Dissertations / Theses on the topic 'Virus-based particles'

In this dissertation the investigation of potential applications of hamster polyomavirus (HaPyV) major capsid protein VP1 based chimeric virus-like particles (VLPs) harboring CTL epitopes for anticancer vaccine development is presented. The objective of this study was to investigate the potential of recombinant HaPyV VP1 based VLPs for anticancer vaccine generation in model systems, including investigation of VP1 applicability for heterologous CTL epitopes insertions, VLPs assembly and ability to induce insert specific immune response in vivo. HaPyV VP1 VLPs carrying CLT epitopes derived from different proteins were generated, most suitable positions for insertion into VP1 protein were selected, the ways to improve assembly and yield of the chimeric VLPs were determined and new VLPs purification procedure was created allowing to purify VLPs cheaper, faster and more efficiently. HaPyV VP1 based VLPs ability to induce CTL immune response in vivo was evaluated for the first time. It was demonstrated that model chimeric VLPs were able to stimulate antigen specific CTL cells in vitro and in vivo, induced insert specific humoral and CTL immune response in vivo and protected mice from insert specific virus infection and antigen-specific tumor growth. Presented data confirmed that HaPyV protein VP1 is universal carrier for CTL epitopes, capable to tolerate insertions, to form VLPs and to induce effective, long lasting immune response against inserted antigens in vivo.
Disertacijoje yra aprašomas perspektyvų panaudoti žiurkėno poliomos viruso (HaPyV) pagrindinio struktūrinio baltymo VP1 formuojamas į virusus panašias daleles priešvėžinių vakcinų kūrimui tyrimas. Pagrindinis disertacijos darbo tikslas buvo modelinėse sistemose parodyti rekombinantinių HaPyV VP1 baltymų formuojamų į virusus panašių dalelių panaudojimo priešvėžinių vakcinų kūrimui galimybes, įvertinant svetimų CTL epitopų įterpimo į VP1 baltymą toleravimą, VPD formavimosi efektyvumą bei sukeltą įterptam antigenui specifinį imuninį atsaką. Disertacijoje atlikta tyrimo srities literatūros apžvalga, smulkiai aprašomi darbe naudoti metodai, atlikti eksperimentai, pateikiami bei analizuojami gauti rezultatai. Darbe pirmą kartą buvo nuodugniai ištirtos HaPyV viruso VP1 baltymo formuojamų VPD savybės, parodytas jų tinkamumas būti CTL epitopų nešikliais, ištirtos įterpimui palankiausios VP1 baltymo vietos, išbandyti nauji VPD gavimo ir gryninimo būdai, pagerinantys chimerinių VPD formavimąsi bei išeigas. Panaudojant modelines chimerines VPD in vivo buvo ištirtas chimerinių HaPyV VP1 pagrindu sukonstruotų VPD sukeliamas humoralinis ir ląstelinis imuninis atsakas. Gauti rezultatai parodė, kad HaPyV VP1 baltymas yra vienas iš nedaugelio virusų struktūrinių baltymų, kurie ne tik formuoja VPD, bet pasižymi ir universaliomis baltymo – nešiklio savybėmis, o in vivo sukelia efektyvų, ilgalaikį, įterptam epitopui specifinį imuninį atsaką.

Les virus-like particles (VLPs) de VIH han sorgit com una prometedora alternativa per al desenvolupament de nous candidats vacunals, però també per al disseny de teràpies avançades en el camp de la nanomedicina. En els últims anys, s’han desenvolupat diferents estratègies d’optimització per la producció de VLPs de VIH en cultius de cèl·lules animals. Malgrat aquests avanços, la manca d’informació sobre el procés de producció de les VLPs a nivell intracel·lular, la necessitat de mètodes analítics adients per la quantificació de les VLPs de VIH i la seua diferenciació d’altres estructures vesiculars, conegudes com extracellular vesicles (EVs), conjuntament amb la falta de mètodes de purificació, han limitat l’ús d’aquestes nanopartícules a la clínica. Per aquesta raó, la motivació d’aquesta tesis doctoral és aprofundir en els diferents paràmetres que participen en la generació de VLPs de VIH, així com el desenvolupament de nous mètodes d’anàlisi i purificació amb l’objectiu d’establir una plataforma de producció per la seua aplicació en l’àmbit de la biotecnologia.
Las virus-like particles (VLPs) derivadas del VIH han surgido como una potente alternativa para el desarrollo de nuevos candidatos vacunales, pero también para el diseño de terapias avanzadas en el campo de la nanomedicina. En los últimos años, se han optimizado diferentes estrategias para la producción de estas VLPs en cultivos de células animales. No obstante, el desconocimiento acerca de los diferentes pasos que acontecen a su producción a nivel intracelular, y que afectan al rendimiento de producción, la falta de métodos analíticos para su correcta caracterización y cuantificación, así como de su diferenciación de otras estructuras vesiculares, conocidas como extracelular vesicles (EVs), y la carencia de métodos de purificación adecuados, dificultan su aplicación en la clínica. Por todo ello, el objetivo de la presente tesis es investigar el proceso de producción de VLPs de VIH, así como desarrollar nuevos métodos analíticos y de purificación con el objetivo de establecer una plataforma de producción de estas nanopartículas para su uso en aplicaciones biotecnológicas.
HIV-1 virus-like particles (VLPs) have emerged as an interesting alternative for the development of novel vaccine candidates and delivery strategies of different cargos into different cells and tissues. Great efforts have been undertaken to optimize the generation of these nanoparticles in animal cell cultures. However, the limited understanding of its production at intracellular level, the need for analytical tools allowing its specific quantification over extracellular vesicles (EVs), and the few purification processes available hamper their clinical application. The aim of this thesis is to gain insight into the process parameters affecting HIV-1 Gag VLP production, and the development of analytical and purification methods to establish a complete platform for its clinical-grade production.

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine epidemic diarrhea virus (PEDV) are two of the most prevalent swine pathogens that have impacted the global swine industry for decades. Both are RNA viruses with increasing heterogeneity over the years, making a vaccine solution ever so challenging. Modified live-attenuated vaccines (MLVs) have been the most common approach, but the long-term safety regarding their potential for pathogenic reversion still needs to be addressed. Subunit based vaccines have been the focus of numerous development studies around the world with renewed interest in their promising prospects in both safety and efficacy. Our lab has developed a unique approach to use hepatitis B virus core capsid protein (HBcAg) as a vaccine delivery vehicle for either PRRSV or PEDV viral epitope antigens. Recombinantly produced HBcAg forms an icosahedral capsid virus-like particle (VLP) that has 240 repeats in a single assembled particle. By inserting different epitope antigens from these porcine pathogens into the particle, we can achieve repetitive antigen presentation to the host's immune system by taking advantage of the polymeric nature of VLP. The first animal study evaluated the efficacy of 4 VLP based vaccine candidates against PRRSV in mice. These 4 vaccines incorporated 2 B-cell epitopes (61QAAIEVYEPGRS72 and 89ELGFVVPPGLSS100) and 2 T-cell epitopes (117LAALICFVIRLAKNC131 and 149KGRLYRWRSPVIIEK163) from PRRSV structural proteins GP3 and GP5 respectively. Candidate GP3-4 was able to stimulate a significant viral neutralizing response in mouse sera against two PRRSV strains, one being heterologous, demonstrating its potential of cross-protection against PRRSV. The second animal study took an optimized VLP vaccine candidate against PEDV from previous development studies in mice, and assessed its efficacy through a comprehensive pregnant gilt vaccination and neonatal piglet challenge model. The vaccine candidate incorporated B-cell epitope 748YSNIGVCK755 from the PEDV spike protein. It was able to elicit significant viral neutralization antibody titer in gilt milk at 3 days post-farrowing (DPF), and provided nursing piglets with clinical relief in terms of morbidity, viral shedding, small intestinal lesions, and 10 days post-challenge (DPC) survival rate.
Doctor of Philosophy
Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine epidemic diarrhea virus (PEDV) are two pathogens that infect pigs, resulting in immense economic losses to the global pork production industry every year. Both viruses have large diversity with various strains due to mutations that have occurred over the years. This makes vaccine development that aims at combating the pathogens even more challenging. One common vaccine strategy has been immunizing animals with modified live viruses with decreased pathogenicity. Naturally, long term safety of this option has been a concern. A much safer vaccine approach that is purely protein based has attracted renewed interest around the world. Protein based vaccines lack genetic materials from the viruses and are not able to replicate inside the host. Our lab has developed a platform that uses protein-based particles (VLPs) originated from the hepatitis B virus (HBV), and incorporates short pieces of proteins from either PRRSV or PEDV to train host's immune system to recognize these pathogens, and hopefully to prevent future infection. For the first animal study, we tested 4 VLP vaccine candidates against PRRSV in mice and discovered that mouse serum from one candidate GP3-4 was able to prevent infection of 2 distinct PRRSV strains in petri dishes, paving the way for further development. For the second animal study, we took an optimized VLP vaccine candidate against PEDV from previous mouse studies, and evaluated its performance in pigs. We immunized pregnant mother pigs with the vaccine before they gave birth, then experimentally infected newborn piglets with the virus. Piglets from the vaccinated mothers showed improved clinical signs and faster recovery from the infection.

Nanotechnology and its potential for biomedical applications has become an area of increasing interest over the last few decades. Specifically, ultrasmall nanoparticles, ranging in size from 5 to 50 nm, are highly sought after for their physical and chemical properties and their ability to be easily transmitted though the bloodstream. By adjusting the material properties, size, surface potential, morphology, surface modifications, and more, of nanoparticles, it is possible to tailor them to a specific use in biomedical areas such as drug and gene delivery, biodetection of pathogens or proteins, and tissue engineering. The aim of this study was to fabricate ultrasmall poly-(lactic-co-glycolic acid) nanoparticles (PLGA NPs) using a quick and easy nanoprecipitation method1, with some modifications, for general use in various biomedical areas. Nanoprecipitation of two solutions – PLGA dissolved in acetonitrile and aqueous poly(vinyl alcohol) (PVA) – at varying concentrations produced ultrasmall nanoparticles that range in size, on average, from 10 to 30 nm. By the data collected from this study, a selection method can be used to choose a desired PLGA nanoparticle size given a potential biomedical application. The desired nanoparticle can be fabricated using specific concentrations of the two nanoprecipitation solutions. Size of the ultrasmall PLGA NPs was characterized by dynamic light scattering (DLS) and confirmed by transmission electron microscopy (TEM). Spherical morphology of the PLGA NPs was also proved by TEM. By generalizing the ultrasmall PLGA NP fabrication process, the idea is that these NPs will be able to be used in various biomedical applications depending on the goal of the furthered study. As an example of potential application, ~15 to 20 nm PLGA NPs were consistently fabricated for use as virus-like particle (VLP) scaffolds. Following formation, PLGA NPs were introduced to modified human papillomavirus (HPV) protein during protein refolding and assembly into virus-like particles (VLPs) via buffer exchange. The size of the VLPs was monitored with and without PLGA nanoparticles present in solution during the refolding process and TEM images were collected to confirm encapsulation.
Master of Science
Nanotechnology, the manipulation of materials on an atomic or molecular scale, and its potential for biomedical applications has become an area of increasing interest over the last few decades. Nanoparticles, spherical or non-spherical entities of sizes approximately one-billionth of a meter, have been used to solve a wide variety of biomedical problems. For reference, a human hair is about 80,000 to 100,000 nm in size and the nanoscale typically ranges in size from 1 to 1000 nm. This size range is not visible to the naked eye, so methods of analysis via scientific equipment becomes paramount. Specifically, this study aims to fabricate ultrasmall nanoparticles, ranging in size from 5 to 50 nm, which are highly sought after for their physical and chemical properties and their ability to easily travel though the bloodstream. By adjusting the material properties, size, shape, surface charge, surface modifications, and more, of nanoparticles, it is possible to tailor them to a specific use in biomedical areas such as drug delivery, detection of viruses, and tissue engineering. The specific aim of this study was to fabricate ultrasmall poly-(lactic-co-glycolic acid) nanoparticles (PLGA NPs), a type of polymer, using a quick and easy nanoprecipitation method1, with some modifications. Nanoprecipitation occurs by combining two liquid solutions – PLGA and aqueous poly(vinyl alcohol) (PVA) – which interact chemically to form a solid component – a polymer nanoparticle. These two solutions, at varying concentrations, produced ultrasmall nanoparticles that range in size, on average, from 10 to 30 nm. Data collected from this study can be used to select a desired nanoparticle size given a potential application. The desired nanoparticle can be fabricated using specific concentrations of the two nanoprecipitation solutions. By generalizing the ultrasmall PLGA NP fabrication process, the idea is that these NPs can be used for a variety of biomedical applications depending on the goal of the furthered study. Two PLGA NP example applications are tested for in this work – in DNA loading and in encapsulation of virus-like particles (VLPs), which are synthetically produced proteins that can be neatly folded to resemble a virus. These VLPs can be used to as an alternative to live vaccines and they can be designed to stimulate the immune system. Positive initial results from this study confirm the potential of these nanoparticles to have a wide impact on the biomedical field depending on specific tailoring to a given application.

Rift Valley Fever (RVF) is an emerging infectious disease found in both livestock and humans. RVF is associated with high abortion and mortality rates in livestock and can be fatal in humans. As such, RVF is economically and socially significant to affected smallholder and subsistence farmers, those infected, and national livestock industries. However, Rift Valley Fever virus (RVFV) vaccines are not commercially available outside of endemic areas or for humans, and current vaccines are limited in their safety and efficacy. A plant-based, viral nanoparticle vaccine offers a more affordable alternative to conventional vaccines that is safe, rapidly producible, and easily scalable, better meeting the needs of impacted communities. This project focuses on assessing the potential of using a Nicotiana benthamiana plant expression system to generate recombinant tobacco mosaic virus (TMV) nanoparticles displaying RVFV glycoprotein epitopes. Eight TMV-RVFV glycoprotein constructs were designed. Five TMV-RVFV constructs were successfully cloned, and four recombinant TMV constructs were successfully expressed in planta. The antigenicity of these constructs was examined for their possible use in RVFV vaccine development.

This dissertation reports on the technology of cell-free protein synthesis (CFPS) including 1) stabilized lyophilized cell-free systems and 2) enhanced heterogeneous cell extracts. This work further considers applications of CFPS systems in 1) rapid vaccine development, 2) functional virus-based nanoparticles, 3) site-specific protein immobilization, and 4) expanding the language of biology using unnatural amino acids. CFPS technology is a versatile protein production platform that has many features unavailable in in vivo expression systems. The primary benefit cell-free systems provide is the direct access to the reaction environment, which is no longer hindered by the presence of a cell-wall. The “openness” of the system makes it a compelling candidate for many technologies. One limitation of CFPS is the necessity of freezing for long-term viable storage. We demonstrate that a lyophilized CFPS system is more stable against nonideal storage than traditional CFPS reagents. The Escherichia coli-based CFPS system in this work is limited by the biocatalytic machinery found natively in E. coli. To combat these limitations, exogenous biocatalysts can be expressed during fermentation of cells prepared into extract. We demonstrate that simple adjustments in the fermentation conditions can significantly increase the activity of the heterogeneous extract. Towards virus-based particles and vaccines, we demonstrate that the open nature of CFPS can be utilized for coexpression of virus proteins and self-assembly of virus particles. This technique allows for the rapid production of potential vaccines and novel functional virus-based nanoparticles. Unnatural amino acids expand the effective language of protein biology. Utilizing CFPS as an expression system, we demonstrated that the incorporation of a single specific unnatural amino acid allows for site-specific immobilization, thus stabilizing the protein against elevated temperatures and chemical denaturants. Current unnatural amino acid incorporation technologies are limited to one or few simultaneous incorporations and suffer from low efficiency. This work proposes a system that could potentially allow for upwards of 40 unnatural amino acids to be simultaneously incorporated, effectively tripling the protein code. These projects demonstrate the power and versatility of CFPS technologies while laying the foundation for promising technologies in the field of biotechnology.

Recombinant hepatitis B virus (HBV) core particles have been successfully used as particulate carriers exposing viral and bacterial antigens on their surface. The objective of this research was to explore the use of recombinant core particles from HBV and its close relative the woodchuck hepatitis virus for edible vaccine technology. This was accomplished in two parts. Part 1 was the transformation of a truncated HBV core protein gene into transgenic tobacco plants and characterization of the gene's expression with respect to mRNA levels, protein levels, and particle self-assembly. Part 2 was the construction of a "universal antigen carrier" based on the woodchuck hepatitis virus (WHV) core protein and generation and characterization of chimeric WHV core proteins carrying two different epitopes from the hepatitis C virus (HCV) core protein. In conclusion, it appears that a different approach may be required to express core proteins from HBV-like viruses in transgenic tobacco plants. (Abstract shortened by UMI.)

"Virus based biopharmaceuticals are considered the most dynamic adopted tools in modern therapeutic medicine. Their use is increasing in the fields of vaccination and gene therapy. Membrane chromatography is becoming an attractive alternative t ool that can be used for virus purification due to its scalability, potential for optimization and economical features, allowing higher productivities with lower DSP costs. (…)"
N/A

HPV is a major infectious cause of human cancers including cervical cancers of which 70% of all cases are caused by HPV types 16 and 18. Worldwide, about 500,000 cervical cancers are diagnosed annually with 250,000 deaths every year (Jenkins 2008). Consequently, the field of HPV research has witnessed many important recent advances as in the fields of epidemiology, diagnosis and prevention.

Existing therapeutic options for the alleviation of nicotine addiction have been largely ineffective at stemming the tide of tobacco use. Immunopharmacotherapy, or vaccination, is a promising, alternate therapy that is currently being explored. Results from previous studies indicate that nicotine vaccines (NVs) are effective in subjects that achieve high drug-specific antibody titers, though overall efficacy has not been observed. Consequently, improvement of these vaccines is necessary before they can achieve approval for human use. In this report, three separate approaches towards NV potentiation are explored. The first approach applied physiologically-based pharmacokinetic (PBPK) modeling to better assess NV potential. Rat and human physiological and pharmacological parameters were obtained from literature and used to construct compartmentalized models for nicotine and cotinine distribution. These models were then calibrated and validated using data obtained from literature. The final models verified the therapeutic potential of the NV concept, identified four key parameters associated with vaccine success, and established correlates for success that could be used to evaluate future NVs prior to clinical trials. In the second approach, conjugate NV scaffoldings were engineered by using wild-type (WT) and chimeric human papilloma (HPV) 16 L1 protein virus-like particles (VLPs). The chimeric protein was created by removing the last 34 C-terminal residues from the WT protein and then incorporating a multi-epitope insert that could universally target major histocompatibility complex (MHC) class II molecules. The proteins were subsequently expressed in E. coli and purified using a multi-step process. Comparisons between the separation outcomes revealed that the insert was able to modulate individual process outcomes and improve overall yield without inhibiting VLP assembly. In the third approach, commonly used carrier proteins were computationally mined for their MHC class II epitope content using human leukocyte antigen (HLA) population frequency data and MHC epitope prediction software. The most immunogenic epitopes were concatenated with interspacing cathepsin cleavage sequences and the resulting protein was re-evaluated using the earlier methods. This work represents the first ever in silico design of chimeric antigens that could potentially target all of the major HLA DQ and HLA DR allotypes found in humans.
Doctor of Philosophy
Existing treatment options for addressing nicotine addiction have been largely ineffective at preventing tobacco use. Vaccination, on the other hand, is a promising, alternate treatment option that is currently being explored. Previous studies have shown that nicotine vaccines (NVs) are effective in the subjects that respond well to the vaccine. Effectiveness in the majority of vaccine recipients, however, has not been observed. Consequently, improvement of these vaccines is necessary before they can be used in humans. In this report, three separate approaches for improving NV effectiveness are explored. The first approach applied physiologically-based pharmacokinetic (PBPK) modeling to better assess NV potential. Parameters were obtained from literature and used to construct models that could predict NV effectiveness in rats and humans. These models were then calibrated and validated using data obtained from literature. The final models verified that NVs could work if certain conditions were met, identified four key parameters associated with vaccine success, and allowed for estimation of NV efficacy prior to their evaluation in humans. In the second approach, protein carriers for conjugate NVs were constructed using the human papilloma (HPV) 16 L1 protein. This protein is known for its ability to form virus-like particles (VLPs). Both a modified and an unmodified (wild-type) protein were constructed. The modified HPV 16 L1 protein was created by replacing the last 34 C-terminal amino acids with a polypeptide insert that could enhance the immunogenicity of the vaccine. The modified and unmodified proteins were then expressed in E. coli and purified. Results indicated that the insert was able to modulate individual process outcomes and improve overall process yield without preventing VLP assembly. In the third approach, commonly used carrier proteins were computationally mined for their MHC class II epitope content using human gene frequency data and MHC epitope prediction software. The epitopes that were predicted to be the most immunogenic were linked together with interspacing protease recognition sequences and the immunogenicity of the resulting protein was re-evaluated using the prediction software. This work represents the first computational design of antigens that could potentially allow a vaccine to be effective in a large portion of human population regardless of the genetic variability.

碩士
國立臺灣大學
應用力學研究所
99
The thesis reports on an approach for detecting Odontoglossum ringspot virus (ORSV) by using micro-particle-tracking-velocimetry (micro-PTV). The ORSV is one of the major viruses in orchids, for example, Phalaenopsis, which is the most important economic plant and export good in Taiwan. To sensing the ORSV, the anti-ORSV proteins are modified on the 300 nm and 500 nm fluorescent beads and then separately mixed with the coat proteins of the ORSV and ORSV samples. The nanobeads suspended in samples were traced and statistically analyzed their Brownian velocity in real-time by micro-PTV. Through observing the variation of Brownian velocity of the nanobeads, the number of anti-ORSV and ORSV conjugation can be quantitatively estimated. Moreover, the image of the ORSV and coat proteins conjugating with nanobeads were captured through transmission electron microscope (TEM) to demonstrate the approach. In the experiment, the detection limit is exhibited to the ratio nanobeads to coat proteins of 1 to 1.5 in 8 minute. Based on our current Brownian motion measurement technique using micro-PTV, the detection limit of the concentration of 0.1 μg/ml can be found. The novel method provides higher sensitivity than enzyme-linked immunosorbent assay (ELISA) and easier operating than reverse transcription polymerase chain reaction (RT-PCR) technique, which are currently major orchid virus detection methods. Further, because of the potential that less device fabrication request, it shows higher capability on integrating with any microfluidic device for making the portable device with disposable chips.

碩士
國立臺灣大學
獸醫學研究所
104
Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals worldwide. Serum neutralization test (SN test), a gold standard for evaluating the protection rate against FMDV, is still performed for disease control in Taiwan. However, SN test is laborious, expensive and requires a high-containment biosafety lab. Based on the current study on vaccinated animals, antigenic site 2 of FMDV is the most immuno-dominant neutralizing site. We aim to establish a blocking ELISA (bELISA) based on FMD virus-like particles (VLPs) and site 2 monoclonal antibody (MAb) to detect antibodies against site 2 from vaccinated animals and replace the SN test. VLPs were expressed by eukaryotic transient expression assay with co-transfect strategy and examined by sucrose gradient centrifugation accompanied with sandwich ELISA. For mapping MAb against site 2 from 41 anti-FMDV MAbs prepared previously, we performed knock-out mutagenesis with VLPs and mVLPs (site 2 mutated) by immunofluorescence assay (IFA) and indirect ELISA. Combined with neutralization assay, results indicated that 6 MAbs recognized site 2. Based on the results of sandwich ELISA and bELISA with experimental serum, S11E-9 was regarded as the best tracer to establish bELISA with VLPs.

博士
國立臺灣大學
生物科技研究所
105
Influenza viruses impact public health due to their continual evolution. Vaccination is an effective countermeasure for influenza intervention. To prepare for possible threats caused by avian and human influenza viruses, we investigated the drift-tolerance of virus-like particles (VLPs) as an improved vaccine candidate. Prime-boost intramuscular immunization in mice with unadjuvanted H6N1-VLPs or H3N2-VLPs engineered using mammalian cells induced long-lasting neutralizing antibody responses against the homologous viruses. Both vaccines were able to confer superior cross-clade humoral immunity against the heterologous viruses belonging to distinct representative antigenic clusters in comparison with the cognate conventional whole-inactivated virus (WIV) vaccines, indicating the antibody repertoire induced by VLP vaccine formulations was insensitive to viral antigenic drift. Furthermore, the H6N1-VLPs and H3N2-VLPs also elicited significantly higher levels of anti-stalk antibodies contributing to a broadened neutralization effect. This advantageous effect was attributed to the uncleaved precursor of their HA proteins. These results uncover a mechanism for induction of wide-range immunity that better tolerates the evolutionary dynamics of influenza viruses and point to the possible use of a VLP as a next-generation vaccine candidate. Collectively, our strategy that combines favorable features of mammalian cell culture-based manufacture, VLP structure and the full-recombinant precursor HA trimer thus builds proof of concept of a new vaccine and may fit into the larger picture of universal/broadly cross-reactive vaccination approach to enhance cross-protection against the dynamically evolving influenza viruses.

Tese de mestrado. Biologia (Microbiologia Aplicada ). Universidade de Lisboa, Faculdade de Ciências, 2010
The high level of immunogenicity of peptides displayed in dense repetitive arrays on virus-like particles (VLPs) makes engineered VLPs promising vaccine carriers. Here, we describe a platform for vaccine development based on a confidential VLP named “X”. Towards this goal, X VLPs were produced by the baculovirus expression system (BEV) and examined for attachment of foreign antigenic peptides. Two distinct gene versions of the major capsid protein, viral protein 2 (VP2), were separately cloned into pFastBac1 to construct both wild type (WT) and mutant (MT) recombinant donor plasmids. These plasmids were transformed into DH10Bac E. coli competent cells for transposition and recombinant bacmids (rbacmids) were generated. The bacmids plasmid DNA were then purified and transfected into Sf9 insect cells to produce WT and MT X VLPs. X VLP VP2 expression was analyzed by SDSPAGE, X VLPs were identified by mass spectrometry and particle assembly was confirmed through transmission electron microscopy (TEM) and high performance liquid chromatography (HPLC). Both WT and MT VP2 were expressed in insect cells and shown to assemble. An original VLP’ purification method was developed in which two separate, sequential, anion exchange and size-exclusion chromatography processes were applied. In order to covalently attach small (ca. 16 aminoacids in length) antigen peptides to the outer surface of VLP, a chemical conjugation technique was employed using a heterobifunctional chemical linker succinimidyl-6-[(ß-maleimidopropionamido)hexanoate] (SMPH). VLPs were purified and successfully conjugated to antigenic peptides. Antibody sera obtained from murine immunization against Y001-conjugated to an alternative VLP platform, showed antigenicity recognising the same antigenic peptide, Y001, covalently linked to X VLP. In this work, we explored whether X VLP expression, purification and bioconjugation may be used in a future biotherapeutic application. Together, these results suggest X VLP can potentially be exploited as an antigen carrier for the development of new VLP-based vaccines.
Os vírus constituem excelentes modelos à nanotecnologia representando por excelência plataformas de interacção comuns a todos os sistemas biológicos. Uma das suas aplicações mais relevantes assenta na utilização única do seu invólucro exterior, formando partículas do tipo viral semelhantes aos vírus de que derivam, normalmente de constituição proteica e sem potencial patogénico. As partículas do tipo viral são já actualmente usadas como vacinas. Neste trabalho, descrevemos o desenvolvimento de uma partícula do tipo viral, composta a partir de um vírus X (confidencial), e a sua possível aplicação como partícula apresentadora de antigénios, vacina modificada. Os processos de expressão, purificação e transformação final por bioconjugação dos X VLPs (do inglês, virus-like particles) são analisados neste trabalho. Contextualização teórica Algumas das aplicações virais mais importantes sustentam-se no facto dos vírus serem entidades dinâmicas e de metastabilidade, propriedades estas que são conferidas, em parte, pelo seu invólucro ou cápside externa de arquitecturas homogéneas e simétricas. De facto, a manipulação química e genética das superfícies externas dos VLPs torna possível a inclusão de novas funcionalidades à arquitectura proteica da partícula e às propriedades que lhes são inerentes. Assim, o princípio que demonstra a possibilidade de modificação de grupos reactivos e ligação de péptidos às superfícies exteriores das cápsides virais foi já provado e desenvolvido em diferentes estudos. Os blocos estruturais virais de construção, as subunidades ou monómeros constituintes, permitem, com relativa facilidade, tanto modificações químicas como genéticas (e.g. bioconjugação química). A modificação de resíduos de aminoácidos, ou outros grupos reactivos ou ligandos, em pontos específicos das partículas do tipo viral, têm sido utilizados para ligações sítio-específicas de pequenas moléculas, incluindo nanopartículas de ouro, fluoróforos, hidratos de carbono, ácidos nucleicos e péptidos. Desta forma, a cápside é o local primário de interface exterior viral que define muitas das suas propriedades moleculares, e de interacção imunológicas, útil a diferentes aplicações baseadas em VLPs para vacinas ou imunoterapêuticos. Partículas do tipo viral Os VLPs são compostos, pelo menos, por uma proteína viral e resultam da montagem (assembly) das diferentes subunidades, em ordem repetitiva, em estruturas geralmente icosaédricas ou bacilares. Estas partículas, mesmo não possuindo qualquer potencial patogénico e não comportando em si qualquer informação genética com capacidade replicativa, conseguem induzir respostas imunitárias potentes, devido ao facto de se assemelharem a uma partícula viral infecciosa e de apresentarem proteínas com a mesma conformação que um vírus infeccioso. Assim sendo, os VLPs podem activar tanto uma resposta imunitária (i) à produção de anticorpos neutralizantes, de suporte a uma acção memória profiláctica, (ii) como uma resposta de efeito terapêutico, associada a acção citotóxica e predominantemente dependente de linfócitos T. Muitos VLPs têm sido adquiridos a partir de vários vírus, desde vírus animais e de plantas, bacteriófagos, Ty retrotransposões de levedura e podem ser expressos em diferentes modelos hospedeiros incluindo bactérias, leveduras, plantas e células de insecto e mamífero. VLPs como plataformas de apresentação antigénica Os VLPs em vacinação são geralmente explorados como partículas subvirais naturais, ou seja, são empregues para despoletar uma resposta imunitária contra as proteínas que constituem o próprio vírus de que derivam. Contudo, estes podem igualmente ser usados como plataformas de apresentação antigénica, levando ao desenvolvimento de respostas imunitárias contra qualquer antigénio alvo apresentado, associado a diferentes alvos terapêuticos ou agentes infecciosos. Os VLPs podem ser empregues como veículos de apresentação antigénica de epítopos tanto para as células B como T. Diferentes métodos podem ser utilizados no processo de apresentação de antigénios à superfície de VLPs, sendo os mais comuns a ligação química covalente ou fusão genética. A fusão genética é normalmente útil para pequenos epitopos péptidicos, enquanto que a conjugação covalente química é geralmente usada para antigénios maiores, em particular pequenas proteínas. Por exemplo, VLPs derivados do bacteriófago Qβ conjugados com nicotina, ou angiotensina II, foram já desenvolvidos por bioconjugação química. Por fusão genética, assembled vírus do papiloma bovino, VLPs quimeras estáveis, foram já modificados para a apresentação de epítopos do vírus do papiloma humano. Recentemente tem sido demonstrado que uma das formas mais eficientes de carregar e conjugar um VLP com antigénios estranhos para exposição ao sistema imunitário é através de ligação química covalente. Essa ligação é feita com o uso de ligandos heterobifuncionais, contendo duas extremidades funcionais diferentes. Um lado do ligando liga-se ao VLP e o outro ao antigénio de interesse. Como exemplo, grupos amina de resíduos de lisinas, expostos à superfície de VLPs existentes no bacteriófago Qβ, são primariamente reactivos com um ester amina reactivo de Nhydroxysuccinimide (NHS) do ligando heterobifunctional succinimidyl-6-((β-maleimidopropionamido) hexonoate)) (SMPH) preservando a actividade do seu grupo maleimida, sulfidril reactivo, mantendose assim livre para se ligar a um outro antigénio, sulfidril reactivo, por exemplo, um alvo cisteína presente numa pequena sequência péptidica. Para assegurar que o antigénio está acoplado directamente ao VLP e que se apresenta numa forma ordenada, os antigénios peptídicos podem ser modificados para conter tanto um grupo terminal amina como um grupo terminal carboxilo na sua sequência contendo um grupo livre de resíduo de cisteína. De forma semelhante, os VLPs provenientes de qualquer outra origem podem ser mutados para incorporar residuos de aminoácidos reactivos para conjugação. Objectivos: Este trabalho teve como objectivo a expressão e produção de X VLPs, a sua purificação e posterior modificação da interface externa para conjugação de péptidos de potencial antigénico, ligados covalentemente à superfície externa do VLP e expostos ao exterior para reconhecimento imunológico. Material e Métodos: Duas versões distintas da proteína viral 2 (VP2) do vírus X, foram separadamente clonadas em pFastBac1 para construir tanto a versão selvagem, WT (do inglês, wild type) como uma versão modificada, MT (do inglês, mutant) de X VLPs. Estes plasmídeos foram transformados numa estirpe competente DH10Bac de E. coli para transposição e formação de bacmids recombinantes. O ADN plasmídico dos rbacmids purificados foi transfectado para células de insecto Sf9 para a expressão de duas versões distintas de X VLPs. A expressão de VP2 foi analisada por SDS-PAGE, os VLPs foram identificados por espectrometria de massa e o seu assembly confirmado através de microscopia electrónica de transmissão e cromatografia líquida de alta performance. Um método original de purificação de X VLPs foi desenvolvido no qual dois processos cromatográficos sequenciais de cromatografia de troca iónica e de exclusão molecular foram empregues. Para a ligação covalente, conjugação de X VLPs com péptidos antigénicos de 16 aminoácidos, uma técnica de bioconjugação química foi usada com a utilização do ligando heterobifuncional succinimidyl-6-[(ß-maleimidopropionamido)hexanoate] (SMPH). Resultados: As duas versões de X VLPs, WT e MT, foram produzidas com sucesso em células de insecto. X VLPs foram purificados e conjugados com o péptido antigénico desejado. Soro obtido a partir da imunização com um outro tipo de VLPs conjugados, mas para o mesmo péptido antigénico, continham anticorpos reactivos para ambas as versões conjugadas de X VLPs, evidenciando o êxito da conjugação, (ligação dos péptidos à superfície externa dos VLPs). Conclusão: Neste trabalho é pela primeira vez descrito um método original para a purificação de X VLPs e é demonstrada a capacidade de ligação, à superfície externa dos X VLPs, de pequenos péptidos antigénicos de 16 aminoácidos de comprimento, por ligações covalentes. Assim, estes resultados sugerem que os X VLPs podem ser explorados como veículos de apresentação antigénica e demonstram potencial para diferentes abordagens terapêuticas para reconhecimento ao sistema imunológico.

碩士
國立宜蘭大學
生物技術與動物科學系
103
Progressive atrophic rhinitis （PAR） is an important upper respiratory disease in swine, clinical symptoms include atrophy of the nasal turbinate bones, deformation of the snout, and growth retardation. PAR is mainly caused by Bodetella bronchiseptica（B. bronchiseptica）and Pasteurella multocida（P. multocida）colonization. The P. multocida toxin（PMT）, isolated from P. multocida, has been shown to be the central etiological agent of this disease. In 1991, Peterson et al. has developed deletion derivatives of recombinant PMT（rPMT）which showed little toxicity but induced effective protection against P. multocida infection. Therefore, PMT has been considered as a good candidate for subunit vaccine development. A novel PMT fragments fused with bio-adjuvants was constructed and was highly expressed in E. coli expression system. We generated the mouse polyclonal anti-PMT and anti-PMT recombinant subunit protein antibodies to identify the antigenicity and immunogenicity of the PMT subunit protein. Results from dot blots revealed that anti-PMT subunit antibody reacted with full-length PMT protein and vice versa, which implies that the PMT subunit protein is both antigenic and immunogenic. In this study, we have developed a novel PAR subunit vaccine which is composed of inactivated P. multocida, B. bronchiseptica, PMT subunit protein and bio-adjuvant. The safety results of the vaccine revealed that no adverse skin reaction and weight loss observed in mice. The survival rate of mice was increased after toxin challenged. To further investigate the immunogenicity of PAR subunit vaccine, the enzyme-linked immunosorbent assay was carried out. Mice immunized with the PAR subunit vaccine showed higher antibody titer. Also, the neutralizing antibodies of PMT and agglutination antibodies of B. bronchiseptica showed higher antibody titer. From the above results, it is suggested that this novel PAR subunit vaccine has its potential in progressive atrophic rhinitis prevention.

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.