, alginate coated ChiPs (AlgChiPs), used for oral vaccination, were negatively charged and had a mean diameter close to 1.5 μm. GPs were electrically neutral and measured between 2 μm and 4 μm. Pol and GluPol were highly positive with a mean diameter of 180 nm. Regarding the oral vaccination study (Chapter 2), although both AlgChiPs and GPs were efficiently internalized by intestinal Peyer’s patches, the oral vaccination schedule resulted in 60 % mice seroconversion, easily surpassed by a SC priming prior the oral boosts. The presence of HBsAg-specific IgA on mucosal surfaces and IFN-γ in the liver were the major advantages found. Interestingly, in vitro studies showed that only ChiPs were able to induce mast cell activation, evaluated by cell degranulation and β- hexosaminidase release. Concerning pDNA vaccination study (Chapter 3), although the excellent Pol transfection results, further enhanced by the combination with GPs (GPsPol) in fibroblast and macrophage cell lines, the SC vaccination either with Pol, GluPol or GPsPol resulted in only 40 % seroconversion and low antibody titers. The mechanistic study with GenChiPs, GenGluChiPs and GPs showed that the increased TNF-α secretion from mice spleen cells was associated to β-glucan (GenGluChiPs and GPs), while RANTES secretion was associated to chitosan (GenChiPs and GenGluChiPs), suggesting an immunological advantage of the newly developed GenGluChiPs. However, in the human monocyte study, the TNF-α production was consistently observed for all the particles. The mice immunization study with HBsAg to validate GenGluChiPs adjuvant (Chapter 4) showed high serum anti-HBsAg IgG, mostly subtype IgG1 followed by IgG3. No signs of cell-mediated immunity were found after two vaccine doses. However, in another study with three vaccine doses (Chapter 5), the GPs adjuvant induced a strong and varied HBsAg-specific cell-mediated immunity observed by the secretion of cytokines related with Th1, Th2, Th17, Th22 and Treg-biased immune responses. For the first time, these studies allowed the validation of GPs as great adjuvant to include in HBsAg vaccines, also revealing a therapeutic value against viral infections. Overall, the work herein developed and described represents an important contribution to the knowledge of both β-glucan and chitosan/β-glucan particle adjuvant mechanisms, with a great impact for future studies.
O vírus da hepatite B (HBV) originou 887 000 mortes em 2015. A Organização Mundial de Saúde (OMS) estabeleceu o objetivo de eliminar o HBV como uma ameaça de saúde pública até 2030. Os principais problemas incluem a elevada prevalência nos países em vias de desenvolvimento devido à ineficiente cobertura de vacinação e elevada transmissão mãe-filho, e a dificuldade em eliminar o HBV dos hepatócitos com as terapias antivirais disponíveis. A nova geração de vacinas deverá ter em conta tanto o sistema imunitário imaturo dos recém-nascidos como a exaustão do sistema imunitário dos portadores crónicos. Além disso, novas vacinas podem promover a estabilidade do antigénio perante variações de temperatura, frequente nos países em vias de desenvolvimento, onde cadeias de transporte adequadas são ainda escassas. Nesse sentido, esta tese teve como objetivo desenvolver novos e poderosos adjuvantes para incluir em novas vacinas, de modo a ir ao encontro das necessidades de controlo da hepatite B. Partículas à base de β-glucano foram selecionadas por simularem as propriedades químicas e tridimensionais dos patogénios. O objetivo principal foi desenvolver uma vacina com a capacidade de induzir uma resposta celular específica para o HBsAg, e para isso foram desenvolvidas e testadas várias estratégias. Curiosamente, esta foi a primeira vez que β-glucanos naturais não modificados foram usados como adjuvante para o HBsAg. Vacinas com o HBsAg foram testadas pelas vias subcutânea (SC) e oral, e vacinas com o plasmídeo (pDNA) foram testadas pela via SC. Foram desenvolvidos vários adjuvantes compostos por β-glucano, tais como partículas de β-glucano (GPs) preparadas por tratamento ácido/alcalino da Saccharomyces cerevisiae, partículas de β- glucano/chitosano (GenGluChiPs) produzidas por um método de precipitação/reticulação com genipina e poliplexos preparados por complexação do pDNA com os polímeros catiónicos PβAE e PDMAEMA (Pol) na presença de β-glucano (GluPol) ou posteriormente combinados com as GPs (GPsPol). Notavelmente, as GenGluChiPs foram produzidas por um método inovador para a combinação de dois polímeros que não interagem entre si. As GPs foram testadas em todas as estratégias de vacinação, enquanto que as GenGluChiPs foram apenas testadas pela via SC com o HBsAg e os GluPol pela via SC com o pDNA. Partículas de quitosano foram desenvolvidas tanto por precipitação (ChiPs) como por precipitação/reticulação (GenChiPs), como termo de comparação. Ambas apresentaram carga positiva e um diâmetro médio de 900 nm. As GenGluChiPs, também positivas, mediam cerca de 1,3 μm. Por outro lado, ChiPs revestidas com alginato (AlgChiPs) para vacinação oral apresentaram carga negativa e um tamanho médio de 1,5 μm. As GPs, neutras, possuíam um tamanho entre 2 μm e 4 μm. Os Pol e os GluPol apresentaram uma carga fortemente positiva e um diâmetro médio de 180 nm. Quanto ao estudo de vacinação oral (Capítulo 2), apesar de as AlgChiPs e as GPs terem sido internalizadas pelas placas de Peyer do intestino de murganhos, três doses pela via oral resultaram em apenas 60 % de eficácia, enquanto que a presença de um priming pela via SC levou a 100 % de eficácia. O mais interessante, não observado pela via SC, foi a indução de anti-HBsAg IgA nas mucosas e aumento de IFN-γ no fígado. Curiosamente, apenas as ChiPs ativaram uma linha celular de mastócitos, verificado pela desgranulação e libertação de β-hexosaminidase. Quanto ao estudo de vacinação com o pDNA (Capítulo 3), apesar da excelente transfecção dos Pol, aumentada na presença de GPs (GPsPol) em fibroblastos e em macrófagos, a vacinação pela via SC resultou em apenas 40 % de eficácia e baixos títulos de anticorpos para todas as formulações (Pol, GluPol e GPsPol). Nos estudos mecanísticos, o aumento da secreção de TNF-α em células do baço de murganhos foi associada com o β-glucano (GenGluChiPs and GPs) e a secreção de RANTES com o quitosano (GenChiPs e GenGluChiPs), sugerindo uma vantagem para as GenGluChiPs aqui desenvolvidas. Por outro lado, o aumento da produção de TNF-α em monócitos humanos, foi observada após a incubação com os três tipos de partículas. A vacinação pela via SC para a validação da GenGluChiPs como adjuvante (Capítulo 4) levou ao aumento de IgG específica para o HBsAg no soro de murganhos, maioritariamente do subtipo IgG1 e também IgG3. As duas doses da vacina mostraram-se ineficazes na indução da resposta celular. Por outro lado, num segundo estudo com três doses da vacina (Capítulo 5), apenas as GPs levaram numa elevada e ampla secreção de citocinas relacionadas com respostas Th1, Th2, Th17, Th22 e Treg, específica para o HBsAg. Pela primeira vez, este estudo consagrou o valor de incluir as GPs em vacinas com o HBsAg, bem como o seu potencial terapêutico para estas infeções virais. No geral, o trabalho desenvolvido providenciou uma importante contribuição para o conhecimento do mecanismo adjuvante de partículas de β-glucano e β- glucano/quitosano, relevante para estudos futuros.

碩士
國立臺灣海洋大學
食品科學系
100
Helicobacter pylori is recognized as the main cause of chronic gastritis, ulcers and gastric cancer in humans. With increased incidence of treatment failure and antibiotic resistance, development of prophylactic or therapeutic vaccination is a desirable alternative. This study focused on the gastric and systemic immune responses induced by DNA vaccination combination with the multi-CpG adjuvant in the H. pylori infection mice. Vaccine-induced protection was correlated with the increased proportions of CD4+ T cells, IL-17, neutrophils and the decreased percentage of Treg in the gastric mucosa. The C3H/HeN mice orally immunized by H. pylori DNA vaccine/chitosan complexes can induce specific and protective antibodies against H. pylori challenge. Analysis of serum cytokines and the percentage of splenocytes revealed that DNA vaccination combined with the adjuvant can induced a better Th1-biased immune response than that without adjuvant. The prophylactically oral-immunized mice with chitosan-pCJ-3/UreB or pCJ-3/Hsp60 DNA vaccine reached 31.25% and 37% protective rates, respectivity. When combining with the adjuvant, the protective rate increased to 75%. These results demonstrate that DNA vaccine through oral immunization exerts its prophylaxis against the pathogen.

碩士
國防醫學院
微生物及免疫學研究所
91
According to the statistical incidences in Taiwan, cervical carcinoma is the first common malignancy in recent years and remains the fifth cause of deaths due to malignancies in women. Besides,it always belongs to one of the usual cancers all over the world. The reasons of cervical carcinoma are numerous inclusive of multiparity,and multiple sexual partners；the major one is the infection of human papillomavirus ( HPV ),especially the types 16 and 18. Many documents indicate that if patients with certain cervical carcinoma reveal positive reactions of human papillomavirus infection,the type 16 accounts for 50%. HPV owns eight early genes and two late genes. Among them, the late genes can translate into outer sheath proteins；they can divide into major capsid protein ( L1 protein ) and minor capsid protein ( L2 protein ).Papers point out that L1 protein can self-assemble into the structure of virus-like particles ( VLPs )；the routes of infection and infective activities are similar to those of nature HPV. In addition, VLPs can induce high titers of neutralizing antibodies in experimental animals. So,we utilized the characteristics of L1 protein to reseach the prophylactic HPV vaccine. We used adeno-associated virus ( AAV ) as a vector and combined three different plasmids including pXX6-YP,pXX2-YP and pXX-16 L1 to cotransfect the 293 cells in order to produce the recombinant AAV-16 L1.There are also papers showed that AAV-L1 can cause immune responses to induce neutralizing antibodies. As to L1 DNA, it can give rise to the same responses as well. Our laboratory used to take AAV-L1 and L1 DNA as prophylactic HPV vaccines to give mice and evaluated the activities to evoke L1-specific antibodies.The results revealed that the expression of AAV-L1 was superior to that of L1 DNA.So,we followed the previous research and integrated three prophylactic HPV vaccines including AAV-L1,L1 protein and L1 DNA to give mice via muscular and venous injections. Then, we made use of ELISA (enzyme-linked immunosorbent assay) to compare their differences of inducing the neutralizing antibodies. The outcome showed that the expression of L1 protein was better than that of AAV-L1 and the latter was better than the expression of L1 DNA. Based on research reports,GM-CSF can enhance the activity of antigen presentation by promoting the increase of macrophages and dendritic cells, and cause an environment to strengthen the immune responses. So, we utilized adenovirus as a vector to carry GM-CSF gene to produce Ad-GM-CSF；then,we combined AAV-L1 with Ad-GM-CSF to be vaccines to give mice and tested the neutralizing antibodies. The results illustrated that the expression of combination was superior to that of L1 protein alone.Besides, we checked the concentrations of IL-4 and IFN- to further investigate if the immune response would go on Th1 or Th2 pathway. Currently, the treatments of cervical carcinoma in clinical are surgery,radiotherapy and chemical therapy.But, there was no truly satisfying achievements in progress and prevention of metastasis.So, we compared the above mentioned vaccines to research which one was really available,and we hoped to find out a pontential prophylactic HPV vaccine for prevention of cervical carcinoma.